U.S. patent application number 17/478016 was filed with the patent office on 2022-05-12 for reversible modification of nucleotides.
The applicant listed for this patent is Pacific Biosciences of California, Inc.. Invention is credited to Aaron W. Feldman, Denis Malyshev.
Application Number | 20220144875 17/478016 |
Document ID | / |
Family ID | 1000006093748 |
Filed Date | 2022-05-12 |
United States Patent
Application |
20220144875 |
Kind Code |
A1 |
Malyshev; Denis ; et
al. |
May 12, 2022 |
REVERSIBLE MODIFICATION OF NUCLEOTIDES
Abstract
Disclosed herein, inter alia, are methods for modifying a
nucleotide, for example including reacting a nucleotide having a
3'-O-oxime moiety such as ##STR00001## with a reagent having an
--ONH.sub.2 moiety to produce a nucleotide having a 3'-O--NH.sub.2
moiety such as ##STR00002## wherein the reagent having the
--ONH.sub.2 moiety further comprises alkyl, alkenyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, or (heteroalicyclyl)alkyl.
Inventors: |
Malyshev; Denis; (San Diego,
CA) ; Feldman; Aaron W.; (San Diego, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Pacific Biosciences of California, Inc. |
Menlo Park |
CA |
US |
|
|
Family ID: |
1000006093748 |
Appl. No.: |
17/478016 |
Filed: |
September 17, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17016992 |
Sep 10, 2020 |
11180520 |
|
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17478016 |
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62898152 |
Sep 10, 2019 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07H 1/00 20130101; C07H
19/10 20130101 |
International
Class: |
C07H 1/00 20060101
C07H001/00; C07H 19/10 20060101 C07H019/10 |
Claims
1.-30. (canceled)
31. A method for modifying a nucleotide, comprising reacting a
nucleotide comprising a 3'-O-oxime moiety with a reagent to produce
a nucleotide comprising a 3'-O--NH.sub.2 moiety, wherein the
nucleotide that comprises the 3'-O-oxime moiety is: ##STR00160##
wherein the nucleotide that comprises the 3'-O--NH.sub.2 moiety is:
##STR00161## wherein B is a nucleobase; R1 is halogen, OCH.sub.3, H
or OH; Q is monophosphate, diphosphate, or triphosphate; and R3 and
R4 are H, CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, or (heteroalicyclyl)alkyl, wherein either
R3 or R4, or both R3 and R4 are not H; and; wherein the reagent is
an alkoxyamine of formula R2-ONH.sub.2, wherein R2 is independently
alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,
aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl,
(heteroalicyclyl)alkyl, or an optionally substituted variant
thereof, wherein R2 has a molecular weight greater than 36
g/mol.
32. The method of claim 31, wherein R3 is CH.sub.3.
33. The method of claim 31, wherein R4 is CH.sub.3.
34. The method of claim 31, wherein Q is triphosphate.
35. The method of claim 31, wherein the reagent is:
##STR00162##
36. The method of claim 35, wherein the reagent is:
##STR00163##
37. The method of claim 35, wherein the reagent is:
##STR00164##
38. The method of claim 31, wherein R2 has a molecular weight
greater than 57 g/mol.
39. The method of claim 38, wherein R2 has a molecular weight
greater than 100 g/mol.
40. The method of claim 38, wherein R2 has a molecular weight
greater than 200 g/mol.
41. The method of claim 31, wherein R2 comprises a hydrocarbon
ring.
42. The method of claim 31, wherein R2 comprises a heterocyclic
ring.
43. The method of claim 31, wherein no more than 5% of the
nucleotide is degraded.
44. The method of claim 31, wherein no more than 3% of the
nucleotide is degraded.
45. The method of claim 31, wherein no more than 2% or no more than
1% of the nucleotide is degraded.
46. The method of claim 31, wherein no more than 3% of the
nucleotide is degraded when the nucleotide comprises a cytosine
nucleobase.
47. The method of claim 31, wherein the degradation of the
nucleotide is less than half the degradation that would be obtained
if R2 was a methyl group.
48. The method of claim 31 or 43, wherein at least 95% of the
nucleotide comprising a 3'-O-oxime moiety is consumed.
49. The method of claim 31, wherein at least 97% or at least 98% of
the nucleotide comprising a 3'-O-oxime moiety is consumed.
50. The method of claim 49, wherein the method adds a blocking
group in place of a 3' protecting group to at least 98% of a
population of nucleotides having a 3' protecting group in no more
than 3 hours.
51. The method of claim 31, wherein R2-ONH.sub.2 has a formula
selected from the group consisting of the following: ##STR00165##
Description
CROSS-REFERENCES TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 17/016,992, filed Sep. 10, 2020, which claims
the benefit of U.S. Provisional Application No. 62/898,152, filed
Sep. 10, 2019, which are incorporated herein by reference in their
entireties and for all purposes.
BACKGROUND
[0002] The present disclosure relates generally to reversible
blocking of nucleotides and nucleic acids to inhibit enzyme
catalyzed modification of their 3' hydroxyl moieties, and has
specific applicability to nucleic acid sequencing processes that
employ cyclic reversible termination.
[0003] Genomes have been analogized as operating manuals containing
instructions for development and functioning of individual
organisms. Genomes have been shown to guide growth and development,
direct the function of vital organs, influence mood and behavior,
etc. The uniqueness of every individual is derived in significant
part from the unique composition of their genome. The differences
between individuals can be observed as differences in the sequence
of nucleotides in the DNA that constitutes their genome. Great
strides have been made in identifying how those sequence
differences predict risk of disease, chances of having adverse
reactions to certain therapeutic drugs, and chances of having
certain abilities and personality traits, to name a few.
[0004] Recently, the diagnostic potential for genomic information
has accelerated due to commercialization of DNA sequencing
platforms that are capable of sequencing at the scale of the
typical human genome, which has a sequence of 3 billion
nucleotides. Although the throughput and cost for sequencing have
improved vastly over the last few decades, there are still many
diseases for which diagnostic sequence signatures have not been
identified and the cost of sequencing is still out of reach for
many clinical applications.
[0005] Many sequencing technologies that have been developed,
including several that are in wide commercial use, employ a cyclic
reversible terminator (CRT) process. The CRT process extends a
primer along a DNA template of interest such that each cycle
results in detection of a single position in the template. The
cycles are repeated to effectively walk down the template in DNA
while reading the sequence in single nucleotide steps. Performing
100 CRT cycles produces a sequence read of 100 nucleotides. Single
nucleotide step size, and as a result single nucleotide resolution
for the resulting sequence read, is achieved by incrementing the
primer with a nucleotide having a 3' reversible terminator moiety.
The primer, after adding the reversibly terminated nucleotide,
cannot be further extended until it is treated with an appropriate
deblocking reagent to remove the reversible terminator moiety. The
accuracy and read length of most CRT processes, including those
that are in commercial use today, are known to be compromised due
to inefficiencies in the steps that incorporate reversible
terminators into primers and in the steps used for deblocking the
primers. Moreover, many CRT processes use deblocking reagents that
can damage the very DNA that is to be sequenced. Because sequencing
is a cumulative process, even small inefficiencies in primer
extension, primer deblocking and resistance to DNA damage can add
up to a substantial adverse impact over the many cycles of a
sequencing process.
[0006] Thus, there exists a need for improved reagents and methods
for reversible termination of nucleotides and nucleic acids. The
present invention satisfies this need and provides related
advantages as well.
BRIEF SUMMARY
[0007] The present disclosure provides a composition that includes
solution containing a nucleotide having a protecting group such as
a 3'-O-oxime moiety and a reagent to remove the protecting group
and deliver to the protecting group's position a blocking group,
such as a reagent having the structure R2-ONH.sub.2. A number of R2
moieties are consistent with the present disclosure. In some cases,
R2 is independently alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally
substituted variant thereof. Alternately or in combination, R2
comprises a moiety having a molecular weight of at least 24, at
least 25, at least 26, at least 27, at least 28, at least 29, at
least 30, at least 31, at least 32, at least 33, at least 34, at
least 35, at least 36, or greater than 36 g/mol. Similarly, an R2
moiety as contemplated herein has a molecular weight of no more
than 1000, no more than 500, no more than 400, no more than 300, no
more than 250, no more than 200, no more than 150, or no more than
100 g/mol. Alternately or in combination, various R2 embodiments
comprise one or more of the following: at least two carbon nuclei,
fluorine nucleus, a phosphorous nucleus, a nitrogen nucleus, a
silicon nucleus, an oxygen nucleus, a hydroxy moiety, heterocyclic
ring, a phenyl ring, or other moiety, alone or in combination with
one another.
[0008] In particular configurations, a representative nucleotide
starting material for methods or compositions herein has the
structure:
##STR00003##
[0009] B can be a nucleobase. R1 can independently be a halogen,
OCH.sub.3, H or OH, or may carry an alternate modification at the
2' R1 position, such that the nucleotide is variously a
ribonucleotide or a deoxyribonucleotide. Q can be independently
monophosphate, diphosphate, triphosphate, nucleic acid, hydrogen,
or other moiety, such as a surface or linker bonded to a surface.
R3 and R4 can be independently H, CH.sub.3, alkyl, alkenyl,
alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,
heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or
an optionally substituted variant thereof. By way of example, the
protecting group moiety at the 3' position of the nucleotide can be
an aldoxime (such that one of R3 or R4 is hydrogen) or a ketoxime
(wherein neither R3 nor R4 is hydrogen). Other protecting group
moieties are contemplated herein and consistent with the present
disclosure.
[0010] The present disclosure also provides methods for modifying a
nucleotide. The methods can be carried out by reacting a nucleotide
having a protecting group such as a 3'-O-oxime moiety with a
reagent to remove the protecting group and deliver to the
protecting group's position a blocking group, such as a reagent
having the structure R2-ONH.sub.2 to produce a nucleotide having a
3'-O--NH.sub.2 moiety, wherein R2 is independently R2 is
--C(R6)(R7)(R8), --C(O)(R6), --P(O)(R6)(R7),
--C(R6)(.dbd.C(R7)(R8)), --Si(R6)(R7)(R8), --S O.sub.2(R6),
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C5-C6), substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6), substituted or unsubstituted cycloalkynyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6), substituted
or unsubstituted heteroalicyclyl (e.g., 3 to 8 membered, 3 to 6
membered, or 5 to 6 membered), substituted or unsubstituted aryl
(e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl), or
substituted or unsubstituted heteroaryl (e.g., 5 to 10 membered
heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered
heteroaryl). In some cases R2 is independently alkyl, alkenyl,
alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,
heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or
an optionally substituted variant thereof. R2 moieties may comprise
a number of constituents, alone or in combination, to covey the
benefits disclosed herein. Some R2 moieties variously comprise at
least 2, at least 3, at least 4, at least 5, at least 6, or more
than 6 carbon nuclei. Similarly, some R2 comprise at least one of
the following: at least 1 nitrogen nucleus, at least one silicon
nucleus, at least one phosphorous nucleus, at least one fluorine
nucleus or at least one other constituent. R2 moieties are in some
cases partially or fully fluorinated, or partially or fully
hydrogenated. In some cases an R2 moiety comprises at least one
component to facilitate solubility. Reagents having structures to
deliver alternate blocking groups or reversible terminator
moieties, such as ONHCH.sub.3, O--CH.sub.2N.sub.3, or other moiety
such as those consistent with reversible termination of nucleic
acid extension, are also contemplated herein. In some
configurations, the nucleotide that has the 3'-O-oxime moiety can
have the structure:
##STR00004##
and the nucleotide that has the 3'-O--NH.sub.2 moiety can have the
structure:
##STR00005##
[0011] B can be a nucleobase. R1 can independently be a halogen,
OCH.sub.3, H or OH. Q can be independently monophosphate,
diphosphate, triphosphate, nucleic acid, or other moiety, such as a
surface or linker bonded to a surface. R3 and R4 can be
independently H, CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally
substituted variant thereof. By way of example, the moiety at the
3' position of the nucleotide can be an aldoxime (such that one of
R3 or R4 is hydrogen) or a ketoxime (neither of R3 or R4 is
hydrogen). In particular configurations, a nucleotide starting
material for methods or compositions herein has the structure:
##STR00006##
B can be a nucleobase. R1 can independently be a halogen,
OCH.sub.3, H or OH. Q can be independently monophosphate,
diphosphate, triphosphate, nucleic acid, or other moiety, such as a
surface or linker bonded to a surface. R3 and R4 can be
independently H, CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally
substituted variant thereof. By way of example, the protecting
group moiety at the 3' position of the nucleotide can be an
aldoxime (such that one of R3 or R4 is hydrogen) or a ketoxime
(wherein neither R3 nor R4 is hydrogen). Other protecting group
moieties are contemplated herein and consistent with the present
disclosure. The present disclosure also provides methods for
modifying a nucleotide. The methods can be carried out by reacting
a nucleotide having a protecting group such as a 3'-O-oxime moiety
with a reagent to deliver a blocking group, such as a reagent
having the structure R2-ONH.sub.2 to produce a nucleotide having a
3'-O--NH.sub.2 moiety, wherein R2 is independently alkyl, alkenyl,
alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,
heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or
an optionally substituted variant thereof. Reagents having
structures to deliver alternate blocking groups, such as
ONHCH.sub.3, O--CH.sub.2N.sub.3, or other moiety consistent with
reversible termination of nucleic acid extension, are also
contemplated herein.
[0012] The present disclosure further provides a kit that includes
a first vessel containing a nucleotide having a 3'-O-oxime moiety
and a second vessel containing a reagent having the structure
R2-ONH.sub.2, wherein R2 is independently alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,
heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or
an optionally substituted variant thereof. In some configurations,
the nucleotide can have the structure:
##STR00007##
[0013] B can be a nucleobase. R1 can independently be a halogen,
OCH.sub.3, H or OH. Q can be independently monophosphate,
diphosphate, triphosphate, nucleic acid, or other moiety, such as a
surface or linker bonded to a surface. R3 and R4 can be
independently H, CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally
substituted variant thereof. By way of example, the moiety at the
3' position of the nucleotide can be an aldoxime (such that one of
R3 or R4 is hydrogen) or a ketoxime (neither of R3 or R4 is
hydrogen).
BRIEF DESCRIPTION OF THE DRAWING
[0014] FIG. 1A shows a plot of percent nucleotide degradation vs.
percent consumption of the 3'-O--(N-acetone oxime) moiety for
3'-O--(N-acetone oxime)-dCTP when treated with various candidate
DRT (`deblocking reversible terminator`) aminooxy reagents sharing
an --O--NH.sub.2 reversible terminator moiety tethered to various
R2 groups. Reactions were performed as described in Example 1 and
assayed after 1 hour for a 25 C reaction. FIG. 1A illustrates that
as R2 increases in molecular weight, one sees a decrease in dC
degradation. Similarly, as R2 increases in molecular weight, one
sees an increase in starting material consumption. FIG. 1B lists
the chemical identities of the DRT reagents assayed in FIG. 1A,
indicating their various R2 moieties.
[0015] FIG. 2 presents examples of deblocking reagents. All
examples in this figure share a common --O-- NH.sub.2 reversible
terminator moiety (at right in each molecule as presented) tethered
to various R2 groups (at left in each molecule as presented)
ranging in molecular weight and chemical structure. Similar
deblocking reagents having R2 groups as disclosed herein tethered
to alternate reversible terminator moieties, such as
--O--NHCH.sub.3, --O--CH.sub.2N.sub.3, or other moiety such as
those consistent with reversible termination of nucleic acid
extension, are also contemplated herein in combination with the R2
moieties presented here or otherwise disclosed herein.
[0016] FIG. 3. Example of a deblocking side reaction with cytidine.
This side reaction accounts for at least some of the dC degradation
quantified in FIG. 1A. As indicated in FIG. 1A, with increasing R2
size one sees an decrease in the side reaction depicted herein.
DETAILED DESCRIPTION
[0017] The present disclosure provides compositions and methods
that are useful for synthesis of nucleotides having moieties at the
3' position, such as moieties that protect the 3' position from
modifications such as chemical modifications and enzymatic
modifications, such as addition of a nucleotide as effected by
template directed DNA polymerase enzymes. A nucleotide that is
protected in this way can be referred to as being blocked or
terminated. For example, when a nucleotide having a protected 3'
position is incorporated into an extending nucleic acid strand as
the 3' nucleotide moiety of a nucleic acid primer, the extended
primer is effectively blocked from being further extended by a
polymerase or ligase. In other words, primer extension has been
terminated.
[0018] In particular configurations or embodiments, a blocked or
terminated nucleotide can have a reversible terminator moiety such
as a 3'-ONH.sub.2 moiety that, unlike the native hydroxyl found at
the 3' position of deoxyribonucleotides and ribonucleotides, is
resistant to modification by certain enzymes and chemical reagents.
Under certain conditions the 3'-ONH.sub.2 moiety can be chemically
converted to a hydroxyl, for example when the blocked nucleotide is
treated with an acid such as nitrous acid. Replacement of the
--O--NH.sub.2 moiety as a whole with an --OH moiety or replacement
of the --NH.sub.2 moiety with an --H both effectively result in
deblocking, and the fate of the 3' O in deblocking is immaterial,
so long as the final product has a 3' OH available for
polymerase-mediated nucleic acid extension. As such the
3'-ONH.sub.2 moiety functions as a reversible terminator when
coupled with the deblocking reagents of the present disclosure.
Other reversible terminators that are readily removed to recover a
3' hydroxy moiety compatible with enzymatic strand extension are
consistent with the disclosure herein, such that not all
embodiments herein are limited to the preparation of a nucleotide
having a 3'-O--NH.sub.2 moiety. Exemplary alternatives include
include 3'-O--NHCH.sub.3, --O--CH.sub.2N.sub.3, and others known in
the art that block strand extension when attached at the 3'
position of the 3' end of a nucleic acid strand, while at the same
time do not preclude binding by a DNA or other nucleic acid
polymerase such that nucleotides carrying such reversible
terminators may be added, at their 5' ends, to the 3' hydroxy
groups of unblocked nucleotides or nucleotide polymers to produce
3' terminated nucleic acid strands.
[0019] Reversible termination of nucleotides are useful for a
variety of applications. For example, a variety of nucleic acid
sequencing platforms utilize a cyclic reversible terminator (CRT)
process in which the sequence of a template nucleic acid is read
while incrementally extending a primer nucleic acid along the
template. Incremental extension can be achieved in CRT processes by
structuring each cycle to include a step of extending the primer
through addition of a reversibly terminated nucleotide (or
oligonucleotide reversibly terminated at its 3' end) followed by
deblocking the extended primer. In each cycle, a detection step is
also carried out in order to identify the type of nucleotide that
is present at a particular position that has been aligned with
respect to the extended primer.
[0020] The reversibly terminated nucleotides, and methods for their
synthesis, set forth herein provide advantages of very efficiently
replacing 3' protecting groups such as 3'-O-oximes or other groups
added to facilitate the chemical manipulation of other regions of a
nucleic acid, such as addition of a triphosphate moiety to a
nucleotide 5' position. Without being bound by theory, it is
predicted that because CRT is a generally cumulative process, any
errors or inefficiencies in blocking and deblocking steps will
produce a cumulatively adverse effect. For example, phasing is the
phenomenon whereby individual nucleic acid molecules within a
population lose sync with each other during the course of a CRT
process, for example due to a defect in removal of a blocking
group, leading to failure to incorporate an additional moiety
during one or more cycles of a CRT process. Phasing can manifest as
extension of one or more primer molecules `falling behind` the
others in the population, or alternately as a defect leading to one
or more primer molecules `going ahead` of others in the population.
A phasing rate of, for example, only 0.5% per cycle may result in
an accumulated loss of roughly half of true signal for a sequencing
or other CRT reaction after 120 cycles. The problem is exacerbated
by the proportional increase in background noise due to false
signals that arise from members of the population that are out of
phase. The accumulated loss in signal-to-noise results in a
limitation on read length (which, in turn, results in reduced
sequencing throughput) and increased errors especially for later
cycles as noise overwhelms signal. The reversibly terminated
nucleotides set forth herein can be used to inhibit disruptions in
extension phase, thereby improving accuracy and read length
reactions such as in nucleic acid sequencing processes. Through
practice of the methods and use of the compositions disclosed
herein, nucleotide compositions are generated having a very high
degree of replacement of a protecting group such as --O-oxime by a
reversible group such as an --O--NH.sub.2, with a low rate of dC
degradation, as depicted in FIG. 1A and as is applicable generally
to the R2 moieties disclosed herein. Consequently, reagents
generated using the compositions herein or through the methods
disclosed herein are in some cases suitable for addition into a
nucleic acid sequencing reagent, such as direct addition without
further clean-up or purification, to produce a sequencing reaction
run having a high degree of accuracy over a substantial length of a
run, as depicted in Example 2, below. Such a reagent may further
comprise a polymerase surfactant, buffer or other reagent suitable
for direct addition into a sequencing device. Without being bound
to theory, the performance of a run such as that presenteded in
Example 2 is at least in part due to decreases in phasing defects
which may otherwise result from inefficient replacement of a
protecting group such as an --O-oxime group with a reversible
terminator such as --O--NH.sub.2, resulting in either retained
protecting groups, unblocked 3' OH moieties, or degraded dC bases
along the lines of those presented in FIG. 3.
[0021] Without being bound by theory, one possible advantage of the
synthetic methods and compositions set forth herein for blocking
the 3' position of a nucleotide is that the methods are relatively
inert to unwanted side reactions with other moieties of the
nucleotide. Accordingly, reversibly terminated nucleotides that are
made by methods or involving the compositions set forth herein may
not be substantially destroyed or modified in ways that carry over
into adverse effects during blocking and deblocking steps that
occur during a CRT process or other biochemical process that
utilizes the reversibly terminated nucleotides. In various cases,
no more than 6%, no more than 5%, no more than 4%, no more than 3%,
no more than 2%, no more than 1% or less than 1% of the
nucleotides, such as cytosine nucleotides, used in a reaction or
represented in a composition herein are modified or degraded
through the practice of the disclosure. Accordingly, the methods
and compositions set forth herein provide improved sequencing
accuracy by providing a sequencing starting material that is
relatively free of degraded or modified bases such as degraded or
modified cytosine that may interfere with the accuracy, phase or
read length of a sequencing reaction. Similarly, the methods and
compositions set forth herein facilitate the production of nucleic
acid sequencing reagents by generating reversibly terminated
nucleotides of a purity that can be, in some cases, directly added
to a sequencing reagent without further purification or
isolation.
[0022] Accordingly, compositions as disclosed herein or produced
through methods disclosed herein facilitate accurate sequencing by
being or by generating reversibly terminated nucleotides that are
relatively clear of degraded or modified bases such as degraded or
modified cytosine bases, while being relatively clear of retained
3' protecting groups such as --O-oximes, and having a relatively
low concentration of unblocked 3' OH groups. These features, alone
or in combination, facilitate sequencing accuracy, phasing or read
length of a sequencing reaction. In addition, the higher efficiency
of methods and purity of compositions resulting from these methods
facilitates more cost-effective sequencing because the compositions
require less purification prior to their employment in sequencing
reactions, and are in some cases added directly into a sequencing
reagent or kit without further isolation or purification.
Definitions
[0023] Terms used herein will be understood to take on their
ordinary meaning in the relevant art unless specified otherwise.
Several terms used herein and their meanings are set forth
below.
[0024] Where substituent groups are specified by their conventional
chemical formulae, written from left to right, they equally
encompass the chemically identical substituents that would result
from writing the structure from right to left, e.g., --CH.sub.2O--
is equivalent to --OCH.sub.2--. Where a substituent group or moiety
is written with internal bonds depicted, it will be understood that
the substituent group or moiety may also be depicted without
internal bonds explicitly drawn and both representation will be
understood to be the same substituent group or moiety, for example
--ONH.sub.2 and --O--NH.sub.2 are the same group.
[0025] As used herein, "alkenyl" refers to an alkyl that contains
in the straight or branched hydrocarbon chain one or more double
bonds. An alkenyl moiety may be unsubstituted or substituted. An
alkenyl moiety can, optionally, have a number of carbon atoms that
is in a range exemplified herein for alkyl moieties.
[0026] As used herein, "alkoxy" refers to the formula --OR wherein
R is an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl
or a cycloalkynyl. A non-limiting list of alkoxys is methoxy,
ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy,
iso-butoxy, sec-butoxy, or tert-butoxy. An alkoxy moiety may be
substituted or unsubstituted.
[0027] As used herein, "alkoxyamine" refers to the formula
R'--ONR.sub.2 wherein R' is an alkyl, an alkenyl, an alkynyl, a
cycloalkyl, a cycloalkenyl or a cycloalkynyl. A non-limiting list
of alkoxys is methoxy, ethoxy, n-propoxy, 1-methylethoxy
(isopropoxy), n-butoxy, iso-butoxy, sec-butoxy, or tert-butoxy,
wherein R' is not hydrogen, and wherein one or both R groups that
are bonded to nitrogen is independently a hydrogen, an alkyl, an
alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl or a
cycloalkynyl. A non-limiting list of alkoxys is methoxy, ethoxy,
n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, iso-butoxy,
sec-butoxy, or tert-butoxy. An alkoxyamine moiety may be
substituted or unsubstituted.
[0028] As used herein, "alkyl" refers to a straight or branched
hydrocarbon chain that comprises a fully saturated (no double or
triple bonds) hydrocarbon moiety. Alkyl is an uncyclized chain. In
some embodiments, the alkyl moiety may have 1 to 20 carbon atoms
(whenever it appears herein, a numerical range such as "1 to 20"
refers to each integer in the given range inclusive of the
endpoints; e.g., "1 to 20 carbon atoms" means that the alkyl moiety
may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc.,
up to and including 20 carbon atoms, although the present
definition also covers the occurrence of the term "alkyl" where no
numerical range is designated). The alkyl moiety may also be a
medium size alkyl having about 7 to about 10 carbon atoms. The
alkyl moiety can also be a lower alkyl having 1 to 6 carbon atoms.
The alkyl moiety of the compounds may be designated as
"C.sub.1-C.sub.4 alkyl" or similar designations. By way of example
only, "C.sub.1-C.sub.4 alkyl" indicates that there are one to four
carbon atoms in the alkyl chain, i.e., the alkyl chain is selected
from methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl,
sec-butyl, and t-butyl. Typical alkyl moieties include, but are in
no way limited to, methyl, ethyl, propyl, isopropyl, butyl,
isobutyl, tertiary butyl, pentyl, and hexyls. The alkyl moiety may
be substituted or unsubstituted. An alkyl moiety having at least
two carbon atoms can be particularly useful, for example, when a
methyl moiety is less effective than a longer chain alkyl
moiety.
[0029] As used herein, "alkynyl" refers to an alkyl that contains
in the straight or branched hydrocarbon chain one or more triple
bonds. An alkynyl moiety may be unsubstituted or substituted. An
alkynyl moiety can, optionally, have a number of carbon atoms that
is in a range exemplified herein for alkyl moieties.
[0030] The term "alkylene," by itself or as part of another
substituent, means, unless otherwise stated, a divalent radical
derived from an alkyl, as exemplified, but not limited by,
--CH.sub.2CH.sub.2CH.sub.2CH.sub.2--. Typically, an alkyl (or
alkylene) group will have from 1 to 24 carbon atoms, with those
groups having 10 or fewer carbon atoms being preferred herein. A
"lower alkyl" or "lower alkylene" is a shorter chain alkyl or
alkylene group, generally having eight or fewer carbon atoms. The
term "alkenylene," by itself or as part of another substituent,
means, unless otherwise stated, a divalent radical derived from an
alkene. The term "alkynylene" by itself or as part of another
substituent, means, unless otherwise stated, a divalent radical
derived from an alkyne.
[0031] The term "heteroalkyl," by itself or in combination with
another term, means, unless otherwise stated, a stable straight or
branched chain, or combinations thereof, including at least one
carbon atom and at least one heteroatom (e.g., O, N, P, Si, and S),
and wherein the nitrogen and sulfur atoms may optionally be
oxidized, and the nitrogen heteroatom may optionally be
quaternized. The heteroatom(s) (e.g., O, N, S, Si, or P) may be
placed at any interior position of the heteroalkyl group or at the
position at which the alkyl group is attached to the remainder of
the molecule. Heteroalkyl is an uncyclized chain. Examples include,
but are not limited to: --CH.sub.2--CH.sub.2--O--CH.sub.3,
--CH.sub.2--CH.sub.2--NH--CH.sub.3,
--CH.sub.2--CH.sub.2--N(CH.sub.3)--CH.sub.3,
--CH.sub.2--S--CH.sub.2--CH.sub.3, --CH.sub.2--S--CH.sub.2,
--S(O)--CH.sub.3, --CH.sub.2--CH.sub.2--S(O).sub.2--CH.sub.3,
--CH.dbd.CHO--CH.sub.3, --Si(CH.sub.3).sub.3,
--CH.sub.2--CH.dbd.N--OCH.sub.3,
--CH.dbd.CH--N(CH.sub.3)--CH.sub.3, --O--CH.sub.3,
--O--CH.sub.2--CH.sub.3, and --CN. Up to two or three heteroatoms
may be consecutive, such as, for example, --CH.sub.2--NH--OCH.sub.3
and --CH.sub.2--O--Si(CH.sub.3).sub.3. A heteroalkyl moiety may
include one heteroatom (e.g., O, N, S, Si, or P). A heteroalkyl
moiety may include two optionally different heteroatoms (e.g., O,
N, S, Si, or P). A heteroalkyl moiety may include three optionally
different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl
moiety may include four optionally different heteroatoms (e.g., O,
N, S, Si, or P). A heteroalkyl moiety may include five optionally
different heteroatoms (e.g., O, N, S, Si, or P). A heteroalkyl
moiety may include up to 8 optionally different heteroatoms (e.g.,
O, N, S, Si, or P). The term "heteroalkenyl," by itself or in
combination with another term, means, unless otherwise stated, a
heteroalkyl including at least one double bond. A heteroalkenyl may
optionally include more than one double bond and/or one or more
triple bonds in additional to the one or more double bonds. The
term "heteroalkynyl," by itself or in combination with another
term, means, unless otherwise stated, a heteroalkyl including at
least one triple bond. A heteroalkynyl may optionally include more
than one triple bond and/or one or more double bonds in additional
to the one or more triple bonds. In embodiments, the carbon-carbon
bonds of a heteroalkyl may be fully saturated (no double or triple
carbon-carbon bonds).
[0032] Similarly, the term "heteroalkylene," by itself or as part
of another substituent, means, unless otherwise stated, a divalent
radical derived from heteroalkyl, as exemplified, but not limited
by, --CH.sub.2--CH.sub.2--S--CH.sub.2--CH.sub.2-- and
--CH.sub.2--S--CH.sub.2--CH.sub.2--NH--CH.sub.2--. For
heteroalkylene groups, heteroatoms can also occupy either or both
of the chain termini (e.g., alkyleneoxy, alkylenedioxy,
alkyleneamino, alkylenediamino, and the like). Still further, for
alkylene and heteroalkylene linking groups, no orientation of the
linking group is implied by the direction in which the formula of
the linking group is written. For example, the formula
--C(O).sub.2R'-- represents both --C(O).sub.2R'-- and
--R'C(O).sub.2--. As described above, heteroalkyl groups, as used
herein, include those groups that are attached to the remainder of
the molecule through a heteroatom, such as --C(O)R', --C(O)NR',
--NR'R'', --OR', --SR', and/or --SO.sub.2R'. Where "heteroalkyl" is
recited, followed by recitations of specific heteroalkyl groups,
such as --NR'R'' or the like, it will be understood that the terms
heteroalkyl and --NR'R'' are not redundant or mutually exclusive.
Rather, the specific heteroalkyl groups are recited to add clarity.
Thus, the term "heteroalkyl" should not be interpreted herein as
excluding specific heteroalkyl groups, such as --NR'R'' or the
like. The term "heteroalkenylene," by itself or as part of another
substituent, means, unless otherwise stated, a divalent radical
derived from a heteroalkene. The term "heteroalkynylene" by itself
or as part of another substituent, means, unless otherwise stated,
a divalent radical derived from a heteroalkyne.
[0033] As used herein, "amine" refers to a --NR.sub.2 moiety
wherein one or more R group can be independently hydrogen, alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. In
embodiments, each R group is independently substituted or
unsubstituted.
[0034] As used herein, "aminooxy" refers to --O--NR.sub.2 moiety,
wherein one or more R group can be independently hydrogen, alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heteroaryl, heteroalicyclyl, aralkyl, or (heteroalicyclyl)alkyl. In
embodiments, each R group is independently substituted or
unsubstituted.
[0035] As used herein, "array" refers to a population of molecules
attached to one or more solid support such that the molecules at
one site can be distinguished from molecules at other sites. An
array can include different molecules that are each located at
different addressable sites on a solid support. Alternatively, an
array can include separate solid supports each functioning as a
site that bears a different molecule, wherein the different
molecules can be identified according to the locations of the solid
supports on a surface to which the solid supports are attached, or
according to the locations of the solid supports in a liquid such
as a fluid stream. The molecules of the array can be, for example,
nucleotides, nucleic acids, nucleic acid primers, nucleic acid
templates, primed template nucleic acids, or nucleic acid enzymes
such as polymerases, ligases, exonucleases or combinations
thereof.
[0036] As used herein, "aryl" refers to a carbocyclic (all carbon)
monocyclic or multicyclic aromatic ring system (including, e.g.,
fused, bridged, or spiro ring systems where two carbocyclic rings
share a chemical bond, e.g., one or more aryl rings with one or
more aryl or non-aryl rings) that has a fully delocalized
pi-electron system throughout at least one of the rings. The number
of carbon atoms in an aryl moiety can vary. For example, in some
embodiments, the aryl moiety can be a C.sub.6-C.sub.14 aryl moiety,
a C.sub.6-C.sub.10 aryl moiety, or a C.sub.6 moiety. Examples of
aryl moieties include, but are not limited to, benzene,
naphthalene, and azulene. An aryl moiety may be substituted or
unsubstituted. In various embodiments, an aryl moiety is
independently a phenyl or naphthyl. In embodiments, a fused ring
aryl refers to multiple rings fused together wherein at least one
of the fused rings is an aryl ring and wherein the multiple rings
are attached to the parent molecular moiety through any carbon atom
contained within an aryl ring of the multiple rings. An "arylene",
alone or as part of another substituent, means a divalent radical
derived from an aryl.
[0037] As used herein, "aralkyl" and "aryl(alkyl)" refer to an aryl
connected, as a substituent, via a lower alkylene moiety. The lower
alkylene and aryl moiety of an aralkyl may be substituted or
unsubstituted. Examples include but are not limited to benzyl,
2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.
[0038] As used herein, "attached" refers to the state of two things
being joined, fastened, adhered, connected or bound to each other.
For example, a nucleic acid can be attached to a solid phase
support by a covalent or non-covalent bond. Similarly, two nucleic
acids can be attached to each other by a covalent (e.g.
phosphodiester) bond or by a non-covalent bond (e.g. hydrogen
bonding between bases of the two nucleic acids). A covalent bond is
characterized by the sharing of pairs of electrons between atoms. A
non-covalent bond is a chemical bond that does not involve the
sharing of pairs of electrons and can include, for example,
hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic
interactions and hydrophobic interactions.
[0039] As used herein, "blocking moiety," when used in reference to
a nucleotide, means a part of the nucleotide that inhibits or
prevents the 3' oxygen of the nucleotide from forming a covalent
linkage to another nucleotide or to an oligonucleotide. For
example, a blocking moiety can inhibit or prevent polymerase
catalyzed formation of a covalent linkage between the 3' position
of the nucleotide and a next correct nucleotide during a nucleic
acid polymerization reaction. Similarly, a blocking moiety can
inhibit or prevent ligase catalyzed formation of a covalent linkage
between the 3' position of the nucleotide and an oligonucleotide
during a nucleic acid ligation reaction. Notably, blocking moieties
as contemplated herein do not interfere with incorporation of the
blocked base into an extending primer strand (e.g., catalyzed by a
polymerase); rather, once incorporated, a blocked nucleotide
interferes with subsequent elongation because the 3' end of the
nucleotide is inaccessible to the 5' phosphate of a subsequent
nucleotide. The blocking moiety of a "reversibly terminated"
nucleotide can be removed from the nucleotide analog, or otherwise
modified, without substantial harm to the extending primer strand
or its complement, to allow the 3'-oxygen of the nucleotide to be
available to covalently link to another nucleotide or to an
oligonucleotide. Such a blocking moiety may be referred to herein
as a "reversible terminator moiety" because the blocking of primer
extension is reversed through removal of the blocking or terminator
moiety. A nucleotide that has a blocking moiety such as a
reversible terminator moiety can be at the 3' end of a nucleic
acid, such as a primer, or can be a monomeric molecule that is not
covalently attached to a nucleic acid. Exemplary blocking moieties
include 3' ONH.sub.2, as well as 3' ONHCH.sub.3 and 3' O
methylazide (3'-OCH.sub.2N.sub.3). In various embodiments, a
protecting moiety may also be a blocking moiety (e.g., a 3'-O-oxime
moiety may be a blocking moiety). As used herein, R2-block is R2
bonded to a blocking moiety such as a reversible terminator moiety.
R2-moieties in solution are in some cases used to stabilize
blocking moieties so as to prevent their unwanted removal from
nucleotides to form 3' OH groups compatible with
polymerase-mediated extension.
[0040] As used herein, a "protecting moiety" is a moiety that
protects a molecule to which it is attached from cross-reaction or
misdirected reaction during a chemical process, such as a
nonenzymatic process or a nonspecific chemical process. Unlike
blocking moieties such as reversible terminating moieties,
protecting moieties are not necessarily readily removed without
risk of cross-reaction or degradation to the protected molecule.
Exemplary moieties include 3'-O-oxime moieties such as
3'-O-alkoximes or 3' O-ketoximes, which protect ribose 3' positions
from cross reaction during 5' phosphorylation reactions, for
example. In various embodiments, a protecting moiety is a
3'-O-oxime (e.g., 3'-O-alkoxime or 3'-O-ketoxime). In embodiments,
the protecting moiety prevents reactivity of the 3' carbon or 3'
oxygen of a nucleotide or nucleoside during a phosphorylation
reaction of the nucleotide or nucleoside, such as may occur during
the addition of a triphosphate moiety to a nucleotide 5'
position.
[0041] The term "comprising" is intended herein to be open-ended,
including recited elements, but also leaving open the possibility
of including additional, unrecited elements.
[0042] As used herein, "cycloalkyl" refers to a completely
saturated (no double or triple bonds) mono- or multi-cyclic
hydrocarbon ring system. When composed of two or more rings, the
rings may be joined together in a fused fashion. A cycloalkyl
moiety can contain 3 to 10 atoms in the ring(s). In some molecules,
a cycloalkyl moiety can contain 3 to 8 atoms in the ring(s). A
cycloalkyl moiety may be unsubstituted or substituted. Typical
cycloalkyl moieties include, but are not limited to, cyclopropyl,
cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and
cyclooctyl.
[0043] As used herein, "cycloalkenyl" refers to a mono- or
multi-cyclic hydrocarbon ring system that contains one or more
double bonds in at least one ring; although, if there is more than
one, the double bonds do not form a fully delocalized pi-electron
system throughout all the rings (otherwise the moiety would be
"aryl," as defined herein). When composed of two or more rings, the
rings may be connected together in a fused fashion. A cycloalkenyl
moiety may be unsubstituted or substituted. A "cycloalkylene",
alone or as part of another substituent, means a divalent radical
derived from a cycloalkyl.
[0044] As used herein, "cycloalkynyl" refers to a mono- or
multi-cyclic hydrocarbon ring system that contains one or more
triple bonds in at least one ring. If there is more than one triple
bond, the triple bonds do not form a fully delocalized pi-electron
system throughout all the rings. When composed of two or more
rings, the rings may be joined together in a fused fashion. A
cycloalkynyl moiety may be unsubstituted or substituted.
[0045] As used herein, "deblock" means to remove or modify a
blocking moiety such as a reversible terminator moiety of a
nucleotide to render the nucleotide extendable, such as through
exposure of or generation of a 3' OH. Such a deblocked nucleotide
or nucleic acid strand is capable of forming a phosphodiester bond
with a second nucleotide, rendering the nucleotide capable of
forming a phosphodiester bond involving the 3' carbon of the
nucleotide and the 5' carbon of a second nucleotide). For example,
a reversibly terminated nucleotide can be present at the 3' end of
a primer such that deblocking renders the primer extendable.
Alternatively, the nucleotide can be in monomeric form when
deblocked.
[0046] As used herein, the term "each," when used in reference to a
collection of items, is intended to identify an individual item in
the collection but does not necessarily refer to every item in the
collection. Exceptions can occur if explicit disclosure or context
clearly dictates otherwise.
[0047] As used herein, the phrase "at least one element selected
from the list of" A, B, and C is intended to read on sets including
one element from the list, two elements form the list or all
elements of the list, and may but is not necessarily intended to
require a representative of each element A, B, and C unless
otherwise designated.
[0048] As used herein, "exogenous," when used in reference to a
moiety of a molecule, means a chemical moiety that is not present
in a natural analog of the molecule. For example, an exogenous
label of a nucleotide is a label that is not present on a naturally
occurring nucleotide. Similarly, an exogenous linker that is
present on a nucleic acid is not found on nucleic acids in their
native milieu.
[0049] As used herein, "extension," when used in reference to a
nucleic acid, means a process of adding at least one nucleotide to
the 3' end of the nucleic acid. The term "polymerase extension,"
when used in reference to a nucleic acid, refers to a polymerase
catalyzed process of adding one or more individual nucleotides to
the 3' end of the nucleic acid. The term "ligase extension," when
used in reference to a nucleic acid, refers to a ligase catalyzed
process of adding at least one oligonucleotide to the 3' end of the
nucleic acid. A nucleotide or oligonucleotide that is added to a
nucleic acid by extension is said to be incorporated into the
nucleic acid. Accordingly, the term "incorporating" can be used to
refer to the process of joining a nucleotide or oligonucleotide to
the 3' end of a nucleic acid by formation of a phosphodiester
bond.
[0050] As used herein, "extendable," when used in reference to a
nucleotide, means that the nucleotide has an oxygen or hydroxyl
moiety at the 3' position, and is capable of forming a covalent
linkage to a next correct nucleotide (or to an oligonucleotide) if
and when incorporated into a nucleic acid. An extendable nucleotide
can be at the 3' position of a primer or it can be a monomeric
nucleotide. A nucleotide that is extendable will lack blocking
moieties such as reversible terminator moieties, and a deblocked
nucleotide or nucleic acid strand is often extendable.
[0051] As used herein, a "flow cell" is a vessel that includes one
or more channels that direct fluid to a detection zone. The
detection zone can be coupled to a detector such that a reaction
occurring in the vessel can be observed. For example, a flow cell
can contain primed template nucleic acid molecules tethered to a
solid phase support, to which nucleotides and ancillary reagents
are iteratively applied and washed away. The flow cell can include
a transparent material that permits the sample to be imaged after a
desired reaction occurs. For example, a flow cell can include a
glass or plastic slide containing small fluidic channels through
which polymerases, dNTPs and buffers can be flowed. The glass or
plastic inside the channels can be decorated with one or more
primed template nucleic acid molecules to be detected. An external
imaging system can be positioned to detect the molecules at a
detection zone. Exemplary flow cells, methods for their manufacture
and methods for their use are described in US Pat. App. Publ. Nos.
2010/0111768 A1, published May 6, 2010 or 2012/0270305 A1,
published Oct. 25, 2012; or WO 05/065814, published Jul. 25, 2005,
each of which hereby is incorporated by reference in its entirety
herein.
[0052] As used herein, "fluid" refers to a liquid or a gas, that is
capable of flowing and that changes its shape to fill a vessel. In
many conditions, a fluid will change shape at a steady rate when
acted upon by a force tending to change its shape. An exemplary
fluid is a fluid foam which is a liquid that contains bubbles of
gas. Exemplary liquids that can be used, for example, in a fluid
foam include those that contain reagents or products of a reaction
such as a binding reaction, nucleic acid sequencing reaction or
reaction used in an analytical assay. Aqueous liquids can be
particularly useful. Exemplary gases include inert gases such as
nitrogen (N.sub.2) or noble gases. Useful noble gases include, for
example, helium (He), neon (Ne), argon (Ar), krypton (Kr) and xenon
(Xe). Another useful gas is atmospheric air of planet earth.
Further examples of fluid foams and methods for making and using
fluid foams are set forth in U.S. Pat. App. Ser. No. 62/883,276,
which is incorporated herein by reference.
[0053] As used herein, "halogen atom", "halogen" or "halo" means
any one of the radio-stable atoms of column 7 of the Periodic Table
of the Elements, such as, fluorine, chlorine, bromine, and iodine.
Additionally, terms such as "haloalkyl" are meant to include
monohaloalkyl and polyhaloalkyl. For example, the term
"halo(C.sub.1-C.sub.4)alkyl" includes, but is not limited to,
fluoromethyl, difluoromethyl, trifluoromethyl,
2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the
like.
[0054] As used herein, "heterocyclyl" refers to a ring system
including at least one heteroatom (e.g., O, N, S). Such systems can
be unsaturated, can include some unsaturation, or can contain some
aromatic portion, or be all aromatic. A heterocyclyl moiety may be
unsubstituted or substituted.
[0055] As used herein, "heteroalicyclic" or "heteroalicyclyl"
refers to three-, four-, five-, six-, seven-, eight-, nine-, ten-,
or more up to 18-membered monocyclic, bicyclic, and tricyclic ring
system wherein carbon atoms together with from 1 to 5 heteroatoms
constitute the ring system. A heterocycle may optionally contain
one or more unsaturated bonds situated in such a way, however, that
a fully delocalized pi-electron system does not occur throughout
all the rings. The heteroatoms are independently selected from
oxygen, sulfur, and nitrogen. A heterocycle may further contain one
or more carbonyl or thiocarbonyl functionalities, and as such the
definition includes oxo-systems and thio-systems such as lactams,
lactones, cyclic imides, cyclic thioimides, and cyclic carbamates.
When composed of two or more rings, the rings may be joined
together in a fused fashion. Additionally, any nitrogens in a
heteroalicyclic moiety may be quaternized. Heteroalicyclyl or
heteroalicyclic moieties may be unsubstituted or substituted.
Examples of such "heteroalicyclic" or "heteroalicyclyl" moieties
include but are not limited to, 1,3-dioxin, 1,3-dioxane,
1,4-dioxane, 1,2-dioxolane, 1,3-dioxolane, 1,4-dioxolane,
1,3-oxathiane, 1,4-oxathiin, 1,3-oxathiolane, 1,3-dithiole,
1,3-dithiolane, 1,4-oxathiane, tetrahydro-1,4-thiazine,
2H-1,2-oxazine, maleimide, succinimide, barbituric acid,
thiobarbituric acid, dioxopiperazine, hydantoin, dihydrouracil,
trioxane, hexahydro-1,3,5-triazine, imidazoline, imidazolidine,
isoxazoline, isoxazolidine, oxazoline, oxazolidine, oxazolidinone,
thiazoline, thiazolidine, morpholine, oxirane, piperidine N-Oxide,
piperidine, piperazine, pyrrolidine, pyrrolidone, pyrrolidione,
4-piperidone, pyrazoline, pyrazolidine, 2-oxopyrrolidine,
tetrahydropyran, 4H-pyran, tetrahydrothiopyran, thiamorpholine,
thiamorpholine sulfoxide, thiamorpholine sulfone, and their
benzo-fused analogs (e.g., benzimidazolidinone,
tetrahydroquinoline, 3,4-methylenedioxyphenyl). In embodiments, a
fused ring heteroalicyclyl refers to multiple rings fused together
wherein at least one of the fused rings is a heteroalicyclyl ring
and wherein the multiple rings are attached to the parent molecular
moiety through any atom contained within a heteroalicyclyl ring of
the multiple rings. A "heteroalicyclylene," alone or as part of
another substituent, means a divalent radical derived from a
heteroalicyclyl.
[0056] As used herein, "heteroaryl" refers to a monocyclic or
multicyclic aromatic ring system (a ring system having a least one
ring with a fully delocalized pi-electron system) that contain(s)
one or more heteroatoms, that is, an element other than carbon,
including but not limited to, nitrogen, oxygen, and sulfur, and at
least one aromatic ring. The number of atoms in the ring(s) of a
heteroaryl moiety can vary. For example, in some embodiments, a
heteroaryl moiety can contain 4 to 14 atoms in the ring(s), 5 to 10
atoms in the ring(s) or 5 to 6 atoms in the ring(s). Furthermore,
the term "heteroaryl" includes fused ring systems where two rings,
such as at least one aryl ring and at least one heteroaryl ring, or
at least two heteroaryl rings, share at least one chemical bond.
Examples of heteroaryl rings include, but are not limited to,
furan, furazan, thiophene, benzothiophene, phthalazine, pyrrole,
oxazole, benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole,
1,2,3-thiadiazole, 1,2,4-thiadiazole, benzothiazole, imidazole,
benzimidazole, indole, indazole, pyrazole, benzopyrazole,
isoxazole, benzoisoxazole, isothiazole, triazole, benzotriazole,
thiadiazole, tetrazole, pyridine, pyridazine, pyrimidine, pyrazine,
purine, pteridine, quinoline, isoquinoline, quinazoline,
quinoxaline, cinnoline, and triazine. A heteroaryl moiety may be
substituted or unsubstituted. In embodiments, the term "heteroaryl"
includes fused ring heteroaryl groups wherein multiple rings are
fused together and wherein at least one of the fused rings is a
heteroaromatic ring and wherein the multiple rings are attached to
the parent molecular moiety through any atom contained within a
heteroaromatic ring of the multiple rings. A "heteroarylene," alone
or as part of another substituent, means a divalent radical derived
from a heteroaryl.
[0057] As used herein, "(heteroalicyclyl)alkyl" is a heterocyclic
or a heteroalicyclic moiety connected, as a substituent, via a
lower alkylene moiety. The lower alkylene and heterocyclic or a
heterocyclyl of a (heteroalicyclyl)alkyl may be substituted or
unsubstituted. Examples include but are not limited to
tetrahydro-2H-pyran-4-yl)methyl, (piperidin-4-yl)ethyl,
(piperidin-4-yl)propyl, (tetrahydro-2H-thiopyran-4-yl)methyl, and
(1,3-thiazinan-4-yl)methyl.
[0058] As used herein, "heteroaralkyl" and "heteroaryl(alkyl)"
refer to a heteroaryl connected, as a substituent, via a lower
alkylene moiety. The lower alkylene and heteroaryl moiety of
heteroaralkyl may be substituted or unsubstituted. Examples include
but are not limited to 2-thienylalkyl, 3-thienylalkyl, furylalkyl,
thienylalkyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and
imidazolylalkyl, and their benzo-fused analogs.
[0059] As used herein, "hydroxy" refers to a --OH moiety.
Similarly, "hydroxylated" refers to a molecule to which --OH
moieties have been added, for example to facilitate solubility.
[0060] As used herein, "label" refers to a molecule, or moiety
thereof, that provides a detectable characteristic. The detectable
characteristic can be, for example, an optical signal such as
absorbance of radiation, fluorescence emission, luminescence
emission, fluorescence lifetime, fluorescence polarization, or the
like; Rayleigh and/or Mie scattering; binding affinity for a ligand
or receptor, such as an antibody or streptavidin; magnetic
properties; electrical properties; conductivity; charge; mass;
radioactivity or the like. Exemplary labels include, without
limitation, a fluorophore, luminophore, chromophore, nanoparticle
(e.g., gold, silver, carbon nanotubes), heavy atoms, radioactive
isotope, mass label, charge label, spin label, receptor, ligand, or
the like.
[0061] As used herein, "linker" and "linker moiety" refer to an
atom or moiety that joins two different entities. Typically, a
linker will provide or comprise a covalent bond, or a series of
covalent bonds, between the two entities. However, a linker can
include non-covalent bonds such as those that form between a
receptor and ligand. Exemplary entities that can be linked include,
but are not limited to, a solid support, moiety (e.g. a nucleotide
monomer of a nucleic acid or a base of a nucleotide) or molecule
(e.g. a nucleic acid or label).
[0062] As used herein, "next correct nucleotide" refers to the
nucleotide type that will bind and/or incorporate at the 3' end of
a primer to complement a base in a template strand to which the
primer is hybridized when correctly basepaired. The base in the
template strand is referred to as the "next base" and is
immediately 5' of the base in the template that is hybridized to
the 3' end of the primer. The next correct nucleotide can be
referred to as the "cognate" of the next base and vice versa.
Cognate nucleotides that interact with each other in a ternary
complex or in a double stranded nucleic acid are said to "pair"
with each other. In accordance with Watson-Crick pairing rules
adenine (A) pairs with thymine (T) or uracil (U), and guanine (G)
pairs with cytosine (C) or uracil (U). A nucleotide having a base
that is not complementary to the next template base is referred to
as an "incorrect", "mismatch" or "non-cognate" nucleotide.
[0063] As used herein, "nucleobase" (also referred to as a
"nitrogenous base" or a "base") refers to a purine or purine
derivative or pyrimidine or pyrimidine derivative that forms the
differentiating unit of a nucleotide. The five primary or canonical
nucleobases are adenine (A), cytosine (C), guanine (G), thymine
(T), and uracil (U). Adenine and guanine are purine bases.
Cytosine, uracil, and thymine are pyrimidine bases. Each of the
base pairs in a typical double-helix includes a purine and a
pyrimidine: either an A paired with a T, an A paired with a U, or a
G paired with a C or a G paired with a U. These purine-pyrimidine
pairs, are referred to as `base complements` or are said to be
"complementary" to each other. Cytosine nucleobases, in particular,
are subject to modification or degradation pursuant to replacing
some protecting groups with blocking groups.
[0064] As used herein, "nucleic acid" refers to at least two
nucleotides covalently linked together. Thus, an exemplary "nucleic
acid" is a polynucleotide, such as DNA, RNA, or any combination
thereof. Typical nucleic acids can be acted upon by a polymerase
during nucleic acid synthesis or detection. The term "nucleic acid"
includes single-, double-, or multiple-stranded DNA, RNA and
analogs (derivatives) thereof.
[0065] As used herein, "nucleotide" can be used to refer to a
native nucleotide or analog thereof. Generally, a nucleotide has
three moieties: a nucleobase (e.g. purine or pyrimidine), a
five-carbon sugar (e.g. ribose or deoxyribose), and at least one
phosphate. Examples include, but are not limited to, nucleotide
triphosphates (NTPs) such as ribonucleotide triphosphates (rNTPs),
deoxyribonucleotide triphosphates (dNTPs), or non-natural analogs
thereof such as dideoxyribonucleotide triphosphates (ddNTPs) or
reversibly terminated nucleotide triphosphates (rtNTPs). A
nucleotide can be in a monomeric form, for example, as a nucleotide
triphosphate, nucleotide diphosphate or nucleotide monophosphate. A
nucleotide can also be a moiety of a nucleic acid polymer, for
example, being identified according to its position in the polymer
such as the 3' nucleotide (i.e. the nucleotide moiety that is
present at the 3' end of the polymer) or the 5' nucleotide (i.e.
the nucleotide moiety that is present at the 5' end of the
polymer). A nucleoside contains a nucleobase and a 5-carbon sugar.
Thus, a nucleotide is a nucleoside having a phosphate moiety.
[0066] As used herein, "optionally substituted," when used in
reference to a moiety of a molecule, means that the moiety may be
unsubstituted or substituted with one or more of the indicated
substituents. Likewise, when a moiety is described as being
"substituted or unsubstituted" or "unsubstituted or substituted,"
the substituent may be selected from one or more the indicated
substituents. If no substituents are indicated, it is meant that
the indicated "optionally substituted" or "substituted" moiety may
be individually and independently substituted with one or more
moieties individually and independently selected from a group of
functionalities including, but not limited to, alkyl, alkenyl,
alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,
heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl,
hydroxy, protected hydroxyl, alkoxy, aryloxy, acyl, mercapto,
alkylthio, arylthio, cyano, halogen, thiocarbonyl, O-carbamyl,
N-carbamyl, O-thiocarbamyl, N-thiocarbamyl, C-amido, N-amido,
S-sulfonamido, N-sulfonamido, C-carboxy, protected C-carboxy,
O-carboxy, isocyanato, thiocyanato, isothiocyanato, nitro, silyl,
sulfenyl, sulfinyl, sulfonyl, haloalkyl, haloalkoxy,
trihalomethanesulfonyl, trihalomethanesulfonamido, amino,
mono-substituted amino group, di-substituted amino group, and
protected derivatives thereof. If no substituents are indicated in
reference to an "unsubstituted" moiety, the omitted moiety(ies) may
individually and independently be selected from a group of
functionalities including, but not limited to, those set forth in
the previous sentence or elsewhere herein.
[0067] Each of the above terms (e.g., "alkyl," "alkenyl,"
"alkynyl," "heteroalkyl," "cycloalkyl," "cycloalkenyl,"
"cycloalkynyl," "heteroalicyclyl," "aryl," "heteroaryl",
"alkylene," "alkenylene," "alkynylene," "heteroalkylene,"
"cycloalkylene," "cycloalkenylene," "cycloalkynylene,"
"heteroalicyclylene," "arylene," and "heteroarylene") includes both
substituted and unsubstituted forms of the indicated radical.
Preferred substituents for each type of radical are provided
below.
[0068] Substituents for the alkyl and heteroalkyl radicals can be
one or more of a variety of groups selected from, but not limited
to, --OR', .dbd.O, .dbd.NR', .dbd.N--OR', --NR'R'', --SR',
-halogen, --SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R',
--CONR'R'', --OC(O)NR'R'', --NR''C(O)R', --NR'C(O)NR''R''',
--NR''C(O).sub.2R', --NRC(NR'R''R''').dbd.NR'''',
--NRC(NR'R'').dbd.NR''', --S(O)R', --S(O).sub.2R',
--S(O).sub.2NR'R'', --NRSO.sub.2R', --NR'NR''R''', --ONR'R'',
--NR'C(O)NR''NR'''R'''', --CN, --NO.sub.2, --NR'SO.sub.2R'',
--NR'C(O)R'', --NR'C(O)OR'', --NR'OR'', --N.sub.3, in a number
ranging from zero to (2m'+1), where m' is the total number of
carbon atoms in such radical. R, R', R'', R''', and R'''' each
preferably independently refer to hydrogen, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted heteroalicyclyl, substituted or
unsubstituted aryl (e.g., aryl substituted with 1-3 halogens),
substituted or unsubstituted heteroaryl, substituted or
unsubstituted alkyl, alkoxy, or thioalkoxy groups, or arylalkyl
groups. When a compound described herein includes more than one R
group, for example, each of the R groups is independently selected
as are each R', R'', R''', and R'''' group when more than one of
these groups is present. When R' and R'' are attached to the same
nitrogen atom, they can be combined with the nitrogen atom to form
a 4-, 5-, 6-, or 7-membered ring. For example, --NR'R'' includes,
but is not limited to, 1-pyrrolidinyl and 4-morpholinyl. From the
above discussion of substituents, one of skill in the art will
understand that the term "alkyl" is meant to include groups
including carbon atoms bound to groups other than hydrogen groups,
such as haloalkyl (e.g., --CF.sub.3 and --CH.sub.2CF.sub.3) and
acyl (e.g., --C(O)CH.sub.3, --C(O)CF.sub.3,
--C(O)CH.sub.2OCH.sub.3, and the like).
[0069] Similar to the substituents described for the alkyl radical,
substituents for the aryl and heteroaryl groups are varied and are
selected from, for example: --OR', --NR'R'', --SR', -halogen,
--SiR'R''R''', --OC(O)R', --C(O)R', --CO.sub.2R', --CONR'R'',
--OC(O)NR'R'', --NR''C(O)R', --NR'C(O)NR''R''', --NR''C(O).sub.2R',
--NRC(NR'R''R''').dbd.NR'''', --NRC(NR'R'').dbd.NR''', --S(O)R',
--S(O).sub.2R', --S(O).sub.2NR'R'', --NRSO.sub.2R', --NR'NR''R''',
--ONR'R'', --NR'C(O)NR''NR'''R'''', --CN, --NO.sub.2, --R',
--N.sub.3, --CH(Ph).sub.2, fluoro(C.sub.1-C.sub.4)alkoxy, and
fluoro(C.sub.1-C.sub.4)alkyl, --NR'SO.sub.2R'', --NR'C(O)R'',
--NR'C(O)--OR'', --NR'OR'', in a number ranging from zero to the
total number of open valences on the aromatic ring system; and
where R', R'', R''', and R'''' are preferably independently
selected from hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
cycloalkenyl, substituted or unsubstituted cycloalkynyl,
substituted or unsubstituted heteroalicyclyl, substituted or
unsubstituted aryl, and substituted or unsubstituted heteroaryl.
When a compound described herein includes more than one R group,
for example, each of the R groups is independently selected as are
each R', R'', R''', and R'''' groups when more than one of these
groups is present.
[0070] Substituents for rings (e.g., cycloalkyl, cycloalkenyl,
cycloalkynyl, heteroalicyclyl, aryl, or heteroaryl) may be depicted
as substituents on the ring rather than on a specific atom of a
ring (commonly referred to as a floating substituent). In such a
case, the substituent may be attached to any of the ring atoms
(obeying the rules of chemical valency) and in the case of fused
rings or spirocyclic rings, a substituent depicted as associated
with one member of the fused rings or spirocyclic rings (a floating
substituent on a single ring), may be a substituent on any of the
fused rings or spirocyclic rings (a floating substituent on
multiple rings). When a substituent is attached to a ring, but not
a specific atom (a floating substituent), and a subscript for the
substituent is an integer greater than one, the multiple
substituents may be on the same atom, same ring, different atoms,
different fused rings, different spirocyclic rings, and each
substituent may optionally be different. Where a point of
attachment of a ring to the remainder of a molecule is not limited
to a single atom (a floating substituent), the attachment point may
be any atom of the ring and in the case of a fused ring or
spirocyclic ring, any atom of any of the fused rings or spirocyclic
rings while obeying the rules of chemical valency. Where a ring,
fused rings, or spirocyclic rings contain one or more ring
heteroatoms and the ring, fused rings, or spirocyclic rings are
shown with one more floating substituents (including, but not
limited to, points of attachment to the remainder of the molecule),
the floating substituents may be bonded to the heteroatoms. Where
the ring heteroatoms are shown bound to one or more hydrogens
(e.g., a ring nitrogen with two bonds to ring atoms and a third
bond to a hydrogen) in the structure or formula with the floating
substituent, when the heteroatom is bonded to the floating
substituent, the substituent will be understood to replace the
hydrogen, while obeying the rules of chemical valency.
[0071] Two or more substituents may optionally be joined to form
aryl, heteroaryl, cycloalkyl, cycloalkenyl, cycloalkynyl, or
heteroalicyclyl groups. Such so-called ring-forming substituents
are typically, though not necessarily, found attached to a cyclic
base structure. In one embodiment, the ring-forming substituents
are attached to adjacent members of the base structure. For
example, two ring-forming substituents attached to adjacent members
of a cyclic base structure create a fused ring structure. In
another embodiment, the ring-forming substituents are attached to a
single member of the base structure. For example, two ring-forming
substituents attached to a single member of a cyclic base structure
create a spirocyclic structure. In yet another embodiment, the
ring-forming substituents are attached to non-adjacent members of
the base structure.
[0072] As used herein, the terms "heteroatom" or "ring heteroatom"
are meant to include oxygen (O), nitrogen (N), sulfur (S), selenium
(Se), phosphorus (P), and silicon (Si).
[0073] A "substituent group," as used herein, means a group
selected from the following moieties: [0074] (A) oxo, [0075]
halogen, --CCl.sub.3, --CBr.sub.3, --CF.sub.3, --CI.sub.3,
--CHCl.sub.2, --CHBr.sub.2, --CHF.sub.2, --CHI.sub.2, --CH.sub.2Cl,
--CH.sub.2Br, --CH.sub.2F, --CH.sub.2I, --OCCl.sub.3, --OCF.sub.3,
--OCBr.sub.3, --OCI.sub.3, --OCHCl.sub.2, --OCHBr.sub.2, --OCHI 2,
--OCHF.sub.2, --OCH.sub.2Cl, --OCH.sub.2Br, --OCH.sub.2I,
--OCH.sub.2F, --CN, --OH, --NH.sub.2, --COOH, --C ONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --OSO.sub.3H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2, --NHC(O)NH.sub.2,
--NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH, --N.sub.3,
unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6
alkyl, or C.sub.1-C.sub.4 alkyl), unsubstituted alkenyl (e.g.,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.6 alkenyl, or
C.sub.2-C.sub.4 alkenyl), unsubstituted alkynyl (e.g.,
C.sub.2-C.sub.8 alkynyl, C.sub.2-C.sub.6 alkynyl, or
C.sub.2-C.sub.4 alkynyl), unsubstituted heteroalkyl (e.g., 2 to 8
membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4
membered heteroalkyl), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8 cycloalkyl, C.sub.3-C.sub.6 cycloalkyl, or
C.sub.5-C.sub.6 cycloalkyl), unsubstituted cycloalkenyl (e.g.,
C.sub.3-C.sub.8 cycloalkenyl, C.sub.3-C.sub.6 cycloalkenyl, or
C.sub.5-C.sub.6 cycloalkenyl), unsubstituted cycloalkynyl (e.g.,
C.sub.3-C.sub.8 cycloalkynyl, C.sub.3-C.sub.6 cycloalkynyl, or
C5-C6 cycloalkynyl), unsubstituted heteroalicyclyl (e.g., 3 to 8
membered heteroalicyclyl, 3 to 6 membered heteroalicyclyl, or 5 to
6 membered heteroalicyclyl), unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl), or unsubstituted
heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered
heteroaryl, or 5 to 6 membered heteroaryl), and [0076] (B) alkyl
(e.g., C.sub.1-C.sub.20, C.sub.1-C.sub.12, C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2), alkenyl
(e.g., C.sub.2-C.sub.20, C.sub.2-C.sub.12, C.sub.2-C.sub.8,
C.sub.2-C.sub.6, or C.sub.2-C.sub.4), alkynyl (e.g.,
C.sub.2-C.sub.20, C.sub.2-C.sub.12, C.sub.2-C.sub.8,
C.sub.2-C.sub.6, or C.sub.2-C.sub.4), heteroalkyl (e.g., 2 to 20
membered, 2 to 12 membered, 2 to 8 membered, 2 to 6 membered, 4 to
6 membered, 2 to 3 membered, or 4 to 5 membered), cycloalkyl (e.g.,
C.sub.3-C.sub.10, C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C5-C6), cycloalkenyl (e.g., C.sub.3-C.sub.10,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), cycloalkynyl (e.g., C.sub.3-C.sub.10,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), heteroalicyclyl (e.g., 3 to 10 membered, 3 to 8
membered, 3 to 6 membered, 4 to 6 membered, 4 to 5 membered, or 5
to 6 membered), aryl (e.g., C.sub.6-C.sub.12, C.sub.6-C.sub.10, or
phenyl), or heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5
to 9 membered, or 5 to 6 membered), substituted with at least one
substituent selected from: [0077] (i) oxo, [0078] halogen,
--CCl.sub.3, --CBr.sub.3, --CF.sub.3, --CI.sub.3, --CHCl.sub.2,
--CHBr.sub.2, --CHF.sub.2, --CHI.sub.2, --CH.sub.2Cl, --C
H.sub.2Br, --CH.sub.2F, --CH.sub.2I, --OCCl.sub.3, --OCF.sub.3,
--OCBr.sub.3, --OCI.sub.3, --OCHCl.sub.2, --OCHBr.sub.2, --O
CHI.sub.2, --OCHF.sub.2, --OCH.sub.2Cl, --OCH.sub.2Br,
--OCH.sub.2I, --OCH.sub.2F, --CN, --OH, --NH.sub.2, --CO OH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --OSO.sub.3H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2,
--NHC(O)NH.sub.2, --NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH,
--N.sub.3, unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4 alkyl), unsubstituted
alkenyl (e.g., C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.6 alkenyl, or
C.sub.2-C.sub.4 alkenyl), unsubstituted alkynyl (e.g.,
C.sub.2-C.sub.8 alkynyl, C.sub.2-C.sub.6 alkynyl, or
C.sub.2-C.sub.4 alkynyl), unsubstituted heteroalkyl (e.g., 2 to 8
membered heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4
membered heteroalkyl), unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8 cycloalkyl, C.sub.3-C.sub.6 cycloalkyl, or
C.sub.5-C.sub.6 cycloalkyl), unsubstituted cycloalkenyl (e.g.,
C.sub.3-C.sub.8 cycloalkenyl, C.sub.3-C.sub.6 cycloalkenyl, or
C.sub.5-C.sub.6 cycloalkenyl), unsubstituted cycloalkynyl (e.g.,
C.sub.3-C.sub.8 cycloalkynyl, C.sub.3-C.sub.6 cycloalkynyl, or
C.sub.5-C.sub.6 cycloalkynyl), unsubstituted heteroalicyclyl (e.g.,
3 to 8 membered heteroalicyclyl, 3 to 6 membered heteroalicyclyl,
or 5 to 6 membered heteroalicyclyl), unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl), or unsubstituted
heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered
heteroaryl, or 5 to 6 membered heteroaryl), and [0079] (ii) alkyl
(e.g., C.sub.1-C.sub.20, C.sub.1-C.sub.12, C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2), alkenyl
(e.g., C.sub.2-C.sub.20, C2-C12, C.sub.2-C.sub.8, C.sub.2-C.sub.6,
or C.sub.2-C.sub.4), alkynyl (e.g., C.sub.2-C.sub.20, C2-C12,
C.sub.2-C.sub.8, C.sub.2-C.sub.6, or C.sub.2-C.sub.4), heteroalkyl
(e.g., 2 to 20 membered, 2 to 12 membered, 2 to 8 membered, 2 to 6
membered, 4 to 6 membered, 2 to 3 membered, or 4 to 5 membered),
cycloalkyl (e.g., C.sub.3-C.sub.10, C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C5-C6), cycloalkenyl (e.g.,
C.sub.3-C.sub.10, C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6), cycloalkynyl (e.g.,
C.sub.3-C.sub.10, C.sub.3-C.sub.8, C.sub.3-C.sub.6,
C.sub.4-C.sub.6, or C.sub.5-C.sub.6), heteroalicyclyl (e.g., 3 to
10 membered, 3 to 8 membered, 3 to 6 membered, 4 to 6 membered, 4
to 5 membered, or 5 to 6 membered), aryl (e.g., C.sub.6-C.sub.12,
C.sub.6-C.sub.10, or phenyl), or heteroaryl (e.g., 5 to 12
membered, 5 to 10 membered, 5 to 9 membered, or 5 to 6 membered),
substituted with at least one substituent selected from: [0080] (a)
oxo, [0081] halogen, --CCl.sub.3, --CBr.sub.3, --CF.sub.3,
--CI.sub.3, --CHCl.sub.2, --CHBr.sub.2, --CHF.sub.2, --CHI.sub.2,
--CH.sub.2Cl, --CH.sub.2Br, --CH.sub.2F, --CH.sub.2I, --OCCl.sub.3,
--OCF.sub.3, --OCBr.sub.3, --OCI.sub.3, --OCHCl.sub.2,
--OCHBr.sub.2, --OCHI.sub.2, --OCHF.sub.2, --OCH.sub.2Cl,
--OCH.sub.2Br, --OCH.sub.2I, --OCH.sub.2F, --CN, --OH, --NH 2,
--COOH, --CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --OSO.sub.3H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2,
--NHC(O)NH.sub.2, --NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH,
--N.sub.3, unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4 alkyl), unsubstituted
alkenyl (e.g., C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.6 alkenyl, or
C.sub.2-C.sub.4 alkenyl), unsubstituted alkynyl (e.g.,
C.sub.2-C.sub.8 alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4
alkynyl), unsubstituted heteroalkyl (e.g., 2 to 8 membered
heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered
heteroalkyl), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8
cycloalkyl, C.sub.3-C.sub.6 cycloalkyl, or C.sub.5-C.sub.6
cycloalkyl), unsubstituted cycloalkenyl (e.g., C.sub.3-C.sub.8
cycloalkenyl, C.sub.3-C.sub.6 cycloalkenyl, or C.sub.5-C.sub.6
cycloalkenyl), unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8
cycloalkynyl, C.sub.3-C.sub.6 cycloalkynyl, or C.sub.5-C.sub.6
cycloalkynyl), unsubstituted heteroalicyclyl (e.g., 3 to 8 membered
heteroalicyclyl, 3 to 6 membered heteroalicyclyl, or 5 to 6
membered heteroalicyclyl), unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl), or unsubstituted
heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered
heteroaryl, or 5 to 6 membered heteroaryl), and [0082] (b) alkyl
(e.g., C.sub.1-C.sub.20, C.sub.1-C.sub.12, C.sub.1-C.sub.8,
C.sub.1-C.sub.6, C.sub.1-C.sub.4, or C.sub.1-C.sub.2), alkenyl
(e.g., C.sub.2-C.sub.20, C.sub.2-C.sub.12, C.sub.2-C.sub.8,
C.sub.2-C.sub.6, or C.sub.2-C.sub.4), alkynyl (e.g.,
C.sub.2-C.sub.20, C2-C12, C.sub.2-C.sub.8, C.sub.2-C.sub.6, or
C.sub.2-C.sub.4), heteroalkyl (e.g., 2 to 20 membered, 2 to 12
membered, 2 to 8 membered, 2 to 6 membered, 4 to 6 membered, 2 to 3
membered, or 4 to 5 membered), cycloalkyl (e.g., C.sub.3-C.sub.10,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or
C.sub.5-C.sub.6), cycloalkenyl (e.g., C.sub.3-C.sub.10,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C5-C6),
cycloalkynyl (e.g., C.sub.3-C.sub.10, C.sub.3-C.sub.8,
C.sub.3-C.sub.6, C.sub.4-C.sub.6, or C.sub.5-C.sub.6),
heteroalicyclyl (e.g., 3 to 10 membered, 3 to 8 membered, 3 to 6
membered, 4 to 6 membered, 4 to 5 membered, or 5 to 6 membered),
aryl (e.g., C.sub.6-C.sub.12, C.sub.6-C.sub.10, or phenyl), or
heteroaryl (e.g., 5 to 12 membered, 5 to 10 membered, 5 to 9
membered, or 5 to 6 membered), substituted with at least one
substituent selected from: oxo, halogen, --CCl.sub.3, --CBr.sub.3,
--CF.sub.3, --CI.sub.3, --CHCl.sub.2, --CHBr.sub.2, --CHF.sub.2,
--CHI.sub.2, --CH.sub.2Cl, --CH.sub.2Br, --CH.sub.2F, --CH.sub.2I,
--OCCl.sub.3, --OCF.sub.3, --OCBr.sub.3, --OCI.sub.3,
--OCHCl.sub.2, --OCHBr 2, --OCHI.sub.2, --OCHF.sub.2,
--OCH.sub.2Cl, --OCH.sub.2Br, --OCH.sub.2I, --OCH.sub.2F, --CN,
--OH, --NH 2, --COOH, --CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H,
--OSO.sub.3H, --SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2,
--NHC(O)NHNH.sub.2, --NHC(O)NH.sub.2, --NHSO.sub.2H, --NHC(O)H,
--NHC(O)OH, --NHOH, --N.sub.3, unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4
alkyl), unsubstituted alkenyl (e.g., C.sub.2-C.sub.8 alkenyl,
C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.4 alkenyl), unsubstituted
alkynyl (e.g., C.sub.2-C.sub.8 alkynyl, C.sub.2-C.sub.6 alkynyl, or
C2-C4 alkynyl), unsubstituted heteroalkyl (e.g., 2 to 8 membered
heteroalkyl, 2 to 6 membered heteroalkyl, or 2 to 4 membered
heteroalkyl), unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8
cycloalkyl, C.sub.3-C.sub.6 cycloalkyl, or C.sub.5-C.sub.6
cycloalkyl), unsubstituted cycloalkenyl (e.g., C.sub.3-C.sub.8
cycloalkenyl, C.sub.3-C.sub.6 cycloalkenyl, or C.sub.5-C.sub.6
cycloalkenyl), unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8
cycloalkynyl, C.sub.3-C.sub.6 cycloalkynyl, or C.sub.5-C.sub.6
cycloalkynyl), unsubstituted heteroalicyclyl (e.g., 3 to 8 membered
heteroalicyclyl, 3 to 6 membered heteroalicyclyl, or 5 to 6
membered heteroalicyclyl), unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl), or unsubstituted
heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered
heteroaryl, or 5 to 6 membered heteroaryl).
[0083] A "size-limited substituent" or "size-limited substituent
group," as used herein, means a group selected from all of the
substituents described above for a "substituent group," wherein
each substituted or unsubstituted alkyl is a substituted or
unsubstituted C.sub.1-C.sub.20 alkyl, each substituted or
unsubstituted alkenyl is a substituted or unsubstituted
C.sub.2-C.sub.20 alkenyl, each substituted or unsubstituted alkynyl
is a substituted or unsubstituted C.sub.2-C.sub.20 alkynyl, each
substituted or unsubstituted heteroalkyl is a substituted or
unsubstituted 2 to 20 membered heteroalkyl, each substituted or
unsubstituted cycloalkyl is a substituted or unsubstituted
C.sub.3-C.sub.8 cycloalkyl, each substituted or unsubstituted
cycloalkenyl is a substituted or unsubstituted C.sub.3-C.sub.8
cycloalkenyl, each substituted or unsubstituted cycloalkynyl is a
substituted or unsubstituted C.sub.3-C.sub.8 cycloalkynyl, each
substituted or unsubstituted heteroalicyclyl is a substituted or
unsubstituted 3 to 8 membered heteroalicyclyl, each substituted or
unsubstituted aryl is a substituted or unsubstituted
C.sub.6-C.sub.10 aryl, and each substituted or unsubstituted
heteroaryl is a substituted or unsubstituted 5 to 10 membered
heteroaryl.
[0084] A "lower substituent" or "lower substituent group," as used
herein, means a group selected from all of the substituents
described above for a "substituent group," wherein each substituted
or unsubstituted alkyl is a substituted or unsubstituted
C.sub.1-C.sub.8 alkyl, each substituted or unsubstituted alkenyl is
a substituted or unsubstituted C.sub.2-C.sub.8 alkenyl, each
substituted or unsubstituted alkynyl is a substituted or
unsubstituted C.sub.2-C.sub.8 alkynyl, each substituted or
unsubstituted heteroalkyl is a substituted or unsubstituted 2 to 8
membered heteroalkyl, each substituted or unsubstituted cycloalkyl
is a substituted or unsubstituted C.sub.3-C.sub.7 cycloalkyl, each
substituted or unsubstituted cycloalkenyl is a substituted or
unsubstituted C.sub.3-C.sub.7 cycloalkenyl, each substituted or
unsubstituted cycloalkynyl is a substituted or unsubstituted
C.sub.3-C.sub.7 cycloalkynyl, each substituted or unsubstituted
heteroalicyclyl is a substituted or unsubstituted 3 to 7 membered
heteroalicyclyl, each substituted or unsubstituted aryl is a
substituted or unsubstituted C.sub.6-C.sub.10 aryl, and each
substituted or unsubstituted heteroaryl is a substituted or
unsubstituted 5 to 9 membered heteroaryl.
[0085] In some embodiments, each substituted group described in the
compounds herein is substituted with at least one substituent
group. More specifically, in some embodiments, each substituted
alkyl, substituted alkenyl, substituted alkynyl, substituted
heteroalkyl, substituted cycloalkyl, substituted cycloalkenyl,
substituted cycloalkynyl, substituted heteroalicyclyl, substituted
aryl, substituted heteroaryl, substituted alkylene, substituted
heteroalkylene, substituted cycloalkylene, substituted
heteroalicyclylene, substituted arylene, and/or substituted
heteroarylene described in the compounds herein are substituted
with at least one substituent group. In other embodiments, at least
one or all of these groups are substituted with at least one
size-limited substituent group. In other embodiments, at least one
or all of these groups are substituted with at least one lower
substituent group.
[0086] In other embodiments of the compounds herein, each
substituted or unsubstituted alkyl may be a substituted or
unsubstituted C.sub.1-C.sub.20 alkyl, each substituted or
unsubstituted alkenyl may be a substituted or unsubstituted
C.sub.2-C.sub.20 alkenyl, each substituted or unsubstituted alkynyl
may be a substituted or unsubstituted C.sub.2-C.sub.20 alkynyl,
each substituted or unsubstituted heteroalkyl is a substituted or
unsubstituted 2 to 20 membered heteroalkyl, each substituted or
unsubstituted cycloalkyl is a substituted or unsubstituted
C.sub.3-C.sub.8 cycloalkyl, each substituted or unsubstituted
cycloalkenyl is a substituted or unsubstituted C.sub.3-C.sub.8
cycloalkenyl, each substituted or unsubstituted cycloalkynyl is a
substituted or unsubstituted C.sub.3-C.sub.8 cycloalkynyl, each
substituted or unsubstituted heteroalicyclyl is a substituted or
unsubstituted 3 to 8 membered heteroalicyclyl, each substituted or
unsubstituted aryl is a substituted or unsubstituted
C.sub.6-C.sub.10 aryl, and/or each substituted or unsubstituted
heteroaryl is a substituted or unsubstituted 5 to 10 membered
heteroaryl. In some embodiments of the compounds herein, each
substituted or unsubstituted alkylene is a substituted or
unsubstituted C.sub.1-C.sub.20 alkylene, each substituted or
unsubstituted alkenylene is a substituted or unsubstituted
C.sub.2-C.sub.20 alkenylene, each substituted or unsubstituted
alkynylene is a substituted or unsubstituted C.sub.2-C.sub.20
alkynylene, each substituted or unsubstituted heteroalkylene is a
substituted or unsubstituted 2 to 20 membered heteroalkylene, each
substituted or unsubstituted cycloalkylene is a substituted or
unsubstituted C.sub.3-C.sub.8 cycloalkylene, each substituted or
unsubstituted cycloalkenylene is a substituted or unsubstituted
C.sub.3-C.sub.8 cycloalkenylene, each substituted or unsubstituted
cycloalkynylene is a substituted or unsubstituted C.sub.3-C.sub.8
cycloalkynylene, each substituted or unsubstituted
heteroalicyclylene is a substituted or unsubstituted 3 to 8
membered heteroalicyclylene, each substituted or unsubstituted
arylene is a substituted or unsubstituted C.sub.6-C.sub.10 arylene,
and/or each substituted or unsubstituted heteroarylene is a
substituted or unsubstituted 5 to 10 membered heteroarylene.
[0087] In some embodiments, each substituted or unsubstituted alkyl
is a substituted or unsubstituted C.sub.1-C.sub.8 alkyl, each
substituted or unsubstituted alkenyl is a substituted or
unsubstituted C.sub.2-C.sub.8 alkenyl, each substituted or
unsubstituted alkynyl is a substituted or unsubstituted
C.sub.2-C.sub.8 alkynyl, each substituted or unsubstituted
heteroalkyl is a substituted or unsubstituted 2 to 8 membered
heteroalkyl, each substituted or unsubstituted cycloalkyl is a
substituted or unsubstituted C.sub.3-C.sub.7 cycloalkyl, each
substituted or unsubstituted cycloalkenyl is a substituted or
unsubstituted C.sub.3-C.sub.7 cycloalkenyl, each substituted or
unsubstituted cycloalkynyl is a substituted or unsubstituted
C.sub.3-C.sub.7 cycloalkynyl, each substituted or unsubstituted
heteroalicyclyl is a substituted or unsubstituted 3 to 7 membered
heteroalicyclyl, each substituted or unsubstituted aryl is a
substituted or unsubstituted C.sub.6-C.sub.10 aryl, and/or each
substituted or unsubstituted heteroaryl is a substituted or
unsubstituted 5 to 9 membered heteroaryl. In some embodiments, each
substituted or unsubstituted alkylene is a substituted or
unsubstituted C.sub.1-C.sub.8 alkylene, each substituted or
unsubstituted alkenylene is a substituted or unsubstituted
C.sub.2-C.sub.8 alkenylene, each substituted or unsubstituted
alkynylene is a substituted or unsubstituted C.sub.2-C.sub.8
alkynylene, each substituted or unsubstituted heteroalkylene is a
substituted or unsubstituted 2 to 8 membered heteroalkylene, each
substituted or unsubstituted cycloalkylene is a substituted or
unsubstituted C.sub.3-C.sub.7 cycloalkylene, each substituted or
unsubstituted cycloalkenylene is a substituted or unsubstituted
C.sub.3-C.sub.7 cycloalkenylene, each substituted or unsubstituted
cycloalkynylene is a substituted or unsubstituted C.sub.3-C.sub.7
cycloalkynylene, each substituted or unsubstituted
heteroalicyclylene is a substituted or unsubstituted 3 to 7
membered heteroalicyclylene, each substituted or unsubstituted
arylene is a substituted or unsubstituted C.sub.6-C.sub.10 arylene,
and/or each substituted or unsubstituted heteroarylene is a
substituted or unsubstituted 5 to 9 membered heteroarylene. In some
embodiments, the compound is a chemical species set forth in the
application (e.g., Examples section, figures, or tables below).
[0088] In embodiments, a substituted or unsubstituted moiety (e.g.,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted cycloalkenyl, substituted or
unsubstituted cycloalkynyl, substituted or unsubstituted
heteroalicyclyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted alkylene,
substituted or unsubstituted alkenylene, substituted or
unsubstituted alkynylene, substituted or unsubstituted
heteroalkylene, substituted or unsubstituted cycloalkylene,
substituted or unsubstituted cycloalkenylene, substituted or
unsubstituted cycloalkynylene, substituted or unsubstituted
heteroalicyclylene, substituted or unsubstituted arylene, and/or
substituted or unsubstituted heteroarylene) is unsubstituted (e.g.,
is an unsubstituted alkyl, unsubstituted alkenyl, unsubstituted
alkynyl, unsubstituted heteroalkyl, unsubstituted cycloalkyl,
unsubstituted cycloalkenyl, unsubstituted cycloalkynyl,
unsubstituted heteroalicyclyl, unsubstituted aryl, unsubstituted
heteroaryl, unsubstituted alkylene, unsubstituted alkenylene,
unsubstituted alkynylene, unsubstituted heteroalkylene,
unsubstituted cycloalkylene, unsubstituted cycloalkenylene,
unsubstituted cycloalkynylene, unsubstituted heteroalicyclylene,
unsubstituted arylene, and/or unsubstituted heteroarylene,
respectively). In embodiments, a substituted or unsubstituted
moiety (e.g., substituted or unsubstituted alkyl, substituted or
unsubstituted alkenyl, substituted or unsubstituted alkynyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
cycloalkenyl, substituted or unsubstituted cycloalkynyl,
substituted or unsubstituted heteroalicyclyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl,
substituted or unsubstituted alkylene, substituted or unsubstituted
alkenylene, substituted or unsubstituted alkynylene, substituted or
unsubstituted heteroalkylene, substituted or unsubstituted
cycloalkylene, substituted or unsubstituted cycloalkenylene,
substituted or unsubstituted cycloalkynylene, substituted or
unsubstituted heteroalicyclylene, substituted or unsubstituted
arylene, and/or substituted or unsubstituted heteroarylene) is
substituted (e.g., is a substituted alkyl, substituted alkenyl,
substituted alkynyl, substituted heteroalkyl, substituted
cycloalkyl, substituted cycloalkenyl, substituted cycloalkynyl,
substituted heteroalicyclyl, substituted aryl, substituted
heteroaryl, substituted alkylene, substituted alkenylene,
substituted alkynylene, substituted heteroalkylene, substituted
cycloalkylene, substituted cycloalkenylene, substituted
cycloalkynylene, substituted heteroalicyclylene, substituted
arylene, and/or substituted heteroarylene, respectively).
[0089] In embodiments, a substituted moiety (e.g., substituted
alkyl, substituted alkenyl, substituted alkynyl, substituted
heteroalkyl, substituted cycloalkyl, substituted cycloalkenyl,
substituted cycloalkynyl, substituted heteroalicyclyl, substituted
aryl, substituted heteroaryl, substituted alkylene, substituted
alkenylene, substituted alkynylene, substituted heteroalkylene,
substituted cycloalkylene, substituted cycloalkenylene, substituted
cycloalkynylene, substituted heteroalicyclylene, substituted
arylene, and/or substituted heteroarylene) is substituted with at
least one substituent group, wherein if the substituted moiety is
substituted with a plurality of substituent groups, each
substituent group may optionally be different. In embodiments, if
the substituted moiety is substituted with a plurality of
substituent groups, each substituent group is different.
[0090] In embodiments, a substituted moiety (e.g., substituted
alkyl, substituted alkenyl, substituted alkynyl, substituted
heteroalkyl, substituted cycloalkyl, substituted cycloalkenyl,
substituted cycloalkynyl, substituted heteroalicyclyl, substituted
aryl, substituted heteroaryl, substituted alkylene, substituted
alkenylene, substituted alkynylene, substituted heteroalkylene,
substituted cycloalkylene, substituted cycloalkenylene, substituted
cycloalkynylene, substituted heteroalicyclylene, substituted
arylene, and/or substituted heteroarylene) is substituted with at
least one size-limited substituent group, wherein if the
substituted moiety is substituted with a plurality of size-limited
substituent groups, each size-limited substituent group may
optionally be different. In embodiments, if the substituted moiety
is substituted with a plurality of size-limited substituent groups,
each size-limited substituent group is different.
[0091] In embodiments, a substituted moiety (e.g., substituted
alkyl, substituted alkenyl, substituted alkynyl, substituted
heteroalkyl, substituted cycloalkyl, substituted cycloalkenyl,
substituted cycloalkynyl, substituted heteroalicyclyl, substituted
aryl, substituted heteroaryl, substituted alkylene, substituted
alkenylene, substituted alkynylene, substituted heteroalkylene,
substituted cycloalkylene, substituted cycloalkenylene, substituted
cycloalkynylene, substituted heteroalicyclylene, substituted
arylene, and/or substituted heteroarylene) is substituted with at
least one lower substituent group, wherein if the substituted
moiety is substituted with a plurality of lower substituent groups,
each lower substituent group may optionally be different. In
embodiments, if the substituted moiety is substituted with a
plurality of lower substituent groups, each lower substituent group
is different.
[0092] In embodiments, a substituted moiety (e.g., substituted
alkyl, substituted alkenyl, substituted alkynyl, substituted
heteroalkyl, substituted cycloalkyl, substituted cycloalkenyl,
substituted cycloalkynyl, substituted heteroalicyclyl, substituted
aryl, substituted heteroaryl, substituted alkylene, substituted
alkenylene, substituted alkynylene, substituted heteroalkylene,
substituted cycloalkylene, substituted cycloalkenylene, substituted
cycloalkynylene, substituted heteroalicyclylene, substituted
arylene, and/or substituted heteroarylene) is substituted with at
least one substituent group, size-limited substituent group, or
lower substituent group; wherein if the substituted moiety is
substituted with a plurality of groups selected from substituent
groups, size-limited substituent groups, and lower substituent
groups; each substituent group, size-limited substituent group,
and/or lower substituent group may optionally be different. In
embodiments, if the substituted moiety is substituted with a
plurality of groups selected from substituent groups, size-limited
substituent groups, and lower substituent groups; each substituent
group, size-limited substituent group, and/or lower substituent
group is different.
[0093] Certain compounds of the present disclosure possess
asymmetric carbon atoms (optical or chiral centers) or double
bonds; the enantiomers, racemates, diastereomers, tautomers,
geometric isomers, stereoisometric forms that may be defined, in
terms of absolute stereochemistry, as (R)- or (S)- or, as (D)- or
(L)- for amino acids, and individual isomers are encompassed within
the scope of the present disclosure. The compounds of the present
disclosure do not include those that are known in art to be too
unstable to synthesize and/or isolate. The present disclosure is
meant to include compounds in racemic and optically pure forms.
Optically active (R)- and (S)-, or (D)- and (L)-isomers may be
prepared using chiral synthons or chiral reagents, or resolved
using conventional techniques. When the compounds described herein
contain olefinic bonds or other centers of geometric asymmetry, and
unless specified otherwise, it is intended that the compounds
include both E and Z geometric isomers.
[0094] As used herein, the term "isomers" refers to compounds
having the same number and kind of atoms, and hence the same
molecular weight, but differing in respect to the structural
arrangement or configuration of the atoms.
[0095] The term "tautomer," as used herein, refers to one of two or
more structural isomers which exist in equilibrium and which are
readily converted from one isomeric form to another.
[0096] It will be apparent to one skilled in the art that certain
compounds of this disclosure may exist in tautomeric forms, all
such tautomeric forms of the compounds being within the scope of
the disclosure.
[0097] Unless otherwise stated, structures depicted herein are also
meant to include all stereochemical forms of the structure; i.e.,
the R and S configurations for each asymmetric center. Therefore,
single stereochemical isomers as well as enantiomeric and
diastereomeric mixtures of the present compounds are within the
scope of the disclosure.
[0098] Unless otherwise stated, structures depicted herein are also
meant to include compounds which differ only in the presence of one
or more isotopically enriched atoms. For example, compounds having
the present structures except for the replacement of a hydrogen by
a deuterium or tritium, or the replacement of a carbon by .sup.13C-
or .sup.14C-enriched carbon are within the scope of this
disclosure.
[0099] The compounds of the present disclosure may also contain
unnatural proportions of atomic isotopes at one or more of the
atoms that constitute such compounds. For example, the compounds
may be radiolabeled with radioactive isotopes, such as for example
tritium (.sup.3H), iodine-125 (.sup.125I), or carbon-14 (.sup.14C).
All isotopic variations of the compounds of the present disclosure,
whether radioactive or not, are encompassed within the scope of the
present disclosure.
[0100] It should be noted that throughout the application that
alternatives are written in Markush groups, for example, each amino
acid position that contains more than one possible amino acid. It
is specifically contemplated that each member of the Markush group
should be considered separately, thereby comprising another
embodiment, and the Markush group is not to be read as a single
unit.
[0101] "Analog" or "analogue" is used in accordance with its plain
ordinary meaning within Chemistry and Biology and refers to a
chemical compound that is structurally similar to another compound
(i.e., a so-called "reference" compound) but differs in
composition, e.g., in the replacement of one atom by an atom of a
different element, or in the presence of a particular functional
group, or the replacement of one functional group by another
functional group, or the absolute stereochemistry of one or more
chiral centers of the reference compound. Accordingly, an analog is
a compound that is similar or comparable in function and appearance
but not in structure or origin to a reference compound.
[0102] The terms "a" or "an", as used in herein means one or more.
In addition, the phrase "substituted with a[n]", as used herein,
means the specified group may be substituted with one or more of
any or all of the named substituents. For example, where a group,
such as an alkyl or heteroaryl group, is "substituted with an
unsubstituted C.sub.1-C.sub.20 alkyl, or unsubstituted 2 to 20
membered heteroalkyl", the group may contain one or more
unsubstituted C.sub.1-C.sub.20 alkyls, and/or one or more
unsubstituted 2 to 20 membered heteroalkyls.
[0103] As used herein, the term "about" means a range of values
including the specified value, which a person of ordinary skill in
the art would consider reasonably similar to the specified value.
In embodiments, about means within a standard deviation using
measurements generally acceptable in the art. In embodiments, about
means a range extending to +/-10% of the specified value. In
embodiments, about includes the specified value.
[0104] In this disclosure, "comprises", "comprising", "containing",
and "having" and the like can have the meaning ascribed to them in
U.S. Patent law and can mean "includes", "including", and the like.
"Consisting essentially of" or "consists essentially" likewise has
the meaning ascribed in U.S. Patent law and the term is open-ended,
allowing for the presence of more than that which is recited so
long as basic or novel characteristics of that which is recited is
not changed by the presence of more than that which is recited, but
excludes prior art embodiments.
[0105] Moreover, where a moiety is substituted with an R
substituent, the group may be referred to as "R-substituted." Where
a moiety is R-substituted, the moiety is substituted with at least
one R substituent and each R substituent is optionally different.
Where a particular R group is present in the description of a
chemical genus (such as Formula (I)), a Roman alphabetic symbol may
be used to distinguish each appearance of that particular R group.
For example, where multiple R.sup.13 substituents are present, each
R.sup.13 substituent may be distinguished as R.sup.13A, R.sup.13B,
R.sup.13C, R.sup.13D, etc., wherein each of R.sup.13A, R.sup.13B,
R.sup.13C, R.sup.13D, etc. is defined within the scope of the
definition of R.sup.13 and optionally differently.
[0106] A "detectable agent" or "detectable moiety" is a substance,
element, compound, or composition; or moiety thereof, detectable by
appropriate means such as spectroscopic, photochemical,
biochemical, immunochemical, chemical, magnetic resonance imaging,
or other physical means. For example, useful detectable agents
include .sup.18F, .sup.32P, .sup.33P, .sup.45Ti, .sup.47Sc,
.sup.52Fe, .sup.9Fe .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.67Ga
.sup.68Ga, .sup.77As, .sup.86Y, .sup.90Y, .sup.89Sr, .sup.89Zr,
.sup.94Tc, .sup.94Tc, .sup.99mTc, .sup.99Mo, .sup.105Pd,
.sup.105Ph, .sup.111Ag, .sup.111In, .sup.123I, .sup.124I,
.sup.125I, .sup.131I, .sup.142Pr, .sup.143Pr, .sup.149Pm,
.sup.153Sm, .sup.154-1581Gd, .sup.161Tb, .sup.166Dy, .sup.166Ho,
.sup.169Er, .sup.175Lu, .sup.177Lu, .sup.186Re, .sup.88Re,
.sup.189Re, .sup.194Ir, .sup.198Au, .sup.199Au, .sup.211At,
.sup.211Pb, .sup.212Bi, .sup.212Pb, .sup.213Bi, .sup.223Ra,
.sup.225Ac, Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm, Eu,
Gd, Tb, Dy, Ho, Er, Tm, Yb, Lu, .sup.32P, fluorophore (e.g.,
fluorescent dyes), electron-dense reagents, enzymes (e.g., as
commonly used in an ELISA), biotin, digoxigenin, paramagnetic
molecules, paramagnetic nanoparticles, ultrasmall superparamagnetic
iron oxide ("USPIO") nanoparticles, USPIO nanoparticle aggregates,
superparamagnetic iron oxide ("SPIO") nanoparticles, SPIO
nanoparticle aggregates, monochrystalline iron oxide nanoparticles,
monochrystalline iron oxide, nanoparticle contrast agents,
liposomes or other delivery vehicles containing Gadolinium chelate
("Gd-chelate") molecules, Gadolinium, radioisotopes, radionuclides
(e.g., carbon-11, nitrogen-13, oxygen-15, fluorine-18,
rubidium-82), fluorodeoxyglucose (e.g., fluorine-18 labeled), any
gamma ray emitting radionuclides, positron-emitting radionuclide,
radiolabeled glucose, radiolabeled water, radiolabeled ammonia,
biocolloids, microbubbles (e.g., including microbubble shells
including albumin, galactose, lipid, and/or polymers; microbubble
gas core including air, heavy gas(es), perfluorcarbon, nitrogen,
octafluoropropane, perflexane lipid microsphere, perflutren, etc.),
iodinated contrast agents (e.g., iohexol, iodixanol, ioversol,
iopamidol, ioxilan, iopromide, diatrizoate, metrizoate, ioxaglate),
barium sulfate, thorium dioxide, gold, gold nanoparticles, gold
nanoparticle aggregates, fluorophores, two-photon fluorophores, or
haptens and proteins or other entities which can be made
detectable, e.g., by incorporating a radiolabel into a peptide
specifically reactive with a target peptide. A detectable moiety is
a monovalent detectable agent or a detectable agent capable of
forming a bond with another composition.
[0107] Radioactive substances (e.g., radioisotopes) that may be
used as imaging and/or labeling agents in accordance with the
embodiments of the disclosure include, but are not limited to,
.sup.18F, .sup.32P, .sup.33P, .sup.45Ti, .sup.47Sc, .sup.52Fe,
.sup.59Fe, .sup.62Cu, .sup.64Cu, .sup.67Cu, .sup.67Ga .sup.68Ga,
.sup.77As, .sup.86Y, .sup.90Y, .sup.89Sr, .sup.89Zr, .sup.94Tc,
.sup.94Tc, .sup.99mTc, .sup.99Mo, .sup.105Pd, .sup.105Ph,
.sup.111Ag, .sup.111In, .sup.123I, .sup.124I, .sup.125I, .sup.131I,
.sup.142Pr, .sup.143Pr, .sup.149Pm, .sup.153Sm, .sup.154-158Gd,
.sup.161Tb, .sup.166Dy, .sup.166Ho, .sup.169Er, .sup.175Lu,
.sup.177Lu, .sup.186Re, .sup.188Re .sup.189Re, .sup.194Ir,
.sup.198Au, .sup.199Au, .sup.211At, .sup.211Pb, .sup.212Bi,
.sup.212Pb, .sup.213Bi, .sup.223Ra, and .sup.225Ac. Paramagnetic
ions that may be used as additional imaging agents in accordance
with the embodiments of the disclosure include, but are not limited
to, ions of transition and lanthanide metals (e.g., metals having
atomic numbers of 21-29, 42, 43, 44, or 57-71). These metals
include ions of Cr, V, Mn, Fe, Co, Ni, Cu, La, Ce, Pr, Nd, Pm, Sm,
Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu.
[0108] The terms "polypeptide", "peptide", and "protein" are used
interchangeably herein to refer to a polymer of amino acid
residues, wherein the polymer may optionally be conjugated to a
moiety that does not consist of amino acids. The terms apply to
amino acid polymers in which one or more amino acid residue is an
artificial chemical mimetic of a corresponding naturally occurring
amino acid, as well as to naturally occurring amino acid polymers
and non-naturally occurring amino acid polymer.
[0109] As used herein, "oxime" refers to a moiety having the
following structure:
##STR00008##
[0110] The oxime moiety can be attached to another moiety via the
oxygen atom in which case the moiety can be referred to as an
"O-oxime" moiety. An aldoxime moiety will have a hydrogen at one of
R or R'. A ketoxime will have hydrogen at neither R nor R'. Oximes
are exemplary protecting groups in some embodiments disclosed
herein. A 3'-O-oxime is an oxime moiety attached to the 3' carbon
of a nucleotide or nucleoside through the oxime oxygen.
[0111] As used herein, "polymerase" can be used to refer to a
nucleic acid synthesizing enzyme, including but not limited to, DNA
polymerase, RNA polymerase, reverse transcriptase, primase and
transferase. Typically, the polymerase has one or more active sites
at which nucleotide binding and/or catalysis of nucleotide
polymerization may occur. The polymerase may catalyze the
polymerization of nucleotides to the 3' end of the first strand of
the double stranded nucleic acid molecule. For example, a
polymerase can catalyze the addition of a next correct nucleotide
to the 3' oxygen group of the first strand of the double stranded
nucleic acid molecule via a phosphodiester bond, thereby covalently
incorporating the nucleotide to the first strand of the double
stranded nucleic acid molecule. Optionally, a polymerase need not
be capable of nucleotide incorporation under one or more conditions
used in a method set forth herein. For example, a mutant polymerase
may be capable of forming a ternary complex but incapable of
catalyzing nucleotide incorporation.
[0112] As used herein, "primed template nucleic acid" or "primed
template" refers to a nucleic acid having a double stranded region
such that one of the strands is a primer and the other strand is a
template. The two strands can be parts of a contiguous nucleic acid
molecule (e.g. a hairpin structure) or the two strands can be
separable molecules that are not covalently attached to each
other.
[0113] As used herein, "primer" refers to a nucleic acid having a
sequence that binds to a nucleic acid at or near a template
sequence. In particular configurations, the primer binds in a
configuration that allows replication of the template, for example,
via polymerase extension of the primer or ligase extension of the
primer. The primer can be a first portion of a nucleic acid
molecule that binds to a second portion of the nucleic acid
molecule, the first portion being a primer sequence and the second
portion being a primer binding sequence (e.g. a hairpin primer).
Alternatively, the primer can be a first nucleic acid molecule that
binds to a second nucleic acid molecule having the template
sequence. A primer can consist of DNA, RNA or analogs thereof. A
primer can have an extendible 3' end or a 3' end that is blocked,
such as reversibly terminated, so as to preclude primer extension
until removal of the blocking moiety.
[0114] As used herein, "site," when used in reference to an array,
means a location in an array where a particular molecule is
present. A site can contain only a single molecule or it can
contain a population of several molecules of the same species (e.g.
an ensemble of the molecules). Alternatively, a site can include a
population of molecules that are different species (e.g. a
population of different template sequences). Sites of an array are
typically discrete. The discrete sites can be contiguous or they
can have spaces between each other. An array useful herein can
have, for example, sites that are separated by less than 100
microns, 50 microns, 10 microns, 5 microns, 1 micron, or 0.5
micron. Alternatively or additionally, an array can have sites that
are separated by greater than 0.5 micron, 1 micron, 5 microns, 10
microns, 50 microns or 100 microns. The sites can each have an area
of less than 1 square millimeter, 500 square microns, 100 square
microns, 25 square microns, 1 square micron or less. The density of
sites of sites in an array can be, for example, at least about 10
sites/cm.sup.2, 100 sites/cm.sup.2, 1.times.10.sup.3
sites/cm.sup.2, 1.times.10.sup.4 sites/cm.sup.2, 1.times.10.sup.5
sites/cm.sup.2, 1.times.10.sup.6 sites/cm.sup.2, or higher.
[0115] As used herein, "solid support" or "solid phase support"
refers to a rigid substrate that is insoluble in aqueous liquid.
The substrate can be non-porous or porous. The substrate can
optionally be capable of taking up a liquid (e.g. due to porosity)
but will typically be sufficiently rigid that the substrate does
not swell substantially when taking up the liquid and does not
contract substantially when the liquid is removed by drying. A
nonporous solid support is generally impermeable to liquids or
gases. Exemplary solid supports include, but are not limited to,
glass and modified or functionalized glass, plastics (including
acrylics, polystyrene and copolymers of styrene and other
materials, polypropylene, polyethylene, polybutylene,
polyurethanes, Teflon.TM., cyclic olefins, polyimides etc.), nylon,
ceramics, resins, Zeonor, silica or silica-based materials
including silicon and modified silicon, carbon, metals, inorganic
glasses, optical fiber bundles, and polymers.
[0116] As used herein, the term "surface" refers to a portion of a
solid support that contacts a fluid. The fluid can be gas or
liquid. The surface can be substantially flat or planar.
Alternatively, the surface can be rounded or contoured. Exemplary
contours that can be included on a surface are wells, depressions,
pillars, ridges, channels or the like. Exemplary materials that can
be used as a solid support include, but are not limited to, those
set forth above.
[0117] As used herein, "ternary complex" refers to an
intermolecular association between a polymerase, a double stranded
nucleic acid and a nucleotide. Typically, the polymerase
facilitates interaction between a next correct nucleotide and a
template strand of the primed nucleic acid. A next correct
nucleotide can interact with the template strand via Watson-Crick
hydrogen bonding. The term "stabilized ternary complex" means a
ternary complex having promoted or prolonged existence or a ternary
complex for which disruption has been inhibited. Generally,
stabilization of the ternary complex prevents covalent
incorporation of the nucleotide component of the ternary complex
into the primed nucleic acid component of the ternary complex.
[0118] As used herein, "type" is used to identify molecules that
share the same chemical structure. For example, a mixture of
nucleotides can include several dCTP molecules. The dCTP molecules
will be understood to be the same type of nucleotide as each other,
but a different type of nucleotide compared to dATP, dGTP, dTTP
etc. Similarly, individual DNA molecules that have the same
sequence of nucleotides are the same type, whereas DNA molecules
with different sequences are different types. The term "type" can
also identify moieties that share the same chemical structure. For
example, the cytosine bases in a template nucleic acid will be
understood to be the same type of base as each other independent of
their position in the template sequence.
[0119] As used herein, a "vessel" is a container that functions to
isolate one chemical process (e.g., a binding event; an
incorporation reaction; addition of a nucleotide to an extending
chain, removal of a reversible terminator from a 3' end of a
blocked nucleotide or extending primer; or replacement of a
protecting moiety by a blocking moiety; or other reaction such as a
reaction involving nucleic acid chemistry) from another, or to
provide a space in which a chemical process can take place.
Non-limiting examples of vessels useful in connection with the
disclosed technique include: flow cells, wells of a multi-well
plate; microscope slides; tubes (e.g., capillary tubes); droplets,
vesicles, test tubes, trays, centrifuge tubes, features in an
array, tubing, channels in a substrate or other volumes used in
nucleic acid chemistry. As used herein, a "manufactured vessel" is
a container that is human-made or human-modified and that functions
to isolate one chemical process (e.g., a binding event; an
incorporation reaction; addition or removal of a protecting moiety,
addition or removal of a blocking moiety such as a reversible
terminator) from another, or to provide a space in which a chemical
process can take place.
[0120] As used herein, the terms "degrade" and "degradation", when
referring to a nucleotide, or a portion of the nucleotide, mean
chemical modification of the nucleotide, or a portion of the
nucleotide, resulting in a different molecule having at least one
modulated function or activity relative to the original (i.e.,
undegraded) nucleotide, or portion of the nucleotide. In some
embodiments the terms "degrade" and "degradation", when referring
to a nucleobase, mean chemical modification of the nucleobase
resulting in a modulated activity or function of the nucleobase or
the nucleotide or nucleoside including the nucleobase. In
embodiments, degradation of a nucleobase may be referred to as
"nucleotide degradation". In embodiments degradation of a specific
type of nucleobase or nucleotide may be referred to as degradation
of the specific nucleobase type or nucleotide type, for example "dC
degradation" refers to degradation of a deoxycytidine (e.g.,
degradation of a cytosine nucleobase in a deoxycytidine,
degradation of a deoxycytidine in a deoxycytidine triphosphate,
degradation of a cytosine nucleobase in an DNA nucleic acid). FIG.
3 depicts an example of a reaction resulting in a degraded dC.
[0121] The embodiments set forth below and recited in the claims
can be understood in view of the above definitions.
Compositions and Methods
[0122] The present disclosure provides compositions that comprise a
solution containing a nucleotide having a protecting group such as
a 3'-O-oxime moiety and a reagent having the structure R2 tethered
to a reversible terminator such as R2-ONH.sub.2, wherein R2 is
--C(R6)(R7)(R8), --C(O)(R6), --P(O)(R6)(R7),
--C(R6)(.dbd.C(R7)(R8)), --Si(R6)(R7)(R8), --S O.sub.2(R6),
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C5-C6), substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6), substituted or unsubstituted cycloalkynyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6), substituted
or unsubstituted heteroalicyclyl (e.g., 3 to 8 membered, 3 to 6
membered, or 5 to 6 membered), substituted or unsubstituted aryl
(e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl), or
substituted or unsubstituted heteroaryl (e.g., 5 to 10 membered
heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered
heteroaryl). In embodiments, R2 has a molecular weight greater than
methyl. In embodiments, R2 has a molecular weight greater than
ethyl. In embodiments, R2 has a molecular weight greater than 36
g/mol. In embodiments, R2 has a molecular weight greater than 57
g/mol. In embodiments, R2 has a molecular weight greater than 100
g/mol. In embodiments, R2 has a molecular weight greater than 200
g/mol. In embodiments, R2 has a molecular weight greater than 300
g/mol. In embodiments, R2 has a molecular weight greater than 400
g/mol. In embodiments, R2 has a molecular weight greater than 500
g/mol. In embodiments, R2 has a molecular weight greater than 600
g/mol. In embodiments, R2 has a molecular weight greater than 700
g/mol. In embodiments, R2 has a molecular weight greater than 800
g/mol. In embodiments, R2 has a molecular weight greater than 900
g/mol. In embodiments, R2 has a molecular weight less than or equal
to 1000 g/mol. In embodiments, R2 has a molecular weight less than
1000 g/mol. Although not wishing to be limited by mechanism, it is
believed that R2 moieties having more steric bulk than hydrogen and
methyl yield improved synthetic results compared to smaller
moieties, and produce fewer unwanted modifications to nucleic acids
and nucleotides. See the results of Example 1A below. In
embodiments, R2 is --C(R6)(R7)(R8) and R6, R7, and R8 are not all
hydrogen. Similarly, in some cases R2 is independently alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl,
(heteroalicyclyl)alkyl, or an optionally substituted variant
thereof. In particular configurations, the nucleotide has the
structure:
##STR00009##
[0123] B can be a nucleobase. R1 can independently be a halogen,
OCH.sub.3, H or OH. Q can be independently monophosphate,
diphosphate, triphosphate, hydrogen, or nucleic acid. R3 and R4 can
be independently H, CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally
substituted variant thereof, or an exogenous label moiety. In
embodiments, R3 and R4 can be independently H, CH.sub.3, alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl,
(heteroalicyclyl)alkyl, or an optionally substituted variant
thereof. By way of example, the moiety at the 3' position of the
nucleotide can be an aldoxime (i.e. one of R3 or R4 is hydrogen) or
a ketoxime (neither of R3 or R4 is hydrogen).
[0124] Substituted and unsubstituted B moieties are both consistent
with the disclosure herein. In embodiments, B is substituted. In
embodiments, B is unsubstituted. In embodiments, a substituted B
(e.g., a substituted nucleobase) is substituted with at least one
substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted B is substituted with
a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
B is substituted, it is substituted with at least one substituent
group. In embodiments, when B is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when B is substituted, it is substituted with at least one lower
substituent group. In embodiments, B is a substituted adenine
moiety. In embodiments, B is a substituted cytosine moiety. In
embodiments, B is a substituted guanine moiety. In embodiments, B
is a substituted thymine moiety. In embodiments, B is a substituted
uracil moiety. In embodiments, B is an unsubstituted adenine
moiety. In embodiments, B is an unsubstituted cytosine moiety. In
embodiments, B is an unsubstituted guanine moiety. In embodiments,
B is an unsubstituted thymine moiety. In embodiments, B is an
unsubstituted uracil moiety.
[0125] Similarly, substituted and unsubstituted Q moieties are
consistent with the disclosure herein. In embodiments, Q is
substituted. In embodiments, Q is unsubstituted. In embodiments, a
substituted Q (e.g., a substituted monophosphate, substituted
diphosphate, substituted triphosphate, hydrogen atom, and/or
substituted nucleic acid) is substituted with at least one
substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted Q is substituted with
a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
Q is substituted, it is substituted with at least one substituent
group. In embodiments, when Q is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when Q is substituted, it is substituted with at least one lower
substituent group.
[0126] In embodiments, Q is deoxyribonucleic acid. In embodiments,
Q is ribonucleic acid. In embodiments, Q is independently
monophosphate, diphosphate, triphosphate, or nucleic acid.
[0127] In embodiments, R1 is H. In embodiments, R1 is OH.
[0128] In embodiments, a substituted R3 (e.g., substituted alkyl,
substituted alkenyl, substituted alkynyl, substituted cycloalkyl,
substituted cycloalkenyl, substituted cycloalkynyl, substituted
aryl, substituted heteroaryl, substituted heteroalicyclyl,
substituted aralkyl, substituted heteroaralkyl, and/or substituted
(heteroalicyclyl)alkyl) is substituted with at least one
substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted R3 is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
R3 is substituted, it is substituted with at least one substituent
group. In embodiments, when R3 is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when R3 is substituted, it is substituted with at least one lower
substituent group.
[0129] In embodiments, R3 is independently H, CH.sub.3, alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl,
(heteroalicyclyl)alkyl, or an optionally substituted variant
thereof, or an exogenous label moiety. In embodiments, R3 is
independently H, CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally
substituted variant thereof. In embodiments, R3 is independently an
exogenous label moiety.
[0130] In embodiments, a substituted R4 (e.g., substituted alkyl,
substituted alkenyl, substituted alkynyl, substituted cycloalkyl,
substituted cycloalkenyl, substituted cycloalkynyl, substituted
aryl, substituted heteroaryl, substituted heteroalicyclyl,
substituted aralkyl, substituted heteroaralkyl, and/or substituted
(heteroalicyclyl)alkyl) is substituted with at least one
substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted R4 is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
R4 is substituted, it is substituted with at least one substituent
group. In embodiments, when R4 is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when R4 is substituted, it is substituted with at least one lower
substituent group.
[0131] In embodiments, R4 is independently H, CH.sub.3, alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl,
(heteroalicyclyl)alkyl, or an optionally substituted variant
thereof, or an exogenous label moiety. In embodiments, R4 is
independently H, CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally
substituted variant thereof. In embodiments, R4 is independently an
exogenous label moiety.
[0132] In embodiments, the solution is an aqueous solution. In
embodiments, the nucleotide is attached to a solid support and
wherein the solid support is in contact with the solution. In
embodiments, the reagent that comprises the structure R2-ONH.sub.2
is attached to a solid support in contact with the aqueous
solution. In embodiments, the nucleobase comprises an exogenous
label moiety.
[0133] The present disclosure also provides methods for modifying a
nucleotide. The methods can be carried out by reacting a nucleotide
having a protecting moiety such as a 3'-O-oxime moiety to a reagent
having the structure R2-block, wherein block is a blocking moiety
such as the reversible terminator moiety --ONH.sub.2, to produce a
nucleotide having a 3'-O--NH.sub.2 moiety or other moiety as a
blocking moiety or a reversible terminator. In some cases R2 is
independently alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof. R2 can be an alkyl other than methyl. In
embodiments, R2 is not --CH.sub.3. In embodiments, R2 is not
hydrogen. In embodiments, R2 is not hydrogen, or --CH.sub.3. The
present disclosure also provides a method for modifying a
nucleotide. The method can be carried out by reacting a nucleotide
having a 3'-O-oxime moiety with a reagent having the structure
R2-ONH.sub.2 to produce a nucleotide having a 3'-O--NH.sub.2
moiety, wherein R2 is independently alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,
heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or
an optionally substituted variant thereof.
[0134] In embodiments, a substituted R2 (e.g., substituted alkyl,
substituted alkenyl, substituted alkynyl, substituted cycloalkyl,
substituted cycloalkenyl, substituted cycloalkynyl, substituted
aryl, substituted heteroaryl, substituted heteroalicyclyl,
substituted aralkyl, substituted heteroaralkyl, and/or substituted
(heteroalicyclyl)alkyl) is substituted with at least one
substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted R2 is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
R2 is substituted, it is substituted with at least one substituent
group. In embodiments, when R2 is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when R2 is substituted, it is substituted with at least one lower
substituent group.
[0135] In embodiments, R2 is --C(R6)(R7)(R8), --C(O)(R6),
--P(O)(R6)(R7), --C(R6)(.dbd.C(R7)(R8)), --Si(R6)(R7)(R8), --S
O.sub.2(R6), substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C5-C6), substituted or
unsubstituted cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
or C.sub.5-C.sub.6), substituted or unsubstituted cycloalkynyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6),
substituted or unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered), substituted or
unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or
phenyl), or substituted or unsubstituted heteroaryl (e.g., 5 to 10
membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered
heteroaryl).
[0136] In embodiments, R2 is --C(R6)(R7)(R8). In embodiments, R2 is
--C(O)(R6). In embodiments, R2 is --P(O)(R6)(R7). In embodiments,
R2 is --C(R6)(.dbd.C(R7)(R8)). In embodiments, R2 is
--Si(R6)(R7)(R8). In embodiments, R2 is --SO.sub.2(R6). In
embodiments, R2 is substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is substituted or unsubstituted cycloalkenyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is substituted or unsubstituted cycloalkynyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is substituted or unsubstituted heteroalicyclyl
(e.g., 3 to 8 membered, 3 to 6 membered, or 5 to 6 membered). In
embodiments, R2 is substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In embodiments,
R2 is substituted or unsubstituted heteroaryl (e.g., 5 to 10
membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered
heteroaryl). In embodiments, R2 is unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is unsubstituted cycloalkenyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is unsubstituted cycloalkynyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered). In embodiments, R2
is unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl,
or phenyl). In embodiments, R2 is unsubstituted heteroaryl (e.g., 5
to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6
membered heteroaryl). In embodiments, R2 is not unsubstituted
methyl. In embodiments, R2 is not unsubstituted ethyl. In
embodiments, R2 is not unsubstituted propyl.
[0137] R6, R7, and R8 are independently hydrogen, halogen,
--CCl.sub.3, --CBr.sub.3, --CF.sub.3, --CI.sub.3, --CHCl.sub.2,
--CHBr.sub.2, --CHF.sub.2, --CHI.sub.2, --CH.sub.2Cl, --CH.sub.2Br,
--CH.sub.2F, --CH.sub.2I, --CN, --OH, --NH.sub.2, --COOH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2,
--NHC(O)NH.sub.2, --NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH,
--OCCl.sub.3, --OCF.sub.3, --OCBr.sub.3, --OCI.sub.3,
--OCHCl.sub.2, --OCHBr.sub.2, --OCHI.sub.2, --OCHF.sub.2,
--OCH.sub.2Cl, --OCH.sub.2Br, --OCH.sub.2I, --OCH.sub.2F,
--SF.sub.5, --N.sub.3, substituted or unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or C1-C4 alkyl),
substituted or unsubstituted alkenyl (e.g., C.sub.2-C.sub.8
alkenyl, C.sub.2-C.sub.6 alkenyl, or C2-C4 alkenyl), substituted or
unsubstituted alkynyl (e.g., C.sub.2-C.sub.8 alkynyl,
C.sub.2-C.sub.6 alkynyl, or C.sub.2-C.sub.4 alkynyl), substituted
or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2
to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl),
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6), substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6), substituted or unsubstituted cycloalkynyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6), substituted
or unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl,
or phenyl), substituted or unsubstituted heteroaryl (e.g., 5 to 10
membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered
heteroaryl), substituted or unsubstituted heteroalicyclyl (e.g., 3
to 8 membered, 3 to 6 membered, or 5 to 6 membered), substituted or
unsubstituted aralkyl, substituted or unsubstituted heteroaralkyl,
substituted or unsubstituted (heteroalicyclyl)alkyl, or an
exogenous label moiety. In embodiments, R6, R7, and R8 are not all
hydrogen. In embodiments, R6 is not hydrogen. In embodiments, R7 is
not hydrogen. In embodiments, R8 is not hydrogen. In embodiments,
R2 is --C(R6)(R7)(R8) and R6, R7, and R8 are not all hydrogen.
[0138] In embodiments, R6, R7, and R8 are independently hydrogen,
halogen, --CCl.sub.3, --CBr.sub.3, --CF.sub.3, --CI.sub.3,
--CHCl.sub.2, --CHBr.sub.2, --CHF.sub.2, --CHI.sub.2, --CH.sub.2Cl,
--CH.sub.2Br, --CH.sub.2F, --CH.sub.2I, --CN, --OH, --NH.sub.2,
--COOH, --CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2,
--NHC(O)NH.sub.2, --NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH,
--OCCl.sub.3, --OCF.sub.3, --OCBr.sub.3, --OCI.sub.3,
--OCHCl.sub.2, --OCHBr.sub.2, --OCHI.sub.2, --OCHF.sub.2,
--OCH.sub.2Cl, --OCH.sub.2Br, --OCH.sub.2I, --OCH.sub.2F,
--SF.sub.5, --N.sub.3, substituted or unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4
alkyl), substituted or unsubstituted alkenyl (e.g., C.sub.2-C.sub.8
alkenyl, C.sub.2-C.sub.6 alkenyl, or C2-C4 alkenyl), substituted or
unsubstituted alkynyl (e.g., C.sub.2-C.sub.8 alkynyl,
C.sub.2-C.sub.6 alkynyl, or C.sub.2-C.sub.4 alkynyl), substituted
or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2
to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl),
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6), substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6), substituted or unsubstituted cycloalkynyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6), substituted
or unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl,
or phenyl), substituted or unsubstituted heteroaryl (e.g., 5 to 10
membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered
heteroaryl), substituted or unsubstituted heteroalicyclyl (e.g., 3
to 8 membered, 3 to 6 membered, or 5 to 6 membered), substituted or
unsubstituted aralkyl, substituted or unsubstituted heteroaralkyl,
or substituted or unsubstituted (heteroalicyclyl)alkyl. In
embodiments, R6, R7, and R8 are not all hydrogen. In embodiments,
R6 is not hydrogen. In embodiments, R7 is not hydrogen. In
embodiments, R8 is not hydrogen. In embodiments, R2 is
--C(R6)(R7)(R8) and R6, R7, and R8 are not all hydrogen.
[0139] In embodiments, a substituted R6 (e.g., substituted alkyl,
substituted alkenyl, substituted alkynyl, substituted heteroalkyl,
substituted cycloalkyl, substituted cycloalkenyl, substituted
cycloalkynyl, substituted aryl, substituted heteroaryl, substituted
heteroalicyclyl, substituted aralkyl, substituted heteroaralkyl,
and/or substituted (heteroalicyclyl)alkyl) is substituted with at
least one substituent group, size-limited substituent group, or
lower substituent group; wherein if the substituted R6 is
substituted with a plurality of groups selected from substituent
groups, size-limited substituent groups, and lower substituent
groups; each substituent group, size-limited substituent group,
and/or lower substituent group may optionally be different. In
embodiments, when R6 is substituted, it is substituted with at
least one substituent group. In embodiments, when R6 is
substituted, it is substituted with at least one size-limited
substituent group. In embodiments, when R6 is substituted, it is
substituted with at least one lower substituent group.
[0140] In embodiments, R6 is independently hydrogen. In
embodiments, R6 is independently halogen. In embodiments, R6 is
independently --CCl.sub.3. In embodiments, R6 is independently
--CBr.sub.3. In embodiments, R6 is independently --CF.sub.3. In
embodiments, R6 is independently --CI.sub.3. In embodiments, R6 is
independently --CHCl.sub.2. In embodiments, R6 is independently
--CHBr.sub.2. In embodiments, R6 is independently --CHF.sub.2. In
embodiments, R6 is independently --CHI.sub.2. In embodiments, R6 is
independently --CH.sub.2Cl. In embodiments, R6 is independently
--CH.sub.2Br. In embodiments, R6 is independently --CH.sub.2F. In
embodiments, R6 is independently --CH.sub.2I. In embodiments, R6 is
independently --CN. In embodiments, R6 is independently --OH. In
embodiments, R6 is independently --NH.sub.2. In embodiments, R6 is
independently --COOH. In embodiments, R6 is independently
--CONH.sub.2. In embodiments, R6 is independently --NO.sub.2. In
embodiments, R6 is independently --SH. In embodiments, R6 is
independently --SO.sub.3H. In embodiments, R6 is independently
--SO.sub.4H. In embodiments, R6 is independently
--SO.sub.2NH.sub.2. In embodiments, R6 is independently
--NHNH.sub.2. In embodiments, R6 is independently --ONH.sub.2. In
embodiments, R6 is independently --NHC(O)NHNH.sub.2. In
embodiments, R6 is independently --NHC(O)NH.sub.2. In embodiments,
R6 is independently --NHSO.sub.2H. In embodiments, R6 is
independently --NHC(O)H. In embodiments, R6 is independently
--NHC(O)OH. In embodiments, R6 is independently --NHOH. In
embodiments, R6 is independently --OCCl.sub.3. In embodiments, R6
is independently --OCF.sub.3. In embodiments, R6 is independently
--OCBr.sub.3. In embodiments, R6 is independently --OCI.sub.3. In
embodiments, R6 is independently --OCHCl.sub.2. In embodiments, R6
is independently --OCHBr.sub.2. In embodiments, R6 is independently
--OCHI.sub.2. In embodiments, R6 is independently --OCHF.sub.2. In
embodiments, R6 is independently --OCH.sub.2Cl. In embodiments, R6
is independently --OCH.sub.2Br. In embodiments, R6 is independently
--OCH.sub.2I. In embodiments, R6 is independently --OCH.sub.2F. In
embodiments, R6 is independently --SF.sub.5. In embodiments, R6 is
independently --N.sub.3. In embodiments, R6 is independently
substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4 alkyl). In embodiments,
R6 is independently substituted or unsubstituted alkenyl (e.g.,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.6 alkenyl, or
C.sub.2-C.sub.4 alkenyl). In embodiments, R6 is independently
substituted or unsubstituted alkynyl (e.g., C.sub.2-C.sub.8
alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4 alkynyl). In
embodiments, R6 is independently substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R6 is
independently substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R6 is independently substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R6 is independently substituted
or unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R6 is
independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In embodiments,
R6 is independently substituted or unsubstituted heteroaryl (e.g.,
5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6
membered heteroaryl). In embodiments, R6 is independently
substituted or unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered). In embodiments, R6
is independently substituted or unsubstituted aralkyl. In
embodiments, R6 is independently substituted or unsubstituted
heteroaralkyl. In embodiments, R6 is independently substituted or
unsubstituted (heteroalicyclyl)alkyl. In embodiments, R6 is
independently an exogenous label moiety.
[0141] In embodiments, R6 is independently unsubstituted alkyl
(e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or
C.sub.1-C.sub.4 alkyl). In embodiments, R6 is independently
unsubstituted alkenyl (e.g., C.sub.2-C.sub.8 alkenyl,
C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.4 alkenyl). In
embodiments, R6 is independently unsubstituted alkynyl (e.g.,
C.sub.2-C.sub.8 alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4
alkynyl). In embodiments, R6 is independently unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R6 is
independently unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R6 is
independently unsubstituted cycloalkenyl (e.g., C.sub.3-C.sub.8,
C3-C6, or C.sub.5-C.sub.6). In embodiments, R6 is independently
unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
or C.sub.5-C.sub.6). In embodiments, R6 is independently
unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or
phenyl). In embodiments, R6 is independently unsubstituted
heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered
heteroaryl, or 5 to 6 membered heteroaryl). In embodiments, R6 is
independently unsubstituted heteroalicyclyl (e.g., 3 to 8 membered,
3 to 6 membered, or 5 to 6 membered). In embodiments, R6 is
independently unsubstituted aralkyl. In embodiments, R6 is
independently unsubstituted heteroaralkyl. In embodiments, R6 is
independently unsubstituted (heteroalicyclyl)alkyl.
[0142] In embodiments, a substituted R7 (e.g., substituted alkyl,
substituted alkenyl, substituted alkynyl, substituted heteroalkyl,
substituted cycloalkyl, substituted cycloalkenyl, substituted
cycloalkynyl, substituted aryl, substituted heteroaryl, substituted
heteroalicyclyl, substituted aralkyl, substituted heteroaralkyl,
and/or substituted (heteroalicyclyl)alkyl) is substituted with at
least one substituent group, size-limited substituent group, or
lower substituent group; wherein if the substituted R7 is
substituted with a plurality of groups selected from substituent
groups, size-limited substituent groups, and lower substituent
groups; each substituent group, size-limited substituent group,
and/or lower substituent group may optionally be different. In
embodiments, when R7 is substituted, it is substituted with at
least one substituent group. In embodiments, when R7 is
substituted, it is substituted with at least one size-limited
substituent group. In embodiments, when R7 is substituted, it is
substituted with at least one lower substituent group.
[0143] In embodiments, R7 is independently hydrogen. In
embodiments, R7 is independently halogen. In embodiments, R7 is
independently --CCl.sub.3. In embodiments, R7 is independently
--CBr.sub.3. In embodiments, R7 is independently --CF.sub.3. In
embodiments, R7 is independently --CI.sub.3. In embodiments, R7 is
independently --CHCl.sub.2. In embodiments, R7 is independently
--CHBr.sub.2. In embodiments, R7 is independently --CHF.sub.2. In
embodiments, R7 is independently --CHI.sub.2. In embodiments, R7 is
independently --CH.sub.2Cl. In embodiments, R7 is independently
--CH.sub.2Br. In embodiments, R7 is independently --CH.sub.2F. In
embodiments, R7 is independently --CH.sub.2I. In embodiments, R7 is
independently --CN. In embodiments, R7 is independently --OH. In
embodiments, R7 is independently --NH.sub.2. In embodiments, R7 is
independently --COOH. In embodiments, R7 is independently
--CONH.sub.2. In embodiments, R7 is independently --NO.sub.2. In
embodiments, R7 is independently --SH. In embodiments, R7 is
independently --SO.sub.3H. In embodiments, R7 is independently
--SO.sub.4H. In embodiments, R7 is independently
--SO.sub.2NH.sub.2. In embodiments, R7 is independently
--NHNH.sub.2. In embodiments, R7 is independently --ONH.sub.2. In
embodiments, R7 is independently --NHC(O)NHNH.sub.2. In
embodiments, R7 is independently --NHC(O)NH.sub.2. In embodiments,
R7 is independently --NHSO.sub.2H. In embodiments, R7 is
independently --NHC(O)H. In embodiments, R7 is independently
--NHC(O)OH. In embodiments, R7 is independently --NHOH. In
embodiments, R7 is independently --OCCl.sub.3. In embodiments, R7
is independently --OCF.sub.3. In embodiments, R7 is independently
--OCBr.sub.3. In embodiments, R7 is independently --OCI.sub.3. In
embodiments, R7 is independently --OCHCl.sub.2. In embodiments, R7
is independently --OCHBr.sub.2. In embodiments, R7 is independently
--OCHI.sub.2. In embodiments, R7 is independently --OCHF.sub.2. In
embodiments, R7 is independently --OCH.sub.2Cl. In embodiments, R7
is independently --OCH.sub.2Br. In embodiments, R7 is independently
--OCH.sub.2I. In embodiments, R7 is independently --OCH.sub.2F. In
embodiments, R7 is independently --SF.sub.5. In embodiments, R7 is
independently --N.sub.3. In embodiments, R7 is independently
substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.6 alkyl, or C1-C4 alkyl). In embodiments, R7 is
independently substituted or unsubstituted alkenyl (e.g.,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.6 alkenyl, or
C.sub.2-C.sub.4 alkenyl). In embodiments, R7 is independently
substituted or unsubstituted alkynyl (e.g., C.sub.2-C.sub.8
alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4 alkynyl). In
embodiments, R7 is independently substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R7 is
independently substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R7 is independently substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R7 is independently substituted
or unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R7 is
independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In embodiments,
R7 is independently substituted or unsubstituted heteroaryl (e.g.,
5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6
membered heteroaryl). In embodiments, R7 is independently
substituted or unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered). In embodiments, R7
is independently substituted or unsubstituted aralkyl. In
embodiments, R7 is independently substituted or unsubstituted
heteroaralkyl. In embodiments, R7 is independently substituted or
unsubstituted (heteroalicyclyl)alkyl. In embodiments, R7 is
independently an exogenous label moiety.
[0144] In embodiments, R7 is independently unsubstituted alkyl
(e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or
C.sub.1-C.sub.4 alkyl). In embodiments, R7 is independently
unsubstituted alkenyl (e.g., C.sub.2-C.sub.8 alkenyl,
C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.4 alkenyl). In
embodiments, R7 is independently unsubstituted alkynyl (e.g.,
C.sub.2-C.sub.8 alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4
alkynyl). In embodiments, R7 is independently unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R7 is
independently unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R7 is
independently unsubstituted cycloalkenyl (e.g., C.sub.3-C.sub.8,
C3-C6, or C.sub.5-C.sub.6). In embodiments, R7 is independently
unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
or C.sub.5-C.sub.6). In embodiments, R7 is independently
unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or
phenyl). In embodiments, R7 is independently unsubstituted
heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered
heteroaryl, or 5 to 6 membered heteroaryl). In embodiments, R7 is
independently unsubstituted heteroalicyclyl (e.g., 3 to 8 membered,
3 to 6 membered, or 5 to 6 membered). In embodiments, R7 is
independently unsubstituted aralkyl. In embodiments, R7 is
independently unsubstituted heteroaralkyl. In embodiments, R7 is
independently unsubstituted (heteroalicyclyl)alkyl.
[0145] In embodiments, a substituted R8 (e.g., substituted alkyl,
substituted alkenyl, substituted alkynyl, substituted heteroalkyl,
substituted cycloalkyl, substituted cycloalkenyl, substituted
cycloalkynyl, substituted aryl, substituted heteroaryl, substituted
heteroalicyclyl, substituted aralkyl, substituted heteroaralkyl,
and/or substituted (heteroalicyclyl)alkyl) is substituted with at
least one substituent group, size-limited substituent group, or
lower substituent group; wherein if the substituted R8 is
substituted with a plurality of groups selected from substituent
groups, size-limited substituent groups, and lower substituent
groups; each substituent group, size-limited substituent group,
and/or lower substituent group may optionally be different. In
embodiments, when R8 is substituted, it is substituted with at
least one substituent group. In embodiments, when R8 is
substituted, it is substituted with at least one size-limited
substituent group. In embodiments, when R8 is substituted, it is
substituted with at least one lower substituent group.
[0146] In embodiments, R8 is independently hydrogen. In
embodiments, R8 is independently halogen. In embodiments, R8 is
independently --CCl.sub.3. In embodiments, R8 is independently
--CBr.sub.3. In embodiments, R8 is independently --CF.sub.3. In
embodiments, R8 is independently --CI.sub.3. In embodiments, R8 is
independently --CHCl.sub.2. In embodiments, R8 is independently
--CHBr.sub.2. In embodiments, R8 is independently --CHF.sub.2. In
embodiments, R8 is independently --CHI.sub.2. In embodiments, R8 is
independently --CH.sub.2Cl. In embodiments, R8 is independently
--CH.sub.2Br. In embodiments, R8 is independently --CH.sub.2F. In
embodiments, R8 is independently --CH.sub.2I. In embodiments, R8 is
independently --CN. In embodiments, R8 is independently --OH. In
embodiments, R8 is independently --NH.sub.2. In embodiments, R8 is
independently --COOH. In embodiments, R8 is independently
--CONH.sub.2. In embodiments, R8 is independently --NO.sub.2. In
embodiments, R8 is independently --SH. In embodiments, R8 is
independently --SO.sub.3H. In embodiments, R8 is independently
--SO.sub.4H. In embodiments, R8 is independently
--SO.sub.2NH.sub.2. In embodiments, R8 is independently
--NHNH.sub.2. In embodiments, R8 is independently --ONH.sub.2. In
embodiments, R8 is independently --NHC(O)NHNH.sub.2. In
embodiments, R8 is independently --NHC(O)NH.sub.2. In embodiments,
R8 is independently --NHSO.sub.2H. In embodiments, R8 is
independently --NHC(O)H. In embodiments, R8 is independently
--NHC(O)OH. In embodiments, R8 is independently --NHOH. In
embodiments, R8 is independently --OCCl.sub.3. In embodiments, R8
is independently --OCF.sub.3. In embodiments, R8 is independently
--OCBr.sub.3. In embodiments, R8 is independently --OCI.sub.3. In
embodiments, R8 is independently --OCHCl.sub.2. In embodiments, R8
is independently --OCHBr.sub.2. In embodiments, R8 is independently
--OCHI.sub.2. In embodiments, R8 is independently --OCHF.sub.2. In
embodiments, R8 is independently --OCH.sub.2Cl. In embodiments, R8
is independently --OCH.sub.2Br. In embodiments, R8 is independently
--OCH.sub.2I. In embodiments, R8 is independently --OCH.sub.2F. In
embodiments, R8 is independently --SF.sub.5. In embodiments, R8 is
independently --N.sub.3. In embodiments, R8 is independently
substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4 alkyl). In embodiments,
R8 is independently substituted or unsubstituted alkenyl (e.g.,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.6 alkenyl, or
C.sub.2-C.sub.4 alkenyl). In embodiments, R8 is independently
substituted or unsubstituted alkynyl (e.g., C.sub.2-C.sub.8
alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4 alkynyl). In
embodiments, R8 is independently substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R8 is
independently substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R8 is independently substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R8 is independently substituted
or unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R8 is
independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In embodiments,
R8 is independently substituted or unsubstituted heteroaryl (e.g.,
5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6
membered heteroaryl). In embodiments, R8 is independently
substituted or unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered). In embodiments, R8
is independently substituted or unsubstituted aralkyl. In
embodiments, R8 is independently substituted or unsubstituted
heteroaralkyl. In embodiments, R8 is independently substituted or
unsubstituted (heteroalicyclyl)alkyl. In embodiments, R8 is
independently an exogenous label moiety.
[0147] In embodiments, R8 is independently unsubstituted alkyl
(e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or
C.sub.1-C.sub.4 alkyl). In embodiments, R8 is independently
unsubstituted alkenyl (e.g., C.sub.2-C.sub.8 alkenyl,
C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.4 alkenyl). In
embodiments, R8 is independently unsubstituted alkynyl (e.g.,
C.sub.2-C.sub.8 alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4
alkynyl). In embodiments, R8 is independently unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R8 is
independently unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R8 is
independently unsubstituted cycloalkenyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R8 is
independently unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R8 is
independently unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl,
C.sub.10 aryl, or phenyl). In embodiments, R8 is independently
unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9
membered heteroaryl, or 5 to 6 membered heteroaryl). In
embodiments, R8 is independently unsubstituted heteroalicyclyl
(e.g., 3 to 8 membered, 3 to 6 membered, or 5 to 6 membered). In
embodiments, R8 is independently unsubstituted aralkyl. In
embodiments, R8 is independently unsubstituted heteroaralkyl. In
embodiments, R8 is independently unsubstituted
(heteroalicyclyl)alkyl.
Turning to FIG. 1A, one sees that with increasing R2 size, one
observes a decreasing percentage of dC degradation in final
reaction product as observed after 60 minutes of a 25 C reaction.
One also observes that R2 moieties having at least two carbon atoms
perform better in terms of starting material consumption than do
smaller R2 moieties.
[0148] In some configurations, the nucleotide that has the
3'-O-oxime moiety can have the structure:
##STR00010##
and the nucleotide that has the 3'-O--NH.sub.2 moiety can have the
structure:
##STR00011##
[0149] B can be a nucleobase. R1 can independently be a halogen,
OCH.sub.3, H or OH. Q can be independently monophosphate,
diphosphate, triphosphate, or nucleic acid. R3 and R4 can be
independently H, CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally
substituted variant thereof. By way of example, the moiety at the
3' position of the nucleotide can be an aldoxime (i.e. one of R3 or
R4 is hydrogen) or a ketoxime (neither of R3 or R4 is
hydrogen).
[0150] In embodiments, the reaction occurs in an aqueous solution.
In embodiments, the nucleotide is attached to a solid support in
contact with the aqueous solution.
[0151] In embodiments, the nucleotide that is produced comprises a
deoxynucleotide. In embodiments, the nucleotide that is produced
includes:
##STR00012##
wherein Base is the nucleobase, and wherein DNA is independently a
single stranded deoxyribonucleic acid or double stranded
deoxyribonucleic acid. In embodiments, Base has a value of B.
[0152] In embodiments, the reagent is independently one of:
##STR00013##
Additional individual examples of the reagent include the molecules
listed in FIG. 1B and the molecules listed in FIG. 2. The present
disclosure also provides a method for modifying a nucleotide. The
method can be carried out by reacting a nucleotide having a
3'-O-oxime moiety with a reagent having the structure R2-ONH.sub.2
to produce a nucleotide having a 3'-O--NH.sub.2 moiety, wherein R2
is independently alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof. In some configurations, the nucleotide that has
the 3'-O-oxime moiety can have the structure:
##STR00014##
and the nucleotide that has the 3'-O--NH.sub.2 moiety can have the
structure:
##STR00015##
B can be a nucleobase. R1 can independently be a halogen,
OCH.sub.3, H or OH. Q can be independently monophosphate,
diphosphate, triphosphate, or nucleic acid. R3 and R4 can be
independently H, CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally
substituted variant thereof. By way of example, the moiety at the
3' position of the nucleotide can be an aldoxime (i.e. one of R3 or
R4 is hydrogen) or a ketoxime (neither of R3 or R4 is
hydrogen).
[0153] A method or composition of the present disclosure can
include any of a variety of nucleotides. The nucleotide can be a
native nucleotide, nucleotide analog or modified nucleotide as
desired to suit a particular application or configuration set forth
herein. Optionally, a nucleotide has a nitrogenous base,
five-carbon sugar, and phosphate group, wherein any moiety of the
nucleotide may be modified, removed and/or replaced as compared to
a native nucleotide. For example, the 5' oxygen of a nucleotide
need not have a phosphate moiety and can instead be protected with
an exogenous moiety such as dimethoxytrityl (DMT) moiety. Reactive
atoms in the base moiety of a nucleotide can be protected with
exogenous moieties if desired. Often, methods and compositions
disclosed herein comprise cytosine bases, and the cytosine bases
are preserved in an undegraded or unmodified form at a higher rate
than comparable methods or compositions in the prior art, as
represented for example by the point DRT 1 in FIG. 1A.
[0154] Nucleotides that are used in a composition, method,
apparatus or system herein can include terminators that reversibly
prevent subsequent nucleotide incorporation at the 3'-end of the
primer after the analog has been incorporated into the primer. For
example, U.S. Pat. No. 7,544,794 (published Jun. 9, 2009), U.S.
Pat. No. 8,034,923 (published Oct. 11, 2011) and U.S. Pat. No.
10,472,383 (published Nov. 12, 2019) (the disclosures of each of
which being incorporated herein by reference in their respective
entireties) describe reversible terminators in which the 3'-OH
group is replaced by a 3'-ONH.sub.2 moiety. These reversible
terminators are resistant to modification by nucleic acid enzymes
such as polymerases and ligases such that they prevent addition of
a subsequent base at the 3' blocked position, thereby terminating
extension of the nucleic acid strand when they are incorporated.
The amino moiety can be removed from a nucleotide (for example,
when the nucleotide is at the 3' end of a primer) by nitrous acid
(HONO), thereby deblocking the nucleotide. The deblocked nucleotide
will have a 3'OH that can be acted upon by a polymerase such that
the 3' oxygen forms a covalent bond with the 5' phosphate of
another nucleotide in a reaction known as an incorporation reaction
or extension reaction. Removal of the amino moiety from a
nucleotide via a deblocking step can similarly allow the nucleotide
to be acted upon by a ligase such that the 3' oxygen forms a
covalent bond with the 5' phosphate of an oligonucleotide in an
incorporation or extension reaction.
[0155] A number of blocking moieties are consistent with the
disclosure herein. Some blocking moieties share the characteristic
that they do not prohibit the nucleotide to which they are bound
from being incorporated at their 5' ends to an available 3' OH
moiety by a nucleotide polymerizing enzyme, so as to incorporate
the nucleotide that blocks further extension. Generally, larger
moieties such as protecting moieties are often too large to be
incorporated by nucleic acid polymerase enzyme. In embodiments,
3'-reversible terminators and 3' blocking moieties include
--ONH.sub.2, --ONHCH.sub.3, --OCH.sub.2N.sub.3, and
--ONHCOCH.sub.3. In embodiments, a 3'-blocking moiety is
--ONH.sub.2. In embodiments, a 3'-blocking moiety is --ONHCH.sub.3.
In embodiments, a 3'-blocking moiety is --OCH.sub.2N.sub.3. In
embodiments, a 3'-blocking moiety is and --ONHCOCH.sub.3. In
embodiments, a 3'-blocking moiety is a 3'-O-oxime moiety. In
embodiments, a 3'-reversible terminator moiety is a 3'-O-oxime
moiety.
[0156] The present disclosure provides new and useful reagents and
methods for synthesizing reversibly terminated nucleotides, such as
those that have a 3'-ONH.sub.2 moiety. Such reversibly terminated
nucleotides can be synthesized by reacting a precursor nucleotide,
having a 3'-O-oxime moiety, with an alkoxyamine reagent having the
structure R2-ONH.sub.2. Particularly useful reagents include those
in which R2 is a moiety other than hydrogen. For example, R2 can be
alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,
aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl,
(heteroalicyclyl)alkyl, or an optionally substituted variant
thereof. Particularly useful R2 moieties include, for example,
--CH.sub.2CH.sub.3, --CH.sub.2CHCH.sub.2, and --C(CH.sub.3).sub.3.
In embodiments, R2 is as described herein, including in
embodiments, claims, and figures. Thus, useful alkoxyamine reagents
can include one or more of the following molecules:
##STR00016##
as well as any of the molecules listed in FIG. 1A or FIG. 2.
[0157] Any of a variety of substitutions can be made to the above
R2 moieties. For example, one or more of the hydrogens in the
--CH.sub.2CH.sub.3 or --CH.sub.2CHCH.sub.2 moieties can be
substituted with a carbonyl (--CO.sub.2), alcohol (--OH), amine
(--NH.sub.2) or halogen (e.g. Bromine, Fluorine, Chlorine, or
Iodine). Similarly, one or more of the hydrogens in one or more of
the methyls of the --C(CH.sub.3).sub.3 moiety can be substituted in
this way. In particular, applications the additional steric bulk
provided by these types of substitutions can beneficially favor
conversion of a 3'-O-oxime moiety of a nucleotide to a
3'-O--NH.sub.2 moiety over unwanted side reactions that modify the
nucleobase moiety of the nucleotide.
[0158] In embodiments, R2 is independently,
##STR00017##
[0159] In embodiments, R2 is independently
##STR00018##
In embodiments, R2 is independently
##STR00019##
In embodiments, R2 is independently
##STR00020##
In embodiments, R2 is independently
##STR00021##
In embodiments, R2 is independently
##STR00022##
In embodiments, R2 is independently
##STR00023##
In embodiments, R2 is independently
##STR00024##
In embodiments, R2 is independently
##STR00025##
In embodiments, R2 is independently
##STR00026##
In embodiments, R2 is independently
##STR00027##
In embodiments, R2 is independently
##STR00028##
In embodiments, R2 is independently
##STR00029##
In embodiments, R2 is independently
##STR00030##
In embodiments, R2 is independently
##STR00031##
In embodiments, R2 is independently
##STR00032##
[0160] In some configurations of the methods and compositions set
forth herein, R2 in the formula R2-ONH.sub.2 can be CH.sub.3.
However, R2 need not be CH.sub.3. Rather, R2 can be a higher order
alkyl than a methyl (i.e. R2 can have more than one carbon).
Accordingly, R2 can be an alkyl other than methyl. In embodiments,
R2 is not --CH.sub.3. In embodiments, R2 is not hydrogen. In
embodiments, R2 is not hydrogen, or --CH.sub.3. In embodiments, R2
is --C(R6)(R7)(R8), --C(O)(R6), --P(O)(R6)(R7),
--C(R6)(.dbd.C(R7)(R8)), --Si(R6)(R7)(R8), --S O.sub.2(R6),
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C5-C6), substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6), substituted or unsubstituted cycloalkynyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6), substituted
or unsubstituted heteroalicyclyl (e.g., 3 to 8 membered, 3 to 6
membered, or 5 to 6 membered), substituted or unsubstituted aryl
(e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl), or
substituted or unsubstituted heteroaryl (e.g., 5 to 10 membered
heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered
heteroaryl). In embodiments, R2 has a molecular weight greater than
methyl. In embodiments, R2 has a molecular weight greater than
ethyl. In embodiments, R2 has a molecular weight greater than 36
g/mol. In embodiments, R2 has a molecular weight greater than 57
g/mol. In embodiments, R2 has a molecular weight greater than 100
g/mol. In embodiments, R2 has a molecular weight greater than 200
g/mol. In embodiments, R2 has a molecular weight greater than 300
g/mol. In embodiments, R2 has a molecular weight greater than 400
g/mol. In embodiments, R2 has a molecular weight greater than 500
g/mol. In embodiments, R2 has a molecular weight greater than 600
g/mol. In embodiments, R2 has a molecular weight greater than 700
g/mol. In embodiments, R2 has a molecular weight greater than 800
g/mol. In embodiments, R2 has a molecular weight greater than 900
g/mol. In embodiments, R2 has a molecular weight less than or equal
to 1000 g/mol. In embodiments, R2 has a molecular weight less than
1000 g/mol. Although not wishing to be limited by mechanism, it is
believed that R2 moieties having more steric bulk than hydrogen and
methyl yield improved synthetic results compared to smaller
moieties, and produce fewer unwanted modifications to nucleic acids
and nucleotides. See the results of Example 1 below. In
embodiments, R2 is --C(R6)(R7)(R8) and R6, R7, and R8 are not all
hydrogen. In embodiments, R2 is not unsubstituted methyl. In
embodiments, R2 is not unsubstituted ethyl. In embodiments, R2 is
not unsubstituted propyl.
[0161] Through the disclosure herein, one observes the following.
R2 moieties as disclosed herein perform substantially better than
do prior art reagents in driving reactions to near completion. In a
60 minute reaction performed at 25.degree. C., for example, one
observes that R2 moieties as disclosed herein exhibit starting
material consumption of at least 92%, at least 93%, at least 94%,
at least 95%, at least 96%, at least 97%, at least 98%, at least
99% or greater than 99% consumption of nucleotide starting
material, whereas prior art approaches achieve less than 92%.
[0162] Similarly, one observes that with increasing R2 size, one
sees a decreasing amount of nucleotide such as dC base degradation,
such that R2 moieties as disclosed herein come to the
above-mentioned completion percentages with no more than 6%, no
more than 5%, no more than 4%, no more than 3%, no more than 2%, no
more than 1%, or less than 1% degradation of dC.
[0163] Similarly, in some cases one sees a correlation between R2
size and reduction of nucleotide such as dC degradation.
Accordingly, disclosed herein are R2 moieties having molecular
weights of at least any of the following values: 24, 25, 26, 27,
28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 39, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,
62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78,
79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,
96, 97, 98, 99, 100, or greater than 100.
In embodiments, R2 moieties are in some cases limited by
solubility, such that in embodiments larger moieties become
insoluble. Accordingly, in embodiments, R2 moieties are variously
limited to no more than any of the following molecular weights:
1000, 900, 800, 700, 650, 600, 550, 500, 450, 400, 350, 300, 290,
280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160,
150, 140, 130, 120, 110, 100, or less than 100 g/mol.
[0164] R2 moieties may comprise a number of constituents, alone or
in combination, to covey the benefits disclosed herein. Some R2
moieties variously comprise at least 2, at least 3, at least 4, at
least 5, at least 6, or more than 6 carbon nuclei. Similarly, some
R2 comprise at least one of the following: at least 1 nitrogen
nucleus, at least one silicon nucleus, at least one phosphorous
nucleus, at least one fluorine nucleus or at least one other
constituent. R2 moieties are in some cases partially or fully
fluorinated, or partially or fully hydrogenated. In some cases an
R2 moiety comprises at least one component to facilitate
solubility.
[0165] Alkoxyamine reagents having the structure R2-ONH.sub.2,
wherein R2 is --CH.sub.2CH.sub.3, --CH.sub.2CHCH.sub.2, and
--C(CH.sub.3).sub.3 are soluble in aqueous solution making them
particularly useful for nucleotide synthesis and modification.
Other R2 moieties that provide aqueous solubility can also be used.
In some configurations, synthesis can be carried out using
alkoxyamine reagents that are not soluble, or only sparingly
soluble, in aqueous solutions. For example, synthesis can be
carried out in non-aqueous solutions or in liquids formed by
mixtures of aqueous and organic solvents. Exemplary alkoxyamine
reagents that can be useful in non-aqueous or partially aqueous
liquids include those having phenyl moieties and/or halogen
moieties, such as:
##STR00033##
Alternately, some moieties that provide aqueous solubility might
include hydroxyls, amines, carboxyls, or other hydrophilic or polar
moieties.
[0166] The above alkoxyamine reagents have a primary amine in the
aminooxy moiety. An alkoxyamine reagent can have a secondary amine
in the aminooxy moiety. For example, an alkoxyamine reagent can
have the structure
##STR00034##
[0167] In particular configurations of the methods or compositions
set forth herein, an alkoxyamine reagent having the structure
R2-ONH.sub.2 can be attached to a solid support. In embodiments,
the reagent that comprises the structure R2-ONH.sub.2 is attached
to a solid support in contact with the aqueous solution. For
example, the reagent can be attached via the nitrogen atom by
substituting one of the hydrogens with a linkage to the solid
support. A benefit of using solid support attached reagent is that
the reaction will yield a solid support attached product that can
be purified from reactants and other reaction components by
separating the solid support from the liquid where the reaction
occurred. Attachment of the reagent to the solid support can also
occur via a linkage to the R2 moiety.
[0168] Generally, a solution that is used to synthesize a
nucleotide having a 3'-ONH.sub.2 moiety using an alkoxyamine
reagent set forth herein can have a pH of 4 to 8. As the pH
decreases in this range, the rate of converting the 3'-O-oxime
moiety of a nucleotide to 3'-O--NH.sub.2 moiety increases. However,
as pH approaches the lower end of the range the risk of side
reactions that cause unwanted modification to other moieties of the
nucleotide also increases. Accordingly, pH for a synthetic reaction
set forth herein can be in a range from 5 to 8, 6 to 8, or 7 to 8.
If desired the pH can be in a range of 4 to 7, 4 to 6, or 4 to 5,
as well as 5 to 7, 5 to 6, or 6 to 7, or any upper and lower range
value encompassed by the range of lower and upper pH values
disclosed herein. Similarly pH for a synthetic reaction set forth
herein can be at least 4, at least 5, at least 6, at least 7, or no
more than 8, no more than 7, no more than 6, or no more than 5.
[0169] Nucleotides having reversible terminators other than
3'-O-oxime or 3'-ONH.sub.2 can be used in a method or composition
herein. For example, nucleotides having different types of
reversible terminators can be useful when it is desired to block a
population of primers such that one or more subpopulation of the
primers can be selectively deblocked. More specifically, a first
subpopulation of primers can be blocked with a 3'-O-oxime or
3'-ONH.sub.2 moiety and a second population can be blocked with a
second type of blocking moiety. The second type of blocking moiety
can, advantageously be deblocked by a method that is orthogonal to
the deblocking methods set forth herein for the 3'-O-oxime or
3'-ONH.sub.2 moiety. Orthogonal methods are those that do not
substantially cross react. Accordingly, the 3'-O-oxime or
3'-ONH.sub.2 moiety can be deblocked by a method that does not
substantially deblock the second blocking moiety, and the second
blocking moiety can be deblocked by a method that does not
substantially deblock the 3'-O-oxime or 3'-ONH.sub.2 moiety,
respectively.
[0170] Examples of other types of reversible terminator moieties
and their deblocking methods include, but are not limited to, the
following. Useful reversible terminator moieties include those that
can be chemically deblocked such as 3' azidomethyl which can be
removed by phosphines, esters which can be deblocked by base
hydrolysis or metal catalysts, and others set forth in U.S. Pat.
Nos. 7,427,673; 7,414,116; 7,057,026; 7,544,794 or 8,034,923; or
PCT publications WO 91/06678 or WO 07/123744, each of which is
incorporated herein by reference. Other useful reversible
terminators include 3' photoreactive moieties that can be removed
by light such as those described in U.S. Pat. Nos. 7,964,352;
9,399,798 and 10,041,115 (each of which is incorporated herein by
reference in its entirety), or enzymatically removable moieties
such as 3' phosphate moieties that can be removed by phosphatases
such as those set forth in U.S. Pat. No. 10,378,051 (which is
incorporated herein by reference in its entirety).
[0171] Nucleotides having irreversible terminators can be used in a
method or composition herein. For example, nucleotides having
irreversible terminators can be useful when it is desired to block
a population of primers such that a first subpopulation of the
primers can be deblocked and a second subpopulation is resistant to
deblocking. More specifically, a first subpopulation of primers can
be blocked with a 3'-ONH.sub.2 moiety and a second population can
be blocked with an irreversible blocking moiety. Irreversible
nucleotides include 2', 3'-dideoxynucleotides (ddNTPs such as
ddGTP, ddATP, ddTTP, ddCTP). Dideoxynucleotides lack the 3'-OH
group of dNTPs that would otherwise participate in
polymerase-mediated or ligase-mediated primer extension. Thus, the
3' position has a hydrogen moiety instead of the native hydroxyl
moiety.
[0172] Nucleotides that are used for particular configurations of
the methods and compositions herein, for example, to participate in
stabilized ternary complexes, need not include blocking groups
(e.g. reversible terminators) that prevent subsequent nucleotide
incorporation at the 3'-end of the primer after the analog has been
incorporated into the primer. This can be the case whether or not
an extension step is carried out using nucleotide(s) having a
blocking group such as a reversible terminator.
[0173] A nucleotide that is a constituent of compositions herein or
produced through method herein, for example, during synthesis
procedures or during a sequencing process, can optionally include
an exogenous label. An exogenously labeled nucleotide can include a
reversible or irreversible terminator moiety, an exogenously
labeled nucleotide can be non-incorporable, an exogenously labeled
nucleotide can lack terminator moieties, an exogenously labeled
nucleotide can be incorporable or an exogenously labeled nucleotide
can be both incorporable and non-terminated. An exogenously labeled
nucleotide can be a moiety in a nucleic acid, for example, being
the 3' nucleotide of a primer. Exogenously labeled nucleotides can
be particularly useful in a Sequencing By Binding.TM. process when
used to form a stabilized ternary complex with a non-labeled
polymerase. Exogenously labeled nucleotides can be useful in a
sequencing by synthesis process when incorporated into a primer by
polymerase.
[0174] Examples of useful exogenous labels include, but are not
limited to, radiolabel moieties, luminophore moieties, fluorophore
moieties, quantum dot moieties, chromophore moieties, enzyme
moieties, electromagnetic spin labeled moieties, nanoparticle light
scattering moieties, and any of a variety of other signal
generating moieties known in the art. Suitable enzyme moieties
include, for example, horseradish peroxidase, alkaline phosphatase,
beta-galactosidase, or acetylcholinesterase. Exemplary fluorophore
labels include, but are not limited to rhodols; resorufins;
coumarins; xanthenes; acridines; fluoresceins; rhodamines;
erythrins; cyanins; phthalaldehydes; naphthylamines;
fluorescamines; benzoxadiazoles; stilbenes; pyrenes; indoles;
borapolyazaindacenes; quinazolinones; eosin; erythrosin; Malachite
green; CY dyes (GE Biosciences), including Cy3 (and its
derivatives), Cy5 (and its derivatives) and Cy7 (and its
derivatives); DYOMICS and DYLIGHT dyes (Dyomics) including DY-547,
DY-630, DY-631, DY-632, DY-633, DY-634, DY-635, DY-647, DY-649,
DY-652, DY-678, DY-680, DY-682, DY-701, DY-734, DY-752, DY-777 and
DY-782; Lucifer Yellow; CASCADE BLUE; TEXAS RED; BODIPY
(boron-dipyrromethene) (Molecular Probes) dyes including BODIPY
630/650 and BODIPY 650/670; ATTO dyes (Atto-Tec) including ATTO
390, ATTO 425, ATTO 465, ATTO 610 611X, ATTO 610, ATTO 635; ALEXA
FLUORS including ALEXA FLUOR 633, ALEXA FLUOR 647, ALEXA FLUOR 660,
ALEXA FLUOR 700, ALEXA FLUOR 750, and ALEXA FLUOR 680 (Molecular
Probes); DDAO (7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one
or any derivatives thereof) (Molecular Probes); QUASAR dyes
(Biosearch); IRDYES dyes (LiCor) including IRDYE 700DX (NHS ester),
IRDYE 800RS (NHS ester) and IRDYE 800CW (NHS ester); EVOBLUE dyes
(Evotech Biosystems); JODA 4 dyes (Applied Biosystems); HILYTE dyes
(AnaSpec); MR121 and MR200 dyes (Roche); Hoechst dyes 33258 and
33242 (Invitrogen); FAIR OAKS RED (Molecular Devices); SUNNYVALE
RED (Molecular Devices); LIGHT CYCLER RED (Roche); EPOCH (Glen
Research) dyes including EPOCH REDMOND RED, EPOCH YAKIMA YELLOW,
EPOCH GIG HARBOR GREEN; Tokyo green (M. Kamiya, et al., 2005 Angew.
Chem. Int. Ed. 44:5439-5441); and CF dyes including CF 647 and
CF555 (Biotium), and others known in the art such as those
described in Principles of Fluorescence Spectroscopy, Joseph R.
Lakowicz (Editor), Plenum Pub Corp, 2nd edition (July 1999) and the
6th Edition of Molecular Probes Handbook by Richard P. Hoagland. In
embodiments, an exogenous label is a detectable moiety. In
embodiments, an exogenous label is a detectable agent.
[0175] An exogenous label can be attached to a nucleotide or
nucleic acid via a linker. A linker that is present in a nucleotide
or nucleic acid can be, but need not be, cleavable. For example,
the exogenous label can be attached to the nucleobase of a
nucleotide or nucleic acid via a linker and the linker can be inert
to an alkoxyamine reagent having formula R2-ONH.sub.2 and or the
linker can be inert to nitrous acid (HONO). The linker can be
stable to conditions used in methods set forth herein such that the
covalent structure of the linker is not changed during any
particular step, or throughout all steps, of a method set forth
herein. Alternatively, the linker that attaches an exogenous label
to a nucleobase can be reactive with an alkoxyamine reagent having
formula R2-ONH.sub.2 such that the reaction detaches the exogenous
label from the nucleobase. For example, the linker can include a
moiety having the structure:
##STR00035##
wherein at least one of R3, R4 or R5 is attached to the exogenous
label directly or via a second moiety of the linker and wherein at
least one of R3, R4 or R5 is attached to the nucleobase directly or
via a third moiety of the linker. Another useful linker moiety is a
hydrazone. For example, the linker can include a moiety having the
structure:
##STR00036##
wherein at least one of R3, R4 or R5 is attached to the exogenous
label directly or via a second moiety of the linker and wherein at
least one of R3, R4 or R5 is attached to the nucleobase directly or
via a third moiety of the linker. These linker moieties can be
reacted with an R2-ONH.sub.2 molecule to cleave the exogenous label
from the nucleobase. In embodiments, B, Base, R3, R4, R5, R6, R7,
or R8 may independently include an exogenous label. In embodiments,
B, Base, R3, R4, R5, R6, R7, or R8 may independently include a
label. In embodiments, B, Base, R3, R4, R5, R6, R7, or R8 may
independently include a detectable moiety. In embodiments, B, Base,
R3, R4, R5, R6, R7, or R8 may independently include a fluorphore
moiety. In embodiments, R3, R4, R5, R6, R7, or R8 independently is
an exogenous label. In embodiments, R3, R4, R5, R6, R7, or R8
independently is a label. In embodiments, R3, R4, R5, R6, R7, or R8
independently is a detectable moiety. In embodiments, R3, R4, R5,
R6, R7, or R8 independently is a fluorphore moiety. In embodiments,
the blocking moiety is an exogenous label moiety. In embodiments,
the blocking moiety includes an exogenous label moiety. In
embodiments, the protecting moiety is an exogenous label moiety. In
embodiments, the protecting moiety includes an exogenous label
moiety.
[0176] R5 is independently H, CH.sub.3, alkyl, alkenyl, alkynyl,
cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,
heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or
an optionally substituted variant thereof, or an exogenous label
moiety.
[0177] In embodiments, a substituted R5 (e.g., substituted alkyl,
substituted alkenyl, substituted alkynyl, substituted cycloalkyl,
substituted cycloalkenyl, substituted cycloalkynyl, substituted
aryl, substituted heteroaryl, substituted heteroalicyclyl,
substituted aralkyl, substituted heteroaralkyl, and/or substituted
(heteroalicyclyl)alkyl) is substituted with at least one
substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted R5 is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
R5 is substituted, it is substituted with at least one substituent
group. In embodiments, when R5 is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when R5 is substituted, it is substituted with at least one lower
substituent group.
[0178] In embodiments, R5 is independently H, CH.sub.3, alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl,
(heteroalicyclyl)alkyl, or an optionally substituted variant
thereof. In embodiments, R5 is independently an exogenous label
moiety.
[0179] In alternative embodiments, a nucleotide, nucleic acid,
reactant for a method set forth herein or product of a method set
forth herein can lack exogenous labels. For example, a nucleic
acid, nucleotide, polymerase and/or stabilized ternary complex can
lack one, several or all of the exogenous labels described herein
or in the references that are cited and incorporated herein. For
example, a non-labeled ternary complex can be detected based on
intrinsic properties of the stabilized ternary complex, such as
mass, charge, intrinsic optical properties or the like. Exemplary
methods for detecting non-labeled ternary complexes are set forth
in commonly owned U.S. Pat. App. Pub. Nos. 2017/0191125 A1 or
2017/0022553 A1; or U.S. Pat. No. 10,246,744, each of which is
incorporated herein by reference.
[0180] In embodiments, the nucleobase includes an exogenous label
moiety (e.g., connected to the nucleobase through a linker, a
fluorophore moiety, an antigen for antibody recognition). In
embodiments, the nucleobase is substituted with an exogenous label
moiety (e.g., connected to the nucleobase through a linker, a
fluorophore moiety, an antigen for antibody recognition). In
embodiments, the nucleobase is covalently bonded to the exogenous
label moiety through a linker. In embodiments, the linker
includes
##STR00037##
In embodiments, the linker includes
##STR00038##
In embodiments, the linker is
##STR00039##
In embodiments, the linker is
##STR00040##
L3 and L5 are each independently a covalent linker. In embodiments,
L5 is bonded to a nucleobase. In embodiments, L3 is bonded to a
nucleobase. In embodiments L5 is bonded to an exogenous label
moiety. In embodiments, L3 is bonded to an exogenous label moiety.
In embodiments, L5 is-L5A-L5B-L5C-L5D-L5E-. In embodiments, L3 is
-L3A-L3B-L3C-L3D-L3E-. In embodiment, the linker connecting the
nucleobase to the exogenous label moiety is -L6-L7-L8-L9-L10-. L3A,
L3B, L3C, L3D, L3E, L5A, L5B, L5C, L5D, L5E, L6, L7, L8, L9, and
L10 are independently a bond, --NH--, --O--, --S--, --SO.sub.2--,
--C(O)--, --C(O)NH--, --NHC(O)--, --NHC(O)NH--, --C(O)O--, --O
C(O)--, --SO.sub.2NH--, --NHSO.sub.2--, substituted or
unsubstituted alkylene (e.g., C.sub.1-C.sub.8, C.sub.1-C.sub.6, or
C.sub.1-C.sub.4), substituted or unsubstituted alkenylene (e.g.,
C.sub.2-C.sub.8, C.sub.2-C.sub.6, or C2-C4), substituted or
unsubstituted alkynylene (e.g., C.sub.2-C.sub.8, C.sub.2-C.sub.6,
or C.sub.2-C.sub.4), substituted or unsubstituted heteroalkylene
(e.g., 2 to 8 membered, 2 to 6 membered, or 2 to 4 membered),
substituted or unsubstituted cycloalkylene (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C5-C6), substituted or unsubstituted
cycloalkenylene (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6), substituted or unsubstituted cycloalkynylene
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6),
substituted or unsubstituted arylene (e.g., C.sub.6-C.sub.10 aryl,
C.sub.10 aryl, or phenylene), substituted or unsubstituted
heteroarylene (e.g., 5 to 10 membered, 5 to 9 membered, or 5 to 6
membered), substituted or unsubstituted heteroalicyclylene (e.g., 3
to 8 membered, 3 to 6 membered, or 5 to 6 membered), substituted or
unsubstituted aralkylene, substituted or unsubstituted
heteroaralkylene, or substituted or unsubstituted
(heteroalicyclyl)alkylene.
[0181] In embodiments, L3 is substituted. In embodiments, L3 is
unsubstituted. In embodiments, a substituted L3 (e.g., a
substituted covalent linker) is substituted with at least one
substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted L3 is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
L3 is substituted, it is substituted with at least one substituent
group. In embodiments, when L3 is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when L3 is substituted, it is substituted with at least one lower
substituent group.
[0182] In embodiments, L5 is substituted. In embodiments, L5 is
unsubstituted. In embodiments, a substituted L5 (e.g., a
substituted covalent linker) is substituted with at least one
substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted L5 is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
L5 is substituted, it is substituted with at least one substituent
group. In embodiments, when L5 is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when L5 is substituted, it is substituted with at least one lower
substituent group.
[0183] In embodiments, a substituted L3A (e.g., substituted
alkylene, substituted alkenylene, substituted alkynylene,
substituted heteroalkylene, substituted cycloalkylene, substituted
cycloalkenylene, substituted cycloalkynylene, substituted arylene,
substituted heteroarylene, substituted heteroalicyclylene,
substituted aralkylene, substituted heteroaralkylene, and/or
substituted (heteroalicyclyl)alkylene) is substituted with at least
one substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted L3A is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
L3A is substituted, it is substituted with at least one substituent
group. In embodiments, when L3A is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when L3A is substituted, it is substituted with at least one lower
substituent group.
[0184] In embodiments, a substituted L3B (e.g., substituted
alkylene, substituted alkenylene, substituted alkynylene,
substituted heteroalkylene, substituted cycloalkylene, substituted
cycloalkenylene, substituted cycloalkynylene, substituted arylene,
substituted heteroarylene, substituted heteroalicyclylene,
substituted aralkylene, substituted heteroaralkylene, and/or
substituted (heteroalicyclyl)alkylene) is substituted with at least
one substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted L3B is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
L3B is substituted, it is substituted with at least one substituent
group. In embodiments, when L3B is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when L3B is substituted, it is substituted with at least one lower
substituent group.
[0185] In embodiments, a substituted L3C (e.g., substituted
alkylene, substituted alkenylene, substituted alkynylene,
substituted heteroalkylene, substituted cycloalkylene, substituted
cycloalkenylene, substituted cycloalkynylene, substituted arylene,
substituted heteroarylene, substituted heteroalicyclylene,
substituted aralkylene, substituted heteroaralkylene, and/or
substituted (heteroalicyclyl)alkylene) is substituted with at least
one substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted L3C is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
L3C is substituted, it is substituted with at least one substituent
group. In embodiments, when L3C is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when L3C is substituted, it is substituted with at least one lower
substituent group.
[0186] In embodiments, a substituted L3D (e.g., substituted
alkylene, substituted alkenylene, substituted alkynylene,
substituted heteroalkylene, substituted cycloalkylene, substituted
cycloalkenylene, substituted cycloalkynylene, substituted arylene,
substituted heteroarylene, substituted heteroalicyclylene,
substituted aralkylene, substituted heteroaralkylene, and/or
substituted (heteroalicyclyl)alkylene) is substituted with at least
one substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted L3D is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
L3D is substituted, it is substituted with at least one substituent
group. In embodiments, when L3D is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when L3D is substituted, it is substituted with at least one lower
substituent group.
[0187] In embodiments, a substituted L3E (e.g., substituted
alkylene, substituted alkenylene, substituted alkynylene,
substituted heteroalkylene, substituted cycloalkylene, substituted
cycloalkenylene, substituted cycloalkynylene, substituted arylene,
substituted heteroarylene, substituted heteroalicyclylene,
substituted aralkylene, substituted heteroaralkylene, and/or
substituted (heteroalicyclyl)alkylene) is substituted with at least
one substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted L3E is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
L3E is substituted, it is substituted with at least one substituent
group. In embodiments, when L3E is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when L3E is substituted, it is substituted with at least one lower
substituent group.
[0188] In embodiments, a substituted L5A (e.g., substituted
alkylene, substituted alkenylene, substituted alkynylene,
substituted heteroalkylene, substituted cycloalkylene, substituted
cycloalkenylene, substituted cycloalkynylene, substituted arylene,
substituted heteroarylene, substituted heteroalicyclylene,
substituted aralkylene, substituted heteroaralkylene, and/or
substituted (heteroalicyclyl)alkylene) is substituted with at least
one substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted L5A is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
L5A is substituted, it is substituted with at least one substituent
group. In embodiments, when L5A is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when L5A is substituted, it is substituted with at least one lower
substituent group.
[0189] In embodiments, a substituted L5B (e.g., substituted
alkylene, substituted alkenylene, substituted alkynylene,
substituted heteroalkylene, substituted cycloalkylene, substituted
cycloalkenylene, substituted cycloalkynylene, substituted arylene,
substituted heteroarylene, substituted heteroalicyclylene,
substituted aralkylene, substituted heteroaralkylene, and/or
substituted (heteroalicyclyl)alkylene) is substituted with at least
one substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted L5B is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
L5B is substituted, it is substituted with at least one substituent
group. In embodiments, when L5B is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when L5B is substituted, it is substituted with at least one lower
substituent group.
[0190] In embodiments, a substituted L5C (e.g., substituted
alkylene, substituted alkenylene, substituted alkynylene,
substituted heteroalkylene, substituted cycloalkylene, substituted
cycloalkenylene, substituted cycloalkynylene, substituted arylene,
substituted heteroarylene, substituted heteroalicyclylene,
substituted aralkylene, substituted heteroaralkylene, and/or
substituted (heteroalicyclyl)alkylene) is substituted with at least
one substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted L5C is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
L5C is substituted, it is substituted with at least one substituent
group. In embodiments, when L5C is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when L5C is substituted, it is substituted with at least one lower
substituent group.
[0191] In embodiments, a substituted L5D (e.g., substituted
alkylene, substituted alkenylene, substituted alkynylene,
substituted heteroalkylene, substituted cycloalkylene, substituted
cycloalkenylene, substituted cycloalkynylene, substituted arylene,
substituted heteroarylene, substituted heteroalicyclylene,
substituted aralkylene, substituted heteroaralkylene, and/or
substituted (heteroalicyclyl)alkylene) is substituted with at least
one substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted L5D is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
L5D is substituted, it is substituted with at least one substituent
group. In embodiments, when L5D is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when L5D is substituted, it is substituted with at least one lower
substituent group.
[0192] In embodiments, a substituted L5E (e.g., substituted
alkylene, substituted alkenylene, substituted alkynylene,
substituted heteroalkylene, substituted cycloalkylene, substituted
cycloalkenylene, substituted cycloalkynylene, substituted arylene,
substituted heteroarylene, substituted heteroalicyclylene,
substituted aralkylene, substituted heteroaralkylene, and/or
substituted (heteroalicyclyl)alkylene) is substituted with at least
one substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted L5E is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
L5E is substituted, it is substituted with at least one substituent
group. In embodiments, when L5E is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when L5E is substituted, it is substituted with at least one lower
substituent group.
[0193] In embodiments, a substituted L6 (e.g., substituted
alkylene, substituted alkenylene, substituted alkynylene,
substituted heteroalkylene, substituted cycloalkylene, substituted
cycloalkenylene, substituted cycloalkynylene, substituted arylene,
substituted heteroarylene, substituted heteroalicyclylene,
substituted aralkylene, substituted heteroaralkylene, and/or
substituted (heteroalicyclyl)alkylene) is substituted with at least
one substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted L6 is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
L6 is substituted, it is substituted with at least one substituent
group. In embodiments, when L6 is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when L6 is substituted, it is substituted with at least one lower
substituent group.
[0194] In embodiments, a substituted L7 (e.g., substituted
alkylene, substituted alkenylene, substituted alkynylene,
substituted heteroalkylene, substituted cycloalkylene, substituted
cycloalkenylene, substituted cycloalkynylene, substituted arylene,
substituted heteroarylene, substituted heteroalicyclylene,
substituted aralkylene, substituted heteroaralkylene, and/or
substituted (heteroalicyclyl)alkylene) is substituted with at least
one substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted L7 is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
L7 is substituted, it is substituted with at least one substituent
group. In embodiments, when L7 is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when L7 is substituted, it is substituted with at least one lower
substituent group.
[0195] In embodiments, a substituted L8 (e.g., substituted
alkylene, substituted alkenylene, substituted alkynylene,
substituted heteroalkylene, substituted cycloalkylene, substituted
cycloalkenylene, substituted cycloalkynylene, substituted arylene,
substituted heteroarylene, substituted heteroalicyclylene,
substituted aralkylene, substituted heteroaralkylene, and/or
substituted (heteroalicyclyl)alkylene) is substituted with at least
one substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted L8 is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
L8 is substituted, it is substituted with at least one substituent
group. In embodiments, when L8 is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when L8 is substituted, it is substituted with at least one lower
substituent group.
[0196] In embodiments, a substituted L9 (e.g., substituted
alkylene, substituted alkenylene, substituted alkynylene,
substituted heteroalkylene, substituted cycloalkylene, substituted
cycloalkenylene, substituted cycloalkynylene, substituted arylene,
substituted heteroarylene, substituted heteroalicyclylene,
substituted aralkylene, substituted heteroaralkylene, and/or
substituted (heteroalicyclyl)alkylene) is substituted with at least
one substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted L9 is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
L9 is substituted, it is substituted with at least one substituent
group. In embodiments, when L9 is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when L9 is substituted, it is substituted with at least one lower
substituent group.
[0197] In embodiments, a substituted L10 (e.g., substituted
alkylene, substituted alkenylene, substituted alkynylene,
substituted heteroalkylene, substituted cycloalkylene, substituted
cycloalkenylene, substituted cycloalkynylene, substituted arylene,
substituted heteroarylene, substituted heteroalicyclylene,
substituted aralkylene, substituted heteroaralkylene, and/or
substituted (heteroalicyclyl)alkylene) is substituted with at least
one substituent group, size-limited substituent group, or lower
substituent group; wherein if the substituted L10 is substituted
with a plurality of groups selected from substituent groups,
size-limited substituent groups, and lower substituent groups; each
substituent group, size-limited substituent group, and/or lower
substituent group may optionally be different. In embodiments, when
L10 is substituted, it is substituted with at least one substituent
group. In embodiments, when L10 is substituted, it is substituted
with at least one size-limited substituent group. In embodiments,
when L10 is substituted, it is substituted with at least one lower
substituent group.
[0198] Alternatively, a nucleotide that is used herein, for
example, for extension of a primer or to participate in forming a
ternary complex can lack exogenous labels (i.e. the nucleotide can
be "non-labeled"). A non-labeled nucleotide can include a
reversible terminator such as a 3'-ONH.sub.2 moiety or an
irreversible terminator moiety. A non-labeled nucleotide can be
non-incorporable, a non-labeled nucleotide can lack terminator
moieties, a non-labeled nucleotide can be incorporable, or a
non-labeled nucleotide can be both incorporable and non-terminated.
Non-labeled nucleotides can be useful, for example, in a Sequencing
By Binding.TM. (SBB.sup.M) process when a label on a polymerase is
used to detect a stabilized ternary complex or when label-free
detection is used. Non-labeled nucleotides can also be useful in an
extension step of a SBB.TM. method or other method set forth
herein. It will be understood that absence of a moiety or function
for a nucleotide refers to the nucleotide having no such function
or moiety. However, it will also be understood that one or more of
the functions or moieties set forth herein for a nucleotide, or
analog thereof, or otherwise known in the art for a nucleotide, or
analog thereof, can be specifically omitted in a method or
composition set forth herein.
[0199] The nucleobase of a nucleotide can be a purine or
pyrimidine. For example, the nucleobase can be a canonical or
primary nucleobase such as adenine, cytosine, guanine, thymine, or
uracil. Other nucleobases can be useful such as those that are
complementary to one or more of the primary or canonical
nucleobases. In embodiments, the nucleobase is complementary to
adenine, cytosine, guanine, thymine, or uracil. Modified
nucleotides can be used, for example, methylated nucleobases such
as 5-methylcytosine or 7-methylguanosine, or hydroxylated and
methylated nucleobases such as 5-hydroxymethylcytosine. Other
useful nucleobases include, for example, hypoxanthine, xanthine,
inosine, isoguanine or isocytosine. Isoguanine and isocytosine can
be particularly useful since they are complementary to each other,
and specific for each other so as to form a third base pair when
compared to the two native base pairs, Adenine:Thymine and
Cytosine:Guanine. A particularly useful nucleobase is
7-deazaguanine, which can provide a convenient nucleobase for
synthetic procedures that attach a label to the nucleobase while
also being capable of pairing with cytosine when the labeled
nucleobase is present in a nucleic acid strand. In embodiments, the
nucleobase is independently 5-methylcytosine, hypoxanthine,
xanthine, inosine, isoguanine, isocytosine, or 7-deazaguanine. In
embodiments, the nucleobase (e.g., B, Base) includes an exogenous
label (e.g., detectable moiety, fluorophore moiety, antigen)
optionally bonded directly to the nucleobase or optionally bonded
through a linker (e.g., L5).
[0200] The sugar moiety of a nucleotide can be a deoxyribose,
ribose or analog thereof. For example, a nucleotide can have one of
the following structures:
##STR00041##
wherein R1 is H, B is a nucleobase and R3 and R4 are independently
H, CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof. This deoxyribonucleotide can be in a monomeric
form whereby Q is a hydrogen, exogenous protecting moiety, or at
least one phosphate (e.g. monophosphate, diphosphate, triphosphate
or greater than three phosphates, any of the preceding which can be
substituted or unsubstituted). Alternatively, the nucleotide can be
a moiety of a nucleic acid, for example, wherein Q is a
deoxyribonucleic acid (DNA). The DNA can be single stranded or
double stranded. In configurations in which the DNA is double
stranded the nucleotide can be a moiety of a primer strand, for
example, being at the 3' end of the primer, or the nucleotide
moiety can be in the template strand. The nucleotide can be a
moiety of a nucleic acid that is a mixed hybrid formed by strands
having different backbones such as a DNA:RNA hybrid, DNA:PNA
hybrid, RNA:PNA hybrid or the like (wherein PNA is peptide nucleic
acid).
[0201] Alternatively, the sugar moiety of a nucleotide can have one
of the following structures:
##STR00042##
wherein R1 is OH, B is a nucleobase and R3 and R4 are independently
H, CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof. This ribonucleotide can be in a monomeric form
whereby Q is at least one phosphate (e.g. monophosphate,
diphosphate, triphosphate or greater than three phosphates, any of
the preceding which can be substituted or unsubstituted).
Alternatively, the nucleotide can be a moiety of a nucleic acid,
for example, wherein Q is a ribonucleic acid (RNA). The RNA can be
single stranded or double stranded. In configurations in which the
RNA is double stranded the nucleotide can be a moiety of a primer
strand, for example, being at the 3' end of the primer, or the
nucleotide moiety can be in the template strand. The nucleotide can
be a moiety of a nucleic acid that is a mixed hybrid formed by
strands having different backbones such as a DNA:RNA hybrid,
DNA:PNA hybrid, RNA:PNA hybrid or the like (wherein PNA is peptide
nucleic acid).
[0202] Nucleic acids that are used in a method or composition
herein can be DNA such as genomic DNA, synthetic DNA, amplified
DNA, complementary DNA (cDNA) or the like. RNA can also be used
such as mRNA, ribosomal RNA, tRNA or the like. Nucleic acid analogs
such as PNA or others known in the art can also be used herein.
Nucleic acid primers, probes or templates can be DNA, RNA or
analogs thereof.
[0203] A nucleic acid may be single stranded or double stranded. A
double stranded nucleic acid may possess a single-stranded nick or
a gap. Where double-stranded, a nucleic acid molecule can be
blunt-ended or have an overhang (e.g. 5' overhang or 3' overhang).
Such nucleic acid molecules can be produced by any chemical or
enzymatic method known in the art. Examples of such methods include
phosphoramidite synthesis or replication in vivo or in vitro. A
nucleic acid of the present disclosure can be linear (e.g. having
free 3' and 5' ends) or it can be circular (e.g. lacking 5' and 3'
ends)
[0204] Deoxyribonucleic acid (DNA) is a particularly useful nucleic
acid. The DNA can be single- or double-stranded deoxyribonucleic
acid. Deoxyribonucleotides are typically joined by phosphodiester
bonds, although in some cases, nucleic acid analogs are included
that may have alternate backbones, such as, phosphoramide (e.g.
Beaucage, et al., Tetrahedron, 49 (10):1925 (1993) and references
therein; Letsinger, J. Org. Chem., 35:3800 (1970); Sprinzl, et al.,
Eur. J. Biochem., 81:579 (1977); Letsinger, et al., Nucl. Acids
Res., 14:3487 (1986); Sawai, et al., Chem. Lett., 805 (1984),
Letsinger, et al., J. Am. Chem. Soc., 110:4470 (1988); and Pauwels,
et al., Chemica Scripta, 26:141 (1986)), phosphorothioate (e.g.
Mag, et al., Nucleic Acids Res., 19:1437 (1991); and U.S. Pat. No.
5,644,048), phosphorodithioate (e.g. Briu, et al., J. Am. Chem.
Soc., 111:2321 (1989)), O-methylphophoroamidite linkages (e.g.
Eckstein, Oligonucleotides and Analogues: A Practical Approach,
Oxford University Press), and peptide nucleic acid (PNA) backbones
and linkages (e.g. Egholm, J. Am. Chem. Soc., 114:1895 (1992);
Meier, et al., Chem. Int. Ed. Engl., 31:1008 (1992); Nielsen,
Nature, 365:566 (1993); Carlsson, et al., Nature, 380:207 (1996)).
Other analog nucleic acids include those with positive backbones
(e.g. Denpcy, et al., Proc, Natd. Acad. Sci. USA, 92:6097 (1995));
non-ionic backbones (e.g. U.S. Pat. Nos. 5,386,023; 5,637,684;
5,602,240; 5,216,141; and U.S. Pat. No. 4,469,863; Kiedrowshi, et
al., Angew. Chem. Intl. Ed. English, 30:423 (1991); Letsinger, et
al., J Am. Chem. Soc., 110:4470 (1988); Letsinger, et al.,
Nucleosides & Nucleotides, 13:1597 (1994); Chapters 2 and 3,
ASC Symposium Series 580, "Carbohydrate Modifications in Antisense
Research", Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker, et al.,
Bioorganic & Medicinal Chem. Lett., 4:395 (1994); Jeffs, et
al., J. Biomolecular NMR, 34:17 (1994); Tetrahedron Lett., 37:743
(1996)) and non-ribose backbones, including for example, those
described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6
and 7, ACS Symposium Series 580, "Carbohydrate Modifications in
Antisense Research", Ed. Y. S. Sanghui and P. Dan Cook. Nucleic
acids containing one or more carbocyclic sugars are also included
within the definition of nucleic acids (e.g. Jenkins, et al., Chem.
Soc. Rev., (1995) pp. 169 176). Several nucleic acid analogs are
described in Rawls, C & E News, Jun. 2, 1997, page 35. All of
these references are hereby expressly incorporated by reference.
These modifications of the ribose-phosphate backbone may be present
to facilitate the addition of exogenous moieties such as labels, or
to increase the stability and half-life of such molecules in
physiological environments. In addition, mixtures of naturally
occurring nucleic acids and analogs can be made. Alternatively,
mixtures of different nucleic acid analogs, and mixtures of
naturally occurring nucleic acid analogs and non-natural analogs
may be made.
[0205] Exemplary organisms from which nucleic acids can be derived
include, for example, those from a mammal such as a rodent, mouse,
rat, rabbit, guinea pig, ungulate, horse, sheep, pig, goat, cow,
cat, dog, primate, human or non-human primate; a plant such as
Arabidopsis thaliana, corn, sorghum, oat, wheat, rice, canola, or
soybean; an algae such as Chlamydomonas reinhardtii; a nematode
such as Caenorhabditis elegans; an insect such as Drosophila
melanogaster, mosquito, fruit fly, honey bee or spider; a fish such
as zebrafish; a reptile; an amphibian such as a frog or Xenopus
laevis; a Dictyostelium discoideum; a fungi such as Pneumocystis
carinii, Takifugu rubripes, yeast, Saccharamoyces cerevisiae or
Schizosaccharomyces pombe; an aplicomplexan alveolate pathogen such
as Plasmodium falciparum; an environmental sample; or any other
eukaryotic, eubacterial or archaeal source. Nucleic acids can also
be derived from a eubacterial prokaryote such as a bacterium,
Escherichia coli, Staphylococci or Mycoplasma pneumoniae; an
archaean prokaryote; a virus such as Hepatitis C virus, a
coronavirus, or human immunodeficiency virus; or a viroid. Nucleic
acids can be derived from a homogeneous culture or population of
the above organisms or alternatively from a collection of several
different organisms, for example, in a community or ecosystem.
Nucleic acids can be isolated using methods known in the art
including, for example, those described in Sambrook et al.,
Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring
Harbor Laboratory, New York (2001) or in Ausubel et al., Current
Protocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.
(1998), each of which is incorporated herein by reference. Cells,
tissues, biological fluids, proteins and other samples can be
obtained from these organisms and used in a composition, apparatus
or method set forth herein.
[0206] A nucleic acid can be obtained from a preparative method
such as genome isolation, genome fragmentation, gene cloning and/or
amplification. The nucleic acid can be obtained from an
amplification technique such as polymerase chain reaction (PCR),
rolling circle amplification (RCA), multiple displacement
amplification (MDA) or the like. Amplification can also be carried
out using a method set forth in Sambrook et al., Molecular Cloning:
A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory,
New York (2001) or in Ausubel et al., Current Protocols in
Molecular Biology, John Wiley and Sons, Baltimore, Md. (1998), each
of which is incorporated herein by reference.
[0207] In some configurations of the methods and compositions set
forth herein, a single nucleotide or single nucleic acid molecule
can be manipulated, modified or detected. The single nucleotide or
single nucleic acid molecule can be in solution phase, or solid
phase (e.g. attached to a surface). In some configurations, the
single molecule is subjected to detection under conditions wherein
individual molecules are resolved one from the other (e.g. single
molecule sequencing). Alternatively, multiple nucleotides of a
particular type or multiple copies of a nucleic acid can be made or
used. The resulting ensemble can be manipulated, modified or
detected, for example, in a nucleic acid sequencing protocol. For
example, a nucleic acid can be amplified in solution or on a
surface (e.g. on the inner wall of a flow cell) using techniques
set forth in further detail below.
[0208] In multiplex embodiments, multiple different nucleic acid
molecules (i.e. a population having a variety of different
sequences) are manipulated or detected. The molecules can
optionally be attached to a surface in a flow cell or other vessel.
The nucleic acids can be attached at unique sites on the surface
and single nucleic acid molecules that are spatially
distinguishable one from the other can be manipulated, modified or
detected in parallel. Alternatively, the nucleic acids can be
amplified on the surface to produce a plurality of surface attached
ensembles. The ensembles can be spatially distinguishable from each
other and manipulated, modified or detected in parallel.
[0209] A method set forth herein can use any of a variety of
nucleic acid amplification techniques. Exemplary techniques that
can be used include, but are not limited to, polymerase chain
reaction (PCR), rolling circle amplification (RCA), multiple
displacement amplification (MDA), bridge amplification, or random
prime amplification (RPA). In particular configurations, one or
more primers used for amplification can be attached to a surface in
a flow cell. In such embodiments, extension of the surface-attached
primers along template nucleic acids will result in complementary
copies of the templates being attached to the surface. Such
amplification methods can be used for analytical purposes such as
real time PCR or quantitative PCR. Alternatively, amplification can
be used to prepare nucleic acids for downstream applications such
as nucleic acid sequencing. Preparative amplification methods that
result in one or more sites on a solid support, where each site is
attached to multiple copies of a particular nucleic acid template,
can be referred to as "clustering" methods. Primers that are used
for DNA amplification techniques can include a 3'-O-oxime moiety to
be converted to a reversible terminator moiety as disclosed herein,
or a 3'-ONH.sub.2 or other reversible terminator moiety as
disclosed herein such that deprotection methods set forth herein
can be used to generate a reversibly terminated primer suitable for
deblocking so as to initiate extension or to control extension at
one or more points in an amplification reaction.
[0210] In PCR techniques, one or both primers used for
amplification can be attached to a surface. Formats that utilize
two species of attached primer are often referred to as bridge
amplification because double stranded amplicons form a bridge-like
structure between the two attached primers that flank the template
sequence that has been copied. Exemplary reagents and conditions
that can be used for bridge amplification are described, for
example, in U.S. Pat. Nos. 5,641,658 or 7,115,400; U.S. Patent Pub.
Nos. 2002/0055100 A1, 2004/0096853 A1, 2004/0002090 A1,
2007/0128624 A1 or 2008/0009420 A1, each of which is incorporated
herein by reference. PCR amplification can also be carried out with
one of the amplification primers attached to the surface and the
second primer in solution. An exemplary format that uses a
combination of one solid phase-attached primer and a solution phase
primer is known as primer walking and can be carried out as
described in U.S. Pat. No. 9,476,080, which is incorporated herein
by reference. Another example is emulsion PCR which can be carried
out as described, for example, in Dressman et al., Proc. Natl.
Acad. Sci. USA 100:8817-8822 (2003), WO 05/010145, or U.S. Patent
Pub. Nos. 2005/0130173 A1 or 2005/0064460 A1, each of which is
incorporated herein by reference. One or both of the primers used
in a PCR technique can include a 3'-O-oxime moiety or 3'-ONH.sub.2
moiety, for example, prior to initiation of amplification or at a
point where amplification is to be stopped or paused. Amplification
can proceed by deblocking the 3' position using a method set forth
herein.
[0211] RCA techniques can be used in a method set forth herein.
Exemplary reagents that can be used in an RCA reaction and methods
by which RCA can produce amplicons are described, for example, in
Lizardi et al., Nat. Genet. 19:225-232 (1998) or US Pat. App. Pub.
No. 2007/0099208 A1, each of which is incorporated herein by
reference. Primers used for RCA can be in solution or attached to a
surface in a flow cell. The primers can optionally include a
3'-O-oxime moiety or 3'-ONH.sub.2 moiety, for example, prior to
initiation of amplification or at a point where amplification is to
be stopped or paused. Amplification can proceed by deblocking the
3' position using a method set forth herein.
[0212] MDA techniques can also be used in a method of the present
disclosure. Some reagents and useful conditions for MDA are
described, for example, in Dean et al., Proc Natl. Acad. Sci. USA
99:5261-66 (2002); Lage et al., Genome Research 13:294-307 (2003);
Walker et al., Molecular Methods for Virus Detection, Academic
Press, Inc., 1995; Walker et al., Nucl. Acids Res. 20:1691-96
(1992); or U.S. Pat. Nos. 5,455,166; 5,130,238; or 6,214,587, each
of which is incorporated herein by reference. Primers used for MDA
can be in solution or attached to a surface in a flow cell. The
primers can optionally include a 3'-O-oxime moiety or 3'-ONH.sub.2
moiety, for example, prior to initiation of amplification or at a
point where amplification is to be stopped or paused. Amplification
can proceed by deblocking the 3' position using a method set forth
herein.
[0213] Nucleic acid templates that are used in a method or
composition herein can be DNA such as genomic DNA, synthetic DNA,
amplified DNA, complementary DNA (cDNA) or the like. RNA can also
be used such as mRNA, ribosomal RNA, tRNA or the like. Nucleic acid
analogs can also be used as templates herein. Primers used herein
can be DNA, RNA or analogs thereof.
[0214] A nucleic acid that is used in a method or composition
herein can be linear, for example, being flanked by a 3' end and a
5' end. Alternatively, a nucleic acid can be circular, thereby
lacking a 3' and 5' end. Whether linear, circular or in any other
conformation, a nucleic acid that is used herein can have a size
that is desired for a particular use or that is a result of
manipulations carried out on the nucleic acid. For example, a
nucleic acid can have a length that is at least 50 bases, 100
bases, 1.times.10.sup.3 bases, 1.times.10.sup.4 bases,
1.times.10.sup.5 bases, 1.times.10.sup.6 bases or longer.
Alternatively or additionally, the nucleic acid length can be at
most 1.times.10.sup.6 bases, 1.times.10.sup.5 bases,
1.times.10.sup.4 bases, 1.times.10.sup.3 bases, 100 bases, 50 bases
or shorter. When a population of nucleic acids is used, the average
length for the population can have a lower and/or upper limit
selected from those ranges.
[0215] A nucleotide can be attached to a solid support. The
nucleotide can be in monomeric form or it can be a moiety of a
nucleic acid. Typically, the nucleotide will be attached to the
solid support via a moiety other than the 3' position. As such the
3' position can be blocked or deblocked as set forth herein.
Accordingly, the nucleotide although being attached to a solid
support can be in contact with a solution. The solution can contain
reagents for blocking or deblocking the solid-phase nucleotide. By
way of example, the attached nucleotide can have one of the
following structures:
##STR00043##
wherein Base is a nucleobase, and wherein DNA is independently a
single stranded deoxyribonucleic acid or double stranded
deoxyribonucleic acid. Optionally, the nucleotide can be attached
to the solid support via the DNA. Alternatively, the sugar can be a
ribose instead of deoxyribose, and the nucleotide can be at the 3'
end of RNA instead of DNA. In this case the RNA can be single
stranded or double stranded. Optionally, the nucleotide can be
attached to the solid support via the RNA.
[0216] In embodiments, a substituted Base (e.g., a substituted
nucleobase) is substituted with at least one substituent group,
size-limited substituent group, or lower substituent group; wherein
if the substituted Base is substituted with a plurality of groups
selected from substituent groups, size-limited substituent groups,
and lower substituent groups; each substituent group, size-limited
substituent group, and/or lower substituent group may optionally be
different. In embodiments, when Base is substituted, it is
substituted with at least one substituent group. In embodiments,
when Base is substituted, it is substituted with at least one
size-limited substituent group. In embodiments, when Base is
substituted, it is substituted with at least one lower substituent
group.
[0217] A particularly useful solid support is an array. Arrays
provide an advantage of multiplex processing of analytes, whereby
the multiple different types of analytes are manipulated or
detected in parallel. Although it is also possible to serially
process different types of analytes using methods or compositions
set forth herein, parallel processing can provide cost savings,
time savings and uniformity of assay conditions. An array can
include at least 2, 10, 100, 1.times.10.sup.3, 1.times.10.sup.4,
1.times.10.sup.5, 1.times.10.sup.6, 1.times.10.sup.9, or more
different analyte sites. Alternatively or additionally, an array
can include at most 1.times.10.sup.9, 1.times.10.sup.6,
1.times.10.sup.5, 1.times.10.sup.4, 1.times.10.sup.3, 100, 10, 2 or
fewer, different analyte sites.
[0218] An array can be attached to an inner surface of a vessel
(e.g. the inner wall of a flow cell) or to a solid support inside
of a vessel (e.g. a bead or other solid support inside of a flow
cell). The vessel or solid support can be made from any of a
variety of materials used for analytical biochemistry. Suitable
materials may include glass, polymeric materials, silicon, quartz
(fused silica), borofloat glass, silica, silica-based materials,
carbon, metals, an optical fiber or bundle of optical fibers,
sapphire, or plastic materials. The material can be selected based
on properties desired for a particular use. For example, materials
that are transparent to a desired wavelength of radiation are
useful for analytical techniques that will utilize radiation at
that wavelength. Conversely, it may be desirable to select a
material that does not pass radiation of a certain wavelength (e.g.
being opaque, absorptive or reflective). Other properties of a
material that can be exploited are inertness or reactivity to
reagents used in a downstream process, such as those set forth
herein, or ease of manipulation, or low cost of manufacture.
[0219] A particularly useful solid support for use in a flow cell
or other vessel is a particle such as a bead or microsphere.
Populations of beads can be used for attachment of populations of
analytes such as nucleotides, nucleic acids, reversibly terminated
primers, deblocked primers, extendable primers, polymerases,
stabilized ternary complexes or the like. In some configurations,
each bead has a single type of stabilized ternary complex, a single
type of component capable of forming the complex, or a single type
of some other analyte set forth herein or in references cited
herein. For example, an individual bead can be attached to a single
type of ternary complex, a single type of template allele, a single
type of template locus, a single type of primer, single type of
reversibly terminated primer, single type of deblocked primer,
single type of extendable primer, or a single type of nucleotide.
Alternatively, different types of components need not be separated
on a bead-by-bead basis. As such, a single bead can bear multiple
different types of: ternary complexes, template nucleic acids,
primers (whether blocked or extendable), primed template nucleic
acids (whether the primers are blocked or extendable) and/or
nucleotides (whether blocked or extendable). The composition of a
bead can vary, depending for example, on the format, chemistry
and/or method of attachment to be used. Exemplary bead compositions
include solid supports, and chemical functionalities thereon, used
in protein and nucleic acid capture methods. Such compositions
include, for example, plastics, ceramics, glass, polystyrene,
melamine, methylstyrene, acrylic polymers, paramagnetic materials,
thoria sol, carbon graphite, titanium dioxide, controlled pore
glass (CPG), latex or cross-linked dextrans such as Sepharose.TM.,
cellulose, nylon, cross-linked micelles and Teflon.TM., as well as
other materials set forth in "Microsphere Detection Guide" from
Bangs Laboratories, Fishers Ind., which is incorporated herein by
reference.
[0220] Beads can have a symmetrical shape, such as spherical,
polyhedral, cylindrical or the like. Alternatively, beads can have
an irregular or non-symmetric shape. Exemplary sizes for beads used
herein can have average diameter that is at least about 10 nm, 100
nm, 1 .mu.m, 5 .mu.m, 10 .mu.m, 100 .mu.m, 1 mm or larger.
Alternatively or additionally, beads used herein can have average
diameter that is at most about 1 mm, 100 .mu.m, 10 .mu.m, 5 .mu.m,
1 .mu.m, 100 nm, 10 nm, 1 nm or smaller. Beads in these size ranges
can be used as array features or as particles in a fluid.
[0221] Exemplary compositions and techniques that can be used to
make an array of beads include, without limitation, those used for
BeadChip.TM. Arrays available from Illumina, Inc. (San Diego,
Calif.) those described in U.S. Pat. Nos. 6,266,459; 6,355,431;
6,770,441; 6,859,570; or 7,622,294; or PCT Publication No. WO
00/63437, each of which is incorporated herein by reference. Beads
can be located at discrete locations, such as wells, on a solid
support, whereby each location accommodates a single bead.
Alternatively, discrete locations where beads reside can each
include a plurality of beads as described, for example, in U.S.
Pat. App. Pub. Nos. 2004/0263923 A1, 2004/0233485 A1, 2004/0132205
A1, or 2004/0125424 A1, each of which is incorporated herein by
reference.
[0222] Other useful arrays include those that are used in nucleic
acid sequencing applications. For example, arrays that are used to
immobilize amplicons of genomic fragments (often referred to as
clusters) can be particularly useful. Examples of nucleic acid
sequencing arrays that can be used herein include those described
in Bentley et al., Nature 456:53-59 (2008), PCT Pub. Nos. WO
91/06678; WO 04/018497 or WO 07/123744; U.S. Pat. Nos. 7,057,026;
7,211,414; 7,315,019; 7,329,492 or 7,405,281; or U.S. Pat. App.
Pub. No. 2008/0108082, each of which is incorporated herein by
reference. Arrays formed on flow cells used for commercial
sequencing platforms, such as those developed by Illumina (San
Diego, Calif.), Life Technologies (a subsidiary of Thermo Fisher,
Waltham Mass.), Pacific Biosciences (Menlo Park, Calif.), MGI (a
subsidiary of BGI, Shenzhen, Guangdong, China), can also be
useful.
[0223] A nucleic acid or other analyte can be attached to a support
in a way that provides detection at a single molecule level or at
an ensemble level. For example, a plurality of different nucleic
acids can be attached to a solid support in a way that an
individual nucleic acid molecule that is in a blocked or deblocked
state on the support can be distinguished from all neighboring
nucleic acid molecules on the support. As such, one or more
different templates can be attached to a solid support in a format
where each single molecule template is physically isolated and
detected in a way that the single molecule is resolved from all
other molecules on the solid support, for example, in a sequencing
process set forth herein.
[0224] Alternatively, a composition or method of the present
disclosure can employ one or more ensembles, an ensemble being a
population of analytes of the same type such as a population of
nucleic acids having a common template sequence. Cluster methods
can be used to attach one or more nucleic acid ensembles to a solid
support. As such, an array can have a plurality of ensembles, each
of the ensembles being referred to as a cluster or array site in
that format. Clusters can be formed using methods known in the art
such as bridge amplification, emulsion PCR or other methods set
forth herein. The nucleic acids in a cluster can include a
reversibly terminated nucleotide, deblocked nucleotide or
extendable nucleotide
[0225] A system of the present disclosure can be configured to use
a flow cell. The flow cell can include a detection channel where an
analytical reaction of interest can be observed. The analytical
reaction can occur in solution phase within the flow cell.
Alternatively, an analytical reaction can occur on a solid support
within the detection channel. For example, a reagent solution can
be flowed over a solid support that is attached to analytes of
interest, such as nucleic acids, and a resulting reaction can be
observed on the solid support. Nucleic acids that are present in a
flow cell, whether surface attached or not, can include a 3'
nucleotide that is reversibly terminated, deblocked or extendable.
A flow cell allows convenient fluidic manipulation by passing
solutions through an ingress opening, into the detection channel
and out of the interior via an egress opening. For example, the
detection channel can have an observation area or volume such as an
optically transparent window through which optical signals can be
observed, an electrical contact through which electronic signals
can be observed or the like. A particularly useful flow cell has a
window that is transparent to excitation radiation and emission
radiation used for luminescence detection. Exemplary flow cells
that can be used herein are described, for example, in US Pat. App.
Pub. No. 2010/0111768 A1, WO 05/065814 or US Pat. App. Pub. No.
2012/0270305 A1, each of which is incorporated herein by
reference.
[0226] The present disclosure provides a method for sequencing a
nucleic acid. The method can be carried out on a primed template
nucleic acid in which the primer has a 3' ONH.sub.2 reversible
terminator moiety that is reacted with a deblocking reagent having
nitrous acid (HONO), thereby converting the primer to an extendable
primer having a 3' hydroxyl moiety. In some configurations, the
reversibly terminated primer can have the following structure:
##STR00044##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH; and Q is a nucleic acid primer.
[0227] Alternatively, a sequencing method can be carried out on a
primed template nucleic acid in which the primer has a 3'-O-oxime
protecting moiety that is removed by treatment with a reagent
having the structure R2-ONH.sub.2 or other reversible terminator
and, thereby converting the primer to a reversibly terminated
primer which can then be converted to an extendable primer having a
3' hydroxyl moiety by, for example, reaction with nitrous acid
(HONO). Alternatively, a sequencing method can be carried out on a
primed template nucleic acid in which the primer has a 3'-O-oxime
reversible terminator moiety that is deblocked by treatment with a
reagent having the structure R2-ONH.sub.2 and with nitrous acid
(HONO), thereby converting the primer to an extendable primer
having a 3' hydroxyl moiety. R2 can independently be alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl,
(heteroalicyclyl)alkyl, or an optionally substituted variant
thereof.
[0228] In embodiments, R2 is --C(R6)(R7)(R8), --C(O)(R6),
--P(O)(R6)(R7), --C(R6)(.dbd.C(R7)(R8)), --Si(R6)(R7)(R8), --S
O2(R6), substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C5-C6), substituted or
unsubstituted cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
or C.sub.5-C.sub.6), substituted or unsubstituted cycloalkynyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6),
substituted or unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered), substituted or
unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or
phenyl), or substituted or unsubstituted heteroaryl (e.g., 5 to 10
membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered
heteroaryl).
[0229] In embodiments, R2 is --C(R6)(R7)(R8). In embodiments, R2 is
--C(O)(R6). In embodiments, R2 is --P(O)(R6)(R7). In embodiments,
R2 is --C(R6)(.dbd.C(R7)(R8)). In embodiments, R2 is
--Si(R6)(R7)(R8). In embodiments, R2 is --SO2(R6). In embodiments,
R2 is substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is substituted or unsubstituted cycloalkenyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is substituted or unsubstituted cycloalkynyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is substituted or unsubstituted heteroalicyclyl
(e.g., 3 to 8 membered, 3 to 6 membered, or 5 to 6 membered). In
embodiments, R2 is substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In embodiments,
R2 is substituted or unsubstituted heteroaryl (e.g., 5 to 10
membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered
heteroaryl). In embodiments, R2 is unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is unsubstituted cycloalkenyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is unsubstituted cycloalkynyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered). In embodiments, R2
is unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl,
or phenyl). In embodiments, R2 is unsubstituted heteroaryl (e.g., 5
to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6
membered heteroaryl). In embodiments, R2 is not unsubstituted
methyl. In embodiments, R2 is not unsubstituted ethyl. In
embodiments, R2 is not unsubstituted propyl.
[0230] R6, R7, and R8 are independently hydrogen, halogen,
--CCl.sub.3, --CBr.sub.3, --CF.sub.3, --CI.sub.3, --CHCl.sub.2,
--CHBr.sub.2, --CHF.sub.2, --CHI.sub.2, --CH.sub.2Cl, --CH.sub.2Br,
--CH.sub.2F, --CH.sub.2I, --CN, --OH, --NH.sub.2, --COOH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2,
--NHC(O)NH.sub.2, --NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH,
--OCCl.sub.3, --OCF.sub.3, --OCBr.sub.3, --OCI.sub.3,
--OCHCl.sub.2, --OCHBr.sub.2, --OCHI.sub.2, --OCHF.sub.2,
--OCH.sub.2Cl, --OCH.sub.2Br, --OCH.sub.2I, --OCH.sub.2F,
--SF.sub.5, --N.sub.3, substituted or unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or C1-C4 alkyl),
substituted or unsubstituted alkenyl (e.g., C.sub.2-C.sub.8
alkenyl, C.sub.2-C.sub.6 alkenyl, or C2-C4 alkenyl), substituted or
unsubstituted alkynyl (e.g., C.sub.2-C.sub.8 alkynyl,
C.sub.2-C.sub.6 alkynyl, or C.sub.2-C.sub.4 alkynyl), substituted
or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2
to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl),
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6), substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6), substituted or unsubstituted cycloalkynyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6), substituted
or unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl,
or phenyl), substituted or unsubstituted heteroaryl (e.g., 5 to 10
membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered
heteroaryl), substituted or unsubstituted heteroalicyclyl (e.g., 3
to 8 membered, 3 to 6 membered, or 5 to 6 membered), substituted or
unsubstituted aralkyl, substituted or unsubstituted heteroaralkyl,
or substituted or unsubstituted (heteroalicyclyl)alkyl. In
embodiments, R6, R7, and R8 are not all hydrogen. In embodiments,
R2 is --C(R6)(R7)(R8) and R6, R7, and R8 are not all hydrogen.
[0231] In embodiments, R6 is independently hydrogen. In
embodiments, R6 is independently halogen. In embodiments, R6 is
independently --CCl.sub.3. In embodiments, R6 is independently
--CBr.sub.3. In embodiments, R6 is independently --CF.sub.3. In
embodiments, R6 is independently --CI.sub.3. In embodiments, R6 is
independently --CHCl.sub.2. In embodiments, R6 is independently
--CHBr.sub.2. In embodiments, R6 is independently --CHF.sub.2. In
embodiments, R6 is independently --CHI.sub.2. In embodiments, R6 is
independently --CH.sub.2Cl. In embodiments, R6 is independently
--CH.sub.2Br. In embodiments, R6 is independently --CH.sub.2F. In
embodiments, R6 is independently --CH.sub.2I. In embodiments, R6 is
independently --CN. In embodiments, R6 is independently --OH. In
embodiments, R6 is independently --NH.sub.2. In embodiments, R6 is
independently --COOH. In embodiments, R6 is independently
--CONH.sub.2. In embodiments, R6 is independently --NO.sub.2. In
embodiments, R6 is independently --SH. In embodiments, R6 is
independently --SO.sub.3H. In embodiments, R6 is independently
--SO.sub.4H. In embodiments, R6 is independently
--SO.sub.2NH.sub.2. In embodiments, R6 is independently
--NHNH.sub.2. In embodiments, R6 is independently --ONH.sub.2. In
embodiments, R6 is independently --NHC(O)NHNH.sub.2. In
embodiments, R6 is independently --NHC(O)NH.sub.2. In embodiments,
R6 is independently --NHSO.sub.2H. In embodiments, R6 is
independently --NHC(O)H. In embodiments, R6 is independently
--NHC(O)OH. In embodiments, R6 is independently --NHOH. In
embodiments, R6 is independently --OCCl.sub.3. In embodiments, R6
is independently --OCF.sub.3. In embodiments, R6 is independently
--OCBr.sub.3. In embodiments, R6 is independently --OCI.sub.3. In
embodiments, R6 is independently --OCHCl.sub.2. In embodiments, R6
is independently --OCHBr.sub.2. In embodiments, R6 is independently
--OCHI.sub.2. In embodiments, R6 is independently --OCHF.sub.2. In
embodiments, R6 is independently --OCH.sub.2Cl. In embodiments, R6
is independently --OCH.sub.2Br. In embodiments, R6 is independently
--OCH.sub.2I. In embodiments, R6 is independently --OCH.sub.2F. In
embodiments, R6 is independently --SF.sub.5. In embodiments, R6 is
independently --N.sub.3. In embodiments, R6 is independently
substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4 alkyl). In embodiments,
R6 is independently substituted or unsubstituted alkenyl (e.g.,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.6 alkenyl, or
C.sub.2-C.sub.4 alkenyl). In embodiments, R6 is independently
substituted or unsubstituted alkynyl (e.g., C.sub.2-C.sub.8
alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4 alkynyl). In
embodiments, R6 is independently substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R6 is
independently substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R6 is independently substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R6 is independently substituted
or unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R6 is
independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In embodiments,
R6 is independently substituted or unsubstituted heteroaryl (e.g.,
5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6
membered heteroaryl). In embodiments, R6 is independently
substituted or unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered). In embodiments, R6
is independently substituted or unsubstituted aralkyl. In
embodiments, R6 is independently substituted or unsubstituted
heteroaralkyl. In embodiments, R6 is independently substituted or
unsubstituted (heteroalicyclyl)alkyl.
[0232] In embodiments, R6 is independently unsubstituted alkyl
(e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or
C.sub.1-C.sub.4 alkyl). In embodiments, R6 is independently
unsubstituted alkenyl (e.g., C.sub.2-C.sub.8 alkenyl,
C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.4 alkenyl). In
embodiments, R6 is independently unsubstituted alkynyl (e.g.,
C.sub.2-C.sub.8 alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4
alkynyl). In embodiments, R6 is independently unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R6 is
independently unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R6 is
independently unsubstituted cycloalkenyl (e.g., C.sub.3-C.sub.8,
C3-C6, or C.sub.5-C.sub.6). In embodiments, R6 is independently
unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
or C.sub.5-C.sub.6). In embodiments, R6 is independently
unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or
phenyl). In embodiments, R6 is independently unsubstituted
heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered
heteroaryl, or 5 to 6 membered heteroaryl). In embodiments, R6 is
independently unsubstituted heteroalicyclyl (e.g., 3 to 8 membered,
3 to 6 membered, or 5 to 6 membered). In embodiments, R6 is
independently unsubstituted aralkyl. In embodiments, R6 is
independently unsubstituted heteroaralkyl. In embodiments, R6 is
independently unsubstituted (heteroalicyclyl)alkyl.
[0233] In embodiments, R7 is independently hydrogen. In
embodiments, R7 is independently halogen. In embodiments, R7 is
independently --CCl.sub.3. In embodiments, R7 is independently
--CBr.sub.3. In embodiments, R7 is independently --CF.sub.3. In
embodiments, R7 is independently --CI.sub.3. In embodiments, R7 is
independently --CHCl.sub.2. In embodiments, R7 is independently
--CHBr.sub.2. In embodiments, R7 is independently --CHF.sub.2. In
embodiments, R7 is independently --CHI.sub.2. In embodiments, R7 is
independently --CH.sub.2Cl. In embodiments, R7 is independently
--CH.sub.2Br. In embodiments, R7 is independently --CH.sub.2F. In
embodiments, R7 is independently --CH.sub.2I. In embodiments, R7 is
independently --CN. In embodiments, R7 is independently --OH. In
embodiments, R7 is independently --NH.sub.2. In embodiments, R7 is
independently --COOH. In embodiments, R7 is independently
--CONH.sub.2. In embodiments, R7 is independently --NO.sub.2. In
embodiments, R7 is independently --SH. In embodiments, R7 is
independently --SO.sub.3H. In embodiments, R7 is independently
--SO.sub.4H. In embodiments, R7 is independently
--SO.sub.2NH.sub.2. In embodiments, R7 is independently
--NHNH.sub.2. In embodiments, R7 is independently --ONH.sub.2. In
embodiments, R7 is independently --NHC(O)NHNH.sub.2. In
embodiments, R7 is independently --NHC(O)NH.sub.2. In embodiments,
R7 is independently --NHSO.sub.2H. In embodiments, R7 is
independently --NHC(O)H. In embodiments, R7 is independently
--NHC(O)OH. In embodiments, R7 is independently --NHOH. In
embodiments, R7 is independently --OCCl.sub.3. In embodiments, R7
is independently --OCF.sub.3. In embodiments, R7 is independently
--OCBr.sub.3. In embodiments, R7 is independently --OCI.sub.3. In
embodiments, R7 is independently --OCHCl.sub.2. In embodiments, R7
is independently --OCHBr.sub.2. In embodiments, R7 is independently
--OCHI.sub.2. In embodiments, R7 is independently --OCHF.sub.2. In
embodiments, R7 is independently --OCH.sub.2Cl. In embodiments, R7
is independently --OCH.sub.2Br. In embodiments, R7 is independently
--OCH.sub.2I. In embodiments, R7 is independently --OCH.sub.2F. In
embodiments, R7 is independently --SF.sub.5. In embodiments, R7 is
independently --N.sub.3. In embodiments, R7 is independently
substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4 alkyl). In embodiments,
R7 is independently substituted or unsubstituted alkenyl (e.g.,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.6 alkenyl, or
C.sub.2-C.sub.4 alkenyl). In embodiments, R7 is independently
substituted or unsubstituted alkynyl (e.g., C.sub.2-C.sub.8
alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4 alkynyl). In
embodiments, R7 is independently substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R7 is
independently substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R7 is independently substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R7 is independently substituted
or unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R7 is
independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In embodiments,
R7 is independently substituted or unsubstituted heteroaryl (e.g.,
5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6
membered heteroaryl). In embodiments, R7 is independently
substituted or unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered). In embodiments, R7
is independently substituted or unsubstituted aralkyl. In
embodiments, R7 is independently substituted or unsubstituted
heteroaralkyl. In embodiments, R7 is independently substituted or
unsubstituted (heteroalicyclyl)alkyl.
[0234] In embodiments, R7 is independently unsubstituted alkyl
(e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or
C.sub.1-C.sub.4 alkyl). In embodiments, R7 is independently
unsubstituted alkenyl (e.g., C.sub.2-C.sub.8 alkenyl,
C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.4 alkenyl). In
embodiments, R7 is independently unsubstituted alkynyl (e.g.,
C.sub.2-C.sub.8 alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4
alkynyl). In embodiments, R7 is independently unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R7 is
independently unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R7 is
independently unsubstituted cycloalkenyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R7 is
independently unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R7 is
independently unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl,
C.sub.10 aryl, or phenyl). In embodiments, R7 is independently
unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9
membered heteroaryl, or 5 to 6 membered heteroaryl). In
embodiments, R7 is independently unsubstituted heteroalicyclyl
(e.g., 3 to 8 membered, 3 to 6 membered, or 5 to 6 membered). In
embodiments, R7 is independently unsubstituted aralkyl. In
embodiments, R7 is independently unsubstituted heteroaralkyl. In
embodiments, R7 is independently unsubstituted
(heteroalicyclyl)alkyl.
[0235] In embodiments, R8 is independently hydrogen. In
embodiments, R8 is independently halogen. In embodiments, R8 is
independently --CCl.sub.3. In embodiments, R8 is independently
--CBr.sub.3. In embodiments, R8 is independently --CF.sub.3. In
embodiments, R8 is independently --CI.sub.3. In embodiments, R8 is
independently --CHCl.sub.2. In embodiments, R8 is independently
--CHBr.sub.2. In embodiments, R8 is independently --CHF.sub.2. In
embodiments, R8 is independently --CHI.sub.2. In embodiments, R8 is
independently --CH.sub.2Cl. In embodiments, R8 is independently
--CH.sub.2Br. In embodiments, R8 is independently --CH.sub.2F. In
embodiments, R8 is independently --CH.sub.2I. In embodiments, R8 is
independently --CN. In embodiments, R8 is independently --OH. In
embodiments, R8 is independently --NH.sub.2. In embodiments, R8 is
independently --COOH. In embodiments, R8 is independently
--CONH.sub.2. In embodiments, R8 is independently --NO.sub.2. In
embodiments, R8 is independently --SH. In embodiments, R8 is
independently --SO.sub.3H. In embodiments, R8 is independently
--SO.sub.4H. In embodiments, R8 is independently
--SO.sub.2NH.sub.2. In embodiments, R8 is independently
--NHNH.sub.2. In embodiments, R8 is independently --ONH.sub.2. In
embodiments, R8 is independently --NHC(O)NHNH.sub.2. In
embodiments, R8 is independently --NHC(O)NH.sub.2. In embodiments,
R8 is independently --NHSO.sub.2H. In embodiments, R8 is
independently --NHC(O)H. In embodiments, R8 is independently
--NHC(O)OH. In embodiments, R8 is independently --NHOH. In
embodiments, R8 is independently --OCCl.sub.3. In embodiments, R8
is independently --OCF.sub.3. In embodiments, R8 is independently
--OCBr.sub.3. In embodiments, R8 is independently --OCI.sub.3. In
embodiments, R8 is independently --OCHCl.sub.2. In embodiments, R8
is independently --OCHBr.sub.2. In embodiments, R8 is independently
--OCHI.sub.2. In embodiments, R8 is independently --OCHF.sub.2. In
embodiments, R8 is independently --OCH.sub.2Cl. In embodiments, R8
is independently --OCH.sub.2Br. In embodiments, R8 is independently
--OCH.sub.2I. In embodiments, R8 is independently --OCH.sub.2F. In
embodiments, R8 is independently --SF.sub.5. In embodiments, R8 is
independently --N.sub.3. In embodiments, R8 is independently
substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.6 alkyl, or C1-C4 alkyl). In embodiments, R8 is
independently substituted or unsubstituted alkenyl (e.g.,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.6 alkenyl, or
C.sub.2-C.sub.4 alkenyl). In embodiments, R8 is independently
substituted or unsubstituted alkynyl (e.g., C.sub.2-C.sub.8
alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4 alkynyl). In
embodiments, R8 is independently substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R8 is
independently substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R8 is independently substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R8 is independently substituted
or unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R8 is
independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In embodiments,
R8 is independently substituted or unsubstituted heteroaryl (e.g.,
5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6
membered heteroaryl). In embodiments, R8 is independently
substituted or unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered). In embodiments, R8
is independently substituted or unsubstituted aralkyl. In
embodiments, R8 is independently substituted or unsubstituted
heteroaralkyl. In embodiments, R8 is independently substituted or
unsubstituted (heteroalicyclyl)alkyl.
[0236] In embodiments, R8 is independently unsubstituted alkyl
(e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or
C.sub.1-C.sub.4 alkyl). In embodiments, R8 is independently
unsubstituted alkenyl (e.g., C.sub.2-C.sub.8 alkenyl,
C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.4 alkenyl). In
embodiments, R8 is independently unsubstituted alkynyl (e.g.,
C.sub.2-C.sub.8 alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4
alkynyl). In embodiments, R8 is independently unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R8 is
independently unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R8 is
independently unsubstituted cycloalkenyl (e.g., C.sub.3-C.sub.8,
C3-C6, or C.sub.5-C.sub.6). In embodiments, R8 is independently
unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
or C.sub.5-C.sub.6). In embodiments, R8 is independently
unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or
phenyl). In embodiments, R8 is independently unsubstituted
heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered
heteroaryl, or 5 to 6 membered heteroaryl). In embodiments, R8 is
independently unsubstituted heteroalicyclyl (e.g., 3 to 8 membered,
3 to 6 membered, or 5 to 6 membered). In embodiments, R8 is
independently unsubstituted aralkyl. In embodiments, R8 is
independently unsubstituted heteroaralkyl. In embodiments, R8 is
independently unsubstituted (heteroalicyclyl)alkyl.
[0237] The deprotecting treatment can be carried out by reacting
the 3'-O-oxime reversible terminator moiety with R2-ONH.sub.2 to
produce 3'-ONH.sub.2 reversible terminator moiety and then treating
the 3'-ONH.sub.2 moiety with HONO to produce a 3' hydroxyl moiety.
In some configurations, the protected primer can have the following
structure:
##STR00045##
[0238] wherein B is a nucleobase; R1 is independently halogen,
OCH.sub.3, H or OH; and Q is a nucleic acid primer. R3 and R4 can
independently be H, CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally
substituted variant thereof. The deblocking treatment can be
carried out by reacting the 3'-O-oxime reversible terminator moiety
with R2-ONH.sub.2 to produce 3'-ONH.sub.2 moiety and then treating
the 3'-ONH.sub.2 moiety with HONO to produce a 3' hydroxyl moiety.
In some configurations, the reversibly terminated primer can have
the following structure:
##STR00046##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH; and Q is a nucleic acid primer. R3 and R4 can
independently be H, CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally
substituted variant thereof.
[0239] In embodiments, R2 is --C(R6)(R7)(R8), --C(O)(R6),
--P(O)(R6)(R7), --C(R6)(.dbd.C(R7)(R8)), --Si(R6)(R7)(R8), --S
O2(R6), substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C5-C6), substituted or
unsubstituted cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
or C.sub.5-C.sub.6), substituted or unsubstituted cycloalkynyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6),
substituted or unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered), substituted or
unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or
phenyl), or substituted or unsubstituted heteroaryl (e.g., 5 to 10
membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered
heteroaryl).
[0240] In embodiments, R2 is --C(R6)(R7)(R8). In embodiments, R2 is
--C(O)(R6). In embodiments, R2 is --P(O)(R6)(R7). In embodiments,
R2 is --C(R6)(.dbd.C(R7)(R8)). In embodiments, R2 is
--Si(R6)(R7)(R8). In embodiments, R2 is --SO.sub.2(R6). In
embodiments, R2 is substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is substituted or unsubstituted cycloalkenyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is substituted or unsubstituted cycloalkynyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is substituted or unsubstituted heteroalicyclyl
(e.g., 3 to 8 membered, 3 to 6 membered, or 5 to 6 membered). In
embodiments, R2 is substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In embodiments,
R2 is substituted or unsubstituted heteroaryl (e.g., 5 to 10
membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered
heteroaryl). In embodiments, R2 is unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is unsubstituted cycloalkenyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is unsubstituted cycloalkynyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered). In embodiments, R2
is unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl,
or phenyl). In embodiments, R2 is unsubstituted heteroaryl (e.g., 5
to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6
membered heteroaryl). In embodiments, R6, R7, and R8 are not all
hydrogen. In embodiments, R2 is --C(R6)(R7)(R8) and R6, R7, and R8
are not all hydrogen. In embodiments, R2 is not unsubstituted
methyl. In embodiments, R2 is not unsubstituted ethyl. In
embodiments, R2 is not unsubstituted propyl.
[0241] R6, R7, and R8 are independently hydrogen, halogen,
--CCl.sub.3, --CBr.sub.3, --CF.sub.3, --CI.sub.3, --CHCl.sub.2,
--CHBr.sub.2, --CHF.sub.2, --CHI.sub.2, --CH.sub.2Cl, --CH.sub.2Br,
--CH.sub.2F, --CH.sub.2I, --CN, --OH, --NH.sub.2, --COOH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2,
--NHC(O)NH.sub.2, --NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH,
--OCCl.sub.3, --OCF.sub.3, --OCBr.sub.3, --OCI.sub.3,
--OCHCl.sub.2, --OCHBr.sub.2, --OCHI.sub.2, --OCHF.sub.2,
--OCH.sub.2Cl, --OCH.sub.2Br, --OCH.sub.2I, --OCH.sub.2F,
--SF.sub.5, --N.sub.3, substituted or unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or C1-C4 alkyl),
substituted or unsubstituted alkenyl (e.g., C.sub.2-C.sub.8
alkenyl, C.sub.2-C.sub.6 alkenyl, or C2-C4 alkenyl), substituted or
unsubstituted alkynyl (e.g., C.sub.2-C.sub.8 alkynyl,
C.sub.2-C.sub.6 alkynyl, or C.sub.2-C.sub.4 alkynyl), substituted
or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2
to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl),
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6), substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6), substituted or unsubstituted cycloalkynyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6), substituted
or unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl,
or phenyl), substituted or unsubstituted heteroaryl (e.g., 5 to 10
membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered
heteroaryl), substituted or unsubstituted heteroalicyclyl (e.g., 3
to 8 membered, 3 to 6 membered, or 5 to 6 membered), substituted or
unsubstituted aralkyl, substituted or unsubstituted heteroaralkyl,
or substituted or unsubstituted (heteroalicyclyl)alkyl.
[0242] In embodiments, R6 is independently hydrogen. In
embodiments, R6 is independently halogen. In embodiments, R6 is
independently --CCl.sub.3. In embodiments, R6 is independently
--CBr.sub.3. In embodiments, R6 is independently --CF.sub.3. In
embodiments, R6 is independently --CI.sub.3. In embodiments, R6 is
independently --CHCl.sub.2. In embodiments, R6 is independently
--CHBr.sub.2. In embodiments, R6 is independently --CHF.sub.2. In
embodiments, R6 is independently --CHI.sub.2. In embodiments, R6 is
independently --CH.sub.2Cl. In embodiments, R6 is independently
--CH.sub.2Br. In embodiments, R6 is independently --CH.sub.2F. In
embodiments, R6 is independently --CH.sub.2I. In embodiments, R6 is
independently --CN. In embodiments, R6 is independently --OH. In
embodiments, R6 is independently --NH.sub.2. In embodiments, R6 is
independently --COOH. In embodiments, R6 is independently
--CONH.sub.2. In embodiments, R6 is independently --NO.sub.2. In
embodiments, R6 is independently --SH. In embodiments, R6 is
independently --SO.sub.3H. In embodiments, R6 is independently
--SO.sub.4H. In embodiments, R6 is independently
--SO.sub.2NH.sub.2. In embodiments, R6 is independently
--NHNH.sub.2. In embodiments, R6 is independently --ONH.sub.2. In
embodiments, R6 is independently --NHC(O)NHNH.sub.2. In
embodiments, R6 is independently --NHC(O)NH.sub.2. In embodiments,
R6 is independently --NHSO.sub.2H. In embodiments, R6 is
independently --NHC(O)H. In embodiments, R6 is independently
--NHC(O)OH. In embodiments, R6 is independently --NHOH. In
embodiments, R6 is independently --OCCl.sub.3. In embodiments, R6
is independently --OCF.sub.3. In embodiments, R6 is independently
--OCBr.sub.3. In embodiments, R6 is independently --OCI.sub.3. In
embodiments, R6 is independently --OCHCl.sub.2. In embodiments, R6
is independently --OCHBr.sub.2. In embodiments, R6 is independently
--OCHI.sub.2. In embodiments, R6 is independently --OCHF.sub.2. In
embodiments, R6 is independently --OCH.sub.2Cl. In embodiments, R6
is independently --OCH.sub.2Br. In embodiments, R6 is independently
--OCH.sub.2I. In embodiments, R6 is independently --OCH.sub.2F. In
embodiments, R6 is independently --SF.sub.5. In embodiments, R6 is
independently --N.sub.3. In embodiments, R6 is independently
substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4 alkyl). In embodiments,
R6 is independently substituted or unsubstituted alkenyl (e.g.,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.6 alkenyl, or
C.sub.2-C.sub.4 alkenyl). In embodiments, R6 is independently
substituted or unsubstituted alkynyl (e.g., C.sub.2-C.sub.8
alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4 alkynyl). In
embodiments, R6 is independently substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R6 is
independently substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R6 is independently substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R6 is independently substituted
or unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R6 is
independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In embodiments,
R6 is independently substituted or unsubstituted heteroaryl (e.g.,
5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6
membered heteroaryl). In embodiments, R6 is independently
substituted or unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered). In embodiments, R6
is independently substituted or unsubstituted aralkyl. In
embodiments, R6 is independently substituted or unsubstituted
heteroaralkyl. In embodiments, R6 is independently substituted or
unsubstituted (heteroalicyclyl)alkyl.
[0243] In embodiments, R6 is independently unsubstituted alkyl
(e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or
C.sub.1-C.sub.4 alkyl). In embodiments, R6 is independently
unsubstituted alkenyl (e.g., C.sub.2-C.sub.8 alkenyl,
C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.4 alkenyl). In
embodiments, R6 is independently unsubstituted alkynyl (e.g.,
C.sub.2-C.sub.8 alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4
alkynyl). In embodiments, R6 is independently unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R6 is
independently unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R6 is
independently unsubstituted cycloalkenyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R6 is
independently unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R6 is
independently unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl,
C.sub.10 aryl, or phenyl). In embodiments, R6 is independently
unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9
membered heteroaryl, or 5 to 6 membered heteroaryl). In
embodiments, R6 is independently unsubstituted heteroalicyclyl
(e.g., 3 to 8 membered, 3 to 6 membered, or 5 to 6 membered). In
embodiments, R6 is independently unsubstituted aralkyl. In
embodiments, R6 is independently unsubstituted heteroaralkyl. In
embodiments, R6 is independently unsubstituted
(heteroalicyclyl)alkyl.
[0244] In embodiments, R7 is independently hydrogen. In
embodiments, R7 is independently halogen. In embodiments, R7 is
independently --CCl.sub.3. In embodiments, R7 is independently
--CBr.sub.3. In embodiments, R7 is independently --CF.sub.3. In
embodiments, R7 is independently --CI.sub.3. In embodiments, R7 is
independently --CHCl.sub.2. In embodiments, R7 is independently
--CHBr.sub.2. In embodiments, R7 is independently --CHF.sub.2. In
embodiments, R7 is independently --CHI.sub.2. In embodiments, R7 is
independently --CH.sub.2Cl. In embodiments, R7 is independently
--CH.sub.2Br. In embodiments, R7 is independently --CH.sub.2F. In
embodiments, R7 is independently --CH.sub.2I. In embodiments, R7 is
independently --CN. In embodiments, R7 is independently --OH. In
embodiments, R7 is independently --NH.sub.2. In embodiments, R7 is
independently --COOH. In embodiments, R7 is independently
--CONH.sub.2. In embodiments, R7 is independently --NO.sub.2. In
embodiments, R7 is independently --SH. In embodiments, R7 is
independently --SO.sub.3H. In embodiments, R7 is independently
--SO.sub.4H. In embodiments, R7 is independently
--SO.sub.2NH.sub.2. In embodiments, R7 is independently
--NHNH.sub.2. In embodiments, R7 is independently --ONH.sub.2. In
embodiments, R7 is independently --NHC(O)NHNH.sub.2. In
embodiments, R7 is independently --NHC(O)NH.sub.2. In embodiments,
R7 is independently --NHSO.sub.2H. In embodiments, R7 is
independently --NHC(O)H. In embodiments, R7 is independently
--NHC(O)OH. In embodiments, R7 is independently --NHOH. In
embodiments, R7 is independently --OCCl.sub.3. In embodiments, R7
is independently --OCF.sub.3. In embodiments, R7 is independently
--OCBr.sub.3. In embodiments, R7 is independently --OCI.sub.3. In
embodiments, R7 is independently --OCHCl.sub.2. In embodiments, R7
is independently --OCHBr.sub.2. In embodiments, R7 is independently
--OCHI.sub.2. In embodiments, R7 is independently --OCHF.sub.2. In
embodiments, R7 is independently --OCH.sub.2Cl. In embodiments, R7
is independently --OCH.sub.2Br. In embodiments, R7 is independently
--OCH.sub.2I. In embodiments, R7 is independently --OCH.sub.2F. In
embodiments, R7 is independently --SF.sub.5. In embodiments, R7 is
independently --N.sub.3. In embodiments, R7 is independently
substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.6 alkyl, or C1-C4 alkyl). In embodiments, R7 is
independently substituted or unsubstituted alkenyl (e.g.,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.6 alkenyl, or
C.sub.2-C.sub.4 alkenyl). In embodiments, R7 is independently
substituted or unsubstituted alkynyl (e.g., C.sub.2-C.sub.8
alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4 alkynyl). In
embodiments, R7 is independently substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R7 is
independently substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R7 is independently substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R7 is independently substituted
or unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R7 is
independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In embodiments,
R7 is independently substituted or unsubstituted heteroaryl (e.g.,
5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6
membered heteroaryl). In embodiments, R7 is independently
substituted or unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered). In embodiments, R7
is independently substituted or unsubstituted aralkyl. In
embodiments, R7 is independently substituted or unsubstituted
heteroaralkyl. In embodiments, R7 is independently substituted or
unsubstituted (heteroalicyclyl)alkyl.
[0245] In embodiments, R7 is independently unsubstituted alkyl
(e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or
C.sub.1-C.sub.4 alkyl). In embodiments, R7 is independently
unsubstituted alkenyl (e.g., C.sub.2-C.sub.8 alkenyl,
C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.4 alkenyl). In
embodiments, R7 is independently unsubstituted alkynyl (e.g.,
C.sub.2-C.sub.8 alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4
alkynyl). In embodiments, R7 is independently unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R7 is
independently unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R7 is
independently unsubstituted cycloalkenyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R7 is
independently unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R7 is
independently unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl,
C.sub.10 aryl, or phenyl). In embodiments, R7 is independently
unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9
membered heteroaryl, or 5 to 6 membered heteroaryl). In
embodiments, R7 is independently unsubstituted heteroalicyclyl
(e.g., 3 to 8 membered, 3 to 6 membered, or 5 to 6 membered). In
embodiments, R7 is independently unsubstituted aralkyl. In
embodiments, R7 is independently unsubstituted heteroaralkyl. In
embodiments, R7 is independently unsubstituted
(heteroalicyclyl)alkyl.
[0246] In embodiments, R8 is independently hydrogen. In
embodiments, R8 is independently halogen. In embodiments, R8 is
independently --CCl.sub.3. In embodiments, R8 is independently
--CBr.sub.3. In embodiments, R8 is independently --CF.sub.3. In
embodiments, R8 is independently --CI.sub.3. In embodiments, R8 is
independently --CHCl.sub.2. In embodiments, R8 is independently
--CHBr.sub.2. In embodiments, R8 is independently --CHF.sub.2. In
embodiments, R8 is independently --CHI.sub.2. In embodiments, R8 is
independently --CH.sub.2Cl. In embodiments, R8 is independently
--CH.sub.2Br. In embodiments, R8 is independently --CH.sub.2F. In
embodiments, R8 is independently --CH.sub.2I. In embodiments, R8 is
independently --CN. In embodiments, R8 is independently --OH. In
embodiments, R8 is independently --NH.sub.2. In embodiments, R8 is
independently --COOH. In embodiments, R8 is independently
--CONH.sub.2. In embodiments, R8 is independently --NO.sub.2. In
embodiments, R8 is independently --SH. In embodiments, R8 is
independently --SO.sub.3H. In embodiments, R8 is independently
--SO.sub.4H. In embodiments, R8 is independently
--SO.sub.2NH.sub.2. In embodiments, R8 is independently
--NHNH.sub.2. In embodiments, R8 is independently --ONH.sub.2. In
embodiments, R8 is independently --NHC(O)NHNH.sub.2. In
embodiments, R8 is independently --NHC(O)NH.sub.2. In embodiments,
R8 is independently --NHSO.sub.2H. In embodiments, R8 is
independently --NHC(O)H. In embodiments, R8 is independently
--NHC(O)OH. In embodiments, R8 is independently --NHOH. In
embodiments, R8 is independently --OCCl.sub.3. In embodiments, R8
is independently --OCF.sub.3. In embodiments, R8 is independently
--OCBr.sub.3. In embodiments, R8 is independently --OCI.sub.3. In
embodiments, R8 is independently --OCHCl.sub.2. In embodiments, R8
is independently --OCHBr.sub.2. In embodiments, R8 is independently
--OCHI.sub.2. In embodiments, R8 is independently --OCHF.sub.2. In
embodiments, R8 is independently --OCH.sub.2Cl. In embodiments, R8
is independently --OCH.sub.2Br. In embodiments, R8 is independently
--OCH.sub.2I. In embodiments, R8 is independently --OCH.sub.2F. In
embodiments, R8 is independently --SF.sub.5. In embodiments, R8 is
independently --N.sub.3. In embodiments, R8 is independently
substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.6 alkyl, or C1-C4 alkyl). In embodiments, R8 is
independently substituted or unsubstituted alkenyl (e.g.,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.6 alkenyl, or
C.sub.2-C.sub.4 alkenyl). In embodiments, R8 is independently
substituted or unsubstituted alkynyl (e.g., C.sub.2-C.sub.8
alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4 alkynyl). In
embodiments, R8 is independently substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R8 is
independently substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R8 is independently substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R8 is independently substituted
or unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R8 is
independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In embodiments,
R8 is independently substituted or unsubstituted heteroaryl (e.g.,
5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6
membered heteroaryl). In embodiments, R8 is independently
substituted or unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered). In embodiments, R8
is independently substituted or unsubstituted aralkyl. In
embodiments, R8 is independently substituted or unsubstituted
heteroaralkyl. In embodiments, R8 is independently substituted or
unsubstituted (heteroalicyclyl)alkyl.
[0247] In embodiments, R8 is independently unsubstituted alkyl
(e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or
C.sub.1-C.sub.4 alkyl). In embodiments, R8 is independently
unsubstituted alkenyl (e.g., C.sub.2-C.sub.8 alkenyl,
C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.4 alkenyl). In
embodiments, R8 is independently unsubstituted alkynyl (e.g.,
C.sub.2-C.sub.8 alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4
alkynyl). In embodiments, R8 is independently unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R8 is
independently unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R8 is
independently unsubstituted cycloalkenyl (e.g., C.sub.3-C.sub.8,
C3-C6, or C.sub.5-C.sub.6). In embodiments, R8 is independently
unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
or C.sub.5-C.sub.6). In embodiments, R8 is independently
unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or
phenyl). In embodiments, R8 is independently unsubstituted
heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered
heteroaryl, or 5 to 6 membered heteroaryl). In embodiments, R8 is
independently unsubstituted heteroalicyclyl (e.g., 3 to 8 membered,
3 to 6 membered, or 5 to 6 membered). In embodiments, R8 is
independently unsubstituted aralkyl. In embodiments, R8 is
independently unsubstituted heteroaralkyl. In embodiments, R8 is
independently unsubstituted (heteroalicyclyl)alkyl.
[0248] Sequencing is commonly carried out using a DNA template and
DNA primer. Accordingly, the primer used in a sequencing process
can have the following structure:
##STR00047##
wherein Base is a nucleobase, and wherein DNA is the
deoxyribonucleic acid primer. It will be understood that RNA can be
used in place of DNA for a sequencing process. R3 and R4 can
independently be H, CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally
substituted variant thereof.
[0249] A sequencing method of the present disclosure can include an
extension step, whereby a nucleotide is added to the 3' end of a
deblocked primer. The nucleotide is typically added via polymerase
catalysis. The nucleotide can include a reversible terminator
moiety so that only a single nucleotide is added to the primer in
the extension step. Similarly, a sequencing method can include an
extension step, whereby an oligonucleotide is added to the 3' end
of the deblocked primer. The oligonucleotide is typically added via
ligase catalysis. The nucleotide at the 3' end of the
oligonucleotide can include a reversible terminator moiety so that
only a single oligonucleotide is added to the primer in the
extension step. A reversibly terminated nucleotide or
oligonucleotide can include an exogenous label or, alternatively,
the reversibly terminated nucleotide or oligonucleotide can lack
exogenous labels. By way of example, the reversibly terminated
nucleotide that is used for primer extension can have the
structure:
##STR00048##
and can be produced through deblocking of a nucleotide such as
##STR00049##
wherein Base is the nucleobase. R3 and R4 can independently be H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0250] In some sequencing processes, a nucleotide or
oligonucleotide that is added to a primer in an extension step can
have an exogenous label moiety such that the exogenous label is
attached to the extended primer. Labelling an extended primer can
be useful for a sequencing by synthesis method or sequencing by
ligation method such as those set forth in further detail in the
description and cited references below. In alternative sequencing
processes the nucleotide or oligonucleotide that is added in an
extension step will lack exogenous label moieties such that the
extended primer will not have an exogenous label. Non-labelled
extended primers can be useful for a Sequencing By Synthesis.TM.
method such as those set forth in further detail in the description
and cited references below.
[0251] The use of reversibly terminated nucleotides and deblocking
methods set forth herein can beneficially facilitate cyclical
techniques that employ repeated cycles of reagent delivery and
product detection. Each cycle can include one step or multiple
steps. For example, each cycle can include all steps needed to
detect signals that are indicative of a single nucleotide position
in a template nucleic acid. Such sequencing processes can be
referred to as cyclical reversible terminator (CRT) techniques.
Each cycle in a CRT technique can include steps for (i) adding a
single reversibly terminated nucleotide (or single reversibly
terminated oligonucleotide) to increment a nascent primer to a
nucleotide position that is to be detected; (ii) detecting a
nucleotide at the single nucleotide position or at an adjacent
position, and (iii) deblocking the nascent primer to allow a return
to step (i) to start a subsequent cycle.
[0252] A specific example of a useful CRT nucleic acid sequencing
process is a Sequencing By Binding.TM. (SBB.TM.) reaction, for
example, as described in commonly owned US Pat. App. Pub. Nos.
2017/0022553 A1; 2018/0044727 A1; 2018/0187245 A1; or 2018/0208983
A1, each of which is incorporated herein by reference. SBB.TM.
methods for determining the sequence of a template nucleic acid
molecule can optionally be based on formation of a ternary complex
(between polymerase, primed nucleic acid and next correct
nucleotide) under specified conditions. The method can include an
examination phase followed by a nucleotide incorporation phase
(also referred to as an extension phase).
[0253] The examination phase of an SBB.TM. process can be carried
out for at least one template nucleic acid molecule that is
hybridized to a primer. The primer can be reversibly terminated.
The primed template can be contacted with a reaction mixture that
includes a polymerase and at least one nucleotide type. Interaction
of polymerase and a nucleotide with the primed template nucleic
acid molecule(s) can be observed under conditions where the
nucleotide is not covalently added to the primer(s); and the next
base in each template nucleic acid can be identified using the
observed interaction of the polymerase and nucleotide with the
primed template nucleic acid molecule(s). The interaction between
the primed template, polymerase and nucleotide can be detected in a
variety of schemes. For example, the nucleotide can contain a
detectable label. Nucleotides with different nucleobase types can
be distinguished by different labels, respectively. Each type of
nucleobase can have a distinguishable label with respect to other
types of nucleobases. Alternatively, some or all of the different
nucleobase types can have the same label and the different
nucleotide types can be distinguished based on separate deliveries
of the respective nucleotide types to the flow cell. In some
embodiments, the polymerase can be labeled. Polymerases that are
associated with different nucleobase types can have unique labels
that distinguish the type of nucleotide to which they are
associated. Alternatively, polymerases can have similar labels and
the different nucleobase types can be distinguished based on
separate deliveries of different nucleotide types to the flow
cell.
[0254] During the examination phase of an SBB.TM. process,
discrimination between correct and incorrect nucleotides can be
facilitated by ternary complex stabilization. A variety of
conditions and reagents can be useful. For example, the primer can
contain a reversible terminator moiety such as those set forth
herein; and/or polymerase cofactors that are required for
extension, such as divalent metal ions, can be absent; and/or
inhibitory divalent cations that inhibit polymerase-based primer
extension can be present; and/or the polymerase that is present in
the examination phase can have a chemical modification and/or
mutation that inhibits primer extension; and/or the nucleotides can
have chemical modifications that inhibit incorporation, such as 5'
modifications that remove or alter the native triphosphate moiety.
The examination phase can include detection using apparatus and
methods set forth herein. Once examination is complete, the SBB.TM.
process can proceed to the next cycle by removing the polymerase
and nucleotides used for examination and subjecting the primed
template nucleic acid to further cycles of deblocking, extension
and examination.
[0255] Washes can be carried out between the various delivery steps
of an SBB.TM. process. Wash steps can be performed between any of a
variety of steps set forth herein. For example, a wash step can be
useful for separating a primed template nucleic acid from other
reagents that were contacted with the primed template nucleic acid
under ternary complex stabilizing conditions during an SBB.TM.
process. Such a wash can remove one or more reagents from
interfering with examination of a mixture or from contaminating a
second mixture that is to be formed on a substrate (or in a vessel)
that had previously been in contact with the first mixture. For
example, a primed template nucleic acid can be contacted with a
polymerase and at least one nucleotide type to form a first mixture
under ternary complex stabilizing conditions, and the first mixture
can be examined. Optionally, a wash can be carried out prior to
examination to remove reagents that are not participating in
formation of a stabilized ternary complex. Alternatively, or
additionally, a wash can be carried out after the examination step
to remove one or more component of the first mixture from the
primed template nucleic acid. Then the primed template nucleic acid
can be contacted with a polymerase and at least one other
nucleotide to form a second mixture under ternary complex
stabilizing conditions, and the second mixture can be examined for
ternary complex formation. As before, an optional wash can be
carried out prior to the second examination to remove reagents that
are not participating in formation of a stabilized ternary
complex.
[0256] Another useful CRT sequencing process is
sequencing-by-synthesis (SBS). SBS generally involves the enzymatic
extension of a nascent primer through the iterative addition of
nucleotides against a template strand to which the primer is
hybridized. Briefly, SBS can be initiated by contacting target
nucleic acids, attached to sites in a flow cell, with one or more
labeled nucleotides, DNA polymerase, etc. Those sites where a
primer is extended using the target nucleic acid as template will
incorporate a labeled nucleotide that can be detected. Optionally,
the labeled nucleotides can further include a reversible terminator
such as any of those set forth herein. For example, a nucleotide
analog having a reversible terminator moiety can be added to a
primer such that subsequent extension cannot occur until a
deblocking reagent of the present disclosure is delivered to remove
the moiety. Thus, for embodiments that use reversible termination,
a deblocking reagent can be delivered to the vessel (before or
after detection occurs). Washes can be carried out between the
various delivery steps. The cycle can be performed n times to
extend the primer by n nucleotides, thereby detecting a sequence of
length n. Exemplary SBS procedures, reagents and detection
components that can be readily modified to employ a reversible
terminator and deblocking reagent of the present disclosure are
described, for example, in Bentley et al., Nature 456:53-59 (2008),
WO 04/018497; WO 91/06678; WO 07/123744; U.S. Pat. Nos. 7,057,026;
7,329,492; 7,211,414; 7,315,019 or 7,405,281, and US Pat. App. Pub.
No. 2008/0108082 A1, each of which is incorporated herein by
reference. Also useful are SBS methods that are commercially
available from Illumina, Inc. (San Diego, Calif.).
[0257] Sequencing-by-ligation reactions are also useful including,
for example, those described in Shendure et al. Science
309:1728-1732 (2005); U.S. Pat. No. 5,599,675; or U.S. Pat. No.
5,750,341, each of which is incorporated herein by reference. Some
embodiments can include sequencing-by-hybridization procedures as
described, for example, in Bains et al., Journal of Theoretical
Biology 135 (3), 303-7 (1988); Drmanac et al., Nature Biotechnology
16, 54-58 (1998); Fodor et al., Science 251 (4995), 767-773 (1995);
or WO 1989/10977, each of which is incorporated herein by
reference. In both sequencing-by-ligation and
sequencing-by-hybridization procedures, primers that are hybridized
to nucleic acid templates are subjected to repeated cycles of
extension by oligonucleotide ligation. Typically, the
oligonucleotides are fluorescently labeled and can be detected to
determine the sequence of the template. The primers and
oligonucleotides used in such processes can include reversible
terminators set forth herein and they can be deblocked using
reagents set forth herein.
[0258] Steps for the above sequencing methods can be carried out
cyclically. For example, examination and extension steps of an
SBB.TM. method can be repeated such that in each cycle a single
next correct nucleotide is examined (i.e. the next correct
nucleotide being a nucleotide that correctly binds to the
nucleotide in a template nucleic acid that is located immediately
5' of the base in the template that is hybridized to the 3'-end of
the hybridized primer) and, subsequently, a single next correct
nucleotide is added to the primer. Any number of cycles of a
sequencing method set forth herein can be carried out including,
for example, at least 1, 2, 10, 25, 50, 100, 150, 250, 500 or more
cycles. Alternatively or additionally, no more than 500, 250, 150,
100, 50, 25, 10, 2 or 1 cycles are carried out.
[0259] Any of a variety of polymerases can be used in a method set
forth herein. Reference to a particular polymerase, such as those
exemplified throughout this disclosure, will be understood to
include functional variants thereof unless indicated otherwise.
Particularly useful functions of a polymerase include formation of
a ternary complex, extension of a primer to introduce a nucleotide
(such as a reversible terminated nucleotide), or catalysis of the
polymerization of a nucleic acid strand using an existing nucleic
acid as a template.
[0260] Polymerases can be classified based on structural homology
such as the classification of polymerases into families identified
as A, B, C, D, X, Y, and RT. DNA Polymerases in Family A include,
for example, T7 DNA polymerase, eukaryotic mitochondrial DNA
Polymerase .gamma., E. coli DNA Pol I, Thermus aquaticus Pol I, and
Bacillus stearothermophilus Pol I. DNA Polymerases in Family B
include, for example, eukaryotic DNA polymerases .alpha., .delta.,
and .epsilon.; DNA polymerase .zeta.; T4 DNA polymerase; Phi29 DNA
polymerase; and RB69 bacteriophage DNA polymerase. Family C
includes, for example, the E. coli DNA Polymerase III alpha
subunit. Family B archaeon DNA polymerases include, for example,
Vent, Deep Vent, Pfu and 9.degree.N (e.g., Therminator.TM. DNA
polymerase from New England BioLabs Inc.; Ipswich, Mass.)
polymerases. Family D includes, for example, polymerases derived
from the Euryarchaeota subdomain of Archaea. DNA Polymerases in
Family X include, for example, eukaryotic polymerases Pol .beta.,
pol .sigma., Pol .lamda., and Pol .mu., and S. cerevisiae Pol4. DNA
Polymerases in Family Y include, for example, Pol .eta., Pol , Pol
.kappa., E. coli Pol IV (DINB) and E. coli Pol V (UmuD'2C). The RT
(reverse transcriptase) family of DNA polymerases includes, for
example, retrovirus reverse transcriptases and eukaryotic
telomerases. Exemplary RNA polymerases include, but are not limited
to, viral RNA polymerases such as T7 RNA polymerase; Eukaryotic RNA
polymerases such as RNA polymerase I, RNA polymerase II, RNA
polymerase III, RNA polymerase IV, and RNA polymerase V; and
Archaea RNA polymerase.
[0261] Further examples of useful DNA polymerases include bacterial
DNA polymerases, eukaryotic DNA polymerases, archaeal DNA
polymerases, viral DNA polymerases and phage DNA polymerases.
Bacterial DNA polymerases include E. coli DNA polymerases I, II and
III, IV and V, the Klenow fragment of E. coli DNA polymerase,
Clostridium stercorarium (Cst) DNA polymerase, Clostridium
thermocellum (Cth) DNA polymerase and Sulfolobus solfataricus (Sso)
DNA polymerase. Eukaryotic DNA polymerases include DNA polymerases
.alpha., .beta., .gamma., .delta., , .eta., .zeta., .lamda.,
.sigma., .mu., and k, as well as the Revl polymerase (terminal
deoxycytidyl transferase) and terminal deoxynucleotidyl transferase
(TdT). Viral DNA polymerases include T4 DNA polymerase, phi-29 DNA
polymerase, GA-1, phi-29-like DNA polymerases, PZA DNA polymerase,
phi-15 DNA polymerase, Cpl DNA polymerase, Cp7 DNA polymerase, T7
DNA polymerase, and T4 polymerase. Other useful DNA polymerases
include thermostable and/or thermophilic DNA polymerases such as
Thermus aquaticus (Taq) DNA polymerase, Thermus filiformis (Tfi)
DNA polymerase, Thermococcus zilligi (Tzi) DNA polymerase, Thermus
thermophilus (Tth) DNA polymerase, Thermus flavusu (Tfl) DNA
polymerase, Pyrococcus woesei (Pwo) DNA polymerase, Pyrococcus
furiosus (Pfu) DNA polymerase and Turbo Pfu DNA polymerase,
Thermococcus litoralis (Tli) DNA polymerase, Pyrococcus sp. GB-D
polymerase, Thermotoga maritima (Tma) DNA polymerase, Bacillus
stearothermophilus (Bst) DNA polymerase, Pyrococcus Kodakaraensis
(KOD) DNA polymerase, Pfx DNA polymerase, Thermococcus sp. JDF-3
(JDF-3) DNA polymerase, Thermococcus gorgonarius (Tgo) DNA
polymerase, Thermococcus acidophilium DNA polymerase; Sulfolobus
acidocaldarius DNA polymerase; Thermococcus sp. go N-7 DNA
polymerase; Pyrodictium occultum DNA polymerase; Methanococcus
voltae DNA polymerase; Methanococcus thermoautotrophicum DNA
polymerase; Methanococcus jannaschii DNA polymerase;
Desulfurococcus strain TOK DNA polymerase (D. Tok Pol); Pyrococcus
abyssi DNA polymerase; Pyrococcus horikoshii DNA polymerase;
Pyrococcus islandicum DNA polymerase; Thermococcus fumicolans DNA
polymerase; Aeropyrum pernix DNA polymerase; and the heterodimeric
DNA polymerase DP1/DP2. Engineered and modified polymerases also
are useful in connection with the disclosed techniques. For
example, modified versions of the extremely thermophilic marine
archaea Thermococcus species 9.degree. N (e.g., Therminator.TM. DNA
polymerase from New England BioLabs Inc.; Ipswich, Mass.) can be
used.
[0262] Useful RNA polymerases include, but are not limited to,
viral RNA polymerases such as T7 RNA polymerase, T3 polymerase, SP6
polymerase, and Kll polymerase; Eukaryotic RNA polymerases such as
RNA polymerase I, RNA polymerase II, RNA polymerase III, RNA
polymerase IV, and RNA polymerase V; and Archaea RNA
polymerase.
[0263] Another useful type of polymerase is a reverse
transcriptase. Exemplary reverse transcriptases include, but are
not limited to, HIV-1 reverse transcriptase from human
immunodeficiency virus type 1 (PDB 1HMV), HIV-2 reverse
transcriptase from human immunodeficiency virus type 2, M-MLV
reverse transcriptase from the Moloney murine leukemia virus, AMV
reverse transcriptase from the avian myeloblastosis virus, and
Telomerase reverse transcriptase that maintains the telomeres of
eukaryotic chromosomes.
[0264] A polymerase having an intrinsic 3'-5' proofreading
exonuclease activity can be useful for some applications of the
methods and systems set forth herein. For example, the 3'-5'
exonuclease activity can be used to remove a reversibly terminated
nucleotide from the 3' end of a primer. Polymerases that
substantially lack 3'-5' proofreading exonuclease activity are also
useful in some configurations, for example, in most sequencing
systems and methods. Absence of exonuclease activity can be a wild
type characteristic or a characteristic imparted by a variant or
engineered polymerase structure. For example, exo minus Klenow
fragment is a mutated version of Klenow fragment that lacks 3'-5'
proofreading exonuclease activity. Klenow fragment and its exo
minus variant can be useful in a method or composition set forth
herein.
[0265] Polymerases that may be used in a method or composition set
forth herein include naturally occurring polymerases and modified
variations thereof, including, but not limited to, mutants,
recombinants, fusions, genetic modifications, chemical
modifications, synthetics, and analogs. Useful polymerases for
ternary complex formation and detection are not limited to
polymerases that have the ability to catalyze a polymerization
reaction. Optionally, a useful polymerase will have the ability to
catalyze a polymerization reaction in at least one condition that
is not used during formation or examination of a stabilized ternary
complex. Exemplary polymerases that can be used to form a
stabilized ternary complex include, for example, wild type and
mutant polymerases set forth in US Pat. App. Pub. Nos. 2017/0314072
A1 or 2018/0155698 A1, or U.S. Pat. App. Ser. No. 62/732,510, each
of which is incorporated herein by reference.
[0266] Polymerases that contain an exogenous label moiety (e.g., an
exogenous luminophore), which can be used to detect the polymerase
(e.g. during an SBB.TM. examination step), can be useful in some
embodiments. Optionally, the exogenous label moiety can be
chemically linked to the polymerase, for example, using a free
sulfhydryl or a free amine moiety of the polymerase. An exogenous
label moiety can also be attached to a polymerase via protein
fusion. Exemplary label moieties that can be attached via protein
fusion include, for example, green fluorescent protein (GFP),
phycobiliprotein (e.g. phycocyanin and phycoerythrin) or
wavelength-shifted variants of GFP or phycobiliprotein.
[0267] Also provided is a nucleic acid detection apparatus that
includes a fluid reagent delivery system, a detection system, and a
flow cell, wherein the fluid reagent delivery system is configured
to deliver a fluid reagent to the flow cell, wherein the detection
system is configured to detect a signal in the flow cell, wherein
the flow cell is attached to a first molecule having the
structure:
##STR00050##
wherein Base is a nucleobase, wherein DNA is independently a single
stranded deoxyribonucleic acid or double stranded deoxyribonucleic
acid, and wherein the fluid reagent comprises a second molecule
comprising the structure: R2-ONH.sub.2, wherein R2, R3 and R4 is
independently alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0268] An apparatus of the present disclosure can be configured for
detection of luminescence or fluorescence. Accordingly, the
apparatus can include a detection system having an optical
excitation apparatus, such as a laser, LED or lamp, and a
luminescence detector such as a camera (e.g. complementary metal
oxide semiconductor (CMOS) camera or charge coupled device (CCD)
camera). Examples of detection apparatus and components thereof
that can be used in a system or method herein are described, for
example, in US Pat. App. Pub. No. 2010/0111768 A1 or U.S. Pat. Nos.
7,329,860; 8,951,781 or 9,193,996, each of which is incorporated
herein by reference. Other detection apparatus include those
commercialized for nucleic acid sequencing such as those provided
by Illumina.TM., Inc. (e.g. HiSeq.TM., MiSeq.TM., NextSeq.TM., or
NovaSeq.TM. systems), Life Technologies.TM. (e.g. ABI PRISM.TM., or
SOLiD.TM. systems), Pacific Biosciences (e.g. systems using
SMRT.TM. Technology such as the Sequel.TM. or RS II.TM. systems),
or Qiagen (e.g. Genereader.TM. system). Other useful detectors are
described in U.S. Pat. Nos. 5,888,737; 6,175,002; 5,695,934;
6,140,489; or 5,863,722; or US Pat. Pub. Nos. 2007/007991 A1,
2009/0247414 A1, or 2010/0111768; or WO2007/123744, each of which
is incorporated herein by reference in its entirety.
[0269] Although the compositions and methods of the present
disclosure are illustrated in the context of optical detection in
several exemplary configurations herein, it will be understood that
other detection modalities can be used in addition or instead. For
example, the detector can be an electronic detector used for
detection of protons or pyrophosphate (see, for example, US Pat.
App. Pub. Nos. 2009/0026082 A1; 2009/0127589 A1; 2010/0137143 A1;
or 2010/0282617 A1, each of which is incorporated herein by
reference in its entirety, or the Ion Torrent.TM. systems
commercially available from ThermoFisher, Waltham, Mass.) or as
used in detection of nanopores such as those commercialized by
Oxford Nanopore.TM., Oxford UK (e.g. MinION.TM. or PromethION.TM.
systems) or set forth in U.S. Pat. No. 7,001,792; Soni &
Meller, Clin. Chem. 53, 1996-2001 (2007); Healy, Nanomed. 2,
459-481 (2007); or Cockroft, et al. J. Am. Chem. Soc. 130, 818-820
(2008), each of which is incorporated herein by reference. A FET
detector can be used such as one or more of those described in U.S.
Pat. App. Ser. No. 62/767,712; US Pat. App. Pub. Nos. 2017/0240962
A1, 2018/0051316 A1, 2018/0112265 A1, 2018/0155773 A1 or
2018/0305727 A1; or U.S. Pat. Nos. 9,164,053, 9,829,456,
10,036,064, or 10,125,391, each of which is incorporated herein by
reference.
[0270] Other detection techniques that can be used in a method set
forth herein include, for example, mass spectrometry which can be
used to perceive mass; surface plasmon resonance which can be used
to perceive binding to a surface; absorbance which can be used to
perceive the wavelength of the energy a label absorbs; calorimetry
which can be used to perceive changes in temperature due to
presence of a label; electrical conductance or impedance which can
be used to perceive electrical properties of a label, or other
known analytic techniques.
[0271] A useful detection apparatus can include a fluid reagent
delivery system. The fluid delivery system can include a reaction
vessel (e.g. flow cell) that is configured to carry out chemical
reactions set forth herein. The reaction vessel can also be
configured to allow detection of the reactions or products of the
reactions. The fluid delivery system can include reservoirs for
storing reagents. The fluids from the reagents can be delivered to
a reaction vessel (e.g. a flow cell) via the fluid delivery system.
Spent reagents and products or waste from a reaction can be
evacuated from the reaction vessel to a waste reservoir.
[0272] In particular configurations, a fluid reagent delivery
system is configured to deliver a primer extension reagent to the
flow cell, wherein the primer extension reagent comprises a
polymerase and a nucleotide having a 3' ONH.sub.2 moiety. For
example, a nucleotide that is stored or delivered by a fluid
reagent delivery system can have the following structure:
##STR00051##
wherein Base is a nucleobase. R3 and R4 can independently be H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof. Other reagents set forth herein, for example,
those that are useful for a CRT sequencing process can be stored or
delivered via a fluid reagent delivery system. For example, the
fluid reagent delivery system can be configured to deliver a
ternary complex formation reagent to a reaction vessel, wherein the
ternary complex formation reagent includes a polymerase and a
nucleotide that is the next correct nucleotide for a primed
template nucleic acid.
[0273] A detection apparatus of the present disclosure can include
a program module configured to direct the apparatus to perform a
nucleic acid sequencing process, wherein the nucleic acid
sequencing process comprises repeated deliveries of the fluid
reagent to a reaction vessel via a fluid reagent delivery system
and repeated detection of the reaction vessel by the detection
system. Control of apparatus components can utilize a general
purpose processor, a Digital Signal Processor (DSP), an Application
Specific Integrated Circuit (ASIC), a Field Programmable Gate Array
(FPGA) or other programmable logic device, discrete gate or
transistor logic, discrete hardware components, or any combination
thereof designed to perform the functions described herein. A
general purpose processor may be a microprocessor, but in the
alternative, the processor may be any conventional processor,
controller, microcontroller, or state machine. A processor may also
be implemented as a combination of computing devices, e.g., a
combination of a DSP and a microprocessor, a plurality of
microprocessors, one or more microprocessors in conjunction with a
DSP core, or any other such configuration. The same or different
processor that is used to control fluids can interact with the
system to acquire, store and process signals (e.g. signals detected
in a method set forth herein). In particular embodiments, a
processor can be used to determine, from the signals, the identity
of the nucleotide that is present at a particular location in a
template nucleic acid. In some cases, the processor will identify a
sequence of nucleotides for the template from the signals that are
detected.
[0274] A useful processor can include one or more of a personal
computer system, server computer system, thin client, thick client,
hand-held or laptop device, multiprocessor system,
microprocessor-based system, set top box, programmable consumer
electronic, network PC, minicomputer system, mainframe computer
system, smart phone, and distributed cloud computing environments
that include any of the above systems or devices, and the like. The
processor can include one or more processors or processing units, a
memory architecture that may include RAM and non-volatile memory.
The memory architecture may further include
removable/non-removable, volatile/non-volatile computer system
storage media. Further, the memory architecture may include one or
more readers for reading from and writing to a non-removable,
non-volatile magnetic media, such as a hard drive, a magnetic disk
drive for reading from and writing to a removable, non-volatile
magnetic disk, and/or an optical disk drive for reading from or
writing to a removable, non-volatile optical disk such as a CD-ROM
or DVD-ROM. The processor may also include a variety of computer
system readable media. Such media may be any available media that
is accessible by a cloud computing environment, such as volatile
and non-volatile media, and removable and non-removable media.
[0275] The memory architecture may include at least one program
product having at least one program module implemented as
executable instructions that are configured to carry out one or
more steps of a method set forth herein. For example, executable
instructions may include an operating system, one or more
application programs, other program modules, and program data.
Generally, program modules may include routines, programs, objects,
components, logic, data structures, and so on, that perform
particular tasks set forth herein.
[0276] The components of a processor or other programmable device
may be coupled by an internal bus that may be implemented as one or
more of any of several types of bus structures, including a memory
bus or memory controller, a peripheral bus, an accelerated graphics
port, and a processor or local bus using any of a variety of bus
architectures. By way of example, and not limitation, such
architectures include Industry Standard Architecture (ISA) bus,
Micro Channel Architecture (MCA) bus, Enhanced ISA (EISA) bus,
Video Electronics Standards Association (VESA) local bus, and
Peripheral Component Interconnects (PCI) bus.
[0277] A processor can optionally communicate with one or more
external devices such as a keyboard, a pointing device (e.g. a
mouse), a display, such as a graphical user interface (GUI), or
other device that facilitates interaction of a user with a system
of the present disclosure. Similarly, the processor can communicate
with other devices (e.g., via network card, modem, etc.). Such
communication can occur via I/O interfaces. Still yet, a processor
of a system herein may communicate with one or more networks such
as a local area network (LAN), a general wide area network (WAN),
and/or a public network (e.g., the Internet) via a suitable network
adapter.
[0278] This disclosure further provides kits that can be used, for
example, for characterizing nucleic acids. A kit can include
reagents for carrying out one or more of the methods set forth
herein or using or generating a composition disclosed herein. For
example, a kit can include one or more reagents set forth herein
for deblocking a reversibly terminated nucleotide or nucleic acid.
Alternatively or additionally, a kit of the present disclosure can
include one or more reagents for carrying out a primer extension
step including, for example, a reversibly terminated nucleotide or
reversibly terminated nucleic acid, and/or polymerase. A polymerase
that is used for an extension step or for an examination step can
also be included in a kit. A kit of the invention can optionally
include reagents for carrying out other steps of a sequencing
process including, for example, an examination steps used to create
and detect a stabilized ternary complex, a wash step, a step for
producing nucleic acid clusters or the like.
[0279] Accordingly, the present disclosure provides a kit that
includes a first vessel containing a nucleotide comprising a
3'-O-oxime moiety and a second vessel containing a reagent
comprising the structure R2-ONH.sub.2, wherein R2 is independently
alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl,
aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl,
(heteroalicyclyl)alkyl, or an optionally substituted variant
thereof.
[0280] In embodiments, R2 is --C(R6)(R7)(R8), --C(O)(R6),
--P(O)(R6)(R7), --C(R6)(.dbd.C(R7)(R8)), --Si(R6)(R7)(R8), --S
O.sub.2(R6), substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C5-C6), substituted or
unsubstituted cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
or C.sub.5-C.sub.6), substituted or unsubstituted cycloalkynyl
(e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6),
substituted or unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered), substituted or
unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or
phenyl), or substituted or unsubstituted heteroaryl (e.g., 5 to 10
membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered
heteroaryl).
[0281] In embodiments, R2 is --C(R6)(R7)(R8). In embodiments, R2 is
--C(O)(R6). In embodiments, R2 is --P(O)(R6)(R7). In embodiments,
R2 is --C(R6)(.dbd.C(R7)(R8)). In embodiments, R2 is
--Si(R6)(R7)(R8). In embodiments, R2 is --SO.sub.2(R6). In
embodiments, R2 is substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is substituted or unsubstituted cycloalkenyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is substituted or unsubstituted cycloalkynyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is substituted or unsubstituted heteroalicyclyl
(e.g., 3 to 8 membered, 3 to 6 membered, or 5 to 6 membered). In
embodiments, R2 is substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In embodiments,
R2 is substituted or unsubstituted heteroaryl (e.g., 5 to 10
membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered
heteroaryl). In embodiments, R2 is unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is unsubstituted cycloalkenyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is unsubstituted cycloalkynyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R2 is unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered). In embodiments, R2
is unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl,
or phenyl). In embodiments, R2 is unsubstituted heteroaryl (e.g., 5
to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6
membered heteroaryl). In embodiments, R2 is not unsubstituted
methyl. In embodiments, R2 is not unsubstituted ethyl. In
embodiments, R2 is not unsubstituted propyl.
[0282] R6, R7, and R8 are independently hydrogen, halogen,
--CCl.sub.3, --CBr.sub.3, --CF.sub.3, --CI.sub.3, --CHCl.sub.2,
--CHBr.sub.2, --CHF.sub.2, --CHI.sub.2, --CH.sub.2Cl, --CH.sub.2Br,
--CH.sub.2F, --CH.sub.2I, --CN, --OH, --NH.sub.2, --COOH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2,
--NHC(O)NH.sub.2, --NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH,
--OCCl.sub.3, --OCF.sub.3, --OCBr.sub.3, --OCI.sub.3,
--OCHCl.sub.2, --OCHBr.sub.2, --OCHI.sub.2, --OCHF.sub.2,
--OCH.sub.2Cl, --OCH.sub.2Br, --OCH.sub.2I, --OCH.sub.2F,
--SF.sub.5, --N.sub.3, substituted or unsubstituted alkyl (e.g.,
C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or C1-C4 alkyl),
substituted or unsubstituted alkenyl (e.g., C.sub.2-C.sub.8
alkenyl, C.sub.2-C.sub.6 alkenyl, or C2-C4 alkenyl), substituted or
unsubstituted alkynyl (e.g., C.sub.2-C.sub.8 alkynyl,
C.sub.2-C.sub.6 alkynyl, or C.sub.2-C.sub.4 alkynyl), substituted
or unsubstituted heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2
to 6 membered heteroalkyl, or 2 to 4 membered heteroalkyl),
substituted or unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6), substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6), substituted or unsubstituted cycloalkynyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6), substituted
or unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl,
or phenyl), substituted or unsubstituted heteroaryl (e.g., 5 to 10
membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6 membered
heteroaryl), substituted or unsubstituted heteroalicyclyl (e.g., 3
to 8 membered, 3 to 6 membered, or 5 to 6 membered), substituted or
unsubstituted aralkyl, substituted or unsubstituted heteroaralkyl,
or substituted or unsubstituted (heteroalicyclyl)alkyl. In
embodiments, R6, R7, and R8 are not all hydrogen. In embodiments,
R2 is --C(R6)(R7)(R8) and R6, R7, and R8 are not all hydrogen.
[0283] In embodiments, R6 is independently hydrogen. In
embodiments, R6 is independently halogen. In embodiments, R6 is
independently --CCl.sub.3. In embodiments, R6 is independently
--CBr.sub.3. In embodiments, R6 is independently --CF.sub.3. In
embodiments, R6 is independently --CI.sub.3. In embodiments, R6 is
independently --CHCl.sub.2. In embodiments, R6 is independently
--CHBr.sub.2. In embodiments, R6 is independently --CHF.sub.2. In
embodiments, R6 is independently --CHI.sub.2. In embodiments, R6 is
independently --CH.sub.2Cl. In embodiments, R6 is independently
--CH.sub.2Br. In embodiments, R6 is independently --CH.sub.2F. In
embodiments, R6 is independently --CH.sub.2I. In embodiments, R6 is
independently --CN. In embodiments, R6 is independently --OH. In
embodiments, R6 is independently --NH.sub.2. In embodiments, R6 is
independently --COOH. In embodiments, R6 is independently
--CONH.sub.2. In embodiments, R6 is independently --NO.sub.2. In
embodiments, R6 is independently --SH. In embodiments, R6 is
independently --SO.sub.3H. In embodiments, R6 is independently
--SO.sub.4H. In embodiments, R6 is independently
--SO.sub.2NH.sub.2. In embodiments, R6 is independently
--NHNH.sub.2. In embodiments, R6 is independently --ONH.sub.2. In
embodiments, R6 is independently --NHC(O)NHNH.sub.2. In
embodiments, R6 is independently --NHC(O)NH.sub.2. In embodiments,
R6 is independently --NHSO.sub.2H. In embodiments, R6 is
independently --NHC(O)H. In embodiments, R6 is independently
--NHC(O)OH. In embodiments, R6 is independently --NHOH. In
embodiments, R6 is independently --OCCl.sub.3. In embodiments, R6
is independently --OCF.sub.3. In embodiments, R6 is independently
--OCBr.sub.3. In embodiments, R6 is independently --OCI.sub.3. In
embodiments, R6 is independently --OCHCl.sub.2. In embodiments, R6
is independently --OCHBr.sub.2. In embodiments, R6 is independently
--OCHI.sub.2. In embodiments, R6 is independently --OCHF.sub.2. In
embodiments, R6 is independently --OCH.sub.2Cl. In embodiments, R6
is independently --OCH.sub.2Br. In embodiments, R6 is independently
--OCH.sub.2I. In embodiments, R6 is independently --OCH.sub.2F. In
embodiments, R6 is independently --SF.sub.5. In embodiments, R6 is
independently --N.sub.3. In embodiments, R6 is independently
substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4 alkyl). In embodiments,
R6 is independently substituted or unsubstituted alkenyl (e.g.,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.6 alkenyl, or
C.sub.2-C.sub.4 alkenyl). In embodiments, R6 is independently
substituted or unsubstituted alkynyl (e.g., C.sub.2-C.sub.8
alkynyl, C.sub.2-C.sub.6 alkynyl, or C.sub.2-C.sub.4 alkynyl). In
embodiments, R6 is independently substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R6 is
independently substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R6 is independently substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R6 is independently substituted
or unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R6 is
independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In embodiments,
R6 is independently substituted or unsubstituted heteroaryl (e.g.,
5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6
membered heteroaryl). In embodiments, R6 is independently
substituted or unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered). In embodiments, R6
is independently substituted or unsubstituted aralkyl. In
embodiments, R6 is independently substituted or unsubstituted
heteroaralkyl. In embodiments, R6 is independently substituted or
unsubstituted (heteroalicyclyl)alkyl.
[0284] In embodiments, R6 is independently unsubstituted alkyl
(e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or
C.sub.1-C.sub.4 alkyl). In embodiments, R6 is independently
unsubstituted alkenyl (e.g., C.sub.2-C.sub.8 alkenyl,
C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.4 alkenyl). In
embodiments, R6 is independently unsubstituted alkynyl (e.g.,
C.sub.2-C.sub.8 alkyl, C.sub.2-C.sub.6 alkyl, or C.sub.2-C.sub.4
alkyl). In embodiments, R6 is independently unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R6 is
independently unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R6 is
independently unsubstituted cycloalkenyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R6 is
independently unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R6 is
independently unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl,
C.sub.10 aryl, or phenyl). In embodiments, R6 is independently
unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9
membered heteroaryl, or 5 to 6 membered heteroaryl). In
embodiments, R6 is independently unsubstituted heteroalicyclyl
(e.g., 3 to 8 membered, 3 to 6 membered, or 5 to 6 membered). In
embodiments, R6 is independently unsubstituted aralkyl. In
embodiments, R6 is independently unsubstituted heteroaralkyl. In
embodiments, R6 is independently unsubstituted
(heteroalicyclyl)alkyl.
[0285] In embodiments, R7 is independently hydrogen. In
embodiments, R7 is independently halogen. In embodiments, R7 is
independently --CCl.sub.3. In embodiments, R7 is independently
--CBr.sub.3. In embodiments, R7 is independently --CF.sub.3. In
embodiments, R7 is independently --CI.sub.3. In embodiments, R7 is
independently --CHCl.sub.2. In embodiments, R7 is independently
--CHBr.sub.2. In embodiments, R7 is independently --CHF.sub.2. In
embodiments, R7 is independently --CHI.sub.2. In embodiments, R7 is
independently --CH.sub.2Cl. In embodiments, R7 is independently
--CH.sub.2Br. In embodiments, R7 is independently --CH.sub.2F. In
embodiments, R7 is independently --CH.sub.2I. In embodiments, R7 is
independently --CN. In embodiments, R7 is independently --OH. In
embodiments, R7 is independently --NH.sub.2. In embodiments, R7 is
independently --COOH. In embodiments, R7 is independently
--CONH.sub.2. In embodiments, R7 is independently --NO.sub.2. In
embodiments, R7 is independently --SH. In embodiments, R7 is
independently --SO.sub.3H. In embodiments, R7 is independently
--SO.sub.4H. In embodiments, R7 is independently
--SO.sub.2NH.sub.2. In embodiments, R7 is independently
--NHNH.sub.2. In embodiments, R7 is independently --ONH.sub.2. In
embodiments, R7 is independently --NHC(O)NHNH.sub.2. In
embodiments, R7 is independently --NHC(O)NH.sub.2. In embodiments,
R7 is independently --NHSO.sub.2H. In embodiments, R7 is
independently --NHC(O)H. In embodiments, R7 is independently
--NHC(O)OH. In embodiments, R7 is independently --NHOH. In
embodiments, R7 is independently --OCCl.sub.3. In embodiments, R7
is independently --OCF.sub.3. In embodiments, R7 is independently
--OCBr.sub.3. In embodiments, R7 is independently --OCI.sub.3. In
embodiments, R7 is independently --OCHCl.sub.2. In embodiments, R7
is independently --OCHBr.sub.2. In embodiments, R7 is independently
--OCHI.sub.2. In embodiments, R7 is independently --OCHF.sub.2. In
embodiments, R7 is independently --OCH.sub.2Cl. In embodiments, R7
is independently --OCH.sub.2Br. In embodiments, R7 is independently
--OCH.sub.2I. In embodiments, R7 is independently --OCH.sub.2F. In
embodiments, R7 is independently --SF.sub.5. In embodiments, R7 is
independently --N.sub.3. In embodiments, R7 is independently
substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.6 alkyl, or C1-C4 alkyl). In embodiments, R7 is
independently substituted or unsubstituted alkenyl (e.g.,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.6 alkenyl, or
C.sub.2-C.sub.4 alkenyl). In embodiments, R7 is independently
substituted or unsubstituted alkynyl (e.g., C.sub.2-C.sub.8
alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4 alkynyl). In
embodiments, R7 is independently substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R7 is
independently substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R7 is independently substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R7 is independently substituted
or unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R7 is
independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In embodiments,
R7 is independently substituted or unsubstituted heteroaryl (e.g.,
5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6
membered heteroaryl). In embodiments, R7 is independently
substituted or unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered). In embodiments, R7
is independently substituted or unsubstituted aralkyl. In
embodiments, R7 is independently substituted or unsubstituted
heteroaralkyl. In embodiments, R7 is independently substituted or
unsubstituted (heteroalicyclyl)alkyl.
[0286] In embodiments, R7 is independently unsubstituted alkyl
(e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or
C.sub.1-C.sub.4 alkyl). In embodiments, R7 is independently
unsubstituted alkenyl (e.g., C.sub.2-C.sub.8 alkenyl,
C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.4 alkenyl). In
embodiments, R7 is independently unsubstituted alkynyl (e.g.,
C.sub.2-C.sub.8 alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4
alkynyl). In embodiments, R7 is independently unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R7 is
independently unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R7 is
independently unsubstituted cycloalkenyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R7 is
independently unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R7 is
independently unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl,
C.sub.10 aryl, or phenyl). In embodiments, R7 is independently
unsubstituted heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9
membered heteroaryl, or 5 to 6 membered heteroaryl). In
embodiments, R7 is independently unsubstituted heteroalicyclyl
(e.g., 3 to 8 membered, 3 to 6 membered, or 5 to 6 membered). In
embodiments, R7 is independently unsubstituted aralkyl. In
embodiments, R7 is independently unsubstituted heteroaralkyl. In
embodiments, R7 is independently unsubstituted
(heteroalicyclyl)alkyl.
[0287] In embodiments, R8 is independently hydrogen. In
embodiments, R8 is independently halogen. In embodiments, R8 is
independently --CCl.sub.3. In embodiments, R8 is independently
--CBr.sub.3. In embodiments, R8 is independently --CF.sub.3. In
embodiments, R8 is independently --CI.sub.3. In embodiments, R8 is
independently --CHCl.sub.2. In embodiments, R8 is independently
--CHBr.sub.2. In embodiments, R8 is independently --CHF.sub.2. In
embodiments, R8 is independently --CHI.sub.2. In embodiments, R8 is
independently --CH.sub.2Cl. In embodiments, R8 is independently
--CH.sub.2Br. In embodiments, R8 is independently --CH.sub.2F. In
embodiments, R8 is independently --CH.sub.2I. In embodiments, R8 is
independently --CN. In embodiments, R8 is independently --OH. In
embodiments, R8 is independently --NH.sub.2. In embodiments, R8 is
independently --COOH. In embodiments, R8 is independently
--CONH.sub.2. In embodiments, R8 is independently --NO.sub.2. In
embodiments, R8 is independently --SH. In embodiments, R8 is
independently --SO.sub.3H. In embodiments, R8 is independently
--SO.sub.4H. In embodiments, R8 is independently
--SO.sub.2NH.sub.2. In embodiments, R8 is independently
--NHNH.sub.2. In embodiments, R8 is independently --ONH.sub.2. In
embodiments, R8 is independently --NHC(O)NHNH.sub.2. In
embodiments, R8 is independently --NHC(O)NH.sub.2. In embodiments,
R8 is independently --NHSO.sub.2H. In embodiments, R8 is
independently --NHC(O)H. In embodiments, R8 is independently
--NHC(O)OH. In embodiments, R8 is independently --NHOH. In
embodiments, R8 is independently --OCCl.sub.3. In embodiments, R8
is independently --OCF.sub.3. In embodiments, R8 is independently
--OCBr.sub.3. In embodiments, R8 is independently --OCI.sub.3. In
embodiments, R8 is independently --OCHCl.sub.2. In embodiments, R8
is independently --OCHBr.sub.2. In embodiments, R8 is independently
--OCHI.sub.2. In embodiments, R8 is independently --OCHF.sub.2. In
embodiments, R8 is independently --OCH.sub.2Cl. In embodiments, R8
is independently --OCH.sub.2Br. In embodiments, R8 is independently
--OCH.sub.2I. In embodiments, R8 is independently --OCH.sub.2F. In
embodiments, R8 is independently --SF.sub.5. In embodiments, R8 is
independently --N.sub.3. In embodiments, R8 is independently
substituted or unsubstituted alkyl (e.g., C.sub.1-C.sub.8 alkyl,
C.sub.1-C.sub.6 alkyl, or C.sub.1-C.sub.4 alkyl). In embodiments,
R8 is independently substituted or unsubstituted alkenyl (e.g.,
C.sub.2-C.sub.8 alkenyl, C.sub.2-C.sub.6 alkenyl, or
C.sub.2-C.sub.4 alkenyl). In embodiments, R8 is independently
substituted or unsubstituted alkynyl (e.g., C.sub.2-C.sub.8
alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4 alkynyl). In
embodiments, R8 is independently substituted or unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R8 is
independently substituted or unsubstituted cycloalkyl (e.g.,
C.sub.3-C.sub.8, C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In
embodiments, R8 is independently substituted or unsubstituted
cycloalkenyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6, or
C.sub.5-C.sub.6). In embodiments, R8 is independently substituted
or unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R8 is
independently substituted or unsubstituted aryl (e.g.,
C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or phenyl). In embodiments,
R8 is independently substituted or unsubstituted heteroaryl (e.g.,
5 to 10 membered heteroaryl, 5 to 9 membered heteroaryl, or 5 to 6
membered heteroaryl). In embodiments, R8 is independently
substituted or unsubstituted heteroalicyclyl (e.g., 3 to 8
membered, 3 to 6 membered, or 5 to 6 membered). In embodiments, R8
is independently substituted or unsubstituted aralkyl. In
embodiments, R8 is independently substituted or unsubstituted
heteroaralkyl. In embodiments, R8 is independently substituted or
unsubstituted (heteroalicyclyl)alkyl.
[0288] In embodiments, R8 is independently unsubstituted alkyl
(e.g., C.sub.1-C.sub.8 alkyl, C.sub.1-C.sub.6 alkyl, or
C.sub.1-C.sub.4 alkyl). In embodiments, R8 is independently
unsubstituted alkenyl (e.g., C.sub.2-C.sub.8 alkenyl,
C.sub.2-C.sub.6 alkenyl, or C.sub.2-C.sub.4 alkenyl). In
embodiments, R8 is independently unsubstituted alkynyl (e.g.,
C.sub.2-C.sub.8 alkynyl, C.sub.2-C.sub.6 alkynyl, or C2-C4
alkynyl). In embodiments, R8 is independently unsubstituted
heteroalkyl (e.g., 2 to 8 membered heteroalkyl, 2 to 6 membered
heteroalkyl, or 2 to 4 membered heteroalkyl). In embodiments, R8 is
independently unsubstituted cycloalkyl (e.g., C.sub.3-C.sub.8,
C.sub.3-C.sub.6, or C.sub.5-C.sub.6). In embodiments, R8 is
independently unsubstituted cycloalkenyl (e.g., C.sub.3-C.sub.8,
C3-C6, or C.sub.5-C.sub.6). In embodiments, R8 is independently
unsubstituted cycloalkynyl (e.g., C.sub.3-C.sub.8, C.sub.3-C.sub.6,
or C.sub.5-C.sub.6). In embodiments, R8 is independently
unsubstituted aryl (e.g., C.sub.6-C.sub.10 aryl, C.sub.10 aryl, or
phenyl). In embodiments, R8 is independently unsubstituted
heteroaryl (e.g., 5 to 10 membered heteroaryl, 5 to 9 membered
heteroaryl, or 5 to 6 membered heteroaryl). In embodiments, R8 is
independently unsubstituted heteroalicyclyl (e.g., 3 to 8 membered,
3 to 6 membered, or 5 to 6 membered). In embodiments, R8 is
independently unsubstituted aralkyl. In embodiments, R8 is
independently unsubstituted heteroaralkyl. In embodiments, R8 is
independently unsubstituted (heteroalicyclyl)alkyl.
[0289] Optionally, the kit can include nitrous acid (HONO). For
example, in a third vessel. In particular configurations, the
nucleotide can have the structure:
##STR00052##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 can independently be
H, CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0290] In embodiments, Q is independently a single stranded
deoxyribonucleic acid or double stranded deoxyribonucleic acid.
[0291] Any of the components or articles used in performing the
methods set forth herein can be usefully packaged into a kit. For
example, the kits can be packed to include some, many or all of the
components or articles used in performing the methods set forth
herein. Exemplary components include, for example, labeled
nucleotides (e.g. extendible labeled nucleotides); polymerases
(labeled or unlabeled); nucleotides having terminator moieties such
as a --ONH.sub.2 moiety (e.g. unlabeled, reversibly terminated
nucleotides); reagents such as N-alkoxyamine reagents set forth
herein; and the like as set forth herein and in references cited
herein. Any of such reagents can include, for example, some, many
or all of the fluids, components and/or articles used for
performing one or more of the subsequent steps for analysis of a
primed template nucleic acid. A kit need not include a primer or
template nucleic acid. Rather, a user of the kit can provide a
primed template nucleic acid which is to be combined with
components of the kit. Similarly, a kit can exclude one or more of
the components set forth herein (such as a flow cell or other
vessel) and, optionally, such excluded components can be provided
by an end user.
[0292] One or more ancillary reagents also can be included in a
kit. Such ancillary reagents can include any of the reagents
exemplified above and/or other types of reagents useful in
performing the methods set forth herein. Instructions can further
be included in a kit. The instructions can include, for example,
procedures for making any components used in the methods set forth
herein, performing one or more steps of any embodiment of the
methods set forth herein and/or instructions for performing any of
the subsequent analysis steps employing a primed template nucleic
acid. For example, a kit can include instructions for extending a
primer with a reversibly terminated nucleotide or a reversibly
terminated oligonucleotide, or instructions for deblocking a
reversibly terminated nucleotide or primer.
[0293] Optionally a kit can include identifying information in the
form of a label that can be read by a human user. Labels that are
machine readable such as bar codes, microchips or radio-frequency
identification (RFID) tags can also be useful.
[0294] In particular embodiments, a kit includes a cartridge having
reservoirs to contain the reagents and further having fluidic
components for transferring reagents from the reservoirs to a
detection instrument. For example, the fluidic components can be
configured to transfer reagents to a flow cell where stabilized
ternary complexes are detected. The cartridge can be configured for
non-permanent introduction to a nucleic acid analysis system such
as a nucleic acid sequencing platform. Accordingly, the cartridge
can be configured for installation with, and removal from, a
detection apparatus. An exemplary fluidic cartridge that can be
included in a kit (or system) of the present disclosure is
described in US Pat. App. Pub. Nos. 2018/0280975 A1 or 2019/0055596
A1, each of which is incorporated herein by reference.
[0295] In an aspect is provided a method of adding a 3'-O--NH.sub.2
moiety to a nucleotide having a 3' protecting group, including
contacting a nucleotide having a 3' protecting group to a reagent
including the structure R2-O--NH.sub.2, wherein R2 has a molecular
weight of at least 24. In embodiments, the nucleotide having a 3'
protecting group includes ribose or deoxyribose. In embodiments,
the nucleotide having a 3' protecting group includes ribose. In
embodiments, the nucleotide having a 3' protecting group includes
2' deoxyribose. In embodiments, the nucleotide having a 3'
protecting group includes phosphate. In embodiments, the nucleotide
having a 3' protecting group includes triphosphate. In embodiments,
the nucleotide having a 3' protecting group includes the
structure:
##STR00053##
wherein the nucleotide that includes the 3'-O--NH.sub.2 moiety
includes the structure:
##STR00054##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0296] In embodiments, the 3' blocking group includes 3'-O-Oxime.
In embodiments, the 3' blocking group includes 3'-O-aldoxime. In
embodiments, the 3' blocking group includes 3'-O-ketoxime.
[0297] In embodiments, the nucleotide having a 3' protecting group
includes the structure:
##STR00055##
wherein the nucleotide that includes the 3'-O--NH.sub.2 moiety
includes the structure:
##STR00056##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0298] In embodiments, the method adds a 3' ONH.sub.2 in place of a
3' protecting group to at least 94% of a population of nucleotides
having a 3' protecting group in no more than 3 hours. In
embodiments, the method adds a 3' ONH.sub.2 in place of a 3'
protecting group to at least 96% of a population of nucleotides
having a 3' protecting group in no more than 3 hours. In
embodiments, the method adds a 3' ONH.sub.2 in place of a 3'
protecting group to at least 98% of a population of nucleotides
having a 3' protecting group in no more than 3 hours. In
embodiments, the method adds a 3' ONH.sub.2 in place of a 3'
protecting group to at least 98% of a population of nucleotides
having a 3' protecting group in no more than 1 hour.
[0299] In embodiments, the nucleotide having a 3' protecting group
includes cytosine, and wherein method degrades no more than 6% of
cytosine bases from a population including a plurality of the
nucleotide having a 3' protecting group including cytosine. In
embodiments, the nucleotide having a 3' protecting group includes
cytosine, and wherein method degrades no more than 5% of cytosine
bases from a population including a plurality of the nucleotide
having a 3' protecting group including cytosine. In embodiments,
the nucleotide having a 3' protecting group includes cytosine, and
wherein method degrades no more than 4% of cytosine bases from a
population including a plurality of the nucleotide having a 3'
protecting group including cytosine. In embodiments, the nucleotide
having a 3' protecting group includes cytosine, and wherein method
degrades no more than 3% of cytosine bases from a population
including a plurality of the nucleotide having a 3' protecting
group including cytosine. In embodiments, the nucleotide having a
3' protecting group includes cytosine, and wherein method degrades
no more than 2% of cytosine bases from a population including a
plurality of the nucleotide having a 3' protecting group including
cytosine. In embodiments, the nucleotide having a 3' protecting
group includes cytosine, and wherein method degrades no more than
1% of cytosine bases from a population including a plurality of the
nucleotide having a 3' protecting group including cytosine.
[0300] In an aspect is provided a method of making a 3'-blocked
nucleotide, the method including contacting a 3'-protected
nucleotide with a reagent having the structure R2-O--NH.sub.2,
R2-O--NHCH.sub.3, R2-O--NHCOCH.sub.3, or R2-O--CH.sub.2N.sub.3
wherein R2 has a molecular weight of at least 24 g/mol and thereby
making a 3'-blocked nucleotide from a 3'-protected nucleotide.
[0301] In embodiments, R2 has a molecular weight of at least 36. In
embodiments, R2 includes a fluorine nucleus. In embodiments, R2
includes at least two carbon nuclei. In embodiments, R2 includes a
carbon-carbon double bond. In embodiments, R2 includes a tertiary
carbon. In embodiments, R2 includes a tert-butyl moiety. In
embodiments, R2 includes a cyclic moiety. In embodiments, R2
includes a silicon nucleus. In embodiments, R2 includes a phenyl
ring. In embodiments, R2 includes a nitrogen nucleus. In
embodiments, R2 includes an oxygen nucleus. In embodiments, R2
includes a hydroxylate moiety.
[0302] In an aspect is provided a method of making a 3'-blocked
nucleotide, the method including contacting a 3'-protected
nucleotide with a reagent having the structure R2-O--NH.sub.2 or
R2-O--CH.sub.2N.sub.3 wherein R2 has a molecular weight of at least
24 g/mol and thereby making a 3'-blocked nucleotide from a
3'-protected nucleotide.
[0303] In embodiments, the 3'-blocked nucleotide includes a
blocking moiety bonded directly to the ribose 3' carbon of the
nucleotide; and the blocking moiety is --ONH.sub.2,
--ONHCOCH.sub.3, --ONHCH.sub.3, --OCH.sub.2N.sub.3. In embodiments,
the 3'-blocked nucleotide includes a blocking moiety bonded
directly to the ribose 3' carbon of the nucleotide; and the
blocking moiety is --ONH.sub.2 or --OCH.sub.2N.sub.3. In
embodiments, the 3'-blocked nucleotide includes a blocking moiety
bonded directly to the ribose 3' carbon of the nucleotide; and the
blocking moiety is --ONH.sub.2.
[0304] In embodiments, the 3'-protected nucleotide comprises a
protecting moiety directly covalently bonded to the ribose 3'
carbon of the nucleotide; and the protecting moiety is a 3'-O-oxime
moiety.
[0305] In embodiments, the --O-oxime moiety has the formula:
--O--N.dbd.C(R3)(R4); and R3 and R4 are independently hydrogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted cycloalkenyl, substituted or
unsubstituted cycloalkynyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heteroalicyclyl, substituted or unsubstituted
aralkyl, substituted or unsubstituted heteroaralkyl, or substituted
or unsubstituted (heteroalicyclyl)alkyl.
[0306] In embodiments, the --O-oxime moiety has the formula:
--O--N.dbd.C(R3)(R4); R3 is hydrogen; and R4 is substituted or
unsubstituted alkyl, substituted or unsubstituted alkenyl,
substituted or unsubstituted alkynyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted cycloalkenyl, substituted or unsubstituted
cycloalkynyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heteroalicyclyl, substituted or unsubstituted aralkyl, substituted
or unsubstituted heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl.
[0307] In embodiments, the --O-oxime moiety has the formula:
--O--N.dbd.C(R3)(R4); and R3 and R4 are independently substituted
or unsubstituted alkyl, substituted or unsubstituted alkenyl,
substituted or unsubstituted alkynyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted cycloalkenyl, substituted or unsubstituted
cycloalkynyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heteroalicyclyl, substituted or unsubstituted aralkyl, substituted
or unsubstituted heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl.
[0308] In embodiments, the 3'-blocked nucleotide and the
3'-protected nucleotide include an adenine moiety or derivative
thereof, guanine moiety or derivative thereof, cytosine moiety or
derivative thereof, thymine moiety or derivative thereof, or uracil
moiety or derivative thereof. In embodiments, the adenine
derivative moiety, guanine derivative moiety, cytosine derivative
moiety, thymine derivative moiety, and uracil derivative moiety
include a detectable moiety.
[0309] In embodiments, the 3'-blocked nucleotide and the
3'-protected nucleotide include a monophosphate moiety or
derivative thereof, diphosphate moiety or derivative thereof,
triphosphate moiety or derivative thereof, or monovalent nucleic
acid moiety or derivative thereof. In embodiments, the 3'-blocked
nucleotide and the 3'-protected nucleotide include a monophosphate
moiety, diphosphate moiety, triphosphate moiety, or monovalent
nucleic acid moiety. In embodiments, the 3'-blocked nucleotide and
the 3'-protected nucleotide comprise a triphosphate moiety. In
embodiments, the 3'-blocked nucleotide and the 3'-protected
nucleotide comprise a monovalent nucleic acid moiety. In
embodiments, the 3'-blocked nucleotide and the 3'-protected
nucleotide comprise a ribose moiety. In embodiments, the 3'-blocked
nucleotide and the 3'-protected nucleotide comprise a 2'
deoxyribose moiety.
[0310] In embodiments, R2 has a molecular weight of 1000 g/mol or
less. In embodiments, R2 has a molecular weight of at least 36
g/mol. In embodiments, R2 has a molecular weight of at least 57
g/mol.
[0311] In embodiments, R2 includes at least two carbon nuclei, a
fluorine nucleus, carbon-carbon double bond, tertiary carbon,
tert-butyl moiety, cyclic moiety, silicon nucleus, phenyl ring,
nitrogen nucleus, oxygen nucleus, or hydroxylate moiety.
[0312] In embodiments, R2 is substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
cycloalkenyl, substituted or unsubstituted cycloalkynyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heteroalicyclyl,
substituted or unsubstituted aralkyl, substituted or unsubstituted
heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl. In embodiments, R2 is not unsubstituted
methyl. In embodiments, R2 is not unsubstituted ethyl. In
embodiments, R2 is not unsubstituted propyl.
[0313] In embodiments, R2 is --C(R6)(R7)(R8), --C(O)(R6),
--P(O)(R6)(R7), --C(R6)(.dbd.C(R7)(R8)), --Si(R6)(R7)(R8), --S
O.sub.2(R6), substituted or unsubstituted cycloalkyl, substituted
or unsubstituted cycloalkenyl, substituted or unsubstituted
cycloalkynyl, substituted or unsubstituted heteroalicyclyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl; and R6, R7, and R8 are independently hydrogen, halogen,
--CCl.sub.3, --CBr.sub.3, --CF.sub.3, --CI.sub.3, --CHCl.sub.2,
--CHBr.sub.2, --CHF.sub.2, --CHI.sub.2, --CH.sub.2Cl, --CH.sub.2Br,
--CH.sub.2F, --CH.sub.2I, --CN, --OH, --NH.sub.2, --COOH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2,
--NHC(O)NH.sub.2, --NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH,
--OCCl.sub.3, --OCF.sub.3, --OCBr.sub.3, --OCI.sub.3,
--OCHCl.sub.2, --OCHBr.sub.2, --OCHI.sub.2, --OCHF.sub.2,
--OCH.sub.2Cl, --OCH.sub.2Br, --OCH.sub.2I, --OCH.sub.2F,
--SF.sub.5, --N.sub.3, substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
cycloalkenyl, substituted or unsubstituted cycloalkynyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heteroalicyclyl,
substituted or unsubstituted aralkyl, substituted or unsubstituted
heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl. In embodiments, R2 is --C(R6)(R7)(R8) and
R6, R7, and R8 are not all hydrogen.
[0314] In embodiments, R2 is --C(R6)(R7)(R8), --C(O)(R6),
--P(O)(R6)(R7), --C(R6)(.dbd.C(R7)(R8)), --Si(R6)(R7)(R8), --S
O.sub.2(R6), unsubstituted 6 membered heteroalicyclyl,
unsubstituted phenyl, substituted or unsubstituted heteroaryl; and
R6, R7, and R8 are independently hydrogen, halogen, --CCl.sub.3,
--CBr.sub.3, --CF.sub.3, --CI.sub.3, --CHCl.sub.2, --CHBr.sub.2,
--CHF.sub.2, --CHI.sub.2, --CH.sub.2Cl, --CH.sub.2Br, --CH.sub.2F,
--CH.sub.2I, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2, --NHC(O)NH.sub.2,
--NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH, --OCCl.sub.3,
--OCF.sub.3, --OCBr.sub.3, --OCI.sub.3, --OCHCl.sub.2,
--OCHBr.sub.2, --OCHI.sub.2, --OCHF.sub.2, --OCH.sub.2Cl,
--OCH.sub.2Br, --OCH.sub.2I, --OCH.sub.2F, --SF.sub.5, --N.sub.3,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted cycloalkenyl, substituted or
unsubstituted cycloalkynyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heteroalicyclyl, substituted or unsubstituted
aralkyl, substituted or unsubstituted heteroaralkyl, or substituted
or unsubstituted (heteroalicyclyl)alkyl. In embodiments, R6, R7,
and R8 are not all hydrogen.
[0315] In embodiments, the reagent has the formula:
##STR00057##
[0316] In embodiments, the reagent has the formula:
##STR00058##
[0317] In embodiments, the 3'-protected nucleotide has the
structure:
##STR00059##
the 3'-blocked nucleotide has the structure:
##STR00060##
B is a nucleobase; R1 is hydrogen, --OH, halogen, --OCH.sub.3; Q is
independently a nucleic acid moiety, triphosphate moiety,
diphosphate moiety, or monophosphate moiety; R3 and R4 are
independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
cycloalkenyl, substituted or unsubstituted cycloalkynyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heteroalicyclyl,
substituted or unsubstituted aralkyl, substituted or unsubstituted
heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl.
[0318] In embodiments, the method adds a 3' ONH.sub.2 in place of a
3' protecting group to at least 94% of a population of nucleotides
having a 3' protecting group in no more than 3 hours. In
embodiments, the method adds a 3' ONH.sub.2 in place of a 3'
protecting group to at least 98% of a population of nucleotides
having a 3' protecting group in no more than 1 hour.
[0319] In embodiments, the nucleotide having a 3' protecting group
includes cytosine, and wherein method degrades no more than 6% of
cytosine bases from a population including a plurality of said
nucleotide having a 3' protecting group including cytosine. In
embodiments, the nucleotide having a 3' protecting group includes
cytosine, and wherein method degrades no more than 1% of cytosine
bases from a population including a plurality of said nucleotide
having a 3' protecting group including cytosine.
[0320] In embodiments, the method further includes making a first
plurality of 3'-blocked nucleotides, the method including
contacting a second plurality of 3'-protected nucleotides with a
third plurality of the reagent and thereby making a first plurality
of 3'-blocked nucleotides from a second plurality of 3'-protected
nucleotides. In embodiments, the first plurality of 3'-blocked
nucleotides are made for 3 hours or less. In embodiments, the first
plurality of 3'-blocked nucleotides are made for 2 hours or less.
In embodiments, the first plurality of 3'-blocked nucleotides are
made for 1 hour or less.
[0321] In embodiments, at least 94% of the first plurality of
3'-protected nucleotide is made into the second plurality of
3'-blocked nucleotides. In embodiments, at least 96% of the first
plurality of 3'-protected nucleotide is made into the second
plurality of 3'-blocked nucleotides. In embodiments, at least 98%
of the first plurality of 3'-protected nucleotide is made into the
second plurality of 3'-blocked nucleotides.
[0322] In embodiments, the first plurality of 3'-blocked
nucleotides includes a fourth plurality of 3'-blocked nucleotides
including a cytosine; and 6% or less of the cytosines are
chemically modified by the third plurality of the reagent. In
embodiments, the first plurality of 3'-blocked nucleotides includes
a fourth plurality of 3'-blocked nucleotides including a cytosine;
and 5% or less of the cytosines are chemically modified by the
third plurality of the reagent. In embodiments, the first plurality
of 3'-blocked nucleotides includes a fourth plurality of 3'-blocked
nucleotides including a cytosine; and 4% or less of the cytosines
are chemically modified by the third plurality of the reagent. In
embodiments, the first plurality of 3'-blocked nucleotides includes
a fourth plurality of 3'-blocked nucleotides including a cytosine;
and 3% or less of the cytosines are chemically modified by the
third plurality of the reagent. In embodiments, the first plurality
of 3'-blocked nucleotides includes a fourth plurality of 3'-blocked
nucleotides including a cytosine; and 2% or less of the cytosines
are chemically modified by the third plurality of the reagent. In
embodiments, the first plurality of 3'-blocked nucleotides includes
a fourth plurality of 3'-blocked nucleotides including a cytosine;
and 1% or less of the cytosines are chemically modified by the
third plurality of the reagent.
[0323] In an aspect is provided a composition including a
population of cytosine nucleotides wherein at least 94% of the
population of cytosine nucleotides include a 3' --O--NH.sub.2,
wherein no more than 8% of the population of cytosine nucleotides
include a 3' moiety other than --O--NH.sub.2, and wherein no more
than 6% of the population of cytosine nucleotides include a
modified cytosine base.
[0324] In embodiments, at least 96% of the population of cytosine
nucleotides include a 3'-O--NH.sub.2. In embodiments, at least 98%
of the population of cytosine nucleotides include a 3'-O--NH.sub.2.
In embodiments, at least 99% of the population of cytosine
nucleotides include a 3'-O--NH.sub.2.
[0325] In embodiments, no more than 6% of the population of
cytosine nucleotides include a 3' moiety other than --O--NH.sub.2.
In embodiments, no more than 4% of the population of cytosine
nucleotides include a 3' moiety other than --O--NH.sub.2. In
embodiments, no more than 2% of the population of cytosine
nucleotides include a 3' moiety other than --O--NH.sub.2. In
embodiments, no more than 1% of the population of cytosine
nucleotides include a 3' moiety other than --O--NH.sub.2.
[0326] In embodiments, no more than 4% of the population of
cytosine nucleotides include a modified cytosine base. In
embodiments, no more than 2% of the population of cytosine
nucleotides include a modified cytosine base. In embodiments, no
more than 1% of the population of cytosine nucleotides include a
modified cytosine base.
[0327] In embodiments, at least 98% of the population of cytosine
nucleotides include a 3'-O--NH.sub.2, wherein no more than 1% of
the population of cytosine nucleotides include a 3' moiety other
than --O--NH.sub.2, and wherein no more than 1% of the population
of cytosine nucleotides include a modified cytosine base.
[0328] In embodiments, the composition is an aqueous solution. In
embodiments, the pH of the solution is no less than 4 and no
greater than 8.
[0329] In embodiments, the composition includes a reagent including
the structure R2-O--NH.sub.2. In embodiments, R2 includes a
molecular weight of at least 24, 26, 57, or 100. In embodiments, R2
includes a molecular weight of at least 24. In embodiments, R2
includes a molecular weight of at least 36. In embodiments, R2
includes at least two carbon nuclei, a fluorine nucleus,
carbon-carbon double bond, tertiary carbon, tert-butyl moiety,
cyclic moiety, silicon nucleus, phenyl ring, nitrogen nucleus,
oxygen nucleus, or hydroxylate moiety. In embodiments, R2 includes
at least two carbon nuclei. In embodiments, R2 includes a fluorine
nucleus. In embodiments, R2 includes a carbon-carbon double bond.
In embodiments, R2 includes a tertiary carbon. In embodiments, R2
includes a tert-butyl moiety. In embodiments, R2 includes a cyclic
moiety. In embodiments, R2 includes a silicon nucleus. In
embodiments, R2 includes a phenyl ring. In embodiments, R2 includes
a nitrogen nucleus. In embodiments, R2 includes a phenyl ring. In
embodiments, R2 includes an oxygen nucleus. In embodiments, R2
includes a hydroxylated moiety.
[0330] In embodiments, the reagent including the structure
R2-O--NH.sub.2 is selected from the group consisting of:
##STR00061##
[0331] In embodiments, the reagent including the structure
R2-O--NH.sub.2 is selected from the group consisting of:
##STR00062##
In embodiments, the reagent including the structure R2-O--NH.sub.2
is
##STR00063##
In embodiments, the reagent including the structure R2-O--NH.sub.2
is
##STR00064##
In embodiments, the reagent including the structure R2-O--NH.sub.2
is
##STR00065##
[0332] In embodiments, the 3' moiety other than --O--NH.sub.2
includes a 3' O-oxime. In embodiments, the 3' moiety other than
--O--NH.sub.2 includes a 3' O-aldoxime. In embodiments, the 3'
moiety other than --O--NH.sub.2 includes a 3' O-ketoxime.
[0333] In embodiments, the composition includes a reagent including
the structure R2-O--NHCH.sub.3 or R2-O--CH.sub.2N.sub.3. In
embodiments, the composition includes a reagent including the
structure R2-O--NHCH.sub.3. In embodiments, the composition
includes a reagent including the structure
R2-O--CH.sub.2N.sub.3.
[0334] In embodiments, at least some of the cytosine nucleotides
having a 3' moiety other than --O--NH.sub.2 nucleotide include the
structure:
##STR00066##
wherein B is a cytosine nucleobase; R1 is independently halogen,
OCH.sub.3, H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0335] In an aspect is provided a method of formulating a
sequencing reagent, including contacting a composition including a
3' protected nucleotide to a reagent including a reversible
terminator moiety to produce a composition including a nucleotide
including a reversible terminator moiety, and formulating the
composition including a nucleotide including a reversible
terminator moiety as a sequencing reagent, wherein formulating the
composition including a reversible terminator moiety does not
include further purification of the composition including a
reversible terminator moiety.
[0336] In embodiments, the method further includes applying the
sequencing reagent to a sequencing reagent to yield a sequencing
run of at least 100 bases having a quality score of at least Q30.
In embodiments, the reagent including a reversible terminator
moiety has a structure R2-O--NH.sub.2.
[0337] In embodiments, R2 is selected from the group consisting of
--C(R6)(R7)(R8), --C(O)(R6), --P(O)(R6)(R7),
--C(R6)(.dbd.C(R7)(R8)), --Si(R6)(R7)(R8), --S O2(R6), substituted
or unsubstituted cycloalkyl, substituted or unsubstituted
cycloalkenyl, substituted or unsubstituted cycloalkynyl,
substituted or unsubstituted heteroalicyclyl, substituted or
unsubstituted aryl, substituted or unsubstituted heteroaryl; and
R6, R7, and R8 are independently hydrogen, halogen, --CCl.sub.3,
--CBr.sub.3, --CF.sub.3, --CI.sub.3, --CHCl.sub.2, --CHBr.sub.2,
--CHF.sub.2, --CHI.sub.2, --CH.sub.2Cl, --CH.sub.2Br, --CH.sub.2F,
--CH.sub.2I, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2, --NHC(O)NH.sub.2,
--NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH, --OCCl.sub.3,
--OCF.sub.3, --OCBr.sub.3, --OCI.sub.3, --OCHCl.sub.2,
--OCHBr.sub.2, --OCHI.sub.2, --OCHF.sub.2, --OCH.sub.2Cl,
--OCH.sub.2Br, --OCH.sub.2I, --OCH.sub.2F, --SF.sub.5, --N.sub.3,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted cycloalkenyl, substituted or
unsubstituted cycloalkynyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heteroalicyclyl, substituted or unsubstituted
aralkyl, substituted or unsubstituted heteroaralkyl, or substituted
or unsubstituted (heteroalicyclyl)alkyl. In embodiments, R2 is
--C(CH.sub.3).sub.3. In embodiments, R2 is --C(R6)(R7)(R8) and R6,
R7, and R8 are not all hydrogen.
[0338] In embodiments, R2 includes at least two carbon nuclei, a
fluorine nucleus, carbon-carbon double bond, tertiary carbon,
tert-butyl moiety, cyclic moiety, silicon nucleus, phenyl ring,
nitrogen nucleus, oxygen nucleus, or hydroxylate moiety. In
embodiments, R2 includes at least two carbon nuclei, carbon-carbon
double bond, tertiary carbon, tert-butyl moiety, cyclic moiety, or
phenyl ring. In embodiments, R2 includes at least two carbon
nuclei. In embodiments, R2 includes a fluorine nucleus, silicon
nucleus, nitrogen nucleus, oxygen nucleus, or hydroxylate moiety.
In embodiments, R2 is --CH.sub.2CH.sub.3, --CH.sub.2CHCH.sub.2, or
--C(CH.sub.3).sub.3. In embodiments, R2 is-C(CH.sub.3).sub.3. In
embodiments, R2 is not unsubstituted methyl. In embodiments, R2 is
not unsubstituted ethyl. In embodiments, R2 is not unsubstituted
propyl.
[0339] In embodiments, R2 has a molecular weight greater than 24,
36, 57, or 100. In embodiments, R2 has a molecular weight of at
least 24, 36, 57, 100, 200, 300, 400, 500, 600, 700, 800, or 900.
In embodiments, R2 has a molecular weight of at least 24. In
embodiments, R2 has a molecular weight of at least 36. In
embodiments, R2 has a molecular weight of at least 57. In
embodiments, R2 has a molecular weight of at least 100. In
embodiments, R2 has a molecular weight of at least 200. In
embodiments, R2 has a molecular weight of at least 300. In
embodiments, R2 has a molecular weight of at least 400. In
embodiments, R2 has a molecular weight of at least 500. In
embodiments, R2 has a molecular weight of at least 600. In
embodiments, R2 has a molecular weight of at least 700. In
embodiments, R2 has a molecular weight of at least 800. In
embodiments, R2 has a molecular weight of at least 900.
[0340] In embodiments, the 3' protected nucleotide includes a
3'-O-oxime moiety. In embodiments, the oxime is an aldoxime or
ketoxime. In embodiments, the oxime is an aldoxime. In embodiments,
the oxime is a ketoxime.
[0341] In embodiments, the composition including a nucleotide
including a reversible terminator moiety includes no more than 6%
of the 3' protected nucleotide relative to 3' protected nucleotide
present initially. In embodiments, the composition including a
nucleotide including a reversible terminator moiety includes no
more than 5%, 4%, 3%, 2%, or 1% of the 3' protected nucleotide
relative to 3' protected nucleotide present initially. In
embodiments, the composition including a nucleotide including a
reversible terminator moiety includes no more than 5% of the 3'
protected nucleotide relative to 3' protected nucleotide present
initially. In embodiments, the composition including a nucleotide
including a reversible terminator moiety includes no more than 4%
of the 3' protected nucleotide relative to 3' protected nucleotide
present initially. In embodiments, the composition including a
nucleotide including a reversible terminator moiety includes no
more than 3% of the 3' protected nucleotide relative to 3'
protected nucleotide present initially. In embodiments, the
composition including a nucleotide including a reversible
terminator moiety includes no more than 2% of the 3' protected
nucleotide relative to 3' protected nucleotide present initially.
In embodiments, the composition including a nucleotide including a
reversible terminator moiety includes no more than 1% of the 3'
protected nucleotide relative to 3' protected nucleotide present
initially.
[0342] In embodiments, the composition including a nucleotide
including a reversible terminator moiety includes degraded or
modified dC nucleobases at a concentration of no more than 6% of
total dC nucleobases in the composition including a nucleotide
including a reversible terminator moiety. In embodiments, the
composition including a nucleotide including a reversible
terminator moiety includes degraded or modified dC nucleobases at a
concentration of no more than 5%, 4%, 3%, 2%, or 1% of total dC
nucleobases in the composition including a nucleotide including a
reversible terminator moiety. In embodiments, the composition
including a nucleotide including a reversible terminator moiety
includes degraded or modified dC nucleobases at a concentration of
no more than 5% of total dC nucleobases in the composition
including a nucleotide including a reversible terminator moiety. In
embodiments, the composition including a nucleotide including a
reversible terminator moiety includes degraded or modified dC
nucleobases at a concentration of no more than 4% of total dC
nucleobases in the composition including a nucleotide including a
reversible terminator moiety. In embodiments, the composition
including a nucleotide including a reversible terminator moiety
includes degraded or modified dC nucleobases at a concentration of
no more than 3% of total dC nucleobases in the composition
including a nucleotide including a reversible terminator moiety. In
embodiments, the composition including a nucleotide including a
reversible terminator moiety includes degraded or modified dC
nucleobases at a concentration of no more than 2% of total dC
nucleobases in the composition including a nucleotide including a
reversible terminator moiety. In embodiments, the composition
including a nucleotide including a reversible terminator moiety
includes degraded or modified dC nucleobases at a concentration of
no more than 1% of total dC nucleobases in the composition
including a nucleotide including a reversible terminator
moiety.
[0343] In an aspect is provided a method for modifying a
nucleotide, including reacting a nucleotide including a 3'-O-oxime
moiety with a reagent including the structure R2-O--NH.sub.2 to
produce a nucleotide including a 3'-O--NH.sub.2 moiety, wherein R2
is independently alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0344] In embodiments, the nucleotide that includes the 3'-O-oxime
moiety includes the structure:
##STR00067##
wherein the nucleotide that includes the 3'-O--NH.sub.2 moiety
includes the structure:
##STR00068##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0345] In embodiments, the nucleotide that is produced includes a
deoxynucleotide. In embodiments, the nucleotide that is produced
includes:
##STR00069##
wherein Base is the nucleobase, and wherein DNA is independently a
single stranded deoxyribonucleic acid or double stranded
deoxyribonucleic acid.
[0346] In embodiments, the nucleobase is a purine or pyrimidine. In
embodiments, the nucleobase is independently adenine, cytosine,
guanine, thymine, or uracil. In embodiments, the nucleobase is
independently 5-methylcytosine, hypoxanthine, xanthine, inosine,
isoguanine, isocytosine, or 7-deazaguanine.
[0347] In embodiments, the reagent is independently one of:
##STR00070##
[0348] In embodiments, the reaction occurs in an aqueous solution.
In embodiments, the nucleotide is attached to a solid support in
contact with the aqueous solution. In embodiments, the reagent that
includes the structure R2-O--NH.sub.2 is attached to a solid
support in contact with the aqueous solution. In embodiments, the
pH of the solution is no less than 4 and no greater than 8.
[0349] In an aspect is provided a solution including a nucleotide
including a 3'-O-oxime moiety and a reagent including the structure
R2-O--NH.sub.2, wherein R2 is independently alkyl, alkenyl,
alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,
heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or
an optionally substituted variant thereof. In embodiments, R2 is
not unsubstituted methyl. In embodiments, R2 is not unsubstituted
ethyl. In embodiments, R2 is not unsubstituted propyl.
[0350] In embodiments, the nucleotide includes the structure:
##STR00071##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0351] In embodiments, the nucleobase is a purine or pyrimidine. In
embodiments, the nucleobase is independently adenine, cytosine,
guanine, thymine, or uracil. In embodiments, the nucleobase is
complementary to adenine, cytosine, guanine, thymine, or uracil. In
embodiments, the nucleobase is independently 5-methylcytosine,
hypoxanthine, xanthine, inosine, isoguanine, isocytosine, or
7-deazaguanine. In embodiments, the nucleobase includes an
exogenous label moiety.
[0352] In embodiments, R1 is H. In embodiments, Q is
deoxyribonucleic acid. In embodiments, R1 is OH. In embodiments, Q
is ribonucleic acid.
[0353] In embodiments, the R2-O--NH.sub.2 molecule is independently
one of:
##STR00072##
[0354] In embodiments, the solution is an aqueous solution. In
embodiments, the nucleotide is attached to a solid support and
wherein the solid support is in contact with the solution. In
embodiments, the reagent that includes the structure R2-O--NH.sub.2
is attached to a solid support in contact with the aqueous
solution. In embodiments, the pH of the solution is no less than 4
and no greater than 8.
[0355] In an aspect is provided a method of sequencing a nucleic
acid including the steps: [0356] A. making a 3'-blocked nucleotide
using a method described herein, including in embodiments, wherein
the method of making does not include isolating the 3'-blocked
nucleotide from the composition including the reactants and
products of contacting the 3'-protected nucleotide with the
reagent; [0357] B. contacting the composition including the
3'-blocked nucleotide of step A with a nucleic acid template,
primer, and polymerase in a reaction vessel; [0358] C. identifying
the 3'-blocked nucleotide that simultaneously contacts the template
nucleic acid, 3' terminus of the primer, and polymerase; and
optionally covalently binding the 3'-blocked nucleotide to the 3'
terminus of the primer, making an extended primer; and thereby
identifying the cognate nucleotide of the template nucleic acid to
the 3'-blocking nucleotide; [0359] D. removing the 3'-blocking
moiety from the 3'-blocked nucleotide of the extended primer of
step C; and [0360] E. repeating steps B to D and thereby sequencing
the template nucleic acid.
[0361] In embodiments, step E is executed at least 99 times. In
embodiments, the composition includes reagent R2-ONH.sub.2. In
embodiments, the reagent R2 is unsubstituted t-butyl. In
embodiments, the reagent R2 is not hydrogen. In embodiments, the
reagent R2 is not unsubstituted methyl. In embodiments, the reagent
R2 is not unsubstituted ethyl. In embodiments, the reagent R2 is
not unsubstituted propyl. In embodiments, the composition does not
include HONH.sub.2. In embodiments, the composition does not
include CH.sub.3ONH.sub.2.
[0362] In embodiments, the quality score of the sequencing is at
least Q20. In embodiments, the quality score of the sequencing is
at least Q30. In embodiments, the quality score of the sequencing
is at least Q50.
[0363] In an aspect is provided a kit including a first vessel
containing a 3'-blocked nucleotide and a second vessel containing a
reagent having the formula R2-O--NH.sub.2, R2-O--NHCH.sub.3, or
R2-O--CH.sub.2N.sub.3 wherein R2 has a molecular weight of at least
24 g/mol.
[0364] In embodiments, the 3'-blocked nucleotide includes a
blocking moiety bonded directly to the ribose 3' carbon of the
nucleotide; and the blocking moiety is --ONH.sub.2, --ONHCH.sub.3,
or --OCH.sub.2CH.sub.3. In embodiments, the 3'-blocked nucleotide
includes a blocking moiety bonded directly to the ribose 3' carbon
of the nucleotide; and the blocking moiety is --ONH.sub.2.
[0365] In embodiments, the 3'-protected nucleotide includes a
protecting moiety directly covalently bonded to the ribose 3'
carbon of the nucleotide; and the protecting moiety is a 3' oxime
moiety.
[0366] In embodiments, the --O-oxime moiety has the formula:
--O--N.dbd.C(R3)(R4); and R3 and R4 are independently hydrogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted cycloalkenyl, substituted or
unsubstituted cycloalkynyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heteroalicyclyl, substituted or unsubstituted
aralkyl, substituted or unsubstituted heteroaralkyl, or substituted
or unsubstituted (heteroalicyclyl)alkyl.
[0367] In embodiments, the --O-oxime moiety has the formula:
--O--N.dbd.C(R3)(R4); R3 is hydrogen; and R4 is substituted or
unsubstituted alkyl, substituted or unsubstituted alkenyl,
substituted or unsubstituted alkynyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted cycloalkenyl, substituted or unsubstituted
cycloalkynyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heteroalicyclyl, substituted or unsubstituted aralkyl, substituted
or unsubstituted heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl.
[0368] In embodiments, the --O-oxime moiety has the formula:
--O--N.dbd.C(R3)(R4); and R3 and R4 are independently substituted
or unsubstituted alkyl, substituted or unsubstituted alkenyl,
substituted or unsubstituted alkynyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted cycloalkenyl, substituted or unsubstituted
cycloalkynyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heteroalicyclyl, substituted or unsubstituted aralkyl, substituted
or unsubstituted heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl.
[0369] In embodiments, the 3'-blocked nucleotide and the
3'-protected nucleotide include an adenine moiety or derivative
thereof, guanine moiety or derivative thereof, cytosine moiety or
derivative thereof, thymine moiety or derivative thereof, or uracil
moiety or derivative thereof. In embodiments, the adenine
derivative moiety, guanine derivative moiety, cytosine derivative
moiety, thymine derivative moiety, and uracil derivative moiety
include a detectable moiety. In embodiments, the 3'-blocked
nucleotide and the 3'-protected nucleotide include a monophosphate
moiety or derivative thereof, diphosphate moiety or derivative
thereof, triphosphate moiety or derivative thereof, or monovalent
nucleic acid moiety or derivative thereof. In embodiments, the
3'-blocked nucleotide and the 3'-protected nucleotide include a
monophosphate moiety, diphosphate moiety, triphosphate moiety, or
monovalent nucleic acid moiety. In embodiments, the 3'-blocked
nucleotide and the 3'-protected nucleotide include a triphosphate
moiety. In embodiments, the 3'-blocked nucleotide and the
3'-protected nucleotide include a monovalent nucleic acid moiety.
In embodiments, the 3'-blocked nucleotide and the 3'-protected
nucleotide include a ribose moiety. In embodiments, the 3'-blocked
nucleotide and the 3'-protected nucleotide include a 2' deoxyribose
moiety.
[0370] In embodiments, R2 has a molecular weight of 1000 g/mol or
less. In embodiments, R2 has a molecular weight of at least 36
g/mol. In embodiments, R2 has a molecular weight of at least 57
g/mol.
[0371] In embodiments, R2 is substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted cycloalkenyl, substituted or unsubstituted
cycloalkynyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heteroalicyclyl, substituted or unsubstituted aralkyl, substituted
or unsubstituted heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl. In embodiments, R2 is not unsubstituted
methyl. In embodiments, R2 is not unsubstituted ethyl. In
embodiments, R2 is not unsubstituted propyl.
[0372] In embodiments, R2 is --C(R6)(R7)(R8), --C(O)(R6),
--P(O)(R6)(R7), --C(R6)(.dbd.C(R7)(R8)), --Si(R6)(R7)(R8), --S
O.sub.2(R6), substituted or unsubstituted cycloalkyl, substituted
or unsubstituted cycloalkenyl, substituted or unsubstituted
cycloalkynyl, substituted or unsubstituted heteroalicyclyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl; and R6, R7, and R8 are independently hydrogen, halogen,
--CCl.sub.3, --CBr.sub.3, --CF.sub.3, --CI.sub.3, --CHCl.sub.2,
--CHBr.sub.2, --CHF.sub.2, --CHI.sub.2, --CH.sub.2Cl, --CH.sub.2Br,
--CH.sub.2F, --CH.sub.2I, --CN, --OH, --NH.sub.2, --COOH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2,
--NHC(O)NH.sub.2, --NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH,
--OCCl.sub.3, --OCF.sub.3, --OCBr.sub.3, --OCI.sub.3,
--OCHCl.sub.2, --OCHBr.sub.2, --OCHI.sub.2, --OCHF.sub.2,
--OCH.sub.2Cl, --OCH.sub.2Br, --OCH.sub.2I, --OCH.sub.2F,
--SF.sub.5, --N.sub.3, substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
cycloalkenyl, substituted or unsubstituted cycloalkynyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heteroalicyclyl,
substituted or unsubstituted aralkyl, substituted or unsubstituted
heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl. In embodiments, R2 is --C(R6)(R7)(R8) and
R6, R7, and R8 are not all hydrogen.
[0373] In embodiments, R2 is --C(R6)(R7)(R8), --C(O)(R6),
--P(O)(R6)(R7), --C(R6)(.dbd.C(R7)(R8)), --Si(R6)(R7)(R8), --S
O.sub.2(R6), unsubstituted 6 membered heteroalicyclyl,
unsubstituted phenyl, substituted or unsubstituted heteroaryl; and
R6, R7, and R8 are independently hydrogen, halogen, --CCl.sub.3,
--CBr.sub.3, --CF.sub.3, --CI.sub.3, --CHCl.sub.2, --CHBr.sub.2,
--CHF.sub.2, --CHI.sub.2, --CH.sub.2Cl, --CH.sub.2Br, --CH.sub.2F,
--CH.sub.2I, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NIC(O)NHNH.sub.2, --NHC(O)NH.sub.2,
--NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH, --OCCl.sub.3,
--OCF.sub.3, --OCBr.sub.3, --OCI.sub.3, --OCHCl.sub.2,
--OCHBr.sub.2, --OCHI.sub.2, --OCHF.sub.2, --OCH.sub.2Cl,
--OCH.sub.2Br, --OCH.sub.2I, --OCH.sub.2F, --SF.sub.5, --N.sub.3,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted cycloalkenyl, substituted or
unsubstituted cycloalkynyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heteroalicyclyl, substituted or unsubstituted
aralkyl, substituted or unsubstituted heteroaralkyl, or substituted
or unsubstituted (heteroalicyclyl)alkyl. In embodiments, R2 is
--C(R6)(R7)(R8) and R6, R7, and R8 are not all hydrogen.
[0374] In embodiments, the reagent has the formula:
##STR00073##
[0375] In embodiments, the reagent has the formula:
##STR00074##
[0376] In embodiments, the 3'-protected nucleotide has the
structure:
##STR00075##
the 3'-blocked nucleotide has the structure:
##STR00076##
B is a nucleobase; R1 is hydrogen, --OH, halogen, --OCH.sub.3; Q is
independently a nucleic acid moiety, triphosphate moiety,
diphosphate moiety, or monophosphate moiety; and R3 and R4 are
independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted cycloalkenyl, substituted or unsubstituted
cycloalkynyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heteroalicyclyl, substituted or unsubstituted aralkyl, substituted
or unsubstituted heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl.
[0377] In an aspect is provided a kit including a first vessel
containing a nucleotide including a 3'-O-oxime moiety and a second
vessel containing a reagent including the structure R2-O--NH.sub.2,
wherein R2 is independently alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally
substituted variant thereof.
[0378] In embodiments, the nucleotide includes the structure:
##STR00077##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0379] In embodiments, the nucleobase is a purine or pyrimidine. In
embodiments, the nucleobase is independently adenine, cytosine,
guanine, thymine, or uracil. In embodiments, the nucleobase is
complementary to adenine, cytosine, guanine, thymine, or uracil. In
embodiments, the nucleobase is independently 5-methylcytosine,
hypoxanthine, xanthine, inosine, isoguanine, isocytosine, or
7-deazaguanine. In embodiments, the nucleobase includes an
exogenous label moiety.
[0380] In embodiments, Q is independently a single stranded
deoxyribonucleic acid or double stranded deoxyribonucleic acid.
[0381] In embodiments, the R2-O--NH.sub.2 molecule is independently
one of:
##STR00078##
[0382] In embodiments, the kit includes an RFID tag or bar
code.
[0383] In embodiments, the nucleotide is attached to a solid
support. In embodiments, the reagent that includes the structure
R2-O--NH.sub.2 is attached to a solid support.
[0384] In an aspect is provided a method of replacing a protecting
group by a blocking group at a 3' position of a nucleotide,
including contacting the nucleotide and a reagent including the
structure R2-block, wherein R2 has a molecular weight of at least
24, wherein R2-block is water soluble, and wherein block includes a
blocking group moiety. In embodiments, block comprises a reversible
terminator moiety. In embodiments, the reversible terminator moiety
does not preclude assembly of a nucleotide reversibly terminated
thereby into a ternary complex.
[0385] In embodiments, R2-block is selected from the group
consisting of R2-O--NH.sub.2, R2-O--NHCH.sub.3, R2-O--NHCOCH.sub.3,
or R2-O--CH.sub.2N.sub.3. In embodiments, R2-block is
R2-O--NH.sub.2. In embodiments, R2-block is R2-O--NHCH.sub.3. In
embodiments, R2-block is R2-O--NHCOCH.sub.3. In embodiments,
R2-block is R2-O--CH.sub.2N.sub.3.
[0386] In embodiments, the nucleotide including a 3' protecting
group is:
##STR00079##
wherein the nucleotide including a 3' blocking group is:
##STR00080##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0387] In embodiments, the protecting group includes an O-oxime. In
embodiments, the oxime moiety has the formula:
--O--N.dbd.C(R3)(R4); and R3 and R4 are independently hydrogen,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted cycloalkenyl, substituted or
unsubstituted cycloalkynyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heteroalicyclyl, substituted or unsubstituted
aralkyl, substituted or unsubstituted heteroaralkyl, or substituted
or unsubstituted (heteroalicyclyl)alkyl. In embodiments, R3 is
Hydrogen. In embodiments, R4 is Hydrogen. In embodiments, the
nucleotide comprises a triphosphate moiety.
[0388] In embodiments, R2 is --C(R6)(R7)(R8), --C(O)(R6),
--P(O)(R6)(R7), --C(R6)(.dbd.C(R7)(R8)), --Si(R6)(R7)(R8), --S
O.sub.2(R6), substituted or unsubstituted cycloalkyl, substituted
or unsubstituted cycloalkenyl, substituted or unsubstituted
cycloalkynyl, substituted or unsubstituted heteroalicyclyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl; and R6, R7, and R8 are independently hydrogen, halogen,
--CCl.sub.3, --CBr.sub.3, --CF.sub.3, --CI.sub.3, --CHCl.sub.2,
--CHBr.sub.2, --CHF.sub.2, --CHI.sub.2, --CH.sub.2Cl, --CH.sub.2Br,
--CH.sub.2F, --CH.sub.2I, --CN, --OH, --NH.sub.2, --COOH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2,
--NHC(O)NH.sub.2, --NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH,
--OCCl.sub.3, --OCF.sub.3, --OCBr.sub.3, --OCI.sub.3,
--OCHCl.sub.2, --OCHBr.sub.2, --OCHI.sub.2, --OCHF.sub.2,
--OCH.sub.2Cl, --OCH.sub.2Br, --OCH.sub.2I, --OCH.sub.2F,
--SF.sub.5, --N.sub.3, substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
cycloalkenyl, substituted or unsubstituted cycloalkynyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heteroalicyclyl,
substituted or unsubstituted aralkyl, substituted or unsubstituted
heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl. In embodiments, R2 is --C(R6)(R7)(R8) and
R6, R7, and R8 are not all hydrogen.
[0389] In embodiments, R2-block has a formula selected from the
group consisting of the following:
##STR00081##
[0390] In embodiments, R2-block has a formula selected from the
group consisting of:
##STR00082##
[0391] In embodiments, R2-block has a formula
##STR00083##
In embodiments, R2-block has a formula
##STR00084##
In embodiments, R2-block has a formula
##STR00085##
In embodiments, R2-block has a formula
##STR00086##
In embodiments, R2-block has a formula
##STR00087##
In embodiments, R2-block has a formula
##STR00088##
In embodiments, R2-block has a formula
##STR00089##
In embodiments, R2-block has a formula
##STR00090##
In embodiments, R2-block has a formula
##STR00091##
In embodiments, R2-block has a formula
##STR00092##
In embodiments, R2-block has a formula
##STR00093##
In embodiments, R2-block has a formula
##STR00094##
In embodiments, R2-block has a formula
##STR00095##
In embodiments, R2-block has a formula
##STR00096##
In embodiments, R2-block has a formula
##STR00097##
[0392] In embodiments, R2 comprises a hydroxylated moiety. In
embodiments, R2 comprises at least two carbon nuclei, a fluorine
nucleus, carbon-carbon double bond, tertiary carbon, tert-butyl
moiety, cyclic moiety, silicon nucleus, phenyl ring, nitrogen
nucleus, oxygen nucleus, or hydroxylate moiety.
[0393] In embodiments, R2 has a molecular weight of no more than
280 g/mol. In embodiments, R2 has a molecular weight of no more
than 100 g/mol.
[0394] In embodiments, the method adds a blocking group in place of
a 3' protecting group to at least 94% of a population of
nucleotides having a 3' protecting group in no more than 3 hours at
25.degree. C. In embodiments, the method adds a blocking group in
place of a 3' protecting group to at least 98% of a population of
nucleotides having a 3' protecting group in no more than 3 hours at
25.degree. C.
[0395] In embodiments, the method degrades no more than 6% of
cytosine nucleobases in a population of nucleotides each comprising
a cytosine nucleobase. In embodiments, the method degrades no more
than 1% of cytosine bases from the population of nucleotides having
a cytosine nucleobase.
[0396] In embodiments, the composition includes
(CH.sub.3).sub.3C--ONH.sub.2. In embodiments, the solution includes
(CH.sub.3).sub.3C--ONH.sub.2. In embodiments, the aqueous solution
includes (CH.sub.3).sub.3C--ONH.sub.2. In embodiments, the vessel
includes (CH.sub.3).sub.3C--ONH.sub.2. In embodiments, the kit
includes (CH.sub.3).sub.3C--ONH.sub.2. In embodiments, the
composition does not include CH.sub.3--ONH.sub.2. In embodiments,
the solution does not include CH.sub.3--ONH.sub.2. In embodiments,
the aqueous solution does not include CH.sub.3--ONH.sub.2. In
embodiments, the vessel does not include CH.sub.3--ONH.sub.2. In
embodiments, the kit does not include CH.sub.3--ONH.sub.2. In
embodiments, the composition does not include H--ONH.sub.2. In
embodiments, the solution does not include H--ONH.sub.2. In
embodiments, the aqueous solution does not include H--ONH.sub.2. In
embodiments, the vessel does not include H--ONH.sub.2. In
embodiments, the kit does not include H--ONH.sub.2.
Embodiments
[0397] Embodiment P1. A method for modifying a nucleotide,
comprising reacting a nucleotide comprising a 3'-G-oxime moiety
with a reagent comprising the structure R2-ONH.sub.2 to produce a
nucleotide comprising a 3'-G-NH.sub.2 moiety, wherein R2 is
independently alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0398] Embodiment P2. The method of embodiment P1, wherein the
nucleotide that comprises the 3'-O-oxime moiety comprises the
structure:
##STR00098##
wherein the nucleotide that comprises the 3'-O--NH.sub.2 moiety
comprises the structure:
##STR00099##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0399] Embodiment P3. The method of embodiment P1, wherein the
reaction occurs in an aqueous solution.
[0400] Embodiment P4. The method of embodiment P3, wherein the
nucleotide is attached to a solid support in contact with the
aqueous solution.
[0401] Embodiment P5. The method of embodiment P3, wherein the
reagent that comprises the structure R2-ONH.sub.2 is attached to a
solid support in contact with the aqueous solution.
[0402] Embodiment P6. The method of any one of embodiments P1
through P5, wherein the nucleobase is a purine or pyrimidine.
[0403] Embodiment P7. The method of embodiment P6, wherein the
nucleobase is independently adenine, cytosine, guanine, thymine, or
uracil.
[0404] Embodiment P8. The method of embodiment P6, wherein the
nucleobase is complementary to adenine, cytosine, guanine, thymine,
or uracil.
[0405] Embodiment P9. The method of embodiment P6, wherein the
nucleobase is independently 5-methylcytosine, hypoxanthine,
xanthine, inosine, isoguanine, isocytosine, or 7-deazaguanine.
[0406] Embodiment P10. The method of any one of embodiments P1
through P9, wherein the pH of the solution is between 4 and 8.
[0407] Embodiment P11. The method of any one of embodiments P1
through P10, wherein the nucleotide that is produced comprises a
deoxynucleotide.
[0408] Embodiment P12. The method of embodiment P11, wherein the
nucleotide that is produced comprises:
##STR00100##
wherein Base is the nucleobase, and wherein DNA is independently a
single stranded deoxyribonucleic acid or double stranded
deoxyribonucleic acid.
[0409] Embodiment P13. The method of any one of embodiments P1
through P12, wherein the reagent is independently one of:
##STR00101##
[0410] Embodiment P14. A solution comprising a nucleotide
comprising a 3'-O-oxime moiety and a reagent comprising the
structure R2-ONH.sub.2, wherein R2 is independently alkyl, alkenyl,
alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,
heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or
an optionally substituted variant thereof.
[0411] Embodiment P15. The solution of embodiment P14, wherein the
nucleotide comprises the structure:
##STR00102##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0412] Embodiment P16. The solution of embodiment P15, wherein R1
is H.
[0413] Embodiment P17. The solution of embodiment P16, wherein Q is
deoxyribonucleic acid.
[0414] Embodiment P18. The solution of embodiment P15, wherein R1
is OH.
[0415] Embodiment P19. The solution of embodiment P18, wherein Q is
ribonucleic acid.
[0416] Embodiment P20. The solution of embodiment P14, wherein the
solution is an aqueous solution.
[0417] Embodiment P21. The solution of embodiment P20, wherein the
nucleotide is attached to a solid support and wherein the solid
support is in contact with the solution.
[0418] Embodiment P22. The solution of embodiment P20, wherein the
reagent that comprises the structure R2-ONH.sub.2 is attached to a
solid support in contact with the aqueous solution.
[0419] Embodiment P23. The solution of any one of embodiments P14
through P2A3, wherein the nucleobase is a purine or pyrimidine.
[0420] Embodiment P24. The solution of embodiment P23, wherein the
nucleobase is independently adenine, cytosine, guanine, thymine, or
uracil.
[0421] Embodiment P25. The solution of embodiment P23, wherein the
nucleobase is complementary to adenine, cytosine, guanine, thymine,
or uracil.
[0422] Embodiment P26. The solution of embodiment P23, wherein the
nucleobase is independently 5-methylcytosine, hypoxanthine,
xanthine, inosine, isoguanine, isocytosine, or 7-deazaguanine.
[0423] Embodiment P27. The solution of any one of embodiments P14
through P26, wherein the nucleobase comprises an exogenous label
moiety.
[0424] Embodiment P28. The solution of any one of embodiments P14
through P27, wherein the pH of the solution is between 4 and 8.
[0425] Embodiment P29. The solution of any one of embodiments P14
through P28, wherein the R2-ONH.sub.2 molecule is independently one
of:
##STR00103##
[0426] Embodiment P30. A kit comprising a first vessel containing a
nucleotide comprising a 3'-O-oxime moiety and a second vessel
containing a reagent comprising the structure R2-ONH.sub.2, wherein
R2 is independently alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally
substituted variant thereof.
[0427] Embodiment P31. The kit of embodiment P30, wherein the
nucleotide comprises the structure:
##STR00104##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0428] Embodiment P32. The kit of embodiment P30, wherein the kit
comprises an RFID tag or bar code.
[0429] Embodiment P33. The kit of embodiment P31 or P32, wherein Q
is independently a single stranded deoxyribonucleic acid or double
stranded deoxyribonucleic acid.
[0430] Embodiment P34. The kit of embodiment P33, wherein the
nucleotide is attached to a solid support.
[0431] Embodiment P35. The kit of embodiment P30, wherein the
reagent that comprises the structure R2-ONH.sub.2 is attached to a
solid support.
[0432] Embodiment P36. The kit of any one of embodiments P30
through P35, wherein the nucleobase is a purine or pyrimidine.
[0433] Embodiment P38. The kit of embodiment P36, wherein the
nucleobase is independently adenine, cytosine, guanine, thymine, or
uracil.
[0434] Embodiment P39. The kit of embodiment P36, wherein the
nucleobase is complementary to adenine, cytosine, guanine, thymine,
or uracil.
[0435] Embodiment P40. The kit of embodiment P36, wherein the
nucleobase is independently 5-methylcytosine, hypoxanthine,
xanthine, inosine, isoguanine, isocytosine, or 7-deazaguanine.
[0436] Embodiment P41. The kit of any one of embodiments P30
through P40, wherein the nucleobase comprises an exogenous label
moiety.
[0437] Embodiment P42. The kit of any one of embodiments P30
through P41, wherein the R2-ONH.sub.2 molecule is independently one
of:
##STR00105##
[0438] Embodiment PP1. A method for modifying a nucleotide,
comprising reacting a nucleotide comprising a 3'-O-oxime moiety
with a reagent comprising the structure R2-ONH.sub.2 to produce a
nucleotide comprising a 3'-O--NH.sub.2 moiety, wherein R2 is
independently alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0439] Embodiment PP2. The method of embodiment PP1, wherein the
nucleotide that comprises the 3'-O-oxime moiety comprises the
structure:
##STR00106##
wherein the nucleotide that comprises the 3'-O--NH.sub.2 moiety
comprises the structure:
##STR00107##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0440] Embodiment PP3. The method of embodiment PP1, wherein the
reaction occurs in an aqueous solution.
[0441] Embodiment PP4. The method of embodiment PP3, wherein the
nucleotide is attached to a solid support in contact with the
aqueous solution.
[0442] Embodiment PP5. The method of embodiment PP3, wherein the
reagent that comprises the structure R2-ONH.sub.2 is attached to a
solid support in contact with the aqueous solution.
[0443] Embodiment PP6. The method of any one of embodiments PP1
through PP5, wherein the nucleobase is a purine or pyrimidine.
[0444] Embodiment PP7. The method of embodiment PP6, wherein the
nucleobase is independently adenine, cytosine, guanine, thymine, or
uracil.
[0445] Embodiment PP8. The method of embodiment PP6, wherein the
nucleobase is complementary to adenine, cytosine, guanine, thymine,
or uracil.
[0446] Embodiment PP9. The method of embodiment PP6, wherein the
nucleobase is independently 5-methylcytosine, hypoxanthine,
xanthine, inosine, isoguanine, isocytosine, or 7-deazaguanine.
[0447] Embodiment PP10. The method of any one of embodiments PP1
through PP9, wherein the pH of the solution is between 4 and 8.
[0448] Embodiment PP11. The method of any one of embodiments PP1
through PP10, wherein the nucleotide that is produced comprises a
deoxynucleotide.
[0449] Embodiment PP12. The method of embodiment PP11, wherein the
nucleotide that is produced comprises:
##STR00108##
wherein Base is the nucleobase, and wherein DNA is independently a
single stranded deoxyribonucleic acid or double stranded
deoxyribonucleic acid.
[0450] Embodiment PP13. The method of any one of embodiments PP1
through PP12, wherein the reagent is independently one of:
##STR00109##
[0451] Embodiment PP14. A solution comprising a nucleotide
comprising a 3'-O-oxime moiety and a reagent comprising the
structure R2-ONH.sub.2, wherein R2 is independently alkyl, alkenyl,
alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,
heteroalicyclyl, aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or
an optionally substituted variant thereof.
[0452] Embodiment PP15. The solution of embodiment PP14, wherein
the nucleotide comprises the structure:
##STR00110##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0453] Embodiment PP16. The solution of embodiment PP15, wherein R1
is H.
[0454] Embodiment PP17. The solution of embodiment PP16, wherein Q
is deoxyribonucleic acid.
[0455] Embodiment PP18. The solution of embodiment PP15, wherein R1
is OH.
[0456] Embodiment PP19. The solution of embodiment PP18, wherein Q
is ribonucleic acid.
[0457] Embodiment PP20. The solution of embodiment PP14, wherein
the solution is an aqueous solution.
[0458] Embodiment PP21. The solution of embodiment PP20, wherein
the nucleotide is attached to a solid support and wherein the solid
support is in contact with the solution.
[0459] Embodiment PP22. The solution of embodiment PP20, wherein
the reagent that comprises the structure R2-ONH.sub.2 is attached
to a solid support in contact with the aqueous solution.
[0460] Embodiment PP23. The solution of any one of embodiments PP14
through PP22, wherein the nucleobase is a purine or pyrimidine.
[0461] Embodiment PP24. The solution of embodiment PP23, wherein
the nucleobase is independently adenine, cytosine, guanine,
thymine, or uracil.
[0462] Embodiment PP25. The solution of embodiment PP23, wherein
the nucleobase is complementary to adenine, cytosine, guanine,
thymine, or uracil.
[0463] Embodiment PP26. The solution of embodiment PP23, wherein
the nucleobase is independently 5-methylcytosine, hypoxanthine,
xanthine, inosine, isoguanine, isocytosine, or 7-deazaguanine.
[0464] Embodiment PP27. The solution of any one of embodiments PP14
through PP26, wherein the nucleobase comprises an exogenous label
moiety.
[0465] Embodiment PP28. The solution of any one of embodiments PP14
through PP27, wherein the pH of the solution is between 4 and
8.
[0466] Embodiment PP29. The solution of any one of embodiments PP14
through PP28, wherein the R2-ONH.sub.2 molecule is independently
one of:
##STR00111##
[0467] Embodiment PP30. A kit comprising a first vessel containing
a nucleotide comprising a 3'-O-oxime moiety and a second vessel
containing a reagent comprising the structure R2-ONH.sub.2, wherein
R2 is independently alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally
substituted variant thereof.
[0468] Embodiment PP31. The kit of embodiment PP30, wherein the
nucleotide comprises the structure:
##STR00112##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0469] Embodiment PP32. The kit of embodiment PP30, wherein the kit
comprises an RFID tag or bar code.
[0470] Embodiment PP33. The kit of embodiment PP31 or PP32, wherein
Q is independently a single stranded deoxyribonucleic acid or
double stranded deoxyribonucleic acid.
[0471] Embodiment PP34. The kit of embodiment PP33, wherein the
nucleotide is attached to a solid support.
[0472] Embodiment PP35. The kit of embodiment PP30, wherein the
reagent that comprises the structure R2-ONH.sub.2 is attached to a
solid support.
[0473] Embodiment PP36. The kit of any one of embodiments PP30
through PP35, wherein the nucleobase is a purine or pyrimidine.
[0474] Embodiment PP38. The kit of embodiment PP36, wherein the
nucleobase is independently adenine, cytosine, guanine, thymine, or
uracil.
[0475] Embodiment PP39. The kit of embodiment PP36, wherein the
nucleobase is complementary to adenine, cytosine, guanine, thymine,
or uracil.
[0476] Embodiment PP40. The kit of embodiment PP36, wherein the
nucleobase is independently 5-methylcytosine, hypoxanthine,
xanthine, inosine, isoguanine, isocytosine, or 7-deazaguanine.
[0477] Embodiment PP41. The kit of any one of embodiments PP30
through PP40, wherein the nucleobase comprises an exogenous label
moiety.
[0478] Embodiment PP42. The kit of any one of embodiments PP30
through PP41, wherein the R2-ONH.sub.2 molecule is independently
one of:
##STR00113##
Further Embodiments
[0479] Embodiment Si. A method of adding a 3'-O--NH.sub.2 moiety to
a nucleotide having a 3' protecting group, comprising contacting a
nucleotide having a 3' protecting group to a reagent comprising the
structure R2-O--NH.sub.2, wherein R2 has a molecular weight of at
least 24.
[0480] Embodiment S2. The method of embodiment S1, wherein the
nucleotide having a 3' protecting group comprises ribose or
deoxyribose.
[0481] Embodiment S3. The method of embodiment S2, wherein the
nucleotide having a 3' protecting group comprises triphosphate.
[0482] Embodiment S4. The method of embodiment S1, wherein the
nucleotide having a 3' protecting group comprises the
structure:
##STR00114##
wherein the nucleotide that comprises the 3'-O--NH.sub.2 moiety
comprises the structure:
##STR00115##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0483] Embodiment S5. A method of making a 3'-blocked nucleotide,
the method comprising contacting a 3'-protected nucleotide with a
reagent having the structure R2-O--NH.sub.2, R2-O--NHCH.sub.3,
R2-O--NHCOCH.sub.3, or R2-O--CH.sub.2N.sub.3 wherein R2 has a
molecular weight of at least 24 g/mol and thereby making a
3'-blocked nucleotide from a 3'-protected nucleotide.
[0484] Embodiment S6. A method of making a 3'-blocked nucleotide,
the method comprising contacting a 3'-protected nucleotide with a
reagent having the structure R2-O--NH.sub.2 or
R2-O--CH.sub.2N.sub.3 wherein R2 has a molecular weight of at least
24 g/mol and thereby making a 3'-blocked nucleotide from a
3'-protected nucleotide.
[0485] Embodiment S7. The method of embodiment S5, wherein the
3'-blocked nucleotide comprises a blocking moiety bonded directly
to the ribose 3' carbon of the nucleotide; and the blocking moiety
is --ONH.sub.2, --ONHCOCH.sub.3, --ONHCH.sub.3,
--OCH.sub.2N.sub.3.
[0486] Embodiment S8. The method of one of embodiments S5 to S6,
wherein the 3'-blocked nucleotide comprises a blocking moiety
bonded directly to the ribose 3' carbon of the nucleotide; and the
blocking moiety is --ONH.sub.2 or --OCH.sub.2N.sub.3.
[0487] Embodiment S9. The method of one of embodiments S5 to S6,
wherein the 3'-blocked nucleotide comprises a blocking moiety
bonded directly to the ribose 3' carbon of the nucleotide; and the
blocking moiety is --ONH.sub.2.
[0488] Embodiment S10. The method of one of embodiments S5 to S9,
wherein the 3'-protected nucleotide comprises a protecting moiety
directly covalently bonded to the ribose 3' carbon of the
nucleotide; and the protecting moiety is a 3'-O-oxime moiety.
[0489] Embodiment S11. The method of embodiment S10, wherein the
--O-oxime moiety has the formula:
--O--N.dbd.C(R3)(R4); and
R3 and R4 are independently hydrogen, substituted or unsubstituted
alkyl, substituted or unsubstituted alkenyl, substituted or
unsubstituted alkynyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted cycloalkenyl, substituted or unsubstituted
cycloalkynyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heteroalicyclyl, substituted or unsubstituted aralkyl, substituted
or unsubstituted heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl.
[0490] Embodiment S12. The method of embodiment S10, wherein the
--O-oxime moiety has the formula:
--O--N.dbd.C(R3)(R4);
R3 is hydrogen; and R4 is substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
cycloalkenyl, substituted or unsubstituted cycloalkynyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heteroalicyclyl,
substituted or unsubstituted aralkyl, substituted or unsubstituted
heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl.
[0491] Embodiment S13. The method of embodiment S10, wherein the
--O-oxime moiety has the formula:
--O--N.dbd.C(R3)(R4); and
[0492] R3 and R4 are independently substituted or unsubstituted
alkyl, substituted or unsubstituted alkenyl, substituted or
unsubstituted alkynyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted cycloalkenyl, substituted or unsubstituted
cycloalkynyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heteroalicyclyl, substituted or unsubstituted aralkyl, substituted
or unsubstituted heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl.
[0493] Embodiment S14. The method of one of embodiments S5 to S13,
wherein the 3'-blocked nucleotide and the 3'-protected nucleotide
comprise an adenine moiety or derivative thereof, guanine moiety or
derivative thereof, cytosine moiety or derivative thereof, thymine
moiety or derivative thereof, or uracil moiety or derivative
thereof.
[0494] Embodiment S15. The method of embodiment S14, wherein the
adenine derivative moiety, guanine derivative moiety, cytosine
derivative moiety, thymine derivative moiety, and uracil derivative
moiety comprise a detectable moiety.
[0495] Embodiment S16. The method of one of embodiments S5 to S15,
wherein the 3'-blocked nucleotide and the 3'-protected nucleotide
comprise a monophosphate moiety or derivative thereof, diphosphate
moiety or derivative thereof, triphosphate moiety or derivative
thereof, or monovalent nucleic acid moiety or derivative
thereof.
[0496] Embodiment S17. The method of embodiment S16, wherein the
3'-blocked nucleotide and the 3'-protected nucleotide comprise a
monophosphate moiety, diphosphate moiety, triphosphate moiety, or
monovalent nucleic acid moiety.
[0497] Embodiment S18. The method of embodiment S16, wherein the
3'-blocked nucleotide and the 3'-protected nucleotide comprise a
triphosphate moiety.
[0498] Embodiment S19. The method of embodiment S16, wherein the
3'-blocked nucleotide and the 3'-protected nucleotide comprise a
monovalent nucleic acid moiety.
[0499] Embodiment S20. The method of one of embodiments S5 to S19,
wherein the 3'-blocked nucleotide and the 3'-protected nucleotide
comprise a ribose moiety.
[0500] Embodiment S21. The method of one of embodiments S5 to S19,
wherein the 3'-blocked nucleotide and the 3'-protected nucleotide
comprise a 2' deoxyribose moiety.
[0501] Embodiment S22. The method of one of embodiments S1 to S21,
wherein R2 has a molecular weight of 1000 g/mol or less.
[0502] Embodiment S23. The method of one of embodiments S1 to S22,
wherein R2 has a molecular weight of at least 36 g/mol.
[0503] Embodiment S24. The method of one of embodiments S1 to S22,
wherein R2 has a molecular weight of at least 57 g/mol.
[0504] Embodiment S25. The method of one of embodiments S1 to S22,
wherein R2 comprises at least two carbon nuclei, a fluorine
nucleus, carbon-carbon double bond, tertiary carbon, tert-butyl
moiety, cyclic moiety, silicon nucleus, phenyl ring, nitrogen
nucleus, oxygen nucleus, or hydroxylate moiety.
[0505] Embodiment S26. The method of one of embodiments S1 to S22,
wherein R2 is substituted or unsubstituted alkyl, substituted or
unsubstituted alkenyl, substituted or unsubstituted alkynyl,
substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
cycloalkenyl, substituted or unsubstituted cycloalkynyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heteroalicyclyl,
substituted or unsubstituted aralkyl, substituted or unsubstituted
heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl.
[0506] Embodiment S27. The method of one of embodiments S1 to S22,
wherein R2 is --C(R6)(R7)(R8), --C(O)(R6), --P(O)(R6)(R7),
--C(R6)(.dbd.C(R7)(R8)), --Si(R6)(R7)(R8), --S O.sub.2(R6),
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted cycloalkenyl, substituted or unsubstituted
cycloalkynyl, substituted or unsubstituted heteroalicyclyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl; and
R6, R7, and R8 are independently hydrogen, halogen, --CCl.sub.3,
--CBr.sub.3, --CF.sub.3, --CI.sub.3, --CHCl.sub.2, --CHBr.sub.2,
--CHF.sub.2, --CHI.sub.2, --CH.sub.2Cl, --CH.sub.2Br, --CH.sub.2F,
--CH.sub.2I, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2, --NHC(O)NH.sub.2,
--NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH, --OCCl.sub.3,
--OCF.sub.3, --OCBr.sub.3, --OCI.sub.3, --OCHCl.sub.2,
--OCHBr.sub.2, --OCHI.sub.2, --OCHF.sub.2, --OCH.sub.2Cl,
--OCH.sub.2Br, --OCH.sub.2I, --OCH.sub.2F, --SF.sub.5, --N.sub.3,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted cycloalkenyl, substituted or
unsubstituted cycloalkynyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heteroalicyclyl, substituted or unsubstituted
aralkyl, substituted or unsubstituted heteroaralkyl, or substituted
or unsubstituted (heteroalicyclyl)alkyl.
[0507] Embodiment S28. The method of embodiment S27, wherein R2 is
--C(R6)(R7)(R8) and R6, R7, and R8 are not all hydrogen.
[0508] Embodiment S29. The method of embodiment S27, wherein R2 is
not unsubstituted methyl.
[0509] Embodiment S30. The method of embodiment S27, wherein R2 is
not unsubstituted ethyl.
[0510] Embodiment S31. The method of embodiment S27, wherein R2 is
not unsubstituted propyl.
[0511] Embodiment 532. The method of one of embodiments S1 to S22,
wherein R2 is --C(R6)(R7)(R8), --C(O)(R6), --P(O)(R6)(R7),
--C(R6)(.dbd.C(R7)(R8)), --Si(R6)(R7)(R8), --S O.sub.2(R6),
unsubstituted 6 membered heteroalicyclyl, unsubstituted phenyl,
substituted or unsubstituted heteroaryl; and
R6, R7, and R8 are independently hydrogen, halogen, --CCl.sub.3,
--CBr.sub.3, --CF.sub.3, --CI.sub.3, --CHCl.sub.2, --CHBr.sub.2,
--CHF.sub.2, --CHI.sub.2, --CH.sub.2Cl, --CH.sub.2Br, --CH.sub.2F,
--CH.sub.2I, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2, --NHC(O)NH.sub.2,
--NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH, --OCCl.sub.3,
--OCF.sub.3, --OCBr.sub.3, --OCI.sub.3, --OCHCl.sub.2,
--OCHBr.sub.2, --OCHI.sub.2, --OCHF.sub.2, --OCH.sub.2Cl,
--OCH.sub.2Br, --OCH.sub.2I, --OCH.sub.2F, --SF.sub.5, --N.sub.3,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted cycloalkenyl, substituted or
unsubstituted cycloalkynyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heteroalicyclyl, substituted or unsubstituted
aralkyl, substituted or unsubstituted heteroaralkyl, or substituted
or unsubstituted (heteroalicyclyl)alkyl.
[0512] Embodiment S33. The method of embodiment S32, wherein R2 is
--C(R6)(R7)(R8) and R6, R7, and R8 are not all hydrogen.
[0513] Embodiment S34. The method of embodiment S32, wherein R2 is
not unsubstituted methyl.
[0514] Embodiment S35. The method of embodiment S32, wherein R2 is
not unsubstituted ethyl.
[0515] Embodiment S36. The method of embodiment S32, wherein R2 is
not unsubstituted propyl.
[0516] Embodiment S37. The method of one of embodiments S1 to S22,
wherein the reagent has the formula:
##STR00116##
[0517] Embodiment S38. The method of one of embodiments S1 to S22,
wherein the reagent has the formula:
##STR00117##
[0518] Embodiment S39. The method of one of embodiments S5 to S11,
wherein the 3'-protected nucleotide has the structure:
##STR00118##
the 3'-blocked nucleotide has the structure:
##STR00119##
B is a nucleobase; R1 is hydrogen, --OH, halogen, --OCH.sub.3; Q is
independently a nucleic acid moiety, triphosphate moiety,
diphosphate moiety, or monophosphate moiety; R3 and R4 are
independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
cycloalkenyl, substituted or unsubstituted cycloalkynyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heteroalicyclyl,
substituted or unsubstituted aralkyl, substituted or unsubstituted
heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl.
[0519] Embodiment S40. The method of one of embodiments S1 to S4,
wherein the method adds a 3' ONH.sub.2 in place of a 3' protecting
group to at least 94% of a population of nucleotides having a 3'
protecting group in no more than 3 hours.
[0520] Embodiment S41. The method of one of embodiments S1 to S4,
wherein the method adds a 3' ONH.sub.2 in place of a 3' protecting
group to at least 98% of a population of nucleotides having a 3'
protecting group in no more than 1 hour.
[0521] Embodiment S42. The method of one of embodiments S1 to S4,
wherein the nucleotide having a 3' protecting group comprises
cytosine, and wherein method degrades no more than 6% of cytosine
bases from a population comprising a plurality of said nucleotide
having a 3' protecting group comprising cytosine.
[0522] Embodiment S43. The method of one of embodiments S1 to S4,
wherein the nucleotide having a 3' protecting group comprises
cytosine, and wherein method degrades no more than 1% of cytosine
bases from a population comprising a plurality of said nucleotide
having a 3' protecting group comprising cytosine.
[0523] Embodiment S44. The method of one of embodiments S5 to S39,
further comprising making a first plurality of 3'-blocked
nucleotides, the method comprising contacting a second plurality of
3'-protected nucleotides with a third plurality of the reagent and
thereby making a first plurality of 3'-blocked nucleotides from a
second plurality of 3'-protected nucleotides.
[0524] Embodiment S45. The method of embodiment S44, wherein the
first plurality of 3'-blocked nucleotides are made for 3 hours or
less.
[0525] Embodiment S46. The method of embodiment S44, wherein the
first plurality of 3'-blocked nucleotides are made for 2 hours or
less.
[0526] Embodiment S47. The method of embodiment S44, wherein the
first plurality of 3'-blocked nucleotides are made for 1 hour or
less.
[0527] Embodiment S48. The method of one of embodiments S44 to S47,
wherein at least 94% of the first plurality of 3'-protected
nucleotide is made into the second plurality of 3'-blocked
nucleotides.
[0528] Embodiment S49. The method of one of embodiments S44 to S47,
wherein at least 96% of the first plurality of 3'-protected
nucleotide is made into the second plurality of 3'-blocked
nucleotides.
[0529] Embodiment S50. The method of one of embodiments S44 to S47,
wherein at least 98% of the first plurality of 3'-protected
nucleotide is made into the second plurality of 3'-blocked
nucleotides.
[0530] Embodiment S51. The method of one of embodiments S44 to S50,
wherein the first plurality of 3'-blocked nucleotides comprises a
fourth plurality of 3'-blocked nucleotides comprising a cytosine;
and 6% or less of the cytosines are chemically modified by the
third plurality of the reagent.
[0531] Embodiment S52. The method of one of embodiments S44 to S50,
wherein the first plurality of 3'-blocked nucleotides comprises a
fourth plurality of 3'-blocked nucleotides comprising a cytosine;
and 5% or less of the cytosines are chemically modified by the
third plurality of the reagent.
[0532] Embodiment S53. The method of one of embodiments S44 to S50,
wherein the first plurality of 3'-blocked nucleotides comprises a
fourth plurality of 3'-blocked nucleotides comprising a cytosine;
and 4% or less of the cytosines are chemically modified by the
third plurality of the reagent.
[0533] Embodiment S54. The method of one of embodiments S44 to S50,
wherein the first plurality of 3'-blocked nucleotides comprises a
fourth plurality of 3'-blocked nucleotides comprising a cytosine;
and 3% or less of the cytosines are chemically modified by the
third plurality of the reagent.
[0534] Embodiment S55. The method of one of embodiments S44 to S50,
wherein the first plurality of 3'-blocked nucleotides comprises a
fourth plurality of 3'-blocked nucleotides comprising a cytosine;
and 2% or less of the cytosines are chemically modified by the
third plurality of the reagent.
[0535] Embodiment S56. The method of one of embodiments S44 to S50,
wherein the first plurality of 3'-blocked nucleotides comprises a
fourth plurality of 3'-blocked nucleotides comprising a cytosine;
and 1% or less of the cytosines are chemically modified by the
third plurality of the reagent.
[0536] Embodiment S57. A composition comprising a population of
cytosine nucleotides wherein at least 94% of the population of
cytosine nucleotides comprise a 3'-O--NH.sub.2, wherein no more
than 8% of the population of cytosine nucleotides comprise a 3'
moiety other than --O--NH.sub.2, and wherein no more than 6% of the
population of cytosine nucleotides comprise a modified cytosine
base.
[0537] Embodiment S58. The composition of embodiment S57, wherein
at least 98% of the population of cytosine nucleotides comprise a
3'-O--NH.sub.2, wherein no more than 1% of the population of
cytosine nucleotides comprise a 3' moiety other than --O--NH.sub.2,
and wherein no more than 1% of the population of cytosine
nucleotides comprise a modified cytosine base.
[0538] Embodiment S59. The composition of embodiment S57, wherein
the composition is an aqueous solution.
[0539] Embodiment S60. The composition of embodiment S57, wherein
the pH of the solution is no less than 4 and no greater than 8.
[0540] Embodiment S61. The composition of embodiment S57, wherein
the composition comprises a reagent comprising the structure
R2-O--NH.sub.2.
[0541] Embodiment S62. The composition of embodiment S61, wherein
R2 comprises a molecular weight of at least 24, 26, 57, or 100.
[0542] Embodiment S63. The composition of embodiment S61, wherein
R2 comprises at least two carbon nuclei, a fluorine nucleus,
carbon-carbon double bond, tertiary carbon, tert-butyl moiety,
cyclic moiety, silicon nucleus, phenyl ring, nitrogen nucleus,
oxygen nucleus, or hydroxylate moiety.
[0543] Embodiment S64. The composition of embodiment S61, wherein
the reagent comprising the structure R2-O--NH.sub.2 is selected
from the group consisting of:
##STR00120##
[0544] Embodiment S65. The composition of one of embodiments S57 to
S64, wherein the 3' moiety other than --O--NH.sub.2 comprises a 3'
O-oxime.
[0545] Embodiment S66. The composition of embodiment S57, wherein
the composition comprises a reagent comprising the structure
R2-O--NHCH.sub.3 or R2-O--CH.sub.2N.sub.3.
[0546] Embodiment S67. The composition of one of embodiments S57 to
S66, wherein at least some of the cytosine nucleotides having a 3'
moiety other than --O--NH.sub.2 nucleotide comprise the
structure:
##STR00121##
wherein B is a cytosine nucleobase; R1 is independently halogen,
OCH.sub.3, H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0547] Embodiment S68. A method of formulating a sequencing
reagent, comprising contacting a composition comprising a 3'
protected nucleotide to a reagent comprising a reversible
terminator moiety to produce a composition comprising a nucleotide
comprising a reversible terminator moiety, and formulating the
composition comprising a nucleotide comprising a reversible
terminator moiety as a sequencing reagent, wherein formulating the
composition comprising a reversible terminator moiety does not
comprise further purification of the composition comprising a
reversible terminator moiety.
[0548] Embodiment S69. The method of embodiment S68, further
comprising applying the sequencing reagent to a sequencing reagent
to yield a sequencing run of at least 100 bases having a quality
score of at least Q30.
[0549] Embodiment S70. The method of embodiment S68, wherein the
reagent comprising a reversible terminator moiety has a structure
R2-O--NH.sub.2.
[0550] Embodiment S71. The method of embodiment S70, wherein R2 is
selected from the group consisting of --C(R6)(R7)(R8), --C(O)(R6),
--P(O)(R6)(R7), --C(R6)(.dbd.C(R7)(R8)), --Si(R6)(R7)(R8), --S
O.sub.2(R6), substituted or unsubstituted cycloalkyl, substituted
or unsubstituted cycloalkenyl, substituted or unsubstituted
cycloalkynyl, substituted or unsubstituted heteroalicyclyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl; and R6, R7, and R8 are independently hydrogen, halogen,
--CCl.sub.3, --CBr.sub.3, --CF.sub.3, --CI.sub.3, --CHCl.sub.2,
--CHBr.sub.2, --CHF.sub.2, --CHI.sub.2, --CH.sub.2Cl, --CH.sub.2Br,
--CH.sub.2F, --CH.sub.2I, --CN, --OH, --NH.sub.2, --COOH,
--CONH.sub.2, --NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H,
--SO.sub.2NH.sub.2, --NHNH.sub.2, --ONH.sub.2, --NHC(O)NHINH.sub.2,
--NHC(O)NH.sub.2, --NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH,
--OCCl.sub.3, --OCF.sub.3, --OCBr.sub.3, --OCI.sub.3,
--OCHCl.sub.2, --OCHBr.sub.2, --OCHI.sub.2, --OCHF.sub.2,
--OCH.sub.2Cl, --OCH.sub.2Br, --OCH.sub.2I, --OCH.sub.2F,
--SF.sub.5, --N.sub.3, substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
cycloalkenyl, substituted or unsubstituted cycloalkynyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heteroalicyclyl,
substituted or unsubstituted aralkyl, substituted or unsubstituted
heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl.
[0551] Embodiment S72. The method of embodiment S71, wherein R2 is
--C(R6)(R7)(R8) and R6, R7, and R8 are not all hydrogen.
[0552] Embodiment S73. The method of embodiment S71, wherein R2 is
not unsubstituted methyl.
[0553] Embodiment S74. The method of embodiment S71, wherein R2 is
not unsubstituted ethyl.
[0554] Embodiment S75. The method of embodiment S71, wherein R2 is
not unsubstituted propyl.
[0555] Embodiment S76. The method of embodiment S70, wherein R2 is
--C(CH.sub.3).sub.3.
[0556] Embodiment S77. The method of embodiment S70, wherein R2 has
a molecular weight greater than 24, 36, 57, or 100.
[0557] Embodiment S78. The method of embodiment S70, wherein R2
comprises at least two carbon nuclei, a fluorine nucleus,
carbon-carbon double bond, tertiary carbon, tert-butyl moiety,
cyclic moiety, silicon nucleus, phenyl ring, nitrogen nucleus,
oxygen nucleus, or hydroxylate moiety.
[0558] Embodiment S79. The method of embodiment S68, wherein the 3'
protected nucleotide comprises a 3'-O-oxime moiety.
[0559] Embodiment S80. The method of embodiment S68, wherein the
composition comprising a nucleotide comprising a reversible
terminator moiety comprises no more than 6% of the 3' protected
nucleotide relative to 3' protected nucleotide present
initially.
[0560] Embodiment S81. The method of embodiment S68, wherein the
composition comprising a nucleotide comprising a reversible
terminator moiety comprises degraded or modified dC nucleobases at
a concentration of no more than 6% of total dC nucleobases in the
composition comprising a nucleotide comprising a reversible
terminator moiety.
[0561] Embodiment S82. A method for modifying a nucleotide,
comprising reacting a nucleotide comprising a 3'-O-oxime moiety
with a reagent comprising the structure R2-O--NH.sub.2 to produce a
nucleotide comprising a 3'-O--NH.sub.2 moiety, wherein R2 is
independently alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0562] Embodiment S83. The method of embodiment S82, wherein the
nucleotide that comprises the 3'-O-oxime moiety comprises the
structure:
##STR00122##
wherein the nucleotide that comprises the 3'-O--NH.sub.2 moiety
comprises the structure:
##STR00123##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0563] Embodiment S84. The method of one of embodiments S82 to S83,
wherein the nucleotide that is produced comprises a
deoxynucleotide.
[0564] Embodiment S85. The method of embodiment S82, wherein the
nucleotide that is produced comprises:
##STR00124##
wherein Base is the nucleobase, and wherein DNA is independently a
single stranded deoxyribonucleic acid or double stranded
deoxyribonucleic acid.
[0565] Embodiment S86. The method of any one of embodiments S82 to
S85, wherein the reagent is independently one of:
##STR00125##
[0566] Embodiment S87. A solution comprising a nucleotide
comprising a 3'-O-oxime moiety and a reagent comprising the
structure R2-O--NH.sub.2, wherein R2 is independently alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl,
(heteroalicyclyl)alkyl, or an optionally substituted variant
thereof.
[0567] Embodiment S88. The solution of embodiment S87, wherein the
nucleotide comprises the structure:
##STR00126##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0568] Embodiment S89. The solution of one of embodiments S87 to
S88, wherein the nucleobase comprises an exogenous label
moiety.
[0569] Embodiment S90. The solution of one of embodiments S87 to
S89, wherein the R2-O--NH.sub.2 molecule is independently one
of:
##STR00127##
[0570] Embodiment S91. A method of sequencing a nucleic acid
comprising the steps: [0571] A. making a 3'-blocked nucleotide
using the method of one of embodiments S5 to S56, wherein the
method of making does not comprise isolating the 3'-blocked
nucleotide from the composition comprising the reactants and
products of contacting the 3'-protected nucleotide with the
reagent; [0572] B. contacting the composition comprising the
3'-blocked nucleotide of step A with a nucleic acid template,
primer, and polymerase in a reaction vessel; [0573] C. identifying
the 3'-blocked nucleotide that simultaneously contacts the template
nucleic acid, 3' terminus of the primer, and polymerase; and
optionally covalently binding the 3'-blocked nucleotide to the 3'
terminus of the primer, making an extended primer; and thereby
identifying the cognate nucleotide of the template nucleic acid to
the 3'-blocking nucleotide; [0574] D. removing the 3'-blocking
moiety from the 3'-blocked nucleotide of the extended primer of
step C; and [0575] E. repeating steps B to D and thereby sequencing
the template nucleic acid.
[0576] Embodiment S92. The method of embodiment S91, wherein step E
is executed at least 99 times.
[0577] Embodiment S93. The method of one of embodiments S91 to S92,
wherein the quality score of the sequencing is at least Q20.
[0578] Embodiment S94. The method of one of embodiments S91 to S92,
wherein the quality score of the sequencing is at least Q30.
[0579] Embodiment S95 The method of one of embodiments S91 to S92,
wherein the quality score of the sequencing is at least Q50.
[0580] Embodiment S96. A kit comprising a first vessel containing a
3'-blocked nucleotide and a second vessel containing a reagent
having the formula R2-O--NH.sub.2, R2-O--NHCH.sub.3, or
R2-O--CH.sub.2N.sub.3 wherein R2 has a molecular weight of at least
24 g/mol.
[0581] Embodiment S97. The kit of embodiment S96, wherein the
3'-blocked nucleotide comprises a blocking moiety bonded directly
to the ribose 3' carbon of the nucleotide; and the blocking moiety
is --ONH.sub.2, --ONHCH.sub.3, or --OCH.sub.2CH.sub.3.
[0582] Embodiment S98. The kit of embodiment S96, wherein the
3'-blocked nucleotide comprises a blocking moiety bonded directly
to the ribose 3' carbon of the nucleotide; and the blocking moiety
is --ONH.sub.2.
[0583] Embodiment S99. The kit of one of embodiments S96 to 598,
wherein the 3'-protected nucleotide comprises a protecting moiety
directly covalently bonded to the ribose 3' carbon of the
nucleotide; and the protecting moiety is a 3' oxime moiety.
[0584] Embodiment S100. The kit of embodiment S99, wherein the
--O-oxime moiety has the formula:
--O--N.dbd.C(R3)(R4); and
R3 and R4 are independently hydrogen, substituted or unsubstituted
alkyl, substituted or unsubstituted alkenyl, substituted or
unsubstituted alkynyl, substituted or unsubstituted heteroalkyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted cycloalkenyl, substituted or unsubstituted
cycloalkynyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heteroalicyclyl, substituted or unsubstituted aralkyl, substituted
or unsubstituted heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl.
[0585] Embodiment S101. The kit of embodiment S99, wherein the
--O-oxime moiety has the formula:
--O--N.dbd.C(R3)(R4);
R3 is hydrogen; and R4 is substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
cycloalkenyl, substituted or unsubstituted cycloalkynyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heteroalicyclyl,
substituted or unsubstituted aralkyl, substituted or unsubstituted
heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl.
[0586] Embodiment S102. The kit of embodiment S99, wherein the
--O-oxime moiety has the formula:
--O--N.dbd.C(R3)(R4); and
R3 and R4 are independently substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
cycloalkenyl, substituted or unsubstituted cycloalkynyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heteroalicyclyl,
substituted or unsubstituted aralkyl, substituted or unsubstituted
heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl.
[0587] Embodiment S103. The kit of one of embodiments S96 to S102,
wherein the 3'-blocked nucleotide and the 3'-protected nucleotide
comprise an adenine moiety or derivative thereof, guanine moiety or
derivative thereof, cytosine moiety or derivative thereof, thymine
moiety or derivative thereof, or uracil moiety or derivative
thereof.
[0588] Embodiment S104. The kit of embodiment S103, wherein the
adenine derivative moiety, guanine derivative moiety, cytosine
derivative moiety, thymine derivative moiety, and uracil derivative
moiety comprise a detectable moiety.
[0589] Embodiment S105. The kit of one of embodiments S96 to S104,
wherein the 3'-blocked nucleotide and the 3'-protected nucleotide
comprise a monophosphate moiety or derivative thereof, diphosphate
moiety or derivative thereof, triphosphate moiety or derivative
thereof, or monovalent nucleic acid moiety or derivative
thereof.
[0590] Embodiment S106. The kit of embodiment S105, wherein the
3'-blocked nucleotide and the 3'-protected nucleotide comprise a
monophosphate moiety, diphosphate moiety, triphosphate moiety, or
monovalent nucleic acid moiety.
[0591] Embodiment S107. The kit of embodiment S105, wherein the
3'-blocked nucleotide and the 3'-protected nucleotide comprise a
triphosphate moiety.
[0592] Embodiment S108. The kit of embodiment S105, wherein the
3'-blocked nucleotide and the 3'-protected nucleotide comprise a
monovalent nucleic acid moiety.
[0593] Embodiment S109. The kit of one of embodiments S96 to S108,
wherein the 3'-blocked nucleotide and the 3'-protected nucleotide
comprise a ribose moiety.
[0594] Embodiment S110. The kit of one of embodiments S96 to S108,
wherein the 3'-blocked nucleotide and the 3'-protected nucleotide
comprise a 2' deoxyribose moiety.
[0595] Embodiment S111. The kit of one of embodiments S96 to S110,
wherein R2 has a molecular weight of 1000 g/mol or less.
[0596] Embodiment S112. The kit of one of embodiments S96 to S111,
wherein R2 has a molecular weight of at least 36 g/mol.
[0597] Embodiment S113. The kit of one of embodiments S96 to S111,
wherein R2 has a molecular weight of at least 57 g/mol.
[0598] Embodiment S114. The kit of one of embodiments S96 to S111,
wherein R2 is substituted or unsubstituted alkyl, substituted or
unsubstituted alkenyl, substituted or unsubstituted alkynyl,
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted cycloalkenyl, substituted or unsubstituted
cycloalkynyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heteroalicyclyl, substituted or unsubstituted aralkyl, substituted
or unsubstituted heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl.
[0599] Embodiment S115. The kit of one of embodiments S96 to S111,
wherein R2 is --C(R6)(R7)(R8), --C(O)(R6), --P(O)(R6)(R7),
--C(R6)(.dbd.C(R7)(R8)), --Si(R6)(R7)(R8), --S O.sub.2(R6),
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted cycloalkenyl, substituted or unsubstituted
cycloalkynyl, substituted or unsubstituted heteroalicyclyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl; and
R6, R7, and R8 are independently hydrogen, halogen, --CCl.sub.3,
--CBr.sub.3, --CF.sub.3, --CI.sub.3, --CHCl.sub.2, --CHBr.sub.2,
--CHF.sub.2, --CHI.sub.2, --CH.sub.2Cl, --CH.sub.2Br, --CH.sub.2F,
--CH.sub.2I, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2, --NHC(O)NH.sub.2,
--NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH, --OCCl.sub.3,
--OCF.sub.3, --OCBr.sub.3--OCI.sub.3, --OCHCl.sub.2, --OCHBr.sub.2,
--OCHI.sub.2, --OCHF.sub.2, --OCH.sub.2Cl, --OCH.sub.2Br,
--OCH.sub.2I, --OCH.sub.2F, --SF.sub.5, --N.sub.3, substituted or
unsubstituted alkyl, substituted or unsubstituted alkenyl,
substituted or unsubstituted alkynyl, substituted or unsubstituted
heteroalkyl, substituted or unsubstituted cycloalkyl, substituted
or unsubstituted cycloalkenyl, substituted or unsubstituted
cycloalkynyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heteroalicyclyl, substituted or unsubstituted aralkyl, substituted
or unsubstituted heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl.
[0600] Embodiment S116. The kit of embodiment S115, wherein R2 is
--C(R6)(R7)(R8) and R6, R7, and R8 are not all hydrogen.
[0601] Embodiment S117. The kit of embodiment S115, wherein R2 is
not unsubstituted methyl.
[0602] Embodiment S118. The kit of embodiment S115, wherein R2 is
not unsubstituted ethyl.
[0603] Embodiment S119. The kit of embodiment S115, wherein R2 is
not unsubstituted propyl.
[0604] Embodiment S120. The kit of one of embodiments S96 to S111,
wherein R2 is --C(R6)(R7)(R8), --C(O)(R6), --P(O)(R6)(R7),
--C(R6)(.dbd.C(R7)(R8)), --Si(R6)(R7)(R8), --S O2(R6),
unsubstituted 6 membered heteroalicyclyl, unsubstituted phenyl,
substituted or unsubstituted heteroaryl; and
R6, R7, and R8 are independently hydrogen, halogen, --CCl.sub.3,
--CBr.sub.3, --CF.sub.3, --CI.sub.3, --CHCl.sub.2, --CHBr.sub.2,
--CHF.sub.2, --CHI.sub.2, --CH.sub.2Cl, --CH.sub.2Br, --CH.sub.2F,
--CH.sub.2I, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2, --NHC(O)NH.sub.2,
--NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH, --OCCl.sub.3,
--OCF.sub.3, --OCBr.sub.3, --OCI.sub.3, --OCHCl.sub.2,
--OCHBr.sub.2, --OCHI.sub.2, --OCHF.sub.2, --OCH.sub.2Cl,
--OCH.sub.2Br, --OCH.sub.2I, --OCH.sub.2F, --SF.sub.5, --N.sub.3,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted cycloalkenyl, substituted or
unsubstituted cycloalkynyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heteroalicyclyl, substituted or unsubstituted
aralkyl, substituted or unsubstituted heteroaralkyl, or substituted
or unsubstituted (heteroalicyclyl)alkyl.
[0605] Embodiment S121. The kit of embodiment S120, wherein R2 is
--C(R6)(R7)(R8) and R6, R7, and R8 are not all hydrogen.
[0606] Embodiment S122. The kit of embodiment S120, wherein R2 is
not unsubstituted methyl.
[0607] Embodiment S123. The kit of embodiment S120, wherein R2 is
not unsubstituted ethyl.
[0608] Embodiment S124. The kit of embodiment S120, wherein R2 is
not unsubstituted propyl.
[0609] Embodiment S125. The kit of one of embodiments S96 to S111,
wherein the reagent has the formula:
##STR00128##
[0610] Embodiment S126. The kit of one of embodiments S96 to S111,
wherein the reagent has the formula:
##STR00129##
[0611] Embodiment S127. The kit of one of embodiments S96 to S100,
wherein the 3'-protected nucleotide has the structure:
##STR00130##
the 3'-blocked nucleotide has the structure:
##STR00131##
B is a nucleobase; R1 is hydrogen, --OH, halogen, --OCH.sub.3; Q is
independently a nucleic acid moiety, triphosphate moiety,
diphosphate moiety, or monophosphate moiety; and R3 and R4 are
independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted cycloalkyl, substituted or
unsubstituted cycloalkenyl, substituted or unsubstituted
cycloalkynyl, substituted or unsubstituted aryl, substituted or
unsubstituted heteroaryl, substituted or unsubstituted
heteroalicyclyl, substituted or unsubstituted aralkyl, substituted
or unsubstituted heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl.
[0612] Embodiment S128. A kit comprising a first vessel containing
a nucleotide comprising a 3'-O-oxime moiety and a second vessel
containing a reagent comprising the structure R2-O--NH.sub.2,
wherein R2 is independently alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally
substituted variant thereof.
[0613] Embodiment S129. The kit of embodiment S128, wherein the
nucleotide comprises the structure:
##STR00132##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0614] Embodiment S130. The kit of one of embodiments S128 to S129,
wherein the nucleobase comprises an exogenous label moiety.
[0615] Embodiment S131. The kit of one of embodiments S128 to S130,
wherein the R2-O--NH.sub.2 molecule is independently one of:
##STR00133##
[0616] Embodiment T1. A method of adding a 3'-O--NH.sub.2 moiety to
a nucleotide having a 3' protecting group, comprising contacting a
nucleotide having a 3' protecting group to a reagent comprising the
structure R2-O--NH.sub.2, wherein R2 has a molecular weight of at
least 24.
[0617] Embodiment T2. The method of embodiment T1, wherein the
nucleotide having a 3' protecting group comprises ribose.
[0618] Embodiment T3. The method of embodiment T1, wherein the
nucleotide having a 3' protecting group comprises 2'
deoxyribose.
[0619] Embodiment T4. The method of embodiment T2, wherein the
nucleotide having a 3' protecting group comprises phosphate.
[0620] Embodiment T5. The method of embodiment T4, wherein the
nucleotide having a 3' protecting group comprises triphosphate.
[0621] Embodiment T6. The method of embodiment T3, wherein the
nucleotide having a 3' protecting group comprises phosphate.
[0622] Embodiment T7. The method of embodiment T6, wherein the
nucleotide having a 3' protecting group comprises triphosphate.
[0623] Embodiment T8. The method of embodiment T1, wherein R2 has a
molecular weight of at least 36.
[0624] Embodiment T9. The method of embodiment T1, wherein R2
comprises a fluorine nucleus.
[0625] Embodiment T10. The method of embodiment T1, wherein R2
comprises at least two carbon nuclei.
[0626] Embodiment T11. The method of embodiment T1, wherein R2
comprises a carbon-carbon double bond.
[0627] Embodiment T12. The method of embodiment T1, wherein R2
comprises a tertiary carbon.
[0628] Embodiment T13. The method of embodiment T12, wherein R2
comprises a tert-butyl moiety.
[0629] Embodiment T14. The method of embodiment T1, wherein R2
comprises a cyclic moiety.
[0630] Embodiment T15. The method of embodiment T1, wherein R2
comprises a silicon nucleus.
[0631] Embodiment T16. The method of embodiment T1, wherein R2
comprises a phenyl ring.
[0632] Embodiment T17. The method of embodiment T1, wherein R2
comprises a nitrogen nucleus.
[0633] Embodiment T18. The method of embodiment T1, wherein R2
comprises an oxygen nucleus.
[0634] Embodiment T19. The method of embodiment T1, wherein R2
comprises a hydroxylate moiety.
[0635] Embodiment T20. The method of embodiment T1, wherein the
reagent comprising the structure R2-O--NH.sub.2 is selected from
the group consisting of:
##STR00134##
[0636] Embodiment T21. The method of embodiment T20, wherein the
reagent comprising the structure R2-O--NH.sub.2 is
##STR00135##
[0637] Embodiment T22. The method of embodiment T20, wherein the
reagent comprising the structure R2-O--NH.sub.2 is
##STR00136##
[0638] Embodiment T23. The method of embodiment T1, wherein the
reagent comprising the structure R2-O--NH.sub.2 is selected from
the group consisting of.
##STR00137##
[0639] Embodiment T24. The method of any one of embodiments T8 to
T23, wherein the 3' blocking group comprises 3'-O-Oxime.
[0640] Embodiment T25. The method of any one of embodiments T8 to
T23, wherein the 3' blocking group comprises 3'-O-aldoxime.
[0641] Embodiment T26. The method of any one of embodiments T8 to
T23, wherein the 3' blocking group comprises 3'-O-ketoxime.
[0642] Embodiment T27. The method of any one of embodiments T8 to
T23, wherein the nucleotide having a 3' protecting group comprises
the structure:
##STR00138##
wherein the nucleotide that comprises the 3'-O--NH.sub.2 moiety
comprises the structure:
##STR00139##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0643] Embodiment T28. The method of any one of embodiments T1 to
T27, wherein the method adds a 3' ONH.sub.2 in place of a 3'
protecting group to at least 94% of a population of nucleotides
having a 3' protecting group in no more than 3 hours.
[0644] Embodiment T29. The method of any one of embodiments T1 to
T27, wherein the method adds a 3' ONH.sub.2 in place of a 3'
protecting group to at least 96% of a population of nucleotides
having a 3' protecting group in no more than 3 hours.
[0645] Embodiment T30. The method of any one of embodiments T1 to
T27, wherein the method adds a 3' ONH.sub.2 in place of a 3'
protecting group to at least 98% of a population of nucleotides
having a 3' protecting group in no more than 3 hours.
[0646] Embodiment T31. The method of any one of embodiments T1 to
T27, wherein the method adds a 3' ONH.sub.2 in place of a 3'
protecting group to at least 98% of a population of nucleotides
having a 3' protecting group in no more than 2 hours.
[0647] Embodiment T32. The method of any one of embodiments T1 to
T27, wherein the method adds a 3' ONH.sub.2 in place of a 3'
protecting group to at least 98% of a population of nucleotides
having a 3' protecting group in no more than 1 hour.
[0648] Embodiment T33. The method of any one of embodiments T1 to
T32, wherein the nucleotide having a 3' protecting group comprises
cytosine, and wherein method degrades no more than 6% of cytosine
bases from a population comprising a plurality of said nucleotide
having a 3' protecting group comprising cytosine.
[0649] Embodiment T34. The method of any one of embodiments T1 to
T32, wherein the nucleotide having a 3' protecting group comprises
cytosine, and wherein method degrades no more than 5% of cytosine
bases from a population comprising a plurality of said nucleotide
having a 3' protecting group comprising cytosine.
[0650] Embodiment T35. The method of any one of embodiments T1 to
T32, wherein the nucleotide having a 3' protecting group comprises
cytosine, and wherein method degrades no more than 4% of cytosine
bases from a population comprising a plurality of said nucleotide
having a 3' protecting group comprising cytosine.
[0651] Embodiment T36. The method of any one of embodiments T1 to
T32, wherein the nucleotide having a 3' protecting group comprises
cytosine, and wherein method degrades no more than 3% of cytosine
bases from a population comprising a plurality of said nucleotide
having a 3' protecting group comprising cytosine.
[0652] Embodiment T37. The method of any one of embodiments T1 to
T32, wherein the nucleotide having a 3' protecting group comprises
cytosine, and wherein method degrades no more than 2% of cytosine
bases from a population comprising a plurality of said nucleotide
having a 3' protecting group comprising cytosine.
[0653] Embodiment T38. The method of any one of embodiments T1 to
T32, wherein the nucleotide having a 3' protecting group comprises
cytosine, and wherein method degrades no more than 1% of cytosine
bases from a population comprising a plurality of said nucleotide
having a 3' protecting group comprising cytosine.
[0654] Embodiment T39. A composition comprising a population of
cytosine nucleotides wherein at least 94% of the population of
cytosine nucleotides comprise a 3'-O--NH.sub.2, wherein no more
than 8% of the population of cytosine nucleotides comprise a 3'
moiety other than --O--NH.sub.2, and wherein no more than 6% of the
population of cytosine nucleotides comprise a modified cytosine
base.
[0655] Embodiment T40. The composition of embodiment T39, wherein
at least 96% of the population of cytosine nucleotides comprise a
3'-O--NH.sub.2.
[0656] Embodiment T41. The composition of embodiment T39, wherein
at least 98% of the population of cytosine nucleotides comprise a
3'-O--NH.sub.2.
[0657] Embodiment T42. The composition of embodiment T39, wherein
at least 99% of the population of cytosine nucleotides comprise a
3'-O--NH.sub.2.
[0658] Embodiment T43. The composition of embodiment T39, wherein
no more than 6% of the population of cytosine nucleotides comprise
a 3' moiety other than --O--NH.sub.2.
[0659] Embodiment T44. The composition of embodiment T39, wherein
no more than 4% of the population of cytosine nucleotides comprise
a 3' moiety other than --O--NH.sub.2.
[0660] Embodiment T45. The composition of embodiment T39, wherein
no more than 2% of the population of cytosine nucleotides comprise
a 3' moiety other than --O--NH.sub.2.
[0661] Embodiment T46. The composition of embodiment T39, wherein
no more than 1% of the population of cytosine nucleotides comprise
a 3' moiety other than --O--NH.sub.2.
[0662] Embodiment T47. The composition of embodiment T39, wherein
no more than 4% of the population of cytosine nucleotides comprise
a modified cytosine base.
[0663] Embodiment T48. The composition of embodiment T39, wherein
no more than 2% of the population of cytosine nucleotides comprise
a modified cytosine base.
[0664] Embodiment T49. The composition of embodiment T39, wherein
no more than 1% of the population of cytosine nucleotides comprise
a modified cytosine base.
[0665] Embodiment T50. The composition of embodiment T39, wherein
at least 98% of the population of cytosine nucleotides comprise a
3'-O--NH.sub.2, wherein no more than 1% of the population of
cytosine nucleotides comprise a 3' moiety other than --O--NH.sub.2,
and wherein no more than 1% of the population of cytosine
nucleotides comprise a modified cytosine base.
[0666] Embodiment T51. The composition of embodiment T39, wherein
the composition is an aqueous solution.
[0667] Embodiment T52. The composition of embodiment T51, wherein
the pH of the solution is no less than 4 and no greater than 8.
[0668] Embodiment T53. The composition of embodiment T39, wherein
the composition comprises a reagent comprising the structure
R2-O--NH.sub.2.
[0669] Embodiment T54. The composition of embodiment T53, wherein
R2 comprises a molecular weight of at least 24.
[0670] Embodiment T55. The composition of embodiment T53, wherein
R2 comprises a molecular weight of at least 36.
[0671] Embodiment T56. The composition of embodiment T53, wherein
R2 comprises a fluorine nucleus.
[0672] Embodiment T57. The composition of embodiment T53, wherein
R2 comprises at least two carbon nuclei.
[0673] Embodiment T58. The composition of embodiment T53, wherein
R2 comprises a carbon-carbon double bond.
[0674] Embodiment T59. The composition of embodiment T53, wherein
R2 comprises a tertiary carbon.
[0675] Embodiment T60. The composition of embodiment T59, wherein
R2 comprises a tert-butyl moiety.
[0676] Embodiment T61. The composition of embodiment T53, wherein
R2 comprises a cyclic moiety.
[0677] Embodiment T62. The composition of embodiment T53, wherein
R2 comprises a silicon nucleus.
[0678] Embodiment T63. The composition of embodiment T53, wherein
R2 comprises a phenyl ring.
[0679] Embodiment T64. The composition of embodiment T53, wherein
R2 comprises a nitrogen nucleus.
[0680] Embodiment T65. The composition of embodiment T53, wherein
R2 comprises an oxygen nucleus.
[0681] Embodiment T66. The composition of embodiment T53, wherein
R2 comprises a hydroxylated moiety.
[0682] Embodiment T67. The composition of embodiment T53, wherein
the reagent comprising the structure R2-O--NH.sub.2 is selected
from the group consisting of:
##STR00140##
[0683] Embodiment T68. The composition of embodiment T53, wherein
the reagent comprising the structure R2-O--NH.sub.2 is
##STR00141##
[0684] Embodiment T69. The composition of embodiment T53, wherein
the reagent comprising the structure R2-O--NH.sub.2 is
##STR00142##
[0685] Embodiment T70. The composition of embodiment T53, wherein
the reagent comprising the structure R2-O--NH.sub.2 is selected
from the group consisting of:
##STR00143##
[0686] Embodiment T71. The composition of any one of embodiments
T39 to T70, wherein the 3' moiety other than --O--NH.sub.2
comprises a 3' O-oxime.
[0687] Embodiment T72. The composition of any one of embodiments
T39 to T70, wherein the 3' moiety other than --O--NH.sub.2
comprises a 3' O-aldoxime.
[0688] Embodiment T73. The composition of any one of embodiments
T39 to T70, wherein the 3' moiety other than --O--NH.sub.2
comprises a 3' O-ketoxime.
[0689] Embodiment T74. The composition of embodiment T39, wherein
the composition comprises a reagent comprising the structure
R2-O--NHCH.sub.3.
[0690] Embodiment T75. The composition of embodiment T39, wherein
the composition comprises a reagent comprising the structure
R2-O--CH.sub.2N.sub.3.
[0691] Embodiment T76. The composition of any one of embodiments
T39 to T70, wherein at least some of the cytosine nucleotides
having a 3' moiety other than --O--NH.sub.2 nucleotide comprise the
structure:
##STR00144##
wherein B is a cytosine nucleobase; R1 is independently halogen,
OCH.sub.3, H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0692] Embodiment T77. A method of formulating a sequencing
reagent, comprising contacting a composition comprising a 3'
protected nucleotide to a reagent comprising a reversible
terminator moiety to produce a composition comprising a nucleotide
comprising a reversible terminator moiety, and formulating the
composition comprising a nucleotide comprising a reversible
terminator moiety as a sequencing reagent, wherein formulating the
composition comprising a reversible terminator moiety does not
comprise further purification of the composition comprising a
reversible terminator moiety.
[0693] Embodiment T78. The method of embodiment T77, further
comprising applying the sequencing reagent to a sequencing reagent
to yield a sequencing run of at least 100 bases having a quality
score of at least Q30.
[0694] Embodiment T79. The method of embodiment T77, wherein the
reagent comprising a reversible terminator moiety has a structure
R2-O--NH.sub.2.
[0695] Embodiment T80. The method of embodiment T79, wherein R2
comprises at least two carbon nuclei, a fluorine nucleus,
carbon-carbon double bond, tertiary carbon, tert-butyl moiety,
cyclic moiety, silicon nucleus, phenyl ring, nitrogen nucleus,
oxygen nucleus, or hydroxylate moiety.
[0696] Embodiment T81. The method of embodiment T80, wherein R2
comprises at least two carbon nuclei, carbon-carbon double bond,
tertiary carbon, tert-butyl moiety, cyclic moiety, or phenyl
ring.
[0697] Embodiment T82. The method of embodiment T81, wherein R2 is
--CH.sub.2CH.sub.3, --CH.sub.2CHCH.sub.2, or
--C(CH.sub.3).sub.3.
[0698] Embodiment T83. The method of embodiment T82, wherein R2 is
--C(CH.sub.3).sub.3.
[0699] Embodiment T84. The method of embodiment T79, wherein R2 has
a molecular weight of at least 24.
[0700] Embodiment T85. The method of embodiment T84, wherein R2 has
a molecular weight of at least 36.
[0701] Embodiment T86. The method of embodiment T84, wherein R2 has
a molecular weight of at least 57.
[0702] Embodiment T87. The method of embodiment T84, wherein R2 has
a molecular weight of at least 100.
[0703] Embodiment T88. The method of embodiment T84, wherein R2 has
a molecular weight of at least 200.
[0704] Embodiment T89. The method of embodiment T84, wherein R2 has
a molecular weight of at least 300.
[0705] Embodiment T90. The method of embodiment T84, wherein R2 has
a molecular weight of at least 400.
[0706] Embodiment T91. The method of embodiment T84, wherein R2 has
a molecular weight of at least 500.
[0707] Embodiment T92. The method of embodiment T84, wherein R2 has
a molecular weight of at least 600.
[0708] Embodiment T93. The method of embodiment T84, wherein R2 has
a molecular weight of at least 700.
[0709] Embodiment T94. The method of embodiment T84, wherein R2 has
a molecular weight of at least 800.
[0710] Embodiment T95. The method of embodiment T84, wherein R2 has
a molecular weight of at least 900.
[0711] Embodiment T96. The method of embodiment T79, wherein R2
comprises at least two carbon nuclei.
[0712] Embodiment T97. The method of embodiment T79, wherein R2
comprises a fluorine nucleus, silicon nucleus, nitrogen nucleus,
oxygen nucleus, or hydroxylate moiety.
[0713] Embodiment T98. The method of embodiment T79, wherein the 3'
protected nucleotide comprises a 3'-O-oxime moiety.
[0714] Embodiment T99. The method of embodiment T98, wherein the
oxime is an aldoxime.
[0715] Embodiment T100. The method of embodiment T98, wherein the
oxime is a ketoxime.
[0716] Embodiment T101. The method of embodiment T79, wherein the
composition comprising a nucleotide comprising a reversible
terminator moiety comprises no more than 6% of the 3' protected
nucleotide relative to 3' protected nucleotide present
initially.
[0717] Embodiment T102. The method of embodiment T101, wherein the
composition comprising a nucleotide comprising a reversible
terminator moiety comprises no more than 5% of the 3' protected
nucleotide relative to 3' protected nucleotide present
initially.
[0718] Embodiment T103. The method of embodiment T101, wherein the
composition comprising a nucleotide comprising a reversible
terminator moiety comprises no more than 4% of the 3' protected
nucleotide relative to 3' protected nucleotide present
initially.
[0719] Embodiment T104. The method of embodiment T101, wherein the
composition comprising a nucleotide comprising a reversible
terminator moiety comprises no more than 3% of the 3' protected
nucleotide relative to 3' protected nucleotide present
initially.
[0720] Embodiment T105. The method of embodiment T101, wherein the
composition comprising a nucleotide comprising a reversible
terminator moiety comprises no more than 2% of the 3' protected
nucleotide relative to 3' protected nucleotide present
initially.
[0721] Embodiment T106. The method of embodiment T101, wherein the
composition comprising a nucleotide comprising a reversible
terminator moiety comprises no more than 1% of the 3' protected
nucleotide relative to 3' protected nucleotide present
initially.
[0722] Embodiment T107. The method of embodiment T79, wherein the
composition comprising a nucleotide comprising a reversible
terminator moiety comprises degraded or modified dC nucleobases at
a concentration of no more than 6% of total dC nucleobases in the
composition comprising a nucleotide comprising a reversible
terminator moiety.
[0723] Embodiment T108. The method of embodiment T79, wherein the
composition comprising a nucleotide comprising a reversible
terminator moiety comprises degraded or modified dC nucleobases at
a concentration of no more than 5% of total dC nucleobases in the
composition comprising a nucleotide comprising a reversible
terminator moiety.
[0724] Embodiment T109. The method of embodiment T79, wherein the
composition comprising a nucleotide comprising a reversible
terminator moiety comprises degraded or modified dC nucleobases at
a concentration of no more than 4% of total dC nucleobases in the
composition comprising a nucleotide comprising a reversible
terminator moiety.
[0725] Embodiment T110. The method of embodiment T79, wherein the
composition comprising a nucleotide comprising a reversible
terminator moiety comprises degraded or modified dC nucleobases at
a concentration of no more than 3% of total dC nucleobases in the
composition comprising a nucleotide comprising a reversible
terminator moiety.
[0726] Embodiment T111. The method of embodiment T79, wherein the
composition comprising a nucleotide comprising a reversible
terminator moiety comprises degraded or modified dC nucleobases at
a concentration of no more than 2% of total dC nucleobases in the
composition comprising a nucleotide comprising a reversible
terminator moiety.
[0727] Embodiment T112. The method of embodiment T79, wherein the
composition comprising a nucleotide comprising a reversible
terminator moiety comprises degraded or modified dC nucleobases at
a concentration of no more than 1% of total dC nucleobases in the
composition comprising a nucleotide comprising a reversible
terminator moiety.
[0728] Embodiment T113. A method for modifying a nucleotide,
comprising reacting a nucleotide comprising a 3'-O-oxime moiety
with a reagent comprising the structure R2-O--NH.sub.2 to produce a
nucleotide comprising a 3'-O--NH.sub.2 moiety, wherein R2 is
independently alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0729] Embodiment T114. The method of embodiment T113, wherein the
nucleotide that comprises the 3'-O-oxime moiety comprises the
structure:
##STR00145##
wherein the nucleotide that comprises the 3'-O--NH.sub.2 moiety
comprises the structure:
##STR00146##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0730] Embodiment T115. The method of embodiment T113, wherein the
reaction occurs in an aqueous solution.
[0731] Embodiment T116. The method of embodiment T115, wherein the
nucleotide is attached to a solid support in contact with the
aqueous solution.
[0732] Embodiment T117. The method of embodiment T115, wherein the
reagent that comprises the structure R2-O--NH.sub.2 is attached to
a solid support in contact with the aqueous solution.
[0733] Embodiment T118. The method of any one of embodiments T113
to T117, wherein the nucleobase is a purine or pyrimidine.
[0734] Embodiment T119. The method of embodiment T118, wherein the
nucleobase is independently adenine, cytosine, guanine, thymine, or
uracil.
[0735] Embodiment T120. The method of embodiment T118, wherein the
nucleobase is complementary to adenine, cytosine, guanine, thymine,
or uracil.
[0736] Embodiment T121. The method of embodiment T118, wherein the
nucleobase is independently 5-methylcytosine, hypoxanthine,
xanthine, inosine, isoguanine, isocytosine, or 7-deazaguanine.
[0737] Embodiment T122. The method of any one of embodiments T113
to T121, wherein the pH of the solution is no less than 4 and no
greater than 8.
[0738] Embodiment T123. The method of any one of embodiments T113
to T122, wherein the nucleotide that is produced comprises a
deoxynucleotide.
[0739] Embodiment T124. The method of embodiment T123, wherein the
nucleotide that is produced comprises:
##STR00147##
wherein Base is the nucleobase, and wherein DNA is independently a
single stranded deoxyribonucleic acid or double stranded
deoxyribonucleic acid.
[0740] Embodiment T125. The method of any one of embodiments T113
to T124, wherein the reagent is independently one of.
##STR00148##
[0741] Embodiment T126. A solution comprising a nucleotide
comprising a 3'-O-oxime moiety and a reagent comprising the
structure R2-O--NH.sub.2, wherein R2 is independently alkyl,
alkenyl, alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl,
heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl,
(heteroalicyclyl)alkyl, or an optionally substituted variant
thereof.
[0742] Embodiment T127. The solution of embodiment T126, wherein
the nucleotide comprises the structure:
##STR00149##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0743] Embodiment T128. The solution of embodiment T127, wherein R1
is H.
[0744] Embodiment T129. The solution of embodiment T128, wherein Q
is deoxyribonucleic acid.
[0745] Embodiment T130. The solution of embodiment T127, wherein R1
is OH.
[0746] Embodiment T131. The solution of embodiment T130, wherein Q
is ribonucleic acid.
[0747] Embodiment T132. The solution of embodiment T126, wherein
the solution is an aqueous solution.
[0748] Embodiment T133. The solution of embodiment T132, wherein
the nucleotide is attached to a solid support and wherein the solid
support is in contact with the solution.
[0749] Embodiment T134 The solution of embodiment T132, wherein the
reagent that comprises the structure R2-O--NH.sub.2 is attached to
a solid support in contact with the aqueous solution.
[0750] Embodiment T135. The solution of any one of embodiments T126
to T134, wherein the nucleobase is a purine or pyrimidine.
[0751] Embodiment T136. The solution of embodiment T135, wherein
the nucleobase is independently adenine, cytosine, guanine,
thymine, or uracil.
[0752] Embodiment T137. The solution of embodiment T135, wherein
the nucleobase is complementary to adenine, cytosine, guanine,
thymine, or uracil.
[0753] Embodiment T138. The solution of embodiment T135, wherein
the nucleobase is independently 5-methylcytosine, hypoxanthine,
xanthine, inosine, isoguanine, isocytosine, or 7-deazaguanine.
[0754] Embodiment T139. The solution of any one of embodiments T126
to T138, wherein the nucleobase comprises an exogenous label
moiety.
[0755] Embodiment T140. The solution of any one of embodiments T126
to T139, wherein the pH of the solution is no less than 4 and no
greater than 8.
[0756] Embodiment T141. The solution of any one of embodiments T126
to T140, wherein the R2-O--NH.sub.2 molecule is independently one
of:
##STR00150##
[0757] Embodiment T142. A kit comprising a first vessel containing
a nucleotide comprising a 3'-O-oxime moiety and a second vessel
containing a reagent comprising the structure R2-O--NH.sub.2,
wherein R2 is independently alkyl, alkenyl, alkynyl, cycloalkyl,
cycloalkenyl, cycloalkynyl, aryl, heteroaryl, heteroalicyclyl,
aralkyl, heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally
substituted variant thereof.
[0758] Embodiment T143. The kit of embodiment T142, wherein the
nucleotide comprises the structure:
##STR00151##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0759] Embodiment T144. The kit of embodiment T142, wherein the kit
comprises an RFID tag or bar code.
[0760] Embodiment T145. The kit of embodiment T143 or T144, wherein
Q is independently a single stranded deoxyribonucleic acid or
double stranded deoxyribonucleic acid.
[0761] Embodiment T146. The kit of embodiment T145, wherein the
nucleotide is attached to a solid support.
[0762] Embodiment T147. The kit of embodiment T142, wherein the
reagent that comprises the structure R2-O--NH.sub.2 is attached to
a solid support.
[0763] Embodiment T148. The kit of any one of embodiments T142 to
T147, wherein the nucleobase is a purine or pyrimidine.
[0764] Embodiment T149. The kit of embodiment T148, wherein the
nucleobase is independently adenine, cytosine, guanine, thymine, or
uracil.
[0765] Embodiment T150. The kit of embodiment T148, wherein the
nucleobase is complementary to adenine, cytosine, guanine, thymine,
or uracil.
[0766] Embodiment T151. The kit of embodiment T148, wherein the
nucleobase is independently 5-methylcytosine, hypoxanthine,
xanthine, inosine, isoguanine, isocytosine, or 7-deazaguanine.
[0767] Embodiment T152. The kit of any one of embodiments T142 to
T151, wherein the nucleobase comprises an exogenous label
moiety.
[0768] Embodiment T153. The kit of any one of embodiments T142 to
T152, wherein the R2-O--NH.sub.2 molecule is independently one
of:
##STR00152##
Additional Embodiments
[0769] Embodiment 1. A method of replacing a protecting group by a
blocking group at a 3' position of a nucleotide, comprising
contacting the nucleotide and a reagent comprising the structure
R2-block, wherein R2 has a molecular weight of at least 24, wherein
R2-block is water soluble, and wherein block comprises a blocking
group moiety.
[0770] Embodiment 2. The method of embodiment 1, wherein block
comprises a reversible terminator moiety.
[0771] Embodiment 3. The method of embodiment 2, wherein the
reversible terminator moiety does not preclude assembly of a
nucleotide reversibly terminated thereby into a ternary
complex.
[0772] Embodiment 4. The method of any one of embodiments 1 to 3,
wherein R2-block is selected from the group consisting of
R2-O--NH.sub.2, R2-O--NHCH.sub.3, R2-O--NHCOCH.sub.3, or
R2-O--CH.sub.2N.sub.3.
[0773] Embodiment 5. The method of any one of embodiments 1 to 4,
wherein R2-block is R2-O--CH.sub.2N.sub.3.
[0774] Embodiment 6. The method of any one of embodiments 1 to 4,
wherein R2-block is R2-O--NH.sub.2.
[0775] Embodiment 7. The method of any one of embodiments 1 to 4,
wherein the nucleotide comprising a 3' protecting group is:
##STR00153##
wherein the nucleotide comprising a 3' blocking group is:
##STR00154##
wherein B is a nucleobase; R1 is independently halogen, OCH.sub.3,
H or OH, Q is independently monophosphate, diphosphate,
triphosphate, or nucleic acid, and R3 and R4 are independently H,
CH.sub.3, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl,
cycloalkynyl, aryl, heteroaryl, heteroalicyclyl, aralkyl,
heteroaralkyl, (heteroalicyclyl)alkyl, or an optionally substituted
variant thereof.
[0776] Embodiment 8. The method of any one of embodiments 1 to 7,
wherein the protecting group comprises an --O-oxime.
[0777] Embodiment 9. The method of embodiment 8, wherein the
--O-oxime moiety has the formula: --O--N.dbd.C(R3)(R4); and R3 and
R4 are independently hydrogen, substituted or unsubstituted alkyl,
substituted or unsubstituted alkenyl, substituted or unsubstituted
alkynyl, substituted or unsubstituted heteroalkyl, substituted or
unsubstituted cycloalkyl, substituted or unsubstituted
cycloalkenyl, substituted or unsubstituted cycloalkynyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl, substituted or unsubstituted heteroalicyclyl,
substituted or unsubstituted aralkyl, substituted or unsubstituted
heteroaralkyl, or substituted or unsubstituted
(heteroalicyclyl)alkyl.
[0778] Embodiment 10. The method of embodiment 9, wherein R3 is
Hydrogen.
[0779] Embodiment 11. The method of any one of embodiments 9 to 10,
wherein R4 is Hydrogen.
[0780] Embodiment 12. The method of any one of embodiments 1 to 11,
wherein the nucleotide comprises a triphosphate moiety.
[0781] Embodiment 13. The method of any one of embodiments 1 to 4,
wherein R2 is --C(R6)(R7)(R8), --C(O)(R6), --P(O)(R6)(R7),
--C(R6)(.dbd.C(R7)(R8)), --Si(R6)(R7)(R8), --S O.sub.2(R6),
substituted or unsubstituted cycloalkyl, substituted or
unsubstituted cycloalkenyl, substituted or unsubstituted
cycloalkynyl, substituted or unsubstituted heteroalicyclyl,
substituted or unsubstituted aryl, substituted or unsubstituted
heteroaryl; and
R6, R7, and R8 are independently hydrogen, halogen, --CCl.sub.3,
--CBr.sub.3, --CF.sub.3, --CI.sub.3, --CHCl.sub.2, --CHBr.sub.2,
--CHF.sub.2, --CHI.sub.2, --CH.sub.2Cl, --CH.sub.2Br, --CH.sub.2F,
--CH.sub.2I, --CN, --OH, --NH.sub.2, --COOH, --CONH.sub.2,
--NO.sub.2, --SH, --SO.sub.3H, --SO.sub.4H, --SO.sub.2NH.sub.2,
--NHNH.sub.2, --ONH.sub.2, --NHC(O)NHNH.sub.2, --NHC(O)NH.sub.2,
--NHSO.sub.2H, --NHC(O)H, --NHC(O)OH, --NHOH, --OCCl.sub.3,
--OCF.sub.3, --OCBr.sub.3, --OCI.sub.3, --OCHCl.sub.2,
--OCHBr.sub.2, --OCHI.sub.2, --OCHF.sub.2, --OCH.sub.2Cl,
--OCH.sub.2Br, --OCH.sub.2I, --OCH.sub.2F, --SF.sub.5, --N.sub.3,
substituted or unsubstituted alkyl, substituted or unsubstituted
alkenyl, substituted or unsubstituted alkynyl, substituted or
unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl,
substituted or unsubstituted cycloalkenyl, substituted or
unsubstituted cycloalkynyl, substituted or unsubstituted aryl,
substituted or unsubstituted heteroaryl, substituted or
unsubstituted heteroalicyclyl, substituted or unsubstituted
aralkyl, substituted or unsubstituted heteroaralkyl, or substituted
or unsubstituted (heteroalicyclyl)alkyl.
[0782] Embodiment 14. The method of embodiment 13, wherein R2 is
--C(R6)(R7)(R8) and R6, R7, and R8 are not all hydrogen.
[0783] Embodiment 15. The method of embodiment 13, wherein R2 is
not unsubstituted methyl.
[0784] Embodiment 16. The method of embodiment 13, wherein R2 is
not unsubstituted ethyl.
[0785] Embodiment 17. The method of embodiment 13, wherein R2 is
not unsubstituted propyl.
[0786] Embodiment 18. The method of embodiment 13, wherein R2-block
has a formula selected from the group consisting of the
following:
##STR00155##
[0787] Embodiment 19. The method of embodiment 18, wherein R2-block
has a formula selected from the group consisting of:
##STR00156##
[0788] Embodiment 20. The method of embodiment 19, wherein R2-block
has a formula
##STR00157##
[0789] Embodiment 21. The method of embodiment 19, wherein R2-block
has a formula
##STR00158##
[0790] Embodiment 22. The method of embodiment 19, wherein R2-block
has a formula
##STR00159##
[0791] Embodiment 23. The method of embodiment 13, wherein R2
comprises a hydroxylated moiety.
[0792] Embodiment 24. The method of embodiment 13, wherein R2
comprises at least two carbon nuclei, a fluorine nucleus,
carbon-carbon double bond, tertiary carbon, tert-butyl moiety,
cyclic moiety, silicon nucleus, phenyl ring, nitrogen nucleus,
oxygen nucleus, or hydroxylate moiety.
[0793] Embodiment 25. The method of embodiment 13, wherein R2 has a
molecular weight of no more than 280 g/mol.
[0794] Embodiment 26. The method of embodiment 13, wherein R2 has a
molecular weight of no more than 100 g/mol.
[0795] Embodiment 27 The method of any one of embodiments 1 to 4,
wherein the method adds a blocking group in place of a 3'
protecting group to at least 94% of a population of nucleotides
having a 3' protecting group in no more than 3 hours at 25.degree.
C.
[0796] Embodiment 28. The method of embodiment 27, wherein the
method adds a blocking group in place of a 3' protecting group to
at least 98% of a population of nucleotides having a 3' protecting
group in no more than 3 hours at 25.degree. C.
[0797] Embodiment 29. The method of embodiment 28, wherein the
method degrades no more than 6% of cytosine nucleobases in a
population of nucleotides each comprising a cytosine
nucleobase.
[0798] Embodiment 30. The method of embodiment 29, wherein the
method degrades no more than 1% of cytosine bases from the
population of nucleotides having a cytosine nucleobase.
[0799] Embodiment 31. The method of any one of embodiments 1 to 19,
wherein the method adds a blocking group in place of a 3'
protecting group to at least 94% of a population of nucleotides
having a 3' protecting group in no more than 3 hours at 25.degree.
C.
[0800] Embodiment 32. The method of embodiment 31, wherein the
method adds a blocking group in place of a 3' protecting group to
at least 98% of a population of nucleotides having a 3' protecting
group in no more than 3 hours at 25.degree. C.
[0801] Embodiment 33. The method of embodiment 32, wherein the
method degrades no more than 6% of cytosine nucleobases in a
population of nucleotides each comprising a cytosine
nucleobase.
[0802] Embodiment 34. The method of embodiment 33, wherein the
method degrades no more than 1% of cytosine nucleobases from the
population of nucleotides having a cytosine nucleobase.
EXAMPLES
[0803] Example 1. This example demonstrates the efficiency of
nucleotide deblocking using various alkoxyamine reagents and
inertness of the deblocking reagents to unwanted modification and
degradation of nucleotide structure. Each aminooxy deblocking
reagent was formulated as a 3.2M solution in water, and the pH was
adjusted to 5.25 with 10M sodium hydroxide. Each of these aminooxy
solutions was then mixed 1:1 with a 100 mM solution of dCTP oxime
triphosphate and incubated at 25 C for 1 hr. At this time, the
reaction was diluted 50-fold with 10% isopropyl methyl ketone in
50% isopropanol/water. Percent consumption and degradation was
analyzed by HPLC using a Phenomenex Gemini C18 column (5 .mu.m
NX-X18 110 .ANG., LC Column 250.times.4.6 mm).
[0804] FIG. 1A shows a plot of percent nucleotide degradation vs.
percent consumption of 3' oxime for dCTP when treated with various
candidate reagents. The results indicate that when reacting
dCTP-3'-oxime with reagents having structure R2-ONH.sub.2, the
species having the R2 moiety of --CH.sub.3, --CH.sub.2CH.sub.3,
--CH.sub.2CHCH.sub.2, or --C(CH.sub.3).sub.3 produce a higher yield
of 3'-ONH.sub.2 nucleotide compared to reaction with hydroxylamine.
The candidate reagent having R2 moiety of --C(CH.sub.3).sub.3
produced the highest yield of 3'-ONH.sub.2 nucleotide.
[0805] The results of FIG. 1A showed that the species having the R2
moiety of --CH.sub.3, --CH.sub.2CHCH.sub.2, or --C(CH.sub.3).sub.3
yielded substantially less unwanted degradation of the nucleotide
compared to treatment with hydroxylamine. Species having the R2
moiety of --CH.sub.2CH.sub.3 demonstrated a degradation level that
was roughly comparable to treatment with hydroxylamine. The reagent
having R2 moiety of --C(CH.sub.3).sub.3 demonstrated the lowest
level of unwanted nucleotide degradation among the reagents tested
in FIG. 1A.
[0806] Example 2. The composition of FIG. 1 produced through the
use of tert-butyl-O--NH.sub.2 was used to formulate a sequencing
reagent without further purification. The sequencing reagent, which
further comprised a polymerase was used in a sequencing run, and a
read quality of at least Q30 was observed for at least 100 bases of
the read.
[0807] This example demonstrates that practice of the method herein
produced nucleotide compositions that are suitable to be formulated
into sequencing reagents without further purification, such as
sequencing reagents comprising a polymerase, surfactant, or other
reagent suitable for a sequencing reaction.
[0808] Throughout this application various publications, patents
and/or patent applications have been referenced. The disclosures of
these documents in their entireties are hereby incorporated by
reference in this application.
[0809] A number of embodiments have been described. Nevertheless,
it will be understood that various modifications may be made.
Accordingly, other embodiments are within the scope of the
following claims.
* * * * *