U.S. patent application number 17/229406 was filed with the patent office on 2021-10-14 for tagged nucleotides useful for nanopore detection.
This patent application is currently assigned to Roche Sequencing Solutions, Inc.. The applicant listed for this patent is Roche Sequencing Solutions, Inc.. Invention is credited to Ashwini Bhat, Peter Crisalli, Dmitriy Gremyachinskiy, Andrew Trans.
Application Number | 20210317521 17/229406 |
Document ID | / |
Family ID | 1000005669475 |
Filed Date | 2021-10-14 |
United States Patent
Application |
20210317521 |
Kind Code |
A1 |
Gremyachinskiy; Dmitriy ; et
al. |
October 14, 2021 |
TAGGED NUCLEOTIDES USEFUL FOR NANOPORE DETECTION
Abstract
The present disclosure relates to compounds comprising a
negatively-charged polymer moiety which is capable of entering a
nanopore and upon entering a nanopore in the presence of positive
ions results in an increased flow of the positive ions through the
nanopore. The present disclosure provides methods of preparing the
compounds and for their use as nanopore-detectable tags, in
particular, for nanopore-based nucleic acid detection and
sequencing.
Inventors: |
Gremyachinskiy; Dmitriy;
(San Francisco, CA) ; Crisalli; Peter; (Sunnyvale,
CA) ; Trans; Andrew; (Mountain View, CA) ;
Bhat; Ashwini; (Milpitas, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Roche Sequencing Solutions, Inc. |
Pleasanton |
CA |
US |
|
|
Assignee: |
Roche Sequencing Solutions,
Inc.
Pleasanton
CA
|
Family ID: |
1000005669475 |
Appl. No.: |
17/229406 |
Filed: |
April 13, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16825086 |
Mar 20, 2020 |
11008613 |
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17229406 |
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15684726 |
Aug 23, 2017 |
10669580 |
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16825086 |
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62380059 |
Aug 26, 2016 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07H 19/10 20130101;
C07H 19/06 20130101; C07H 21/04 20130101; C07K 14/00 20130101; C12Q
1/6823 20130101; C12Q 1/6869 20130101 |
International
Class: |
C12Q 1/6869 20060101
C12Q001/6869; C12Q 1/6823 20060101 C12Q001/6823; C07H 21/04
20060101 C07H021/04; C07H 19/10 20060101 C07H019/10; C07H 19/06
20060101 C07H019/06; C07K 14/00 20060101 C07K014/00 |
Claims
1. A method for determining the sequence of a nucleic acid
comprising: (a) providing a nanopore sequencing composition
comprising: (a1) a membrane with a cis side and a trans side, (a2)
an electrode on the cis side and the trans side of the membrane,
(a3) an electrolyte solution comprising positive ions in contact
with both electrodes, (a4) a nanopore comprising a channel
extending through the membrane, wherein the channel provide a path
through which the positive ions may flow across the membrane, (a5)
a set of tagged nucleoside-5'-oligophosphate (N5OP) disposed in the
electrolyte solution, wherein at least one of the tagged N5OPs is
covalently linked to a tag that comprises a negatively-charged
polymer moiety that is capable of increasing the flow of the
positive ions through the channel, and (a6) an active polymerase
situated adjacent to the nanopore and complexed with a primed
strand of the nucleic acid, wherein the active polymerase
catalytically incorporates the tagged N5OPs into a complementary
strand of the primed nucleic acid, wherein the tag of the N5OP
incorporated into the complementary strand enters into the nanopore
channel and changes the flow of the positive ions through the
channel of the pore; (b) detecting the different flows of positive
ions resulting from the entry of the different tags in the nanopore
over time and correlating to each of the different compounds
incorporated by the polymerase which are complementary to the
nucleic acid sequence, and thereby determining the nucleic acid
sequence.
2. The method of claim 1, wherein the negatively-charged polymer
comprises a covalently linked chain of from 20 to 50 monomer units
selected from the monomer unit structures of formula (1a), (1 b),
(1c), (1d), (1e), (1f), (1g), (1 h), (1 i), (1 j), (1 k), (1 l), (1
m), (2a), (2b), (2c), (3a), (3b), (3c), and any combination thereof
##STR00094## ##STR00095##
3. The method of claim 2, wherein the monomer units are selected
from the group consisting of the structures of formula (2a), (2b),
(2c), (3a), (3b), (3c), and any combination thereof.
4. The method of claim 1, wherein the increased flow of the
positive ions results in a measured current across the nanopore
that is greater than an open channel (O.C.) current.
5. The method of claim 4, wherein the increased flow of the
positive ions results in a measured current across the nanopore
that is at least 5% greater than O.C. current.
6. The method of claim 1, wherein the negatively-charged polymer
moiety has an overall negative charge of from (-25) to (-50).
7. The method of claim 1, wherein the negatively-charged polymer
moiety comprises a covalently linked chain of from 25 to 40 monomer
units.
8. The method of claim 1, wherein the tagged N50Ps have the
structure of formula I: N--P-L-T (I) wherein: N is a nucleoside; P
is an oligophosphate covalently attached to a 5'-O group of the
nucleoside, wherein the oligophosphate consists of 3 to 12
phosphate groups; L is a linker covalently attached to a terminal
phosphate group of the oligophosphate; and T is the tag, wherein
the tag is covalently attached to the linker.
9. The method of claim 1, wherein the tagged N5OP comprising the
negatively-charged polymer moiety is a compound of structural
formula (II) ##STR00096## Wherein, Base is selected from adenosine,
cytidine, guanosine, thymidine, and uridine; R is selected from H
and OH; N is from 1 to 4; Linker is a linker comprising a
covalently bonded chain of 2 to 100 atoms; and Tag is the tag.
10. The method of claim 9, wherein the linker comprises a chemical
group selected from the group consisting of: ester, ether,
thioether, amine, amide, imide, carbonate, carbamate, squarate,
thiazole, thiazolidine, hydrazone, oxime, triazole,
dihydropyridazine, phosphodiester, polyethylene glycol (PEG), and
any combination thereof.
11. The method of claim 1, wherein the tagged N5OP comprising the
negatively-charged polymer moiety is a compound of structural
formula (III) ##STR00097## Wherein, Base is selected from
adenosine, cytidine, guanosine, thymidine, and uridine; R is
selected from H and OH; N is from 1 to 4; L.sub.B-X-L.sub.A is the
linker, wherein (a) L.sub.A and L.sub.B each independently
comprises a chemical moiety selected from the group consisting of:
linear (C.sub.1-C.sub.12) alkyl, linear (C.sub.1-C.sub.12) alkene,
linear (C.sub.1-C.sub.12) alkyne, ester, ether, thioether, amine,
amide, imide, carbonate, carbamate, squarate, thiazole,
thiazolidine, hydrazone, oxime, triazole, dihydropyridazine,
phosphodiester, polyethylene glycol (PEG), and combinations
thereof; and (b) X comprises a chemical moiety selected from the
group consisting of ester, ether, thioether, amine, amide, imide,
carbonate, carbamate, squarate, thiazole, thiazolidine, hydrazone,
oxime, triazole, and dihydropyridazine; and Tag is the tag.
12. The method of claim 1, wherein the tagged N5OP comprising the
negatively-charged polymer moiety is a compound of structural
formula (IIIa) ##STR00098## wherein, Base is selected from
adenosine, cytidine, guanosine, thymidine, and uridine; R is
selected from H and OH; N is from 1 to 4; P is from 2 to 10; and
Tag is the tag comprising the negatively-charged polymer
moiety.
13. The method of claim 12, wherein R=H, n=4, and p=5.
14. a composition having the structure of formula I: N--P-L-T (I)
wherein: N is a nucleoside; P is an oligophosphate covalently
attached to a 5'-O group of the nucleoside, wherein the
oligophosphate consists of 3 to 12 phosphate groups; L is a linker
covalently attached to a terminal phosphate group of the
oligophosphate; and T is a tag covalently attached to the linker,
wherein the tag comprises a negatively-charged polymer moiety which
is capable of entering a nanopore and upon entering a nanopore in
the presence of positive ions results in an increased flow of the
positive ions through the nanopore.
15. The composition of claim 14, wherein the composition is a
compound of structural formula (II) ##STR00099## wherein, Base is
selected from adenosine, cytidine, guanosine, thymidine, and
uridine; R is selected from H and OH; N is from 1 to 4; Linker is a
linker comprising a covalently bonded chain of 2 to 100 atoms; and
Tag is the tag.
16. The composition of claim 14, wherein the linker comprises a
chemical group selected from the group consisting of: ester, ether,
thioether, amine, amide, imide, carbonate, carbamate, squarate,
thiazole, thiazolidine, hydrazone, oxime, triazole,
dihydropyridazine, phosphodiester, polyethylene glycol (PEG), and
any combination thereof.
17. The composition of claim 14, wherein the composition is a
compound of structural formula (III) ##STR00100## wherein, Base is
selected from adenosine, cytidine, guanosine, thymidine, and
uridine; R is selected from H and OH; N is from 1 to 4;
L.sub.B-X-L.sub.A is the linker, wherein (a) L.sub.A and L.sub.B
each independently comprises a chemical moiety selected from the
group consisting of: linear (C.sub.1-C.sub.12) alkyl, linear
(C.sub.1-C.sub.12) alkene, linear (C.sub.1-C.sub.12) alkyne, ester,
ether, thioether, amine, amide, imide, carbonate, carbamate,
squarate, thiazole, thiazolidine, hydrazone, oxime, triazole,
dihydropyridazine, phosphodiester, polyethylene glycol (PEG), and
combinations thereof; and (b) X comprises a chemical moiety
selected from the group consisting of ester, ether, thioether,
amine, amide, imide, carbonate, carbamate, squarate, thiazole,
thiazolidine, hydrazone, oxime, triazole, and dihydropyridazine;
and Tag is the tag.
18. The composition of claim 14, wherein the composition is a
compound of structural formula (IIIa) ##STR00101## Wherein, Base is
selected from adenosine, cytidine, guanosine, thymidine, and
uridine; R is selected from H and OH; N is from 1 to 4; P is from 2
to 10; and Tag is the tag comprising the negatively-charged polymer
moiety.
19. The composition of claim 18, wherein R=H, n=4, and p=5.
20. The composition of claim 14, wherein the negatively-charged
polymer comprises a covalently linked chain of from 20 to 50
monomer units selected from the monomer unit structures of formula
(2a), (2b), (2c), (3a), (3b), (3c), and any combination thereof
##STR00102##
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 16/825,086, filed Mar. 20, 2020, which is a
continuation of U.S. patent application Ser. No. 15/684,726, filed
Aug. 23, 2017, which issued as U.S. Pat. No. 10,669,580 on Jun. 2,
2020, which claims priority to U.S. Provisional Application No.
62/380,059, filed Aug. 26, 2016, each of which is incorporated
herein in their entirety by reference.
FIELD
[0002] This application relates to compounds comprising a
negatively-charged polymer moiety which is capable of entering a
nanopore and upon entering a nanopore in the presence of positive
ions results in an increased flow of the positive ions through the
nanopore. The present disclosure provides methods of preparing the
compounds and for their use as nanopore-detectable tags, in
particular, for nanopore-based nucleic acid detection and
sequencing.
REFERENCE TO SEQUENCE LISTING
[0003] The official copy of the Sequence Listing is submitted
concurrently with the specification as an ASCII formatted text file
with a file name of "04338-542WO1_SL_ST25.txt", a creation date of
Aug. 11, 2017, and a size of 10,771 bytes. The Sequence Listing
filed herewith is part of the specification and is incorporated in
its entirety by reference herein.
BACKGROUND
[0004] Numerous methods for using nanopores to detect nucleic acids
(e.g., DNA) or other molecules are known in the art. One common
method involves applying an electric field across the nanopore to
induce the nucleic acid to enter and partially block the nanopore,
and measuring the current level and duration of the current
blockage as the molecule rapidly enters and translocates through
the pore. Both the current level and the duration of the blockage
can reveal information about the molecule (typically, a polymeric
molecule such as DNA). This type of nanopore detection method has
also been carried out using polymeric polyethylene glycol (PEG)
molecules and the length was of the polymer was found to affect
both the current level and dwell time. See e.g., Joseph W. F.
Robertson, Claudio G. Rodrigues, Vincent M. Stanford, Kenneth A.
Rubinson, Oleg V. Krasilnikov, and John J. Kasianowicz, Proc.
Nat'l. Acad. Sci. USA, 104; 8207 (2007).
[0005] Another method of observing a molecule using a nanopore is
to attach a bulky moiety to the molecule so that it cannot pass, or
cannot quickly pass, through the pore. An example is the use of the
relatively bulky protein streptavidin that tightly binds biotin and
Biotin can easily be covalently attached to DNA. With the DNA held
between the pull of the electric field and the bulky protein, it
can remain in a fixed position in the nanopore long enough to make
an accurate measurement of pore current (milliseconds to seconds).
It can then be released (e.g. by turning off or reversing the
electric field) and the pore used again for another measurement. In
addition to streptavidin, other proteins and molecules can be used
as translocation blockers. For instance, antibodies which bind
specific ligands or enzymes like DNA polymerase can be used. Even
double-stranded DNA may be too large to pass through
.alpha.-hemolysin pores, and it too can be used to hold DNA (or
other polymers) in a fixed position in a nanopore under the pull of
an electric field.
[0006] Nucleic acid sequencing is the process for determining the
nucleotide sequence of a nucleic acid. Such sequence information
may be helpful in diagnosing and/or treating a subject. For
example, the sequence of a nucleic acid of a subject may be used to
identify, diagnose, and potentially develop treatments for genetic
diseases. As another example, research into pathogens may lead to
treatment for contagious diseases. Since some diseases are
characterized by as little as one nucleotide difference in a chain
of millions of nucleotides, highly accurate sequencing is
essential.
[0007] Single-molecule sequencing-by-synthesis (SBS) techniques
using nanopores have been developed. See e.g., US Pat. Publ. Nos.
2013/0244340 A1, 2013/0264207 A1, 2014/0134616 A1. Nanopore SBS
involves using a DNA polymerase (or other strand-extending enzyme)
to synthesize a DNA strand complementary to a target sequence
template and concurrently determining the identity of each
nucleotide monomer as it is added to the growing strand, thereby
determining the target sequence. Each added nucleotide monomer is
detected by monitoring signals due to ion flow through a nanopore
located adjacent to the polymerase active site over time as the
strand is synthesized. Obtaining an accurate signal requires proper
positioning of the polymerase active site near a nanopore, and the
use of a tag on each added nucleotide which can enter the nanopore
and provide an identifiable change in the ion flow through the
pore. It also requires controlling the parameters of DNA polymerase
strand extension reaction, including nucleotide monomer on-rate,
processivity, transition rate, and overall read length. In order to
provide for accurate nanopore sequencing, it is important for the
tag to enter and reside in the nanopore for a sufficient amount of
time (i.e., "dwell time"), and while residing in the nanopore,
provide for a sufficiently detectable, and identifiable signal
associated with the ion flow through the nanopore, such that the
specific nucleotide associated with the tag can be distinguished
unambiguously from the other tagged nucleotides.
[0008] Kumar et al., (2012) "PEG-Labeled Nucleotides and Nanopore
Detection for Single Molecule DNA Sequencing by Synthesis,"
Scientific Reports, 2:684; DOI: 10.1038/srep00684, describes using
a nanopore to distinguish four different length PEG-coumarin tags
attached via a terminal 5'-phosphoramidate to a dG nucleotide, and
separately demonstrates efficient and accurate incorporation of
these four PEG-coumarin tagged dG nucleotides by DNA polymerase.
See also, US Patent Application Publications US 2013/0244340 A1,
published Sep. 19, 2013, US 2013/0264207 A1, published Oct. 10,
2013, and US 2014/0134616 A1, published May 14, 2014.
[0009] WO 2013/154999 and WO 2013/191793 describe the use of tagged
nucleotides for nanopore SBS, and disclose the possible use of a
single nucleotide attached to a single tag comprising branched PEG
chains.
[0010] WO 2015/148402 describes the use of tagged nucleotides for
nanopore SBS comprising a single nucleotide attached to a single
tag, wherein the tag comprises any or a range of oligonucleotides
(or oligonucleotide analogues) that have lengths of 30 monomer
units or longer.
[0011] The above-described prior disclosures teach tagged
nucleotide structures having a single nucleotide moiety attached to
a single tag, or a branched tag. The general approach of these
disclosures is to increase the size and structural variability of
the tag and thereby facilitate better nanopore detection for SBS.
The increased size these prior disclosed tagged nucleotides however
creates a further obstacle to their utility for SBS by decreasing
the substrate concentrations that can be achieved.
[0012] The above-described prior disclosures fail to teach specific
tagged nucleotide structures that can provide high enough substrate
concentrations to drive the polymerase extension reaction at rates
desirable for efficient SBS, particularly in a nanopore setting
where solution volumes are minimal and molecular concentrations
critical. Accordingly, there remains a need for tagged nucleotide
compositions and methods that can be used to improve efficiency and
throughput in nanopore SBS and other sequencing techniques.
SUMMARY
[0013] The present disclosure provides compounds comprising a
negatively-charged polymer moiety capable of entering a nanopore
and upon entering the nanopore in the presence of positive ions
results in an increased flow of the positive ions through the
nanopore. Because of their ability to increase the flow of positive
ions through a nanopore the compounds can be used as
nanopore-detectable tags. Moreover, the ability of the compounds to
increase the flow of positive ions provides for nanopore detectable
signals that are above the "open channel" ("O.C.") current signal
of a nanopore with no tag present, or the "blocking" or "blockade"
signals that are below the O.C. current signal, and which is
typical of known nanopore-detectable tags. This ability of the ion
amplifying compounds of the present disclosure to provide
nanopore-detectable signals above O.C. current allows for improved
sensitivity and dynamic range for detection, and thereby increased
accuracy in nanopore detection and sequencing applications.
[0014] Accordingly, the present disclosure also provides tagged
nucleotide compounds comprising a nucleoside-5'-oligophosphate
moiety capable of being a substrate for a polymerase and a tag,
wherein the tag comprises a negatively-charged polymer moiety
capable of entering a nanopore and upon entering the nanopore in
the presence of positive ions results in an increased flow of the
positive ions through the nanopore. The ion flow amplifying tagged
nucleotide compounds of the present disclosure are well-suited for
use in any nucleic acid sequencing-by-synthesis system that
utilizes tagged nucleotides as polymerase substrates and identifies
the unknown sequence by nanopore detection of the tagged
by-products of the polymerase extension reaction. Additionally, the
present disclosure also provides processes for preparing and using
such ion flow amplifying compounds, the tagged nucleotide
compounds, and methods for using these compounds in nanopore
detection and nanopore sequencing of nucleic acids.
[0015] In some embodiments, the present disclosure provides a
compound comprising a negatively-charged polymer moiety which is
capable of entering a nanopore and upon entering a nanopore in the
presence of positive ions results in an increased flow of the
positive ions through the nanopore. In some embodiments, the
compound further comprises a nucleoside-5'-oligophosphate moiety
capable of being a substrate for a polymerase covalently linked to
the polymer moiety.
[0016] In some embodiments, the disclosure provides a compound of
structural formula (I)
N--P-L-T (I)
wherein, N is a nucleoside; P is an oligophosphate covalently
attached to a 5'-O group of the nucleoside, wherein the
oligophosphate consists of 3 to 12 phosphate groups; L is a linker
covalently attached to a terminal phosphate group of the
oligophosphate; and T is a tag covalently attached to the linker,
wherein the tag comprises a negatively-charged polymer moiety which
is capable of entering a nanopore and upon entering a nanopore in
the presence of positive ions results in an increased flow of the
positive ions through the nanopore.
[0017] In some embodiments, the compound of formula (I) has a
structural formula (II)
##STR00001##
wherein, Base is selected from adenosine, cytidine, guanosine,
thymidine, and uridine; R is selected from H and OH; n is from 1 to
4; Linker is a linker comprising a covalently bonded chain of 2 to
100 atoms; and Tag is a negatively-charged polymer moiety which is
capable of entering a nanopore and upon entering a nanopore in the
presence of positive ions results in an increased flow of the
positive ions through the nanopore. In some embodiments, of the
compounds of formula (I) or (II), the linker comprises a chemical
group selected from the group consisting of: ester, ether,
thioether, amine, amide, imide, carbonate, carbamate, squarate,
thiazole, thiazolidine, hydrazone, oxime, triazole,
dihydropyridazine, phosphodiester, polyethylene glycol (PEG), and
any combination thereof.
[0018] In some embodiments, the compound of formula (II) has a
structural formula (III)
##STR00002##
wherein, Base is selected from adenosine, cytidine, guanosine,
thymidine, and uridine; R is selected from H and OH; n is from 1 to
4; L.sub.B-X-L.sub.A is the linker, wherein (a) L.sub.A and L.sub.B
each independently comprises a chemical moiety selected from the
group consisting of: linear (C.sub.1-C.sub.12) alkyl, linear
(C.sub.1-C.sub.12) alkene, linear (C.sub.1-C.sub.12) alkyne, ester,
ether, thioether, amine, amide, imide, carbonate, carbamate,
squarate, thiazole, thiazolidine, hydrazone, oxime, triazole,
dihydropyridazine, phosphodiester, polyethylene glycol (PEG), and
combinations thereof; and (b) X comprises a chemical moiety
selected from the group consisting of ester, ether, thioether,
amine, amide, imide, carbonate, carbamate, squarate, thiazole,
thiazolidine, hydrazone, oxime, triazole, and dihydropyridazine;
and Tag is a negatively-charged polymer moiety which is capable of
entering a nanopore and upon entering a nanopore in the presence of
positive ions results in an increased flow of the positive ions
through the nanopore.
[0019] In some embodiments, the compound of structural formula
(III) has a structural formula (IIIa)
##STR00003##
[0020] wherein, Base is selected from adenosine, cytidine,
guanosine, thymidine, and uridine; R is selected from H and OH; n
is from 1 to 4; p is from 2 to 10; and Tag is a negatively-charged
polymer moiety which is capable of entering a nanopore and upon
entering a nanopore in the presence of positive ions results in an
increased flow of the positive ions through the nanopore. In some
embodiment, the compound of structural formula (IIIa) is a compound
wherein R=H, n=4, and p=5.
[0021] Generally, the ion-flow amplifying compounds of the present
disclosure share the feature of a negatively-charged polymer moiety
which is capable of entering a nanopore and upon entering a
nanopore in the presence of positive ions results in an increased
flow of the positive ions through the nanopore. The present
disclosure provides a range of structural and functional parameters
that characterize the negatively-charged polymer moiety of the
compounds disclosed herein.
[0022] In some embodiments, the negatively-charged polymer moiety
comprises a covalently linked chain of from 150 to 900 atoms with a
negatively charged group attached to an atom of the chain at an
interval of every 3 to 20 atoms, optionally an interval of every 3
to 10 atoms. In some embodiments, the covalently linked chain of
from 150 to 900 atoms comprises chemical groups selected from
phosphodiester, peptide, ester, ether, alkyl, alkenyl, alkynyl,
perfluorinated alkyl, and any combination thereof. In some
embodiments, the negatively-charged polymer moiety has a molecular
length of between about 80 angstroms and about 250 angstroms and a
molecular diameter of less than about 15 angstroms, optionally a
molecular diameter of less than about 12 angstroms, or optionally a
molecular diameter of less than about 10 angstroms. In some
embodiments, the negatively-charged polymer moiety has at least one
negative charge per 10 angstroms of molecular length, optionally at
least one negative charge per 7.5 angstroms of molecular length, or
at least one negative charge per 3.5 angstroms of molecular length.
In some embodiments, the negatively-charged polymer moiety has an
overall negative charge of from (-25) to (-50), optionally an
overall negative charge of from (-30) to (-40), or optionally an
overall negative charge of from (-31) to (-37).
[0023] In some embodiments, the negatively-charged polymer moiety
comprises a covalently linked chain of from 20 to 50 monomer units,
optionally from 25 to 40 monomer units, or optionally from 27 to 35
monomer units. In some embodiments, the monomer units comprise a
negatively-charged group attached to a covalently linked chain of
from 3 to 20 atoms, optionally a chain of from 3 to 10 atoms. In
some embodiments, the monomer units comprise covalent links
selected from the group consisting of phosphodiester, peptide,
ester, ether, alkyl, alkenyl, alkynyl, perfluoroalkyl, and any
combination thereof. In some embodiments, the monomer units
comprise a negatively charged group selected from the group
consisting of phosphate, sulfate, carboxylate, and any combination
thereof. In some embodiments, the monomer units are selected from
the group consisting of the structures of formula (1a), (1b), (1c),
(1d), (1e), (1f), (1g), (1h), (1i), (1j), (1k), (1l), (1m), (2a),
(2b), (2c), (3a), (3b), (3c), and any combination thereof
##STR00004## ##STR00005##
[0024] In some embodiments, the negatively-charged polymer moiety
comprises an oligonucleotide of formula (A).sub.n, wherein A is a
monomer unit independently selected from the monomer unit
structures of formula (1a), (1b), (1c), (1d), (1e), (1f), (1g),
(1h), (1i), (1j), (1k), (1l), and (1m), and n is from 10 to 40,
optionally n is from 20 to 40, n is from 25 to 35, or n is from 27
to 35. In some embodiments, the negatively-charged polymer moiety
comprises an oligonucleotide of formula
(A).sub.m-(B).sub.n-(C).sub.p-(D).sub.q, wherein A, B, C, and D,
are monomer units independently selected from the monomer unit
structures of formula (1a), (1b), (1c), (1d), (1e), (1f), (1g),
(1h), (1i), (1j), (1k), (1l), and (1m), and each of m, n, p, and q
is from 0 to 40, and m+n+p+q is from 10 to 40, optionally m+n+p+q
is from 20 to 40, m+n+p+q is from 25 to 35, or m+n+p+q is from 27
to 35.
[0025] In some embodiments, the negatively-charged polymer moiety
comprises an oligonucleotide selected from the group consisting of:
-TT-(SpC2).sub.28- (SEQ ID NO: 1), -TT-(SpC3).sub.28- (SEQ ID NO:
2), -(SpC3).sub.31- (SEQ ID NO: 3), -TT-(dSp).sub.26-TT- (SEQ ID
NO: 4), -TT-(SpC4-F4).sub.28- (SEQ ID NO: 5),
-TT-(SpC7-Pra2).sub.28- (SEQ ID NO: 6),
-(SpC2).sub.8-T.sub.6-(SpC2).sub.16- (SEQ ID NO: 12),
-(SpC2).sub.8-(N3CEdT).sub.7-(SpC2).sub.15- (SEQ ID NO: 13),
-(SpC2).sub.6-TT-(BHEB)-T-(SpC2).sub.20-(SEQ ID NO: 14,
-(SpC2).sub.9-T-(BHEB).sub.2-T-(SpC2).sub.17- (SEQ ID NO: 15), and
-(SpC2).sub.8-(S500).sub.3-(SpC2).sub.17- (SEQ ID NO: 16).
[0026] In some embodiments, the increased ion flow induced by the
negatively-charged polymer moiety results in a measured current
across the nanopore that is greater than O.C. current, optionally
at least 5% greater than O.C. current, at least 10% greater than
O.C. current, at least 25% greater than O.C. current, or at least
50% greater than O.C. current.
[0027] In some embodiments, the present disclosure also provides
compositions comprising at least one of the ion-flow amplifying
compounds. In some embodiments, the composition comprises a set of
compounds, wherein each compound of the set comprises a different
tag which results in a different flow of positive ions through a
nanopore when the tag enters the nanopore, and at least one of the
compounds of the set is a compound comprising a negatively-charged
polymer moiety (as provided herein) which is capable of entering a
nanopore and upon entering a nanopore in the presence of positive
ions results in an increased flow of the positive ions through the
nanopore, and thereby resulting in a measured current across the
nanopore that is greater than O.C. current. In one embodiment of
the composition, the set of compounds comprises the tagged
nucleotide compounds dA6P-(Linker)-T.sub.30-C3;
dC6P-(Linker)-TT-(dSp).sub.26-TT-C3;
dG6P-(Linker)-TT-(SpC2).sub.28-biotin;
dT6P-(Linker)-TT-(SpC3).sub.28-biotin; wherein, "(Linker)" refers
to a linker of formula (XVd)
##STR00006##
[0028] The present disclosure also provides methods of using the
ion-flow amplifying compounds. Accordingly, in some embodiments,
the disclosure provides method for determining the sequence of a
nucleic acid comprising: (a) providing a nanopore sequencing
composition comprising: a membrane, an electrode on the cis side
and the trans side of the membrane, a nanopore with its pore
extending through the membrane, an electrolyte solution comprising
positive ions in contact with both electrodes, an active polymerase
situated adjacent to the nanopore, and a primer strand complexed
with the polymerase; (b) contacting the nanopore sequencing
composition with (i) a strand of the nucleic acid; and (ii) a set
of compounds each comprising a different
nucleoside-5'-oligophosphate moiety covalently linked to a tag,
wherein each member of the set of compounds has a different tag
which results in a different flow of positive ions through a
nanopore when the tag enters the nanopore, and at least one of the
different tags comprises a negatively-charged polymer moiety which
upon entering a nanopore in the presence of positive ions results
in an increased flow of the positive ions through the nanopore; and
(c) detecting the different flows of positive ions resulting from
the entry of the different tags in the nanopore over time and
correlating to each of the different compounds incorporated by the
polymerase which are complementary to the nucleic acid sequence,
and thereby determining the nucleic acid sequence.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1 depicts a plot of a series of ion flow amplifying tag
signals (above open channel current) detected over time in a
sequencing by synthesis experiment using a nanopore array device
comprising Pol6 polymerase conjugated to an .alpha.-hemolysin
nanopore as described in Example 2.
DETAILED DESCRIPTION
[0030] For the descriptions herein and the appended claims, the
singular forms "a", and "an" include plural referents unless the
context clearly indicates otherwise. Thus, for example, reference
to "a protein" includes more than one protein, and reference to "a
compound" refers to more than one compound. The use of "comprise,"
"comprises," "comprising" "include," "includes," and "including"
are interchangeable and not intended to be limiting. It is to be
further understood that where descriptions of various embodiments
use the term "comprising," those skilled in the art would
understand that in some specific instances, an embodiment can be
alternatively described using language "consisting essentially of"
or "consisting of."
[0031] Where a range of values is provided, unless the context
clearly dictates otherwise, it is understood that each intervening
integer of the value, and each tenth of each intervening integer of
the value, unless the context clearly dictates otherwise, between
the upper and lower limit of that range, and any other stated or
intervening value in that stated range, is encompassed within the
invention. The upper and lower limits of these smaller ranges may
independently be included in the smaller ranges, and are also
encompassed within the invention, subject to any specifically
excluded limit in the stated range. Where the stated range includes
one or both of the limits, ranges excluding (i) either or (ii) both
of those included limits are also included in the invention. For
example "1 to 50" includes "2 to 25", "5 to 20", "25 to 50", "1 to
10", etc.
[0032] It is to be understood that both the foregoing general
description, including the drawings, and the following detailed
description are exemplary and explanatory only and are not
restrictive of this disclosure.
Definitions
[0033] "Nucleic acid," as used herein, refers to a molecule of one
or more nucleic acid subunits which comprise one of the
nucleobases, adenine (A), cytosine (C), guanine (G), thymine (T),
and uracil (U), or variants thereof. Nucleic acid can refer to a
polymer of nucleotides (e.g., dAMP, dCMP, dGMP, dTMP), also
referred to as a polynucleotide or oligonucleotide, and includes
DNA, RNA, in both single and double-stranded form, and hybrids
thereof.
[0034] "Nucleotide," as used herein refers to a
nucleoside-5'-oligophosphate compound, or structural analog of a
nucleoside-5'-oligophosphate, which is capable of acting as a
substrate or inhibitor of a nucleic acid polymerase. Exemplary
nucleotides include, but are not limited to,
nucleoside-5'-triphosphates (e.g., dATP, dCTP, dGTP, dTTP, and
dUTP); nucleosides (e.g., dA, dC, dG, dT, and dU) with
5'-oligophosphate chains of 4 or more phosphates in length (e.g.,
5'-tetraphosphosphate, 5'-pentaphosphosphate, 5'-hexaphosphosphate,
5'-heptaphosphosphate, 5'-octaphosphosphate); and structural
analogs of nucleoside-5'-triphosphates that can have a modified
base moiety (e.g., a substituted purine or pyrimidine base), a
modified sugar moiety (e.g., an O-alkylated sugar), and/or a
modified oligophosphate moiety (e.g., an oligophosphate comprising
a thio-phosphate, a methylene, and/or other bridges between
phosphates).
[0035] "Nucleoside," as used herein, refers to a molecular moiety
that comprises a naturally occurring or non-naturally occurring
nucleobase attached to a sugar moiety (e.g., ribose or
deoxyribose).
[0036] "Oligophosphate," as used herein, refers to a molecular
moiety that comprises an oligomer of phosphate groups. For example,
an oligophosphate can comprise an oligomer of from 2 to 20
phosphates, an oligomer of from 3 to 12 phosphates, an oligomer of
from 3 to 9 phosphates.
[0037] "Polymerase," as used herein, refers to any natural or
non-naturally occurring enzyme or other catalyst that is capable of
catalyzing a polymerization reaction, such as the polymerization of
nucleotide monomers to form a nucleic acid polymer. Exemplary
polymerases that may be used in the compositions and methods of the
present disclosure include the nucleic acid polymerases such as DNA
polymerase (e.g., enzyme of class EC 2.7.7.7), RNA polymerase
(e.g., enzyme of class EC 2.7.7.6 or EC 2.7.7.48), reverse
transcriptase (e.g., enzyme of class EC 2.7.7.49), and DNA ligase
(e.g., enzyme of class EC 6.5.1.1).
[0038] "Moiety," as used herein, refers to part of a molecule.
[0039] "Linker," as used herein, refers to any molecular moiety
that provides a bonding attachment with some space between two or
more molecules, molecular groups, and/or molecular moieties.
[0040] "Tag," as used herein, refers to a moiety or part of a
molecule that enables or enhances the ability to detect and/or
identify, either directly or indirectly, a molecule or molecular
complex, which is coupled to the tag. For example, the tag can
provide a detectable property or characteristic, such as steric
bulk or volume, electrostatic charge, electrochemical potential,
optical and/or spectroscopic signature.
[0041] "Nanopore," as used herein, refers to a pore, channel, or
passage formed or otherwise provided in a membrane or other barrier
material that has a characteristic width or diameter of about 1
angstrom to about 10,000 angstroms. A nanopore can be made of a
naturally-occurring pore-forming protein, such as .alpha.-hemolysin
from S. aureus, or a mutant or variant of a wild-type pore-forming
protein, either non-naturally occurring (i.e., engineered) such as
.alpha.-HL-C46, or naturally occurring. A membrane may be an
organic membrane, such as a lipid bilayer, or a synthetic membrane
made of a non-naturally occurring polymeric material. The nanopore
may be disposed adjacent or in proximity to a sensor, a sensing
circuit, or an electrode coupled to a sensing circuit, such as, for
example, a complementary metal-oxide semiconductor (CMOS) or field
effect transistor (FET) circuit.
[0042] "Nanopore-detectable tag" as used herein refers to a tag
that can enter into, become positioned in, be captured by,
translocate through, and/or traverse a nanopore and thereby result
in a detectable change in current through the nanopore. Exemplary
nanopore-detectable tags include, but are not limited to, natural
or synthetic polymers, such as polyethylene glycol,
oligonucleotides, polypeptides, carbohydrates, peptide nucleic acid
polymers, locked nucleic acid polymers, any of which may be
optionally modified with or linked to chemical groups, such as dye
moieties, or fluorophores, that can result in detectable nanopore
current changes.
[0043] "Ion flow," as used herein, refers to the movement of ions,
typically in a solution, due to an electromotive force, such as the
potential between an anode and a cathode. Ion flow typically can be
measured as current or the decay of an electrostatic potential.
[0044] "Ion flow amplifying," or "ion flow enhancing," as used
herein in the context of nanopore detection, refers to the
characteristic of resulting in an increase in ion flow through a
nanopore greater than the ion flow through the nanopore in its
"open channel" (O.C.) state.
[0045] "Open channel current," "O.C. current," or "Background
current" as used herein refers to the current level measured across
a nanopore when a potential is applied and the nanopore is open
(e.g., no tag is present in the nanopore).
[0046] "Tag current" as used herein refers to the current level
measured across a nanopore when a potential is applied and a tag is
present the nanopore. Typically, the presence of a tag in a
nanopore results in a blocking of ion flow through the nanopore and
a result decrease in measure tag current level. The ion flow
amplifying tags of the present disclosure, however, when present in
a nanopore result in an increased ion flow indicated in a tag
current level measured across a nanopore greater than O.C.
current.
[0047] "Dwell time" as used herein in the context of capture of a
tag in a nanopore refers to the time that the tag spends in the
nanopore as detected by a tag current.
[0048] "Molecular length," as used herein, refers to the average
length of a molecule (or a moiety) at its longest axial dimension
when the molecule (or moiety) is in its average solution
conformation.
[0049] "Molecular diameter," as used herein, refers to the average
length of a molecule (or a moiety) at its the shortest axial
dimension when the molecule (or moiety) is in its average solution
conformation.
[0050] "Overall charge," as used herein, refers to the sum of the
positively and negatively charged chemical groups that make up a
molecule (or a moiety). For example, a tag moiety comprising a
polymer of 30 monomer units with 30 phosphodiester groups (each of
which has a charge of (-1) at pH 7) is a negatively-charged polymer
with an overall charge of (-30).
[0051] "Negatively-charged," as used herein in the context of a tag
moiety refers to a tag moiety having an overall charge that is
negative.
Detailed Description of Various Embodiments
[0052] Overview: Ion-Flow Amplifying Tags and Nanopore
Sequencing
[0053] The present disclosure describes compositions of ion flow
amplifying tagged nucleotide compounds and related methods,
devices, and systems that are useful for nanopore detection and
sequencing of nucleic acids. The ion flow amplifying tagged
nucleotide compounds can be used in methods to accurately detect
individual nucleotide incorporation by a nucleic acid polymerase
into a growing strand that is complementary to a template nucleic
acid strand.
[0054] Generally, nanopore-based nucleotide acid sequencing uses a
mixture of four nucleotide analogs (e.g., dA6P, dC6P, dG6P, and
dT6P) that can be incorporated by an enzyme into a growing strand.
Each nucleotide analog has a covalently attached tag moiety that
provides an identifiable, and distinguishable signature when
detected with a nanopore. The strand extending enzyme (e.g., DNA
polymerase) specifically binds the tagged nucleotide compound that
is complimentary to a template nucleic acid strand which is
hybridized to the growing nucleic acid strand at its active site.
The strand extending enzyme then catalytically couples (i.e.,
incorporates) the complimentary nucleotide moiety of the tagged
nucleotide compound to the end of the growing nucleic acid strand.
Completion of the catalytic incorporation event results in the
release of the tag moiety and the oligophosphate moiety (minus the
one phosphate incorporated into the growing strand) which then
passes through the adjacent nanopore. Even before it undergoes
catalytic process that releases it from the incorporated nucleotide
however, the tag moiety of a tagged nucleotide compound enters the
pore of the nanopore under an applied potential and thereby alters
the background positive ion flow through the nanopore. Generally,
the presence of a tag moiety in a nanopore results in decreasing
(or blocking) the flow of positive ions through the nanopore. This
"blocking current" is detected as signal that is a percentage of
(or below) the "open channel" (or "O.C.") current resulting from
positive ion flow through the nanopore with no tag moiety present.
To date, various molecular properties of tag moieties have been
modified (e.g., mass, volume, 3-D structure, electrostatic charge)
and found to affect the interaction with the pore, thereby allowing
for nanopore detection to distinguish different tag moieties each
of which can correspond to a different nucleotide. A variety of
nanopore systems and methods for using them to detect tagged
molecules including tagged nucleotides for nucleic acid sequencing
are known in the art. See, for example, U.S. patent application
Ser. No. 12/308,091, Ju et al., filed May 18, 2009; U.S. patent
application Ser. No. 13/994,431, Ju et al., filed Jun. 14, 2013; US
Patent Application Publications US 2013/0244340 A1, published Sep.
19, 2013, US 2013/0264207 A1, published Oct. 10, 2013, and US
2014/0134616 A1, published May 14, 2014; PCT Appl. No.
PCT/US13/35635, Ju et al., filed Apr. 8, 2013; and PCT Appl. No.
PCT/US13/35640, Ju et al., filed Apr. 8, 2013, PCT International
Publication No. WO2015/148402, US Provisional Patent Appl. Nos.
62/235,551, filed Sep. 30, 2015, and 62/216,634, filed Sep. 10,
2015, each of which is hereby incorporated herein by reference in
its entirety.
[0055] All prior known tag moieties used for nanopore detection,
however, result in a "blocking" current signal resulting from a
decrease in positive ion flow through the nanopore. The surprising
result provided by the present disclosure is that certain tag
moiety structures comprising negatively-charged polymers are
capable of entering a nanopore and upon entering the nanopore in
the presence of positive ions result in an increased flow of the
positive ions through the nanopore. These ion flow amplifying tag
moiety structures provide a whole new range nanopore detectable
signals that occur above the open channel current and below
"blocking" signal range for detection known in the art.
[0056] Tagged Nucleotide Compound Structures
[0057] The present disclosure provides ion flow amplifying
compounds useful as tags, and ion flow amplifying tagged compound
embodiments (e.g., tagged nucleotides) that can be characterized by
a range of structures and sub-structures. Generally, the ion flow
amplifying tagged compound of the present disclosure comprises a
negatively-charged polymer moiety which is capable of entering a
nanopore and upon entering a nanopore in the presence of positive
ions results in an increased flow of the positive ions through the
nanopore.
[0058] Generally, the ion flow amplifying tagged nucleotide
compound of the present disclosure comprises an ion flow amplifying
tag moiety covalently linked to a nucleoside-5'-oligophosphate
moiety, wherein the tag moiety comprises a negatively-charged
polymer moiety which is capable of entering a nanopore and upon
entering a nanopore in the presence of positive ions results in an
increased flow of the positive ions through the nanopore. In some
embodiments, the ion flow amplifying tagged nucleotide comprises an
ion flow amplifying compound (e.g., ion flow amplifying tag) and
further comprises a nucleoside-5'-oligophosphate moiety capable of
being a substrate for a polymerase covalently linked to the polymer
moiety
[0059] As described elsewhere herein, the ion flow amplifying tag
moieties of the present disclosure result in technical advantages
including increased dynamic range and sensitivity for nanopore
detection of the tagged compounds (e.g., tagged nucleotides) due to
their ability to result in a measured current signal that is
greater than the O.C. signal. In some embodiments, the increased
ion flow results in an increased measured current across a nanopore
that is at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 75%, 90%, or
greater than the O.C. signal across the nanopore.
[0060] Although the present disclosure describes numerous
embodiments where the ion flow amplifying tag moieties of the
present disclosure can be used in tagged nucleotide compounds for
nanopore based sequencing-by-synthesis (SBS) methods, it is also
contemplated that the ion flow amplifying tag moieties can be
conjugated to other types of compounds and used as tags in any
method that involves nanopore detection of a tagged compound. It is
contemplated that any of assay using nanopore detection of a tagged
compound could be easily adapted to use an ion flow amplifying tag
moiety. Thus, the ordinary artisan can use the synthesis methods
disclosed herein to prepare ion flow amplifying tagged nucleotide
compounds to prepare other tagged compound using the ion amplifying
tag moiety structures disclosed herein.
[0061] In some embodiments, the present disclosure provides an ion
flow amplifying tagged nucleotide compound of structural formula
(I)
N--P-L-T (I)
wherein, N is a nucleoside; P is an oligophosphate covalently
attached to a 5'-O group of the nucleoside, wherein the
oligophosphate consists of 3 to 12 phosphate groups; L is a linker
covalently attached to a terminal phosphate group of the
oligophosphate; and T is a tag covalently attached to the linker,
wherein the tag comprises a negatively-charged polymer moiety which
is capable of entering a nanopore and upon entering a nanopore in
the presence of positive ions results in an increased flow of the
positive ions through the nanopore.
[0062] The nucleoside (N) can be any nucleoside capable of being
incorporated by a strand-extending enzyme, such as a polymerase,
when the nucleoside is covalently coupled to an oligophosphate (P),
such as a triphosphate. The nucleoside can comprise a naturally
occurring or non-naturally occurring nucleobase, and a naturally
occurring or non-naturally occurring sugar moiety, such as a ribose
or deoxyribose group. In some embodiments, the nucleobase is
selected from group consisting of adenosine, cytidine, guanosine,
thymidine, and uridine. The sugar moiety should provide a free
hydroxyl group at a position (e.g., a 3'-OH group) that can form a
phosphodiester bond with a growing polynucleotide strand when
catalytically incorporated by a strand extending enzyme. The
nucleoside sugar moiety should also provide a group allowing
covalent attachment of an oligophosphate moiety (e.g., a 5'-O
group).
[0063] In some embodiments, the present disclosure provides an ion
flow amplifying tagged nucleotide compound of structural formula
(II)
##STR00007##
wherein, Base is selected from adenosine, cytidine, guanosine,
thymidine, and uridine; R is selected from H and OH; n is from 1 to
4; Linker is a linker comprising a covalently bonded chain of 2 to
100 atoms; and Tag is a tag comprising a negatively-charged polymer
moiety which is capable of entering a nanopore and upon entering a
nanopore in the presence of positive ions results in an increased
flow of the positive ions through the nanopore.
[0064] In some embodiments, the nucleobase ("Base") can be any
naturally or non-naturally occurring (e.g., chemically modified)
base which is capable of being incorporated by a strand-extending
enzyme, such as a polymerase. In some embodiments, the nucleobase
is selected from group consisting of adenosine, cytidine,
guanosine, thymidine, and uridine.
[0065] The oligophosphate (P) moiety of the ion flow amplifying
tagged nucleotide compounds can be any oligophosphate which, when
attached to the 5'-O of the nucleoside, allows the resulting
nucleotide to still be capable of being incorporated by a
strand-extending enzyme, such as a polymerase. Generally,
strand-extending enzymes, such as polymerase, are capable of
incorporating nucleotides comprising oligophosphates having chains
of from 3 to 12 phosphate groups. Accordingly, in an ion flow
amplifying tagged nucleotide compound of the present disclosure
(e.g., the compound of structural formula (I) or (II)) the
oligophosphate (P) group can comprise 3 to 12 phosphate groups.
[0066] As depicted in in the compound of structural formula (II),
the oligophosphate of 3 to 12 phosphate groups would be represented
by values of n=1 to n=10. Thus, in some embodiments of the present
disclosure, the ion flow amplifying tagged nucleotide compound
comprises an oligophosphate (P) group comprising 3 to 9 phosphate
groups (or n=1 to 7 for formula (II)). In some embodiments, the
oligophosphate group comprises 4 to 6 phosphate groups (or n=2 to 4
for formula (II)). In some embodiments, the oligophosphate group
comprises 6 phosphate groups (or n=4 for formula (II)).
[0067] In other embodiments, the ion flow amplifying tagged
nucleotide compounds of the present disclosure can comprise
oligophosphate chains of 4 to 20 phosphates, 4 to 12 phosphates, 4
to 9 phosphates, 4 to 6 phosphates, wherein the chain is attached
at the 5' position of the nucleoside (e.g., 5'-tetraphosphate,
5'-pentaphosphate, 5'-hexaphosphate, 5'-heptaphosphate,
5'-octaphosphate, 5'-nonaphosphate, 5'-decaphosphate, etc.).
[0068] It is further contemplated that the ion flow amplifying
tagged nucleotide compounds of the present disclosure, can include
oligophosphate moieties comprising modified phosphate groups,
phosphate analogs, or other non-phosphate chemical groups, provided
that the inclusion of such phosphate groups does not prevent the
resulting ion flow amplifying tagged nucleotide from being
incorporated by a strand-extending enzyme when the oligophosphate
is attached to the 5'-O of the nucleoside. Typically, incorporation
by a strand-extending enzyme requires a naturally occurring
phosphate group at the a-position and a phosphodiester bond between
the .alpha.-position and .beta.-positions of the oligophosphate.
Thus, in some embodiments, the oligophosphate can comprise a
thiophosphate group. Additionally, it is contemplated that the
oligophosphate can include an oligomer of phosphate or
phosphate-analog groups with one or more non-phosphate groups, such
as a methylene, and/or a bridging group between two or more
phosphate groups.
[0069] Linkers
[0070] It is contemplated that a wide range of linkers can be used
to covalently couple the ion flow amplifying tag moieties of the
present disclosure to a compound desired to be tagged for nanopore
detection (e.g., tagged nucleotide compound). Generally, the linker
can comprise any molecular moiety that is capable of providing a
covalent coupling and a spacing or structure between the compound
and the tag moiety that is desired for the particular nanopore
detection method. Such linker parameters can be routinely
determined by the ordinary artisan using methods known in the
art.
[0071] In some embodiments, the desired spacing or structure can be
selected and optimized for the specific use of the ion flow
amplifying tagged nucleotide compound. For example, a linker can be
selected that provides a spacing that allows the ion flow
amplifying tag moiety to enter and reside in the nanopore when any
one of the multiple tagged nucleotides forms a ternary complex with
an adjacent polymerase. Generally, the negatively charged polymer
structure of the ion flow amplifying tag moiety should adopt a
narrow and elongated rod-like conformation such that it capable of
entering the nanopore having its length approximately spanning the
length of the nanopore. Depending on how the polymerase is coupled
to the nanopore, a linker of a slightly shorter or longer length
may be selected so as to allow the ion flow amplifying tag moiety
to attain this nanopore spanning position (and provide the optimal
ion flow enhancement associate signal) when the tagged nucleotide
forms a proper ternary complex at the polymerase active site.
[0072] Generally, the linkers useful with the ion flow amplifying
tagged nucleotide compounds of the present disclosure (e.g.,
compounds of formulas (I) and (II)) comprise a covalently bonded
chain of 2 to 100 atoms. In some embodiments, the linker chain of 2
to 100 atoms comprises one or more chemical moieties selected from
the group consisting of: linear (C.sub.1-C.sub.12) alkyl, linear
(C.sub.1-C.sub.12) alkene, linear (C.sub.1-C.sub.12) alkyne, ester,
ether, thioether, amine, amide, imide, carbonate, carbamate,
squarate, thiazole, thiazolidine, hydrazone, oxime, triazole,
dihydropyridazine, phosphodiester, polyethylene glycol (PEG), and
combinations thereof. A variety of linkers comprising a range of
chemical moieties that are useful in the ion flow amplifying tagged
compounds of the present disclosure are described and exemplified
herein.
[0073] Typically, in the ion flow amplifying tagged nucleotide
embodiments of the present disclosure, the linker is formed during
the preparation of a compound of structural formula (I) or (II), in
a chemical reaction that covalent couples the terminal phosphate
(or phosphate analog) of the oligophosphate moiety to the tag, or
to a linker moiety that is attached to, or can be covalently
attached to the tag. More specifically, this chemical reaction
typically involves a tag moiety modified with a reactive
linker-forming group and a nucleotide comprising an oligophosphate
moiety, wherein the terminus of the oligophosphate is also modified
with a reactive linker-forming group. This type of linker forming
chemical reaction can be depicted in a tagged nucleotide compound
of formula (III) as in Scheme 1.
##STR00008##
[0074] As depicted in Scheme 1, X.sub.A and X.sub.B are reactive
linker forming groups, and L.sub.A and L.sub.B, are precursor
linker moieties to the finally formed linkers of structure
-L.sub.B-X-L.sub.A- found in the tagged nucleotide compound of
formula (III). Thus, X.sub.A and X.sub.B are chemical moieties
which are capable of undergoing a chemical reaction that results in
a covalent coupling between one of the multiple nucleotide and the
tag. The product of each covalent coupling reaction between the
linker forming groups, X.sub.A and X.sub.B, is a linker comprising
a general structure -L.sub.B-X-L.sub.A-. Thus, in some embodiments
of the present disclosure, the linker "L" or "Linker" as in the
compounds of formula (I) and (II) is a linker of structural formula
"-L.sub.B-X-L.sub.A-" as depicted in Scheme 1.
[0075] The chemical moiety, "X" (of the "-L.sub.B-X-L.sub.A-") of
structural formula (III) is the new chemical linker moiety produced
in the linker forming reaction. Often, the name of the particular
chemical group X is used to denote the type of linker, although the
other parts of the linker provided by L.sub.A and L.sub.B may
contribute substantially to the overall structure of the linker. In
some embodiments, the linker comprises a chemical moiety, X,
produced in the linker forming reaction between the linker forming
reagents, X.sub.A and X.sub.B, wherein X is a chemical moiety
selected from the group consisting of ester, ether, thioether,
amine, amide, imide, carbonate, carbamate, squarate, thiazole,
thiazolidine, hydrazone, oxime, triazole, dihydropyridazine,
phosphodiester, and polyethylene glycol (PEG).
[0076] The chemical moieties, L.sub.A and L.sub.B are chemical
groups which can effectively act as linkers or spacers between the
nucleoside-5'-oligophosphate moiety or the tag moiety and their
linker forming reagent groups, X.sub.A and X.sub.B. Typically,
L.sub.A and L.sub.B are chemical moieties that do not react in the
linker forming reaction but which provide additional spacing or
structure for the final formed linker. The L.sub.A and L.sub.B
moieties can be the same or different. In some embodiments, L.sub.A
or L.sub.B can be much longer or shorter than the other, and/or
provide different structural features, for example features that
result in more or less conformational flexibility. Accordingly, in
some embodiments, L.sub.A and L.sub.B moieties useful in the ion
flow amplifying tagged nucleotide compounds of the present
disclosure comprise a covalently bonded chain of 2 to 100 atoms,
and optionally, one or more chemical moieties selected from the
group consisting of: linear (C.sub.1-C.sub.12) alkyl, linear
(C.sub.1-C.sub.12) alkene, linear (C.sub.1-C.sub.12) alkyne, ester,
ether, thioether, amine, amide, imide, carbonate, carbamate,
squarate, thiazole, thiazolidine, hydrazone, oxime, triazole,
dihydropyridazine, phosphodiester, polyethylene glycol (PEG), and
combinations thereof.
[0077] Thus, in some embodiments, the present disclosure provides
an ion flow amplifying tagged nucleotide compound of structural
formula (III)
##STR00009##
[0078] wherein, Base is selected from adenosine, cytidine,
guanosine, thymidine, and uridine; R is selected from H and OH; n
is from 1 to 4; L.sub.B-X-L.sub.A is the linker, wherein (a)
L.sub.A and L.sub.B each independently comprises a chemical moiety
selected from the group consisting of: linear (C.sub.1-C.sub.12)
alkyl, linear (C.sub.1-C.sub.12) alkene, linear (C.sub.1-C.sub.12)
alkyne, ester, ether, thioether, amine, amide, imide, carbonate,
carbamate, squarate, thiazole, thiazolidine, hydrazone, oxime,
triazole, dihydropyridazine, phosphodiester, polyethylene glycol
(PEG), and combinations thereof; and (b) X comprises a chemical
moiety selected from the group consisting of ester, ether,
thioether, amine, amide, imide, carbonate, carbamate, squarate,
thiazole, thiazolidine, hydrazone, oxime, triazole, and
dihydropyridazine; In some embodiments, L.sub.A and L.sub.B; and
Tag is a tag comprising a negatively-charged polymer moiety which
is capable of entering a nanopore and upon entering a nanopore in
the presence of positive ions results in an increased flow of the
positive ions through the nanopore.
[0079] In some embodiments, the characteristic linker moiety X is a
triazole group. Such a triazole group can be formed in a "click"
reaction between an azide linker forming reagent, and an alkyne
linker forming reagent. In addition, it is contemplated that a
linker in a compound of structural formula (III) comprising a
triazole group X can further comprise include a linear alkyl and/or
amide groups on one or both sides of the triazole group.
Accordingly, in one embodiment, the present disclosure provides an
ion flow amplifying tagged nucleotide compound of structural
formula (IIIa)
##STR00010##
[0080] wherein, Base is selected from adenosine, cytidine,
guanosine, thymidine, and uridine; R is selected from H and OH; n
is from 1 to 4; p is from 2 to 10; and Tag is a tag comprising a
negatively-charged polymer moiety which is capable of entering a
nanopore and upon entering a nanopore in the presence of positive
ions results in an increased flow of the positive ions through the
nanopore. In some embodiments of the tagged nucleotide compound of
structural formula (IIIa), the compound comprises the structural
parameters R is H, n=4, and p=5, resulting in the compound of
formula (IIId)
##STR00011##
[0081] Exemplary linker forming groups, X.sub.A and X.sub.B, linker
precursor moieties, L.sub.A and L.sub.B and the resulting linker
that they form, of formula -L.sub.A-X-L.sub.B-, are shown in Table
1, below.
TABLE-US-00001 TABLE 1 R.sub.1--L.sub.A--X--L.sub.B--R.sub.2*
R.sub.1--L.sub.A--X.sub.A* X.sub.B--L.sub.B--R.sub.2* (or
R.sub.1-Linker-R.sub.2)* ##STR00012## (IVa) ##STR00013## (IVb)
##STR00014## (IVc) ##STR00015## (Va) ##STR00016## (Vb) ##STR00017##
(Vc) ##STR00018## (VIa) ##STR00019## (VIb) ##STR00020## (VIc)
##STR00021## (VIIa) ##STR00022## (VIIb) wherein, Z is a suitable
leaving group, e.g., F, Cl, Br, or I ##STR00023## (VIIc)
##STR00024## (VIIIa) ##STR00025## (VIIIb) ##STR00026## (VIIIc)
##STR00027## (IXa) ##STR00028## (IXb) wherein, Z is a suitable
leaving group, e.g., F, Cl, Br, or I. ##STR00029## (IXc)
##STR00030## (Xa) ##STR00031## ##STR00032## (Xc) ##STR00033## (XIa)
##STR00034## (XIb) ##STR00035## (XIc) ##STR00036## (XIIa)
##STR00037## (XIIb) ##STR00038## (XIIc) ##STR00039## (XIIIa)
##STR00040## (XIIIb) ##STR00041## (XIIIc) ##STR00042## (XIVa)
wherein, Z is a suitable leaving group, e.g., --OSu, --OBt, or
--OAt ##STR00043## (XIVb) ##STR00044## (XIVc) ##STR00045## (XVa)
##STR00046## (XVb) ##STR00047## (XVc) ##STR00048## (XVIa)
##STR00049## (XVIb) ##STR00050## (XVIc) ##STR00051## (XVIIa)
##STR00052## (XVIIb) wherein, X.sub.1 and X.sub.2 are atoms
independently selected from C and N; and R.sub.3 is a chemical
group selected from the group consisting of: H, F, Cl, Br, I,
CH.sub.3, CF.sub.3, NH.sub.2, NO.sub.2, OH, C(O)OH, C(O)OCH.sub.3,
C(O)NH.sub.2, linear or branched (C.sub.2-C.sub.5) alkyl, linear or
branched (C.sub.2-C.sub.5) alkenyl, linear or branched
(C.sub.2-C.sub.5) alkynyl, unsubstituted or para-substituted
6-membered aryl ring, and unsubstituted or para-substituted
6-membered heteroaryl ring. ##STR00053## (XVIIc) wherein, X.sub.1
and X.sub.2 are atoms independently selected from C and N; and
R.sub.3 is a chemical group selected from the group consisting of:
H, F, Cl, Br, I, CH.sub.3, CF.sub.3, NH.sub.2, NO.sub.2, OH,
C(O)OH, C(O)OCH.sub.3, C(O)NH.sub.2, linear or branched (C.sub.2-
C.sub.5) alkyl, linear or branched (C.sub.2- C.sub.5) alkenyl,
linear or branched (C.sub.2- C.sub.5) alkynyl, unsubstituted or
para- substituted 6-membered aryl ring, and unsubstituted or para-
substituted 6-membered heteroaryl ring. *R.sub.1 and R.sub.2 are a
tag and nucleoside-5'-oligophosphate, respectively, or R.sub.1 and
R.sub.2 are a nucleoside-5'-oligophosphate and tag,
respectively
[0082] Table 1 exemplifies range of linkers and the corresponding
reactive linker-forming groups that undergo a reaction that results
in the covalent coupling linker. These various linkers and
reactions are well-known in the art. The ordinary artisan will be
able to identify the reagents needed for these reactions and either
synthesize them or obtain them commercially. For example, reagents
for conjugating or cross-linking polypeptide (or proteins) to other
biomolecules can be used as linker forming groups to prepare the
ion flow amplifying tagged nucleotide structures of the present
disclosure. (See e.g., catalog of "crosslinking reagents" available
from Thermo Scientific, USA at www.piercenet.com or Sigma-Aldrich,
USA at www.sigmaaldrich.com). Similarly, terminal phosphate
modified nucleosides and/or reagents for such modification with
azide or alkyne groups (or other linker forming groups) are
commercially available (see e.g., Jena Bioscience Gmbh, Jena,
Germany). Additionally, a wide range of FMOC-protected amino acid
residues modified with azide or alkyne groups (or other linker
forming groups) that can be used in the automated solid-phase
synthesis of polypeptides are commercially available (see e.g.,
AnaSpec, Fremont, Calif., USA).
[0083] It is contemplated that any of the pairs of linker forming
groups of structural formulae (IVa) -(XVIIa) and (IVb)-(XVIIb) can
be used in either configuration in preparing a linker useful in an
ion flow amplifying tagged nucleotide compounds of the present
disclosure (e.g., compound of formula (III)). That is, any of the
linker forming groups, X.sub.A and X.sub.B can be used on either
the tag or the nucleotide, as long as the linker forming groups are
paired to provide the linker reaction forming the linker moiety X.
Thus, any of the linker forming groups of structural formulae (IVa)
-(XVIIa) could be attached to either the tag or the nucleotide, and
the conjugate linker forming group of structural formulae (IVb)
-(XVIIb) would be attached to the other. Thus, the groups R.sub.1
and R.sub.2 as depicted in the linkers of form
R.sub.1-L.sub.A-X-L.sub.B-R.sub.2 in Table 1, can represent either
the tag and the nucleotide, or the nucleotide and the tag,
respectively. Accordingly, in some embodiments, the present
disclosure provides ion flow amplifying tagged nucleotide compounds
of formula (III), wherein the compound comprises a compound of
formula R.sub.1-L.sub.A-X-L.sub.B-R.sub.2, wherein R.sub.1 and
R.sub.2 are the nucleotide and the tag, or R.sub.1 and R.sub.2 are
the tag and the nucleotide, respectively, and -L.sub.A-X-L.sub.B-
comprises a chemical moiety selected from the moieties of
structural formula (IVc) -(XVIIc) in Table 1.
[0084] As described above, the chemical moieties L.sub.A and
L.sub.B which make up the linker can each independently comprise
chemical moieties including linear (C.sub.1-C.sub.12) alkyl, ester,
ether, thioether, amine, amide, imide, carbonate, carbamate,
polyethylene glycol (PEG), and combinations thereof. Similar to the
linker forming groups X.sub.A and X.sub.B, it is contemplated that
any of the chemical moieties L.sub.A and L.sub.B, which make up the
linker, can each independently be used with any of the linker
forming groups, and can be used on either the tag or the
nucleotide. Additionally, it is contemplated that the chemical
moieties L.sub.A and L.sub.B can be the same or different.
[0085] In one embodiment, the linker used in a tagged nucleotide
compound the formula (III) is a linker in formula (XVc) comprising
a triazole group X with moieties L.sub.A and L.sub.B on either side
resulting in the linker of formula (XVd) shown below.
##STR00054##
[0086] In some embodiments of the ion flow amplifying tagged
nucleotide compounds of formula (III), the L.sub.A and L.sub.B
chemical moieties comprise chemical moieties independently selected
from the group consisting of moiety structures of formula (XVIIIa)
-formula (XVIIId) as in Table 2.
TABLE-US-00002 TABLE 2 ##STR00055## (XVIIIa) wherein, n = 1 to 50,
and q, r, and s each independently = 0, 1, 2, or 3; ##STR00056##
(XVIIIb) wherein, n = 1 to 50, and q, r, and s each independently =
0, 1, 2, or 3; ##STR00057## (XVIIIc) wherein, n = 1 to 50, and q,
r, and s each independently = 0, 1, 2, or 3; ##STR00058## (XVIIId)
wherein, n = 1 to 50, and q, r, and s each independently = 0, 1, 2,
or 3.
[0087] Although Scheme 1 depicts the "-L.sub.B-X-L.sub.A-" linker
that is formed as a moiety separate from the tag, it is
contemplated that in some embodiments, the linker can be formed in
a reaction with a linker forming group that can comprise part of
the tag. For example, the tag can comprise an oligonucleotide
wherein the oligonucleotide includes a monomer unit modified with a
propargyl or other alkynyl group which can be covalently coupled to
a desired nucleotide (or nucleotide analog) via an azide-alkyne
"click" reaction. This propargyl group which could also be
considered part of the tag can act as a linker forming group (i.e.,
"X.sub.B") and undergoes a linker forming reaction with a linker
forming group attached to a nucleotide.
[0088] Branched or Dendrimeric Linkers
[0089] In addition to the wide range of linkers having two reactive
ends capable of covalent coupling to a plurality of molecular
moieties, the ion flow amplifying tagged nucleotides of the present
disclosure generally include at least one "branched" or
"dendrimeric" linker, which is a type of linker moiety that has
three or more reactive ends. The use of linkers comprising a
branched or dendrimeric linker moiety facilitate the covalent
coupling of a single tag to two or more nucleotides. The use of
such linkers in tagged nucleotide compounds with improved
properties as polymerase substrates are described in U.S.
provisional appl. 62/342,796, filed May 27, 2016, entitled "Tagged
Multi-Nucleotides Useful For Nucleic Acid Sequencing," which is
hereby incorporated by reference herein.
[0090] Branched or dendrimeric linker moieties capable of providing
three or more reactive ends that can be used in the tagged
nucleotide compounds of the present disclosure are well-known in
the art. See e.g., Shchepinov et al., "Oligonucleotide dendrimers:
synthesis and use as polylabelled DNA probes," Nucleic Acids
Research, 1997, Vol. 25, No. 22, 4447-4454. Branched or dendrimeric
linker moieties providing three or more reactive ends useful in the
compounds of the present disclosure are commercially available from
various vendors of DNA synthesis reagents, e.g., Glen Research
(Virginia, USA; www.glenresearch.com).
[0091] Accordingly, in some embodiments the ion flow amplifying
tagged nucleotide compounds of the present disclosure (e.g.,
structural formula (I) and (II) can comprise a linker, wherein the
linker comprises a branched or dendrimeric moiety capable of
forming covalent linkages with three or more molecular
moieties.
[0092] Exemplary reagents useful for preparing ion flow amplifying
tagged nucleotide compound of the present disclosure wherein the
linker comprises a branched or dendrimeric moiety include the
protected phosphoramidite reagent compounds (19) and (20) shown
below.
##STR00059##
[0093] The branched or dendrimeric phosphoramidite "doubler" and
"trebler" units of compounds (19) and (20) are easily attached to
the end of oligonucleotide chains to generate a linker end on the
oligonucleotide capable of attached 2 or more molecular moieties.
Accordingly, an oligonucleotide comprising natural and/or
non-natural monomer units can be used as a tag for ion flow
amplifying tagged nucleotide.
[0094] In some embodiments of the present disclosure, the ion flow
amplifying tagged nucleotide compound comprises a branched or
dendrimeric "doubler" linker moiety and has a structural formula
(IIIb):
##STR00060##
wherein, "Base" is a naturally occurring or non-naturally occurring
nucleobase; R is selected from H and OH; m is from 2 to 12; p is
from 2-10; and Tag is a tag comprising a negatively-charged polymer
moiety which is capable of entering a nanopore and upon entering a
nanopore in the presence of positive ions results in an increased
flow of the positive ions through the nanopore.
[0095] In some embodiments of the present disclosure, the ion flow
amplifying tagged nucleotide compound comprises a branched or
dendrimeric "trebler" linker moiety and has a structural formula
(IIIc):
##STR00061##
wherein, "Base" is a naturally occurring or non-naturally occurring
nucleobase; R is selected from H and OH; m is from 2 to 12; p is
from 2-10; and Tag is a tag comprising a negatively-charged polymer
moiety which is capable of entering a nanopore and upon entering a
nanopore in the presence of positive ions results in an increased
flow of the positive ions through the nanopore.
[0096] Additionally, the branched or dendrimeric phosphoramidite
"doubler" unit of compound (19) and the "trebler" unit of compound
(20) can be easily combined to create linkers capable of covalent
coupling a single molecular moiety (e.g., a tag) to 4, 6, 8, 9, 12,
or more nucleotides. For example, a tag can be linked to compound
(19) and then compound (20) via standard phosphoramidite synthesis
methods to generate compound (21), which is capable of further
linking to at least six additional molecular moieties, such as six
nucleotides.
##STR00062##
[0097] The three-ended phosphoramidite "doubler" unit of compound
(19) can also be prepared (or commercially obtained) with one DMT
protecting group and one FMOC protecting group. This "doubler" unit
with two different protecting groups can then be used to attach
subsequently two different branched or dendrimeric units. For
example, a "doubler" unit of compound (19) and a "trebler" unit of
compound (20) may be covalently attached in a serial fashion to a
"doubler" unit having DMT and Fmoc protecting groups that was
previously attached to a single tag. Such a combination provides a
single tag with a linker moiety capable of further linking to at
least five additional molecular moieties, such as five
nucleotides.
[0098] The ordinary artisan will immediately recognize that the
branched or dendrimeric phosphoramidite units of compounds (19) and
(20), or other such branched or dendrimeric linker moieties can be
combined in numerous ways to generate ion flow amplifying tagged
nucleotide compounds of the present disclosure.
[0099] Ion Flow Amplifying Tags
[0100] As noted elsewhere herein, prior known tag moieties used for
nanopore detection upon entering a nanopore cause a decrease in
positive ion flow through the nanopore which results in "blocking"
current signal measured across the nanopore that is below the O.C.
signal. The tag moieties of the present disclosure upon entering a
nanopore result in the surprising technical effect of an increased
flow of the positive ions through the nanopore. This increased flow
of positive ions results in a measured tag current signal across
the nanopore that is above the O.C. signal of the nanopore without
any tag present. Thus, the ion flow amplifying tag moieties
disclosed herein do not result in a "blocking" current signal but
an ion flow amplifying tag current signal. This surprising
technical effect of ion flow amplification opens up a whole new
range nanopore detectable signals above the O.C. signal. In this
detection range there generally is less background noise than the
range below O.C. signal. Thus, the ion flow amplifying tags exhibit
the advantages of both increasing the dynamic range to both above
and below the O.C. signal, and providing higher sensitivity due to
lower background noise in detecting the tag current signals in this
range. Accordingly, in some embodiments of the ion flow amplifying
tags useful in the compounds and tagged nucleotides of the present
disclosure, the increased positive ion flow results in a measured
current across the nanopore that is greater than O.C. current,
optionally at least 5% greater than O.C. current, at least 10%
greater than O.C. current, at least 25% greater than O.C. current,
or at least 50% greater than O.C. current.
[0101] Generally, the structures of the ion flow amplifying tags of
the present disclosure comprise a negatively-charged polymer moiety
which is capable of entering a nanopore and upon entering a
nanopore in the presence of positive ions results in an increased
flow of the positive ions through the nanopore. Without intending
to be bound by a proposed mechanism, it is believed that the long,
extended, thin polyanionic structure of such tags is able to
rapidly bind numerous positive ions on the cis side of a nanopore
device and then as the tag is pulled into the nanopore by an
applied potential it effectively delivers the positive ions to the
trans side of the device--more quickly than they can move through
the nanopore by themselves.
[0102] As noted above, the ion flow amplifying tag moieties should
have structures comprising a negatively charged polymer that can
adopt a narrow, elongated, rod-like conformation in aqueous
solution, and which has a molecular length capable of approximately
spanning the length of the nanopore. Generally, a standard DNA
strands has 3.4 nm (or 34 angstroms) per 10 nucleotides, or a
molecular length of about 102 angstroms per 30 nucleotides. The
natural nucleotide T has a molecular diameter of about 18
angstroms, whereas a non-natural abasic nucleotide such as "Sp2,"
"Sp3," "dSp," (also referred to in the oligonucleotide synthesis
art as a "spacer") has a narrower molecular diameter of about 10
angstroms (or less).
[0103] Accordingly, ion flow amplifying tags useful in the
compounds and tagged nucleotides of the present disclosure,
generally comprise a negatively-charged polymer moiety that has a
molecular length of about 80 angstroms to about 250 angstroms, and
a molecular diameter of about 8 angstroms to about 18 angstroms. In
some embodiments, the tagged nucleotides comprise a
negatively-charged polymer moiety that has a molecular length of
between about 80 angstroms and about 250 angstroms, and a molecular
diameter of less than about 15 angstroms, optionally, a molecular
diameter of less than about 12 angstroms, or less than about 10
angstroms.
[0104] The ion flow amplifying tags of the present disclosure have
negative charges distributed regularly along their length to allow
suitably efficient transport of positive ions through the nanopore.
Accordingly, in some embodiments the ion flow amplifying tags
useful in the compounds and tagged nucleotides of the present
disclosure, generally comprise a negatively-charged polymer moiety,
wherein the negatively-charged polymer moiety has at least one
negative charge per 10 angstroms of molecular length, optionally at
least one negative charge per 7.5 angstroms of molecular length, or
at least one negative charge per 3.5 angstroms of molecular
length.
[0105] The density of negative charge on the ion flow amplifying
tag structure can alternatively be described in term of negative
charge per number of atoms in the polymer chain. Thus, in some
embodiments the ion flow amplifying tags useful in the compounds
and tagged nucleotides of the present disclosure comprises a
negatively-charged polymer moiety comprising a covalently linked
chain of from 150 to 900 atoms with a negatively charged group
attached to an atom of the chain at an interval of every 3 to 20
atoms, optionally an interval of every 3 to 10 atoms. In some
embodiments, the covalently linked chain of from 150 to 900 atoms
comprises chemical groups selected from phosphodiester, peptide,
ester, ether, alkyl, alkenyl, alkynyl, perfluorinated alkyl, and
any combination thereof. In some embodiments, the negatively
charged group is selected from group consisting of phosphate,
phosphonate, phosphorothioate, sulfate, sulfonate, carboxylate, and
any combination thereof.
[0106] Generally, the overall negative charge of the ion amplifying
tag can be varied based on the length and charge distribution over
the length of the tag. Such parameters can be varied in order to
alter the tag current signal. In some embodiments of the ion flow
amplifying tags useful in the compounds and tagged nucleotides of
the present disclosure the negatively-charged polymer moiety has an
overall negative charge of from (-20) to (-60), optionally, an
overall negative charge of from (-25) to (-50), optionally, an
overall negative charge of from (-30) to (-40), or optionally an
overall negative charge of from (-31) to (-37).
[0107] The ion flow amplifying tags generally comprise a negatively
charged polymer structure. That polymeric structure can be further
described in terms of the number and type of its constituent
monomer units. Generally, monomer units useful in the negatively
charged polymer moiety of the tags of the present disclosure
comprise a negatively-charged group attached to a covalently linked
chain of from 3 to 20 atoms, optionally a chain of from 3 to 10
atoms. Accordingly, in some embodiments of the ion flow amplifying
tags useful in the compounds and tagged nucleotides of the present
disclosure the negatively-charged polymer moiety comprises a
covalently linked chain of from 10 to 60 monomer units, optionally
from 20 to 50 monomer units, optionally from 25 to 40 monomer
units, or optionally from 27 to 35 monomer units.
[0108] Generally, the monomer units are described in terms of their
covalent linking structure within the polymer moiety. Accordingly,
in some embodiments the monomer units of the polymer moiety
comprise covalent links selected from the group consisting of
phosphodiester, peptide, ester, ether, alkyl, alkenyl, alkynyl,
perfluoroalkyl, and any combination thereof. In some embodiments,
the monomer units comprise a negatively charged group selected from
the group consisting of phosphate, sulfate, carboxylate, and any
combination thereof.
[0109] Specific monomer units useful as constituents of the
negatively charged polymer moiety of the ion flow amplifying tags
of the present disclosure are provided in Table 3. Accordingly, in
some embodiments the ion flow amplifying tags useful in the
compounds and tagged nucleotides of the present disclosure
comprises a negatively-charged polymer moiety comprises monomer
units, wherein the monomer units are independently selected from
the group consisting of the structures of formula (1a), (1b), (1c),
(1d), (1e), (1f), (1g), (1h), (1i), (1j), (1 k), (1l), (1m), (2a),
(2b), (2c), (3a), (3b), (3c), and any combination thereof.
TABLE-US-00003 TABLE 3 Monomer Units ##STR00063## (1a) ##STR00064##
(1b) ##STR00065## (1c) ##STR00066## (1d) ##STR00067## (1e)
##STR00068## (1f) ##STR00069## (1g) ##STR00070## (1h) ##STR00071##
(1i) ##STR00072## (1j) ##STR00073## (1k) ##STR00074## (1l)
##STR00075## (1m) ##STR00076## (2a) ##STR00077## (2b) ##STR00078##
(2c) ##STR00079## (3a) ##STR00080## (3b) ##STR00081## (3c)
[0110] Oligonucleotide Tags
[0111] As noted in the Background and elsewhere herein, a wide
range of oligonucleotide have been prepared and used tag moieties
for nanopore detection. These prior known oligonucleotide tags,
however, are known only to produce tag current signals below O.C.
signal indicating that they result in a decrease in positive ion
flow. The present disclosure provides oligonucleotide tags that are
capable of amplifying ion flow. Generally, the ion flow amplifying
oligonucleotide tags comprise polymers of 25 to 35 monomer units
which are non-natural abasic nucleotide analog structures. These
amidite reagent are easily synthesized into polymers via standard
automated amidite coupling chemistry, and used to prepare ion flow
amplifying tagged nucleotides of the present disclosure. Such
monomer unit structures are shown in Table 3 as structures of
formula (1a), (1b), (1c), (1d), (1e), (1f), (1g), (1 h), (1i),
(1j), (1 k), (1l), and (1m). Thus, in some embodiments, the ion
flow amplifying tags useful in the compounds and tagged nucleotides
of the present disclosure comprise a negatively-charged polymer,
wherein the polymer comprises monomer units selected from the
structures of formula (1a), (1b), (1c), (1d), (1e), (1f), (1g),
(1h), (1i), (1j), (1k), (1l), (1m), and any combination
thereof.
[0112] The monomer structures comprising phosphodiester linkages of
Table 3, and a wide variety of other such non-natural abasic
nucleotide analogs (often also referred to as spacer units) are
available commercially in amidite reagent form (e.g.,
phosphoramidite or phosphonamidite) (from e.g., Glen Research,
22825 Davis Drive, Sterling, Va., USA). It is contemplated that
additional ion flow amplifying tags comprising this range of
phosphodiester linked monomer units can be used prepared using
these amidite reagents and standard automated amidite synthesis
methods.
[0113] Amidite reagents corresponding to the monomer units with
phosphodiester linkages of Table 3 can be used to prepare an ion
flow amplifying tag having a polymeric structure via standard
amidite coupling chemistry. That is, each of the phosphoramidite
(or phosphonamidite) reagents will react in an amidite coupling
reaction with a nucleotide polymer (e.g., oligonucleotide) to
insert a monomer unit with its particular structure into the
polymer. This resulting polymeric structure will have phosphate (or
phosphonate) linkage to the adjacent monomer units in the polymer.
Such polymers can then be used to prepare an ion flow amplifying
tagged nucleotide of the present disclosure via linking chemistry
disclosed herein, and well-known to the skilled artisan.
Accordingly, the present disclosure provides an ion flow amplifying
tagged nucleotide compound (e.g., having structural formula (I),
(II), or (III)), wherein the tag comprises a negatively charged
polymeric structure having at least one monomer unit resulting from
the reaction of an amidite reagent corresponding to a monomer unit
of Table 3.
[0114] Generally, in any of the embodiments of ion flow amplifying
tagged nucleotide compounds disclosed herein, the Tag can comprise
an oligonucleotide of at least 10-mer, 15-mer, 20-mer, 25-mer,
30-mer, 35-mer, 40-mer, 50-mer, 60-mer or more monomer units in
length; optionally, wherein the oligonucleotide comprises monomer
units selected from Table 3.
[0115] The ordinary artisan will recognize that some of the monomer
units disclosed in Table 3 are also referred to in commercial
oligonucleotide synthesis catalogs as "spacers" (e.g., "SpC3",
"dSp"). The ordinary artisan will also recognize that some of the
oligonucleotide tags described herein (including in the Examples)
are referred to using well-known oligonucleotide synthesis
nomenclature (see e.g., the web-site of Integrated DNA Technologies
at www.idtdna.com for further description of commonly used
oligonucleotide synthesis nomenclature). For example, in the
context of the oligonucleotide embodiments disclosed elsewhere
herein, the phosphodiester linked monomer unit structures of Table
3, structures of formula (1a), (1b), (1c), (1d), (1e), (1f), (1g),
(1h), (1i), (1j), (1k), (1l), and (1m), are referred to herein by
the oligonucleotide synthesis related names of Table 4.
TABLE-US-00004 TABLE 4 Oligonucleotide Oligonucleotide Synthesis
Related Monomer Unit Structure Name of Monomer Unit (1a) SpC2 (1b)
SpC3 (1c) SpC6 (1d) SpC9 (1e) SpC12 (1f) dSp (1g) Sp9 (1h) Sp18
(1i) SpC4-F4 (1j) SpC7-Pra2 (1k) SpC6-Bz (1l) BHEB (1m) S500
[0116] In some embodiments, the present disclosure provides an ion
flow amplifying tagged nucleotide compound (e.g., of structural
formula (I), (II), or (III)), wherein the negatively-charged
polymer moiety comprises an oligonucleotide of formula
(A).sub.m-(B).sub.n-(C).sub.p-(D).sub.q, wherein A, B, C, and D,
are monomer units independently selected from the monomer unit
structures of formula (1a), (1b), (1c), (1d), (1e), (1f), (1g),
(1h), (1i), (1j), (1k), (1l), and (1m), and each of m, n, p, and q
is from 0 to 40, and m+n+p+q is from 10 to 40, optionally m+n+p+q
is from 20 to 40, m+n+p+q is from 25 to 35, or m+n+p+q is from 27
to 35.
[0117] In some embodiments, the present disclosure provides an ion
flow amplifying tagged nucleotide compound (e.g., of structural
formula (I), (II), or (III)), wherein the negatively-charged
polymer moiety comprises an oligonucleotide of formula (A).sub.n,
wherein A is a monomer unit independently selected from the monomer
unit structures of formula (1a), (1b), (1c), (1d), (1e), (1f),
(1g), (1h), (1i), (1j), (1k), (1l), and (1m), and n is from 10 to
40, optionally n is from 20 to 40, n is from 25 to 35, or n is from
27 to 35.
[0118] In some embodiments, the present disclosure provides an ion
flow amplifying tagged nucleotide compound (e.g., of structural
formula (I), (II), or (III)), wherein the negatively-charged
polymer moiety comprises an oligonucleotide selected from Table
5.
TABLE-US-00005 TABLE 5 Exemplary Negatively-Charged Polymer
Moieties (Oligonucleotides) -TT-(SpC2).sub.28- (SEQ ID NO: 1)
-TT-(SpC3).sub.28- (SEQ ID NO: 2) -(SpC3).sub.31- (SEQ ID NO: 3)
-TT-(dSp).sub.26-TT- (SEQ ID NO: 4) -TT-(SpC4-F4).sub.28- (SEQ ID
NO: 5) -TT-(SpC7-Pra2).sub.28- (SEQ ID NO: 6)
-(SpC2).sub.8-T.sub.6-(SpC2).sub.16- (SEQ ID NO: 12)
-(SpC2).sub.8-(N3CEdT).sub.7-(SpC2).sub.15- (SEQ ID NO: 13)
-(SpC2).sub.6-TT-(BHEB)-T-(SpC2).sub.20- (SEQ ID NO: 14
-(SpC2).sub.9-T-(BHEB).sub.2-T-(SpC2).sub.17- (SEQ ID NO: 15)
-(SpC2).sub.8-(S500).sub.3-(SpC2).sub.17- (SEQ ID NO: 16)
[0119] Ion flow amplifying tagged nucleotide compounds, wherein the
tag comprises an oligonucleotide include compounds also are
disclosed and exemplified in the Examples.
[0120] The exemplary negatively charged polymer moieties useful as
ion flow amplifying compounds generally have molecular lengths of
about 80 angstroms to about 150 angstroms, and molecular diameters
of less than about 15 angstroms.
[0121] It is contemplated that the negatively-charged polymer
moiety comprising an oligonucleotide can further comprise a 3' end
group. Such 3' end groups are known to act to protect the tag from
digestion in the presence of polymerase enzymes. See e.g.,
protective 3' end groups as disclosed in WO 2015/148402. Exemplary
3' end groups useful in the ion flow amplifying tags of the present
disclosure include propanol ("C.sub.3") and biotin, however, the
ordinary artisan will recognize that numerous other groups are
available.
[0122] The present disclosure provides the ordinary artisan with
tools to prepare ion flow amplifying tagged nucleotides with tag
moieties that provide ion flow amplification characteristics useful
across a wide range of nanopore devices and detection systems.
[0123] Methods of Preparing Ion Flow Amplifying Tagged
Nucleotides
[0124] Standard synthetic methods can be used in preparing the ion
flow amplifying tagged nucleotide compounds of the present
disclosure (e.g., compounds of structural formulas (I), (II),
(III)). The standard azido-alkyne click reaction is described above
(e.g., compounds of (XIX), (XX), (XXI), or (XXII)) and in the
Examples. Tables 1 and 2 illustrate a range of linkers and linker
forming group reactions that can be used in preparing the ion flow
amplifying tagged nucleotides of the present disclosure. In one
embodiment, any of the linker forming groups of structural formulas
(IVa) -(XVIIa) shown in Table 1 can be attached to a branched or
dendrimeric linker attached to a tag, or to a terminal phosphate of
a nucleotide, and the corresponding conjugate linker forming group
of structural formulae (IVb) -(XVIIb) would be attached to other.
The resulting covalent linker structures forming the
multi-nucleotide-oligophosphate-linker-tag compound are exemplified
by structural formulae (IVc) -(XVIIc) in Table 1. The covalent
linkage structure and include the dihydropyrazidine group structure
(XVIIc) that results from the click reaction of trans-cyclooctene
(XVIIa) and tetrazine (XVIIb) linker forming groups.
[0125] Accordingly, the present disclosure provides a method of
preparing a ion flow amplifying tagged nucleotide comprising: (a)
providing (i) a nucleotide with from 3 to 12 phosphates attached to
its 5'-position, wherein the terminal phosphate is coupled to a
first linker forming group (e.g., X.sub.A or X.sub.B); and (ii) an
ion flow amplifying tag, wherein the tag is coupled to a second
linker forming group (e.g., X.sub.B or X.sub.A) that is capable of
reacting with the first linker forming group to form a linker
(e.g., --X--); and (b) reacting the first linker forming group with
the second linker forming group to form the ion flow amplifying
tagged nucleotide. First and second linker forming groups that are
capable of reacting to form a linker are exemplified in Table 1
above. Thus, in some embodiments of the method, the first linker
forming group is selected from the compounds of structural formulas
(IVa) -(XVIIa) and the second linker forming group is the
corresponding reactive compound of structural formulas (IVb)
-(XVIIb); or alternatively, the first linker forming group is
selected from the compounds of structural formulas (IVb) -(XVIIb)
and the second linker forming group is the corresponding reactive
compound of structural formulas (IVa) -(XVIIa).
[0126] In some embodiments, the disclosure provides method of
preparing the ion flow amplifying tapped nucleotide compound of
structural formula (II)
##STR00082## [0127] wherein, Base is selected from adenosine,
cytidine, guanosine, thymidine, and uridine; R is selected from H
and OH; n is from 1 to 4; Linker is a linker comprising a
covalently bonded chain of 2 to 100 atoms; m is from 2 to 12; and
Tag is an ion flow amplifying tag comprising a negatively-charged
polymer moiety which is capable of entering a nanopore and upon
entering a nanopore in the presence of positive ions results in an
increased flow of the positive ions through the nanopore; and the
method comprises the steps of: [0128] (a) providing (i) a
nucleotide with from 3 to 12 phosphates attached to its
5'-position, wherein the terminal phosphate is coupled to a first
linker forming group; and (ii) an ion flow amplifying tag, wherein
the tag comprises a molecular moiety which is capable of producing
a detectable signal, comprising at least a second linker forming
group capable of reacting with the first linker forming group to
form a covalent linker between the nucleotide and the tag; [0129]
wherein [0130] (1) the first linker forming group is selected from
the compounds of structural formulas (IVa) -(XVIIa) and the second
linker forming group is the corresponding reactive compound of
structural formulas (IVb) -(XVIIb); or [0131] (2) the first linker
forming group is selected from the compounds of structural formulas
(IVb) -(XVIIb) and the second linker forming group is the
corresponding reactive compound of structural formulas (IVa)
-(XVIIa); [0132] and [0133] (b) reacting the first linker forming
group with the second linker forming group, thereby forming a
covalent linkage between the nucleotide and ion flow amplifying
tag.
[0134] In some embodiments of the methods of preparing the ion flow
amplifying tagged nucleotide compound, the first linker forming
group attached to the terminal phosphate is an azide group and the
second linker forming group attached to the tag is an alkyne. In
other embodiments, the first linker forming group attached to the
terminal phosphate is an alkyne group and the second linker forming
group attached to the tag is an azide.
[0135] In some embodiments of the methods of preparing the ion flow
amplifying tagged nucleotide, the first linker forming group
attached to the terminal phosphate is a tetrazine and the second
linker forming group attached to the tag is a trans-cyclooctene. In
other embodiments, the first linker forming group attached to the
terminal phosphate is a trans-cyclooctene and the second linker
forming group attached to the tag is a tetrazine.
[0136] As described elsewhere herein, in some embodiments of the
methods of preparing tagged nucleotides, a branched or dendrimeric
linker structure can be used to form a multi-nucleotide tagged with
a single ion flow amplifying tag. For example, the linker structure
can be generated using the doubler or trebler linker units of
compounds (19) or (20). In some embodiments, the doubler or trebler
linker units can be linked in a serial fashion to generate branched
or dendrimeric linkers have four or more reactive linker forming
groups available (e.g., as in compound (21)).
[0137] Use of Tagged Nucleotides in Nanopore Sequencing
[0138] The ion flow amplifying tagged nucleotide compounds of the
present disclosure can be used in the known nanopore sequencing
methods wherein a nanopore detects the presence of a tag attached
to a complementary nucleotide as it is incorporated (or after it is
incorporated and released) by a strand-extending enzyme (e.g.,
polymerase, ligase) located proximal to the nanopore and which is
extending a primer complementary of a target nucleic acid sequence.
General methods, materials, devices, and systems for carrying out
nanopore sequencing using tagged nucleotides are described in US
Pat. Publ. Nos. 2013/0244340 A1, 2013/0264207 A1, 2014/0134616 A1,
2015/0119259 A1, and U.S. Ser. No. 14/666,124, filed Mar. 23, 2015,
each of which is hereby incorporated by reference herein. The ion
flow amplifying tagged nucleotides of the present disclosure can be
employed in these general methods for using tagged-nucleotides for
nanopore sequencing of nucleic acids. Indeed, as illustrated in the
Examples herein, the ion flow amplifying tagged nucleotide
compounds of the present disclosure have the improved
characteristic of increasing ion flow and resulting in nanopore
detectable tag currents above the open channel (O.C.) current of
the nanopore along. These above O.C. signals are in a detection
range that exhibits lower noise and thereby allow for more accurate
sequence reads in nanopore sequencing than the corresponding
non-ion-flow amplifying tagged nucleotide compounds.
[0139] Thus, in one embodiment, the present disclosure provides a
method for determining the sequence of a nucleic acid comprising:
(a) providing a nanopore sequencing composition comprising: a
membrane, an electrode on the cis side and the trans side of the
membrane, a nanopore with its pore extending through the membrane,
an electrolyte solution in contact with both electrodes, an active
polymerase situated adjacent to the nanopore, and a primer strand
complexed with the polymerase; (b) contacting the nanopore
sequencing composition with (i) a strand of the nucleic acid; and
(ii) a set of compounds each comprising a different
nucleoside-5'-oligophosphate moiety covalently linked to a tag,
wherein each member of the set of compounds has a different tag
which results in a different flow of positive ions through a
nanopore when the tag enters the nanopore, and at least one of the
different tags comprises a negatively-charged polymer moiety which
upon entering a nanopore in the presence of positive ions results
in an increased flow of the positive ions through the nanopore; and
(c) detecting the different flows of positive ions resulting from
the entry of the different tags in the nanopore over time and
correlating to each of the different compounds incorporated by the
polymerase which are complementary to the nucleic acid sequence,
and thereby determining the nucleic acid sequence.
[0140] In some embodiments, the present disclosure provides a
method for determining the sequence of a nucleic acid comprising:
(a) providing a nanopore sequencing composition comprising: a
membrane, an electrode on the cis side and the trans side of the
membrane, a nanopore with its pore extending through the membrane,
an electrolyte solution in contact with both electrodes, an active
polymerase situated adjacent to the nanopore, and a primer strand
complexed with the polymerase; (b) contacting the nanopore
sequencing composition with (i) a strand of the nucleic acid; and
(ii) a set of tagged nucleotides each with a different tag, wherein
each different tag causes a different tag current level across the
electrodes when it is situated in the nanopore, and the set
comprises at least one compound of structural formula (I)
N--P-L-T (I)
wherein, N is a nucleoside; P is an oligophosphate covalently
attached to a 5'-O group of the nucleoside, wherein the
oligophosphate consists of 3 to 12 phosphate groups; L is a linker
covalently attached to a terminal phosphate group of the
oligophosphate; m is from 2 to 12 and indicates the number of
N--P-L moieties; and T is a ion flow amplifying tag covalently
attached the N--P-L moieties, wherein the tag comprises a
negatively-charged polymer moiety which upon entering a nanopore in
the presence of positive ions results in an increased flow of
positive ions through the nanopore; and (d) detecting the flow of
positive ions through the nanopore by detecting current levels
across the electrodes over time and correlating to each of the
different tagged nucleotides incorporated by the polymerase which
are complimentary to the nucleic acid sequence, and thereby
determining the nucleic acid sequence.
[0141] In some embodiments of the method for determining the
sequence of a nucleic acid, the set of tagged nucleotides each with
a different tag, comprises at least one ion flow amplifying tagged
compound that comprises a structure of formula (II):
##STR00083##
wherein, Base is selected from adenosine, cytidine, guanosine,
thymidine, and uridine; R is selected from H and OH; n is from 1 to
4; Linker is a linker comprising a covalently bonded chain of 2 to
100 atoms; m is from 2 to 12; and Tag is a tag comprising a
negatively-charged polymer moiety which is capable of entering a
nanopore and upon entering a nanopore in the presence of positive
ions results in an increased flow of the positive ions through the
nanopore.
[0142] When used in the methods for determining the sequence of a
nucleic acid the ion flow amplifying tagged nucleotide compounds
comprising structures of formula (I) or (II) can include any of the
ranges of compound embodiments disclosed elsewhere herein. For
example, the nucleoside (N) of formula (I) can be any nucleoside
capable of being incorporated by a strand-extending enzyme, such as
a polymerase, when the nucleoside is covalently coupled to an
oligophosphate (P), such as a triphosphate; and the nucleoside can
comprise a naturally occurring or non-naturally occurring
nucleobase, and a naturally occurring or non-naturally occurring
sugar moiety, such as a ribose or deoxyribose group.
[0143] Sets of Tagged Nucleotides
[0144] As described elsewhere herein, methods for determining the
sequence of a nucleic acid using nanopore detection generally
require a set of tagged nucleotide compounds each capable of being
a substrate for a strand-extending enzyme and each comprising a
different tag associated with a nucleotide that is desired to be
detected. In standard embodiments for sequencing DNA strands, the
method requires a set of at least the four standard
deoxy-nucleotides dA, dC, dG, and dT, wherein each different
nucleotide is attached to a different tag capable of being detected
upon the nucleotide being incorporated by a proximal strand
extending enzyme, and furthermore wherein the each tag's nanopore
detectable signal (e.g., tag current) is distinguishable from the
nanopore detectable signals of each of the other three tags,
thereby allowing identification of the specific nucleotide
incorporated by the enzyme. Generally, each of the different tagged
nucleotides in a set is distinguished by the distinctive detectable
signal the tag produces when it is incorporated into a new
complementary strand by a strand-extending enzyme.
[0145] Among the detectable signal characteristics, alone or in
combination, that can be used to distinguish the tagged nucleotides
in a nanopore detection method is the change in ion flow caused by
the presence of the tag in the nanopore, which in turn results in a
change in the current level measure across the electrodes of the
nanopore detection system (under either DC or AC potential).
Accordingly, in some embodiments, the present disclosure provides a
set of tagged nucleotides each with a different tag, wherein each
different tag causes a different ion flow through the pore
resulting in a different detectable tag current level across the
electrodes when it is situated in the nanopore, and the set
comprises at least one compound of structural formula (I)
N--P-L-T (I)
wherein, N is a nucleoside; P is an oligophosphate covalently
attached to a 5'-O group of the nucleoside, wherein the
oligophosphate consists of 3 to 12 phosphate groups; L is a linker
covalently attached to a terminal phosphate group of the
oligophosphate; m is from 2 to 12 and indicates the number of
N--P-L moieties; and T is a tag covalently attached the N--P-L
moieties, T is a ion flow amplifying tag covalently attached the
N--P-L moieties, wherein the tag comprises a negatively-charged
polymer moiety which upon entering a nanopore in the presence of
positive ions results in an increased flow of positive ions through
the nanopore.
[0146] In some embodiments of the set of ion flow amplifying tagged
nucleotides each with a different tag, the set comprises at least
one compound that comprises a structure of formula (II):
##STR00084##
wherein, Base is selected from adenosine, cytidine, guanosine,
thymidine, and uridine; R is selected from H and OH; n is from 1 to
4; Linker is a linker comprising a covalently bonded chain of 2 to
100 atoms; m is from 2 to 12; and Tag is a tag comprising a
negatively-charged polymer moiety which is capable of entering a
nanopore and upon entering a nanopore in the presence of positive
ions results in an increased flow of the positive ions through the
nanopore.
[0147] It is contemplated that the ion flow amplifying tagged
nucleotides of the present disclosure may be used in sets of tagged
nucleotides that also include tagged nucleotides that block rather
than amplify ion flow through the nanopore, and/or sets with tagged
nucleotides having different types of tags, such as both
oligonucleotide tags and polypeptide tags. For example, in some
embodiments, the set of ion flow amplifying tagged nucleotides can
comprise an ion flow amplifying tagged nucleotide of structural
formula (I), (II), or (III), and the other tagged nucleotides in
the set can comprise nucleotides attached to oligonucleotide tags
that do not amplify ion flow and which result in tag current signal
below the nanopore O.C. signal level. Such oligonucleotide-tagged
nucleotides that result in tag current signal below the nanopore
O.C. signal levels are known in the art and can be used in sets
along with the ion flow amplifying tagged nucleotides disclosed
herein. (See e.g., US Pat. Publ. Nos. 2013/0244340 A1, 2013/0264207
A1, 2014/0134616 A1, 2015/0119259 A1, and U.S. Ser. No. 14/666,124,
filed Mar. 23, 2015, each of which is hereby incorporated by
reference herein).
[0148] Alternatively, in some embodiments, a set tagged nucleotides
can include ion flow amplifying tagged nucleotides from across a
range of negatively charge polymer moiety structures, such as those
available using the monomer units of Table 3--e.g., an
oligonucleotide tag, a polypeptide tag, and/or a polyethylene
glycol tag.
[0149] In some embodiments, the set of tagged nucleotides comprises
at least two, at least three, or at least four ion flow amplifying
tagged nucleotide compounds of structural formula (I), (II), or
(III) wherein each of the different tags of the at least two, at
least three, or at least four of the ion flow amplifying tagged
nucleotide compounds in the set produces a nanopore detectable
signal that is distinguishable from the others in the set. Methods
and techniques for determining the nanopore detectable signal
characteristics, such as tag current and/or dwell time, are known
in the art. (See e.g., US Pat. Publ. Nos. 2013/0244340 A1,
2013/0264207 A1, 2014/0134616 A1, 2015/0119259 A1, and U.S. Ser.
No. 14/666,124, filed Mar. 23, 2015, each of which is hereby
incorporated by reference herein.) Such methods include nanopore
sequencing experiments under AC voltage potentials using a nanopore
array as described in the Examples herein.
[0150] Accordingly, in some embodiments, the present disclosure
provides a set of tagged nucleotides comprising at least two
different ion flow amplifying tagged nucleotides each having a
different tag, wherein the at least two different tags exhibit
distinguishable levels of amplified positive ion flow resulting
different measurable tag current levels above the nanopore O.C.
current level. In some embodiments, the at least two different ion
flow amplifying tagged nucleotides comprise a compound of structure
(I), structure (II), or structure (III). In some embodiments, the
at least two different ion flow amplifying tagged nucleotides each
comprises a different negatively charged polymer moiety,
optionally, each polymer moiety comprising a different sequence of
monomer units selected from Table 3 of Table 4. In some
embodiments, the at least two different tags exhibit tag current
levels above O.C. current level that differ by at least 10%, at
least 25%, at least 50%, or at least 75%. The measurement of the
difference between tag current levels can be made using any
suitable nanopore detection method. For example, the tag current
levels of each of the at least two different ion flow amplifying
tagged nucleotides can be measured in a nanopore sequencing
experiment, as is generally described in the Examples herein.
[0151] In some embodiments, the at least two different ion flow
amplifying tagged nucleotides comprise the following set of four
tagged nucleotide compounds: dA6P-(Linker)-T.sub.30-C3;
dC6P-(Linker)-TT-(dSp).sub.26-TT-C3;
dG6P-(Linker)-TT-(SpC2).sub.28-biotin; and
dT6P-(Linker)-TT-(SpC3).sub.28-biotin; wherein, "(Linker)" refers
to a linker of formula (XVd) (see above).
[0152] Nanopore Devices
[0153] Nanopore devices and methods for making and using them in
nanopore detection applications, such as nanopore sequencing using
ion flow amplifying tagged nucleotides of the present disclosure,
are known in the art (See e.g., U.S. Pat. Nos. 7,005,264 B2;
7,846,738; 6,617,113; 6,746,594; 6,673,615; 6,627,067; 6,464,842;
6,362,002; 6,267,872; 6.015,714; 5,795,782; and U.S. Publication
Nos. 2015/0119259, 2014/0134616, 2013/0264207, 2013/0244340,
2004/0121525, and 2003'0104428, each of which are hereby
incorporated by reference in their entirety). Nanopore devices
useful for measuring nanopore detection are also described in the
Examples disclosed herein. Generally, the nanopore devices comprise
a pore-forming protein embedded in a lipid-bilayer membrane,
wherein the membrane is immobilized or attached to a solid
substrate which comprises a well or reservoir. The pore of the
nanopore extends through the membrane creating a fluidic connection
between the cis and trans sides of the membrane. Typically, the
solid substrate comprises a material selected from the group
consisting of polymer, glass, silicon, and a combination thereof.
Additionally, the solid substrate comprises adjacent to the
nanopore, a sensor, a sensing circuit, or an electrode coupled to a
sensing circuit, optionally, a complementary metal-oxide
semiconductor (CMOS), or field effect transistor (FET) circuit.
Typically, there are electrodes on the cis and trans sides of the
membrane that allow for a DC or AC voltage potential to be set
across the membrane which generates a baseline current flow (or
O.C. current level) through the pore of the nanopore. The presence
of a tag, such as an ion flow amplifying tag of the present
disclosure, results in change in positive ion flow through the
nanopore and thereby generates a measurable change in current level
across the electrodes relative to the O.C. current of the
nanopore.
[0154] It is contemplated that the ion flow amplifying tag
compounds of the present disclosure can be used with a wide range
nanopore devices comprising nanopores generated by both
naturally-occurring, and non-naturally occurring (e.g., engineered
or recombinant) pore-forming proteins. A wide range of pore-forming
proteins are known in the art that can be used to generate
nanopores useful for nanopore detection of the ion flow amplifying
tags of the present disclosure. Representative pore forming
proteins include, but are not limited to, .alpha.-hemolysin,
.beta.-hemolysin, .gamma.-hemolysin, aerolysin, cytolysin,
leukocidin, melittin, MspA porin and porin A. The pore-forming
protein, .alpha.-hemolysin from Staphyloccocus aureus (also
referred to herein as ".alpha.-HL"), is one of the most-studied
members of the class of pore-forming proteins, and has been used
extensively in creating nanopore devices. (See e.g., U.S.
Publication Nos. 2015/0119259, 2014/0134616, 2013/0264207, and
2013/0244340.) .alpha.-HL also has been sequenced, cloned,
extensively characterized structurally and functionally using a
wide range of techniques including site-directed mutagenesis and
chemical labelling (see e.g., Valeva et al. (2001), and references
cited therein). It is contemplated that the compounds described
herein can be used with a-HL heptameric nanopores that are formed
from naturally occurring a-HL pore forming proteins, non-naturally
occurring a-HL pore forming proteins, or combinations thereof. In
one embodiment, the amino acid sequence of the naturally occurring
(i.e., wild type) a-HL pore forming protein subunit is shown as SEQ
ID NO: 22 (which is a mature sequence suitable for determining the
location of substitutions described herein and therefore does not
include the initial methionine residue). Non-naturally occurring
a-HL pore forming proteins include, without limitation, variant
pore polypeptides comprising one or more of the following
substitutions: H35G, H144A, E111N, M113A, D127G, D128G, T129G,
K131G, K147N, and V149K. Other suitable .alpha.-HL pore
polypeptides are described in U.S. Published Patent Application
Nos. 2017-0088588, as well as U.S. patent application Ser. Nos.
15/492,214, 15/274,770, and 15/638,273 each of which is hereby
incorporated by reference herein.
[0155] A heptameric complex of .alpha.-HL monomers spontaneously
forms a nanopore that embeds in and creates a pore through a lipid
bilayer membrane. It has been shown that heptamers of .alpha.-HL
comprising a ratio of 6:1 native .alpha.-HL to mutant .alpha.-HL
can form nanopores (see e.g., Valeva et al. (2001) "Membrane
insertion of the heptameric staphylococcal alpha-toxin pore--A
domino-like structural transition that is allosterically modulated
by the target cell membrane," J. Biol. Chem. 276(18): 14835-14841,
and references cited therein). Further, .alpha.-HL has been
engineered with cysteine residue substitutions inserted at numerous
positions allowing for covalent modification of the protein through
maleimide linker chemistry (Ibid.) For example, the engineered
.alpha.-hemolysin-C46 (".alpha.-HL-C46"), comprises a K46C amino
acid residue substitution that allows for modification with a
linker that can be used to covalently attach a strand-extending
enzyme, such as polymerase, using common click reaction chemistry.
Alternatively, the .alpha.-HL heptamer can be modified covalently
with a DNA-polymerase using a SpyCatcher/SpyTag conjugation method
as described in WO 2015/148402, which is hereby incorporated by
reference herein (see also, Zakeri and Howarth (2010), J. Am. Chem.
Soc. 132:4526-7.
[0156] In one embodiment, the heptameric .alpha.-HL nanopore
comprises at least one naturally occurring protein (i.e., wild
type), and at least one non-naturally occurring protein. In another
embodiment, the non-naturally occurring protein comprises an H144A
amino acid substitution. In one other embodiment, the nanopore
comprises one wild type protein and six proteins having the H144A
substitution. In other embodiments, the non-naturally occurring
protein comprises an H144A amino acid substitution and an H35G
substitution. In some embodiments, the nanopore comprises one wild
type protein and six proteins having the H144A and H35G
substitution. In another embodiment, the heptameric .alpha.-HL
nanopore comprises more than one non-naturally occurring protein.
In one embodiment, the nanopore comprises a first and a second
non-naturally occurring protein. In one other embodiment, the first
non-naturally occurring protein comprises an H144A, an H35G, an
E111N, an M113A, a K147N, a D127G, a D128G, a T129G, a K131G, and a
V149K substitution. In another embodiment, the second non-naturally
occurring protein comprises an E111N, an M113A, a K147N, a D127G, a
D128G, a T129G, and a K131G substitution. In some embodiments, the
nanopore comprises six of the first non-naturally occurring
proteins and one of the first non-naturally occurring proteins.
[0157] Accordingly, in some embodiments, the ion flow amplifying
tag compounds of the present disclosure can be used with a nanopore
device, wherein the nanopore comprises a heptameric .alpha.-HL
complex, which has 6:1 native .alpha.-HL to a modified, or an
engineered version of .alpha.-HL, wherein the modified .alpha.-HL
is conjugated covalently to a strand-extending enzyme, such as DNA
polymerase. For example, the engineered .alpha.-HL-C46 can be
modified with a linker allowing the use of
tetrazine-trans-cyclooctene click chemistry to covalently attach a
Bst2.0 variant of DNA polymerase to the heptameric 6:1 nanopore.
Such an embodiment is described in U.S. Provisional Application No.
62/130,326, filed Mar. 9, 2015, and U.S. Published Patent
Application No. 2017-0175183, each of which is hereby incorporated
by reference herein.
[0158] The ion flow amplifying tagged nucleotides, associated
compositions, and methods provided herein can be used with a wide
range of strand-extending enzymes such as the polymerases and
ligases known in the art.
[0159] DNA polymerases are a family of enzymes that use
single-stranded DNA as a template to synthesize the complementary
DNA strand. DNA polymerases add free nucleotides to the 3' end of a
newly-forming strand resulting in extension of the new strand in
the 5'-to-3' direction. Most DNA polymerases also possess
exonucleolytic activity. For example, many DNA polymerases have
3'.fwdarw.5' exonuclease activity. Such multifunctional DNA
polymerases can recognize an incorrectly incorporated nucleotide
and use the 3'.fwdarw.5' exonuclease activity to excise the
incorrect nucleotide, an activity known as proofreading. Following
nucleotide excision, the polymerase can re-insert the correct
nucleotide and strand extension can continue. Some DNA polymerases
also have 5'.fwdarw.3' exonuclease activity.
[0160] DNA polymerases are used in many DNA sequencing
technologies, including nanopore-based sequencing-by-synthesis.
However, a DNA strand can move rapidly through the nanopore (e.g.,
at a rate of 1 to 5 .mu.s per base), which can make nanopore
detecting of each polymerase-catalyzed incorporation event
difficult to measure and prone to high background noise, which can
result in difficulties in obtaining single-nucleotide resolution.
The ability to control the rate of DNA polymerase activity, as well
as, increase the signal level from correct incorporation is
important during sequencing-by-synthesis, particular when using
nanopore detection. As shown in the Examples, the ion flow
amplifying tagged nucleotide compounds of the present disclosure
provide for a wider range of detectable tag current signals above
the O.C. signal that provide better signal separation and lower
noise levels, and thereby allow for more accurate nanopore-based
nucleic acid detection and sequencing.
[0161] Exemplary polymerases that may be used with the ion flow
amplifying tagged nucleotide compounds and methods of the present
disclosure include the nucleic acid polymerases such as DNA
polymerase (e.g., enzyme of class EC 2.7.7.7), RNA polymerase
(e.g., enzyme of class EC 2.7.7.6 or EC 2.7.7.48), reverse
transcriptase (e.g., enzyme of class EC 2.7.7.49), and DNA ligase
(e.g., enzyme of class EC 6.5.1.1).
[0162] In some embodiments, the polymerase useful with ion flow
amplifying tagged nucleotides is 9.degree. N polymerase, E. coli
DNA Polymerase I, Bacteriophage T4 DNA polymerase, Sequenase, Taq
DNA polymerase, 9.degree. N polymerase (exo-)A485L/Y409V or Phi29
DNA polymerase .PHI.29 DNA Polymerase).
[0163] In some embodiments, the strand extending enzyme that
incorporates the ion flow amplifying tagged nucleotides comprises a
DNA polymerase from Bacillus stearothermophilus. In some
embodiments, the large fragment of DNA polymerase from B.
stearothermophilus. In one embodiment, the polymerase is DNA
polymerase Bst 2.0 (commercially available from New England
BioLabs, Inc., Massachusetts, USA).
[0164] In some embodiments, the polymerase is a Pol6 DNA
polymerase, or an exonuclease deficient variant of a Pol6, such as
Pol6 having the mutation D44A. A range of additional Pol6 variants
useful with the ion flow amplifying tagged nucleotides of the
present disclosure are described in US patent publication no.
2016/0333327 A1, published Nov. 17, 2016, which is hereby
incorporated by reference herein.
EXAMPLES
[0165] Various features and embodiments of the disclosure are
illustrated in the following representative examples, which are
intended to be illustrative, and not limiting. Those skilled in the
art will readily appreciate that the specific examples are only
illustrative of the invention as described more fully in the claims
which follow thereafter. Every embodiment and feature described in
the application should be understood to be interchangeable and
combinable with every embodiment contained within.
Example 1: Preparation of Ion Flow Amplifying Tagged Nucleotide
Compounds
[0166] This example illustrates a general method for preparation of
a tagged nucleotide compound of structural formula (IIId), wherein
the compound comprises nucleoside-5'-hexaphosphate, wherein the
terminal phosphate group is linked to an ion flow amplifying tag
comprising an oligonucleotide. More specifically, this example
describes the preparation of the following three ion flow
amplifying tagged nucleotide compounds shown in Table 6.
TABLE-US-00006 TABLE 6 Ion Flow Amplifying Tag Tagged Nucleotide
(Sequence identifier) dC6P-(Linker).sup.1-TT-(dSp).sub.26-TT-C3
-TT-(dSp).sub.26-TT-C3 (SEQ ID NO: 7)
dG6P-(Linker).sup.1-TT-(SpC2).sub.28-biotin
-TT-(SpC2).sub.28-biotin (SEQ ID NO: 8)
dT6P-(Linker).sup.1-TT-(SpC3).sub.28-biotin
-TT-(SpC3).sub.28-biotin (SEQ ID NO: 9) .sup.1"(Linker)" refers to
the triazole linker of formula (XVd) ##STR00085##
[0167] The synthesis method uses a standard Cu catalyzed
azido-alkyne click reaction to covalently couple the 5'-end of an
ion flow amplifying tag that is an oligonucleotide to the terminal
phosphate group of the nucleotide hexaphosphate (dN6P) as shown
generally in Scheme 2.
##STR00086##
[0168] This synthesis method of Scheme 2 is described below for the
preparation of the specific ion flow amplifying tagged nucleotide,
dT6P-(Linker)-TT-(SpC3).sub.28-biotin (i.e., compound of formula
(IIIa), where Base is T and Tag is the oligonucleotide,
TT-(SpC3).sub.28-biotin. This same method is used to prepare the
other tagged nucleotides listed Table 6.
[0169] A. Synthesis dT6P-Azide (Compound (1))
##STR00087##
[0170] Preparation of 11-azido-1-undecanol: 11-azido-1-undecanol is
prepared according to the reaction Scheme 3 and procedure
below.
##STR00088##
[0171] In a dried round bottom flask, sodium azide (1.44 g, 22 mM)
was added to a solution of 11-Bromo-1-undecanol (1.84 g, 7.38 mmol)
in anhydrous DMF (40 mL). The resulting white suspension was
stirred under nitrogen atmosphere at ambient temperature overnight.
The suspension was filtered and rinsed with DCM (50 mL). The
solution was concentrated under vacuum to give yellowish oil. The
compound can be used in the following steps without further
purification.
[0172] Preparation of 11-azido-1-undecanyl triphosphate:
11-azido-1-undecanyl triphosphate is prepared according to the
reaction Scheme 4 and procedure below.
##STR00089##
[0173] In a dried round bottom flask, 11-azido-1-undecanol (0.20 g,
0.94 mmol) was dissolved in anhydrous DMF (2.0 mL). Salicyl
chlorophosphite (0.20 g, 1.03 mmol) was added in one portion. The
resulting solution was stirred at ambient temperature under
nitrogen for 45 minutes. In another flask, a solution of
pyrophosphate tributylamine (0.566, 1.03 mmol) in anhydrous DMF and
tributylamine (1.39 g, 7.51 mmol) was prepared and then added to
the reaction solution. The resulting mixture was stirred for an
hour and was oxidized with 20 mM iodine solution (80 mL, 1.55
mmol), giving cyclic meta-triphosphate intermediate that can be
analyzed by mass spectrometer. After another hour of stirring, the
reaction was quenched first with Na.sub.2SO.sub.3 (10%, 4 mL),
allowed to stir for 20 minutes, followed by TEAB (0.10 M, 20 mL).
The resulting mixture was stirred at ambient temperature overnight.
The crude product was purified by TeleDyne CombiFlash RF+ column
system using 30 g HP C18 column eluting with CH.sub.3CN/0.1 TEAA
(0% to 50% CH.sub.3CN in 16 minutes). The product is concentrated
under vacuum and dried on a lyophilizer.
[0174] Preparation of dT6P-azide (compound (1)): dT6P-azide is
prepared according to the reaction Scheme 5 and procedure
below.
##STR00090##
[0175] 11-azido-1-undecanyl triphosphate (0.091 g, 0.12 mmol) was
dissolved in anhydrous DMF (1.5 mL) and was activated with carbonyl
diimidazole ("CDI") (0.078 g, 0.48 mmol) for 4 hours at ambient
temperature. The excess CDI was quenched with methanol (0.029 mL,
0.72 mmol), stirring additional 30 minutes. Then a solution of
dTTP+3Bu4N (0.20 g, 0.17 mmol) in anhydrous DMF (2.0 mL) was added,
followed by MgCl.sub.2 (0.114 g, 1.20 mmol). The resulting slurry
solution was stirred for 24-36 hours at ambient temperature. The
reaction was quenched with TEAB 0.1 M (20 mL), stirring for 30
minutes. The crude compound (1) was purified by ion-exchange
chromatography (0.1 M to 1 M in 30 minutes), followed by RP-C18
HPLC (10-45% CH.sub.3CN in 35 minutes) to yield 15-30 .mu.mol of
product. The formation of the compound (1) was confirmed by mass
spectrometry (cal. 917.06, observed 916.03 for negative ion).
[0176] B. Synthesis of 5'-Propargyl-TT-(Sp3).sub.28-Biotin
Oligonucleotide (Compound (2))
##STR00091##
[0177] The 5'-propargyl-TT-(SpC3).sub.28-biotin oligonucleotide
used as a tag was synthesized on an ABI 3900 DNA Synthesizer using
standard solid phase phosphoramidite chemistry protocols and
commercially available reagents, including the abasic 3 carbon
spacer amidite, "SpC3," and the biotin amidite reagents (available
from e.g., Glen Research, 22825 Davis Drive, Sterling, Va., USA).
In the final automated oligonucleotide synthesis step a
propargyl-C.sub.5-phosphoramidite linker was added to the
5'-phosphate of the penultimate T nucleotide resulting in the
propargyl-modified oligonucleotide of compound (2).
[0178] C. Click-Reaction to Form Tagged Nucleotide,
dT6P-(Linker)-TT-(SpC3).sub.28-Biotin
[0179] The click reaction forming ion flow amplifying tagged
nucleotide compound, dT6P-(Linker)-TT-(SpC3).sub.28-biotin is
carried out according to the reaction of Scheme 2 and the following
procedures. The dT6P-azide (compound (1)) (300 nmol) and the
5'-propargyl-TT-(Sp3).sub.28-biotin oligonucleotide (compound (2))
(100 nmol) are mixed in DI-water (100 .mu.L). The copper-catalyzed
azido-alkyne click-reaction is initiated according to the standard
literature procedure using Cu(I) bromide (6000 nmol) and THPTA
(4000 nmol) in a mixture solution of DMSO/t-Butanol (3:1). The
reaction solution is mixed at ambient temperature overnight on a
shaker. The crude mixture is purified by RP C18-HPLC (0.1M
TEAA/CH.sub.3CN). Formation of the desired conjugated product,
dT6P-(Linker)-TT-(SpC3).sub.28-biotin, is confirmed by mass
spectrometer (MS calc. 5939.8; MS obsd. 5940.9 for negative
ion).
[0180] Formation of the other two tagged nucleotides of Table 6 are
also confirmed by mass spectrometer as follows:
dC6P-(Linker)-TT-(dSp).sub.26-TT-C3 (MS calc. 7037.7; MS obsd.
7038.0 for negative ion); dG6P-(Linker)-TT-(SpC2).sub.28-biotin (MS
calc. 5571.9; MS obsd. 5573.0 for negative ion).
Example 2: Use of Ion Flow Amplifying Tagged Nucleotides for
Nanopore Sequencing
[0181] This example illustrates the improved nanopore detection
characteristics of a set of four different tagged nucleotide
compounds (shown in Table 7) corresponding to each of the four
canonical nucleotides, A, C, T, and G, which can be incorporated by
a polymerase.
TABLE-US-00007 TABLE 7 Tagged Nucleotides Oligonucleotide Tag
dA6P-(Linker)-T.sub.30-C3 SEQ ID NO: 10
dC6P-(Linker)-TT-(dSp).sub.26-TT-C3 SEQ ID NO: 7
dG6P-(Linker)-TT-(SpC2).sub.28-biotin SEQ ID NO: 8
dT6P-(Linker)-TT-(SpC3).sub.28-biotin SEQ ID NO: 9 Abbreviations
are those commonly used for oligonucleotide synthesis: "dSp" =
abasic furan spacer; "SpC2" = abasic 2 carbon spacer; "SpC3" =
abasic 3 carbon spacer; "C3" = 3`-propanol. "(Linker)" refers to
the triazole linker of formula (XVd).
[0182] In the set, the nucleotide hexaphosphate compounds, dC6P,
dG6P, and dT6P, each is tagged with a different ion-flow enhancing
tag. Each of these three comprises a negatively-charged polymer of
30 phosphodiester-linked monomer units (i.e., an oligonucleotide)
covalently coupled to the terminal phosphate of the nucleotide
hexaphosphate via a triazole linker formed as described in Example
1. The fourth tagged nucleotide hexaphosphate in the set is a dA6P
tagged with a T30-03 oligonucleotide. This tagged nucleotide is
known to not result in increased ion flow through a nanopore but
rather results in a blocking current below O.C. indicated decreased
ion flow.
[0183] Briefly, the nanopore detection of the set of four tagged
nucleotides is carried out using an array of .alpha.-HL nanopores
each conjugated to Pol6 polymerase. The .alpha.-HL-Pol6 nanopore
conjugates are embedded in membranes formed over an array of
individually addressable integrated circuit chips. This
.alpha.-HL-Pol6 nanopore array is exposed to a DNA template and a
set of the three different nucleotide substrates tagged with
different ion-flow enhancing tags shown in Table 7. As the specific
tagged nucleotide that is complementary to the DNA template is
captured and bound to the Pol6 polymerase active site, the ion-flow
enhancing tag moiety becomes positioned in the .alpha.-HL nanopore
conjugated nearby. Under the applied AC potential, the presence of
the tag in the pore causes an increase in positive ion flow through
the nanopore resulting in a distinctive current measured at the
nanopore device electrodes that is greater than the O.C. current
(i.e., current with no tag in the nanopore). The distinctive
above-O.C. currents measured as the different tag moieties enter
the nanopore during Pol6 synthesis of a complementary DNA extension
strand can be used to detect and identify the DNA template.
[0184] Nanopore detection system: The nanopore ion-flow
measurements are performed using a nanopore array microchip
comprising a CMOS microchip that has an array of 128,000 silver
electrodes within shallow wells (chip fabricated by Genia
Technologies, Mountain View, Calif., USA). Methods for fabricating
and using such nanopore array microchips can also be found in U.S.
Patent Application Publication Nos. 2013/0244340 A1, US
2013/0264207 A1, and US2014/0134616 A1 each of which is hereby
incorporated by reference herein. Each well in the array is
manufactured using a standard CMOS process with surface
modifications that allow for constant contact with biological
reagents and conductive salts. Each well can support a phospholipid
bilayer membrane with a nanopore-polymerase conjugate embedded
therein. The electrode at each well is individually addressable by
computer interface. All reagents used are introduced into a simple
flow cell above the array microchip using a computer-controlled
syringe pump. The chip supports analog to digital conversion and
reports electrical measurements from all electrodes independently
at a rate of over 1000 points per second. Nanopore blocking current
measurements can be made asynchronously at each of 128K addressable
nanopore-containing membranes in the array at least once every
millisecond (msec) and recorded on the interfaced computer.
[0185] Formation of lipid bilayer on chip: The phospholipid bilayer
membrane on the chip is prepared using
1,2-diphytanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids).
The lipid powder is dissolved in decane at 15 mM and then painted
in a layer across the wells on the chip. A thinning process then is
initiated by pumping air through the cis side of the array wells,
thus reducing multi-lamellar lipid membranes to a single
bilayer.
[0186] Insertion of .alpha.-HL-Pol6 conjugate in membrane: After
the lipid bilayer forms on the wells of the array chip, 0.1 .mu.M
of a 6:1 .alpha.-HL-Pol6 nanopore-polymerase conjugate, 0.4 .mu.M
of the desired DNA template, all in a buffer solution of 3 mM
MgCl.sub.2, 20 mM HEPES, and 300 mM K-Glu, pH 8, at 20.degree. C.
is added to the cis side of the chip. The nanopore-polymerase
conjugate in the mixture either is electroporated or spontaneously
inserts into the lipid bilayer. The non-polymerase modified
.alpha.-HL nanopores (i.e., the 6 subunits of the 6:1 heptamer)
include the H144A mutation.
[0187] The DNA template is a dumb-bell circular template which has
the sequence:
TABLE-US-00008 (SEQ ID NO: 11)
CAGTCAGTAGAGAGAGATATCTCTCTCAAAAACGGAGGAGGAGGACAGTCA
GTAGAGAGAGATATCTCTCTCAAAAACGGAGGAGGAGGA.
[0188] Nanopore ion flow measurements: After insertion of the
complex into the membrane, the solution on the cis side is replaced
by 300 mM potassium glutamate, pH 8, 3 mM MgCl.sub.2, 10 mM LiCl,
20 mM HEPES, 5 mM TCEP, 10 .mu.M of the set of the 4 differently
tagged nucleotides (3 of which are tagged with different ion-flow
enhancing tags), 10 .mu.M of the one nucleotide tagged with ion
blocking tag, at 20.degree. C. The trans side buffer solution is:
380 mM potassium glutamate, 3 mM MgCl.sub.2, 10 mM LiCl, 20 mM
HEPES, pH 8. These buffer solutions are used as the electrolyte
solutions for the nanopore ion flow measurements. A Pt/Ag/AgCl
electrode setup is used and an AC current of a -10 mV to 200 mV
square waveform applied at 50 Hz. AC current has certain advantages
for nanopore detection as it allows for the tag to be repeatedly
directed into and then expelled from the nanopore thereby providing
more opportunities to measure signals resulting from the ion flow
through the nanopore. Also, the ion flow during the positive and
negative AC current cycles counteract each other to reduce the net
rate of ion depletion from the cis side, and possible detrimental
effects on signals resulting from this depletion.
[0189] Tag current signals representing four distinct ion-flow
events are observed from the set of four tagged nucleotides as they
are captured by the .alpha.-HL-Pol6 nanopore-polymerase conjugates
primed with the DNA template. Plots of these events are recorded
over time and analyzed. Generally, events that last longer than 10
ms indicate productive nucleotide capture coincident with
polymerase incorporation of the correct base complementary to the
template strand.
[0190] Results
[0191] As shown in FIG. 1, signals representing three distinct
ion-flow enhancement events and an ion-flow blocking event are
observed from the set of four tagged nucleotides as they are
captured by the .alpha.-HL-Pol6 nanopore-polymerase conjugates
primed with the DNA template. Average tag current values measure
for relevant nanopore array carried out with the set of tagged
nucleotides are shown in Table 8.
TABLE-US-00009 TABLE 8 Tag Current Tagged Nucleotide (% O.C.)
dA6P-(Linker)-T.sub.30-C3 50 dC6P-(Linker)-TT-(dSp).sub.26-TT-C3
125 dG6P-(Linker)-TT-(SpC2).sub.28-biotin 175
dT6P-(Linker)-TT-(SpC3).sub.28-biotin 150 "(Linker)" refers to the
triazole linker of formula (XVd).
[0192] As shown by plot of FIG. 1 and the results in Table 8, the
three ion flow amplifying tagged nucleotides
(dC6P-(Linker)-TT-(dSp).sub.26-TT-C3;
dT6P-(Linker)-TT-(SpC3).sub.28-biotin; and
dG6P-(Linker)-TT-(SpC2).sub.28-biotin) exhibit tag current signal
levels above the open channel current of the nanopore indicating
that the presence of the tag in the nanopore results in increase
positive ion flow. The fourth tagged nucleotide present,
dA6P-(Linker)-T.sub.30-C3, exhibits a tag current signal level
below open channel current (i.e., a blocking current) indicating
the presence of the tag in the nanopore results in decreased
positive ion flow through the nanopore. The ability to provide tag
currents signals above open channel provides a wider dynamic range
for nanopore detection of tags. The wide range and broad separation
between the ion flow events resulting from this set of tags allows
for more accuracy in detecting and/or sequencing nucleic acids
using nanopore devices.
Example 3: Ion Flow Amplifying Tagged Nucleotides
[0193] Nanopore detection experiments can be used to identify
additional tagged nucleotide compounds which have ion flow
amplifying characteristics that allow for detection above open
channel.
[0194] Nanopore detection experiments carried out according to the
general methods described in Example 2 identified the tagged
nucleotides shown in Table 9. These tagged nucleotides can be
incorporated by a polymerase and provide a tag current signal above
open channel.
TABLE-US-00010 TABLE 9 Tag Sequence Tag Current Tagged Nucleotide
(SEQ ID NO:) (% O.C.)
dG6P-(Linker)-(SpC2).sub.8-T.sub.6-(SpC2).sub.16-C3 17 120.sup.1
dC6P-(Linker)-(SpC2).sub.8-(N3CEdT).sub.7-(SpC2).sub.15-C3 18
120.sup.2 dA6P-(Linker)-(SpC2).sub.6-TT-(BHEB)-T-(SpC2).sub.20-C3
19 155.sup.1
dC6P-(Linker)-(SpC2).sub.9-T-(BHEB).sub.2-T-(SpC2).sub.17-C3 20
189.sup.1 dT6P-(Linker)-(SpC2).sub.8-(S500).sub.3-(SpC2).sub.17-C3
21 170.sup.2 .sup.1Cis side buffer solution: 300 mM K-Glu, 10 mM
MgCl.sub.2, 15 mM LiCl, 20 mM HEPES, pH 8; trans side buffer
solution: 380 mM K-Glu, 5 mM MgCl.sub.2, 15 mM LiCl, 5 mM TCEP, 20
mM HEPES, pH 8; AC current: 10 to 140 mV at 50 Hz. .sup.2Cis side
buffer solution: 300 mM K-Glu, 10 mM MgCl.sub.2, 15 mM LiOAc, 5 mM
TCEP, 20 mM HEPES, pH 7.8; trans side buffer solution: 380 mM
K-Glu, 10 mM MgCl.sub.2, 15 mM LiOAc, 20 mM HEPES, pH 7.5; AC
current: 10 to 140 mV at 50 Hz. Abbreviations: "dSp" = abasic furan
spacer; "SpC2" = abasic 2 carbon spacer; "SpC3" = abasic 3 carbon
spacer; "N3CET" = 3-N-cyanoethyl-dT amidite (dT with a cyanoethyl
group at position N3 of the base); "BHEB" =
bis-hydroxyethylbenzene, which is a spacer that provides the
following 1,4-bis-ethyl-benzene phosphodiester structure in an
oligonucleotide: ##STR00092## "S500" =
1,4-bis(3-butyn-1-ol)benzene, which is a spacer that provides the
following 1,4-bis-butyn-benzene phosphodiester structure in an
oligonucleotide: ##STR00093## "C3" = 3'-propanol; "(Linker)" refers
to the triazole linker of formula (XVd).
[0195] All publications, patents, patent applications and other
documents cited in this application are hereby incorporated by
reference in their entireties for all purposes to the same extent
as if each individual publication, patent, patent application or
other document were individually indicated to be incorporated by
reference for all purposes.
[0196] While various specific embodiments have been illustrated and
described, it will be appreciated that various changes can be made
without departing from the spirit and scope of the invention(s).
Sequence CWU 1
1
22130DNAArtificial Sequencesynthetic
sequencemisc_feature(3)..(30)ethyl amidite 1ttnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 30230DNAArtificial Sequencesynthetic
sequencemisc_feature(3)..(30)propyl amidite 2ttnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 30331DNAArtificial Sequencesynthesized
oligonucleotidemisc_feature(1)..(31)propyl amidite 3nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn n 31430DNAArtificial Sequencesynthesized
oligonucleotidemisc_feature(3)..(28)furan amidite 4ttnnnnnnnn
nnnnnnnnnn nnnnnnnntt 30530DNAArtificial sequencesynthesized
oligonucleotidemisc_feature(3)..(30)2,2,3,3-tetrafluorobutyl
amidite 5ttnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30630DNAArtificial
Sequencesynthesized
oligonucleotidemisc_feature(3)..(30)hepta-2,4-diyn amidite
6ttnnnnnnnn nnnnnnnnnn nnnnnnnnnn 30730DNAArtificial
Sequencesynthesized oligonucleotidemisc_feature(3)..(28)furan
amiditemisc_feature(30)..(30)T-propanol 7ttnnnnnnnn nnnnnnnnnn
nnnnnnnntt 30830DNAArtificial Sequencesynthesized
oligonucleotidemisc_feature(3)..(30)ethyl
amiditemisc_feature(30)..(30)3'-biotin 8ttnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 30930DNAArtificial Sequencesynthesized
oligonucleotidemisc_feature(3)..(30)propyl
amiditemisc_feature(30)..(30)3' biotin 9ttnnnnnnnn nnnnnnnnnn
nnnnnnnnnn 301030DNAArtificial Sequencesynthesized
oligonucleotidemisc_feature(30)..(30)T-propanol 10tttttttttt
tttttttttt tttttttttt 301190DNAArtificial SequenceSynthesized
oligonucleotide 11cagtcagtag agagagatat ctctctcaaa aacggaggag
gaggacagtc agtagagaga 60gatatctctc tcaaaaacgg aggaggagga
901230DNAArtificial Sequencesynthesized
oligonucleotidemisc_feature(1)..(8)ethyl
amiditemisc_feature(15)..(30)ethyl amidite 12nnnnnnnntt ttttnnnnnn
nnnnnnnnnn 301330DNAArtificial sequencesynthesized
oligonucleotidemisc_feature(1)..(8)ethyl
amiditemodified_base(9)..(15)3-N-cyanoethyl-dTmisc_feature(9)..(15)n
is a, c, g, or tmisc_feature(16)..(30)ethyl amidite 13nnnnnnnnnn
nnnnnnnnnn nnnnnnnnnn 301430DNAArtificial Sequencesynthesized
oligonucleotidemisc_feature(1)..(6)ethyl
amiditemisc_feature(9)..(9)1,4-bis-ethyl-benzenemisc_feature(11)..(30)eth-
yl amidite 14nnnnnnttnt nnnnnnnnnn nnnnnnnnnn 301530DNAArtificial
Sequencesynthesized oligonucleotidemisc_feature(1)..(9)ethyl
amiditemisc_feature(11)..(12)1,4-bis-ethyl-benzenemisc_feature(14)..(30)e-
thyl amidite 15nnnnnnnnnt nntnnnnnnn nnnnnnnnnn 301628DNAArtificial
Sequencesynthesized oligonucleotidemisc_feature(1)..(8)ethyl
amiditemisc_feature(9)..(11)1,4-bis-butyn-benzenemisc_feature(12)..(28)et-
hyl amidite 16nnnnnnnnnn nnnnnnnnnn nnnnnnnn 281731DNAArtificial
Sequencesynthesized oligonucleotidemisc_feature(1)..(8)ethyl
amiditemisc_feature(15)..(30)ethyl amiditemisc_feature(31)..(31)3'
propanol 17nnnnnnnntt ttttnnnnnn nnnnnnnnnn n 311831DNAArtificial
Sequencesynthesized oligonucleotidemisc_feature(1)..(8)ethyl
amiditemodified_base(9)..(15)3-N-cyanoethyl-dTmisc_feature(9)..(15)n
is a, c, g, or tmisc_feature(16)..(30)ethyl
amiditemisc_feature(31)..(31)3'-propanol 18nnnnnnnnnn nnnnnnnnnn
nnnnnnnnnn n 311931DNAArtificial Sequencesynthesized
oligonucleotidemisc_feature(1)..(6)ethyl
amiditemisc_feature(9)..(9)1,4-bis-ethyl-benzenemisc_feature(11)..(30)eth-
yl amiditemisc_feature(31)..(31)3'-propanol 19nnnnnnttnt nnnnnnnnnn
nnnnnnnnnn n 312031DNAArtificial Sequencesynthesized
oligonucleotidemisc_feature(1)..(9)ethyl
amiditemisc_feature(11)..(12)1,4-bis-ethyl-benzenemisc_feature(14)..(30)e-
thyl amiditemisc_feature(31)..(31)3'-propanol 20nnnnnnnnnt
nntnnnnnnn nnnnnnnnnn n 312129DNAArtificial Sequencesynthesized
oligonucleotidemisc_feature(1)..(8)ethyl
amiditemisc_feature(9)..(11)1,4-bis-butyn-benzenemisc_feature(12)..(28)et-
hyl amiditemisc_feature(29)..(29)3'-propanol 21nnnnnnnnnn
nnnnnnnnnn nnnnnnnnn 2922305PRTStaphylococcus aureus 22Ala Asp Ser
Asp Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly Ser1 5 10 15Asn Thr
Thr Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu Asn 20 25 30Gly
Met His Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Lys Asn His 35 40
45Asn Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly Gln
50 55 60Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly Leu Ala
Trp65 70 75 80Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn
Glu Val Ala 85 90 95Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp
Thr Lys Glu Tyr 100 105 110Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly
Asn Val Thr Gly Asp Asp 115 120 125Thr Gly Lys Ile Gly Gly Leu Ile
Gly Ala Asn Val Ser Ile Gly His 130 135 140Thr Leu Lys Tyr Val Gln
Pro Asp Phe Lys Thr Ile Leu Glu Ser Pro145 150 155 160Thr Asp Lys
Lys Val Gly Trp Lys Val Ile Phe Asn Asn Met Val Asn 165 170 175Gln
Asn Trp Gly Pro Tyr Asp Arg Asp Ser Trp Asn Pro Val Tyr Gly 180 185
190Asn Gln Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys Ala Ala Asp
195 200 205Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser Ser
Gly Phe 210 215 220Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp Arg
Lys Ala Ser Lys225 230 235 240Gln Gln Thr Asn Ile Asp Val Ile Tyr
Glu Arg Val Arg Asp Asp Tyr 245 250 255Gln Leu His Trp Thr Ser Thr
Asn Trp Lys Gly Thr Asn Thr Lys Asp 260 265 270Lys Trp Thr Asp Arg
Ser Ser Glu Arg Tyr Lys Ile Asp Trp Glu Lys 275 280 285Glu Glu Met
Thr Asn Gly Leu Ser Ala Trp Ser His Pro Gln Phe Glu 290 295
300Lys305
* * * * *
References