U.S. patent application number 16/826035 was filed with the patent office on 2020-11-05 for biochemically activated electronic device.
The applicant listed for this patent is ILLUMINA, INC.. Invention is credited to Jingwei BAI, Boyan BOYANOV, Cheng-Yao CHEN, Kevin L. GUNDERSON, Jeffrey G. MANDELL, Michel PERBOST.
Application Number | 20200348257 16/826035 |
Document ID | / |
Family ID | 1000004969599 |
Filed Date | 2020-11-05 |
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United States Patent
Application |
20200348257 |
Kind Code |
A1 |
BOYANOV; Boyan ; et
al. |
November 5, 2020 |
BIOCHEMICALLY ACTIVATED ELECTRONIC DEVICE
Abstract
A kit includes an array of charge sensors attached to a
solid-phase substrate , wherein individual charge sensors in the
array respectively comprise a channel and a polymerase and are
located at different addressable locations on the solid-phase
substrate; and a liquid comprising a plurality of different nucleic
acids, each of the different nucleic acids including a unique label
to be detected by a charge sensor of the array.
Inventors: |
BOYANOV; Boyan; (San Diego,
CA) ; MANDELL; Jeffrey G.; (San Diego, CA) ;
BAI; Jingwei; (San Diego, CA) ; GUNDERSON; Kevin
L.; (Encinitas, CA) ; CHEN; Cheng-Yao; (San
Diego, CA) ; PERBOST; Michel; (San Diego,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ILLUMINA, INC. |
San Diego |
CA |
US |
|
|
Family ID: |
1000004969599 |
Appl. No.: |
16/826035 |
Filed: |
March 20, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14798762 |
Jul 14, 2015 |
10605766 |
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16826035 |
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62171523 |
Jun 5, 2015 |
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62161709 |
May 14, 2015 |
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62069198 |
Oct 27, 2014 |
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62024856 |
Jul 15, 2014 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B82Y 15/00 20130101;
G01N 27/4145 20130101; B01J 2219/00722 20130101; C12Q 1/6869
20130101; C12Q 1/6825 20130101; B01J 2219/00653 20130101 |
International
Class: |
G01N 27/414 20060101
G01N027/414; C12Q 1/6869 20060101 C12Q001/6869; C12Q 1/6825
20060101 C12Q001/6825 |
Claims
1. A kit, comprising: an array of charge sensors attached to a
solid-phase substrate , wherein individual charge sensors in the
array respectively comprise a channel and a polymerase and are
located at different addressable locations on the solid-phase
substrate; and a liquid comprising a plurality of different nucleic
acids, each of the different nucleic acids including a unique label
to be detected by a charge sensor of the array.
2. The kit of claim 1, wherein the polymerase is immobilized to a
respective individual charge sensor.
3. The kit of claim 2, wherein the polymerase is immobilized by
covalent attachment.
4. The kit of claim 2, wherein the polymerase is immobilized by
noncovalent attachment.
5. The kit of claim 1, wherein the unique labels comprise charge
labels.
6. The kit of claim 5, wherein the charge labels comprise phosphate
moieties.
7. The kit of claim 1, wherein at least some of the individual
charge sensors in the array each comprises its own respective
single polymerase.
8. The kit of claim 7, wherein greater than 50% of the individual
charge sensors in the array each comprises its own respective
single polymerase.
9. The kit of claim 8, further comprising an amplifier coupled to
each of the individual charge sensors in the array, the amplifier
to acquire signal from the individual charge sensors that comprise
their own respective single polymerases, and the amplifier not to
acquire signal from individual charge sensors that do not comprise
any polymerase or that comprise more than one of their own
respective polymerases.
10. The kit of claim 1, wherein each polymerase is attached to a
repellant moiety that occupies a volume of space that sterically
hinders more than one of the polymerases from attaching to an
individual charge sensor.
11. The kit of claim 10, wherein the repellant moiety comprises a
bead or particle, the bead or particle comprising a solid support
material, gel, or a combination thereof.
12. The kit of claim 1, wherein the channel is a carbon
nanotube.
13. The kit of claim 1, wherein the channel is a nanowire.
14. The kit of claim 1, wherein the charge sensor comprises a
pinched region having a diameter that is less than a diameter of
other regions of the charge sensor.
15. The kit of claim 1, further comprising electrodes attached to
the solid substrate for producing an electrophoretic force to
position the polymerase.
16. A composition comprising the kit of claim 1, and further
comprising: a plurality of nucleic acid templates; and a plurality
of nascent nucleic acid strands, wherein the polymerase of each of
the individual charge sensors in the array binds one of the nucleic
acid templates, one of the nascent nucleic acid strands, and one of
the different nucleic acids and brings the unique label of that
nucleic acid within sufficient proximity to the channel of that
charge sensor to create a distinguishable signal.
17. The composition of claim 16, wherein the charge sensors are
functionalized with oligonucleotides and the unique labels
hybridize to one or more of the oligonucleotides.
18. The composition of claim 16, wherein the unique label is
cleavable from that nucleic acid by the polymerase leaving the
unique label free to diffuse away from the charge sensor.
19. The composition of claim 16, wherein the unique labels comprise
oligonucleotide moieties.
20. The composition of claim 19, wherein the oligonucleotide
moieties hybridize to respectively complementary sequences
immobilized to the charge sensor.
21. The composition of claim 19, wherein each of the
oligonucleotide moieties comprises a plurality of inosines.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/798,762, filed on Jul. 14, 2015, which is
based on, and claims the benefit of, U.S. Provisional Application
No. 62/171,523, filed Jun. 5, 2015; U.S. Provisional Application
No. 62/161,709, filed May 14, 2015; U.S. Provisional Application
No. 62/069,198, filed Oct. 27, 2014; and U.S. Provisional
Application No. 62/024,856, filed Jul. 15, 2014, the entire
contents of each of which are incorporated herein by reference.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Aug. 25, 2015, is named IP-1285-US_SL.txt and is 15,955 bytes in
size.
BACKGROUND
[0003] This disclosure relates generally to biosensor-based
detection, and more specifically to biosensors that can be used for
nucleic acid sequencing.
[0004] Currently available commercial platforms for sequencing DNA
are relatively costly. The majority of these platforms use a
`sequencing-by-synthesis` approach, so called because DNA polymers
are synthesized while detecting the addition of each monomer (i.e.
nucleotide) to the growing polymer structure. Because a template
DNA strand strictly directs synthesis of a new DNA polymer, one can
infer the sequence of the template DNA from the series of
nucleotide monomers that were added to the growing strand during
the synthesis. Monitoring the reaction uses relatively expensive
hardware such as lasers, detection optics and complex fluid
delivery systems. The most successful commercial platforms to date
also require expensive reagents and hardware to amplify the DNA
templates before sequencing-by-synthesis can even begin. The
complexity and expense of these platforms has hindered their use in
some clinical and research contexts where there is a clear need for
DNA sequencing technology.
[0005] Thus, there exists a need for improvements to nucleic acid
sequencing platforms to make them more cost effective, rapid and
convenient. The present disclosure addresses this need and provides
other advantages as well.
BRIEF SUMMARY
[0006] The present disclosure provides a first method of nucleic
acid sequencing. The method can include the steps of (a) providing
a polymerase tethered to a solid support charge sensor; (b)
providing one or more labeled nucleotides, whereby the presence of
the label can be detected by the charge sensor when the label is in
proximity to the charge sensor; and (c) detecting incorporation of
the labeled nucleotide into a nascent strand complementary to a
template nucleic acid.
[0007] The present disclosure also provides a method for attaching
reaction components to charge sensors. The method can include the
steps of (a) providing a solid support including a plurality of
charge sensors, wherein each of the charge sensors has a capacity
to attach a plurality of reaction components; (b) providing a fluid
containing a plurality of reaction components of a particular type;
and (c) contacting the solid support with the fluid under
conditions wherein (i) the plurality of reaction components of the
particular type are in fluid communication with the plurality of
charge sensors, (ii) a greater number of reaction components of the
particular type is in the fluid than the number of charge sensors
on the solid support; and (iii) reaction components of the
particular type from the fluid attach to the charge sensors under
conditions that result in a solid support where each of the charge
sensors is attached to a single one of the reaction components.
[0008] In some embodiments the method for attaching reaction
components to charge sensors can include the steps of (a) providing
a solid support including a plurality of charge sensors, wherein
each of the charge sensors has a capacity to attach a plurality of
reaction components; (b) providing a fluid containing a plurality
of reaction components of a particular type, wherein each of the
reaction components of the particular type is bound to a repellant
moiety; and (c) contacting the solid support with the fluid under
conditions wherein (i) the plurality of reaction components of the
particular type are in fluid communication with the plurality of
charge sensors, (ii) a greater number of reaction components of the
particular type is in the fluid than the number of charge sensors
on the solid support; (iii) reaction components from the fluid
attach to the charge sensors, and (iv) the repellant moiety bound
to each of the reaction components prevents more than one of the
reaction components in the plurality of reaction components from
attaching to each of the charge sensors.
[0009] A method for attaching reaction components to charge sensors
can include the steps of (a) providing a solid support including a
plurality of charge sensors, wherein each of the charge sensors has
a capacity to attach a plurality of reaction components; (b)
providing a fluid containing a plurality of reaction components of
a particular type; (c) contacting the solid support with the fluid
under conditions wherein (i) the plurality of reaction components
of the particular type are in fluid communication with the
plurality of charge sensors, (ii) a greater number of reaction
components of the particular type is in the fluid than the number
of charge sensors on the solid support; and (iii) reaction
components of the particular type from the fluid attach to the
charge sensors, thereby forming modified charge sensors that are
attached to multiple reaction components of the particular type
from the fluid; and (d) removing one or more of the reaction
components of the particular type from each of the modified charge
sensors to leave a single one of the reaction components of the
particular type attached to each of the modified charge
sensors.
[0010] The present disclosure provides a method of detecting a
nucleotide. The method can include the steps of (a) providing a
nucleotide binding protein (e.g. a polymerase) tethered to a solid
support charge sensor; (b) providing one or more labeled
nucleotides, whereby the presence of the label can be detected by
the charge sensor when the label is in proximity to the charge
sensor; and (c) detecting binding of the labeled nucleotide to the
protein using the charge sensor.
[0011] In particular embodiments, a method of nucleic acid
sequencing can be performed by (a) providing a polymerase tethered
to a solid support charge sensor; (b) providing one or more labeled
nucleotides, whereby the presence of the label can be detected by
the charge sensor when the label is in proximity to the charge
sensor; and (c) detecting incorporation of the labeled nucleotide
into a nascent strand complementary to a template nucleic acid
using the charge sensor.
[0012] A method of nucleic acid sequencing provided by the present
disclosure can include the steps of (a) providing a polymerase
tethered to a solid support charge sensor; (b) providing one or
more labeled nucleotides, whereby the presence of the label can be
detected by the charge sensor when the label is in proximity to the
charge sensor, wherein the one or more labeled nucleotides have
reversible terminator moieties; (c) detecting incorporation of the
one or more labeled nucleotides into a nascent strand complementary
to a template nucleic acid using the charge sensor, thereby forming
a reversibly terminated nascent strand; (d) modifying the
reversible terminated nascent strand to render the nascent strand
capable of further incorporation of nucleotide; and (e) repeating
(b) through (d) to obtain a sequence of the template nucleic
acid.
[0013] Also provided is a method of nucleic acid sequencing that
includes the steps of (a) providing a polymerase tethered to a
solid support charge sensor; (b) contacting the polymerase with a
template nucleic acid and one or more different nucleotide types
under conditions wherein the polymerase catalyzes addition of the
one or more nucleotide types to form a nucleic acid complement of
the nucleic acid template, and wherein the addition of one or more
different nucleotide types produces a conformational signal change
from the polymerase that is detected by the charge sensor; (c)
detecting a change in the signal from the polymerase using the
charge sensor; and (d) determining the rate, polarity, amplitude or
time duration for the change in the signal for the addition of the
one or more different nucleotide type, thereby determining a
sequence of nucleotides for the template nucleic acid.
BRIEF DESCRIPTION OF THE DRAWINGS
[0014] FIG. 1 shows a polymerase attached to a charge sensor via a
tether.
[0015] FIG. 2 shows polymerases attached to charge sensors via
nucleic acid tethers and bound to nucleotides that can be
distinguished based on charge or proximity to the sensor.
[0016] FIG. 3 shows polymerases attached to charge sensors via
nucleic acid tethers and bound to nucleotides that can be
distinguished based on charge.
[0017] FIG. 4 shows a polymerase tethered to a charge sensor,
wherein the charge sensor is also attached to a plurality of
oligonucleotides capable of binding to labels on nucleotides.
[0018] FIG. 5 shows a nucleotide label having two negatively
charged oxygens at the end of an oligonucleotide moiety of the
label.
[0019] FIG. 6 shows charge tags that can be detected using a charge
sensor.
[0020] FIG. 7 shows aligned sequences (SEQ ID NOS 1-3,
respectively, in order of appearance) of Klenow fragment (PolIxxo)
and Bsu-LF (Bsuxxx1), in which the location of the O-helix finger
domain of Klenow fragment and the aligned location in Bsu-LF is
boxed. Also shown in the inset at the right is a model showing the
three dimensional locations of several residues of the Klenow
fragment O-helix finger juxtaposed with a model of a nanotube.
[0021] FIG. 8 shows a charge sensor having a pinched region and a
polymerase tethered to the charge sensor at the pinched region. The
polymerase is complexed with a template strand and nascent strand
of a nucleic acid.
[0022] FIG. 9 shows a diagrammatic representation for a method of
introducing a pinch into silicon using local oxidation.
[0023] FIG. 10 shows a charge sensor that is attached to a
polymerase (Pol) via a tether having a nucleic acid sequence
(generically represented as a sequence of 10 Ns). The polymerase is
complexed to a target nucleic acid and binding sites for labels
associated with four different nucleotides (ATP, GTP, CTP and TTP,
respectively) are indicated.
[0024] FIG. 11 shows a charge sensor that is attached to a
polymerase (Pol) via a tether having a nucleic acid sequence
(generically represented as a sequence of 10 Ns). The polymerase is
complexed to a target nucleic acid and a labeled CTP analog. The
label on the CTP analog includes a nucleic acid region having
inosines (I) and a specificity region (A'B'C') that hybridizes to a
complementary region on the tether (ABC).
[0025] FIG. 12 shows a tethered polymerase in four different
positional states relative to the charge sensor due to the binding
of four different nucleotide analogs. Each of the nucleotide
analogs has an oligonucleotide moiety of the same length as the
other 3 nucleotide analogs, but each nucleotide analog has a
specific binding sequence that binds to a different region of the
tether compared to the regions where the other nucleotide analogs
bind.
DETAILED DESCRIPTION
[0026] Embodiments of the present disclosure relate generally to
apparatus, compositions and methods for single molecule detection
useful in applications such as nucleotide incorporation events
detected in nucleic acid sequencing procedures. There is a need for
improved detection systems which provide long sequencing reads in
high-throughput manner. Embodiments of the invention set forth
herein satisfy this need and provide other advantages as well.
[0027] Complementary metal-oxide-semiconductor (CMOS)-based sensing
schemes have been used for nucleic acid sequencing. Current
CMOS-based sensing schemes exploit electrochemical detection of the
by-products of the DNA polymerization that occurs in an SBS
reaction (e.g. either protons or pyrophosphate). These methods
provide advantages of being lightless and label-free. Being
lightless, the reactions do not require expensive optics for
detection. Cost and complexity of preparing reagents is typically
reduced when label free reagents are used. However, disadvantages
of current CMOS-based sensing schemes are that the reaction
by-products, that are to be detected, are mobile and have a natural
tendency to diffuse away from the reaction zone, which can result
in cross-talk between neighboring sites when attempting to perform
multiplexed sequencing reactions. Given the large size of most
genomes and the limited read length for typical sequencing
reactions, multiplexing is very important to achieve desired
coverage levels for research and clinical applications of
sequencing technology.
[0028] The present disclosure provides a unique detection modality
that can be used for nucleic acid sequencing and for detection of
nucleic acids and other analytes in general. An exemplary
embodiment is shown in FIG. 1. Briefly, polymerase 1 is immobilized
on the gate 5 of a silicon nanowire field-effect transistor (FET) 2
with a tether 3. Optionally, the nanowire can be made of material
other than silicon or the nanowire can be replaced with a nanotube.
Optionally, the tether 3 can be a conductive polymer strand, as
indicated by the positive charge 6 at the end of the tether that is
proximal to the polymerase and the negative charge 7 at the end of
the tether that is distal to the polymerase and attached to the
gate 5. The ssDNA 4 to be sequenced is bound to polymerase 1 after
having been introduced in solution along with nucleotides and other
reactants. As the complimentary strand is synthesized, disturbances
in the charge distribution in the vicinity of the FET 2 are
generated, either as a result of conformational changes of the
polymerase 1, or due to presence of the nucleotides, possibly
modified with an electrically active tag in the vicinity of the FET
2. Those modifications in the charge distribution are sensed by the
nanowire-FET 2 and detected as a modulation in the FET
transconductance current.
[0029] Some advantages of the FET-based apparatus and methods set
forth herein are: (1) single-molecule sensitivity can be achieved
with a properly scaled FET (2) high degree of parallelization (also
called "multiplexability") is facilitated since the detected charge
disturbance is localized in the vicinity of the polymerase, thereby
avoiding cross-talk between neighboring FET sites (3) the optional
use of a conducting tether assists in transmitting the charge
disturbance to the gate and minimizes the undesirable effects of
screening from the biological solution and (4) silicon nanowire FET
can be conveniently manufactured using processes that are
compatible with semiconductor manufacturing facilities.
[0030] Terms used herein will be understood to take on their
ordinary meaning unless specified otherwise. Examples of several
terms used herein and their definitions are set forth below.
[0031] As used herein, the term "array" refers to a population of
charge sensors or molecules that are attached to one or more
solid-phase substrates such that the charge sensors or molecules
can be differentiated from each other according to their relative
location. An array can include different molecules that are each
located at a different addressable location (e.g. at different
charge sensors) on a solid-phase substrate. Alternatively, an array
can include separate solid-phase substrates each bearing a
different molecule, wherein the different probe molecules can be
identified according to the locations of the solid-phase substrates
on a surface to which the solid-phase substrates are attached or
according to the locations of the solid-phase substrates in a
liquid such as a fluid stream. The molecules of the array can be
nucleic acid primers, nucleic acid probes, nucleic acid templates
or nucleic acid enzymes such as polymerases and exonucleases.
[0032] As used herein, the term "attached" refers to the state of
two things being joined, fastened, adhered, connected or bound to
each other. For example, a reaction component, such as a
polymerase, can be attached to a solid phase component, such as a
charge sensor, by a covalent or non-covalent bond. A covalent bond
is characterized by the sharing of pairs of electrons between
atoms. A non-covalent bond is a chemical bond that does not involve
the sharing of pairs of electrons and can include, for example,
hydrogen bonds, ionic bonds, van der Waals forces, hydrophilic
interactions and hydrophobic interactions.
[0033] As used herein, the term "charge sensor" is intended to mean
a detection device that translates perturbations at its surface or
in its surrounding electrical field into an electrical signal. For
example, a charge sensor can translate the arrival or departure of
a reaction component into an electrical signal. A charge sensor can
also translate interactions between two reaction components, or
conformational changes in a single reaction component, into an
electrical signal. An exemplary charge sensor is a field effect
transistor (FET) such as a carbon nanotube (CNT), single-walled
carbon nanotube (SWNT) based FET, silicon nanowire (SiNW) FET,
graphene nanoribbon FET (and related nanoribbon FETs fabricated
from 2D materials such as MoS.sub.2, silicene, etc), tunnel FET
(TFET), and steep subthreshold slope devices (see, for example,
Swaminathan et al., Proceedings of the 51st Annual Design
Automation Conference on Design Automation Conference, pg 1-6,
ISBN: 978-1-4503-2730-5 (2014) and Ionescu et al., Nature 479,
329-337 (2011)). Examples of FET and SWNT sensors that can be used
in the methods and apparatus of the present disclosure are set
forth in US Pat. App. Pub. No. 2013/0078622 A1, which is
incorporated herein by reference in its entirety.
[0034] As used herein, the term "cleavable tether" is intended to
mean a chemical linker having a bond that can be selectively broken
by a chemical or physical treatment. Generally, the bond breakage
is selective with respect to the chemical or physical treatment not
having a substantial adverse effect on other reaction components
that are present. A cleavable tether can be susceptible to
selective bond breakage with agents such as, but not limited to,
light, base, acid, heat, enzymes and chemical reagents. Electric
field and physical agitation can also be used to cleave the tether.
In a preferred embodiment the linker is a nucleotide linker. In
some cases the linker comprises a site for cleavage by a sequence
specific restriction endonuclease. However, sequence specific
cleavage sites need not be present in some linkers used in
accordance with methods set forth herein.
[0035] As used herein, the term "concatameric repeat" is intended
to mean a serially repeating string of a particular nucleotide
sequence in a single nucleic acid molecule. Concatameric repeats
can be produced, for example, by rolling circle amplification (RCA)
whereby the repeated nucleotide sequence is the complement of the
template that is replicated by RCA.
[0036] As used herein, the term "conducting tether" is intended to
mean a chemical linker through which electricity can be conducted
or through which the electrical effects of an electric field can be
transmitted. A conducting tether can be used to chemically link a
reaction component to a charge sensor and to conduct electricity
between the reaction component and the charge sensor. Exemplary
conducting tethers include, but are not limited to, those having a
doped polythiophene, poly(3,4-ethylenedioxythiophene),
polyacetylene, polypyrrole, polyaniline, polyfluorene,
polyphenylene, polypyrene, polyazulene, polynaphthalenes,
polycarbazole, polyindole, or polyazepine structure. Linkers that
can be useful as conducting tethers are also set forth in US Pat.
App. Pub. No. 2010/0073847 A1, which is incorporated herein by
reference in its entirety.
[0037] As used herein, the term "conformational signal change"
means the appearance, disappearance, or alteration of a detectable
signal from a molecule in response to a change in the structure,
shape or arrangement of parts of the molecule. For example, the
signal change can be due to a change in the interaction of a label
with a first portion of the molecule to interact with a second
portion of the molecule. The term, when specifically recited, is
intended to distinguish from changes in signal that arise from a
label of a molecule due to a change in the interaction of the label
with a reactant that binds specifically to the molecule or a change
in the interaction of the label with a product that results from
catalytic activity of the molecule.
[0038] As used herein, the term "conformationally labeled," when
used in reference to a molecule, means having at least one label
that is responsive to a change in the structure of the molecule, a
change in the shape of the molecule or a change in the arrangement
of parts of the molecule. The molecule can be, for example, a
polymerase, reverse transcriptase, exonuclease or other nucleic
acid enzyme such as those set forth herein below. The parts of the
molecule can be, for example, atoms that change relative location
due to rotation about one or more chemical bonds occurring in the
molecular structure between the atoms. The parts of the molecule
can be domains of a macromolecule such as those commonly known in
the relevant art. For example, polymerases include domains referred
to as the finger, palm and thumb domains. In the case of proteins
the parts can be regions of secondary, tertiary or quaternary
structure. The label(s) can be attached to the molecule, for
example, via a covalent linkage. However, the label(s) need not be
attached to the molecule, being, for example, located in proximity
to the molecule. In particular embodiments the label is not
attached to a reactant or product of the molecule such as a
nucleotide or nucleic acid.
[0039] As used herein, the term "different", when used in reference
to nucleic acids, means that the nucleic acids have nucleotide
sequences that are not the same as each other. Two or more
different nucleic acids can have nucleotide sequences that are
different along their entire length. Alternatively, two or more
different nucleic acids can have nucleotide sequences that are
different along a substantial portion of their length. For example,
two or more different nucleic acids can have target nucleotide
sequence portions that are different for the two or more molecules
while also having a universal sequence portion that is the same on
the two or more molecules. The term "different" can be similarly
applied to other molecules, such as polymerases and nucleic acid
enzymes.
[0040] As used herein, the term "each," when used in reference to a
collection of items, is intended to identify an individual item in
the collection but does not necessarily refer to every item in the
collection. Exceptions can occur if explicit disclosure or context
clearly dictates otherwise.
[0041] As used herein, the term "fluidic communication," when used
in reference to a molecule in a fluid and a site in contact with
the fluid, refers to the ability of the molecule to move in or
through the fluid to contact or enter the site. The term can also
refer to the ability of the molecule to separate from or exit the
site to enter the solution. Fluidic communication can occur when
there are no barriers that prevent the molecule from entering the
site, contacting the site, separating from the site and/or exiting
the site. However, fluidic communication is understood to exist
even if diffusion is retarded, reduced or altered so long as access
is not absolutely prevented.
[0042] As used herein, the term "label," when used in reference to
a reaction component, is intended to mean a detectable reaction
component or detectable moiety of a reaction component. A useful
label is a charge label (also called a charge tag) that can be
detected by a charge sensor. A label can be intrinsic to a reaction
component that is to be detected (e.g. a charged amino acid of a
polymerase) or the label can be extrinsic to the reaction component
(e.g. a non-naturally occurring modification of an amino acid). In
some embodiments a label can include multiple moieties having
separate functions. For example a label can include a linker
component (such as a nucleic acid) and a charge tag component.
[0043] As used herein, the term "non-natural," when used in
reference to a moiety of a molecule, is intended to refer to a
moiety that is not found attached to the molecule in its natural
milieu or in a biological system unperturbed by human, technical
intervention. Typically, non-natural moieties are synthetic
modifications of molecules that render the molecules structurally
or chemically distinct from the unmodified molecule or from
molecules having natural modifications. As used herein, the term
"non-natural," when used in reference to an analog used for a
process, is intended to mean an analog that is not found in the
natural milieu where the process occurs. Typically, non-natural
analogs are synthetic analogs that are structurally or chemically
distinct from other types of molecules in the class to which the
analog belongs.
[0044] As used herein, the term "nucleic acid" is intended to be
consistent with its use in the art and includes naturally occurring
nucleic acids or functional analogs thereof. Particularly useful
functional analogs are capable of hybridizing to a nucleic acid in
a sequence specific fashion or capable of being used as a template
for replication of a particular nucleotide sequence. Naturally
occurring nucleic acids generally have a backbone containing
phosphodiester bonds. An analog structure can have an alternate
backbone linkage including any of a variety of those known in the
art such as peptide nucleic acid (PNA) or locked nucleic acid
(LNA). Naturally occurring nucleic acids generally have a
deoxyribose sugar (e.g. found in deoxyribonucleic acid (DNA)) or a
ribose sugar (e.g. found in ribonucleic acid (RNA)).
[0045] A nucleic acid can contain any of a variety of analogs of
these sugar moieties that are known in the art. A nucleic acid can
include native or non-native bases. In this regard, a native
deoxyribonucleic acid can have one or more bases selected from the
group consisting of adenine, thymine, cytosine or guanine and a
ribonucleic acid can have one or more bases selected from the group
consisting of uracil, adenine, cytosine or guanine. Useful
non-native bases that can be included in a nucleic acid are known
in the art.
[0046] As used herein, the term "nucleotide" is intended to include
natural nucleotides, analogs thereof, ribonucleotides,
deoxyribonucleotides, dideoxyribonucleotides and other molecules
known as nucleotides. The term can be used to refer to a monomeric
unit that is present in a polymer, for example to identify a
subunit present in a DNA or RNA strand. The term can also be used
to refer to a molecule that is not necessarily present in a
polymer, for example, a molecule that is capable of being
incorporated into a polynucleotide in a template dependent manner
by a polymerase. The term can refer to a nucleoside unit having,
for example, 0, 1, 2, 3 or more phosphates on the 5' carbon. For
example, tetraphosphate nucleotides and pentaphosphate nucleotides
can be particularly useful. Exemplary natural nucleotides include,
without limitation, ATP, UTP, CTP, GTP, ADP, UDP, CDP, GDP, AMP,
UMP, CMP, GMP, dATP, dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP,
dAMP, dTMP, dCMP, and dGMP.
[0047] Non-natural nucleotides also referred to herein as
nucleotide analogs, include those that are not present in a natural
biological system or not substantially incorporated into
polynucleotides by a polymerase in its natural milieu, for example,
in a non-recombinant cell that expresses the polymerase.
Particularly useful non-natural nucleotides include those that are
incorporated into a polynucleotide strand by a polymerase at a rate
that is substantially faster or slower than the rate at which
another nucleotide, such as a natural nucleotide that base-pairs
with the same Watson-Crick complementary base, is incorporated into
the strand by the polymerase. For example, a non-natural nucleotide
may be incorporated at a rate that is at least 2 fold different, 5
fold different, 10 fold different, 25 fold different, 50 fold
different, 100 fold different, 1000 fold different, 10000 fold
different or more when compared to the incorporation rate of a
natural nucleotide. A non-natural nucleotide can be capable of
being further extended after being incorporated into a
polynucleotide. Examples include, nucleotide analogs having a 3'
hydroxyl or nucleotide analogs having a reversible terminator
moiety at the 3' position that can be removed to allow further
extension of a polynucleotide that has incorporated the nucleotide
analog. Examples of reversible terminator moieties that can be used
are described, for example, in U.S. Pat Nos. 7,427,673; 7,414,116;
and 7,057,026 and PCT publications WO 91/06678 and WO 07/123744,
each of which is incorporated herein by reference in its entirety.
It will be understood that in some embodiments a nucleotide analog
having a 3' terminator moiety or lacking a 3' hydroxyl (such as a
dideoxynucleotide analog) can be used under conditions where the
polynucleotide that has incorporated the nucleotide analog is not
further extended. In some embodiments, the nucleotide(s) will not
include a reversible terminator moiety, or the nucleotides(s) will
not include a non-reversible terminator moiety or the nucleotide(s)
will not include any terminator moiety at all. Nucleotide analogs
with modifications at the 5' position are also useful.
[0048] As used herein, the term "protection moiety" is intended to
mean a compound or portion thereof that is attached to a reaction
component to prevent the reaction component from undergoing a
particular reaction. For example, a nucleic acid molecule can be
bound to a nucleic acid enzyme such that the nucleic acid molecule
prevents the nucleic acid enzyme from degradation or modification
by a treatment that would otherwise cause degradation or
modification of the enzyme. An antibody can also serve to bind a
reaction component to protect the reaction component from
degradation, inactivation or other reaction.
[0049] As used herein, the term "reaction component" is intended to
mean a molecule that takes part in a reaction. Examples include,
reactants that are consumed in a reaction, products that are
created by a reaction, catalysts such as enzymes, that facilitate a
reaction, solvents, salts, buffers and other molecules.
[0050] As used herein, the term "repellant moiety" is intended to
mean a molecule or portion thereof that will occupy a space to
prevent or inhibit occupancy of another molecule at the space or to
inhibit juxtaposition of another molecule near the space. A
repellant moiety can act via steric exclusion, charge repulsion,
hydrophobic-hydrophilic repulsion or other forces.
[0051] As used herein, the term "terminator moiety," when used in
reference to a nucleotide, means a part of the nucleotide that
inhibits or prevents the nucleotide from forming a covalent linkage
to a second nucleotide. For example, in the case of nucleotides
having a pentose moiety, a terminator moiety can prevent formation
of a phosphodiester bond between the 3' oxygen of the nucleotide
and the 5' phosphate of the second nucleotide. The terminator
moiety can be part of a nucleotide that is a monomer unit present
in a nucleic acid polymer or the terminator moiety can be a part of
a free nucleotide (e.g. a nucleotide triphosphate). The terminator
moiety that is part of a nucleotide can be reversible, such that
the terminator moiety can be modified to render the nucleotide
capable of forming a covalent linkage to a second nucleotide. In
particular embodiments, a terminator moiety, such as a reversible
terminator moiety, can be attached to the 3' position or 2'
position of a pentose moiety of a nucleotide analog.
[0052] As used herein, the term "solid support" refers to a rigid
substrate that is insoluble in aqueous liquid. The substrate can be
non-porous or porous. The substrate can optionally be capable of
taking up a liquid (e.g. due to porosity) but will typically be
sufficiently rigid that the substrate does not swell substantially
when taking up the liquid and does not contract substantially when
the liquid is removed by drying. A nonporous solid support is
generally impermeable to liquids or gases. Exemplary solid supports
include, but are not limited to, glass and modified or
functionalized glass, plastics (including acrylics, polystyrene and
copolymers of styrene and other materials, polypropylene,
polyethylene, polybutylene, polyurethanes, Teflon.TM., cyclic
olefins, polyimides etc.), nylon, ceramics, resins, Zeonor, silica
or silica-based materials including silicon and modified silicon,
carbon, metals, inorganic glasses, optical fiber bundles, and
polymers. Particularly useful solid supports for some embodiments
are located within a flow cell apparatus. Exemplary flow cells are
set forth in further detail below.
[0053] As used herein, the term "type" (or "species") is used to
identify molecules that share the same chemical structure. For
example, a mixture of nucleotides can include several dCTP
molecules. The dCTP molecules will be understood to be the same
type or species as each other. Similarly, individual DNA molecules
that have the same sequence of nucleotides are the same type or
species.
[0054] The embodiments set forth below and recited in the claims
can be understood in view of the above definitions.
[0055] The present disclosure provides apparatus, compositions and
methods useful for single molecule detection in applications such
as nucleotide incorporation events detected in nucleic acid
sequencing procedures. The apparatus, compositions and methods set
forth herein are particularly useful, for example, in single
molecule nucleic acid sequencing reactions, such as sequencing by
synthesis. However, it will be appreciated that the apparatus,
compositions and methods set forth herein can be used for any other
suitable detection schemes, including, but not limited to single
molecule detection.
[0056] The present disclosure provides a first method of nucleic
acid sequencing. The method can include the steps of (a) providing
a polymerase tethered to a solid support charge sensor; (b)
providing one or more labeled nucleotides, whereby the presence of
the label can be detected by the charge sensor when the label is in
proximity to the charge sensor; and (c) detecting incorporation of
the labeled nucleotide into a nascent strand complementary to a
template nucleic acid.
[0057] The polymerase used in the first method of nucleic acid
sequencing can be tethered to the solid support charge sensor with
a tether comprising nucleic acid. For example, the tether can
comprise deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or
protein nucleic acid (PNA).
[0058] In some embodiments of the first method of nucleic acid
sequencing, the labeled nucleotide is labeled at the y-phosphate
position of the nucleotide. The label can comprise an
oligonucleotide and, optionally, the oligonucleotide is capable of
hybridizing to an immobilized nucleic acid.
[0059] Further optionally, the immobilized nucleic acid used in the
first method of nucleic acid sequencing is part of a tether, the
tether immobilizing a polymerase to the solid support charge
sensor.
[0060] In some configurations of the first method of nucleic acid
sequencing, the polymerase is held in proximity of less than 10, 9,
8, 7, 6, 5, 4, 3, 2, or 1 nm to the charge sensor.
[0061] In some embodiments of the first method of nucleic acid
sequencing, the label is cleaved from the nucleotide after
incorporation, for example, by the polymerase.
[0062] Optionally in the first method of nucleic acid sequencing,
the one or more labeled nucleotides comprise a plurality of charge
tags. For example, the one or more labeled nucleotides can comprise
a unique charge tag for each of four types of nucleotides. The
charge tag can be a negative charge tag, for example, comprising
one or more of: a phosphate group, DMT and/or FMOC. Alternatively,
the charge tag can be a positive charge tag, optionally comprising
a primary amine.
[0063] The one or more labeled nucleotides in the first method of
nucleic acid sequencing can comprise a plurality of different
oligonucleotides (i.e. oligonucleotide moieties) capable of
hybridizing to a plurality of immobilized tether sequences.
Alternatively or additionally, a plurality of different
oligonucleotides can be capable of hybridizing to a plurality of
different locations within a particular tether used to immobilize a
polymerase.
[0064] In some embodiments of the first method of nucleic acid
sequencing, the one or more labeled nucleotides comprise 4
different charge tags and each of said labeled nucleotides
comprises an oligonucleotide capable of hybridizing to the same
immobilized tether sequence.
[0065] In some embodiments of the first method of nucleic acid
sequencing, the polymerase tether and the labels on the labeled
nucleotides contain deoxyribonucleotides that bind complementarily
to each other, ribonucleotides that bind complementarily to each
other, or deoxyribonucleotides that bind complementarily to
ribonucleotides. For example, the polymerase tether and the labels
on the labeled nucleotides can form a DNA:DNA duplex, RNA:RNA
duplex or DNA:RNA heteroduplex. The deoxyribonucleotides or
ribonucleotides can be nucleotide analogs that increase or modify
duplex stability. For example, 2'-O-Methyl (2'-O-Me) or 2'-Fluoro
(2'-F) modified ribonucleotides can be used. The 2'-O-me and 2'-F
RNA modifications are known to increase the melting temperature of
RNA:RNA duplexes by 0.5.degree. C. to 1.degree. C. per base pair,
but result in only small changes in RNA:DNA stability (Majlessi et
al., Nucleic Acids Res. 26:2224-9 (1998), incorporated herein by
reference in its entirety). The increase in stability of the
modified RNA:RNA base pairs can be used to accurately position the
tag along the RNA tether as discussed below.
[0066] Moreover, the charge sensor can be functionalized with
capture oligonucleotides and the labeled nucleotide can comprise an
oligonucleotide label which hybridizes to one or more of the
capture oligonucleotides in the first method of nucleic acid
sequencing.
[0067] The charge sensor used in the first method of nucleic acid
sequencing can comprise a nanowire FET. Optionally, the charge
sensor comprises a carbon nanotube.
[0068] The charge sensor in the first method of nucleic acid
sequencing can be part of an array of charge sensors.
[0069] The detecting step of the first method of nucleic acid
sequencing can comprise detecting a plurality of incorporation
events in succession.
[0070] The methods and apparatus set forth herein can provide long
nucleic acid sequencing reads; fast reads; high throughput
capability for sequencing; and a scalable platform for sequencing.
In some embodiments, any compromises in single read accuracy can be
mitigated by performing multiple overlapping reads due to the
ability of the methods and apparatus set forth herein to provide
throughput in the number of reads performed in parallel.
[0071] An exemplary sensor is shown in FIG. 1. Here a polymerase 1
creates a reaction site where nucleotides can be incorporated into
a primed DNA template 4. The polymerase 1 is attached to a nanowire
FET 2 via a tether 3. The apparatus provides single molecule
sensitivity. Changes in charge distribution at the reaction site
(e.g. polymerase conformation changes, nucleotide incorporation,
arrival or departure of charged tags, changes in proximity of the
polymerase to the charge sensor etc.) transmit to the gate and can
be detected.
[0072] In particular embodiments, an apparatus or method of the
present disclosure uses deeply scaled FinFET transistors as
single-molecule charge sensors. FinFet sensors benefit from
technology already under development by leading edge semiconductor
manufacturers. Furthermore, previously published components can be
used, including but not limited to (1) those used for
immobilization of lysozyme on CNT to observe enzyme processivity in
real time as described in Choi et al, Science, 335, 319 (2012), (2)
those used to immobilize the Pol 1 Klenow fragment on CNT and
observe DNA processivity in real time as described in Olsen et al,
J. Amer. Chem. Soc., 135, 7885 (2013), (3) those used to elucidate
a transduction mechanism as moving charged residues due to protein
allosteric motion as described in Chi et al, NanoLett 13, 625
(2013). The present methods can also employ the apparatus,
components of the apparatus, and methods set forth in US Pat. App.
Pub. No. 2013/0078622 A1. Each of the above references is
incorporated herein by reference in its entirety.
[0073] The apparatus and methods set forth in US Pat. App. Pub. No.
2013/0078622 A1 provide a Debye screening length of 1-2 nm in
50-100 mM NaCl. In this apparatus the allosteric motion must be
near the attachment point of the polymerase to the transistor.
Also, allosteric motion must be base-dependent to enable real-time
discrimination of different types of nucleotides. Such resolution
has not been previously demonstrated.
[0074] An embodiment that can be used to overcome limitations of
some apparatus that utilize allosteric-based detection is
diagrammed in FIG. 2. Here polymerase can be immobilized to a
charge sensor such as a single walled carbon nanotube, silicon
nanowire or FinFET. Immobilization can be via tethers that include
DNA, RNA, PNA or analogs thereof. For convenience of demonstration
the diagram shows four polymerases tethered to the charge sensor,
each polymerase also being bound to a different gamma-phosphate
labeled nucleotide type. As shown, nucleotides have an
oligonucleotide moiety attached to the gamma-phosphate. A beta- or
gamma-phosphate-labeled nucleotide that is properly matched to a
template strand of a target nucleic acid will be held in place by a
polymerase that is also bound to the template long enough to
temporarily hybridize the oligonucleotide moiety to the tether
(e.g. via Watson-Crick base complementarity). The hybridization
causes the oligonucleotide moiety to perturb the field around the
charge sensor which produces a detectable signal due to the change
in transistor current through the charge sensor. The diagram shows
the oligonucleotide moiety entering a field that is within 1-2 nm
of the charge sensor. The properly matched beta- or
gamma-phosphate-labeled nucleotide will be incorporated into a
nascent strand that is hybridized to the template nucleic acid.
This will, in turn, break the bond between the beta phosphate and
the newly incorporated nucleotide. As a result, the oligonucleotide
moiety (whether attached at the beta- or gamma-position of the
nucleotide) is free to dehybridize from the tether and diffuse away
from the charge sensor, thereby returning the field around the
sensor to its unperturbed state. The appearance and disappearance
of signal as the field around the charge sensor is perturbed and
returned to the unperturbed state, respectively, can be correlated
with incorporation of a nucleotide into the nascent strand of the
target nucleic acid.
[0075] Particular embodiments can exploit synergistic binding of
the gamma-phosphate labeled nucleotide to the polymerase and to the
tether. The stability of the oligonucleotide moiety:tether complex
can be relatively low such that the complex does not form for
gamma-phosphate labeled nucleotide that are not also bound to
polymerase (i.e. gamma-phosphate labeled nucleotides that are free
in solution do not substantially bind to the tether). However, the
synergistic effect of the affinities of the nucleotide moiety for
the polymerase and the oligonucleotide moiety for the tether add up
to allow substantial binding affinity overall. In some embodiments,
the synergistic effect can exploit a combination of specific
binding affinity between the nucleotide label and tether along with
weak affinity produced by non-specific binding interactions. For
example, specific binding can result from standard Watson-Crick
base pairing and non-specific binding interactions can result from
interactions of promiscuous bases (e.g. inosine) with native
nucleotides. Thus, when the gamma-phosphate labeled nucleotide is
bound to polymerase during incorporation, synergistic binding
occurs which greatly increases the stability of the interaction
between oligonucleotide moiety and tether. After the gamma
phosphate is cleaved by the polymerase, the synergistic effect is
lost and the oligonucleotide moiety will dissociate from the
tether.
[0076] The type of nucleotide that is incorporated into the nascent
strand at each position of the template strand can be determined
based on unique properties of labels incorporated into each type of
nucleotide. For example, four types of dNTPs can be distinguished
by the position where the oligonucleotide moiety hybridizes to the
tether, the length of the oligonucleotide moiety and/or the
presence of a charged moiety on the label. FIG. 2 provides an
example where four-state discrimination is achieved using 2 charge
tags (other than the negatively charged phosphates of the
oligonucleotide moiety) and two tether hybridization positions.
Specifically, dCTP is uniquely labeled with a negatively charged
extrinsic moiety, dTTP is uniquely labeled with a positively
charged extrinsic moiety, dATP and dGTP are distinguished from the
other two nucleotide types based on absence of any extrinsic charge
moiety, and dATP is distinguished from dGTP based on differential
proximity of the oligonucleotide moieties to the charge sensor when
they are hybridized to the tether.
[0077] It will be understood that different nucleotide types can be
distinguished based on any of a variety of combinations of positive
charge moieties, negative charge moieties and/or tether
hybridization locations. Alternatively or additionally, the charge
moieties used to distinguish different types of nucleotides can
differ in the strengths of the charges, even if the charges have
the same sign. The exemplary configuration shown in FIG. 3 provides
four-state discrimination based on a single tether hybridization
position and four different charge moieties. Specifically, dGTP and
dCTP both contain negatively charged moieties that distinguish them
from dATP and dTTP, and dGTP can be distinguished from dCTP due to
charge that is distinguishably higher than the charge on dCTP.
Similarly, dATP and dTTP can be distinguished from each other due
to the higher positive charge on the dATP moiety compared to the
dTTP moiety.
[0078] As noted previously herein, the precision of tag placement
at specific hybridization positions along a tether can be enhanced
through the use of a tether having ribonucleotides and a nucleotide
label having 2'-O-Me and 2'F modified RNA bases. Alternative
configurations can use a tether that contains 2'-O-Me and 2'F
modified ribonucleotides with label having ribonucleotides, or both
the tether and label can include a mixture of native
ribonucleotides and 2'-O-Me and 2'F modified ribonucleotides.
Although it is possible to use a tether and/or oligonucleotide
moiety that is primarily composed of RNA, it may be desirable to
use a DNA-based or PNA-based tether and/or oligonucleotide to avoid
nuclease sensitivity that is associated with RNA. For example, a
DNA-based or PNA-based tether and/or oligonucleotide can include
native ribonucleotides or non-native ribonucleotide analogs to
achieve binding advantages set forth herein while reducing risk of
unwanted nuclease digestion. In further embodiments, the tether can
include one or more deoxyribonucleotides that are complementary to
ribonucleotides in a nucleotide label or alternatively the tether
can include ribonucleotides that are complementary to
deoxyribonucleotides in a nucleotide label.
[0079] A tether that attaches a polymerase to a charge sensor can
have different binding positions for different nucleotide analogs
as set forth in several exemplary embodiments herein. The binding
positions for two or more nucleotide analogs can overlap or they
can be discrete with no overlap. For example, as shown in FIG. 10,
the binding sites for ATP and GTP analogs overlap on the tether by
2 nucleotides (i.e. there is a 1 nucleotide offset between the two
binding sites). For purposes of illustration, the tether sequence
is depicted as a series of generic "N" nucleotides. Any of a
variety of sequences can be used in accordance with rules of
complementarity and desired hybridization strengths and
specificities. As also shown in FIG. 10, the binding sites for ATP
and TTP on the tether have no overlap, being discrete and separated
by 1 nucleotide. Depending on the length of the tether, length of
the binding sites and length of the oligonucleotide moieties on the
nucleotide analogs, some, all or none of the binding sites on the
tether can overlap.
[0080] The oligonucleotide moiety of a nucleotide analog can have a
sequence of nucleotides that hybridizes specifically to a
complementary sequence on a tether. In some embodiments the
oligonucleotide moiety can also include promiscuous nucleotide
positions that bind non-specifically to a tether. Such positions
can provide a weak interaction between the oligonucleotide moiety
and tether that facilitates the formation of a specific hybrid
structure. For example, as shown in FIG. 11, an oligonucleotide
moiety can include several inosines (I) that are known to bind
promiscuously, albeit weakly, with all four native nucleotides of
DNA. The oligonucleotide moiety and tether can form a weak complex
via interactions between the inosines in the oligonucleotide moiety
and the native nucleotides in the tether. This can allow the
specific portions of the sequence (e.g. indicated as ABC and its
complement A'B'C' in the figure) to associate more rapidly than
they would have if required to diffuse absent formation of a weak
complex. Furthermore, once a specific complex has formed the
inosines can provide further stability.
[0081] The exemplary oligonucleotide moiety in FIG. 11 includes
promiscuous nucleotide positions flanking both sides of the
specific sequence. However, it will be understood that one or more
promiscuous nucleotide positions can be located on only the 5' or
3' side of the specific sequence. Other examples of promiscuous
nucleotide positions include those formed by degenerate
oligonucleotide synthesis or those formed with other nucleotide
analogs known in the art to hybridize promiscuously with 2 or more
types of nucleotides.
[0082] In the examples shown in FIG. 10 and FIG. 11, as well as
other examples set forth herein, the tether is a nucleic acid that
hybridizes to an oligonucleotide moiety of a nucleotide analog. It
will be understood that other binding partners can be used as
tether and label moiety instead of the nucleic acids. The binding
sites can be discrete or overlapping as exemplified above for
nucleic acids. Also, the binding sites can include a combination of
weak, non-specific interacting partners along with stronger,
specific interacting partners.
[0083] Several embodiments set forth herein have exemplified the
use of a plurality of different nucleotide analogs having
oligonucleotide moieties of differing lengths. In such embodiments,
the different nucleotide types can be distinguished based on the
different lengths of the oligonucleotide moieties. Alternatively,
different nucleotide analogs can have oligonucleotide moieties of
the same length. However, each nucleotide analog can have a
specific binding sequence that binds to a different region of a
tether compared to the regions where the other nucleotide analogs
bind. An exemplary configuration is shown in FIG. 12 where binding
of the polymerase to different nucleotide analogs places the
polymerase in one of four distinguishable states. The
oligonucleotide moiety for the ATP analog binds to a location on
the tether that is nearest to the attachment point of the tether to
the polymerase, the oligonucleotide moiety for the TTP analog binds
to a location on the tether that is furthest from the attachment
point of the tether to the polymerase, and the oligonucleotide
moieties for the GTP and CTP analogs bind to respectively distinct
locations on the tether that are at intermediate distances from the
binding sites for the other two nucleotide analogs. As such,
binding of the different nucleotide analogs to the polymerase will
position the polymerase at different distances from the charge
sensor (e.g. causing different size loops to form in the tether as
shown in the figure). The different nucleotide types can be
distinguished based on the differences in signals produced for the
different distances of the polymerase from the sensor. In
embodiments where one or more of the nucleotide analogs includes a
charge tag or other detectable moiety (e.g. attached at the end of
the oligonucleotide moiety that is distal to the nucleotide
moiety), the binding between the oligonucleotide moiety and tether
will position the detectable moiety at different distances from the
charge sensor. In this case, the different nucleotide types can be
distinguished based on the differences in signals produced for the
different distances of the detectable moieties from the sensor.
[0084] As demonstrated by the embodiment diagrammed in FIG. 4, the
tether that attaches polymerase to the charge sensor need not be
capable of hybridizing to the tags present on the nucleotides.
Rather, the charge sensor can be functionalized to attach one or
more oligonucleotides that are complementary to one or more of the
nucleotide types being detected. Discrimination of the different
nucleotides can be achieved based on sign of the charge, strength
of the charge, length of the oligonucleotide moiety that hybridizes
to the surface attached oligonucleotide(s), or proximity/location
on the surface attached oligonucleotide(s) where the
oligonucleotide moiety hybridizes, or a combination thereof.
[0085] Advantages of several configurations set forth above
include, for example, overcoming screening issues by placing
charges within 1-2 nm of the gate with atomic precision, ability to
achieve a higher level of current modulation through the use of
charge tags, and opening up of the space of available polymerases
since base-specific allosteric motion is not required for
detection.
[0086] An exemplary charge tag that can be useful in the apparatus
and methods set forth herein is a phosphate moiety, for example,
located at the 5' end of a nucleic acid moiety. This moiety can be
readily added during available oligonucleotide synthesis protocols
and will result in two negatively charged oxygens at the end of the
oligonucleotide moiety as shown in FIG. 5. Chemical phosphorylation
during oligonucleotide synthesis can be achieved by converting a
DMT protecting group into a 5' phosphate group using 2-[2-(4,4'
Dimethoxytrityloxy)ethylsulfonyl]ethyl-(2-cyanoethyl)-(N,N-diisopropyl)-p-
hosphoramidite (available from Glen Research, Sterling Va., catalog
No. CPR 10-1900). A series of charge tags having different numbers
of negative charges can be made using
Tris-2,2,2-[3-(4,4'-dimethoxytrityloxy)propyloxymethyl]ethyl-[(2-cyanoeth-
yl)-(N,N-diisopropyl)]-phosphoramidite (available from Glen
Research, Sterling Va., catalog No. 10-1922-xx),
1,3-bis-[5-(4,4'-dimethoxytrityloxy)pentylamido]propyl-2-[(2-cyanoethyl)--
(N,N-diisopropyl)]-phosphoramidite (available from Glen Research,
Sterling Va., catalog No. 10-1920-xx),
1-[5-(4,4'-dimethoxytrityloxy)pentylamido]-3-[5-fluorenomethoxycarbonylox-
ypentylamido]-propyl-2-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite
(available from Glen Research, Sterling Va., catalog No.
10-1921-xx), or oligonucleotide dendrimers which contain various
numbers of DMT (4,4'-dimethoxytrityl) or Fmoc
(Fluorenylmethyloxycarbonyl) moieties, such as those available from
Glen Research or shown in FIG. 6. A useful positively charged tag
is
2-[2-(4-Monomethoxytrityl)aminoethoxy]ethyl-(2-cyanoethyl)-N,N-diisopropy-
l)-phosphoramidite (available from Glen Research, Sterling Va.,
catalog No. 10-1905-xx). Another useful positively charged moiety
is a 5' primary amine which would have a single positive charge at
the appropriate pH.
[0087] Table I provides a listing of useful modifications and
charges that can be used as labels in an apparatus or method set
forth herein.
TABLE-US-00001 TABLE I 5' Terminus Reagents Final Charge State 5'
OH N/A Neutral 5' Phosphate CPR 10-1900 (Glen Res.) -2 5' Phosphate
(x2) CPR 10-1900 -4 and symmetric doubler (Glen Res.) 5' Phosphate
(x3) CPR 10-1900 -6 and symmetric trebler (Glen Res.) 5' primary
amine 5' amino-modifier 5 +1
[0088] The present disclosure provides a method for attaching
reaction components to charge sensors. The method can include the
steps of (a) providing a solid support including a plurality of
charge sensors, wherein each of the charge sensors has a capacity
to attach a plurality of reaction components; (b) providing a fluid
containing a plurality of reaction components of a particular type;
and (c) contacting the solid support with the fluid under
conditions wherein (i) the plurality of reaction components of the
particular type are in fluid communication with the plurality of
charge sensors, (ii) a greater number of reaction components of the
particular type is in the fluid than the number of charge sensors
on the solid support; and (iii) reaction components of the
particular type from the fluid attach to the charge sensors under
conditions that result in a solid support where each of the charge
sensors is attached to a single one of the reaction components.
[0089] In some embodiments the method for attaching reaction
components to charge sensors can include the steps of (a) providing
a solid support including a plurality of charge sensors, wherein
each of the charge sensors has a capacity to attach a plurality of
reaction components; (b) providing a fluid containing a plurality
of reaction components of a particular type, wherein each of the
reaction components of the particular type is bound to a repellant
moiety; and (c) contacting the solid support with the fluid under
conditions wherein (i) the plurality of reaction components of the
particular type are in fluid communication with the plurality of
charge sensors, (ii) a greater number of reaction components of the
particular type is in the fluid than the number of charge sensors
on the solid support; (iii) reaction components from the fluid
attach to the charge sensors, and (iv) the repellant moiety bound
to each of the reaction components prevents more than one of the
reaction components in the plurality of reaction components from
attaching to each of the charge sensors.
[0090] A method for attaching reaction components to charge sensors
can include the steps of (a) providing a solid support including a
plurality of charge sensors, wherein each of the charge sensors has
a capacity to attach a plurality of reaction components; (b)
providing a fluid containing a plurality of reaction components of
a particular type; (c) contacting the solid support with the fluid
under conditions wherein (i) the plurality of reaction components
of the particular type are in fluid communication with the
plurality of charge sensors, (ii) a greater number of reaction
components of the particular type is in the fluid than the number
of charge sensors on the solid support; and (iii) reaction
components of the particular type from the fluid attach to the
charge sensors, thereby forming modified charge sensors that are
attached to multiple reaction components of the particular type
from the fluid; and (d) removing one or more of the reaction
components of the particular type from each of the modified charge
sensors to leave a single one of the reaction components of the
particular type attached to each of the modified charge
sensors.
[0091] Useful charge devices include analytical devices that can
incorporate a reaction component in direct spatial contact with a
transduction element in a way to allow the rapid and convenient
conversion of reaction events to detectable signals. Devices based
on field-effect transistors (FETs) can directly translate
interactions between reaction components (e.g., polymerases) and
the transistor surface into readable electrical signals. In a
standard FET, current flows along a conducting path (the channel)
that is connected to two electrodes, (the source and the drain).
The channel conductance between the source and the drain is
switched on and off by a third (gate) electrode that can be
capacitively coupled through a thin dielectric layer.
[0092] In particular embodiments, FETs are configured to accomplish
single molecule detection. More particularly, these charge sensors
can be configured to monitor the dynamics of a single molecule
reaction. Any type of conduction channel that is generally found in
field effect transistors can be used in an apparatus or method set
forth herein. Exemplary conduction channels are formed from metals,
metal oxides, semiconductors, or nanometer-scale conductors such as
nanowires, or graphene.
[0093] Particularly useful charge sensors for single molecule
detection are single-walled carbon nanotubes (SWNTs). See, for
example, Star et al., Nano. Lett. 3, 459 (2003); Star et al., Org.
Lett. 6, 2089 (2004); Besterman et al., Nano. Lett. 3, 727 (2003);
Gruner, Anal. Biooanal. Chem. 384, 322 (2005); Chen et al. Proc.
Natl. Acad. Sci. U.S.A. 100, 4984 (2003) and US Pat App. Pub. No.
2013/0078622 A1, each of which is incorporated herein by reference
in its entirety. SWNTs are extremely small conductors, typically on
the order of about 1 nanometer in diameter.
[0094] A SWNT can be coated with a chemoselective polymer, metal or
metal oxide nanoparticle, or reaction components like proteins,
nucleic acids or antibodies. See for example, Besterman et al.,
Nano. Lett. 3, 727 (2003); and Chen et al. Proc. Natl. Acad. Sci.
U.S.A. 100, 4984 (2003). Single reaction components can be attached
to these SWNT and other charge sensors using methods set forth
herein.
[0095] In some embodiments a single reaction component can be
attached to a charge sensor by creating one single covalent defect
on the charge sensor, for example, using techniques set forth in
Goldsmith et al. Science 315, 77 (2007), which is incorporated
herein by reference in its entirety. For example a SWNT can be
produced having a single defect such that a variety of attachment
chemistries can be used to link a single reaction component to the
reactive defect site selectively, without coating the rest of the
SWNT with additional reaction components. SWNTs can also be
attached to reaction components by non-covalent means, for example,
using techniques set forth in Chen et al, J. Am. Chem. Soc. 123,
3838 (2001), which is incorporated herein by reference in its
entirety. These methods can be modified as set forth herein to
reliably bind a single reaction component non-covalently to a
SWNT.
[0096] SWNTs are semiconductors with electronic bandgaps that can
vary from 1 electron volt to effectively zero. SWNTs are useful as
conduction channels because single molecule sensing devices can be
fabricated from SWNT wires of any type, with or without gate
electrodes, and on glass, plastic, or silicon substrates. Useful
SWNTs and their configurations for single molecule detection are
set forth in US Pat App. Pub. No. 2013/0078622 A1, which is
incorporated herein by reference in its entirety.
[0097] Other charge sensors that can be modified for use in an
apparatus or method set forth herein include, without limitation,
silicon nanowire (SiNW) FET, FET made of III-V materials, silicon
FinFET, graphene nanoribbon FETs as well as nanoribbon FETs from
other 2D materials such as MoS.sub.2 and silicene, tunnel FET
(TFET), and steep subthreshold slope devices (see, for example,
Swaminathan et al., Proceedings of the 51st Annual Design
Automation Conference on Design Automation Conference, pg 1-6,
ISBN: 978-1-4503-2730-5 (2014) and Ionescu et al., Nature 479,
329-337 (2011)). Carbon nanotubes can also be useful.
[0098] A plurality of charge sensors can be provided in the form of
an array of charge sensors. The array can include at least 10, 100,
1.times.10.sup.3, 1.times.10.sup.4, 1.times.10.sup.4,
1.times.10.sup.4 or more charge sensors. Each individual charge
sensor can be located at a discrete location in the array that is
separated from the other charge sensors in the array. For example,
each charge sensor can reside in a well or depression in a solid
support. The locations, even when separated from each other, can
optionally be in fluid contact with a bulk solution. In such a
configuration, multiplex reactions can occur on the array of charge
sensors by delivering common reagents to all of the charge sensors
via bulk fluid delivery. Taking nucleic acid sequencing reactions
as an example, nucleotides can be delivered via bulk solution to an
array of wells (or other features), each well (or other feature)
hosting an individual sequencing reaction. The nucleotide delivery
will result in parallel sequencing reactions at the wells (or other
features).
[0099] A charge sensor, such as a Si nanowire can have dimensions
that are less than 10 nm wide and greater than 100 nm long. A Si
nanowire or other charge sensor can be placed in a well that is 10
nm.times.10 nm, 50 nm.times.100 nm or larger. For example, a well
within which a charge sensor resides can have an opening on a
surface that is at least 100 nm.sup.2, 1000 nm.sup.2, 5000
nm.sup.2, 1.times.10.sup.4 nm.sup.2, or larger. The circuitry to
read out the signal from the charge sensing element can occupy an
area of the solid support that is 1 micron.times.1 micron or
larger.
[0100] In some embodiments, a charge sensor will have a uniform
width along the entire length. Alternatively, a charge sensor can
have substantial variable width along its length. For example, a
charge sensor can include a region of relatively narrow width, akin
to a `pinched` region. The width of the pinch region can have a
diameter that is, for example, 75%, 50%, 40%, 30%, 20% or 10% of
the diameter of the relatively larger width regions of the charge
sensor. The pinch can provide the advantage of increasing the
effect of a polymerase's charged residue motion on the sensor. A
further benefit is relaxed tolerances for channel fabrication and
opening/alignment of the first layer dielectric passivation of the
FET sensor. For example a charge sensor having a 20 nm diameter
with a 10 nm pinched area can be particularly useful. Other
dimensions for a charge sensor with a pinched region include, for
example diameters of at least 25, 30, 35, or 40 nm or more with a
pinched region that is at most 10, 20, 30, 40, or 50 nm long.
Sensors having smaller diameters can also be used for example those
having diameters of at least 5, 10, 15, or 20 nm can have a pinched
region with a length in the exemplified range. It will be
understood that in some embodiments the maximum diameter of a
charge sensor will be 50 nm, 40 nm, 30 nm, 20 nm 10 nm or less.
[0101] A tether for a polymerase or other enzyme can be attached to
a pinched region or at a point that is proximal to the pinched
region. Thus, the tether can be placed to localize a polymerase or
other enzyme in proximity to the pinched region.
[0102] An exemplary implementation of a charge sensor having a
pinched region is shown in FIG. 8. The pinched region can be
fabricated via appropriate lithographic mask design where the shape
of the mask is such that it produces the desired pinch.
Alternatively, proximity effects during lithographic patterning may
be used to locally pinch the charge sensor by placing electrodes
orthogonal to the sensor in close proximity to the region to be
pinched. In addition to producing the desired pinch, the proximity
electrodes can be used to produce an electrophoretic force that
positions the enzyme at the pinch region.
[0103] Another exemplary implementation is shown in FIG. 9. Here a
pinch is introduced into silicon using local oxidation. In a first
step a silicon wire is coated with an oxidation barrier, such as
Silicon Nitride (SiNx). A region of the silicon wire can be exposed
using local lithography to etch away the oxidation barrier. Then
the partially coated wire can be oxidized to reduce the width of
the silicon wire at the exposed region. Further etching can be
carried out to expose the silicon pinch for chemical
conjugation.
[0104] Those skilled in the art will recognize that the above
methods for producing a pinch in a charge sensor are exemplary and
not limiting; a pinch may be produced with a number of other
methods common in semiconductor manufacturing, e.g., through the
use of sacrificial layers, selective deposition and etch, or
additional lithographic masks, among others.
[0105] The density of an array can be from 2 to as many as a
billion or more different reaction sites per square cm. Very high
density arrays are useful in the invention including, for example,
those having at least about 10,000,000 reaction sites/cm.sup.2,
including, for example, at least about 100,000,000 reaction
sites/cm.sup.2, 1,000,000,000 reaction sites/cm.sup.2, up to about
2,000,000,000 reaction sites/cm.sup.2or higher. High density arrays
can also be used including, for example, those in the range from
about 100,000 reaction sites/cm.sup.2 to about 10,000,000 reaction
sites/cm.sup.2. Moderate density arrays useful in the invention can
range from about 10,000 reaction sites/cm.sup.2 to about 100,000
reaction sites/cm.sup.2. Low density arrays are generally less than
about 10,000 reaction sites/cm.sup.2.
[0106] Any of a variety of reaction components can be attached to a
charge sensor. For example, a receptor, such as an antibody or
lectin; a ligand, such as a nucleotide, epitope, carbohydrate, or
drug candidate; a nucleic acid such as target nucleic acid, tether
nucleic acid or other nucleic acid set forth in connection with a
reaction set forth herein; or an enzyme, such as a polymerase or
other nucleic acid binding enzyme, a kinase, phosphatase,
exonuclease, protease, or metabolic enzyme. Other useful reaction
components include, but are not limited to, components of reactions
set forth herein or known in the art of molecular biology or
biochemistry.
[0107] Multiplex embodiments, including, for example, those that
employ an array of charge sensors can be configured such that a
single type of reaction component is attached to each charge
sensor. For example, the charge sensors in a multiplex embodiment
can substantially all be attached to a polymerase. Furthermore, the
same species of polymerase can be attached to each of the charge
sensor. This configuration can provide an expected uniform output
from each charge sensor, but for differences in the other reaction
components that come into contact with each respective charge
sensor. Such a configuration can be achieved by providing a fluid
that is homogeneous with respect to having a single type of
polymerase when attaching the polymerases to the charge
sensors.
[0108] Any of a variety of polymerases can be used in a method or
composition set forth herein including, for example, protein-based
enzymes isolated from biological systems and functional variants
thereof. Reference to a particular polymerase, such as those
exemplified below, will be understood to include functional
variants thereof unless indicated otherwise. A particularly useful
function of a polymerase is to catalyze the polymerization of a
nucleic acid strand using an existing nucleic acid as a template.
Other functions that are useful are described elsewhere herein.
Examples of useful polymerases include DNA polymerases and RNA
polymerases. Exemplary DNA polymerases include those that have been
classified by structural homology into families identified as A, B,
C, D, X, Y, and RT. DNA Polymerases in Family A include, for
example, T7 DNA polymerase, eukaryotic mitochondrial DNA Polymerase
.gamma., E. coli DNA Pol I, Thermus aquaticus Pol I, and Bacillus
stearothermophilus Pol I. DNA Polymerases in Family B include, for
example, eukaryotic DNA polymerases .alpha., .delta., and
.epsilon.; DNA polymerase .zeta.; T4 DNA polymerase, Phi29 DNA
polymerase, and RB69 bacteriophage DNA polymerase. Family C
includes, for example, the E. coli DNA Polymerase III alpha
subunit. Family D includes, for example, polymerases derived from
the Euryarchaeota subdomain of Archaea. DNA Polymerases in Family X
include, for example, eukaryotic polymerases Pol .beta., pol
.sigma., Pol .lamda., and Pol .mu., and S. cerevisiae Pol4. DNA
Polymerases in Family Y include, for example, Pol .eta., Pol iota,
Pol kappa, E. coli Pol IV (DINB) and E. coli Pol V (UmuD'2C). The
RT (reverse transcriptase) family of DNA polymerases includes, for
example, retrovirus reverse transcriptases and eukaryotic
telomerases. Exemplary RNA polymerases include, but are not limited
to, viral RNA polymerases such as T7 RNA polymerase; Eukaryotic RNA
polymerases such as RNA polymerase I, RNA polymerase II, RNA
polymerase III, RNA polymerase IV, and RNA polymerase V; and
Archaea RNA polymerase.
[0109] The above classifications are provided for illustrative
purposes. It will be understood that variations in the
classification system are possible. For example, in at least one
classification system Family C polymerases have been categorized as
a subcategory of Family X. Furthermore, polymerases can be
classified according to other characteristics, whether functional
or structural, that may or may not overlap with the structural
characteristics exemplified above. Some exemplary characteristics
are set forth in further detail below.
[0110] A polymerase having an intrinsic 3'-5' proofreading
exonuclease activity can be useful for some embodiments.
Polymerases that substantially lack 3'-5' proofreading exonuclease
activity are also useful in some embodiments, for example, in most
sequencing embodiments. Absence of exonuclease activity can be a
wild type characteristic or a characteristic imparted by a variant
or engineered polymerase structure. For example, exo minus Klenow
fragment is a mutated version of Klenow fragment that lacks 3'-5'
proofreading exonuclease activity. Klenow fragment and its exo
minus variant can be useful in a method or composition set forth
herein. On the other hand, the large fragment of A-family DNA
polymerases, such as Bsu DNA polymerase I (Bsu-LF), naturally lack
a 3' to 5' exonuclease function.
[0111] Polymerases having 3' to 5' exonuclease activity undergo
intramolecular and intermolecular switching as described, for
example, in Lamichhane et al. J. Am. Chem. Soc. 135:4735-4742
(2013), which is incorporated herein by reference in its entirety.
In some embodiments, it is desirable to use a polymerase that lacks
3' to 5' exonuclease activity. For example, in some embodiments the
switching can cause a conformational change that is difficult to
distinguish from one or more of the conformational changes that
occur due to polymerase activity and that are utilized for
sequencing or other analyses. The 3' to 5' exonuclease activity can
be removed by removing all or part of the 3' to 5' exonuclease
domain or by introducing loss of function mutations into the 3' to
5' exonuclease domain.
[0112] The large fragment of A-family DNA polymerases, such as Bsu
DNA polymerase I (Bsu-LF), can be further modified for use in a
method set forth herein. Native Bsu-LF has no cysteine (Cys)
residues. One or more Cys residue can be engineered at a surface
accessible location of Bsu-LF to allow for a convenient attachment
site for a tether or other linker having a sulfhydryl reactive
moiety. Candidate sites for introduction of a Cys mutation can be
determined from inspection of crystal structures of Bsu-LF or other
homologous A-family polymerases.
[0113] It may also be desirable to engineer Bsu-LF to introduce
charged residues that will interact favorably with a charge sensor.
For example, a beneficial modification is to introduce one or more
residues from the O-helix finger domain of Klenow fragment into
comparable position(s) of Bsu-LF, the net result of which is to
introduce more charged amino acids into the mutant Bsu-LF. See FIG.
7 for the location of the O-helix finger domain of Klenow fragment
(PolIxxo in the figure) and the location to be replaced in Bsu-LF
(Bsuxxx1) in the figure. Similar changes can be made to other
A-family polymerases such as Taq DNA polymerase or Bst DNA
polymerase
[0114] Polymerases can be characterized according to their
processivity. A polymerase can have an average processivity that is
at least about 50 nucleotides, 100 nucleotides, 1,000 nucleotides,
10,000 nucleotides, 100,000 nucleotides or more. Alternatively or
additionally, the average processivity for a polymerase used as set
forth herein can be, for example, at most 1 million nucleotides,
100,000 nucleotides, 10,000 nucleotides, 1,000 nucleotides, 100
nucleotides or 50 nucleotides. Polymerases can also be
characterized according to their rate of processivity or nucleotide
incorporation. For example, many native polymerases can incorporate
nucleotides at a rate of at least 1,000 nucleotides per second. In
some embodiments a slower rate may be desired. For example, an
appropriate polymerase and reaction conditions can be used to
achieve an average rate of at most 500 nucleotides per second, 100
nucleotides per second, 10 nucleotides per second, 1 nucleotide per
second, 1 nucleotide per 10 seconds, 1 nucleotide per minute or
slower. As set forth in further detail elsewhere herein, nucleotide
analogs can be used that have slower or faster rates of
incorporation than naturally occurring nucleotides. It will be
understood that polymerases from any of a variety of sources can be
modified to increase or decrease their average processivity or
their average rate of processivity (e.g. average rate of nucleotide
incorporation) or both. Accordingly, a desired reaction rate can be
achieved using appropriate polymerase(s), nucleotide analog(s),
nucleic acid template(s) and other reaction conditions.
[0115] Depending on the embodiment that is to be used, a polymerase
can be either thermophilic or heat inactivatable. Thermophilic
polymerases are typically useful for high temperature conditions or
in thermocycling conditions such as those employed for polymerase
chain reaction (PCR) techniques. Examples of thermophilic
polymerases include, but are not limited to 9.degree. N DNA
Polymerase, Taq DNA polymerase, Phusion.RTM. DNA polymerase, Pfu
DNA polymerase, RB69 DNA polymerase, KOD DNA polymerase, and
VentR.RTM. DNA polymerase. Most polymerases isolated from
non-thermophilic organisms are heat inactivatable. Examples are DNA
polymerases from phage. It will be understood that polymerases from
any of a variety of sources can be modified to increase or decrease
their tolerance to high temperature conditions. A heat spike (i.e.
brief time period of increased temperature) can be used to
inactivate one or more heat inactivatable polymerases in an array
while leaving thermophilic polymerases in an active state for
subsequent reactions or for subsequent cycles of a sequencing
reaction.
[0116] In an alternative embodiment, several different types of
reaction component can be attached across the multiplex collection
of charge sensors. For example, a first subset of charge sensors in
an array can be attached to a first species of polymerase and a
second subset of charge sensors in the array can be attached to a
second species of polymerase. Two, three, or more species of
polymerase can be used. The use of different species of polymerase
can be useful when the different polymerases have different
specificity or sensitivity for different types of nucleotides or
different template sequences. For example, the different types of
polymerases can produce mutually distinguishable signals detectable
by the charge sensors when incorporating the same type of
nucleotide into a nascent strand of a nucleic acid. In another
example, the different types of polymerases can include at least
one DNA polymerase and at least one RNA polymerase. Such a
configuration can be achieved by providing a fluid that is
heterogeneous with respect to having multiple type of polymerase
when attaching the polymerases to the charge sensors.
[0117] In some configurations, a charge sensor will have a capacity
to attach more than one reaction component of a particular type.
For example, a charge sensor may have a capacity to attach more
than one polymerase. Depending on the size of the charge sensor and
volume occupied by the polymerase, the charge sensor may have a
capacity to attach at least 2, 3, 4, 5, 10, 15, 25 or more
polymerases. This can be the case for a plurality of charge sensors
in an array. As will be set forth in further detail below,
conditions can be temporarily imposed to decrease the capacity of a
charge sensor, increase the steric bulk of a polymerase (or other
reaction component), or otherwise favor attachment of a single
polymerase (or other reaction component) to an individual charge
sensor. Again, the conditions can be applied to an array of charge
sensors.
[0118] A reaction component can be attached to a charge sensor
using any of a variety of chemistries known in the art. For
example, chemical linkers can be used. In many embodiments, the
surface of the charge sensor is one of SiO.sub.2, Al.sub.2O.sub.3,
HfO.sub.2, Ta.sub.2O.sub.5. Other oxides can also be used, for
example from the lanthanide group. Nitrides and oxinytrides are
also possible. The attachment to a linker can conveniently be made
through a surface hydroxyl. In particular embodiments, the linker
molecule includes at least a first and a second functional group.
Generally, the first functional group interacts with the charge
sensor and the second functional group interacts with the reaction
component. Exemplary first functional groups include a pyrene, a
benzene, a cyclohexane, and
2,3-dichloro-5,6-dicyano-1,4-benzoquinone. An exemplary second
functional group is maleimide. Other chemistries known to
covalently link proteins to surfaces or other moieties can be used
such as those sold by Thermo Fisher (Waltham, Mass.), or Sigma
Aldrich (St. Louis, Mo.). The chemical group on the polymerase
attached to the tethers can be thiol, amine or carboxylic group. In
certain embodiments in which the conduction channel is a SWNT, the
surface of a SWNT is a roughly one atom thick layer of graphite.
The linker molecule can be covalently linked to one or few carbon
atom, or it can interact with a sidewall of the SWNT through pi-pi
stacking.
[0119] A reaction component can be attached to a charge sensor by a
non-covalent linkage such as one formed between a receptor and a
ligand. Particularly useful linkages are those between streptavidin
(or variants or analogs thereof) and biotin (or its analogs), those
between complementary nucleic acids, those between antibodies and
epitopes and the like. Members of the above pairs can be linked to
a reaction component and charge sensor, respectively, such that
contacting a fluid containing the reaction components with a solid
support having the charge sensor will result in formation of the
noncovalent bond that tethers the reaction component to the charge
sensor.
[0120] In some embodiments, the reaction components from the fluid
attach to the charge sensors to form a conducting tether. Exemplary
conducting tethers include those having a structure that includes
doped polythiophene, poly(3,4-ethylenedioxythiophene),
polyacetylenes, polypyrroles, polyanilines, polyfluorenes,
polyphenylenes, polypyrenes, polyazulenes, polynaphthalenes,
polycarbazoles, polyindoles, or polyazepines. Charge doping of
these tether structures can be achieved by oxidation of the
polymer. Exemplary conducting tethers and methods for their
creation are set forth in Vernitskaya et al. Russ. Chem. Rev.
66:443ff (1997); MacDiarmid, Angew. Chem., Int. Ed. 40:2581-2590
(2001); or McNeill et al., Aust. J. Chem. 16:1056-75 (1963), each
of which is incorporated herein by reference in its entirety.
[0121] In particular embodiments, a solid support can be within or
part of a vessel such as a well, tube, channel, cuvette, Petri
plate, bottle or the like. A particularly useful vessel is a
flow-cell, for example, as described in US 2010/0111768 A1 or
Bentley et al., Nature 456:53-59 (2008), each of which is
incorporated herein by reference in its entirety. Exemplary
flow-cells are those that are commercially available from Illumina,
Inc. (San Diego, Calif.). Flow cells are convenient for delivering
bulk reagents to an array of charge sensors during attachment of
reaction components to the charge sensors or during subsequent
reactions carried out with the reaction components on the charge
sensors. Cyclic processes such as nucleic acid sequencing reactions
are particularly well suited for flow cell devices. Another
particularly useful vessel is a well in a multiwell plate or
microtiter plate.
[0122] A method set forth herein can include steps of contacting a
plurality of charge sensors with a fluid containing a plurality of
reaction components of a particular type under conditions wherein
the reaction components are in fluid communication with the
plurality of charge sensors and the reaction components attach to
the charge sensors. The result can be that each of the charge
sensors becomes attached to a single one of the reaction component
even when number of reaction components in the fluid is greater
than the number of charge sensors that are contacted by the fluid.
The fraction of the charge sensors that attach to one and only one
of the reaction components of the particular type would be expected
to conform to the Poisson distribution. The Poisson distribution
sets a maximum of 37% occupancy for the fraction of charge sensors
that would attach to only a single reaction component of a
particular type when those reaction components were delivered to
the charge sensors in a bulk fluid. However, in accordance with the
methods set forth herein, bulk fluid delivery of reaction
components of a particular type (e.g. polymerase or other nucleic
acid enzyme) can result in greater than 35%, 40%, 50%, 75%, 90%,
95% or 99% of the charge sensors in the plurality being occupied by
a single reaction component of the particular type.
[0123] In some embodiments of the methods, reaction components can
be transported from bulk solution to charge sensors, for example,
by diffusion or other passive process. Attachment of the reaction
components to the charge sensors can thus occur, for example, in
accordance with chemistries set forth herein or known in the art.
Alternatively, reaction components can be actively transported to
the charge sensors, for example, via electric field (e-field)
assisted transport. Again, attachment of the reaction components to
the charge sensor can result using chemistries set forth herein or
known in the art.
[0124] A method set forth herein can be modified to use electric
field (e-field) assisted transport of reaction components to sites
that contain charge sensors. For example, each charge sensor on a
solid support can be present at a site that is electrically coupled
to a power source to produce an electric charge that attracts
polymerases or other reaction components to that site and into
proximity with the charge sensor at that site. Exemplary methods
and apparatus for using e-field assist to attract analytes to sites
of an array are described in US 2009/0032401 A1, which is
incorporated herein by reference in its entirety. E-field assist
can be used in a method of the present disclosure, for example,
under conditions where a plurality of different polymerases (or
other reaction components) is in solution such that the polymerases
are in fluidic communication with the plurality of charge sensors.
The charge at the site of each charge sensor can be adjusted to
achieve a desired rate or amount of transport for the polymerase
(or any other particular type of reaction component).
[0125] In particular embodiments that utilize e-field assisted
transport, the e-field can be consistently applied throughout the
course of the reaction that is used to attach a polymerase (or any
other particular type of reaction component) to a charge sensor.
Alternatively, the e-field can be changed (e.g. increased or
decreased) as the attachment reaction progresses and charge sensors
fill with polymerase (or any other particular type of reaction
component). For example, increasing the e-field can provide the
benefit of increasing the number of charge sensors that attach to a
polymerase. The rate at which the e-field is increased, and the
amplitude range for the increase, can be selected to balance the
increasing rate of reaction component transport to charge sensors
over time with the increasing number of charge sensors that have
become attached to polymerase over that same period of time. The
rate of change for the e-field can be based on a predicted or
expected rate of polymer attachment. Alternatively, the e-field can
be changed in response to empirical detection of polymerase
attachment to the charge sensors as set forth in further detail
herein.
[0126] In particular embodiments, an e-field can be applied
substantially uniformly to all of the sites of an array that have
charge sensors. Thus, polymerases (or other reaction components)
that are in solution will have an equal probability of being
transported to any given charge sensor. In an alternative
embodiment, an e-field can be applied to only a subset of the
charge sensor sites that are present in an array. In this way,
e-field assist can be used to selectively attach polymerase (or
other reaction component) to some charge sensors over others.
Furthermore, if desired, an attractive charge can be applied at a
first subset of charge sensor sites in order to transport
polymerase to the first subset of sites and in the meantime a
repellant charge can be applied to a second subset of charge sensor
sites to inhibit polymerases from being transported to those sites
or to remove (e.g. via desorption or degradation) polymerase from
the second subset of sites. Similarly a repellant charge can be
applied to interstitial regions of an array that do not contain
charge sensors in order to inhibit polymerases from being
transported to the interstitial regions or to remove (e.g. via
desorption or degradation) polymerases from the interstitial
regions.
[0127] In many configurations an amplifier that is used for a
charge sensor will occupy substantially more space than the sensor
itself. For example, readout circuitry may occupy an area of a
detection device that is anywhere from 2.times.2 microns to
20.times.20 microns, whereas a single nanowire transistor may
occupy as little as 100.times.500 nanometers. The size and
dimensions of an array of charge sensors can in some embodiments be
limited by the space occupied by multiple amplifiers, for example,
if an amplifier is present for each charge sensor. The limitation
can be overcome in particular embodiments of the present apparatus
and methods by configuring an array of charge sensors such that a
each amplifier is operationally connected to several charge
sensors. For example, 2, 3, 4, 5, 6, 8, 10 or more charge sensors
can be connected to the same amplifier. Thus a higher density of
charge sensors can be present on an array than in a configuration
where there is a one to one connectivity between amplifiers and
charge sensors. In operation, an amplifier can be assigned to
amplify signal from only one of many charge sensors to which it is
(or was at one time) connected. For example, the array of charge
sensors can be loaded by contacting it with a fluid containing a
plurality of reaction components of a particular type. This loading
technique may result in a substantial number of the charge sensors
being attached to more than one reaction component of a particular
type and others not being loaded with any of that type of reaction
component at all. Among several charge sensors that are connected
to a common amplifier, a single sensor that has attached to only a
single reaction component can be distinguished from the others that
are overloaded or unloaded and the amplifier can be assigned to
acquire signal from the single loaded charge sensors while not
acquiring signal from the other charge sensors to which it is (or
was) connected.
[0128] In some embodiments, an individual charge sensor will have
capacity for greater than one reaction component of a particular
type. Taking a polymerase as an example, each charge sensor may
have capacity to attach several polymerase molecules at once. In
such cases, the polymerase can be attached to a repellant moiety
that occupies a volume of space that sterically hinders more than
one of the polymerases that is bound to another repellant moiety
from attaching to an individual charge sensor. Similarly, the
repellant moiety can have a charge polarity that electrostatically
hinders more than one of the polymerases that is bound to another
repellant moiety from attaching to the same charge sensor.
[0129] A particularly useful repellant moiety is nucleic acid. A
repellant nucleic acid can provide both steric and electrostatic
hindrance to limit occupancy. A repellant nucleic acid is well
suited to polymerases, nucleic acid enzymes and other reaction
components that have a binding affinity for nucleic acids. However,
it will be understood that this type of affinity is not necessary
because synthetic methods can be used to attach repellant nucleic
acids to reaction components, for example, using the linkers,
tethers and attachment chemistries set forth herein or known in the
art. The length, sequence composition, or secondary structure of
the repellant nucleic acid can be modulated to achieve a desired
occupancy. For example, larger nucleic acids can be used when the
charge sensors have a relatively high capacity for the reaction
component to which the charge sensor will be attached. Smaller
nucleic acids can be sufficient for limiting loading of smaller
charge sensors (or when relatively large reaction components or
highly charged reaction components are used thus requiring only a
small increase in repellant properties). Nucleic acids are also
useful as repellant moieties since the sequence of the nucleic acid
can be selected to achieve desired binding properties to a
polymerase or other nucleic acid enzyme.
[0130] In some embodiments, a repellant nucleic acid can be
compacted into a nanoball structure. Methods of compacting nucleic
acids are known in the art (for example, as described by
Bloomfield, Curr. Opin. Struct. Biol. 6(3): 334-41 (1996), and US
Pat. App. Pub. No. 2007/0099208 A1, each of which is incorporated
herein by reference in its entirety). For example, an alcohol or
polyamine such as spermine or spermidine can be used. A compacted
nucleic acid will have a structure that is more densely packed than
the structure of the nucleic acid in the absence of a compacting
agent or compacting condition and the structure will typically
resemble a ball or globule. The generation of such compacted
nucleic acid balls is useful for creating repellant moieties.
Various methods can be used to generate balls of a desired size,
for example, using various compacting techniques and/or varying the
number of copies in an amplicon. Generally, the compacted amplicons
have an average diameter or width ranging from about 0.1 .mu.m to
about 5 .mu.m, for example, about 0.1 .mu.m, about 0.2 .mu.m, about
0.5 .mu.m, about 1 .mu.m, 2 .mu.m, about 3 .mu.m, about 4 .mu.m and
about 5 .mu.m.
[0131] Other polymeric molecules are also useful as repellant
moieties, including without limitation, polyethylene glycol,
polythenes, polypropylene, polyvinyl chloride, Teflon, nylon,
polyamides, polyacetals, polyesters, Buna rubbers, polyacrylates,
polystyrene, and polychlorotrifluoroethene. Beads or particles made
of solid support materials or gels can also function as repellant
moieties.
[0132] A repellant moiety can remain attached to a reaction
component while the reaction component participates in a particular
reaction that is to be detected by a charge sensor to which the
reaction component is attached. For example, a repellant moiety
that is bound to a polymerase while the polymerase is loaded onto
and attached to a charge sensor can remain attached to the
polymerase in a subsequent nucleotide addition reaction that is
detected by the charge sensor. Alternatively, the repellant moiety
can be removed from the reaction component after the reaction
component has been attached to a charge sensor. Taking again the
example of a polymerase, a repellant moiety such as repellant
nucleic acid can be removed from the polymerase prior to the
polymerase participating in a detected reaction with a target
nucleic acid. Repellant moieties that are bound to a polymerase or
other reaction component can be removed by techniques known to
those skilled in the art to result in removal. For example,
non-covalently bound moieties can be removed by washing, or
competitive displacement using other moieties that bind to the
reaction component. Covalently bound moieties can be removed by
chemical or physical means such as those set forth herein in regard
to cleaving tethers. Repellant moieties once removed from a
reaction component can be washed away from the charge sensor to
which the reaction component remains attached.
[0133] In some embodiments, a charge sensor that has a capacity for
more than one reaction component of a particular type, can be
overloaded such that an individual charge sensor is attached to
several of the reaction components and then reaction components can
be removed (or inactivated or degraded) from the charge sensor
leaving only a single active reaction component attached to the
individual charge sensor. For example, a method of attaching
reaction components to charge sensors can be carried out to create
cleavable tethers between each reaction component and the charge
sensor to which it is attached. In some cases the reaction
components that are delivered in a fluid can include precursors to
cleavable tethers, the charge sensors can include precursors to
cleavable tethers or both the reaction component and charge sensor
can have precursors that react together to form a cleavable
tether.
[0134] Cleavable tethers can be cleaved by bond breakage due to
physical or chemical processes. For example, a method set forth
herein can include a step of cleaving a cleavable tether by
photochemical cleavage, electrochemical cleavage, electric field,
mechanical agitation, chemical cleavage or heat. Useful cleavable
tethers and their precursors include those used for modification of
proteins and are commercially available, for example, from Thermo
Fisher (Waltham, Mass.), or Sigma Aldrich (St. Louis, Mo.).
[0135] Other methods can be used to remove one or more reaction
components from a charge sensor. For example, removal can be
achieved by degrading one or more reaction components from each
charge sensor. Degradation can be achieved by physical methods such
as heat, photo-oxidation, sonication or the like. Chemical
degradation is also possible for example using pH changes, chemical
denaturants, proteases or the like. In some cases, the extent of
degradation can be modulated by contacting the sensor-attached
reaction components with a protection moiety. The amount of
protection moiety supplied to an array of charge sensors can be
titrated to result in binding to a single reaction component, on
average, per charge sensor. When degradation is subsequently
carried out all but the protected reaction component attached to
each charge sensor will be degraded. This will leave a single
reaction component attached to each charge sensor. The protection
moiety can remain bound to the reaction component as it
participates in a reaction that is detected by the charge sensor,
or the protection moiety can be removed. In some cases, the
degradation can happen by binding the polymerase active site with a
chemically or photochemically active oligonucleotide analog. A
chemical or photochemical treatment can be applied to crosslink the
oligonucleotide analog to the polymerase. As a result, these
polymerase can be rendered incapable of accepting a target nucleic
acid, or primer, or the polymerase may be rendered incapable of
conformational changes (e.g. open-close conformation changes) that
would have been detected by charge sensor.
[0136] An example of a useful protection moiety is a nucleic acid
such as a DNA nanoball. For example, a nucleic acid can be used to
bind to a polymerase or other nucleic acid enzyme to provide
stability against denaturation or chemical modification. A nucleic
acid can also provide a steric block preventing proteases from
having access to a polymerase that is attached to a charge sensor.
Another example is a primer hybridized single stranded DNA; in this
case the 3' end of the primer will be bound to the catalytic center
of the polymerase, preventing binding of reactive polymerase
deactivation moieties. A further example of a protection moiety is
a protein such as an antibody that specifically targets a
polymerase. The protein or antibody can protect against chemical
modification or provide a steric block preventing proteases from
having access to the polymerase. If it is desirable to remove the
antibody, the antibody could be removed using, for example, heat.
An appropriate temperature would be one at which the antibody is no
longer stably bound to the polymerase, but at which the polymerase
is stable. Other materials that have been exemplified herein for
use as repellant moieties can serve as protection moieties.
[0137] Degradation, inactivation or removal of excess reaction
components from charge sensors can be carried out with or without
monitoring of the charge sensors to determine the extent of
degradation or removal. For example, in particular embodiments a
process of removing one or more reaction components of a particular
type from modified charge sensors can include steps of (i) removing
one or more of the reaction components from each of the modified
charge sensors, (ii) monitoring the charge sensors to distinguish
the presence of multiple reaction components from the presence of a
single reaction component, and (iii) discontinuing the removing to
leave a single one of the reaction components attached to each of
the modified charge sensors. The status of the charge sensor can be
monitored by detecting changes in signal from the charge sensor.
Alternatively or additionally, a different sensor can be used to
detect presence or absence of reaction components at the surface of
the charge sensor. Any of a variety of detection modalities can be
used including for example, fluorometry to detect fluorescent
labels on reaction components, optical scatter methods, absorbance
methods to detect chromophores and other analytical detection
methods known in the art pertaining to the detection of proteins
and other reaction components.
[0138] A method of the present disclosure can include a step of
providing one or more target nucleic acids to a solid support that
comprises at least one charge sensor. In particular embodiments the
charge sensor will have been previously attached to another
reaction component that will react with the nucleic acid(s) in a
desired reaction, examples of which include nucleic acid enzymes
such as polymerases. In other embodiments target nucleic acid(s)
can be delivered to a charge sensor before or at the same time that
other reaction components (e.g. nucleic acid enzymes or
polymerases) are delivered to the charge sensor(s).
[0139] Target nucleic acids used in a method or apparatus of the
present disclosure can be composed of DNA, RNA or analogs thereof.
The source of the target nucleic acids can be genomic DNA,
messenger RNA, or other nucleic acids from native sources. In some
cases the target nucleic acids that are derived from such sources
can be amplified prior to use in a method or composition herein.
Any of a variety of known amplification techniques can be used
including, but not limited to, polymerase chain reaction (PCR),
rolling circle amplification (RCA), multiple displacement
amplification (MDA), or random prime amplification (RPA). It will
be understood that amplification of target nucleic acids prior to
use in a method or apparatus set forth herein is optional. As such,
target nucleic acids will not be amplified prior to use in some
embodiments of the methods and compositions set forth herein.
Target nucleic acids can optionally be derived from synthetic
libraries. Synthetic nucleic acids can have native DNA or RNA
compositions or can be analogs thereof.
[0140] Exemplary biological samples from which target nucleic acids
can be derived include, for example, those from a mammal such as a
rodent, mouse, rat, rabbit, guinea pig, ungulate, horse, sheep,
pig, goat, cow, cat, dog, primate, human or non-human primate; a
plant such as Arabidopsis thaliana, corn, sorghum, oat, wheat,
rice, canola, or soybean; an algae such as Chlamydomonas
reinhardtii; a nematode such as Caenorhabditis elegans; an insect
such as Drosophila melanogaster, mosquito, fruit fly, honey bee or
spider; a fish such as zebrafish; a reptile; an amphibian such as a
frog or Xenopus laevis; a dictyostelium discoideum; a fungi such as
pneumocystis carinii, Takifugu rubripes, yeast, Saccharamoyces
cerevisiae or Schizosaccharomyces pombe; or a plasmodium
falciparum. Target nucleic acids can also be derived from a
prokaryote such as a bacterium, Escherichia coli, staphylococci or
mycoplasma pneumoniae; an archae; a virus such as Hepatitis C
virus, ebola virus or human immunodeficiency virus; or a viroid.
Target nucleic acids can be derived from a homogeneous culture or
population of the above organisms or alternatively from a
collection of several different organisms, for example, in a
community or ecosystem.
[0141] Target nucleic acids need not be derived from natural
sources and can instead be synthesized using known techniques. For
example, gene expression probes or genotyping probes can be
synthesized and used in the methods and apparatus set forth
herein.
[0142] In some embodiments, target nucleic acids can be obtained as
fragments of one or more larger nucleic acids. Fragmentation can be
carried out using any of a variety of techniques known in the art
including, for example, nebulization, sonication, chemical
cleavage, enzymatic cleavage, or physical shearing. Fragmentation
may also result from use of a particular amplification technique
that produces amplicons by copying only a portion of a larger
nucleic acid. For example, PCR amplification produces fragments
having a size defined by the length of the nucleotide sequence on
the original template that is between the locations where flanking
primers hybridize during amplification.
[0143] A population of target nucleic acids, or amplicons thereof,
can have an average strand length that is desired or appropriate
for a particular application of the methods or apparatus set forth
herein. For example, the average strand length can be less than
about 100,000 nucleotides, 50,000 nucleotides, 10,000 nucleotides,
5,000 nucleotides, 1,000 nucleotides, 500 nucleotides, 100
nucleotides, or 50 nucleotides. Alternatively or additionally, the
average strand length can be greater than about 10 nucleotides, 50
nucleotides, 100 nucleotides, 500 nucleotides, 1,000 nucleotides,
5,000 nucleotides, 10,000 nucleotides, 50,000 nucleotides, or
100,000 nucleotides. The average strand length for a population of
target nucleic acids, or amplicons thereof, can be in a range
between a maximum and minimum value set forth above.
[0144] In some cases a population of target nucleic acids can be
produced under conditions or otherwise configured to have a maximum
length for its members. For example, the maximum length for the
members that are used in one or more steps of a method set forth
herein or that are present in a particular composition can be less
than about 100,000 nucleotides, 50,000 nucleotides, 10,000
nucleotides, 5,000 nucleotides, 1,000 nucleotides, 500 nucleotides,
100 nucleotides or 50 nucleotides. Alternatively or additionally, a
population of target nucleic acids, or amplicons thereof, can be
produced under conditions or otherwise configured to have a minimum
length for its members. For example, the minimum length for the
members that are used in one or more steps of a method set forth
herein or that are present in a particular composition can be more
than about 10 nucleotides, 50 nucleotides, 100 nucleotides, 500
nucleotides, 1,000 nucleotides, 5,000 nucleotides, 10,000
nucleotides, 50,000 nucleotides, or 100,000 nucleotides. The
maximum and minimum strand length for target nucleic acids in a
population can be in a range between a maximum and minimum value
set forth above.
[0145] The present disclosure provides a method of detecting a
nucleotide. The method can include the steps of (a) providing a
nucleotide binding protein (e.g. a polymerase) tethered to a solid
support charge sensor; (b) providing one or more labeled
nucleotides, whereby the presence of the label can be detected by
the charge sensor when the label is in proximity to the charge
sensor; and (c) detecting binding of the labeled nucleotide to the
protein using the charge sensor.
[0146] The binding of a nucleotide to a nucleic acid binding
enzyme, such as a polymerase, can be detected based on the
recruitment of a charge label to the enzyme which in turn causes a
detectable perturbation in the field around the charge sensor to
which the enzyme is attached. Exemplary charge labels are set forth
previously herein. In particular embodiments, the charge label is
attached to the .beta.- or .gamma.-phosphate position of the
nucleotide. An advantage of attaching the label at the beta- or
gamma-phosphate position of the nucleotide is that the label can be
removed by the catalytic activity of polymerase when incorporating
the nucleotide into a nascent strand. However, the label need not
be removed by polymerase activity. Thus, the label can be attached
at any of a variety of positions on a nucleotide including for
example via a linker to the base moiety of a nucleotide (see, for
example, the nucleotide positions and linkers set forth in U.S.
Pat. Nos. 7,427,673; 7,414,116; and 7,057,026 and PCT publications
WO 91/06678 and WO 07/123744, each of which is incorporated herein
by reference in its entirety). A label can also be attached at the
alpha-phosphate position of the nucleotide or at the ribose moiety
of the nucleotide. A label attached to any of a variety of moieties
of a nucleotide can optionally be cleaved from the nucleotide after
being detected, for example, via cleavage of a charge linker.
[0147] A label used in a method or apparatus set forth herein can
further include an oligonucleotide moiety. Exemplary
oligonucleotide moieties include DNA, RNA, and PNA as set forth
previously herein. As exemplified previously herein, an
oligonucleotide moiety can be useful for hybridizing to a nucleic
acid tether or other nucleic acid so as to localize electric field
perturbation to occur with a desired distance of a charge sensor.
The oligonucleotide moiety can occur as an intermediary structure
between the nucleotide and a charged moiety. However, the charge
moiety is optional and need not be located at an end of the
oligonucleotide moiety that is distal to the point of attachment to
the nucleotide. Although several embodiments are exemplified herein
with reference to nucleotide analogs having oligonucleotide
moieties that interact with nucleic acid tethers, it will be
understood that nucleotide analogs can have other linker components
in place of the oligonucleotide moieties. The linker components can
comprise one member of a binding pair that interacts with another
member that is part of a tether.
[0148] In some embodiments, different nucleotide types can be
attached to different labels. Thus, the differences in signal
arising from the labels can be used to distinguish different
nucleotide types. This can be particularly useful for nucleic acid
sequencing methods where several different types of nucleotides are
delivered to a polymerase in a way that the several different types
of nucleotides are present in parallel during a detection event.
For example, four different nucleotides having four different
charge moieties can be used as exemplified previously herein with
regard to FIG. 2 and FIG. 3. Alternatively or additionally, the
label moieties can contain oligonucleotide moieties that hybridize
to different tether sequences to produce mutually distinct signals
at a charge sensor. In some embodiments several labeled nucleotides
used in a method set forth herein will have different charge
labels, respectively, but each of said labeled nucleotides will
have an oligonucleotide moiety that is capable of hybridizing to
the same immobilized tether sequence. As set forth previously
herein, different nucleotide analogs can have oligonucleotide
moieties with different lengths, or alternatively, two or more of
the nucleotide analogs can have oligonucleotide moieties that are
the same length.
[0149] Different nucleotides need not have different labels. The
different nucleotides can be delivered to a reaction separately
such that the nucleotides are distinguished based on knowledge of
when and where they are delivered. For example, a sequencing
reaction can include sequential additions of four separate
nucleotides per cycle with washes between nucleotide additions.
This separate delivery of nucleotides can be done whether the
different nucleotides are uniquely labeled or uniformly
labeled.
[0150] In particular embodiments, a method of nucleic acid
sequencing can be performed by (a) providing a polymerase tethered
to a solid support charge sensor; (b) providing one or more labeled
nucleotides, whereby the presence of the label can be detected by
the charge sensor when the label is in proximity to the charge
sensor; and (c) detecting incorporation of the labeled nucleotide
into a nascent strand complementary to a template nucleic acid
using the charge sensor. A plurality of incorporation events can be
detected in succession to determine the sequence. Alternatively,
only a single incorporation event is detected for each nascent
strand and this information is combined with knowledge of the
sequence for the nascent strand (or the template to which it is
hybridized) to arrive at the sequence.
[0151] In multiplex embodiments, the solid support may include a
plurality of charge sensors that are tethered to polymerases, and
the method includes a step of detecting incorporation of a labeled
nucleotide into a nascent strand complementary to a template
nucleic acid at each polymerase in the plurality of polymerases.
The plurality of polymerases used in a multiplex embodiment can
optionally include at least two different types of polymerases. The
different types of polymerases can be selected to produce mutually
distinguishable signals detectable by the charge sensors when
incorporating the same type of nucleotide into a nascent strand of
nucleic acid. In this way, an array of charge sensors having
different attached polymerases (e.g. one per charge sensor) can
distinguish a greater variety of nucleic acid sequences or provide
greater sensitivity than would be available using an array having
only one type of polymerase attached to the charge sensors. For
example, the same template when sequenced by the action of two
different polymerases will produce two different series of signals.
The two series of signals can be compared or otherwise used in
combination to provide a more accurate nucleotide sequence than
would be derivable from only one series of signals from only one
type of polymerase.
[0152] An array of the present disclosure, for example, having been
produced by a method set forth herein, can be used for any of a
variety of applications. A particularly useful application is
nucleic acid sequencing. One example is sequencing-by-synthesis
(SBS). In SBS, extension of a nucleic acid primer along a nucleic
acid template (e.g. a target nucleic acid or amplicon thereof) is
monitored to determine the sequence of nucleotides in the template.
The underlying chemical process can be polymerization (e.g. as
catalyzed by a polymerase enzyme). In a particular polymerase-based
SBS embodiment, nucleotides are added to a primer (thereby
extending the primer) in a template dependent fashion such that
detection of the order and type of nucleotides added to the primer
can be used to determine the sequence of the template. A plurality
of different templates at different charge sensors of an array set
forth herein can be subjected to an SBS technique under conditions
where events occurring for different templates can be distinguished
due to their location at a specific charge sensor of the array.
[0153] Flow cells provide a convenient format for housing an array
that is produced by the methods of the present disclosure and that
is subjected to an SBS or other detection technique that involves
repeated delivery of reagents in cycles. For example, to initiate a
first SBS cycle, one or more nucleotides (optionally having charge
labels), can be flowed into/through a flow cell that houses an
array of charge sensors each having an attached polymerase to which
a nucleic acid template is bound. Those sites of an array where
primer extension causes a nucleotide to be incorporated can be
detected. Optionally, the nucleotides can further include a
reversible termination property that terminates further primer
extension once a nucleotide has been added to a primer. For
example, a nucleotide analog having a reversible terminator moiety
can be added to a primer such that subsequent extension cannot
occur until a deblocking agent is delivered to remove the moiety.
Thus, for embodiments that use reversible termination, a deblocking
reagent can be delivered to the flow cell (before or after
detection occurs). Washes can be carried out between the various
delivery steps. The cycle can then be repeated n times to extend
the primer by n nucleotides, thereby detecting a sequence of length
n. Exemplary SBS procedures and fluidic systems that can be readily
adapted for use with an array produced by the methods of the
present disclosure are described, for example, in Bentley et al.,
Nature 456:53-59 (2008), WO 04/018497; U.S. Pat. No. 7,057,026; WO
91/06678; WO 07/123744; U.S. Pat. Nos. 7,329,492; 7,211,414;
7,315,019; 7,405,281, and US 2008/0108082, each of which is
incorporated herein by reference in its entirety.
[0154] Sequencing-by-ligation reactions are also useful including,
for example, those described in Shendure et al. Science
309:1728-1732 (2005); U.S. Pat. Nos. 5,599,675; and 5,750,341, each
of which is incorporated herein by reference in its entirety. Some
embodiments can include sequencing-by-hybridization procedures as
described, for example, in Bains et al., Journal of Theoretical
Biology 135(3), 303-7 (1988); Drmanac et al., Nature Biotechnology
16, 54-58 (1998); Fodor et al., Science 251(4995), 767-773 (1995);
and WO 1989/10977, each of which is incorporated herein by
reference in its entirety. In both Sequencing-by-ligation and
sequencing-by-hybridization procedures, target nucleic acids (or
amplicons thereof) are subjected to repeated cycles of
oligonucleotide delivery and detection. Such methods can be readily
modified to detect ligase conformational changes or to detect
charge labeled oligonucleotides in place of the fluorescent
detection of optical labels described in the published methods.
[0155] Another useful application for an array of the present
disclosure, for example, having been produced by a method set forth
herein, is gene expression analysis. Gene expression can be
detected or quantified using RNA sequencing techniques, such as
those, referred to as digital RNA sequencing. RNA sequencing
techniques can be carried out using sequencing methodologies known
in the art such as those set forth above except that fluorescence
detection of optically labeled nucleotides can be replaced with the
charge-based detection methods set forth herein. Gene expression
can also be detected or quantified using hybridization techniques
carried out by direct hybridization to an array or using a
multiplex assay, the products of which are detected on an array.
Exemplary molecular biological assays that can be used for
array-based expression and genotyping analysis are described in
U.S. Pat. Nos. 7,582,420; 6,890,741; 6,913,884 or 6,355,431 or US
Pat. Pub. Nos. 2005/0053980 A1; 2009/0186349 A1 or US 2005/0181440
A1, each of which is incorporated herein by reference in its
entirety. These methods can be readily adapted by replacing optical
labels and fluorescence detection with the charge-based detection
techniques, and optionally the charge labels, set forth herein.
[0156] A method of nucleic acid sequencing provided by the present
disclosure can include the steps of (a) providing a polymerase
tethered to a solid support charge sensor; (b) providing one or
more labeled nucleotides, whereby the presence of the label can be
detected by the charge sensor when the label is in proximity to the
charge sensor, wherein the one or more labeled nucleotides have
reversible terminator moieties; (c) detecting incorporation of the
one or more labeled nucleotides into a nascent strand complementary
to a template nucleic acid using the charge sensor, thereby forming
a reversibly terminated nascent strand; (d) modifying the
reversible terminated nascent strand to render the nascent strand
capable of further incorporation of nucleotide; and (e) repeating
(b) through (d) to obtain a sequence of the template nucleic
acid.
[0157] The use of reversibly terminated nucleotides in a sequencing
reaction provides advantages of step control to the polymerase
extension process that would otherwise be continuous. This step
control can be useful for increasing the amount of time that a
newly extended nucleic acid strand spends in a detectable state.
For example, a charge label that is on a reversibly terminated
nucleotide can be maintained on a nascent strand after being added
by a polymerase and until a desired amount of signal is accumulated
by the charge sensor. This can allow for increased signal
collection. Then the sequencing process can proceed by addition of
deblocking agent followed by subsequent cycles of nucleotide
addition.
[0158] Another advantage of the step control conferred by the use
of reversibly terminated nucleotides is the ability to synchronize
an ensemble of reaction components that undergo the same reaction.
Thus, a sequencing method set forth herein can be carried out for
multiple copies of the same nucleic acid bound to multiple
polymerases at the site where a charge sensor is located (e.g. the
multiple polymerases can be attached to a single charge sensor).
The detection step used to identify the nucleotide added during
each cycle of polymerase activity can be effectively synchronized
by use of reversibly terminated nucleotides. Although reversibly
terminated nucleotides provide advantages for ensemble detection,
it will be understood that sequencing methods employing reversibly
terminated nucleotides can be used when a single polymerase is
attached to an individual charge sensor.
[0159] An ensemble of sequencing reactions can be set up to include
a plurality of polymerases attached to a common charge sensor,
wherein the polymerases are bound to target nucleic acids having
the same template sequence and a primer (or nascent strand) with
the same sequence. The polymerases can be attached to the charge
sensor as set forth previously herein. Then one or more target
nucleic acids comprising multiple repeats of the same template
sequence can be contacted with the polymerases. For example, a
target nucleic acid molecule having concatameric repeats of the
template sequence can be delivered to the polymerases at the
particular site. In this case, the subunit of sequence that forms
each repeat can function as an individual template sequence.
Optionally, the nucleic acid encoding the concatameric repeat can
be cleaved to form individual molecules each having a single
template sequence. These individual molecules can then be sequenced
by polymerases at the site. The nucleic acid encoding the
concatameric repeat can be created by rolling circle amplification
(RCA), for example, as described in Lizardi et al., Nat. Genet.
19:225-232 (1998) and US Pat App. Pub. No. 2007/0099208 A1, each of
which is incorporated herein by reference in its entirety.
[0160] Also provided is a method of nucleic acid sequencing that
includes the steps of (a) providing a polymerase tethered to a
solid support charge sensor; (b) contacting the polymerase with a
template nucleic acid and one or more different nucleotide types
under conditions wherein the polymerase catalyzes addition of the
one or more nucleotide types to form a nucleic acid complement of
the nucleic acid template, and wherein the addition of one or more
different nucleotide types produces a conformational signal change
from the polymerase that is detected by the charge sensor; (c)
detecting a change in the signal from the polymerase using the
charge sensor; and (d) determining the rate, polarity, amplitude or
time duration for the change in the signal for the addition of the
one or more different nucleotide type, thereby determining a
sequence of nucleotides for the template nucleic acid.
[0161] In particular embodiments, a sequence of nucleotides for a
nucleic acid template can be determined based on conformational
changes occurring in a nucleic acid enzyme such as a polymerase.
Distinguishing the conformational changes that occur for each type
of nucleotide that the enzyme interacts with and determining the
sequence of those changes can be used to determine the sequence of
the nucleic acid. For example, a polymerase that sequentially adds
of nucleotides to a nascent nucleic acid strand undergoes
conformational changes with each nucleotide addition. As set forth
in further detail herein, the conformational changes that occur for
each type of nucleotide that is added can be distinguished using a
charge sensor (or based on knowledge of which nucleotide(s) is/are
fluidically delivered to the substrate where sequencing is being
monitored) and the sequence of those changes can be detected to
determine the sequence of the nucleic acid.
[0162] A nucleic acid enzyme can be labeled to produce one or more
signals indicative of a conformational change in the enzyme as it
interacts with one or more reactants such as a nucleic acid or
nucleotide. A polymerase can be conformationally labeled such that
activity of the polymerase can be monitored by detection of
allosteric charge movement. For example, a polymerase can be
conformationally labeled to allow detection of a signal indicative
of nucleotide binding, a signal indicative of addition of a
nucleotide to a growing nucleic acid molecule, or a signal
indicative of an intermediate change in the conformation of the
polymerase between binding and catalysis. Accordingly, a polymerase
can include at least one non-natural label moiety that is detected
by the charge sensor. Polymerase can be engineered to have negative
and positive charges that maximize the charge change per unit
volume through allosteric movements. If this unit volume is close
to the charge sensor, this movement can easily be detected. In
particular embodiments, a signal detected by a charge sensor from a
conformationally labeled polymerase can distinguish a binding event
from a catalytic event. However, such a distinction may not be
necessary for some embodiments and the signal can be merely
indicative of the overall addition of a nucleotide. Alternatively
or additionally, the signal can distinguish binding of a correctly
base-paired nucleotide from binding of an incorrectly base-paired
nucleotide.
[0163] A particularly useful label moiety that can be attached to a
polymerase or other nucleic acid enzyme used in a method or
apparatus set forth herein is a negative charge label, examples of
which include, but are not limited to, a phosphate group, carboxyl
group, amino acid, DMT and/or FMOC. Also useful are positive charge
labels including, for example, a primary amine. Intrinsic labels
such as amino acid side chains or naturally occurring post
translational modifications (e.g. phosphorylation, addition of
flavin, reduction of disulfides or the like) can also provide
useful moieties for detection in a method or apparatus set forth
herein.
[0164] Some embodiments can employ a nucleotide analog that is
incorporated into a polynucleotide strand by a polymerase at a rate
that is measurably different than the rate at which another
nucleotide is incorporated into the strand by the polymerase.
Another useful nucleotide analog is one that is bound to a
polymerase at a rate that is measurably different than the rate at
which another nucleotide is bound to the polymerase. A nucleotide
analog that causes a conformational change of a polymerase at a
rate that is measurably different than another nucleotide is also
useful. The relative rate of binding, incorporation or polymerase
conformational change for a nucleotide analog can be measured
relative to a natural nucleotide having the same Watson-Crick base
pairing partner or relative to other nucleotides that are used in a
nucleic acid synthesis reaction. The relative rate can be faster or
slower for the nucleotide analog.
[0165] According to particular embodiments, a polymerase or other
nucleic acid enzyme can be conformationally labeled. Conformational
labeling of nucleic acid enzymes provides advantages for nucleic
acid sequence analysis. Conformationally labeled molecules, and
methods for making and using them, will be exemplified below with
regard to labeled polymerases. It will be understood that other
nucleic acid enzymes such as exonucleases and reverse
transcriptases can be made and used similarly.
[0166] Polymerases undergo conformational changes in the course of
synthesizing a nucleic acid polymer. For example, polymerases
undergo a conformational change from an open conformation to a
closed conformation upon binding of a nucleotide. Thus, a
polymerase that is bound to a nucleic acid template and growing
primer is in what is referred to in the art as an "open"
conformation. A polymerase that is bound to a nucleic acid
template, primer and a correctly base paired nucleotide is in what
is referred to in the art as a "closed" conformation. At a more
detailed structural level, the transition from the open to closed
conformation is characterized by relative movement within the
polymerase resulting in the "thumb" domain and "fingers" domain
being closer to each other. In the open conformation the thumb
domain is further from the fingers domain, akin to the opening and
closing of the palm of a hand. In various polymerases, the distance
between the tip of the finger and the thumb can change up to 30
angstroms between the "open" and "closed" conformations. The
distance between the tip of the finger and the rest of the protein
domains can also change up to 10 angstroms. It will be understood
that larger changes may also occur and can be exploited in a method
set forth herein such that a change that is greater than 10
angstroms can be detected. Furthermore, smaller changes can be
detected including those that are less than about 10, 8, 6, 4, or 2
angstroms so long as the change in distance is sufficient to be
detectable using a charge sensor.
[0167] In particular embodiments, a charge label that is attached
to a finger domain can be attached to a residue at position 376 or
residues within 5 angstroms radius from position 376 of the Phi29
DNA polymerase and a label that is attached to the thumb or other
domain can be attached to a residue at position 535, 203, 510, 564,
or residues within 5 angstroms radius from these positions of the
Phi29 DNA polymerase. Labels can be attached at positions and using
methods set forth in US Pat. App. Pub. No. 2011/0312529 A1; U.S.
Pat. No. 6,908,763 or WO 2010/068884 A2, each of which is
incorporated herein by reference in its entirety.
[0168] A change in conformation of a polymerase, for example, from
an open conformation to a closed conformation, can be detected
using a conformational label. Any label can be used that produces a
charge signal that is responsive to a change in the structure,
shape or arrangement of amino acid residues such as the changes
that occur between the open and closed conformations of a
polymerase.
[0169] A charge label can be attached to a polymerase, for example,
via covalent linkage. Alternatively or additionally, a probe can be
attached to another molecule that is in proximity to a polymerase,
such that a conformational change in the polymerase causes a change
in signal from the probe. For example, the polymerase can be
attached to a charge sensor and the charge sensor can have a probe
that is capable of interacting with the polymerase in a way that
signals from the probe change in response to conformational changes
of the polymerase. In a particular embodiment, a charge label can
be attached site specifically to a polymerase by introducing
cysteine residue at a desired location in the polymerase and then
modifying the polymerase with a label having a moiety that reacts
specifically with the sulfur group of cysteine, an exemplary
reactive moiety being a reactive maleimide moiety. Labels can also
be introduced to a polymerase or other nucleic acid enzyme by split
inteins as described in Yang et al. J. Am. Chem. Soc.,
131:11644-11645 (2009), which is incorporated herein by reference
in its entirety. Labels can also be introduced to nucleic acid
enzymes by genetically encoded unnatural amino acids. One example
is described in Fleissner et al. Proc. Nat'l. Acad. Sci. USA
106:21637-42 (2009), which is incorporated herein by reference in
its entirety.
[0170] In some embodiments, one or more tethers can be attached to
a polymerase at locations on the polymerase where conformational
changes are transmitted to a charge sensor to which the tethers are
also attached. For example, conformational changes can cause a
perturbation that is transmitted by one or more conducting tethers
to a charge sensor. In some cases a single polymerase can be
attached to a charge sensor via two or more tethers. In this
configuration, changes in the conformation of the polymerase can
alter the relative positions of the two or more tethers, which can
in turn produce field perturbations that can be detected by the
charge sensor. One or more tethers can be site selectively attached
to a polymerase using known methods of mutagenesis, chemical
modification, or both. For example, one or more cysteine can be
introduced as site specific mutation(s) in an engineered polymerase
allowing attachment of a sulfur reactive tether to the cysteine(s).
Exemplary attachment points include those set forth herein and in
US Pat. App. Pub. No. 2011/0312529 A1 (which is incorporated herein
by reference in its entirety) for attachment of conformational
labels.
[0171] In addition to the conformational changes set forth herein
and otherwise known in the art, polymerases undergo several
transitions in the course of adding a nucleotide to a nascent
strand. The transitions can be distinguished from each other, for
example, by kinetic characterization. Distinguishable transitions
include, for example, those set forth in US Pat. App. Pub. No.
2011/0312529 A1. One or more of the transitions that a polymerase
undergoes when adding a nucleotide to a nucleic acid can be
detected by a charge sensor, for example, using a polymerase that
is optionally conformationally labeled. Time based or kinetic
measurement of signals detected by a charge sensor to which a
polymerase is attached can be used to distinguish one transition
from another.
[0172] In particular embodiments, time-based or kinetic
measurements of a polymerase attached to a charge sensor can be
used to distinguish the species of nucleotide that is added to a
nucleic acid. For example, a time based or kinetic measurement can
be used to distinguish the species of nucleotide that is bound to a
polymerase to form one or more of the complexes set forth in US
Pat. App. Pub. No. 2011/0312529 A1. Alternatively or additionally,
time-based or kinetic measurements of a charge sensor-attached
polymerase can be used to distinguish the binding and/or
incorporation of a correctly Watson-Crick base-paired nucleotide
from one that is incorrectly base-paired to the template nucleic
acid.
[0173] Methods that use time-based or kinetic discrimination of
nucleotides, can be facilitated by use of very fast mixing of
reagents at the charge sensors coupled with real time detection.
The mixing can occur on the sub-milliseconds timescale in
accordance with available stopped-flow instrumentation. The fast
mixing of reagents can be achieved using fast fluidics, active or
passive mixing, and proper confinement (e.g. mix blousing) of the
reaction to overcome limitations by diffusion. Stopped-flow
delivery is particularly useful. Stopped flow delivery provides
delivery of fluid to a detection site using rapid flow of the fluid
followed by abrupt stoppage of the flow. The fluid that is
delivered typically displaces an equal volume of fluid from the
detection site. The fluid can mix with a solid-phase analyte such
as a polymerase attached to a charge sensor. The dead time for
stopped-flow fluid delivery can be, for example, less than 2
milliseconds (msec). Accordingly, the dead time can be no longer
than 2 msec, 1.5 msec, 1 msec, 0.8 msec, 0.6 msec, 0.5 msec or 0.4
msec. For useful stopped flow and rapid mixing fluidic systems see,
for example, Chance, B. J. Frank. Inst., 229, 613 (1940), and US
Pat. App. Pub. No. US 2013/0165328 A1, each of which is
incorporated herein by reference in its entirety.
[0174] A sequence of time-based or kinetic measurements for a
charge-sensor attached polymerase can be used to determine the
sequence of a template nucleic acid being used by the polymerase to
synthesize a complementary strand. It will be understood that the
sequence of the template strand can be inferred from the sequence
of nucleotides incorporated into the strand that is being extended.
As such, determination of the sequence of one strand will be
understood to include determination of the sequence of its
complementary strand.
[0175] Any of a variety of nucleotide species can be useful in a
method or composition set forth herein. For example, naturally
occurring nucleotides can be used such as ATP, UTP, CTP, GTP, ADP,
UDP, CDP, GDP, AMP, UMP, CMP, GMP, dATP, dTTP, dCTP, dGTP, dADP,
dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP. Typically, dNTP
nucleotides are incorporated into a DNA strand by DNA polymerases
and NTP nucleotides are incorporated into an RNA strand by RNA
polymerases. In particular embodiments, NTP nucleotides or analogs
thereof can be incorporated into DNA by a DNA polymerase, for
example, in cases where the NTP, or analog thereof, is capable of
being incorporated into the DNA by the DNA polymerase and where the
rate or time duration for a DNA polymerase transition using the
NTP, or analog thereof, can be distinguished from the rate or time
duration for the DNA polymerase transition using another
nucleotide. Alternatively, dNTP nucleotides or analogs thereof can
be incorporated into RNA by an RNA polymerase, for example, in
cases where the dNTP, or analog thereof, is capable of being
incorporated into the RNA by the RNA polymerase and where the rate
or time duration for an RNA polymerase transition using the dNTP,
or analog thereof, can be distinguished from the rate or time
duration for the RNA polymerase transition using another
nucleotide. Additionally, dNTP nucleotides or analogs thereof can
be incorporated into DNA from an RNA template by a reverse
transcriptase, for example, in cases where the dNTP, or analog
thereof, is capable of being incorporated into the DNA from an RNA
template by a reverse transcriptase and where the rate or time
duration for a reverse transcriptase transition using the dNTP, or
analog thereof, can be distinguished from the rate or time duration
for the reverse transcriptase transition using another nucleotide.
The relative difference in rate or time duration can be a relative
increase in the rate, a relative increase in duration, a relative
decrease in rate or a relative decrease in duration.
[0176] Non-natural nucleotide analogs are also useful. Particularly
useful non-natural nucleotide analogs include, but are not limited
to, those that produce a detectably different rate or time duration
for a polymerase transition that can be distinguished from the rate
or time duration for a polymerase transition with another
nucleotide. For example, a non-natural nucleotide analog may
usefully produce a detectably different rate or time duration for a
polymerase transition that can be distinguished from the rate or
time duration for the same transition of the polymerase with
another nucleotide such as a naturally occurring nucleotide.
Exemplary nucleotide analogs that can be used include, but are not
limited to, dNTPaS; NTPaS; nucleotides having unnatural nucleobases
identified in Hwang et al., Nucl. Acids Res. 34:2037-2045 (2006)
(incorporated herein by reference in its entirety) as ICS, 3MN,
7AI, BEN, DM5, TM, 2Br, 3Br, 4Br, 2CN, 3CN, 4CN, 2FB, 3FB, MM1, MM2
and MM3; or nucleotides having other non-natural nucleobases such
as those described in Patro et al. Biochem. 48:180-189 (2009)
(incorporated herein by reference in its entirety) which include
2-amino-1-deazapurine, 1-deazapurine, 2-pyridine, hypoxanthine,
purine, 6-Cl-purine, 2-amino-dA, 2-amino purine or
6-Cl-2-amino-purine or nucleotides having non-natural nucleobases
such as those described in Krueger et al. Chem Biol. 16:242-8
(2009) (incorporated herein by reference in its entirety) which
include iso-G, iso-C, 5SICS, MMO2, Ds, Pa, FI, FB, dZ, DNB, thymine
isosteres, 5-NI, dP, azole-carboxamide, xA, Im-No, Im-ON, J, A*,
T*.
[0177] Non-natural nucleotide analogs having 5' modifications are
particularly useful. The non-natural nucleotide analog will
typically have a triphosphate but can have more or fewer
phosphates. In particular embodiments, one or more of the alpha
phosphate, beta phosphate or gamma phosphate of a non-natural
nucleotide is covalently attached to a moiety other than oxygen. A
moiety that is attached to a phosphate or otherwise present at the
5' position can provide a negative charge, a positive charge,
metal-chelating activity or steric bulk. Exemplary moieties
include, but are not limited to, amino acids, in the L-enantiomer
form or R-enantiomer form, such as histidine, aspartate, glutamate,
tryptophan, phenylalanine, methionine, tyrosine, cysteine, glycine
alanine, or proline; an amino group; a chelated metal such as
magnesium or manganese; a methyl group; a halogen such as bromine,
chlorine or iodine; a thiol group; an electron withdrawing group;
an electron donating group; an aromatic amine; or an aliphatic
amine. These and other moieties may be advantageous in embodiments
where they provide an interaction with a polymerase, or other
nucleic acid enzyme, that differs from the interaction that the
enzyme has with a nucleotide lacking the moiety. As such, the
presence and absence of the moiety on respective nucleotide species
can be exploited to distinguish the nucleotide species in a
sequencing method, for example, based on the rate, time duration
and/or intensity for a conformational signal change in a nucleic
acid enzyme acting on the nucleotide species.
[0178] A reaction composition or method can include one or more
nucleotide species. For example, a reaction composition or method
used for sequence analysis can include four different nucleotide
species capable of forming Watson-Crick base pairs with four
respective nucleotide species in a nucleic acid template being
synthesized. Particular embodiments can include at least two
different nucleotide species, at least three different nucleotide
species, at least four different nucleotide species, or more. At
least two of the nucleotide species can be non-natural nucleotide
analogs, at least three of the nucleotide species can be
non-natural nucleotide analogs, or at least four of the nucleotide
species can be non-natural nucleotide analogs. Thus a reaction
composition or method can include a mixture of natural nucleotides
and non-natural nucleotide analogs. Alternatively, a reaction
composition can lack natural nucleotides having instead only
non-natural nucleotide analogs. The reaction can be carried out
under conditions in which only non-natural nucleotide analogs are
incorporated into a growing nucleic acid by a polymerase or other
nucleic acid enzyme.
[0179] In some embodiments, a reaction composition or method can
include nucleotide species that base-pair with no more than one
nucleotide species in a nucleic acid template. For example, a
method can be carried out under conditions wherein different
nucleotide species are contacted with a polymerase and nucleic acid
in separate, sequential reactions. Specifically, a nucleotide
species that base-pairs with A can be added in a first reaction, a
nucleotide species that base-pairs with C can be added in a second
reaction, a nucleotide species that base-pairs with T can be added
in a third reaction, and a nucleotide species that base-pairs with
G can be added in a fourth reaction. The reactions are referred to
as first, second, third and fourth merely to illustrate that the
reactions are separate but this does not necessarily limit the
order by which the species can be added in a method set forth
herein. Rather, nucleotide species that base-pair with A, C, T or G
can be added in any order desired or appropriate for a particular
embodiment of the methods. Typically in a sequencing method
nucleotide species that base-pair with four different nucleotide
species in a given template nucleic acid are added sequentially to
complete a cycle of the sequencing method. However, it will be
understood that fewer than four nucleotide additions can be used in
some embodiments. Furthermore, it will be understood that mixtures
of nucleotides that base-pair with more than one but no more than
2, 3 or 4 nucleotide species can be used. Similarly, mixtures of
nucleotides that base-pair with more than two but no more than 3 or
4 nucleotide species can be used, or mixtures of nucleotides that
base-pair with more than three but no more than 4 nucleotide
species can be used.
[0180] Multiplex methods are also possible. For example, a solid
support used in a method of the present disclosure can include a
plurality of charge sensors that are tethered to polymerases, the
polymerases can be contacted with template nucleic acids and at
least four different nucleotide types under conditions wherein the
polymerases catalyze sequential addition of the nucleotide types to
form nucleic acid complements of the nucleic acid templates; a
series of changes in the signal from the polymerases can be
detected using the charge sensors; and the sequences of nucleotides
can be determined for the template nucleic acids. The plurality of
polymerases present on the solid support can include at least two
different types of polymerases. The different types of polymerases
can optionally produce mutually distinguishable signals detectable
by the charge sensors when incorporating the same type of
nucleotide into a nascent nucleic acid strand. Distinguishing these
mutually distinguishable signals can be used as a basis for
determining the sequences for the plurality of nucleic acids.
[0181] Throughout this application various publications, patent
applications or patents have been referenced. The disclosures of
these publications in their entireties are hereby incorporated by
reference in this application in order to more fully describe the
state of the art to which this invention pertains.
[0182] The term "comprising" is intended herein to be open-ended,
including not only the recited elements, but further encompassing
any additional elements.
[0183] Although the invention has been described with reference to
the examples provided above, it should be understood that various
modifications can be made without departing from the invention.
Accordingly, the invention is limited only by the claims.
Sequence CWU 1
1
31272PRTEscherichia coli 1Thr Ser Tyr His Gln Ala Val Thr Ala Thr
Gly Arg Leu Ser Ser Thr1 5 10 15Asp Pro Asn Leu Gln Asn Ile Pro Val
Arg Asn Glu Glu Gly Arg Arg 20 25 30Ile Arg Gln Ala Phe Ile Ala Pro
Glu Asp Tyr Val Ile Val Ser Ala 35 40 45Asp Tyr Ser Gln Ile Glu Leu
Arg Ile Met Ala His Leu Ser Arg Asp 50 55 60Lys Gly Leu Leu Thr Ala
Phe Ala Glu Gly Lys Asp Ile His Arg Ala65 70 75 80Thr Ala Ala Glu
Val Phe Gly Leu Pro Leu Glu Thr Val Thr Ser Glu 85 90 95Gln Arg Arg
Ser Ala Lys Ala Ile Asn Phe Gly Leu Ile Tyr Gly Met 100 105 110Ser
Ala Phe Gly Leu Ala Arg Gln Leu Asn Ile Pro Arg Lys Glu Ala 115 120
125Gln Lys Tyr Met Asp Leu Tyr Phe Glu Arg Tyr Pro Gly Val Leu Glu
130 135 140Tyr Met Glu Arg Thr Arg Ala Gln Ala Lys Glu Gln Gly Tyr
Val Glu145 150 155 160Thr Leu Asp Gly Arg Arg Leu Tyr Leu Pro Asp
Ile Lys Ser Ser Asn 165 170 175Gly Ala Arg Arg Ala Ala Ala Glu Arg
Ala Ala Ile Asn Ala Pro Met 180 185 190Gln Gly Thr Ala Ala Asp Ile
Ile Lys Arg Ala Met Ile Ala Val Asp 195 200 205Ala Trp Leu Gln Ala
Glu Gln Pro Arg Val Arg Met Ile Met Gln Val 210 215 220His Asp Glu
Leu Val Phe Glu Val His Lys Asp Asp Val Asp Ala Val225 230 235
240Ala Lys Gln Ile His Gln Leu Met Glu Asn Cys Thr Arg Leu Asp Val
245 250 255Pro Leu Leu Val Glu Val Gly Ser Gly Glu Asn Trp Asp Gln
Ala His 260 265 2702273PRTBacillus subtilis 2Thr Arg Phe Asn Gln
Ala Leu Thr Gln Thr Gly Arg Leu Ser Ser Thr1 5 10 15Asp Pro Asn Leu
Gln Asn Ile Pro Ile Arg Leu Glu Glu Gly Arg Lys 20 25 30Ile Arg Gln
Ala Phe Val Pro Ser Glu Lys Asp Trp Leu Ile Phe Ala 35 40 45Ala Asp
Tyr Ser Gln Ile Glu Leu Arg Val Leu Ala His Ile Ser Lys 50 55 60Asp
Glu Asn Leu Ile Glu Ala Phe Thr Asn Asp Met Asp Ile His Thr65 70 75
80Lys Thr Ala Met Asp Val Phe His Val Ala Lys Asp Glu Val Thr Ser
85 90 95Ala Met Arg Arg Gln Ala Lys Ala Val Asn Phe Gly Ile Val Tyr
Gly 100 105 110Ile Ser Asp Tyr Gly Leu Ser Gln Asn Leu Gly Ile Thr
Arg Lys Glu 115 120 125Ala Gly Ala Phe Ile Asp Arg Tyr Leu Glu Ser
Phe Gln Gly Val Lys 130 135 140Ala Tyr Met Glu Asp Ser Val Gln Glu
Ala Lys Gln Lys Gly Tyr Val145 150 155 160Thr Thr Leu Met His Arg
Arg Arg Tyr Ile Pro Glu Leu Thr Ser Arg 165 170 175Asn Phe Asn Ile
Arg Ser Phe Ala Glu Arg Thr Ala Met Asn Thr Pro 180 185 190Ile Gln
Gly Ser Ala Ala Asp Ile Ile Lys Lys Ala Met Ile Asp Met 195 200
205Ala Ala Lys Leu Lys Glu Lys Gln Leu Lys Ala Arg Leu Leu Leu Gln
210 215 220Val His Asp Glu Leu Ile Phe Glu Ala Pro Lys Glu Glu Ile
Glu Ile225 230 235 240Leu Glu Lys Leu Val Pro Glu Val Met Glu His
Ala Leu Ala Leu Asp 245 250 255Val Pro Leu Lys Val Asp Phe Ala Ser
Gly Pro Ser Trp Tyr Asp Ala 260 265 270Lys3273PRTArtificial
SequenceDescription of Artificial Sequence Synthetic consensus
polypeptideMOD_RES(2)..(2)Ser or ArgMOD_RES(3)..(3)Tyr or
PheMOD_RES(4)..(4)His or AsnMOD_RES(7)..(7)Val or
LeuMOD_RES(9)..(9)Ala or GlnMOD_RES(25)..(25)Val or
IleMOD_RES(27)..(27)Asn or LeuMOD_RES(32)..(32)Arg or
LysMOD_RES(38)..(38)Ile or ValMOD_RES(39)..(39)Ala or
ProMOD_RES(40)..(40)Pro or SerMOD_RES(41)..(41)Glu or not
presentMOD_RES(42)..(42)Glu or LysMOD_RES(44)..(44)Tyr or
TrpMOD_RES(45)..(45)Val or LeuMOD_RES(47)..(47)Val or
PheMOD_RES(48)..(48)Ser or AlaMOD_RES(58)..(58)Ile or
ValMOD_RES(59)..(59)Met or LeuMOD_RES(62)..(62)Leu or
IleMOD_RES(64)..(64)Arg or LysMOD_RES(66)..(66)Lys or
GluMOD_RES(67)..(67)Gly or AsnMOD_RES(69)..(69)Leu or
IleMOD_RES(70)..(70)Thr or GluMOD_RES(73)..(73)Ala or
ThrMOD_RES(74)..(74)Glu or AsnMOD_RES(75)..(75)Gly or
AspMOD_RES(76)..(76)Lys or MetMOD_RES(80)..(80)Arg or
ThrMOD_RES(81)..(81)Ala or LysMOD_RES(84)..(84)Ala or
MetMOD_RES(85)..(85)Glu or AspMOD_RES(88)..(88)Gly or
HisMOD_RES(89)..(89)Leu or ValMOD_RES(90)..(90)Pro or
AlaMOD_RES(91)..(91)Leu or LysMOD_RES(92)..(92)Glu or
AspMOD_RES(93)..(93)Thr or GluMOD_RES(97)..(97)Glu or
AlaMOD_RES(98)..(98)Gln or MetMOD_RES(101)..(101)Ser or
GlnMOD_RES(105)..(105)Ile or ValMOD_RES(109)..(109)Leu or
IleMOD_RES(110)..(110)Ile or ValMOD_RES(113)..(113)Met or
IleMOD_RES(115)..(115)Ala or AspMOD_RES(116)..(116)Phe or
TyrMOD_RES(119)..(119)Ala or SerMOD_RES(120)..(120)Arg or
GlnMOD_RES(121)..(121)Gln or AsnMOD_RES(123)..(123)Asn or
GlyMOD_RES(125)..(125)Pro or ThrMOD_RES(130)..(130)Gln or
GlyMOD_RES(131)..(131)Lys or AlaMOD_RES(132)..(132)Tyr or
PheMOD_RES(133)..(133)Met or IleMOD_RES(135)..(135)Leu or
ArgMOD_RES(137)..(137)Phe or LeuMOD_RES(139)..(139)Arg or
SerMOD_RES(140)..(140)Tyr or PheMOD_RES(141)..(141)Pro or
GlnMOD_RES(144)..(144)Leu or LysMOD_RES(145)..(145)Glu or
AlaMOD_RES(149)..(149)Arg or AspMOD_RES(150)..(150)Thr or
SerMOD_RES(151)..(151)Arg or ValMOD_RES(152)..(152)Ala or
GlnMOD_RES(153)..(153)Gln or GluMOD_RES(156)..(156)Glu or
GlnMOD_RES(157)..(157)Gln or LysMOD_RES(161)..(161)Glu or
ThrMOD_RES(164)..(164)Asp or MetMOD_RES(165)..(165)Gly or
HisMOD_RES(168)..(168)Leu or ArgMOD_RES(170)..(170)Leu or
IleMOD_RES(172)..(172)Asp or GluMOD_RES(173)..(173)Ile or
LeuMOD_RES(174)..(174)Lys or ThrMOD_RES(176)..(176)Ser or
ArgMOD_RES(178)..(178)Gly or PheMOD_RES(179)..(179)Ala or
AsnMOD_RES(180)..(180)Arg or IleMOD_RES(182)..(182)Ala or
SerMOD_RES(183)..(183)Ala or PheMOD_RES(187)..(187)Ala or
ThrMOD_RES(189)..(189)Ile or MetMOD_RES(191)..(191)Ala or
ThrMOD_RES(193)..(193)Met or IleMOD_RES(196)..(196)Thr or
SerMOD_RES(203)..(203)Arg or LysMOD_RES(207)..(207)Ala or
AspMOD_RES(208)..(208)Val or MetMOD_RES(209)..(209)Asp or
AlaMOD_RES(211)..(211)Trp or LysMOD_RES(213)..(213)Gln or
LysMOD_RES(214)..(214)Ala or GluMOD_RES(215)..(215)Glu or
LysMOD_RES(217)..(217)Pro or LeuMOD_RES(218)..(218)Arg or
LysMOD_RES(219)..(219)Val or AlaMOD_RES(221)..(221)Met or
LeuMOD_RES(222)..(222)Ile or LeuMOD_RES(223)..(223)Met or
LeuMOD_RES(230)..(230)Val or IleMOD_RES(233)..(233)Val or
AlaMOD_RES(234)..(234)His or ProMOD_RES(236)..(236)Asp or
GluMOD_RES(237)..(237)Asp or GluMOD_RES(238)..(238)Val or
IleMOD_RES(239)..(239)Asp or GluMOD_RES(240)..(240)Ale or
IleMOD_RES(241)..(241)Val or LeuMOD_RES(242)..(242)Ala or
GluMOD_RES(244)..(244)Gln or LeuMOD_RES(245)..(245)Ile or
ValMOD_RES(246)..(246)His or ProMOD_RES(247)..(247)Gln or
GluMOD_RES(248)..(248)Leu or ValMOD_RES(251)..(251)Asn or
HisMOD_RES(252)..(252)Cys or AlaMOD_RES(253)..(253)Thr or
LeuMOD_RES(254)..(254)Arg or AlaMOD_RES(260)..(260)Leu or
LysMOD_RES(262)..(262)Glu or AspMOD_RES(263)..(263)Val or
PheMOD_RES(264)..(264)Gly or AlaMOD_RES(267)..(267)Glu or
ProMOD_RES(268)..(268)Asn or SerMOD_RES(270)..(270)Asp or
TyrMOD_RES(271)..(271)Gln or AspMOD_RES(273)..(273)His or Lys 3Thr
Xaa Xaa Xaa Gln Ala Xaa Thr Xaa Thr Gly Arg Leu Ser Ser Thr1 5 10
15Asp Pro Asn Leu Gln Asn Ile Pro Xaa Arg Xaa Glu Glu Gly Arg Xaa
20 25 30Ile Arg Gln Ala Phe Xaa Xaa Xaa Glu Xaa Asp Xaa Xaa Ile Xaa
Xaa 35 40 45Ala Asp Tyr Ser Gln Ile Glu Leu Arg Xaa Xaa Ala His Xaa
Ser Xaa 50 55 60Asp Xaa Xaa Leu Xaa Xaa Ala Phe Xaa Xaa Xaa Xaa Asp
Ile His Xaa65 70 75 80Xaa Thr Ala Xaa Xaa Val Phe Xaa Xaa Xaa Xaa
Xaa Xaa Val Thr Ser 85 90 95Xaa Xaa Arg Arg Xaa Ala Lys Ala Xaa Asn
Phe Gly Xaa Xaa Tyr Gly 100 105 110Xaa Ser Xaa Xaa Gly Leu Xaa Xaa
Xaa Leu Xaa Ile Xaa Arg Lys Glu 115 120 125Ala Xaa Xaa Xaa Xaa Asp
Xaa Tyr Xaa Glu Xaa Xaa Xaa Gly Val Xaa 130 135 140Xaa Tyr Met Glu
Xaa Xaa Xaa Xaa Xaa Ala Lys Xaa Xaa Gly Tyr Val145 150 155 160Xaa
Thr Leu Xaa Xaa Arg Arg Xaa Tyr Xaa Pro Xaa Xaa Xaa Ser Xaa 165 170
175Asn Xaa Xaa Xaa Arg Xaa Xaa Ala Glu Arg Xaa Ala Xaa Asn Xaa Pro
180 185 190Xaa Gln Gly Xaa Ala Ala Asp Ile Ile Lys Xaa Ala Met Ile
Xaa Xaa 195 200 205Xaa Ala Xaa Leu Xaa Xaa Xaa Gln Xaa Xaa Xaa Arg
Xaa Xaa Xaa Gln 210 215 220Val His Asp Glu Leu Xaa Phe Glu Xaa Xaa
Lys Xaa Xaa Xaa Xaa Xaa225 230 235 240Xaa Xaa Lys Xaa Xaa Xaa Xaa
Xaa Met Glu Xaa Xaa Xaa Xaa Leu Asp 245 250 255Val Pro Leu Xaa Val
Xaa Xaa Xaa Ser Gly Xaa Xaa Trp Xaa Xaa Ala 260 265 270Xaa
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