U.S. patent application number 14/516785 was filed with the patent office on 2015-02-05 for dna sequencing by nanopore using modified nucleotides.
This patent application is currently assigned to THE TRUSTEES OF COLUMBIA UNIVERSITY IN THE CITY OF NEW YORK. The applicant listed for this patent is Jingyue Ju. Invention is credited to Jingyue Ju.
Application Number | 20150037788 14/516785 |
Document ID | / |
Family ID | 38832077 |
Filed Date | 2015-02-05 |
United States Patent
Application |
20150037788 |
Kind Code |
A1 |
Ju; Jingyue |
February 5, 2015 |
DNA SEQUENCING BY NANOPORE USING MODIFIED NUCLEOTIDES
Abstract
This invention provides a process for sequencing single-stranded
DNA by employing a nanopore and modified nucleotides.
Inventors: |
Ju; Jingyue; (Englewood
Cliffs, NJ) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Ju; Jingyue |
Englewood Cliffs |
NJ |
US |
|
|
Assignee: |
THE TRUSTEES OF COLUMBIA UNIVERSITY
IN THE CITY OF NEW YORK
NEW YORK
NY
|
Family ID: |
38832077 |
Appl. No.: |
14/516785 |
Filed: |
October 17, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12308091 |
May 18, 2009 |
8889348 |
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PCT/US2007/013559 |
Jun 7, 2007 |
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14516785 |
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60811912 |
Jun 7, 2006 |
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Current U.S.
Class: |
435/6.1 |
Current CPC
Class: |
C12Q 1/6869 20130101;
C12Q 1/6869 20130101; C12Q 2565/631 20130101; C12Q 2565/101
20130101; C12Q 2565/631 20130101; C12Q 2525/101 20130101; C12Q
1/6869 20130101 |
Class at
Publication: |
435/6.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Goverment Interests
[0001] The invention disclosed herein was made with government
support under grant no. 1R21HG003718-01 from the National Human
Genome Research Institute. Accordingly, the U.S. Government has
certain rights in this invention.
[0002] Throughout this application, various publications are
referenced in parentheses by number. Full citations for these
references may be found at the end of the specification immediately
preceding the claims. The disclosures of these publications in
their entireties are hereby incorporated by reference into this
application to more fully describe the state of the art to which
this invention pertains.
Claims
1-17. (canceled)
18. A method for determining the nucleotide sequence of a
single-stranded DNA comprising the steps of: (a) synthesizing a
double-stranded DNA using the single-stranded DNA as template, a
primer, a DNA polymerase, and all four nucleotides A, G, C and T;
(b) determining using a nanopore an electronic signature
corresponding to each A, G, C and T being polymerized using the
single-stranded DNA as a template; and (c) comparing each
electronic signature determined in (b) with electronic signatures
corresponding to each of A, G, C and T so as to determine the
identity of each such nucleotide, thereby determining the
nucleotide sequence of the single-stranded DNA.
19. The method of claim 18, wherein the nanopore has a diameter of
from about 1 nm to about 5 mm.
20. The method of claim 18, wherein the nanopore has a diameter of
from about 1 nm to about 3 nm.
21. The method of claim 18, wherein the nanopore has a diameter of
about 1 nm, 2 nm, 3 nm, 4 nm or 5 nm.
22. The method of claim 18, wherein each A and each T nucleotide
comprises a modifying group.
23. The method of claim 18, wherein each G and each C nucleotide
comprises a modifying group.
24. The method of claim 18, wherein each A and each C nucleotide or
each G and each T nucleotide comprises a modifying group.
25. The method of claim 18, wherein A, G, C and T nucleotides of
said single-stranded DNA have an order of bulkiness that is
T>A>G>C.
Description
BACKGROUND OF THE INVENTION
[0003] DNA sequencing is a fundamental technology for biology.
Several analytical methods have been developed to detect DNA or RNA
at the single molecule level using chemical or physical microscopic
technologies (15, 16, 21 and 23). In the past few years, the ion
channel has been explored for detecting individual DNA or RNA
strands, with nanopore being a candidate for high rate sequencing
and analysis of DNA [9, 10, 4, 3 and 7].
[0004] In 1996, Kasianowicz et al. first demonstrated that the
.alpha.-hemolysin channel, an exotoxin secreted by a bacterium,
could be used to detect nucleic acids at the single molecule level
[8]. The monomeric polypeptide self-assembles in a lipid bilayer
membrane to form a heptameric pore, with a 2.6 nm-diameter
vestibule and 1.5 nm-diameter limiting aperture (namely, the
narrowest point of the pore) [1, 14 and 15]. In an aqueous ionic
salt solution such as KCl, the pore formed by the .alpha.-hemolysin
channel conducts a sufficiently strong and steady ionic current
when an appropriate voltage is applied across the membrane. The
limiting aperture of the nanopore allows linear single-stranded but
not double-stranded nucleic acid molecules (diameter .about.2.0 nm)
to pass through. The polyanionic nucleic acids are driven through
the pore by the applied electric field, which blocks or reduces the
ionic current that would be otherwise unimpeded. This process of
passage generates an electronic signature (FIG. 1) [23 and 5]. A
particular nucleic acid molecule, when entering and passing through
the nanopore, will generate a characteristic signature that
distinguishes it from others. The duration of the blockade is
proportional to the length of the nucleic acid, and its signal
strength is related to the steric and electronic properties of the
nucleotides, namely the identity of the four bases (A, C, G and
T).
[0005] A specific event diagram is constructed which is the plot of
translocation time versus blockade current. This specific event
diagram (also referred to as an electronic signature) is used to
distinguish the lengths and the compositions of polynucleotides by
single-channel recording techniques based on characteristic
parameters such as translocation current, translocation duration,
and their corresponding dispersions in the diagram [14].
[0006] Although the nanopore approach is known as a DNA detection
method, this approach for base-to-base sequencing has not yet been
achieved.
SUMMARY OF THE INVENTION
[0007] This invention provides a method for determining the
nucleotide sequence of a single-stranded DNA comprising the steps
of: [0008] (a) passing the single-stranded DNA through a pore of
suitable diameter by applying an electric field to the DNA, wherein
at least each A or each G residue and at least each C, each T or
each U residue comprises a modifying group bound to its respective
base so that each type of nucleotide in the DNA has an electronic
signature which is distinguishable from the electronic signature of
each other type of nucleotide in the DNA; [0009] (b) for each
nucleotide of the DNA which passes through the pore, determining an
electronic signature for such nucleotide; and [0010] (c) comparing
each electronic signature determined in step (b) with electronic
signatures corresponding to each of A, G, C and T modified as per
the nucleotides in the single-stranded DNA, so as to determine the
identity of each such nucleotide, thereby determining the
nucleotide sequence of the single-stranded DNA.
[0011] This invention also provides a method for determining the
nucleotide sequence of a single-stranded RNA comprising the steps
of: [0012] (a) passing the single-stranded RNA through a pore of
suitable diameter by applying an electric field to the RNA, wherein
at least each A or each G residue and at least each C or each U
residue comprises a modifying group bound to its respective base so
that each type of nucleotide in the RNA has an electronic signature
which is distinguishable from the electronic signature of each
other type of nucleotide in the RNA; [0013] (b) for each nucleotide
of the RNA which passes through the pore, determining an electronic
signature for such nucleotide; and [0014] (c) comparing each
electronic signature determined in step (b) with electronic
signatures corresponding to each of A, G, C and U modified as per
the nucleotides in the single-stranded RNA, so as to determine the
identity of each such nucleotide, thereby determining the
nucleotide sequence of the single-stranded RNA.
[0015] This invention also provides a nucleotide having an azido
group covalently bound to its base.
[0016] This invention also provides a method for making a modified
nucleotide comprising contacting the instant nucleotide with an
alkyne-containing compound under conditions permitting reaction
between the azido and the alkyne groups, thereby making the
modified nucleotide.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1. .alpha.-Hemolysin protein self-assembles in a lipid
bilayer to form an ion channel and a nucleic acid stretch passes
through it (left), with the corresponding electronic signatures
generated (right) [23 and 5].
[0018] FIG. 2. Structures of nucleotides dATP, dGTP, dCTP and
dTTP.
[0019] FIG. 3. Nucleotide bulkiness in ascending order: (a)
5'-C.sub.60T-3', (b) 5'-G*.sub.60T-3', (c) 5'-A*.sub.60T-3', and
(d) 5'-T*.sub.60T-3'.
[0020] FIG. 4. Structures of dCTP and dGTP, and modified
nucleotides (dATP-NH.sub.2 and dUTP-NH.sub.2).
[0021] FIG. 5. Modification of dATP-NH.sub.2 and dUTP-NH.sub.2.
[0022] FIG. 6. DNA-extension reaction using modified nucleotides
(dATP-NHCOR.sub.1 and dUTP-NHCOR.sub.2) to generate a modified
single-stranded DNA chain. (SEQ ID NOs. 1 and 2 for template and
primer, respectively).
[0023] FIG. 7. Steps of verifying sequencing capacity via nanopore
using various DNA sequences. (SEQ ID NOs. 3-6 for part (i), top to
bottom, respectively; SEQ ID NO:7 for part (ii); SEQ ID NO:8 for
part (iii); and SEQ ID NO:9 for part (iv)).
[0024] FIG. 8. Structures of unmodified nucleotides (dCTP and dGTP)
and hook-labeled nucleotides (dATP-NH.sub.2 and dUTP-N.sub.3). The
amino and the azido groups function as hooks to conjugate with
bulky groups after the nucleotides are incorporated into the DNA
strand.
[0025] FIG. 9. Synthesis of dUTP-N.sub.3.
[0026] FIG. 10. DNA-extension reaction using hook-labeled
nucleotides (dATP-NH.sub.2 and dUTP-N.sub.3) to generate a modified
single-stranded DNA chain, which will then react with large
functional groups (R1 and R3) selectively for distinct detection by
nanopore. (SEQ ID NOs. 1 and 2 for template and primer,
respectively)
DETAILED DESCRIPTION OF THE INVENTION
Terms
[0027] As used herein, and unless stated otherwise, each of the
following terms shall have the definition set forth below.
TABLE-US-00001 A- Adenine; C- Cytosine; DNA- Deoxyribonucleic acid;
G- Guanine; RNA- Ribonucleic acid; T- Thymine; and U- Uracil.
[0028] "Electronic signature" of a nucleotide passing through a
pore via application of an electronic field shall include, for
example, the duration of the nucleotide's passage through the pore
together with the observed amplitude of current during that
passage. Electronic signatures can be visualized, for example, by a
plot of current (e.g. pA) versus time. Electronic signature for a
DNA is also envisioned and can be, for example, a plot of current
(e.g. pA) versus time for the DNA to pass through the pore via
application of an electric field.
[0029] "Nanopore" includes, for example, a structure comprising (a)
a first and a second compartment separated by a physical barrier,
which barrier has at least one pore with a diameter, for example,
of from about 1 to 10 nm, and (b) a means for applying an electric
field across the barrier so that a charged molecule such as DNA can
pass from the first compartment through the pore to the second
compartment. The nanopore ideally further comprises a means for
measuring the electronic signature of a molecule passing through
its barrier. The nanopore barrier may be synthetic or naturally
occurring in part. Barriers can include, for example, lipid
bilayers having therein .alpha.-hemolysin, oligomeric protein
channels such as porins, and synthetic peptides and the like.
Barriers can also include inorganic plates having one or more holes
of a suitable size. Herein "nanopore", "nanopore barrier" and the
"pore" in the nanopore barrier are sometimes used equivalently.
[0030] "Nucleic acid" shall mean any nucleic acid molecule,
including, without limitation, DNA, RNA and hybrids thereof. The
nucleic acid bases that form nucleic acid molecules can be the
bases A, C, G, T and U, as well as derivatives thereof. Derivatives
of these bases are well known in the art, and are exemplified in
PCR Systems. Reagents and Consumables (Perkin Elmer Catalogue
1996-1997, Roche Molecular Systems, Inc., Branchburg, N.J.,
USA).
[0031] "Type" of nucleotide refers to A, G, C, T or U.
EMBODIMENTS OF THE INVENTION
[0032] This invention provides a method for determining the
nucleotide sequence of a single-stranded DNA comprising the steps
of: [0033] (a) passing the single-stranded DNA through a pore of
suitable diameter by applying an electric field to the DNA, wherein
at least each A or each G residue and at least each C, each T or
each U residue comprises a modifying group bound to its respective
base so that each type of nucleotide in the DNA has an electronic
signature which is distinguishable from the electronic signature of
each other type of nucleotide in the DNA; [0034] (b) for each
nucleotide of the DNA which passes through the pore, determining an
electronic signature for such nucleotide; and [0035] (c) comparing
each electronic signature determined in step (b) with electronic
signatures corresponding to each of A, (, C and T modified as per
the nucleotides in the single-stranded DNA, so as to determine the
identity of each such nucleotide, thereby determining the
nucleotide sequence of the single-stranded DNA.
[0036] In an embodiment of the instant method, the single-stranded
DNA is obtained by (a) synthesizing double-stranded DNA using a
single-stranded template, a DNA polymerase and nucleotides, wherein
at least each A or each G residue and at least each C or each T
residue comprises a modifying group bound to its respective bass so
that each type of nucleotide in the DNA has an electronic signature
which is distinguishable from the electronic signature of each
other type nucleotide in the DNA, and (b) removing from the
resulting double-stranded DNA the single-stranded DNA containing
modified nucleotides.
[0037] In another embodiment of the instant method, the
single-stranded DNA is obtained by (a) synthesizing double-stranded
DNA using a single-stranded template, a DNA polymerase and
nucleotides, wherein at least each A, each G, each C, each U or
each T residue comprises an azido group bound to its base, and at
least each A, each G, each C, each U and each T comprises an amino
group bound to its base, whereby the azido and amino groups do not
reside on the same type of base, (b) removing from the resulting
double-stranded DNA the single-stranded. DNA containing the azido
and amino group-containing nucleotides and (c) reacting the
resulting single-stranded DNA with a first modifying group which
forms a bond with the azido group and a second modifying group
which forms a bond with the amino group so as to obtain the
single-stranded DNA.
[0038] This invention also provides a method for determining the
nucleotide sequence of a single-stranded RNA comprising the steps
of: [0039] (a) passing the single-stranded RNA through a pore of
suitable diameter by applying an electric field to the RNA, wherein
at least each A or each G residue and at least each C or each U
residue comprises a modifying group bound to its respective base so
that each type of nucleotide in the RNA has an electronic signature
which is distinguishable from the electronic signature of each
other type of nucleotide in the RNA; [0040] (b) for each nucleotide
of the RNA which passes through the pore, determining an electronic
signature for such nucleotide; and [0041] (c) comparing each
electronic signature determined in step (b) with electronic
signatures corresponding to each of A, G, C and U modified as per
the nucleotides in the single-stranded RNA, so as to determine the
identity of each such nucleotide, thereby determining the
nucleotide sequence of the single-stranded RNA.
[0042] In an embodiment of the instant method, the single-stranded
RNA is obtained by (a) synthesizing double-stranded RNA using a
single-stranded template, an RNA polymerase and nucleotides,
wherein at least each A, each G, each C or each U residue comprises
an azido group bound to its base, and at least each A, each G, each
C and each U comprises an amino group bound to its base, whereby
the azido and amino groups do not reside on the same type of base,
(b) removing from the resulting double-stranded RNA the
single-stranded RNA containing the azido and amino group-containing
nucleotides and (c) reacting the resulting single-stranded RNA with
a first modifying group which forms a bond with the azido group and
a second modifying group which forms a bond with the amino group so
as to obtain the single-stranded RNA.
[0043] In another embodiment of the instant method, the
single-stranded RNA is obtained by (a) synthesizing double-stranded
RNA using a single-stranded template, an RNA polymerase and
nucleotides, wherein at least each A or each G residue and at least
each C or each U residue comprises a modifying group bound to its
respective base so that each type of nucleotide in the RNA has an
electronic signature which is distinguishable from the electronic
signature of each other type nucleotide in the RNA, and (b)
removing from the resulting double-stranded RNA the single-stranded
RNA containing modified nucleotides.
[0044] In one embodiment of the instant methods, the pore has a
diameter of from about 1 nm to about 5 nm. In a further embodiment
of the instant methods, the pore has a diameter of from about 1 nm
to about 3 nm. In embodiments of the instant methods, the pore has
a diameter of about 1 nm, 2 nm, 3 nm, 4 nm or 5 nm. In further
embodiments, the pore is, for example, about 1.0, 1.1, 1.2, 1.3,
1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6,
2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9,
4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9 or 5.0 nm in
diameter.
[0045] In one embodiment, a single pore is employed. In another
embodiment, multiple pores are employed.
[0046] Nanopore devices are known in the art. See, for example,
references [24] through [34]. Nanopores and methods employing them
are disclosed in U.S. Pat. Nos. 7,005,264 B2 and 6,617,113 which
are hereby incorporated by reference in their entirety.
[0047] In one embodiment of the instant methods, each A and each T
or each U residue comprises a modifying group; each A and each U
residue comprises a modifying group; and/or each G and each C
residue comprises a modifying group.
[0048] Moieties used to modify nucleotides can differ in size
and/or charge, so long as each type of nucleotide in a nucleic acid
whose sequence is being determined by the instant methods has an
electronic signature which differs from each other type.
[0049] DNA polymerases which can be used in the instant invention
include, for example E. Coli DNA polymerase I, Bacteriophage T4 DNA
polymerase, Sequenase.TM., Taq DNA polymerase and 9.degree. N
polymerase (exo-) A485L/Y409V.RNA polymerases which can be used in
the instant invention include, for example, Bacteriophage SP6, T7
and T3 RNA polymerases.
[0050] This invention also provides a nucleotide having an azido
group covalently bound to its base. In one embodiment, the
nucleotide is dUTP and the azido group is bound to the base at the
5-position. In one embodiment, the nucleotide is dATP and the azido
group is bound to the base at the 8-position. In another
embodiment, the nucleotide is dGTP and the azido group is bound to
the base at the 8-position. The azido and amino groups can also be
any other groups which permit binding of a unique moiety to each
type of nucleotide.
[0051] This invention also provides a method for making a modified
nucleotide comprising contacting the instant nucleotide with an
alkyne-containing compound under conditions permitting reaction
between the azido and the alkyne groups, thereby making the
modified nucleotide.
[0052] This invention will be better understood by reference to the
Experimental Details which follow, but those skilled in the art
will readily appreciate that the specific experiments detailed are
only illustrative of the invention as described more fully in the
claims which follow thereafter.
EXPERIMENTAL DETAILS
[0053] The structures of the four nucleotides are shown in FIG. 2.
A and G are purines, while C and T are pyrimidines. The overall
molecular sizes of A and G are very similar, while the sizes of C
and T are similar. Thus, nanopore has been shown to be able to
differentiate between purines and pyrimidines [1 and 14], but not
to be able to distinguish between individual purines, A and G, or
between individual pyrimidines, C and T.
[0054] Disclosed here is the design of modified nucleotides to
enhance discrimination of each nucleotide by modifying A and T.
Since A and G are bulky purines similar in size, they will generate
similar blocking current signatures (also called electronic
signatures) in the nanopore. Likewise C and T, both pyrimidines,
will generate similar signatures. The site selected for
modification is on the 7-position of A and the 5-position of T
nucleotide molecules. The 7-position of A and the 5-position of T
have been shown to be chemically modified with bulky groups while
not affecting basic DNA properties, such as forming the
double-stranded DNA structure and being able to carry out
polymerase reactions [2, 13 and 17]. These modifications will
enlarge the discrimination of the bases by nanopore due to the
increased size differences between the four nucleotides (A, G, C
and T). In addition, the DNA translocation rate through the
nanopore is expected to slow down due to the bulkiness of the
modified nucleotides. Thus, achieving the accuracy and reliability
required for the base-to-base sequencing is envisioned. The overall
analytical parameters in the nanopore sequencing, such as
concentration of the polynucleotide, magnitude of applied voltage,
temperature and pH value of the solution, are optimized in order to
get the most accurate and reliable results for the detection and
analysis of the DNA chain.
[0055] Use of Synthetic DNA Carrying Bulky Groups for Detection by
Nanopore
[0056] In order to investigate the effect of nucleotide bulkiness
on electronic blockade signals generated by the nanopore, various
polynucleotides are synthesized with different bulky groups
attached to the base of the nucleotide by a DNA synthesizer.
Initially, regular C's and G's are used to synthesize a series of
polynucleotides (FIGS. 3a and 3b). In addition, a series of
polynucleotides using modified A's (6-amino-hexylamino attached to
the 8-position of the base) and modified T's (BIOTIN attached to
the 5-position of the base) (FIGS. 3c and 3d), which increase the
bulkiness of the nucleotides, are synthesized. The order of the
bulkiness of the nucleotides in FIG. 3 is as follows:
T*>A*>G>C. These polynucleotides are then passed through
the nanopore to identify the relationship between the bulky groups
attached to the base and the difference in electronic blockade
signal between the different bases.
[0057] Attachment of Bulky Groups to Nucleotides for Nanopore
Detection
(1) Design and Synthesis of Modified Nucleotides (dATP-NHCOR.sub.1
and dUTP-NHCOR.sub.2).
[0058] Synthesized dATP-NH.sub.2 and dUTP-NH.sub.2 are used as
starting materials for further nucleotide modification while
unmodified dCTP and dGTP are used directly (FIG. 4). The routes of
nucleotide modification are shown in FIG. 5. The commercially
available carboxylic acids 1.about.10 will be converted into the
corresponding N-hydroxysuccinimidyl (NHS) esters conveniently using
N-hydroxysuccinimide and DCC [20 and 22]. Then the nucleotides for
modification (dATP-NH.sub.2 and dUTP-NH.sub.2) will be connected
with the modification groups R.sub.1 and R.sub.2 respectively in
DMF and NaHCO.sub.3/Na.sub.3CO.sub.3 buffer solution [13 and 17].
After modification, the order of nucleotide bulkiness will be:
A*>U*>G>C, as purines (A and G) are larger than
pyrimidines (C and U) and in general the modification group R.sub.1
is larger than R.sub.2.
(2) DNA-Extension Reaction Using Modified Nucleotides
(dATP-NHCOR.sub.1 and dUTP-NHCOR.sub.2).
[0059] The modified dATP and dUTP, and the unmodified dCTP and
dGTP, are then be used in a polymerase reaction to generate
single-stranded DNA. As shown in FIG. 6, after the polymerase
reaction, the single-stranded DNA chain is obtained after being
denatured from the template chain, which is composed of the
modified dATP and dUTP ad well as unmodified dCTP and dGTP. The
5'-end of the primer chain is modified on the base by a biotin
moiety to isolate only DNA product that has incorporated the
modified nucleotides. These modified single-stranded chains are
then used in the nanopore by single-channel recording techniques
for sequencing sensitivity and accuracy evaluation.
[0060] DNA-Sequencing Study by Nanopore
[0061] To validate nanopore's ability to distinguish the four
different nucleotides in DNA, a series of tests are conducted as
shown in FIG. 7. First, a polynucleotide stretch composed of only
50 identical nucleotides (i) is prepared by polymerase reaction as
described above. Each DNA sequence is expected to generate
different electronic blockade signatures due to the larger size
difference of the nucleotides. The modification effects of R.sub.1
and R.sub.2 for A and T can be compared for preliminary
optimization. Next, a polynucleotide stretch composed of 30
modified A's and 30 modified T's (ii) is prepared and then tested
in nanopore to demonstrate that the electronic blockade signatures
differ in magnitude between A and T and are easily
distinguishable.
[0062] Based on the signatures generated, the candidates for
R.sub.1 and R.sub.2 groups are selected to achieve the best
discrimination in signal. Third, a shorter polynucleotide stretch
composed of 10 A's, 10 C's, 10 G's and 10 T's (iii) are prepared
and tested in nanopore for further confirmation on the electronic
blockade signatures (also called electronic signatures). Finally, a
polynucleotide stretch composed of three consecutive A-C-G-T
sequence (iv) is prepared and tested in nanopore. The detailed
sequencing conditions can be optimized according to known methods.
Based on these results, random DNA chain with modified A and T and
unmodified C and G is evaluated for accurate detection and
discrimination by the nanopore. These procedures allow
characterization of the signals from each of the nucleotide and the
transitions between nucleotide of different identities. The
magnitude and duration of the blockade signatures on the event
diagram are then analyzed and compared with known diagrams for
validation. The schematic of the predicted blockade signals from
DNA molecules (ii), (iii) and (iv) are shown in FIG. 7. Thus, with
these rational chemical designs and modifications of the building
blocks of DNA, this invention envisions using nanopore to decipher
DNA sequences at the single molecule level with single base
resolution.
[0063] Attach Small Hooks to the Nucleotides for Synthesis of DNA
in Polymerase Reaction for Nanopore Detection
[0064] If a DNA polymerase is not able to synthesize a long strand
of DNA due to the bulkiness of the functional groups introduced, an
alternative strategy is to introduce small `hooks` to the
nucleotides, then perform polymerase reaction to produce DNA
products with hook-labeled nucleotides incorporated in them.
[0065] The DNA products are then linked with the large functional
groups through the hook for distinct detection by nanopore.
(1) Design and Synthesis of Hook-Labeled Nucleotide
dUTP-N.sub.3.
[0066] The available dCTP, dGTP and dATP-NH.sub.2 are used as
starting materials directly (FIG. 8), while dUTP-N.sub.3 is
synthesized from 5-iodo-2'-deoxyuridine as shown in FIG. 9.
5-Iodo-2'-deoxyuridine is first coupled with propargylamine in the
presence of palladium(0) and copper(I) catalysts. Then the amino
group is converted into azido group by the diazo transfer method
[11]. Finally triphosphate is introduced to the 5'-hydroxy group of
the nucleoside to yield dUTP-N, (6.
(2) DNA-Extension Reaction Using Hook-Labeled Nucleotides
(dATP-NH.sub.2 and dUTP-N.sub.3).
[0067] The dATP-NH.sub.2 and dUTP-N.sub.3, and the unmodified dCTP
and dGTP, are used in polymerase reaction on the single-stranded
nucleic acid template to obtain hook-labeled DNA products. Due to
the small sizes of the azido and amino groups, these nucleotides
are expected to be good substrates of commonly used DNA
polymerases. After isolation of the single stranded DNA carrying
the hook, the azido groups on these modified DNA chains will be
further modified by Huisgen 1,3-dipolar cycloaddition with terminal
alkynes (R.sub.3C.ident.CH) in the presence of copper(I) catalyst
(FIG. 10) [18 and 19]. The amino groups on the "A" nucleotides of
these modified DNA chains are connected with the modification
groups R.sub.1 in DMF and NaHCO.sub.3/Na.sub.2CO.sub.3 buffer
solution [13 and 17]. After modification, the order of nucleotides
bulkiness on the chain will be: A*>U*>G>C since in general
the modification group R.sub.1 is larger than R.sub.3.
[0068] Nanopore Construction and Detection of DNA
[0069] Based on information in the art, nanopores are constructed
with different configurations and modifications for characterizing
DNA containing nucleotides of different sizes.
[0070] Synthetic nanopores are described in references [24] through
[28] which are hereby incorporated by reference in their entirety.
The mechanics and kinetics of DNA passage through the pores are
described in references [291 and (30], respectively.
[0071] Natural nanopores are described in references [31] through
[34] which are hereby incorporated by reference in their
entirety.
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Sequence CWU 1
1
9110DNAArtificial SequenceChemically Synthesized Sequence
1tcgagctgca 10210RNAARTIFICIAL SEQUENCECHEMICALLY SYNTHESIZED
SEQUENCE 2ugcagcucga 10350DNAARTIFICIAL SEQUENCECHEMICALLY
SYNTHESIZED SEQUENCE 3aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa
aaaaaaaaaa 50450DNAARTIFICIAL SEQUNCECHEMICALLY SYNTHESIZED
SEQUENCE 4cccccccccc cccccccccc cccccccccc cccccccccc cccccccccc
50550DNAARTIFICIAL SEQUENCECHEMICALLY SYNTHESIZED SEQUENCE
5gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg
50650DNAARTIFICIAL SEQUENCECHEMICALLY SYNTHESIZED SEQUENCE
6tttttttttt tttttttttt tttttttttt tttttttttt tttttttttt
50760DNAARTIFICIAL SEQUENCECHEMICALLY SYNTHESIZED SEQUENCE
7aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa tttttttttt tttttttttt tttttttttt
60840DNAARTIFICIAL SEQUENCECHEMICALLY SYNTHESIZED SEQUENCE
8aaaaaaaaaa cccccccccc gggggggggg tttttttttt 40924DNAARTIFICIAL
SEQUENCECHEMICALLY SYNTHESIZED SEQUENCE 9aaacccgggt ttcccgggaa attt
24
* * * * *