U.S. patent application number 16/882353 was filed with the patent office on 2020-09-10 for polymer tagged nucleotides for single molecule electronic snp assay.
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 The Trustees of Columbia University in the City of New York. Invention is credited to Minchen Chien, Youngjin Cho, Jingyue Ju, Sergey Kalachikov, Shiv Kumar, James J. Russo, Chuanjuan Tao.
Application Number | 20200283833 16/882353 |
Document ID | / |
Family ID | 1000004856613 |
Filed Date | 2020-09-10 |
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United States Patent
Application |
20200283833 |
Kind Code |
A1 |
Ju; Jingyue ; et
al. |
September 10, 2020 |
POLYMER TAGGED NUCLEOTIDES FOR SINGLE MOLECULE ELECTRONIC SNP
ASSAY
Abstract
This invention provides methods of using labeled nucleotide
polyphosphate analogues to detect the identity or presence of a
nucleotide at certain positions in nucleic acid sequences with
single molecule sensitivity using nanopore detection, nucleotides
and primer-conjugated nanopore proteins for use in such methods,
and processes for producing such nucleotides and primer-conjugated
nanopore proteins.
Inventors: |
Ju; Jingyue; (Englewood
Cliffs, NJ) ; Cho; Youngjin; (New York, NY) ;
Kumar; Shiv; (Belle Mead, NJ) ; Kalachikov;
Sergey; (New York, NY) ; Tao; Chuanjuan; (New
York, NY) ; Chien; Minchen; (Tenafly, NJ) ;
Russo; James J.; (New York, NY) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Trustees of Columbia University in the City of New
York |
New York |
NY |
US |
|
|
Assignee: |
The Trustees of Columbia University
in the City of New York
New York
NY
|
Family ID: |
1000004856613 |
Appl. No.: |
16/882353 |
Filed: |
May 22, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15560820 |
Sep 22, 2017 |
10662463 |
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PCT/US16/23607 |
Mar 22, 2016 |
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16882353 |
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62137014 |
Mar 23, 2015 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12Q 1/6869 20130101;
G01N 33/48721 20130101; C12Q 2565/631 20130101; C12Q 1/6827
20130101 |
International
Class: |
C12Q 1/6827 20060101
C12Q001/6827; G01N 33/487 20060101 G01N033/487; C12Q 1/6869
20060101 C12Q001/6869 |
Claims
1. A method for identifying a single nucleotide residue of interest
at a position within a stretch of consecutive nucleotide residues
in a nucleic acid, comprising the steps of: a) incubating the
nucleic acid, under an applied voltage, with (1) a nanopore; (2) an
oligonucleotide primer conjugated to the nanopore and hybridized to
the nucleotides in the nucleic acid immediately 3' to the single
nucleotide residue of interest; (3) at least one labeled
terminating nucleotide polyphosphate (NPP) analogue, wherein the
label is attached to either the base or the terminal phosphate of
the NPP analogue; and (4) a nucleic acid polymerase; so that a NPP
analogue is incorporated into the primer if it is complementary to
the single nucleotide residue of interest, and the label attached
to the incorporated NPP analogue is drawn into the nanopore; b)
detecting by nanopore the signature of the label of the NPP
analogue incorporated into the primer, so as to identify the
incorporated NPP analogue; thereby identifying the single
nucleotide residue of interest.
2. The method of claim 1, wherein in step (a) the nucleic acid is
incubated with at least two NPP analogues, each comprising a
different base or analogue of a base, and a distinct label.
3. The method of claim 2, wherein in step (a) the nucleic acid is
incubated with at least four NPP analogues, each comprising a
different base or analogue of a base, and a distinct label.
4. The method of claim 3, wherein in step (a) the nucleic acid is
incubated with exactly four NPP analogues, each comprising a
different base or analogue of a base, and a distinct label.
5. The method of claim 1, wherein in step (a) the nucleic acid is
incubated with one NPP analogue, and if the NPP analogue is not
incorporated, iteratively repeating the incubating with a different
NPP analogue until a NPP analogue is incorporated and its label
detected by nanopore.
6. A method for identifying a single nucleotide residue of interest
at a position within a stretch of consecutive nucleotide residues
in a nucleic acid, comprising the steps of: (a) incubating the
nucleic acid, under an applied voltage, with (1) a nanopore; (2) an
oligonucleotide primer conjugated to the nanopore and hybridized to
the nucleotides in the nucleic acid immediately 3' to the single
nucleotide of interest; (3) at least one labeled nucleotide
polyphosphate (NPP) analogue, wherein the label is attached to
either the base or the terminal phosphate of the NPP analogue; (4)
a nucleic acid polymerase; and (5) a non-catalytic ion which
permits transient binding of a complementary labeled NPP analogue
to the nucleic acid polymerase but inhibits incorporation of the
bound NPP analogue; so that a NPP analogue is transiently bound to
the nucleic acid polymerase if it is complementary to the single
nucleotide of interest, and the label attached to the transiently
bound NPP analogue is drawn into the nanopore; (b) detecting by
nanopore the signature of the label of the NPP analogue transiently
bound to the primed template by polymerase, so as to identify the
NPP analogue; thereby identifying the single nucleotide residue of
interest.
7. The method of claim 6, wherein in step (a) the nucleic acid is
incubated with at least two NPP analogues, each comprising a
different base or analogue of a base, and a distinct label.
8. The method of claim 7, wherein in step (a) the nucleic acid is
incubated with at least four NPP analogues, each comprising a
different base or analogue of a base, and a distinct label.
9. The method of claim 8, wherein in step (a) the nucleic acid is
incubated with exactly four NPP analogues, each comprising a
different base or analogue of a base, and a distinct label.
10. The method of claim 6, wherein in step (a) the nucleic acid is
incubated with one NPP analogue, and if the NPP analogue does not
transiently bind to the nucleic acid polymerase, iteratively
repeating the incubating with a different NPP analogue until a NPP
analogue is transiently bound and its label detected by
nanopore.
11. The method of any one of claims 6-10, wherein the non-catalytic
metal ion is Sr.sup.2+ or Ca.sup.2+.
12. The method of any one of claims 6-11, wherein each NPP is a
deoxyribonucleotide polyphosphate (dNPP).
13. The method of any one of claims 1-11, wherein each NPP is a
dideoxyribonucleotide polyphosphate (ddNPP).
14. The method of claim 13, wherein each ddNPP comprises a label
having a coumarin-PEG moiety.
15. The method of claim 13, wherein each ddNPP comprises a label
having an oligonucleotide-based tag.
16. The method of any one of claims 6-11, wherein each NPP is a
ribonucleotide polyphosphate (rNPP).
17. The method of any one of claims 1-16, wherein the nucleic acid
is single-stranded DNA.
18. The method of any one of claims 1-16, wherein the nucleic acid
is double-stranded DNA.
19. The method of any one of claims 1-16, wherein the nucleic acid
is single-stranded RNA.
20. The method of any one of claims 1-16, wherein the nucleic acid
is double-stranded RNA.
21. The method of any one of claims 12-13, wherein the nucleic acid
is single-stranded DNA or double-stranded DNA and the nucleic acid
polymerase is DNA polymerase.
22. The method of any one of claims 12-13, wherein the nucleic acid
is RNA and the nucleic acid polymerase is reverse
transcriptase.
23. The method of any one of claims 12-13 and 16, wherein the
nucleic acid polymerase is RNA polymerase.
24. The method of any one of claims 1-23, wherein the label is
attached to the base.
25. The method of any one of claims 1-23, wherein the label is
attached to the terminal phosphate.
26. The method of any one of claims 1-25, wherein the label
comprises one or more of ethylene glycol, an amino acid, a
carbohydrate, a peptide, a dye, a chemiluminescent compound, a
mononucleotide, a dinucleotide, a trinucleotide, a tetranucleotide,
a pentanucleotide, a hexanucleotide, an oligonucleotide, an
aliphatic acid, an aromatic acid, an alcohol, a thiol group, a
cyano group, a nitro group, an alkyl group, an alkenyl group, an
alkynyl group, an azido group, or a combination thereof.
27. The method of any one of claims 1-26, wherein the label is a
polymeric label.
28. The method of any one of claims 1-27, wherein the labels are
polyethylene glycol (PEG) labels.
29. The method of claim 28, wherein the PEG labels each have a
different length from each other.
30. The method of any one of claims 1-29, wherein the labels are
oligonucleotide labels.
31. The method of any one of claims 1-30, wherein the signature is
an electronic signature.
32. The method of claim 31, wherein the electronic signature is an
electrical current blockade signature.
33. The method of claim 32, wherein the electrical current blockade
signature is a stuttering current blockade signature.
34. The method of any one of claims 1-33, wherein the nanopore is a
solid-state nanopore.
35. The method of any one of claims 1-33, wherein the nanopore is
in a solid state membrane.
36. The method of any one of claims 1-33, wherein the nanopore is a
biological pore.
37. The method of any one of claims 1-33, wherein the nanopore is
proteinaceous.
38. The method of claim 37, wherein the nanopore comprises alpha
hemolysin.
39. The method of claim 38, wherein each nanopore comprises seven
alpha hemolysin monomers, any or all of which are conjugated to an
identical primer.
40. The method of claim 38, wherein each nanopore comprises eight
MspA monomers, any or all of which are conjugated to an identical
primer.
41. The method of claim 38, wherein each nanopore comprises nine
CsgG monomers, any or all of which are conjugated to an identical
primer.
42. The method of claim 14, wherein the nucleic acid is incubated
with four coumarin-PEG-ddNPPs, each comprising a different base,
and each comprising a coumarin-PEG-label of a different length.
43. The method of claim 14, wherein the nucleic acid is incubated
with four oligonucleotide-tagged ddNPPs, each comprising a
different base, and each comprising an oligonucleotide of different
length and/or composition.
44. The method of claim 43, wherein the four coumarin-PEG labels
are coumarin-PEG16, coumarin-PEG20, coumarin-PEG24, and
coumarin-PEG36.
45. The method of claim 44, wherein each coumarin-PEG label is
attached to the terminal phosphate of the ddNPP.
46. The method of claim 44, wherein each coumarin-PEG label is a
coumarin-PEG-aminopropargyl label and is attached to the 5-position
of the base if the ddNPP is ddCPP, ddUPP, or ddTPP, and to the
7-position of the base if the ddNPP is ddAPP or ddGPP.
47. The method of any one of claims 1-46, wherein the sequence of
the primer is 10-40 nucleotides long.
48. The method of claim 47, wherein the sequence of the primer is
18-24 nucleotides long.
49. An assay for performing the method of any one of claims
1-48.
50. A dideoxynucleotide tetraphosphate (ddN4P) analogue, comprising
a coumarin-polyethylene glycol (PEG)-aminopropargyl label attached
to the terminal phosphate thereof.
51. The ddN4P analogue of claim 50, having the structure
##STR00011## wherein n is 16, 20, 24, or 36, and wherein B is a
base selected from the group consisting of adenine, cytosine,
thymine, guanine, and uracil.
52. The NPP analogue of claim 50, having the structure ##STR00012##
wherein BASE is selected from the group adenine, cytosine, thymine,
uracil, guanine, 7-deaza-adenine, and 7-deaza-guanine or analog
thereof; R and R' can be independently H, OH, O-alkyl, F, Cl, Br,
N.sub.3, NH.sub.2, O--NH.sub.2, O-allyl, O--CH.sub.2N.sub.3, 2',
3'-isopropylidine or groups which only allow a single nucleotide to
be incorporated by DNA polymerase; TAG can be any polymeric
molecule that can be detected by nanopore and may be selected from
the group oligonucleotides, peptides, carbohydrates, and PEGs of
different length.
53. A composition comprising four ddN4P analogues of claim 46 or
claim 51, wherein each ddN4P comprises a different base and a
distinct label, and each has a different value of n.
54. The composition of claim 53, wherein the four ddN4P analogues
are a ddA4P analogue, a ddG4P analogue, a ddC4P analogue, and
either a ddT4P analogue or a ddU4P analogue.
55. The ddN4P analogue of claim 50, wherein the base is
guanine.
56. A process for producing the ddN4P analogue of claim 50,
comprising: a) contacting a ddN4P with diaminoheptane in
carbodiimide (EDAC) and imidazole buffer under conditions
permitting the diaminoheptane to attach to the terminal phosphate;
b) contacting a 6-methoxycoumarin N-hydroxysuccinimidyl ester (NHS)
with an amino-PEG.sub.n-acid moiety in dimethylformamide, wherein n
is the number of ethylene glycol monomers in the PEG, under
conditions permitting the production of a coumarin-PEG.sub.n-acid
compound; c) reacting the product of step b) with
N,N-disuccinimidyl carbonate in dimethylformamide, under conditions
permitting the production of a coumarin-PEG.sub.n-NHS compound; and
d) reacting the products of steps a) and c) to produce a
coumarin-PEG.sub.n-ddN4P analogue.
57. The process of claim 56, further comprising, prior to step a),
reacting a ddNTP with tributylammonium phosphate in order to obtain
the ddN4P.
58. A process for producing the ddG4P analogue of claim 55,
comprising: a) contacting a ddG4P with diaminoheptane in
carbodiimide (EDAC) and imidazole buffer under conditions
permitting the diaminoheptane to attach to the terminal phosphate;
b) contacting a 6-methoxycoumarin N-hydroxysuccinimidyl ester (NHS)
with an amino-PEG.sub.n-acid moiety in dimethylformamide, wherein n
is the number of ethylene glycol monomers in the PEG, under
conditions permitting the production of a coumarin-PEG.sub.n-acid
compound; c) reacting the product of step b) with
N,N-disuccinimidyl carbonate in dimethylformamide, under conditions
permitting the production of a coumarin-PEG.sub.n-NHS compound; and
d) reacting the products of steps a) and c) to produce a
coumarin-PEG.sub.n-ddG4P analogue.
59. The process of claim 58, further comprising, prior to step a),
reacting a ddGTP with tributylammonium phosphate in order to obtain
the ddG4P.
60. A dideoxynucleotide triphosphate (ddNTP) analogue, comprising a
coumarin-polyethylene glycol (PEG)-aminopropargyl label attached to
the base thereof.
61. The ddNTP analogue of claim 60 having the structure
##STR00013## wherein B is a base selected from the group adenine,
cytosine, thymine, guanine, and uracil; and n is 16, 20, 24, or
36.
62. A composition comprising four ddNTP analogues of claim 60 or
61, wherein each ddNTP comprises a different base, and each has a
different value of n.
63. The composition of claim 62, wherein the four ddNTP analogues
are a ddATP analogue, a ddGTP analogue, a ddCTP analogue, and
either a ddTTP analogue or a ddUTP analogue.
64. A process for producing a ddNTP analogue of claim 60 or 61,
comprising: a) contacting a 6-methoxycoumarin N-hydroxysuccinimidyl
ester (NHS) with an amino-PEG.sub.n-acid moiety in
dimethylformamide, wherein n is the number of ethylene glycol
monomers in the PEG, under conditions permitting the production of
a coumarin-PEG-acid compound; b) reacting the product of step a)
with N,N-disuccinimidyl carbonate in dimethylformamide, under
conditions permitting the production of a coumarin-PEG.sub.n-NHS
compound; and c) reacting the product of step b) with an
aminopropargyl-ddNTP, wherein the aminopropargyl moiety is attached
to the base, in dimethylformamide, to produce a
coumarin-PEG.sub.n-aminopropargyl-ddNTP.
65. The process of claim 64, wherein the aminopropargyl-ddNTP in
step c) is a 5-aminopropargyl-ddNTP if the ddNTP comprises a
cytosine, uracil, or thymidine base, and wherein the
aminopropargyl-ddNTP in step c) is a 7-aminopropargyl-ddNTP if the
ddNTP comprises a adenine or guanine base.
66. The process of any one of claims 56-59, 63, and 65, wherein n
is 16, 20, 24, or 36.
67. An alpha hemolysin protein, having a primer conjugated
thereto.
68. The alpha hemolysin protein of claim 67, wherein the alpha
hemolysin comprises a C46 mutation, and the primer is conjugated to
the cysteine residue at position 46.
69. A process for producing the primer-conjugated .alpha.-hemolysin
of claim 68, comprising: a) contacting a
sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate
(sSMCC) hetero bifunctional crosslinker, comprising an
amino-reactive N-hydrosysuccinimide (NHS) ester and a
thiol-reactive maleimide group at opposite ends, with a primer
comprising a terminal amino group, under conditions permitting the
terminal amino group to react with the amino-reactive NHS ester; b)
removing residual free sSMCCs from the solution; and c) contacting
the product resulting from step a) with an .alpha.-hemolysin,
wherein the .alpha.-hemolysin comprises a C46 mutation, under
conditions permitting the thiol-reactive maleimide group to react
with the cysteine residue at position 46; thereby conjugating the
primer to the .alpha.-hemolysin.
70. The process of claim 69, wherein the removal of residual free
sSMCCs in step b) is performed via purification by gel
filtration.
71. A conductance measurement system comprising: a) an electrically
resistive barrier separating at least a first and a second
electrolyte solution; said electrically resistive barrier comprises
at least one pore with a diameter on nanometer scale; said at least
one pore being configured to allow an ionic current to be driven
across said first and second electrolyte solutions by an applied
potential; b) at least one labeled terminating nucleotide
polyphosphate (NPP) analogue, wherein the label is attached to
either the base or the terminal phosphate of the NPP analogue, in
at least one of said first and second electrolyte solutions; and c)
a means of measuring the ionic current and a means of recording its
time course as a time series, including time periods when the at
least one pore is unobstructed by the label and also time periods
when a label causes pulses of reduced-conductance.
72. The system of claim 71, wherein said at least one pore
comprises features configured to: (1) capture the polymer label on
the primer-incorporated nucleotide that has the label attached to
the base to produce a unique electronic signature, and (2) detect
the unique electronic signature of the polymer label while the
labeled nucleotide is complexed with the polymerase and the primed
template in a ternary complex before incorporation.
73. The system of claim 71 or 72, wherein the labeled terminating
NPP analogue is a ddNPP analogue selected from the group consisting
of ##STR00014## wherein B is a base selected from the group
adenine, cytosine, thymine, guanine, and uracil; and n is 16, 20,
24, or 36.
74. The system of claim 73, comprising four ddNPP analogues having
the structure ##STR00015## wherein each ddNTP comprises a different
base, and each has a different value of n.
75. The system of claim 73, comprising four ddNPP analogues having
the structure ##STR00016## wherein each ddNTP comprises a different
base, and each has a different value of n.
76. The system of claim 74 or 75, wherein the four ddNTP analogues
are a ddATP analogue, a ddGTP analogue, a ddCTP analogue, and
either a ddTTP analogue or a ddUTP analogue.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/137,014, filed Mar. 23, 2015, which is
incorporated herein by reference in its entirety.
[0002] Throughout this application, various publications and
patents are referenced. Full citations for these references may be
found at the end of the specification immediately preceding the
claims. The disclosures of these publications and patents in their
entirety are hereby incorporated by reference into this application
to more fully describe the state of the art to which this invention
pertains.
[0003] This application incorporates-by-reference nucleotide and
amino acid sequences that are present in the file named
"160322_0575_87413-A_Sequence_Listing_RBR.txt", which is 2
kilobytes in size, and which was created Mar. 21, 2016 in the
IBM-PC machine format, having an operating system computability
with MS-Windows, which is contained in the text file filed Mar. 22,
2016 as part of this application.
BACKGROUND OF THE INVENTION
[0004] A single nucleotide polymorphism (SNP) is a single base
variation in the genome of a living organism. SNPs may occur in
coding sequences of genes and non-coding regions of genes,
including regulatory regions. SNPs in the coding sequences of the
genome are classified as two types: synonymous and nonsynonymous.
Synonymous SNPs do not alter the protein sequence due to the
degeneracy of the genetic code, while nonsynonymous SNPs change the
amino acid sequence of the encoded protein. The nonsynonymous SNPs
are further divided into two types: missense and nonsense. A
missense mutation is a single nucleotide point mutation leading to
a codon that codes for a different amino acid compared to the
wild-type, whereas a nonsense mutation is a point mutation that
results in a premature stop codon. SNPs that are not in
protein-coding regions can impact the function of the genes by
altering splicing sequences and binding activity of transcription
factors as well as gene expression. Among all the genetic
variations, SNPs are the most common genetic differences between
human beings. Over 3.1 million SNPs have been characterized from
the human genome in a second-generation human haplotype map (Frazer
et al. 2007). Thus, SNPs are important biomarkers for investigating
the molecular basis underlying the mechanism for disease
development, laying a foundation for precision medicine.
[0005] The Human Genome Project and the construction of a
comprehensive human genome sequence map (Lander et al. 2001, Venter
et al. 2001, and Wheeler et al. 2008) provide valuable resources
for the study of genetic variations. These genetic differences
include SNPs, gene copy number variations, insertions and
deletions. SNPs have been established as unique biomarkers for the
discovery and characterization disease genes (Kwok 2000 and Roses
2000). These research efforts require the characterization of large
number of SNPs with technologies that are cost-effective and
high-throughput with high-accuracy. The following DNA sequencing
platforms are widely used for characterizing genetic variations:
(1) 4-color fluorescent Sanger method (Smith et al. 1986, Ju et al.
1995, Ju et al. 1996, Salas-Solano et al. 1998, and Kheterpal et
al. 1996), (2) sequencing by synthesis (SBS) using cleavable
fluorescent nucleotide reversible terminators (Ju et al. 2006 and
Bentley et al. 2008), (3) SBS with detection of the
chemiluminescent signals caused by a cascade of enzymatic reactions
following the release of pyrophosphate during polymerase reaction
(pyrosequencing) (Margulies et al. 2005), (4) SBS with electronic
detection of the released proton during polymerase reaction (ion
torrent sequencing) (Rothberg et al. 2011), and (5) single molecule
fluorescent SBS methods (Harris et al. 2008 and Eid et al. 2009).
However, these sequencing technologies are not designed for
pinpoint detection of SNPs, and are still too costly for performing
large scale SNP studies. Matrix assisted laser
desorption/ionization time-of-flight mass spectrometry (MALDI-TOF
MS) and fluorescence emission are two dominant detection methods
for SNP analysis. SNP assay approaches using the above two
detection methods are reviewed below.
SNP Analysis by MALDI-TOP MS Detection
[0006] MALDI-TOF MS measures the mass of the target molecules with
highly accurate results in a digital format. It has been used for
SNP detection by single base extension (SBE) (Haff et al. 1997,
Tang et al. 1999, Ross et al. 1998, Fei et al. 1998, and Griffin et
al. 2000), hybridization (Stoerker et al. 2000 and Ross et al.
1997), and invasive cleavage (Griffin et al. 1999 and Lyamichev et
al. 1999). MALDI-TOF MS has also been used for gene expression
analysis and single-copy DNA haplotyping in the context of
nucleotide extension by polymerase (Ding et al., PNAS
100:3059-3064, 2003 and Ding et al., PNAS 100:7449-7453 2003).
[0007] Most multiplex SNP analyses make use of the specificity of
the SBE reaction catalyzed by polymerase. One of the widely used
SNP characterization method utilizes SBE and MALDI-TOF MS
detection. In this approach, oligonucleotide primers are designed
and synthesized based on the genetic variation in the target gene.
The 3'-end of the primer anneals immediately next to a SNP site of
the DNA template. A single dideoxynucleotide that is complementary
to the SNP site is then incorporated into the primer by DNA
polymerase. The identity of the SNP is determined by the mass of
the resulting primer extension product obtained from the MALDI-TOF
MS spectrum.
SNP Analysis by Fluorescence Detection
[0008] Numerous SNP genotyping methods have been developed using
fluorescence labeling and detection, including microarray (Hartmann
et al. 2009), PCR-RFLP analysis (Chowdhury et al. 2007), and TaqMan
real-time genotyping (Bai et al. 2004). There are several
advantages to using fluorescence labeling and detection, which
include a variety of robust chemical coupling methods to tag the
target molecules, high detection sensitivity of several
photophysical parameters (fluorescence life time, emission and
polarization) and the capability of multiplexing. The molecular
inversion probe (MIP) approach has been developed for SNP detection
(Hardenbol et al. 2003). In this method, successive extension and
ligation of locus-specific DNA probes yields a circular shape at
polymorphic sites of the target gene. The linear probes are then
selectively degraded, whereas the circular DNA probes that contain
allelic information are amplified and analyzed using a microarray
with fluorescence detection. Using this approach, Hardenbol et al.
(2003) performed genotyping of more than 1,000 SNPs per assay. The
MIP method has the advantage of a very high level of multiplexing.
However, many enzymatic reaction steps and complicated probe design
are required for MIP.
[0009] Prior multiplex SNP assays primarily used either mass
spectrometric detection or fluorescent tags and optical detection.
None of these previous assays offer single molecule detection
sensitivity and all require bulky instruments. None used nanopores
to identify molecular or polymer tags corresponding to nucleotides
of interest or SNPs, so as to identify the nucleotides of interest
or SNPs.
SUMMARY OF THE INVENTION
[0010] The present invention is directed to a method for
identifying a single nucleotide residue of interest at a position
within a stretch of consecutive nucleotide residues in a nucleic
acid, comprising the steps of: [0011] (a) incubating the nucleic
acid, under an applied voltage, with [0012] (1) a nanopore; [0013]
(2) an oligonucleotide primer conjugated to the nanopore and
hybridized to the nucleotides in the nucleic acid immediately 3' to
the single nucleotide residue of interest; [0014] (3) at least one
labeled terminating nucleotide polyphosphate (NPP) analogue,
wherein the label is attached to either the base or the terminal
phosphate of the NPP analogue; and [0015] (4) a nucleic acid
polymerase; [0016] so that a NPP analogue is incorporated into the
primer if it is complementary to the single nucleotide residue of
interest, and the label attached to the incorporated NPP analogue
is drawn into the nanopore; [0017] (b) detecting by nanopore the
signature of the label of the NPP analogue incorporated into the
primer, so as to identify the incorporated NPP analogue; [0018]
thereby identifying the single nucleotide residue of interest.
[0019] The invention is further directed to a method for
identifying a single nucleotide residue of interest at a position
within a stretch of consecutive nucleotide residues in a nucleic
acid, comprising the steps of: [0020] (a) incubating the nucleic
acid, under an applied voltage, with [0021] (1) a nanopore; [0022]
(2) an oligonucleotide primer conjugated to the nanopore and
hybridized to the nucleotides in the nucleic acid immediately 3' to
the single nucleotide of interest; [0023] (3) at least one labeled
nucleotide polyphosphate (NPP) analogue, wherein the label is
attached to either the base or the terminal phosphate of the NPP
analogue; [0024] (4) a nucleic acid polymerase; and [0025] (5) a
non-catalytic compound which permits transient binding of a
complementary NPP analogue to the nucleic acid polymerase and the
primed template but inhibits incorporation of the bound NPP
analogue; [0026] so that a NPP analogue is transiently bound to the
nucleic acid polymerase and the primed template if the NPP is
complementary to the single nucleotide of interest, and the label
attached to the transiently bound NPP analogue is drawn into the
nanopore; [0027] (b) detecting by nanopore the signature of the
label of the NPP analogue transiently bound to the polymerase and
the primed template, so as to identify the NPP analogue; [0028]
thereby identifying the single nucleotide residue of interest.
[0029] The invention also provides for an assay for performing any
of the methods of the invention.
[0030] The invention also provides for a dideoxynucleotide
polyphosphate (ddNPP) analogue, comprising a label attached to the
terminal phosphate thereof, where the polyphosphate comprises 3-10
phosphate units.
[0031] The invention also provides for a deoxynucleotide
polyphosphate (dNPP) analogue, comprising a label attached to the
terminal phosphate thereof, where the polyphosphate comprises 3-10
phosphate units.
[0032] The invention also provides for a ribonucleotide
polyphosphate (rNPP) analogue, comprising a label attached to the
terminal phosphate thereof, where the polyphosphate comprises 3-10
phosphate units.
[0033] The invention also provides for a dideoxynucleotide
tetraphosphate (ddN4P) analogue, comprising a coumarin-polyethylene
glycol (PEG)-aminopropargyl label attached to the terminal
phosphate thereof.
[0034] The invention also provides for a composition comprising
four ddN4P analogues of the invention, wherein each ddN4P comprises
a different base and a distinct label formed by a polymer, each
with a different number (n) of monomers.
[0035] The invention also provides for a process for producing a
ddN4P analogue of the invention, comprising: [0036] a) contacting a
ddN4P with diaminoheptane in carbodiimide (EDAC) and imidazole
buffer under conditions permitting the diaminoheptane to attach to
the terminal phosphate; [0037] b) contacting a 6-methoxycoumarin
N-hydroxysuccinimidyl ester (NHS) with an amino-PEG.sub.n-acid
moiety in dimethylformamide, wherein n is the number of ethylene
glycol monomers in the PEG, under conditions permitting the
production of a coumarin-PEG.sub.n-acid compound; [0038] c)
reacting the product of step b) with N,N-disuccinimidyl carbonate
in dimethylformamide, under conditions permitting the production of
a coumarin-PEG.sub.n-NHS compound; and [0039] d) reacting the
products of steps a) and c) to produce a coumarin-PEG.sub.n-ddN4P
analogue.
[0040] The invention also provides for a process for producing the
ddG4P analogue of the invention, comprising: [0041] a) contacting a
ddG4P with diaminoheptane in carbodiimide (EDAC) and imidazole
buffer under conditions permitting the diaminoheptane to attach to
the terminal phosphate; [0042] b) contacting a 6-methoxycoumarin
N-hydroxysuccinimidyl ester (NHS) with an amino-PEG-acid moiety in
dimethylformamide, wherein n is the number of ethylene glycol
monomers in the PEG, under conditions permitting the production of
a coumarin-PEG-acid compound; [0043] c) reacting the product of
step b) with N,N disuccinimidyl carbonate in dimethylformamide,
under conditions permitting the production of a coumarin-PEG-NHS
compound; and [0044] d) reacting the products of steps a) and c) to
produce a coumarin-PEG.sub.n-ddG4P analogue.
[0045] The invention also provides for a dideoxynucleotide
triphosphate (ddNTP) analogue, comprising a coumarin-polyethylene
glycol (PEG)-aminopropargyl label attached to the base thereof.
[0046] The invention also provides for a composition comprising
four ddNTP analogues of the invention, wherein each ddNTP comprises
a different base and a distinct label formed by a polymer, each
with a different number (n) of monomers.
[0047] The invention also provides for a dideoxynucleotide
polyphosphate (ddNPP) analogue, comprising a label attached to the
base thereof, where the polyphosphate comprises 3-10 phosphate
units.
[0048] The invention also provides for a deoxynucleotide
polyphosphate (dNPP) analogue, comprising a label attached to the
base thereof, where the polyphosphate comprises 3-10 phosphate
units.
[0049] The invention also provides for a ribonucleotide
polyphosphate (rNPP) analogue, comprising a label attached to the
base thereof, where the polyphosphate comprises 3-10 phosphate
units.
[0050] The invention also provides for a process for producing a
ddNTP analogue of the invention, comprising: [0051] a) contacting a
6-methoxycoumarin N-hydroxysuccinimidyl ester (NHS) with an
amino-PEG.sub.n-acid moiety in dimethylformamide, wherein n is the
number of ethylene glycol monomers in the PEG, under conditions
permitting the production of a coumarin-PEG-acid compound; [0052]
b) reacting the product of step a) with N,N-disuccinimidyl
carbonate in dimethylformamide, under conditions permitting the
production of a coumarin-PEG.sub.n-NHS compound; and [0053] c)
reacting the product of step b) with an aminopropargyl-ddNTP,
wherein the aminopropargyl moiety is base-attached, in
dimethylformamide, to produce a
coumarin-PEG.sub.n-aminopropargyl-ddNTP.
[0054] The invention also provides for a process for producing a
polymer tagged ddNTP analogue, comprising: [0055] a) contacting a
5(7)-propargylamino-ddNTP with amino-protected caproic acid NHS
ester; [0056] b) reacting the product of step a) with ammonium
hydroxide to produce 5(7)-propargylamidoaminocaproyl-ddNTPs; [0057]
c) reacting the product of step b) with azidobutyric acid NHS
ester; and [0058] d) reacting the product of step c) with
5'-alkynyl-oligonucleotide tag to produce oligonucleotide tagged
ddNTPs.
[0059] In some cases the label or tag attached to the base moiety
of the nucleoside polyphosphate can be an oligonucleotide or
peptide. The oligonucleotide can be of any length from 10-100
monomeric units of thymidine, cytidine, adenosine, guanosine, or
derivatives thereof, abasic units (deoxyribose units), and
non-hydrogen bond forming modified base units.
[0060] The oligonucleotide may consists of phosphodiester linkage,
phosphorothioate linkage, borano-phosphate, and methyl phosphonate
linkage between the two monomeric units. The examples of
oligonucleotide-tags have been disclosed in an earlier published
patent application "Chemical methods for producing tagged
nucleotides" US 2015/0368710 which is incorporated herein as a
reference.
[0061] Connection of the nucleotides to the tag can also be
achieved by the formation of a disulfide, formation of an amide,
formation of an ester, by alkylation (e.g., using a substituted
iodoacetamide reagent) or forming adducts using aldehydes and
amines or hydrazines, azide-alkyn coupling or tetrazine-diene
coupling. Numerous conjugation chemistries can be found in
Bioconjugate Techniques by Greg T. Hermanson, (2008), which is
incorporated herein by reference in its entirety.
[0062] Specific examples of reactive groups on the nucleotides or
the Oligonucleotide Tags and groups with which groups can react are
provided in Table 1. These reactive groups with which they can
react can be present either on the linker or on the tag
TABLE-US-00001 TABLE 1 Possible Reactive Substituent and Functional
Groups Reactive Therewith Reactive Groups Functional Groups
Succinimidyl esters Primary amino, secondary amino Anhydrides, acid
halides Amino and Hydroxyl groups Carboxyl Amino, Hydroxy, Thiols
Aldehyde, Amino groups Isothiocyanate & Isocyanates Vinyl
sulphone & Amino groups Dichlorotriazine Haloacetamides Thiols,
Imidazoles Maleimides Thiols, Hydroxy, Amino Thiols Thiols,
Maleimide, Haloacetamides Phosphoramidites, Hydroxy, Amino, Thiol
groups Activated Phosphates Azide Alkyne Tetrazine Diones
[0063] The invention also provides for an alpha hemolysin protein,
having a primer conjugated thereto.
[0064] The invention also provides for a process for producing the
primer-conjugated .alpha.-hemolysin of the invention, comprising:
[0065] a) contacting a sulfosuccinimidyl-4-(N-maleimidomethyl)
cyclohexane-1-carboxylate (sSMCC) hetero bifunctional crosslinker,
comprising an amido-reactive N-hydroxysuccinimide (NHS) ester and a
thiol-reactive maleimide group at opposite ends, with a primer
comprising a terminal amino group, under conditions permitting the
terminal amino group to react with the amido-reactive NHS ester;
[0066] b) removing residual unreacted sSMCCs from the solution; and
[0067] c) contacting the product resulting from step a) with an
.alpha.-hemolysin, wherein the .alpha.-hemolysin comprises a
cysteine mutation at position 46, under conditions permitting the
thiol-reactive maleimide group to react with the cysteine residue
at position 46; thereby conjugating the primer to the
.alpha.-hemolysin.
[0068] The invention also provides for a conductance measurement
system comprising: [0069] a) an electrically resistive barrier
separating at least a first and a second electrolyte solution;
[0070] said electrically resistive barrier comprises at least one
pore with a diameter on nanometer scale; [0071] said at least one
pore being configured to allow an ionic current to be driven across
said first and second electrolyte solutions by an applied
potential; [0072] b) at least one labeled terminating nucleotide
polyphosphate (NPP) analogue, wherein the label is attached to
either the base or the terminal phosphate of the NPP analogue, in
at least one of said first and second electrolyte solutions; and
[0073] c) a means of measuring the ionic current and a means of
recording its time course as a time series, including time periods
when at least one pore is unobstructed by the label and also time
periods when a label causes pulses of reduced conductance.
BRIEF DESCRIPTION OF THE FIGURES
[0074] FIG. 1. Exemplary scheme of single molecule electronic SNP
genotyping using polymer-labeled ddNTPs (in this example, the tags
are attached to the base of the ddNTPs) in a primer-conjugated
nanopore array. Nanopores that are conjugated with SNP primers
(i.e. primers which are substantially fully complementary to the
nucleotides in the DNA immediately 3' to the single nucleotide of
interest, identified as the queried nucleotide) are prepared.
Single base extension is performed by adding 4 differently tagged
ddNTPs, DNA polymerase and DNA templates. The primers having
complementary sequences to the added templates are extended by the
polymerase with tagged ddNTPs. Under an applied voltage, the tags
are pulled into the pore and the current signal from the pore is
read, revealing the specific genotype of the template.
[0075] FIG. 2. Exemplary scheme of single molecule electronic SNP
genotyping using 4 tagged nucleotides in a primer-conjugated
nanopore array in the presence of non-catalytic metal ions such as
Sr.sup.2+ (in this example, the tags can be attached to either the
base or the terminal phosphate of the dNTPs or ddNTPs). Nanopores
that are conjugated with SNP primers are prepared. The closed
ternary complex of DNA polymerase, a matched template-primer pair
and the complementary tagged nucleotide to the queried site in the
template are formed and the complex is temporarily paused in the
presence of non-catalytic Sr.sup.2+ ion. During this frozen period,
the tags on the incoming nucleotide in the ternary complex are
pulled into the pore under an applied voltage and the current
signal from the pore is read, revealing the specific genotype of
the template.
[0076] FIG. 3. Single molecule electronic SNP genotyping scheme and
the expected current signals by using 4 tagged nucleotides. Four
tagged nucleotides are added to the primer-template moiety attached
to the nanopore in solution containing non-catalytic Sr.sup.2+ ion
and polymerase such that one of the tag-nucleotides complementary
to the next base on the template forms a ternary complex, but is
not incorporated, and current blockade signal is recorded multiple
times for SNP detection.
[0077] FIG. 4. Design and synthesis of the four
coumarin-PEG.sub.n-dideoxyguanosine-5'-tetraphosphates. The
specific synthesis shown is for a ddG4P, but the same process can
be used, mutatis mutandis, to produce other ddN4Ps.
[0078] FIG. 5. Design and synthesis of the
coumarin-PEG.sub.n-aminopropargyl-ddNTPs.
[0079] FIG. 6. Design and synthesis of oligonucleotide-tagged
aminopropargyl-ddNTPs.
[0080] FIG. 7. MALDI-TOF MS measurements and structures of
oligonucleotide-tagged aminopropargyl-ddTTP
(ddTTP-Cy3-T.sub.4-dSp.sub.3-T.sub.23-C3) and -ddCTP
(ddCTP-Cy3-T.sub.2-dSp.sub.8-T.sub.20-C3).
[0081] FIG. 8. Single-base polymerase extension reactions with
oligonucleotide-tagged aminopropargyl-ddTTP and -ddCTP
characterized by gel electrophoresis. Extension reactions were
conducted in the presence of tagged ddCTP (top gel) and different
enzymes including Klenow, Sequenase and Thermo Sequenase. Reactions
are performed with 10 pmol of self-primed looped template
(5'-GATAGCGCCGCGCCTTGGCGCGGCGC-3') (SEQ ID No: 1), 200 pmol of
tagged ddNTPs and 5 units of each enzyme incubated for 1 hr at
37.degree. C. In the bottom gel, lanes 1-4 are extension reactions
with tagged ddTTP and lanes 5-8 are reactions with tagged ddCTP
using Thermo Sequenase. Lane 9 is a negative control extension
reaction without the enzyme. Lanes 10 and 12 are tagged ddCTPs and
primer-template controls respectively.
[0082] FIG. 9. Scheme for characterizing current signatures of
different tags by extending biotinylated primers with tagged
ddNTPs. Primers having a single biotin molecule attached to the
base of the 3'-nucleotide and complementary template DNA are
incubated with Thermo Sequenase, Mg.sup.2+ and tagged ddNTPs. The
DNA polymerase extends the primers with a single tagged ddNTP that
is complementary to the next base in the template DNA. After the
incubation, streptavidin molecules that have strong affinity for
biotin are added to the extension reaction and both the unextended
and extended primers are captured by the streptavidin. The reaction
mixture is then applied to the nanopore electronic detection
system. Although streptavidin itself cannot fit through the pore
due to its much larger size, it holds the primer extension product
in a position whereby the tag can enter the pore, generating a
unique current signature specific for the tag on each nucleotide.
In this way, the current signatures of different tags can be
confirmed.
[0083] FIG. 10. Nanopore current blockade levels generated by the
tags on the primer extension products by incorporating tagged ddCTP
(ddCTP-Cy3-T.sub.2-dSp.sub.8-T.sub.20-C3) and tagged ddTTP
(ddTTP-Cy3-T.sub.4-dSp.sub.3-T.sub.23-C3). The tag on the primer
extension product incorporating a tagged ddCTP displayed a current
blockade of about 26-30% of the open pore current level and the tag
on the primer extension product incorporating a tagged ddTTP gave a
46-50% blockade.
[0084] FIG. 11. Synthesis of primer-conjugated .alpha.HL. A primer
with a terminal amino group is conjugated to
sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate
(sSMCC) hetero bifunctional crosslinker which contains an
amino-reactive N-hydroxysuccinimide (NHS ester) and a
thiol-reactive maleimide group at opposite ends. The
crosslinker-attached primer is then reacted with an .alpha.HL
mutant having a cysteine residue at position 46, so that the
maleimide group in the crosslinker reacts with the thiol group in
the cysteine residue. Primary structure of recombinant hemolysin
mutant 46C:
TABLE-US-00002 (SEQ ID No: 2)
MADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDCNHN
KKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQIS
DYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTLKYV
QPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKT
RNGSMKAAENFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIY
ERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTNKGHHHH HH.
[0085] FIG. 12. Two possible scenarios after the extension
reaction. The left figure shows an example of a homozygous genotype
where the SNP primers are all extended by the same nucleotide
representing the two identical alleles. The right figure indicates
the case for a heterozygous genotype where each primer is extended
by one of two different nucleotides each carrying a unique tag
representing the two different alleles.
DETAILED DESCRIPTION OF THE INVENTION
[0086] While various embodiments of the invention have been shown
and described herein, it will be obvious to those skilled in the
art that such embodiments are provided by way of example only.
Numerous variations, changes, and substitutes may occur to those
skilled in the art without departing from the invention. It should
be understood that various alternatives to the embodiments of the
invention described herein may be employed.
Terms
[0087] As used herein, and unless stated otherwise, each of the
following terms shall have the definition set forth below.
A--Adenine;
C--Cytosine;
[0088] DNA--Deoxyribonucleic acid;
G--Guanine;
[0089] RNA--Ribonucleic acid;
T--Thymine; and
U--Uracil.
[0090] The articles "a", "an" and "the" are non-limiting. For
example, "the method" includes the broadest definition of the
meaning of the phrase, which can be more than one method.
[0091] "Signature" of a compound in a pore shall include, for
example, a signal or change occurring when the compound passes
through, resides in, or otherwise interacts with the pore. One such
change may be an electronic signature.
[0092] "Electronic signature" of a nucleotide or other molecules,
such as labels and polymer tags captured by the pore or passing
through a pore via application of an electric field shall include,
for example, the duration of the nucleotide's or molecule's capture
by or 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
be captured by or pass through the pore via application of an
electric field.
[0093] "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,
nucleotide, nucleotide analogue, or tag can be captured by the pore
or 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 captured by the
pore or passing through its barrier. The nanopore barrier may be
synthetic or naturally occurring, or both, in part. Barriers can,
for example, be biological, comprising naturally-occurring
compounds or materials derived from such compounds. This includes,
for example, lipid bilayers having therein .alpha.-hemolysin,
oligomeric protein channels such as porins, and synthetic peptides
and the like. Barriers can also be, for example, solid state
nanopores including, for example, 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.
[0094] "Detection via nanopore" or "detection by a nanopore"
includes, for example, detecting a change in ionic current through
a nanopore caused by a molecule captured by the pore or entering,
translocating through, or otherwise interacting with a nanopore.
For example, in an aqueous ionic salt solution such as KCl, when an
appropriate voltage is applied across the membrane, the pore formed
by an .alpha.-hemolysin channel conducts a sufficiently strong and
steady ionic current. A charged molecule can then be driven through
the pore by the applied electric field, thus blocking or reducing
the ionic current that would be otherwise unimpeded. This process
of passage generates an electronic signature. A particular
molecule, when captured by the pore or entering and passing through
the nanopore, generates a characteristic signature that
distinguishes it from other molecules. The duration of the blockade
and the signal strength is related to the steric, electronic, and
other physical and chemical properties of the molecule. Thus a
specific event diagram, which is a plot of translocation time
versus blockade current, is obtained and used to identify the
molecule by single channel recording techniques based on
characteristic parameters such as translocation current,
translocation duration, and their corresponding dispersion in the
diagram.
[0095] "Nucleic acid" shall mean any nucleic acid molecule,
including, without limitation, DNA, RNA and hybrids thereof as well
as their analogues. 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).
[0096] "Hybridize" shall mean the annealing of one single-stranded
nucleic acid (such as primer) to another nucleic acid based on the
well-understood principle of sequence complementarity. In an
embodiment the other nucleic acid is a single-stranded nucleic
acid. The propensity for hybridization between nucleic acids
depends on the temperature and ionic strength of their miliu, the
length of the nucleic acids and the degree of complementarity. The
effect of these parameters on hybridization is described in, for
example, Sambrook J, Fritsch E F, Maniatis T., Molecular cloning: a
laboratory manual, Cold Spring Harbor Laboratory Press, New York
(1989). As used herein, hybridization of a primer, or of a DNA
extension product, respectively, is extendable by creation of a
phosphodiester bond with an available nucleotide or nucleotide
analogue capable of forming a phosphodiester bond, therewith.
[0097] "Primer" as used herein (a primer sequence) is a short,
usually chemically synthesized oligonucleotide, of appropriate
length, for example about 10-30 bases, sufficient to hybridize to a
target DNA (e.g. a single stranded DNA) and permit the addition of
a nucleotide residue thereto, or oligonuclectide or polynucleotide
synthesis therefrom, under suitable conditions well-known in the
art. In an embodiment the primer is a DNA primer, i.e. a primer
consisting of, or largely consisting of, deoxyribonucleotide
residues or their analogues. The primers are designed to have a
sequence that is the complement of a region of template/target DNA
to which the primer hybridizes. The addition of a nucleotide
residue to the 3' end of a primer by formation of a phosphodiester
bond results in a DNA extension product. The addition of a
nucleotide residue to the 3' end of the DNA extension product by
formation of a phosphodiester bond results in a further DNA
extension product.
[0098] "Nucleotide," as used herein, refers to a
nucleoside-5'-polyphosphate compound, or a structural analog
thereof, which can be incorporated by a nucleic acid polymerase to
extend a growing nucleic acid chain (such as a primer). Nucleotides
may comprise bases such as A, C, G, T, U, or analogues thereof, and
may comprise 3, 4, 5, 6, 7, 8, or more phosphates in the phosphate
group. Nucleotides may be modified at one or more of the base,
sugar, or phosphate group. A nucleotide may have a label or tag
attached (a "tagged nucleotide" or "labeled nucleotide").
[0099] "Terminating nucleotide" shall mean any modified or
unmodified nucleotide which, when incorporated into a nucleotide
strand, prevents or severely hampers further elongation of the
nucleotide strand. One example is a dideoxynucleotide. "Terminating
nucleotide" also comprises any nucleotide which will form a ternary
complex with the primed template and DNA polymerase in the presence
of non-catalytic metal ions preventing incorporation of the
nucleotide (Vander Horn et al, 2014). A terminating nucleotide may
have a label or tag attached.
[0100] "Polymer", as used herein, refers to any molecule or moiety
comprised of a plurality of repeating units. This includes both
homopolymers, polymers comprised of identical repeating units, such
as polyethylene glycol, and heteropolymers, polymers comprised of
similar but different repeating units, such as certain
oligonucleotides.
[0101] "Polymerase," as used herein, refers to any natural or
non-naturally occurring enzyme or other catalyst that is capable of
catalyzing a polymerization reaction, such as the polymerization of
nucleotide monomers to form a nucleic acid polymer. Exemplary
polymerases that may be used in the compositions and methods of the
present disclosure include the nucleic acid polymerases such as DNA
polymerase, DNA- or RNA-dependent RNA polymerase, and reverse
transcriptase.
[0102] As used herein, "substantially identical" or "substantially
fully complementary" sequences have at least about 80% sequence
identity or complementarity, respectively, to a nucleotide
sequence. Substantially identical sequences or substantially fully
complementary sequences may have at least about 85%, 90%, 95% or
100% sequence identity or complementarity, respectively.
Principle of Single Molecule Electronic SNP Assay
[0103] The invention disclosed herein pertains to methods of using
labeled nucleotide polyphosphate analogues to detect the identity
or presence of a nucleotide at certain positions in DNA (or RNA,
mutatis mutandis) sequences with single molecule sensitivity using
nanopore detection, nucleotides and primer-conjugated nanopore
proteins for use in such methods, and processes for producing such
nucleotides and primer-conjugated nanopore proteins.
[0104] Four ddNTPs are labeled with different length PEG molecules
or other polymer tags, such that each nucleotide can be well
discriminated by detecting the tags in a nanopore system after the
nucleotides are incorporated into the primer to determine the
genotype of the complementary nucleotide of the template. For
example, first, four different PEGs with 16, 20, 24 and 36 ethylene
glycol units that have been shown to display distinct nanopore
blockade signals for potential DNA sequencing by synthesis (Kumar
et al. 2012) are selected as tags to label ddNTPs. To avoid
interrupting the active sites of DNA polymerase, two positions in
ddNTPs are tested for the tag attachment: either the terminal
phosphate or the specific position of the base in each nucleotide.
Second, in order to perform the analysis, a homogeneous SNP
genotyping platform is prepared by conjugating a primer to the cap
of alpha hemolysin (.alpha.HL). Third, using these unique reagents,
the tagged-ddNTPs and primer-conjugated nanopores, single molecule
electronic SNP genotyping is performed in the nanopore system.
Specifically, primer-conjugated nanopores are reconstituted in the
lipid bilayers. After confirming the currents that indicate
insertion of the pore, single base extension (SBE) is performed by
adding a specific circularized template which has complementary
sequences to the primer, DNA polymerase and four differently tagged
ddNTPs to the pores. Under an applied voltage, the tags from each
extended primer are captured in the pore and the distinct current
signatures from each pore are analyzed, decoding the nucleotide
incorporated into the extended primer. This, in turn, reveals the
genotype of the template (FIG. 1).
[0105] Alternatively, the DNA polymerase can be momentarily stalled
in the closed ternary complex by adding a high concentration of
non-catalytic Sr.sup.2+ ion which permits transient-binding of a
complementary nucleotide and a primed template to DNA polymerase
but inhibits the incorporation of the bound nucleotide as described
by Vander Horn et al. (2014). Thus, the addition of Sr.sup.2+
temporally pauses DNA polymerase complex and extend the time span
of the nucleotide binding in the closed form of DNA polymerase
without the incorporation step. This prolonged time allows the tags
to be captured and read in the pore multiple times generating
stuttering current blockade signatures. In addition, both tagged
dideoxynucleotides and deoxynucleotides can be employed for
recognition of the inquired SNP site and are expected to generate
the same results since the nucleotide in this approach is not in
fact incorporated into the primer but complexed with the polymerase
in the ternary complex in the presence of Sr.sup.2+. Schemes of the
alternative assay using Sr.sup.2+ ion are provided in FIG. 2 and
FIG. 3. Overall, the demonstration of these configurations lays a
foundation for the development of a complete system for the single
molecule electronic SNP genotyping assay.
Embodiments of the Invention
[0106] The present invention is directed to a method for
identifying a single nucleotide residue of interest at a position
within a stretch of consecutive nucleotide residues in a nucleic
acid, comprising the steps of: [0107] (a) incubating the nucleic
acid, under an applied voltage, with [0108] (1) a nanopore; [0109]
(2) an oligonucleotide primer conjugated to the nanopore and
hybridized to the nucleotides in the nucleic acid immediately 3' to
the single nucleotide residue of interest; [0110] (3) at least one
labeled terminating nucleotide polyphosphate (NPP) analogue,
wherein the label is attached to either the base or the terminal
phosphate of the NPP analogue; and [0111] (4) a nucleic acid
polymerase; [0112] so that a NPP analogue is incorporated into the
primer if it is complementary to the single nucleotide residue of
interest, and the label attached to the incorporated NPP analogue
is drawn into the nanopore; [0113] (b) detecting by nanopore the
signature of the label of the NPP analogue incorporated into the
primer, so as to identify the incorporated NPP analogue; [0114]
thereby identifying the single nucleotide residue of interest.
[0115] In an embodiment of the invention, in step (a) the nucleic
acid is incubated with at least two NPP analogues. In a further
embodiment, the nucleic acid is incubated with at least four NPP
analogues. In a further embodiment, the nucleic acid is incubated
with exactly four NPP analogues.
[0116] In an embodiment of the invention, in step (a) the nucleic
acid is incubated with one NPP analogue, and if the NPP analogue is
not incorporated, iteratively repeating the incubating with a
different NPP analogue until a NPP analogue is incorporated.
[0117] The invention is further directed to a method for
identifying a single nucleotide residue of interest at a position
within a stretch of consecutive nucleotide residues in a nucleic
acid, comprising the steps of: [0118] a) incubating the nucleic
acid, under an applied voltage, with [0119] (1) a nanopore; [0120]
(2) an oligonucleotide primer conjugated to the nanopore and
hybridized to the nucleotides in the nucleic acid immediately 3' to
the single nucleotide of interest; [0121] (3) at least one labeled
nucleotide polyphosphate (NPP) analogue, wherein the label is
attached to either the base or the terminal phosphate of the NPP
analogue; [0122] (4) a nucleic acid polymerase; and [0123] (5) a
non-catalytic compound which permits transient binding of a
complementary NPP analogue to the nucleic acid polymerase and the
primed template but inhibits incorporation of the bound NPP
analogue; [0124] so that a NPP analogue is transiently bound to the
nucleic acid polymerase and the primed template if the NPP is
complementary to the single nucleotide of interest, and the label
attached to the transiently bound NPP analogue is drawn into the
nanopore; [0125] b) detecting by nanopore the signature of the
label of the NPP analogue transiently bound to the polymerase and
the primed template, so as to identify the NPP analogue; [0126]
thereby identifying the single nucleotide residue of interest.
[0127] In an embodiment of the invention, in step (a) the nucleic
acid is incubated with at least two NPP analogues. In a further
embodiment, the nucleic acid is incubated with at least four NPP
analogues. In a further embodiment, the nucleic acid is incubated
with exactly four NPP analogues.
[0128] In an embodiment of the invention, in step (a) the nucleic
acid is incubated with one NPP analogue, and if the NPP analogue
does not transiently bind to the nucleic acid polymerase,
iteratively repeating the incubating with a different NPP analogue
until a NPP analogue is transiently bound.
[0129] In an embodiment of the invention, the non-catalytic
compound is Sr.sup.2+.
[0130] In an embodiment of the invention, each NPP is a
deoxyribonucleotide polyphosphate (dNPP) or its analogue.
[0131] In another embodiment of the invention, each NPP is a
dideoxyribonucleotide polyphosphate (ddNPP) or its analogue.
[0132] In an embodiment of the invention, each ddNPP or its
analogue comprises a label having a coumarin-PEG moiety.
[0133] In an embodiment of the invention, each ddNPP or its
analogue comprises a label having a oligonucleotide-based tag
varying from about 10-50 monomeric units.
[0134] In an embodiment of the invention, each NPP is a
ribonucleotide polyphosphate (rNPP) or its analogue.
[0135] In an embodiment of the invention, the nucleic acid is
single-stranded DNA. In another embodiment, the nucleic acid is
double-stranded DNA. In another embodiment, the nucleic acid is
single-stranded RNA. In another embodiment, the nucleic acid is
double-stranded RNA.
[0136] In an embodiment of the invention, the nucleic acid is
single-stranded DNA or double-stranded DNA and the nucleic acid
polymerase is DNA polymerase. In another embodiment, the nucleic
acid is RNA and the nucleic acid polymerase is reverse
transcriptase.
[0137] In an embodiment of the invention, the nucleic acid
polymerase is RNA polymerase.
[0138] In an embodiment of the invention, the label is attached to
the base. In another embodiment, the label is attached to the
terminal phosphate.
[0139] In an embodiment of the invention, the label comprises one
or more of ethylene glycol, an amino acid, a carbohydrate, a
peptide, a dye, a chemiluminescent compound, a mononucleotide, a
dinucleotide, a trinucleotide, a tetranucleotide, a
pentanucleotide, a hexanucleotide, an oligonucleotide, an aliphatic
acid, an aromatic acid, an alcohol, a thiol group, a cyano group, a
nitro group, an alkyl group, an alkenyl group, an alkynyl group, an
azido group, or a combination thereof.
[0140] In an embodiment of the invention, the label is a polymeric
label. In a further embodiment, the labels are polyethylene glycol
(PEG) labels. In a further embodiment, the PEG labels each have a
different length from each other. In another embodiment, the labels
are oligonucleotide labels (Fuller et al 2015).
[0141] In an embodiment of the invention, the signature is an
electronic signature. In a further embodiment, the electronic
signature is an electrical current blockade signature. In a further
embodiment, the electrical current blockade signature is a
stuttering current blockade signature.
[0142] In an embodiment of the invention, the nanopore is a
solid-state nanopore. In another embodiment, the nanopore is in a
solid state membrane. In another embodiment, the nanopore is a
biological pore. In another embodiment, the nanopore is
proteinaceous. In another embodiment, the nanopore comprises alpha
hemolysin. In another embodiment, each nanopore comprises seven
alpha hemolysin monomers, any or all of which are conjugated to an
identical primer. In another embodiment, the nanopore comprises
MspA (Manrao et al 2012). In another embodiment, each nanopore
comprises eight alpha MspA monomers, any or all of which are
conjugated to an identical primer. In another embodiment, the
nanopore comprises CsgG (Goyal et al 2014). In another embodiment,
each nanopore comprises nine alpha CsgG monomers, any or all of
which are conjugated to an identical primer.
[0143] In an embodiment of the invention, the nucleic acid is
incubated with four coumarin-PEG-ddNPPs, each comprising a
different base, and each comprising a coumarin-PEG-label of a
different length. In a further embodiment, the four coumarin-PEG
labels are coumarin-PEG16, coumarin-PEG20, coumarin-PEG24, and
coumarin-PEG36. In an embodiment, each coumarin-PEG label is
attached to the terminal phosphate of the ddNPP. In another
embodiment, each coumarin-PEG label is a
coumarin-PEG-aminopropargyl label and is attached to the 5-position
of the base if the ddNPP is ddCPP, ddUPP, or ddTPP, and to the
7-position of the base if the ddNPP is ddAPP or ddGPP.
[0144] In an embodiment of the invention, the nucleic acid is
incubated with four oligonucleotide-tagged ddNPPs, each comprising
a different base, and each comprising an oligonucleotide tag of
different lengths and compositions. In a further embodiment, the
four oligonucleotide tags are as described in Fuller et al (2015).
In an embodiment, each oligonucleotide tag is attached to the
terminal phosphate of the ddNPP. In another embodiment, each
oligonucleotide tag is attached to the 5-position of the base if
the ddNPP is ddCPP, ddUPP, or ddTPP, and to the 7-position of the
base if the ddNPP is ddAPP or ddGPP.
[0145] In an embodiment of the invention the sequence of the primer
is 10-40 nucleotides long. In another embodiment, the sequence of
the primer is 18-24 nucleotides long.
[0146] The invention also provides for an assay for performing any
of the methods of the invention.
[0147] The invention also provides for a dideoxynucleotide
tetraphosphate (ddN4P) analogue, comprising a coumarin-polyethylene
glycol (PEG)-aminopropargyl label attached to the terminal
phosphate thereof.
[0148] In an embodiment of the invention, the ddN4P analogue has
the structure
##STR00001## [0149] wherein n is 16, 20, 24, or 36, and wherein B
is a base selected from the group consisting of adenine, cytosine,
thymine, guanine, and uracil.
[0150] In an embodiment, the base is guanine.
[0151] The invention also provides for a composition comprising
four ddN4P analogues of the invention, wherein each ddN4P comprises
a different base and a unique polymeric tag, and each tag has a
different composition and number of monomers.
[0152] In an embodiment of the invention, wherein the four ddN4P
analogues are a ddA4P analogue, a ddG4P analogue, a ddC4P analogue,
and either a ddT4P analogue or a ddU4P analogue.
[0153] The invention also provides for a process for producing a
ddN4P analogue of the invention, comprising: [0154] a) contacting a
ddN4P with diaminoheptane in carbodiimide (EDAC) and imidazole
buffer under conditions permitting the diaminoheptane to attach to
the terminal phosphate; [0155] b) contacting a 6-methoxycoumarin
N-hydroxysuccinimidyl ester (NHS) with an amino-PEG.sub.n-acid
moiety in dimethylformamide, wherein n is the number of ethylene
glycol monomers in the PEG, under conditions permitting the
production of a coumarin-PEG.sub.n-acid compound; [0156] c)
reacting the product of step b) with N,N disuccinimidyl carbonate
in dimethylformamide, under conditions permitting the production of
a coumarin-PEG.sub.n-NHS compound; and [0157] d) reacting the
products of steps a) and c) to produce a coumarin-PEG.sub.n-ddN4P
analogue.
[0158] In an embodiment, the process further comprises, prior to
step a), reacting a ddNTP with tributylammonium phosphate in order
to obtain the ddN4P.
[0159] The invention also provides for a process for producing the
ddG4P analogue of the invention, comprising: [0160] a) contacting a
ddG4P with diaminoheptane in carbodiimide (EDAC) and imidazole
buffer under conditions permitting the diaminoheptane to attach to
the terminal phosphate; [0161] b) contacting a 6-methoxycoumarin
N-hydroxysuccinimidyl ester (NHS) with an amino-PEG.sub.n-acid
moiety in dimethylformamide, wherein n is the number of ethylene
glycol monomers in the PEG, under conditions permitting the
production of a coumarin-PEG.sub.n-acid compound; [0162] c)
reacting the product of step b) with N,N-disuccinimidyl carbonate
in dimethylformamide, under conditions permitting the production of
a coumarin-PEG.sub.n-NHS compound; and [0163] d) reacting the
products of steps a) and c) to produce a coumarin-PEG.sub.n-ddG4P
analogue.
[0164] In an embodiment, the process further comprises, prior to
step a), reacting a ddGTP with tributylammonium phosphate in order
to obtain the ddG4P.
[0165] The invention also provides for a dideoxynucleotide
triphosphate (ddNTP) analogue, comprising a coumarin-polyethylene
glycol (PEG)-aminopropargyl label attached to the base thereof.
[0166] In an embodiment, the ddNTP analogue has the structure
##STR00002## [0167] wherein B is a base selected from the group
adenine, cytosine, thymine, guanine, and uracil or derivatives
thereof; and n is 16, 20, 24, or 36.
[0168] The invention also provides for a composition comprising
four ddNTP analogues of the invention, wherein each ddNTP comprises
a different base and a unique polymeric tag, and each tag has a
different composition and number of monomers.
[0169] In a further embodiment, the four ddNTP analogues are a
ddATP analogue, a ddGTP analogue, a ddCTP analogue, and either a
ddTTP analogue or a ddUTP analogue.
[0170] The invention also provides for a process for producing a
ddNTP analogue of the invention, comprising: [0171] a) contacting a
6-methoxycoumarin N-hydroxysuccinimidyl ester (NHS) with an
amino-PEG.sub.n-acid moiety in dimethylformamide, wherein n is the
number of ethylene glycol monomers in the PEG, under conditions
permitting the production of a coumarin-PEG.sub.n-acid compound;
[0172] b) reacting the product of step a) with N,N disuccinimidyl
carbonate in dimethylformamide, under conditions permitting the
production of a coumarin-PEG.sub.n-NHS compound; and [0173] c)
reacting the product of step b) with an aminopropargyl-ddNTP,
wherein the aminopropargyl moiety is base-attached, in
dimethylformamide, to produce a
coumarin-PEG.sub.n-aminopropargyl-ddNTP.
[0174] In an embodiment, the aminopropargyl-ddNTP in step c) is a
5-aminopropargyl-ddNTP if the ddNTP comprises a cytosine, uracil,
or thymidine base, and wherein the aminopropargyl-ddNTP in step c)
is a 7-aminopropargyl-ddNTP if the ddNTP comprises an adenine or
guanine base.
[0175] In an embodiment of the invention, n is 16, 20, 24, or
36.
[0176] In a further embodiment, the labeled terminating NPP
analogue is a nucleotide analogue selected from the groups
consisting of
##STR00003## [0177] wherein BASE is selected from the group
adenine, cytosine, thymine, uracil, guanine, 7-deaza-adenine, and
7-deaza-guanine or analog thereof; [0178] R and R' can be
independently H, OH, O-alkyl, F, Cl, Br, N.sub.3, NH, O--NH.sub.2,
O-allyl, O--CH.sub.2N.sub.3, 2', 3'-isopropylidine or groups which
only allow a single nucleotide to be incorporated by DNA
polymerase; [0179] TAG can be any polymeric molecule that can be
detected by nanopore and may be selected from the group
oligonucleotides, peptides, carbohydrates, and PEGs of different
length.
[0180] The invention also provides for a process for producing a
polymer tagged ddNTP analogue, comprising: [0181] a) contacting a
5(7)-propargylamino-ddNTP with amino-protected caproic acid NHS
ester; [0182] b) reacting the product of step a) with ammonium
hydroxide to produce 5(7)-propargylamidoaminocaproyl-ddNTPs; [0183]
c) reacting the product of step b) with azidobutyric acid NHS
ester; and [0184] d) reacting the product of step c) with
5'-alkynyl-oligonucleotide tag to produce oligonucleotide tagged
ddNTPs
[0185] The invention also provides for a dideoxynucleotide
tetraphosphate (ddN4P) analogue, comprising a coumarin-polyethylene
glycol (PEG)-aminopropargyl label attached to the terminal
phosphate thereof.
[0186] In an embodiment of the invention, the ddN4P analogue has
the structure
##STR00004## [0187] wherein n is 16, 20, 24, or 36, and wherein B
is a base selected from the group consisting of adenine, cytosine,
thymine, guanine, and uracil.
[0188] In an embodiment, the base is guanine.
[0189] The invention also provides for a composition comprising
four ddN4P analogues of the invention, wherein each ddN4P comprises
a different base and a unique polymeric tag, and each tag has a
different composition and number of monomers.
[0190] In an embodiment of the invention, wherein the four ddN4P
analogues are a ddA4P analogue, a ddG4P analogue, a ddC4P analogue,
and either a ddT4P analogue or a ddU4P analogue.
[0191] The invention also provides for a process for producing a
ddN4P analogue of the invention, comprising: [0192] a) contacting a
ddN4P with diaminoheptane in carbodiimide (EDAC) and imidazole
buffer under conditions permitting the diaminoheptane to attach to
the terminal phosphate; [0193] b) contacting a 6-methoxycoumarin
N-hydroxysuccinimidyl ester (NHS) with an amino-PEG.sub.n-acid
moiety in dimethylformamide, wherein n is the number of ethylene
glycol monomers in the PEG, under conditions permitting the
production of a coumarin-PEG-acid compound; [0194] c) reacting the
product of step b) with N,N disuccinimidyl carbonate in
dimethylformamide, under conditions permitting the production of a
coumarin-PEG.sub.n-NHS compound; and [0195] d) reacting the
products of steps a) and c) to produce a coumarin-PEG.sub.n-ddN4P
analogue.
[0196] In an embodiment, the process further comprises, prior to
step a), reacting a ddNTP with tributylammonium phosphate in order
to obtain the ddN4P.
[0197] The invention also provides for a process for producing the
ddG4P analogue of the invention, comprising: [0198] a) contacting a
ddG4P with diaminoheptane in carbodiimide (EDAC) and imidazole
buffer under conditions permitting the diaminoheptane to attach to
the terminal phosphate; [0199] b) contacting a 6-methoxycoumarin
N-hydroxysuccinimidyl ester (NHS) with an amino-PEG.sub.n-acid
moiety in dimethylformamide, wherein n is the number of ethylene
glycol monomers in the PEG, under conditions permitting the
production of a coumarin-PEG.sub.n-acid compound; [0200] c)
reacting the product of step b) with N,N-disuccinimidyl carbonate
in dimethylformamide, under conditions permitting the production of
a coumarin-PEG.sub.n-NHS compound; and [0201] d) reacting the
products of steps a) and c) to produce a coumarin-PEG.sub.n-ddG4P
analogue.
[0202] In an embodiment, the process further comprises, prior to
step a), reacting a ddGTP with tributylammonium phosphate in order
to obtain the ddG4P.
[0203] The invention also provides for a dideoxynucleotide
triphosphate (ddNTP) analogue, comprising a coumarin-polyethylene
glycol (PEG)-aminopropargyl label attached to the base thereof.
[0204] In an embodiment, the ddNTP analogue has the structure:
##STR00005## [0205] wherein B is a base selected from the group
adenine, cytosine, thymine, guanine, and uracil or derivatives
thereof; and n is 16, 20, 24, or 36.
[0206] The invention also provides for a composition comprising
four ddNTP analogues of the invention, wherein each ddNTP comprises
a different base and a unique polymeric tag, and each tag has a
different composition and number of monomers.
[0207] In a further embodiment, the four ddNTP analogues are a
ddATP analogue, a ddGTP analogue, a ddCTP analogue, and either a
ddTTP analogue or a ddUTP analogue.
[0208] The invention also provides for a process for producing a
ddNTP analogue of the invention, comprising: [0209] a) contacting a
6-methoxycoumarin N-hydroxysuccinimidyl ester (NHS) with an
amino-PEG.sub.n-acid moiety in dimethylformamide, wherein n is the
number of ethylene glycol monomers in the PEG, under conditions
permitting the production of a coumarin-PEG.sub.n-acid compound;
[0210] b) reacting the product of step a) with N,N disuccinimidyl
carbonate in dimethylformamide, under conditions permitting the
production of a coumarin-PEG.sub.n-NHS compound; and [0211] c)
reacting the product of step b) with an aminopropargyl-ddNTP,
wherein the aminopropargyl moiety is base-attached, in
dimethylformamide, to produce a
coumarin-PEG.sub.n-aminopropargyl-ddNTP.
[0212] In an embodiment, the aminopropargyl-ddNTP in step c) is a
5-aminopropargyl-ddNTP if the ddNTP comprises a cytosine, uracil,
or thymidine base, and wherein the aminopropargyl-ddNTP in step c)
is a 7-aminopropargyl-ddNTP if the ddNTP comprises an adenine or
guanine base.
[0213] In an embodiment of the invention, n is 16, 20, 24, or
36.
[0214] In a further embodiment, the labeled terminating NPP
analogue is a nucleotide analogue selected from the groups
consisting of:
##STR00006## [0215] wherein BASE is selected from the group
adenine, cytosine, thymine, uracil, guanine, 7-deaza-adenine, and
7-deaza-guanine or analog thereof;
[0216] R and R' can be independently H, OH, O-alkyl, F, Cl, Br,
N.sub.3, NH.sub.2, O--NH.sub.2, O-allyl, O--CH.sub.2N.sub.3, 2',
3'-isopropylidine or groups which only allow a single nucleotide to
be incorporated by DNA polymerase;
[0217] TAG can be any polymeric molecule that can be detected by
nanopore and may be selected from the group oligonucleotides,
peptides, carbohydrates, and PEGs of different length.
[0218] The invention also provides for a process for producing a
polymer tagged ddNTP analogue, comprising: [0219] a) contacting a
5(7)-propargylamino-ddNTP with amino-protected caproic acid NHS
ester; [0220] b) reacting the product of step a) with ammonium
hydroxide to produce 5(7)-propargylamidoaminocaproyl-ddNTPs; [0221]
c) reacting the product of step b) with azidobutyric acid NHS
ester; and [0222] d) reacting the product of step c) with
5'-alkynyl-oligonucleotide tag to produce oligonucleotide tagged
ddNTPs.
[0223] The invention also provides for a process for producing a
dNTP analogue with the tag attached on the base, comprising: [0224]
a) contacting a 5(7)-propargylamino-dNTP with amino-protected
caproic acid NHS ester; [0225] b) reacting the product of step a)
with ammonium hydroxide to produce
5(7)-propargylamidoaminocaproyl-dNTPs; [0226] c) reacting the
product of step b) with azidobutyric acid NHS ester; and [0227]
d)reacting the product of step c) with 5'-alkynyl-oligonucleotide
tag to produce oligonucleotide tagged dNTPs
[0228] The invention also provides for a process of using dNTP
analogues with distinct tags attached to the terminal phosphate
(Fuller et al. 2015) for SNP detection by the heretofore described
approach.
[0229] The invention also provides for an alpha hemolysin protein,
having a primer conjugated thereto.
[0230] In an embodiment, the alpha hemolysin comprises a C46
mutation, and the primer is conjugated to the cysteine residue at
position 46.
[0231] The invention also provides for a process for producing the
primer-conjugated .alpha.-hemolysin of the invention, comprising:
a) contacting a sulfosuccinimidyl-4-(N-maleimidomethyl)
cyclohexane-1-carboxylate (sSMCC) hetero bifunctional crosslinker,
comprising an amido-reactive N-hydroxysuccinimide (NHS) ester and a
thiol-reactive maleimide group at opposite ends, with a primer
comprising a terminal amino group, under conditions permitting the
terminal amino group to react with the amido-reactive NHS ester;
[0232] b) removing residual unreacted sSMCCs from the solution; and
[0233] c) contacting the product resulting from step a) with an
.alpha.-hemolysin, wherein the .alpha.-hemolysin comprises a
cysteine mutation at position 46, under conditions permitting the
thiol-reactive maleimide group to react with the cysteine residue
at position 46; thereby conjugating the primer to the
.alpha.-hemolysin.
[0234] In an embodiment, the removal of unreacted free sSMCCs in
step b) is performed via purification by gel filtration.
[0235] The invention also provides for a conductance measurement
system comprising: [0236] a) an electrically resistive barrier
separating at least a first and a second electrolyte solution;
[0237] said electrically resistive barrier comprises at least one
pore with a diameter on nanometer scale; [0238] said at least one
pore being configured to allow an ionic current to be driven across
said first and second electrolyte solutions by an applied
potential; [0239] b) at least one at least one labeled terminating
nucleotide polyphosphate (NPP) analogue, wherein the label is
attached to either the base or the terminal phosphate of the NPP
analogue, in at least one of said first and second electrolyte
solutions; and [0240] c) a means of measuring the ionic current and
a means of recording its time course as a time series, including
time periods when at least one pore is unobstructed by the label
and also time periods when a label causes pulses of
reduced-conductance.
[0241] In a further embodiment, the labeled terminating NPP
analogue is a ddNPP analogue selected from the group consisting
of
##STR00007## [0242] wherein B (BASE) is a base selected from the
group adenine, cytosine, thymine, guanine, and uracil or
derivatives thereof; and n is 16, 20, 24, or 36.
[0243] TAG can be any polymeric molecule that can be detected by
nanopore and may be selected from the group oligonucleotides,
peptides, carbohydrates, and PEGs of different length.
[0244] In an embodiment, the system comprises four ddNPP analogues
having the structure
##STR00008## [0245] wherein each ddNTP comprises a different base,
and each has a different value of n.
[0246] In an embodiment, the system comprises four ddNPP analogues
having the structure
##STR00009## [0247] wherein each ddNTP comprises a different base,
and each has a different value of n.
[0248] In an embodiment, the system comprises four ddNTP analogues
which are a ddATP analogue, a ddGTP analogue, a ddCTP analogue, and
either a ddTTP analogue or a ddUTP analogue.
[0249] As used herein, "alkyl" includes both branched and
straight-chain saturated aliphatic hydrocarbon groups having the
specified number of carbon atoms and may be unsubstituted or
substituted. Thus, C.sub.1-C.sub.n as in "C.sub.1-C.sub.n alkyl" is
defined to include groups having 1, 2, . . . , n- or n carbons in a
linear or branched arrangement. For example, a "C.sub.1-C.sub.5
alkyl" is defined to include groups having 1, 2, 3, 4, or 5 carbons
in a linear or branched arrangement, and specifically includes
methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, and
pentyl.
[0250] As used herein, "alkenyl" refers to a non-aromatic
hydrocarbon group, straight or branched, containing at least 1
carbon to carbon double bond, and up to the maximum possible number
of non-aromatic carbon-carbon double bonds may be present, and may
be unsubstituted or substituted. For example, "C.sub.2-C.sub.5
alkenyl" means an alkenyl group having 2, 3, 4, or 5, carbon atoms,
and up to 1, 2, 3, or 4, carbon-carbon double bonds respectively.
Alkenyl groups include ethenyl, propenyl, and butenyl.
[0251] The term "alkynyl" refers to a hydrocarbon group, straight
or branched, containing at least 1 carbon to carbon triple bond,
and up to the maximum possible number of non-aromatic carbon-carbon
triple bonds may be present, and may be unsubstituted or
substituted. Thus, "C.sub.2-C.sub.5 alkynyl" means an alkynyl group
having 2 or 3 carbon atoms and 1 carbon-carbon triple bond, or
having 4 or 5 carbon atoms and up to 2 carbon-carbon triple bonds.
Alkynyl groups include ethynyl, propynyl and butynyl.
[0252] The term "substituted" refers to a functional group as
described above such as an alkyl, or a hydrocarbyl, in which at
least one bond to a hydrogen atom contained therein is replaced by
a bond to non-hydrogen or non-carbon atom, provided that normal
valencies are maintained and that the substitution(s) result(s) in
a stable compound. Substituted groups also include groups in which
one or more bonds to a carbon(s) or hydrogen(s) atom are replaced
by one or more bonds, including double or triple bonds, to a
heteroatom. Non-limiting examples of substituents include the
functional groups described above, and for example, N, so as to
form --CN.
Exemplary Labels
[0253] A label (also referred to herein as a tag) may be any
chemical group or molecule that is capable of being detected in a
nanopore. In one embodiment, a label provides an electronic
signature by blocking or impeding ionic current through a
nanopore.
[0254] In some cases, a label comprises one or more of ethylene
glycol, an amino acid, a carbohydrate, a peptide, a dye, a
chemiluminescent compound, a mononucleotide, a dinucleotide, a
trinucleotide, a tetranucleotide, a pentanucleotide, a
hexanucleotide, an oligonucleotide (modified or unmodified), an
aliphatic acid, an aromatic acid, an alcohol, a thiol group, a
cyano group, a nitro group, an alkyl group, an alkenyl group, an
alkynyl group, an azido group, or a combination thereof.
[0255] In some cases, the label is a polymer. Polyethylene glycol
(PEG) is an example of a polymer and has the following
structure:
##STR00010##
[0256] Any number of ethylene glycol units (W) may be used. In some
cases, each label is a PEG label comprising a different number of
ethylene glycol units.
Nanopore Detection of Labels
[0257] Previously, Kasianowicz et al. (1996) discovered that the
.alpha.-hemolysin (.alpha.HL) protein nanopore, which has a 1.5
nm-diameter limiting aperture (Song et al. 1996, Bezrukov et al.
1996, Krasilnikov 2002, and Kasianowicz et al. 1995), could be used
to electronically detect nucleic acids at the single molecule
level. Thus, the .alpha.HL nanopore has been investigated widely
for the development of a single molecule electronic DNA sequencing
technology (Kasianowicz et al. 1996, Kasianowicz et al. 2002,
Kasianowicz et al. 1998, and Clarke et al. 2009). The majority of
these research efforts involve strand DNA sequencing by nanopore,
which aim at sequencing DNA by threading it through the nanopore
and detecting the electrical current blockade from the 4
nucleotides (A, C, G, T) (Cherf et al. 2012 and Manrao et al.
2012).
[0258] The native .alpha.HL nanopore has an intrinsic property for
high-resolution discrimination of molecules and ions, which enables
the discrimination between aqueous H.sup.+ and D.sup.+ ions
(Kasianowicz et al. 1995). Robertson et al. (2007) have
demonstrated that the .alpha.HL nanopore can easily separate more
than 20 different PEG polymers at single monomer level. This study
indicates that the mean residence time of the PEG polymer in the
pore increases with its size (Reiner et al. 2010). Recently, Kumar
et al. (2012) have reported the use of 4 PEGs of distinct size to
label the terminal phosphate of nucleotides for single molecule
electronic DNA sequencing by synthesis with nanopore detection.
Based on these previous investigations, the single molecule
electronic multiplex SNP assay described herein will be capable of
detecting multiple genetic variations simultaneously using PEGs of
different sizes to tag nucleotides.
[0259] 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 are only
illustrative of the invention as described more fully in the claims
which follow thereafter. Every embodiment and feature described in
the application should be understood to be interchangeable and
combinable with every embodiment contained within.
EXPERIMENTAL DETAILS AND DISCUSSION
Review of SNP Analysis Via MALDI-TOF MS
[0260] The early SBE method for multiplex SNP analysis using
MALDI-TOF MS detected both primers and their extension products,
because both were loaded to the MS analyzer. This requires the
unambiguous simultaneous detection of multiplex primers and their
extension products. However, for longer biopolymers, such as DNA,
MALDI-TOF MS analyzer has limitations in resolution and
sensitivity. As a result, larger DNA molecules could not be
resolved by MALDI-TOF MS. To address this issue, Kim et al.
developed a multiplex SNP assay (SPC-SBE) using solid phase
capturable (SPC) biotinylated dideoxynucleotide terminators
(biotin-ddNTPs) in SBE by detection with MALDI-TOF MS (Kim et al.
2002 and Kim et al. 2003).
[0261] In the SPC-SBE method, a library of oligonucleotide primers
corresponding to the multiple SNP sites are designed to have
different molecular mass. These primers are then annealed to the
SNP sites of the target gene and extended with a specific
biotin-ddNTP by DNA polymerase, producing 3'-biotinylated DNA
products. Treatment of the polymerase reaction mixtures by
streptavidin-coated magnetic beads leads to the capture of the DNA
products that carry a biotin moiety at the 3'-end. The excess
primers, DNA polymerase and salts in the reaction are washed away.
The pure DNA extension products are subsequently released from the
magnetic beads by denaturing the biotin-streptavidin interaction
with formamide at 95.degree. C., and characterized with MALDI-TOF
MS for SNP determination. In the SPC-SBE approach, the accuracy and
scope of multiplexing in SNP analysis is significantly increased,
because only the isolated primer extension products are loaded into
the MALDI-TOF MS analyzer. Consequently, the resulting mass
spectrum is free of the non-extended primer peaks and their
associated dimers, which do not carry a biotin moiety and are
removed during SPC. SPC also facilitates desalting of the captured
DNA products and therefore enhances the accuracy and the overall
quality of the MS data for SNP analysis.
[0262] In summary, in the SPC-SBE multiplex SNP assay with
MALDI-TOF MS (Kim et al. 2002), multiplex PCR products are produced
as templates from genomic DNA for carrying out SBE reactions using
SNP specific primers with different mass. Only the DNA extension
products extended by a specific biotin-ddNTP are captured while the
other components of the reaction are removed. The captured DNA
products are then released and loaded on to the MALDI-TOF MS
analyzer to identify nucleotide variation. It has been shown that
unextended primers occupy the effective mass range in the mass
spectrum reducing the ability for multiplexing. The excess primers
can form a dimer, producing false peaks in the mass spectrum
(Roskey et al. 1996). All the above complications are completely
removed by the SPC-SBE. Due to the large molecular weight
difference of the four biotin-ddNTPs, polymerase extension products
from these dideoxynucleotides are unambiguously detected with well
resolved molecular weights. The molecular weight of the primer
extension products in comparison to the masses of the corresponding
primers reveal the identity of each nucleotide at the polymorphic
site. The SPC-SBE method is particularly beneficial in determining
heterozygous genotypes. In this case, two peaks, one corresponding
to each allele, will be clearly discernible in the resulting mass
spectrum.
[0263] MALDI-TOF MS, when used for characterizing SNPs, can
simultaneously measure the masses of DNA molecules over a certain
range. To make best use of this feature for the analysis of
multiple SNPs in a single MS spectrum, if excess primers are not
removed, masses of all primers and their extension products have to
be sufficiently different to produce peaks that can be adequately
resolved in the mass spectrum. For example, Ross et al. (1998)
performed simultaneous detection of multiple SNPs by tuning the
masses of all primers and their extension products so that they
would lie in the range of 4.5 kDa and 7.6 kDa with no overlapping.
In contrast, by eliminating the unextended primers that occupy the
valuable mass range in the mass spectrum, the SPC-SBE approach
significantly increases the scope of multiplexing in
characterization of SNPs. Genetic variations (C282Y and H63D) in
the human hereditary hemochromatosis gene were successfully and
accurately characterized by SPC-SBE (Kim et al. 2002). Thirty
polymorphic sites in exons 5, 7 and 8 of the tumor suppressor gene
p53, which are most frequently mutated in human cancer (Hollstein
et al. 1991 and Bardelli et al. 2003), from Wilms' tumors, head and
neck squamous carcinomas as well as colorectal carcinomas, were
also precisely determined with the SPC-SBE method (Kim et al.
2004). Using the SPC-SBE approach, Misra et al. performed
concurrent analysis of 40 SNPs of CYP2C9 and 50 SNPs of CYP2A13 in
the cytochrome P450 (CYP450) genes (Misra et al. 2007).
[0264] DNA purification exploiting the strong interaction of a
small molecule, biotin, and a protein, streptavidin on solid
surfaces such as magnetic beads is extensively used in
biotechnology. However, the affinity between biotin and
streptavidin is among the strongest known non-covalent bonds. The
denaturing of the biotin-streptavidin interaction requires
treatment with formamide at 95.degree. C. and the reaction yield is
often low. To further optimize the condition for SPC and release of
the DNA extension products from streptavidin-coated magnetic beads,
Qiu et al. (Anal. Biochem, 427:193-201, 2012) developed a set of
chemically cleavable biotinylated dideoxynucleotides,
ddNTPs-N.sub.3-Biotin (ddATP-N.sub.3-Biotin, ddGTP-N.sub.3-Biotin,
ddCTP-N.sub.3-Biotin and ddUTP-N.sub.3-Biotin), for application in
DNA sequencing and SNP analysis by MALDI-TOF MS. These cleavable
biotinylated dideoxynucleotides have been successfully used in
SPC-SBE to characterize mitochondrial SNPs (Qiu et al., Anal.
Biochem, 427:202-210, 2012).
[0265] Several alternative methods for multiplex SNP analysis that
use mass spectrometry have been developed. For example, the
commercially available MASSARRAY.TM. assay (Rodi et al. 2002) from
Sequenom Inc. is widely used for characterizing genetic variations,
including mitochondrial SNPs for population studies (Cerezo et al.
2009) and detection of heteroplasmy (Xiu-Cheng et al. 2008). The
MASSARRAY.TM. assay is automated with high throughput. In one form
of this approach, the primer is extended by DNA polymerase in the
presence of three dideoxynucleotides and one deoxynucleotide that
corresponds to one of the two alleles. At the end of the reaction,
single nucleotide primer extension products and the primers
extended with two or more nucleotides as well as the unextended
primers are all loaded on to the MALDI-TOF MS analyzer and detected
in the mass spectrum. Since no labeling of any reaction components
is required, the MASSARRAY.TM. assay is simple to perform. However,
it is limited in performing simultaneous high level multiplex
analysis of SNPs, because all reaction products and all unextended
primers are both loaded into the MS analyzer.
Review of SNP Analysis by Fluorescence Detection
[0266] The fluorescence polarization--template-directed
dye-terminator incorporation (FP-TDI) SNP assay (Chen et al. 1999)
uses single nucleotide polymerase extension with allele-specific
fluorescence-labeled dideoxynucleotide terminators. The genotypes
of the extension products are characterized by monitoring the
unique change in fluorescence polarization. The FP-TDI approach
offers a simple SNP detection method but with a limited scope of
multiplexing.
[0267] The BEAMing (beads, emulsion, amplification, and magnetics)
approach (Dressman et al. 2003) has been developed for detecting
genetic variations with the aim of high-sensitivity and
high-throughput. In this method, each individual DNA template is
discretely amplified by a large number of oligonucleotide primers
that are immobilized on a magnetic bead in a water-oil emulsion,
the target SNPs are distinguished by unique fluorescent dye-labeled
probes and characterized using flow cytometry. The BEAMing approach
not only allows the identification of allelic variations, but also
offers the ability to quantify these variations. In addition, the
DNA sample can be recovered from the flow cytometer for further
analysis. The disadvantage of the BEAMing method is that multiple
steps of manipulation are required, which can lead to difficulties
in accurate characterization of allele frequency.
[0268] Tong et al. have developed a multiplex fluorescent SNP assay
using SBE and combinatorial fluorescence energy transfer (CFET)
tags (Tong et al. 2002). A larger number of CFET tags with unique
fluorescence signatures have been constructed using a small number
of fluorophores with distinct emissions by exploiting fluorescence
energy transfer and the combinatorial concept. The CFET tags can
all be excited at a single wavelength of 488 nm and detected and
differentiated by a simple optical system. The principle of the
approach is outlined as follows. A library of CFET-labeled
oligonucleotide primers are designed and synthesized so that the
nucleotide at the 3'-end is complementary to a particular SNP in
the template. In a single tube reaction, the CFET-labeled
oligonucleotide primers and biotin-ddNTPs are used to perform SBE
on the DNA templates containing the SNPs. CFET-labeled primer that
perfectly matches with the DNA template is extended with a
biotin-ddNTP by DNA polymerase. The 3'-biotinylated DNA products
are isolated by capture with streptavidin-coated magnetic beads,
while the unextended primers and other components in the reaction
are not captured and eliminated by washing. A multicolor
laser-induced fluorescence electrophoresis instrument is used to
analyze the biotinylated fluorescent DNA products. The SNPs are
determined by the distinct fluorescence signature and
electrophoretic mobility of each DNA extension product in the
electropherogram. Using oligonucleotide ligation, Tong et al.
(2001) have used CFET tags to detect multiplex nucleotide
variations simultaneously from the retinoblastoma tumor suppressor
gene. None of the mass spectroscopy or fluorescence based
approaches reviewed above offer detection of SNPs with single
molecule sensitivity.
Experiment 1
[0269] Design and Synthesis of PEG-Labeled ddNTP Analogues for DNA
Polymerase Extension
[0270] In an embodiment of single molecule, single-base extension
electronic SNP genotyping scheme described herein, current blockade
signals in the nanopore are generated by the capture of PEG
molecules that are attached to the nucleotides. Hence, the design
and synthesis of PEG-labeled ddNTPs and test these molecules as
substrates for DNA polymerases are required. It is necessary to
find a unique position in the ddNTPs to attach the tags without
interrupting the active sites of DNA polymerases and the native DNA
structure during the nucleotide incorporation. To achieve this, two
possible positions in ddNTPs are tested for the tag attachment:
either the terminal phosphate or the specific position on the base
in each nucleotide.
[0271] First, considering that pyrophosphate is released from the
enzyme complex during the polymerase reaction, we reasoned that the
terminal phosphate of each of the four nucleotides might be
available for the attachment of longer polymer tags. Indeed, Kumar
et al. (2012) have attached PEG molecules of different lengths to
the terminal phosphate position of deoxyguanosine and demonstrated
that these modified nucleotides are incorporated into primers with
100% efficiency for DNA sequencing. In detail, they added an extra
phosphate to the deoxyguanosine triphosphate (dGTP) to serve as a
linker between the .gamma.-phosphate of dGTP and the tag,
preventing the tag from interfering with the active site of the DNA
polymerase during the incorporation. Then, four distinct PEG tags
were attached, generating PEG tagged deoxyguanosine tetraphosphates
(PEG-dG4P). They showed that these PEG-dG4P nucleotides are
efficient substrates for DNA polymerase extension (Kumar et al.
2012). This result indicates that DNA polymerase can tolerate
sizable modification at the terminal phosphate position of
deoxynucleotide triphosphates (dNTPs), including the additional
phosphate and the PEG tag, and utilize such nucleotide analogues as
competent substrates for primer extension by polymerase. An earlier
study had shown that DNA polymerases recognize tetra- or longer
polyphosphates with improved efficiency (up to 50-fold) than the
corresponding tri-phosphates (Kumar et al. 2005).
[0272] Thus, taking advantage of the above strategy, as an example,
the present approach includes different length PEG molecules being
attached to the terminal phosphate position in each ddNTP. The
synthesis process is as follows. First, dideoxynucleoside
triphosphates (ddNTPs) are converted to dideoxynucleoside
tetraphosphates (ddN4Ps) by treatment with tributylammonium
phosphate. Then, a diaminoheptane linker is added to the terminal
phosphate of the tetraphosphate to produce ddN4P-heptyl-NH.sub.2
(Product A) for attaching different length PEG tags. In a separate
set of reactions, 6-methoxy-coumarin N-hydroxysuccinimidyl ester is
reacted with one of four amino-PEG.sub.n-COOH molecules, with n
corresponding to 16, 20, 24, or 36 ethylene glycol units, to yield
coumarin-PEG.sub.n-COOH molecules, which are subsequently converted
to the corresponding NHS-esters (Product B). The coumarin moiety is
employed to track the purification of intermediates and the final
nucleotide analogues. Finally, coupling of ddN4P-heptyl-NH.sub.2
(Product A) with the coumarin-PEG.sub.n-NHS esters (Product B)
produces the final tagged nucleotide analogues. The PEG molecules
with 16, 20, 24, or 36 ethylene glycol units are attached to A, C,
G or T, respectively. An example of the synthesis scheme for the
proposed coumarin-PEG.sub.n-ddG4P molecule is provided in FIG. 4.
Following the same scheme, a complete set of ddN4Ps (A, C, G, T),
each of which is tagged with a unique PEG polymer, is
synthesized.
[0273] Second, it has also been demonstrated that certain modified
DNA polymerases can tolerate nucleotides with extensive
modifications containing bulky groups at the 5-position of
pyrimidines (C and U) and the 7-position of purines (A and G)
(Rosenblum et al. 1997). Indeed, several nucleotides labeled with
fluorescent dyes at the 5-position of pyrimidines or 7-position of
purines have been created and employed for various genomic
applications including DNA labeling and sequencing (Ju et al.
2006). Based on these previous studies, different length PEG tags
are attached to the 5-position of C/U and the 7-position of A/G as
an alternative design. In order to synthesize these molecules,
6-methoxycoumarin-NHS ester is reacted with one of the four
amino-PEG.sub.n-COOH molecules with 16, 20, 24, or 36 ethylene
glycol units to yield coumarin-PEG.sub.r-COOH molecules, which is
subsequently converted to the corresponding NHS-esters. Then, the
resulting coumarin-PEG.sub.n-NHS-ester is coupled with
5-aminopropargyl-ddNTPs for C/U and 7-aminopropargyl-ddNTPs for
A/G. The general synthesis scheme for the
coumarin-PEG.sub.n-aminopropargyl-ddNTPs is provided in FIG. 5.
Additionally, different types of tags such as polymers of
oligosaccharides, nucleotides, or oligopeptides can be synthesized
and tested for improved nanopore signal resolution.
[0274] After synthesizing these molecules, their capability as
substrates for DNA polymerase extension reactions are examined
using a self-primed loop template DNA. If each compound is
recognized and incorporated by DNA polymerase into the primer, the
molecular weight of the primer will be increased by the size of the
expected nucleoside monophosphate in the case of 5' phosphate
tagging, or tagged nucleoside monophosphate in base-tagged
molecules, and this can be detected by MALDI-TOF mass
spectroscopy.
Synthesis of Oligonucleotide-Tagged Nucleoside Polyphosphates
[0275] The general overall synthetic scheme is presented in FIG. 6.
Synthesis of the oligonucleotide-tagged nucleotides involves the
coupling
5(7)-alkynylamino-2'-3'-dideoxynucleoside-5'-triphosphate-azides
(ddNTP-N3) with 5'-hexynyl oligonucleotides.
Synthesis of 5 or 7-Propargylamidoaminocaproyl-ddNTPs (5-8).
[0276] The 5(7)-propargylamino-dideoxynucleotides (1-4) were
prepared following the procedure described by Hobbs and Cocuzza
(1991) and the longer linker arm dideoxynucleotides (5-8) were
prepared according to Duthie et al. (2002) and purified by reverse
phase HPLC.
Addition of Azido group to 5 or 7-Propargylamidoaminocaproyl-ddNTPs
(9-12)
[0277] The extended ddNTP-NH2 nucleotides (5-8, 5 .mu.mol) were
each dissolved in 0.1 M bicarbonate-carbonate buffer (200 .mu.l, pH
8.7) and azidobutyric acid-NHS (15 pmol) in 100 .mu.l DMF was
added. The reaction mixture was stirred overnight at room
temperature and was purified by HPLC using 0.1 M TEAC buffer (pH
7.5) and an acetonitrile gradient to yield products 9-12. MALDI TOF
MS data: ddATP-N3: 750 (calculated 751); ddUTP-N3: 726 (calculated
729); ddGTP-N3: 765 (calculated 767); ddCTP-N3: 725 (calculated
728).
Click Reaction Between ddNTP-N3 Nucleotides (9-12) and 5'-Hexynyl
Oligonucleotide Tags to Produce Polymer-Tagged ddNTP
Nucleotides
[0278] To each 5'-hexynyl-oligonucleotide tag (custom synthesized
by TriLink, 500 nmol in 200 .mu.l H.sub.2O) was added a solution of
the corresponding ddNTP-N.sub.3 nucleotide (750 nmol) followed by
the addition of copper bromide (50 .mu.l, 0.1 M solution in 3:1
DMSO/t-BuOH) and tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine
(TBTA) (100 .mu.l, 0.1 M solution in 3:1 DMSO/t-BuOH). The reaction
mixture was stirred at 40.degree. C. for 16 hr followed by HPLC
purification using 0.1 M TEAC buffer (pH 7.5) and an acetonitrile
gradient. The tagged nucleotides were characterized by MALDI-TOF
mass spectroscopy and single base polymerase extension reaction.
MALDI-TOF mass data for two of the tagged nucleotides are shown in
FIG. 7: ddCTP-Cy3-dT.sub.2-dSp.sub.8-dT.sub.20-C3, 9601 (calculated
9605); ddTTP-Cy3-dT.sub.4-dSp.sub.3-dT.sub.23-C3, 10226 (calculated
10225).
Single Base Extension Reactions with Oligonucleotide-Tagged ddCTP
and Oligonucleotide-Tagged ddTTP
[0279] Extension reactions were carried out using a template-loop
primer in which the next complementary base on the template was
either A or G, (allowing extension by a single complementary
nucleotide. Reactions were performed with 10 pmol of self-primed
looped template, 200 pmole of tagged ddNTPs and 5 units of each
enzyme (Klenow, Sequenase and Thermo Sequenase) incubated for 1 hr
at 37.degree. C. DNA extension products were characterized by gel
electrophoresis as shown in FIG. 8.
Method of Detection of SNPs Using Streptavidin-Biotin Capture
[0280] A scheme for characterizing current signatures of different
tags by extending biotinylated primers with tagged ddNTPs is
presented in FIG. 9. Primers having a single biotin molecule
attached to the base of the 3'-nucleotide and complementary
template DNA are incubated with Thermo Sequenase, Mg.sup.2 and
tagged ddNTPs. The DNA polymerase extends the primers with a single
tagged ddNTP that is complementary to the next base in the template
DNA. After the incubation, streptavidin molecules that have strong
affinity for biotin are added to the extension reaction and both
the unextended and extended primers are captured by the
streptavidin. The reaction mixture is then applied to the nanopore
electronic detection system. Although streptavidin itself cannot
fit through the pore due to its much larger size, it holds the
primer extension product in a position whereby the tag can enter
the pore, generating a unique current signature specific for the
tag on each nucleotide. In this way, the current signatures of
different tags can be confirmed.
Nanopore Current Blockade Levels of Tagged Nucleotides
[0281] Nanopore current blockade levels generated by the tags on
the primer extension products by incorporating tagged ddCTP
(ddCTP-Cy3-T.sub.2-dSp.sub.8-T.sub.20-C3) and tagged ddTTP
(ddTTP-Cy3-T.sub.4-dSp.sub.3-T.sub.23-C3) are shown in FIG. 10. The
tag on the primer extension product incorporating a tagged ddCTP
displayed a current blockade of about 26-30% of the open pore
current level and the tag on the primer extension product
incorporating a tagged ddTTP gave a 46-50% blockade.
Experiment 2
Conjugation of Primers to .alpha.HL Monomers
[0282] By conjugating a primer to monomeric .alpha.HL, allele
discrimination and allele detection can be performed in the same
environment. In order to conjugate a primer to the .alpha.HL, an
engineered .alpha.HL construct, in which a lysine residue is
mutated to a cysteine at position 46 (a C46 mutation) for
site-specific labeling of the protein and a hexahistidine tag is
added at the C-terminus, is employed for the primer attachment.
Using this recombinant DNA construct, first, the C46 monomer is
expressed in Escherichia coli strain BL21 (DE3) and purified by
nickel affinity chromatography. In a separate reaction, the primer
with a terminal amino group is conjugated to a
sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate
(sSMCC) hetero bifunctional crosslinker which contains an
amino-reactive N-hydroxysuccinimide (NHS ester) and a
thiol-reactive maleimide group at opposite ends. Then, the sSMCC
crosslinker-conjugated primers are purified from the residual
unreacted crosslinkers in the solution by Sephadex G50. Finally,
the C46 monomer is reacted with the linker-attached primers via the
thiol group. This generates single primer-conjugated C46 monomers
via the reaction between the thiol group in the cysteine residue in
the mutant monomer and the maleimide group in the crosslinker. This
scheme is shown in FIG. 11.
Experiment 3
Confirmation of Electronic Signatures
[0283] As described hereinabove, four distinct ionic current
blockade patterns have been shown to be produced by the four
distinct PEG.sub.n labels (n=16, 20, 24, 36) in an .alpha.HL
channel at the single molecule level for DNA sequencing (Kumar et
al. 2012). First, in order to confirm the above results and
establish a control electronic signature for each PEG label,
synthetic versions of the expected released tags
(coumarin-PEG.sub.n-NH.sub.2) are analyzed for their nanopore
current blockade effects in a nanopore. Initially, IM KCl is
introduced to the system. Then,
1,2-diphytanoyl-sn-glycero-3-phosphocholine is added to form a
lipid bilayer. Once the membrane seals the orifice, the wild type
.alpha.HL monomer is injected into the solution immediately
adjacent to the membrane and an electrical potential is applied to
the system. The ionic current is monitored until a sudden increase
in current amplitude occurs indicating formation of a single
channel in the membrane. After confirming the correct current
signature for the single heptameric channel insertion,
coumarin-PEG.sub.n-NH.sub.2 molecules are added to the pores and
the ionic current is recorded. From this experiment, the blockade
depth and residence time distributions of each PEG.sub.n molecule
over a wide range of transmembrane potentials are analyzed and
conditions such as salt concentration or temperature-voltage
profile are optimized for the highest signal intensities and lowest
background noise. This generates the reference current signatures
for the different repeat unit PEG molecules. Nucleotides labeled by
tags based on modified oligonucleotides have been shown to display
distinct electronic signatures in nanopores (Fuller et al.
2015).
Experiment 4
Perform Single Molecule Electronic SNP Genotyping in the
Primer-Conjugated Nanopore Format
[0284] After establishing the reference current signatures for each
tag, the single molecule electronic SNP genotyping assay described
heretofore is performed. After adding about 0.2 M KCl and forming a
lipid bilayer in the system, the purified primer conjugated mutant
C46 .alpha.HL described hereinabove is introduced to the lipid
bilayer. Once one monomer binds to the membrane, it associates with
neighboring monomers in the solution to form a homoheptameric
transmembrane pore. Since the monomer described hereinabove is
engineered to possess a single primer, this forms a pore bearing 7
identical primers. In order to confirm the formation of such a
pore, electrical potential is applied across each individual lipid
bilayer in the nanopore chip system (Fuller et al. 2015). Once the
current signature that indicates the insertion of the pore is
confirmed, SBE is performed by adding DNA templates containing
specific SNP loci, 4 different length PEG-labeled ddNPPs (with
labels attached to the base of each ddNTP) and DNA polymerase to
the pore. This extends the primers by a single base with one of the
4 PEG-labeled ddNTPs if the added templates have complementarity to
the attached primers in the pore.
[0285] By having several identical primers on a single pore, tags
are read multiple times from a single pore, increasing the
reliability efficiency of event detection. Moreover, two different
current signatures should be detected from a single pore if the
tested template is heterozygous while only one current signature
would appear in the system if the template is homozygous (FIG. 12).
In order to test this, two different templates having identical
sequences except at the SNP site are designed, mixed together, and
tested in the system. If the two different templates bind to
primers on a single pore, the primers are extended by different tag
attached ddNPPs. As a result, two different current signatures are
detected from a single pore.
Experiment 5
[0286] Perform Single Molecule Electronic SNP Genotyping in the
Primer-Conjugated Nanopore Format with Terminal Phosphate-Labeled
Nucleotides
[0287] In the case where the ddNPPs or dNPPs having polymer tags
attached at their terminal phosphate positions are selected as
substrates for DNA polymerase extension reaction, the tags would be
expected to be released along with polyphosphates as the
nucleotides are incorporated into the primers. Although the passage
of individual polymer molecules through nanopores has been
successfully detected and demonstrated in previous studies (Kumar
et al. 2012), it might still be challenging to obtain reliable
current signals from each released polymer tag molecule at the
single molecule level due to their rapid translocation. In order to
overcome this potential issue and ensure sufficient dwell time of
the tags in the nanopore system, a high concentration of
non-catalytic metal ions (Vander Horn et al. 2014) such as
Sr.sup.2+ or Ca.sup.2+, which permit transient binding of a
complementary nucleotide to DNA polymerase but inhibit
incorporation of the bound nucleotide, is added. Competing for the
magnesium (Mg.sup.2+) that catalyzes complete incorporation of
ddNPPs or dNPPs to the primers, the addition of Sr.sup.2+ or
Ca.sup.2+ extends the time between binding of the nucleotide in the
polymerase closed ternary complex and release of the polyphosphate.
This extended period of time allows the polymer tags to be captured
and held longer in the pore while the nucleotide is still
interacting with the closed form of DNA polymerase but not yet
fully incorporated. Also, the rate of nucleotide incorporation by
DNA polymerases in vitro can be tuned to about 100 milliseconds per
base addition while the translocation of the polymer tags through
the nanopore happens in microseconds. Thus, trapping the polymer
tags from the nucleotides in the pore long enough to generate
signals allows more accurate and reliable genotyping detection than
by just letting the released polymer tags pass through the
pore.
[0288] In the case where the nucleotides having polymer tags
attached to the base position are chosen as substrates for the
extension, there is no need to add Sr.sup.2+ since the polymer tags
are permanently incorporated in the primers that are conjugated to
the pores. Thus, the long polymer tag is stably captured from the
extended primer in each pore after the SBE step.
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Sequence CWU 1
1
2126DNAArtificial Sequencetemplate 1gatagcgccg cgccttggcg cggcgc
262302PRTArtificial SequenceHemolysin mutant 2Met Ala Asp Ser Asp
Ile Asn Ile Lys Thr Gly Thr Thr Asp Ile Gly1 5 10 15Ser Asn Thr Thr
Val Lys Thr Gly Asp Leu Val Thr Tyr Asp Lys Glu 20 25 30Asn Gly Met
His Lys Lys Val Phe Tyr Ser Phe Ile Asp Asp Cys Asn 35 40 45His Asn
Lys Lys Leu Leu Val Ile Arg Thr Lys Gly Thr Ile Ala Gly 50 55 60Gln
Tyr Arg Val Tyr Ser Glu Glu Gly Ala Asn Lys Ser Gly Leu Ala65 70 75
80Trp Pro Ser Ala Phe Lys Val Gln Leu Gln Leu Pro Asp Asn Glu Val
85 90 95Ala Gln Ile Ser Asp Tyr Tyr Pro Arg Asn Ser Ile Asp Thr Lys
Glu 100 105 110Tyr Met Ser Thr Leu Thr Tyr Gly Phe Asn Gly Asn Val
Thr Gly Asp 115 120 125Asp Thr Gly Lys Ile Gly Gly Leu Ile Gly Ala
Asn Val Ser Ile Gly 130 135 140His Thr Leu Lys Tyr Val Gln Pro Asp
Phe Lys Thr Ile Leu Glu Ser145 150 155 160Pro Thr Asp Lys Lys Val
Gly Trp Lys Val Ile Phe Asn Asn Met Val 165 170 175Asn Gln Asn Trp
Gly Pro Tyr Asp Arg Asp Ser Trp Asn Pro Val Tyr 180 185 190Gly Asn
Gln Leu Phe Met Lys Thr Arg Asn Gly Ser Met Lys Ala Ala 195 200
205Glu Asn Phe Leu Asp Pro Asn Lys Ala Ser Ser Leu Leu Ser Ser Gly
210 215 220Phe Ser Pro Asp Phe Ala Thr Val Ile Thr Met Asp Arg Lys
Ala Ser225 230 235 240Lys Gln Gln Thr Asn Ile Asp Val Ile Tyr Glu
Arg Val Arg Asp Asp 245 250 255Tyr Gln Leu His Trp Thr Ser Thr Asn
Trp Lys Gly Thr Asn Thr Lys 260 265 270Asp Lys Trp Thr Asp Arg Ser
Ser Glu Arg Tyr Lys Ile Asp Trp Glu 275 280 285Lys Glu Glu Met Thr
Asn Lys Gly His His His His His His 290 295 300
* * * * *