METHOD OF PREPARATION OF NANOPORE AND USES THEREOF

• US 20150111759A1
• Filed: 04/08/2013
• Published: 04/23/2015
• Est. Priority Date: 04/09/2012
• Status: Active Grant

• Current Assignee: THE GOVERNMENT OF THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY OF COMMERCE
• Original Assignee: Trustees Of Columbia University In The City Of New York (Columbia University), THE GOVERNMENT OF THE UNITED STATES OF AMERICA, AS REPRESENTED BY THE SECRETARY OF COMMERCE
• Inventors: Ju, Jingyue, Kumar, Shiv, Tao, Chuanjuan, Chien, Minchen, Russo, James J., Kasianowicz, John J., Robertson, Joseph W.F.
• Granted Patent: US 10,246,479 B2
• Time in Patent Office: Days
• US Class Current: 506/2
• CPC Class Codes: B82Y 5/00 Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C12Q 1/42 involving phosphatase
• CPC Class Codes: C12Q 1/48 involving transferase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2521/525 Phosphatase
• CPC Class Codes: C12Q 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2535/107 Maxam and Gilbert method, i.e. sequential release and detection of nucleotides
• CPC Class Codes: C12Q 2537/157 A reaction step characterised by the number of molecules incorporated or released
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• CPC Class Codes: C12Q 2600/158 Expression markers
• CPC Class Codes: G01N 2333/9125 with a definite EC number (2.7.7.-)
• CPC Class Codes: G01N 33/48721 Investigating individual macromolecules, e.g. by translocation through nanopores Coulter counters in general G01N15/12; fabrication methods for nanoscale apertures B81B1/00; sequencing of nucleic acids C12Q1/68

Abstract

This disclosure provides systems and methods for sequencing nucleic acids using nucleotide analogues and translocation of tags from incorporated nucleotide analogues through a nanopore. In aspects, this disclosure is related to composition, method, and system for sequencing a nucleic acid using tag molecules and detection of translocation through a nanopore of tags released from incorporation of the molecule.

Claims

1. A conductance measurement system comprising:

(a) a first and a second compartment with a first and a second electrolyte solution separated by a physical barrier, which barrier has at least one pore with diameter on nanometer scale;

(b) a means for applying an electric field across the barrier;

(c) a means for measuring change in the electric field;

(d) at least one polymerase attached to the pore; and

(e) more than one phosphatase enzyme attached to the pore.

2. The conductance measurement system of claim 1, wherein the pore has a diameter of from about 1 to 10 nm.

3. The conductance measurement system of claim 1, wherein the polymerase and the phosphatase enzymes are covalently attached to the pore.

4. The conductance measurement system of claim 1, wherein more phosphatase enzymes than polymerases are attached to the pore.

5. The conductance measurement system of any one of claims 1-4, wherein the phosphatase enzymes are positioned such that polyphosphate produced by the polymerase in a polymerase reaction interacts with the phosphatase enzymes before entering the pore.

6. The conductance measurement system of claim 5, wherein the rate of interaction between the phosphatase enzymes and the polyphosphate is faster than, or equal to, the rate of the polymerase producing the polyphosphate.

7. The conductance measurement system of any one of claims 1-6, wherein each of the first and the second compartments has an electrical charge.

8. The conductance measurement system of any one of claims 1-7, wherein the interior of the pore has a negative charge.

9. The conductance measurement system of any one of claims 1-8, wherein the pore is biological or synthetic.

10. The conductance measurement system of claim 9, wherein the pore is proteinaceous.

11. The conductance measurement system of claim 10, wherein the pore is an alpha hemolysin protein.

12. The conductance measurement system of any one of claims 1-8, wherein the pore is a solid-state pore.

13. The conductance measurement system of any one of claims 1-12, wherein the pore is formed of graphene.

14. The conductance measurement system of any one of claims 1-13, further comprising an array of pores each having substantially identical features.

15. The conductance measurement system of any one of claims 1-13, further comprising an array of pores of different diameters.

16. The conductance measurement system of any one of claims 1-13, further comprising an array of pores, wherein the pores are configured to apply different electrical fields across the barrier.

17. The conductance measurement system of any one of claims 1-16, wherein the conductance measurement system is integrated with CMOS electronics.

18. The conductance measurement system of claim 17, wherein the pore or array of pores is integrated directly into a CMOS die as shown in FIG. 46.

71. The conductance measurement system of claim 1, wherein the first and second electrolyte solutions are the same.

19. A compound having the structure:

20. The compound of claim 19, wherein the magnitude of the charge on the tag is the same as the magnitude of the charge on the remainder of the compound.

21. The compound of claim 19 or 20, wherein the tag comprising multiple ethylene glycol units.

22. The compound of any one of claims 19-21, wherein the tag comprises 16, 20, 24, or 36 ethylene glycol units.

23. The compound of any one of claims 19-22, wherein the tag comprises an additional identifiable moiety.

24. The compound of claim 23, wherein the additional identifiable moiety is a coumarin based dye.

25. The compound of any one of claims 19-24, wherein the tag has a positive charge.

26. The compound of any one of claims 19-25, wherein removal of the tag changes the charge of the compound.

27. The compound of any one of claims 19-26, wherein the tag further comprises appropriate number of lysines or arginines to balance the number of phosphates.

28. A composition comprising four different types of a compound having the structure:

29. The composition of claim 28, further comprising a fifth type of compound which differs from each of the four types of compound in the base and in the tag, wherein the base of the fifth type of compound is uracil or a derivative thereof if the base of the fourth type of compound is thymine or a derivative thereof, or wherein the base of the fifth type of compound is thymine or a derivative thereof if the base of the fourth type of compound is uracil or a derivative thereof.

30. A method for determining the identity of a compound comprising:

(a) contacting the compound with a conductance measurement system comprising;

(i) a first and a second compartment with a first and a second electrolyte solution separated by a physical barrier, which barrier has at least one pore with diameter on nanometer scale;

(ii) a means for applying an electric field across the barrier;

(iii) a means for measuring change in the electric field;

(b) recording the change in the electric field when the compound translocates through the pore wherein the change in the electric field is the result of interaction between the compound, the electrolyte, and the pore, and is indicative of the size, charge, and composition of the compound,

thereby allowing correlation between the change and predetermined values to determine the identity of the compound.

31. The method of claim 30, further comprising a step of treating the compound with a phosphatase enzyme before step (a).

33. The method of any one of claims 30-32, further comprising a step of adjusting current bias of the electric field in step (a).

63. The method of any one of claims 30-53 and 56-62, further comprising a step of calibrating the conductance measurement system.

64. The method of any one of claims 30-53 and 56-63, wherein the accuracy is greater than 4σ

.

65. The method of any one of claims 30-53 and 56-63, wherein the accuracy is greater than 5σ

.

66. The method of any one of claims 30-53 and 56-63, wherein the accuracy is greater than 6σ

.

32. A method for determining whether a compound is a tag or a precursor of the tag comprising:

(a) contacting the compound with a conductance measurement system comprising;

(i) a first and a second compartment with a first and a second electrolyte solution separated by a physical barrier, which barrier has at least one pore with diameter on nanometer scale;

(ii) a means for applying an electric field across the barrier;

(iii) a means for measuring change in the electric field;

(b) recording the change in the electric field when the compound translocates through the pore; and

(c) comparing the change in the electric field with pre-determined values corresponding to the tag and the precursor of the tag,

thereby determining whether the compound is the tag or the precursor thereof.

34. A method for determining the nucleotide sequence of a single-stranded DNA, which method comprising:

(a) contacting the single-stranded DNA with

a conductance measurement system comprising;

(i) a first and a second compartment with a first and a second electrolyte solution separated by a physical barrier, which barrier has at least one pore with diameter on nanometer scale;

(ii) a means for applying an electric field across the barrier;

(iii) a means for measuring change in the electric field;

(iv) at least one polymerase attached to the pore; and

(v) more than one phosphatase enzyme attached to the pore, and

a composition comprising four different types of a compound having the structure;

38. The method of any one of claims 34-37, wherein more phosphatase enzymes than polymerases are attached to the pore.

39. The method of any one of claims 34-37, wherein the single-stranded DNA or RNA is obtained by denaturing a double-stranded DNA or RNA, whichever is applicable.

40. The method of any one of claims 34-37, wherein multiple copies of the same single-stranded DNA or RNA are immobilized on a bead.

41. The method of claim 40, wherein the nucleotide sequence of the single-stranded DNA or RNA is determined using multiple copies of the same single-stranded DNA or RNA.

42. The method of any one of claims 34-41, further comprising a washing step after each iteration of step (b) to remove unincoporated compound from contact with the single-stranded DNA or RNA.

43. The method of any one of claims 34-42, further comprising a step after each iteration of step (b) to determine the identity of an additional identifiable moiety attached to the tag.

44. The method of any one of claims 34-43, wherein at least 85-99% of the released tags translocate through the pore.

45. The method of any one of claims 34-44, wherein the compound further comprises a reversible terminator.

46. The method of claim 45, further comprises a step of removing the reversible terminator after each iteration of step (b).

47. The method of claim 46, wherein the reversible terminator is removed by biological means, chemical means, physical means, or by light irradiation.

48. The method of any one of claims 34-47, wherein the interior of the pore has a charge which is reverse in sign relative to the charge of the tag or of the polyphosphate having the tag attached thereto.

49. The method of any one of claims 34-48, wherein each of the first and the second compartments of the conductance measurement system has a charge.

50. The method of claim 49, wherein the charges of the first and the second compartments are opposite in polarity.

51. The method of claim 49, wherein the charges of the first and the second compartments are adjustable.

52. The method of any one of claims 34-51, wherein the rate of the tag translocating through the pore in step (b) is determined based on the charge of tag and the charges of the first and the second compartments.

53. The method of any one of claims 34-52, wherein each of the first and the second compartments has a charge such that in step (b) the tag translocates through the pore at a rate which is faster than, or equal to, the rate at which the tag or the polyphosphate having the tag attached thereto is being released in step (a).

35. A method for determining the nucleotide sequence of a single-stranded DNA, the method comprising:

(a) contacting the single-stranded DNA with

a conductance measurement system comprising;

(i) a first and a second compartment with a first and a second electrolyte solution separated by a physical barrier, which barrier has at least one pore with diameter on nanometer scale;

(ii) a means for applying an electric field across the barrier;

(iii) a means for measuring change in the electric field;

(iv) at least one polymerase attached to the pore; and

(v) more than one phosphatase enzyme attached to the pore, and

a compound having the structure;

36. A method for determining the nucleotide sequence of a single-stranded RNA, which method comprising:

(a) contacting the single-stranded RNA with

a conductance measurement system comprising;

(i) a first and a second compartment with a first and a second electrolyte solution separated by a physical barrier, which barrier has at least one pore with diameter on nanometer scale;

(ii) a means for applying an electric field across the barrier;

(iii) a means for measuring change in the electric field;

(iv) at least one polymerase attached to the pore; and

(v) more than one phosphatase enzyme attached to the pore, and

a composition comprising four different types of a compound having the structure;

37. A method for determining the nucleotide sequence of a single-stranded RNA, the method comprising:

(a) contacting the single-stranded RNA with

a conductance measurement system comprising;

(i) a first and a second compartment with a first and a second electrolyte solution separated by a physical barrier, which barrier has at least one pore with diameter on nanometer scale;

(ii) a means for applying an electric field across the barrier;

(iii) a means for measuring change in the electric field;

(iv) at least one polymerase attached to the pore; and

(v) more than one phosphatase enzyme attached to the pore, and

a compound having the structure;

54. A conductance measurement system comprising:

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;

at least one compound with a tag in at least one of said first and second electrolyte solutions;

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;

said at least one pore comprising a feature configured to cleave the tag from the compound to release the tag; and

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 tag and also time periods when the tag causes pulses of reduced-conductance.

55. The conductance measurement system of claim 54 wherein the tag has a residence time in the pore which is greater than limitations of ionic current bandwidth and current shot noise of said means of measuring the ionic current.

72. conductance measurement system of claim 54, wherein the first and the second electrolyte solutions are the same.

56. A method to delineate segments of a conductance time series into regions statistically consistent with the unobstructed pore conductance level, and pulses of reduced-conductance, and also statistically stationary segments within individual pulses of reduced-conductance, said conductance time series being generated with a conductance measurement system comprising:

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;

at least one compound with a tag in at least one of said first and second electrolyte solutions;

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;

said at least one pore comprising a feature configured to cleave the tag from the compound to release the tag; and

a means of measuring the ionic current and a means of recording said conductance time series, including time periods when the at least one pore is unobstructed by said tag and also time periods when said tag causes pulses of reduced-conductance;

said method to delineate segments of a conductance time series being selected from the group consisting of;

(a) a Viterbi decoding of the maximum likelihood state sequence of a Continuous Density of a Hidden Markov Model estimated from the raw conductance time series;

(b) a delineation of the regions of pulses of reduced-conductance via comparison to a threshold for deviation from the open-pore conductance level; and

(c) a means to characterize pulses of reduced-conductance by estimating the central tendencies of the ionic current levels for each segment, or by measure of central tendencies and segment duration together, the measure of segment central tendency being selected from the group consisting of;

(i) a mean parameter of a Gaussian component of a first GMM estimated from the conductance time series as part of a Continuous Density Hidden Markov Model;

(ii) an arithmetic mean;

(iii) a trimmed mean;

(iv) a median; and

(v) a Maximum A Posteriori estimator of sample location, or a maximum likelihood estimator of sample location.

57. The method of claim 56 further comprising at least one:

(a) a maximum likelihood estimate of a second Gaussian Mixture Model based upon the measures of central tendency of conductance segments;

(b) a peak finding by means of interpolation and smoothing of the empirical probability density of the estimates of central tendencies of segments of the conductance times series and finding roots of the derivatives of the interpolating functions; and

(c) another means of locating the modes of multimodal distribution estimator.

59. The method of any one of claims 56-58, wherein the compound is treated with phosphatase before measuring the reduction in the ionic current.

60. The method of any one of claims 56-59, wherein the compound is an alternatively charged compound which has a first net charge and, after a chemical, physical or biological reaction, a different second net charge.

61. The method of any one of claims 56-60, wherein the mathematical analysis is selected from the group consisting of GMM, threshold detection and averaging, and sliding window analysis.

62. The method of any one of claims 56-61, wherein the at least one parameter is selected from the group consisting of the concentration, size, charge, and composition of the compound.

73. The method of claim 56, wherein the first and the second electrolyte solutions are the same.

58. A method for determining at least one parameter of a compound in a solution comprising the steps of:

placing a first fluid in a first reservoir;

placing a second fluid in a second reservoir;

at least one of said first and said second fluid comprising at least one compound, wherein the compound is a tagged nucleotide or a tag cleaved from a tagged nucleotide;

said first fluid in said first reservoir being separated from said second fluid in said second reservoir with an electrically resistive barrier;

said electrically resistive barrier comprising at least one pore;

passing an ionic current through said first fluid, said at least one pore, and said second fluid with an electrical potential between said first and said second fluid;

measuring the ionic current passing through said at least one pore and the duration of changes in the ionic current;

the measuring of the ionic current being carried out for a period of time sufficient to measure a reduction in the ionic current caused by the compound interacting with said at least one pore;

and determining at least one parameter of the compound by mathematically analyzing the changes in the ionic current and the duration of the changes in the ionic current over the period of time;

said mathematical analysis comprising at least one step selected from the group consisting of;

i) a mean parameter of a Gaussian component of a first GMM estimated from the conductance time series as part of a Continuous Density Hidden Markov Model;

ii) an Event-Mean Extraction;

iii) Maximum Likelihood Event State Assignment;

iv) threshold detection and averaging;

v) sliding window analysis;

vi) an arithmetic mean;

vii) a trimmed mean;

viii) a median; and

ix) a Maximum A Posteriori estimator of sample location, or a maximum likelihood estimator of sample location.

74. The method of claim 58, wherein the first fluid and the second fluid are the same.

67. A tagged nucleotide, wherein the nucleotide comprises a tag capable of being cleaved in a nucleotide polymerization event and detected with the aid of a nanopore.

68. The nucleotide of claim 67, wherein the tag is attached to the 5′

-phosphate of the nucleotide.

69. The nucleotide of claim 67, wherein the tag is not a fluorophore.

70. The nucleotide of claim 67, wherein the tag is detectable by its charge, shape, size, or any combination thereof.

75. A tagged nucleotide, wherein the nucleotide comprises a tag capable of being cleaved in a nucleotide polymerization event and detected with the aid of a nanopore.

76. The nucleotide of claim 75, wherein the tag is attached to the 5′

-phosphate of the nucleotide.

77. The nucleotide of claim 75, wherein the tag is not a fluorophore.

78. The nucleotide of claim 75, wherein the tag is detectable by its charge, shape, size, or any combination thereof.

79. A method for nucleic acid sequencing, the method comprising providing an array of individually addressable sites, each site having a nanopore attached to a nucleic acid polymerase, and, at a given site of said array, polymerizing tagged nucleotides with a polymerase, wherein a tag is released and detected by a nanopore at said given site.