Compositions, systems, and methods for sequencing polynucleotides using tethers anchored to polymerases adjacent to nanopores

• US 10,648,022 B2
• Filed: 06/02/2016
• Issued: 05/12/2020
• Est. Priority Date: 06/03/2015
• Status: Active Grant

• Current Assignee: Illumina Incorporated
• Original Assignee: Illumina Incorporated
• Inventors: Gunderson, Kevin L., Mandell, Jeffrey G.
• Primary Examiner(s): Forman, Betty J
• Application Number:
  US15/577,728
• Publication Number:
  US 20180155774A1
• Time in Patent Office: 1,440 Days
• CPC Class Codes: C12Q 1/6839 Triple helix formation or other higher order conformations in hybridisation assays
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2521/543 Immobilised enzyme(s)
• CPC Class Codes: C12Q 2525/197 incorporating a spacer/coupling moiety
• CPC Class Codes: C12Q 2565/607 being a sensor, e.g. electrode
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore
• 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

A composition includes a nanopore including first and second sides and an aperture, nucleotides each including an elongated tag, and a first polynucleotide that is complementary to a second polynucleotide. A polymerase can be disposed adjacent to the first side of the nanopore and configured to add nucleotides to the first polynucleotide based on a sequence of the second polynucleotide. A permanent tether can include a head region anchored to the polymerase, a tail region, and an elongated body disposed therebetween that occurs in the aperture of the nanopore. A first moiety can be disposed on the elongated body that binds to the elongated tag of a first nucleotide upon which the polymerase is acting. A reporter region can be disposed on the elongated body that indicates when the first nucleotide is complementary or is not complementary to a next nucleotide in the sequence of the second polynucleotide.

Claims

1. A composition including:

a nanopore including a first side, a second side, and an aperture extending through the first and second sides;

a plurality of nucleotides, wherein each of the nucleotides comprises an elongated tag;

first and second polynucleotides, the first polynucleotide being complementary to the second polynucleotide;

a polymerase disposed adjacent to the first side of the nanopore, the polymerase configured to add nucleotides of the plurality of nucleotides to the first polynucleotide based on a sequence of the second polynucleotide;

a permanent tether including a head region, a tail region, and an elongated body disposed therebetween, the head region being anchored to the polymerase, wherein the elongated body is disposed in the aperture of the nanopore;

a first moiety disposed on the elongated body, wherein the first moiety is configured to bind to the elongated tag of a first nucleotide upon which the polymerase is acting; and

a reporter region disposed on the elongated body, wherein the reporter region is configured to indicate when the first nucleotide is complementary or is not complementary to a next nucleotide in the sequence of the second polynucleotide.

2. The composition of claim 1, the first moiety being configured to generate a first signal state responsive to the polymerase acting upon the first nucleotide, the first nucleotide being identifiable based on the first signal state.

3. The composition of claim 2, wherein the polymerase acting upon the first nucleotide comprises the polymerase binding the first nucleotide.

4. The composition of claim 2, the first signal state including an electrical or optical signal.

5. The composition of claim 2, the reporter region being configured to generate a second signal state when the first nucleotide is complementary or is not complementary to the next nucleotide of the second polynucleotide.

6. The composition of claim 5, the reporter region being configured to generate the second signal state responsive to the polymerase successfully incorporating the first nucleotide into the first polynucleotide.

7. The composition of claim 6, the reporter region being configured to generate the second signal state responsive to release of pyrophosphate responsive to the polymerase successfully incorporating the first nucleotide into the first polynucleotide.

8. The composition of claim 7, the polymerase being modified so as to delay release of the pyrophosphate responsive to incorporation of the first nucleotide into the first polynucleotide.

9. The composition of claim 8, wherein the polymerase comprises a modified recombinant .PHI.29, B103, GA-1, PZA, .PHI.15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase.

10. The composition of claim 8, wherein the polymerase comprises a modified recombinant .PHI.29 DNA polymerase having at least one amino acid substitution or combination of substitutions selected from the group consisting of:

an amino acid substitution at position 484, an amino acid substitution at position 198, and an amino acid substitution at position 381.

11. The composition of claim 8, wherein the polymerase comprises a modified recombinant .PHI.29 DNA polymerase having at least one amino acid substitution or combination of substitutions selected from the group consisting of E374Y, K512Y, T368F, A484E, A484Y, N387L, T372Q, T372L, K478Y, 1370W, F198W, and L381A.

12. The composition of claim 5, the reporter region being configured to generate the second signal state responsive to a conformational change of the polymerase.

13. The composition of claim 12, a magnitude or a time duration of the conformational change of the polymerase being responsive to the first nucleotide being complementary or not complementary to the next nucleotide of the second polynucleotide, a magnitude or a time duration, or both, of the second signal state being based upon the conformational change of the polymerase.

14. The composition of claim 12, the measurement circuitry further being configured to measure a second current or flux state through the aperture.

15. The composition of claim 14, wherein the second current or flux state is based on a position of the reporter region within the aperture, it being determinable based on the second current or flux state whether the first nucleotide is complementary or is not complementary to the next nucleotide in the second polynucleotide.

16. The composition of claim 5, wherein the second signal state includes an electrical signal.

17. The composition of claim 5, wherein the second signal state includes an optical signal.

18. The composition of claim 17, wherein the first moiety and a second moiety of the tether are configured to hybridize with one another so as to form a hairpin structure.

19. The composition of claim 18, further including a voltage source configured to apply a voltage across the first and second sides.

20. The composition of claim 19, wherein the first moiety and the second moiety of the tether are configured to dehybridize from one another responsive to the voltage in a two-step process.

21. The composition of claim 1, wherein the elongated tag comprises a first nucleotide sequence and the first moiety comprises a second nucleotide sequence that is complementary to the first nucleotide sequence.

22. A system including the composition of claim 21, and further including measurement circuitry configured to measure a first current or flux state through the aperture.

23. The composition of claim 22, wherein the first current or flux state is based on the elongated tag, the first nucleotide being identifiable based on the first current or flux state.

24. A method including:

(a) providing a nanopore including a first side, a second side, and an aperture extending through the first and second sides;

(b) providing a plurality of nucleotides, wherein each of the nucleotides comprises an elongated tag;

(c) providing first and second polynucleotides, the first polynucleotide being complementary to the second polynucleotide;

(d) providing a polymerase disposed adjacent to the first side of the nanopore, the polymerase configured to add nucleotides of the plurality of nucleotides to the first polynucleotide based on a sequence of the second polynucleotide, wherein the polymerase is anchored to a permanent tether including a head region, a tail region, and an elongated body disposed therebetween, the elongated body is disposed in the aperture of the nanopore;

(e) determining that a first nucleotide is being acted upon by the polymerase based on binding of the elongated tag to a first moiety disposed on the elongated body; and

(f) with a reporter region disposed on the elongated body, indicating when the first nucleotide is complementary or is not complementary to a next nucleotide in the sequence of the second polynucleotide.

25. The method of claim 24, said determining comprising generating a first signal state responsive to the polymerase acting upon the first nucleotide and identifying the first nucleotide based on the first signal state.

26. The method of claim 25, wherein the polymerase acting upon the first nucleotide comprises the polymerase binding the first nucleotide.

27. The method of claim 26, the first signal state including an electrical or optical signal.

28. The method of claim 26, said indicating comprising detecting a second signal state and determining based upon the second signal state that the first nucleotide is complementary or is not complementary to the next nucleotide of the second polynucleotide.

29. The method of claim 28, comprising generating the second signal state responsive to the polymerase incorporating the first nucleotide into the first polynucleotide.

30. The method of claim 29, comprising generating the second signal state responsive to release of pyrophosphate responsive to the polymerase incorporating the first nucleotide into the first polynucleotide.

31. The method of claim 30, the polymerase being modified so as to delay release of the pyrophosphate responsive to incorporation of the first nucleotide into the first polynucleotide.

32. The method of claim 31, wherein the polymerase comprises a modified recombinant .PHI.29, B103, GA-1, PZA, .PHI.15, BS32, M2Y, Nf, G1, Cp-1, PRD1, PZE, SF5, Cp-5, Cp-7, PR4, PR5, PR722, or L17 polymerase.

33. The method of claim 31, wherein the polymerase comprises a modified recombinant .PHI.29 DNA polymerase having at least one amino acid substitution or combination of substitutions selected from the group consisting of:

an amino acid substitution at position 484, an amino acid substitution at position 198, and an amino acid substitution at position 381.

34. The method of claim 33, wherein the polymerase comprises a modified recombinant .PHI.29 DNA polymerase having at least one amino acid substitution or combination of substitutions selected from the group consisting of E374Y, K512Y, T368F, A484E, A484Y, N387L, T372Q, T372L, K478Y, 1370W, F198W, and L381A.

35. The method of claim 28, comprising generating the second signal state responsive to a conformational change of the polymerase.

36. The method of claim 35, a magnitude or a time duration of the conformational change of the polymerase being responsive to the first nucleotide being complementary or not complementary to the next nucleotide of the polynucleotide, a magnitude or a time duration, or both, of the second signal state being based upon the conformational change of the polymerase.

37. The method of claim 29, wherein the second signal state includes an electrical signal.

38. The method of claim 29, wherein the second signal state includes an optical signal.

39. The method of claim 24, wherein the elongated tag comprises a first nucleotide sequence and the first moiety comprises a second nucleotide sequence that is complementary to the first nucleotide sequence.

40. The method of claim 39, said determining further comprising measuring a first current or flux state through the aperture.

41. The method of claim 40, wherein the first current or flux state is based on the elongated tag, said determining further including identifying the first nucleotide based on the first current or flux state.

42. The method of claim 41, said indicating including moving the reporter region within the aperture responsive to the polymerase acting upon the first nucleotide.

43. The method of claim 42, said indicating further including measuring a second current or flux state through the aperture.

44. The method of claim 43, wherein the second current or flux state is based on a position of the reporter region within the aperture, said indicating further including determining based on the second current or flux state whether the first nucleotide is complementary or is not complementary to the next nucleotide in the second polynucleotide.

45. The method of claim 39, the first moiety and a second moiety of the tether hybridizing with one another so as to form a hairpin structure.

46. The method of claim 45, further including applying a voltage across the first and second sides.

47. The method of claim 46, wherein the first moiety and the second moiety of the tether dehybridize from one another responsive to the voltage in a two-step process.

48. The method of claim 24, further comprising (g) determining that a subsequent nucleotide is being acted upon by the polymerase based on binding of the elongated tag to the first moiety disposed on the elongated body;

and (h) with a reporter region disposed on the elongated body, indicating when the subsequent nucleotide is complementary or is not complementary to a nucleotide that is subsequent to the next nucleotide in the sequence of the second polynucleotide.

49. The method of claim 48, further comprising repeating steps (g) and (h) for a plurality of subsequent nucleotides that are complementary or not complementary to a plurality of nucleotides that are subsequent to the next nucleotide.