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1. (WO2018100370) METHODS AND SYSTEMS FOR CHARACTERIZING ANALYTES USING NANOPORES
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CLAIMS

1. A method of characterizing a polynucleotide, the method comprising:

(i) combining in a solution:

a) a construct comprising a double-stranded polynucleotide, having a template strand and a complement strand, wherein the template strand and the complement strand are not covalently linked, with

b) a nanopore, wherein one or more tags that bind to a portion of the construct is conjugated to the nanopore,

wherein the construct and the nanopore are combined under conditions in which the construct binds to the nanopore;

(ii) providing a condition so as to permit the template strand of the construct to enter the nanopore, so as to permit separation of the template strand and translocation of at least a portion of the template strand through the nanopore;

(iii) measuring a change in a property indicative of translocation of the template strand through the nanopore; and

(iv) characterizing the polynucleotide based on the measured change in the property as the template strand translocates through the nanopore.

2. The method of claim 1, wherein the solution is ionic and the measured property is ion current flow through the nanopore.

3. The method of claim 1 or 2, wherein an adaptor is attached to one or both of the two ends of the double-stranded polynucleotide, each adaptor comprising a duplex stem and a first single strand extending from the duplex stem, wherein the first single strand of one adaptor is contiguous with the template strand and the first single strand of the other adaptor is contiguous with the complement strand.

4. The method of any one of the preceding claims, wherein the one or more tags is conjugated to an outer rim of the nanopore.

5. The method of any one of the preceding claims, wherein the condition is a potential difference across the nanopore.

6. The method of any one of the preceding claims, wherein the nanopore is disposed in a membrane.

7. The method of any one of claims 3 to 6, wherein one or more tags that bind to a portion of the adaptor is conjugated to the nanopore.

8. The method of claim 7, wherein the one or more tags are conjugated to the outer rim of the nanopore.

9. The method of any one of the claims 3 to 8, wherein step ii) comprises: applying a potential difference across the membrane so as to permit the first single strand contiguous with the template strand of the construct to enter the nanopore, maintaining the potential difference across the nanopore for a sufficient period of time so as to permit separation of the template strand and translocation of at least a portion of the template strand through the nanopore.

10. The method of any one of the preceding claims wherein step iii) comprises measuring a change in ionic current flow through the nanopore as the template strand

translocates through the nanopore.

11. The method of claim 10, wherein step iv) comprises characterizing the polynucleotide based on the change in ionic current flow through the nanopore measured as the template strand translocates through the nanopore.

12. The method of any one of the preceding claims, wherein a polynucleotide unwinding enzyme is present in the solution on the cz's-opening side of the nanopore.

13. The method of any one of the preceding claims, wherein one or more

polynucleotide unwinding enzymes is prebound to the polynucleotide.

14 The method of any one of claims 3 to 13, wherein a polynucleotide unwinding enzyme is prebound to one or each of both adaptors.

15. The method of any one of the preceding claims, wherein a polynucleotide unwinding enzyme is provided within the lumen of the nanopore.

16. The method of any one of the preceding claims, wherein the nanopore also functions to unwind the polynucleotide.

17. The method of any one of the preceding claims, wherein the nanopore is a motor protein nanopore, optionally which is phi29.

18. The method of any one of claims 3 to 17, wherein, for each adaptor, a

polynucleotide unwinding enzyme is bound to the first single strand extending from the duplex stem.

19. The method of claim 18, wherein the unwinding of the template strand is facilitated by its corresponding bound polynucleotide unwinding enzyme.

20. The method of any one of the preceding claims, wherein at least one of the one or more tags that bind to a portion of the construct is a nucleic acid having sequence

complementarity to the portion of the construct.

21. The method of any one of claims 3 to 20, wherein at least one of the one or more tags that bind to a portion of the adaptor is a nucleic acid having sequence complementarity to the portion of the adaptor.

22. The method of claim 20 or 21, wherein the nucleic acid is uncharged.

23. The method of claim 22, wherein the nucleic acid is PNA or morpholino.

24. The method of any one of the preceding claims, wherein the polynucleotide comprises RNA and/or DNA.

25. The method of any one of the preceding claims, wherein unwinding of the polynucleotide reveals a portion of the complement strand for hybridization with a tag.

26. The method of any one of claims 9 to 25, wherein the portion of the adaptor to which the oligonucleotide has complementarity is within the duplex stem on a strand contiguous with the first single strand, and wherein the potential difference is maintained for a sufficient time to permit unwinding of the polynucleotide to an extent that the portion of the adaptor that has its first single strand contiguous with the complement strand is available for hybridization with a tag.

27. The method of claim 25 or 26 further comprising maintaining the conditions for a sufficient time to permit the complement strand to enter and translocate the nanopore following translocation of the template strand through the nanopore.

28. The method of claim 26 further comprising maintaining the potential difference for a sufficient time to permit the first single strand contiguous with the complement strand to enter the nanopore and to permit translocation of the complement strand through the nanopore, following translocation of the template strand through the nanopore.

29. The method of claim 28 further comprising measuring a change in a property indicative of translocation of the complement strand through the nanopore.

30. The method of claim 29, wherein the property is ionic current flow through the nanopore as the complement strand translocates through the nanopore.

31. The method of claim 29 or 30 further comprising characterizing the

polynucleotide based further on the change in a measured property indicative of translocation of the complement strand through the nanopore.

32. The method of claim 31, wherein data indicative of the measured properties indicative of translocation of both the complement and template strands through the nanopore is obtained and used to characterize the polynucleotide.

33. The method of claim 32, wherein the template strand data is compared or combined with the complement strand data to characterize the polynucleotide.

34. The method of any of any one of the preceding claims, wherein the nanopore comprises a first tag and a second tag, and the first tag and the second tag bind to a portion of the first single strand of the adaptor that is contiguous with the template strand and to a portion of the first single strand of the adaptor that is contiguous with the complement strand, respectively.

35. The method of any one of claims 3 to 34, wherein each adaptor comprises a second single strand extending from the duplex stem, wherein the second single strand of the one adaptor is contiguous with the complement strand and/or the second single strand of the other adaptor is contiguous with the template strand.

36. The method of claim 35, wherein at least one of the one or more tags that bind to a portion of the adaptor is an oligonucleotide having sequence complementarity to a portion of the adaptor within the second single strand.

37. The method of claim 36, wherein two or more of the one or more tags that bind to a portion of the adaptor are oligonucleotides having sequence complementarity to a portion of the adaptor within the second single strand.

38. The method of any one of claims 1 to 37, further comprising

determining a sequence of the template strand based on measurements of changes in the measured property as the template strand translocates through the pore,

determining a sequence of the complement strand based on measurements of changes in the measured property as the complement strand translocates through the pore, and

comparing the sequence of the template strand with the sequence of the complement strand to establish a sequence of the polynucleotide.

39. A system for characterizing a polynucleotide, the system comprising:

(i) a construct comprising a polynucleotide having a template strand and a complement strand, and

(ii) a nanopore disposed in a membrane, the nanopore comprising an outer rim to which is conjugated at least one nucleic acid having sequence complementarity with a portion of the adaptor.

40. The system of claim 39,wherein the template strand and the complement strand are not covalently linked,

wherein an adaptor is attached at each of two ends of the polynucleotide,

each adaptor comprising a duplex stem and a first single strand extending from the duplex stem,

wherein the first single strand of one adaptor is contiguous with the template strand and the first single strand of the other adaptor is contiguous with the complement strand, and wherein, for each adaptor, a polynucleotide unwinding enzyme is bound to the first single strand extending from the duplex stem,

41. The system of claim 40, wherein the portion of the adaptor is within the duplex stem on a strand contiguous with the first single strand.

42. The system of claim 40, wherein each adaptor comprises a second single strand extending from the duplex stem, wherein the second single strand of the one adaptor is contiguous with the complement strand and the second single strand of the other adaptor is contiguous with the template strand.

43. The system of claim 40, wherein the portion of the adaptor is within the second single strand.

44. The system of claim 42, wherein the at least one nucleic acid:

(a) has sequence complementarity with a portion of the adaptor that is within the duplex stem on a strand contiguous with the first single strand, and

(b) has further sequence complementarity with a portion of the adaptor that is within the second single strand.

45. The system of claim 42, wherein at least two nucleic acids are conjugated to the nanopore, wherein one of the at least two nucleic acids has sequence complementarity with a portion of the adaptor that is within the duplex stem on a strand contiguous with the first single strand, and wherein the other of the at least two nucleic acids has sequence complementarity with a portion of the adaptor that is within the second single strand.

46. The system of claim 45, wherein the at least two nucleic acids are conjugated to the outer rim of the nanopore.

47. A method for preparing a system for characterizing a polynucleotide, the method comprising:

(i) obtaining a construct comprising a polynucleotide having a template strand and a complement strand, wherein the template strand and the complement strand are not covalently linked, and

(ii) combining the construct with a nanopore disposed in a membrane under conditions in which the construct is exposed to an outer rim of the nanopore, wherein at least one nucleic acid having sequence complementarity with a portion of the adaptor is conjugated to the outer rim of the nanopore.

48. The method of claim 47, wherein an adaptor is attached at each of two ends of the polynucleotide,

each adaptor comprising a duplex stem and a first single strand extending from the duplex stem,

wherein the first single strand of one adaptor is contiguous with the template strand and the first single strand of the other adaptor is contiguous with the complement strand,

wherein, for each adaptor, a polynucleotide unwinding enzyme is bound to the first single strand extending from the duplex stem.

49. A complex comprising:

i. a nanopore having a tag,

ii. a complement polynucleotide strand bound to the nanopore via the tag, and iii. a template polynucleotide strand partially hybridized with the complement polynucleotide strand, wherein the template polynucleotide strand is partially disposed within the lumen of the nanopore.

50. A complex comprising:

i. a nanopore having two or more tags, and

ii. a double stranded polynucleotide comprising template and complement strands wherein each strand is bound to one of the two or more tags.

51. A complex according to claim 49 or 50 wherein the tag is at an outer rim external to its lumen.

52. A system for characterizing analytes, the system comprising:

(i) a nanopore disposed in a membrane, the nanopore comprising an outer rim to which is present at least one common tag; and

(ii) a plurality of different analytes, each different analyte being attached to a binding partner of the at least one tag.

53. The system of claim 52, wherein each analyte is a biopolymer.

54. The system of claim 52, wherein the biopolymer is selected from: a

polynucleotide, a polypeptide, a polysaccharide, and a lipid.

55. The system of claim 54, wherein each analyte is a polynucleotide.

56. The system of claim 55, wherein the binding partner is a nucleotide sequence of an adaptor that is attached to the polynucleotide, and wherein the at least one common tag is a nucleic acid having sequence complementarity with the nucleotide sequence of the adaptor.

57. A method for determining a characteristic of an analyte using a nanopore, the method comprising:

i. providing analyte;

ii. causing one or more analytes to bind to a nanopore an outer rim of a nanopore external to the lumen of the nanopore;

iii. obtaining measurements of the analyte that has bound to the nanopore while moving the analyte with respect to the nanopore, wherein the measurements are indicative of one or more characteristics of the analyte; and

iv. characterizing the analyte based on the measurements obtained in step iii.

58. The method of claim 57, wherein the one or more analytes bind an outer rim of a nanopore external to the lumen of the nanopore.

59. The method of claim 58, wherein the more than one analytes bind to respectively more than one tags conjugated to the nanopore.

60. The method of claim 59, wherein the one or more tags are conjugated to an outer rim of the nanopore external to the lumen of the nanopore.

61. The method of claim any one of claims 57 to 60, wherein the first analyte is a polynucleotide.

62. The method according to any one of claims 57 to 61 further comprising:

obtaining measurements of a second analyte that has bound to the nanopore while moving the second analyte with respect to the nanopore, wherein the measurements obtained of the second analyte are indicative of one or more characteristics of the second analyte, and

characterizing the second analyte based on the obtained measurements of the second analyte.

63. The method of claim 57, wherein a second analyte is bound to the nanopore during movement of the first analyte with respect to the nanopore.

64. The method of claim 62 or 63, wherein the second analyte is a polynucleotide.

65. A complex comprising:

a nanopore having a plurality of tags, wherein a first analyte that is partially within the lumen of the nanopore and a second analyte is bound to one of the capture moieties.

66. The complex of claim 65, wherein the plurality of tags are on an outer rim external to its lumen.

67. The complex of claim 65 or 66, wherein the first analyte and the second analyte are polynucleotides.

68. A method for sequentially translocating two non-covalently bound molecules through a nanopore, the method comprising:

contacting a pair of non-covalently bound molecules to a nanopore under conditions that promote translocation of a first member of the pair of non-covalently bound molecules through the nanopore,

wherein a binding site on a second member of the pair is exposed during translocation of the first member through the nanopore,

and wherein the binding site reversibly binds to a tag that is present on the nanopore.

69. The method of claim 68, wherein the non-covalently bound molecules are complementary nucleic acid strands.

70. The method of claim 68, wherein the tag on the nanopore is an oligonucleotide, and the binding site on the second member is a portion of a nucleic acid that has a sequence that is complementary to the tag.

71. The method of claim 68, wherein the pair of non-covalently bound molecules comprise a target nucleic acid attached to an adaptor, and wherein the binding site is present on the adaptor.

72. A method of characterizing a polynucleotide comprising:

contacting a pair of non-covalently bound molecules to a nanopore under conditions that promote translocation of a first member of the pair of non-covalently bound molecules through the nanopore sequentially followed by translocation of the second member of the pair of non-covalently bound molecules,

measuring a property indicative of the translocation of the first and second members of the pair, and obtaining data indicative of the measured property, and

determining the characteristic based upon the obtained data of both the first and second members.

73. A method of sequencing a target polynucleotide, comprising:

(a) contacting a transmembrane pore with:

(i) a double stranded polynucleotide comprising the target polynucleotide and a polynucleotide complementary to the target polynucleotide, wherein the target polynucleotide and the polynucleotide complementary to the target polynucleotide each comprise a single stranded leader sequence; and

(ii) a polynucleotide binding protein capable of separating the strands of a double stranded polynucleotide and controlling the movement of a polynucleotide through a transmembrane pore;

(b) detecting a signal corresponding to ion flow through the pore to detect

polynucleotides translocating through the pore;

(c) identifying a signal corresponding to translocation of the target polynucleotide and a sequential signal corresponding to the separate translocation of the polynucleotide complementary to the target polynucleotide; and

(d) analyzing the signals identified in (c),

thereby sequencing the target polynucleotide.