MUTANT PORES

• US 20170306398A1
• Filed: 10/06/2015
• Published: 10/26/2017
• Est. Priority Date: 10/07/2014
• Status: Active Grant

• Current Assignee: Oxford Nanopore Technologies Limited
• Original Assignee: Oxford Nanopore Technologies Limited
• Inventors: Jayasinghe, Lakmal, Wallace, Elizabeth Jayne, Pugh, Jonathan Bankes, Hambley, Richard George, Wood, Neil Roger, Heron, Andrew John, White, James
• Granted Patent: US 10,266,885 B2
• Time in Patent Office: Days
• CPC Class Codes: B01L 2200/0663 Stretching or orienting elongated molecules or particles
• CPC Class Codes: B01L 2300/0636 Integrated biosensor, microarrays
• CPC Class Codes: B01L 2300/0896 Nanoscaled
• CPC Class Codes: B01L 3/502761 specially adapted for handling suspended solids or molecules independently from the bulk fluid flow, e.g. for trapping or sorting beads, for physically stretching molecules investigating characteristics of particles G01N15/00
• CPC Class Codes: C07K 14/35 from Mycobacteriaceae (F)
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• 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

The invention relates to mutant forms of Msp. The invention also relates to polynucleotide characterisation using Msp.

Claims

1. A mutant Msp monomer comprising a variant of the sequence shown in SEQ ID NO:

2, wherein in the variant (a) 2, 4, 6, 8 or 10 of the amino acids at positions 72 to 82 of SEQ ID NO;

2 have been deleted and (b) 2, 4, 6, 8 or 10 of the amino acids at positions 111 to 121 of SEQ ID NO;

2 have been deleted.

2. A mutant monomer according to claim 1, wherein the same number of amino acids have been deleted in (a) and (b).

3. A mutant according to claim 1, wherein the positions of the amino acids that have been deleted in (a) correspond to the positions of the amino acids that have been deleted in (b) as shown in Table 1.

4. A mutant according to claim 1, wherein one or more positions of the amino acids that have been deleted in (a) do not correspond to the one or more positions of the amino acids that have been deleted in (b) as shown in Table 1.

5. A mutant according to claim 4, wherein the positions of the amino acids that have been deleted in (a) do not correspond to the positions of the amino acids that have been deleted in (b) as shown in Table 1.

6. A mutant monomer according to claim 1, wherein the amino acids that have been deleted in (a) and/or (b) are consecutive.

7. A mutant monomer according to claim 1,

wherein (i) L74, G75, D118 and L119 have been deleted, (ii) G75, V76, A117 and D118 have been deleted, (iii) V76, G77, S116 and A117 have been deleted, (iv) G77, I78, I115 and S116 have been deleted, (v) I78, N79, S114 and I115 have been deleted, (vi) N79, F80, V113 and S114 have been deleted or (vii) F80, S81, G112 and V113 have been deleted;

or

wherein L74, G75, V76, G77, S116, A117, D118 and L119 have been deleted.

8. A mutant monomer according to claim 1, wherein the variant does not comprise aspartic acid (D) at position 90 of SEQ ID NO:

2 and/or does not comprise aspartic acid (D) at position 91 of SEQ ID NO;

2.

9. A mutant monomer according to claim 1, wherein the variant comprises serine (S), glutamine (Q), leucine (L), methionine (M), isoleucine (I), alanine (A), valine (V), glycine (G), phenylalanine (F), tryptophan (W), tyrosine (Y), histidine (H), threonine (T), arginine (R), lysine (K), asparagine (N) or cysteine (C) at position 90 and/or position 91.

10. A mutant monomer according to claim 1, wherein the variant comprises aspartic acid (D) or glutamic acid (E) at position 93 of SEQ ID NO:

2.

11. A mutant monomer according to claim 1, wherein the variant does not comprise aspartic acid (D) or glutamic acid (E) at position 93 of SEQ ID NO:

2.

12. A mutant monomer according to claim 1, wherein the variant comprises one or more modifications which decrease the net negative charge of the inward facing amino acids in the cap forming region and/or the barrel forming region of the monomer.

13. A mutant monomer according to claim 12, wherein the cap forming region comprises amino acids 1 to 72 and 122 to 184 of SEQ ID NO:

2.

14. A mutant monomer according to claim 12, wherein the barrel forming region comprises amino acids 72 to 82 and 112 to 121 of SEQ ID NO:

2.

15. A mutant monomer according to claim 1, wherein the inward facing amino acids in the cap forming region are V9, Q12, D13, R14, T15, W40, 149, P53, G54, D56, E57, E59, T61, E63, Y66, Q67, I68, F70, P123, I125, Q126, E127, V128, A129, T130, F131, S132, V133, D134, S136, G137, E139, V144, H148, T150, V151, T152, F163, R165, I167, S169, T170 and S173.

16. A mutant monomer according to claim 1, wherein the inward facing amino acids in the barrel forming region are S73, G75, G77, N79, S81, G112, S114, S116, D118 and G120.

17. A mutant monomer according to claim 1, wherein the one or more modifications reduce the net negative charge at one or more of positions 118, 126, 134 and 139.

18. A mutant monomer according to claim 1, wherein the variant does not comprise aspartic acid (D) or glutamic acid (E) at one or more of positions 118, 126, 134 and 139.

19. A mutant monomer according to claim 1, wherein the variant comprises arginine (R), glycine (G) or asparagine (N) at one or more of positions 118, 126, 134 and 139.

20. A mutant monomer according to claim 1, wherein the variant comprises D118R, Q126R, D134R and E139K.

21. A mutant monomer according to claim 1, wherein the variant comprises one or more of:

(e) serine (S) at position 75;

(f) serine (S) at position 77; and

(g) asparagine (N) or lysine (K) at position 88.

22. A mutant monomer according to claim 21, wherein the variant comprises all of (e) to (g) or G75S, G77S and L88N.

23. A mutant monomer according to claim 1, wherein the variant comprises (a) D90N, D91N, D93N, D118R, D134R and E139K, (b) L88N, D90N, D91N, D93N, D118R, D134R and E139K, (c) G75S, G77S, L88N, D90N, D91N, D93N, D118R, Q126R, D134R and E139K or (d) G75S, G77S, L88N, D90N, D91N, D118R, Q126R, D134R and E139K.

24. A mutant monomer according to claim 1, wherein the variant does not comprise aspartic acid (D) or glutamic acid (E) at one or more of positions 56, 57 and 59 or comprises one or more of (a) D56N or D56R, (b) E57N or E57R and (c) E59N or E59R.

25. A mutant monomer according to claim 1, wherein the variant further comprises one or more of:

(i) phenylalanine (F) at position 89;

(j) glutamic acid (E) at position 95 and lysine (K) at position 98;

(l) aspartic acid (D) at position 96;

(m) glycine (G) at position 102;

(n) alanine (A) at position 103; and

(o) alanine (A), serine (S) or proline (P) at position 108.

26. A mutant monomer according to claim 1, wherein the variant comprises D56N, E59R, L88N, D90N, D91N, Q126R, D134R and E139K and L74, G75, D118 and L119 have been deleted from the variant.

27. A construct comprising two or more covalently attached MspA monomers, wherein at least one of the monomers is a mutant monomer according to claim 1.

28. A construct according to claim 27, wherein the two or more mutant monomers are the same or different.

29. A construct according to claim 27, wherein the two or more mutant monomers are genetically fused.

30. A construct according to claim 27, wherein the two or more mutant monomers are attached via one or more linkers.

31. A construct according to claim 27, wherein the construct comprises two mutant monomers claim 1,

32. A polynucleotide which encodes a mutant monomer according to claim 1.

33. A homo-oligomeric pore derived from Msp comprising identical mutant monomers according to claim 1.

34. A homo-oligomeric pore according to claim 33, wherein the pore comprises eight identical mutant monomers as defined in claim 1.

35. A hetero-oligomeric pore derived from Msp comprising at least one mutant monomer according to claim 1.

36. A hetero-oligomeric pore according to claim 35, wherein the pore comprises (a) eight mutant monomers as defined in claim 1 and wherein at least one of them differs from the others.

37. A method of characterising a target polynucleotide, comprising:

a) contacting the polynucleotide with a pore according to claim 33 such that the polynucleotide moves through the pore; and

b) taking one or more measurements as the polynucleotide moves with respect to the pore, wherein the measurements are indicative of one or more characteristics of the polynucleotide, and thereby characterising the target polynucleotide.

38. A method according to claim 37, wherein the one or more characteristics are selected from (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide and (v) whether or not the polynucleotide is modified.

39. A method according to claim 37, wherein the one or more characteristics of the polynucleotide are measured by electrical measurement and/or optical measurement.

40. A method according to claim 39, wherein the electrical measurement is a current measurement, an impedance measurement, a tunnelling measurement or a field effect transistor (FET) measurement.

41. A method according to claim 37, wherein step a) further comprises contacting the polynucleotide with a polynucleotide binding protein such that the protein controls the movement of the polynucleotide through the pore.

42. A method according to claim 41, wherein the method comprises:

a) contacting the polynucleotide with the pore and the polynucleotide binding protein such that the polynucleotide moves through the pore and the protein controls the movement of the polynucleotide through the pore; and

b) measuring the current passing through the pore as the polynucleotide moves with respect to the pore wherein the current is indicative of one or more characteristics of the polynucleotide and thereby characterising the target polynucleotide.

43. A method according to claim 41, wherein the polynucleotide binding protein is a helicase or is derived from a helicase.

44. A method according to claim 37, wherein the pore is in a membrane.

45. A method according to claim 44, wherein membrane is an amphiphilic layer or a solid state layer.

46. A method according to claim 44, wherein the polynucleotide is coupled to the membrane before it is contacted with the pore.

47. A kit for characterising a target polynucleotide comprising (a) a pore according to claim 33 and (b) the components of a membrane.

48. An apparatus for characterising target polynucleotides in a sample, comprising (a) a plurality of pores according to claim 33 and (b) a plurality of membranes.

49. An apparatus according to claim 48, wherein the apparatus comprises:

a sensor device that is capable of supporting the plurality of pores and membranes being operable to perform polynucleotide characterisation using the pores and membranes; and

at least one port for delivery of the material for performing the characterisation.

50. An apparatus according to claim 49, wherein the apparatus comprises:

a sensor device that is capable of supporting the plurality of pores and membranes being operable to perform polynucleotide characterisation using the pores and membranes; and

at least one reservoir for holding material for performing the characterisation.

51. An apparatus according to claim 49, wherein the apparatus further comprises:

a fluidics system configured to controllably supply material from the at least one reservoir to the sensor device; and

a plurality of containers for receiving respective samples, the fluidics system being configured to supply the samples selectively from the containers to the sensor device.

52. A method of characterising a target polynucleotide, comprising:

a) contacting the polynucleotide with a pore according to claim 33, a polymerase and labelled nucleotides such that phosphate labelled species are sequentially added to the target polynucleotide by the polymerase, wherein the phosphate species contain a label specific for each nucleotide; and

b) detecting the phosphate labelled species using the pore and thereby characterising the polynucleotide.

53. A method of producing a mutant monomer according to claim 1, comprising expressing a polynucleotide which encodes the mutant monomer in a suitable host cell and thereby producing the mutant monomer.