Mutant pores

• US 10,266,885 B2
• Filed: 10/06/2015
• Issued: 04/23/2019
• 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
• Primary Examiner(s): Carlson, Karen Cochrane
• Application Number:
  US15/517,671
• Publication Number:
  US 20170306398A1
• Time in Patent Office: 1,295 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 characterization using Msp.

Claims

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

3. The 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), wherein the corresponding deletions are selected from the group consisting of:

W72 and N121;

S73 and G120;

L74 and L119;

G75 and D118;

V76 and A117;

G77 and S116;

I78 and I115;

N79 and S114;

F80 and V113;

S81 and G112; and

Y82 and P111.

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

5. The 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,

(vii) F80, S81, G112 and V113 have been deleted;

or

(viii) L74, G75, V76, G77, S116, A117, D118 and L119 have been deleted.

6. The mutant monomer according to claim 1, wherein the variant further comprises an amino acid substitution at position D90 and/or position D91 of SEC) ID NO:

2.

7. The mutant monomer according to claim 6, wherein the variant comprises serine (S), glutamine (G), 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 D90 and/or position D91.

8. The mutant monomer according to claim 1, wherein the variant further comprises amino acid substitution D93E of SEQ ID NO:

2.

9. The mutant monomer according to claim 1, wherein the variant further comprises one or more amino acid substitutions which decrease the net negative charge of the inward facing amino acids in the cap forming region comprising amino acids 1 to 72 and 122 to 184 of SEQ ID NO:

2 and/or the barrel forming region comprising amino acids 72-82 and 112 to 121 of SEQ ID NO;

2 of the monomer.

10. The mutant monomer according to claim 9, wherein the inward facing amino acids in the cap forming region are V9, Q12, D13, R14, T15, W40, I49, P53, G54, D56, E57, E59, T61, E63, Y66, Q67, 168, F70, P123, I125, Q126, E127, V128, A129, T130, F131, S132, V133, D134, S136, G137, E139, V144, H148, T150, V151, T152, F163, R165, 1167, S169, T170 and S173.

11. The mutant monomer according claim 9, wherein the inward facing amino acids in the barrel forming region are S73, G75, G77, N79, S81, G112, S114, S116, D118 and G120.

12. The mutant monomer according claim 9, wherein the one or more amino add substitutions reduce the net negative charge at one or more of positions D118, Q126, D134, and E139.

13. The mutant monomer according to claim 12, wherein the variant comprises arginine (R), glycine (C) or asparagine (N) at one or more of positions D118, Q126, D134, and E139.

14. The mutant monomer according to claim 13, wherein the variant comprises D118R, Q126R, D134R and E139K.

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

(i) serine (S) at position G75;

(ii) serine (S) at position G77; and

(iii) asparagine (N) or lysine (K) at position L88.

16. The mutant monomer according to claim 15, wherein the variant comprises G75S, G77S and L88N.

17. The mutant monomer according to claims claim 1, wherein the variant further comprises

(i) a D90N, D91N, D93N, D118R, D134R and E139K,

(ii) L88N, D90N, D91N, D93N, D118R, D134R and E139K,

(iii) G75S, G77S, L88N, D90N, D91N, D93N, D118R, Q126R, D134R and E139K or

(iv) G75S, G77S, L88N, D90N, D91N, D118R, Q126R, D134R and E139K.

18. The mutant monomer according to claims claim 1, wherein the variant further comprises one or more of amino acid substitutions at positions D56, E57, and E59 or comprises one or more of:

(i) D56N or D56R,

(ii) E57N or E57R and,

(iii) E59N or E59R.

19. The mutant monomer according claim 1, wherein the variant further comprises one or more of:

(i) phenylalanine (F) at position I89;

(ii) glutamic acid (E) at position T95 and lysine (K) at position P98;

(iii) aspartic acid (D) at position A96;

(iv) glycine (G) at position N102;

(v) alanine (A) at position S103; and

(vi) alanine (A), serine (S) or proline (P) at position N108.

20. The mutant monomer according to claims claim 1, wherein the variant comprises amino acid deletions at L74, G75, D118 and L119 and further comprises amino acid substitutions D56N, E59R, L88N, D90N, D91N, Q126R, D134R and E139K.

21. 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.

22. The construct according to claim 21, wherein the construct comprises two or more mutant monomers that are the same or different.

23. The construct according to claim 22, wherein the two or more mutant monomers are genetically fused.

24. The construct according to claim 22, wherein the two or more mutant monomers are attached via one or more linkers.

25. The construct according to claim 21 wherein the construct comprises two mutant monomers.

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

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

28. The homo-oligomeric pore according to claim 27, wherein the pore comprises eight identical mutant monomers.

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

30. The hetero-oligomeric pore according to claim 29, wherein the pore comprises eight mutant monomers and wherein at least one of them differs from the others.

31. A method of characterizing a target polynucleotide, comprising:

a) contacting the polynucleotide with a pore according to claim 27 or 29 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 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;

wherein the measurements are indicative of one or more characteristics of the polynucleotide,

and thereby characterizing the target polynucleotide.

32. The method according to claim 31, wherein the one or more characteristics of the polynucleotide are measured by electrical measurement and/or optical measurement.

33. The method according to claim 32, wherein the electrical measurement is a current measurement;

an impedance measurement, a tunnelling measurement, or a field effect transistor (FET) measurement.

34. The method according to claim 31, 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.

35. The method according to claim 34, 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 characterizing the target polynucleotide.

36. The method according to claim 34, wherein the polynucleotide binding protein is a helicase or is derived from a helicase.

37. The method according to claim 31, wherein the pore is in a membrane.

38. The method according to claim 37, wherein membrane is an amphiphilic layer or a solid state layer.

39. The method according to claim 37, wherein the polynucleotide is coupled to the membrane before it is contacted with the pore.

40. A kit for characterizing a target polynucleotide comprising (a) a pore according to claim 27 or 29 and (b) the components of a membrane.

41. An apparatus for characterizing target polynucleotides in a sample, comprising (a) a plurality of pores according to claim 27 or 29 and (b) a plurality of membranes.

42. The apparatus according to claim 41, wherein the apparatus comprises:

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

at least one port for delivery of the material for performing the polynucleotide characterization.

43. The apparatus according to claim 42, further comprising at least one reservoir for holding material for performing the polynucleotide characterization.

44. The apparatus according to claim 42, 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.

45. A method for the characterization of a target polynucleotide, wherein said characterization is 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;

said method comprising;

a) contacting the polynucleotide with a pore according to claim 27 or 29, 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 characterizing the polynucleotide.

46. 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.