Hetero-pores

• US 10,472,673 B2
• Filed: 02/17/2016
• Issued: 11/12/2019
• Est. Priority Date: 02/19/2015
• Status: Active Grant

• Current Assignee: Oxford Nanopore Technologies Limited
• Original Assignee: Oxford Nanopore Technologies Limited
• Inventors: Brown, Clive Gavin, Clarke, James Anthony, Hambley, Richard George, Heron, Andrew John, Jayasinghe, Lakmal, Milton, John, Pugh, Jonathan Bankes, Wallace, Elizabeth Jayne, White, James
• Primary Examiner(s): Carlson, Karen Cochrane
• Application Number:
  US15/551,953
• Publication Number:
  US 20180030526A1
• Time in Patent Office: 1,364 Days
• CPC Class Codes: C07K 14/35 from Mycobacteriaceae (F)
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

The invention relates to hetero-oligomeric pores derived from Msp. The invention also relates to polynucleotide characterisation using the hetero-oligomeric pores.

Claims

1. A hetero-oligomeric pore derived from Msp which comprises a narrowing having a net negative charge, wherein the hetero-oligomeric pore comprises at least one monomer comprising a narrowing forming region comprising an amino acid sequence having at least one amino acid substitution from a non-negatively charged amino acid to a negatively charged amino acid compared with an amino acid sequence of a reference narrowing forming region, wherein the reference narrowing forming region corresponds to amino acids 83 to 111 of SEQ ID NO:

2 and has the following substitutions;

D90N, D91N, and D93N, and wherein none of the remaining monomers of the hetero-oligomeric pore has a sequence as set forth in SEQ ID NO;

2.

2. A hetero-oligomeric pore according to claim 1, wherein the narrowing of the hetero-oligomeric pore comprises one, two, three or four negatively charged amino acids.

3. A hetero-oligomeric pore according to claim 1, wherein the narrowing of the hetero-oligomeric pore comprises one, two, three or four negatively charged amino acids and the two, three or four negatively charged amino acids are in different monomers.

4. A hetero-oligomeric pore according to claim 1, wherein the narrowing of the hetero-oligomeric pore comprises one, two, three or four negatively charged amino acids and the remaining amino acids in the narrowing are not charged.

5. A hetero-oligomeric pore according to claim 1, wherein at least one remaining monomer differs from the rest of the remaining monomers by comprising a negatively charged amino acid at one or more of the positions which correspond to positions 88, 90, 91, 92, 93, 102, 103 and 105 of SEQ ID NO:

2 and wherein the rest of the monomers comprise an amino acid which is not charged at the position(s) which correspond(s) to position(s) 90 and/or 91 of SEQ ID NO;

2.

6. A hetero-oligomeric pore according to claim 1, wherein the pore comprises eight monomers and wherein the at least one monomer comprises a negatively charged amino acid at one or more of the positions which correspond to positions 88, 92, 102, 103 and 105 of SEQ ID NO:

2, and wherein the remaining monomers do not contain a negatively charged amino acid at one or more positions 90, 91, or 93 of SEQ ID NO;

2.

7. A hetero-oligomeric pore according to claim 1, wherein the hetero-oligomeric pore comprises eight monomers and wherein at least one remaining monomer differs from the rest of the remaining monomers by comprising a negatively charged amino acid at the position(s) which correspond(s) to position(s) (i) 90;

(ii) 91;

(iii) 93;

(iii) 90 and 91;

(iv) 90 and 93;

(v) 91 and 93;

(vi) 105;

(vii) 90 and 105;

or (viii) 90, 91 and 105 of SEQ ID NO;

2.

8. A hetero-oligomeric pore according to claim 1, wherein the hetero-oligomeric pore comprises eight monomers and wherein only one remaining monomer differs from the rest of the remaining monomers by comprising a negatively charged amino acid at the position(s) which correspond(s) to position(s) 90 and/or 91 of SEQ ID NO:

2, optionally wherein the negatively charged amino acid is aspartic acid (D) or glutamic acid (E).

9. A hetero-oligomeric pore according to claim 1, wherein the hetero-oligomeric pore comprises eight monomers and wherein one or more of the remaining monomers comprise a mutation or substitution at one or more of the positions which correspond to positions G75, G77, L88, D118, Q126, D134 and E139 of SEQ ID NO:

2, optionally wherein the one or more monomers comprise one or more of G75S, G77S, L88N, D118R, Q126R, D134R and E139K.

10. A hetero-oligomeric pore according to claim 1, wherein the hetero-oligomeric pore comprises eight monomers and wherein one or more of the remaining monomers comprise proline (P) at the position which corresponds to position 108 in SEQ ID NO:

2.

11. A hetero-oligomeric pore according to claim 1, wherein the hetero-oligomeric pore comprises eight monomers and wherein one or more of the remaining monomers comprise (a) 2, 4, 6, 8 or 10 of the residues which correspond to positions 72 to 82 of SEQ ID NO:

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

2 have been deleted.

12. A hetero-oligomeric pore according to claim 1, wherein the hetero-oligomeric pore comprises eight monomers and wherein:

(a) the remaining monomers comprise an asparagine (N) at the position which corresponds to position 90 of SEQ ID NO;

2;

(b) the remaining monomers comprise an asparagine (N) at the position which corresponds to position 91 of SEQ ID NO;

2;

(c) the remaining monomers comprise an asparagine (N) at the positions which correspond to positions 90 and 91 of SEQ ID NO;

2;

(d) the remaining monomers comprise a glutamine (Q) at the position which corresponds to position 90 of SEQ ID NO;

2;

(e) the remaining monomers comprise a glutamine (Q) at the position which corresponds to position 91 of SEQ ID NO;

2;

(f) the remaining monomers comprise a glutamine (Q) at the positions which correspond to positions 90 and 91 of SEQ ID NO;

2;

(g) the at least one monomer comprises an aspartic acid (D) at the position which corresponds to position 88 of SEQ ID NO;

2 and the remaining monomers comprise an asparagine (N) at the position which corresponds to position 88 of SEQ ID NO;

2;

(h) the at least one monomer comprises an aspartic acid (D) at the position which corresponds to position 88 of SEQ ID NO;

2 and the remaining monomers comprise a glutamine (Q) at the position which corresponds to position 88 of SEQ ID NO;

2;

(i) the at least one monomer comprises an aspartic acid (D) at the position which corresponds to position 103 of SEQ ID NO;

2 and the remaining monomers comprise a serine (S) at the position which corresponds to position 103 of SEQ ID NO;

2;

(j) the at least one monomer comprises an aspartic acid (D) at the position which corresponds to position 105 of SEQ ID NO;

2 and the remaining monomers comprise an isoleucine (I) at the position which corresponds to position 105 of SEQ ID NO;

2;

(k) the at least one monomer comprises an aspartic acid (D) at the position which corresponds to position 88 of SEQ ID NO;

2 and the remaining monomers comprise an asparagine (N) at the positions which correspond to positions 88 and 90 of SEQ ID NO;

2;

(l) the at least one monomer comprises an aspartic acid (D) at the position which corresponds to position 88 of SEQ ID NO;

2 and the remaining monomers comprise a glutamine (Q) at the positions which correspond to positions 88 and 90 of SEQ ID NO;

2;

(m) the at least one monomer comprises an aspartic acid (D) at the position which corresponds to position 103 of SEQ ID NO;

2 and the remaining monomers comprise an asparagine (N) and serine (S) at the positions which correspond to positions 90 and 103 of SEQ ID NO;

2 respectively;

(n) the at least one monomer comprises an aspartic acid (D) at the position which corresponds to position 103 of SEQ ID NO;

2 and the remaining monomers comprise a glutamine (Q) and serine (S) at the positions which correspond to positions 90 and 103 of SEQ ID NO;

2 respectively;

(o) the at least one monomer comprises an aspartic acid (D) at the position which corresponds to position 105 of SEQ ID NO;

2 and the remaining monomers comprise an asparagine (N) and isoleucine (I) at the positions which correspond to positions 90 and 105 of SEQ ID NO;

2 respectively;

(p) the at least one monomer comprises an aspartic acid (D) at the position which corresponds to position 105 of SEQ ID NO;

2 and the remaining monomers comprise a glutamine (Q) and isoleucine (I) at the positions which correspond to positions 90 and 105 of SEQ ID NO;

2 respectively;

(q) the remaining monomers comprise an asparagine (N) at the positions which correspond to positions 90 and 93 of SEQ ID NO;

2;

(r) the remaining monomers comprise a glutamine (Q) at the positions which correspond to positions 90 and 93 of SEQ ID NO;

2;

(s) the remaining monomers comprise an asparagine (N) at the positions which correspond to positions 91 and 93 of SEQ ID NO;

2;

(t) the remaining monomers comprise a glutamine (Q) at the positions which correspond to positions 91 and 93 of SEQ ID NO;

2;

(u) the remaining monomers comprise an asparagine (N) at the position which corresponds to position 93 of SEQ ID NO;

2;

(v) the remaining monomers comprise a glutamine (Q) at the position which corresponds to position 93 of SEQ ID NO;

2;

(w) the remaining monomers comprise an aspartic acid (D) at the position which corresponds to position 93 of SEQ ID NO;

2;

(x) the at least one monomer comprises an aspartic acid (D) at the positions which correspond to positions 88 and 105 of SEQ ID NO;

2 and the remaining monomers comprise an asparagine (N) and isoleucine (I) at the positions which correspond to positions 88 and 105 of SEQ ID NO;

2, respectively;

or

(y) the at least one monomer comprises an aspartic acid (D) at the positions which correspond to positions 88 and 105 of SEQ ID NO;

2 and the remaining monomers comprise a glutamine (Q) and isoleurine (I) at the positions which correspond to positions 88 and 105 of SEQ ID NO;

2, respectively.

13. A kit for characterising a target polynucleotide comprising (a) the hetero-oligomeric pore of claim 1, and (b) the components of a membrane.

14. A sensor for characterising a target polynucleotide, comprising a complex between the hetero-oligomeric pore of claim 1, and a polynucleotide binding protein.

15. An apparatus for characterising target polynucleotides in a sample, comprising (a) a plurality of the hetero-oligomeric pores of claim 1 and (b) a plurality of membranes, wherein the apparatus further comprises:

a sensor device that is capable of supporting the plurality of hetero-oligomeric 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.

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

a) contacting the polynucleotide with the hetero-oligomeric pore of claim 1, 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.

17. A method according to claim 16, 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.

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

a) contacting the polynucleotide with the hetero-oligomeric pore of claim 1, 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.