Mutant pores

• US 10,167,503 B2
• Filed: 05/01/2015
• Issued: 01/01/2019
• Est. Priority Date: 05/02/2014
• Status: Active Grant

• Current Assignee: Oxford Nanopore Technologies Limited
• Original Assignee: Oxford Nanopore Technologies Limited
• Inventors: Clarke, James Anthony, Heron, Andrew John, Jayasinghe, Lakmal, Wallace, Elizabeth Jayne
• Primary Examiner(s): Saidha, Tekchand
• Application Number:
  US15/308,206
• Publication Number:
  US 20170058337A1
• Time in Patent Office: 1,341 Days
• Field of Search: 435183
• CPC Class Codes: C07K 14/35 from Mycobacteriaceae (F)
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2563/157 Nanotubes or nanorods
• CPC Class Codes: C12Q 2565/631 being a biochannel or pore

Abstract

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

Claims

1. A mutant Mycobacterium smegmatis porin (Msp) monomer comprising a variant of the sequence that is at least 90% identical to the sequence of SEQ ID NO:

2, which comprises a cap forming region and a barrel forming region, wherein the variant;

(a) does not comprise aspartic add (D) at position 90;

(b) does not comprise aspartic acid (D) at position 91;

(c) comprises aspartic add (D) or glutamic add (E) at position 93; and

(d) comprises one or more amino acid modifications in the cap forming region and/or the barrel forming region of SEQ ID NO;

2 such that, when the mutant Msp monomer forms a pore, the net negative charge of inward facing amino acids is decreased, and wherein the cap forming region comprises amino acids 1 to 72 and 122 to 184 of SEQ ID NO;

2.

2. The mutant monomer according to claim 1, wherein the barrel forming region comprises amino acids 73 to 82 and 112 to 121 of SEQ ID NO:

2.

3. The mutant monomer according to claim 1, 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, 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.

4. The 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.

5. The mutant monomer according to claim 1, wherein the one or more modifications are one or more deletions of negatively charged amino acids or one or more substitutions of negatively charged amino acids with one or more positively charged, uncharged, non-polar and/or aromatic amino acids.

6. The mutant monomer according to claim 5, wherein the one or more negatively charged amino acids are substituted with alanine (A), valine (V), asparagine (N) or glycine (G).

7. The mutant monomer according to claim 1, wherein the one or more modifications are one or more introductions of positively charged amino acids.

8. The mutant monomer according to claim 5, wherein the one or more positively charged amino acids are histidine (H), lysine (K) and/or arginine (R).

9. The mutant monomer according to claim 1, wherein the one or more modifications are one or more chemical modifications of one or more negatively charged amino acids which neutralise their negative charge.

10. The 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.

11. The mutant monomer according to claim 1, wherein

(i) the variant comprises a positively charged amino acid at one or more of positions 114, 116, 120, 123, 70, 73, 75, 77 and 79,

(ii) the variant comprises a positively charged amino acid at one or more of positions 123, 125, 127 and 128;

(iii) the variant comprises a positively charged amino acid at one or more of positions 129, 132, 136, 137, 59, 61 and 63;

(iv) the variant comprises a positively charged amino acid at one or more of positions 137, 138, 141, 143, 45, 47, 49 and 51;

(v) the variant does not comprise aspartic acid (D) or glutamic acid (E) at one or more of positions 118, 126, 134 and 139;

(vi) the variant comprises arginine (R), glycine (G) or asparagine (N) at one or more of positions 118, 126, 134 and 139;

(vii) the variant comprises D118R, Q126R, D134R and E139K;

(viii) 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;

(ix) the variant comprises asparagine (N) at position 90 and/or position 91;

(x) 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;

(xi) the variant comprises G75S, G77S and L88K or G75S, G77S and L88N;

(xii) the variant comprises G75S, G77S, L88N, D90N, D91N, D118R, Q126R, D134R and E139K; and

/or

(xiii) the variant further comprises one or more of;

(h) phenylalanine (F) at position 89;

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

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

(k) glycine (G) at position 102;

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

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