Mutant pores

Patent metadata

Field	Value
Publication number	US-9751915-B2
Application number	US-201213984628-A
Country	US
Kind code	B2
Filing date	Feb 10, 2012
Priority date	Feb 11, 2011
Publication date	Sep 5, 2017
Grant date	Sep 5, 2017

Abstract

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

First claim

The invention claimed is:

1. A mutant Msp monomer comprising a variant of the sequence shown in SEQ ID NO: 2, wherein the variant comprises a mutation at least one of the following positions of SEQ ID NO: 2: position 75, position 77, position 88 and position 126.

2. A mutant Msp monomer according to claim 1 , wherein the variant comprises at least one of the following substitutions: G75S, G77S, L88N and Q126R.

3. A mutant Msp monomer according to claim 2 , wherein the variant comprises the following substitutions: G75S, G77S and Q126R.

4. A mutant Msp monomer according to claim 3 , wherein the variant further comprises a mutation at position 88.

5. A mutant according to claim 4 , wherein the mutation at position 88 is L88N.

6. A mutant Msp monomer according to claim 1 , wherein the variant further comprises at least one of the following mutations:
(a) serine (S), glutamine (Q) or tyrosine (Y) at position 90;
(b) leucine (L) or serine (S) at position 105;
(c) arginine (R) at position 59;
(d) leucine (L) at position 78;
(e) asparagine (N) at position 81;
(f) asparagine (N) at position 83;
(g) serine (S) or threonine (T) at position 86;
(h) phenylalanine (F), valine (V) or leucine (L) at position 87;
(i) phenylalanine (F), valine (V) or leucine (L) at position 89;
(j) leucine (L), phenylalanine (F), tryptophan (W), histidine (H), threonine (T), glycine (G), alanine (A), valine (V), arginine (R), lysine (K), asparagine (N) or cysteine (C) at position 90;
(k) 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 91;
(l) alanine (A) or serine (S) at position 92;
(m) serine (S), alanine (A), threonine (T), glycine (G) at position 93;
(n) leucine (L) at position 94;
(o) valine (V) at position 95;
(p) arginine (R), aspartic acid (D), valine (V), asparagine (N), serine (S) or threonine (T) at position 96;
(q) serine (S) at position 97;
(r) serine (S) at position 98;
(s) serine (S) at position 99;
(t) serine (S) at position 100;
(u) phenylalanine (F) at position 101;
(v) lysine (K), serine (S) or threonine (T) at position 102;
(w) alanine (A), glutamine (Q), asparagine (N), glycine (G) or threonine (T) at position 103;
(x) isoleucine at position 104;
(y) tyrosine (Y), alanine (A), glutamine (Q), asparagine (N), threonine (T), phenylalanine (F), tryptophan (W), histidine (H), glycine (G), valine (V), arginine (R), lysine (K), proline (P), or cysteine (C) at position 105;
(z) phenylalanine (F), isoleucine (I), valine (V) or serine (S) at position 106;
(aa) proline (P) or serine (S) at position 108;
(bb) asparagine (N) at position 118;
(cc) serine (S) or cysteine (C) at position 103; and
(dd) cysteine at one or more of positions 10 to 15, 51 to 60, 136 to 139 and 168 to 172.

7. A mutant according to claim 1 , wherein the variant further comprises one or more of the following substitutions at the other positions:
(a) serine (S) at position 75, serine (S) at position 77, asparagine (N) at position 88, glutamine (Q) at position 90 and arginine (R) at position 126;
(b) one or more of (i) glutamine (Q) at position 90 and (ii) alanine (A) at position 105;
(c) one or more of (i) serine (S) at position 90 and (ii) serine (S) at position 92;
(d) one or more of (i) glutamine (Q) at position 87 and (ii) serine (S) at position 90;
(e) one or more of (i) tyrosine (Y) at position 89 and (ii) serine (S) at position 90;
(f) one or more of (i) serine (S) at position 90 and (ii) alanine (A) at position 92;
(g) one or more of (i) serine (S) at position 90 and (ii) asparagine (N) at position 94;
(h) one or more of (i) serine (S) at position 90 and (ii) isoleucine (I) at position 104;
(i) one or more of (i) glutamine (Q) at position 90, (ii) serine (S) at position 93 and (iii) alanine (A) at position 105;
(j) one or more of (i) phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H) at position 90, (ii) phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H) at position 91 and (iii) phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H) at position 105;
(k) one or more of (i) serine (S), threonine (T), glycine (G), alanine (A) or valine (V) at position 90, (ii) serine (S), threonine (T), glycine (G), alanine (A) or valine (V) at position 91 and (iii) serine (S), threonine (T), glycine (G), alanine (A) or valine (V) at position 105;
(l) serine (S), arginine (R), lysine (K) or histidine (H) at position 90 and/or serine (S), arginine (R), lysine (K) or histidine (H) at position 91;
(m) serine (S), threonine (T), asparagine (N), glutamine (Q), tyrosine (Y) or histidine (H) at position 90 and/or serine (S), threonine (T), asparagine (N), glutamine (Q), tyrosine (Y) or histidine (H) at position 91; and
(n) cysteine at one or more of positions 90, 91 and 103.

8. A mutant according to claim 1 , wherein the variant further comprises at least one of the following substitution(s) at the other positions:

(i)
D90S;

(ii)
D90Q;

(iii)
D90Y;

(iv)
I105L;

(v)
I105S;

(vi)
G75S, G77S,

L88N, D90Q

and Q126R;

(vii)
E59R;

(viii)
I78L;

(ix)
S81N;

(x)
T83N;

(xi)
N86S;

(xii)
N86T;

(xiii)
I87F;

(xiv)
I87V;

(xv)
I87L;

(xvi)
I89F;

(xvii)
I89V;

(xviii)
I89L;

(xix)
N90S;

(xx)
N90Q;

(xxi)
N90L;

(xxii)
N90Y;

(xxiii)
N91S;

(xxiv)
N91Q;

(xxv)
N91L;

Assignees

• Clarke James
• Heron Andrew John
• Jayasinghe Lakmal
• Wallace Elizabeth Jayne
• White James
• Oxford Nanopore Tech Ltd

Inventors

• Clarke James
• Heron Andrew John
• Jayasinghe Lakmal
• Wallace Elizabeth Jayne
• White James

Classifications

• C12Q1/6869

  Methods for sequencing · CPC title

• C07K14/35 Primary

  from Mycobacteriaceae (F) · CPC title

• A61K39/04

  Mycobacterium, e.g. Mycobacterium tuberculosis · CPC title

• A61K39/00

  Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53) · CPC title

• C12Q2521/101

  DNA polymerase · CPC title