# US 9,751,915 B2 · Mutant pores

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 9,751,915 B2
- Document ID: US9751915B2
- Status: Granted
- Kind: B2
- Application number: 13/984,628
- Filed: 2012-02-10
- Published: 2014-07-03
- Granted: 2017-09-05
- Earliest claimed: 2011-02-11
- Assignees: Oxford Nanopore Technologies PLC
- Applicants: James Clarke; Andrew John Heron; Lakmal Jayasinghe; Elizabeth Jayne Wallace; James White
- Inventors: James Clarke; Andrew John Heron; Lakmal Jayasinghe; Elizabeth Jayne Wallace; James White
- Web page: https://patentgrep.com/patent/US9751915B2

## CPC

- C07K 14/35: from Mycobacteriaceae (F)
- A61K 39/04: Mycobacterium, e.g. Mycobacterium tuberculosis
- C12Q 1/6869: Methods for sequencing
- A61K 39/00: Medicinal preparations containing antigens or antibodies (materials for immunoassay G01N33/53)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES

## Abstract

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

## Claims

### Claim 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.

### Claim 2 (depends on 1)

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

### Claim 3 (depends on 2)

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

### Claim 4 (depends on 3)

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

### Claim 5 (depends on 4)

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

### Claim 6 (depends on 1)

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.

### Claim 7 (depends on 1)

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.

### Claim 8 (depends on 1)

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;
(xxvi)
N91M;
(xxvii)
N91I;
(xxviii)
N91A;
(xxix)
N91V;
(xxx)
N91G;
(xxxi)
G92A;
(xxxii)
G92S;
(xxxiii)
N93S;
(xxxiv)
N93A;
(xxxv)
N93T;
(xxxvi)
I94L;
(xxxvii)
T95V;
(xxxviii)
A96R;
(xxxix)
A96D;
(xl)
A96V;
(xli)
A96N;
(xlii)
A96S;
(xliii)
A96T;
(xliv)
P97S;
(xlv)
P98S;
(xlvi)
F99S;
(xlvii)
G100S;
(xlviii)
L101F;
(xlix)
N102K;
(l)
N102S;
(li)
N102T;
(lii)
S103A;
(liii)
S103Q;
(liv)
S103N;
(lv)
S103G;
(lvi)
S103T;
(lvii)
V104I;
(lviii)
I105Y;
(lix)
I105L;
(lx)
I105A;
(lxi)
I105Q;
(lxii)
I105N;
(lxiii)
I105S;
(lxiv)
I105T;
(lxv)
T106F;
(lxvi);
T106I;
(lxvii)
T106V;
(lxviii)
T106S;
(lxix)
N108P;
(lxx)
N108S;
(lxxi)
D90Q and
I105A;
(lxxii)
D90S and
G92S;
(lxxiii)
I87Q and
D90S;
(lxxiv)
I89Y and
D90S;
(lxxv)
D90S and
G92A;
(lxxvi)
D90S and
I94N;
(lxxvii)
D90S and
V104I;
(lxxviii)
D90Q,
D93S and
I105A;
(lxxix)
N91Y;
(lxxx)
N90Y and
N91G;
(lxxxi)
N90G and
N91Y;
(lxxxii)
N90G and
N91G;
(lxxxiii)
I05G;
(lxxxiv)
N90R;
(lxxxv)
N91R;
(lxxxvi)
N90R
and N91R;
(lxxxvii)
N90K;
(lxxxviii)
N91K;
(lxxxix)
N90K
and N91K;
(xc)
N90Q and
N91G;
(xci)
N90G and
N91Q;
(xcii)
N90Q and
N91Q;
(xciii)
R118N;
(xciv)
N91C;
(xcv)
N90C;
(xcvi)
N90W;
(xcvii)
N91W;
(xcviii)
N90K;
(xcix)
N91K;
(c)
N90R;
(ci)
N91R;
(cii)
N90S and
N91S;
(ciii)
N90Y and
I105A;
(civ)
N90G and
I105A;
(cv)
N90Q and
I105A;
(cvi)
N90S and
I105A;
(cvii)
N90G and
N93G;
(cviii)
N90G;
(cix)
N93G;
(cx)
N90G and
N91A;
(cxi)
I105K;
(cxii)
I105R;
(cxiii)
I105V;
(cxiv)
I105P;
(cxv)
I105W;
(cxvi)
N90R and
I105A;
(cxvii)
N90S and
I105A;
(cxviii)
S103C; and
(cxix)
I105C.

### Claim 9 (depends on 1)

A mutant according to claim 1, wherein the mutant is chemically modified.

### Claim 10 (depends on 9)

A mutant according to claim 9, wherein (i) the mutant is chemically modified by attachment of a molecule to one or more cysteines, attachment of a molecule to one or more lysines, attachment of a molecule to one or more non-natural amino acids, enzyme modification of an epitope or modification of a terminus; (ii) the mutant is chemically modified by attachment of a molecule to one or more cysteines and the one or more cysteines have been introduced to the mutant by substitution; (iii) the mutant is chemically modified by attachment of a molecule to one or more cysteines, attachment of a molecule to one or more lysines or attachment of a molecule to one or more non-natural amino acids and the molecule is (a) a molecular adaptor that facilitates the interaction between a pore comprising the monomer and a target nucleotide or target nucleic acid sequence or (b) a nucleic acid binding protein; (iv) the mutant is chemically modified by attachment of a molecule to one or more cysteines, attachment of a molecule to one or more lysines or attachment of a molecule to one or more non-natural amino acids and the attachment is via a linker or (v) the mutant is chemically modified by attachment of a molecule to one or more cysteines, attachment of a molecule to one or more lysines or attachment of a molecule to one or more non-natural amino acids and the molecule is attached to one or more of positions 90, 91 and 103 of SEQ ID NO: 2.

### Claim 11

A construct comprising two or more covalently attached Msp monomers.

### Claim 12 (depends on 1)

A construct comprising two or more covalently attached Msp monomers, wherein (a) the two or more monomers are the same or different; (b) at least one monomer comprises the sequence shown in SEQ ID NO: 2; (c) at least one of the monomers is a mutant monomer as defined in claim 1; (d) the construct comprises two monomers and at least one of the monomers is a mutant as defined in claim 1; (e) the monomers are genetically fused; or (f) the monomers are attached via a linker.

### Claim 13 (depends on 1, 11)

A polynucleotide which encodes a mutant according to claim 1 or a construct according to claim 11.

### Claim 14 (depends on 1)

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

### Claim 15 (depends on 1)

A homo-oligomeric Msp pore comprising eight identical mutant monomers according to claim 1.

### Claim 16 (depends on 1)

A hetero-oligomeric Msp pore comprising at least one mutant monomer according to claim 1, wherein at least one of the eight monomers differs from the others.

### Claim 17 (depends on 1)

A hetero-oligomeric Msp pore, wherein
(i) the pore comprises eight mutant monomers according to claim 1 and at least one of them differs from the others;
(ii) the pore comprises at least one monomer comprising the sequence shown in SEQ ID NO: 2;
(iii) the pore comprises (a) one mutant monomer and (b) seven identical monomers, wherein the mutant monomer in (a) is different from the identical monomers in (b); or
(iv) the pore comprises (a) seven monomers comprising the sequence shown in SEQ ID NO: 2 and one mutant monomer according to claim 1 comprising the substitution N90R, N90K, N90Y, N9OQ, N90W or N90C, (b) seven monomers comprising the sequence shown in SEQ ID NO: 2 and one mutant monomer according to claim 1 comprising the substitution N91R, N91K, N91Y, N91Q, N91W or N91C or (c) seven monomers comprising the sequence shown in SEQ ID NO: 2 and one mutant monomer according to claim 1 comprising the substitution L88C, S103C or I105C.

### Claim 18 (depends on 15)

A pore according to claim 15, wherein at least one of the mutant monomers is chemically-modified.

### Claim 19 (depends on 17)

A pore according to claim 17, wherein at least one of the mutant monomers is chemically-modified.

### Claim 20 (depends on 11)

A pore comprising at least one construct according to claim 11.

### Claim 21 (depends on 1)

A pore comprising two or more covalently attached Msp monomers, further comprising:
four constructs each comprising two monomers and at least one of the monomers is a mutant as defined in claim 1; or
one construct comprising a mutant monomer as defined in claim 1 and six monomers each comprising (i) the sequence shown in SEQ ID NO: 2 or (ii) a variant of SEQ ID NO: 2 as defined in claim 1.

### Claim 22 (depends on 20)

A pore according to claim 20, wherein at least one of the constructs is chemically-modified.

### Claim 23 (depends on 14, 16, 20)

A method of characterising a target nucleic acid sequence, comprising:
(a) contacting the target nucleic acid sequence with a pore according to claim 14, 16 or 20 and a nucleic acid binding protein so that the nucleic acid binding protein controls the movement of the target nucleic acid sequence through the pore, wherein the nucleic acid binding protein is selected from the group consisting of nucleases, polymerases, topoisomerases, ligases, helicases, and single strand binding proteins (SSB); and
(b) measuring, upon application of a potential across the pore, a current passing through the pore as the target nucleic acid sequence moves through the pore, wherein the current flow through the pore is distinctive for nucleotides of the target nucleic acid and thereby characterising the target nucleic acid sequence based on the current measurements.

### Claim 24 (depends on 14, 16, 20)

A kit for characterising a target nucleic acid sequence comprising (a) a pore according to claim 14, 16 or 20 and (b) a nucleic acid handling enzyme, wherein the nucleic acid handling enzyme is selected from the group consisting of nucleases, polymerases, topoisomerases, ligases, helicases, and single strand binding proteins (SSB).

### Claim 25 (depends on 14, 16, 20)

An apparatus for characterising target nucleic acid sequences in a sample, comprising (a) a plurality of pores according to claim 14, 16 or 20 and (b) a plurality of nucleic acid handling enzymes, wherein the nucleic acid handling enzyme is selected from the group consisting of nucleases, polymerases, topoisomerases, ligases, helicases, and single strand binding proteins (SSB).

### Claim 26 (depends on 25)

An apparatus according to claim 25, wherein the apparatus comprises:
a sensor device that is capable of supporting the plurality of pores and being operable to perform nucleic acid characterisation using the pores and enzymes;
at least one reservoir for holding material for performing the characterisation;
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.