Mutant pores

• US 9,751,915 B2
• Filed: 02/10/2012
• Issued: 09/05/2017
• Est. Priority Date: 02/11/2011
• Status: Active Grant

• Current Assignee: Oxford Nanopore Technologies Limited
• Original Assignee: Oxford Nanopore Technologies Limited
• Inventors: Clarke, James, Heron, Andrew John, Jayasinghe, Lakmal, Wallace, Elizabeth Jayne, White, James
• Primary Examiner(s): Swartz, Rodney P
• Application Number:
  US13/984,628
• Publication Number:
  US 20140186823A1
• Time in Patent Office: 2,034 Days
• Field of Search: 4242341, 4242481
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: A61K 39/04 Mycobacterium, e.g. Mycobacterium tuberculosis
• CPC Class Codes: C07K 14/35 from Mycobacteriaceae (F)
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins
• CPC Class Codes: C12Q 2525/101 incorporating non-naturally occurring nucleotides, e.g. inosine
• CPC Class Codes: C12Q 2563/116 electrical properties of nucleic acids, e.g. impedance, conductivity or resistance
• 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 nucleic acid characterization using Msp.

Claims

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:

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

10. 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.

12. 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.

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

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

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

16. 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.

17. 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.

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

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

21. 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.

23. 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.

24. 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).

25. 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).

26. 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.

11. A construct comprising two or more covalently attached Msp monomers.

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

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