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1. (WO2013153359) MUTANT LYSENIN PORES
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CLAIMS

1. A mutant lysenin monomer comprising a variant of the sequence shown in SEQ ID NO: 2, wherein the monomer is capable of forming a pore and wherein the variant comprises one or more modifications within the region of from about position 44 to about position 126 of SEQ ID NO: 2 which alter the ability of the monomer to interact with a polynucleotide.

2. A mutant lysenin monomer according to claim 1, wherein the one or more modifications (a) alter the steric effect of the monomer, (b) alter the net charge of the monomer, (c) alter the ability of the monomer to hydrogen bond with the polynucleotide, (d) introduce or remove chemical groups that interact through delocalized electron pi systems and/or (e) alter the structure of the monomer.

3. A mutant lysenin monomer according to claim 2, wherein the one or more modifications increase or decrease the net positive charge.

4. A mutant lysenin monomer according to claim 3, wherein the net positive charge is increased by introducing one or more positively charged amino acids and/or neutralising one or more negative charges.

5. A mutant lysenin monomer according to claim 4, wherein the one or more negative charges are neutralised by substituting one or more negatively charged amino acids with one or more uncharged amino acids, non-polar amino acids and/or aromatic amino acids or by introducing one or more positive charged amino acids adjacent to one or more negatively charged amino acids.

6. A mutant lysenin monomer according to any one of the preceding claims, wherein the variant comprises a substitution at one or more of the following positions of SEQ ID NO: 2: M44, N46, N48, E50, R52, H58, D68, F70, E71, S74, E76, S78, Y79, S80, H81, S82, E84, E85, S86, Q87, S89, M90, E92, E94, E97, E102, H103, T104, T106, R115, Ql 17, Nl 19, D121 and D126. .

7. A mutant lysenin monomer according to claim 6, wherein the amino acid(s) substituted into the variant are selected from asparagine (N), serine (S), glutamine (Q), arginine (R), glycine (G), tyrosine (Y), aspartic acid (D), leucine (L), lysine (K) or alanine (A).

8. A mutant lysenin monomer according to any one of the preceding claims, wherein the variant comprises at least one of the following mutations:

(a) serine (S) at position 44;

(b) serine (S) at position 46;

(c) serine (S) at position 48;

(d) serine (S) at position 52;

(e) serine (S) at position 58;

(f) serine (S) at position 68;

(g) serine (S) at position 70;

GO serine (S) at position 71;

(i) serine (S) at position 76;

0) serine (S) at position 79;

(k) serine (S) at position 81;

(1) serine (S), aspartic acid (D) or glutamine (Q) at position 84;

(m) serine (S) or lysine (K) at position 85;

(n) serine (S) at position 87;

(o) serine (S) at position 90;

(P) asparagine (N) or glutamine (Q) at position 92;

(q) serine (S) or asparagine (N) at position 94;

(r) serine (S) or asparagine (N) at position 97;

(s) serine (S) at position 102;

(t) serine (S) at position 103;

(u) asparagine (N) or serine (S) at position 121;

(v) serine (S) at position 50;

(w) asparagine (N) or serine (S) at position 94;

(x) asparagine (N) or serine (S) at position 97;

(y) serine (S) or asparagine (N) at position 121;

00 asparagine (N) or glutamine (Q) or glycine (G) at position 126.; and

(aa) serine (S) or asparagine (N) at position 128.

9. A mutant lysenin monomer according to claim 8, wherein the variant compri following substitutions:

i. one or more of E84D and E85K;

ii. one or more of E84Q, E85K, E92Q, E97S, D126G and E167A;

iii. one or more of E92N, E94N, E97N, D121N and D126N;

iv. one or more of E92N, E94N, E97N, D121N, D126N and E128N;

v. one or more of E76S, E84Q, E85K, E92Q, E97S, D126G and E167A; vi. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E50S; vii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E71 S; viii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E94S; ix. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E102S; x. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E128S; xi. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E135S; xii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and D68S; xiii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and D121 S; xiv. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and D134S; or xv. one or more of E84D, E85K and E92Q;

xvi. one or more of E84Q, E85K, E92Q, E97S, D126G and E135S;

xvii . one or more of E85K, E92Q, E94 S, E97 S and D 126G;

xviii. one or more of E76S, E85K, E92Q, E97S and D126G;

xix. one or more of E71 S, E85K, E92Q, E97S and D126G;

xx. one or more of D68S, E85K, E92Q, E97S and D126G;

xxi. one or more of E85K, E92Q, E97S and D126G;

xxii. one or more of E84Q, E85K, E92Q, E97S, H103S and D126G;

xxiii. one or more of E84Q, E85K, M90S, E92Q, E97S and D126G;

xxiv. one or more of E84Q, Q87S, E85K, E92Q, E97S and D126G;

xxv. one or more of E84Q, E85S, E92Q, E97S and D126G;

xxvi. one or more of E84S, E85K, E92Q, E97S and D126G;

xxvii. one or more of H81 S, E84Q, E85K, E92Q, E97S and D126G;

xxviii. one or more of Y79S, E84Q, E85K, E92Q, E97S and D126G;

xxix. one or more of F70S, E84Q, E85K, E92Q, E97S and D126G;

xxx. one or more of H58S, E84Q, E85K, E92Q, E97S and D126G;

xxxi. one or more of R52S, E84Q, E85K, E92Q, E97S and D126G;

xxxii. one or more of N48S, E84Q, E85K, E92Q, E97S and D126G;

xxxiii. one or more of N46S, E84Q, E85K, E92Q, E97S and D126G;

xxxiv. one or more of M44S, E84Q, E85K, E92Q, E97S and D126G;

xxxv. one or more of E92Q and E97S;

xxxvi. one or more of E84Q, E85K, E92Q and E97S;

xxxvii. one or more of E84Q and E85K;

xxxviii. one or more of E84Q, E85K and D126G;

xxxix. one or more of E84Q, E85K, D126G and E167A;

xl. one or more of E92Q, E97S and D126G;

xli. one or more of E84Q, E85K, E92Q, E97S and D126G;

xlii. one or more of E84Q, E85K, E92Q, E97S and E167A;

xliii. one or more of E84Q, E85K, E92Q, D126G and E167A;

xliv. one or more of E84Q, E85K, E97S, D126G and E167A;

xlv. one or more of E84Q, E92Q, E97S, D126G and E167A;

xlvi. one or more of E85K, E92Q, E97S, D126G and E167A;

xlvii. one or more of E84D, E85K and E92Q;

xlviii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and D121 S;

xlix. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and D68S;

1. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E135S;

li. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E128S;

Hi. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E102S;

liii. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E94S;

liv. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E71 S;

lv. one or more of E84Q, E85K, E92Q, E97S, D126G, E167A and E50S;

lvi. one or more of E76S, E84Q, E85K, E92Q, E97S, D126G and E167A;

lvii. one or more of E92N, E94N, E97N, D121N, D126N and E128N;

lviii. one or more of E92N, E94N, E97N, D121N and D126N; or

lix. one or more of E84Q, E85K, E92Q, E97S, D126G and E167A.

10. A mutant lysenin monomer according to claim 9, wherein the variant comprises all of the substitutions in any one of i to lix.

11. A mutant lysenin monomer according to any one of the preceding claims, wherein the mutant is chemically modified.

12. A mutant lysenin monomer according to claim 11, wherein 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.

13. A mutant lysenin monomer according to claim 12, wherein the one or more cysteines or one or more non-natural amino acids have been introduced to the mutant by substitution.

14. A mutant lysenin monomer according to claim 12 or 13, wherein the molecule is (a) a molecular adaptor that facilitates the interaction between a pore comprising the monomer and a target analyte, target nucleotide or target polynucleotide or (b) a polynucleotide binding protein.

15. A mutant lysenin monomer according to any one or claims 12 to 14, wherein the attachment is via a linker.

16. A mutant lysenin monomer according to any one of claims 12 to 15, wherein the molecule is attached to one or more of the positions in the variant that correspond to from about positions 1 to about position 43 and from about position 127 to about position 297 of SEQ ID NO: 2.

17. A construct comprising two or more covalently attached monomers derived from lysenin, wherein at least one of the monomers is a mutant lysenin monomer as defined in any one of claims 1 to 16.

18. A construct according to claim 17, wherein the two or more monomers are the same or different.

19. A construct according to claim 17 or 18, wherein at least one monomer comprises the sequence shown in SEQ ID NO: 2.

20. A construct according to any one of claims 17 to 19, wherein the construct comprises two monomers.

21. A construct according to any one of claims 17 to 20, wherein the monomers are genetically fused.

22. A construct according to any one of claims 17 to 21, wherein the monomers are attached via a linker.

23. A polynucleotide which encodes a mutant lysenin monomer according to any one of claims 1 to 10 or a construct according to claim 21.

24. A homo-oligomeric pore derived from lysenin comprising a sufficient number of mutant lysenin monomers according to any one of claims 1 to 10.

25. A hetero-oligomeric pore derived from lysenin comprising at least one mutant lysenin monomer according to any one of claims 1 to 10.

26. A hetero-oligomeric pore according to claim 25, wherein the pore comprises at least one monomer comprising the sequence shown in SEQ ID NO: 2 or a paralogue, a homologue or a variant thereof.

27. A hetero-oligomeric pore according to claim 25 or 26, wherein the pore comprises (a) one mutant lysenin monomer according to any one of claims 1 to 9 and (b) a sufficient number of identical monomers to form the pore, wherein the mutant monomer in (a) is different from the identical monomers in (b).

28. A hetero-oligomeric pore according to any one of claims 25 to 27, wherein the pore comprises only one mutant lysenin monomer according to any one of claims 1 to 10.

29. A pore according to any one of claims 24 to 28, wherein at least one of the mutant lysenin monomers is chemically-modified as defined in claims 10 to 15.

30. A pore comprising at least one construct according to any one of claims 17 to 22.

31. A pore according to claim 30, which comprises (a) one construct as defined in claim 18 and (b) a sufficient number of monomers to form the pore each comprising (i) the sequence shown in SEQ ID NO: 2 or (ii) a variant of SEQ ID NO: 2 as defined in any one of claims 1 to 10.

32. A pore according to any one of claims 24 to 32, wherein at least one of the constructs is chemically-modified as defined in claims 11 to 16.

A method of characterising a target analyte, compri

(a) contacting the target polynucleotide with a pore according to any one of claims 24 to 32 such that the target polynucleotide moves through the pore; and

(b) taking one or more measurements as the analyte moves with respect to the pore wherein the measurements are indicative of one or more characteristics of the target analyte and thereby characterising the target analyte.

34. A method according to claim 33, wherein the target analyte is a metal ion, an inorganic salt, a polymer, an amino acid, a peptide, a polypeptide, a protein, a nucleotide, an

oligonucleotide, a polynucleotide, a dye, a bleach, a pharmaceutical, a diagnostic agent, a recreational drug, an explosive or an environmental pollutant.

35. A method according to claim 34, wherein the target analyte is a target polynucleotide.

36. A method according to claim 35, wherein step (a) comprises contacting the target polynucleotide with the pore and a polynucleotide binding protein and the protein controls the movement of the target polynucleotide through the pore.

37. A method according to claim 35 or 36, wherein characterising the target polynucleotide comprises estimating the sequence of or sequencing the target polynucleotide.

38. A method of forming a sensor for characterising a target polynucleotide, comprising forming a complex between a pore according to any one of claims 24 to 32 and a polynucleotide binding protein and thereby forming a sensor for characterising the target polynucleotide.

39. A method according to claim 38, wherein the complex is formed by (a) contacting the pore and the polynucleotide binding protein in the presence of the target polynucleotide and (a) applying a potential across the pore.

40. A method according to claim 39, wherein the potential is a voltage potential or a chemical potential.

41. A method according to claim 38, wherein the complex is formed by covalently attaching the pore to the protein.

42. A sensor for characterising a target polynucleotide, comprising a complex between a pore according to any one of claim 24 to 32 and a polynucleotide binding protein.

43. Use of a pore according to any one of claims 24 to 32 to characterise a target analyte.

44. A kit for characterising a target polynucleotide comprising (a) a pore according to any one of claims 24 to 32 and (b) a polynucleotide binding protein.

45. A kit according to claim 44, wherein the kit further comprises a chip comprising an amphiphilic layer.

46. An apparatus for characterising target polynucleotides in a sample, comprising (a) a plurality of pores according to any one of claims 24 to 32 and (b) a plurality of polynucleotide binding proteins.

47. An apparatus according to claim 46, wherein the apparatus comprises:

a sensor device that is capable of supporting the plurality of pores and being operable to perform polynucleotide characterisation using the pores and proteins;

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.

48. A method of improving the ability of a lysenin monomer comprising the sequence shown in SEQ ID NO: 2 to characterise a polynucleotide, comprising making one or more modifications within the region of from about position 44 to about position 126 of SEQ ID NO: 2 which alter the ability of the monomer to interact with a polynucleotide and do not affect the ability of the monomer to form a pore.

49. A method of producing a construct according to any one of claims 17 to 22, comprising covalently attaching at least one mutant lysenin monomer according to any one of claim 1 to 16 to one or more monomers derived from lysenin.

50. A method of forming a pore according to any one of claims 24 to 32, comprising allowing at least one mutant monomer according to any one of 1 to 16 or at least one construct according to any one of claims 17 to 22 to oligomerise with a sufficient number of monomers according to any one of claims 1 to 16, constructs according to any one of claims 17 to 22 or monomers derived from lysenin to form a pore.