METHODS OF ENHANCING TRANSLOCATION OF CHARGED ANALYTES THROUGH TRANSMEMBRANE PROTEIN PORES

1 - 26 . (canceled)

27 . A method of enhancing translocation of a charged analyte through a transmembrane protein pore, comprising:

(a) increasing the net opposing charge of the barrel or channel and/or entrance of the pore; and

(b) determining whether or not translocation of the analyte through the resulting pore is enhanced.

28 . A method according to claim 27 , wherein increasing the net opposing charge:

(a) increases the frequency of translocation of the analyte through the pore;

(b) decreases the threshold voltage for translocation of the analyte through the pore;

(c) decreases the translocation speed of the analyte through the pore; or

(d) decreases the number of non-translocation interactions between the analyte and the pore.

29 . A method according to claim 27 , wherein the analyte is a polymer or a nucleic acid sequence.

30 . A method according to claim 27 , wherein the pore comprises:

(a) seven subunits each comprising SEQ ID NO: 2 or a variant thereof; or

(b) seven subunits each comprising SEQ ID NO: 4 or a variant thereof.

31 . A method according to claim 27 , wherein the analyte is negatively charged and step (a) involves increasing the net positive charge.

32 . A method according to claim 31 , wherein the net positive charge is increased by introducing one or more positively charged amino acids into the barrel or channel and/or entrance of the pore.

33 . A method according to claim 32 , wherein the introducing is carried out by substitution.

34 . A method according to claim 33 , wherein the one or more positively-charge amino acids are histidine (H), lysine (K) and/or arginine (R).

35 . A method according to claim 31 , wherein the net positive charge is increased substituting one or more negatively charged amino acids in the barrel or channel and/or entrance of the pore with one or more uncharged amino acids, non-polar amino acids and/or aromatic amino acids.

36 . A method according to claim 27 , wherein the entrance is the portion of the pore through which the analyte enters the barrel or channel.

37 . A transmembrane protein pore enhanced by a method according to claim 27 .

38 . A transmembrane protein pore in which the net opposing charge of its barrel or channel and/or entrance has been increased to enhance the translocation of a charged analyte through the pore.

39 . A transmembrane protein pore according to claim 37 or 38 , wherein the pore comprises:

(a) at least one subunit comprising SEQ ID NO: 6 or a variant thereof;

(b) at least one subunit comprising SEQ ID NO: 8 or a variant thereof; or

(c) at least one subunit comprising SEQ ID NO: 10 or a variant thereof.

40 . A transmembrane protein pore according to claim 39 , wherein the pore is a homoheptamer comprising seven subunits of SEQ ID NO: 6, 8 or 10 or a variant thereof.

41 . A polynucleotide encoding a transmembrane protein pore according to claim 37 or 38 .

42 . A polynucleotide encoding a transmembrane protein pore subunit having the sequence of SEQ ID NO: 6, 8 or 10 or a variant thereof.

43 . A polynucleotide according to claim 42 , wherein the polynucleotide comprises the sequence shown in SEQ ID NO: 5, 7 or 9 or a variant thereof

44 . A method of determining presence or absence of an analyte in a sample, comprising:

(a) contacting the sample with a transmembrane protein pore according to claim 37 or 38 under conditions that allow the analyte, if present, to translocate through and interact with the pore; and

(b) measuring the current passing through the pore during the interaction and thereby determining the presence or absence of the analyte.

45 . A method of sequencing a target nucleic acid sequence, comprising:

(a) pushing or pulling the target sequence through a transmembrane protein pore according to claims 37 to 38 so that a proportion of the nucleotides in the target sequence interact with the pore; and

(b) measuring the current passing through the pore during each interaction and thereby determining the sequence of the target sequence.

46 . A kit for sequencing a nucleic acid, comprising a transmembrane protein pore according to claim 37 or 38 and a nucleic acid handling enzyme.