Method for sequencing a heteropolymeric target nucleic acid sequence

1. A method for sequencing a heteropolymeric target nucleic acid sequence, comprising:

(a) passing the heteropolymeric target nucleic acid sequence through a transmembrane protein pore so that the nucleotides in the heteropolymeric target nucleic acid sequence interact with two or more distinct sites of amino acids within the sequence of the transmembrane protein pore that are capable of discriminating between different nucleotides, such that the overall current passing through the pore is influenced by the interactions between each of the two or more sites and the nucleotides located at each of the sites;

wherein: (1) the pore has been determined to comprise at least two of said distinct sites of amino acids, (2) at least one of the distinct site of amino acids is modified by amino acid substitution to alter its ability to discriminate between different nucleotides, and (3) the heteropolymeric target nucleic acid sequence is not cleaved or digested to form individual nucleotides before the heteropolymeric target nucleic acid sequence is sequenced; and

(b) measuring the overall current passing through the pore, thereby determining the sequence of the heteropolymeric target nucleic acid sequence.

2. A method according to claim 1 , wherein;

(a) the two or more distinct sites each discriminate between different nucleotides in a different manner;

(b) the interaction of a selected nucleotide with each of the two or more distinct sites results in a different current passing through the transmembrane protein pore; or

(c) the interaction of different nucleotides with each of the two or more distinct sites results in differing currents passing through the transmembrane protein pore, wherein the separation between the mean values of the differing currents differs between each of the two or more distinct sites.

3. A method according to claim 1 , wherein the transmembrane protein pore is modified to alter the current flowing through the transmembrane protein pore when a selected nucleotide interacts with the two or more distinct sites.

4. A method according to claim 1 , wherein;

(a) the heteropolymeric target nucleic acid sequence comprises three or more different nucleotides;

(b) the heteropolymeric target nucleic acid sequence comprises four different nucleotides; or;

(c) the heteropolymeric target nucleic acid sequence comprises four different nucleotides and the four different nucleotides comprise the nucleobases (a) adenine, (b) guanine, (c) thymine or uracil and (d) cytosine.

5. A method according to claim 1 , wherein the heteropolymeric target nucleic acid sequence is passed through the transmembrane protein pore using a nucleic acid handling enzyme.

6. A method according to claim 5 , wherein;

(a) the nucleic acid handling enzyme is derived from a nuclease;

(b) the nucleic acid handling enzyme is derived from a nuclease, wherein the nuclease is a member of any one of the Enzyme Classification groups 3.1.11, 3.1.13, 3.1.14, 3.1.15, 3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30 or 3.1.31;

(c) the nucleic acid handling enzyme is derived from an exonuclease;

(d) the nucleic acid handling enzyme is derived from an exonuclease, wherein the exonuclease comprises a sequence set forth in any one of SEQ ID NOs: 6, 8, 10 and 12, or a variant thereof;

(e) the nucleic acid handling enzyme is derived from a polymerase or helicase;

(f) the nucleic acid handling enzyme is derived from a polymerase or helicase and

(i) the polymerase is member of any one of the Enzyme Classification groups 2.7.7.6, 2.7.7.7, 2.7.7.19, 2.7.7.48 or 2.7.7.49; or

(ii) the helicase is member of any one of the Enzyme Classification groups 3.6.1. or 2.7.7.;

(g) the nucleic acid handling enzyme is derived from a polymerase, wherein the polymerase is a DNA-dependent DNA polymerase, RNA-dependent DNA polymerase, DNA-dependent RNA polymerase, or RNA-dependent RNA polymerase; or

(h) the helicase is an ATP-dependent DNA helicase, ATP-dependent RNA helicase, or ATP-independent RNA helicase.

7. A method according to claim 1 , wherein;

(a) the transmembrane protein pore is derived from α-hemolysin;

(b) the transmembrane protein pore is derived from α-hemolysin and comprises seven subunits comprising the sequence set forth in SEQ ID NO: 2 or a variant thereof; or

(c) the transmembrane protein pore is derived from α-hemolysin, comprises the sequence set forth in SEQ ID NO: 2 or a variant thereof, and all seven subunits have an asparagine at position 111 of SEQ ID NO: 2 and an asparagine at position 147 of SEQ ID NO: 2.

8. The method according to claim 1 , wherein the pore does not contain a covalently linked exogenous moiety that facilitates an interaction between the pore and nucleotides.

9. A method for sequencing a heteropolymeric target nucleic acid sequence, comprising:

(a) passing the heteropolymeric target nucleic acid sequence through a transmembrane protein pore so that the nucleotides in the heteropolymeric target nucleic acid sequence interact with two or more distinct sites of amino acids within the sequence of the pore that are capable of discriminating between different nucleotides, such that the overall current passing through the pore is influenced by the interactions between each of the two or more sites and the nucleotides located at each of the sites;

wherein: (1) at least two of the distinct sites of amino acids are modified by amino acid substitution to alter their ability to discriminate between different nucleotides, (2) the pore does not contain a covalently linked exogenous moiety that facilitates an interaction between the pore and nucleotides, and (3) the heteropolymeric target nucleic acid sequence is not cleaved or digested to form individual nucleotides before the heteropolymeric target nucleic acid sequence is sequenced; and

(b) measuring the overall current passing through the pore, thereby determining the sequence of the heteropolymeric target nucleic acid sequence.

10. A method according to claim 9 , wherein;

(a) the two or more distinct sites each discriminate between different nucleotides in a different manner;

(b) the interaction of a selected nucleotide with each of the two or more distinct sites results in a different current passing through the transmembrane protein pore; or

(c) the interaction of different nucleotides with each of the two or more distinct sites results in differing currents passing through the transmembrane protein pore, wherein the separation between the mean values of the differing currents differs between each of the two or more distinct sites.

11. A method according to claim 9 , wherein the transmembrane protein pore is modified to alter the current flowing through the transmembrane protein pore when a selected nucleotide interacts with the two or more distinct sites.

12. A method according to claim 9 , wherein;

(a) the heteropolymeric target nucleic acid sequence comprises three or more different nucleotides;

(b) the heteropolymeric target nucleic acid sequence comprises four different nucleotides; or;

(c) the heteropolymeric target nucleic acid sequence comprises four different nucleotides and the four different nucleotides comprise the nucleobases (a) adenine, (b) guanine, (c) thymine or uracil and (d) cytosine.

13. A method according to claim 9 , wherein the heteropolymeric target nucleic acid sequence is passed through the transmembrane protein pore using a nucleic acid handling enzyme.

14. A method according to claim 13 , wherein;

(a) the nucleic acid handling enzyme is derived from a nuclease;

(b) the nucleic acid handling enzyme is derived from a nuclease, wherein the nuclease is a member of any one of the Enzyme Classification groups 3.1.11, 3.1.13, 3.1.14, 3.1.15, 3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30 or 3.1.31;

(c) the nucleic acid handling enzyme is derived from an exonuclease;

(d) the nucleic acid handling enzyme is derived from an exonuclease, wherein the exonuclease comprises a sequence set forth in any one of SEQ ID NOs: 6, 8, 10 and 12, or a variant thereof;

(e) the nucleic acid handling enzyme is derived from a polymerase or helicase;

(f) the nucleic acid handling enzyme is derived from a polymerase or helicase and

(i) the polymerase is member of any one of the Enzyme Classification groups 2.7.7.6, 2.7.7.7, 2.7.7.19, 2.7.7.48 or 2.7.7.49; or

(ii) the helicase is member of any one of the Enzyme Classification groups 3.6.1. or 2.7.7.;

(g) the nucleic acid handling enzyme is derived from a polymerase, wherein the polymerase is a DNA-dependent DNA polymerase, RNA-dependent DNA polymerase, DNA-dependent RNA polymerase, or RNA-dependent RNA polymerase; or

(h) the helicase is an ATP-dependent DNA helicase, ATP-dependent RNA helicase, or ATP-independent RNA helicase.

15. A method according to claim 9 , wherein;

(a) the transmembrane protein pore is derived from α-hemolysin;

(b) the transmembrane protein pore is derived from α-hemolysin and comprises seven subunits comprising the sequence set forth in SEQ ID NO: 2 or a variant thereof; or

(c) the transmembrane protein pore is derived from α-hemolysin, comprises the sequence set forth in SEQ ID NO: 2 or a variant thereof, and all seven subunits have an asparagine at position 111 of SEQ ID NO: 2 and an asparagine at position 147 of SEQ ID NO: 2.