mutant pores

Abstract

The present invention relates to mutant forms of cytolysins. The present invention also relates to the use of said mutant forms of cytolysins for analyte characterization.

Description

[0001] This application is a divisional application of the divisional application 202310422087.0. The divisional application 202310422087.0 is a divisional application of the application number 201780022553.9, filed on April 6, 2017, with the title “Mutant Pore”. TECHNICAL FIELD

[0002] The present invention relates to mutant forms of lysenin. The present invention also relates to the use of said mutant forms of lysenin for analyte characterization. BACKGROUND

[0003] Nanopore sensing is a sensing method that relies on the observation of individual binding or interaction events between an analyte molecule and a receptor. A nanopore sensor can be created by placing a nanometer-sized single pore in an insulating membrane and measuring the voltage-driven ion transport through the pore in the presence of an analyte molecule. The identity of the analyte is revealed by its unique current signature, especially the duration and extent of current block and changes in current level. Such nanopore sensors are commercially available, for example, the MinION® MinION by Oxford Nanopore Technologies Ltd TM An apparatus comprising an array of nanopores integrated within an electronic chip.

[0004] There is a need for fast and inexpensive nucleic acid (e.g., DNA or RNA) sequencing technology in a wide range of applications. Existing technologies are slow and expensive, primarily because they rely on amplification techniques to produce large amounts of nucleic acid and require large amounts of specialized fluorescent chemicals for signal detection. Nanopore sensing has the potential to provide fast and inexpensive nucleic acid sequencing by reducing the amount of nucleotides and reagents required.

[0005] One of the basic elements for sequencing nucleic acids using nanopore sensing is to control the movement of the nucleic acid through the pore. Another element is to distinguish the nucleotides as the nucleic acid polymer moves through the pore. In the past, to achieve nucleotide discrimination, the nucleic acid has been passed through a mutant of lysenin. This has provided current signatures that have been shown to be sequence dependent. It has also been shown that, when using lysenin pores, a large number of nucleotides contribute to the observed current, making a direct relationship between the observed current and the polynucleotide challenging.

[0006] While the range of currents used for nucleotide discrimination has been improved by hemolysin pore mutations, the sequencing system would have higher performance if the difference in current between nucleotides could be further improved. In addition, it has been observed that some current states show very high variation as the nucleic acid moves through the pore. It has also been shown that some mutant hemolysin pores exhibit higher variation than others. While the variation in these states can contain sequence-specific information, it is desirable to produce pores with low variation to simplify the system. It is also desirable to reduce the number of nucleotides that contribute to the observed current.

[0007] Cytolysin (also known as efLl) is a pore-forming toxin purified from the coelomic fluid of the earthworm Eisenia fetida. It specifically binds to sphingomyelin, which inhibits cytolysin-induced hemolysis (Yamaji et al., J. Biol. Chem., 1998, vol. 273, no. 9, pp. 5300-5306). The crystal structure of the cytolysin monomer is disclosed in De Colbis et al., Structure, 2012, vol. 20, pp. 1498-1507. SUMMARY

[0008] The present inventors have surprisingly discovered new mutant cytolysin monomers in which one or more modifications have been made to improve the ability of the monomers to interact with a polynucleotide. The present inventors have also surprisingly demonstrated that pores comprising the novel mutant monomers have enhanced ability to interact with a polynucleotide and thus exhibit improved properties for estimating characteristics of a polynucleotide such as its sequence. The mutant pores surprisingly exhibit improved nucleotide discrimination. In particular, the mutant pores surprisingly exhibit increased current range, which makes it easier to discriminate between different nucleotides, and reduced state variation, which increases the signal-to-noise ratio. In addition, the number of nucleotides contributing to the current as the polynucleotide moves through the pore is reduced. This makes it easier to identify a direct relationship between the observed current as the polynucleotide moves through the pore and the polynucleotide sequence.

[0009] Unless stated to the contrary, all amino acid substitutions, deletions, and / or additions disclosed herein are with reference to a mutant cytolysin monomer comprising the sequence set forth in SEQ ID NO: 2.

[0010] Reference to a mutant lysenin monomer comprising a variant of the sequence shown in SEQ ID NO: 2 encompasses a mutant lysenin monomer comprising a variant of the sequence as set out in SEQ ID NO: 14 to 16. Amino acid substitutions, deletions and / or additions equivalent to those disclosed herein with reference to SEQ ID NO: 2 can be made to a lysenin monomer comprising a variant of the sequence shown in SEQ ID NO: 2.

[0011] A mutant monomer can be considered to be an isolated monomer.

[0012] Thus, the present application provides 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 a modification at one or more of the following positions: K37, G43, K45, V47, S49, T51, H83, V88, T91, T93, V95, Y96, S98, K99, V100, 101, P108, P109, T110, S111, K112 and T114.

[0013] The present application also provides 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 of the following substitutions:

[0014] D35N / S;

[0015] S74K / R;

[0016] E76D / N;

[0017] S78R / K / N / Q;

[0018] S80K / R / N / Q;

[0019] S82K / R / N / Q;

[0020] E84R / K / N / A;

[0021] E85N;

[0022] S86K / Q;

[0023] S89K;

[0024] M90K / I / A;

[0025] E92D / S;

[0026] E94D / Q / G / A / K / R / S / N;

[0027] E102N / Q / D / S;

[0028] T104R / K / Q;

[0029] T106R / K / Q;

[0030] R115S;

[0031] Q117S; and

[0032] N119S.

[0033] The present application also provides a mutant cytolysin monomer comprising a variant of the sequence set forth in SEQ ID NO: 2, wherein the monomer is capable of forming a pore, and wherein the variant comprises a mutation at one or more of the following:

[0034] D35 / E94 / T106;

[0035] K37 / E94 / E102 / T106;

[0036] K37 / E94 / T104 / T106;

[0037] K37 / E94 / T106;

[0038] K37 / E94 / E102 / T106;

[0039] G43 / E94 / T106;

[0040] K45 / V47 / E92 / E94 / T106;

[0041] K45 / V47 / E94 / T106;

[0042] K45 / S49 / E92 / E94 / T106;

[0043] K45 / S49 / E94 / T106;

[0044] K45 / E94 / T106;

[0045] K45 / T106;

[0046] V47 / E94 / T106;

[0047] V47 / V88 / E94 / T106;

[0048] S49 / E94 / T106;

[0049] T51 / E94D / T106;

[0050] S74 / E94;

[0051] E76 / E94;

[0052] S78 / E94;

[0053] Y79 / E94;

[0054] S80 / E94;

[0055] S82 / E94;

[0056] S82 / E94 / T106;

[0057] H83 / E94;

[0058] H83 / E94 / T106;

[0059] E85 / E94 / T106;

[0060] S86 / E94;

[0061] V88 / M90 / E94 / T106;

[0062] S89 / E94;

[0063] M90 / E94 / T106;

[0064] T91 / E94 / T106;

[0065] E92 / E94 / T106;

[0066] T93 / E94 / T106;

[0067] E94 / Y96 / T106;

[0068] E94 / S98 / K99 / T106;

[0069] E94 / K99 / T106;

[0070] E94 / E102;

[0071] E94 / T104;

[0072] E94 / T106;

[0073] E94 / P108;

[0074] E94 / P109;

[0075] E94 / T110;

[0076] E94 / S111;

[0077] E94 / T114;

[0078] E94 / R115;

[0079] E94 / Q117; and

[0080] E94 / E119.

[0081] The present application also provides a mutant cytolysin monomer comprising a variant of the sequence set forth in SEQ ID NO: 2, wherein the monomer is capable of forming a pore, and wherein the variant comprises one or more of the following substitutions:

[0082] E84R / E94D;

[0083] E84K / E94D;

[0084] E84N / E94D;

[0085] E84A / E94Q;

[0086] E84K / E94Q and

[0087] E94Q / D121S.

[0088] The present application also provides a mutant cytolysin monomer comprising a variant of the sequence set forth in SEQ ID NO: 2, wherein the variant comprises one of the following combinations of substitutions:

[0089] - E84Q / E85K / E92Q / E94D / E97S / D126G;

[0090] - E84Q / E85K / E92Q / E94Q / E97S / D126G; or

[0091] - E84Q / E85K / E92Q / E94D / E97S / T106K / D126G.

[0092] The present application also provides a mutant cytolysin monomer comprising a variant of the sequence set forth in SEQ ID NO: 2, wherein in the variant (a) 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 of the amino acids at positions 34 to 70 of SEQ ID NO: 2, or corresponding to those positions, have been deleted, and (b) 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 of the amino acids at positions 71 to 107 of SEQ ID NO: 2, or corresponding to those positions, have been deleted.

[0093] The present application also provides:

[0094] - a construct comprising two or more covalently linked monomers derived from a cytolysin, wherein at least one of the monomers is a mutant cytolysin monomer of the invention;

[0095] - a polynucleotide encoding a mutant cytolysin monomer of the invention or a genetic fusion construct of the invention;

[0096] - a homo-oligomeric pore derived from a cytolysin comprising a sufficient number of mutant cytolysin monomers of the invention;

[0097] - a hetero-oligomeric pore derived from a cytolysin comprising at least one mutant cytolysin monomer of the invention;

[0098] - a pore comprising at least one construct of the invention;

[0099] - a method of characterizing a target analyte comprising: (a) contacting the target analyte with a pore of the invention such that the target analyte moves through the pore; (b) taking one or more measurements as the analyte moves relative to the pore, wherein the measurements are indicative of one or more properties of the target analyte, and therefrom characterizing the target analyte;

[0100] - a method of forming a sensor for characterizing a target polynucleotide comprising forming a complex between a pore of the invention and a polynucleotide binding protein and therefrom forming a sensor for characterizing the target polynucleotide;

[0101] - a sensor for characterizing a target polynucleotide comprising a complex between a pore of the invention and a polynucleotide binding protein;

[0102] - use of a pore of the invention for characterizing a target analyte;

[0103] - a kit for characterizing a target polynucleotide comprising (a) a pore of the invention and (b) a membrane;

[0104] - an apparatus for characterizing a target polynucleotide in a sample comprising (a) a plurality of pores of the invention and (b) a plurality of polynucleotide binding proteins;

[0105] - a method of improving the ability of a cytolysin monomer comprising the sequence set forth in SEQ ID NO: 2 to characterize a polynucleotide comprising making one or more modifications and / or substitutions of the invention;

[0106] - a method of producing a construct of the invention comprising: covalently linking at least one mutant cytolysin monomer of the invention to one or more monomers derived from a cytolysin; and

[0107] - a method of forming a pore of the invention comprising: allowing at least one mutant monomer of the invention or at least one construct of the invention to oligomerize with a sufficient number of monomers of the invention, constructs of the invention, or monomers derived from a lysenin to form a pore. BRIEF DESCRIPTION OF DRAWINGS

[0108] Figure 1 A median plot of lysenin mutant 1 is shown.

[0109] Figure 2 A median plot of lysenin mutant 10 is shown.

[0110] Figure 3 A median plot of lysenin mutant - lysenin - (E84Q / E85K / E92Q / E94D / E97S / T106K / D126G / C272A / C283A) 9 (SEQ ID NO: 2 with mutations E84Q / E85K / E92Q / E94D / E97S / T106K / D126G / C272A / C283A) is shown.

[0111] Figure 4 A median plot of lysenin mutant - lysenin - (E84Q / E85K / E92Q / E94C / E97S / T106K / D126G / C272A / C283A) 9 (SEQ ID NO: 2 with mutations E84Q / E85K / E92Q / E94C / E97S / T106K / D126G / C272A / C283A) with 2-iodo-N-(2,2,2-trifluoroethyl)acetamide attached through E94C is shown.

[0112] Figure 5 Adaptors used in examples are shown. A corresponds to 30 iSpC3. B corresponds to SEQ IN NO: 19. C corresponds to 4 iSp18. D corresponds to SEQ ID NO: 20. E corresponds to SEQ ID NO: 21 with 5BNA-G / / iBNA-G / / iBNA-T / / iBNA-T / / i-BNA-A attached to its 5' end. F corresponds to SEQ ID NO: 22 with 5' phosphate. G corresponds to SEQ ID NO: 24. H corresponds to Cholesterol.

[0113] Figure 6The 3D structure of a lysenin monomer is shown. Upon interaction with a membrane containing sphingomyelin, lysenin monomers assemble together through an intermediate pre-pore to form a nonamer pore. The polypeptide segment shown in black (corresponding to amino acids 65 to 74 of SEQ ID NO: 2) transforms into the bottom ring of the beta barrel shown in Figure 7 during the assembly process. The two beta strands on either side of the polypeptide segment shown in black, as well as the polypeptide segments that link those beta strands to the polypeptide segment shown in black (corresponding to amino acids 34 to 64 and 75 to 107 of SEQ ID NO: 2) extend to form the beta barrel of the pore as shown in Figure 7. This large structural change makes it difficult to predict the beta barrel region of the lysenin pore by studying the monomer structure.

[0114] Figure 7 depicts a region of the lysenin pore. Figure 7A The 3D structure of a nonamer pore of lysenin is shown, and Figure 7B The structure of a monomer taken from the lysenin pore is shown. Each monomer contributes two beta strands to the barrel of the lysenin pore. The beta strands (containing amino acids corresponding to amino acids 34 to 64 and 75 to 107 of SEQ ID NO: 2) are linked by an unstructured loop at the bottom of the pore (amino acids corresponding to positions 65 to 74 of SEQ ID NO: 2).

[0115] Figure 8 An alignment of the amino acid sequence of lysenin (SEQ ID NO: 2) with the amino acid sequences of three lysenin-related proteins (SEQ ID NOs: 14 to 16) is shown. Three lysenin homologues with sequences closely related to lysenin were identified by performing a BLAST search using a database of non-redundant protein sequences. The protein sequences of lysenin-related protein 1 (LRP1), lysenin-related protein 2 (LRP2), and lysenin-related protein 3 (LRP3) were aligned with the sequence of lysenin to show the similarity of the four proteins. Dark gray shading indicates that an identical amino acid is present in the position in all four sequences. LRP1 is about 75% identical to lysenin, LRP2 is about 88% identical to lysenin, and LRP3 is about 79% identical to lysenin.

[0116] SEQUENCE LISTING

[0117] SEQ ID NO: 1 shows a polynucleotide sequence encoding a lysenin monomer.

[0118] SEQ ID NO: 2 shows an amino acid sequence of a lysenin monomer.

[0119] SEQ ID NO: 3 shows a polynucleotide sequence encoding Phi29 DNA polymerase.

[0120] SEQ ID NO: 4 sets forth the amino acid sequence of Phi29 DNA polymerase.

[0121] SEQ ID NO: 5 sets forth a codon-optimized polynucleotide sequence derived from the sbcB gene from E. coli. It encodes exonuclease I enzyme from E. coli (EcoExo I).

[0122] SEQ ID NO: 6 sets forth the amino acid sequence of exonuclease I enzyme from E. coli (EcoExo I).

[0123] SEQ ID NO: 7 sets forth a codon-optimized polynucleotide sequence derived from the xthA gene from E. coli. It encodes exonuclease III enzyme from E. coli.

[0124] SEQ ID NO: 8 sets forth the amino acid sequence of exonuclease III enzyme from E. coli. This enzyme performs distributive digestion of 5' monophosphate nucleotides from one strand of double-stranded DNA (dsDNA) in the 3' to 5' direction. Enzyme priming on a strand requires a 5' overhang of about 4 nucleotides.

[0125] SEQ ID NO: 9 sets forth a codon-optimized polynucleotide sequence derived from the recJ gene from Thermus. It encodes RecJ enzyme from Thermus (TthRecJ-cd).

[0126] SEQ ID NO: 10 sets forth the amino acid sequence of RecJ enzyme from Thermus (TthRecJ-cd). This enzyme performs processive digestion of 5' monophosphate nucleotides from ssDNA in the 5' to 3' direction. Enzyme priming on a strand requires at least 4 nucleotides.

[0127] SEQ ID NO: 11 sets forth a codon-optimized polynucleotide sequence derived from the bacteriophage lambda exo (redX) gene. It encodes bacteriophage lambda exonuclease.

[0128] SEQ ID NO: 12 sets forth the amino acid sequence of bacteriophage lambda exonuclease. The sequence is one of three identical subunits that assemble into a trimer. The enzyme performs highly processive digestion of nucleotides from one strand of dsDNA in the 5' to 3' direction (http: / / www.neb.com / nebecomm / products / productM0262.asp). Enzyme priming on a strand preferentially requires a 5' overhang of about 4 nucleotides with 5' phosphate.

[0129] SEQ ID NO: 13 sets forth the amino acid sequence of Hel308 Mbu.

[0130] SEQ ID NO: 14 shows the amino acid sequence of lysenin-related protein (LRP) 1.

[0131] SEQ ID NO: 15 shows the amino acid sequence of lysenin-related protein (LRP) 2.

[0132] SEQ ID NO: 16 shows the amino acid sequence of lysenin-related protein (LRP) 3.

[0133] SEQ ID NO: 17 shows the amino acid sequence of the activated version of parasporin-2. The full-length protein is cleaved at its amino and carboxy termini to form the activated version capable of forming pores.

[0134] SEQ ID NO: 18 shows the amino acid sequence of Dda 1993.

[0135] SEQ ID NOs: 19-24 show polynucleotide sequences used in examples. DETAILED DESCRIPTION

[0136] It should be understood that different applications of the disclosed products and methods can be tailored to specific needs in the art. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments of the application only and is not intended to be limiting.

[0137] Also, as used in the specification and the appended claims, the singular "a," "an" and "the" shall include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a mutation monomer" includes "multiple mutation monomers," reference to "a substitution" includes two or more such substitutions, reference to "a pore" includes two or more such pores, reference to "a polynucleotide" includes two or more such polynucleotides, and the like.

[0138] In the present specification, where different amino acids are separated by the symbol " / " at a particular location, the " / " symbol means "or". For example, P108R / K means P108R or P108K. In the present specification, where different positions or different substitutions are separated by the symbol " / " the " / " symbol means "and". For example, E94 / P108 means E94 and P108, or E94D / P108K means E94D and P108K.

[0139] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

[0140] Mutant cytolysin monomer

[0141] In one aspect, the present application provides mutant cytolysin monomers. Mutant cytolysin monomers can be used to form pores of the present application. Mutant cytolysin monomers are monomers whose sequence differs from the sequence of a wild-type cytolysin monomer (e.g., SEQ ID NO: 2, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16). Mutant cytolysin monomers generally retain the ability to form pores in the presence of other monomers of the present application or other monomers from cytolysins or derived from cytolysins. Thus, mutant monomers generally are capable of forming pores. Methods for confirming the ability of mutant monomers to form pores are well known in the art and are described in the examples. For example, pore formation can be determined by electrophysiology. Pores are generally inserted into a membrane, which can be, for example, a lipid membrane or a block copolymer membrane. Electrical or optical measurements can be obtained from a cytolysin pore inserted into a membrane, such as a pore comprising one or more monomers of the present application. An electrical potential difference can be applied across the membrane, and current through the membrane can be detected. Current can be detected by any appropriate method, such as by electrical or optical means. The ability of a pore to translocate a polynucleotide, Optionally single stranded polynucleotide Modifications of the invention

[0142] When present in a pore, a mutant monomer has an altered ability to interact with a polynucleotide. Thus, a pore comprising one or more of the mutant monomers has improved nucleotide reading properties, for example, showing (1) improved polynucleotide capture and (2) improved polynucleotide recognition or discrimination. In particular, pores constructed from mutant monomers more readily capture nucleotides and polynucleotides than wild-type. In addition, pores constructed from mutant monomers show increased current ranges and reduced state changes, the increased current ranges making it easier to discriminate between different nucleotides, and the reduced state changes increasing the signal-to-noise ratio. In addition, the number of nucleotides contributing to the current as a polynucleotide moves through a pore constructed from a mutant is reduced. This makes it easier to recognize a direct relationship between the observed current as a polynucleotide moves through a pore and the polynucleotide sequence. The improved nucleotide reading properties of the mutants are achieved through five main mechanisms, namely through changes in:

[0143] • steric hindrance (increasing or decreasing the size of an amino acid residue);

[0144] • charge (e.g., introducing or removing -ve charge and / or introducing or removing +ve charge);

[0145] ​• hydrogen bonding (e.g., introduction of amino acids that can hydrogen bond to base pairs);

[0146] • pi stacking (e.g., introduction of amino acids that interact through delocalized electron pi systems); and / or

[0147] • pore structure alteration (e.g., introduction of amino acids that increase the size of the barrel or channel).

[0148] Any one or more of these five mechanisms can be responsible for the improved properties of the pores formed by the mutant monomers of the application. For example, due to altered steric hindrance, altered hydrogen bonding, and altered structure, the pores including the mutant monomers of the application can exhibit improved nucleotide reading properties.

[0149] The mutant monomers of the application include variants of the sequence set forth in SEQ ID NO: 2. SEQ ID NO: 2 is the wild-type sequence of a cytolysin monomer. A variant of SEQ ID NO: 2 is a polypeptide whose amino acid sequence differs from that of SEQ ID NO: 2. Typically, the variant retains its ability to form a pore.

[0150] Pores including one or more of the mutant monomers comprising substitutions at S80, T106, T104 exhibit improved polynucleotide capture. Specific examples of such substitutions include S80K / R, T104R / K, and T106R / K. Other substitutions that increase the positive charge of the amino acid side chain at any one or more, such as 2, 3, 4, or 5, of these positions can be used to improve the properties of the pores including the mutant monomers, i.e., to improve capture of polynucleotides compared to the wild-type pore or to pores including mutant monomers of other capture-enhancing mutations, such as E84Q / E85K / E92Q / E97S / D126G, e.g., pores including mutant monomers comprising only those mutations or mutant monomers including the following mutations E84Q / E85K / E92Q / E94D / E97S / D126G. Typically, where an improvement is determined relative to a pore including other mutations, such as E84Q / E85K / E92Q / E97S / D126G or E84Q / E85K / E92Q / E94D / E97S / D126G, those mutations are also present in the mutant monomer being tested, i.e., the effect of one or more of the mutations or the combination of mutations is determined relative to a baseline monomer / pore that is consistent with the monomer / pore being tested but not at the one or more test positions. The properties of the pores including the mutant monomers or control monomers can be determined using hetero-oligomeric pores or, more preferably, homo-oligomeric pores. Examples of preferred combinations of mutations are described throughout the specification, e.g., in Table 9.

[0151] Pores comprising one or more of a mutant monomer comprising substitutions at D35, K37, K45, V47, S49, E76, S78, S82, V88, S89, M90, T91, E92, E94, Y96, S98, V100, T104 show improved polynucleotide recognition or discrimination. Particular examples of such substitutions include D35N, K37N / S, K45R / K / D / T / Y / N, V47K / R, S49K / R / L, T51K E76S / N, S78N, S82N, V88I, S89Q, M90I / A, T91S, E92D / E, E94D / Q / N, Y96D, S98Q, V100S, and T104K. As described in Table 9, each of these mutations can reduce noise, increase current range, and / or reduce channel gating. Other mutations that increase or decrease the size of the amino acid side chain, increase or decrease charge, result in the same hydrogen bonding, and / or affect pi stacking in the same way as any one or more of these exemplary mutations made to the indicated positions in SEQ ID NO: 2 or corresponding positions in a variant of SEQ ID NO: 2 are made. Mutations can be introduced individually or in combination. For example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or 18 of these positions can be mutated to improve the properties of a pore comprising the mutant monomer, i.e., to improve signal to noise, increase range, and / or reduce channel gating, such that polynucleotide recognition and discrimination is improved in a pore comprising a mutant monomer comprising mutations E84Q / E85K / E92Q / E97S / D126G, such as a monomer comprising only those mutations, a mutant monomer comprising mutations E84Q / E85K / E92Q / E94D / E97S / D126G, a mutant monomer comprising mutations E84Q / E85K / E92Q / E94Q / E97S / D126G, and / or a mutant monomer comprising mutations E84Q / E85K / E92Q / E94D / E97S / T106K / D126G, relative to a wild-type pore, a pore comprising other mutations, such as E84Q / E85K / E92Q / E97S / D126G, E84Q / E85K / E92Q / E94D / E97S / D126G, E84Q / E85K / E92Q / E94Q / E97S / D126G, or E84Q / E85K / E92Q / E94D / E97S / T106K / D126G. Generally, where an improvement is determined relative to a pore comprising other mutations, such as E84Q / E85K / E92Q / E97S / D126G, E84Q / E85K / E92Q / E94D / E97S / D126G, E84Q / E85K / E92Q / E94Q / E97S / D126G, or E84Q / E85K / E92Q / E94D / E97S / T106K / D126G, those mutations are also present in the mutant monomer being tested, i.e., the effect of one or more of the mutations or the combination of mutations is determined relative to a baseline monomer / pore that is consistent with the monomer / pore being tested but not at the one or more test positions. The properties of a pore comprising the mutant monomer or a control monomer can be determined using a hetero-oligomeric pore or, more preferably, a homo-oligomeric pore.Examples of preferred combinations of mutations are described throughout the specification, for example in Table 9.

[0152] Compared to a wild-type pore or a pore comprising a mutant monomer comprising the mutations E84Q / E85K / E92Q / E97S / D126G, a pore comprising one or more of the mutant monomers comprising a substitution at E94 and / or Y96 can reduce the number of nucleotides contributing to the current when a polynucleotide moves through the pore. For example, the substitution Y96D / E, preferably in combination with E94Q / D, can be made to reduce the size of the read head. The reduction in the number of nucleotides contributing to the current when a polynucleotide moves through the pore can also be achieved compared to a wild-type pore or a pore comprising a mutant monomer comprising the mutations E84Q / E85K / E92Q / E97S / D126G by deleting an even number of amino acids from each of the two beta strands that form part of the barrel of the pore, i.e. positions corresponding to amino acids 34 to 65 and 74 to 107 of SEQ ID NO: 2, from each of the two beta strands that form part of the barrel of the pore, i.e. positions corresponding to amino acids 34 to 65 and 74 to 107 of SEQ ID NO: 2, as described herein.

[0153] Figure 8

[0154] The present invention provides a mutant cytolysin monomer wherein the amino acid sequence of the beta sheet contributing to the structure of the barrel in the cytolysin pore is modified compared to a wild-type cytolysin and compared to cytolysin mutants disclosed in the art, for example in WO 2013 / 153359. The modifications of the present invention are made in the region of the cytolysin monomer corresponding to amino acids 34 to 107 of SEQ ID NO: 2, in particular amino acids 34 to 65 and 74 to 107 of SEQ ID NO: 2. The corresponding regions of the LR1, LR2 and LR3 monomers are shown in the alignment of Bucket deletion .

[0155] Thus, the present application provides a mutant cytolysin 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, such as 2 to 22, 3 to 20, 4 to 15, 5 to 10, 6, 7, 8 or 9 modifications at the following positions: K37, G43, K45, V47, S49, T51, H83, V88, T91, T93, V95, Y96, S98, K99, V100, 101, P108, P109, T110, S111, K112 and T114. The variant can comprise modifications at any number of the positions and at any combination of the positions. In an aspect, the modifications can be substitutions, deletions or additions of amino acids, and are preferably substitution or deletion mutations. Preferred modifications are discussed below under the heading "further modifications". The mutant cytolysin monomer can comprise modifications at other positions of SEQ ID NO: 2. For example, in addition to one or more, such as 2 to 20, 3 to 15, 4 to 10 or 6 to 8 modifications of the present application, the mutant cytolysin monomer can have one or more, such as 2 to 20, 3 to 15, 4 to 10 or 6 to 8 amino acid substitutions or deletions in the sequence of SEQ ID NO: 2 described in the art, for example in WO 2013 / 153359.

[0156] The variant preferably comprises modifications at one or more of the following positions T91, V95, Y96, S98, K99, V100, 101 and K112. The variant can have modifications at any number of the positions and at any combination of the positions. The modifications are preferably substitutions with serine (S) or glutamine (Q). The variant preferably comprises one or more of the substitutions T91 S, V95S, Y96S, S98Q, K99S, V100S, 101 S and K112S. The variant can comprise any number of these substitutions and any combination of these substitutions.

[0157] The variant preferably comprises a modification at one or more of the following positions: K37, G43, K45, V47, S49, T51, H83, V88, T91, T93, Y96, S98, K99, P108, P109, T110, S111, and T114. The variant can comprise a modification at any number of the positions and any combination of the positions. The modification is preferably a substitution with asparagine (N), tryptophan (W), serine (S), glutamine (Q), lysine (K), aspartic acid (D), arginine (R), threonine (T), tyrosine (Y), leucine (L), or isoleucine (I). The variant preferably comprises one or more of the following substitutions: K37N / W / S / Q, G43K, K45D / R / N / Q / T / Y, V47K / S / N, S49K / L, T51K, H83S / K, V88I / T, T91K, T93K, Y96D, S98K, K99Q / L, P108K / R, P109K, T110K / R, S111K, and T114K. The variant preferably comprises a modification at one or more of the following positions:

[0158]

[0159] The variant preferably comprises one or more of the following substitutions:

[0160]

[0161] The present application also provides a mutant cytolysin monomer comprising a variant of the sequence set forth in SEQ ID NO: 2, wherein the monomer is capable of forming a pore, and wherein the variant comprises one or more of the following substitutions:

[0162] D35N / S;

[0163] S74K / R;

[0164] E76D / N;

[0165] S78R / K / N / Q;

[0166] S80K / R / N / Q;

[0167] S82K / R / N / Q;

[0168] E84R / K / N / A;

[0169] E85N;

[0170] S86K / Q;

[0171] S89K;

[0172] M90K / I / A;

[0173] E92D / S;

[0174] E94D / Q / G / A / K / R / S / N;

[0175] E102N / Q / D / S;

[0176] T104R / K / Q;

[0177] T106R / K / Q;

[0178] R115S;

[0179] Q117S; and

[0180] N119S.

[0181] The variant can include any number of these substitutions and any combination of these substitutions. The variant preferably includes one or more of the following substitutions: E94D / Q / G / A / K / R / S, S86Q, and E92S, such as E94D / Q / G / A / K / R / S; S86Q; E92S; E94D / Q / G / A / K / R / S and S86Q; E94D / Q / G / A / K / R / S and E92S; S86Q and E92S; or E94D / Q / G / A / K / R / S, S86Q, and E92S.

[0182] The variant preferably includes one or more of the following substitutions:

[0183] D35N / S;

[0184] S74K / R;

[0185] E76D / N;

[0186] S78R / K / N / Q;

[0187] S80K / R / N / Q;

[0188] S82K / R / N / Q;

[0189] E84R / K / N / A;

[0190] E85N;

[0191] S86K;

[0192] S89K;

[0193] M90K / I / A;

[0194] E92D;

[0195] E94D / Q / K / N;

[0196] E102N / Q / D / S;

[0197] T104R / K / Q;

[0198] T106R / K / Q;

[0199] R115S;

[0200] Q117S; and

[0201] N119S.

[0202] The variant can comprise any number of these substitutions and combinations of these substitutions.

[0203] The variant preferably comprises one or more of the following substitutions:

[0204]

[0205]

[0206] The variant can comprise any number of these substitutions and any combination of these substitutions.

[0207] The present application also provides a mutant cytolysin monomer comprising a variant of the sequence set forth in SEQ ID NO: 2, wherein the monomer is capable of forming a pore, and wherein the variant comprises a mutation at one or more of the following:

[0208] D35 / E94 / T106;

[0209] K37 / E94 / E102 / T106;

[0210] K37 / E94 / T104 / T106;

[0211] K37 / E94 / T106;

[0212] K37 / E94 / E102 / T106;

[0213] G43 / E94 / T106;

[0214] K45 / V47 / E92 / E94 / T106;

[0215] K45 / V47 / E94 / T106;

[0216] K45 / S49 / E92 / E94 / T106;

[0217] K45 / S49 / E94 / T106;

[0218] K45 / E94 / T106;

[0219] K45 / T106;

[0220] V47 / E94 / T106;

[0221] V47 / V88 / E94 / T106;

[0222] S49 / E94 / T106;

[0223] T51 / E94D / T106;

[0224] S74 / E94;

[0225] E76 / E94;

[0226] S78 / E94;

[0227] Y79 / E94;

[0228] S80 / E94;

[0229] S82 / E94;

[0230] S82 / E94 / T106;

[0231] H83 / E94;

[0232] H83 / E94 / T106;

[0233] E85 / E94 / T106;

[0234] S86 / E94;

[0235] V88 / M90 / E94 / T106;

[0236] S89 / E94;

[0237] M90 / E94 / T106;

[0238] T91 / E94 / T106;

[0239] E92 / E94 / T106;

[0240] T93 / E94 / T106;

[0241] E94 / Y96 / T106;

[0242] E94 / S98 / K99 / T106;

[0243] E94 / K99 / T106;

[0244] E94 / E102;

[0245] E94 / T104;

[0246] E94 / T106;

[0247] E94 / P108;

[0248] E94 / P109;

[0249] E94 / T110;

[0250] E94 / S111;

[0251] E94 / T114;

[0252] E94 / R115;

[0253] E94 / Q117; and

[0254] E94 / E119.

[0255] The variant preferably comprises one or more of the following substitutions: D35N / E94D / T106K;

[0256] D35S / E94D / T106K;

[0257] K37Q / E94D / E102N / T106K;

[0258] K37S / E94D / E102S / T106K;

[0259] K37S / E94D / T104K / T106K;

[0260] K37N / E94D / T106K;

[0261] K37W / E94D / T106K;

[0262] K37S / E94D / T106K;

[0263] G43K / E94D / T106K;

[0264] K45N / V47K / E92D / E94N / T106K;

[0265] K45T / V47K / E94D / T106K;

[0266] K45N / S49K / E94N / E92D / T106K;

[0267] K45Y / S49K / E94D / T106K;

[0268] K45D / E94K / T106K;

[0269] K45R / E94D / T106K;

[0270] K45N / E94N / T106K;

[0271] K45Q / E94Q / T106K;

[0272] K45R / T106K;

[0273] V47S / E94D / T106K;

[0274] V47K / E94D / T106K;

[0275] V47N / V88T / E94D / T106K;

[0276] S49L / E94D / T106K;

[0277] T51K / E94D / T106K;

[0278] S74K / E94D;

[0279] S74R / E94D;

[0280] E76D / E94D;

[0281] E76N / E94D;

[0282] E76S / E94Q;

[0283] E76N / E94Q;

[0284] S78R / E94D;

[0285] S78K / E94D;

[0286] S78N / E94D;

[0287] S78Q / E94Q;

[0288] Y79S / E94Q;

[0289] S80K / E94D;

[0290] S80R / E94D;

[0291] S80N / E94D;

[0292] S80Q / E94Q;

[0293] S82K / E94D;

[0294] S82R / E94D;

[0295] S82N / E94D;

[0296] S82Q / E94Q;

[0297] S82K / E94D / T106K;

[0298] H83S / E94Q;

[0299] H83K / E94D / T106K;

[0300] E85N / E94D / T106K;

[0301] S86K / E94D;

[0302] V88I / M90A / E94D / T106K;

[0303] S89K / E94D;

[0304] M90K / E94D / T106K;

[0305] M90I / E94D / T106K;

[0306] T91K / E94D / T106K;

[0307] E92D / E94Q / T106K;

[0308] T93K / E94D / T106K;

[0309] E94Q / Y96D / T106K;

[0310] E94D / S98K / K99L / T106K;

[0311] E94D / K99Q / T106K;

[0312] E94D / E102N;

[0313] E94D / E102Q;

[0314] E94D / E102D;

[0315] E94D / T104R;

[0316] E94D / T104K;

[0317] E94Q / T104Q;

[0318] E94D / T106R;

[0319] E94D / T106K;

[0320] E94Q / T106Q;

[0321] E94Q / T106K;

[0322] E94D / P108K;

[0323] E94D / P108R;

[0324] E94D / P109K;

[0325] E94D / T110K;

[0326] E94D / T110R;

[0327] E94D / S111K;

[0328] E94D / T114K;

[0329] E94Q / R115S;

[0330] E94Q / Q117S; and

[0331] E94Q / N119S.

[0332] The variant can include any number of these substitutions and any combination of these substitutions.

[0333] The present application also provides a mutant lysenin monomer comprising a variant of the sequence set forth in SEQ ID NO: 2, wherein the monomer is capable of forming a pore, and wherein the variant includes one or more of the following substitutions:

[0334] E84R / E94D;

[0335] E84K / E94D;

[0336] E84N / E94D;

[0337] E84A / E94Q;

[0338] E84K / E94Q and

[0339] E94Q / D121S.

[0340] The variant can include any number of these substitutions and any combination of these substitutions.

[0341] The mutant monomers of the present application preferably include any combination of the modifications and / or substitutions described above. Exemplary combinations are disclosed in the Examples.

[0342] Chemical modification

[0343] In another embodiment, the present application also provides a mutant cytolysin monomer comprising a variant of the sequence set forth in SEQ ID NO: 2, wherein in the variant (a) 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 of the amino acids at positions 34 to 70 of SEQ ID NO: 2 have been deleted, or wherein the amino acids corresponding to positions 34 to 70 of SEQ ID NO: 2 have been deleted, and (b) 2, 4, 6, 8, 10, 12, 14, 16, 18, or 20 of the amino acids at positions 71 to 107 of SEQ ID NO: 2 have been deleted, or wherein the amino acid residues at positions corresponding to positions 71 to 107 of SEQ ID NO: 2 have been deleted.

[0344] The number of amino acids deleted from positions 34 to 70 can be different from the number of amino acids deleted from positions 71 to 107. Preferably, the number of amino acids deleted from positions 34 to 70 is the same as the number of amino acids deleted from positions 71 to 107.

[0345] Amino acids from positions 34 to 70 and amino acids from positions 71 to 107 can be deleted. The positions of the amino acids that have been deleted are preferably shown in one row of Table 1 or Table 2 or in more than one row of Table 1 and / or Table 2. For example, if D35 and V34 are deleted from positions 34 to 70, then T104 and I105 can be deleted from positions 71 to 107. Similarly, D35, V34, K37, and I38 can be deleted from positions 34 to 70, and E102, H103, T104, and I105 can be deleted from positions 71 to 107. This ensures that the beta-sheet structure lining of the barrel of the pore is maintained.

[0346] Table 1

[0347]

[0348]

[0349] Table 2

[0350]

[0351]

[0352]

[0353] Amino acids deleted from positions 34 to 70 and positions 71 to 107 need not be in one row of Table 1 or 2. For example, if D35 and V34 are deleted from positions 34 to 70, then I72 and E71 can be deleted from positions 71 to 107.

[0354] The amino acids deleted from positions 34 to 70 are preferably consecutive. The amino acids deleted from positions 71 to 107 are preferably consecutive. The amino acids deleted from positions 34 to 70 and from positions 71 to 107 are preferably consecutive.

[0355] The present invention preferably provides a mutant monomer, wherein the following are missing:

[0356] (i) N46 / V47 / T91 / T92; or

[0357] (ii)N48 / S49 / T91 / T92.

[0358] Those skilled in the art can identify other combinations of amino acids that may be omitted according to the present invention. The following discussion uses the residue numbers in SEQ ID NO: 2 (i.e., before any amino acid is omitted as described above).

[0359] The barrel deletion variant further preferably includes any modifications and / or substitutions discussed above or below, where appropriate. "Where appropriate" means whether the position remains in the mutant monomer after the barrel deletion.

[0360] Figure 1

[0361] In another aspect, the present invention provides chemically modified mutant cytosolic monomers. The mutant monomer can be any of the mutant monomers discussed above or below. Therefore, the mutant monomers of the present invention, such as variants of SEQ ID NO: 2 including modifications at one or more of the following positions: K37, G43, K45, V47, S49, T51, H83, V88, T91, T93, V95, Y96, S98, K99, V100, I101, P108, P109, T110, S111, K112, and T114, or variants including barrel deletions of the above, can be chemically modified according to the present invention, as discussed below.

[0362] The mutant monomer can be chemically modified to include any of the further modifications discussed below, i.e., one or more modifications that alter the ability of the monomer or, preferably, the region of the polynucleotide with which the region interacts, within the region including from about position 44 to about position 126 of SEQ ID NO: 2. These chemically modified monomers need not include the modifications of the present invention, i.e., need not include modifications at one or more of the following positions: K37, G43, K45, V47, S49, T51, H83, V88, T91, T93, V95, Y96, S98, K99, V100, 1101, P108, P109, T110, S111, K112, and T114. The chemically modified mutant monomer preferably includes a variant of SEQ ID NO: 2 that includes a substitution at one or more of the following positions of SEQ ID NO: 2: (a) E84, E85, E92, E97, and D126; (b) E85, E97, and D126 or (c) E84 and E92. Any number of the substitutions discussed below or any combination thereof can be made.

[0363] The mutant monomer can be chemically modified in any manner that reduces or shrinks the diameter of the barrel or channel of the pore formed by the monomer. This is discussed in more detail below.

[0364] The chemical modification is such that a chemical molecule is covalently attached to the mutant monomer, preferably. Any method known in the art can be used to covalently attach a chemical molecule to the mutant monomer. The chemical molecule is typically attached by chemical linkage.

[0365] The mutant monomer is chemically modified, preferably by attaching a molecule to one or more cysteines (cysteine linkage), attaching a molecule to one or more lysines, attaching a molecule to one or more unnatural amino acids, enzymatic modification of an epitope. If the chemical modifier is attached by a cysteine linkage, the one or more cysteines have been introduced to the mutant monomer, preferably by substitution. Suitable methods for performing such modifications are well known in the art. Suitable unnatural amino acids include, but are not limited to, 4-azido-L-phenylalanine (Faz) and Liu C.C. and Schultz P.G., Annu. Rev. Biochem., 2010, vol. 79, pp. 413-444 Further modifications any of the amino acids numbered 1 to 71 in

[0366] The mutant monomers can be chemically modified by attaching any molecule that has the effect of reducing or shrinking the diameter of the barrel with the pores formed by the monomers at any location or site. The mutant monomers can be chemically modified by attaching: (i) maleimides such as: 4-azidomaleimide, 1. N-(2-hydroxyethyl)maleimide, N-cyclohexylmaleimide, 1.3-maleimidopropionic acid, 1.1-4-aminophenyl-1H-pyrrole, 2,5, dione, 1.1-4-hydroxyphenyl-1H-pyrrole, 2,5, dione, N-ethylmaleimide, N-methoxycarbonylmaleimide, N-tert-butylmaleimide, N-(2-aminoethyl)maleimide, 3-maleimidyl-PROXYL, N-(4-chlorophenyl)maleimide, 1-[4-(dimethylamino)-3,5-dinitrophenyl]-1H-pyrrole-2,5-dione, N-[4-(2-benzimidazolyl)phenyl]maleimide, N-[4-(2-benzoxazolyl)phenyl]maleimide, N-(1-naphthyl)maleimide, N-(2,4-dimethylphenyl)maleimide, N-(2,4-difluorophenyl)maleimide, N-(3-chloro-p-tolyl)-maleimide, 1-(2-amino-ethyl)-pyrrole-2,5-dione hydrochloride, 1-cyclopentyl-3-methyl-2,5-dihydro-1H-pyrrole-2,5-dione, 1-(3-aminopropyl)-2,5-dihydro-1H-pyrrole-2,5-dione hydrochloride, 3-methyl-1-[2-oxo-2-(piperazin-1-yl)ethyl]-2,5-dihydro-1H-pyrrole-2,5-dione hydrochloride, 1-benzyl-2,5-dihydro-1H-pyrrole-2,5-dione, 3-methyl-1-(3,3,3-trifluoropropyl)-2,5-dihydro-1H-pyrrole-2,5-dione, 1-[4-(methylamino)cyclohexyl]-2,5-dihydro-1H-pyrrole-2,5-dione trifluoroacetate, SMILES O=C1C=CC(=O)N1CC=2C=CN=CC2, SMILES O=C1C=CC(=O)N1CN2CCNCC2, 1-benzyl-3-methyl-2,5-dihydro-1H-pyrrole-2,5-dione, 1-(2-fluorophenyl)-3-methyl-2,5-dihydro-1H-pyrrole-2,5-dione, N-(4-phenoxyphenyl)maleimide, N-(4-nitrophenyl)maleimide;(ii) iodoacetamides, such as 3-(2-iodoacetamido)-PROXYL, N-(cyclopropylmethyl)-2- iodoacetamide, 2-iodo-N-(2-phenylethyl)acetamide, 2-iodo-N-(2,2,2- trifluoroethyl)acetamide, N-(4-acetylphenyl)-2-iodoacetamide, N-(4- (aminosulfonyl)phenyl)-2-iodoacetamide, N-(l,3-benzothiazol-2-yl)-2- iodoacetamide, N-(2,6-diethylphenyl)-2-iodoacetamide, N-(2-benzoyl-4- chlorophenyl)-2-iodoacetamide; (iii) bromoacetamides, such as N-(4- (acetylamino)phenyl)-2-bromoacetamide, N-(2-acetylphenyl)-2- bromoacetamide, 2-bromo-N-(2-cyanophenyl)acetamide, 2-bromo-N-(3- (trifluoromethyl)phenyl)acetamide, N-(2-benzoylphenyl)-2-bromoacetamide, 2-bromo-N-(4-fluorophenyl)-3-methylbutanamide, N-benzyl 2-bromo-N- phenylpropanamide, N-(2-bromo-butyryl)-4-chloro-benzenesulfonamide, 2-bromo-N-methyl-N-phenylacetamide, 2-bromo-N-phenethyl-acetamide, 2- adamant- 1 -yl-2-bromo-N-cyclohexyl-acetamide, 2-bromo-N-(2- methylphenyl)butanamide, acetanilide p-bromoaniline; (iv) disulfides, such as: ALDRITHIOL-2, ALDRITHIOL-4, isopropyl disulfide, 1- (isobutyl disulfanyl)-2-methylpropane, dibenzyl disulfide, 4- aminophenyl disulfide, 3-(2-pyridyl disulfide)propionic acid, 3-(2-pyridyl disulfide)propionic acid hydrazide, 3-(2-pyridyl disulfide)propionic acid N- succinimidyl ester, am6amPDP1-βCD; and (v) thiols, such as: 4-phenylthiazole-2- thiol, Pulpald, 5,6,7,8-tetrahydro-quinazoline-2-thiol.

[0367] The mutant monomers can be chemically modified by attachment of polyethylene glycol (PEG), nucleic acids such as DNA, dyes, fluorophores, or chromophores. In some embodiments, the mutant monomers are chemically modified with molecular adapters that facilitate interactions between the pores comprising the monomers and target analytes, target nucleotides, or target polynucleotide sequences. The presence of the adapters improves the host-guest chemistry of the pores and nucleotides or polynucleotides, and thereby improves the sequencing ability of the pores formed by the mutant monomers.

[0368] The chemically modified mutant monomer preferably comprises a variant of the sequence set forth in SEQ ID NO: 2. Variants are defined as follows. The variants typically comprise one or more substitutions, wherein one or more residues are substituted with cysteine, lysine, or a non-natural amino acid.Non-natural amino acids include, but are not limited to: 4-azido-L-phenylalanine (Faz), 4-acetyl-L-phenylalanine, 3-acetyl-L-phenylalanine, 4-acetoacetyl-L-phenylalanine, O-allyl-L-tyrosine, 3-(phenylvinyl)-L-alanine, O-2-propyn-l-yl-L-tyrosine, 4-(dihydroxyboryl)-L-phenylalanine, 4-[(ethylsulfanyl)carbonyl]-L-phenylalanine, (2S)-2-amino-3-4-[(prop-2-ylsulfanyl)carbonyl]phenyl; propionic acid, (2S)-2-amino-3-4-[(2-amino-3-sulfanylpropionyl)amino]phenyl; propionic acid, O-methyl-L-tyrosine, 4-amino-L-phenylalanine, 4-cyano-L-phenylalanine, 3-cyano-L-phenylalanine, 4-fluoro-L-phenylalanine, 4-iodo-L-phenylalanine, 4-bromo-L-phenylalanine, O-(trifluoromethyl)tyrosine, 4-nitro-L-phenylalanine, 3-hydroxy-L-tyrosine, 3-amino-L-tyrosine, 3-iodo-L-tyrosine, 4-isopropyl-L-phenylalanine, 3-(2-naphthyl)-L-alanine, 4-phenyl-L-phenylalanine, (2S)-2-amino-3-(naphthalen-2-ylamino)propanoic acid, 6-(methylsulfanyl)norleucine, 6-oxo-L-lysine, D-tyrosine, (2R)-2-hydroxy-3-(4-hydroxyphenyl)propanoic acid, (2R)-2-aminooctanoate 3-(2,2'-bipyridin-5-yl)-D-alanine, 2-amino-3-(8-hydroxy-3-quinolinyl)propanoic acid, 4-benzoyl-L-phenylalanine, S-(2-nitrobenzyl)cysteine, (2R)-2-amino-3-[(2-nitrobenzyl)sulfanyl]propanoic acid, (2S)-2-amino-3-[(2-nitrobenzyl)oxy]propanoic acid, O-(4,5-dimethoxy-2-nitrobenzyl)-L-serine, (2S)-2-amino-6-([(2-nitrobenzyl)oxy]carbonyl; amino)hexanoic acid, O-(2-nitrobenzyl)-L-tyrosine, 2-nitrophenylalanine, 4-[(E)-phenyldiazenyl]-L-phenylalanine, 4-[3-(trifluoromethyl)-3H-diazepin-3-yl]-D-phenylalanine, 2-amino-3-[[5-(dimethylamino)-l-naphthyl]sulfonylamino]propanoic acid, (2S)-2-amino-4-(7-hydroxy-2-oxo-2H-chromen-4-yl)butanoic acid, (2S)-3-[(6-acetylnaphthaleneacetamide-2-yl)amino]-2-aminopropanoic acid, 4-(carboxymethyl)phenylalanine, 3-nitro-L-tyrosine, O-sulfo-L-tyrosine, (2R)-6-acetylamino-2-aminohexanoic acid, 1-methylhistidine, 2-aminononanoic acid, 2-aminodecanoic acid, -L-homocysteine, 5-sulfanylnorvaline, 6-sulfanyl-L-norleucine, 5-(methylthio)-L-norvaline, N.6 -[(2R,3R)-3-methyl-3,4-dihydro-2H-pyrrolo-2-yl]carbonyl; -L-lysine, N 6 -[(benzyloxy)carbonyl]lysine, (2S)-2-amino-6-[(cyclopentylcarbonyl)amino]hexanoic acid, N 6 -[(cyclopentyloxy)carbonyl]-L-lysine, (2S)-2-amino-6-[(2R)-tetrahydrofuran-2-ylcarbonyl]amino; hexanoic acid, (2S)-2-amino-8-[(2R,3S)-3-ethynyltetrahydrofuran-2-yl]-8-oxooctanoic acid, N 6 -(tert-Butoxycarbonyl)-L-lysine, (2S)-2-hydroxy-6-([(2-methyl-2-propyl)oxy]carbonyl;amino)hexanoic acid, N 6 -[(allyloxy)carbonyl]lysine, (2S)-2-amino-6-([(2-azidobenzyl)oxy]carbonyl;amino)hexanoic acid, N 6 -L-prolyl-L-lysine, (2S)-2-amino-6-[(prop-2-yn-1-yloxy)carbonyl]amino; hexanoic acid and N 6 -[(2-azidoethoxy)carbonyl]-L-lysine. The preferred non-natural amino acid is 4-azido-L-phenylalanine (Faz).

[0369] The mutant monomer can be chemically modified by attaching any molecule to any of the following positions in SEQ ID NO: 2: K37, V47, S49, T55, S86, E92, and E94. More preferably, the mutant monomer can be chemically modified by attaching any molecule to position E92 and / or E94. In one embodiment, the mutant monomer is chemically modified by attaching molecules to one or more cysteine ​​residues (cysteine ​​linkages), one or more lysine residues, or one or more non-natural amino acids at these positions. The mutant monomer preferably comprises a variant of the sequence shown in SEQ ID NO: 2, which includes one or more of K37C, V47C, S49C, T55C, S86C, E92C, and E94C, wherein one or more molecules are attached to the one or more introduced cysteine ​​residues. The mutant monomer more preferably comprises a variant of the sequence shown in SEQ ID NO: 2, which includes E92C and / or E94C, wherein one or more molecules are attached to the one or more introduced cysteine ​​residues. In each of these two preferred embodiments, the one or more cysteines (Cs) may be replaced by one or more lysines or one or more non-natural amino acids such as one or more Faz.

[0370] The reactivity of cysteine residues can be enhanced by modification of the adjacent residues. For example, the basic groups flanking arginine, histidine or lysine residues will change the pKa of the cysteine thiol to a more reactive S - The reactivity of cysteine residues can be protected by thiol protecting groups such as dTNB. These can be reacted with one or more cysteine residues of the mutant monomer prior to attachment of the linker.

[0371] The molecule can be attached directly to the mutant monomer. Preferably, the molecule is attached to the mutant monomer using a linker such as a chemical crosslinker or a peptide linker. Suitable chemical crosslinkers are well known in the art. Preferred crosslinkers include 3-(pyridin-2- yldisulfanyl)propanoic acid 2,5-dioxopyrrolidin-1 -yl ester, 4-(pyridin-2- yldisulfanyl)butanoic acid 2,5-dioxopyrrolidin-1 -yl ester and 8-(pyridin-2- yldisulfanyl)octanoic acid 2,5-dioxopyrrolidin-1 -yl ester. The most preferred crosslinker is 3-(2-pyridyldithio)propionic acid succinimidyl ester (SPDP). Typically, the molecule is covalently linked to the bifunctional crosslinker before the molecule / crosslinker complex is covalently linked to the mutant monomer, but it is also possible to covalently link the bifunctional crosslinker to the monomer before the bifunctional crosslinker / monomer complex is attached to the molecule.

[0372] Preferably, the linker is resistant to dithiothreitol (DTT). Suitable linkers include, but are not limited to, iodoacetamide-based and maleimide-based linkers.

[0373] The advantages of pores comprising chemically modified mutant monomers of the application are discussed in more detail below.

[0374] Further chemical modifications that can be made in accordance with the application are discussed below.

[0375] Figure 1

[0376] Any of the mutant monomers discussed above can include further modifications within the region of about position 44 to about position 126 of SEQ ID NO: 2 (i.e. where the relevant amino position remains in the mutant monomer or is not modified / substituted by another amino acid) where appropriate. At least part of this region typically contributes to the transmembrane region of the cytolysin. At least part of this region typically contributes to the barrel or channel of the cytolysin. At least part of this region typically contributes to the inner wall or lining of the cytolysin.

[0377] The transmembrane region of cytolysin has been identified as positions 44 to 67 of SEQ ID NO: 2 (De Colbis et al., Structure, 2012, vol. 20, pages 1498 to 1507).

[0378] The variant preferably comprises one or more modifications within the region from about position 44 to about position 126 of SEQ ID NO: 2 that alter the ability of the monomer or preferably the region to interact with a polynucleotide. The interaction between the monomer and the polynucleotide can be increased or decreased. Increasing the interaction between the monomer and the polynucleotide will, for example, facilitate capture of the polynucleotide through a pore comprising the mutant monomer. Decreasing the interaction between the region and the polynucleotide will, for example, improve recognition or discrimination of the polynucleotide. Recognition or discrimination of the polynucleotide can be improved by decreasing the variability in the state of the pore comprising the mutant monomer, which increases the signal-to-noise ratio, and / or decreasing the number of nucleotides in the polynucleotide that contribute to the current when the polynucleotide moves through the pore comprising the mutant monomer.

[0379] The ability of the monomer to interact with a polynucleotide can be determined using methods well known in the art. The monomer can interact with the polynucleotide in any manner, for example, through non-covalent interactions such as hydrophobic interactions, hydrogen bonding, van der Waals forces, pi (pi)-cation interactions, or electrostatic forces. For example, the ability of the region to bind to a polynucleotide can be measured using a conventional binding assay. Suitable assays include, but are not limited to, fluorescence-based binding assays, nuclear magnetic resonance (NMR), isothermal titration calorimetry (ITC), or electron spin resonance (ESR) spectroscopy. Alternatively, the ability of a pore comprising one or more of the mutant monomers to interact with a polynucleotide can be determined using any of the methods discussed above or below. Preferred assays are described in the examples.

[0380] One or more modifications can be made within the region from about position 44 to about position 126 of SEQ ID NO: 2. Preferably, the one or more modifications are made within any of the following regions: from about position 40 to about position 125, from about position 50 to about position 120, from about position 60 to about position 110, and from about position 70 to about position 100. If the one or more modifications are made to improve polynucleotide capture, more preferably the modifications are made within any of the following regions: from about position 44 to about position 103, from about position 68 to about position 103, from about position 84 to about position 103, from about position 44 to about position 97, from about position 68 to about position 97, or from about position 84 to about position 97. If the one or more modifications are made to improve polynucleotide recognition or discrimination, more preferably the modifications are made within any of the following regions: from about position 44 to about position 109, from about position 44 to about position 97, or from about position 48 to about position 88. Preferably, the region is from about position 44 to about position 67 of SEQ ID NO: 2.

[0381] If the one or more modifications are intended to improve polynucleotide recognition or discrimination, preferably the modifications are made in addition to the one or more modifications for improving polynucleotide capture. This allows the pore formed by the mutant monomer to efficiently capture a polynucleotide and then to characterize the polynucleotide, such as to estimate its sequence, as discussed below.

[0382] Modifications of protein nanopores that alter the ability of the protein nanopore to interact with a polynucleotide, particularly to improve its ability to capture and / or recognize or discriminate a polynucleotide, are well documented in the art. For example, such modifications are disclosed in WO 2010 / 034018 and WO 2010 / 055307. Similar modifications can be made to the cytolysin monomers according to the present application.

[0383] Any number of modifications can be made, such as 1, 2, 5, 10, 15, 20, 30, or more modifications. Any one or more modifications can be made so long as the ability of the monomer to interact with a polynucleotide is altered. Suitable modifications include, but are not limited to, amino acid substitutions, amino acid additions, and amino acid deletions. Preferably, the one or more modifications is one or more substitutions. This is discussed in more detail below.

[0384] The one or more modifications preferably (a) alter the steric effect of the monomer or preferably the region; (b) alter the net charge of the monomer or preferably the region; (c) alter the ability of the monomer or preferably the region to hydrogen bond with a polynucleotide; (d) introduce or remove a chemical group that interacts through a delocalized electronic pi system and / or (e) alter the structure of the monomer or preferably the region. The one or more modifications more preferably results in any combination of (a) through (e), such as (a) and (b); (a) and (c); (a) and (d); (a) and (e); (b) and (c); (b) and (d); (b) and (e); (c) and (d); (c) and (e); (d) and (e), (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (c) and (d); (a), (c) and (e); (a), (d) and (e); (b), (c) and (d); (b), (c) and (e); (b), (d) and (e); (c), (d) and (e); (a), (b), (c) and (d); (a), (b), (c) and (e); (a), (b), (d) and (e); (a), (c), (d) and (e); (b), (c), (d) and (e); and (a), (b), (c) and (d).

[0385] For (a), the steric effect of the monomer can be increased or decreased. Any method of altering the steric effect can be used in accordance with the present application. The introduction of bulky residues such as phenylalanine (F), tryptophan (W), tyrosine (Y), or histidine (H) increases the steric effect of the monomer. The one or more modifications are preferably the introduction of one or more of F, W, Y, and H. Any combination of F, W, Y, and H can be introduced. The one or more of F, W, Y, and H can be introduced by addition. The one or more of F, W, Y, and H are preferably introduced by substitution. Suitable positions for introducing such residues are discussed in more detail below.

[0386] The removal of bulky residues such as phenylalanine (F), tryptophan (W), tyrosine (Y), or histidine (H) conversely decreases the steric effect of the monomer. The one or more modifications are preferably the removal of one or more of F, W, Y, and H. Any combination of F, W, Y, and H can be removed. The one or more of F, W, Y, and H can be removed by deletion. The one or more of F, W, Y, and H are preferably removed by substitution with residues having smaller side groups such as serine (S), threonine (T), alanine (A), and valine (V).

[0387] For (b), the net charge can be altered in any manner. Preferably, the net positive charge is increased or decreased. The net positive charge can be increased in any manner. Preferably, the net positive charge is increased by the introduction, preferably by substitution, of one or more positively charged amino acids and / or neutralization, preferably by substitution, of one or more negative charges.

[0388] Preferably, the net positive charge is increased by the introduction of one or more positively charged amino acids. The one or more positively charged amino acids can be introduced by addition. Preferably, the one or more positively charged amino acids are introduced by substitution. A positively charged amino acid is one having a net positive charge. The one or more positively charged amino acids can be naturally occurring or non-naturally occurring. The positively charged amino acid can be synthetic or modified. For example, a modified amino acid having a net positive charge can be specifically designed for the present application. Various different types of modifications to amino acids are well known in the art.

[0389] Preferred naturally occurring positively charged amino acids include, but are not limited to, histidine (H), lysine (K), and arginine (R). The one or more modifications are preferably the introduction of one or more of H, K, and R. Any number of H, K, and R and any combination thereof can be introduced. The one or more of H, K, and R can be introduced by addition. The one or more of H, K, and R are preferably introduced by substitution. Suitable positions for introducing such residues are discussed in more detail below.

[0390] Methods for adding or substituting non-naturally occurring amino acids are also well known in the art. For example, non-naturally occurring amino acids can be introduced by including synthetic aminoacyl-tRNAs in the IVTT system used to express the pore. Alternatively, non-naturally occurring amino acids can be introduced by expressing the mutant monomer in E. coli that is auxotrophic for particular amino acids in the presence of synthetic (i.e., non-naturally occurring) analogs of those particular amino acids. If the pore is produced using partial peptide synthesis, non-naturally occurring amino acids can also be produced by naked ligation.

[0391] Methods for adding or substituting non-naturally occurring amino acids are also well known in the art. For example, non-naturally occurring amino acids can be introduced by including synthetic aminoacyl-tRNAs in the IVTT system used to express the pore. Alternatively, non-naturally occurring amino acids can be introduced by expressing the mutant monomer in E. coli that is auxotrophic for particular amino acids in the presence of synthetic (i.e., non-naturally occurring) analogs of those particular amino acids. If the pore is produced using partial peptide synthesis, non-naturally occurring amino acids can also be produced by naked ligation.

[0392] Any amino acid can be substituted with a positively charged amino acid. One or more uncharged amino acids, non-polar amino acids, and / or aromatic amino acids can be substituted with one or more positively charged amino acids. Uncharged amino acids have no net charge. Suitable uncharged amino acids include, but are not limited to, cysteine (C), serine (S), threonine (T), methionine (M), asparagine (N), and glutamine (Q). Non-polar amino acids have non-polar side chains. Suitable non-polar amino acids include, but are not limited to, glycine (G), alanine (A), proline (P), isoleucine (I), leucine (L), and valine (V). Aromatic amino acids have aromatic side chains. Suitable aromatic amino acids include, but are not limited to, histidine (H), phenylalanine (F), tryptophan (W), and tyrosine (Y). Preferably, one or more negatively charged amino acids are substituted with one or more positively charged amino acids. Suitable negatively charged amino acids include, but are not limited to, aspartic acid (D) and glutamic acid (E).

[0393] Preferred substitutions include, but are not limited to, using K for E, using R for M, using H for M, using K for M, using R for D, using H for D, using K for D, using R for E, using H for E, using R for N, using R for T, and using R for G. Most preferably, K is used for E.

[0394] Any number of positively charged amino acids can be introduced or substituted. For example, 1, 2, 5, 10, 15, 20, 25, 30, or more positively charged amino acids can be introduced or substituted.

[0395] More preferably, the net positive charge is increased by neutralizing one or more negative charges. The one or more negative charges can be neutralized by substituting one or more negatively charged amino acids with one or more uncharged, nonpolar, and / or aromatic amino acids. Removing a negative charge increases the net positive charge. The uncharged, nonpolar, and / or aromatic amino acids can be naturally occurring or non-naturally occurring. They can be synthetic or modified. Suitable uncharged, nonpolar, and aromatic amino acids are discussed above. Preferred substitutions include, but are not limited to: substituting E with Q, substituting E with S, substituting E with A, substituting D with Q, substituting E with N, substituting D with N, substituting D with G, and substituting D with S.

[0396] Any number of uncharged, nonpolar, and / or aromatic amino acids and any combination thereof can be substituted. For example, 1, 2, 5, 10, 15, 20, 25, or 30 or more uncharged, nonpolar, and / or aromatic amino acids can be substituted. The negatively charged amino acid can be substituted with: (1) an uncharged amino acid; (2) a nonpolar amino acid; (3) an aromatic amino acid; (4) an uncharged and nonpolar amino acid; (5) an uncharged and aromatic amino acid; and (5) a nonpolar and aromatic amino acid; or (6) an uncharged, nonpolar, and aromatic amino acid.

[0397] The one or more negative charges can be neutralized by introducing one or more positively charged amino acids near, such as within 1, 2, 3, or 4 amino acids of, or adjacent to, the one or more negatively charged amino acids. Examples of positively charged and negatively charged amino acids are discussed above. The positively charged amino acids can be introduced in any of the manners discussed above, such as by substitution.

[0398] Preferably, the net positive charge is decreased by introducing one or more negatively charged amino acids and / or neutralizing one or more positive charges. Ways in which this can be accomplished will become clear in light of the discussion above with reference to increasing the net positive charge. All of the embodiments discussed above with reference to increasing the net positive charge are equally applicable to decreasing the net positive charge, except that the charges are changed in the opposite manner. In particular, the one or more positive charges are preferably neutralized by substituting one or more positively charged amino acids with one or more uncharged, nonpolar, and / or aromatic amino acids and / or by introducing one or more negatively charged amino acids near, such as within 1, 2, 3, or 4 amino acids of, or adjacent to, the one or more positively charged amino acids.

[0399] Preferably the net negative charge is increased or decreased. All of the above embodiments discussed above with reference to increasing or decreasing the net positive charge are equally applicable to decreasing or increasing the net negative charge, respectively.

[0400] For (c), the ability of the monomer to hydrogen bond can be altered in any way. The introduction of serine (S), threonine (T), asparagine (N), glutamine (Q), tyrosine (Y) or histidine (H) increases the hydrogen bonding ability of the monomer. Preferably the one or more modifications are the introduction of one or more of S, T, N, Q, Y and H. Any combination of S, T, N, Q, Y and H can be introduced. The one or more of S, T, N, Q, Y and H can be introduced by addition. Preferably the one or more of S, T, N, Q, Y and H is introduced by substitution. Suitable positions for introducing such residues are discussed in more detail below.

[0401] The removal of serine (S), threonine (T), asparagine (N), glutamine (Q), tyrosine (Y) or histidine (H) decreases the hydrogen bonding ability of the monomer. Preferably the one or more modifications are the removal of one or more of S, T, N, Q, Y and H. Any combination of S, T, N, Q, Y and H can be removed. The one or more of S, T, N, Q, Y and H can be removed by deletion. Preferably the one or more of S, T, N, Q, Y and H is removed by substitution with other amino acids that are less good at hydrogen bonding such as alanine (A), valine (V), isoleucine (I) and leucine (L).

[0402] For (d), the introduction of aromatic residues such as phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H) also increases pi-stacking in the monomer. The removal of aromatic residues such as phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H) also decreases pi-stacking in the monomer. Such amino acids can be introduced or removed as discussed above with reference to (a).

[0403] For (e), one or more modifications can be made to the structure of the monomer in accordance with the application. For example, one or more loop regions can be removed, shortened or amplified. This generally facilitates the entry or exit of the polynucleotide from the pore. The one or more loop regions can be on the cis side of the pore, on the trans side of the pore or on both sides of the pore. Alternatively, one or more regions of the amino and / or carboxy termini of the pore can be amplified or deleted. This generally changes the size and / or charge of the pore.

[0404] It will be clear from the above discussion that the introduction of certain amino acids will enhance the ability of the monomer to interact with the polynucleotide through more than one mechanism. For example, the use of H in place of E will increase the net positive charge (by neutralizing the negative charge) according to (b) and will increase the ability of the monomer to hydrogen bond according to (c).

[0405] The variant preferably 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, Q117, N119, D121, and D126. The variant preferably comprises a substitution at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, or 34 of those positions. The variant preferably comprises a substitution at one or more of the following positions of SEQ ID NO: 2: D68, E71, S74, E76, S78, S80, S82, E84, E85, S86, Q87, S89, E92, E102, T104, T106, R115, Q117, N119, and D121. The variant preferably comprises a substitution at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of those positions.

[0406] The variant preferably comprises a substitution at one or more of the following positions of SEQ ID NO: 2: (a) E84, E85, E92, E97, and D126; (b) E85, E97, and D126 or (c) E84 and E92. The amino acid substituted into the variant can be a naturally occurring derivative or a non-naturally occurring derivative. The amino acid substituted into the variant can be a D-amino acid. Each of the positions listed above can be substituted with an asparagine (N), a serine (S), a glutamine (Q), an arginine (R), a glycine (G), a tyrosine (Y), an aspartic acid (D), a leucine (L), a lysine (K), or an alanine (A).

[0407] The variant preferably comprises at least one of the following mutations of SEQ ID NO: 2:

[0408] (a) serine (S) at position 44;

[0409] (b) serine (S) at position 46;

[0410] (c) serine (S) at position 48;

[0411] (d) serine (S) at position 52;

[0412] (e) serine (S) at position 58;

[0413] (f) serine (S) at position 68;

[0414] (g) serine (S) at position 70;

[0415] (h) serine (S) at position 71;

[0416] (i) serine (S) at position 76;

[0417] (j) serine (S) at position 79;

[0418] (k) serine (S) at position 81;

[0419] (l) serine (S), aspartic acid (D), or glutamine (Q) at position 84;

[0420] (m) serine (S) or lysine (K) at position 85;

[0421] (n) serine (S) at position 87;

[0422] (o) serine (S) at position 90;

[0423] (p) asparagine (N) or glutamine (Q) at position 92;

[0424] (q) serine (S) or asparagine (N) at position 94;

[0425] (r) serine (S) or asparagine (N) at position 97;

[0426] (s) serine (S) at position 102;

[0427] (t) serine (S) at position 103;

[0428] (u) asparagine (N) or serine (S) at position 121;

[0429] (v) serine (S) at position 50;

[0430] (w) Asparagine (N) or Serine (S) at position 94;

[0431] (x) Asparagine (N) or Serine (S) at position 97;

[0432] (y) Serine (S) or Asparagine (N) at position 121 ;

[0433] (z) Asparagine (N) or Glutamine (Q) at position 126; and

[0434] (aa) Serine (S) or Asparagine (N) at position 128.

[0435] The variant can comprise any number of mutations (a) to (aa), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 or 27 of the mutations. Preferred combinations of mutations are discussed below. The amino acids introduced into the variant can be naturally occurring derivatives or non-naturally occurring derivatives. The amino acids introduced into the variant can be D-amino acids.

[0436] The variant preferably includes at least one of the following mutations of SEQ ID NO: 2:

[0437] (a) Serine (S) at position 68;

[0438] (b) Serine (S) at position 71 ;

[0439] (c) Serine (S) at position 76;

[0440] (d) Aspartic acid (D) or Glutamine (Q) at position 84;

[0441] (e) Lysine (K) at position 85;

[0442] (f) Asparagine (N) or Glutamine (Q) at position 92;

[0443] (g) Serine (S) at position 102;

[0444] (h) Asparagine (N) or Serine (S) at position 121 ;

[0445] (i) Serine (S) at position 50;

[0446] (j) Asparagine (N) or Serine (S) at position 94;

[0447] (k) Asparagine (N) or Serine (S) at position 97; and

[0448] (l) Asparagine (N) or Glutamine (Q) at position 126.

[0449] The variant can comprise any number of mutations (a) through (1), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 of the mutations. Preferred combinations of mutations are discussed below. The amino acids introduced into the variant can be naturally-occurring derivatives or non-naturally-occurring derivatives. The amino acids introduced into the variant can be D-amino acids.

[0450] The variant can comprise one or more additional modifications outside the region of about position 44 to about position 126 of SEQ ID NO: 2 that, in combination with the modifications in the region discussed above, improve polynucleotide capture and / or improve polynucleotide recognition or discrimination. Suitable modifications include, but are not limited to, substitutions at one or more of D35, E128, E135, E134, and E167. In particular, removing negative charge by substituting E at one or more of positions 128, 135, 134, and 167 improves polynucleotide capture. The E at one or more of these positions can be substituted in any of the ways discussed above. Preferably, all of E128, E135, E134, and E167 are substituted as discussed above. Preferably, the E is substituted with an A. In other words, the variant preferably includes one or more or all of E128A, E135A, E134A, and E167A. Another preferred substitution is D35Q.

[0451] In preferred embodiments, the variant includes the following substitutions in SEQ ID NO: 2:

[0452] i. one or more, such as both, of E84D and E85K;

[0453] ii. one or more, such as 2, 3, 4, 5, or 6, of E84Q, E85K, E92Q, E97S, D126G, and E167A;

[0454] iii. one or more, such as 2, 3, 4, or 5, of E92N, E94N, E97N, D121N, and D126N;

[0455] iv. one or more, such as 2, 3, 4, 5, or 6, of E92N, E94N, E97N, D121N, D126N, and E128N;

[0456] v. one or more, such as 2, 3, 4, 5, 6, or 7 of E76S, E84Q, E85K, E92Q, E97S, D126G, and E167A;

[0457] vi. one or more, such as 2, 3, 4, 5, 6, or 7 of E84Q, E85K, E92Q, E97S, D126G, E167A, and E50S;

[0458] vii. one or more, such as 2, 3, 4, 5, 6, or 7 of E84Q, E85K, E92Q, E97S, D126G, E167A, and E71S;

[0459] viii. one or more, such as 2, 3, 4, 5, 6, or 7 of E84Q, E85K, E92Q, E97S, D126G, E167A, and E94S;

[0460] ix. one or more, such as 2, 3, 4, 5, 6, or 7 of E84Q, E85K, E92Q, E97S, D126G, E167A, and E102S;

[0461] x. one or more, such as 2, 3, 4, 5, 6, or 7 of E84Q, E85K, E92Q, E97S, D126G, E167A, and E128S;

[0462] xi. one or more, such as 2, 3, 4, 5, 6, or 7 of E84Q, E85K, E92Q, E97S, D126G, E167A, and E135S;

[0463] xii. one or more, such as 2, 3, 4, 5, 6, or 7 of E84Q, E85K, E92Q, E97S, D126G, E167A, and D68S;

[0464] xiii. one or more, such as 2, 3, 4, 5, 6, or 7 of E84Q, E85K, E92Q, E97S, D126G, E167A, and D121S;

[0465] xiv. one or more, such as 2, 3, 4, 5, 6, or 7 of E84Q, E85K, E92Q, E97S, D126G, E167A, and D134S;

[0466] xv. one or more, such as 2 or 3 of E84D, E85K, and E92Q;

[0467] xvi. one or more, such as 1, 2, 3, 4, 5, or 6 of E84Q, E85K, E92Q, E97S, D126G, and E135S;

[0468] xvii. one or more, such as 1, 2, 3, 4, or 5 of E85K, E92Q, E94S, E97S, and D126G;

[0469] xviii. one or more, such as 1, 2, 3, 4, or 5 of E76S, E85K, E92Q, E97S, and D126G;

[0470] xix. one or more, such as 1, 2, 3, 4, or 5 of E71S, E85K, E92Q, E97S, and D126G;

[0471] xx. one or more, such as 1, 2, 3, 4, or 5 of D68S, E85K, E92Q, E97S, and D126G;

[0472] xxi. one or more, such as 1, 2, 3, or 4 of E85K, E92Q, E97S, and D126G;

[0473] xxii. one or more, such as 1, 2, 3, 4, 5, or 6 of E84Q, E85K, E92Q, E97S, H103S, and D126G;

[0474] xxiii. one or more, such as 1, 2, 3, 4, 5, or 6 of E84Q, E85K, M90S, E92Q, E97S, and D126G;

[0475] xxiv. one or more, such as 1, 2, 3, 4, 5, or 6 of E84Q, Q87S, E85K, E92Q, E97S, and D126G;

[0476] xxv. one or more, such as 1, 2, 3, 4, or 5 of E84Q, E85S, E92Q, E97S, and D126G;

[0477] xxvi. one or more, such as 1, 2, 3, 4, or 5 of E84S, E85K, E92Q, E97S, and D126G;

[0478] xxvii. one or more, such as 1, 2, 3, 4, 5, or 6 of H81S, E84Q, E85K, E92Q, E97S, and D126G;

[0479] xxviii. one or more, such as 1, 2, 3, 4, 5, or 6, of Y79S, E84Q, E85K, E92Q, E97S, and D126G;

[0480] xxix. one or more, such as 1, 2, 3, 4, 5, or 6, of F70S, E84Q, E85K, E92Q, E97S, and D126G;

[0481] xxx. one or more, such as 1, 2, 3, 4, 5, or 6, of H58S, E84Q, E85K, E92Q, E97S, and D126G;

[0482] xxxi. one or more, such as 1, 2, 3, 4, 5, or 6, of R52S, E84Q, E85K, E92Q, E97S, and D126G;

[0483] xxxii. one or more, such as 1, 2, 3, 4, 5, or 6, of N48S, E84Q, E85K, E92Q, E97S, and D126G;

[0484] xxxiii. one or more, such as 1, 2, 3, 4, 5, or 6, of N46S, E84Q, E85K, E92Q, E97S, and D126G;

[0485] xxxiv. one or more, such as 1, 2, 3, 4, 5, or 6, of M44S, E84Q, E85K, E92Q, E97S, and D126G;

[0486] xxxv. one or more, such as 1 or 2, of E92Q and E97S;

[0487] xxxvi. one or more, such as 1, 2, 3, or 4, of E84Q, E85K, E92Q, and E97S;

[0488] xxxvii. one or more, such as 1 or 2, of E84Q and E85K;

[0489] xxxviii. one or more, such as 1, 2, or 3, of E84Q, E85K, and D126G;

[0490] xxxix. one or more, such as 1, 2, 3, or 4, of E84Q, E85K, D126G, and E167A;

[0491] xl. one or more, such as 1, 2, or 3, of E92Q, E97S, and D126G;

[0492] xli. one or more, such as 1, 2, 3, 4, or 5 of E84Q, E85K, E92Q, E97S, and D126G;

[0493] xlii. one or more, such as 1, 2, 3, 4, or 5 of E84Q, E85K, E92Q, E97S, and E167A;

[0494] xliii. one or more, such as 1, 2, 3, 4, or 5 of E84Q, E85K, E92Q, D126G, and E167A;

[0495] xliv. one or more, such as 1, 2, 3, 4, or 5 of E84Q, E85K, E97S, D126G, and E167A;

[0496] xlv. one or more, such as 1, 2, 3, 4, or 5 of E84Q, E92Q, E97S, D126G, and E167A;

[0497] xlvi. one or more, such as 1, 2, 3, 4, or 5 of E85K, E92Q, E97S, D126G, and E167A;

[0498] xlvii. one or more, such as 1, 2, or 3 of E84D, E85K, and E92Q;

[0499] xlviii. one or more, such as 1, 2, 3, 4, 5, 6, or 7 of E84Q, E85K, E92Q, E97S, D126G, E167A, and D121S;

[0500] xlix. one or more, such as 1, 2, 3, 4, 5, 6, or 7 of E84Q, E85K, E92Q, E97S, D126G, E167A, and D68S;

[0501] l. one or more, such as 1, 2, 3, 4, 5, 6, or 7 of E84Q, E85K, E92Q, E97S, D126G, E167A, and E135S;

[0502] li. one or more, such as 1, 2, 3, 4, 5, 6, or 7 of E84Q, E85K, E92Q, E97S, D126G, E167A, and E128S;

[0503] lii. one or more, such as 1, 2, 3, 4, 5, 6, or 7 of E84Q, E85K, E92Q, E97S, D126G, E167A, and E102S;

[0504] liii. one or more, such as 1, 2, 3, 4, 5, 6, or 7 of E84Q, E85K, E92Q, E97S, D126G, E167A, and E94S;

[0505] liv. one or more, such as 1, 2, 3, 4, 5, 6, or 7 of E84Q, E85K, E92Q, E97S, D126G, E167A, and E71S;

[0506] lv. one or more, such as 1, 2, 3, 4, 5, 6, or 7 of E84Q, E85K, E92Q, E97S, D126G, E167A, and E50S;

[0507] lvi. one or more, such as 1, 2, 3, 4, 5, 6, or 7 of E76S, E84Q, E85K, E92Q, E97S, D126G, and E167A;

[0508] lvii. one or more, such as 1, 2, 3, 4, 5, or 6 of E92N, E94N, E97N, D121N, D126N, and E128N;

[0509] lviii. one or more, such as 1, 2, 3, 4, or 5 of E92N, E94N, E97N, D121N, and D126N; or

[0510] lix. one or more, such as 1, 2, 3, 4, 5, or 6 of E84Q, E85K, E92Q, E97S, D126G, and E167A

[0511] In the foregoing, the first letter refers to the amino acid in SEQ ID NO: 2 that is being replaced, the number is the position in SEQ ID NO: 2, and the second letter refers to the amino acid that will be used to replace the first. Thus, E84D means using an aspartic acid (D) to replace the glutamic acid (E) at position 84.

[0512] The variant can comprise any number of the substitutions in any one of i through Ix, such as 1, 2, 3, 4, 5, 6, or 7. The variant preferably comprises all of the substitutions indicated in any one of i through Ix, above.

[0513] In preferred embodiments, the variant comprises a substitution in any of i to xv above. The variant can comprise any number of substitutions in any of i to xv, such as 1, 2, 3, 4, 5, 6 or 7. The variant preferably comprises all of the substitutions set out in any of i to xv above.

[0514] If the one or more modifications are intended to improve the ability of the monomer to recognize or discriminate between polynucleotides, preferably the one or more modifications can be made in addition to the modifications discussed above to improve polynucleotide capture such as E84Q, E85K, E92Q, E97S, D126G and E167A.

[0515] The one or more modifications made to the identified region can involve substituting one or more amino acids in the region with the amino acid present at the corresponding position in the homolog or paralog of the lysenin. Four examples of homologs of lysenin are set out in SEQ ID NOs: 14 to 17. An advantage of such substitutions is that they can result in a pore-forming mutant monomer, as the homolog monomers also form pores. For example, mutations can be made at any one or more of the positions in SEQ ID NO: 2 that are different between SEQ ID NO: 2 and any of SEQ ID NOs: 14 to 17. Such mutations can be a substitution of an amino acid in SEQ ID NO: 2 with the amino acid from the corresponding position in any of SEQ ID NOs: 14 to 17, preferably SEQ ID NOs: 14 to 16. Alternatively, the mutation at any of these positions can be a substitution with any amino acid, or can be a deletion or insertion mutation, such as a substitution, deletion or insertion of 1 to 30 amino acids, such as 2 to 20, 3 to 10 or 4 to 8 amino acids. In addition to the mutations disclosed herein and the mutations disclosed in the prior art, for example in 2013 / 153359, the amino acids that are conserved or identical between SEQ ID NO: 2 and all of SEQ ID NOs: 14 to 17, more preferably all of SEQ ID NOs: 14 to 16, are preferably conserved or present in the variants of the application. However, a conservative mutation can be made at any one or more of these positions that are conserved or identical between SEQ ID NO: 2 and all of SEQ ID NOs: 14 to 17 or more preferably SEQ ID NOs: 14 to 16.

[0516] The present invention provides a cytolysin mutant monomer comprising any one or more of the amino acids substituted into the particular position of SEQ ID NO: 2 at a position described herein as corresponding to a particular position in the structure of a cytolysin monomer. The corresponding position can be determined by standard techniques in the art. For example, the PILEUP and BLAST algorithms mentioned above can be used to align the sequence of a cytolysin monomer with SEQ ID NO: 2 and thus identify the corresponding residue.

[0517] The mutant monomer typically retains the ability to form the same 3D structure as the wild type cytolysin monomer, such as the same 3D structure as a cytolysin monomer having the sequence of SEQ ID NO: 2. The 3D structure of a cytolysin monomer is known in the art and is disclosed in, for example, De Colbis et al., Structure, 2012, vol. 20, pp. 1498-1507. The mutant monomer typically retains the ability to form a homo-oligomeric pore and / or a hetero-oligomeric pore with other cytolysin monomers. When present in a pore, the mutant monomer typically retains the ability to refold to form the same 3D structure as the wild type cytolysin monomer. The 3D structure of a cytolysin monomer in a cytolysin pore is shown in Figure 7 herein. Any number of mutations can be made in the wild type cytolysin sequence in addition to the mutations described herein, such as 2 to 100, 3 to 80, 4 to 70, 5 to 60, 10 to 50, or 20 to 40, provided that the cytolysin mutant monomer retains one or more of the improved properties conferred on it by the mutations of the present invention.

[0518] Typically, when a cytolysin monomer assembles with other identical mutant monomers or with different cytolysin mutant monomers to form a pore, the cytolysin monomer will retain the ability to contribute two beta sheets to the barrel of the cytolysin pore.

[0519] The variant further preferably comprises one or more or, where appropriate, all of E84Q / E85K / E92Q / E97S / D126G. "Where appropriate" means whether these positions are still present in the mutant monomer or are not modified by a different amino acid.

[0520] In addition to the specific mutations discussed above, variants can comprise other mutations. These mutations do not necessarily enhance the ability of the monomer to interact with the polynucleotide. Mutations can facilitate, for example, expression and / or purification. Throughout the length of the amino acid sequence of SEQ ID NO: 2, a variant will preferably be at least 50% homologous to the sequence based on amino acid similarity or identity. More preferably, a variant can be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and more preferably at least 95%, 97%, or 99% homologous to the amino acid sequence of SEQ ID NO: 2 throughout the sequence based on amino acid similarity or identity. There can be at least 80%, for example, at least 85%, 90%, or 95% amino acid similarity or identity ("hard homology") over a stretch of 100 or more, for example, 125, 150, 175, or 200 or more contiguous amino acids.

[0521] Standard methods in the art can be used to determine homology. For example, the UWGCG package provides the BESTFIT program which can be used to calculate homology, for example using its default settings (Devereux et al. (1984) Nucleic Acids Research 12, pp. 387-395). The PILEUP and BLAST algorithms can be used to calculate homology or to align sequences (recognizing identical or corresponding sequences, generally according to their default settings), as described in Altschul S.F. (1993) J Mol Evol, 36, pp. 290-300; Altschul, S.F. et al. (1990) J Mol Biol, 215, pp. 403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ). Similarity can be measured using pairwise identity or by applying a scoring matrix such as BLOSUM62 and translating to equivalent identity. Positions of deliberate mutations will be masked out when determining homology as they represent functional rather than evolutionary changes. Position-specific scoring matrices can be applied more sensitively by running, for example, PSIBLAST on a comprehensive database of protein sequences. Different scoring matrices reflecting substitution frequencies within the chemical-physical properties of amino acids (e.g. charge) rather than evolutionary time scales can be used.

[0522] Amino acid substitutions can be made in the amino acid sequence of SEQ ID NO: 2 in addition to the substitutions discussed above, for example up to 1, 2, 3, 4, 5, 10, 20, or 30 substitutions. Conservative substitutions use other amino acids with similar chemical structures, similar chemical properties, or similar side chain volumes in place of an amino acid. The introduced amino acid can have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, electro neutrality, or charge as the amino acid it replaces. Alternatively, a conservative substitution can introduce another aromatic or aliphatic amino acid in place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and can be selected according to the properties of the 20 primary amino acids as defined in Table 3 below. Where amino acids have similar polarity, this can also be determined with reference to the hydrophilicity scale of the side chains of the amino acids in Table 4.

[0523] Table 3 - Chemical properties of amino acids

[0524]

[0525] Table 4 - Hydrophilicity scale

[0526]

[0527]

[0528] Variants can include one or more substitutions outside the regions specified above, where the amino acid is replaced with the amino acid at one or more corresponding positions in homologs and paralogs of cytolysin. Four examples of homologs of cytolysin are shown in SEQ ID NOs: 14-17.

[0529] Additionally, one or more amino acid residues of the amino acid sequence of SEQ ID NO: 2 can be deleted from the variants described above. Up to 1, 2, 3, 4, 5, 10, 20, or 30 or more residues can be deleted.

[0530] A variant can comprise a fragment of SEQ ID NO: 2. Such a fragment retains pore-forming activity. This can be determined as described above. The fragment can be at least 50, 100, 150, 200, or 250 amino acids in length. Such a fragment can be used to produce a pore of the application. The fragment need not contain the entire region of about position 44 to about position 126 of SEQ ID NO: 2 because the region can be modified by one or more deletions according to the application. Thus, the application contemplates fragments that are shorter in length than the unmodified region. The fragment preferably includes the pore-forming domain of SEQ ID NO: 2. The fragment more preferably includes the region of about position 44 to about position 126 of SEQ ID NO: 2 that is modified according to the application.

[0531] Alternatively or additionally, one or more amino acids can be added to the variants described above. The additions can be provided at the amino or carboxy terminus of the amino acid sequence of SEQ ID NO: 2, variants including fragments thereof. The additions can be very short, e.g., 1 to 10 amino acids in length. Alternatively, the additions can be longer, e.g., up to 50 or 100 amino acids in length. Carrier proteins can be fused to the amino acid sequences according to the application. Other fusion proteins are discussed in more detail below.

[0532] As discussed above, a variant is a polypeptide having an amino acid sequence that differs from that of SEQ ID NO: 2 and retains its ability to form pores. The variant typically contains the region of SEQ ID NO: 2 responsible for pore formation, i.e., about position 44 to about position 126, and this region is modified according to the application as discussed above. It can contain a fragment of this region, as discussed above. In addition to the modifications of the application, the variant of SEQ ID NO: 2 can comprise one or more additional modifications, such as substitutions, additions, or deletions. These modifications are preferably located in extensions of the variant corresponding to about position 1 to about position 43 and about position 127 to about position 297 of SEQ ID NO: 2 (i.e., outside the region modified according to the application).

[0533] The mutant monomer can be modified to aid in its recognition or purification, for example, by the addition of a histidine residue (his tag), an aspartic acid residue (asp tag), a streptavidin tag or a flag tag or by the addition of a signal sequence for promoting secretion of the mutant monomer from cells in which the polypeptide does not naturally contain such a sequence. An alternative to introducing a genetic tag is to chemically react a tag to a native or engineered position on the pore. An example of such a procedure would be to react a gel shifting reagent to an engineered cysteine on the outside of the pore. This has been shown to be a method for isolating hemolysin hetero-oligomers (Chem Biol, July 1997, vol. 4, no. 7, pages 497 to 505).

[0534] The mutant monomer can be labelled using a revealing tag. The revealing tag can be any suitable tag that allows the pore to be detected. Suitable labels include, but are not limited to, fluorescent molecules; radioisotopes, for example, 125 I, 35 S, enzymes, antibodies, antigens, polynucleotides, polyethylene glycol (PEG), peptides and ligands, such as biotin.

[0535] D-amino acids can also be used to produce the mutant monomer. For example, the mutant monomer can comprise a mixture of L-amino acids and D-amino acids. This is routine in the art for producing such proteins or peptides.

[0536] The mutant monomer contains one or more specific modifications for facilitating interaction with a polynucleotide. The mutant monomer can also contain other non-specific modifications, so long as the modifications do not interfere with pore formation. A variety of non-specific side chain modifications are known in the art and can be made to the side chains of the mutant monomer. Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH4, amidination using methyl acetylimidate or acylation using acetic anhydride.

[0537] The mutant monomer can be produced using standard methods known in the art. The monomer can be prepared synthetically or by recombinant means. For example, the monomer can be synthesised by in vitro translation and transcription (IVTT). Suitable methods for producing pore monomers are discussed in International Application No. PCT / GB09 / 001690 (published as WO 2010 / 004273), PCT / GB09 / 001679 (published as WO 2010 / 004265) or PCT / GB10 / 000133 (published as WO 2010 / 086603). Methods for inserting pores into membranes are discussed below.

[0538] The polynucleotide sequence encoding the mutant monomer can be derived or replicated using standard methods in the field. This sequence is discussed in more detail below. The polynucleotide sequence encoding the mutant monomer can be expressed in bacterial host cells using standard techniques in the field. The mutant monomer can be generated from a recombinant expression vector via in situ expression of the polypeptide in cells. The expression vector optionally carries an inducible promoter for controlling polypeptide expression.

[0539] Mutant monomers can be produced on a large scale after purification from the pore-generating organism using any protein liquid chromatography system, or after recombinant expression, as described below. Typical protein liquid chromatography systems include FPLC, AKTA systems, Bio-Cad systems, Bio-Rad biosystems, and Gilson HPLC systems. The mutant monomers can then be inserted into naturally occurring or artificial membranes for use according to the present invention. Methods for inserting pores into membranes are discussed below.

[0540] In some embodiments, the mutant monomer is chemically modified. The mutant monomer can be chemically modified in any manner and at any site. Preferably, the mutant monomer is chemically modified by attaching the molecule to one or more cysteine ​​residues (cysteine ​​linkage), attaching the molecule to one or more lysine residues, attaching the molecule to one or more unnatural amino acids, enzymatic modification of the epitope, or terminal modification. Suitable methods for performing such modifications are well known in the art. Suitable unnatural amino acids include, but are not limited to, 4-azido-L-phenylalanine (Faz) and Liu CC and Schultz PG, *Annu. Rev. Biochem.*, 2010, Vol. 79, pp. 413–444. Preparation of mutant cytolysin monomer Any amino acid numbered 1 to 71. Mutant monomers can be chemically modified by attaching any molecule. For example, mutant monomers can be chemically modified by attaching polyethylene glycol (PEG), nucleic acids such as DNA, dyes, fluorophores, or chromophores.

[0541] In some embodiments, the mutant monomer is chemically modified using a molecular adaptor that promotes the interaction between the pore, comprising the monomer, and the target analyte, target nucleotide, or target polynucleotide sequence. The presence of the adaptor improves the host-guest chemistry between the pore and the nucleotide or polynucleotide, and thereby enhances the sequencing capability of the pore formed from the mutant monomer. The principles of host-guest chemistry are well known in the art. The adaptor affects the physical or chemical properties of the pore, which enhances its interaction with the nucleotide or polynucleotide sequence. The adaptor may alter the charge of the pore's barrel or channel, or specifically interact with or bind to a nucleotide or polynucleotide, thereby promoting its interaction with the pore.

[0542] The molecular adaptor is preferably a cyclic molecule such as a cyclodextrin, a species capable of hybridization, a DNA binding agent or intercalator, a peptide or peptide analog, a synthetic polymer, an aromatic planar molecule, a small molecule with a positive charge, or a small molecule capable of hydrogen bonding.

[0543] The adaptor can be cyclic. The cyclic adaptor preferably has the same symmetry as the pore.

[0544] The adaptor typically interacts with the analyte, nucleotide, or polynucleotide through host-guest chemistry. The adaptor is typically capable of interacting with the nucleotide or polynucleotide. The adaptor includes one or more chemical groups capable of interacting with the nucleotide or polynucleotide. The one or more chemical groups preferably interact with the nucleotide or polynucleotide through non-covalent interactions such as hydrophobic interactions, hydrogen bonding, van der Waals forces, pi-cation interactions, and / or electrostatic forces. The one or more chemical groups capable of interacting with the nucleotide or polynucleotide are preferably positively charged. The one or more chemical groups capable of interacting with the nucleotide or polynucleotide are more preferably include an amino group. The amino group can be attached to a primary, secondary, or tertiary carbon atom. The adaptor is even more preferably includes an amino ring such as a ring composed of 6, 7, 8, or 9 amino groups. The adaptor is most preferably includes a ring composed of 6 or 9 amino groups. The protonated amino ring can interact with a negatively charged phosphate group in the nucleotide or polynucleotide.

[0545] The correct positioning of the adaptor within the pore can be facilitated by host-guest chemistry between the adaptor and the pore including a mutant monomer. The adaptor preferably includes one or more chemical groups capable of interacting with one or more amino acids in the pore. The adaptor more preferably includes one or more chemical groups capable of interacting with one or more amino acids in the pore through non-covalent interactions such as hydrophobic interactions, hydrogen bonding, van der Waals forces, pi-cation interactions, and / or electrostatic forces. The chemical groups capable of interacting with one or more amino acids in the pore are typically hydroxyl groups or amines. The hydroxyl groups can be attached to primary, secondary, or tertiary carbon atoms. The hydroxyl groups can form hydrogen bonds with uncharged amino acids in the pore. Any adaptor that facilitates the interaction between the pore and the nucleotide or polynucleotide can be used.

[0546] Suitable adaptors include, but are not limited to, cyclodextrins, cyclotripeptides, and cucurbiturils. The adaptor is preferably a cyclodextrin or derivative thereof. The cyclodextrin or derivative thereof can be any of the cyclodextrins or derivatives thereof disclosed in Eliseev, A. V. and Schneider, H-J. (1994), J. Am. Chem. Soc., Vol. 116, pp. 6081-6088. The adaptor is more preferably hepta-6-amino-β-cyclodextrin (am7-βCD), 6-mono-deoxy-6-monoamino-β-cyclodextrin (am1-βCD), or hepta-(6-deoxy-6-guanidino)-cyclodextrin (gu7-βCD). The guanidino group in gu7-βCD has a much higher pKa than the primary amine in am7-βCD, and thus it carries more positive charge. This gu7-βCD adaptor can be used to increase the residence time of the nucleotides in the pore, increase the accuracy of the measured residual current, and increase the rate of base detection at high temperatures or low data acquisition rates.

[0547] If a 3-(2-pyridyldithio)propionic acid succinimidyl ester (SPDP) crosslinker is used as discussed in more detail below, the adaptor is preferably hepta(6-deoxy-6-amino)-6-N-mono(2-pyridyl)dithio-propionyl-β-cyclodextrin (am6amPDP1-βCD).

[0548] More suitable adaptors include γ-cyclodextrin, which includes 8 sugar units (and thus has eight-fold symmetry). The γ-cyclodextrin can contain a linker molecule, or can be modified to include all or more of the modified sugar units used in the examples of β-cyclodextrin discussed above.

[0549] The molecular adaptor is preferably covalently linked to the mutant monomer. The adaptor can be covalently linked to the pore using any method known in the art. The adaptor is typically attached by chemical linkage. If the molecular adaptor is attached by a cysteine linkage, the one or more cysteines have preferably been introduced into the mutant by substitution. The mutant monomer of the present invention can of course include a cysteine residue at one or both of positions 272 and 283. The mutant monomer can be chemically modified by attaching a molecular adaptor to one or both of these cysteines. Alternatively, the mutant monomer can be chemically modified by attaching a molecule to one or more cysteines or unnatural amino acids such as FAz introduced at other positions.

[0550] The reactivity of a cysteine residue can be enhanced by modifying the adjacent residues. For example, the basic groups of flanking arginine, histidine, or lysine residues will change the pKa of the cysteine thiol to be more reactive -The pKa of the base. The reactivity of cysteine residues can be protected by a thiol protecting group such as dTNB. These can be reacted with one or more cysteine residues of the mutant monomer prior to attachment of the linker. The molecule can be attached directly to the mutant monomer. Preferably, the molecule is attached to the mutant monomer using a linker such as a chemical crosslinker or a peptide linker.

[0551] Suitable chemical crosslinkers are well known in the art. Preferred crosslinkers include 3-(pyridin-2-yl disulfanyl)propanoic acid 2,5-dioxopyrrolidin-1-yl ester, 4-(pyridin-2-yl disulfanyl)butanoic acid 2,5-dioxopyrrolidin-1-yl ester and 8-(pyridin-2-yl disulfanyl)octanoic acid 2,5-dioxopyrrolidin-1-yl ester. The most preferred crosslinker is 3-(2-pyridyl dithio)propionic acid succinimidyl ester (SPDP). Typically, the molecule is covalently linked to the bifunctional crosslinker before the molecule / crosslinker complex is covalently linked to the mutant monomer, but it is also possible to covalently link the bifunctional crosslinker to the monomer before the bifunctional crosslinker / monomer complex is attached to the molecule.

[0552] Preferably, the linker is resistant to dithiothreitol (DTT). Suitable linkers include, but are not limited to, iodoacetamide-based and maleimide-based linkers.

[0553] In other embodiments, the monomer can be attached to a polynucleotide binding protein. This forms a modular sequencing system that can be used in the methods of the application. Polynucleotide binding proteins are discussed below.

[0554] The polynucleotide binding protein can be covalently linked to the mutant monomer. The protein can be covalently linked to the pore using any method known in the art. The monomer and the protein can be chemically or genetically fused. The monomer and the protein are genetically fused if the entire construct is expressed from a single polynucleotide sequence. Genetic fusion of the monomer to the polynucleotide binding protein is discussed in International Application No. PCT / GB09 / 001679 (published as WO 2010 / 004265).

[0555] If the polynucleotide binding protein is attached by a cysteine linkage, the one or more cysteines have preferably been introduced into the mutant by substitution. This substitution is typically made in a loop region that has low conservation in the homologues, indicating that mutations or insertions can be tolerated. Thus, it is suitable for attachment of the polynucleotide binding protein. This substitution is typically made in residues 1 to 43 and 127 to 297 of SEQ ID NO: 2. The reactivity of the cysteine residues can be enhanced by modification as described above.

[0556] The polynucleotide binding protein can be attached directly to the mutant monomer or attached via one or more linkers. The polynucleotide binding protein can be attached to the mutant monomer using the hybrid linker described in International Application No. PCT / GB10 / 000132 (published as WO 2010 / 086602). Alternatively, a peptide linker can be used. A peptide linker is an amino acid sequence. The length, flexibility and hydrophilicity of the peptide linker are generally designed such that it does not interfere with the function of the monomer and molecule. Preferred flexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10 or 16, serine and / or glycine. More preferred flexible linkers comprise (SG)i, (SG)2, (SG)3, (SG)4, (SG)5and (SG)8, where S is serine and G is glycine. Preferred rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or 24, proline. More preferred rigid linkers comprise (P) 12 where P is proline.

[0557] The mutant monomer can be chemically modified using a molecular linker and a polynucleotide binding protein.

[0558] Construct

[0559] The present application also provides a method of improving the ability of a characterization polynucleotide comprising a lysenin monomer to characterize a polynucleotide. The method comprises making one or more modifications and / or substitutions in SEQ ID NO: 2. Any of the embodiments discussed above with reference to the mutant lysenin monomer and below with reference to the characterization polynucleotide are equally applicable to this method of the present application.

[0560] Polynucleotide

[0561] The present application also provides a construct comprising two or more covalently linked monomers derived from lysenin, wherein at least one of the monomers is a mutant lysenin monomer of the present application. The construct of the present application retains its ability to form a pore. One or more constructs of the present application can be used to form a pore for characterizing a target analyte. One or more constructs of the present application can be used to form a pore for characterizing a target polynucleotide, such as sequencing a target nucleotide. The construct can comprise 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more monomers. The two or more monomers can be the same or different.

[0562] At least one monomer in the construct is a mutant monomer of the application. Two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more monomers in the construct can be mutant monomers of the application. Preferably all monomers in the construct are mutant monomers of the application. The mutant monomers can be the same or different. In a preferred embodiment, the construct comprises two mutant monomers of the application.

[0563] The mutant monomers of the application in the construct are preferably substantially the same length or the same length. The barrels of the mutant monomers of the application in the construct are preferably substantially the same length or the same length. Length can be measured in number of amino acids and / or length units. The number of amino acids of the mutant monomers of the application in the construct is preferably the same as the number of amino acids deleted from positions 34 to 70 and / or positions 71 to 107 as described above.

[0564] The other monomers in the construct need not be mutant monomers of the application. For example, at least one monomer can comprise the sequence set forth in SEQ ID NO: 2. At least one monomer in the construct can be a paralog or homolog of SEQ ID NO: 2. Suitable homologs are set forth in SEQ ID NOs: 14 to 17.

[0565] Alternatively, at least one monomer can comprise a variant of SEQ ID NO: 2 that is at least 50% homologous over its entire sequence based on amino acid identity to SEQ ID NO: 2, but does not comprise any of the specific mutations required for the mutant monomer of the application, or in which the amino acids have not been deleted as described above. More preferably, the variant can be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and more preferably at least 95%, 97% or 99% homologous over its entire sequence based on amino acid identity to the amino acid sequence of SEQ ID NO: 2. The variant can be a fragment or any other variant discussed above. The construct of the application can also comprise a variant of SEQ ID NO: 14, 15, 16 or 17 that is at least 50% homologous over its entire sequence based on amino acid identity to SEQ ID NO: 14, 15, 16 or 17 or at least any of the other homology levels mentioned above.

[0566] All monomers in the construct can be mutant monomers of the application. The mutant monomers can be the same or different. In a more preferred embodiment, the construct comprises two monomers and at least one of the monomers is a mutant monomer of the application.

[0567] The monomers can be genetically fused. The monomers are genetically fused if the entire construct is expressed from a single polynucleotide sequence. The coding sequences of the monomers can be combined in any way to form a single polynucleotide sequence that encodes the construct. Genetic fusion is discussed in International Application No. PCT / GB09 / 001679 (published as WO 2010 / 004265).

[0568] The monomers can be genetically fused in any configuration. The monomers can be fused by the terminal amino acids of the monomers. For example, the amino terminus of one monomer can be fused to the carboxy terminus of another monomer.

[0569] Two or more monomers can be directly genetically fused together. The monomers are preferably genetically fused using linkers. The linkers can be designed to limit the mobility of the monomers. Preferred linkers are amino acid sequences (i.e. peptide linkers). Any of the peptide linkers discussed above can be used.

[0570] The length, flexibility and hydrophilicity of the peptide linkers are each generally designed such that they do not interfere with the function of the monomers and the molecule. Preferred flexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10 or 16, serine and / or glycine. More preferred flexible linkers include (SG)i, (SG)2, (SG)3, (SG)4, (SG)5and (SG)8, where S is serine and G is glycine. Preferred rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or 24, proline. More preferred rigid linkers include (P) 12 where P is proline.

[0571] In another preferred embodiment, the monomers are chemically fused. The monomers are chemically fused if they are chemically attached, for example by a chemical cross-linking agent. Any of the chemical cross-linking agents discussed above can be used. The linkers can be attached to one or more cysteine residues introduced into the mutant monomers or unnatural amino acids such as those described by Faz et al. Alternatively, the linkers can be attached to the terminus of one of the monomers in the construct. The monomers are typically linked by one or more of residues 1 to 43 and 127 to 297 of SEQ ID NO: 2.

[0572] If the construct contains different monomers, cross-linking of the monomers themselves can be prevented by having the concentration of the linkers greatly exceed the monomers. Alternatively, a "lock and key" arrangement can be used in which two linkers are used. Only one end of each linker can react together to form a longer linker and the other end of the linkers each react with a different monomer. Such linkers are described in International Application No. PCT / GB10 / 000132 (published as WO 2010 / 086602).

[0573] The present application also provides a method of producing a construct of the present application. The method comprises covalently linking at least one mutant cytolysin monomer of the present application to one or more monomers derived from a cytolysin. Any of the embodiments discussed above with reference to the constructs of the present application are equally applicable to the method of producing the construct.

[0574] Figure 1

[0575] The present application also provides polynucleotides encoding the mutant monomers of the present application. The mutant monomers can be any of the mutant monomers discussed above. The polynucleotide sequence preferably comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, or 95% homologous to the sequence of SEQ ID NO: 1 over the entire sequence based on nucleotide identity. There can be at least 80%, for example at least 85%, 90%, or 95% nucleotide identity over stretches of 300 or more, for example 375, 450, 525, or 600 or more contiguous nucleotides ("hard homology"). Homology can be calculated as described above. The polynucleotide sequence can comprise a sequence that differs from SEQ ID NO: 1 on the basis of the degeneracy of the genetic code.

[0576] The present application also provides polynucleotide sequences encoding any of the genetic fusion constructs of the present application. The polynucleotide preferably comprises two or more sequences as set forth in SEQ ID NO: 1 or a variant thereof as described above.

[0577] Standard methods in the art can be used to derive or replicate the polynucleotide sequences. Chromosomal DNA encoding wild-type cytolysins can be extracted from a pore-producing organism such as Eisenia foetida. The gene encoding the pore monomer can be amplified using PCR involving specific primers. The amplified sequence can then be subjected to site-directed mutagenesis. Suitable methods of site-directed mutagenesis are known in the art and include, for example, combinatorial chain reaction. The polynucleotides encoding the constructs of the present application can be prepared using well-known techniques such as those described in Sambrook, J. and Russell, D. (2001), Molecular Cloning: A Laboratory Manual, 3rdedition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.

[0578] The resulting polynucleotide sequence can then be incorporated into a recombinant replicable vector, such as a cloning vector. The vector can be used to replicate the polynucleotide in a compatible host cell. Thus, the polynucleotide sequence can be produced by introducing the polynucleotide into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions that cause the vector to replicate. The vector can be recovered from the host cell. Suitable host cells for cloning polynucleotides are known in the art and are described in more detail below.

[0579] The polynucleotide sequence can be cloned into an appropriate expression vector. In an expression vector, the polynucleotide sequence is typically operably linked to control sequences capable of providing for expression of the coding sequence by a host cell. Such an expression vector can be used to express the pore subunit.

[0580] The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A control sequence that is "operably linked" to a coding sequence is linked in such a way that the expression of the coding sequence is achieved under conditions compatible with the control sequences. Multiple copies of the same or different polynucleotide sequences can be introduced into a vector.

[0581] The expression vector can then be introduced into an appropriate host cell. Thus, a mutant monomer or construct of the application can be produced by inserting the polynucleotide sequence into an expression vector, introducing the vector into a compatible bacterial host cell, and growing the host cell under conditions that cause expression of the polynucleotide sequence. The recombinantly expressed monomer or construct can be self-assembled into a pore in the host cell membrane. Alternatively, the recombinant pore produced in this manner can be removed from the host cell and inserted into another membrane. When a pore comprising at least two different subunits is produced, the different subunits can be expressed individually in different host cells as described above, removed from the host cells, and assembled into a pore in a separate membrane, such as a sheep red blood cell membrane or a liposome containing sphingomyelin.

[0582] For example, the lysenin monomer can be oligomerized by adding a lipid mixture comprising sphingomyelin and one or more of the following lipids: phosphatidylserine; POPE; cholesterol; and Soy PC, and incubating the mixture, for example, at 30°C for 60 minutes. The oligomerized monomers can be purified by any appropriate method, for example, by SDS-PAGE and gel purification as described in WO2013 / 153359.

[0583] The vector can be, for example, a plasmid, viral or bacteriophage vector having an origin of replication, optionally a promoter for expression of the polynucleotide sequence, and optionally a regulator of the promoter. The vector can contain one or more selectable marker genes, such as a tetracycline resistance gene. The promoter and other expression control signals can be selected to be compatible with the host cell for which the expression vector is designed. T7, trc, lac, ara or lambda L Promoters.

[0584] The host cell typically expresses the pore subunit at a high level. The host cell transformed with the polynucleotide sequence can be selected to be compatible with the expression vector used to transform the cell. The host cell is typically a bacterium and is preferably E. coli. Any cell having a lambda DE3 lysogen, such as C41(DE3), BL21(DE3), JM109(DE3), B834(DE3), TUNER, Origami and Origami B can express a vector including a T7 promoter. In addition to the conditions listed above, the lyticus protein can be expressed using any of the methods described in Proc Natl Acad Sci USA, 2008 Dec 30; 105(52): 20647-20642.

[0585] Pores

[0586] The present invention also provides various pores. The pores of the present invention are ideal for characterizing an analyte. The pores of the present invention are particularly ideal for characterizing polynucleotide sequences, such as sequencing polynucleotides, as they can distinguish between different nucleotides with high sensitivity. The pores can be used to characterize nucleic acids, such as DNA and RNA, including sequencing nucleic acids and identifying single base changes. The pores of the present invention can even distinguish between methylated and unmethylated nucleotides. The pores of the present invention have a very high base resolution. The pores show almost complete separation of all four DNA nucleotides. The pores can further be used to distinguish between deoxy cytosine monophosphate (dCMP) and methyl-dCMP based on residence time in the pore and current flow through the pore.

[0587] The pores of the present invention can also distinguish between different nucleotides under a range of conditions. In particular, the pores will distinguish between nucleotides under conditions that favor characterizing the polynucleotide, such as sequencing it. The extent to which the pores of the present invention can distinguish between different nucleotides can be controlled by varying the applied electric potential, salt concentration, buffer, temperature and presence of additives such as urea, betaine and DTT. This allows for fine tuning of the function of the pore, especially when sequencing. This is discussed in more detail below. The pores of the present invention can also be used to identify polynucleotide polymers based on interaction with one or more monomers rather than on a nucleotide-by-nucleotide basis.

[0588] The pores of the application can be isolated, substantially isolated, purified, or substantially purified. A pore of the application is isolated or purified if it is completely free of any other components, such as lipids or other pores. A pore is substantially isolated if it is mixed with a carrier or diluent that will not interfere with its intended use. For example, a pore is substantially isolated or substantially purified if it exists in a form that includes less than 10%, less than 5%, less than 2%, or less than 1% of other components, such as lipids or other pores. Alternatively, the pores of the application can exist in a lipid bilayer.

[0589] The pores of the application can exist as a single pore or a single pore. Alternatively, the pores of the application can exist in a homogenous population or a heterogenous population of two or more pores or a plurality of two or more pores.

[0590] Homooligomeric pores

[0591] The application also provides a homooligomeric pore derived from a cytolysin that includes identical mutant monomers of the application. The monomers are identical in terms of their amino acid sequence. The homooligomeric pores of the application are ideal for characterizing a polynucleotide, such as sequencing it. The homooligomeric pores of the application can have any of the advantages discussed above. Advantages of particular homooligomeric pores of the application are illustrated in the Examples.

[0592] A homooligomeric pore can contain any number of mutant monomers. The pore typically includes two or more mutant monomers. A homooligomeric pore can contain any number of mutant monomers. The pore typically includes at least 6, at least 7, at least 8, at least 9, or at least 10 identical mutant monomers, such as 6, 7, 8, 9, or 10 mutant monomers. The pore preferably includes eight or nine identical mutant monomers. The pore most preferably includes nine identical mutant monomers. This number of monomers is referred to herein as a “sufficient number.”

[0593] One or more of the mutant monomers, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10, are preferably chemically modified as discussed above or below.

[0594] One or more of the mutant monomers are preferably chemically modified as discussed above or below. In other words, as long as the amino acid sequence of each of the monomers is identical, one or more of the monomers that are chemically modified (and the other monomers that are not chemically modified) do not prevent the pore from being a homooligomer.

[0595] Methods for preparing a lysenin pore are described in Yamaji et al., J. Biol. Chem., 1998, vol. 273, no. 9, pages 5300 to 5306.

[0596] Hetero-oligomeric pore

[0597] The present application also provides a hetero-oligomeric pore derived from a lysenin comprising at least one mutant monomer of the present application, wherein at least one of the monomers is different from the other monomers. The monomers are different from the other monomers in terms of their amino acid sequence. The hetero-oligomeric pore of the present application is ideal for characterizing a polynucleotide, such as sequencing thereof. The hetero-oligomeric pore can be prepared using methods known in the art (e.g., Protein Sci, 2002 Jul, vol. 11, no. 7, pages 1813 to 1824).

[0598] The hetero-oligomeric pore contains monomers sufficient to form a pore. The monomers can be of any type, including but not limited to wild-type. The pore typically comprises two or more monomers. The pore typically comprises at least 6, at least 7, at least 8, at least 9, or at least 10 monomers, such as 6, 7, 8, 9, or 10 monomers. The pore preferably comprises eight or nine monomers. The pore most preferably comprises nine monomers. This number of monomers is referred to herein as a "sufficient number".

[0599] The pore comprises at least one monomer comprising the sequence set forth in SEQ ID NO: 2, a paralog thereof, a homolog thereof, or a variant thereof that does not have the mutation required for a mutant monomer of the present application or in which an amino acid has not been deleted, as described above. Suitable variants are any of the variants discussed above with reference to the construct of the present application, including SEQ ID NO: 2, 14, 15, 16, and 17, and variants thereof. In this embodiment, the remaining monomers are preferably mutant monomers of the present application.

[0600] In a preferred embodiment, the pore comprises (a) one mutant monomer of the present application and (b) a uniform number of monomers sufficient to form a pore, wherein the mutant monomer in (a) is different from the uniform monomers in (b). The uniform monomers in (b) preferably comprise the sequence set forth in SEQ ID NO: 2, a paralog thereof, a homolog thereof, or a variant thereof that does not have the mutation required for a mutant monomer of the present application.

[0601] The hetero-oligomeric pore of the present application preferably comprises only one mutant lysenin monomer of the present application.

[0602] In another preferred embodiment, all monomers in the hetero-oligomeric pore are mutant monomers of the invention, and at least one of them is different from the other monomers.

[0603] The mutant monomers of the invention in the pore are preferably of substantially the same or the same length. The barrels of the mutant monomers of the invention in the pore are preferably of substantially the same or the same length. Length can be measured in number of amino acids and / or length units. The number of amino acids of the mutant monomers of the invention in the pore is preferably the same as the number of amino acids missing from position 34 to position 70 and / or position 71 to position 107.

[0604] In all embodiments discussed above, one or more of the mutant monomers is preferably chemically modified as discussed above or below. The presence of a chemical modification on one monomer does not result in a pore that is a hetero-oligomer. The amino acid sequence of at least one monomer must be different from one or more of the sequences of the other monomers. Methods for making pores are discussed in more detail below.

[0605] Pore comprising a construct

[0606] The present invention also provides a pore comprising at least one construct of the invention. A construct of the invention comprises two or more covalently linked monomers derived from a cytolysin, wherein at least one of the monomers is a mutant cytolysin monomer of the invention. In other words, a construct must contain more than one monomer. At least two of the monomers in the pore are in the form of a construct of the invention. The monomers can be of any type.

[0607] A pore typically contains (a) one construct comprising two monomers and (b) a number of monomers sufficient to form a pore. The construct can be any of the constructs discussed above. The monomers can be any of the monomers discussed above, including a mutant monomer of the invention.

[0608] Another typical pore comprises more than one construct of the invention, such as two, three or four constructs of the invention. Such a pore further comprises a number of monomers sufficient to form a pore. The monomers can be any of the mutant monomers discussed above. A further pore of the invention comprises only constructs comprising 2 monomers. A particular pore according to the invention comprises several constructs each comprising two monomers. The constructs can oligomerize into a pore having such that only one monomer from each construct contributes to the structure of the pore. Typically, the other monomers of the constructs, i.e. the monomers that do not form the pore, will be located outside the pore.

[0609] Mutations can be introduced into the construct as discussed above. The mutations can be alternating, i.e. the mutations are different for each monomer within the di-monomer construct and the construct assembles into a homo-oligomer, resulting in alternating modifications. In other words, monomers comprising MutA and MutB are fused and assembled to form an A-B:A-B:A-B:A-B pore. Alternatively, the mutations can be adjacent, i.e. the same mutation is introduced into both monomers of the construct and this is then oligomerised with a different mutation monomer. In other words, monomers comprising MutA are fused, followed by oligomerisation with monomers comprising MutB to form A-A:B:B:B:B:B:B.

[0610] One or more of the monomers of the present application in the pore comprising construct can be chemically modified as discussed above or below.

[0611] Chemically modified pores of the present application

[0612] In another aspect, the present application provides a chemically modified lysenin pore comprising one or more mutant monomers chemically modified such that the open diameter of the barrel / channel of the assembled pore is reduced, narrowed or constricted at one or more sites, such as two, three, four or five sites, along the length of the barrel. The pore can comprise any number of the monomers discussed above with reference to the homo-oligomeric pores and the hetero-oligomeric pores of the present application. The pore preferably comprises nine chemically modified monomers. The chemically modified pore can be homo-oligomeric, as described above. In other words, all of the monomers in the chemically modified pore can have the same amino acid sequence and can be chemically modified in the same way. The chemically modified pore can be hetero-oligomeric, as described above. In other words, the pore can comprise (a) only one chemically modified monomer, (b) more than one, such as two, three, four, five, six, seven or eight chemically modified monomers, wherein at least two, such as three, four, five, six or seven of the chemically modified monomers are different from each other or (c) only chemically modified monomers (i.e. all of the monomers are chemically modified), wherein at least two, such as three, four, five, six, seven, eight or nine of the chemically modified monomers are different from each other. The monomers can differ from each other in terms of their amino acid sequence, their chemical modification or both their amino acid sequence and their chemical modification. The one or more chemically modified monomers can be any of the chemically modified monomers discussed above and / or below.

[0613] The present application also provides a mutant lysenin monomer chemically modified in any of the ways discussed below. The mutant monomer can be any of the mutant monomers discussed above or below. Thus, the mutant monomers of the present application can be chemically modified according to the present application, as discussed below, such as a variant of SEQ ID NO: 2 including a modification at one or more of the following positions: K37, G43, K45, V47, S49, T51, H83, V88, T91, T93, V95, Y96, S98, K99, V100, 101, P108, P109, T110, S111, K112, and T114 or a variant including the barrel deletion described above.

[0614] The mutant monomer can be chemically modified such that the diameter of the barrel of the assembled pore is reduced or narrowed by any reduction factor depending on the size of the analyte to be passed through the pore. The width of the constriction region will generally determine the extent to which the measurement signal is disrupted during analyte transfer due to, for example, the analyte reducing the ionic flow through the pore. The greater the signal disruption, generally the higher the measurement sensitivity. Thus, the constriction region can be selected to be slightly wider than the analyte to be transferred. For transfer of, for example, ssDNA, the width of the constriction region can be selected from a value in the range of 0.8 nm to 3.0 nm.

[0615] The chemical modification can also determine the length of the constriction region, which in turn will determine the number of polymer units, such as nucleotides, that contribute to the measurement signal. The number of nucleotides that contribute to the current signal at any particular time can be referred to as a k-mer, where k is an integer and can be an integer or a fraction. In the case of measuring a polynucleotide having 4 types of nucleobases, a 3-mer will produce 4 3 potential signal levels. Larger values of k will produce a larger number of signal levels. It is generally desirable to provide a short constriction region, as this simplifies the analysis of the measurement signal data.

[0616] The chemical modification is such that the chemical molecule is preferably covalently linked to the mutant monomer or the one or more mutant monomers. The chemical molecule can be covalently linked to the pore, the mutant monomer or the one or more mutant monomers using any method known in the art. The chemical molecule is typically attached by chemical linkage.

[0617] Chemical modification of the mutant monomer or mutant monomers is preferably performed by attaching the molecule to one or more cysteines (cysteine ligation), attaching the molecule to one or more lysines, attaching the molecule to one or more unnatural amino acids, enzymatic modification of the epitope. If the chemical modifier is attached by cysteine ligation, the one or more cysteines have preferably been introduced into the mutant by substitution. Suitable methods for performing such modifications are well known in the art. Suitable unnatural amino acids include, but are not limited to, 4-azido-L-phenylalanine (Faz) and Liu C.C. and Schultz P.G., Annu. Rev. Biochem., 2010, vol. 79, pages 413 to 444 Method of characterising an analyte any of the amino acids numbered 1 to 71 in SEQ ID NO: 1.

[0618] The mutant monomer or mutant monomers can be chemically modified by attaching any molecule that has the effect of reducing or shrinking the diameter of the barrel of the assembly pore at any location or site. The mutant monomer can be chemically modified by attaching: (i) a maleimide such as: 4-phenylazomaleinanil, 1. N-(2-hydroxyethyl)maleimide, N-cyclohexylmaleimide, 1.3-maleimidopropionic acid, 1.1-4-aminophenyl-1H-pyrrole, 2,5, dione, 1.1-4-hydroxyphenyl-1H-pyrrole, 2,5, dione, N-ethylmaleimide, N-methoxycarbonylmaleimide, N-tert-butylmaleimide, N-(2-aminoethyl)maleimide, 3-maleimidyl-PROXYL, N-(4-chlorophenyl)maleimide, 1-[4-(dimethylamino)-3,5-dinitrophenyl]-1H-pyrrole-2,5-dione, N-[4-(2-benzimidazolyl)phenyl]maleimide, N-[4-(2-benzoxazolyl)phenyl]maleimide, N-(1-naphthyl)maleimide, N-(2,4-dimethylphenyl)maleimide, N-(2,4-difluorophenyl)maleimide, N-(3-chloro-p-tolyl)-maleimide, 1-(2-amino-ethyl)-pyrrole-2,5-dione hydrochloride, 1-cyclopentyl-3-methyl-2,5-dihydro-1H-pyrrole-2,5-dione, 1-(3-aminopropyl)-2,5-dihydro-1H-pyrrole-2,5-dione hydrochloride, 3-methyl-1-[2-oxo-2-(piperazin-1-yl)ethyl]-2,5-dihydro-1H-pyrrole-2,5-dione hydrochloride, 1-benzyl-2,5-dihydro-1H-pyrrole-2,5-dione, 3-methyl-1-(3,3,3-trifluoropropyl)-2,5-dihydro-1H-pyrrole-2,5-dione, 1-[4-(methylamino)cyclohexyl]-2,5-dihydro-1H-pyrrole-2,5-dione trifluoroacetate, SMILES O=C1C=CC(=O)N1CC=2C=CN=CC2, SMILES O=C1C=CC(=O)N1CN2CCNCC2, 1-benzyl-3-methyl-2,5-dihydro-1H-pyrrole-2,5-dione, 1-(2-fluorophenyl)-3-methyl-2,5-dihydro-1H-pyrrole-2,5-dione, N-(4-phenoxyphenyl)maleimide, N-(4-nitrophenyl)maleimide;(ii) iodoacetamides, such as 3-(2-iodoacetamido)-PROXYL, N-(cyclopropylmethyl)-2- iodoacetamide, 2-iodo-N-(2-phenylethyl)acetamide, 2-iodo-N-(2,2,2- trifluoroethyl)acetamide, N-(4-acetylphenyl)-2-iodoacetamide, N-(4- (aminosulfonyl)phenyl)-2-iodoacetamide, N-(l,3-benzothiazol-2-yl)-2- iodoacetamide, N-(2,6-diethylphenyl)-2-iodoacetamide, N-(2-benzoyl-4- chlorophenyl)-2-iodoacetamide; (iii) bromoacetamides, such as N-(4- (acetylamino)phenyl)-2-bromoacetamide, N-(2-acetylphenyl)-2- bromoacetamide, 2-bromo-N-(2-cyanophenyl)acetamide, 2-bromo-N-(3- (trifluoromethyl)phenyl)acetamide, N-(2-benzoylphenyl)-2-bromoacetamide, 2-bromo-N-(4-fluorophenyl)-3-methylbutanamide, N-benzyl 2-bromo-N- phenylpropanamide, N-(2-bromo-butyryl)-4-chloro-benzenesulfonamide, 2-bromo-N-methyl-N phenylacetamide, 2-bromo-N-phenethyl-acetamide, 2- adamant- 1 -yl-2-bromo-N-cyclohexyl-acetamide, 2-bromo-N-(2- methylphenyl)butanamide, acetaminobromoaniline; (iv) disulfides, such as: ALDRITHIOL-2, ALDRITHIOL-4, isopropyl disulfide, 1-(isobutyl disulfanyl)-2- methylpropane, dibenzyl disulfide, 4-aminophenyl disulfide, 3-(2- pyridyl disulfide)propionic acid, 3-(2-pyridyl disulfide)propionic acid hydrazide, 3-(2-pyridyl disulfide)propionic acid N-succinimidyl ester, am6amPDP1-βCD;

[0619] and (v) thiols, such as: 4-phenylthiazole-2-thiol, Pulpald, 5,6,7,8-tetrahydro- quinazoline-2-thiol.

[0620] The mutant monomer or mutant monomers can be chemically modified by attachment of polyethylene glycol (PEG), a nucleic acid such as DNA, a dye, a fluorophore, or a chromophore. In some embodiments, the mutant monomer or mutant monomers are chemically modified using a molecular adaptor that facilitates interaction between the pore comprising the monomer and a target analyte, a target nucleotide, or a target polynucleotide sequence. The presence of the adaptor improves the host-guest chemistry of the pore and the nucleotide or polynucleotide, and thereby improves the sequencing ability of the pore formed by the mutant monomer.

[0621] The mutant monomer or mutant monomers can be chemically modified by attaching any molecule with the effect of reducing or narrowing the open diameter of the barrel of the assembly pore at any position. K37, V47, S49, T55, S86, E92, E94. More preferably, the mutant monomer can be chemically modified by attaching any molecule with the effect of reducing or narrowing the open diameter of the barrel of the assembly pore at positions E92 and E94. In one embodiment, the mutant monomer or mutant monomers are chemically modified by attaching a molecule to one or more cysteine residues at these positions (cysteine ligation).

[0622] The reactivity of a cysteine residue can be enhanced by modifying the adjacent residues. For example, the basic groups of flanking arginine, histidine or lysine residues change the pKa of the cysteine thiol to the more reactive S - The reactivity of a cysteine residue can be protected by a thiol protecting group such as dTNB. These can react with one or more cysteine residues of the mutant monomer prior to attachment of the linker.

[0623] The molecule can be attached directly to the mutant monomer or mutant monomers. Preferably, the molecule is attached to the mutant monomer using a linker such as a chemical crosslinker or a peptide linker. Suitable chemical crosslinkers are well known in the art. Preferred crosslinkers include 3-(pyridin-2- yldisulfanyl)propionic acid 2,5-dioxopyrrolidin-1-yl ester, 4-(pyridin-2- yldisulfanyl)butyric acid 2,5-dioxopyrrolidin-1-yl ester and 8-(pyridin-2- yldisulfanyl)octanoic acid 2,5-dioxopyrrolidin-1-yl ester. The most preferred crosslinker is 3-(2-pyridyldithio)propionic acid succinimidyl ester (SPDP). Typically, the molecule is covalently linked to the bifunctional crosslinker before the molecule / crosslinker complex is covalently linked to the mutant monomer, but it is also possible to covalently link the bifunctional crosslinker to the monomer before the bifunctional crosslinker / monomer complex is attached to the molecule.

[0624] Preferably, the linker is resistant to dithiothreitol (DTT). Suitable linkers include but are not limited to iodoacetamide-based and maleimide-based linkers.

[0625] Pores chemically modified in this way show the following particular advantages: (i) improved clarity of the read (ii) improved discrimination of bases and (iii) improved range, i.e. improved signal to noise.

[0626] By using chemical molecules to modify specific positions within the barrel, new readheads can be introduced or old readheads can be modified. Due to the size of the modified molecules, the physical size of the readhead can be significantly altered. Similarly, due to the chemical properties of the modified molecules, the properties of the readhead can be altered. It has been demonstrated that a combination of both effects results in a readhead with increased resolution and better base discrimination. Not only is the relative contribution of the signal for different bases at different positions altered, the readhead positions at the extremes show much less discrimination, meaning that their contribution to the signal is greatly reduced and therefore the length of the K-mer determined at a given moment is shorter. This more distinct readhead makes the process of deconvoluting the K-mer from the raw signal much simpler.

[0627] Generating pores of the invention

[0628] The present invention also provides a method of generating a pore of the invention. The method comprises allowing at least one mutant monomer of the invention or at least one construct of the invention to oligomerise with a sufficient number of mutant cytolysin monomers of the invention, constructs of the invention, cytolysin monomers or monomers derived from cytolysin to form a pore. If the method involves making a homo-oligomeric pore of the invention, all of the monomers used in the method are mutant cytolysin monomers of the invention having the same amino acid sequence. If the method involves making a hetero-oligomeric pore of the invention, at least one of the monomers is different from the other monomers.

[0629] Typically, the monomers are expressed in a host cell as described above, removed from the host cell and assembled into a pore in a separate membrane such as a sheep red blood cell membrane or a liposome containing sphingomyelin.

[0630] For example, the cytolysin monomers can be oligomerised by adding a lipid mixture comprising sphingomyelin and one or more of the following lipids: phosphatidylserine; POPE; cholesterol; and Soy PC and incubating the mixture, for example at 30°C for 60 minutes. The oligomerised monomers can be purified by any appropriate method, for example by SDS-PAGE and gel purification as described in WO2013 / 153359.

[0631] Any of the embodiments discussed above in relation to pores of the invention apply equally to the method of generating a pore.

[0632] Anal Biochem

[0633] The present invention provides a method of characterising a target analyte. The method comprises contacting the target analyte with a pore of the present invention, such that the target analyte moves through the pore. The pore can be any of the pores discussed above. Then, one or more properties of the target analyte are measured using standard methods known in the art, as the analyte moves relative to the pore. The one or more properties of the target analyte are preferably measured as the analyte moves through the pore. Steps (a) and (b) are preferably carried out with an electric potential applied across the pore. As discussed in more detail below, the applied electric potential typically causes a complex to form between the pore and a polynucleotide binding protein. The applied electric potential can be a voltage potential. Alternatively, the applied electric potential can be a chemical potential. An example of such an operation is the use of a salt gradient across an amphiphilic layer. Salt gradients are disclosed in Holden et al, J Am Chem Soc., 11 July 2007, vol. 129, no. 27, pages 8650 to 8655.

[0634] The method of the present invention is for characterising a target analyte. The method is for characterising at least one analyte. The method can involve characterising two or more analytes. The method can comprise characterising any number of analytes, such as 2, 5, 10, 15, 20, 30, 40, 50, 100 or more analytes.

[0635] The target analyte is preferably 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 drug, a diagnostic agent, a recreational drug, an explosive or an environmental pollutant. The method can involve characterising two or more analytes of the same type, such as two or more proteins, two or more nucleotides or two or more drugs. Alternatively, the method can involve characterising two or more analytes of different types, such as one or more proteins, one or more nucleotides and one or more drugs.

[0636] The target analyte can be secreted from a cell. Alternatively, the target analyte can be an analyte that is present inside a cell, such that the analyte must be extracted from the cell before the present invention can be performed.

[0637] The analyte is preferably an amino acid, a peptide, a polypeptide and / or a protein. The amino acid, peptide, polypeptide or protein can be naturally occurring or non-naturally occurring. The polypeptide or protein can comprise synthetic or modified amino acids within it. Various different types of modification to amino acids are known in the art. Suitable amino acids and modifications thereof are as discussed above. For the purposes of the present invention, it will be understood that the target analyte can be modified by any method available in the art.

[0638] The protein can be an enzyme, an antibody, a hormone, a growth factor, or a growth regulatory protein, such as a cytokine. The cytokine can be selected from the group consisting of interleukins, preferably IFN-1, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, and IL-13; interferons, preferably IL-γ; and other cytokines, such as TNF-α. The protein can be a bacterial protein, a fungal protein, a viral protein, or a parasite-derived protein.

[0639] The target analyte is preferably a nucleotide, oligonucleotide, or polynucleotide. The nucleotide typically contains a nucleobase, a sugar, and at least one phosphate group. The nucleobase is typically heterocyclic. The nucleobase includes, but is not limited to, purines and pyrimidines, and more specifically, adenine, guanine, thymine, uracil, and cytosine. The sugar is typically a pentose sugar. The nucleotide sugar includes, but is not limited to, ribose and deoxyribose. The nucleotide is typically a ribonucleotide or a deoxyribonucleotide. The nucleotide typically contains a mono-, di-, or tri-phosphate. The phosphate can be attached on the 5' or 3' side of the nucleotide.

[0640] Nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), 5-methylcytidine monophosphate, 5-methylcytidine diphosphate, 5-methylcytidine triphosphate, 5-hydroxymethylcytidine monophosphate, 5-hydroxymethylcytidine diphosphate, 5-hydroxymethylcytidine triphosphate, cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), and deoxycytidine triphosphate (dCTP), 5-methyl-2'-deoxycytidine monophosphate, 5-methyl-2'-deoxycytidine diphosphate, 5-methyl-2'-deoxycytidine triphosphate, 5-hydroxymethyl-2'-deoxycytidine monophosphate, 5-hydroxymethyl-2'-deoxycytidine diphosphate, and 5-hydroxymethyl-2'-deoxycytidine triphosphate. The nucleotide is preferably selected from AMP, TMP, GMP, UMP, dAMP, dTMP, dGMP, or dCMP. The nucleotide can be abasic (i.e., lack a nucleobase). The nucleotide can contain additional modifications. In particular, suitable modified nucleotides include, but are not limited to, 2' amino purines (such as 2'-amino cytidine and 2'-amino uridine), 2'-hydroxyl purines (such as 2'-fluoro purines (2'-fluoro cytidine and 2'-fluoro uridine), hydroxyl purines (such as 5'-alpha-P-borano uridine), 2'-O-methyl nucleotides (such as 2'-O-methyl adenosine, 2'-O-methyl guanosine, 2'-O-methyl cytidine, and 2'-O-methyl uridine), 4'-thio purines (such as 4'-thio uridine and 4'-thio cytidine), and nucleotides with modifications to the nucleobase (such as 5-pentynyl-2'-deoxyuridine, 5-(3-aminopropyl)-uridine, and 1,6-diaminohexyl-N-5-carbamoylmethyl uridine).

[0641] An oligonucleotide is a short polymer of nucleotides, typically having 50 or fewer nucleotides, such as 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, or 5 or fewer nucleotides. An oligonucleotide can include any of the nucleotides discussed below, including abasic and modified nucleotides. The methods of the present invention are preferably used to characterize a target polynucleotide. A polynucleotide, such as a nucleic acid, is a macromolecule that includes two or more nucleotides. A polynucleotide or nucleic acid can include any combination of any nucleotides. The nucleotides can be naturally occurring or artificial. One or more of the nucleotides in the target polynucleotide can be oxidized or methylated. One or more of the nucleotides in the target polynucleotide can be damaged. For example, the polynucleotide can include a pyrimidine dimer. Such dimers are often associated with damage caused by ultraviolet light and are a major cause of skin melanoma. One or more of the nucleotides in the target polynucleotide can be modified, for example, with a label or tag. Appropriate labels are described below. The target polynucleotide can include one or more spacers.

[0642] A nucleotide is defined above. Nucleotides present in a polynucleotide include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), cytosine nucleotide monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP), and deoxycytidine monophosphate (dCMP). The nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP, and dUMP.

[0643] A nucleotide can be abasic (i.e., lack a nucleobase).

[0644] Nucleotides in a polynucleotide can be attached to one another in any manner. Nucleotides are typically attached by their sugars and phosphate groups, as in nucleic acids. Nucleotides can be linked by their nucleobases, as in pyrimidine dimers.

[0645] A polynucleotide can be single-stranded or double-stranded. At least a portion of a polynucleotide is preferably double-stranded. A single-stranded polynucleotide can have one or more primers hybridized thereto, and thus include one or more short regions of double-stranded polynucleotide. A primer can be the same type of polynucleotide as the target polynucleotide or can be a different type of polynucleotide.

[0646] A polynucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). A polynucleotide can include an RNA strand hybridized to a strand of DNA. A polynucleotide can be any synthetic nucleic acid known in the art, such as a peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), or other synthetic polymer with nucleotide side chains.

[0647] The entire or only a portion of a target polynucleotide can be characterized using this method. A target polynucleotide can be of any length. For example, a polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotide pairs in length. A polynucleotide can be 1000 or more nucleotide pairs, 5000 or more nucleotide pairs, or 100000 or more nucleotide pairs in length.

[0648] A target analyte, such as a target polynucleotide, is present in any appropriate sample. The present application is typically performed on a sample known to contain or suspected of containing a target analyte, such as a target polynucleotide. Alternatively, the present application can be performed on a sample to confirm the identity of one or more target analytes, such as one or more target polynucleotides, whose presence in the sample is known or expected.

[0649] A sample can be a biological sample. The present application can be performed in vitro on a sample obtained or extracted from any organism or microorganism. An organism or microorganism is typically an archaeal, prokaryotic, or eukaryotic microorganism, and is typically a member of one of the following five kingdoms: Plantae, Animalia, Fungi, Monera, and Protista. The present application can be performed in vitro on a sample obtained or extracted from any virus. A sample is preferably a fluid sample. A sample typically includes a body fluid of a patient. A sample can be urine, lymph, saliva, mucus, or amniotic fluid, but is preferably blood, plasma, or serum. Typically, a sample is derived from a human, but can alternatively be from another mammal, such as from a commercially raised animal, such as a horse, cow, sheep, or pig, or alternatively can be a pet, such as a cat or dog. Alternatively, a sample derived from a plant can typically be obtained from an economic crop, such as a cereal, legume, fruit, or vegetable, for example, wheat, barley, oat, rape, maize, soybean, rice, sugar beet, banana, apple, tomato, potato, grape, tobacco, bean, lentil, sugar cane, cocoa, cotton.

[0650] A sample can be a non-biological sample. A non-biological sample is preferably a fluid sample. Examples of non-biological samples include surgical fluids, water such as drinking water, seawater, or river water, and reagents for laboratory testing.

[0651] The sample is typically treated prior to assay, for example by centrifugation or passing through a filter to remove unwanted molecules or membranes of cells such as red blood cells. The sample can be measured immediately after it is taken. The sample can also typically be stored prior to analysis, preferably at below -70°C.

[0652] Pores are typically present in the membrane. Any membrane can be used in accordance with the present application. Suitable membranes are well known in the art. The membrane preferably comprises sphingomyelin. The membrane is preferably an amphiphilic layer. An amphiphilic layer is a layer formed from amphiphilic molecules, such as phospholipids, having at least one hydrophilic portion and at least one lipophilic or hydrophobic portion. The amphiphilic molecules can be synthetic or naturally occurring. Non-naturally occurring amphiphiles and amphiphiles that form monolayers are known in the art and include, for example, block copolymers (Gonzalez-Perez et al, Langmuir, 2009, vol. 25, pp. 10447-10450). A block copolymer is a polymeric material in which two or more monomeric subunits are polymerised together to produce a single polymer chain. Block copolymers typically have properties contributed by each monomeric subunit. However, block copolymers can have unique properties not possessed by polymers formed from the individual subunits. Block copolymers can be engineered such that one of the monomeric subunits is hydrophobic (i.e. lipophilic) in an aqueous medium, while one or more other subunits are hydrophilic. In this case, the block copolymer can possess amphiphilic properties, and can form a structure that mimics a biological membrane. The block copolymer can be a diblock (which consists of two monomeric subunits), but can also be constructed from more than two monomeric subunits to form more complex arrangements that behave as amphiphiles. The copolymer can be a triblock, tetrablock or pentablock copolymer.

[0653] The amphiphilic layer can be a monolayer or a bilayer. The amphiphilic layer is typically a planar lipid bilayer or a supported bilayer.

[0654] The amphiphilic layer is typically a lipid bilayer. Lipid bilayers are models of cell membranes and act as an excellent platform for a range of experimental studies. For example, lipid bilayers can be used to study membrane proteins in vitro by single channel recording. Alternatively, lipid bilayers can be used as biosensors to detect the presence of a range of substances. The lipid bilayer can be any lipid bilayer. Suitable lipid bilayers include, but are not limited to, planar lipid bilayers, supported bilayers or liposomes. The lipid bilayer is preferably a planar lipid bilayer. Suitable lipid bilayers are disclosed in International Application No. PCT / GB08 / 000563 (published as WO 2008 / 102121), International Application No. PCT / GB08 / 004127 (published as WO 2009 / 077734) and International Application No. PCT / GB2006 / 001057 (published as WO 2006 / 100484).

[0655] Methods for forming lipid bilayers are known in the art. Suitable methods are disclosed in examples. Lipid bilayers are typically formed by the method of Montal and Mueller (Proc Natl Acad Sci USA, 1972, vol. 69, pp. 3561-3566), in which a lipid monolayer is carried on a water solution / air interface by an aperture on either side of the interface, which is perpendicular to the interface.

[0656] The method of Montal and Mueller is popular because it is cost effective and is a relatively straightforward method of forming a good quality lipid bilayer suitable for protein pore insertion. Other common methods of bilayer formation include tip-dipping of a liposome bilayer, bilayer painting and patch-clamping.

[0657] In preferred embodiments, the lipid bilayer is formed as disclosed in International Application No. PCT / GB08 / 004127 (published as WO 2009 / 077734). In another preferred embodiment, the membrane is a solid state layer. A solid state layer is not of biological origin. In other words, a solid state layer is not derived from or isolated from a biological environment, such as a synthetic manufactured version of a living organism or cell or biologically obtainable structure. A solid state layer can be formed from both organic and inorganic materials, including but not limited to: microelectronic materials; insulating materials such as Si3N4, A12O3 and SiO; organic and inorganic polymers such as polyamides; plastics such as or elastomers such as two-component addition-cured silicone rubbers; and glasses. A solid state layer can be formed from a monolayer of atoms, such as graphene, or a layer only a few atoms thick. Suitable graphene layers are disclosed in International Application No. PCT / US2008 / 010637 (published as WO 2009 / 035647).

[0658] The method is typically performed using: (i) an artificial amphipathic layer comprising a pore; (ii) a separate naturally occurring lipid bilayer comprising a pore; or (iii) a cell having a pore inserted therein. The method is typically performed using an artificial amphipathic layer, such as an artificial lipid bilayer. The layer can comprise other transmembrane and / or intramembrane proteins as well as other molecules in addition to the pore. Suitable apparatus and conditions are discussed below. The methods of the application are typically performed in vitro.

[0659] An analyte, such as a target polynucleotide, can be coupled to the membrane. This can be done using any known method. If the membrane is an amphipathic layer, such as a lipid bilayer (as discussed in detail above), the analyte, such as a target polynucleotide, is preferably coupled to the membrane by a polypeptide present in the membrane or a hydrophobic anchor present in the membrane. The hydrophobic anchor is preferably a lipid, a fatty acid, a sterol, a carbon nanotube or an amino acid.

[0660] The analyte, such as a target polynucleotide, can be coupled directly to the membrane. The analyte, such as a target polynucleotide, is preferably coupled to the membrane through a linker. Preferred linkers include, but are not limited to, polymers, such as polynucleotides, polyethylene glycol (PEG), and polypeptides. If the polynucleotide is coupled directly to the membrane, some data will be lost because the run characterization cannot continue to the end of the polynucleotide due to the distance between the membrane and the inside of the pore. If a linker is used, the polynucleotide can be fully processed. If a linker is used, the linker can be attached to the polynucleotide at any position. The linker is preferably attached to the polynucleotide at the tail polymer.

[0661] The coupling can be stable or transient. For some applications, the transient nature of the coupling is preferred. If the stable coupling molecule is attached directly to the 5' or 3' end of the polynucleotide, some data will be lost because the run characterization cannot continue to the end of the polynucleotide due to the distance between the bilayer and the inside of the pore. If the coupling is transient, then the polynucleotide can be fully processed when the end of the coupling becomes randomly free of the bilayer. Chemical groups that form stable or transient links with membranes are discussed in more detail below. The analyte, such as a target polynucleotide, can be transiently coupled to an amphiphilic layer, such as a lipid bilayer, using a cholesterol or a fatty acyl chain. Any fatty acyl chain of length 6 to 30 carbon atoms can be used, such as hexadecanoic acid.

[0662] In preferred embodiments, the analyte, such as a target polynucleotide, is coupled to an amphiphilic layer. Coupling of analytes, such as target polynucleotides, to synthetic lipid bilayers has been performed previously using a variety of different tethering strategies. This information is summarized in Table 5 below:

[0663] Table 5

[0664]

[0665] Polynucleotides can be functionalized in a synthetic reaction using modified phosphoramidites that are easily compatible for the addition of reactive groups such as thiol, cholesterol, lipid, and biotin groups. These different attachment chemistries create a range of attachment options for the polynucleotide. Each different modification group tethers the polynucleotide in a slightly different way, and the coupling is not always permanent, thus giving different residence times for the polynucleotide to couple to the bilayer. The advantages of transient coupling are discussed above.

[0666] Coupling of polynucleotides can also be achieved by a variety of other means, provided that a moiety that is reactive to the moiety is added to the polynucleotide. It has been previously reported that reactive groups can be added to either end of DNA. Thiol groups can be added to the 5' of ssDNA using polynucleotide kinase and ATPγS (Grant, G. P. and P. Z. Qin (2007). "A facile method for attaching nitroxide spin labels at the 5' terminus of nucleic acids." Nucleic Acids Res. 35(10): e77). A wider collection of chemical groups such as biotin, thiols, and fluorophores can be added using terminal transferase to incorporate modified oligonucleotides into the 3' of ssDNA (Kumar, A., P. Tchen, et al. (1988). "Nonradioactive labelling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase." Nucleic Acids Res. 16(24): 8577-8585). Method of identifying individual nucleotides , vol. 169, no. 2, pp. 376-382).

[0667] Alternatively, the reactive group can be considered to be an addition to a short piece of DNA that is complementary to the DNA already coupled to the bilayer, such that attachment can be achieved by hybridization. Ligation of short pieces of ssDNA using T4 RNA ligase I has been reported (Troutt, A. B., M. G. McHeyzer-Williams, et al. (1992). "Ligation-anchored PCR: a simple amplification technique with single-sided specificity." Proc Natl Acad Sci U S A 89(20): 9823-5). Alternatively, ssDNA or dsDNA can be ligated to native dsDNA and then the two strands are separated by heat or chemical denaturation. For native dsDNA, a piece of ssDNA can be added to one or both ends of the duplex, or a dsDNA can be added to one or both ends. Then, when the duplex is melted, if the ssDNA is used to ligate or modify at the 5' end, 3' end, each single strand will have either a 5' modification or a 3' modification, or if the dsDNA is used to ligate, each single strand will have both a 5' and 3' modification. If the polynucleotide is a synthetic strand, the coupling chemistry can be incorporated during chemical synthesis of the polynucleotide. For example, the polynucleotide can be synthesized using primers to which a reactive group is attached.

[0668] A common technique for amplifying a segment of genomic DNA is to use the polymerase chain reaction (PCR). Here, using two synthetic oligonucleotide primers, a large number of copies of the same DNA segment can be produced, where for each copy, the 5' of each strand in the duplex will be a synthetic polynucleotide. By using an antisense primer with a reactive group, such as a cholesterol, thiol, biotin, or lipid, each copy of the amplified target DNA will contain a reactive group for coupling.

[0669] The pores used in the methods of the application are pores of the application (i.e. pores comprising at least one mutant monomer of the application or at least one construct of the application). The pores can be chemically modified in any of the ways discussed above. Preferably the pores are modified with covalent adapters that are capable of interacting with target analytes, as discussed above.

[0670] The method is preferably for characterising a target polynucleotide and step (a) comprises: contacting the target polynucleotide with the pore and a polynucleotide binding protein, and the polynucleotide binding protein controls movement of the target polynucleotide through the pore. The polynucleotide binding protein can be any protein that is able to bind to a polynucleotide and control its movement through a pore. It is straightforward in the art to determine whether a polynucleotide binding protein binds to a polynucleotide. The polynucleotide binding protein typically interacts with a polynucleotide and modifies at least one property of the polynucleotide. The polynucleotide binding protein can modify the polynucleotide by cleaving it to form individual nucleotides or shorter chains of nucleotides such as di- or tri-nucleotides. The portion can modify the polynucleotide by orienting it or moving it to a particular location, i.e. controlling its movement.

[0671] The polynucleotide binding protein is preferably a polynucleotide handling enzyme. A polynucleotide handling enzyme is a polypeptide that is able to interact with a polynucleotide and modify at least one property thereof. The enzyme can modify the polynucleotide by cleaving it to form individual nucleotides or shorter chains of nucleotides such as di- or tri-nucleotides. The enzyme can modify the polynucleotide by orienting it or moving it to a particular location. The polynucleotide binding protein typically comprises a polynucleotide binding domain and a catalytic domain. The polynucleotide handling enzyme need not display enzymatic activity, only that it is able to bind to a target sequence and control its movement through a pore. For example, the enzyme can be modified to remove its enzymatic activity, or can be used under conditions that prevent it from acting as an enzyme. Such conditions are discussed in more detail below.

[0672] The polynucleotide handling enzyme is preferably derived from a resolvase. The polynucleotide handling enzyme used in the construct of the enzyme is more preferably derived from a member of any of the following enzyme classification (EC) 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 and 3.1.31. The enzyme can be any of the enzymes disclosed in International Application No. PCT / GB10 / 000133 (published as WO 2010 / 086603).

[0673] Preferred enzymes are polymerases, exonucleases, helicases and topoisomerases such as gyrase. Suitable enzymes include but are not limited to exonuclease I from E. coli (SEQ ID NO: 6), exonuclease III from E. coli (SEQ ID NO: 8), RecJ from Thermus (SEQ ID NO: 10) and bacteriophage lambda exonuclease (SEQ ID NO: 12) and variants thereof. The three subunits comprising the sequence shown in SEQ ID NO: 10 or variants thereof interact to form a trimeric exonuclease. The enzyme can be Phi29 DNA polymerase (SEQ ID NO: 4) or a variant thereof. The enzyme can be a helicase or be derived from a helicase. A typical helicase is Hel308, RecD or XPD, for example, Hel308 Mbu (SEQ ID NO: 13) or a variant thereof.

[0674] The enzyme is most preferably derived from a helicase such as a Hel308 helicase, a RecD helicase such as a TraI helicase or a TrwC helicase, an XPD helicase or a Dda helicase. The helicase can be any of the helicases, modified helicases or helicase constructs disclosed in International Application Nos: PCT / GB2012 / 052579 (published as WO 2013 / 057495); PCT / GB2012 / 053274 (published as WO 2013 / 098562); PCT / GB2012 / 053273 (published as WO 2013 098561); PCT / GB2013 / 051925 (published as WO 2014 / 013260); PCT / GB2013 / 051924 (published as WO 2014 / 013259); PCT / GB2013 / 051928 (published as WO 2014 / 013262) and PCT / GB2014 / 052736.

[0675] The helicase preferably comprises the sequence shown in SEQ ID NO: 18 (Dda) or a variant thereof. The variant can differ from the native sequence in any of the ways discussed below in relation to the transmembrane pore. Preferred variants of SEQ ID NO: 18 include: (a) E94C and A360C; or (b) E94C, A360C, C109A and C136A, and then optionally (AM1) G1G2 (i.e. deletion of M1 and then addition of G1 and G2).

[0676] A variant of SEQ ID NO: 4, 6, 8, 10, 12, 13, or 18 is an enzyme whose amino acid sequence differs from that of SEQ ID NO: 4, 6, 8, 10, 12, 13, or 18 and which retains polynucleotide binding ability. The variant can comprise modifications that facilitate binding of a polynucleotide and / or facilitate activity of the polynucleotide at high salt concentrations and / or at room temperature.

[0677] A variant will preferably be at least 50% homologous to the sequence over the entire length of the amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 13, or 18, based on amino acid identity. More preferably, the variant polypeptide can be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and more preferably at least 95%, 97%, or 99% homologous to the amino acid sequence of SEQ ID NO: 4, 6, 8, 10, 12, 13, or 18 over the entire sequence, based on amino acid identity. There can be at least 80%, for example at least 85%, 90%, or 95% amino acid identity over a stretch of 200 or more, for example 230, 250, 270, or 280 or more, contiguous amino acids (“hard homology”). Homology is determined as described above. The variant can differ from the wild-type sequence in any of the ways discussed above with reference to SEQ ID NO: 2. The enzyme can be covalently linked to the pore, as discussed above.

[0678] There are two main strategies for sequencing polynucleotides using a nanopore, namely strand sequencing and exonuclease sequencing. The methods of the application can involve strand sequencing or exonuclease sequencing.

[0679] In strand sequencing, DNA is transferred through the nanopore by an applied potential or against an applied potential. An exonuclease that acts progressively or gradually on double stranded DNA can be used on the cis side of the pore to feed the remaining single strand under the applied potential or on the trans side under the opposite potential. Likewise, a helicase that unwinds double stranded DNA can also be used in a similar fashion. A polymerase can also be used. There is also the possibility of sequencing applications that require strand transfer against an applied potential, but the DNA must first be “captured” by the enzyme under the opposite or no potential. In the case where the potential is then switched back after binding, the strand will pass through the pore in the cis to trans fashion and held in the elongated conformation by the current. A single stranded DNA exonuclease or a single stranded DNA dependent polymerase can act as a molecular motor to pull the recently transferred single strand back through the pore in a stepwise controlled fashion against the applied potential from trans to cis.

[0680] In one embodiment, the method of characterizing a target polynucleotide involves contacting the target sequence with a pore and a helicase. Any helicase can be used in the method. The helicase can function in two modes relative to the pore. First, the method is preferably performed using a helicase such that the helicase controls the movement of the target sequence through the pore using the field generated by the applied voltage. In this mode, the 5' end of the DNA is first captured in the pore and the enzyme controls the movement of the DNA into the pore such that the target sequence is threaded through the pore using the field until it is eventually transferred through to the opposite side of the bilayer. Alternatively, the method is preferably performed such that the helicase controls the movement of the target sequence through the pore against the field generated by the applied voltage. In this mode, the 3' end of the DNA is first captured in the pore and the enzyme controls the movement of the DNA through the pore such that the target sequence is pulled out of the pore against the applied field until it is eventually pushed back to the same side of the bilayer.

[0681] In exonuclease sequencing, an exonuclease releases individual nucleotides from one end of a target polynucleotide, and these individual nucleotides are identified as discussed below. In another embodiment, the method of characterizing a target polynucleotide involves contacting the target sequence with a pore and an exonuclease. Any of the exonucleases discussed above can be used in the method. The enzyme can be covalently attached to the pore, as discussed above.

[0682] An exonuclease is an enzyme that typically latches onto one end of a polynucleotide and digests the sequence one nucleotide at a time from that end. An exonuclease can digest a polynucleotide in the 5' to 3' direction or the 3' to 5' direction. The end of the polynucleotide to which the exonuclease binds is typically determined by selecting the enzyme used in the art and / or using methods known in the art. A hydroxyl group or cap structure at either end of a polynucleotide can typically be used to prevent or promote the binding of an exonuclease to a particular end of a polynucleotide.

[0683] The method involves contacting the polynucleotide with an exonuclease such that nucleotides are digested from the end of the polynucleotide at a rate that allows for the characterization or identification of a certain proportion of the nucleotides, as discussed above. Methods for doing so are well known in the art. For example, Edman degradation is used to sequentially digest single amino acids from the end of a polypeptide such that the amino acids can be identified using high performance liquid chromatography (HPLC). Homologous methods can be used in the present invention.

[0684] The rate at which the exonuclease acts is typically slower than the optimal rate of wild-type exonuclease. Suitable rates of activity of the exonuclease in the methods of the application include digestion rates of 0.5 to 1000 nucleotides per second, 0.6 to 500 nucleotides per second, 0.7 to 200 nucleotides per second, 0.8 to 100 nucleotides per second, 0.9 to 50 nucleotides per second or 1 to 20 or 10 nucleotides per second. The rate is preferably 1, 10, 100, 500 or 1000 nucleotides per second. Suitable rates of exonuclease activity can be achieved in different ways. For example, a variant exonuclease having a reduced optimal rate of activity can be used in accordance with the application.

[0685] The methods of the application involve measuring one or more properties of a target analyte, such as a target polynucleotide. The methods can involve measuring two, three, four or five or more properties of a target analyte, such as a target polynucleotide. For a target polynucleotide, the one or more properties are preferably selected from: (i) the length of the target polynucleotide; (ii) the identity of the target polynucleotide; (iii) the sequence of the target polynucleotide; (iv) the secondary structure of the target polynucleotide; and (v) whether the target polynucleotide is modified. Any combination of (i) to (v) can be measured in accordance with the application.

[0686] For (i), the length of the polynucleotide can be measured using the number of interactions between the target polynucleotide and the pore.

[0687] For (ii), the identity of the polynucleotide can be measured in a number of ways. The identity of the polynucleotide can be measured in conjunction with measuring the sequence of the target polynucleotide or without measuring the sequence of the target polynucleotide. The former is straightforward; the polynucleotide is sequenced and thereby identified. The latter can be performed in several ways. For example, the presence of a particular motif in the polynucleotide can be measured (without measuring the remainder of the sequence of the polynucleotide). Alternatively, the measurement of a particular electrical and / or optical signal in the method can identify the target polynucleotide as being from a particular source.

[0688] For (iii), the sequence of the polynucleotide can be determined as previously described. Suitable sequencing methods, in particular sequencing methods using electrical measurements, are described in Stoddart D et al, Proc Natl Acad Sci, 2012, vol. 106, no. 19, pp. 7702-7707; Lieberman KR et al, J Am Chem Soc, 2010, vol. 132, no. 50, pp. 17961-17972; and International Application WO 2000 / 28312.

[0689] For (iv), the secondary structure can be measured in a number of ways. For example, if the method involves electrical measurements, then the secondary structure can be measured using changes in residence time or changes in current flow through the pore. This allows regions of single stranded polynucleotides and double stranded polynucleotides to be distinguished.

[0690] For (v), it can be measured whether any modifications are present. The method preferably comprises determining whether the target polynucleotide is modified by methylation, by oxidation, by damage using one or more proteins or using one or more labels, tags or spacers. Particular modifications will cause particular interactions with the pore which can be measured using the methods described below. For example, methylcytosine and cytosine can be distinguished on the basis of the current flow through the pore during interaction of the pore with each nucleotide.

[0691] The application also provides a method of estimating the sequence of a target polynucleotide. The application also provides a method of sequencing a target polynucleotide.

[0692] A variety of different types of measurement can be made. This includes but is not limited to electrical measurements and optical measurements. Possible electrical measurements include current measurements, impedance measurements, tunnelling measurements (Ivanov AP et al, Nano Lett., 12 January 2011, vol. 11, no. 1, pp. 279-285) and FET measurements (International application WO 2005 / 124888). Suitable optical methods involving fluorescence measurements are disclosed in J. Am. Chem. Soc, 2009, vol. 131, pp. 1652 and 1653. Optical measurements can be combined with point measurements (Soni GV et al, Rev Sci Instrum, January 2010, vol. 81, no. 1, p. 014301). The measurements can be transmembrane current measurements, such as measurements of ionic current flow through the pore.

[0693] Application WO 2005 / 124888). Suitable optical methods involving fluorescence measurements are disclosed in J. Am. Chem. Soc, 2009, vol. 131, pp. 1652 and 1653. Optical measurements can be combined with point measurements (Soni GV et al, Rev Sci Instrum, January 2010, vol. 81, no. 1, p. 014301). The measurements can be transmembrane current measurements, such as measurements of ionic current flow through the pore.

[0694] Electrical measurements can be made using standard single channel recording equipment as described in:

[0695] Stoddart D et al, Proc Natl Acad Sci, 2012, vol. 106, no. 19, pp. 7702-7707; Lieberman KR et al, J Am Chem Soc, 2010, vol. 132, no. 50, pp. 17961-17972; and International application WO 2005 / 124888.

[0696] WO-2000 / 28312. Alternatively, electrical measurements can be made using a multi-channel system such as described in, for example:

[0697] International application WO-2009 / 077734 and international application WO-2011 / 067559.

[0698] In preferred embodiments, the method comprises:

[0699] (a) contacting a target polynucleotide with a pore and a polynucleotide binding protein of the application, such that the target polynucleotide moves through the pore and the binding protein controls the movement of the target polynucleotide through the pore; and

[0700] (b) measuring the current through the pore as the polynucleotide moves relative to the pore, wherein the current is indicative of one or more properties of the target polynucleotide, and characterising the target polynucleotide therefrom.

[0701] The method can be performed using any apparatus suitable for a membrane / pore system in which a pore is inserted into a membrane. The method can be performed using any apparatus suitable for transmembrane pore sensing. For example, the apparatus comprises a chamber containing an aqueous solution and a barrier dividing the chamber into two sections. The barrier has an aperture in which a membrane containing a pore is formed.

[0702] The method can be performed using the apparatus described in international application number PCT / GB08 / 000562 (WO 2008 / 102120).

[0703] The method can involve measuring the current through a pore as an analyte, such as a target polynucleotide, moves relative to the pore. Thus, the apparatus can further comprise circuitry capable of applying an electrical potential and measuring an electrical signal across the membrane and pore. The method can be performed using a patch clamp or a voltage clamp. The method preferably involves the use of a voltage clamp.

[0704] The methods of the invention can involve measuring the current through the pore as the analyte, such as a target polynucleotide, moves relative to the pore. Suitable conditions for measuring ion current through a transmembrane protein pore are known in the art and disclosed in the examples. The methods are typically performed using a voltage applied across the membrane and the pore. The voltage used is typically +2 V to -2 V, typically -400 mV to +400 mV. The voltage used is preferably in a range having a lower limit selected from -400 mV, -300 mV, -200 mV, -150 mV, -100 mV, -50 mV, -20 mV, and 0 mV, and an upper limit independently selected from +10 mV, +20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV, and +400 mV. The voltage used is more preferably in a range of 100 mV to 240 mV, and most preferably in a range of 120 mV to 220 mV. Differentiation of different nucleotides through the pore can be increased by using an increased applied potential.

[0705] The methods are typically performed in the presence of any charge carrier, such as a metal salt, e.g., an alkali metal salt; a halide salt, e.g., a chloride salt such as an alkali metal chloride salt. The charge carrier can comprise an ionic liquid or an organic salt, such as tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride, or 1-ethyl-3-methylimidazolium chloride. In the exemplary apparatus discussed above, the salt is present in an aqueous solution in the chamber. Potassium chloride (KCl), sodium chloride (NaCl), or cesium chloride (CsCl) are typically used. KCl is preferred. The salt concentration can be saturated. The salt concentration can be 3 M or less, and is typically 0.1 M to 2.5 M, 0.3 M to 1.9 M, 0.5 M to 1.8 M, 0.7 M to 1.7 M, 0.9 M to 1.6 M, or 1 M to 1.4 M. The salt concentration is preferably 150 mM to 1 M. The methods are preferably performed using a salt concentration of at least 0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M, or at least 3.0 M. High salt concentrations provide high signal-to-noise ratios and allow the identification of current indicative of the presence of a nucleotide against a background of normal current fluctuations.

[0706] The method is typically performed in the presence of a buffer. In the exemplary apparatus discussed above, the buffer is present in the aqueous solution in the chamber. Any buffer can be used in the method of the application. Typically, the buffer is HEPES. Another suitable buffer is Tris-HCl buffer. The method is typically performed at a pH of 4.0 to 12.0, 4.5 to 10.0, 5.0 to 9.0, 5.5 to 8.8, 6.0 to 8.7, or 7.0 to 8.8, or 7.5 to 8.5. The pH used is preferably about 7.5.

[0707] The method can be performed at a temperature of 0 °C to 100 °C, 15 °C to 95 °C, 16 °C to 90 °C, 17 °C to 85 °C, 18 °C to 80 °C, 19 °C to 70 °C, or 20 °C to 60 °C. The method is typically performed at room temperature. The method is optionally performed at a temperature that supports the function of the enzyme, such as about 37 °C.

[0708] The method is typically performed in the presence of free nucleotides or free nucleotide analogs and enzyme cofactors that facilitate the action of polynucleotide binding proteins such as helicases or exonucleases. The free nucleotides can be one or more of any of the individual nucleotides discussed above. The free nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (dGDP), deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), and deoxycytidine triphosphate (dCTP). The free nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, or dCMP. The free nucleotides are preferably adenosine triphosphate (ATP). The enzyme cofactor is a factor that allows a helicase to function. The enzyme cofactor is preferably a divalent metal cation. The divalent metal cation is preferably Mg 2+ , Mn2+ Ca 2+ Co 2+ The enzyme cofactor is most preferably Mg 2+ .

[0709] The target polynucleotide can be contacted with the pore and the polynucleotide binding protein in any order. Preferably, the target polynucleotide is first complexed with the polynucleotide binding protein when the target polynucleotide is contacted with the polynucleotide binding protein and the pore. When a voltage is applied across the pore, the target polynucleotide / protein complex is then complexed with the pore and controls the movement of the polynucleotide through the pore.

[0710] Method of forming a sensor

[0711] The present application also provides a method of characterising an individual nucleotide. In other words, the target analyte is an individual nucleotide. The method comprises: contacting the nucleotide with a pore of the present application, such that the nucleotide interacts with the pore; and measuring the current through the pore during the interaction and thereby characterising the nucleotide. Thus, the present application relates to nanopore sensing of individual nucleotides. The present application also provides a method for identifying an individual nucleotide, comprising: measuring the current through the pore during the interaction and thereby determining the identity of the nucleotide. Any of the pores discussed above can be used. Preferably, the pore is chemically modified with a molecular adaptor, as discussed above.

[0712] A nucleotide is present if the current flows through the pore in a manner specific to the nucleotide (i.e. if a unique current flow associated with the analyte is detected through the pore). A nucleotide is not present if the current does not flow through the pore in a manner specific to the nucleotide.

[0713] The present application can be used to distinguish between nucleotides of the same structure on the basis of their different effects on the current through the pore. Individual nucleotides can be identified at the single molecule level according to the amplitude of the nucleotide current when the nucleotide interacts with the pore. The present application can also be used to determine whether a particular nucleotide is present in a sample. The present application can also be used to measure the concentration of a particular nucleotide in a sample.

[0714] The pore is typically present in a membrane. The method can be performed using any appropriate membrane / pore system described above.

[0715] A single nucleotide is a single nucleotide. A single nucleotide is a nucleotide that is not bound to another nucleotide or polynucleotide by a nucleotide linkage. A nucleotide linkage involves one of the phosphate groups of a nucleotide that is bound to the sugar group of another nucleotide. A single nucleotide is typically a nucleotide that is bound to another polynucleotide by a nucleotide linkage that is composed of at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1000, or at least 5000 nucleotides. For example, a single nucleotide has been digested from a target analyte polynucleotide sequence such as a DNA or RNA strand. The methods of the present invention can be used to identify any nucleotide. The nucleotide can be any of the nucleotides discussed above.

[0716] The nucleotide can be derived from a digestion of a nucleic acid sequence such as a ribonucleic acid (RNA) or a deoxyribonucleic acid (DNA). The nucleic acid sequence can be digested using any method known in the art. Suitable methods include, but are not limited to, methods using enzymes or catalysts. Catalytic digestion of nucleic acids is disclosed in Deck et al., Inorg. Chem., 2002, vol. 41, pp. 669-677.

[0717] The single nucleotide from a single polynucleotide can be contacted with the pore in a sequential manner so as to sequence all or a portion of the polynucleotide. Sequencing a polynucleotide is discussed in more detail above.

[0718] The nucleotide can be contacted with the pore on both sides of the membrane. The nucleotide can be introduced to the pore on both sides of the membrane. The nucleotide can be contacted with a side of the membrane that allows the nucleotide to pass through the pore to the other side of the membrane. For example, the nucleotide is contacted with an end of the pore that in its native environment allows ions or small molecules such as nucleotides to enter the barrel or channel of the pore so that the nucleotide can pass through the pore. In this case, the nucleotide interacts with the pore and / or adapter as it passes through the barrel or channel of the pore across the membrane. Alternatively, the nucleotide can be contacted with a side of the membrane that allows the nucleotide to interact with the pore through or by binding to an adapter, dissociate from the pore and remain on the same side of the membrane. The present invention provides pores in which the location of the adapter is fixed. Thus, the nucleotide is preferably contacted with an end of the pore that allows the adapter to interact with the nucleotide.

[0719] The nucleotide can interact with the pore in any manner and at any site. As discussed above, the nucleotide is preferably reversibly bound to the pore through or by binding to an adapter. The nucleotide is most preferably reversibly bound to the pore through or by binding to an adapter as it passes through the pore across the membrane. The nucleotide can also be reversibly bound to the barrel or channel of the pore through or by binding to an adapter as it passes through the pore across the membrane.

[0720] During the interaction between the nucleotide and the pore, the nucleotide affects the current flowing through the pore in a manner that is specific to the nucleotide. For example, a particular nucleotide will decrease the current flowing through the pore for a particular average period of time and to a particular extent. In other words, the current flowing through the pore is unique for a particular nucleotide. Control experiments can be performed to determine the effect a particular nucleotide has on the current flowing through the pore. The results generated by performing the method of the application on a test sample can then be compared to the results derived from such control experiments to identify a particular nucleotide in the sample or to determine whether a particular nucleotide is present in the sample. The frequency with which the current flowing through the pore is affected in a manner indicative of a particular nucleotide can be used to determine the concentration of the nucleotide in the sample. The ratio of different nucleotides within a sample can also be calculated. For example, the ratio of dCMP to methyl-dCMP can be calculated.

[0721] The method can involve the use of any of the apparatus, samples or conditions discussed above.

[0722] Kit

[0723] The application also provides a method of forming a sensor for characterising a target polynucleotide. The method comprises forming a complex between a pore of the application and a polynucleotide binding protein such as a helicase or an exonuclease. The complex can be formed by contacting the pore and the protein in the presence of the target polynucleotide and then applying an electrical potential across the pore. The applied electrical potential can be a chemical potential or a voltage potential as described above. Alternatively, the complex can be formed by covalently linking the pore to the protein. Methods for covalently linking are known in the art and are disclosed, for example, in International Application No. PCT / GB09 / 001679 (published as WO 2010 / 004265) and PCT / GB10 / 000133 (published as WO 2010 / 086603). The complex is a sensor for characterising a target polynucleotide. The method preferably comprises forming a complex between a pore of the application and a helicase. Any of the embodiments discussed above are equally applicable to this method.

[0724] The application also provides a sensor for characterising a target polynucleotide. The sensor comprises a complex between a pore of the application and a polynucleotide binding protein. Any of the embodiments discussed above are equally applicable to the sensor of the application.

[0725] Apparatus

[0726] The present application also provides a kit for characterising, such as sequencing, a target polynucleotide. The kit comprises (a) a well of the present application and (b) a membrane. The kit preferably further comprises a polynucleotide binding protein such as a helicase or exonuclease. Any of the embodiments discussed above equally apply to the kit of the present application.

[0727] The kit of the present application can additionally comprise one or more other reagents or instruments that enable any of the embodiments mentioned above to be performed. Such reagents or instruments include one or more of: one or more appropriate buffers (aqueous solutions), a device for obtaining a sample from a subject (such as a container or instrument comprising a needle), a device for amplifying and / or expressing a polynucleotide sequence, a membrane as defined above or a voltage clamp or patch clamp apparatus. The reagents can be present in the kit in a dry state, such that a fluid sample re-suspends the reagents. The kit can also optionally comprise instructions enabling the kit to be used in the method of the present application or details about which patients the method can be used for. The kit can optionally comprise nucleotides.

[0728] Example 1

[0729] The present application also provides an apparatus for characterising, such as sequencing, a target polynucleotide in a sample. The apparatus can comprise (a) a plurality of wells of the present application and (b) a plurality of polynucleotide binding proteins such as helicases or exonucleases. The apparatus can be any conventional apparatus for the analysis of an analyte, such as an array or a chip.

[0730] The array or chip typically contains a plurality of wells of a membrane such as a block copolymer membrane, each of which is inserted with a single nanopore. The array can be integrated within an electronic chip.

[0731] The apparatus preferably comprises:

[0732] a sensor device capable of supporting a plurality of wells and operable to perform polynucleotide characterisation or sequencing using the wells and the proteins;

[0733] at least one reservoir for holding material for performing the characterisation or sequencing;

[0734] a fluidics system configured to controllably supply material from the at least one reservoir to the sensor device; and

[0735] a plurality of containers for receiving respective samples, the fluidics system being configured to selectively supply the samples from the containers to the sensor device.

[0736] The device can be any of the devices described in International Application No. PCT / GB10 / 000789 (published as WO 2010 / 122293), International Application No. PCT / GB10 / 002206 (published as WO 2011 / 067559) or International Application No. PCT / US99 / 25679 (published as WO 00 / 28312).

[0737] The following example illustrates the application.

[0738] Materials and methods

[0739] This example describes how to use the helicase-T4 Dda-E94C / C109A / C136A / A360C (SEQ ID NO: 18 with mutations E94C / C109A / C136A / A360C) to control DNA movement through a number of different mutant lysenin nanopores. All of the nanopores tested exhibited a change in current as DNA was transferred through the nanopore. The mutant nanopores tested exhibited: 1) an increased range; 2) a reduced noise; 3) an improved signal to noise ratio; 4) an increased capture compared to the mutant control nanopore; or 5) a changed read size compared to the baseline.

[0740] DNA construct preparation

[0741] Figure 5

[0742] • 70uL of T4 Dda-E94C / C109A / C136A / A360C buffer exchange (using a Zeba column) into 70uL of lx KOAc buffer with 2mM EDTA.

[0743] • 70uL of T4 Dda-E94C / C109A / C136A / A360C buffer exchange mixture was added to 70uL of 2uM DNA adapter (see HS / ATP step for details on the sequence). The sample was then mixed and incubated at room temperature for 5 minutes.

[0744] • 1uL of 140mM TMAD was added and the sample was mixed and incubated at room temperature for 60 minutes. This sample was referred to as sample A. A 2ul aliquot was then removed for Agilent analysis.

[0745] Reagent

[0746] • The reagents in the table below were mixed and incubated at room temperature for 25 minutes. This sample was referred to as sample B.

[0747] Volume Final Sample A (500 nM) 220 nM 139 2x Hs buffer (100 mM Hepes, 2 M KCl, pH 8) 14 mM 150 1x 600 mM MgCl2 7 100 mM rATP 14 mM 4.2 Final SPRI purification 300.2

[0748] Use of enzyme to ligate 10 kb lambda C to adaptor

[0749] • To sample B, add 1.1 mL of SPRI beads, and then mix and incubate the sample for 5 minutes.

[0750] • Pellet the beads and remove the supernatant. Then wash the beads with 50 mM Tris.HCl, 2.5 M NaCl, 20% PEG8000.

[0751] • Elute sample C in 70 uL of 10 mM Tris.HCl, 20 mM NaCl.

[0752] Reagent

[0753] • Incubate the reagents in the table below at 20°C for 10 minutes in a thermocycler.

[0754]

[0755]

[0756] • The reaction mixture (1 x 500 μΐ aliquot) was then treated as follows: SPRI purification using 200 μΐ of 20% SPRI beads; washed in 750 μΐ of wash buffer 1; and eluted in 125 μΐ of elution buffer 1. The final DNA sequence (SEQ ID NO: 24) was hybridized to DNA. This sample was called sample D.

[0757] Components of ligation buffer (5x)

[0758] Volume Final 1 M Tris.HCl pH 8 150 mM 15 50 mM 1M MgCl2 5 100 mM ATP 5 mM 5 40% PEG 8000 Total 75 30％ 100 uL Reagent

[0759] Components of wash buffer 1

[0760] Volume Final Water 1 M Tris.HCl pH 8 1100 50 mM 100 5 M NaCl 750 mM 300 40% PEG 8000 Total 500 10％ 2000 uL Reagent

[0761] Components of elution buffer 1

[0762] Volume Final Water Up to 1000 uL 906.7 0.5 M CAPS pH 10 40 mM 80 3 M KCl 40 mM 13.3 Total 1000 uL Electrophysiology experiments

[0763] Results

[0764] Electrical measurements were taken from single cytolysin nanopores inserted in block copolymer containing buffer (25 mM potassium phosphate buffer, 150 mM potassium ferrocyanide (II), 150 mM potassium ferricyanide (III), pH 8.0). After reaching a single pore inserted in the block copolymer, the buffer (2 mL, 25 mM potassium phosphate buffer, 150 mM potassium ferrocyanide (II), 150 mM potassium ferricyanide (III), pH 8.0) was then flowed through the system to remove any excess cytolysin nanopores. 150 μL of 500 mM KCl, 25 mM potassium phosphate, pH 8.0 was then flowed through the system. 10 minutes later, 150 uL of 500 mM Kcl, 25 mM potassium phosphate, pH 8.0 was flowed through the system and then a T4 Dda-E94C / C109A / C136A / A360C, DNA, fuel (MgCl2, ATP) premix (total 150 μL, sample D) was flowed into the single nanopore experiment system. The experiment was run at 180 mV and the helicase controlled DNA movement was monitored.

[0765] Read analysis

[0766] A number of different nanopores were investigated to determine the effect of mutations on the transmembrane pore region. The following lists the mutant pores investigated along with the baseline nanopores with which they were compared (baseline pores 1 through 4). A number of different parameters were investigated to identify improved nanopores: 1) the average noise of the signal (where noise equals the standard deviation of all events in a trace, calculated over all traces), which will be lower in the improved nanopore than the baseline; 2) the average current range, which is a measure of the range of current levels within the signal, and which will be higher in the improved nanopore than the baseline; 3) the average signal to noise ratio cited in the table is the signal to noise ratio (average current range divided by the average noise of the signal) in all traces, and which will be higher in the improved nanopore than the baseline; 4) the capture rate of DNA, which will be higher in the improved nanopore than the baseline; and 5) the read size, which can be increased or decreased in the improved nanopore depending on the size of the read of the baseline.

[0767] Each of the following tables contains relevant data for the corresponding baseline nanopore. Table 6 = Mutant 1, Table 7 = Mutant 2, Table 8 = Mutant 3, and Table 9 = Mutant 10, which are then compared to the mutant pores.

[0768] Cytolysin Mutant 1 = Cytolysin-(E84Q / E85K / E92Q / E97S / D126G)9 (SEQ ID NO: 2 with mutations E84Q / E85K / E92Q / E97S / D126G). (Baseline 1)

[0769] Cytolysin Mutant 2 = Cytolysin-(E84Q / E85K / E92Q / E94D / E97S / D126G)9 (SEQ ID NO: 2 with mutations E84Q / E85K / E92Q / E94D / E97S / D126G). (Baseline 2)

[0770] Cytolysin Mutant 3 = Cytolysin-(E84Q / E85K / E92Q / E94Q / E97S / D126G)9 (SEQ ID NO: 2 with mutations E84Q / E85K / E92Q / E94Q / E97S / D126G). (Baseline 3)

[0771] Cytolysin Mutant 4 = Cytolysin-(E84Q / E85K / S89Q / E92Q / E97S / D126G)9 (SEQ ID NO: 2 with mutations E84Q / E85K / S89Q / E92Q / E97S / D126G).

[0772] Cytolysin Mutant 5 = Cytolysin-(E84Q / E85K / T91S / E92Q / E97S / D126G)9 (SEQ ID NO: 2 with mutations E84Q / E85K / T91S / E92Q / E97S / D126G).

[0773] Cytolysin Mutant 6 = Cytolysin-(E84Q / E85K / E92Q / E97S / S98Q / D126G)9 (SEQ ID NO: 2 with mutations E84Q / E85K / E92Q / E97S / S98Q / D126G).

[0774] Cytolysin Mutant 7 = Cytolysin-(E84Q / E85K / E92Q / E97S / V100S / D126G)9 (SEQ ID NO: 2 with mutations E84Q / E85K / E92Q / E97S / V100S / D126G).

[0775] Cytolysin Mutant 8 = Cytolysin-(E84Q / E85K / E92Q / E94D / E97S / S80K / D126G)9 (SEQ ID NO: 2 with mutations E84Q / E85K / E92Q / E94D / E97S / S80K / D126G).

[0776] Cytolysin Mutant 9 = Cytolysin-(E84Q / E85K / E92Q / E94D / E97S / T106R / D126G)9 (SEQ ID NO: 2 with mutations E84Q / E85K / E92Q / E94D / E97S / T106R / D126G).

[0777] Cytolysin mutant 10 = Cytolysin-(E84Q / E85K / E92Q / E94D / E97S / T106K / D126G)9 (SEQ ID NO: 2 with mutations E84Q / E85K / E92Q / E94D / E97S / T106K / D126G). (Baseline 4)

[0778] Cytolysin mutant 11 = Cytolysin-(E84Q / E85K / E92Q / E94D / E97S / T104R / D126G)9 (SEQ ID NO: 2 with mutations E84Q / E85K / E92Q / E94D / E97S / T104R / D126G).

[0779] Cytolysin mutant 12 = Cytolysin-(E84Q / E85K / E92Q / E94D / E97S / T104K / D126G)9 (SEQ ID NO: 2 with mutations E84Q / E85K / E92Q / E94D / E97S / T104K / D126G).

[0780] Cytolysin mutant 13 = Cytolysin-(S78N / E84Q / E85K / E92Q / E94D / E97S / D126G)9 (SEQ ID NO: 2 with mutations S78N / E84Q / E85K / E92Q / E94D / E97S / D126G).

[0781] Cytolysin mutant 14 = Cytolysin-(S82N / E84Q / E85K / E92Q / E94D / E97S / D126G)9 (SEQ ID NO: 2 with mutations S82N / E84Q / E85K / E92Q / E94D / E97S / D126G).

[0782] Cytolysin mutant 15 = Cytolysin-(E76N / E84Q / E85K / E92Q / E94Q / E97S / D126G)9 (SEQ ID NO: 2 with mutations E76N / E84Q / E85K / E92Q / E94Q / E97S / D126G).

[0783] Cytolysin mutant 16 = Cytolysin-(E76S / E84Q / E85K / E92Q / E94Q / E97S / D126G)9 (SEQ ID NO: 2 with mutations E76S / E84Q / E85K / E92Q / E94Q / E97S / D126G).

[0784] Cytolysin Mutant 17 = Cytolysin-(E84Q / E85K / E92Q / E94Q / Y96D / D97S / T106K / D126G)9 (SEQ ID NO: 2 with mutations E84Q / E85K / E92Q / E94Q / Y96D / D97S / T106K / D126G).

[0785] Cytolysin Mutant 18 = Cytolysin-(K45D / E84Q / E85K / E92Q / E94K / D97S / T106K / D126G)9 (SEQ ID NO: 2 with mutations K45D / E84Q / E85K / E92Q / E94K / D97S / T106K / D126G).

[0786] Cytolysin Mutant 19 = Cytolysin-(K45R / E84Q / E85K / E92Q / E94D / D97S / T106K / D126G)9 (SEQ ID NO: 2 with mutations K45R / E84Q / E85K / E92Q / E94D / D97S / T106K / D126G).

[0787] Cytolysin Mutant 20 = Cytolysin-(D35N / E84Q / E85K / E92Q / E94D / D97S / T106K / D126G)9 (SEQ ID NO: 2 with mutations D35N / E84Q / E85K / E92Q / E94D / D97S / T106K / D126G).

[0788] Cytolysin Mutant 21 = Cytolysin-(K37N / E84Q / E85K / E92Q / E94D / D97S / T106K / D126G)9 (SEQ ID NO: 2 with mutations K37N / E84Q / E85K / E92Q / E94D / D97S / T106K / D126G).

[0789] Cytolysin Mutant 22 = Cytolysin-(K37S / E84Q / E85K / E92Q / E94D / D97S / T106K / D126G)9 (SEQ ID NO: 2 with mutations K37S / E84Q / E85K / E92Q / E94D / D97S / T106K / D126G).

[0790] Cytolysin Mutant 23 = Cytolysin-(E84Q / E85K / E92D / E94Q / D97S / T106K / D126G)9 (SEQ ID NO: 2 with mutations E84Q / E85K / E92D / E94Q / D97S / T106K / D126G).

[0791] Cytolysin Mutant 24 = Cytolysin-(E84Q / E85K / E92E / E94Q / D97S / T106K / D126G)9 (SEQ ID NO: 2 with mutations E84Q / E85K / E92E / E94Q / D97S / T106K / D126G).

[0792] Cytolysin Mutant 25 = Cytolysin-(K37S / E84Q / E85K / E92Q / E94D / D97S / T104K / T106K / D126G)9 (SEQ ID NO: 2 with mutations K37S / E84Q / E85K / E92Q / E94D / D97S / T104K / T106K / D126G).

[0793] Cytolysin Mutant 26 = Cytolysin-(E84Q / E85K / M90I / E92Q / E94D / E97S / T106K / D126G)9 (SEQ ID NO: 2 with mutations E84Q / E85K / M90I / E92Q / E94D / E97S / T106K / D126G).

[0794] Cytolysin Mutant 27 = Cytolysin-(K45T / V47K / E84Q / E85K / E92Q / E94D / E97S / T106K / D126G)9 (SEQ ID NO: 2 with mutations K45T / V47K / E84Q / E85K / E92Q / E94D / E97S / T106K / D126G).

[0795] Cytolysin Mutant 28 = Cytolysin-(T51K / E84Q / E85K / E92Q / E94D / E97S / T106K / D126G)9 (SEQ ID NO: 2 with mutations T51K / E84Q / E85K / E92Q / E94D / E97S / T106K / D126G).

[0796] Cytolysin Mutant 29 = Cytolysin-(K45Y / S49K / E84Q / E85K / E92Q / E94D / E97S / T106K / D126G)9 (SEQ ID NO: 2 with mutations K45Y / S49K / E84Q / E85K / E92Q / E94D / E97S / T106K / D126G).

[0797] Cytolysin Mutant 30 = Cytolysin-(S49L / E84Q / E85K / E92Q / E94D / E97S / T106K / D126G)9 (SEQ ID NO: 2 with mutations S49L / E84Q / E85K / E92Q / E94D / E97S / T106K / D126G).

[0798] Cytolysin mutant 31 = Cytolysin-(E84Q / E85K / V88I / M90A / E92Q / E94D / E97S / T106K / D126G)9 (SEQ ID NO: 2 with mutations E84Q / E85K / V88I / M90A / E92Q / E94D / E97S / T106K / D126G).

[0799] Cytolysin mutant 32 = Cytolysin-(K45N / S49K / E84Q / E85K / E92D / E94N / E97S / T106K / D126G)9 (SEQ ID NO: 2 with mutations K45N / S49K / E84Q / E85K / E92D / E94N / E97S / T106K / D126G).

[0800] Cytolysin mutant 33 = Cytolysin-(K45N / V47K / E84Q / E85K / E92D / E94N / E97S / T106K / D126G)9 (SEQ ID NO: 2 with mutations K45N / V47K / E84Q / E85K / E92D / E94N / E97S / T106K / D126G).

[0801]

[0802] Table 6

[0803]

[0804] Table 7

[0805]

[0806] Table 8

[0807]

[0808]

[0809] Table 9

[0810] Figure 1

[0811] For Cytolysin mutants 1 and 10, we obtained models of the expected ion current distribution for all possible 9-mer polynucleotides. The models can include the mean and standard deviation of the current distribution for each 9-mer.

[0812] We examined and compared the structure of the models obtained for Cytolysin mutants 1 and 10. The figures (see Figure 2 and Figure 1Examples of this comparison are provided. In the case of each model (i.e. cytolysin 1 or 10), we combined the mean of the distribution of all 9-mers of the form A, x_2, x_3, x_4, x_5, x_6, x_7, x_8, x_9, where x_{i} represents any polynucleotide selected from {A, C, G, T}, the combination being applied to the mean of the median. This median averaging was repeated for all nucleotides {A, C, G, T} in position 1 and for all positions, so that we obtain 36 medians encoding the median effect for each nucleotide when the nucleotide is present in any of the 9 positions of the 9-mer.

[0813] Figure 2 (cytolysin mutant 1) and Figure 1 (cytolysin mutant 2) plot these medians for two different pores. Figure 2 and Figure 2 The plots in (cytolysin mutant 1) and (cytolysin mutant 2) show the degree of discrimination between all bases at each position in the read. The greater the discrimination, the greater the difference between the levels of current contribution at that particular position. If a position is not part of the read, the current contribution at that position will be similar for all four bases. Figure 1 (cytolysin mutant 10) shows similar current contributions for all four bases at positions 6 to 8 of the read. Example 2 (cytolysin mutant 1) does not show similar current contributions for all four bases at any position in the read. Thus, the read of cytolysin mutant 10 is shorter than the read of cytolysin mutant 1. A shorter read can be advantageous because fewer bases contribute to the signal at any one time, which can lead to improved accuracy of base calling.

[0814] Example 3

[0815] This example describes a protocol for producing chemically modified assembled pores with barrels / channels of reduced diameter.

[0816] Monomeric cytolysin samples (about 10 pmol) were first reduced to ensure maximum reactivity of cysteine residues and thus efficient coupling reactions. Monomeric cytolysin samples (about 10 pmol) were incubated with 1 mM dithiothreitol (DTT) for 5 to 15 minutes. Cell debris and suspended aggregates were then pelleted by centrifugation at 20,000 rpm for 10 minutes. The soluble fraction was then recovered and its buffer exchanged into 1 mM Tris, 1 mM EDTA, pH 8.0 using Zeba spin columns (ThermoFisher) with a 7Kd molecular weight cut-off.

[0817] The molecule to be attached (e.g. 2-iodo-N-(2,2,2-trifluoroethyl)acetamide) was dissolved in an appropriate solvent, typically DMSO, to a concentration of 100 mM. This was added to the buffer exchanged cytolysin monomer sample to a final concentration of 1 mM. The resulting solution was incubated at 30°C for 2 hours. The modified sample (100 uL) was then oligomerised by the addition of 20 uL of 5 lipid mix from Encapsula Nanosciences (Phosphatidylserine (0.325 mg / ml): POPE (0.55 mg / ml): Cholesterol (0.45 mg / ml): Soy PC (0.9 mg / ml): Sphingomyelin (0.275 mg / ml)). The sample was incubated at 30°C for 60 minutes. The sample was then subjected to SDS-PAGE and purified from the gel as described in International Application No. PCT / GB2013 / 050667 (published as WO2013 / 153359).

[0818] Materials and methods

[0819] This example compares chemically modified assembled cytolysin pores with reduced diameter barrels / channels (Cytolysin- (E84Q / E85K / E92Q / E94C / E97S / T106K / D126G / C272A / C283A)9 (SEQ ID NO: 2 with mutations E84Q / E85K / E92Q / E94C / E97S / T106K / D126G / C272A / C283A) vs Cytolysin- (E84Q / E85K / E92Q / E94D / E97S / T106K / D126G / C272A / C283A)9 (SEQ ID NO: 2 with mutations E84Q / E85K / E92Q / E94D / E97S / T106K / D126G / C272A / C283A).

[0820] Results

[0821] DNA constructs were prepared as described in Example 1. Electrophysiology experiments were performed as described in Example 1.

[0822] Figure 3

[0823] Electrophysiology experiments showed that chemically modified assembled pore (cytolysin - (E84Q / E85K / E92Q / E94C / E97S / T106K / D126G / C272A / C283A)9 (SEQ ID NO: 2 with mutations E84Q / E85K / E92Q / E94C / E97S / T106K / D126G / C272A / C283A) with 2-iodo-N-(2,2,2-trifluoroethyl)acetamide attached through E94C) showed a median range of 21 pA, which is greater than cytolysin - (E84Q / E85K / E92Q / E94D / E97S / T106K / D126G / C272A / C283A)9, which showed a median range of 12 pA. This increase in median range provides a greater current space for resolution of k-mers.

[0824] Figure 4 (E84Q / E85K / E92Q / E94D / E97S / T106K / D126G / C272A / C283A)9) and Figure 4 ((E84Q / E85K / E92Q / E94C / E97S / T106K / D126G / C272A / C283A)9 (SEQ ID NO: 2 with mutations E84Q / E85K / E92Q / E94C / E97S / T106K / D126G / C272A / C283A) with 2-iodo-N-(2,2,2-trifluoroethyl)acetamide attached through E94C) showed plots of median as described in Example 1. When comparing to Figure 3 to Figure 4 the relative contribution of the signal to different bases at different positions has changed, ​ extreme read positions (positions 7 to 8) in (E84Q / E85K / E92Q / E94D / E97S / T106K / D126G / C272A / C283A)9 show much less discrimination, meaning that their contribution to the signal is greatly reduced and therefore the length of the k-mer determined at a given instant is shorter. This shorter read can be advantageous as fewer bases are contributing to the signal at any one time, which can lead to an increase in base calling accuracy.

[0825] Cys-lysozyme with 2-iodo-N-(2-phenylethyl)acetamide attached through E92C (E84Q / E85S / E92C / E94D / E97S / T106K / D126G / C272A / C283A)9 (SEQ ID NO: 2 with mutations E84Q / E85S / E92C / E94D / E97S / T106K / D126G / C272A / C283A) and Cys-lysozyme with 1-benzyl-2,5-dihydro-1H-pyrrole-2,5-dione attached through E92C (E84Q / E85S / E92C / E94D / E97S / T106K / D126G / C272A / C283A)9 (SEQ ID NO: 2 with mutations E84Q / E85S / E92C / E94D / E97S / T106K / D126G / C272A / C283A) were used to perform experiments similar to those described in Example 3.

Claims

1. A mutant cytolysin monomer, the mutant cytolysin monomer being a variant of the amino acid sequence set forth in SEQ ID NO: 2, wherein the variant is capable of forming a pore comprising nine monomers, the mutation of the variant selected from the group consisting of: E84Q / E85K / E92Q / E94D / E97S / T106R / D126G, E84Q / E85K / E92Q / E94D / E97S / T106K / D126G, E84Q / E85K / E92Q / E94Q / Y96D / D97S / T106K / D126G, K45D / E84Q / E85K / E92Q / E94K / D97S / T106K / D126G, K45R / E84Q / E85K / E92Q / E94D / D97S / T106K / D126G, D35N / E84Q / E85K / E92Q / E94D / D97S / T106K / D126G, K37N / E84Q / E85K / E92Q / E94D / D97S / T106K / D126G, K37S / E84Q / E85K / E92Q / E94D / D97S / T106K / D126G, E84Q / E85K / E92D / E94Q / D97S / T106K / D126G, E84Q / E85K / E92E / E94Q / D97S / T106K / D126G, K37S / E84Q / E85K / E92Q / E94D / D97S / T104K / T106K / D126G, E84Q / E85K / M90I / E92Q / E94D / E97S / T106K / D126G, K45T / V47K / E84Q / E85K / E92Q / E94D / E97S / T106K / D126G, T51K / E84Q / E85K / E92Q / E94D / E97S / T106K / D126G, K45Y / S49K / E84Q / E85K / E92Q / E94D / E97S / T106K / D126G, S49L / E84Q / E85K / E92Q / E94D / E97S / T106K / D126G, E84Q / E85K / V88I / M90A / E92Q / E94D / E97S / T106K / D126G, K45N / S49K / E84Q / E85K / E92D / E94N / E97S / T106K / D126G, and K45N / V47K / E84Q / E85K / E92D / E94N / E97S / T106K / D126G.

2. The mutant cytolysin monomer of claim 1, wherein, the mutant is chemically modified.

3. A polynucleotide encoding the mutant cytolysin monomer of claim 1 or 2.

4. A pore comprising nine mutant cytolysin monomers of claim 1 or 2.

5. A method of characterizing a target analyte, comprising: (a) contacting the target analyte with the aperture of claim 4, such that the target analyte moves through the aperture; and (b) taking one or more measurements as the analyte moves relative to the aperture, wherein the measurements are indicative of one or more properties of the target analyte, and therefrom characterizing the target analyte.

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