MUTANT PORES

Field of the invention

The invention relates to mutant forms of Msp. The invention also relates to nucleic acid characterisation using Msp.

Background of the invention

Nanopore sensing is an approach to sensing that relies on the observation of individual binding events between analyte molecules and a receptor. Nanopore sensors can be created by placing a single pore of nanometer dimensions in an insulating membrane and measuring voltage-driven ionic transport through the pore in the presence of analyte molecules. The identity of an analyte is revealed through its distinctive current signature, notably the duration and extent of current block and the variance of current levels.

There is currently a need for rapid and cheap nucleic acid (e.g. DNA or RNA) sequencing technologies across a wide range of applications. Existing technologies are slow and expensive mainly because they rely on amplification techniques to produce large volumes of nucleic acid and require a high quantity of specialist fluorescent chemicals for signal detection. Nanopore sensing has the potential to provide rapid and cheap nucleic acid sequencing by reducing the quantity of nucleotide and reagents required.

Two of the essential components of sequencing nucleic acids using nanopore sensing are

(1) the control of nucleic acid movement through the pore and (2) the discrimination of nucleotides as the nucleic acid polymer is moved through the pore. In the past, to achieve nucleotide discrimination the nucleic acid has been passed through a mutant of hemolysin. This has provided current signatures that have been shown to be sequence dependent. It has also been shown that a large number of nucleotides contribute to the observed current, making a direct relationship between observed current and nucleic acid sequence challenging.

While the current range for nucleotide discrimination has been improved through mutation of the hemolysin pore, a sequencing system would have higher performance if the current differences between nucleotides could be improved further. In addition, it has been observed that when the nucleic acids are moved through a pore, some current states show high variance. It has also been shown that some mutant hemolysin pores exhibit higher variance than others. While the variance of these states may contain sequence specific information, it is desirable to produce pores that have low variance to simplify the system. It is also desirable to reduce the number of nucleotides that contribute to the observed current.

The different forms of Msp are porins from Mycobacterium smegmatis. MspA is a 157 kDa octameric porin from Mycobacterium smegmatis. The structure of MspA has been well documented by researchers (Gundlach, Proc Natl Acad Sci U S A. 2010 Sep 14; 107(37): 16060-5. Epub 2010 Aug 26). Some key residues have been identified and modified to enhance the properties of the pore. These mutations have been performed to allow DNA to transition through the MspA pore. MspB, C and D are also known forms of Msp.

Summary of the invention

The inventors have surprisingly demonstrated that novel mutants of Msp display improved properties for estimating the characteristics, such as the sequence of nucleic acids.

The mutants surprisingly display improved nucleotide discrimination. In particular, the mutants surprisingly display an increased current range, which makes it easier to discriminate between different nucleotides, and a reduced variance of states, which increases the signal-to-noise ratio. In addition, the number of nucleotides contributing to the current as the nucleic acid moves through the pore is decreased. This makes it easier to identify a direct relationship between the observed current as the nucleic acid moves through the pore and the nucleic acid sequence.

The inventors have also surprisingly shown that Msp shows improved sequencing properties when the movement of the nucleic acid through the pore is controlled by a Phi29 DNA polymerase. In particular, the coupling of Msp and Phi29 DNA polymerase results in three unexpected advantages. First, the nucleic acid moves through the pore at a rate that is commercially viable yet allows effective sequencing. Second, an increased current range is observed as the nucleic acid moves through the pore allowing the sequence to be determined more easily. Third, a decreased current variance is observed thereby increasing the signal-to-noise ratio.

Accordingly, the invention provides a mutant Msp monomer comprising a variant of the sequence shown in SEQ ID NO: 2, wherein the variant comprises at least one of the following mutations:

(a) asparagine (N), serine (S), glutamine (Q) or threonine (T) at position

(b) serine (S), glutamine (Q) or tyrosine (Y) at position 90;

(c) leucine (L) or serine (S) at position 105;

(d) arginine (R) at position 126;

(e) serine (S) at position 75;

(f) serine (S) at position 77;

(g) arginine (R) at position 59;

(h) glutamine (Q) , asparagine (N) or threonine (T) at position 75;

(i) glutamine (Q) , asparagine (N) or threonine (T) at position 77;

(j) leucine (L) at position 78;

(k) asparagine (N) at position 81;

(l) asparagine (N) at position 83;

(m) serine (S) or threonine (T) at position 86;

(n) phenylalanine (F), valine (V) or leucine (L) at position 87;

(o) tyrosine (Y), phenylalanine (F), valine (V), arginine (R), alanine (A), glycine

(G) or cysteine (C) at position 88;

(p) phenylalanine (F), valine (V) or leucine (L) at position 89;

(q) leucine (L), phenylalanine (F), tryptophan (W), histidine (H), threonine (T), glycine (G), alanine (A), valine (V), arginine (R), lysine (K), asparagine (N) or cysteine (C) at position 90;

(r) serine (S), glutamine (Q), leucine (L), methionine (M), isoleucine (I), alanine (A), valine (V), glycine (G), phenylalanine (F), tryptophan (W), tyrosine (Y), histidine (H), threonine (T), arginine (R), lysine (K), asparagine (N) or cysteine (C) at position 91;

(s) alanine (A) or serine (S) at position 92;

(t) serine (S), alanine (A), threonine (T), glycine (G) at position 93;

(u) leucine (L) at position 94;

(v) valine (V) at position 95;

(W) arginine (R), aspartic acid (D), valine (V), asparagine (N), serine (S) or

threonine (T) at position 96;

(x) serine (S) at position 97;

(y) serine (S) at position 98;

(z) serine (S) at position 99;

(aa) serine (S) at position 100;

(bb) phenylalanine (F) at position 101;

(cc) lysine (K), serine (S) or threonine (T) at position 102;

(dd) alanine (A), glutamine (Q), asparagine (N), glycine (G) or threonine (T) at position 103;

(ee) isoleucine at position 104;

(ff) tyrosine (Y), alanine (A), glutamine (Q), asparagine (N), threonine (T),

phenylalanine (F), tryptophan (W), histidine (H), glycine (G), valine (V), arginine (R), lysine (K), proline (P), or cysteine (C) at position 105;

phenylalanine (F), isoleucine (I), valine (V) or serine (S) at position 106; (hh) proline (P) or serine (S) at position 108;

(ii) asparagine (N) at position 118;

(jj) serine (S) or cysteine (C) at position 103; and

(kk) cysteine at one or more of positions 10 to 15, 51 to 60, 136 to 139 and 168 to

172.

The invention also provides:

- a construct comprising two or more covalently attached monomers derived from Msp; - a polynucleotide which encodes a mutant of the invention or a construct of the invention; - a homo-oligomeric pore derived from Msp comprising identical mutant monomers of the invention;

- a hetero-oligomeric pore derived from Msp comprising at least one mutant monomer of the invention, wherein at least one of the eight monomers differs from the others;

- a method of characterising a target nucleic acid sequence, comprising:

(a) contacting the target sequence with a pore of the invention and a nucleic acid binding protein so that the protein controls the movement of the target sequence through the pore and a proportion of the nucleotides in the target sequence interacts with the pore; and

(b) measuring the current passing through the pore during each interaction and thereby characterising the target sequence;

- a kit for sequencing a target nucleic acid sequence comprising (a) a pore of the invention and (b) a nucleic acid handling enzyme;

- an apparatus for sequencing target nucleic acid sequences in a sample, comprising (a) a plurality of pores of the invention and (b) a plurality of nucleic acid handling enzymes;

- a method of characterising a target nucleic acid sequence, comprising:

(a) contacting the target sequence with a pore derived from Msp and a Phi29 DNA polymerase such that the polymerase controls the movement of the target sequence through the pore and a proportion of the nucleotides in the target sequence interacts with the pore; and

(b) measuring the current passing through the pore during each interaction and thereby characterising the target sequence, wherein steps (a) and (b) are carried out with a voltage applied across the pore;

- a method of forming a sensor for characterising a target nucleic acid sequence, comprising:

(a) contacting a pore derived from Msp with a Phi29 DNA polymerase in the presence of the target nucleic acid sequence; and

(b) applying a voltage across the pore to form a complex between the pore and the polymerase; and thereby forming a sensor for characterising the target nucleic acid sequence; - a method of increasing the rate of activity of a Phi29 DNA polymerase, comprising:

(a) contacting the Phi29 DNA polymerase with a pore derived from Msp in the presence of a nucleic acid sequence; and

(b) applying a voltage across the pore to form a complex between the pore and the polymerase; and thereby increasing the rate of activity of a Phi29 DNA polymerase;

- a kit for characterising a target nucleic acid sequence comprising (a) a pore derived from Msp and (b) a Phi29 DNA polymerase; and

- an apparatus for characterising target nucleic acid sequences in a sample, comprising a plurality of pores derived from Msp and a plurality of Phi29 DNA polymerases.

Description of the Figures

Fig. 1 shows the average dwell time of individual current levels as a single DNA strand translocates the nanopore. The data is collated from a number of single molecules and is split into quartiles by current levels.

Fig. 2 shows current levels and variance obtained from using Phi29 in Unzipping mode to move a DNA strand (SEQ ID NO: 15) through the MS-(NNNRRK)8 nanopore.

Fig. 3 shows current levels and variance obtained from using Phi29 in Unzipping mode to move a DNA strand (SEQ ID NO: 15) through the HL-(mutant)7 nanopore.

Fig. 4 shows the current levels for a single MspA channel recorded at a range of applied potentials (-200 mV to 200 mV).

Fig. 5 shows the IV curve of open pore levels for the baseline MspA mutant, MS-(B1)8. Each line represents a single pore.

Fig.6 shows the IV curve of open pore levels for the MspA mutant, MS-(B1-I105Y)8. Each line represents a single pore.

Fig. 7 shows the IV curve of open pore levels for the MspA mutant, MS-(B1-I105N)8.

Each line represents a single pore.

Fig. 8 shows the change in current between a high conductance state (275 pA) and a low conductance state (150 pA) for the MS-(B1-I105A)8 pore at 180 mV.

Fig. 9 shows the current levels produced when DNA is unzipped through the baseline MS-(B1)8 pore. Current range for these events is ~ 30 pA.

Fig. 10 shows the current levels produced when DNA is unzipped through the baseline MS-(B1-I105A)8 pore. Current range for these events is ~ 40 pA.

Fig. 11 shows the DNA substrate design used in Examples 9 and 12 and 15.

Fig. 12 shows the DNA substrate design used in Examples 10 and 11.

Fig. 13 shows how the sequencing profile changes, for the same DNA sequence, when point mutations are made in the MspA monomer sequence. These plots show the average of the profile of the levels obtained from multiple polynucleotides. A) This graph shows the sequencing profile for the MS-(B1)8 pore. B) This graph shows the sequencing profile for the MS-(B1-D90Q-D93S-1105A)8 pore. C) This graph shows the sequencing profile for the MS-(B 1 -D90Q-Q126R)8 pore. D) This graph shows the sequencing profile for the MS-(B1-L88N-D90Q-D91M)8 pore. E) This graph shows the sequencing profile for the MS-(B1-L88N-D90Q-D91S)8 pore. F) This graph shows the sequencing profile for the MS-(B1-G75S-G77S-L88N-Q126R)8 pore.

Fig. 14 shows the DNA substrate design used in Example 13.

Fig. 15 shows an example event trace for the controlled translocation of RNA, mediated by Phi29 DNA polymerase, through the MspA mutant pore MS-(B1)8. An expanded view, of the region highlighted in the upper trace, is shown below.

Fig. 16 shows pore insertion into the lipid bilayer. A) Shows pore insertion of the MS-(B1)8 oligomerised from the monomer. B) Shows pore insertion of the MS-(B1-B1)4 oligomerised from the dimer.

Fig. 17 shows an example event trace for the controlled translocation of DNA, mediated by a helicase, through the MS-(B1)8 mutant pore which was produced by oligomerisation of the monomer. An expanded view, of the region highlighted in the upper trace, is shown below.

Fig. 18 shows an example event trace for the controlled translocation of DNA, mediated by a helicase, through the MS-(B1-B1)4 mutant pore which was produced by oligomerisation of the dimer. An expanded view, of the region highlighted in the upper trace, is shown below.

Fig. 19 shows the DNA substrate design used in Example 16.

Fig. 20 shows an example event trace for the controlled translocation of DNA containing both cytosine and 5-methylcytosine, mediated by a helicase, through the MS-(B1-L88N)8 mutant pore. An expanded view of the region highlighted in the upper trace is shown below.

Description of the Sequence Listing

SEQ ID NO: 1 shows the polynucleotide sequence encoding the NNN-RRK mutant MspA monomer.

SEQ ID NO: 2 (also referred to as "Bl") shows the amino acid sequence of the mature form of the NNN-RRK mutant of the MspA monomer. The mutant lacks the signal sequence and the amino terminal methionine (encoded by the start codon) and includes the following mutations: D90N, D91N, D93N, Dl 18R, D134R and E139K. These mutations allow DNA transition through the MspA pore.

SEQ ID NO: 3 shows the polynucleotide sequence encoding the Phi29 DNA polymerase.

SEQ ID NO: 4 shows the amino acid sequence of the Phi29 DNA polymerase.

SEQ ID NO: 5 shows the codon optimised polynucleotide sequence derived from the sbcB gene from E. coli. It encodes the exonuclease I enzyme (EcoExo I) from E. coli.

SEQ ID NO: 6 shows the amino acid sequence of exonuclease I enzyme (EcoExo I) from

E. coli.

SEQ ID NO: 7 shows the codon optimised polynucleotide sequence derived from the xthA gene from E. coli. It encodes the exonuclease III enzyme from E. coli.

SEQ ID NO: 8 shows the amino acid sequence of the exonuclease III enzyme from ii. coli. This enzyme performs distributive digestion of 5' monophosphate nucleosides from one strand of double stranded DNA (dsDNA) in a 3 ' - 5 ' direction. Enzyme initiation on a strand requires a 5' overhang of approximately 4 nucleotides.

SEQ ID NO: 9 shows the codon optimised polynucleotide sequence derived from the recJ gene from T. thermophilus. It encodes the RecJ enzyme from T. thermophilus (TthRecJ-cd).

SEQ ID NO: 10 shows the amino acid sequence of the RecJ enzyme from T.

thermophilus (TthRecJ-cd). This enzyme performs processive digestion of 5' monophosphate nucleosides from ssDNA in a 5 ' - 3 ' direction. Enzyme initiation on a strand requires at least 4 nucleotides.

SEQ ID NO: 11 shows the codon optimised polynucleotide sequence derived from the bacteriophage lambda exo (redX) gene. It encodes the bacteriophage lambda exonuclease.

SEQ ID NO: 12 shows the amino acid sequence of the 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 a 5'-3'direction (http://www.neb.com/nebecomm/products/productM0262.asp). Enzyme initiation on a strand preferentially requires a 5 ' overhang of approximately 4 nucleotides with a 5 ' phosphate.

SEQ ID NOs: 13 to 15 show the sequences used in Example 2.

SEQ ID NOs: 16 to 18 show the amino acid sequences of the mature forms of the MspB, C and D mutants respectively. The mature forms lack the signal sequence.

SEQ ID NOs: 19 and 20 show the sequences used in Examples 9, 12 and 15.

SEQ ID NOs: 21 to 23 show the sequences used in Examples 10 and 11.

SEQ ID NOs: 24 to 27 show the sequences used in Example 13.

SEQ ID NO: 28 shows the DNA sequence of the dimer of the mature form of the NNN-RRK mutant of the MspA monomer used in Example 14.

SEQ ID NO: 29 shows the protein sequence of the dimer of the mature form of the NNN-RRK mutant of the MspA monomer used in Example 14.

SEQ ID NO: 30, 31 and 32 show the sequences used in Example 16.

SEQ ID NO: 33 shows the linker sequence shown used in the construct shown in SEQ ID NO: 29.

Detailed description of the invention

It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only, and is not intended to be limiting.

In addition as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a mutant" includes "mutants", reference to "a substitution" includes two or more such substitutions, reference to "a pore" includes two or more such pores, reference to "a nucleic acid sequence" includes two or more such sequences, and the like.

All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

Mutant Msp monomers

The present invention provides mutant Msp monomers. The mutant Msp monomers may be used to form the pores of the invention. A mutant Msp monomer is a monomer whose sequence varies from that of a wild-type Msp monomer and which retains the ability to form a pore. Methods for confirming the ability of mutant monomers to form pores are well-known in the art and are discussed in more detail below.

The mutant monomers have improved nucleotide reading properties i.e. display improved nucleotide capture and discrimination. In particular, pores constructed from the mutant monomers capture nucleotides and nucleic acids more easily than the wild type. In addition, pores constructed from the mutant monomers display an increased current range, which makes it easier to discriminate between different nucleotides, and a reduced variance of states, which increases the signal-to-noise ratio. In addition, the number of nucleotides contributing to the current as the nucleic acid moves through pores constructed from the mutants is decreased. This makes it easier to identify a direct relationship between the observed current as the nucleic acid moves through the pore and the nucleic acid sequence. The improved nucleotide reading properties of the mutants are achieved via five main mechanisms, namely by changes in the:

● sterics (increasing or decreasing the size of amino acid residues); ● charge (e.g. introducing +ve charge to interact with the nucleic acid sequence); ● hydrogen bonding (e.g. introducing amino acids that can hydrogen bond to the base pairs);

● pi stacking (e,g, introducing amino acids that interact through delocalised electron pi systems); and/or

● alteration of the structure of the pore (e.g. introducing amino acids that increase the size of the vestibule and/or constriction).

Any one or more of these five mechanisms may be responsible for the improved properties of the pores of the invention. For instance, a pore of the invention may display improved nucleotide reading properties as a result of altered sterics, altered hydrogen bonding and an altered structure.

The introduction of bulky residues, such as phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H), increases the sterics of the pore. The introduction of aromatic residues, such as phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H), also increases the pi staking in the pore. The introduction of bulky or aromatic residues also alters the structure of the pore, for instance by opening up the pore and increasing the size of the vestibule and/or constriction. This is described in more detail below.

A mutant monomer of the invention comprises a variant of the sequence shown in SEQ ID NO: 2. SEQ ID NO: 2 is the NNN-RRK mutant of the MspA monomer. It includes the following mutations: D90N, D91N, D93N, D118R, D134R and E139K. A variant of SEQ ID NO: 2 is a polypeptide that has an amino acid sequence which varies from that of SEQ ID NO: 2 and which retains its ability to form a pore.

The variant comprises at least one of the following mutations:

(a) asparagine (N), serine (S), glutamine (Q) or threonine (T) at position

(b) serine (S), glutamine (Q) or tyrosine (Y) at position 90;

(c) leucine (L) or serine (S) at position 105;

(d) arginine (R) at position 126;

(e) serine (S) at position 75;

(f) serine (S) at position 77;

(g) arginine (R) at position 59;

(h) glutamine (Q) , asparagine (N) or threonine (T) at position 75;

(i) glutamine (Q) , asparagine (N) or threonine (T) at position 77;

(j) leucine (L) at position 78;

(k) asparagine (N) at position 81 ;

(l) asparagine (N) at position 83;

(m) serine (S) or threonine (T) at position 86;

(n) phenylalanine (F), valine (V) or leucine (L) at position 87;

(o) tyrosine (Y), phenylalanine (F), valine (V), arginine (R), alanine (A), glycine (G) or cysteine (C) at position 88;

(p) phenylalanine (F), valine (V) or leucine (L) at position 89;

(q) leucine (L), phenylalanine (F), tryptophan (W), histidine (H), threonine (T), glycine (G), alanine (A), valine (V), arginine (R), lysine (K), asparagine (N) or cysteine (C) at position 90;

(r) serine (S), glutamine (Q), leucine (L), methionine (M), isoleucine (I), alanine (A), valine (V), glycine (G), phenylalanine (F), tryptophan (W), tyrosine (Y), histidine

(H), threonine (T), arginine (R), lysine (K), asparagine (N) or cysteine (C) at position 91 ;

(s) alanine (A) or serine (S) at position 92;

(t) serine (S), alanine (A), threonine (T), glycine (G) at position 93;

(u) leucine (L) at position 94;

(v) valine (V) at position 95;

(w) arginine (R), aspartic acid (D), valine (V), asparagine (N), serine (S) or threonine

(T) at position 96;

(x) serine (S) at position 97;

(y) serine (S) at position 98;

(z) serine (S) at position 99;

(aa) serine (S) at position 100;

(bb) phenylalanine (F) at position 101;

(cc) lysine (K), serine (S) or threonine (T) at position 102;

(dd) alanine (A), glutamine (Q), asparagine (N), glycine (G) or threonine (T) at

position 103;

(ee) isoleucine at position 104;

(ff) tyrosine (Y), alanine (A), glutamine (Q), asparagine (N), threonine (T),

phenylalanine (F), tryptophan (W), histidine (H), glycine (G), valine (V), arginine (R), lysine (K), proline (P), or cysteine (C) at position 105;

(gg) phenylalanine (F), isoleucine (I), valine (V) or serine (S) at position 106;

(hh) proline (P) or serine (S) at position 108;

(ii) asparagine (N) at position 118;

(jj) serine (S) or cysteine (C) at position 103; and

(kk) cysteine at one or more of positions 10 to 15, 51 to 60, 136 to 139 and 168 to 172. In wild-type MspA, residues 88 and 105 in each monomer form a hydrophobic ring in the inner constriction of the pore. The hydrophobic residues at positions L88 and 1105 sit just above the main constriction of the pore, facing into the aqueous channel. Mutation of these residues produces pores that have significantly higher open pore currents to the baseline (SEQ ID NO: 2). The current differences observed when mutations are made at these positions are significantly higher than would be expected from making a single mutation. This surprising result implies that mutations at these positions may have an effect on the structure of the channel rather than just the local environment at these residues. Although the SEQ ID NO: 2 baseline has been reported to exhibit a wide range of pore conductance, the reason for this is not well understood. Mutations to positions L88 and 1105 result in the dominant pore current level being significantly higher than the baseline pore. In addition, this higher conductance state is the dominant conformation of the mutant, which is desirable for a large current range and increased signal to noise.

The introduction of N, S, Q or T at position 88 (i.e. mutation (a) above) introduces into the inner constriction of the pore an amino acid that can hydrogen bond to the nucleotides in a nucleic acid.

Residues 90 and 91 in each monomer also form part of the inner constriction of the pore. Residue 118 in each monomer is present within the vestibule of the pore. Residue 134 in each monomer is part of the entrance to the pore.

The introduction of S, Q or Y at position 90 (i.e. mutation (b) above) introduces into the inner constriction of the pore an amino acid that can hydrogen bond to the nucleotides in a nucleic acid.

The variant may include any number of mutations (a) to (kk), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more of the mutations. Preferred combinations of mutations are discussed below. The amino acids introduced into the variant may be naturally-occuring or non-naturally occurring derivatives thereof. The amino acids introduced into the variant may be D-amino acids.

Any number of cysteines may be introduced into the variant. Cysteines are preferably introduced at one or more, such as two or all of, positions 90, 91 and 103. These positions may be useful for chemical attachment of a molecular adaptor as discussed in more detail below. Any number of cysteines, such as 2, 3, 4, 5, 6 or more cysteines, may be introduced at positions 10 to 15, 51 to 60, 136 to 139 and 168 to 172. These positions are present in non-conserved loop regions of the pore and so are useful for chemically attaching a nucleic acid binding protein to the pore as discussed in more detail below.

In a preferred embodiment, the variant comprises one or more of the substitutions shown in (A) to (Z) below. The variant may include any number of the substitutions in A to Z, such as 1, 2, 3, 4 or 5.

(A) The introduction of one or more of (i) serine (S) at positon 75, (ii) serine (S) at position 77, (iii) asparagine (N) at position 88, (iv) glutamine (Q) at position 90 and (v) arginine (R) at position 126. The variant may include 1 , 2, 3, 4 or 5 of these substitutions. The advantages of homo-octameric pores including all four substitutions in each monomer are shown in Table 3 below.

(B) The introduction of one or more of (i) glutamine (Q) at position 90 and (ii) arginine (R) at position 126. The variant may include 1 or 2 of these substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 3 below.

(C) The introduction of one or more of (i) asparagine (N) at position 88, (ii) glutamine (Q) at position 90 and (iii) arginine (R) at position 126. The variant may include 1, 2 or 3 of these substitutions. The advantages of homo-octameric pores including all three of these substitutions in each monomer are shown in Table 3 below.

(D) The introduction of one or more of (i) serine (S) at position 88 and (ii) glutamine (Q) at position 90. The variant may include 1 or 2 of these substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 3 below.

(E) The introduction of one or more of (i) asparagine (N) at position 88 and (ii) glutamine (Q) at position 90. The variant may include 1 or 2 of these substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 3 below.

(F) The introduction of one or more of (i) glutamine (Q) at position 90 and (ii) alanine (A) at position 105. The variant may include 1 or 2 of these substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.

(G) The introduction of one or more of (i) serine (S) at position 90 and (ii) serine (S) at position 92. The variant may include 1 or 2 of these substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.

(H) The introduction of one or more of (i) threonine (T) at position 88 and (ii) serine (S) at position 90. The variant may include 1 or 2 of these substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.

(I) The introduction of one or more of (i) glutamine (Q) at position 87 and (ii) serine (S) at position 90. The variant may include 1 or 2 of these substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.

(J) The introduction of one or more of (i) tyrosine (Y) at position 89 and (ii) serine

(S) at position 90. The variant may include 1 or 2 of these substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.

(K) The introduction of one or more of (i) asparagine (N) at position 88 and (ii) phenylalanine (F) at position 89. The variant may include 1 or 2 of these substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.

(L) The introduction of one or more of (i) asparagine (N) at position 88 and (ii) tyrosine (Y) at position 89. The variant may include 1 or 2 of these substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.

(M) The introduction of one or more of (i) serine (S) at position 90 and (ii) alanine (A) at position 92. The variant may include 1 or 2 of these substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.

(N) The introduction of one or more of (i) serine (S) at position 90 and (ii) asparagine

(N) at position 94. The variant may include 1 or 2 of these substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.

(O) The introduction of one or more of (i) serine (S) at position 90 and (ii) iso leucine (I) at position 104. The variant may include 1 or 2 of these substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.

(P) The introduction of one or more of (i) aspartic acid (D) at position 88 and (ii) lysine (K) at position 105. The variant may include 1 or 2 of these substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.

(Q) The introduction of one or more of (i) asparagine (N) at position 88 and (ii) arginine (R) at position 126. The variant may include 1 or 2 of these substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.

(R) The one or more of (i) asparagine (N) at position 88, (ii) glutamine (Q) at position 90 and (iii) arginine (R) at position 91. The variant may include 1, 2 or 3 of these substitutions. The advantages of homo-octameric pores including all three substitutions in each monomer are shown in Table 2 below.

(S) The introduction of or more of (i) asparagine (N) at position 88, (ii) glutamine (Q) at position 90 and (iii) serine (S) at position 91. The variant may include 1 , 2 or 3 of these substitutions. The advantages of homo-octameric pores including all three substitutions in each monomer are shown in Table 2 below.

(T) The introduction of one or more of (i) asparagine (N) at position 88, (ii) glutamine (Q) at position 90 and (iii) valine (V) at position 105. The variant may include 1 , 2 or 3 of these substitutions. The advantages of homo-octameric pores including all three substitutions in each monomer are shown in Table 2 below.

(U) The introduction of one or more of (i) glutamine (Q) at position 90, (ii) serine (S) at position 93 and (iii) alaine (A) at position 105. The variant may include 1, 2 or 3 of these substitutions. The advantages of homo-octameric pores including all three substitutions in each monomer are shown in Table 2 below.

(V) The introduction of one or more of (i) phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H) at position 90, (ii) phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H) at position 91 and (iii) phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H) at position 105. The variant may include 1, 2 or 3 of these substitutions. The introduction of these bulky, aromatic residues increases the sterics and pi stacking in the vestibule and/or constriction of the pore. They also increase the size of the vestibule and/or constriction (i.e. open up the pore).

(W) The introduction of one or more of (i) serine (S), threonine (T), glycine (G), alanine (A) or valine (V) at position 90, (ii) serine (S), threonine (T), glycine (G), alanine (A) or valine (V) at position 91 and (iii) serine (S), threonine (T), glycine (G), alanine (A) or valine (V) at position 105. The variant may include 1, 2 or 3 of these substitutions. The introduction of smaller residues decreases the sterics in the vestibule and/or constriction of the pore.

(X) The introduction of serine (S), arginine (R), lysine (K) or histidine (H) at position 90 and/or serine (S), arginine (R), lysine (K) or histidine (H) at position 91. The introduction of positively-charged residues (R, K or H) increases the interactions between the constriction of the pore and the nucleic acid sequence.

(Y) The introduction of serine (S), threonine (T), asparagine (N), glutamine (Q), tyrosine (Y) or histidine (H) at position 90 and/or serine (S), threonine (T), asparagine (N), glutamine (Q), tyrosine (Y) or histidine (H) at position 91. The introduction of these residues increases the hydrogen bonding that occurs between the constriction of the pore and the nucleic acid sequence. They also increase the size of the vestibule and/or constriction (i.e. open up the pore).

(Z) The introduction of cysteine at one or more of positions 90, 9 land 103. This allows chemical groups to be attached to the pore via cysteine linkage. This is discussed in more detail above and below.

Preferred variants include, but are not limited to, those comprising at least one of the following substitution(s): L88N; L88S; L88Q; L88T; D90S; D90Q; D90Y; I105L; I105S;

Q126R; G75S; G77S; G75S, G77S, L88N and Q126R; G75S, G77S, L88N, D90Q and Q126R; D90Q and Q126R; L88N, D90Q and Q126R; L88S and D90Q; L88N and D90Q; E59R; G75Q;

G75N; G75S; G75T; G77Q; G77N; G77S; G77T; I78L; S81N; T83N; N86S; N86T; I87F; I87V;

I87L; L88N; L88S; L88Y; L88F; L88V; L88Q; L88T; I89F; I89V; I89L; N90S; N90Q; N90L;

N90Y; N91S; N91Q; N91L; N91M; N91I; N91A; N91V; N91G; G92A; G92S; N93S; N93A;

N93T; I94L; T95V; A96R; A96D; A96V; A96N; A96S; A96T; P97S; P98S; F99S; G100S; L101F; N102K; N102S; N102T; S103A; S103Q; S103N; S103G; S103T; V104I; I105Y; I105L;

I105A; I105Q; I105N; I105S; I105T; T106F; T106I; T106V; T106S; N108P; N108S; D90Q and

1105 A; D90S and G92S; L88T and D90S; I87Q and D90S; I89Y and D90S; L88N and I89F;

L88N and I89Y; D90S and G92A; D90S and I94N; D90S and V104I; L88D and I105K; L88N and Q126R; L88N, D90Q and D91R; L88N, D90Q and D91S; L88N, D90Q and I105V; D90Q, D93S and I105A; N91Y; N90Y and N91G; N90G and N91Y; N90G and N91G; I05G; N90R;

N91R; N90R and N91R; N90K; N91K; N90K and N91K; N90Q and N91G; N90G and N91Q;

N90Q and N91Q; R118N; N91C; N90C; N90W; N91W; N90K; N91K; N90R; N91R; N90S and

N91S; N90Y and I105A; N90G and I105A; N90Q and I105A; N90S and I105A; L88A and

I105A; L88S and I105S; L88N and I105N; N90G and N93G; N90G; N93G; N90G and N91A; I105K; I105R; I105V; I105P; I105W; L88R; L88A; L88G; L88N; N90R and I105A; N90S and

I105A; L88A and I105A; L88S and I105S; L88N and I105N; L88C; S103C; and I105C.

A particularly preferred variant comprises I105N. Pores constructed from mutant monomers comprising I105N have a residual current that is increased by approximately 80%.

The change in current in relation to different nucleotides is also increased. This reflects a change in structure of pores constructed from mutant monomers comprising I105N. Such pores therefore have an improved ability to discriminate nucleotides.

Preferred single mutants and their advantages when used in homo-octameric pores are shown in Table 1 below.

Preferred multiple mutants and their advantages when used in homo-octameric pores are shown in Table 2 below.

The most preferred mutants and their advantages when used in homo-octameric pores are shown in the Table 3 below.

In addition to the specific mutations discussed above, the variant may include other mutations. Over the entire length of the amino acid sequence of SEQ ID NO: 2, a variant will preferably be at least 50% homologous to that sequence based on amino acid identity. More preferably, the variant may 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 based on amino acid identity to the amino acid sequence of SEQ ID NO: 2 over the entire sequence. There may be at least 80%, for example at least 85%, 90% or 95%, amino acid identity over a stretch of 100 or more, for example 125, 150, 175 or 200 or more, contiguous amino acids ("hard homology").

Standard methods in the art may be used to determine homology. For example the UWGCG Package provides the BESTFIT program which can be used to calculate homology, for example used on its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387- 395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S.F et al (1990) J Mol Biol 215:403-10.

Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighbourhood word score threshold (Altschul et al, supra). These initial neighbourhood word hits act as seeds for initiating searches to find HSP's containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915-10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1 , preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

SEQ ID NO: 2 is the NNN-RRK mutant of the MspA monomer. The variant may comprise any of the mutations in the MspB, C or D monomers compared with MspA. The mature forms of MspB, C and D are show in SEQ ID NOs: 16 to 18. In particular, the variant may comprise the following substitution present in MspB: A138P. The variant may comprise one or more of the following substitutions present in MspC: A96G, N102E and A138P. The variant may comprise one or more of the following mutations present in MspD: Deletion of G1, L2V, E5Q, L8V, D13G, W21A, D22E, K47T, I49H, I68V, D91G, A96Q, N102D, S103T, V104I, S136K and G141A. The variant may comprise combinations of one or more of the mutations and substitutions from Msp B, C and D.

Amino acid substitutions may be made to the amino acid sequence of SEQ ID NO: 2 in addition to those discussed above, for example up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well-known in the art and may be selected in accordance with the properties of the 20 main amino acids as defined in Table 4 below. Where amino acids have similar polarity, this can also be determined by reference to the hydropathy scale for amino acid side chains in Table 5.

One or more amino acid residues of the amino acid sequence of SEQ ID NO: 2 may additionally be deleted from the polypeptides described above. Up to 1, 2, 3, 4, 5, 10, 20 or 30 residues may be deleted, or more.

Variants may include fragments of SEQ ID NO: 2. Such fragments retain pore forming activity. Fragments may be at least 50, 100, 150 or 200 amino acids in length. Such fragments may be used to produce the pores of the invention. A fragment preferably comprises the pore forming domain of SEQ ID NO: 2. Fragments must include one of residues 88, 90, 91, 105, 118 and 134 of SEQ ID NO: 2. Typically, fragments include all of residues 88, 90, 91, 105, 118 and 134 of SEQ ID NO: 2.

One or more amino acids may be alternatively or additionally added to the polypeptides described above. An extension may be provided at the amino terminal or carboxy terminal of the amino acid sequence of SEQ ID NO: 2 or polypeptide variant or fragment thereof. The extension may be quite short, for example from 1 to 10 amino acids in length. Alternatively, the extension may be longer, for example up to 50 or 100 amino acids. A carrier protein may be fused to an amino acid sequence according to the invention. Other fusion proteins are discussed in more detail below. A variant may have a methionine at the amino terminal of SEQ ID NO: 2.

As discussed above, a variant is a polypeptide that has an amino acid sequence which varies from that of SEQ ID NO: 2 and which retains its ability to form a pore. A variant typically contains the regions of SEQ ID NO: 2 that are responsible for pore formation. The pore forming ability of Msp, which contains a β-barrel, is provided by β-sheets in each subunit. A variant of SEQ ID NO: 2 typically comprises the regions in SEQ ID NO: 2 that form β-sheets. One or more modifications can be made to the regions of SEQ ID NO: 2 that form β-sheets as long as the resulting variant retains its ability to form a pore. A variant of SEQ ID NO: 2 preferably includes one or more modifications, such as substitutions, additions or deletions, within its a-helices and/or loop regions.

The mutant monomers may be modified to assist their identification or purification, for example by the addition of histidine residues (a his tag), aspartic acid residues (an asp tag), a streptavidin tag or a flag tag, or by the addition of a signal sequence to promote their secretion from a cell where the polypeptide does not naturally contain such a sequence. An alternative to introducing a genetic tag is to chemically react a tag onto a native or engineered position on the pore. An example of this would be to react a gel-shift reagent to a cysteine engineered on the outside of the pore. This has been demonstrated as a method for separating hemolysin hetero-oligomers (Chem Biol. 1997 Jul;4(7):497-505).

The mutant monomer may be labelled with a revealing label. The revealing label may be any suitable label which allows the pore to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g. 125l, 35S, enzymes, antibodies, antigens, polynucleotides and ligands such as biotin.

The mutant monomer may be made synthetically or by recombinant means. For example, the pore may be synthesized by in vitro translation and transcription (IVTT). The amino acid sequence of the mutant monomer may be modified to include non-naturally occurring amino acids or to increase the stability of the monomer. When the mutant monomer is produced by synthetic means, such amino acids may be introduced during production. The mutant monomer may also be altered following either synthetic or recombinant production.

The mutant monomer may also be produced using D-amino acids. For instance, the mutant monomer may comprise a mixture of L-amino acids and D-amino acids. This is conventional in the art for producing such proteins or peptides.

The mutant monomer contains one or more specific modifications to facilitate nucleotide discrimination. The mutant monomer may also contain other non-specific modifications as long as they do not interfere with pore formation. A number of non-specific side chain modifications are known in the art and may 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 with methylacetimidate or acylation with acetic anhydride.

The mutant monomer can be produced using standard methods known in the art.

Polynucleotide sequences encoding a mutant monomer may be derived and replicated using standard methods in the art. Such sequences are discussed in more detail below. Polynucleotide sequences encoding a mutant monomer may be expressed in a bacterial host cell using standard techniques in the art. The mutant monomer may be produced in a cell by in situ expression of the polypeptide from a recombinant expression vector. The expression vector optionally carries an inducible promoter to control the expression of the polypeptide.

A mutant monomer may be produced in large scale following purification by any protein liquid chromatography system from pore producing organisms or after recombinant expression as described below. Typical protein liquid chromatography systems include FPLC, AKTA systems, the Bio-Cad system, the Bio-Rad BioLogic system and the Gilson HPLC system. The mutant monomer may then be inserted into a naturally occurring or artificial membrane for use in accordance with the invention. Methods for inserting pore into membranes are discussed below.

In some embodiments, the mutant monomer is chemically modified. The mutant monomer can be chemically modified in any way and at any site. The mutant monomer is preferably chemically modified by attachment of a molecule to one or more cysteines (cysteine linkage), attachment of a molecule to one or more lysines, attachment of a molecule to one or more non-natural amino acids, enzyme modification of an epitope or modification of a terminus. Suitable methods for carrying out such modifications are well-known in the art. The mutant monomer may be chemically modified by the attachment of any molecule. For instance, the mutant monomer may be chemically modified by attachment of a dye or a fluorophore.

In some embodiments, the mutant monomer is chemically modified with a molecular adaptor that facilitates the interaction between a pore comprising the monomer and a target nucleotide or target nucleic acid sequence. The presence of the adaptor improves the host-guest chemistry of the pore and the nucleotide or nucleic acid sequence and thereby improves the sequencing ability of pores formed from the mutant monomer. The principles of host-guest chemistry are well-known in the art. The adaptor has an effect on the physical or chemical properties of the pore that improves its interaction with the nucleotide or nucleic acid sequence. The adaptor may alter the charge of the barrel or channel of the pore or specifically interact with or bind to the nucleotide or nucleic acid sequence thereby facilitating its interaction with the pore.

The molecular adaptor is preferably a cyclic molecule, a cyclodextrin, a species that is capable of hybridization, a DNA binder or interchelator, a peptide or peptide analogue, a synthetic polymer, an aromatic planar molecule, a small positively-charged molecule or a small molecule capable of hydrogen-bonding.

The adaptor may be cyclic. A cyclic adaptor preferably has the same symmetry as the pore. The adaptor preferably has eight-fold symmetry since Msp typically has eight subunits around a central axis. This is discussed in more detail below.

The adaptor typically interacts with the nucleotide or nucleic acid sequence via host-guest chemistry. The adaptor is typically capable of interacting with the nucleotide or nucleic acid sequence. The adaptor comprises one or more chemical groups that are capable of interacting with the nucleotide or nucleic acid sequence. The one or more chemical groups preferably interact with the nucleotide or nucleic acid sequence by non-covalent interactions, such as hydrophobic interactions, hydrogen bonding, Van der Waal's forces, π-cation interactions and/or electrostatic forces. The one or more chemical groups that are capable of interacting with the nucleotide or nucleic acid sequence are preferably positively charged. The one or more chemical groups that are capable of interacting with the nucleotide or nucleic acid sequence more preferably comprise amino groups. The amino groups can be attached to primary, secondary or tertiary carbon atoms. The adaptor even more preferably comprises a ring of amino groups, such as a ring of 6, 7 or 8 amino groups. The adaptor most preferably comprises a ring of eight amino groups. A ring of protonated amino groups may interact with negatively charged phosphate groups in the nucleotide or nucleic acid sequence.

The correct positioning of the adaptor within the pore can be facilitated by host-guest chemistry between the adaptor and the pore comprising the mutant monomer. The adaptor preferably comprises one or more chemical groups that are capable of interacting with one or more amino acids in the pore. The adaptor more preferably comprises one or more chemical groups that are capable of interacting with one or more amino acids in the pore via non-covalent interactions, such as hydrophobic interactions, hydrogen bonding, Van der Waal's forces, π-cation interactions and/or electrostatic forces. The chemical groups that are capable of interacting with one or more amino acids in the pore are typically hydroxyls or amines. The hydroxyl groups can be attached to primary, secondary or tertiary carbon atoms. The hydroxyl groups may form hydrogen bonds with uncharged amino acids in the pore. Any adaptor that that facilitates the interaction between the pore and the nucleotide or nucleic acid sequence can be used.

Suitable adaptors include, but are not limited to, cyclodextrins, cyclic peptides and cucurbiturils. The adaptor is preferably a cyclodextrin or a derivative thereof. The cyclodextrin or derivative thereof may be any of those disclosed in Eliseev, A. V., and Schneider, H-J. (1994) J. Am. Chem. Soc. 116, 6081-6088. The adaptor is more preferably heptakis-6-amino-β-cyclodextrin (am7-βCD), 6-monodeoxy-6-monoamino-P-cyclodextrin (am1-βCD) or heptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu7-βCD). The guanidino group in gu7-βCD has a much higher pKa than the primary amines in am7-βCD and so it more positively charged. This gu7- PCD adaptor may be used to increase the dwell time of the nucleotide in the pore, to increase the accuracy of the residual current measured, as well as to increase the base detection rate at high temperatures or low data acquisition rates.

If a succinimidyl 3-(2-pyridyldithio)propionate (SPDP) crosslinker is used as discussed in more detail below, the adaptor is preferably heptakis(6-deoxy-6-amino)-6-N-mono(2-pyridyl)dithiopropanoyl- β-cyclodextrin (amsamPDP1 -PCD) .

More suitable adaptors include γ-cyclodextrins, which comprise 8 sugar units (and therefore have eight-fold symmetry). The γ-cyclodextrin may contain a linker molecule or may be modified to comprise all or more of the modified sugar units used in the β-cyclodextrin examples discussed above.

The molecular adaptor is preferably covalently attached to the mutant monomer. The adaptor can be covalently attached to the pore using any method known in the art. The adaptor is typically attached via chemical linkage. If the molecular adaptor is attached via cysteine linkage, the one or more cysteines have preferably been introduced to the mutant by substitution. The mutant monomers of the invention can of course comprise a cysteine residue at one or more of positions 88, 90, 91, 103 and 105. The mutant monomer may be chemically modified by attachment of a molecular adaptor to one or more, such as 2, 3, 4 or 5, of these cysteines.

Alternatively, the mutant monomer may be chemically modified by attachment of a molecule to one or more cysteines introduced at other positions. The molecular adaptor is preferably attached to one or more of positions 90, 91 and 103 of SEQ ID NO: 2.

The reactivity of cysteine residues may be enhanced by modification of the adjacent residues. For instance, the basic groups of flanking arginine, histidine or lysine residues will change the pKa of the cysteines thiol group to that of the more reactive S- group. The reactivity of cysteine residues may be protected by thiol protective groups such as dTNB. These may be reacted with one or more cysteine residues of the mutant monomer before a linker is attached. The molecule may be attached directly to the mutant monomer. The molecule is preferably 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 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldisulfanyl)propanoate, 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfanyl)butanoate and 2,5-dioxopyrrolidin-1-yl 8-(pyridin-2-yldisulfanyl)octananoate. The most preferred crosslinker is succinimidyl 3-(2-pyridyldithio)propionate (SPDP). Typically, the molecule is covalently attached to the bifunctional crosslinker before the molecule/crosslinker complex is covalently attached to the mutant monomer but it is also possible to covalently attach the bifunctional crosslinker to the monomer before the bifunctional crosslinker/monomer complex is attached to the molecule.

The linker is preferably resistant to dithiothreitol (DTT). Suitable linkers include, but are not limited to, iodoacetamide-based and Maleimide-based linkers.

In other embodiment, the monomer may be attached to a nucleic acid binding protein. This forms a modular sequencing system that may be used in the methods of sequencing of the invention. Nucleic acid binding proteins are discussed below.

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

If the nucleic acid binding protein is attached via cysteine linkage, the one or more cysteines have preferably been introduced to the mutant by substitution. The mutant monomers of the invention can of course comprise cysteine residues at one or more of positions 10 to 15, 51 to 60, 136 to 139 and 168 to 172. These positions are present in loop regions which have low conservation amongst homologues indicating that mutations or insertions may be tolerated. They are therefore suitable for attaching a nucleic acid binding protein. The reactivity of cysteine residues may be enhanced by modification as described above.

The nucleic acid binding protein may be attached directly to the mutant monomer or via one or more linkers. The molecule may be attached to the mutant monomer using the hybridization linkers described in International Application No. PCT/GB 10/000132 (published as WO 2010/086602). Alternatively, peptide linkers may be used. Peptide linkers are amino acid sequences. The length, flexibility and hydrophilicity of the peptide linker are typically designed such that it does not to disturb the functions 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 amino acids. More preferred flexible linkers include (SG)1, (SG)2, (SG)3, (SG)4, (SG)5 and (SG)8 wherein 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 amino acids. More preferred rigid linkers include (P)i2 wherein P is proline.

The mutant monomer may be chemically modified with a molecular adaptor and a nucleic acid binding protein.

Constructs

The invention also provides a construct comprising two or more covalently attached monomers derived from Msp. The construct of the invention retains its ability to form a pore. One or more constructs of the invention may be used to form pores for characterising, such as sequencing, nucleic acids sequences. The construct may comprise 2, 3, 4, 5, 6, 7, 8, 9 or 10 monomers. The two or more monomers may be the same or different.

The monomers do not have to be mutant monomers of the invention. For instance, at least one monomer may comprise the sequence shown in SEQ ID NO: 2. Alternatively, at least one monomer may comprise a variant of SEQ ID NO: 2 which is at least 50% homologous to SEQ ID NO: 2 over its entire sequence based on amino acid identity, but does not include any of the specific mutations required by the mutant monomers of the invention. More preferably, the variant may 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 based on

amino acid identity to the amino acid sequence of SEQ ID NO: 2 over the entire sequence. In a preferred embodiment, at least one monomer in the construct is a mutant monomer of the invention. All of the monomers in the construct may be a mutant monomer of the invention. The mutant monomers may 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 invention.

The monomers are preferably genetically fused. Monomers are genetically fused if the whole construct is expressed from a single polynucleotide sequence. The coding sequences of the monomers may be combined in any way to form a single polynucleotide sequence encoding the construct.

The monomers may be genetically fused in any configuration. The monomers may be fused via their terminal amino acids. For instance, the amino terminus of the one monomer may be fused to the carboxy terminus of another monomer. If the construct is formed from the genetic fusion of two or more monomers each comprising the sequence shown in SEQ ID NO: 2 or a variant thereof, the second and subsequent monomers in the construct (in the amino to carboxy direction) may comprise a methionine at their amino terminal ends (each of which is fused to the carboxy terminus of the previous monomer). For instance, if M is a monomer comprising the sequence shown in SEQ ID NO: 2 or a variant (without an amino terminal methionine) and mM is a monomer comprising the sequence shown in SEQ ID NO: 2 or a variant with an amino terminal methionine, the construct may comprise the sequence M-mM, M-mM-mM or M-mM-mM-mM. The presences of these methionines typically results from the expression of the start codons (i.e. ATGs) at the 5' end of the polynucleotides encoding the second or subsequent monomers within the polynucleotide encoding entire construct. The first monomer in the construct (in the amino to carboxy direction) may also comprise a methionine (e.g. mM-mM, mM-mM-mM or mM-mM-mM-mM).

CLAIMS
1. A mutant Msp monomer comprising a variant of the sequence shown in SEQ ID NO: 2,
I. wherein the variant comprises at least one of the following mutations:
I, (a) asparagine (N), serine (S), glutamine (Q) or threonine (T) at position 88;
(b) serine (S), glutamine (Q) or tyrosine (Y) at position 90;
(c) leucine (L) or serine (S) at position 105;
(d) arginine (R) at position 126;
(e) serine (S) at position 75;
(0 serine (S) at position 77;
(g) arginine (R) at position 59;
(h) glutamine (Q) , asparagine (N) or threonine (T) at position 75;
(i) glutamine (Q) , asparagine (N) or threonine (T) at position 77;
6) leucine (L) at position 78;
I
I (k) asparagine (N) at position 8 1 ;
(1) asparagine (N) at position 83;
(m) serine (S) or threonine (T) at position 86;
1 (n) phenylalanine (F), valine (V) or leucine (L) at position 87;
(0) tyrosine (Y), phenylalanine (F), valine (V), arginine (R), alanine (A), glycine (G) or
cysteine (C) at position 88;
(p) phenylalanine (F), valine (V) or leucine (L) at position 89;
I (q) leucine (L), phenylalanine (F): tryptophan (W), histidine (H), threonine (T), glycine
(G), alanine (A), valine (V), arginine (R), lysine (K), asparagine (N) or cysteine (C) at
position 90;
(r) serine (S), glutamine (Q), Leucine (L), methionine (M), isoleucine (I), alanine (A),
valine (V), glycine (G), phenylalanine (F), tryptophan (W), tyrosine (Y), histidine (H),
threonine (T), arginine (R), lysine (K), asparagine (N) or cysteine (C) at position 9 1 ;
(s) alanine (A) or serine (S) at position 92;
(t) serine (S), alanine (A), threonine (T), glycine (G) at position 93;
(u) leucine (L) at position 94;
(v) valine (V) at position 95;
(w) arginine (R), aspartic acid @), valine (V), asparagine (N), serine (S) or threonine (T)
at position 96;
(x) serine (S) at position 97;
(y) serine (S) at position 98;
(2) serine (S) at position 99;
(aa) serine (S) at position 100;
(bb) phenylalanine (F) at position 10 1 ;
(CC) lysine Q, serine (S) or threonine (T) at position 102;
(dd) alanine (A), glutamine (Q), asparagine (N), glycine (G) or threonine (T) at position
103;
(ee) isoleucine at position 104;
(ff) tyrosine (Y), alanine (A), glutamine (Q), asparagine (N), threonine (T),
phenylalanine (F), tryptophan (W), histidine (H), glycine (G), valine 0, arginine (R),
lysine (K), proline (P), or cysteine (C) at position 105;
(gg) phenylalanine (F), isoleucine (I), valine (V' or serine (S) at position 106;
(hh) proline (P) or serine (S) at position 108;
(ii) asparagine (N) at position 1 18;
(jj) serine (S) or cysteine (C) at position 103; and
(kk) cysteine at one or more ofpositions 10 to 15,51 to 60, 136 to 139 and 168 to 172.
2. A mutant according to claim 1, wherein the variant comprises one or more of the following
substitutions:
(a) one or more of (i) serine (S) at positon 75, (ii) serine (S) at position 77, (iii)
asparagine (N) at position 88 , (iv) glutamine (Q) at position 90 and (v) arginine (R) at
position 126;
(b) one or more of (i) glutamine (Q) at position 90 and (ii) arginine (R) at position 126;
(c) one or more of (i) asparagine (N) at position 88, (ii) glutamine (Q) at position 90 and
(iii) arginine (R) at position 126;
(d) one or more of (i) serine (S) at position 88 and (ii) glutamine (Q) at position 90;
(e) one or more of (i) asparagine (N) at position 88 and (ii) glutamine (Q) at position 90;
(f) one or more of (i) glutamine (Q) at position 90 and (ii) alanine (A) at position 105;
(g) one or more of (i) serine (S) at position 90 and (ii) serine (S) at position 92;
(h) one or more of (i) threonine (T) at position 88 and (ii) serine (S) at position 90;
(i) one or more of (i) glutamine (Q) at position 87 and (ii) serine (S) at position 90;
0) onc or more of (i) tyrosinc (Y) at position 89 and (ii) serine (S) at position 90;
(k) one or more of (i) asparagine (N) at position 88 and (ii) phenylalanine (F) at position
89;
(1) one or more of (i) asparagine (N) at position 88 and (ii) tyrosine (Y) at position 89;
(m) one or more of (i) serine (S) at position 90 and (ii) alanine (A) at position 92;
73
(n) one or more of (i) serine (S) at position 90 and (ii) asparagine (N) at position 94;
(0) one or more of (i) serine (S) at position 90 and (ii) isoleucine (I) at position 104;
(p) one or more of (i) aspartic acid (D) at position 88 and (ii) lysine (K) at position 105;
(q) one or more of (i) asparagine (N) at position 88 and (ii) arginine (R) at position 126;
(r) one or more of (i) asparagine (N) at position 88, (ii) glutamine (Q) at position 90 and
(iii) arginine (R) at position 9 1 ;
(s) one or more of (i) asparagine (N) at position 88, (ii) glutamine (Q) at position 90 and
(iii) serine (S) at 91;
(t) one or more of (i) asparagine (N) at position 88, (ii) glutarnine (Q) at position 90 and
(iii) valine (V) at position 105;
(u) one or more of (i) glutamine (Q) at position 90, (ii) serine (S) at position 93 and (iii)
alaine (A) at position 105;
(v) one or more of (i) phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H) at
position 90, (ii) phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H) at position
91 and (iii) phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H) at position
105;
(w) one or more of (i) serine (S), threonine (T), glycine (G), alanine (A) or valine (V) at
position 90, (ii) serine (S), threonine (T), glycine (G), alanine (A) or valine (V) at position
91 and (iii) serine (S), threonine (T), glycine (G), alanine (A) or valine (V) at position 105;
(x) serine (S), arginine (R), lysine (K) or histidine (H) at position 90 andlor serine (S),
arginine (R), lysine (K) or histidine (H) at position 9 1 ;
(y) serine (S), threonine (T), asparagine (N), glutarnine (Q), tyrosine (Y) or histidine (H)
at position 90 and/or serine (S), threonine (T), asparagine (N), glutamine (Q), tyrosine (Y)
or histidine (H) at position 91; and
(z) cysteine at one or more of positions 90,91 and 103.
3. , A mutant according to claim 1 or 2, wherein the variant comprises at least one of the
following substitution(s):
(vii) D90Y;
(i) L88N; (viii) I 1 05 L;
(ii) L88S; (ix)IlOSS;
(iii)L88Q; (x) Q126R;
(iv)L88T; (xi)G75S;
(v) D90S; (xii) G77S;
(vi)D90Q;
(xiii) G75S,
G77S, L88N
and Q 126R;
(xiv) G75S,
G77S, L88N,
D90Q and
Q126R;
(xv) D90Q and
Q 126R;
(xvi) L88Ny
D90Q and
Q 126R;
(xvii) L88S and
D90Q;
(xviii) L88N and
D90Q;
(xix) E59R;
(xx) G75Q;
(xxi) G75N;
(xxii) G75S;
(xxiii) G75T;
(xxiv) G77Q;
(xxv) G77N;
(xxvi) G77S;
(xxvii) G77T;
(xxviii)I78L;
(xxix) S8 1N;
(xxx) T83N;
(xxxi) N86S;
(xxxii) N86T;
(xxxiii)I87F;
(xxxiv) I87V;
(xxxv) I87L;
(xxxvi)L88N;
(xxxvii) L88
s;
(xxxviii) L88
y ;
(xxxix)L88F;
(xl)L88V;
(xli) L88Q;
(xlii) L88T;
74
(xliii) I89F;
(xliv) I89V;
(xlv) I89L;
(xlvi) N90S;
(xlvii) N90Q;
(xlviii) N90L;
(xlix) N90Y;
(1) N91S;
(li) N91 Q;
(lii)N9 1 L;
(liii) N91 M;
(liv) N91I;
(lv)N9 I A;
(lvi) N91V;
(Ivii) N91G;
(Iviii) G92A,
(lix) G92S;
(Ix)N93S;
(Ixi) N93Ay
(Ixii) N93T;
(lxiii) I94L;
(Ixiv) T95V;
(lxv) A96R;
( h i ) A96D;
(Ixvii) A96V;
(Ixviii) A96N;
(Ixix) A96S;
(Ixx) A96T;
( h i ) P97S;
(lxxii) P98S;
(Ixxiii) F99S;
(lxxiv) G 100s;
(lxxv) L1 O1F;
(Ixxvi) N 102K;
(lxxvii)N 102s;
(Ixxviii) N10
2T;
(lxxix) S 103A;
(lxxx) S 103Q;
(lxxxi) S 103N;
(1xxxii)S 103G;
(Ixxxiii) S10
3T;
(Ixxxiv) V10
41;
(Ixxxv) 11 05Y;
(Ixxxvi) I1 05
L;
(lxxxvii) I1 05
A;
(Ixxxviii) I 105
Q;
(Ixxxix) I105
N;
(xc) 1105s;
(xci) I105T;
(xcii) T106F;
(xciii) T 1061;
(xciv) T106V;
(xcv) T106S;
(xcvi) N108P;
(xcvii) N 1 08s;
(xcviii) D90Q and
I1 05A;
(xcix) D90S and
G92S;
(c) L88T and
D90S;
(ci) I87Q and
D90S;
(cii) I89Yand
D90S;
(ciii) L88N and
I89F;
(civ) L88N and
189Y;
(cv) D90S and
G92A;
(cvi) D90S and
I94N;
(cvii) D90S and '
V 1041;
(cviii) L88D and
I105K;
(cix) L88N and
Q 126R;
(cx) L88N,
D90Q and
D9 1 R,
(cxi) L88N,
D90Q and
D91S;
(cxii) L88N,
D90Q and
I1 05V;
(cxiii) D90Q,
D93S and
I1 05A;
(cxiv) N91Y;
(cxv) N90Y and
N91G;
(cxvi) N90G and
N91Y;
(cxvii) N90G and
N91G;
75
(cxviii) I05G;
(cxix) N90R;
(cxx) N91R;
(cxxi) N90R and
N9 1 R;
(cxxii) N90K;
(cxxiii) N9 1 K;
(cxxiv) N90K and
N9 1 K,
(cxxv) N90Q and
N91G;
(cxxvi) N90G and
N91Q;
(cxxvii) N90
Q and N91Q;
(cxxviii) R11
8N;
(cxxix) N9 1 C;
(cxxx) N90C;
(cxxxi) N90W;
(cxxxii) N9 1
w;
(cxxxiii) N90
K;
(cxxxiv) N91
K;
(cxxxv) N90
R;
(cxxxvi) N91
R;
(cxxxvii) N90
S and N91S;
(cxxxviii) N90
Y and I105A;
(cxxxix) N90
G and I1 O5A;
(cxl) N90Q and
1105A;
(cxli) N90S and
1105A;
(cxlii) L88A and
11 05A;
(cxliii) L88S and
1105s;
(cxliv) L88N and
IlO5N;
(cxlv) N90G and
N93G;
(cxlvi) N90G;
(cxlvii)N93G;
(cxlviii) N90
G and N9 1 A;
(cxlix) I1 05K,
(cl)IlO5R;
(cli) I105V;
(clii) I1 05P;
(cliii) I1 05 W;
(cliv)
(clv) L88R;
(clvi) L88A;
(clvii) L88G;
(clviii) L88N;
(clix) N90R and
I105A;
(clx) N90S and
1105A;
(clxi) L88A and
I105A;
(clxii) L88S and
1105s;
76
(clxiii) L88N and (clxv) S 103C; and
1105N; (clxvi) I1 05C.
(clxiv) L88C;
4. A mutant according to any one of the preceding claims, wherein the mutant is chemically
modified.
5. A mutant according to claim 4, wherein the mutant is chemically modified by attachment
of a molecule to one or more cysteines, attachment of a molecule to one or more lysines,
attachment of a molecule to one or more non-natural amino acids, enzyme modification of an
epitope or modification of a terminus.
6. A mutant according to claim 5, wherein the one or more cysteines have been introduced to
the mutant by substitution.
7. A mutant according to claim 5 or 6, wherein the molecule is (a) a molecular adaptor that
facilitates the interaction between a pore comprising the monomer and a target nucleotide or
target nucleic acid sequence or @) a nucleic acid binding protein..
8. A mutant according to any one or claims 5 to 7, wherein the attachment is via a linker.
9. A mutant according to any one of claims 5 to 8, wherein the molecule is attached to one or
more ofpositions 90,91 and 103 of SEQ ID NO: 2.
10. A construct comprising two or more covalently attached monomers derived from Msp.
1 1. A construct according to claim 10, wherein the two or more monomers are the same or
different.
12. A construct according to claim 10 or 1 1, wherein at least one monomer comprises the
sequence shown in SEQ ID NO: 2.
13. A construct according to any one of claims 10 to 12, wherein at least one of the monomers
is a mutant monomer as defined in any one of claims 1 to 8.
77 e 14. A construct according to any one of claims 10 to 13, wherein the construct comprises two
monomers and at least one of the monomers is a mutant as defined in any one of claims 1 to 8.
15. A construct according to any one of claims 10 to 14, wherein the monomers are genetically
fused.
16. A construct according to any one of claims 10 to 15, wherein the monomers are attached
via a linker.
17. A polynucleotide which encodes a mutant according to any one of claims 1 to 3 or a
construct according to claim 1 5.
18. A homo-oligomeric pore derived from Msp comprising identical mutant monomers
according to any one of claims 1 to 3.
19. A homo-oligomeric pore according to claim 18, wherein the pore comprises eight identical
mutant monomers according to any one of claims 1 to 3.
20. A hetero-oligomeric pore derived from Msp comprising at least one mutant monomer
according to any one of claims 1 to 3, wherein at least one of the eight monomers differs from
the others.
21. A hetero-oligomeric pore according to claim 20, wherein the pore comprises eight mutant
monomers according to claim 1 and at least one of them differs from the others.
22. A hetero-oligomeric pore according to claim 2 1, wherein the pore comprises at least one
monomer comprising the sequence shown in SEQ ID NO: 2.
23. A hetero-oligomeric pore according to claim 21 or 22, wherein the pore comprises (a) one
mutant monomer and (b) seven identical monomers, wherein the mutant monomer in (a) is
different from the identical monomers in (b).
24. A hetero-oligomeric pore according to any one of claims 21 to 23, wherein the pore
comprises:
78
(a) seven monomers comprising the sequence shown in SEQ ID NO: 2 and one mutant
monomer comprising the substitution N90R, N90K, N90Y, N90Q, N90W or N90C;
(b) seven monomers comprising the sequence shown in SEQ ID NO: 2 and one mutant
monomer comprising the substitution N91R, N91K, N91Y, N91Q, N91 W or N91C;
or
(c) seven monomers comprising the sequence shown in SEQ ID NO: 2 and one mutant
monomer comprising the substitution L88C, S103C or 1105C.
25. A pore according to any one of claims 18 to 24, wherein at least one of the mutant
monomers is chemically-modified as defined in claims 4 to 9.
26. A pore comprising at least one construct according to claims 10 to 16.
27. A pore according to claim 26, which comprises (a) one construct as defined in claim 13
and (b) six monomers each comprising (i) the sequence shown in SEQ ID NO: 2 or (ii) a variant
of SEQ ID NO: 2 as defined in claim 1 or 2.
28, A pore according to claim 26, which comprises four contructs as defined in claim 14.
29. A pore according to any one of claims 26 to 28, wherein at least one of the constructs is
chemically-modified as defined in claims 4 to 9.
30. A method of characterising a target nucleic acid sequence, comprising:
(a) contacting the target sequence with a pore according to any one of claims 18 to 29
and a nucleic acid binding protein so that the protein controls the movement of the target
sequence through the pore and a proportion of the nucleotides in the target sequence
interacts with the pore; and
(b) measuring the current passing through the pore during each interaction and thereby
characterising the target sequence.
3 1. A method according to claim 30, wherein characterising the target nucleic acid sequence
comprises estimating the sequence of or sequencing the target nucleic acid sequence.
32. A kit for characterising a target nucleic acid sequence comprising (a) a pore according to
any one of claims 18 to 29 and (b) a nucleic acid handling enzyme.
@ 33. An apparatus for characterising target nucleic acid sequences in a sample, comprising (a) a
plurality of pores according to claims 18 to 29 and (b) a plurality of nucleic acid handling
enzymes.
34. An apparatus according to claim 32, wherein the apparatus comprises:
a sensor device that is capable of supporting the plurality of pores and being operable to
perform nucleic acid characterisation using the pores and enzymes;
at least one reservoir for holding material for performing the characterisation;
a fluidics system configured to controllably supply material from the at least one reservoir
to the sensor device; and
a plurality of containers for receiving respective samples, the fluidics system being
configured to supply the samples selectively from the containers to the sensor device.
35. A method of characterising a target nucleic acid sequence, comprising:
(a) contacting the target sequence with a pore derived from Msp and a Phi29 DNA
polymerase such that the polymerase controls the movement of the target sequence through
the pore and a proportion of the nucleotides in the target sequence interacts with the pore;
and
(b) measuring the current passing through the pore during each interaction and thereby
characterising the target sequence, wherein steps (a) and (b) are carried out with a voltage
applied across the pore.
36. A method according to claim 35, wherein characterising the target nucleic acid sequence
comprises estimating the sequence of or sequencing the target nucleic acid sequence.
37. A method according to claim 35 or 36, wherein steps (a) and @) are carried out in the
presence of free nucleotides and an enzyme cofactor such that the polymerase moves the target
sequence through the pore against the field resulting from the applied voltage.
38. A method according to claim 37, wherein the method further comprises:
(c) removing the free nucleotides such that the polymerase moves the target sequence
through the pore in the opposite direction to that in steps fa) and (b) and a proportion of the
nucleotides in the target sequence interacts with the pore; and
80
(d) measuring the current passing through the pore during each interaction and thereby
proof reading the sequence of the target sequence obtained in step (b), wherein steps (c)
and (d) are also carried out with a voltage applied across the pore.
39. A method according to claim 35 or 36, wherein steps (a) and (b) are carried out in the
absence of free nucleotides and the presence of an enzyme cofactor such that the polymerase
moves the target sequence through the pore with the field resulting from the applied voltage.
40. A method according to claim 39, wherein the method further comprises:
(c) adding free nucleotides such that the polymerase moves the target sequence through
the pore in the opposite direction to that in steps (a) and (b) and a proportion of the
nucleotides in the target sequence interacts with the pore; and
(d) measuring the current passing through the pore during each interaction and thereby
proof reading the sequence of the target sequence obtained in step (b), wherein steps (c)
and (d) are also carried out with a voltage applied across the pore.
41. A method according to claim 35 or 36, wherein steps (a) and (b) are carried out in the
absence of free nucleotides and the absence of an enzyme cofactor such that the polymerase
controls the movement of the target sequence through the pore with the field resulting from the
applied vo It age.
42. A method according to claim 41, wherein the method further comprises:
(c) lowering the voltage applied across the pore such that the target sequence moves
through the pore in the opposite direction to that in steps (a) and (b) and a proportion of the
nucleotides in the target sequence interacts with the pore; and
(d) measuring the cu.ment passing through the pore during each interaction and thereby
proof reading the sequence of the target sequence obtained in step (b), wherein steps (c)
and (d) are also carried out with a voltage applied across the pore.
43. A method of forming a sensor for characterising a target nucleic acid sequence,
comprising:
(a) contacting a pore derived fiom Msp with a Phi29 DNA polymerase in the presence
of the target nucleic acid sequence; and
(b) applying a voltage across the pore to form a complex between the pore and the
polymerase;
8 1
and thereby forming a sensor for characterising the target nucleic acid sequence.
44. A method of increasing the rate of activity of a Phi29 DNA polymerase, comprising:
(a) contacting the Phi29 DNA polymerase with a pore derived from Msp in the presence
of a nucleic acid sequence; and
@) applying a voltage across the pore to form a complex between the pore and the
polymerase;
and thereby increasing the rate of activity of a Phi29 DNA polymerase.
45. A method according to claim 43 or 44, which hrther comprises increasing the applied
voltage across the pore to increase the rate of activity of the Phi29 DNA polymerase.
46. A method according to any one of claims 35 to 45, wherein at least a portion of the nucleic
acid sequence is double stranded.
47. A method according to any one of claims 35 to 46, wherein the pore is as defined in any
one of claims 18 to 29.
48. A method according to any one of claims 35 to 46, wherein the pore comprises eight
monomers comprising the sequence shown in SEQ ID NO: 2 or a variant thereof.
49. A method according to any one of claims 35 to 48, wherein the Phi29 DNA polymerase
comprises the sequence shown in SEQ ID NO: 4 or a variant thereof having at least 50%
homology to SEQ ID NO: 4 based on amino acid identity over the entire sequence and retains
enzyme activity.
50. A kit for characterising a target nucleic acid sequence comprising (a) a pore derived from
Msp and (b) a Phi29 DNA polymerase.
51. An apparatus for characterising target nucleic acid sequences in a sample, comprising a
plurality of pores derived from Msp and a plurality of Phi29 DNA polyrnerases.
52. An apparatus according to claim 5 1, wherein the analysis apparatus is as defined in claim