MUTANT MSP MONOMER, CONSTRUCT, POLYNUCLEOTIDE, PORE, KIT AND APPARATUS TO CHARACTERIZE A TARGET NUCL

专利摘要:
mutant msp monomer, construct, polynucleotide, pore, kit and apparatus for characterizing a target nucleic acid sequence, and, method for characterizing a target nucleic acid sequence. the invention relates to mutant forms of msp. the invention also relates to nucleic acid characterization using msp.
公开号:BR112013020411B1
申请号:R112013020411-7
申请日:2012-02-10
公开日:2021-09-08
发明作者:James Clarke；Andrew John Heron；Lakmal Jayasinghe；Jayne Wallace；James White
申请人:Oxford Nanopore Technologies Limited；
IPC主号:

专利说明:

Field of Invention
[001] The invention concerns the mutant forms of Msp. The invention also concerns the characterization of nucleic acid using Msp. Fundamentals of the Invention
[002] Nanopore detection is an approach to detection that depends on the observation of individual binding events between analyte molecules and a receptor. Nanopore sensors can be generated by placing a single nanometer-sized pore on an insulating membrane, and measuring the stress-driven ion transport across the pore in the presence of analyte molecules. The identity of an analyte is revealed through its distinctive current signature, notably the duration and length of the current block and the variance of current levels.
[003] There is currently a need for fast and inexpensive nucleic acid sequencing technologies (eg DNA or RNA) across a wide range of applications. Existing technologies are slow and expensive primarily because they rely on amplification techniques to produce large volumes of nucleic acid and require a high amount of special-purpose fluorescent chemicals for signal detection. Nanopore detection has the potential to provide fast and inexpensive nucleic acid sequencing by reducing the amount of nucleotide and reagents required.
[004] Two of the essential components of nucleic acid sequencing using nanopore detection are (1) the control of nucleic acid movement through the pore and (2) the discrimination of nucleotides as the nucleic acid polymer moves through the pore. In the past, to obtain nucleotide discrimination the nucleic acid was passed through a hemolysin mutant. This provided current signatures that were 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 the observed current and the challenging nucleic acid sequence.
[005] Although the current range for nucleotide discrimination has been improved through mutation of the hemolysin pore, a sequencing system would have better performance if the current differences between nucleotides could be further improved. Furthermore, it was observed that when nucleic acids moved through a pore, some current states showed high variance. It was also shown that some mutant hemolysin pores exhibited greater variance than others. Although 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.
[006] 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 US A. 2010 Sep 14; 107(37): 16060-5. Epub 2010 Aug 26). Some key residues have been identified and modified to improve pore properties. These mutations were made to allow DNA to migrate through the MspA pore. MspB, C and D are also known forms of Msp. Invention Summary
[007] The inventors have surprisingly demonstrated that novel Msp mutants have better properties for estimating characteristics, such as the nucleic acid sequence. Mutants surprisingly show better nucleotide discrimination. In particular, the mutants surprisingly have a larger current range, which makes it easy to discriminate between different nucleotides, and a smaller state variance, which increases the signal-to-noise ratio. Furthermore, 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 current observed as the nucleic acid moves through the pore and the nucleic acid sequence.
[008] The inventors have also surprisingly shown that Msp shows improved sequencing properties when the movement of 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 surprising advantages. First, nucleic acid moves through the pore at a rate that is commercially viable yet allows for effective sequencing. Second, a greater range of current is observed as the nucleic acid moves through the pore allowing the sequence to be more easily determined. Third, a low current variance is observed, thereby increasing the signal-to-noise ratio.
[009] Thus, 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 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; (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; (11) 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 encoding a mutant of the invention or a construct of the invention; - an Msp-derived homo-oligomeric pore comprising identical mutant monomers of the invention; - an Msp-derived hetero-oligomeric pore comprising at least one mutant monomer of the invention, wherein at least one of the eight monomers differs from the others; - a method for characterizing a target nucleic acid sequence, comprising: (a) contacting the target sequence with a pore of the invention and a nucleic acid binding protein such that the protein controls movement of the target sequence through the pore and a proportion of the nucleotides in the target sequence interact with the pore; and (b) measuring the current passing through the pore during each interaction and thereby characterizing the target sequence; - a kit for sequencing a target nucleic acid sequence comprising (a) a portion 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 for characterizing a target nucleic acid sequence, comprising: (a) contacting the target sequence with a pore derived from Msp and a Phi29 DNA polymerase in such a way that the polymerase controls movement of the target sequence through the pore and a proportion of the nucleotides in the target sequence interact with the pore; and (b) measuring the current passing through the pore during each interaction and thereby characterizing the target sequence, in which steps (a) and (b) are carried out with a voltage applied across the pore; - a method of forming a sensor for characterizing a target nucleic acid sequence, comprising: (a) contacting an Msp-derived pore with a Phi29 DNA polymerase in the presence of the target nucleic acid sequence; and (b) applying tension across the pore to form a complex between the pore and the polymerase; and thereby forming a sensor to characterize the target nucleic acid sequence; - a method for increasing the activity rate 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 tension across the pore to form a complex between the pore and the polymerase; and thereby increasing the activity rate of a Phi29 DNA polymerase; - a kit for characterizing a target nucleic acid sequence comprising (a) a portion derived from Msp and (b) a Phi29 DNA polymerase; and - an apparatus for characterizing target nucleic acid sequences in a sample, comprising a plurality of pores derived from Msp and a plurality of DNA polymerase Phi29s. Description of Figures
[0010] Fig. 1 shows the average dwell time of individual current levels as a single strand of DNA translocates the nanopore. Data is pasted from a number of single molecules and divided into quartiles by current levels.
[0011] Fig. 2 shows current levels and variance obtained from using Phi29 in aperture mode to move a strand of DNA (SEQ ID NO: 15) through the nanopore MS-(NNNRRK)8.
[0012] Fig. 3 shows current levels and variance obtained using Phi29 in aperture mode to move a strand of DNA (SEQ ID NO: 15) through the HL-(mutant)7 nanopore.
[0013] Fig. 4 shows current levels for a single MspA channel recorded at a range of applied potentials (-200 mV to 200 mV).
Fig. 5 shows the open pore levels curve IV for the baseline MspA mutant, MS-(B1)8. Each line represents a single pore.
Fig. 6 shows the open pore levels curve IV for the MspA mutant, MS-(B1-I105Y)8. Each line represents a single pore.
Fig. 7 shows the open pore levels curve IV for the MspA mutant, MS-(B1-I105N)8. Each line represents a single pore.
[0017] Fig. 8 shows the change in current between a state of high conductance (275 pA) and a state of low conductance (150 pA) for pore MS-(B1-I105A)8 at 180 mV.
[0018] Fig. 9 shows the levels of current produced when DNA is opened through the baseline MS-(B1)8 pore. The current range for these events is ~30 pA.
[0019] Fig. 10 shows the current levels produced when DNA is opened through the baseline MS-(B1-I10 A)8 pore. The current range for these events is ~40 pA.
[0020] Fig. 11 shows the design of the DNA substrate used in examples 9 and 12 and 15.
[0021] Fig. 12 shows the design of the DNA substrate used in examples 10 and 11.
[0022] Fig. 13 shows how the sequencing profile changes to the same DNA sequence when point mutations are made in the MspA monomer sequence. These graphs show the average profile of the levels obtained from multiple polynucleotides. A) This graph shows the sequencing profile for pore MS-(B1)8. B) This graph shows the sequencing profile for pore MS-(B1-D90Q-D93S-1105A)8. C) This graph shows the sequencing profile for pore MS-(B 1 -D90Q-Q126R)8. D) This graph shows the sequencing profile for the MS- pore (B1-L88N-D90Q-D91M)8. 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.
[0023] Fig. 14 shows the design of the DNA substrate used in Example 13.
Fig. 15 shows an example event trace for the controlled translocation of RNA, mediated by Phi29 DNA polymerase, through the MS-(B1)8 pore of mutant MspA. An expanded view of the region highlighted in the top trace is shown below.
[0025] Fig. 16 shows pore insertion into the lipid bilayer. A) Shows pore insertion of MS-(BL)8 oligomerized from monomer. B) Shows pore insert of MS-(B1-B1)4 oligomerized from dimer.
Fig. 17 shows an example event trace for the controlled translocation of DNA, mediated by a helicase, through the pore of the mutant MS-(B1)8 that was produced by oligomerization of the monomer. An expanded view of the region highlighted in the top trace is shown below.
Fig. 18 shows an example event trace for the controlled translocation of DNA, mediated by a helicase, through the pore of the mutant MS-(B1-B1)4 that was produced by oligomerization of the dimer. An expanded view of the region highlighted in the top trace is shown below.
[0028] Fig. 19 shows the design of the DNA substrate used in example 16.
[0029] 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 pore of the mutant MS-(B1-L88N)8. An expanded view of the region highlighted in the top trace is shown below. Sequence Listing Description
[0030] SEQ ID NO: 1 shows the polynucleotide sequence encoding the mutant NNN-RR MspA monomer.
SEQ ID NO: 2 (also referred to as "B1") 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 amino terminal methionine (encoded by the start codon) and includes the following mutations: D90N, D91N, D93N, D118R, D134R and E139K. These mutations allow the transition of DNA through the MspA pore.
[0032] SEQ ID NO: 3 shows the polynucleotide sequence encoding DNA polymerase Phi29.
[0033] SEQ ID NO: 4 shows the amino acid sequence of DNA polymerase Phi29.
[0034] SEQ ID NO: 5 shows the codon-optimized polynucleotide sequence derived from the sbcB gene of E. coli. It encodes the E. coli exonuclease I (EcoExo I) enzyme.
SEQ ID NO: 6 shows the amino acid sequence of exonuclease I (EcoExo I) enzyme from E. coli.
[0036] SEQ ID NO: 7 shows the codon-optimized polynucleotide sequence derived from the xthA gene of E. coli. It encodes the E. coli exonuclease III enzyme.
[0037] SEQ ID NO: 8 shows the amino acid sequence of the E. coli exonuclease III enzyme. This enzyme performs distributive 5' monophosphate nucleoside digestion of a double-stranded DNA strand (DNAds) 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-optimized polynucleotide sequence derived from the recJ gene of T. thermophilus. It encodes the T. thermophilus RecJ enzyme (TthRecJ-cd).
[0039] SEQ ID NO: 10 shows the amino acid sequence of the T. thermophilus RecJ enzyme (TthRecJ-cd). This enzyme performs processive 5' nucleoside monophosphate digestion of ss DNAs in a 5' - 3' direction. One-strand enzyme initiation requires at least 4 nucleotides.
[0040] SEQ ID NO: 11 shows the codon-optimized polynucleotide sequence derived from bacteriophage lambda of the exo gene (redX). It encodes bacteriophage lambda exonuclease.
SEQ ID NO: 12 shows 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 single-strand nucleotide digestion of DNA ds, in a 5'-3' direction (http://www.neb.com/nebecomm/products/productM0262.asp). Enzyme initiation on a strand preferably requires a 5' overhang of approximately 4 nucleotides with a 5' phosphate.
[0042] SEQ ID NOs: 13 to 15 show the sequences used in example 2.
[0043] SEQ ID NOs: 16 to 18 show the amino acid sequences of the mature forms of the MspB, C and D mutants respectively. Mature forms lack the signal sequence.
[0044] SEQ ID NOs: 19 and 20 show the sequences used in examples 9, 12 and 15.
[0045] SEQ ID NOs: 21 to 23 show the sequences used in examples 10 and 11.
[0046] SEQ ID NOs: 24 to 27 show the sequences used in example 13.
[0047] 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.
[0048] 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.
[0049] SEQ ID NO: 30, 31 and 32 show the sequences used in example 16.
[0050] SEQ ID NO:33 shows the linker sequence used shown in the construct shown in SEQ ID NO:29. Detailed Description of the Invention
[0051] It should be understood that different applications of the disclosed products and methods can be tailored to specific needs in technology. It is also to be understood that the terminology used herein is for the sole purpose of describing particular embodiments of the invention, and is not to be considered limiting.
[0052] Furthermore, as used in this specification and the appended claims, the singular forms "a", "an", and "the" "a" 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 pores such as these, reference to "an acid sequence nucleic" includes two or more such sequences, and the like.
[0053] 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 relates to mutant Msp monomers. Mutant Msp monomers can 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 to confirm the ability of mutant monomers to form pores are well known in the art and are discussed in more detail below.
[0055] Mutant monomers have better nucleotide reading properties, that is, they have better nucleotide capture and discrimination. In particular, pores constructed from the mutant monomers capture nucleotides and nucleic acids more easily than the wild type. Furthermore, pores constructed from the mutant monomers present a greater current range, which makes it easy to discriminate different nucleotides, and a smaller state variance, which increases the signal-to-noise ratio. Furthermore, the number of nucleotides contributing to the current as the nucleic acid moves through the constructed pores of the mutants is diminished. This makes it easier to identify a direct relationship between the current observed as the nucleic acid moves through the pore and the nucleic acid sequence. The best nucleotide reading properties of mutants are obtained through five main mechanisms, namely, by changes in: • sterics (increasing or decreasing the size of amino acid residues); • charge (for example, introducing +ve charge to interact with the nucleic acid sequence); • hydrogen bonding (for example, introducing amino acids that can hydrogen bond in base pairs); • Pi stacking (eg, introducing interacting amino acids through delocalized pi electron systems); and/or • alteration of pore structure (eg, introducing amino acids that increase vestibule size and/or constriction).
[0056] Any one or more of these five mechanisms may be responsible for the improved pore properties of the invention. For example, a pore of the invention may exhibit improved nucleotide reading properties due to altered sterics, altered hydrogen bonding and an altered structure.
[0057] The introduction of bulky residues, such as phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H), increases pore sterics. The introduction of aromatic residues, such as phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H), also increases pi-stacking in the pore. The introduction of bulky or aromatic residues also alters the pore structure, for example, opening the pore and increasing the size of the vestibule and/or constriction. This is described in more detail below.
[0058] 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, Dl18R, D134R and E139K. A variant of SEQ ID NO: 2 is a polypeptide that has an amino acid sequence that varies from that of SEQ ID NO: 2 and that 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 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; (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.
[0060] In wild-type MspA, residues 88 and 105 in each monomer form a hydrophobic ring in the internal constriction of the pore. The hydrophobic residues at positions L88 and I105 settle just above the main pore constriction, facing the aqueous channel. Mutation of these residues produces pores that have significantly larger open-pore currents at baseline (SEQ ID NO: 2). The current differences observed when mutations are made at these positions are significantly greater than would be expected by making a single mutation. This result surprisingly implies that mutations at these positions can have an effect on the channel structure other than just the local environment at these residues. Although the baseline of SEQ ID NO: 2 has been reported to have a wide range of pore conductance, the reason for this is not well understood. Mutations at positions L88 and I105 cause the dominant pore current level to be significantly greater than the baseline pore. Furthermore, this higher conductance state is the dominant conformation of the mutant, which is desirable for a large current range and higher signal-to-noise ratio.
[0061] The introduction of N, S, Q or T at position 88 (ie, mutation (a) above) introduces into the internal constriction of the pore an amino acid that can hydrogen bond to nucleotides in a nucleic acid.
[0062] Residues 90 and 91 in each monomer also form part of the internal constriction of the pore. Residue 118 in each monomer is present in the vestibule of the pore. Residue 134 in each monomer is part of the entrance to the pore.
[0063] The introduction of S, Q or Y at position 90 (ie, mutation (b) above) introduces into the internal constriction of the pore an amino acid that can hydrogen bond to 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. Amino acids introduced into the variant can be naturally occurring or non-naturally occurring derivatives thereof. Amino acids introduced into the variant can be D-amino acids.
[0065] Any number of cysteines can be introduced in the variant. Cysteines are preferably introduced at one or more, such as two or all of positions 90, 91 and 103. These positions can be used for chemical attachment of a molecular adapter as discussed in more detail below. Any number of cysteines, such as 2, 3, 4, 5, 6 or more cysteines, can be introduced at positions 10 to 15, 51 to 60, 136 to 139 and 168 to 172. These positions are present in non-loop regions. conserved from the pore and are thus used to chemically attach a nucleic acid binding protein to the pore as discussed in more detail below.
[0066] In a preferred embodiment, the variant comprises one or more of the substitutions shown in (a) to (Z) below. The variant can include any number of substitutions in A to Z, such as 1, 2, 3, 4 or 5.
[0067] (A) The introduction of one or more of (i) serine (S) at position 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 such substitutions. The advantages of homo-octameric pores including all four substitutions in each monomer are shown in Table 3 below.
[0068] (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 such substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 3 below.
[0069] (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. 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.
[0070] (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 such substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 3 below.
[0071] (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 such substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 3 below.
[0072] (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 such substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.
[0073] (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 such substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.
[0074] (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 such substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.
[0075] (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 such substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.
[0076] (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 such substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.
[0077] (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 such substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.
[0078] (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 such substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.
[0079] (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 such substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.
[0080] (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 such substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.
[0081] (O) The introduction of one or more of (i) serine (S) at position 90 and (ii) isoleucine (I) at position 104. The variant may include 1 or 2 such substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.
[0082] (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 such substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.
[0083] (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 such substitutions. The advantages of homo-octameric pores including both substitutions in each monomer are shown in Table 2 below.
[0084] (R) One or more of (i) asparagine (N) at position 88, (ii) glutamine (Q) at position 90 and (iii) arginine (R) at position 91. Variant may include 1, 2 or 3 such replacements. The advantages of homo-octameric pores including all three substitutions in each monomer are shown in Table 2 below.
[0085] (S) The introduction of one or more of (i) asparagine (N) at position 88, (ii) glutamine (Q) at position 90 and (iii) serine (S) at position 91. 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.
[0086] (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.
[0087] (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. 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.
[0088] (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. Variant may include 1, 2 or 3 these replacements. The introduction of these bulky aromatic residues increases the steric and Pi stacking in the vestibule and/or constriction of the pore. It also increases the size of the vestibule and/or constriction (ie opens the pore).
[0089] (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 such substitutions. The introduction of smaller residues decreases the sterics in the vestibule and/or constriction of the pore.
[0090] (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 pore constriction and the nucleic acid sequence.
[0091] (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 pore constriction and the nucleic acid sequence. It also increases the size of the vestibule and/or constriction (ie, opens the pore).
[0092] (Z) The introduction of cysteine at one or more of positions 90, 91 and 103. This allows chemical groups to be attached in the pore via cysteine bonding. 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; G77; G77S; G77T; 178L; S81N; T83N; N86S; N86T; 87F; 87V; 87L; 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; 94L; 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 I105A; D90S and G92S; L88T and D90S; 87Q and D90S; I89Y and D90S; L88N and I89F; L88N and I89Y; D90S and G92A; D90S and 94N; 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 91G; N90G and N91Y; N90G and 91G; 105G; N90R; N91R; N90R and N91R; N90K; N91; N90K and N91K; N90Q and 91G; 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 93G; N90G; N93G; N90G and 91A; 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%.
[0095] The change in current with respect to different nucleotides is also increased. This reflects a change in the structure of pores constructed from mutant monomers comprising I105N. Pores like these therefore have a greater ability to discriminate nucleotides.
[0096] Preferred simple mutants and their advantages when used in homo-octameric pores are shown in table 1 below. Table 1


[0097] Preferred multiple mutants and their advantages when used in homo-octameric pores are shown in table 2 below. Table 2

[0098] The above all preferred mutants and their advantages when used in homo-octameric pores are shown in table 3 below. Table 3 - Preferred above all mutants and their advantages

[0099] 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 the 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 minus 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 2 throughout the sequence. There may be at least 80%, eg at least 85%, 90% or 95% amino acid identity over a stretch of 100 or more, eg 125, 150, 175 or 200 or more, contiguous amino acids ("homology" difficult").
[00100] Standard methods in technology can be used to determine homology. For example, the UWGCG package provides the BESTFIT program that can be used to calculate homology, eg used in 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 align sequences (such as identifying equivalent residues or corresponding sequences (typically in their default settings)), for example, in the manner described in Altschul SF (1993) J Mol Evol 36:290 -300; Altschul, S.F et al (1990) J Mol Biol 215:40310.
[00101] Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying a pair of highest-scoring sequences (HSPs) by identifying short words of length W in the query string that both match and satisfy some positive-value T-score threshold when aligned with one of the same length in a base sequence of data. T is referred to as the neighborhood word score threshold (Altschul et al, supra). These initial neighbor word hits act as seeds to initiate searches to find HSP's containing them. Word hits are extended in both directions along each sequence to the point that the cumulative alignment score can be increased. Extensions to the word hits in each direction are halted when: the cumulative alignment score falls off by X quantity from its maximum attained value; the cumulative score goes to zero or less, due to the accumulation of one or more negative-scoring residue alignments; or the end of any sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program defaults to a word length (W) of 11, the BLOSUM62 score matrix (see Henikoff and Henikoff (192) 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.
[00102] The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see, for example, Karlin and Altschul (1993) Proc. Natl. Academic 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 per change. For example, a sequence is considered similar to another sequence if the least probability of summation compared to the first sequence with the second sequence is less than about 1, preferably less than about 0.1, more preferably less than about 0. 01, and above all preferably less than about 0.001.
[00103] SEQ ID NO: 2 is the NNN-RRK mutant of the MspA monomer. The variant can comprise any of the mutations in the MspB, C or D monomers compared to MspA. Mature forms of MspB, C and D are shown 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 Msp B, C and D mutations and substitutions.
[00104] Amino acid substitutions can be made in 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 introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acids they replace. Alternatively, conservative substitution can introduce another amino acid that is aromatic or aliphatic in place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the technology and can be selected according to the properties of the top 20 amino acids as defined in Table 4 below. Where amino acids have similar polarity, this can also be determined by referring to the hydropathy scale for amino acid side chains in table 5. Table 4 - Chemical properties of amino acids
Table 5 - Hydropathy Scale


[00105] One or more amino acid residues of the amino acid sequence of SEQ ID NO: 2 can additionally be deleted from the above-described polypeptides. Up to 1, 2, 3, 4, 5, 10, 20 or 30 or more residues can be deleted.
[00106] Variants may include fragments of SEQ ID NO: 2. Such fragments retain pore-forming activity. Fragments can be at least 50, 100, 150 or 200 amino acids long. Such fragments can be used to produce the pores of the invention. A fragment preferably comprises the pore-forming domain of SEQ ID NO: 2. Fragments should 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.
[00107] One or more amino acids may alternatively or additionally be added to the polypeptides described above. An extension may be provided at the amino terminus or carboxy terminus of the amino acid sequence of SEQ ID NO: 2 or polypeptide variant or fragment thereof. The extension can be quite short, for example 1 to 10 amino acids long. Alternatively, the extension can be greater, for example up to 50 or 100 amino acids. A carrier protein can be fused to an amino acid sequence in accordance with the invention. Other fusion proteins are discussed in more detail below. A variant may have a methionine at the amino terminus of SEQ ID NO:2.
[00108] As discussed above, a variant is a polypeptide that has an amino acid sequence that varies from that of SEQ ID NO:2 and that 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 capacity 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 so 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, in its helix and/or loop regions.
[00109] Mutant monomers can be modified to aid in their identification or purification, for example, by the addition of histidine residues (an his tag), aspartic acid residues (an asp tag), a streptavidin tag or a flag tag, or by adding a signal sequence to promote its secretion from a cell where the polypeptide did not naturally contain such a sequence. An alternative to introducing a genetic tag is to chemically react a tag at a native or developed position in the pore. An example of this would be reacting a gel-shutdown reagent with a cysteine developed from outside the pore. This has been demonstrated as a method to separate hemolysin hetero-oligomers (Chem Biol. 1997 Jul;4(7):497-505).
[00110] The mutant monomer can be marked with a reveal label. The reveal label can be any suitable label that allows the pore to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, for example, 1251, 35S enzymes, antibodies, antigens, polynucleotides and linkers such as biotin.
[00111] The mutant monomer can be made synthetically or through recombinants. For example, the pore can be synthesized by in vitro translation and transcription (IVTT). The amino acid sequence of the mutant monomer can 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 can be introduced during production. The mutant monomer can also be altered after production, either synthetic or recombinant.
[00112] The mutant monomer can also be produced using D-amino acids. For example, the mutant monomer can comprise a mixture of L-amino acids and D-amino acids. This is conventional in the technology for producing such proteins or peptides.
[00113] 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. Numerous 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 aBH4, amidination with methylacetimidate or acylation with acetic anhydride.
[00114] The mutant monomer can be produced using standard methods known in the technology.
[00115] Polynucleotide sequences encoding a mutant monomer can be derived and replicated using standard methods in technology. Sequences like these are discussed in more detail below. Polynucleotide sequences encoding a mutant monomer can be expressed in a bacterial host cell using techniques standard in the technology. The mutant monomer can 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 expression of the polypeptide.
[00116] A mutant monomer can be produced on a large scale after 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, Bio-Cad system, Bio-Rad BioLogic system, and Gilson HPLC system. The mutant monomer can then be inserted into a naturally occurring or artificial membrane for use in accordance with the invention. Methods for inserting pores into membranes are discussed below.
[00117] In some embodiments, the mutant monomer is chemically modified. The mutant monomer can be chemically modified in any way and anywhere. The mutant monomer is preferably chemically modified by attaching a molecule to one or more cysteines (cysteine binding), attaching a molecule to one or more lysines, attaching a molecule to one or more unnatural amino acids, enzymatic modification of an epitope or modification of a terminal. Suitable methods for carrying out such modifications are well known in the art. The mutant monomer can be chemically modified by attaching any molecule. For example, the mutant monomer can be chemically modified by attaching a dye or a fluorophore.
[00118] In some embodiments, the mutant monomer is chemically modified with a molecular adapter that facilitates the interaction between a pore comprising the monomer and a target nucleotide or target nucleic acid sequence. The presence of the adapter improves host-guest pore chemistry and 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 technology. The adapter has an effect on the physical or chemical properties of the pore that improves its interaction with the nucleotide or nucleic acid sequence. The adapter can alter the charge of the barrel or pore channel or specifically interact with or bind to the nucleotide or nucleic acid sequence, thereby facilitating their interaction with the pore.
[00119] The molecular adapter is preferably a cyclic molecule, a cyclodextrin, a species that is capable of hybridization, a DNA linker or interchelant, a peptide or peptide analogue, a synthetic polymer, a planar aromatic molecule, a positively small molecule charged, or a small molecule capable of hydrogen bonding.
[00120] Adapter can be cyclic. A cyclic adapter preferably has the same symmetry as the pore. The adapter preferably has eight times symmetry since Msp typically has eight subunits around a central axis. This is discussed in more detail below.
[00121] The adapter typically interacts with the nucleotide or nucleic acid sequence via host-guest chemistry. The adapter is typically capable of interacting with the nucleotide or nucleic acid sequence. The adapter comprises one or more chemical groups that are capable of interacting with the nucleotide or nucleic acid sequence. 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 forces, n-cation interactions and/or electrostatic forces. One or more chemical groups that are capable of interacting with the nucleotide or nucleic acid sequence are preferably positively charged. One or more chemical groups that are capable of interacting with the nucleotide or nucleic acid sequence more preferably comprise amino groups. Amino groups can be attached at primary, secondary or tertiary carbon atoms. The adapter even more preferably comprises a ring of amino groups, such as a ring of 6, 7 or 8 amino groups. The adapter above all preferably comprises a ring of eight amino groups. A ring of protonated amino groups can interact with negatively charged phosphate groups in the nucleotide or nucleic acid sequence.
[00122] The correct positioning of the adapter within the pore can be facilitated by host-guest chemistry between the adapter and the pore comprising the mutant monomer. The adapter preferably comprises one or more chemical groups that are capable of interacting with one or more amino acids in the pore. The adapter most preferably comprises one or more chemical groups that are capable of interacting with one or more amino acids in the pore through non-covalent interactions, such as hydrophobic interactions, hydrogen bonding, Van der Waal forces, n-cation interactions and/ or electrostatic forces. Chemical groups that are capable of interacting with one or more amino acids in the pore are typically hydroxyls or amines. Hydroxyl groups can be attached to primary, secondary or tertiary carbon atoms. Hydroxyl groups can form hydrogen bonds with uncharged amino acids in the pore. Any adapter that facilitates the interaction between the pore and the nucleotide or nucleic acid sequence can be used.
[00123] Suitable adapters include, but are not limited to, cyclodextrins, cyclic peptides and cucurbituryls. The adapter is preferably a cyclodextrin or a derivative thereof. The cyclodextrin or its derivative can be any of those disclosed in Eliseev, A.V., and Schneider, H-J. (1994) J. Am. Chem. Soc. 116, 6081-6088. The adapter is more preferably heptakis-6-amino-β-cyclodextrin (am7-pCD), 6-monodeoxy-6-monoamino-cyclodextrin (ami-CD) or heptakis-(6-deoxy-6-guanidino)-cyclodextrin ( gu7-pCD). The guanidino group in gu7-pCD has a much higher pKa than the primary amines in am7-pCD and thus it is more positively charged. This gu7-PCD adapter can be used to increase the nucleotide residence time in the pore, to increase the accuracy of the measured residual current, as well as to increase the base detection rate at high temperatures or low data acquisition rates.
[00124] If a succinimidyl 3-(2-pyridylthio)propionate (SPDP) crosslinker is used as discussed in more detail below, the adapter will preferably be heptakis(6-deoxy-6-amino)-6-N-mono (2-pyridyl)dithiopropanoyl-β-cyclodextrin (am6amPDP 1-βCD).
[00125] More suitable adapters include Y-cyclodextrins, which comprise 8 sugar units (and therefore have eight-fold symmetry). The Y-cyclodextrin can contain a linker molecule or can be modified to comprise all or more of the modified sugar moieties used in the β-cyclodextrin examples discussed above.
[00126] The molecular adapter is preferably covalently attached to the mutant monomer. The adapter can be covalently attached to the pore using any method known in the art. The adapter is typically attached via chemical bonding. If the molecular adapter is attached via cysteine binding, one or more cysteines will preferably be introduced into the mutant by substitution. The mutant monomers of the invention may 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 attaching a molecular linker to one or more such as 2, 3, 4 or 5 of these cysteines.
[00127] Alternatively, the mutant monomer can be chemically modified by attaching a molecule to one or more cysteines introduced at other positions. The molecular adapter is preferably attached to one or more of positions 90, 91 and 103 of SEQ ID NO:2.
[00128] The reactivity of cysteine residues can be increased by modifying adjacent residues. For example, the basic groups of flanking arginine, histidine or lysine residues will shift the pKa from the thiol group of cysteines to that of the more reactive S- group. 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 before a linker is attached. The molecule can 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.
[00129] 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-octananoate - dioxopyrrolidin-1-yl 8-(pyridin-2-yldisulfanyl). The most preferred crosslinker is succinimidyl 3-(2-pyridylthio)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 crosslinker/bifunctional monomer complex is attached to the molecule.
[00130] The binder is preferably resistant to dithiothreitol (DTT). Suitable binders include, but are not limited to, iodoacetamide-based and maleimide-based binders.
[00131] In another embodiment, the monomer can be attached to a nucleic acid binding protein. This forms a modular sequencing system that can be used in the sequencing methods 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 can be chemically fused or genetically fused. The monomer and protein are genetically fused if the entire construct is expressed from a single polynucleotide sequence. Genetic fusion of a pore to a nucleic acid binding protein is discussed in international application PCT/GB09/001679 (published as WO 2010/004265).
If the nucleic acid binding protein is attached via cysteine binding, one or more cysteines will preferably be introduced into the mutant by substitution. The mutant monomers of the invention can certainly 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 that have low conservation between homologs indicating that mutations or insertions may be tolerated. They are therefore suitable for attaching a nucleic acid binding protein. The reactivity of cysteine residues can be increased by modification as described above.
The nucleic acid binding protein can be attached directly to the mutant monomer or via one or more linkers. The molecule can be attached to the mutant monomer using the hybridization linkers described in international application PCT/GB 10/000132 (published as WO 2010/086602). Alternatively, peptide linkers can be used. Peptide linkers are the amino acid sequences. The length, flexibility and hydrophilicity of the peptide linker are typically designed in such a way that they do not 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 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, amino acids of proline. More preferred rigid linkers include (P)12 where P is proline.
[00135] The mutant monomer can be chemically modified with a molecular adapter and a nucleic acid binding protein. constructs
The invention also relates to 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 can be used to form pores to characterize, such as sequence, nucleic acid sequences. The construct can comprise 2, 3, 4, 5, 6, 7, 8, 9 or 10 monomers. The two or more monomers can be the same or different.
[00137] The monomers need not be mutant monomers of the invention. For example, at least one monomer can comprise the sequence shown in SEQ ID NO:2. Alternatively, at least one monomer can comprise a variant of SEQ ID NO:2 that is at least 50% homologous with SEQ ID NO:2 throughout their sequence based on amino acid identity, but do 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 minus 95%, 97% or 99% homologous based on amino acid identity with the amino acid sequence of SEQ ID NO: 2 throughout the 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 can be a mutant monomer of the invention. Mutant monomers can be the same or different. In a preferred embodiment, the construct comprises two monomers and at least one of the monomers is a mutant monomer of the invention.
[00138] The monomers are preferably genetically fused. 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.
[00139] Monomers can be genetically fused in any configuration. Monomers can be fused via their terminal amino acids. For example, the amino terminus of one monomer can 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 monomer and subsequent monomers in the construct (in the amino to carboxy direction) may comprise a methionine at its amino terminal ends (each of which is fused to the carboxy terminal of the previous monomer). For example, 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 methionine terminal, the construct may comprise the sequence M-mM, M-mM-mM or M-mM-mM-mM. The presences of these methionines typically result from expression of the start codon (ie, ATGs) at the 5' end of the polynucleotides encoding the second monomer or subsequent monomers in the polynucleotide encoding the entire construct. The first monomer in the construct (in the amino to carboxy direction) may also comprise a methionine (for example, mM-mM, mM-mM-mM or mM-mM-mM-mM).
[00140] The two or more monomers can be genetically fused directly together. The monomers are preferably genetically fused using a linker. The binder can be designed to restrict 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. The construct preferably comprises the sequence shown in SEQ ID NO: 29 or a variant thereof. Each monomer in SEQ ID NO:29 comprises the sequence shown in SEQ ID NO:2 or a variant thereof. The second monomer also comprises a methionine at its amino terminus as described above. The two monomers are linked by a peptide linker. A variant of SEQ ID NO:29 may vary from SEQ ID NO:29 in any of the ways discussed above with reference to variants of SEQ ID NO:2. The linker may also be modified or replaced with a peptide linker discussed above.
[00141] In another preferred embodiment, the monomers are chemically fused. A subunit is chemically fused into an enzyme if the two parts are chemically attached, for example, by means of a chemical crosslinker. Any of the chemical crosslinkers discussed above can be used. The linker can be attached to one or more cysteine residues introduced into a mutant monomer of the invention. Alternatively, the linker can be attached to a terminus of one of the monomers in the construct.
[00142] If a construct contains different monomers, crosslinking of the monomers itself can be prevented by keeping the binder concentration in a large excess of the monomers. Alternatively, a “lock and key” arrangement can be used where two binders are used. Only one end of each binder can react with each other to form a larger binder and the other ends of the binder each react with a different monomer. Such linkers are described in International Application No. PCT/GB 10/000132 (published as WO 2010/086602). Polynucleotides
The present invention also relates to polynucleotide sequences encoding a mutant monomer of the invention. The mutant monomer can be any of those discussed above. The polynucleotide sequence preferably comprises a sequence at least 50%, 60%, 70%, 80%, 90% or 95% homologous based on nucleotide identity with the sequence of SEQ ID NO: 1 throughout the sequence. There may be at least 80%, eg at least 85%, 90% or 95% nucleotide identity over a stretch of 300 or more, eg 375, 450, 525 or 600 or more, contiguous nucleotides ("homology" difficult"). Homology can be calculated as described above. The polynucleotide sequence may comprise a sequence that differs from SEQ ID NO: 1 based on the degeneracy of the genetic code.
[00144] The present invention also relates to polynucleotide sequences encoding any of the genetically fused constructs of the invention. The polynucleotide preferably comprises two or more sequences as shown in SEQ ID NO: 1 or a variant thereof as described above. The polynucleotide sequence preferably comprises the sequence of SEQ ID NO: 28 or a sequence at least 50%, 60%, 70%, 80%, 90% or 95% homologous based on nucleotide identity with the sequence of SEQ ID NO : 28 in the entire sequence. There may be at least 80%, eg at least 85%, 90% or 95% nucleotide identity over a stretch of 600 or more, eg 750, 900, 1,050 or 1200 or more, contiguous nucleotides ("homology" difficult"). Homology can be calculated as described above. The polynucleotide sequence may comprise a sequence that differs from SEQ ID NO: 28 based on the degeneracy of the genetic code.
[00145] Polynucleotide sequences can be derived and replicated using methods standard in technology. Chromosomal DNA encoding wild-type Msp can be extracted from a pore-producing organism such as Mycobacterium smegmatis. The gene encoding the pore subunit can be amplified using PCR involving specific primers. The amplified sequence can then undergo site-directed mutagenesis. Suitable site-directed mutagenesis methods are known in the technology and include, for example, combining chain reaction. Polynucleotides encoding a construct of the invention can be made using well known techniques, such as those described in Sambrook, J. and Russell, D. (2001). Molecular Cloning: a Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
The resulting polynucleotide sequence can then be incorporated into a replicable recombinant vector such as a cloning vector. The vector can be used to replicate the polynucleotide in a compatible host cell. Thus, polynucleotide sequences can be made by introducing a polynucleotide into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions that effect replication of the vector. The vector can be retrieved from the host cell. Suitable host cells for cloning polynucleotides are known in the art and described in more detail below.
[00147] The polynucleotide sequence can be cloned into a suitable expression vector. In an expression vector, the polynucleotide sequence is typically operably linked to a control sequence that is capable of providing expression of the coding sequence by the host cell. Such expression vectors can be used to express a pore subunit.
[00148] The terms "operably linked" refer to a juxtaposition in which the described components are in a relationship that allows them to function in their intended way. A control sequence "operably linked" to a coding sequence which is linked in such a way that expression of the coding sequence is obtained under conditions compatible with the control sequences. Multiple copies of the same or different polynucleotide sequences can be introduced into the vector.
[00149] The expression vector can then be introduced into a suitable host cell. Thus, a mutant monomer or construct of the invention can be produced by inserting a polynucleotide sequence into an expression vector, introducing the vector into a compatible bacterial host cell, and growing the host cell under conditions that effect expression of the polynucleotide sequence. The recombinantly expressed monomer or construct can self-assemble into a pore in the host cell membrane. Alternatively, the recombinant pore produced in this way can be removed from the host cell and inserted into another membrane. When producing pores comprising at least two different subunits, the different subunits can be expressed separately in different host cells as described above, removed from the host cells and assembled into a pore in a separate membrane, such as a rabbit cell membrane.
The vectors can be, for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of said polynucleotide sequence and optionally a promoter regulator. Vectors can contain one or more selectable marker genes, for example, a tetracycline resistant gene. Promoters and other expression regulation signals can be selected to be compatible with the host cell for which the expression vector is designed. A T7, trc, lac, ara or L promoter is typically used.
[00151] The host cell typically expresses the pore subunit at a high level. Host cells transformed with a polynucleotide sequence will be chosen to be compatible with the expression vector used to transform the cell. The host cell is typically bacterial and preferably Escherichia coli. Any cell with a DE3 X lysogen, for example, C41 (DE3), BL21 (DE3), JM109 (DE3), B834 (DE3), TUNER, Origami and Origami B, can express a vector comprising the T7 promoter. In addition to the conditions listed above, any of the methods cited in Proc Natl Acad Sci US A. 2008 Dec 30;105(52):20647-52 can be used to express the Msp protein. pores
[00152] The invention also concerns various pores. The pores of the invention are ideal for characterization, such as sequencing, of nucleic acid sequences because they can discriminate different nucleotides with a high degree of sensitivity. Pores can surprisingly distinguish between the four nucleotides in DNA and RNA. The pores of the invention can further distinguish between methylated and unmethylated nucleotides. The pore base resolution of the invention is surprisingly high. The pores show nearly complete separation of all four DNA nucleotides. The pores further discriminate between deoxycytidine monophosphate (dCMP) and methyl-dCMP based on the residence time in the pore and the current passing through the pore.
[00153] The pores of the invention can also discriminate between different nucleotides in a range of conditions. In particular, the pores will discriminate between nucleotides under conditions that are favorable for characterization, such as nucleic acid sequencing. The extent to which the pores of the invention can discriminate different nucleotides can be controlled by changing the applied potential, the salt concentration, the buffer, the temperature and the presence of additives such as urea, betaine and DTT. This allows pore function to be fine-tuned, particularly during sequencing. This is discussed in more detail below. The pores of the invention can also be used to identify nucleic acid polymers interacting with one or more monomers other than one nucleotide per nucleotide base.
A pore of the invention may be isolated, substantially isolated, purified or substantially purified. A pore of the invention is isolated or purified if it is completely free of any other components, such as lipids or other pores. A pore is substantially insulated if it is mixed with carriers or diluents that will not interfere with its intended use. For example, a pore is substantially isolated or substantially purified if it is in a form that comprises less than 10%, less than 5%, less than 2% or less than 1% of other components, such as lipids or other pores. Alternatively, a pore of the invention can be present in a lipid bilayer.
[00155] A pore of the invention may be present as an individual or single pore. Alternatively, a pore of the invention may be present in a homologous or heterologous population of two or more pores. homo-oligomeric pores
The invention also relates to an Msp-derived homo-oligomeric pore comprising identical mutant monomers of the invention. The homo-oligomeric pore preferably comprises one of the mutants shown in tables 1, 2 and 3. The homo-oligomeric pore of the invention is ideal for characterizing, as well as sequencing, nucleic acids. The homooligomeric pore of the invention may have any of the advantages discussed above. The advantages of specific homo-oligomeric pores of the invention are indicated in tables 1, 2 and 3.
[00157] The homo-oligomeric pore can contain any number of mutant monomers. The pore typically comprises 7, 8, 9 or 10 identical mutant monomers. The pore preferably comprises eight identical mutant monomers. One or more, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the mutant monomers are preferably chemically modified as discussed above.
[00158] Methods for fabricating pores are discussed in more detail below. hetero-oligomeric pores
The invention also concerns 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. The hetero-oligomeric pore of the invention is ideal for characterizing, as well as sequencing, nucleic acids. Heterooligomeric pores can be produced using methods known in the art (eg, Protein Sci. 2002 Jul;11(7):1813-24).
[00160] The hetero-oligomeric pore contains enough monomers to form the pore. Monomers can be of any type. The pore typically comprises 7, 8, 9 or 10 monomers. The pore preferably comprises eight monomers.
The pore may comprise at least one monomer comprising (a) the sequence shown in SEQ ID NO: 2 or (b) a variant thereof which did not have a mutation required by the mutant monomers of the invention. Suitable variants are discussed above. In this embodiment, the remaining monomers are preferably mutant monomers of the invention. Accordingly, the pore may comprise 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutant monomers of the invention.
In a preferred embodiment, 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). Monomers identical in (b) preferably comprise (i) the sequence shown in SEQ ID NO: 2 or (ii) a variant thereof which did not have a mutation present in the mutant monomers of the invention.
Preferred pores include, but are not limited to, any of the following: (a) Seven monomers comprising the sequence shown in SEQ ID NO: 2 and a mutant monomer comprising the substitution N90R, N90, N90Y, N90Q, N90W or N90C. These pores have a single steric amino acid (Y or W), a single charged amino acid (K or R), or a single reactive amino acid (C) introduced into the internal constriction. (b) Seven monomers comprising the sequence shown in SEQ ID NO: 2 and a mutant monomer comprising the substitution N91R, N91, N91Y, N91Q, N91W or N91C. These pores have a single steric amino acid (Y or W), a single charged amino acid (K or R), or a single reactive amino acid (C) introduced into the internal constriction. (c) Seven monomers comprising the sequence shown in SEQ ID NO: 2 and a mutant monomer comprising the substitution L88C, S103C or I105C. These pores have a reactive amino acid introduced into the pore.
[00164] In another preferred embodiment, all monomers (ie 10, 9, 8 or 7 of the monomers) are mutant monomers of the invention and at least one of them differs from the others. In a more preferred embodiment, the pore comprises eight mutant monomers of the invention and at least one of them differs from the others.
In all of the embodiments discussed above, one or more, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the mutant monomers are preferably chemically modified as discussed above. Preferred pores (a) to (c) above are preferably chemically modified by attaching a molecule to one or more of the introduced cysteines.
[00166] Methods for fabricating pores are discussed in more detail below. Pores containing construct
The invention also relates to a pore comprising at least one construct of the invention. A construct of the invention comprises two or more covalently attached monomers derived from Msp. In other words, a construct must contain more than one monomer. The pore contains enough constructs and, if necessary, monomers to form the pore. For example, an octameric pore can comprise (a) two constructs each comprising four monomers or (b) a construct comprising two monomers and six monomers that do not form part of a construct. At least two of the monomers in the pore are in the form of a construct of the invention. Monomers can be of any type. The pore typically comprises 7, 8, 9 or 10 monomers in total (at least two of which must be in one construct). The pore preferably comprises eight monomers (at least two of which must be in one construct).
[00168] A pore typically contains (a) a construct comprising two monomers and (b) 5, 6, 7 or 8 monomers. The construct can be any of those discussed above. The monomers can be any of those discussed above, including mutant monomers of the invention.
[00169] Another typical pore comprises more than one construct of the invention, such as two, three or four constructs of the invention. Pores such as these additionally comprise sufficient monomers to form the pore. The monomer can be any of those discussed above. An additional pore of the invention only comprises constructs comprising 2 monomers, for example a pore may comprise 4, 5, 6, 7 or 8 constructs comprising 2 monomers. A specific pore according to the inventions comprises four constructs each comprising two monomers. The constructs can oligomerize into a pore with a structure such that only one monomer of a construct contributes to the barrel or vestibule of the pore. Typically, the other monomers in the construct will be outside the barrel or vestibule of the pore. For example, pores of the invention may comprise 5, 6, 7 or 8 constructs comprising 2 monomers where the barrel or vestibule comprises 8 monomers.
[00170] Mutations can be introduced into the construct as described above. Mutations can be staggered, that is, the mutations are different for each monomer in one of the monomer constructs and the constructs are assembled as a homo-oligomer resulting in staggered 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 neighbors, ie identical mutations are introduced into two monomers in a construct and this is then oligomerized with different mutant monomers. In other words, monomers comprising MutA are fused followed by oligomerization with monomers containing MutB to form A-A:B:B:B:B:B:B.
[00171] One or more of the monomers of the invention in a pore containing construct may be chemically modified as discussed above. Methods of Identifying an Individual Nucleotide
The present invention is also concerned with methods of characterizing an individual nucleotide. The methods comprise contacting the nucleotide with a pore of the invention so that the nucleotide interacts with the pore and measuring the current passing through the pore during the interaction and thereby characterizing the nucleotide. The invention therefore involves nanopore detection of an individual nucleotide. The invention also concerns methods of identifying an individual nucleotide comprising measuring the current passing through the pore during the interaction and thereby determining the identity of the nucleotide. Any of the pores of the invention can be used. The pore of the invention is preferably chemically modified with a molecular adapter as discussed above.
The nucleotide is present if the current passes through the pore in a nucleotide-specific manner (that is, if a distinctive current associated with the nucleotide passing through the pore is detected). The nucleotide is absent if the current does not pass through the pore in a specific way for the nucleotide.
[00174] The invention can be used to differentiate nucleotides of similar structure based on the different effects they have on current passing through a pore. Individual nucleotides can be identified at the single molecule level of their current amplitude when they interact with the pore. The invention can also be used to determine whether or not a particular nucleotide is present in a sample. The invention can also be used to measure the concentration of a particular nucleotide in a sample.
[00175] The methods can be performed using any suitable membrane/pore system in which a pore of the invention is inserted into a membrane. Methods are typically carried out using (i) an artificial membrane comprising a pore of the invention, (ii) an isolated naturally occurring membrane comprising a pore of the invention, or (iii) a cell expressing a pore that has been modified in accordance with invention. The methods are preferably carried out using an artificial membrane. The membrane may comprise other transmembrane and/or intramembrane proteins as well as other molecules in addition to the pore of the invention.
The membrane forms a barrier to the flow of ions, nucleotides and nucleic acids. Any membrane can be used in accordance with the invention. Suitable membranes are well known in the art. The membrane is preferably an amphiphilic layer. An amphiphilic layer is a layer formed from amphiphilic molecules, such as phospholipids, which have both hydrophilic and lipophilic properties. Amphiphiles can be synthetic or naturally occurring. The amphiphilic layer can be a monolayer or a bilayer. Non-naturally occurring amphiphiles and amphiphiles that form a monolayer are known in the technology and include, for example, block copolymers (Gonzalez-Perez et ah, Langmuir, 2009, 25, 10447-10450).
[00177] The membrane may be a lipid bilayer. Lipid bilayers suitable for use in accordance with the invention can be made using methods known in the art. For example, lipid bilayer membranes can be formed using the method of Montal and Mueller (1972). Lipid bilayers can also be formed using the method described in international application No. PCT/GB08/000563.
The method of the invention can be carried out using lipid bilayers formed from any membrane lipid including, but not limited to, phospholipids, glycolipids, cholesterol, mycolic acid and mixtures thereof. Any of the lipids described in international application No. PCT/GB08/000563 can be used.
[00179] 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 an organism or cell, or a synthetically fabricated version of a biologically available structure. Solid state layers can be formed from both organic and inorganic materials including, but not limited to, microelectronic materials, insulating materials such as Si3N4, Al12O3, and SiO, organic and inorganic polymers such as polyamide, plastics such as Teflon® or elastomers such as two-component addition-curing silicone rubber, and glasses. The solid state layer can be formed from monoatomic layers, such as graphene, or layers that are only a few atoms thick. Suitable graphene layers are disclosed in international application No. PCT/US2008/010637 (published as WO 2009/035647). An amphiphilic layer can be formed through a solid state pore. This can be described in technology as hybrid pore formation (Hall et ah, Nat Nanotechnol., 2010, 5, 874-877).
[00180] Methods are known in technology to insert pores into membranes such as lipid bilayers. For example, the pore can be suspended in a purified form in a solution containing a lipid bilayer in such a way that it diffuses into the lipid bilayer and is inserted binding into the lipid bilayer and assembled into a functional state. Alternatively, the pore can be directly inserted into the membrane using the "pick and place" method described in M.A. Holden, H. Bayley. J. Am. Chem. Soc. 2005, 127, 6502-6503 and International Application No. PCT/GB2006/001057 (published as WO 2006/100484).
The methods of the invention are typically performed in vitro. individual nucleotide
[00182] An individual nucleotide is a single nucleotide. An individual nucleotide is one that is not linked to another nucleotide or nucleic acid by a nucleotide linkage. A nucleotide bond involves one of the phosphate groups of one nucleotide being attached to the sugar group of another nucleotide. An individual nucleotide is typically one that is not linked by a nucleotide linkage to another nucleic acid sequence of at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1,000 or at least 5,000 nucleotides. For example, the individual nucleotide has been digested from a target polynucleotide sequence, such as a strand of DNA or RNA.
The methods of the invention can be used to identify any nucleotide. The nucleotide can be naturally occurring or artificial. A nucleotide typically contains a nucleobase, a sugar and at least one phosphate group. The nucleobase is typically heterocyclic. Suitable nucleobases include purines and pyrimidines and more specifically adenine, guanine, thymine, uracil and cytosine. Sugar is typically a pentose sugar. Suitable sugars include, but are not limited to, ribose and deoxyribose. The nucleotide is typically a ribonucleotide or deoxyribonucleotide. The nucleotide typically contains a monophosphate, diphosphate or triphosphate.
Suitable 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), tripho deoxyuridine phosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP) and deoxycytidine triphosphate (dCTP). The nucleotide is preferably AMP, TMP, GMP, UMP, dAMP, dTMP, dGMP or dCMP.
The nucleotide can be derived from digestion of a nucleic acid sequence such as ribonucleic acid (RA) or deoxyribonucleic acid. Nucleic acid sequences can be digested using any method known in the art. Suitable methods include, but are not limited to, those using enzymes or catalysts. Catalytic digestion of nucleic acids is disclosed in Deck et al., Inorg. Chem, 2002; 41: 669-677.
[00186] Individual nucleotides from a single nucleic acid sequence can be contacted with the pore in a sequential manner in order to sequence all or part of the nucleic acid.
[00187] Nucleic acid sequencing is discussed in more detail below.
The nucleotide is typically unmodified, such as when the nucleotide is derived from digestion of a nucleic acid sequence. Alternatively, the nucleotide can be modified or damaged. The nucleotide is typically methylated or oxidized. The nucleotide can be labeled with a reveal label. The reveal label can be any suitable label that allows the nucleotide to be detected. Suitable labels include fluorescent molecules, radioisotopes, for example 125 I, 35 S, and linkers such as biotin.
[00189] The nucleotide is typically present in any suitable biological sample. Appropriate biological samples are discussed above. Interaction between pore and nucleotide
[00190] The nucleotide can be placed in contact with the pore on either side of the membrane. The nucleotide can be introduced into the pore on either side of the membrane. The nucleotide can be placed in contact with one side of the membrane which allows the nucleotide to pass through the pore to the other side of the membrane. For example, the nucleotide is placed in contact with one end of the pore, which in its native environment allows entry of ions or small molecules, such as nucleotides, into the barrel or pore channel in such a way that the nucleotide can pass through the pore. . In such cases, the nucleotide interacts with the pore and/or adapter as it passes through the membrane through the barrel or pore channel. Alternatively, the nucleotide can be placed in contact with the side of the membrane which allows the nucleotide to interact with the pore through an adapter or together with it, dissociate from the pore and remain on the same side of the membrane. The present invention concerns pores in which the position of the adapter is fixed. As a result, the nucleotide is preferably placed in contact with the end of the pore which allows the adapter to interact with the nucleotide.
[00191] The nucleotide can interact with the pore in any way and anywhere. As discussed above, the nucleotide preferably reversibly binds to the pore via or together with an adapter. The nucleotide above all preferably reversibly binds to the pore via an adapter or together with it as it passes through the pore through the membrane. The nucleotide can also reversibly bind to the barrel or pore channel via an adapter or together with it as it passes through the pore through the membrane.
[00192] During the interaction between the nucleotide and the pore, the nucleotide affects the current that passes through the pore in a way specific to this nucleotide. For example, a particular nucleotide will reduce the current passing through the pore for a particular mean period of time and to a particular extent. In other words, the current passing through the pore is distinctive for a particular nucleotide. Control experiments can be performed to determine the effect a particular nucleotide has on the current passing through the pore. Results of performing the method of the invention on a test sample can then be compared with those derived from such a control experiment in order to identify a particular nucleotide in the sample or determine whether a particular nucleotide is present in the sample. The frequency at which the current passing through the pore is affected in a manner indicative of a particular nucleotide can be used to determine the concentration of that nucleotide in the sample. The ratio of different nucleotides in a sample can also be calculated. For example, the ratio of dCMP to methyl-dCMP can be calculated. Device
[00193] The methods can be performed using any apparatus that is suitable for investigating a membrane/pore system in which a pore of the invention is inserted into a membrane. The method can be performed using any device that is suitable for nanopore detection. For example, an apparatus comprises a chamber comprising an aqueous solution and a barrier that separates the chamber into two sections. The barrier has an opening in which the membrane containing the pore is formed. The nucleotide can be brought into contact with the pore by introducing the nucleotide into the chamber. The nucleotide can be introduced into either of the two sections of the chamber.
[00194] The methods can be performed using an apparatus described in international application No. PCT/GB08/000562.
[00195] The methods of the invention involve measuring the current that passes through the pore during interaction with the nucleotide. Therefore, an apparatus also comprises an electrical circuit capable of applying a potential and measuring an electrical signal across the membrane and pore. Methods can be performed using a patch clamp or a voltage clamp. The methods preferably involve the use of a voltage clamp. Sample
[00196] The nucleotide is present in any suitable sample. The invention is typically performed on a sample that is known to contain or suspected to contain the nucleotide. The invention can be carried out on a sample that contains one or more nucleotides whose identity is unknown. Alternatively, the invention can be performed on a sample to confirm the identity of one or more nucleotides whose presence in the sample is known or expected.
[00197] The sample may be a biological sample. The invention can be carried out in vitro on a sample obtained or extracted from any organism or micro-organism. The organism or micro-organism is typically prokaryotic or eukaryotic and typically belongs to one of five kingdoms: plantae, animalia, fungi, monera and protist. The invention can be carried out in vitro on a sample obtained or extracted from any virus. The sample is preferably a fluid sample. The sample typically comprises a patient's bodily fluid. The sample can be urine, lymph, saliva, mucus or amniotic fluid, but is preferably blood, plasma or serum. Typically, the sample is of human origin, but alternatively it may be from another mammalian animal such as commercially farm-raised animals such as horses, cattle, sheep or pigs or it may alternatively be domestic animals such as cats or dogs. Alternatively a plant source sample is typically obtained from a commercial crop, such as a cereal, vegetable, fruit or vegetable, for example wheat, barley, oats, canola, corn, soybeans, rice, bananas, apples, tomatoes, potatoes , grapes, tobacco, beans, peas, sugar cane, cocoa, cotton, tea, coffee.
[00198] The sample may be a non-biological sample. The non-biological sample is preferably a fluid sample. Examples of a non-biological sample include surgical fluids, water such as drinking water, sea water or river water, and reagents for laboratory testing.
[00199] The sample is typically processed before being tested, for example, by centrifugation or by passing through a membrane that filters out unwanted molecules or cells, such as red blood cells. The sample can be measured immediately when taken. The sample can also typically be stored prior to testing, preferably below -70°C. Conditions
[00200] The methods of the invention involve measuring a current that passes through the pore during interaction with the nucleotide. Conditions suitable for measuring ionic currents through transmembrane protein pores are known in the technology and disclosed in the example. The method is performed with tension applied across the membrane and pore. The voltage used is typically from -400 mV to +400 mV. The voltage used is preferably in a range with a selected lower limit of -400 mV, -300 mV, -200 mV, -150 mV, -100 mV, -50 mV, -20 mV and 0 mV and an independently selected upper limit of +10 mV, +20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV. The voltage used is most preferably in the range 100 mV to 240 mV and above all preferably in the range 160 mV to 240 mV. It is possible to increase discrimination between different nucleotides by a pore of the invention using greater applied potential.
[00201] The methods are typically performed in the presence of any alkali metal chloride salt. In the exemplary apparatus discussed above, salt is present in the aqueous solution in the chamber. Potassium chloride (C1), sodium chloride (NaCl) or cesium chloride (CsCl) are typically used. KCl is preferred. The salt concentration is typically 0.1 to 2.5 M, 0.3 to 1.9 M, 0.5 to 1.8 M, 0.7 to 1.7 M, 0.9 to 1.6M or from 1M to 1.4M. The salt concentration is preferably 10mM to 1M. High concentration of salts provide a high signal-to-noise ratio and allow currents indicative of the presence of a nucleotide to be identified against the background of normal current fluctuations. Lower concentration of salts can be used if nucleotide detection is performed in the presence of an enzyme, such as during nucleic acid sequencing. This is discussed in more detail below.
[00202] Methods are 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 invention. A suitable buffer is Tris-HCl buffer. The methods are typically carried out at a pH of 4.0 to 12.0, from 4.5 to 10.0, from 5.0 to 9.0, from 5.5 to 8.8, from 6.0 to 8 ,7 or from 7.0 to 8.8 or 7.5 to 8.5. The pH used is preferably about 7.5.
[00203] The methods are typically carried out from 0°C to 100°C, from 15°C to 95°C, from 16°C to 90°C, from 17°C to 85°C, from 18°C to 80 °C, 19°C to 70°C, or from 20°C to 60°C. Methods can be performed at room temperature. The methods are preferably carried out at a temperature that supports enzyme function, such as about 37°C. Methods of characterizing nucleic acids
The present invention also relates to methods of characterizing a target nucleic acid sequence. One or more characteristics of the target nucleic acid sequence can be determined. The method may involve measuring two, three, four or five or more characteristics of the target nucleic acid sequence. One or more characteristics are preferably selected from (i) the length of the target nucleic acid sequence, (ii) the identity of the target nucleic acid sequence, (iii) the sequence of the target nucleic acid sequence, (iv) the secondary structure of the target nucleic acid sequence and (v) whether or not the target nucleic acid sequence is modified. Any combination of (i) to (v) can be determined in accordance with the invention.
For (i), the length of the nucleic acid sequence can be measured using the number of interactions between the target nucleic acid sequence and the pore.
For (ii), nucleic acid sequence identity can be measured in a number of ways.
Nucleic acid sequence identity can be measured together with sequence measurements of the target nucleic acid sequence or without sequence measurements of the target nucleic acid sequence. The first is straightforward; the nucleic acid is sequenced and thereby identified. The latter can be done in a number of ways. For example, the presence of a particular motif in the nucleic acid sequence can be measured (without measuring the remaining sequence of the polynucleotide). Alternatively, measurements of a particular electrical signal in the method can identify the target nucleic acid sequence coming from a particular source.
For (iii), the sequence of the nucleic acid sequence can be determined as described previously. Suitable sequencing methods, particularly those using electrical measurements, are described in Stoddart D et al., Proc Natl Acad Sci, 12;106(19):7702-7, Lieberman KR et al, J Am Chem Soc. 2010;132( 50):17961-72, and international application WO 2000/28312.
[00209] For (iv), the secondary structure can be measured in a variety of ways. For example, secondary structure can be measured using a change in residence time or a change in current passing through the pore.
The invention also relates to a method of estimating the sequence of a target nucleic acid sequence. The invention further concerns a method of sequencing a target nucleic acid sequence.
A nucleic acid is a macromolecule comprising two or more nucleotides. Nucleotides can be any of those discussed above.
[00212] In one embodiment, the method comprises (a) contacting the target sequence with a pore of the invention and a nucleic acid binding protein such that the protein controls movement of the target sequence through the pore and a proportion of the nucleotides in the target sequence interact with the pore and (b) measure the current passing through the pore during each interaction and thereby characterize, such as estimate, the sequence or sequencing of the target sequence. Consequently, the method involves nanopore detection of a proportion of the nucleotides in a target nucleic acid sequence as the nucleotides pass through the barrel or channel in order to characterize, such as sequencing, the target sequence.
In another embodiment, the method comprises (a) contacting the target sequence with a pore of the invention and an exonuclease in such a way that the exonuclease digests an individual nucleotide from one end of the target sequence; (b) contacting the nucleotide with the pore so that the nucleotide interacts with the adapter; (c) measure the current passing through the pore during the interaction and thereby characterize the nucleotide; and (d) repeating steps (a) to (c) at the same end of the target sequence and thereby characterizing the target sequence. Consequently, the method involves nanopore detection of a proportion of the nucleotides in a target nucleic acid sequence in a successive manner in order to characterize the target sequence. In a preferred embodiment, the method concerns sequencing the target nucleic acid sequence and step (a) comprises determining the identity of the nucleotide. Individual nucleotides are described above.
[00214] The pores of the invention are particularly suitable for these methods because they exhibit better nucleotide discrimination. In particular, they have a larger current range, which makes it easy to discriminate between different nucleotides, and a smaller state variance, which increases the signal-to-noise ratio. Furthermore, relative to the first modality, 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 current observed as the nucleic acid moves through the pore and the nucleic acid sequence. The pores of the invention are preferably chemically modified with (1) a molecular adapter and/or (2) the nucleic acid binding protein or exonuclease as discussed above.
All or only part of the target nucleic acid sequence can be characterized, as sequenced, using this method. The nucleic acid sequence can be of any length. For example, the nucleic acid sequence 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 nucleotides in length. The nucleic acid sequence can be 1,000 or more nucleotides or 5,000 or more nucleotides in length. The nucleic acid sequence can be naturally occurring or artificial. For example, the method can be used to verify the sequence of a fabricated oligonucleotide. Methods are typically performed in vitro.
The methods can be performed using any suitable membrane/pore system in which a pore is inserted into a membrane. Methods are typically performed using any of the systems, apparatus or conditions disclosed above.
[00217] As mentioned earlier, good nucleotide discrimination can be obtained at low concentration of salts if the temperature is increased. In addition to increasing the solution temperature, there are numerous other strategies that can be employed to increase the solution conductance while maintaining conditions that are suitable for enzymatic activity. One such strategy is to use the lipid bilayer to split two different salt solution concentrations, a low salt salt concentration on the enzyme side and a high salt concentration on the opposite side. An example of this approach is to use 200 mM KC1 in the cis side of the membrane and 500 mM C1 in the trans chamber. Under these conditions, the conductance through the pore is expected to be roughly equivalent to 400 mM C1 under normal conditions, and the enzyme will only experience 200 mM if placed on the cis side. Another possible benefit of using asymmetric salt conditions is the pore-induced osmotic gradient. This liquid stream of water can be used to push nucleotides into the pore for detection. A similar effect can be obtained using a neutral osmolyte such as sucrose, glycerol or PEG. Another possibility is to use a solution with relatively low levels of KC1 and have an additional charge carrying species that is less disruptive to enzyme activity.
[00218] The target sequence being analyzed can be combined with known protective chemicals to prevent the sequence from being acted upon by the binding protein or exonuclease while in bulk solution. The pore can then be used to remove the protective chemical. This can be achieved either by using protecting groups that are not hybridized by the pore, binding protein or enzyme under an applied potential (WO 2008/124107) or by using protecting chemicals that are removed by the binding protein or enzyme when held in close proximity. immediate pore (J Am Chem Soc. 2010 Dec 22;132(50):17961-72).
[00219] Tape sequencing
[00220] Tape sequencing involves the controlled and stepwise translocation of nucleic acid polymers through a pore. Pores of the invention can be used in tape sequencing. One method of the invention uses a nucleic acid binding protein to control movement of the target sequence through the pore. Examples of such proteins include, but are not limited to, nucleic acid handling enzymes such as nucleases, polymerases, topoisomerases, ligases and helicases, and non-catalytic binding proteins such as those classified by SCOP (Structural Classification of Protens) under the superfamily of nucleic acid binding protein (50249). The binding protein can be single-stranded binding protein (SSB).
[00221] A nucleic acid is a macromolecule comprising two or more nucleotides. The protein-bound nucleic acid can comprise any combination of any nucleotide. Nucleotides can be any of those discussed above. Nucleic acid can be deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The nucleic acid can be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threosis nucleic acid (TNA), blocked nucleic acid (LNA) or other synthetic polymers with side chains of nucleotide. The protein-bound nucleic acid can be single-stranded, such as cDNA, RNA, GNA, TNA or LNA, or double-stranded, such as DNA. Proteins that bind single-stranded nucleic acids can be used to sequence double-stranded DNA as long as the double-stranded DNA is dissociated into a single strand before it is bound by the protein.
The nucleic acid binding protein is preferably a nucleic acid handling enzyme. A nucleic acid handling enzyme is a polypeptide that is capable of interacting with and modifying at least one property of a nucleic acid. The enzyme can modify the nucleic acid by cleaving it to form individual nucleotides or short nucleotide chains, such as di- or trinucleotides. The enzyme can modify the nucleic acid by orienting it or moving it to a specific position. The nucleic acid handling enzyme need not exhibit enzymatic activity as long as it is able to bind the target sequence and control its movement through the pore. For example, the enzyme can be modified to remove its enzymatic activity or it can be used under conditions that prevent it from acting as an enzyme. Such conditions are discussed in more detail below.
[00223] The nucleic acid handling enzyme is preferably derived from a nucleolytic enzyme. The nucleic acid handling enzyme used in the enzyme construct is most preferably derived from a member of any of the 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 may be any of those disclosed in international application No. PCT/GB 10/000133 (published as WO 2010/086603).
[00224] Preferred enzymes are polymerases, exonucleases, helicases and topoisomerases such as gyrases. Suitable enzymes include, but are not limited to, E. coli exonuclease I (SEQ ID NO: 6), E. coli exonuclease III enzyme (SEQ ID NO: 8), T. thermophilus RecJ (SEQ ID NO: 10), and bacteriophage lambda exonuclease (SEQ ID NO: 12) and variants thereof. Three subunits comprising the sequence shown in SEQ ID NO: 10 or a variant thereof interact to form an exonuclease trimer. The enzyme is preferably based on Phi29 DNA polymerase (SEQ ID NO: 4).
[00225] A variant of SEQ ID NOs: 4, 6, 8, 10 or 12 is an enzyme that has an amino acid sequence that varies from that of SEQ ID NOs: 4, 6, 8, 10 or 12 and that retains acid capacity nucleic binding. The variant may include modifications that facilitate nucleic acid binding and/or facilitate its activity at high concentration of salts and/or at room temperature.
Over the entire length of the amino acid sequence of SEQ ID NO: 4, 6, 8, 10 or 12, a variant will preferably be at least 50% homologous to the sequence based on amino acid identity. More preferably, the variant polypeptide 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: 4, 6, 8, 10 or 12 throughout the sequence. There may be at least 80%, eg at least 85%, 90% or 95% amino acid identity over a stretch of 200 or more, eg 230, 250, 270 or 280 or more, contiguous amino acids ("homology" difficult"). Homology is determined as described above. The variant may differ from the wild type sequence in any of the ways discussed above with reference to SEQ ID NO: 2. The enzyme may be covalently attached to the pore as discussed above.
[00227] A is not required that the enzyme be in such close proximity to the pore lumen as for individual nucleotide sequencing as there is no potential for disorder in the array in which nucleotides reach the pore protection fraction.
[00228] The two strategies for sequencing single-stranded DNA are the translocation of the DNA through the nanopore, both cis to trans and trans to cis, as with or against an applied potential. The most advantageous mechanism for strand sequencing is the controlled translocation of single-stranded DNA through the nanopore under an applied potential.
[00229] Exonucleases that act progressively or processively on double-stranded DNA can be used on the cis side of the pore to feed the remaining single strand through an applied potential or the translation under a reverse potential. Likewise, a double-stranded DNA uncoiling helicase can also be used in a similar manner. There are also possibilities for sequencing applications that require translocation of the strand against an applied potential, but the DNA must first be “picked up” by the enzyme under a reverse potential or no potential at all. With the potential then changed back after bonding the tape will transition from cis to trans through the pore and will be held in an extended conformation by the current passing. Single-stranded DNA exonucleases or single-stranded DNA-dependent polymerases can act as molecular motors to push the newly translocated single-stranded back through the pore in a controlled stepwise manner, trans to cis, against the applied potential. Exonuclease-based methods
[00230] In one embodiment, the method for characterizing a target nucleic acid sequence involves contacting the target sequence with an exonuclease enzyme. Any of the exonuclease enzymes discussed above can be used in the method. Individual nucleotides release exonuclease from one end of the target sequence. The enzyme can be covalently attached to the pore as discussed above.
[00231] Exonucleases are enzymes that typically take one end of a nucleic acid sequence and digest the sequence one nucleotide at a time from that end. Exonuclease can digest nucleic acid in the 5' to 3' direction or 3' to 5' direction. The end of the nucleic acid to which the exonuclease binds is typically determined through the choice of enzyme used and/or using methods known in the art. Hydroxyl groups or cap structures at either end of the nucleic acid sequence can typically be used to prevent or facilitate exonuclease binding at a particular end of the nucleic acid sequence.
The method involves contacting the nucleic acid sequence with the exonuclease so that the nucleotides are digested from the end of the nucleic acid at a rate that allows for the characterization or identification of a proportion of nucleotides as discussed above. Methods for doing this are well known in the technology. For example, Edman degradation is used to successively digest single amino acids from the end of a polypeptide in such a way that they can be identified using High Performance Liquid Chromatography (HPLC). A homologous method can be used in the present invention.
The rate at which the exonuclease works is typically lower than the optimal rate for a wild-type exonuclease. A suitable rate of exonuclease activity in the method of the invention involves digestion of 0.5 to 1000 nucleotides per second, from 0.6 to 500 nucleotides per second, 0.7 to 200 nucleotides per second, from 0.8 to 100 nucleotides per second second, from 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. An adequate rate of exonuclease activity can be achieved in several ways. For example, variant exonucleases with an optimally reduced rate of activity can be used in accordance with the invention. Msp and Phi29 DNA polymerase
In a preferred embodiment, characterization, such as strand sequencing, is performed using an Msp-derived pore and a Phi29 DNA polymerase. The method comprises (a) contacting the target sequence with an Msp-derived pore and a Phi29 DNA polymerase in such a way that the polymerase controls the movement of the target sequence through the pore and a proportion of the nucleotides in the target sequence interact with the pore and (b) measure the current passing through the pore during each interaction and thereby characterize, such as determining the sequence, the target sequence, in which steps (a) and (b) are performed with an applied voltage through the pore. When the target sequence is contacted with a Phi29 DNA polymerase and an Msp-derived pore, the target sequence first forms a complex with the Phi29 DNA polymerase. When tension is applied across the pore, complex target sequence/DNA polymerase Phi29 forms a complex with the pore and controls movement of the target sequence through the pore.
[00235] This modality has three surprising advantages. First, the target sequence moves through the pore at a rate that is commercially viable and still allows for effective sequencing. The target sequence moves through the Msp pore faster than it moves through a hemolysin pore. Second, a greater range of current is observed as the nucleic acid moves through the pore allowing the sequence to be more easily determined. Thirdly, a smaller current variance is observed when the specific pore and polymerase are used together thereby increasing the signal-to-noise ratio.
Any nucleic acid sequence described above can be characterized or sequenced. At least a portion of the nucleic acid sequence is preferably double-stranded.
[00237] The pore can be any of the pores discussed above. The pore is preferably a pore of the invention. The pore may comprise eight monomers comprising the sequence shown in SEQ ID NO: 2, 16, 17 or 18 or a variant thereof. The pore does not have to include any of the mutations of the invention.
[00238] Phi29 wild-type DNA polymerase has polymerase and exonuclease activity. It can also open double-stranded nucleic acids under the right conditions. Consequently, the enzyme can work in three ways. This is discussed in more detail below.
The Phi29 DNA polymerase may comprise the sequence shown in SEQ ID NO: 4 or a variant thereof. A variant of SEQ ID NOs:4 is an enzyme that has an amino acid sequence that varies from that of SEQ ID NOs:4 and that retains nucleic acid binding activity. The variant must work in at least one of the three ways discussed below. Preferably, the variant works in all three modes. The variant can include modifications that facilitate the handling of the nucleic acid and/or facilitate its activity at high concentration of salts and/or at room temperature.
Over the entire length of the amino acid sequence of SEQ ID NO: 4, a variant will preferably be at least 40% homologous to the sequence based on amino acid identity. More preferably, the variant polypeptide may be at least 50%, 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: 4 throughout the sequence. There may be at least 80%, eg at least 85%, 90% or 95% amino acid identity over a stretch of 200 or more, eg 230, 250, 270 or 280 or more, contiguous amino acids ("homology" difficult"). Homology is determined as described above. The variant may differ from the wild type sequence in any of the ways discussed above with reference to SEQ ID NO: 2. The enzyme may be covalently attached to the pore as discussed above.
[00241] Any of the systems, apparatus or conditions discussed above can be used in accordance with this preferred embodiment. The salt concentration is typically from 0.15M to 0.6M. The salt is preferably KCl.
[00242] The method can be performed in one of three preferred modes based on the three modes of DNA polymerase Phi29. Each mode includes a sequence proof reading method. First, the method is preferably carried out using DNA polymerase P 29 as a polymerase. In this embodiment, steps (a) and (b) 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 stress. The target sequence moves in the 5’ to 3’ direction. Free nucleotides can be one or more of any of the individual nucleotides discussed above. The enzyme cofactor is a factor that allows Phi29 DNA polymerase to function as both a polymerase and an exonuclease. The enzyme cofactor is preferably a divalent metal cation. The divalent metal cation is preferably Mg, Mn, Ca or Co. The enzyme cofactor is above all preferably Mg2+. The method preferably further comprises (c) removing the free nucleotides in such a way that the polymerase moves the target sequence through the pore with the field resulting from the applied stress (i.e., in the 3' to 5' direction) and a proportion of the nucleotides in the target sequence it interacts with the pore and (d) measure the current that passes through the pore during each interaction and thereby carry out the proof reading of the sequence of the target sequence obtained in step (b), in which the steps ( c) and (d) are also carried out with a stress applied across the pore.
[00243] Second, the method is preferably performed using Phi29 DNA polymerase as an exonuclease. In this embodiment, steps (a) and (b) are carried out in the absence of free nucleotides and in the presence of an enzyme cofactor such that the polymerase moves the target sequence through the pore with the field resulting from the applied stress. Target sequence moves in 3' to 5' direction. The method preferably further comprises (c) adding free nucleotides such that the polymerase moves the target sequence through the pore against the field resulting from the applied stress (i.e., in the 5' to 3' direction) and a proportion of the nucleotides in the target sequence interacts with the pore and (d) measure the current that passes through the pore during each interaction and thereby test-read the target sequence sequence obtained in step (b), in which steps (c) ) and (d) are also carried out with a stress applied across the pore.
[00244] Thirdly, the method is preferably performed using DNA polymerase Phi29 in aperture mode. In this modality, steps (a) and (b) are carried out in the absence of free nucleotides and in 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 voltage ( as it is open). In this modality, the polymerase acts as a brake preventing the target sequence from moving through the pore too quickly under the influence of applied stress. The method preferably further comprises (c) reducing the voltage applied across the pore such that the target sequence moves through the pore in the opposite direction from that in steps (a) and (b) (i.e., as it reanneals) and a proportion of the nucleotides in the target sequence interacts with the pore and (d) measure the current passing through the pore during each interaction and thereby proofread the target sequence sequence obtained in step (b), wherein steps (c) and (d) are also carried out with a stress applied across the pore.
The invention also relates to a method for forming a sensor to sequence a target nucleic acid sequence, comprising (a) contacting an Msp-derived pore 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 to sequence the target nucleic acid sequence. The invention further relates to a method of increasing the activity rate of a Phi29 DNA polymerase comprising contacting the Phi29 DNA polymerase with an Msp-derived pore in the presence of a nucleic acid sequence and applying a strain across the pore to form a complex between the pore and the polymerase and thereby increase the activity rate of a Phi29 DNA polymerase. cases
The present invention also relates to kits for characterizing, such as sequencing, a target nucleic acid sequence. A kit comprises (a) a pore of the invention and (b) a nucleic acid handling enzyme. Another kit comprises (a) an Msp-derived portion and (b) a Phi29 DNA polymerase. Any of the embodiments discussed above with reference to the methods of the invention are equally applicable to the kits of the invention.
The kits of the invention may additionally comprise one or more other reagents or instruments that allow any of the aforementioned modalities to be carried out. Such reagents or instruments include one or more of the following: suitable buffer(s) (aqueous solutions), means for obtaining a sample from a subject (such as a vessel or an instrument comprising a needle), means for amplifying and/ or expressing polynucleotide sequences, a membrane as defined above, or strain, or patch clamp apparatus. Reagents may be present in the kit in a dry state such that a fluid sample will resuspend the reagents. The kit may also optionally comprise instructions to enable the kit to be used in the method of the invention or details relating to which patients the method may be used. The kit can optionally comprise nucleotides. Device
The invention also concerns an apparatus for characterizing, such as sequencing, target nucleic acid sequences in a sample. The apparatus may comprise (a) a plurality of pores of the invention and (b) a plurality of nucleic acid handling enzymes. Alternatively, the invention may comprise a plurality of pores derived from Msp and a plurality of DNA polymerase Phi29s. The apparatus may be any conventional apparatus for analyte analysis, such as an array or chip.
[00249] The apparatus preferably comprises: a sensor device that is capable of supporting the plurality of pores and being operable to perform characterization or sequencing of nucleic acid using the pores and enzymes; • at least one reservoir to hold material to carry out characterization or sequencing; • a fluidic system configured to controllably supply material from at least one reservoir to the sensing device; and • a plurality of respective sample receiving containers, the fluidic system being configured to selectively supply the samples from the containers to the sensor device. The apparatus may be any of those described in International Application No. PCT/GB 10/000789 (published as WO 2010/122293), International Application No. PCT/GB 10/002206 (not yet published) or International Application No. PCT/ US99/25679 (published as WO 00/28312). The following examples illustrate the invention: Example 1
[00250] Homo-oligomers are pores where all monomer units are identical. As monomer units will self-assemble, these are the simplest constructs to produce. Our strategies for improving base reader properties can be divided into categories: • Steric (increasing or decreasing the size of amino acid residues) • Charging (introducing +ve charge to interact with DNA) • Hydrogen bonding (residues that can bind hydrogen to base pairs) • Pi stacking (amino acids that interact through delocalized electron pi systems)
[00251] Augmented sterics / Pi stacking (all NNN-RRK background): Steric - substitution of residues with volume (eg, Phenylalanine, Tryptophan, Tyrosine, Histidine) Pi-stacking - substitution of aromatic residues (eg, Phenylalanine, Tryptophan, Tyrosine, Histidine)
[00252] In all the following tables (6-11), the mutations made for SEQ ID NO: 2 are shown. Bl = SEQ ID NO: 2. Table 6

[00253] Decreased Sterics - substitution for residues with smaller size (eg Serine, Threonine, Glycine, Alanine, Valine) Table 7

[00254] Charge-substitution for positively charged residues (eg Arginine, Lysine, Histidine) Table 8

[00255] Hydrogen bonding - substitution for residues with binding capacity (eg Asparagine, Glutamine, Tyrosine, Histidine) Table 9
Table 10

[00256] Homo-oligomers can also be modified to contain a reactive group, which can then be chemically modified. Table 11
Example 2
[00257] Different monomer units can be combined to create unique oligomer pores. When the oligomer contains more than one different subunit (eg, MS-(MutA) 6(MutB) j(MutC) 1), the pore is a hetero-oligomer. Hetero-oligomers typically have only one modified unit (eg MS-(MutA)7(MutB)1). Other hetero-oligomer ratios can also be formed (eg MS-(MutA)6(MutB)2). Subunits may also include SEQ ID NO:2.
[00258] The advantage of hetero-oligomers is that a single chemical change can be made in the pore (without introducing a change for each monomer unit). This is a less drastic change to structure than a homo-oligomer and can allow residues to be introduced into the pore at a position that did not work for a homo-oligomer. A single residue interacting with DNA can be beneficial compared to multiple units (eg, a single Arg in a hetero-octamer, compared to eight Arg in an octamer). Mutants can also be combined to produce different effects on the same residue, an example of this would be to reduce the size by seven units while increasing the size by one (eg MS-(D90 g)8(D90Y)1).
[00259] Mutant design rules will be similar to those previously presented for homo-oligomers.
[00260] Introduction of a Single Steric Residue Table 12

[00261] Introduction of a Single Loaded Waste Table 13

[00262] Introduction of a Single Reactive Waste Table 14
Example 3
[00263] Introduction of a Single Reactive Waste for Chemical Modification. Table 15
Example 4
The following tables summarize the mutant pores of the invention. The first concerns homo-oligomers and the second concerns hetero-oligomers. Table 16
Table 17

Example 5 - MspA compared to HL
[00265] We combine Phi29 DNA polymerase (DNAP) as a molecular engine with a mutant MspA nanopore to allow controlled movement of a strand of DNA through the pore. A voltage was applied across the pore and a current was generated from the movement of ions in a salt solution from either side of the nanopore. As the DNA moved through the pore, the ionic current through the pore changed relative to the DNA. This information was shown to be sequence dependent.
[00266] We compared a mutant form of hemolysin with MspA, in particular MS-(Bl)g. The current range is greater for MspA compared to hemolysin (HL). Furthermore, the current range is also greater for MspA when a strand of DNA is threaded into the pore.
[00267] We show that there are numerous surprising features with MspA that were not predicted by linking MspA and Phi29 DNAP to each other. The main differences are: 1. Faster tape movement (Aperture mode) compared to HL.
[00268] 2. Increased current range when moving a tape through the pore.
[00269] 3. Decreased variance of current levels compared to HL mutants. Faster tape movement
A 134mer DNAss template (SEQ ID NO: 13) was hybridized to an 84mer DNAss (SEQ ID NO: 14) to form an 84mer DNAds template with a 5' overhang of 50mer DNAss. This strand moved through the MspA mutant MS-(B1)8 and the hemolysin mutant using Phi29 DNAP in Open mode. Two races were acquired; one at 400 mM KCl and the other at 600 mM KCl, all at room temperature with 10 mM Hepes, pH 8.0, 1 mM EDTA, 1 mM DTT. The applied potential was optimized for each mutant construct; HL was run at 220 mV and MspA at 180 mV.
[00271] Current levels were extracted as DNA events in the enzyme-bound state, these events were indexed and the current level, duration and variance of the event recorded.
[00272] For the opening of all runs, the opening speed was not consistent across the tape.
[00273] This can be shown by averaging the event duration, divided by event index quarters (Fig. 1). The first quarter provided events that had a much longer duration than the following quarters, this was true for both HL and MspA. For the first quarter, the mean event length was the smallest for MspA at 400 mM KCl and the smallest for HL at 600 mM. However, in Q2, Q3 and Q4, MspA produced small events for both salt conditions. Since the signal to noise is sufficient, small events are desirable as they indicate rapid movement of the DNA strand through the pore, thus increasing the experimental throughput. Largest Current Range and Smallest Variance
[00274] In the nanopore experiments described here, current levels basically depend on salt concentration, applied voltage, and temperature. HL and MS-(B1)8 MspA mutants were compared in open mode using Phi29 DNA polymerase with physical adjustment conditions of: 600 mM KCl, 10 mM Hepes, 1 mM EDTA, 1 mM DTT, pH 8.0, +220 mV. The DNA used in this experiment was a 100mer staple with a 34mer single-stranded 5' overhang (SEQ ID NO: 15). Runs were conducted at room temperature.
[00275] Current levels were extracted from DNA events in the enzyme-bound state, these events were indexed and the current level, duration and variance of the event recorded (Figures 2 and 3).
[00276] It is clear from these experiments that the MspA mutant gives a significantly greater current range of approximately 50 pA compared to the HL mutant where the range is approximately 20 pA (Figures 2 and 3). A large current range is advantageous as it will provide a greater signal to noise and will make it easier to distinguish distinct current states. This is of particular benefit for sequencing applications when N bases can contribute to the current signal, leading to possible 4N current states.
[00277] The variance of states is also reduced for the MspA mutant compared to the HL. This is shown by the standard deviation of the events in the previous traces (Figures 2 and 3). For previous tapes, the mean standard deviation across all events for the MspA tape was 3.6 compared to 4.5 for HL. Low state variance is desirable to allow accurate estimates of the current level of the event. Example 6 - Comparison of MS-(B1)8 baseline open pore current with MS-(B1-I105)8 mutants
[00278] MspA pore current levels can be controlled by mutating position I105 in the protein. We demonstrate that the open pore current can be increased above 80% due to a single MspA monomer mutation. Single channels were inserted into a lipid membrane under the following conditions: 400 mM KCl, 10 mM Hepes, pH 8.0, room temperature. The open pore current level was recorded over an applied potential range of -200 mV to 200 mV to produce an IV curve. The experiment was repeated for numerous pores to assess sample distribution. An example of the data from a curve IV run can be seen (Fig. 4).
[00279] In our experiments, the baseline MS-(B1)8 mutant produces pores that have an open pore current of approximately 150 pA at +160 mV (Fig. 5).
[00280] The experiment was repeated with the mutant MS-(B1-I105Y)8 which exhibited a large number of pores with a higher residual current. For these channels, the open pore current was approximately 200 pA at +160 mV (Fig. 6).
[00281] The experiment was repeated with the mutant MS-(B1-I105N)8 which exhibited two main distributions of current levels. Ten out of sixteen pores gave a greater residual current in a tight distribution. For these channels, the open pore current was approximately 280 pA at +160 mV (Fig. 7). Example 7 - an MS-(B1-I105A)8 pore that spontaneously changes conductance
[00282] It was observed that MspA mutant pores spontaneously change the conductance during electrical recording experiments.
[00283] Electrical measurements were acquired in the manner described in example 6, using the pore of the mutant MS-(B1-II 05 A)8.
[00284] A single MspA mutant pore is able to switch between spontaneously high and low conductance states (Fig. 8). This suggests that mutations to MspA allow for conformational changes that are rarely observed in the baseline MS-(B1)8 pore. It is possible that mutations at position I105 stabilize the high conductance state of the pore. Example 8 - Comparison of DNA strands when moving DNA through the pores of baseline MS-(B1)s compared to the pores of MS-(B1-I1Q5A)s
[00285] Pore of MS-(B1)8 and pores of MS-(Bl-I105N)8 were compared in open mode using Phi29 DNA polymerase with physical adjustment conditions of: 400 mM KCl, 10 mM Hepes, EDTA 1 mM, 1 mM DTT, pH 8.0, +180 mV. The DNA used in this experiment was a 100mer staple with a 34mer single-stranded 5' overhang (SEQ ID NO: 15). Runs were conducted at room temperature.
[00286] Current levels were extracted as DNA events in the enzyme-bound state, these events were indexed and the current level, duration and variance of the event recorded.
[00287] The spread of DNA strand current levels moving through the MS-(BL)8 mutant was ~30 pA under these conditions (Fig. 9). The same experiment was repeated using the mutant MS-(Bl-I105A)8, current levels exhibited a range of ~40 pA for the same strand of DNA (Fig. 10). The higher current range of the MS-(I105A)8 mutant is desirable to discriminate the nucleotide combinations in the nanopore. Example 9 - Baseline Signal Noise Comparison of MS-(B1)s with MS-(B1-L88N)s Mutants
[00288] MspA pore noise levels can be controlled by mutating the L88 position in the MspA monomer sequence. It has been shown that the noise level can be reduced by 19% as a result of producing a single mutation in the MspA monomer.
[00289] This example compares the MS-(B1)8 pore and the MS-(B1-L88N)8 pores in translocation mode, using a helicase to control the movement of intact DNA strands through a nanopore. Materials
[00290] Nucleotide primers were designed to amplify a -400 bp fragment of PhiX174. Each of the 5' ends of these nucleotide primers included a non-complementary region of 50 nucleotides, either a homopolymeric stretch or sections of homopolymeric 10 nucleotide repeating units. These served as the identifiers for controlled translocation of the strand through a nanopore, as well as determining the translocation directionality. Furthermore, the 5'-end of the forward nucleotide primer was capped to include four 2'-0-Methyl-Uracil (mU) nucleotides and the 5'-end of the reverse nucleotide primer was chemically phosphorylated. These nucleotide primer modifications then allow controlled digestion predominantly of the antisense strand only, using lambda exonuclease. The mU cap protects the sense strand from nuclease digestion while the P04 on the 5'- of the antisense strand promotes it. Therefore, after incubation with lambda exonuclease only the sense strand of the duplex remains intact, now as single-stranded DNA (DNAss). The generated ss DNA was then PAGE purified as previously described.
[00291] The design of the DNA substrate used in this experiment is shown in Fig. 11 (SEQ ID NOs: 19 and 20 (sequences and tags shown below)). The DNA substrate consists of a 400base section of PhiX DNAss, with a 50T 5'-reader to aid capture through the nanopore. Annealed to this strand just after the 50T reader is a nucleotide primer containing a 3' cholesterol tag (3' Cholesteryl-TEG) to enrich the DNA on the surface of the bilayer, and thus increase capture efficiency. SEQ ID NO: 19 mUmUmUmUTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTTTTGGTTGTTTCTGTTGGTGCTGATATTGCTTTTGATGCCGA CCCTAAATTTTTTGCCTGTTTGGTTCGCTTTGAGTCTTCTTCGGTTC CGACTACCCTCCCGACTGCCTATGATGTTTATCCTTTGAATGGTCG CCATGATGGTGGTTATTATACCGTCAAGGACTGTGTGACTATTGAC GTCCTTCCCCGTACGCCGGGCAATAACGTTTATGTTGGTTTCATGG TTTGGTCTAACTTTACCGCTACTAAATGCCGCGGATTGGTTTCGCT GAATCAGGTTATTAAAGAGATTATTTGTCTCCAGCCACTTAAGTGA GGTGATTTATGTTTGGTGCTATTGCTGGCGGTATTGCTTCTGCTCTT GCTGGTGGCGCCATGTCTAAATTGTTTGGAGGCGGTC SEQ ID NO: 20 (more 3 'tag cholesteryl-TEG) GCAATATCAGCACCAACAGAAACAACCTTTTTTTTTTTTTTTTTTTT tttttttttt / 3CholTEG / Experimental Method Buffered Solution: 400 mM NaCl, 10 mM Hepes pH 8.0 , 1 mM ATP, 1 mM MgCl 2 , 1 mM DTT Nanopores: MspA MS(BL)8; MspA MS(B1-L88N)8 Enzyme: Helicase
[00292] Electrical measurements were acquired from single MspA nanopores embedded in 1,2-diphytanoyl-glycero-3-phosphocholine lipid bilayers (Avanti Polar Lipids). Bilayers were formed through openings of - 100 μm in diameter in PTFE films of 20 μm thick (in custom Delrin chambers) using the Montal-Mueller technique, separating two 1 mL buffered solutions. All experiments were performed in the given buffered solution. Single-channel currents were measured on Axopatch 200B amplifiers (Molecular Devices) equipped with 1440A digitizers. Ag/AgCl electrodes were connected in the buffered solutions so that the cis compartment (in which both nanopore and enzyme/DNA are added) is connected to the ground of the Axopatch preamplifier, and the trans compartment is connected to the active electrode of the preamplifier .
[00293] After obtaining a single pore of both MS(B1)8 and MS(B1-L88N)8 in the bilayer, DNA polynucleotide (SEQ ID NOs: 19 and 20) and helicase were added to 100 μL of buffer and pre -incubated for 5 minutes (DNA = 1.5 nM, Enzyme = 1 μM). This pre-incubation mix was added to 900 μL of buffer in the cis compartment of the electrophysiology chamber to initiate the capture of helicase-DNA complexes in the MspA nanopore (to give final DNA concentrations = 0.15 nM, Enzyme = 0, 1 µM). Helicase ATPase activity was initiated as required by the addition of divalent metal (1 mM MgCl 2 ) and NTP (1 mM ATP) in the cis compartment. Experiments were carried out at a constant potential of +140 mV. Current levels were extracted as enzyme-linked DNA events, these events were indexed and the current level, duration and variance of the event recorded.
[00294] Using the MspA MS-(B1)8 pore, 31.08% of the detected events had a standard deviation >2.0 at an applied potential of +140 mV (additional data summarized in table 18). The experiment was repeated with the MS-(B1-L88N)8 mutant where only 12.38% of the detected events exhibited a standard deviation of >2.0 at an applied potential of +140 mV (additional data summarized in table 18). Therefore, the point mutation in L88 in the MspA monomer sequence has reduced the observed noise range by 19%. Table 18
Example 10 - Baseline Signal Noise Comparison of MS-(B1)8 with MS-(B1-L88N)8, MS-(B1-L88S)8 and MS-(B1-L88Q)8 Mutants
[00295] MspA pore noise levels can be altered by mutating the position of L88 in the protein. It has been shown that the noise level can be reduced as a result of producing a simple mutation in the MspA monomer.
This example compares the MS-(B1)8 portion with the MS-(B1-L88N)8, MS-(B1-L88S)8 and MS-(B1-L88Q)8 pores in open mode, using Phi29 DNA polymerase to control the movement of intact DNA strands through a nanopore. The design of the DNA substrate used in all the experiments described in this example is shown in Fig. 12 (SEQ ID NOs: 21, 22 and 23). SEQ ID NO: 23 was tagged with an IDT Int Spacer 9 (iSp9) and 3' Cholesteryl-TEG (3CholTEG) as shown below. Runs were conducted at room temperature at an applied potential of +180 mV. SEQ ID NO: 23: CAGCGATGGAGATAC/iSp9//3 Cho 1TEG/ Experimental Method Buffered solution: 400 mM KC1, 10 mM Hepes pH 8.0, 1 mM EDTA, 1 mM DTT Nanopores: MS(B1)8 MspA; MspA MS(B1-L88N)8; MS(B1-L88S)8 MspA; MS(B1-L88Q)8 MspA; Enzyme: Phi29 DNA polymerase SEQ ID NO: 4
[00297] Electrical measurements were acquired in the manner described in example 9. After obtaining a single pore of both MS(B1)8, MS(B1-L88N)8, MS(B1-L88S)8 and MS(B1-L88Q) 8 in the bilayer, DNA polynucleotide (SEQ ID NOs: 21, 22 and 23) and DNA polymerase Phi29 were added to 100 μL of buffer and pre-incubated for 5 minutes. This pre-incubation mixture was added to 900 μL of buffer in the cis compartment of the electrophysiology chamber to initiate capture of the polymerase-DNA complexes in the MspA nanopore (to give final DNA concentrations = 0.5 nM, Enzyme = 0.1 µM). Experiments were carried out at a constant potential of +180 mV. Current levels observed when the DNA is in the enzyme-bound state were indexed and the current level, its duration and variance were recorded.
[00298] In the experiments, the baseline mutant MS-(B1)8 exhibited high noise levels (76.15% standard deviations > 2.0, see table 19) at +180 mV. The other three mutants tested, (MS-(B1-L88N)8, MS-(B1-L88S)8 and MS-(B1-L88Q)8) that had a single point mutation at position L88, all observed more noise levels. lower (see Table 19) than the baseline pore with respect to the same strand of DNA sequence. Therefore, it was possible to reduce signal noise by applying point mutations at position L88 in MspA monomer sequence. Table 19
Example 11 - Comparison of the general baseline signal range of MS-(B1)8 with other MspA mutants
[00299] The MspA pore signal range can be increased by mutating several positions in the sequence of the MspA protein monomer.
This example compares the portion of MS-(B1)8 with the following pores - MS-(B1-D90Q)8, MS-(B1-I105L)8, MS-(B1-I105Y)8, MS-( B1-I89Y-D90S)8, MS-(B1-N86T)8 and MS-(B1-S103G)8 pores in aperture mode, using a Phi29 DNA polymerase to control the movement of intact DNA strands through a nanopore. The DNA substrate design, used in all the experiments described in this example, is shown in Fig. 12 (SEQ ID NOs: 21, 22, and 23). SEQ ID NO: 23, tagged with iSp9 and 3CholTEG is shown above. Runs were conducted at room temperature at an applied potential of +180 mV. Current levels observed when the DNA is in the enzyme-bound state were indexed and the current level, its duration and variance were recorded. Experimental Method Buffered solution: 400 mM KCl, 10 mM Hepes pH 8.0, 1 mM EDTA, 1 mM DTT Nanopores: MS(B1)8 MspA; MS(B1-D90Q)8 MspA; MS-(B1-I105L)8 MspA; MS-(B1-I105Y)8 MspA; MS-(B1-I89Y-D90S)8 MspA; MS-(B1-N86T)8 MspA; MS-(B1-S103G)8 MspA; Enzyme: Phi29 DNA polymerase SEQ ID NO: 4
[00301] Electrical measurements were acquired in the manner described in example 10. After obtaining a single pore both MS(B1)8, MS(B1-D90Q)8, MS(B1-I105L)8, MS(B1-I105Y)8 , MS-(B1-I189Y-D90S)8, MS-(B1-N86T)8 and MS-(B1-S103G)8 in the bilayer, DNA polynucleotide (SEQ ID NOs: 21, 22 and 23) and DNA polymerase Phi29 was added to 100 μL of buffer and pre-incubated for 5 minutes. This pre-incubation mix was added to 900 μL of buffer in the cis compartment of the electrophysiology chamber to initiate capture of the polymerase-DNA complexes in the MspA nanopore (to give final DNA concentrations = 0.5 nM, Enzyme = 0.1 µM). Experiments were carried out at a constant potential of +180 mV. Current levels observed when the DNA is in the enzyme-bound state were indexed and the current level, its duration and variance were recorded.
In the experiments, the MS-(B1) mutant from baseline 8 exhibited a maximum range of 35 pA at +180 mV (Table 20). The other 6 mutants tested (MS-(B1-D90Q)8, MS-(B1-I105L)8, MS-(B1-I105Y)8, MS-(B1-I89Y-D90S)8, MS-(B1-N86T) )8 and MS-(B1-S103G)8) all observed a greater maximum range than the baseline pore (See Table 20) with respect to the same strand of DNA sequence. Therefore, it was possible to increase the signal range by applying point mutations at various locations in the MspA monomer sequence. Table 20
Example 12 - Comparison of general baseline sequencing profile of MS-(B1)8 with other MspA mutants
The pore sequencing profile of MspA can be controlled by producing mutation at a variety of positions in the sequence of the MspA protein monomer.
This example compares the portion of MS-(B1)8 with MS-(B1-D90Q-D93S-I105A)8,MS-(B1-D90Q-Q126R)8, MS-(B1-L88N-D90Q-D91M )8, MS-(B1-L88N-D90Q-D91 S)8 and MS-(B1-G75S-G77S-L88N-Q126R)8 pores in translocation mode, using a helicase to control the movement of intact DNA strands through of a nanopore. Experimental Method Buffered solution: 400 mM NaCl, 10 mM Hepes pH 8.0, 1 mM ATP, 1 mM MgCl 2 , 1 mM DTT Nanopores: MS(B1)8 MspA; MS(B1-D90Q-D93S-I105A)8 MspA; MS(B1-D90Q-Q126R)8 MspA; MS(B1-L88N-D90Q-D91M)8 MspA; MS(B1-L88N-D90Q-D91S)8 MspA; MS(B1-G75S-G77S-L88N-Q126R)8 MspA; Enzyme: Helicase
[00305] The experimental setup was carried out in the manner described in example 9. After obtaining a single pore both of MS-(B1)8, MS-(B1-D90Q-D93S-I105A)8, MS-(B1-D90Q-Q126R ), MS-(B1-L88N-D90Q-D91M)8, MS-(B1-L88N-D90Q-D91 S)8 and MS-(B1-G75S-G77S-L88N-Q126R)8 in bilayer, DNA polynucleotide (SEQ ID NOs: 19 and 20 (sequence and tags shown above)) and helicase were added to 100 µL of buffer and pre-incubated for 5 minutes (DNA = 1.5 nM, Enzyme = 1 µM). This pre-incubation mix was added to 900 μL of buffer in the cis compartment of the electrophysiology chamber to initiate capture of helicase-DNA complexes in the MspA nanopore (to give final DNA concentrations = 0.15 nM, Enzyme = 0.1 µM). Helicase ATPase activity was initiated as required by the addition of divalent metal (1 mM MgCl 2 ) and NTP (1 mM ATP) to the cis compartment. Experiments were carried out at a constant potential of +140 mV. Current levels observed when the DNA was in the enzyme-bound state were indexed and the current level, its duration and variance were recorded.
[00306] In the experiments, the baseline MS-(B 1)8 mutant produced the sequencing profile shown in Fig. 13a. The experiment was repeated with the following mutants MS-(B1-D90Q-D93S-I105A)8, MS-(B1-D90Q-Q126R), MS-(B1-L88N-D90Q-D91M)8, MS-(B1-L88N -D90Q-D91S)8 and MS-(B1-G75S-G77S-L88N-Q126R)8, which exhibited a variety of different sequencing profiles (see Fig. 13 bf). Therefore, by producing point mutations at a variety of positions in the MspA monomer sequence it is possible to alter the sequencing profile that is detected. Example 13 - Analysis of an RNA strand sequence using the MS-(B1)8 baseline pore
This example describes how the baseline portion of MspA MS-(B1)8 combined with Phi29 DNA polymerase can be used to sequence an RNA strand.
[00308] This example uses the MS-(B1)8 pore in aperture mode, using a Phi29 DNA polymerase to control the movement of intact RNA strands through a nanopore. The RNA/DNA hybrid substrate design used in this experiment is shown in Fig. 14 (SEQ ID NOs: 24 and 25). SEQ ID NOs: 24 and 25 will be shown below (RNA in bold). Runs were conducted at room temperature at an applied potential of +180 mV. SEQ ID NO: 24: 5'OH-CCCCCCCCCCCCCCACCCCCCCCCCCCCCCCCCCUAUUCUG UUUAUGUUUCUUGUUUGUGU - 3'OH SEQ ID NO: 25 (plus cholesterol tag): 5'Phos-UAUUCUGUUUAUGUUUCUUGUUUGUUAGCCCUUUGA’UAAGACAAGhol Materials
[00309] In order to synthesize the RNA/DNA hybrid strand (120 mer in length), it was necessary to link SEQ ID NOs: 24 and 25 to each other. This was achieved by using the complementary DNA adapter strand SEQ ID NO: 26 to link the two strands in close proximity, where they were subsequently linked together forming SEQ ID NO: 27 120mer DNA/RNA hybrid. SEQ NO: 27 (plus cholesterol tag; RNA in bold): 5'OH-CCCCCCCCCCCCCCACCCCCCCCCCCCCCCCCCCUAUUCUG UUUAUGUUUCUGUUUGUUAUUCUGUUUAUGUUUCUUGUUUG UUAGCCCCCUUUGAUAGACAAAUACAAAGAACAAA Buffered 1 mM Knol Solution 1 mM, pH, 1 mM, pH, 1 mM, Buffered Solution, pH, 400, 1 mM, Buffered, 1 mM, pH, 8'Col, pH, 1 mM, Buffered Solution: 1 mM KonO : MS(B1)8 MspA; Enzyme: Phi29 DNA polymerase SEQ ID NO: 4
[00310] Electrical measurements were acquired as described in example 10. After obtaining a single pore of MS(B1)8 in the bilayer, DNA polynucleotide (SEQ ID NOs: 24 and 25) and DNA polymerase Phi29 were added to 100 μL of buffer and pre-incubated for 5 minutes. This pre-incubation mix was added to 900 μL of buffer in the cis compartment of the electrophysiology chamber to initiate capture of the polymerase-DNA complexes in the MspA nanopore (to give final DNA concentrations = 0.2 nM, Enzyme = 0.2 μM) . Experiments were carried out at a constant potential of +180 mV. Current levels were extracted as enzyme-linked DNA events, these events were indexed and the current level, duration and variance of the event recorded.
[00311] In the experiments, it was observed that the baseline MS-(B1)8 mutant, combined with Phi29 DNA polymerase as a molecular motor, detects distinct levels of current as the RNA strand was threaded through the pore . These current signals were then used to determine the target sequence. Typical RNA translocation events, in DNA polymerase Phi29 opening mode, are shown in Fig. 15. Example 14 - Dimer and oligomerization of MspA to form pores
[00312] This example describes the preparation and oligomerization of the MspA dimer. Dimer Preparation
NNNRRK MspA monomeric protein consists of 184 amino acid residues. A single polypeptide was designed to produce a dimeric version of the MspA-NNNRRK protein.
The DNA sequence encoding the 184 residue MspA-NNNRRK polypeptide was linked to a second DNA sequence encoding the identical polypeptide chain by means of a short DNA linker sequence. The DNA linker sequence encodes SGSGSGDDDDDDDDSGSGSS (SEQ ID NO: 33; shown as -(SG)3-D8-(SG)2(SS)-). An initiator codon (ATG) was added immediately before the first base and a DNA encoding two stop codons (TAATAG) was added after the last base. Therefore, the entire DNA sequence encoding MspA-NNNRRK-(SG)-D8-(SG)2(SS)-MspA-NNNRRK is shown in SEQ ID NO:28.
[00315] The DNA was synthesized by GenScript USA Inc and cloned into a pT7 vector for expression purposes.
[00316] The protein was generated by in vitro transcription and translation (IVTT) coupled using an E. coli T7-S30 circular DNA extract system (Promega).
[00317] The 1 mM complete amino acid minus cysteine mixture and the 1 mM complete minus methionine amino acid mixture were mixed in equal volumes to obtain the functional amino acid solution to generate high concentrations of the proteins. The amino acid mixture (2.5.0 μL), premix solution (10 μL), [35S]L-methionine (0.5 μL) and rifampicin (2 μL, 50 mg/mL) were mixed with DNA of the plasmid (4 μL,400 ng/mL) and T7 S30 extract (7.5 μL). Synthesis was performed for 90 minutes at 37°C to generate 25 μL of IVTT proteins for MspA-NNNRRK monomer and dimer. After the reaction, samples were centrifuged at 25,000 g for 10 minutes and the supernatant was discarded. The precipitate was washed with 100 µL of MBSA (10 mM MOPS, 150 mM NaCl, pH 7.4 containing 1 mg/mL BSA) and resuspended in 25 µL of Lamellae buffer sample. Samples were subjected to SDS-PAGE on a 10% gel. The gel was dried at 80°C for 45 minutes and exposed to X-ray film for 2 hours. The gel showed 2 distinct bands, one corresponding to the MspA dimer and one to the MspA monomer. Monomer and Dimer Oligomerization
[00318] Expression of the dimer and, separately, the monomer was performed in the presence of synthetic lipid vesicles to facilitate oligomerization. A five-component lipid blend was used (PS: SM: PE: PC: Cholesterol in ratio 10:10:20:30:30, 25 Mg/ml). 50 μL of lipid mixture was centrifuged at 25,000 g for 10 minutes in a 1.5 mL Eppendorf tube and the supernatant was discarded. The 1 mM complete amino acid minus cysteine mix and the 1 mM complete minus methionine amino acid mix were mixed in equal volumes to obtain the functional amino acid solution required to generate high concentrations of the proteins. The membrane precipitate was resuspended with a mixture of amino acid (10.0 μL), premix solution (40 μL), [3 S]L-methionine and rifampicin (2 μL, 50 mg/mL). Plasmid DNA (16 μL, 400 ng/mL) and T7 S30 extract (30.0 μL) were added to initiate synthesis. The synthesis was performed for 90 minutes at 37°C to generate 100 μL of IVTT protein. Sample from the IVTT reaction was centrifuged (25,000 g, 10 minutes) and the resulting membrane precipitate was washed with MBSA and subjected to SDS-polyacrylamide gel electrophoresis on a 7.5% gel. The gel was dried on a 3M watman paper at 50°C for 3 hours and exposed to X-ray film for 2 hours. The gel showed 8 distinct bands for the oligomerized MspA dimer, all of which migrated slower on SDS PAGE than the oligomerized monomer. Protein purification for bilayer experiments
[00319] Three bands of the protein dimer oligomerization experiment were excised from the gel and purified. Using the autoradiogram as the template, bands were cut and rehydrated in buffer (150 to 200 µL of 25 mM Tris.HCl, pH 8.0). The paper was removed and the piece of gel was crushed using a pestle. The slurry was filtered through a QIAshredder column (Qiagen) by centrifugation at 25,000 x g for 10 minutes. The protein resulting from the third band of the monomer level was then used in the electrophysiology experiments described in example 15. Example 15 - Comparison of MS-(B1)8 oligomerized from monomer with MS-(B1-BD4 oligomerized from Dimer
This example compares the pore of MS-(B1)8 oligomerized from the monomer (SEQ ID NO: 2) with the pore of MS-(B1-B1)4 oligomerized from the dimer (SEQ ID NO: 29 ) in translocation mode, using a helicase to control the movement of intact DNA strands (SEQ ID NOs: 19 and 20 (sequence and tags shown above)) through a nanopore. Experimental Method Buffered solution: 400 mM NaCl, 10 mM Hepes pH 8.0, 1 mM ATP, 1 mM MgCl 2 , 1 mM DTT Nanopores: MS-(B1)8; MS-(B1-B1)4 Enzyme: Helicase
[00321] Electrical measurements were acquired using 128-well silicon chips (format 75 μm in diameter, depth 20 μm and 250 μm pitch) that were plated with silver (WO 2009/077734). Chips were initially washed with 20 ml of ethanol, then 20 ml of dH2O, then 20 ml of ethanol before treatment with CF4 plasma. The used chips were then pretreated by dip coating, vacuum sealing and stored at 4°C. Before use the chips were naturally warmed to room temperature for at least 20 minutes.
Bilayers were formed by passing a series of 3.6 mg/mL lipid loads of 1,2-diphytanoyl-glycero-3-phosphocholine (DPhPC, Avanti Polar Lipids, AL, USA) dissolved in 1 M KCl, Tris 10 mM, pH 7.5, at 0.45 µL/β across the chip. Initially, a lipid load (250 µL) flowed through the chip, followed by a 100 µL air load. Two additional loads of 155 µL and 150 µL of lipid solution, each separated by a load of 100 µL of air, were then passed through the chip. After formation of the bilayer, the chamber was flushed with 3 mL of buffer at a flow rate of 3 μL/s. Electrical recording of the bilayer formation was performed at 10 kHz with an integration capacitance of 1.0 pF.
[00323] A biological nanopore solution was prepared using either the MS-(B1)8 pore oligomerized from the monomer or the MS-(B1-B1)4 pore oligomerized from the dimer in 10 mM Tris, EDTA 1 mM, pH 8.0. A holding potential of +180 mV was applied and the solution drained through the chip and the pores naturally entered the bilayers. The sampling rate and integration capacitance were then maintained at 10 kHz and 1.0 pF respectively and the applied potential reduced to zero.
[00324] A control program, which applied a maintenance potential of +180 mV, was run. DNA polynucleotide (SEQ ID NOs: 19 and 20) and helicase were pre-incubated for 5 minutes. This pre-incubation mix (which included MgCl2 and ATP) was then drained through the chip to initiate capture of helicase-DNA complexes in the MspA nanopore (to give final DNA concentrations = 1.5 nM, Enzyme = 10 nM) . Experiments were carried out at a constant potential of +180 mV. Current levels were extracted as DNA events in the enzyme-linked state. These events were indexed and the current level, duration and variance of the event recorded.
[00325] In the experiments, the baseline MS-(B1-B1)4 mutant pore formed from the oligomerization of the dimer inserted into the lipid bilayers as effectively as the MS(B1)8 pore formed from the oligomerization of the monomer (see Fig. 16 showing pore insertion for MS(B1)8 and MS-(B1-B1)4). When the oligomerized pores of the monomer and dimer were combined with a helicase as a molecular motor, it was possible to detect distinct levels of current as the strand of DNA was pushed through the pore. Typical translocation events, in helicase translocation mode, are shown in Fig. 17 for the MS-(B1)8 pore formed from monomer oligomerization and Fig. 18 for the MS-(B1-B1) pore 4 formed from the oligomerization of the dimer. Therefore, it was observed that the pore of the MS-(B1-B1)4 mutant oligomerized from the dimer unit was as good a pore as the pore of the MS-(B1)8 mutant oligomerized from the monomer unit. Example 16 - Use of the MspA pore of the MS-(B1-L88N)s mutant to distinguish 5-methylcytosine from cytosine
This example describes how the MspA portion of the MS- (B1-L88N)8 mutant can be used to distinguish cytosine from its epigenetically modified base 5-methylcytosine. The DNA substrate designs used in this experiment are shown in Fig. 19 and have the following sequences: TTTTTTTTT/idSp/TTTTTTTTmCTTTTTTTTCTTTTTTTT mCGTTTTTTTTCGTTTTTTTTGTATCTCCATCGCTGCCCCCTTTTTTTCC CCCTTTTT (which is SEQ ID NO: 30 with 9 T nucleotides Intpd in an ID 5' end). mC represents 5-methylcytosine GGCAGCGATGGAGATACTTGAGGCGAGCGGTCAA (SEQ ID NO: 31) and 5CholTEG/TTGACCGCTCGCCTC (SEQ ID NO: 32 with a 5' Cholesteryl-TEG tag). Materials
[00327] In order to form the DNA strand construct shown in Fig. 19 it was necessary to hybridize SEQ ID NO: 30, 31 and 32 together. This was accomplished by pre-incubating all three strands at the same time. Experimental Method Buffered solution: 1 M KCl, 10 mM Hepes pH 8.0, 1 mM ATP, 1 mM MgCl 2 , 1 mM DTT Nanopores: MspA MS(B1-L88N)8 Enzyme: Helicase
[00328] The experimental setup was performed as described in example 9. After obtaining a single pore of MS-(B1-L88N)8, in the bilayer, DNA polynucleotide (SEQ ID NOs: 30, 31 and 32) and helicase were added to 50 μL of buffer and pre-incubated for 5 minutes (DNA = 5 nM, Enzyme = 100 nM). This pre-incubation mix was added to 950 µL of buffer in the cis compartment of the electrophysiology chamber to initiate capture of helicase-DNA complexes in the MspA nanopore (to give final DNA concentrations = 5 nM, Enzyme = 100 nM). Helicase ATPase activity was initiated as required by the addition of divalent metal (1 mM MgCl 2 ) and NTP (1 mM ATP) to the cis compartment. Experiments were carried out at a constant potential of +120 mV. Current levels were extracted as DNA events in the enzyme-linked state. These events were indexed and the current level, duration and variance of the event recorded.
[00329] In the experiments it was observed that cytosine and 5-methylcytosine produced different levels of current when translocated through the pore of MS-(B1-L88N)8 under the control of a helicase (see Fig. 20). Therefore, using this mutated form of MspA it was possible to distinguish cytosine from its epigenetically modified base of 5-methylcytosine.

权利要求:
Claims (20)
[0001]
1. Mutant Msp monomer comprising a variant of the sequence shown in SEQ ID NO: 2, characterized in that the variant comprises the following substitution: L88N.
[0002]
2. Mutant Msp monomer according to claim 1, characterized in that the variant further comprises at least one of the following substitutions: G75S, G77S and Q126R.
[0003]
3. Mutant Msp monomer according to claim 2, characterized by the fact that the variant comprises the following mutations: G75S, G77S and Q126R.
[0004]
4. Mutant Msp monomer according to any one of claims 1 to 3, characterized by the fact that the variant further comprises at least one of the following mutations: (a) serine (S), glutamine (Q) or tyrosine (Y) in position 90; (b) leucine (L) or serine (S) at position 105; (c) arginine (R) at position 59; (d) leucine (L) at position 78; (e) asparagine (N) at position 81; (f) asparagine (N) at position 83; (g) serine (S) or threonine (T) at position 86; (h) phenylalanine (F), valine (V) or leucine (L) at position 87; (i) phenylalanine (F), valine (V) or leucine (L) at position 89; (j) 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; (k) 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; (1) alanine (A) or serine (S) at position 92; (m) serine (S), alanine (a), threonine (T), glycine (G) at position 93; (n) leucine (L) at position 94; (o) valine (V) at position 95; (p) arginine (R), aspartic acid (D), valine (V), asparagine (N), serine (S) or threonine (T) at position 96; (q) serine (S) at position 97; (r) serine (S) at position 98; (s) serine (S) at position 99; (t) serine (S) at position 100; (u) phenylalanine (F) at position 101; (v) lysine (K), serine (S) or threonine (T) at position 102; (w) alanine (A), glutamine (Q), asparagine (N), glycine (G) or threonine (T) at position 103; (x) isoleucine at position 104; (y) 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; (z) phenylalanine (F), isoleucine (I), valine (V) or serine (S) at position 106; (aa) proline (P) or serine (S) at position 108; (bb) asparagine (N) at position 118; (cc) serine (S) or cysteine (C) at position 103; and (dd) cysteine at one or more of positions 10 to 15, 51 to 60, 136 to 139 and 168 to 172, wherein the mutant Msp monomer retains the ability to form a pore.
[0005]
5. Mutant Msp monomer according to any one of claims 1 to 4, characterized in that the variant comprises one or more of the following substitutions: (a) serine (S) at position 75, serine (S) at position 77, asparagine (N) at position 88, glutamine (Q) at position 90 and arginine (R) at position 126; (b) one or more of (i) glutamine (Q) at position 90 and (ii) alanine (A) at position 105; (c) one or more of (i) serine (S) at position 90 and (ii) serine (S) at position 92; (d) one or more of (i) glutamine (Q) at position 87 and (ii) serine (S) at position 90; (e) one or more of (i) tyrosine (Y) at position 89 and (ii) serine (S) at position 90; (f) one or more of (i) serine (S) at position 90 and (ii) alanine (A) at position 92; (g) one or more of (i) serine (S) at position 90 and (ii) asparagine (N) at position 94; (h) one or more of (i) serine (S) at position 90 and (ii) isoleucine (I) at position 104; (i) one or more of (i) glutamine (Q) at position 90, (ii) serine (S) at position 93 and (iii) alanine (A) at position 105; (j) 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; (k) 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; (l) 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 ; (m) 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; and (n) cysteine at one or more of positions 90, 91 and 103, and wherein the mutant Msp monomer retains the ability to form a pore.
[0006]
6. Mutant Msp monomer according to any one of claims 1 to 5, characterized in that the variant comprises at least one of the following substitution(s): (i) N90S; (ii) N90Q; (iii) N90Y; (iv) I105L; (v) I105S; (vi) G75S, G77S, L88N, N90Q and Q126R; (vii) E59R; (viii) 178L; (ix) S81N; (x) T83N; (xi) N86S; (xii) N86T; (xiii) I87F; (xiv) I87V; (xv) I87L; (xvi) I89F; (xvii) I89V; (xviii) I89L; (xix) N90L; (xx) N91S; (xxi) N91Q; (xxii) N91L; (xxiii) N91M; (xxiv) N91I; (xxv) N91A; (xxvi) N91V; (xxvii) N91G; (xxviii) G92A; (xxix) G92S; (xxx) N93S; (xxxi) N93A; (xxxii) N93T; (xxxiii) 1994L; (xxxiv) T95V; (xxxv) A96R; (xxxvi) A96D; (xxxvii) A96V; (xxxviii) A96N; (xxxix) A96S; (x1) A96T; (xli) P97S; (xlii) P98S; (xliii) F99S; (xliv) G100S; (xlv) L101F; (xlvi) N102K; (xlvii) N102S; (xlviii) N102T; (xlix) S103A; (1) S103Q; (li) S103N; (lii) S103G; (liii) S103T; (liv) V104I; (lv) I105Y; (lvi) I105L; (lvii) I105A; (lviii) I105Q; (lix) I105N; (1x) I105S; (lxi) I105T; (lxii) T106F; (lxiii) T106I; (lxiv) T106V; (1xv) T106S; (lxvi) N108P; (lxvii) NO 108S; (lxviii) N90Q and I105A; (lxix) N90S and G92S; (1xx) I87Q and N90S; (1xxi) I89Y and N90S; (lxxii) N90S and G92A; (lxxiii) N90S and I94N; (lxxiv) N90S and V104I; (1xxv) N90Q, D93S and I105A; (1xxvi) N91Y; (1xxvii) N90Y and N91G; (lxxviii) N90G and N91Y; (lxxix) N90G and N91G; (lxxx) I105G; (lxxxi) N90R; (lxxxii) N91R; (lxxxiii) N90R and N91R; (lxxxiv) N90K; (lxxxv) N91K; (lxxxvi) N90K and N91K; (lxxxvii) N90Q and N91G; (lxxxviii) N90G and N91Q; (lxxxix) N90Q and N91Q; (xc) R118N; (xci) N91C; (xcii) N90C; (xciii) N90W; (xciv) N91W; (xcv) N90K; (xcvi) N91K; (xcvii) N90R; (xcviii) N91R; (xcix) N90S and N91S; (c) N90Y and I105A; (ci) N90G and I105A; (cii) N90Q and I105A; (ciii) N90S and I105A; (civ) N90G and N93G; (cv) N90G; (cvi) N93G; (cvii) N90G and N91A; (cviii) I105K; (cix) I105R; (cx) I105V; (cxi) I105P; (cxii) I105W; (cxiii) N90R and I105A; (cxiv) N90S and I105A; (cxv) S103C; and (cxvi) I105C and wherein the mutant Msp monomer retains the ability to form a pore.
[0007]
7. Mutant Msp monomer according to any one of claims 1 to 6, characterized in that: (i) the mutant is chemically modified by attaching a molecule to one or more cysteines, attaching a molecule to one or more lysines, attachment of a molecule to one or more unnatural amino acids, enzymatic modification of an epitope or modification of a terminus; (ii) the mutant is chemically modified by attaching a molecule to one or more cysteines and one or more cysteines have been introduced into the mutant by substitution; (iii) the mutant is chemically modified by attaching a molecule to one or more cysteines, attaching a molecule to one or more lysines, or attaching a molecule to one or more unnatural amino acids and the molecule is (a) a molecular adapter which facilitates the interaction between a pore comprising the monomer and a target nucleotide or target nucleic acid sequence or (b) a nucleic acid binding protein; (iv) the mutant is chemically modified by attaching a molecule to one or more cysteines, attaching a molecule to one or more lysines, or attaching a molecule to one or more unnatural amino acids and the attachment is via a linker or (v) the mutant is chemically modified by attaching a molecule to one or more cysteines, attaching a molecule to one or more lysines, or attaching a molecule to one or more unnatural amino acids and the molecule is attached to one or more of the positions 90, 91 and 103 of SEQ ID NO:2.
[0008]
8. Construct, characterized in that it comprises two or more covalently attached monomers derived from Msp, in which at least one of the monomers is a mutant monomer as defined in any one of claims 1 to 7.
[0009]
9. Construct according to claim 8, characterized in that (a) the two or more monomers are the same or different; (b) at least one monomer comprises the sequence shown in SEQ ID NO: 2; (c) the construct comprises two monomers and at least one of the monomers is a mutant monomer as defined in any one of claims 1 to 7; (d) the monomers are genetically fused; or (e) the monomers are attached via a linker.
[0010]
10. Polynucleotide, characterized in that it encodes a mutant as defined in claim 1 or a construct as defined in claim 8, wherein the polynucleotide is selected from the group consisting of SEQ ID NO: 1, wherein the codon for leucine at position 88 of the protein of SEQ ID NO: 2 is replaced by a codon for asparagine, and its degenerate sequences encoding said protein.
[0011]
11. Homo-oligomeric pore, characterized in that it is derived from Msp comprising identical mutant monomers as defined in any one of claims 1 to 7.
[0012]
12. Homo-oligomeric pore according to claim 11, characterized in that the pore comprises eight identical mutant monomers as defined in any one of claims 1 to 7.
[0013]
13. Hetero-oligomeric pore derived from Msp, comprising at least one mutant monomer, as defined in any one of claims 1 to 7, characterized in that at least one of the eight monomers differs from the others.
[0014]
14. Heterooligomeric pore according to claim 13, characterized in that (i) the pore comprises eight mutant monomers as defined in any one of claims 1 to 7 and at least one of them differs from the others; (ii) the pore comprises at least one monomer comprising the sequence shown in SEQ ID NO: 2; (iii) 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); or (iv) the pore comprises (a) seven monomers comprising the sequence shown in SEQ ID NO: 2 and a mutant monomer as defined in any one of claims 1 to 7, further comprising the substitution N90R, N90K, N90Y, N90Q, N90W or N90C; (b) seven monomers comprising the sequence shown in SEQ ID NO: 2 and a mutant monomer as defined in any one of claims 1 to 7 comprising the substitution N91R, N91K, N91Y, N91Q, N91W or N91C; or (c) seven monomers comprising the sequence shown in SEQ ID NO: 2 and a mutant monomer as defined in any one of claims 1 to 7 comprising the substitution L88C, S103C or I105C.
[0015]
15. Pore, characterized in that it comprises at least one construct as defined in claims 8 or 9.
[0016]
16. Pore according to claim 15, characterized in that it comprises - four constructs, each comprising two monomers and at least one of the monomers is a mutant as defined in any one of claims 1 to 7; or - a construct comprising a mutant monomer as defined in any one of claims 1 to 7 and 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 any one of claims 1 to 7.
[0017]
17. Method for characterizing a target nucleic acid sequence, characterized in that it comprises: (a) contacting the target sequence with a pore as defined in any one of claims 11 to 16 and a nucleic acid binding protein such that the protein controls movement of the target sequence through the pore and a proportion of the nucleotides in the target sequence interact with the pore; and (b) measuring the current passing through the pore during each interaction and thereby characterizing the target sequence.
[0018]
18. Method according to claim 17, characterized in that the characterization of the target nucleic acid sequence comprises estimating the sequence or sequencing the target nucleic acid sequence.
[0019]
19. Kit for characterizing a target nucleic acid sequence, characterized in that it comprises (a) a pore as defined in any one of claims 11 to 16 and (b) a nucleic acid handling enzyme.
[0020]
20. Apparatus for characterizing target nucleic acid sequences in a sample, characterized in that the apparatus comprises a chamber comprising an aqueous solution and a barrier that separates the chamber into two sections, wherein the barrier has an opening in which a membrane is formed and wherein a pore as defined in any one of claims 11 to 16 is inserted into the membrane.

类似技术:

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公开号 | 公开日 | 专利标题

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BR112013020411B1|2021-09-08|MUTANT MSP MONOMER, CONSTRUCT, POLYNUCLEOTIDE, PORE, KIT AND APPARATUS TO CHARACTERIZE A TARGET NUCLEIC ACID SEQUENCE, AND METHOD TO CHARACTERIZE A TARGET NUCLEIC ACID SEQUENCE

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同族专利:

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公开号 | 公开日

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JP2014506575A|2014-03-17|

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BR112013020411A2|2017-06-06|

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KR20140049511A|2014-04-25|

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KR101939420B1|2019-01-16|

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AU2012215135B9|2017-03-09|

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JP2017148052A|2017-08-31|

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EP2673638B1|2019-10-30|

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US20140186823A1|2014-07-03|

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法律状态:
2018-04-03| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-08-20| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2021-01-05| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2021-07-27| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-09-08| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 10/02/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161441718P| true| 2011-02-11|2011-02-11|

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US61/441718|2011-02-11|

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PCT/GB2012/050301|WO2012107778A2|2011-02-11|2012-02-10|Mutant pores|

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