Novel hole cellular and hole

Abstract

The present invention relates to novel pore monomer conjugates, pore complexes formed from the conjugates and their use in analyte detection and characterization.

Description

Technical Field

[0001] The present invention relates to novel pore monomer conjugates, pore complexes formed from the conjugates and their use in analyte detection and characterization. Background Art

[0002] Nanopore sensing is an analyte detection and characterization method that relies on the observation of individual binding or interaction events between analyte molecules and ion-conducting channels. Two of the essential components for analyte characterization using nanopore sensing are (1) controlling the movement of the analyte through the pore, and (2) discriminating the constituent building blocks as the analyte moves through the pore. During nanopore sensing, the narrowest portion of the pore forms the most discriminating portion of the nanopore with respect to the current signature as a function of the analyte passing through. CsgG was identified as a non-gated, non-selective protein secretion channel from Escherichia coli (Goyal et al., 2014) and has been used as a nanopore for the detection and characterization of analytes. Mutations of the wild-type CsgG pore that improve the properties of the pore in this context are also disclosed (WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317, WO 2017 / 149318, WO 2018 / 211241 and WO 2019 / 002893, which are incorporated herein by reference in their entirety).

[0003] For polynucleotide analytes, nucleotide discrimination is achieved by measuring the current as the polynucleotide passes through the pore. Multiple nucleotides contribute to the observed current, so the height of the channel constriction and the extent of interaction with the polynucleotide affect the relationship between the observed current and the polynucleotide sequence. Although the current range and signal-to-noise ratio of nucleotide discrimination have been improved by mutations in the CsgG pore, if the current difference between nucleotides can be further improved, the sequencing system will have higher performance. Therefore, it is necessary to identify new ways to improve the sensing characteristics of nanopores. Summary of the invention

[0004] The inventors surprisingly showed that pore complexes formed from pore monomer conjugates exhibited increased current range and / or increased signal-to-noise ratio (SNR) during analyte characterization, wherein a CsgG pore monomer is attached to a CsgF peptide via a specific linker. The inventors also surprisingly showed that pore complexes formed from pore monomer conjugates exhibited increased current range and increased signal-to-noise ratio (SNR) during analyte characterization, wherein a loop region in a CsgG pore monomer is attached to a CsgF peptide. Both the increased current range and the increased SNR improve the ability to discriminate analytes as they pass through the pore. Neither the improvement in range nor the improvement in SNR could have been predicted from previous experiments using CsgG and CsgF. Thus, the present invention provides a pore monomer conjugate comprising a CsgG pore monomer attached to a CsgF peptide, wherein the CsgF peptide is attached to the CsgG pore monomer via a sulfonyl fluoride-containing linker, a sulfonyl triazole-containing linker, a fluorosulfate-containing linker, or a fluoroacetamide-containing linker. The present invention also provides a pore monomer conjugate comprising a CsgG pore monomer attached to a CsgF peptide, wherein the CsgF peptide is attached to a residue in the CsgG pore monomer corresponding to any one of positions 143, 146, 190, 192, 193, 194, 195, 196, 197, 198, 199 or 200 in SEQ ID NO:3.

[0005] The present invention also provides:

[0006] - a construct comprising two or more covalently attached pore monomer conjugates of the invention;

[0007] - a pore complex comprising at least one pore monomer conjugate of the invention or at least one construct of the invention, wherein the CsgF peptide forms a constriction in the pore complex;

[0008] - a pore multimer comprising two or more pores, wherein at least one of the pores is a pore complex of the invention;

[0009] - a membrane comprising a pore complex according to the invention or a pore polymer according to the invention;

[0010] - a method for producing a pore monomer conjugate of the invention, the method comprising attaching a CsgF peptide to a cysteine ​​residue in a CsgG pore monomer;

[0011] - a method for producing a pore complex of the invention or a pore multimer of the invention, the method comprising expressing at least one pore monomer conjugate of the invention or a construct of the invention and sufficient pore monomer or construct to form the pore complex or pore multimer in a host cell, and allowing the pore complex or pore multimer to form in the host cell;

[0012] - a method for producing a pore complex of the invention or a pore multimer of the invention, the method comprising contacting at least one pore monomer conjugate of the invention or a construct of the invention with sufficient pore monomer or construct in vitro and allowing the pore complex or pore multimer to form;

[0013] - A method for determining the presence, absence or one or more characteristics of a target analyte, the method comprising the steps of:

[0014] (i) contacting a target analyte with a pore complex of the invention or a pore multimer of the invention, such that the target analyte moves relative to the pore complex or the pore multimer; and

[0015] (ii) making one or more measurements as the analyte moves relative to the pore complex or pore multimer and thereby determining the presence, absence or one or more characteristics of the analyte.

[0016] - use of a pore complex of the invention or a pore multimer of the invention for determining the presence, absence or one or more characteristics of a target analyte;

[0017] - a polynucleotide encoding a pore monomer conjugate of the invention or a construct of the invention;

[0018] - a kit for characterizing a target analyte, the kit comprising (a) a pore complex according to the invention or a pore multimer according to the invention and (b) components of a membrane;

[0019] - A kit for characterizing a target polynucleotide or a target polypeptide, the kit comprising (a) a pore complex of the invention or a pore multimer of the invention and (b) a polynucleotide binding protein;

[0020] - A device for characterising a target polynucleotide or target polypeptide in a sample, the device comprising (a) a plurality of pore complexes of the invention or a plurality of pore multimers of the invention and (b) a plurality of polynucleotide binding proteins;

[0021] - an array comprising a plurality of membranes according to the invention;

[0022] - A system comprising (a) a membrane according to the invention or an array according to the invention, (b) means for applying an electric potential across the membrane, and (c) means for detecting an electric or optical signal across the membrane;

[0023] - a device comprising a pore complex according to the invention or a pore multimer according to the invention inserted into an extracorporeal membrane; and

[0024] - A device produced by a method comprising the steps of: (i) obtaining a pore complex of the invention or a pore multimer of the invention, and (ii) contacting the pore complex or pore multimer with an extracorporeal membrane such that the pore complex or pore multimer inserts into the extracorporeal membrane. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 : General workflow for cross-linking with sulfonyl fluoride linkers. For example, the CsgF peptide is functionalized with a sulfonyl fluoride linker on Lys30. When the functionalized CsgF is in close proximity to the CsgG pore, the OH group on Y196 in the CsgG pore will replace the fluorine group to covalently link the CsgG pore to the CsgF peptide.

[0026] Figure 2 : Possible examples of functionalized CsgF peptides. A: CsgF peptide with a fluorosulfate linker. BE: CsgF peptide with an arylsulfonyl fluoride linker. FG: CsgF peptide with an alkylsulfonyl fluoride linker. HL: CsgF peptide with an arylsulfonyl fluoride linker. MN: CsgF peptide with a fluoroacetamide linker. Representative reagents and chemistries used for functionalization are as follows: for A, amide bond formation with 3-[(fluorosulfonyl)oxy]benzoic acid (m-OSO2F); for B, 4-(fluorosulfonyl)phenyl]acetic acid (CH2PH-P-SO2F); for C, 4-(fluorosulfonyl)benzoic acid (p-SO2F); for D, 3-(fluorosulfonyl)benzoic acid (m-SO2F); for E, 4-(2-aminoethyl)benzene-1-sulfonyl fluoride; for M, 4-(2-fluoroacetamido)benzoic acid; for N,3-(2-fluoroacetamido)benzoic acid; for F, 1,4-addition of ethylenesulfonyl fluoride. For G, cycloaddition of azide-alkyne with 2-[(prop-2-yn-1-yl)oxy]ethane-1-sulfonyl fluoride. For HL, amide bond formation followed by synthesis of sulfonyltriazoles (such as WO 2020 / 214336, which is incorporated herein by reference in its entirety); for H, substituted 3-phenyl-1H-1,2,4-triazole and 4-(chlorosulfonyl)benzoyl chloride (X=H, OMe, CN, Br, Ph or CF3); for I, substituted 3-phenyl-1H-1,2,4-triazole and 3-(chlorosulfonyl)benzoyl chloride (X=H, OMe, CN, Br, Ph or CF3); for J, 1H-1,2,4-triazole and 4-(chlorosulfonyl)benzoyl chloride; for K, 3-(pyridin-3-yl)-1H-1,2,4-triazole and 4-(chlorosulfonyl)benzoyl chloride; for L; 3-(pyridin-3-yl)-1H-1,2,4-triazole and 3-(chlorosulfonyl)benzoyl chloride. 1,2,4-triazole may be replaced by 1,2,3-triazole.

[0027] Figure 3: SDS-PAGE gel analysis of well controls of CsgG only and CsgG / CsgF complexes as they dissociate into their constituent monomer components upon boiling in the presence of DTT. Lane 2 shows no band shift compared to the CsgG only control in lane 1. This therefore means that the CsgG monomer does not remain bound to the CsgF peptide upon heating in the absence of sulfonyl fluoride. Lanes 3, 4, and 6 show band shifts compared to the CsgG only control in lane 1, indicating a covalent linkage between CsgG and CsgF. In lane 5, most of the band is at the same height as the CsgG only control in lane 1, indicating that CsgF does not remain bound to CsgG, however electrophysiological recordings indicate that the sample is mostly complex and therefore it is believed that the C-terminal CsgF-meta-OSO2F is hydrolyzed upon boiling in DTT. Legend:

[0028] 1.CsgG-F56Q:ONLP20134

[0029] 2.CsgG-F56Q / CsgF-del(S31-F119): ONLP20783

[0030] 3. CsgG-F56Q / CsgF-K30-m-SO2F-del(S31-F119):ONLP20784

[0031] 4.CsgG-F56Q / CsgF-K30-CH2-PH-P-SO2F-del(S31-F119): ONLP20822

[0032] 5. CsgG-F56Q / CsgF-K30-inter-OSO2F-del (S31-F119): ONLP20820

[0033] 6. CsgG-F56Q / CsgF-K30-p-SO2F-del(S31-F119):ONLP20821

[0034] Figure 4: Bar graph showing classification of wells inserted into a minION flow cell. In the absence of CsgF peptide, the majority of wells are CsgG-only wells. Note that a small number of wells are misclassified as CsgG / CsgF complexes. For the well sample with del(S31-F119)CsgF peptide, approximately half of the channels do not retain the CsgF peptide in the absence of any covalent attachment chemistry. However, a high proportion of CsgG / CsgF complexes are observed when the well complex contains CsgF functionalized with sulfonyl fluoride. The thresholds used to classify the inserted well types are as follows: CsgG-only wells = wells with an opening current between 160pA and 200pA; CsgG / CsgF complexes = wells with an opening current between 70pA and 140pA. Both classifications also have an opening noise of <18pA.

[0035] Figure 5 : Bar graph showing the average classification of wells inserted into the minION flow cell. Data are Figure 4 Average values ​​of runs (flow cell) of the same wells shown in .

[0036] Figure 6 : Snapshot of a run report showing ionic current (pA) versus time (s) as ssDNA translocates through a CsgG-only pore. Each individual graph corresponds to a single pore inserted into a minION flow cell. At an applied voltage of -180 mV, the open pore current observed for these CsgG-only pores was approximately 170 pA to 200 pA.

[0037] Figure 7 : Snapshot of a run report showing ionic current (pA) versus time (s) as ssDNA translocates through CsgG containing del(S31-F119) CsgF peptide plus / minus sulfonyl fluoride (p-SO2F). Each individual plot corresponds to a single pore inserted into a minION flow cell. The open pore current observed for the CsgG / CsgF complex is approximately 100 pA at an applied voltage of -180 mV. Note that the pore complex without sulfonyl fluoride is not stable and therefore the CsgF peptide is not retained within CsgG in all channels, e.g. channel 177 is only the pore, while channel 179 is a CsgG / CsgF complex. However, when the complex contains the sulfonyl fluoride peptide variant, it can be seen that stability is improved and the peptide is retained within CsgG, i.e. channel 165 is a CsgG / CsgF complex.

[0038] Figure 8: Snapshot of a run report showing ionic current (pA) versus time (s) for translocation of ssDNA through CsgG containing del(S31-F119) CsgF peptide with sulfonyl fluoride (meta-OSO2F or CH2PH-P-SO2F peptide variants). Each individual graph corresponds to a single pore inserted into a minION flow cell. At an applied voltage of -180 mV, the observed open pore current for the CsgG / CsgF complex was approximately 100 pA.

[0039] Fig. 9 : Snapshot of a run report showing ionic current (pA) versus time (s) as single-stranded DNA translocates through CsgG containing the del(S31-F119)CsgF peptide with a sulfonyl fluoride, m-SO2F. Each individual graph corresponds to a single pore inserted into a minION flow cell. At an applied voltage of -180 mV, the open pore current observed for the CsgG / CsgF complex was approximately 100 pA.

[0040] Fig.10 : Traces of ionic current (pA) versus time (s) as single-stranded DNA translocates through a CsgG-only pore. The original current trace is shown in black, and the event detection signal is shown in red. For each pore, the top row shows the full DNA current trace, while the bottom row shows a zoomed-in view of the first portion of the current trace.

[0041] Fig.11 : Traces of ionic current (pA) versus time (s) as single-stranded DNA translocates through CsgG containing del(S31-F119)CsgF peptide plus / minus sulfonyl fluoride (p-SO2F). The original current trace is shown in black, and the event detection signal is shown in red. For each well, the top row shows the full DNA current trace, while the bottom row shows a magnified view of the first portion of the current trace.

[0042] Fig.12 : Traces of ionic current (pA) versus time (s) when single-stranded DNA is translocated through CsgG containing del(S31-F119)CsgF peptide with sulfonyl fluoride (meta-OSO2F or CH2PH-P-SO2F peptide variants). The original current trace is shown in black and the event detection signal is shown in red. For each well, the top row shows the full DNA current trace, while the bottom row shows a magnified view of the first portion of the current trace.

[0043] Fig.13: Traces of ionic current (pA) versus time (s) as single-stranded DNA translocates through CsgG containing del(S31-F119)CsgF peptide with sulfonyl fluoride, m-SO2F. The original current trace is shown in black, and the event detection signal is shown in red. For each well, the top row shows the full DNA current trace, while the bottom row shows a zoomed-in view of the first portion of the current trace.

[0044] Fig.14 : Box plots showing signal metrics for CsgG-based pores. SNR is the signal-to-noise ratio, which is the range of the ionic current divided by the noise when single-stranded DNA translocates through the pore. The SNR increases in the presence of the sulfonyl fluoride peptide variants, e.g., the median SNR and range of CsgG-WT-F56Q-Q153C / CsgF-WT-K30-meta-SO2F-del(S31-F119) are higher than those of CsgG-WT-F56Q / CsgF-WT-del(S31-F119).

[0045] Fig.15 Figure 3: Structure and size of the wild-type CsgG pore from Escherichia coli strain K12 (database accession code for this structure is 4UV3). The distances shown are measured from backbone to backbone of the amino acids that form the pore structure. The CsgG pore is a tightly interconnected, symmetrical non-polymeric pore that resembles a crown. The total height is And the maximum outer diameter is It defines a central channel and consists of three parts: (A) the cap region, (B) the constriction region, and (C) the transmembrane β-barrel region. The axial length or height of the cap is Its inner diameter is Opening The β barrel has 36 strands and an axial length of And the inner diameter is At the level of the predicted lipid-water interface, the transition between the pore cap and the β-barrel is clear, as is the constriction located between them. The diameter of the constriction is approximately And along the axis of the channel Length.

[0046] Fig.16 .Examples of amino acid residues having reactive groups.

[0047] Fig.17 : (A) Extracellular view of the CsgG:CsgF complex (based on PDB ID: 6L7C). (B) Orientation of the CsgG:CsgF complex within the bacterial membrane. (C) Schematic illustration of the cross-linking between nucleophilic residues on CsgG and SuTide via the proximity-enhanced SuFEx reaction. (D) (1-35)The C-terminal residues of the CsgG target nucleophile Tyr196 α -C α (E) Structures of three SuTide warheads; 3-SO2F-CsgF, 4-SO2F-CsgF, and 4-CH2SO2F-CsgF.

[0048] Fig.18 : (A) Representative SDS-PAGE gel showing the time-dependent formation of complexes between each SuTide and CsgG. The gel shows a gradual decrease in CsgG monomers (corresponding to an apparent molecular weight of 35 kDa), accompanied by a corresponding increase in the intensity of a single covalent CsgG:SuTide complex band over time (corresponding to an apparent molecular weight of 38 kDa). (B) Detailed listing of the time-dependent formation of each CsgG:SuTide complex 1 / 2 Table of contents.

[0049] Fig.19 : (A) Top: Structure of 3-SO2F-CsgF with Tyr196 showing the angle q between the introduced phenolic oxygen and the fluoride leaving group O,S,F As well as the introduction of phenolic oxygen and sulfur O,S Bottom: Examples of 3-SO2F-CsgF with Tyr196 in the top cluster taken from three independent MD simulations of this probe. (B) Top: d O,S Binned radial distribution of . Values ​​are the average of three independent runs. The minimum observed d O,S for Bottom: Alignment of all Tyr196 residues from three independent MD simulations and the respective positions of each sulfur atom in 3-SO2F-CsgF. (C) The apparent molar concentration between each distance bin (C 表观 ). These are calculated by computing the binned radial distribution of the probability per unit volume described in B (D)d O,S distance The binning q of each probe is O,S,F Angle graph. Values ​​are the average of three independent runs. (E) d for each probe O,S With q O,S,F 2D plot of . Values ​​are the average of three independent runs.

[0050] Description of Sequence Listing

[0051] SEQ ID NO: 1 shows the polynucleotide sequence of wild-type E. coli CsgG from strain K12, including the signal sequence (Gene ID: 945619).

[0052] SEQ ID NO: 2 shows the amino acid sequence of wild-type E. coli CsgG, including the signal sequence (Uniprot accession number P0AEA2).

[0053] SEQ ID NO: 3 shows the amino acid sequence of wild-type E. coli CsgG as a mature protein (Uniprot Accession No. P0AEA2).

[0054] SEQ ID NO:4 shows the polynucleotide sequence of wild-type E. coli CsgF from strain K12, including the signal sequence (Gene ID: 945622).

[0055] SEQ ID NO:5 shows the amino acid sequence of wild-type E. coli CsgF, including the signal sequence (Uniprot accession number P0AE98).

[0056] SEQ ID NO: 6 shows the amino acid sequence of wild-type E. coli CsgF as a mature protein (Uniprot accession number P0AE98).

[0057] SEQ ID NO:7 shows the synthetic construct used in Example 1. DETAILED DESCRIPTION

[0058] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. All publications, patents and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. To the extent that publications and patents or patent applications incorporated by reference are inconsistent with the present invention contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0059] The present invention will be described with respect to specific embodiments and with reference to certain figures, but the present invention is not limited thereto and is limited only by the claims. Any reference numerals in the claims should not be construed as limiting the scope. Of course, it should be understood that according to any one particular embodiment of the present invention, not all aspects or advantages need be achieved. Thus, for example, one skilled in the art will recognize that the present invention can be implemented or performed in a manner that achieves or optimizes one advantage or a group of advantages taught herein without having to achieve other aspects or advantages taught or suggested herein.

[0060] In addition, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "polynucleotide" includes two or more polynucleotides, reference to a "polynucleotide binding protein" includes two or more such proteins, reference to a "helicase" includes two or more helicases, reference to a "monomer" refers to two or more monomers, reference to a "pore" includes two or more pores, etc.

[0061] In all discussions herein, the standard single letter codes for amino acids are used. They are as follows: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine (V). Standard substitution symbols are also used, i.e., Q42R means that the Q at position 42 is replaced by R.

[0062] In the paragraphs of this article where different amino acids at a particular position are separated by a / symbol, the / symbol means "or". For example, Q87R / K means Q87R or Q87K. In the paragraphs of this article where different positions are separated by a / symbol, the / symbol means "and", such that Y51 / N55 is Y51 and N55.

[0063] The general definitions in WO 2019 / 002893 are incorporated herein by reference in their entirety.

[0064] Pore ​​Monomer Conjugate

[0065] The present invention provides a pore monomer conjugate comprising a CsgG pore monomer attached to a CsgF peptide. The CsgG pore monomer is preferably covalently attached to the CsgF peptide. Suitable CsgG pore monomers and CsgF peptides are described in more detail below.

[0066] In one embodiment, the CsgF peptide is attached to the CsgG pore monomer via a sulfonyl fluoride-containing linker, a sulfonyltriazole-containing linker, a fluorosulfate-containing linker, or a fluoroacetamide-containing linker.

[0067] The linker can be any linker discussed below with reference to the constructs of the invention. The linker preferably comprises or consists of: (a) a sulfonyl fluoride, sulfonyl triazole, fluorosulfate or fluoroacetamide group and (b) a linear carbon chain of 2, 3, 4, 5, 6 or more carbon atoms and / or a saturated or unsaturated cyclic group containing 3, 5 or 6 carbon atoms. Any linker can be used, including the linkers used in the Examples (see Figure 1 and 2 ).

[0068] The linker is preferably a linker containing sulfonyl fluoride. The linker is preferably CH2PH-P-SO2F, p-SO2F, m-SO2F, 4-(2-aminoethyl)benzene-1-sulfonyl fluoride, ethylenesulfonyl fluoride, or 2-[(prop-2-yn-1-yl)oxy]ethane-1-sulfonyl fluoride.

[0069] The linker is preferably a sulfonyltriazole-containing linker. The linker is preferably a substituted 4-(3-phenyl-1H-1,2,4-triazole-1-sulfonyl)benzoic acid (preferably Figure 2 H in which X=H, OMe, CN, Br, Ph or CF3), substituted 3-(3-phenyl-1H-1,2,4-triazole-1-sulfonyl)benzoic acid, 4-(1H-1,2,4-triazole-1-sulfonyl)benzoic acid, 4-[3-(pyridin-3-yl)-1H-1,2,4-triazole-1-sulfonyl]benzoic acid, or 3-[3-(pyridin-3-yl)-1H-1,2,4-triazole-1-sulfonyl]benzoic acid.

[0070] The linker is preferably a fluorine-containing sulfate linker. The linker is preferably m-OSO2F.

[0071] The linker is preferably a fluoroacetamide-containing linker. The linker is preferably 4-(2-fluoroacetamido)benzoic acid or 3-(2-fluoroacetamido)benzoic acid.

[0072] The linker may be a residue in the CsgF peptide and / or the CsgG pore monomer that is modified to include a sulfonyl fluoride, sulfonyl triazole, fluorosulfate or fluoroacetamide group. The CsgF peptide is preferably covalently attached to the CsgG pore monomer via a linker.

[0073] The reactive groups in the linker can react with residues in the CsgF peptide and / or the CsgG pore monomer to attach the two together, preferably covalently. The linker may comprise two reactive groups, such as two sulfonyl fluoride groups. One can react with the CsgF peptide and the other can react with the CsgG pore monomer. The reactive groups in the linker preferably react with serine, lysine, threonine, tyrosine, histidine or cysteine ​​residues in the CsgF peptide and / or the CsgG pore monomer. These residues may be native residues in the CsgF peptide and / or the CsgG pore monomer. The residues may be introduced into the CsgF peptide and / or the CsgG pore monomer, preferably by substitution or addition.

[0074] SEQ ID NO: 6 shows the amino acid sequence of wild-type E. coli CsgF as a mature protein. The N-terminus or a residue at any one of positions 1 to 40 (such as position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40) in the CsgF peptide is preferably attached to the CsgG pore monomer via a linker. The residue at any one of positions 23 to 40 in the CsgF peptide, such as position 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, is preferably attached to the CsgG pore monomer via a linker. The residue at any one of positions 29 to 35 in the CsgF peptide, such as position 29, 30, 31, 32, 33, 34 or 35, is preferably attached to the CsgG pore monomer via a linker.

[0075] The N-terminus of the CsgF peptide or a residue corresponding to any one of positions 1 to 40 in the CsgF peptide, such as position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40, is preferably attached to the CsgG pore monomer via a linker. The N-terminus or a residue corresponding to any one of positions 1 to 40 in SEQ ID NO: 6 (such as position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40) in the CsgF peptide is preferably attached to the CsgG pore monomer via a linker. The residue corresponding to any one of positions 23 to 40 in SEQ ID NO: 6 (such as corresponding to position 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40) in the CsgF peptide is preferably attached to the CsgG pore monomer via a linker. The residue corresponding to any one of positions 29 to 35 in SEQ ID NO: 6 (such as corresponding to position 29, 30, 31, 32, 33, 34 or 35) is preferably attached to the CsgG pore monomer via a linker.

[0076] The residue at position 30 of the CsgF peptide, or a residue at a position in the CsgF peptide corresponding to position 30 in SEQ ID NO: 6, is more preferably attached to the CsgG pore monomer via a linker.

[0077] The following table shows additional positions in the CsgF peptide that are preferably attached to the CsgG pore monomer via a linker. The positions in the left column can be positions in the CsgF peptide or positions in SEQ ID NO:6 that correspond to preferred positions. The right column shows preferred positions in the CsgG pore monomer to which the positions in the left column are preferably attached via a linker. The CsgF peptide can be attached to any of the positions in the right column via a linker.

[0078]

[0079]

[0080]

[0081] SEQ ID NO:3 shows the amino acid sequence of wild-type E. coli CsgG as a mature protein. The CsgF peptide is preferably attached to residues in the loop-forming region of the CsgG pore monomer. The loop-forming region corresponds to positions 142-146 and 190-200 in SEQ ID NO:3. The CsgF peptide is preferably attached to residues corresponding to positions 142-146 and 190-200 in SEQ ID NO:3. The residue preferably corresponds to position 142, 143, 144, 145, 146, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199 or 200 in SEQ ID NO:3. Additional preferred positions in SEQ ID NO:3 are shown in the right column of the above table.

[0082] The CsgF peptide is preferably attached to a serine, lysine, threonine, tyrosine, histidine or cysteine ​​residue in the CsgG pore monomer via a linker. The sulfonyl fluoride group is capable of reacting with any of these residues. The sulfonyl fluoride group in the linker preferably reacts with a serine, lysine, threonine, tyrosine, histidine or cysteine ​​residue in the CsgG pore monomer. These residues may be natural residues in the CsgG pore monomer. The residues may preferably be introduced into the CsgG pore monomer by substitution or addition.

[0083] In another embodiment, the CsgF peptide is attached to a residue in the CsgG pore monomer corresponding to any of positions 143, 146, 190, 192, 193, 194, 195, 196, 197, 198, 199 or 200 in SEQ ID NO: 3. The CsgF peptide is more preferably attached to a residue in the CsgG pore monomer corresponding to position 196 in SEQ ID NO: 3. The CsgF peptide may be attached, preferably covalently attached, to any of these positions using a linker. The linker may be any of those described above and below. The linker may comprise a sulfonyl fluoride group, a sulfonyltriazole group, a fluorosulfate group or a fluoroacetamide group.

[0084] The residue corresponding to any of positions 143, 146, 190, 192, 193, 194, 195, 196, 197, 198, 199 or 200 in SEQ ID NO: 3 is preferably a serine, lysine, threonine, tyrosine, histidine, cysteine ​​or threonine residue. Any of these residues may be a natural residue. Any of these residues may be introduced into the CsgG pore monomer, preferably by substitution or addition.

[0085] The residue corresponding to any of positions 143, 146, 190, 192, 193, 194, 195, 196, 197, 198, 199 or 200 in SEQ ID NO: 3 is preferably modified with a reactive group. Any of the reactive groups described above or below can be used. For example, the residue is preferably a derivative of diaminopropionic acid, diaminobutyric acid, ornithine, lysine (Lys), homo-Lys, p-aminophenylalanine, p-aminophenylglycine, α-methyllysine or 1,4-diaminocyclohexane-1-carboxylic acid, in which the side chain amino group is covalently attached to a reactive group (e.g., α-chloroacetamide) with or without a linker. Examples include Fig.16 shown.

[0086] The CsgF peptide is preferably attached to the CsgG pore monomer using any reactive group capable of reacting with a residue corresponding to any of positions 143, 146, 190, 192, 193, 194, 195, 196, 197, 198, 199 or 200 in SEQ ID NO: 3, preferably a residue introduced at any of these positions by substitution or addition, or a reactive group introduced at any of these positions.

[0087] The reactive group is preferably an amine reactive group such as a thioester, NHS-ester, pentafluorophenyl ester, benzyl halide, sulfonyl fluoride, fluorosulfate or sulfonyltriazole. Suitable sulfonyl-triazole groups have been discussed above and are shown in Figure 2 The reactive group is preferably an amine reactive group such as a thioester, NHS-ester, pentafluorophenyl ester, benzyl halide, benzyl fluoride, sulfonyl halide, sulfonyl fluoride, halosulfate, fluorosulfate, sulfonyltriazole or boronic acid. Suitable sulfonyl-triazole groups have been discussed above and are shown in Figure 2 In (see HL).

[0088] The reactive group is preferably an oxygen reactive group, such as an alkyl halide, sulfonyl fluoride, fluorosulfate or sulfonyl triazole. The alkyl halide is preferably a chloromethyl ketone. The reactive group is preferably an oxygen reactive group, such as an alkyl halide, alkyl fluoride, sulfonyl halide, sulfonyl fluoride, halosulfate, fluorosulfate or sulfonyl triazole. The alkyl halide is preferably a chloromethyl ketone.

[0089] The reactive group is preferably a fluoroacetamide group. Suitable fluoroacetamide groups have been discussed above and are shown in Figure 2 Medium (see M and N).

[0090] Additional reactive groups are described in Nature Chemistry, 2021, 13, 1081-1092, Cell Chemical Biology, 2020, 27, 970-985, J. Am. Chem. Soc. 2019, 141, 7, 2782–2799, and Current Opinion in Chemical Biology, 2015, 18-26 (each incorporated herein by reference in its entirety).

[0091] Any residue in the CsgF peptide may be attached to any of the residues at positions corresponding to 143, 146, 190, 192, 193, 194, 195, 196, 197, 198, 199 or 200 in SEQ ID NO: 3. The residue in CsgF is preferably any of those discussed above with reference to the sulfonyl fluoride, sulfonyl triazole, fluorosulfate and fluoroacetamide embodiments of the invention.

[0092] The distance between the CsgG pore monomer and the CsgF peptide in the pore monomer conjugate and / or the length of the linker is preferably less than about 3.00 nm, such as less than about 2.90 nm, less than about 2.80 nm, less than about 2.70 nm, less than about 2.60 nm, less than about 2.50 nm, less than about 2.40 nm, less than about 2.30 nm, less than about 2.20 nm, less than about 2.10, less than about 2.00 nm, less than about 1.90 nm, less than about 1.80 nm, less than about 1.70 nm, less than about 1.60 nm, less than about 1.50 nm, less than about 1.40 nm, less than about 1.30 nm, less than about 1.20 nm, less than about 1.10 nm, less than about 1.00 nm, less than about 0.90 nm, less than about 0.80 nm, less than about 0.70 nm, less than about 0.60 nm, less than about 0.50 nm, or less than about 0.40 nm.

[0093] The distance between the CsgG pore monomer and the CsgF peptide in the pore monomer conjugate and / or the length of the linker is preferably from about 0.40 nm to about 3.00 nm, such as from about 0.45 nm to about 2.80 nm, from about 0.50 nm to about 2.50 nm, from about 0.55 nm to about 2.20 nm, from about 0.60 nm to about 2.00 nm, from about 0.65 nm to about 1.50 nm, from about 0.70 nm to about 1.40 nm, from about 0.75 nm to about 1.30 nm, from about 0.80 nm to about 1.20 nm, from about 0.85 nm to about 1.10 nm, and from about 0.90 nm to about 1.00 nm. The distance between the CsgG pore monomer and the CsgF peptide in the pore monomer conjugate and / or the length of the linker is preferably from about 0.50 nm to about 1.50 nm. The distance between the CsgG pore monomer and the CsgF peptide in the pore monomer conjugate and / or the length of the linker is preferably from about 0.60 nm to about 1.20 nm.

[0094] The pore monomer conjugates of the present invention are capable of forming pores or pore complexes. This can be measured using conventional methods, including any of those described in WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317, WO 2017 / 149318, WO 2018 / 211241 and WO 2019 / 002893 (all of which are incorporated herein by reference in their entirety) and the Examples.

[0095] CsgG pore monomer

[0096] A CsgG pore monomer is a monomer capable of forming a CsgG pore. Such monomers are known in the art, in particular from WO2019 / 002893 (incorporated herein by reference in its entirety). A CsgG pore preferably comprises one or more of (a) a cap region, (b) a constriction region, and (c) a transmembrane β-barrel region, such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c). A CsgG pore monomer preferably comprises one or more of (a) a cap-forming region, (b) a constriction-forming region, and (c) a transmembrane β-barrel-forming region, such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c). The residues of SEQ ID NO: 3 forming these regions are defined as follows. The CsgG pore formed by the monomer may have any structure, but preferably has or comprises the structure of a wild-type CsgG pore ( Fig.15 ). The protein structure of CsgG defines a channel, or pore, that allows molecules and ions to translocate from one side of the membrane to the other.

[0097] "Constriction", "pore opening", "constriction region", "channel constriction" or "constriction site" as used interchangeably herein refers to an aperture defined by the lumen surface of a pore or pore complex that functions to allow ions and target molecules (such as but not limited to polynucleotides or single nucleotides) but not other non-target molecules to pass through the pore or pore complex channel. The constriction is typically the narrowest pore within a pore or pore complex or within a channel defined by a pore or pore complex. The constriction can be used to restrict the passage of molecules through the pore. The size of the constriction is typically a key factor in determining the suitability of a pore or pore complex for analyte characterization. If the constriction is too small, the molecule to be characterized will not be able to pass through. However, in order to achieve the maximum effect on the flow of ions through the channel, the constriction should not be too large. For example, the constriction should not be wider than the solvent accessible transverse diameter of the target analyte. Ideally, the diameter of any constriction should be as close as possible to the transverse diameter of the analyte to be passed. The CsgF peptide and the CsgG pore monomer typically each provide at least one constriction, such that the pore complex of the invention comprises two or more constrictions.

[0098] The CsgG pore can be of any size, but preferably has the dimensions of a wild-type CsgG pore ( Fig.15 The CsgG pore preferably has a width of about 100 nm at its widest point. to about The outer diameter of the to about or about to about The CsgG pore preferably has a width of approximately The CsgG pore preferably has an outer diameter of about to about The total length, such as about to about or about to about The CsgG pore preferably has a size of approximately References to "total length" and "length" relate to the length of the hole or hole area when viewed from the side (see, for example, Fig.15 side view in Figure 1).

[0099] The cap region preferably has about to about length, such as about to about or about to about The cap region preferably has about The channel defined by the cap region preferably has a diameter of about to about An opening such as one with a diameter of about to about or about to about The channel defined by the cap region preferably has a diameter of about The diameter of the channel defined by the cap region at its narrowest point is preferably about to about Such as the diameter at its narrowest point is about to about or about to about The diameter of the channel defined by the cap region at its narrowest point is preferably about

[0100] The contraction zone preferably has a to about length, such as about to about or about to about The contraction zone preferably has a The diameter of the channel defined by the constriction at its narrowest point is preferably about to about Such as the diameter at its narrowest point is about to about about to about or about to about The diameter of the channel defined by the constriction is preferably about or The diameter of the channel defined by the constriction is preferably about The contracted diameter is preferably about to about Such as a diameter of about to about about to about or about to about The contracted diameter is preferably about or The contracted diameter is preferably about

[0101] The transmembrane β barrel region preferably has about to about length, such as about to about or about to about The transmembrane β barrel preferably has a The diameter of the channel defined by the transmembrane β barrel region at its narrowest point is preferably about to about Such as the diameter at its narrowest point is about to about or about to about The diameter of the channel defined by the transmembrane β-barrel region at its narrowest point is preferably about

[0102] All of the above measurements are based on measurements from the backbone to the backbone of the amino acids that form the different regions (e.g. Fig.15 shown).

[0103] SEQ ID NO: 3 shows the sequence of wild-type E. coli CsgG as a mature protein. Residues 1 to 41, 64 to 131, 156 to 180, and 212 to 262 of SEQ ID NO: 3 form a cap region. Residues 42 to 63 of SEQ ID NO: 3 form a constriction region. Residues 132 to 155 and 181 to 211 of SEQ ID NO: 3 form a transmembrane β-barrel region.

[0104] The CsgG pore monomer is preferably a variant of SEQ ID NO:3. The variant CsgG monomer may also be referred to as a modified CsgG pore monomer or a mutated CsgG pore monomer. The modifications or mutations in the variant include, but are not limited to, any one or more modifications disclosed herein or a combination of said modifications. The CsgG pore monomer may be a CsgG homologous monomer. A CsgG homologous monomer is a polypeptide having at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% complete sequence identity with wild-type E. coli CsgG as shown in SEQ ID NO:3. CsgG homologs are also referred to as polypeptides containing the PFAM domain PF03783, which is a characteristic of CsgG-like proteins. A list of currently known CsgG homologs and CsgG architectures can be found at http: / / pfam.xfam.org / / family / PF03783 Found everywhere.

[0105] Over the entire length of the amino acid sequence of SEQ ID NO:3, based on amino acid identity, the variant will preferably be at least 40% homologous to the sequence. More preferably, based on amino acid identity to the amino acid sequence of SEQ ID NO:3 over the entire sequence, the variant may be at least 45%, 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. Over the entire length of the amino acid sequence of SEQ ID NO:3, the variant will preferably be at least 40% identical to the sequence. More preferably, the variant may be at least 45%, 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% identical to SEQ ID NO:3 over the entire sequence.

[0106] Sequence identity may also relate to fragments or parts of a CsgG pore monomer. Thus, a sequence may have less than 40% overall sequence homology / identity to SEQ ID NO:3, but the sequence of a particular region, domain or subunit may share at least 80%, 90% or up to 99% sequence homology / identity with the corresponding region of SEQ ID NO:3. There may be at least 80%, such as at least 85%, 90% or 95% amino acid identity over a stretch of 100 or more, such as 125, 150, 175 or 200 or more contiguous amino acids ("hard homology"). The CsgG pore monomer is preferably a variant of SEQ ID NO:3 comprising a sequence that is at least 40% homologous to the cap region (residues 1 to 41, 64 to 131, 156 to 180 and 212 to 262) of SEQ ID NO:3. More preferably, the variant may comprise a sequence that is at least 45%, 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 the amino acid identity to residues 1 to 41, 64 to 131, 156 to 180 and 212 to 262 of SEQ ID NO: 3. The variant preferably comprises a sequence that is at least 40% identical to residues 1 to 41, 64 to 131, 156 to 180 and 212 to 262 of SEQ ID NO: 3. More preferably, the variant may comprise a sequence that is at least 45%, 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% identical to residues 1 to 41, 64 to 131, 156 to 180 and 212 to 262 of SEQ ID NO: 3. Homology and / or identity is typically measured over the entire length of the cap region.

[0107] The CsgG pore monomer is preferably a variant of SEQ ID NO:3 comprising a sequence that is at least 40% homologous to the constriction region (residues 42 to 63) of SEQ ID NO:3. More preferably, the variant may comprise a sequence that is at least 45%, 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 residues 42 to 63 of SEQ ID NO:3. The variant preferably comprises a sequence that is at least 40% identical to residues 42 to 63 of SEQ ID NO:3. More preferably, the variant may comprise a sequence that is at least 45%, 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% identical to residues 42 to 63 of SEQ ID NO: 3. Homology and / or identity is typically measured over the entire length of the constriction region.

[0108] The CsgG pore monomer is preferably a variant of SEQ ID NO:3 comprising a sequence that is at least 40% homologous to the transmembrane β barrel region (residues 132 to 155 and 181 to 211) of SEQ ID NO:3. More preferably, the variant may comprise a sequence that is at least 45%, 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 residues 132 to 155 and 181 to 211 of SEQ ID NO:3. The variant preferably comprises a sequence that is at least 40% identical to residues 132 to 155 and 181 to 211 of SEQ ID NO:3. More preferably, the variant may comprise a sequence that is at least 45%, 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% identical to residues 132 to 155 and 181 to 211 of SEQ ID NO: 3. Homology and / or identity is typically measured over the entire length of the transmembrane β barrel region.

[0109] The CsgG pore monomer is highly conserved (as can be readily appreciated from Figures 45 to 47 of WO 2017 / 149317). Furthermore, based on knowledge of mutations associated with SEQ ID NO: 3, equivalent positions for mutations in CsgG pore monomers other than SEQ ID NO: 3 can be determined.

[0110] Thus, reference to a mutant CsgG pore monomer comprising a variant of the sequence as shown in SEQ ID NO: 3 and specific amino acid mutations thereof as set out in the claims and elsewhere in the specification also encompasses a mutant CsgG pore monomer comprising a variant of any of the sequences shown in SEQ ID NO: 68 to 88 of WO 2019 / 002893 (incorporated herein by reference in its entirety) and corresponding amino acid mutations thereof. The CsgG pore monomer may also be any of the sequences shown in CN 113773373 A, CN 113896776 A, CN113912683A and CN 113754743A or variants thereof. It will be further understood that the present invention extends to other variant CsgG pore monomers showing highly conserved regions not explicitly identified in this specification.

[0111] Homology can be determined using standard methods in the art. For example, the UWGCG package provides the BESTFIT program that can be used to calculate homology, for example, using (Devereux et al. (1984) Nucleic Acids Research 12, pp. 387-395) on its default settings. PILEUP and BLAST algorithms can be used to calculate homology or arrange sequences (such as identifying equivalent residues or corresponding sequences (usually under their default settings)), for example, as described in Altschul SF (1993) J Mol Evol 36: 290-300; Altschul, SF et al. (1990) J Mol Biol 215: 403-10. Software for performing BLAST analysis is publicly available at the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0112] SEQ ID NO:3 is a wild-type CsgG pore monomer from E. coli strain K-12 substrain MC4100. Variants of SEQ ID NO:3 may comprise any substitution present in another CsgG homologue. Preferred CsgG homologues are shown in SEQ ID NOs:68 to 88 of WO 2019 / 002893 (which is incorporated herein by reference in its entirety). Variants may comprise a combination of one or more substitutions present in SEQ ID NOs:68 to 88 WO 2019 / 002893 (which is incorporated herein by reference in its entirety) compared to SEQ ID NO:3, including one or more substitutions, one or more conservative mutations, one or more deletions or one or more insertion mutations, such as a deletion or insertion of 1 to 10 amino acids, such as 2 to 8 or 3 to 6 amino acids.

[0113] The CsgG pore monomer in the pore monomer conjugates of the invention generally retains the ability to form the same 3D structure as a wild-type CsgG pore monomer, such as the same 3D structure as a CsgG pore monomer having the sequence of SEQ ID NO: 3. The 3D structure of CsgG is known in the art and is disclosed, for example, in Goyal et al. (2014) Nature 516(7530):250-3. In addition to the mutations described herein, any number of mutations may be made in the wild-type CsgG sequence, provided that the CsgG pore monomer retains the improved properties conferred to it by the mutations of the invention.

[0114] Typically, a CsgG pore monomer will retain the ability to form a structure comprising five α-helices and five β-strands. It is therefore envisaged that further mutations may be made in any of these regions in any CsgG pore monomer without affecting the ability of the monomer to form a pore that can translocate a polynucleotide. It is also contemplated that deletions of one or more amino acids may be made in any loop region connecting the α-helices and β-strands and / or in the N-terminal and / or C-terminal regions of the CsgG pore monomer without affecting the ability of the monomer to form a pore that can translocate a polynucleotide.

[0115] In addition to those discussed above, amino acid substitutions can also be made to the amino acid sequence of SEQ ID NO:3, for example, up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replace amino acids with other amino acids having similar chemical structures, similar chemical properties or similar side chain volumes. The introduced amino acid can have a polarity, hydrophilicity, hydrophobicity, alkalinity, acidity, neutrality or charge similar to the amino acid it replaces. Alternatively, conservative substitutions can introduce another aromatic or aliphatic amino acid to replace a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art.

[0116] The CsgG pore monomer may be modified to introduce one or more cysteines, one or more hydrophobic amino acids, one or more charged amino acids, one or more unnatural amino acids, one or more polar amino acids, or one or more photoreactive amino acids. Any number and combination of such introductions may be made. The introduction is preferably by substitution or addition.

[0117] One or more amino acid residues of the amino acid sequence of SEQ ID NO: 3 may additionally be deleted from the above polypeptides. Up to 1, 2, 3, 4, 5, 10, 20 or 30 or more residues may be deleted.

[0118] Variants may include fragments of SEQ ID NO:3. Such fragments retain pore-forming activity. The length of the fragment may be at least 50, at least 100, at least 150, at least 200 or at least 250 amino acids. Such fragments may be used to generate pores. The fragment preferably comprises the transmembrane beta barrel region of SEQ ID NO:3, i.e. residues 132 to 155 and 181 to 211, or a variant thereof as described above.

[0119] Alternatively or additionally one or more amino acids can be added to the above polypeptide. Extensions can be provided at the amino terminal or carboxyl terminal of the amino acid sequence of SEQ ID NO:3 or its polypeptide variant or fragment. The extension can be very short, for example 1 to 10 amino acids in length. Alternatively, the extension can be longer, for example up to 50 or 100 amino acids. The carrier protein can be fused with the amino acid sequence according to the present invention. Other fusion proteins will be discussed in more detail below.

[0120] A variant of SEQ ID NO:3 is a polypeptide having an amino acid sequence that differs from the amino acid sequence of SEQ ID NO:3 and that retains its ability to form a pore. The variant generally contains the region of SEQ ID NO:3 that is responsible for pore formation. The pore-forming ability of β-barrel-containing CsgG is provided by the β-strands in the transmembrane β-barrel region of each monomer. A variant of SEQ ID NO:3 generally comprises the β-strand-forming region of SEQ ID NO:3, i.e. residues 132 to 155 and 181 to 211, or a variant thereof as described above. One or more modifications may be made to the β-strand-forming region of SEQ ID NO:3, as long as the resulting variant retains its ability to form a pore.

[0121] The one or more modifications in the CsgG pore monomer preferably improve the ability of the pore complex comprising the pore monomer to characterize an analyte. For example, the modification / mutation / substitution is expected to change the number, size, shape, placement or orientation of constrictions within the channel from the pore monomer conjugate of the invention. The CsgG pore monomer or variant of SEQ ID NO: 3 may have any of the specific modifications or substitutions disclosed in WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317, WO 2017 / 149318, WO 2018 / 211241 and WO 2019 / 002893 (all of which are incorporated herein by reference in their entirety).

[0122] Preferred modifications or substitutions in SEQ ID NO: 3 include, but are not limited to, one or more of the following, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more or all:

[0123] (a) a substitution at position Y51, such as Y51I, Y51L, Y51A, Y51V, Y51T, Y51S, Y51Q or Y51N;

[0124] (b) a substitution at position N55, such as N55I, N55L, N55A, N55V, N55T, N55S or N55Q;

[0125] (c) a substitution at position F56, such as F56I, F56L, F56A, F56V, F56T, F56S, F56Q or F56N;

[0126] (d) a substitution at position L90, such as L90N, L90D, L90E, L90R or L90K;

[0127] (e) a substitution at position N91, such as N91D, N91E, N91R or N91K;

[0128] (f) a substitution at position K94, such as K94R, K94F, K94Y, K94Q, K94W, K94L, K94S or K94N;

[0129] (g) a substitution at position R192, such as R192Q, R192F, R192S, R192D or R192T; and

[0130] (i) a substitution at position C215, such as C215T, C215S, C215I, C215L, C215A, C215V or C215G.

[0131] The variant of SEQ ID NO: 3 may further comprise a deletion of one or more positions, such as a deletion of T104-N109, a deletion of F193-L199, or a deletion of F195-L199.

[0132] Any number of CsgG pore monomers in a pore or pore complex of the invention, such as 6, 7, 8, 9 or 10, may be variants of SEQ ID NO: 3. All six to ten monomers in a pore or pore complex are preferably variants of SEQ ID NO: 3. The variants in a pore complex may be the same or different. The variant is preferably the same in each pore monomer conjugate in a pore complex of the invention.

[0133] CsgF peptide

[0134] The term "CsgF peptide" preferably defines a CsgF peptide that has been truncated from its C-terminus (i.e., is an N-terminal fragment). The CsgF peptide may be a fragment of wild-type E. coli CsgF (SEQ ID NO: 5 or SEQ ID NO: 6), or a fragment of a wild-type homologue of E. coli CsgF, such as, for example, a peptide comprising any of the amino acid sequences shown in WO 2019 / 002893 (incorporated herein by reference in its entirety). A CsgF homologue refers to a polypeptide having at least 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% complete sequence identity with wild-type E. coli CsgF as shown in SEQ ID NO: 6. A CsgF homologue may also be referred to as a polypeptide containing the PFAM domain PF10614, which is characteristic of CsgF-like proteins. A list of currently known CsgF homologues and CsgF structures can be found at http: / / pfam.xfam.org / / family / PF10614 The mature CsgF (as shown in SEQ ID NO:6) can be divided into three main regions: a "CsgF contractile peptide" (FCP), a "neck" region, and a "head" region. The "head" region of the CsgF peptide is different from the contraction of the pore as described herein. The "head" region of the CsgF peptide may also be referred to as the "C-terminal head domain". The structure of CsgF is discussed in detail in WO 2019 / 002893 (incorporated herein by reference in its entirety).

[0135] The CsgF peptide used in the pore monomer conjugate of the present invention is preferably a truncated CsgF peptide that lacks the C-terminal head; lacks the C-terminal head and a portion of the neck domain of CsgF (e.g., the truncated CsgF peptide may comprise only a portion of the neck domain of CsgF); or lacks the C-terminal head and neck domain of CsgF. The CsgF peptide may lack a portion of the neck domain of CsgF, for example, the CsgF peptide may comprise a portion of the neck domain, such as, for example, amino acid residue 36 (see SEQ ID: NO: 6) from the N-terminus of the neck domain (e.g., residues 36-40, 36-41, 36-42, 36-43, 36-45, 36-46 to residues 36-50 or 36-60 of SEQ ID NO: 6). The CsgF peptide preferably comprises a CsgG binding region and a region that forms a constriction in the pore. The CsgG binding region typically comprises residues 1 to 11 and / or 29 to 32 of the CsgF protein (SEQ ID NO: 6 or a homolog from another species) and may include one or more modifications. The region that forms the constriction structure in the pore typically comprises residues 9 to 28 of the CsgF protein (SEQ ID NO: 6 or a homolog from another species) and may include one or more modifications. Residues 9 to 17 comprise the conserved motif N9PXFGGXXX 17and form a turn region. Residues 9 to 28 form an α-helix. 17 (N17 in SEQ ID NO:6) forms the apex of the constriction, corresponding to the narrowest part of the CsgF constriction in the pore. The CsgF constriction also forms stabilizing contacts with the CsgG β-barrel primarily at residues 8, 9, 10, 11, 12, 18, 21, 22, 29, and 30 of SEQ ID NO:6.

[0136] The CsgF peptide typically has a length of 28 to 50 amino acids, such as 29 to 49, 30 to 45 or 32 to 40 amino acids. Preferably, the CsgF peptide comprises 29 to 35 amino acids or 29 to 45 amino acids. The CsgF peptide comprises all or part of the FCP, which corresponds to residues 1 to 35 of SEQ ID NO: 6. In the case where the CsgF peptide is shorter than the FCP, it is preferably truncated at the C-terminus.

[0137] The CsgF peptide may have a length of 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 amino acids.

[0138] The CsgF peptide may comprise the amino acid sequence of SEQ ID NO: 6, from residue 1 to any one of residues 25 to 60, such as 27 to 50, for example 28 to 45, of SEQ ID NO: 6, or the corresponding residues from a homologue of SEQ ID NO: 6 or any variant thereof. More specifically, the CsgF peptide may comprise residues 1 to 29 of SEQ ID NO: 6 or a homologue or variant thereof.

[0139] The CsgF peptide is preferably a truncated CsgF peptide lacking one or more amino acids from CsgF shown in SEQ ID NO: 6. The CsgF peptide is preferably a truncated CsgF peptide lacking a stretch of amino acids starting at any one of positions 15-35 and ending at position 119 of SEQ ID NO: 6. The CsgF peptide is preferably a truncated CsgF peptide lacking amino acids 15-119, 16-119, 17-119, 18-119, 19-119, 20-119, 21-119, 22-119, 23-119, 24-119, 25-119, 26-119, 27-119, 28-119, 29-119, 30-119, 31-119, 32-119, 33-119, 34-119 or 35-119 from SEQ ID NO:6.

[0140] Examples of such CsgF peptides comprise, consist essentially of, or consist of residues 1 to 34 of SEQ ID NO: 6, residues 1 to 30 of SEQ ID NO: 6, residues 1 to 45 of SEQ ID NO: 6, or residues 1 to 35 of SEQ ID NO: 6, and any homologs or variants thereof.

[0141] In the CsgF peptide, one or more residues may be modified. For example, the CsgF peptide may comprise modifications at positions corresponding to one or more of the following positions in SEQ ID NO: 6: G1, T4, F5, R8, N9, N11, F12, N17, A20, N24, A26, Q27, and Q29.

[0142] The CsgF peptide may be modified to introduce, for example, one or more cysteines, one or more hydrophobic amino acids, one or more charged amino acids, one or more unnatural amino acids, one or more polar amino acids, or one or more photoreactive amino acids at positions corresponding to one or more of the following positions in SEQ ID NO: 6: G1, T4, F5, R8, N9, N11, F12, A26, and Q29. Any number and combination of such introductions may be made. The introduction is preferably by substitution or addition.

[0143] For example, the CsgF peptide may comprise a modification at a position corresponding to one or more of the following positions in SEQ ID NO: 6: N15, N17, A20, N24, and A28. The CsgF peptide may comprise a modification at a position corresponding to D34 to stabilize the CsgG-CsgF complex. The CsgF peptide may comprise one or more of the following substitutions: N15S / A / T / Q / G / L / V / I / F / Y / W / R / K / D / C / E, N17S / A / T / Q / G / L / V / I / F / Y / W / R / K / D / C / E, A20S / T / Q / N / G / L / V / I / F / Y / W / R / K / D / C / E, N24S / T / Q / A / G / L / V / I / F / Y / W / R / K / D / C / E, A28S / T / Q / N / G / L / V / I / F / Y / W / R / K / D / C / E, and D34F / Y / W / R / K / N / Q / C / E. The CsgF peptide may, for example, comprise one or more of the following substitutions: G1C, T4C, N17S, and D34Y or D34N.

[0144] CsgF peptides can be produced by cleaving longer proteins (such as full-length CsgF) using an enzyme. Cleavage at specific sites can be performed by modifying longer proteins (such as full-length CsgF) to include an enzyme cleavage site at an appropriate position. Examples of CsgF amino acid sequences that have been modified to include such enzyme cleavage sites are shown in SEQ ID NOs: 56 to 67 of WO 2019 / 002893 (incorporated by reference in its entirety). After cleavage, all or part of the added enzyme cleavage site may be present in the CsgF peptide that associates with CsgG to form a pore. Therefore, the CsgF peptide may further include all or part of the enzyme cleavage site at its C-terminus.

[0145] Some examples of suitable CsgF peptides are shown in Table 3 of WO 2019 / 002893 (incorporated by reference in its entirety).

[0146] The CsgF peptide is preferably a variant of any of the CsgF sequences discussed above, including SEQ ID NO:6, comprising one or more modifications compared to the comparison sequence. Variants will preferably be at least 40% homologous to the amino acid sequence of SEQ ID NO:6 based on amino acid identity over the entire length of the sequence. More preferably, variants may be at least 45%, 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:6 over the entire sequence. Variants will preferably be at least 40% identical to the amino acid sequence of SEQ ID NO:6 over the entire length of the sequence. More preferably, the variant may be at least 45%, 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% identical to SEQ ID NO: 6 over the entire sequence. There may be at least 80%, such as at least 85%, 90% or 95% amino acid identity over a stretch of 100 or more, such as 125, 150, 175 or 200 or more contiguous amino acids ("hard homology"). These homology / identity levels apply equally to any of the other CsgF peptides described above.

[0147] Any number of CsgF peptides in a pore or pore complex of the invention, such as 6, 7, 8, 9 or 10, may contain one or more substitutions compared to SEQ ID NO: 6. All six to ten monomers in a pore or pore complex preferably contain one or more substitutions compared to SEQ ID NO: 6. The CsgF peptides in a pore complex may be the same or different. The CsgF peptide is preferably the same in each pore monomer conjugate in a pore complex of the invention.

[0148] Stabilization and other mutations

[0149] In the pore complex of the invention, the interaction between the CsgF peptide and the CsgG pore may be stabilized, for example, by hydrophobic interactions and / or electrostatic interactions. These may be interactions between one or more of the following pairs of positions of SEQ ID NO:6 and SEQ ID NO:3: 1 and 153, 4 and 133, 5 and 136, 8 and 187, 8 and 203, 9 and 203, 11 and 142, 11 and 201, 12 and 149, 12 and 203, 26 and 191, and 29 and 144, respectively.

[0150] The residues at one or more of the positions listed above in the CsgF peptide and / or the CsgG pore monomer may be modified to enhance the interaction between CsgG and CsgF in the pore complex. Although the CsgG:CsgF complex is very stable, when CsgF is truncated, the stability of the CsgG:CsgF complex is reduced compared to a complex comprising full-length CsgF. Thus, a disulfide bond may be formed between CsgG and CsgF to make the complex more stable, for example after the introduction of a cysteine ​​residue at the position identified herein. The pore complex may be prepared by any of the aforementioned methods, and disulfide bond formation may be induced by the use of an oxidizing agent (e.g., copper-o-phenanthroline). Other interactions (e.g., hydrophobic interactions, charge-charge interactions / electrostatic interactions) may also be used at these positions instead of cysteine ​​interactions.

[0151] Non-natural amino acids can also be incorporated into those positions. Covalent bonds can be formed by click chemistry. For example, non-natural amino acids with azides or alkynes or with dibenzocyclooctyne (DBCO) groups and / or bicyclo [6.1.0] nonyne (BCN) groups can be introduced into one or more of these positions.

[0152] Such stabilizing mutations may be combined with any other modifications to CsgG and / or CsgF, such as those disclosed herein.

[0153] To facilitate such interactions, one or more non-natural or photoreactive amino acids may be included / substituted at one or more positions in the CsgG pore monomer corresponding to one or more of positions 132, 133, 136, 138, 140, 142, 144, 145, 147, 149, 151, 153, 155, 183, 185, 187, 189, 191, 201, 203, 205, 207 and 209 of SEQ ID NO: 3.

[0154] To facilitate such interactions, one or more non-naturally reactive or photoreactive amino acids may be included / substituted at one or more positions corresponding to one or more of positions 1, 4, 5, 8, 9, 11, 12, 26, or 29 of SEQ ID NO:6.

[0155] Preferred exemplary CsgF peptides comprise the following mutations relative to SEQ ID NO:6: N15X1 / N17X2 / A20X3 / N24X4 / A28X5 / D34X6, wherein X1 is N / S / A / T / Q / G / L / V / I / F / Y / W / R / K / D / C / E, X2 is N / S / A / T / Q / G / L / V / I / F / Y / W / R / K / D / C / E, and X3 is A / S / T / Q / N / G / L / V / I / F / Y / W / R / K / D / C / E, X4 is N / S / T / Q / A / G / L / V / I / F / Y / W / R / K / D / C / E, X5 is A / S / T / Q / N / G / L / V / I / F / Y / W / R / K / D / C / E and X6 is D / F / Y / W / R / K / N / Q / C / E. Mutations at positions N15, N17, A20, N24 and A28 are contraction mutations, and the mutation at position 34 affects the interaction of CsgF with the bottom of the CsgG pore monomer to stabilize the interaction.

[0156] Construct

[0157] The invention also provides a construct comprising two or more covalently attached pore monomer conjugates of the invention. The construct may comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more pore monomer conjugates of the invention. The construct may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 pore monomer conjugates of the invention. The two or more pore monomer conjugates may be the same or different. The two or more pore monomer conjugates may differ based on one or more of: (a) the sequence of the CsgG pore monomer, (b) the sequence of the CsgF peptide, (c) a linker, (d) an attachment position on the CsgG pore monomer, and (e) an attachment position on the CsgF peptide. The pore monomer conjugates may differ based on: (a); (b); (c); (d); (e); (a) and (b); (a) and (c); (a) and (d); (a) and (e); (b) and (c); (b) and (d); (b) and (e); (c) and (d); (c) and (e); (d) and (e); (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (c) and (d); (a), (b) and (e); (a), (b), (c), (d), and (e); (b), (c), (d), and (e); (b), (c), (d), and (e); (c), (d), and (e); (a), (b), (c), and (d); (a), (b), (c), and (e); (a), (b), (d), and (e); (a), (c), (d), and (e); (b), (c), (d), and (e); and (a), (b), (c), (d), and (e). Two or more pore monomer conjugates are preferably identical (i.e., identical).

[0158] The construct preferably comprises two pore monomer conjugates. The two or more pore monomer conjugates may be the same or different. The two or more pore monomer conjugates are preferably the same (ie, identical).

[0159] The pore monomer conjugates may optionally be genetically fused via a linker, or chemically fused, for example via a chemical cross-linker. Methods for covalently attaching monomers are disclosed in WO 2017 / 149316, WO 2017 / 149317 and WO 2017 / 149318 (incorporated herein by reference in their entirety).

[0160] The linker is preferably an amino acid sequence and / or a chemical cross-linker. Suitable amino acid linkers, such as peptide linkers, are known in the art. The length, flexibility and hydrophilicity of the amino acid or peptide linker are generally designed so that the CsgF peptide forms a constriction in the pore complex of the invention. 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, (SG)8, (SG) 10 、(SG) 15 or (SG) 20 , wherein S is serine and G is glycine. Preferred rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or 24 proline amino acids. More preferred rigid linkers include (P) 12 , where P is proline.

[0161] Suitable chemical cross-linking agents are well known in the art. Suitable chemical cross-linking agents include, but are not limited to, those containing the following functional groups: maleimide, active esters, succinimides, azides, alkynes (such as dibenzocyclooctynol (DIBO or DBCO), difluorocycloalkynes and linear alkynes), phosphines (such as those used for traceless and non-traceless Staudinger linkages), haloacetyl groups (such as iodoacetamide), phosgene-type reagents, sulfonyl chloride reagents, isothiocyanates, acyl halides, hydrazines, disulfides, vinyl sulfones, aziridines, and photoreactive reagents (such as aryl azides, diaziridines).

[0162] The reaction between amino acids and functional groups may be spontaneous, such as cysteine / maleimide, or may require external reagents, such as Cu(I) for attachment of azides and linear alkynes.

[0163] The linker may comprise any molecule that extends across the desired distance. The length of the linker may vary from one carbon (phosgene-type linker) to many angstroms. Examples of linker molecules include, but are not limited to, polyethylene glycol (PEG), polypeptides, polysaccharides, deoxyribonucleic acid (DNA), peptide nucleic acid (PNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), saturated and unsaturated hydrocarbons, polyamides. These linkers may be inert or reactive, in particular they may be chemically cleaved at defined positions, or they may themselves be modified with fluorophores or ligands. After attachment (such as covalent attachment) of the CsgF peptide to the CsgG pore monomer, the linker is preferably resistant to reducing agents (such as dithiothreitol (DTT)).

[0164] Preferred crosslinking agents include 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldithio) propionate, 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldithio) butyrate, and 2,5-dioxopyrrolidin-1-yl 8-(pyridin-2-yldithio) octanoate, bismaleimide PEG 1k, bismaleimide PEG 3.4k, bismaleimide PEG 5k, bismaleimide PEG 10k, bis(maleimido)ethane (BMOE), bismaleimidohexane (BMH), 1,4-bismaleimidobutane (BMB), 1,4-bismaleimido-2,3-dihydroxybutane (BMDB), BM[PEO]2(1,8-bismaleimidodiglycol), BM[PEO]3(1,11-bismaleimidotriglycol), tris[2-maleimidoethyl]amine (TMEA), DTME disulfide bismaleimidoethane, bismaleimido PEG3, bismaleimido PEG11, DBCO-maleimide, DBCO-PEG4-maleimide, DBCO-PEG4-NH2, DBCO-PEG4-NHS, DBCO-NHS, DBCO-PEG-DBCO 2.8kDa, DBCO-PEG-DBCO 4.0 kDa, DBCO-15 atoms-DBCO, DBCO-26 atoms-DBCO, DBCO-35 atoms-DBCO, DBCO-PEG4-SS-PEG3-biotin, DBCO-SS-PEG3-biotin, DBCO-SS-PEG11-biotin, (succinimidyl 3-(2-pyridyldithio) propionate (SPDP) and maleimide-PEG (2 kDa)-maleimide (α,ω-bismaleimide poly (ethylene glycol)). The most preferred cross-linking agent is maleimide-propyl-SRDFWRS-(1,2-diaminoethane)-propyl-maleimide.

[0165] The linker is preferably resistant to dithiothreitol (DTT). Suitable linkers include, but are not limited to, iodoacetamide-based and maleimide-based linkers.

[0166] The pore monomer conjugates may be connected using two or more linkers, each linker comprising a hybridizable region and a group capable of forming a covalent bond. The hybridizable region in the linker hybridizes and connects the CsgG pore monomer and the CsgF peptide. The connected CsgG pore monomer and CsgF peptide are then coupled via the formation of a covalent bond between the groups. Any of the specific linkers disclosed in WO 2010 / 086602 (incorporated herein by reference in its entirety) may be used according to the present invention.

[0167] The linker may be labeled. Suitable labels include, but are not limited to, fluorescent molecules (such as Cy3 or 555), radioactive isotopes (e.g. 125 I. 35 S. 32 P), enzymes, antibodies, antigens, polynucleotides and ligands (such as biotin). Such labels allow the amount of linker to be quantified. The label can also be a cleavable purification tag, such as biotin, or a specific sequence that is revealed in the identification method, such as a peptide that is not present in the protein itself but is released by trypsin digestion.

[0168] A preferred method of linking the pore monomer conjugates is via cysteine ​​bonds. This can be mediated by bifunctional chemical cross-linkers or by amino acid linkers with terminally present cysteine ​​residues.

[0169] Another preferred attachment method is via 4-azidophenylalanine or Faz connection. This can be mediated by a bifunctional chemical linker or by a polypeptide linker with a terminal 4-azidophenylalanine or Faz residue. Other suitable linkers are discussed in more detail below.

[0170] Pore ​​complex of the present invention

[0171] The terms "pore complex" or "composite pore" are used interchangeably herein and refer to an oligomeric pore complex comprising at least one pore monomer conjugate of the present invention (including, for example, one or more pore monomer conjugates, such as two or more pore monomer conjugates, three or more pore monomer conjugates, etc.). The pore complex of the present invention has the characteristics of a biological pore, i.e., it has a typical protein structure and defines a channel. When the pore complex is provided in an environment having a membrane component, a membrane, a cell or an insulating layer, the pore complex will be inserted into the membrane or the insulating layer and form a "transmembrane pore complex".

[0172] The CsgG portion of the pore complex of the invention (i.e., the portion formed by at least one CsgG pore monomer in at least one conjugate of the invention) preferably has or comprises any structure and / or size of the CsgG pore described above. The CsgG constriction in the pore complex of the invention preferably has or comprises any constriction diameter described above.

[0173] At least one CsgF peptide (in at least one pore monomer conjugate or construct) preferably forms a constriction in the pore complex. The at least one CsgF peptide is preferably inserted into the lumen of the pore complex. The present invention relates to a CsgG pore complexed with a CsgF peptide that introduces an additional channel constriction in the pore complex and surprisingly results in an increased current range and an increased signal-to-noise ratio (SNR). The additional constriction introduced by forming a complex with the CsgF peptide enlarges the contact surface with the analyte passing through and can act as a second constriction for analyte detection and characterization. Pores comprising the pore monomer conjugates of the present invention can improve the characterization of analytes (such as polynucleotides), providing a more discriminating direct relationship between the observed currents as the polynucleotide moves through the pore. In particular, by having two stacked constrictions spaced apart by a defined distance, the pore complex can facilitate the characterization of polynucleotides containing at least one homopolymeric stretch, e.g., several consecutive copies of the same nucleotide, which otherwise exceeds the interaction length of a single CsgG constriction. Additionally, by having two stacked constrictions at a defined distance, small molecule analytes including organic or inorganic drugs and pollutants that pass through the pore complex will pass through both constrictions in succession. The chemical properties of either constriction can be modified independently, each constriction giving unique interaction properties with the analyte, thereby providing additional discrimination capabilities during analyte detection.

[0174] The CsgF contraction formed in the pore complex preferably has a to about Such as to about about to about or about to about The additional CsgF peptide constriction may be about 10 nm or less, such as about 5 nm or less, such as about 1, 2, 3, 4, 5, 6, 7, 8, 9 nm away from the constriction of the CsgG pore. The distance between the CsgF peptide and the CsgG pore monomer is also discussed above with reference to the pore monomer conjugates of the invention.

[0175] The pore complex or transmembrane pore complex of the invention includes a pore complex with two constrictions, i.e., two channel constrictions are positioned in such a way that one constriction does not interfere with the accuracy of the other constriction. The pore complex may include any mutation, CsgG pore monomer or CsgF peptide described in WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317, WO 2019 / 002893, WO 2017 / 149318, WO 2018 / 211241 and WO 2019 / 002893 (all of which are incorporated herein by reference in their entirety). The pore complex or transmembrane pore complex of the invention includes a pore complex with one constriction. For example, the constriction may be removed from the CsgG pore monomer in the conjugate of the invention, such that the pore complex of the invention contains only one constriction provided by the CsgF peptide. The invention provides a pore complex comprising at least one pore monomer conjugate of the invention. The pore complex typically comprises at least 6, 7, 8, 9 or 10 pore monomer conjugates of the invention. The pore complex preferably comprises 8 or 9 pore monomer conjugates of the invention. The pore monomer conjugates are typically identical (ie, identical).

[0176] The pore complex is preferably a homo-oligomer comprising 6 to 10, such as 6, 7, 8, 9 or 10 pore monomer conjugates of the invention. The pore monomer conjugates are usually identical. The pore complex preferably comprises 8 or 9 identical pore monomer conjugates of the invention. The pore monomer conjugate may be any of those discussed above.

[0177] The invention provides a pore complex comprising at least one construct of the invention. The pore complex typically comprises at least 1, 2, 3, 4 or 5 constructs of the invention. The pore complex comprises sufficient CsgG pore monomers to form a pore. For example, an octameric pore may comprise (a) four constructs, each construct comprising two pore monomer conjugates, (b) two constructs, each construct comprising four pore monomer conjugates, (c) one construct comprising two pore monomer conjugates and six pore monomer conjugates that do not form part of the construct, (d) three constructs comprising two pore monomer conjugates and two pore monomer conjugates that do not form part of the construct, and (e) combinations thereof. For example, the same and further possibilities are provided for non-polymeric pores. Other combinations of constructs and monomers can be envisaged by the skilled person. One or more constructs of the invention may be used to form a pore complex for characterizing (such as sequencing) a polynucleotide. The pore complex preferably comprises 4 constructs of the invention, each construct comprising two pore monomer conjugates. The constructs are typically identical (i.e., identical).

[0178] The pore complex is preferably a homo-oligomer comprising 1 to 5, such as 1, 2, 3, 4, 5 constructs of the invention. The constructs are typically identical (i.e., identical). The pore complex preferably comprises 4 identical constructs of the invention, each construct comprising two pore monomer conjugates. The constructs may be any of those discussed above.

[0179] The CsgG pore monomers in a CsgG pore preferably all have about the same length or have the same length. The barrels of the CsgG pore monomers of the invention in a pore preferably have about the same length or have the same length. The length may be measured in terms of number of amino acids and / or length units.

[0180] The pore complexes of the invention may be isolated, substantially isolated, purified or substantially purified. If the pore complexes of the invention are completely free of any other components, such as lipids or other pores, they are isolated or purified. If the pore complexes are mixed with a carrier or diluent that does not interfere with its intended use, they are substantially isolated. For example, if the pore complexes are present in a form containing less than 10%, less than 5%, less than 2% or less than 1% of other components (such as block copolymers, lipids or other pores), they are substantially isolated or substantially purified. Alternatively, the pore complexes of the invention may be present in a membrane. Suitable membranes are discussed below.

[0181] The pore complexes of the invention may exist as individual or single pore complexes. Alternatively, the pore complexes of the invention may exist in two or more pore complexes or homogenous or heterogenous populations of pores. Other forms of pore complexes related to the invention are discussed in more detail below.

[0182] Polymeric pore complex

[0183] The invention also provides a pore polymer comprising two or more pores, wherein at least one of the pores is a pore complex of the invention. The polymer may comprise any number of pores, such as 3, 4, 5, 6, 7 or 8 or more pores. Any number of pores in the polymer, including all of them, may be a pore complex of the invention.

[0184] The pore polymer may be a dual pore complex comprising a first pore complex of the invention and a second pore or complex. The second pore or complex is typically derived from CsgG. The second pore complex may be a complex of the invention. Both the first pore complex and the second pore complex are preferably pore complexes of the invention. In the dual pore complex, the first pore complex may be attached to the second pore (complex) by hydrophobic interactions and / or by one or more disulfide bonds. One or more, such as 2, 3, 4, 5, 6, 8, 9, for example all monomers of the first pore complex and / or the second pore (complex) may be modified to enhance such interactions. This may be achieved in any suitable manner. Specific methods for forming dual pores from CsgG-derived pores are described in WO2019 / 002893 (incorporated herein by reference in its entirety).

[0185] The pore multimers of the invention may be isolated, substantially isolated, purified or substantially purified. These terms are defined above with reference to the pore complexes of the invention.

[0186] Film Examples

[0187] The invention also provides a pore complex of the invention or a pore polymer of the invention contained in a membrane. The invention also provides a membrane comprising a pore complex of the invention or a pore polymer of the invention. These products can be used directly for molecular sensing, such as analyte characterization and polynucleotide sequencing. Suitable membranes are discussed in more detail below.

[0188] Methods for preparing modified proteins

[0189] Methods for introducing or substituting non-naturally occurring amino acids in CsgG pore monomers and CsgF peptides are also well known in the art and are described in WO 2019 / 002893 (incorporated herein by reference in its entirety). Proteins may be modified to aid their identification or purification, for example by adding a streptavidin tag or by adding a signal sequence to facilitate their secretion from cells in which the monomers do not naturally contain such sequences. Proteins may also be produced using D-amino acids or mixtures of L-amino acids and D-amino acids. This is routine in the art for producing such proteins or peptides.

[0190] The CsgG pore monomer, CsgF peptide, pore monomer conjugate, construct, pore complex or pore multimer (i.e., any protein of the invention) may be chemically modified. The protein may be chemically modified in any manner and at any site. The protein may be chemically modified by attachment of a molecule to one or more cysteines (cysteine ​​ligation), attachment of 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. Suitable methods for performing such modifications are well known in the art. The protein may be chemically modified by attachment of any molecule (e.g., a dye or fluorophore).

[0191] The protein can be chemically modified with a molecular adapter that promotes the interaction between the hole containing the monomer and the target nucleotide or target polynucleotide sequence. Suitable adapters, including cyclic molecules, cyclodextrins, substances capable of hybridization, DNA binders or mutual chelators, peptides or peptide analogs, synthetic polymers, aromatic planar molecules, small molecules with positive charges or small molecules capable of hydrogen bonding, are described in WO 2019 / 002893 (incorporated herein in its entirety by reference). Any of the methods and joints discussed above can be used to attach molecular adapters.

[0192] Proteins can be attached to polynucleotide binding proteins. This forms a modular sequencing system that can be used in the sequencing method of the present invention. Polynucleotide binding proteins are discussed below. Proteins can be covalently attached to monomers using any method known in the art. Monomers and proteins can be chemically fused or genetically fused. Genetic fusion of monomers to polynucleotide binding proteins is discussed in WO 2010 / 004265 (incorporated herein by reference in its entirety). Polynucleotide binding proteins can be attached via cysteine ​​bonds using any of the above methods.

[0193] The polynucleotide binding protein may be attached directly to the protein via one or more linkers. The molecule may be attached to the CsgG pore monomer using a hybridization linker described in WO2010 / 086602 (incorporated herein by reference in its entirety). Alternatively, a peptide linker may be used. Suitable peptide linkers are discussed above.

[0194] Any of the proteins can be modified to aid in their identification or purification, for example by adding histidine residues (his tags), aspartic acid residues (asp tags), streptavidin tags, flag tags, SUMO tags, GST tags or MBP tags, or by adding signal sequences to promote their secretion from cells in which the polypeptide does not naturally contain such sequences. An alternative to introducing genetic tags is to chemically react the tags to a native or engineered position on the protein. An example of this is reacting a gel shift reagent with a cysteine ​​engineered on the outside of the protein. This has been shown to be a method for separating hemolysin hetero-oligomers (Chem Biol. 1997 July; 4(7): 497-505).

[0195] Any of the proteins may be labeled with a revealing marker. The revealing marker may be any suitable marker that allows detection of the protein. Suitable markers include, but are not limited to, fluorescent molecules, radioisotopes (e.g., 125I, 35S), enzymes, antibodies, antigens, polynucleotides, and ligands (such as biotin).

[0196] The protein may also contain other non-specific modifications, as long as they do not interfere with the function of the protein. Many non-specific side chain modifications are known in the art and can be performed on the side chains of proteins. Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH4, amidation with methyl acetimidate, or acylation with acetic anhydride.

[0197] Any protein can be produced using standard methods known in the art. Polynucleotide sequences encoding proteins can be derived and replicated using standard methods in the art. Polynucleotide sequences encoding proteins can be expressed in bacterial host cells using standard techniques in the art. Proteins can be produced in cells by in situ expression of polypeptides from recombinant expression vectors. The expression vectors optionally carry inducible promoters to control the expression of polypeptides. These methods are described in Sambrook, J. and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[0198] Proteins can be produced on a large scale after purification from a protein-producing organism by any protein liquid chromatography system or after recombinant expression. Typical protein liquid chromatography systems include FPLC, AKTA system, Bio-Cad system, Bio-Rad BioLogic system, and Gilson HPLC system.

[0199] Methods for producing pore monomer conjugates

[0200] The invention provides a method for producing a pore monomer conjugate of the invention. The method comprises attaching (preferably covalently attaching) a CsgF peptide to a CsgG pore monomer using a sulfonyl fluoride-containing linker, a sulfonyltriazole-containing linker, a fluorosulfate-containing linker or a fluoroacetamide-containing linker. The method may involve the use of any of the above linkers. The linker may attach or covalently attach the CsgF peptide to the CsgG pore monomer at any of the positions discussed above with reference to the pore monomer conjugate of the invention.

[0201] Alternatively, the method comprises attaching, preferably covalently attaching, the CsgF peptide to a residue in the CsgG pore monomer corresponding to any of positions 143, 146, 190, 192, 193, 194, 195, 196, 197, 198, 199 or 200 in SEQ ID NO: 3. The method may involve the use of any of the reactive groups and / or linkers described above.

[0202] The method generally comprises contacting the CsgF peptide and the CsgG pore monomer with a linker. The components may be contacted with the linker in any order, such as first the CsgF peptide and then the CsgG pore monomer, first the CsgG pore monomer and then the CsgF peptide, or both components contacted simultaneously. The linker is preferably first attached to the CsgF peptide or the CsgG pore monomer, and then attached to the other components of the conjugate. The method preferably comprises attaching or covalently attaching the linker to the CsgF peptide, and then contacting the linker and the CsgF peptide with the CsgG pore monomer under conditions where the CsgF peptide is attached or covalently attached to the CsgG pore monomer via the linker. These conditions are well known to those skilled in the art and are discussed in the Examples. The method is generally performed in vitro as defined below.

[0203] Any of the embodiments discussed above with reference to the pore monomer conjugates of the invention are equally applicable to these methods.

[0204] Methods for generating holes

[0205] The present invention also provides a method for producing a pore complex of the present invention or a pore multimer of the present invention.

[0206] The method may involve expressing a pore complex in a host cell. In particular, the method may comprise expressing at least one pore monomer conjugate of the invention or a construct of the invention and sufficient pore monomer or construct to form a pore complex or pore multimer in the host cell, and allowing the pore complex or pore multimer to form in the host cell. The sufficient pore monomer or construct is preferably sufficient pore monomer conjugate of the invention or sufficient construct of the invention. The amount of CsgG pore monomer, pore monomer conjugate or construct required to form a pore complex of the invention or a pore multimer of the invention is discussed above. Suitable host cells and expression systems are known in the art and are discussed in the Examples.

[0207] The method may involve forming a pore complex in a non-cellular or in vitro environment. In particular, the method may comprise contacting at least one pore monomer conjugate of the invention or a construct of the invention with sufficient pore monomer or construct in vitro and allowing the formation of a pore complex or pore multimer. The pore monomer conjugate or construct may be produced separately by in vitro translation and transcription (IVTT) and then incubated with sufficient pore monomer or construct. Sufficient pore monomer or construct is preferably sufficient pore monomer conjugate of the invention or sufficient construct of the invention. The amount of CsgG pore monomer, pore monomer conjugate or construct required to form a pore complex of the invention or a pore multimer of the invention is discussed above. The method may be performed in an "in vitro system", which refers to a system comprising at least the components and environment necessary to perform the method, and utilizing biomolecules, organisms, cells (or parts of cells) outside of their normal naturally occurring environment, allowing for more detailed, more convenient or more efficient analysis than with whole organisms. The in vitro system may also comprise a suitable buffer composition provided in a test tube, to which the protein components forming the complex have been added. The skilled person is aware of the options for providing the system.

[0208] Some or all components of the pore complex or pore multimer may be labeled to facilitate purification. Purification may also be performed when the components are unlabeled. Methods known in the art (e.g., ion exchange, gel filtration, hydrophobic interaction column chromatography, etc.) may be used alone or in various combinations to purify components of the pore.

[0209] The pore complex or pore multimer can be prepared prior to insertion into the membrane or after the components have been inserted into the membrane.

[0210] Methods for preparing the pores and complexes of the present invention and ways of labeling them are disclosed in WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317 and WO 2017 / 149318, WO 2018 / 211241 and WO2019 / 002893 (all of which are incorporated herein by reference in their entirety).

[0211] Methods for characterizing analytes

[0212] The present invention provides a method for determining the presence, absence or one or more characteristics of a target analyte. The method comprises contacting the target analyte with a pore complex of the present invention or a pore multimer of the present invention, allowing the target analyte to move relative to (such as into or through) the pore complex or pore multimer, and taking one or more measurements as the analyte moves relative to the pore complex or pore multimer, and thereby determining the presence, absence or one or more characteristics of the analyte. The target analyte may also be referred to as a template analyte or an analyte of interest.

[0213] The pore complex of the invention or the pore multimer of the invention may be any of those described above.

[0214] The method is used to determine the presence, absence or one or more characteristics of the target analyte. The method can be used to determine the presence, absence or one or more characteristics of at least one analyte. The method can be related to determining the presence, absence or one or more characteristics of two or more analytes. The method can include determining the presence, absence or one or more characteristics of any number of analytes (such as 2, 5, 10, 15, 20, 30, 40, 50, 100 or more analytes). Any number of features of one or more analytes can be determined, such as 1, 2, 3, 4, 5, 10 or more features.

[0215] The binding of molecules in the channel of the pore complex or pore polymer or near any opening of the channel will have an effect on the open channel ion flow through the pore complex or pore polymer, which is the essence of "molecular sensing". In a similar manner to nucleic acid sequencing applications, changes in open channel ion flow can be measured by changes in current using suitable measurement techniques (e.g., WO 2000 / 28312 and D. Stoddart et al., Proc. Natl. Acad. Sci., 2010, 106, 7702-7 or WO2009 / 077734; all incorporated herein by reference in their entirety). The degree of reduction in ion flow measured by the reduction in current is related to the size of the obstacle in or near the pore. Therefore, the binding of the molecule of interest (also referred to as "analyte") in or near the pore provides a detectable and measurable event, thereby forming the basis of a "biosensor". Suitable molecules for nanopore sensing include nucleic acids; proteins; peptides; polysaccharides and small molecules (here refers to low molecular weight (e.g., <900Da or <500Da) organic or inorganic compounds) such as drugs, toxins, cytokines and pollutants. Detecting the presence of biomolecules can be applied to personalized drug development, medicine, diagnostics, life science research, environmental monitoring, and security and / or defense industries.

[0216] Pore ​​complexes or pore polymers can be used as molecular or biological sensors. The analyte molecules to be detected can be bound to either side of the channel, or within the lumen of the channel itself. The location of binding can be determined by the size of the molecule to be sensed.

[0217] The target analyte is preferably a metal ion, an inorganic salt, a polymer, an amino acid, a peptide, a polypeptide, a protein, a nucleotide, an oligonucleotide, a polynucleotide, a monosaccharide, an oligosaccharide, a polysaccharide, a dye, a bleach, a drug, a diagnostic agent, a recreational drug, an explosive, a toxic compound or an environmental pollutant. The analyte may comprise two or more different molecules, such as a peptide and a polypeptide. The method may involve determining the presence, absence or one or more characteristics of two or more analytes of the same type (such as two or more proteins, two or more nucleotides or two or more drugs). Alternatively, the method may involve determining the presence, absence or one or more characteristics of two or more different types of analytes (such as one or more proteins, one or more nucleotides and one or more drugs).

[0218] The target analyte may be secreted from the cell. Alternatively, the target analyte may be an analyte present inside the cell, such that the analyte must be extracted from the cell before the method can be performed.

[0219] The pore complex or pore polymer can be modified by recombinant or chemical methods to increase the binding strength, binding site or binding specificity of the molecule to be sensed. Typical modifications include the addition of a specific binding moiety that is complementary to the structure of the molecule to be sensed. When the analyte molecule comprises a nucleic acid, the binding moiety may comprise a cyclodextrin or an oligonucleotide; for small molecules, this may be a known complementary binding region, such as the antigen binding portion of an antibody or non-antibody molecule, including a single-chain variable fragment (scFv) region or an antigen recognition domain from a T cell receptor (TCR); or for proteins, it may be a known ligand for the target protein. In this way, the pore complexes or pore polymers can act as molecular sensors for detecting the presence of suitable antigens (including epitopes) in a sample, which antigens may include cell surface antigens, including receptors, markers of solid tumors or blood cancer cells (such as lymphomas or leukemias), viral antigens, bacterial antigens, protozoan antigens, allergens, allergy-related molecules, albumins (such as human, rodent or bovine), fluorescent molecules (including fluorescein), blood group antigens, small molecules, drugs, enzymes, catalytic sites of enzymes or enzyme substrates, and transition state analogs of enzyme substrates. As described above, modifications can be achieved using known genetic engineering and recombinant DNA techniques. Any adapted positioning will depend on the properties of the molecule to be sensed, such as size, three-dimensional structure and its biochemical properties. The selection of suitable structures can utilize computational structure design. The determination and optimization of protein-protein interactions or protein-small molecule interactions can be performed using methods such as The studies were conducted using techniques such as BIAcore, which uses surface plasmon resonance to detect molecular interactions (BIAcore, Inc., Piscataway, NJ; see also www.biacore.com).

[0220] The analyte is preferably an amino acid, peptide, polypeptide or protein. The amino acid, peptide, polypeptide or protein may be naturally occurring or non-naturally occurring. The polypeptide or protein may include synthetic or modified amino acids. Several different types of modifications to amino acids are known in the art. Suitable amino acids and modifications thereof are described above. It should be understood that the target analyte may be modified by any method available in the art.

[0221] The analyte is preferably a polynucleotide, such as a nucleic acid, which is defined as a macromolecule containing two or more nucleotides. Nucleic acids are particularly suitable for nanopore sequencing. Naturally occurring nucleic acid bases in DNA and RNA can be distinguished by their physical size. When a nucleic acid molecule or a single base passes through the channel of a nanopore, the size difference between the bases results in a directly related reduction in the ion flow through the channel. Changes in ion flow can be recorded. Suitable electrical measurement techniques for recording changes in ion flow are discussed above. Through appropriate calibration, the characteristic reduction of ion flow can be used to identify specific nucleotides and related bases passing through the channel in real time. In typical nanopore nucleic acid sequencing, due to partial obstruction of the channel by nucleotides, as the individual nucleotides of the nucleic acid sequence of interest pass through the channel of the nanopore in sequence, the open channel ion flow decreases. This reduction in ion flow is measured using the above-mentioned suitable recording techniques. The reduction in ion flow can be calibrated to the reduction in the measured ion flow of known nucleotides passing through the channel, thereby generating a means for determining which nucleotide is passing through the channel, and therefore, when performed sequentially, a way to determine the nucleotide sequence of the nucleic acid passing through the nanopore is provided. In order to accurately determine a single nucleotide, it is generally necessary that the reduction in ion flow through the channel is directly related to the size of the single nucleotide passing through the contraction. It should be understood that the complete nucleic acid polymer that "passes through" the hole via the action of, for example, a related polymerase can be sequenced. Alternatively, the sequence can be determined by the passage of triphosphate nucleotide bases that have been sequentially removed from the target nucleic acid near the hole (see, for example, WO2014 / 187924, which is incorporated herein by reference in its entirety).

[0222] A polynucleotide or nucleic acid may comprise any combination of any nucleotides. Nucleotides may be naturally occurring or artificial. One or more nucleotides in a polynucleotide may be oxidized or methylated. One or more nucleotides in a polynucleotide may be damaged. For example, a polynucleotide may comprise a pyrimidine dimer. Such dimers are often associated with damage caused by ultraviolet light and are the main cause of skin melanoma. One or more nucleotides in a polynucleotide may be modified, for example, with a marker or tag, suitable examples of which are known to the skilled person. A polynucleotide may comprise one or more spacers. Nucleotides typically contain a nucleobase, a sugar, and at least one phosphate group. Nucleobases and sugars form nucleosides. Nucleobases are typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines, and more specifically include adenine (A), guanine (G), thymine (T), uracil (U), and cytosine (C). Sugars are typically pentoses. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. Sugar is preferably deoxyribose. The polynucleotide preferably comprises the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and / or thymidine (dT), deoxyguanosine (dG) and deoxycytidine (dC). Nucleotides are generally ribonucleotides or deoxyribonucleotides. Nucleotides generally contain monophosphate, diphosphate or triphosphate. Nucleotides may contain more than three phosphates, such as 4 or 5 phosphates. Phosphoric acid may be attached to the 5' or 3' side of the nucleotide. The nucleotides in the polynucleotide may be attached to each other in any manner. Nucleotides are generally attached by their sugar and phosphate groups, as in nucleic acids. Nucleotides may be attached by their core bases, as in pyrimidine dimers. Polynucleotides may be single-stranded or double-stranded. At least a portion of the polynucleotide is preferably double-stranded. The polynucleotide is most preferably ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In particular, the method of using a polynucleotide as an analyte alternatively comprises determining one or more characteristics selected from the group consisting of: (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide and (v) whether the polynucleotide is modified.

[0223] Polynucleotides can have any length (i). For example, the length of a polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400 or at least 500 nucleotides or nucleotide pairs. The length of a polynucleotide can be 1000 or more nucleotides or nucleotide pairs, 5000 or more nucleotides or nucleotide pairs or 100000 or more nucleotides or nucleotide pairs. Any number of polynucleotides can be studied. For example, the method can involve characterizing 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more polynucleotides. If characterizing two or more polynucleotides, they can be different polynucleotides or two examples of the same polynucleotide. Polynucleotides can be naturally occurring or artificial. For example, the method can be used to verify the sequence of the oligonucleotides manufactured. The method is usually carried out in vitro.

[0224] The nucleotide may have any identity (ii) and includes, but is not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), 5-methylcytidine monophosphate, 5-hydroxymethylcytidine monophosphate, cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP), deoxycytidine monophosphate (dCMP), and deoxymethylcytidine monophosphate. The nucleotide is preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP, and dUMP. The nucleotide may be abasic (i.e., lack a nucleobase). The nucleotide may also lack a nucleobase and a sugar (i.e., be a C3 spacer). The sequence of nucleotides (iii) is determined by the consecutive identities of subsequent nucleotides attached to each other in the 5' to 3' direction of the chain throughout the polynucleotide strain.

[0225] The pore complexes and pore polymers of the invention are particularly useful in the analysis of homopolymers. For example, they can be used to determine the sequence of a polynucleotide comprising two or more, such as at least 3, 4, 5, 6, 7, 8, 9 or 10 identical consecutive nucleotides. For example, they can be used to sequence a polynucleotide comprising polyA, polyT, polyG and / or polyC regions.

[0226] The CsgG pore constriction consists of residues at positions 51, 55, and 56 of SEQ ID NO:3. The constrictions of CsgG and its constriction mutants are generally sharp. As DNA passes through the constriction, approximately 5 bases of DNA interacting with the constriction of the pore dominate the current signal at any given time. While these sharper constrictions are very good at reading mixed sequence regions of DNA (when A, T, G, and C are mixed), when homopolymeric regions (e.g., polyT, polyG, polyA, polyC) are present within the DNA, the signal becomes flat and lacks information. Because 5 bases dominate the signal of CsgG and its constriction mutants, photopolymers longer than 5 bases are difficult to discern without the use of additional residence time information. However, if the DNA is passing through a second constriction formed by the CsgF peptide, more DNA bases will interact with the combined constriction, increasing the length of homopolymers that can be discerned.

[0227] Preferably, a polynucleotide binding protein is used to control the movement of the polynucleotide relative to the hole (such as through the hole). Suitable proteins are discussed in more detail below. The present invention provides a method for determining the presence, absence or one or more characteristics of a target polynucleotide, the method comprising the following steps:

[0228] (i) contacting a target polynucleotide with a pore complex of the invention or a pore multimer of the invention and a polynucleotide binding protein, such that the polynucleotide binding protein controls movement of the target analyte relative to (such as through) the pore complex or pore multimer; and

[0229] (ii) taking one or more measurements as the polynucleotide moves relative to (such as through) the pore complex or pore polymer,

[0230] And thereby determine the presence, absence or one or more characteristics of the polynucleotide.

[0231] In any of these methods, one or more characteristics of the target analyte are preferably measured by electrical and / or optical measurements. The electrical measurements are current measurements, impedance measurements, tunneling measurements or field effect transistor (FET) measurements. The method preferably comprises measuring the current flowing through the pore complex or pore multimer as the analyte moves relative to (such as through) the pore.

[0232] General conditions for carrying out the methods of the invention are discussed in more detail below with reference to the kits and systems of the invention.

[0233] Polynucleotides of the present invention

[0234] The present invention also provides a polynucleotide encoding a pore monomer conjugate of the present invention or a construct of the present invention. The polynucleotide may be any of those discussed above. The present invention also provides an expression vector comprising the polynucleotide of the present invention. The present invention also provides a host cell comprising the polynucleotide of the present invention or a host cell of the present invention. Suitable vectors and host cells are known in the art.

[0235] Reagent test kit

[0236] The present invention also provides a kit for characterizing a target analyte. In one embodiment, the kit includes (a) a pore complex of the present invention or a pore multimer of the present invention and (b) a component of a membrane. Suitable membranes and components are discussed below.

[0237] In another embodiment, the kit comprises (a) a pore complex of the present invention or a pore polymer of the present invention and (b) a polynucleotide binding protein. The kit preferably further comprises components of a membrane. The kit may comprise components of any type of membrane, such as an amphiphilic layer or a triblock copolymer membrane. Preferred polynucleotide binding proteins are polymerases, exonucleases, helicases and topoisomerases, such as gyrase. Suitable enzymes include, but are not limited to, exonuclease I from Escherichia coli, exonuclease III from Escherichia coli, RecJ from thermophilic Streptococcus and bacteriophage lambda exonucleases, TatD exonucleases and variants thereof. Three subunits comprising the RecJ sequence from thermophilic Streptococcus or a variant thereof interact to form a trimer exonuclease. The polymerase may be 3173 DNA polymerase (which can be obtained from Corporation), SD polymerase (available from The enzyme may be a Phi29 DNA polymerase or a variant thereof. The topoisomerase is preferably a member of any one of the partial classification (EC) groups 5.99.1.2 and 5.99.1.3.

[0238] The enzyme is most preferably derived from a helicase, such as Hel308 Mbu, Hel308 Csy, Hel308 Tga, Hel308 Mhu, TraI Eco, XPD Mbu or a variant thereof. Any helicase can be used in the present invention. The helicase can be or be derived from a Hel308 helicase, a RecD helicase, such as a TraI helicase or a TrwC helicase, an XPD helicase or a Dda helicase. The helicase can be any one of a helicase, a modified helicase or a helicase construct, disclosed in WO 2013 / 057495; WO 2013 / 098562; WO 2013098561; WO 2014 / 013260; WO 2014 / 013259; WO 2014 / 013262 and WO 2015 / 055981. All of these are incorporated by reference in their entirety.

[0239] The kit may further comprise one or more anchors, such as cholesterol, for coupling the target analyte to the membrane. The kit may further comprise one or more polynucleotide adapters, which may be attached to the target polynucleotide to facilitate characterization of the polynucleotide. Anchors, such as cholesterol, are preferably attached to the polynucleotide adapters.

[0240] The test kit may additionally include one or more other reagents or instruments that enable any of the above embodiments to be performed. Such reagents or instruments include one or more of the following: a suitable buffer (aqueous solution), an instrument (such as a container or instrument comprising a needle) for obtaining a sample from a subject, an instrument or voltage or a patch clamp device for amplifying and / or expressing polynucleotides. Reagents may be present in the test kit in a dry state so that the fluid sample resuspends the reagent. The test kit may also optionally include instructions for enabling the test kit to be used for the method of the present invention or details about which organism the method may be used for. Finally, the test kit may also include other components useful in characterizing the analyte.

[0241] equipment

[0242] The present invention also provides a device for characterizing a target analyte in a sample, the device comprising (a) a plurality of pore complexes of the invention or a plurality of pore multimers of the invention, and (b) a plurality of polynucleotide binding proteins. The plurality of pore complexes or the plurality of pore multimers may be any of those described above.

[0243] The invention also provides a device comprising a pore complex according to the invention or a pore multimer according to the invention inserted into an extracorporeal membrane.

[0244] The present invention also provides a device produced by a method comprising the following steps: (i) obtaining the pore complex of the present invention or the pore multimer of the present invention, and (ii) contacting the pore complex or the pore multimer with an extracorporeal membrane such that the pore complex or the pore multimer is inserted into the extracorporeal membrane.

[0245] Any of the specific embodiments discussed above are equally applicable to the apparatus of the present invention.

[0246] Array

[0247] The present invention also provides an array comprising a plurality of membranes of the present invention. Any embodiment discussed above with respect to the membrane of the present invention is also applicable to the array of the present invention. The array can be configured to perform any one of the following methods.

[0248] In preferred embodiments, each membrane in the array comprises one pore complex or pore polymer. Due to the manner in which the array is formed, for example, the array may comprise one or more membranes that do not comprise a pore complex or pore polymer, and / or one or more membranes that comprise two or more pore complexes or polymers. The array may comprise from about 2 to about 1000 layers, such as from about 10 to about 800 layers, from about 20 to about 600 layers, or from about 30 to about 500 layers of membrane.

[0249] system

[0250] The present invention provides a system comprising (a) a membrane of the present invention or an array of the present invention, (b) a device for applying an electric potential across the membrane, and (c) a device for detecting an electric or optical signal across the membrane.

[0251] The pores and membranes may be any pores and membranes as described above and below.

[0252] In one embodiment, the system further comprises a first chamber and a second chamber, wherein the first chamber and the second chamber are separated by a membrane. When used to characterize a target analyte, the system may further comprise a target analyte, wherein the target analyte is transiently located in the continuous channel, and wherein one end of the target analyte is located in the first chamber, and one end of the target analyte is located in the second chamber. The target analyte is preferably a target polypeptide or a target polynucleotide.

[0253] In one embodiment, the system further includes a conductive solution in contact with the hole, an electrode providing a voltage potential across the membrane, and a measurement system for measuring the current through the hole. The voltage applied across the membrane and the hole is preferably +5V to -5V, such as -600mV to +600mV or -400mV to +400mV. The voltage used is preferably in the range of 100mV to 240mV and more preferably in the range of 120mV to 220mV. By using an increased applied potential, the distinction between different amino acids or nucleotides in the hole can be increased. Any suitable conductive solution can be used. For example, the solution can include a charge carrier, such as a metal salt, such as an alkali metal salt, a halide salt, such as a chloride salt, such as an alkali metal chloride salt. The charge carrier can include an ionic liquid or an organic salt, such as tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride or 1-ethyl-3-methylimidazolium chloride. In an exemplary system, the salt is present in the aqueous solution in the chamber. Potassium chloride (KCl), sodium chloride (NaCl), cesium chloride (CsCl) or a mixture of potassium ferrocyanide and potassium ferrocyanide is commonly used. KCl, NaCl and a mixture of potassium ferrocyanide and potassium ferrocyanide are preferred. The charge carriers may be asymmetric across the membrane. For example, the type and / or concentration of charge carriers may be different on each side of the membrane, e.g., in each chamber.

[0254] The salt concentration may be in a saturated state. The salt concentration may be 3 M or less, and 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 1.6 M, or 1 M to 1.4 M. The salt concentration is preferably 150 mM to 1 M. The method is preferably performed using a salt concentration of at least 0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M, or at least 3.0 M. High salt concentrations provide a high signal-to-noise ratio and allow identification of current indicating the presence of amino acids or nucleotides against the background of normal current fluctuations.

[0255] A buffer may be present in the conductive solution. Typically, the buffer is a phosphate buffer. Other suitable buffers are HEPES and Tris-HCl buffers. The pH of the conductive solution may be 4.0 to 12.0, 4.5 to 10.0, 5.0 to 9.0, 5.5 to 8.8, 6.0 to 8.7, or 7.0 to 8.8, or 7.5 to 8.5. The pH used is preferably about 7.5.

[0256] The system can be included in an apparatus. The apparatus can be any conventional apparatus for analyte analysis, such as an array or a chip. The apparatus is preferably configured to perform the disclosed method. For example, the apparatus can include a chamber containing an aqueous solution and a barrier that divides the chamber into two sections. The barrier typically has a hole in which a membrane containing the hole is formed. Alternatively, the barrier forms a membrane in which there are holes.

[0257] The device may also include circuitry capable of applying an electrical potential and measuring the electrical signal across the membrane and pore.

[0258] The apparatus may be any of those described in WO 2008 / 102120, WO 2009 / 077734, WO 2010 / 122293, WO 2011 / 067559 or WO 00 / 28312 (all of which are incorporated herein by reference in their entirety).

[0259] membrane

[0260] Any suitable membrane can be used in the system. The membrane is preferably an amphiphilic layer. The amphiphilic layer is a layer formed by amphiphilic molecules such as phospholipids having hydrophilic and lipophilic properties. The amphiphilic molecules can be synthetic or naturally occurring. Non-naturally occurring amphiphiles and amphiphiles forming a monolayer are known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). Block copolymers are polymeric materials in which two or more monomer subunits are polymerized together to produce a single polymer chain. Block copolymers generally have properties contributed by each monomer subunit. However, block copolymers can have unique properties that polymers formed by individual subunits do not have. Block copolymers can be designed so that one of the monomer subunits is hydrophobic (i.e., lipophilic), while the other subunits are hydrophilic when in an aqueous medium. In this case, block copolymers can have amphiphilic properties and can form a structure that simulates a biofilm. The block copolymers may be diblock (which consists of two monomer subunits), but may also be constructed from more than two monomer subunits to form more complex arrangements that behave as amphiphiles. The copolymers may be triblock, tetrablock or pentablock copolymers. The membrane is preferably a triblock copolymer membrane.

[0261] The membrane is most preferably one of the membranes disclosed in International Application No. WO 2014 / 064443 or WO 2014 / 064444.

[0262] The amphiphilic molecule can be chemically modified or functionalized to facilitate coupling to the polynucleotide. The amphiphilic layer can be a single layer or a double layer. The amphiphilic layer is generally planar. The amphiphilic layer can be curved. The amphiphilic layer can be supported.

[0263] Amphiphilic membranes are usually naturally mobile, essentially acting as a -8 cm s -1 This means that the pore and the coupled polynucleotide can move normally within the amphiphilic membrane.

[0264] The membrane can be a lipid bilayer. The lipid bilayer is a model of a cell membrane and is used as an excellent platform for a range of experimental studies. For example, a lipid bilayer can be used for in vitro studies of membrane proteins by single channel recording. Alternatively, a lipid bilayer can be used as a biosensor for detecting the presence of a range of substances. The lipid bilayer can be any lipid bilayer. Suitable lipid bilayers include, but are not limited to, planar lipid bilayers, supported bilayers or liposomes. The lipid bilayer is preferably a planar lipid bilayer. Suitable lipid bilayers are disclosed in WO 2008 / 102121, WO 2009 / 077734 and WO 2006 / 100484 (all incorporated herein by reference in their entirety).

[0265] The film preferably comprises a solid layer. The solid layer can be formed of organic and inorganic materials, including but not limited to microelectronic materials, insulating materials (such as Si3N4, Al2O3 and SiO), organic and inorganic polymers (such as polyamide), plastics (such as ) or elastomers (such as two-component addition-cured silicone rubber) and glass. The solid layer can be formed of graphene. Suitable graphene layers are disclosed in WO2009 / 035647 (which is incorporated herein by reference in its entirety). If the membrane comprises a solid layer, the pores are typically present in the amphiphilic membrane or layer, which is contained within the solid layer, such as holes, holes, gaps, channels, grooves or gaps within the solid layer. A skilled person can prepare a suitable solid / amphiphilic hybrid system. Suitable systems are disclosed in WO 2009 / 020682 and WO 2012 / 005857 (both of which are incorporated herein by reference in their entirety). Any of the amphiphilic membranes or layers discussed above can be used.

[0266] The method is typically performed using (i) an artificial amphiphilic layer comprising a pore, (ii) an isolated naturally occurring lipid bilayer comprising a pore, or (iii) a cell having a pore inserted therein. The method is typically performed using an artificial amphiphilic layer, such as an artificial triblock copolymer layer. The layer may contain other transmembrane and / or intramembrane proteins and other molecules in addition to the pore. Suitable equipment and conditions are discussed below. The method of the invention is typically performed in vitro.

[0267] Sequence Listing

[0268] SEQ ID NO: 1 (>P0AEA2; coding sequence of WT CsgG from E. coli K12)

[0269] ATGCAGCGCTTATTTCTTTTGGTTGCCGTCATGTTACTGAGCGGATGCTTAACCGCCC

[0270] CGCCTAAAGAAGCCGCCAGACCGACATTAATGCCTCGTGCTCAGAGCTACAAAGATT

[0271] TGACCCATCTGCCAGCGCCGACGGGTAAAATCTTTGTTTCGGTATACAACATTCAGGA

[0272] CGAAACCGGGCAATTTAAACCCTACCCGGCAAGTAACTTCTCCACTGCTGTTCCGCA

[0273] AAGCGCCACGGCAATGCTGGTCACGGCACTGAAAGATTCTCGCTGGTTTATACCGCT

[0274] GGAGCGCCAGGGCTTACAAAACCTGCTTAACGAGCGCAAGATTATTCGTGCGGCAC

[0275] AAGAAAACGGCACGGTTGCCATTAATAACCGAATCCCGCTGCAATCTTTAACGGCGG

[0276] CAAATATCATGGTTGAAGGTTCGATTATCGGTTATGAAAGCAACGTCAAATCTGGCGG

[0277] GGTTGGGGCAAGATATTTTGGCATCGGTGCCGACACGCAATACCAGCTCGATCAGAT

[0278] TGCCGTGAACCTGCGCGTCGTCAATGTGAGTACCGGCGAGATCCTTTCTTCGGTGAA

[0279] CACCAGTAAGACGATACTTTCCTATGAAGTTCAGGCCGGGGTTTTCCGCTTTATTGAC

[0280] TACCAGCGCTTGCTTGAAGGGGAAGTGGGTTACACCTCGAACGAACCTGTTATGCTG

[0281] TGCCTGATGTCGGCTATCGAAACAGGGGTCATTTTCCTGATTAATGATGGTATCGACC

[0282] GTGGTCTGTGGGATTTGCAAAATAAAGCAGAACGGCAGAATGACATTCTGGTGAAAT

[0283] ACCGCCATATGTCGGTTCCACCGGAATCCTGA

[0284] SEQ ID NO:2(>P0AEA2(1:277); WT Pro-CsgG from Escherichia coli K12)

[0285] MQRLFLLVAVMLLSGCLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDET

[0286] GQFKPYPASNFSTAVPQSATAMLVTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTV

[0287] AINNRIPLQSLTAANIMVEGSIIGYESNVKSGGVGARYFGIGADTQYQLDQIAVNLRVVN

[0288] VSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGEVGYTSNEPVMLCLMSAIETGVIF

[0289] LINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPES

[0290] SEQ ID NO:3(>P0AEA2(16:277); Mature CsgG from Escherichia coli K12)

[0291] CLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQFKPYPASNFSTAVP

[0292] QSATAMLVTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIM

[0293] VEGSIIGYESNVKSGGVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILS

[0294] YEVQAGVFRFIDYQRLLEGEVGYTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNK

[0295] AERQNDILVKYRHMSVPPES

[0296] SEQ ID NO:4(>P0AE98; Coding sequence of WT CsgF from Escherichia coli K12)

[0297] ATGCGTGTCAAACATGCAGTAGTTCTACTCATGCTTATTTCGCCATTAAGTTGGGCTG

[0298] GAACCATGACTTTCCAGTTCCGTAATCCAAACTTTGGTGGTAACCCAAATAATGGCGC

[0299] TTTTTTATTAAATAGCGCTCAGGCCCAAAACTCTTATAAAGATCCGAGCTATAACGAT

[0300] GACTTTGGTATTGAAACACCCTCAGCGTTAGATAACTTTACTCAGGCCATCCAGTCAC

[0301] AAATTTTAGGTGGGCTACTGTCGAATATTAATACCGGTAAACCGGGCCGCATGGTGAC

[0302] CAACGATTATATTGTCGATATTGCCAACCGCGATGGTCAATTGCAGTTGAACGTGACA

[0303] GATCGTAAAACCGGACAAACCTCGACCATCCAGGTTTCGGGTTTACAAAATAACTCA

[0304] ACCGATTTT

[0305] SEQ ID NO:5(>P0AE98(1:138); WT Pro-CsgF from Escherichia coli K12)

[0306] MRVKHAVVLLMLISPLSWAGTMTFQFRNPNFGGNPNNGAFLLNSAQAQNSYKDPSYN

[0307] DDFGIETPSALDNFTQAIQSQILGGLLSNINTGKPGRMVTNDYIVDIANRDGQLQLNVTD

[0308] RKTGQTSTIQVSGLQNNSTDF

[0309] SEQ ID NO:6 (>P0AE98 (20:138); WT mature CsgF from E. coli K12)

[0310] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNSYKDPSYNDDFGIETPSALDNFTQAIQSQI

[0311] LGGLLSNINTGKPGRMVTNDYIVDIANRDGQLQLNVTDRKTGQTSTIQVSGLQNNSTDF

[0312] The following examples illustrate the present invention. It should be understood that although specific embodiments, specific configurations, and materials and / or molecules have been discussed herein for engineered cells and methods according to the present invention, various changes or modifications may be made in form and detail without departing from the scope and spirit of the present invention. The following examples are provided to better illustrate specific embodiments and should not be construed as limiting the present application. The present application is limited only by the claims.

[0313] Example 1

[0314] Detailed methods for making and testing mutant CsgG pores and mutant CsgG / CsgF complexes are described in WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317, WO 2017 / 149318, WO 2018 / 211241 and WO 2019 / 002893 (all of which are incorporated herein by reference in their entirety).

[0315] E. coli CsgG pore production

[0316] Recombinant expression vectors encoding CsgG variant nanopores with a C-terminal Strep affinity tag and an ampicillin resistance gene were transformed into chemically competent E. coli cells. The cells were plated onto LB agar plates containing appropriate antibiotics for selection. A single colony from the agar plate was inoculated into LB medium containing antibiotics and grown overnight. The culture was diluted into autoinduction medium plus necessary antibiotics and incubated at 18°C ​​for 68 hours. The cells were harvested by centrifugation and then lysed and extracted into 1x Bugbuster extraction reagent (Merck 70921) and 0.1% DDM. The pores were purified from the supernatant using affinity chromatography, heat treatment and size exclusion chromatography to select oligomeric nanopores as judged by SDS-PAGE.

[0317] CsgG / CsgF complex formation protocol

[0318] CsgG-CsgF complexes were prepared from nanopores purified as described above and chemically synthesized CsgF peptides, either modified with sulfonyl fluoride or not. The nanopores were buffer exchanged into pH 7.0 buffer minus the reducing agent and incubated at 25°C for 1 hour in an 8-fold molar excess of peptide relative to CsgG monomer. The reaction was terminated by heating at 60°C for 15 minutes, followed by centrifugation to remove any precipitate, and DTT was added to 5 mM to prevent any further reaction. Figure 3 :SDS_PAGE analysis – By heating

[0319] 300 ng of the complex and CsgG-only well controls were added to each 0.5 mL Protein LoBind Eppendorf tube (Fisher, 10316752) and made up to 10 μL volume with reaction buffer. The final volume was made up to 20 μL by adding 10 uL of 2x Laemmli buffer. Each sample was loaded in its entirety onto a 4%-20% TGX gel (BioRad, 5671093) run with 1x TGS buffer (Sigma, T7777). Run at 300 V for 21 minutes. To image the gel, Spyro Ruby (Merk, S4942) stain was used according to the manufacturer's instructions. It was then imaged on a GE Typhoon gel imager using a 450 nm laser.

[0320] Figures 4 to 14 : DNA curve (i.e., DNA translocation current trace)

[0321] Electrical measurements were taken from CsgG-only and CsgG / CsgF complexes inserted into MinION flow cells. After the individual pores were inserted into the block copolymer membrane, 1 mL of a buffer containing 25 mM potassium phosphate, 150 mM potassium ferrocyanide (II), 150 mM potassium ferrocyanide (III), pH 8.0 was flowed through the system to remove any excess nanopores.

[0322] As previously described, Y adapters were prepared by annealing DNA oligonucleotides (WO 2016 / 034591, which is incorporated herein in its entirety). The DNA motor was loaded and closed on the adapter. The subsequent material was subjected to HPLC purification. The Y adapter contained a 30C3 leader for easier capture by the nanopore and a side arm for tethering to the membrane.

[0323] The analyte used to assess the DNA profile was a 3.6 kilobase DNA fragment from the 3' end of the lambda genome. Preparation of the analyte, ligation of the analyte to the Y adaptor, SPRI bead cleanup of the ligated analyte, and addition to the minION flow cell were performed using the Oxford Nanopore Technologies Q-SQK-LSK109 protocol.

[0324] Electrical measurements were obtained using a minION Mk1b from Oxford Nanopore Technologies. Standard sequencing scripts were run at -180 mV for 2 to 6 hours with static flicking every 5 minutes to remove extended nanopore blocks. Raw data were collected in batch FAST5 files using MinKNOW software (Oxford Nanopore Technologies). At least 150 wells per flow cell were tested for each well type.

[0325] Fig.14 Summary of data shown:

[0326]

[0327] Synthesis of peptides containing arylsulfonyl fluoride ( Figure 2 B, C, D and E)

[0328] CsgF(NH2-GTMTFQFRNPNFGGNPNNGAFLLNSAQAQ K -CONH2) (SEQ ID NO: 7; K Lys containing sulfonyl fluoride

[0329] Peptides were synthesized on Rink Amide ChemMatrix resin (0.48 mmol / g, 0.1 mmol scale) using an automated microwave peptide synthesizer (Biotage Alstra+Initiator). A standard Fmoc solid phase peptide synthesis protocol was used, except that Fmoc-Lys(Mmt)-OH was used for Lys and Boc-Gly-OH was used for N-terminal Gly. The deprotection step was performed at 70°C with 20% 4-methylpiperidine in DMF (4.5 mL) for 5 minutes, and each coupling step was performed at 75°C with a DMF solution of Fmoc-protected amino acids (5 eq), HCTU (4.98 eq) and DIPEA (10 eq) for 5 minutes. After the synthesis was complete, the Mmt protecting group was selectively removed by treating the resin with a solution of AcOH:TFE:DCM (1:2:7) (5 mL) for 5 minutes, and the process was repeated 4 times. After washing with DCM, the arylsulfonyl fluoride group was introduced according to the literature protocol (Hoppmann, C.; Wang, L., Proximity-enabled bioreactivity togenerate covalent peptide inhibitors of p53-Mdm4. Chem Commun (Camb) 2016, 52 (29), 5140-3). In short, the resin was treated with a DMF (2.5 mL) solution of a carboxylic acid (5 eq) containing arylsulfonyl fluoride at room temperature, followed by treatment with a DMF (5 eq) and DIPEA (10 eq) solution of PyBOP and mixing for 30 minutes, followed by washing with DMF. The final deprotection and cleavage of the peptide from the resin was performed using TFA / H2O / TIS (95 / 2.5 / 2.5, v / v). After TFA was evaporated by a nitrogen stream, the crude peptide was precipitated by adding cold ether and purified on a reversed phase C4 column (Vydac). The composition and purity of the peptides were confirmed by MALDI-TOF and analytical HPLC (Phenomenex Jupiter 5um C18, 4.6x259 mm, 5% to 100% B in 20 min, flow rate: 1 mL / min).

[0330] CsgF-K30-meta-SO2F-del(S31-F119) observed m / z 3426.98, calculated m / z 3427.70

[0331] CsgF-K30-meta-SO2F-del(S31-F119) observed m / z 3427.76, calculated m / z 3427.70

[0332] CsgF-K30-CH2-para-SO2F-del(S31-F119) observed m / z 3442.6, calculated m / z 3442.7 picture Synthesis of peptide A containing aryl fluorosulfate

[0333] CsgF(NH2-GTMTFQFRNPNFGGNPNNGAFLLNSAQAQ K -CONH2))(SEQ ID NO:7; K Lys containing sulfonyl fluoride

[0334] Peptides were synthesized on Rink Amide ChemMatrix resin (0.48 mmol / g, 0.1 mmol scale) using an automated microwave peptide synthesizer (Biotage Alstra+Initiator). A standard Fmoc solid phase peptide synthesis protocol was used, except that Fmoc-Lys(Mmt)-OH was used for Lys and Boc-Gly-OH was used for N-terminal Gly. The deprotection step was performed at 70°C with 20% 4-methylpiperidine in DMF (4.5 mL) for 5 minutes, and each coupling step was performed at 75°C with a DMF solution of Fmoc-protected amino acids (5 eq), HCTU (4.98 eq) and DIPEA (10 eq) for 5 minutes. After the synthesis was complete, the Mmt protecting group was selectively removed by treating the resin with a solution of AcOH:TFE:DCM (1:2:7) (5 mL) for 5 minutes, and the process was repeated 4 times. The introduction of fluorosulfate groups was also carried out according to the literature protocol (Baggio, C.; Udompholkul, P.; Gambini, L.; Salem, AF; Jossart, J.; Perry, JJP; Pellecchia, M., Aryl-fluorosulfate-based Lysine Covalent Pan-Inhibitors of Apoptosis Protein (IAP) Antagonists with Cellular Efficacy. J Med Chem 2019, 62 (20), 9188-9200. Gambini, L.; Baggio, C.; Udompholkul, P.; Jossart, J.; Salem, AF; Perry, JJP; Pellecchia, M., Covalent Inhibitors of Protein-Protein Interactions Targeting Lysine, Tyrosine, or Histidine Residues. J Med Chem 2019, 62(11), 5616-5627). Briefly, after removal of the Mmt group with AcOH:TFE:DCM (1:2:7), the resin was treated with a mixture of 3-hydroxybenzoic acid (10 eq), HCTU (9.8 eq) and DIPEA (20 eq) in DMF (5 mL) for 12 h. After washing with DMF and then DCM, the resin was treated with a solution of AISF (5 eq) and DBU (11 eq) in DCM (5 mL) overnight. Final deprotection and cleavage of the peptide from the resin was performed using TFA / H2O / TIS (95 / 2.5 / 2.5, v / v).After blowing off TFA by nitrogen flow, the crude peptide was precipitated by adding cold ether and purified on a reverse phase C4 column (Vydac). The composition and purity of the peptide were confirmed by MALDI-TOF and analytical HPLC (Phenomenex Jupiter 5um C18, 4.6x259 mm, 5% to 100% B in 20 minutes, flow rate: 1 mL / min).

[0335] CsgF-K30-meta-OSO2F-del(S31-F119) observed m / z 3444.38, calculated m / z 3443.70 Example 2

[0336] In this example, 3-SO2F is the same as m-SO2F, 4-SO2F is the same as p-SO2F, and 4-CH2SO2F is the same as CH2PH-P-SO2F.

[0337] introduce

[0338] Proximity labeling has become a powerful tool for probing molecular interactions and drug design. The method relies on noncovalent binding interactions to position a molecule with a reactive group in close proximity to a second molecule. Because high local concentrations greatly increase reaction rates, relatively non-reactive moieties can be used to ensure that reactions occur with precise regioselectivity and minimal background reactivity with solvents. Early examples such as aspirin were discovered by accident, but chemists now use a wide range of covalent labeling methods to purposefully design drugs or probe protein interactions. Sulfonyl fluorides, first introduced by Roberta Coleman, have proven particularly useful in this context, and sulfonyl fluorides and fluorosulfonates are now widely used as small molecule probes in chemical biology. These SuFEx groups have low reactivity to water, but react with nucleophilic side chains (particularly Tyr and Lys) when kept in close proximity by noncovalent binding interactions with other parts of the molecule. Peptides and proteins can also be modified as active site-directed reagents or molecular probes. For example, Powers and Kettner first introduced irreversible probes such as chloromethyl ketones and reversible covalent probes such as boronic acid to achieve effective and selective inhibition of proteases. Lei Wang and others have expanded the use of reactive proteins for proximity labeling by engineering the biosynthetic machinery of Escherichia coli to incorporate unnatural amino acids containing sulfonyl fluoride and benzyl fluoride into proteins, providing reagents for probing even transient protein-protein interactions and a new class of protein drugs. In addition, Fujimori and co-workers have developed methods to introduce sulfonyl fluoride into peptides as inhibitors of PhD domains in proteins. Proximity labeling in noncovalent assemblies has also been used to probe scientific questions such as the origin of life. However, despite a large number of successful applications in basic research and drug design, we are not aware of proximity labeling that can guide the formation of covalently cross-linked protein assemblies for practical applications in nanotechnology and engineering.

[0339] Here, we use proximity chemistry to stabilize an 18-subunit 300 kDa membrane protein complex with potential use in DNA sequencing. These pore-forming membrane protein complexes have considerable potential for applications ranging from single-molecule detection of small molecules in biology to nucleic acid sequencing in nanotechnology. Specifically, the protein complex is based on the CsgG non-polymeric channel ( Fig.17A to 17B), which is part of the curli biogenesis system and is used for research and translational applications. In this method, the motor protein feeds single-stranded DNA through the channel. When the DNA is translocated through the pore, it modulates the electrically detected ionic conductance in a sequence-specific manner. The CsgG:CsgF complex, together with its natural binding partner CsgF, forms the core apparatus of the curli secretion and assembly channel and consists of 18 proteins (9xCsgG+9xCsgF). The N-terminal region of CsgF is highly conserved and critical for complex formation. Truncation of this region of CsgF to a 30-35-mer peptide results in the formation of a complex that recapitulates the original contacts with CsgG, but then becomes substantially unstable due to the unstructured C-terminal region of the resulting peptide when it is truncated (e.g. CsgG:CsgF (1-35) Complex ratio CsgG:CsgF (1-30) The addition of CsgF subunits to the CsgG pore appears particularly advantageous for DNA sequencing because it extends the length and chemistry of the pore constriction, expanding attractive possibilities for higher fidelity in nucleic acid sequencing and increasing the platform for new sensing applications [Remaut, Nature Biotech 2020]. However, the development of derivatives of the CsgG:CsgF complex for practical applications has been challenged by issues associated with robust assembly in vitro. Covalent linkage of the CsgG and CsgF subunits would provide an attractive approach to increase stability and allow modular assembly of sensing modules. Nature's proximity ligation method involves the formation of a disulfide bond, in which non-covalent forces bring two thiols into close proximity for an oxidative coupling reaction. However, disulfides are unstable to reducing conditions often used in protein assays, and it would also be helpful to avoid reliance on the use of Cys residues, which could provide additional possibilities for orthogonal biocompatible reactions.

[0340] To address these limitations, we designed SuTide-sulfonyl fluoride-modified CsgF peptide derivatives that react fully with CsgG via proximity-enhanced ligation. Given the diversity of amino acid side chains that these probes can interact with, the design of SuTide, including their precise position in CsgF and the choice of the specific probes used, requires exquisite precision. Very high efficiency is necessary for the formation of robust CsgG / CsgF complexes, as even at 95% completion of the reaction, approximately half of the total CsgG pores have only eight CsgF modifications, resulting in complex instability and inhomogeneity in channel currents (based on a multinomial probability distribution). Therefore, the precision of the design approach is extremely demanding.

[0341] Nonetheless, we have successfully designed SuTides that react with CsgG subunits in essentially quantitative yields. The resulting covalent complexes are highly stable and can be inserted into the bilayer at significantly higher yields than the corresponding non-covalent complexes, resulting in up to a 2-fold increase in the fraction of bilayer-embedded stable covalent complexes compared to non-covalent complexes. These findings illustrate the potential of our design approach to achieve proximity-enhanced ligation for precise, high-yield construction of high-molecular-weight protein complexes. Finally, we employed molecular dynamics to explore the high efficiency of ligation, yielding insights that may aid future applications of SuFEx chemistry in a variety of molecular contexts.

[0342] result

[0343] Design and synthesis

[0344] To achieve efficient SuFEx labeling, we selected a benzenesulfonyl fluoride derivative of lysine based on previous random cross-linking studies showing that this linkage offers a compromise between flexibility and reactivity. To form a stable complex between the CsgG and CsgF peptides, we first identified positions that placed the probe at distances and angles that favored proximity-enhanced linkage between the probe and the target nucleophilic residues ( Fig.17 C). In addition, the introduced crosslinks should preserve the native structure of the protein and not block the ion-conducting pore. This requirement eliminated many potential locations for the target nucleophilic amino acid side chains in CsgG and the sulfonyl fluoride in the CsgF:CsgG complex. We selected Tyr196 on CsgG as the target nucleophile and the six C-terminal residues of the CsgF peptide as possible locations for the introduction of the sulfonyl fluoride warhead. The C-terminal residues between these residues and Tyr196 α -C α The distance is to Within the range ( Fig.17 D), which has been suggested as a rough guide to the optimal distance for sulfonyl fluoride-based proximity-enhanced ligation. (1-35) The distances and angles between the hydroxyl oxygen on Tyr196 on each of the six C-terminal residues of the peptide and the sulfonyl thiol of each probe and manual rotamer analysis identified position 30 as the preferred location for placement of the warhead.

[0345] By incorporating each different probe into WT CsgF (1-29)Three types of SuTide were synthesized by introducing a Lys residue at position 30 of the sequence of K30. Each introduced a 3- or 4-substituted sulfonyl-phenyl fluoride or 4-sulfonyl-benzyl fluoride into the primary amine of K30 via an amide bond (referred to as 3-SO2F-CsgF, 4-SO2F-CsgF, and 4-CH2SO2F-CsgF, respectively) ( Fig.17 E). The peptide was synthesized by solid phase peptide synthesis with a trityl protecting group on the C-terminal Lys, which was removed after chain assembly was complete. The trityl group was then deprotected and the resulting Lys30 amine coupled to the appropriate carboxylic acid containing the completed sulfonyl fluoride. The remaining protecting groups and concomitant removal from the resin were performed by treatment with trifluoroacetic acid, and the resulting SuTide was purified to homogeneity. We have not observed problems associated with the stability or hydrolysis of SuTide in acidic aqueous solutions or when stored in DMSO at -20°C. Reaction of SuTide with CsgG

[0346] When they were reacted overnight at an 8-fold molar excess (over CsgG monomer), each SuTide was found to react with preformed non-polymeric CsgG pore complexes in nearly quantitative yield ( Fig.18 A). We used mass spectrometry of tryptic digests of the resulting products to confirm the covalent adducts (data not shown). The intensity of the peptide containing the targeted Tyr196 (CsgG 191-198) was reduced 10- to 100-fold compared to the WT CsgG:CsgF complex (data not shown). This peptide (FIDYQR) also lacks other residues that can readily react with sulfonyl fluoride, confirming attachment to the targeted Tyr residue. Finally, the intensity of other surrounding peptide fragments rich in Lys residues (highly reactive with sulfonyl fluoride) was unaffected before and after trypsin treatment (data not shown). Together, these results demonstrate the regioselectivity of the reaction.

[0347] The time course of the reaction of each SuTide with CsgG was assessed by sampling time points via SDS-PAGE ( Fig.18 A) Reaction half-life of SuTide 3-SO2F-CsgF (t 1 / 2 ) is 0.66 hours; t of 4-SO2F-CsgF 1 / 2 The t of 4-CH2SO2F-CsgF is 1.5 h. 1 / 2 4 hours (corresponding to approximately k1=1, 0.46 and 0.17 hr respectively -1 The first-order rate constant ( Fig.18B). These differences may reflect contributions from both the intrinsic reactivity of the reactive groups and from their effective concentrations. To help dissect these effects, we measured the kinetics of the reaction of acetyl-Tyr-O-methyl ester (Ac-Tyr-OMe) with the n-butylamide form of 3-SO2F-CsgF (3-SO2F-But). In the presence of a large excess of sulfonyl fluoride (5.0 mM 3-SO2F-But, 0.5 mM Ac-Tyr-OMe), we observed a pseudo-first-order rate constant of 0.01 hr for the Ac-Tyr-Ome reaction. -1 (Data not shown). If the concentration of 3-SO2F-But is taken into account, the corresponding secondary rate constant k2 is 2M -1 hr -1 , providing a good indicator of the reactivity of 3-SO2F-bullet. The ratio of the first-order rate constant for the complex reaction of 3-SO2F-CsgF with CsgG to the second-order rate constant for the model reaction of 3-SO2F-But (k1 / k2) gives an effective concentration of 0.5M.

[0348] Although we did not determine the rates for the corresponding models of 4-SO2F-CsgF and 4-CH2SO2F-CsgF, an extensive comparison of the effects of these substituents and substitution patterns on the relative reaction rates of sulfonyl fluorides with Ac-Tyr-OMe is available from [Gilbert et al. ACS Chem. Bio 2023]. Linear scaling as in [Gilbert et al. ACS Chem. Bio 2023] provides an approximate calculation of the secondary rate constant, which is 2.4 M -1 hr -1 and 0.6M -1 hr -1 , allowing one to account for the differences in the intrinsic chemical reactivity of the warheads associated with the three probes. The corresponding C 有效 The pseudo-first-order rate constants of 4-SO2F-CsgF and 4-CH2SO2F-CsgF are 0.19 M and 0.28 M, respectively.

[0349] Molecular dynamics

[0350] Taking into account the differences in chemical reactivity, we next used molecular dynamics (MD) simulations to provide a kinetically defined C 有效 The CsgG-3-SO2F-CsgF complex is consistent with the expected dynamic ensemble of conformers observed in the CsgG-3-SO2F-CsgF complex. 有效Qualitative comparison of . Given that the sulfonyl fluoride is located on a flexible Lys residue at the C-terminus of CsgF, we expect significant local flexibility. MD therefore seems well suited to provide a reasonable estimate of the fraction of time that the reactive groups are in van der Waals contacts and at an angle that favours a displacement reaction. Clearly, more sophisticated calculations will be required to determine the absolute rates, but we expect that classical MD simulations may be able to provide insight into the origin of the high effective concentrations in the pre-reacted complex. All-atom simulations were performed at a simulation temperature of 293 K and the AMBER force field. Three independent 200 nanosecond simulations of the non-polymeric CsgG-SuTide complex were calculated, corresponding to a total simulation time of 3*9*200=5,400 nanoseconds, ensuring good sampling in the high nanosecond to low microsecond time scale. Examples of CsgG Tyr196 and 3-SO2F-CsgF in the top cluster are shown in Figure 2. Fig.19 A. In addition, the distance between the phenolic oxygen of Tyr and the sulfonyl group S (d O,S ) range is To about , indicating good sampling over a wide range of distances. Next, we constructed d O,S The radial distribution of the sulfonyl fluoride sulfur atom to determine the probability of finding a reactive phenol O within the van der Waals distance of the sulfonyl fluoride sulfur atom ( Fig.19 B).

[0351] These distributions were then calculated as probabilities per unit volume, from which the apparent molar concentration (C 表观 ). For 3-SO2F-CsgF, the calculated value corresponds to the close van der Waals contact The distance box C 表观 The value of 4-SO2F-CsgF and 4-CH2SO2F-CsgF is 0.5M and 1.1M respectively. Fig.19 C). These values ​​differ from each other by no more than a factor of three and are in consistent rank order. We next examined the angle θ between the introduced phenolic oxygen and the fluoride leaving group. O,S,F As d O,S In the SuFEx reaction, the nucleophile approaches the trans-fluoride. Therefore, θ O,S,F The value of is approximately between 140° and 180°, which is , it is expected to promote the response ( Fig.19 D) In ​​fact, all three SuTides O,S With θ O,S,F The 2D plots of α and β all show maximum values, and they are the highest for the most reactive peptide 3-SO2F-CsgF ( Fig.19 E).

[0352] Discussion and Conclusion

[0353] The fundamental purpose of this research was to develop methods to enhance the assembly, stability and yield of very large protein complexes. The use of sulfonyl fluorides for this purpose solves practical problems associated with the assembly of nanopores with customized pore linings, which can now be systematically varied to maximize the fidelity of DNA sequencing. In addition, this method provides a useful method for introducing multiple proteins or peptides for single-molecule detection and a potential platform for protein sequencing.

[0354] Several efforts to synthesize sulfonyl fluorides to enhance affinity and stability of peptide-protein complexes have been reported recently. However, placement of the warheads was empirically placed to maximize the yield of protein-peptide complexes. In this case, we performed a more systematic search of sequence positions and rotamers, and ultimately, successful placement depends heavily on our understanding of protein structure and chemical reactivity. However, we also wondered whether molecular dynamics calculations could provide insight into the high C 有效 and regioselectivity. We were pleasantly surprised to find that C 表观 With C 有效 There is reasonable absolute and rank order agreement between the two. Considering the distance and angle of approach of the nucleophilic side chain is very consistent with the observed reactivity. We anticipate that this approach will be useful in cases where there is significant flexibility between reactive groups, in which case MD guidance can be expected to lead to the design of highly reactive proximal positions. However, in cases where the nucleophile and warhead are less flexible, we expect stereoelectronic effects to become more dominant and QM / MM calculations will be required to accurately rank potential designs. While the work reported here focuses on sulfonyl fluorides, a variety of other chemistries, including benzyl fluorides, may be advantageous to explore.

Claims

1. A pore monomer conjugate comprising a CsgG pore monomer attached to a CsgF peptide, wherein the CsgF peptide is attached to the CsgG pore monomer via a sulfonyl fluoride-containing linker, a sulfonyltriazole-containing linker, a fluorosulfate-containing linker, or a fluoroacetamide-containing linker.

2. The pore monomer conjugate according to claim 1, wherein (a) the N-terminus or a residue at any one of positions 1 to 40 in the CsgF peptide is attached to the CsgG pore monomer via a linker, or (b) a residue at position 30 of the CsgF peptide or a residue at a position in the CsgF peptide corresponding to position 30 in SEQ ID NO: 6 is attached to the CsgG pore monomer via the linker.

3. The pore monomer conjugate of claim 1 or 2, wherein the CsgF peptide is attached to a residue in the loop-forming region of the CsgG pore monomer.

4. The pore monomer conjugate of claim 3, wherein the loop-forming region corresponds to positions 142-146 and 190-200 in SEQ ID NO:

3.

5. The pore monomer conjugate according to any one of the preceding claims, wherein the CsgF peptide is attached to a serine, lysine, threonine, tyrosine, histidine or cysteine ​​residue in the CsgG pore monomer via the linker.

6. The pore monomer conjugate according to any one of the preceding claims, wherein the CsgF peptide is covalently attached to the CsgG pore monomer via the linker.

7. A pore monomer conjugate comprising a CsgG pore monomer attached to a CsgF peptide, wherein the CsgF peptide is attached to a residue in the CsgG pore monomer corresponding to any one of positions 143, 146, 190, 192, 193, 194, 195, 196, 197, 198, 199 or 200 in SEQ ID NO:

3.

8. The pore monomer conjugate of claim 7, wherein the residue is a serine, lysine, threonine, tyrosine, histidine or cysteine ​​residue in the CsgG pore monomer and / or is modified with a reactive group.

9. The pore monomer conjugate of claim 7 or 8, wherein the CsgF peptide is attached to the CsgG pore monomer using (a) an amine reactive group such as a thioester, NHS-ester, pentafluorophenyl ester, benzyl halide, benzyl fluoride, sulfonyl halide, sulfonyl fluoride, halosulfate, fluorosulfate, sulfonyltriazole or boronic acid, or (b) an oxygen reactive group such as an alkyl halide, alkyl fluoride, sulfonyl halide, sulfonyl fluoride, halosulfate, fluorosulfate or sulfonyltriazole.

10. The pore monomer conjugate according to any one of claims 7 to 9, wherein the residue in the CsgF peptide defined in claim 2 is attached to the CsgG pore monomer.

11. The pore monomer conjugate according to any one of the preceding claims, wherein the CsgG pore monomer is a variant of SEQ ID NO: 3 and / or the CsgF peptide is a variant of SEQ ID NO:

6.

12. A construct comprising two or more covalently attached pore monomer conjugates according to any one of claims 1 to 11.

13. The construct of claim 12, wherein the pore monomer conjugate is genetically fused and / or attached via a linker.

14. A pore complex comprising at least one pore monomer conjugate according to any one of the preceding claims or at least one construct according to claim 12 or 13, wherein the CsgF peptide forms a constriction in the pore complex.

15. The pore complex of claim 14, wherein the pore complex is a homo-oligomer comprising 6 to 10 pore monomer conjugates according to any one of claims 1 to 11 or 1 to 5 constructs according to claims 12 or 13.

16. The pore complex of claim 14 or 15, wherein the CsgF peptide is inserted into the lumen of the pore complex.

17. A pore multimer comprising two or more pores, wherein at least one of the pores is a pore complex according to any one of claims 14 to 16.

18. The pore complex according to any one of claims 14 to 16 or the pore multimer according to claim 17, comprised in a membrane.

19. A membrane comprising the pore complex according to any one of claims 14 to 16 or the pore polymer according to claim 17.

20. A method for producing a pore monomer conjugate according to any one of claims 1 to 11, the method comprising attaching the CsgF peptide to the CsgG pore monomer.

21. A method for producing a pore complex according to any one of claims 14 to 16 or a pore multimer according to claim 17, the method comprising expressing at least one pore monomer conjugate according to any one of claims 1 to 11 or a construct according to claim 12 or 13 and sufficient pore monomers or constructs to form the pore complex or the pore multimer in a host cell; and allowing the pore complex or the pore multimer to form in the host cell.

22. A method for producing a pore complex according to any one of claims 14 to 16 or a pore multimer according to claim 17, the method comprising contacting at least one pore monomer conjugate according to any one of claims 1 to 11 or a construct according to claim 12 or 13 with sufficient pore monomers or constructs in vitro, and allowing the pore complex or the pore multimer to form.

23. A method for determining the presence, absence or one or more characteristics of a target analyte, the method comprising the steps of: (i) contacting the target analyte with the pore complex according to any one of claims 14 to 16 or the pore multimer according to claim 17, such that the target analyte moves relative to the pore complex or the pore multimer; as well as (ii) making one or more measurements as the analyte moves relative to the pore complex or the pore multimer and thereby determining the presence, absence or one or more characteristics of the analyte.

24. The method of claim 23, wherein the analyte is a peptide, polypeptide, polysaccharide, small organic or inorganic compound, such as a pharmacologically active compound, a toxic compound, and a pollutant.

25. The method of claim 24, wherein the analyte is a polynucleotide.

26. The method of claim 25, wherein the polynucleotide comprises at least one homopolymeric region.

27. The method of claim 25 or 26, comprising determining one or more characteristics selected from: (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide, and (v) whether the polynucleotide is modified.

28. A method of characterising a polynucleotide, a peptide or a polypeptide using the pore complex of any one of claims 14 to 16 or the pore multimer of claim 17.

29. Use of a pore complex according to any one of claims 14 to 16 or a pore multimer according to claim 17 for determining the presence, absence or one or more characteristics of a target analyte.

30. A polynucleotide encoding a pore monomer conjugate according to any one of claims 1 to 11 or a construct according to claim 12 or 13.

31. A kit for characterizing a target analyte, the kit comprising (a) a pore complex according to any one of claims 14 to 16 or a pore multimer according to claim 17, and (b) components of a membrane.

32. A kit for characterizing a target polynucleotide or a target polypeptide, the kit comprising (a) a pore complex according to any one of claims 14 to 16 or a pore multimer according to claim 17, and (b) a polynucleotide binding protein or a polypeptide handling enzyme.

33. A device for characterizing a target polynucleotide or a target polypeptide in a sample, the device comprising (a) a plurality of pore complexes according to any one of claims 14 to 16 or a plurality of pore multimers according to claim 17, and (b) a plurality of polynucleotide binding proteins or a plurality of polypeptide processing enzymes.

34. An array comprising a plurality of the membranes of claim 19.

35. A system comprising (a) a membrane according to claim 19 or an array according to claim 34, (b) a device for applying an electric potential across the membrane, and (c) a device for detecting an electrical signal or an optical signal across the membrane.

36. A device comprising a pore complex according to any one of claims 14 to 16 or a pore multimer according to claim 17 inserted into an extracorporeal membrane.

37. A device produced by a method comprising the following: (i) obtaining a pore complex according to any one of claims 14 to 16 or a pore polymer according to claim 17, and (ii) contacting the pore complex or the pore polymer with an extracorporeal membrane so that the pore complex or the pore polymer is inserted into the extracorporeal membrane.

Images