De novo hole

Abstract

Aspects of the present disclosure relate to protein pore complexes and their use in analyte detection and characterization. The present disclosure is based, in part, on a nanopore complex formed from a CsgG-like pore and one or more helper proteins that form one or more channel contractions in the nanopore complex. In some embodiments, the one or more helper proteins are fusion proteins. The disclosure further relates to methods for designing helper proteins and producing the nanopore complexes and for use in molecular sensing and analyte sequencing applications.

Description

Background Art

[0001] Two important components of polymer characterization using nanopore sensing are (1) controlling the movement of the polymer through the pore, and (2) discerning the constituent building blocks as the polymer moves through the pore. During nanopore sensing, the narrowest portion of the pore forms a constriction, which is the most discriminating portion of the nanopore with respect to the current signature as a function of the analyte passing through it. 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 detecting and characterizing analytes. Mutations of the wild-type CsgG pore that improve the properties of the pore in this context have also been disclosed (WO2016 / 034591, WO2017 / 149316, WO2017 / 149317 and WO2017 / 149318, PCT / GB2018 / 051191, all of which are incorporated herein by reference in their entirety).

[0002] For analytes that are polynucleotides, nucleotide discrimination is achieved by passing through this mutant pore, but the current signature has been shown to be sequence-dependent, and multiple nucleotides contribute to the observed current, so the height of the channel constriction and the extent of the interaction surface with the analyte affect the relationship between the observed current and the polynucleotide sequence. Although the current nucleotide discrimination range has been improved by mutations in the CsgG pore, if the current differences between nucleotides can be further improved, the sequencing system will have higher performance. Summary of the invention

[0003] In some aspects, the present disclosure relates to protein pore complexes and their use in analyte detection and characterization. The present disclosure is based in part on a nanopore complex formed by a CsgG pore and one or more accessory proteins that form one or more channel constrictions in the nanopore complex. In some embodiments, the one or more accessory proteins are fusion proteins. As further described in the Examples, it was surprisingly found that accessory proteins that impart certain desired characteristics to CsgG protein nanopores (e.g., modulation of pore width, extension of the pore lumen, formation of one or more additional constrictions, etc.) can be designed de novo using computer-based structural analysis tools. In some embodiments, the de novo designed accessory proteins (e.g., fusion proteins) form one or more constrictions in the lumen of a CsgG nanopore and improve the discrimination of polymer units as an analyte moves through the nanopore.

[0004] Some aspects of the present disclosure further relate to methods for designing accessory proteins and generating nanopore complexes and for use in molecular sensing and nucleic acid sequencing applications.

[0005] In some aspects, the disclosure provides a protein nanopore complex comprising: a CsgG nanopore comprising an inner cavity; and a fusion polypeptide comprising a first portion comprising a CsgF protein and a second portion comprising a helix formation accessory protein, wherein the fusion protein is attached to the nanopore.

[0006] In some embodiments, the first portion of the fusion protein is attached to the CsgG nanopore. In some embodiments, the first portion of the fusion protein is located inside the lumen of the CsgG nanopore. In some embodiments, the first portion of the fusion protein extends outside the lumen of the CsgG nanopore. In some embodiments, the first portion forms a first constriction in the lumen of the CsgG nanopore.

[0007] In some embodiments, the second portion forms a second constriction.

[0008] In some embodiments, the CsgG nanopore further comprises a constriction.

[0009] In some embodiments, the second portion is not attached to the CsgG nanopore. In some embodiments, the second portion comprises one or more helices (e.g., α-helices, etc.).

[0010] In some embodiments, each of the helices (e.g., α-helices, etc.) of the second portion comprises between 0 and 15 α-helical turns. In some embodiments, the second portion comprises a first α-helix containing one to four α-helical turns and a second α-helix containing three to six α-helical turns. In some embodiments, the second α-helix is ​​stacked relative to the first α-helix. In some embodiments, the second portion comprises between 1 and 55 amino acid residues. In some embodiments, each of the helices comprises 1 to 20 amino acid residues having a Phi angle in the range of about -45° to -90° and a Psi angle in the range of about 0° to -70°. In some embodiments, each of the helices comprises 1 to 30 amino acid residues having a Phi angle in the range of about -45° to -90° and a Psi angle in the range of about 0° to -70°.

[0011] In some embodiments, the distance (e.g., vertical distance) between the first contraction zone and the second contraction zone is about to about (For example, when measured as the α-carbon (C a ) is within the range of ). In some embodiments, the protein nanopore complex has a distance greater than The axial length of to about within the range.

[0012] In some embodiments, the fusion protein is attached to the nanopore via a linker. In some embodiments, the linker comprises a bond, a peptide linker, or a chemical linker. In some embodiments, the linker comprises a bond formed by a sulfur (VI) fluoride exchange (SuFEx) reaction. In some embodiments, the linker comprises one or more maleimide molecules.

[0013] In some embodiments, the fusion protein is cyclized. In some embodiments, the cyclization comprises one or more side chain to side chain cyclization bonds. In some embodiments, at least one of the side chain to side chain cyclization bonds is a disulfide bond.

[0014] In some aspects, the present disclosure provides a protein nanopore complex comprising: a CsgG nanopore comprising an inner cavity and a first constriction formed within the inner cavity of the nanopore; and a fusion protein comprising a first portion comprising a CsgF protein and a second portion comprising a helix formation accessory protein, wherein the fusion protein is attached to the nanopore.

[0015] In some embodiments, the first portion of the fusion protein is attached to the CsgG nanopore. In some embodiments, the first portion of the fusion protein is located inside the lumen of the CsgG nanopore.

[0016] In some embodiments, the second portion of the fusion protein is located outside the lumen of the CsgG nanopore.

[0017] In some embodiments, the first portion forms a second constriction in the lumen of the CsgG nanopore. In some embodiments, the second portion forms a third constriction in the lumen of the CsgG nanopore.

[0018] In some embodiments, the second moiety is not attached to the CsgG nanopore.

[0019] In some embodiments, the second portion comprises one or more helices (e.g., α-helices, etc.). In some embodiments, each of the helices (e.g., α-helices) comprises between 0 and 15 α-helical turns. In some embodiments, the second portion comprises between 1 and 54 amino acid residues. In some embodiments, each of the helices comprises 1 to 36 amino acid residues having a Phi angle in the range of about -45° to -90° and a Psi angle in the range of about 0° to -70°. In some embodiments, each of the helices comprises 1 to 36 amino acid residues having a Phi angle in the range of about -45° to -90° and a Psi angle in the range of about 0° to -70°.

[0020] In some embodiments, the fusion protein is cyclized. In some embodiments, the cyclization comprises one or more side chain to side chain cyclization bonds. In some embodiments, the cyclization comprises one or more side chain to tail (e.g., C-terminal) cyclization bonds. In some embodiments, at least one of the cyclization bonds is a disulfide bond.

[0021] In some aspects, the present disclosure provides a protein nanopore complex comprising a CsgG nanopore comprising an inner cavity and a first constriction formed within the inner cavity of the nanopore; a first accessory protein that is attached to the CsgG nanopore and forms a second constriction in the inner cavity of the nanopore; and a second accessory protein that is attached to the CsgG nanopore or the first accessory protein and forms a third constriction.

[0022] In some embodiments, the first accessory protein is located inside the lumen of the CsgG nanopore. In some embodiments, the first accessory protein comprises a CsgF protein or peptide.

[0023] In some embodiments, the second auxiliary protein comprises one or more helices (e.g., α-helices, etc.). In some embodiments, each of the one or more helices (e.g., α-helices) comprises between 0 and 15 α-helical turns. In some embodiments, the second auxiliary protein comprises two α-helices.

[0024] In some embodiments, one of the α-helices comprises between 1 and 6 α-helical turns. In some embodiments, one of the α-helices comprises between 1 and 10 α-helical turns. In some embodiments, one of the α-helices comprises three α-helical turns and the other α-helix comprises three or four α-helical turns. In some embodiments, each of the helices comprises 1 to 36 amino acid residues having a Phi angle in the range of about -45° to -90° and a Psi angle in the range of about 0° to -70°. In some embodiments, each of the helices comprises 1 to 36 amino acid residues having a Phi angle in the range of about -45° to -90° and a Psi angle in the range of about 0° to -70°.

[0025] In some embodiments, the second accessory protein comprises at least one alpha helix that is stacked relative to the alpha-helical stack of the first accessory protein. In some embodiments, the second accessory protein comprises between 1 and 55 amino acid residues.

[0026] In some embodiments, the distance (e.g., vertical distance) between the first contraction and the second contraction is about to about (For example, when measured as the α-carbon (C a ). In some embodiments, the distance between the second contraction and the third contraction is about to about In some embodiments, the protein nanopore complex has a The axial length of to about within the range.

[0027] In some embodiments, the first auxiliary protein and the second auxiliary protein are attached by a joint. In some embodiments, the joint includes a key, a peptide joint or a chemical joint. In some embodiments, the joint includes a key formed by sulfur (VI) fluoride exchange (SuFEx) reaction. In some embodiments, the joint includes one or more maleimide molecules. In some embodiments, the joint includes one or more cyclization bonds (for example, the first amino acid of the joint can be covalently or non-covalently attached to the second amino acid of the joint, for example, by a cross-linking agent).

[0028] In some embodiments, the first auxiliary protein and the second auxiliary protein comprise one or more side chain to side chain cyclization bonds. In some embodiments, the first auxiliary protein and the second auxiliary protein comprise one or more side chain to tail (e.g., C-terminal) cyclization bonds. In some embodiments, at least one of the cyclization bonds is a disulfide bond.

[0029] In some aspects, the present disclosure provides a system for characterizing a target analyte, the system comprising a protein nanopore complex as described herein inserted into a membrane.

[0030] In some embodiments, the system further comprises a conductive solution in contact with the protein nanopore complex, an electrode providing a voltage potential across the membrane, and a measurement system for measuring a current through the protein nanopore complex.

[0031] In some aspects, the present disclosure provides a method for characterizing a target analyte, comprising the steps of: contacting a system as described herein with a target analyte; applying an electric potential across a membrane so that the target analyte enters a lumen formed by a protein nanopore complex; and performing one or more measurements as the target analyte moves relative to the lumen, thereby characterizing the target analyte.

[0032] In some embodiments, the target analyte comprises a target polynucleotide.

[0033] In some embodiments, making one or more measurements comprises measuring a current through a continuous channel, wherein the current is indicative of the presence and / or one or more characteristics of a target analyte, and thereby detecting and / or characterizing the target analyte.

[0034] In some embodiments, the target analyte is a polynucleotide, and the nucleotides in the polynucleotide interact with the first and second (and optionally, the third) contraction regions within the inner cavity, and wherein each of the first, second (and optionally, the third) contraction regions is capable of discriminating different nucleotides, such that the total current passing through the inner cavity is affected by the interaction between each of the first, second, and third contraction regions and the nucleotides located at each of these regions.

[0035] In some aspects, the present disclosure provides a method of preparing a protein nanopore complex, the protein nanopore complex comprising:

[0036] (a) a CsgG nanopore comprising an inner lumen; and

[0037] (b) a fusion polypeptide comprising a first portion comprising a CsgF protein and a second portion comprising a helix-forming accessory protein, wherein the fusion protein is attached to a nanopore, and wherein at least one domain of the fusion polypeptide is designed using a computer-generated algorithm. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figures 1A to 1C show the workflow for de novo design of fusion proteins. Figure 1A shows the design workflow using the CsgG nanopore. Wild-type CsgF (residues 1-35; left) is shown in orange. Residues 17-30 of wild-type CsgF (red) were selected as targets, and we searched for geometrically matching and designable helices, which were packed and projected onto the pore to generate a nanopore with a diameter between and Figure 1B shows a top view of a non-polymeric CsgG-fusion protein complex, demonstrating the additional constriction achieved by the de novo designed fusion protein.

[0039] Figure 2 Representative data for prioritization of de novo fusion protein sequences designed using Rosetta are shown. Sequences were selected for experimental validation based on the lowest energy score and highest PackStat score.

[0040] Figures 3A to 3DPSIPRED protein secondary structure analysis based on the amino acid sequence of the de novo designed fusion protein is shown. Residues are colored according to whether they are predicted to be a strand, helix, or coil, respectively. Figure 3A The predicted secondary structure of the fusion protein and the mature sequence of wild-type CsgF are shown. Figure 3B Secondary structure analysis of de novo designed fusion proteins ONT1 to ONT10 is shown. Figure 3C Secondary structure analysis of de novo designed fusion proteins ONT11 to ONT20 is shown. Figure 3D Protein secondary structure analysis of de novo designed fusion proteins ONT21 to ONT25 is shown.

[0041] Figures 4A to 4C The predicted 3-dimensional structures of alternative sequences of de novo designed fusion proteins are shown. Figure 4A The predicted structures of the de novo designed fusion proteins ONT1 to ONT10 are shown. Figure 4B The predicted structures of the de novo designed fusion proteins ONT11 to ONT20 are shown. Figure 4C The predicted structures of the de novo designed fusion proteins ONT21 to ONT25 are shown.

[0042] Figure 5 Representative SDS-PAGE gel analysis of CsgG-only pores and CsgG / fusion protein complexes are shown, where the complexes contain CsgF-del(S31-F119) control or de novo designed fusion proteins, with or without maleimide crosslinker. Complexes containing fusion proteins show band shifts, indicating that these samples are pore complexes. Note that samples were not heated prior to loading on the gel.

[0043] Figure 6 Representative SDS-PAGE gel analysis of CsgG-only pores and CsgG / fusion protein complexes containing CsgF-del(S31-F119) control or de novo designed fusion proteins with or without maleimide crosslinkers are shown. The pores were resolved into their constituent monomer components upon boiling in the presence of DTT prior to loading on the gel.

[0044] Figure 7 Representative ionic current (pA) versus time (s) traces are shown 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.

[0045] Figure 8Shown are representative ionic current (pA) versus time (s) traces as single-stranded DNA translocates through CsgG containing the del(S31-F119) CsgF peptide, with or without a maleimide cross-linker.

[0046] Fig. 9 Shown are representative ionic current (pA) versus time (s) traces as single-stranded DNA translocates through CsgG containing de novo designed fusion proteins in the absence of maleimide cross-linker.

[0047] Fig.10 Shown are representative ionic current (pA) versus time (s) traces as single-stranded DNA translocates through CsgG containing de novo designed fusion proteins plus / minus maleimide cross-links.

[0048] Fig.11 Representative ionic current (pA) versus time (s) traces are shown when single-stranded DNA is translocated through CsgG containing de novo designed fusion proteins, with or without a maleimide cross-linker. The fusion protein contains a K37R mutation as well as a cysteine ​​residue to form an internal disulfide bond within the peptide, i.e., a circularized fusion protein.

[0049] Fig.12 Representative curves are shown, demonstrating the position within the pore and its contribution to the overall change in ionic current levels ("discrimination") as a DNA molecule translocates through the pore. The CsgG-only pore (plus / minus Q153C) shows one major discrimination peak at position 0.

[0050] Fig.13 Representative curves are shown, demonstrating the position within the pore and its contribution to the overall change in ionic current levels ("discrimination") as the DNA molecule translocates through the pore. The dashed box shows the region that would be affected by the introduction of a de novo designed fusion protein. CsgG-CsgF-del (S31-F119) pores with or without a maleimide crosslinker show two discrimination peaks. As seen in the CsgG-only pores, the main discrimination peak is at position 0, and an additional discrimination peak is located 4 to 6 nucleotides below the main constriction (positions -4 to -6). This additional discrimination region has less impact on the ionic current than the main discrimination peak at position 0.

[0051] Fig.14Representative curves are shown, demonstrating the position within the pore and its contribution to the overall change in ionic current levels ("discrimination") as the DNA molecule translocates through the pore. Distance within the pore is measured in nucleotide steps relative to the main constriction. Negative values ​​correspond to positions below the main constriction, while positive values ​​correspond to positions above the main constriction (CsgG). The dashed box shows the region that will be affected by the introduction of the de novo designed fusion protein. Complexes composed of CsgG and a de novo designed K37R-containing fusion protein (with or without a maleimide crosslinker; with cyclization) show three discrimination peaks. As seen in the CsgG-only pore, the main discrimination peak is at position 0, and additional peaks are at positions -6 and -9. The peak at position -9 corresponds to the expected constriction produced by the de novo designed fusion protein when folded in the correct orientation.

[0052] Fig.15 An example of two proteins connected by a maleimidopropionic acid linker is shown.

[0053] Fig.16 Examples of porins and ancillaries (eg, fusion proteins) functionalized with reactive modifying agents such as thiol modifying agents are shown.

[0054] Fig.17 Representative ionic current (pA) versus time (s) traces are shown when single-stranded DNA translocates through CsgG containing a de novo designed fusion protein (SEQ ID NO: 61), with (bottom two traces) or without (top two traces) a maleimide cross-linker. 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.

[0055] Fig.18 Representative curves are shown, demonstrating the position within the pore and its contribution to the overall change in ionic current levels ("discrimination") as the DNA molecule translocates through the pore. Distance within the pore is measured in nucleotide steps relative to the main constriction. Negative values ​​correspond to positions below the main constriction, while positive values ​​correspond to positions above the main constriction (CsgG). The dashed box shows the region that will be affected by the introduction of the de novo designed fusion protein. Complexes consisting of CsgG and the de novo designed fusion protein (SEQ ID NO:61), with (bottom curve) or without (top curve) a maleimide cross-linker; neither is cyclized) show three discrimination peaks. As seen in the CsgG-only pore, the main discrimination peak is at position 0, and additional peaks are at positions -5 and -11. The peak at position -11 corresponds to the expected constriction produced by the de novo designed fusion protein when folded in the correct orientation.

[0056] Fig.19The structure and size of the wild-type CsgG pore from E. coli strain K12 are shown (the database accession code for this structure is 4UV3). The distances shown are measured from the backbone to the 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. DETAILED DESCRIPTION

[0057] Aspects of the present disclosure relate to compositions and methods for characterizing analytes using nanopore-based systems. The present disclosure is based, in part, on a protein nanopore complex formed by a CsgG pore and one or more accessory proteins that form one or more channel constrictions in the nanopore complex. In some embodiments, the one or more accessory proteins are fusion proteins. As further described in the Examples, it was surprisingly found that accessory proteins that impart certain desired characteristics to CsgG nanopores (e.g., modulation of pore width, extension of the pore lumen, formation of one or more additional constrictions, etc.) can be designed de novo using computer-based structural analysis tools. In some embodiments, the de novo designed accessory proteins (e.g., fusion proteins) form one or more additional constrictions in the lumen of the CsgG pore and improve discrimination of polymer units as the analyte moves through the nanopore.

[0058] Accessory proteins

[0059] The protein nanopore complex (also interchangeably referred to as the protein pore complex) as described in the present disclosure may include one or more auxiliary proteins. As used herein, the terms "peptide", "polypeptide" or "protein" are used interchangeably herein and refer to two or more amino acids linked together by peptide bonds. In some embodiments, the protein (also referred to as a polypeptide or peptide) comprises between 2 and 2000 amino acids. In some embodiments, the protein comprises between 2 and 10 amino acids, between 2 and 25 amino acids, between 2 and 50 amino acids, between 2 and 100 amino acids, between 2 and 500 amino acids, or between 2 and 1000 amino acids (or any number of amino acids therebetween, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 250, 500, 750, 1000 amino acids, etc.). In some embodiments, the protein comprises more than 2000 amino acids. In some embodiments, the peptide, polypeptide or protein is of synthetic origin (e.g., not present in nature, e.g., not naturally expressed in any living organism). In some embodiments, the peptide, polypeptide or protein is naturally occurring (e.g., naturally expressed in a living organism that has not been genetically modified to express the peptide, polypeptide or protein). In some embodiments, the peptide, polypeptide or protein can be naturally expressed by an organism. In some embodiments, the peptide, polypeptide or protein is heterologously expressed by an organism (e.g., an organism that has been genetically modified to express the peptide, polypeptide or protein). In some embodiments, the peptide, polypeptide or protein is chemically synthesized (e.g., by in vitro transcription, peptide synthesis, etc.). The peptide, polypeptide or protein may comprise one or more naturally occurring amino acids (L-amino acids, D-amino acids, etc.), one or more non-naturally occurring amino acids (e.g., radiolabeled amino acids, non-canonical amino acids, non-natural amino acids, etc.), or a combination of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids.

[0060] In some embodiments, the accessory protein is a fusion protein. The term "fusion protein" refers to a naturally occurring, synthetic, semisynthetic or recombinant single protein molecule that comprises all or part of two or more heterologous polypeptides (e.g., polypeptides that are heterologous to each other) joined by peptide bonds. In some embodiments, the fusion protein comprises all or part of at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 heterologous polypeptides joined by peptide bonds. As used herein, "a portion of a peptide" refers to 2 or more amino acids of a peptide. In some embodiments, a portion of a peptide comprises the entire amino acid sequence of a peptide, or at least: 5, 10, 20, 30, 50, or 100 amino acids (e.g., 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, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, In some embodiments, the fusion protein comprises a first part, a second part, a C-terminus, a N-terminus, a N-terminus, a C-terminus, a C-terminus, a N ... The portions of the fusion protein can be directly joined (e.g., the amino acids of a portion can be directly joined to the amino acids of a second portion via a peptide bond between the terminal amino acids of the portions) or indirectly joined (e.g., the amino acids of a portion of the fusion protein can be bound to a linker, for example, via a first peptide bond, which is bound to the second portion of the fusion protein via a second peptide bond). In some embodiments, the first auxiliary protein is the first portion of the fusion protein and the second auxiliary protein is the second portion of the fusion protein. The use of a linker to connect portions of the fusion protein is further described herein, for example in the section entitled "Linker."

[0061] In some embodiments, the protein nanopore complex comprises a plurality of subunits or monomers (e.g., a plurality of CsgG monomers) arranged around a central cavity or pore (also referred to as the "lumen" of the nanopore). Formation of protein nanopores is further described herein, for example in the section entitled "CsgG pore". In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more) accessory proteins are arranged in the lumen of the nanopore or form a continuous channel (e.g., a continuous lumen) with the lumen of the nanopore. In some embodiments, the protein nanopore complex comprises a ratio of pore monomers (e.g., CsgG pore monomers) to accessory proteins of 9:1, 9:2, 9:3, 9:4, 9:5, 9:6, 9:7, 9:8, 9:9 (e.g., 1:1), 9:10, 9:11, 9:12, 9:13, 9:14, 9:15, 9:16, 9:17, or 9:18 (e.g., 1:2). In some embodiments, the one or more accessory proteins or one or more fusion proteins may have the same symmetry as the nanopore. For example, when the nanopore comprises eight monomers around the central axis, there are eight accessory proteins (or eight fusion proteins), or when the nanopore comprises nine monomers around the central axis, there are nine accessory proteins (or nine fusion proteins), etc. In some embodiments, the one or more accessory proteins (or one or more fusion proteins) may comprise more or fewer monomers than the nanopore, such as one more or one less.

[0062] The lumen of a nanopore or protein nanopore complex may have one or more constrictions. "Constriction", "pore mouth", "constriction region", "channel constriction" or "constriction site" as used interchangeably herein refers to the pore size defined by the lumen surface of a pore or protein pore complex, which 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 protein pore complex channel. The constriction is typically the narrowest pore within a pore or protein 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 ion flow through the channel, each constriction should not be too large. For example, each constriction should not be wider than the solvent accessible lateral diameter of the target analyte. Ideally, the diameter of each constriction should be as close as possible to the lateral diameter of the analyte passing through.

[0063] The number of constrictions in the protein pore complexes described in the present disclosure may vary. In some embodiments, the protein pore complex comprises at least 1, 2, 3, 4, 5, or more constrictions. In some embodiments, the protein pore complex comprises 2 or 3 constrictions. In some embodiments, the protein pore complex comprises 2 constrictions. In some embodiments, the first constriction is formed by a first accessory protein and the second constriction is formed by a second accessory protein. In some embodiments, the first constriction is formed by a portion of a CsgG nanopore and the second constriction is formed by an accessory protein or a fusion protein. In some embodiments, the protein pore complex comprises 3 constrictions. In some embodiments, the first constriction is formed by a portion of a CsgG nanopore, the second constriction is formed by a first accessory protein, and the third constriction is formed by a second accessory protein. In some embodiments, the first constriction is formed by a portion of a CsgG nanopore and the second and third constrictions are formed by a fusion protein.

[0064] The narrowest point of the central cavity or pore generally forms a constriction in the continuous channel. In some embodiments, the α-carbon (C ) of the amino acid residue extending farthest into the lumen of the nanopore to form the constriction is measured. a ). In some embodiments, the diameter of the constriction is calculated by measuring the distance between the Van der Waals radii of the atoms that extend farthest into the lumen of the nanopore to form the constriction. In some embodiments, the minimum diameter of the constriction (e.g., a constriction formed by a portion of the CsgG protein, a constriction formed by an accessory protein, a constriction formed by a fusion protein, etc.) is in the range of about 0.5 nm to about 4.0 nm (e.g., as measured by the distance between the Van der Waals radii). In some embodiments, the minimum diameter of the constriction is in the range of about 0.5 to about 3.0 nm, or about 0.5 to about 2.0 nm, preferably from about 0.7 to about 1.8 nm, about 0.8 to about 1.7 nm, about 0.9 to about 1.6 nm, or about 1.0 to about 1.5 nm, such as about 1.1, 1.2, 1.3, or 1.4 nm. In some embodiments, the minimum diameter of the constriction is about to about within the range, for example or (For example, as through C a To C a In some embodiments, the contracted minimum diameter is about to about within the range (e.g., as determined by C a To C a In some embodiments, the contracted minimum diameter is about to about within the range (e.g., as determined by C a To C a measured).

[0065] The distance between one or more constrictions in the lumen of the protein pore complex may vary. In some embodiments, the distance between the first constriction and the second constriction is about to about In some embodiments, the length of the distance between the first contraction zone and the second contraction zone is about

[0066]

[0067] or In some embodiments, the distance between the first contraction zone and the second contraction zone is greater than (For example, wait).

[0068] In some embodiments, the distance between the second contraction zone and the third contraction zone is about to about In some embodiments, the length of the distance between the first contraction zone and the second contraction zone is about

[0069] or In some embodiments, the distance between the second contraction zone and the third contraction zone is greater than (For example, wait).

[0070] In some embodiments, the distance between the first contraction zone and the third contraction zone is about to about In some embodiments, the length of the distance between the first contraction zone and the second contraction zone is about

[0071] or In some embodiments, the distance between the first contraction zone and the third contraction zone is greater than (For example, wait).

[0072] In certain embodiments, the auxiliary protein (or fusion protein) can be modified from its native state to provide a contraction with a desired minimum diameter. For example, the auxiliary protein can be modified, such as by introducing one or more large residues through targeted mutations, to produce a contraction with a minimum diameter within the above-specified range. In one embodiment, the maximum height of the auxiliary protein is about 3nm to about 20nm, such as about 4nm to about 10nm. In one embodiment, the length of the channel in the auxiliary protein is about 3nm to about 20nm, such as about 4nm to about 10nm. Height is the size of the auxiliary protein in a direction perpendicular to the film.

[0073] In some embodiments, the accessory protein (e.g., the first accessory protein or the second accessory protein) or the fusion protein (e.g., the first portion of the fusion protein or the second portion of the fusion protein) extends outside the lumen of the protein pore complex. The accessory protein or the fusion protein may extend outside the cis or trans side of the lumen of the protein pore complex (e.g., when the protein pore complex is inserted into a membrane). In some embodiments, the accessory protein or the fusion protein is measured by measuring the C a The distance that the accessory protein or fusion protein extends outside the lumen of the protein pore complex is calculated by the distance between the accessory protein or fusion protein and a reference amino acid of the protein pore (e.g., the CsgG pore) (e.g., amino acid residue Phe144 or Tyr196 of a wild-type CsgG monomer). In some embodiments, the accessory protein or fusion protein extends outside the lumen by about About In some embodiments, the auxiliary protein or fusion protein extends outside the lumen to about About In some embodiments, the auxiliary protein or fusion protein extends outside the lumen to about About In some embodiments, the auxiliary protein or fusion protein extends outside the lumen to about

[0074] or about

[0075] The length between the first constriction and the second constriction of the protein pore complex generally affects the axial length of the protein pore complex. In some embodiments, the axial length of the protein pore complex refers to the distance between the top of the lumen of the protein pore complex and the bottom of the lumen of the protein pore complex. In some embodiments, the protein pore complex has a diameter greater than In some embodiments, the axial length of the protein pore complex (e.g., a protein pore complex comprising one or more accessory proteins or one or more fusion proteins) is about to about within the range, for example

[0076] or

[0077] In some embodiments, the auxiliary protein or fusion protein comprises one or more positively charged amino acids, such as arginine, lysine or histidine, or aromatic amino acids, such as tyrosine or tryptophan, which are located at or near (e.g., within about 1, 2, 3, 4 or 5 nm of the contraction) formed by the auxiliary protein or fusion protein. In some embodiments, the auxiliary protein or fusion protein comprises one or more polar amino acids, negative amino acids or hydrophobic amino acids, which are located at or near (e.g., within about 1, 2, 3, 4 or 5 nm of the contraction) formed by the auxiliary protein or fusion protein. In some embodiments, the one or more amino acids located at or near (e.g., within about 1, 2, 3, 4 or 5 nm of the contraction) formed by the auxiliary protein or fusion protein are asparagine, threonine, serine or glutamic acid. These amino acids generally promote the interaction between the hole and the polynucleotide.

[0078] The positioning of one or more accessory proteins (or one or more fusion proteins) of a protein pore complex may vary. In some embodiments, the accessory protein (or fusion protein) is completely located within the lumen of the protein pore complex. In some embodiments, the accessory protein or fusion protein comprises a portion extending beyond the lumen of the protein pore complex, for example extending above the lumen of the protein pore complex (e.g., extending above the cap region on the cis side of the protein pore complex) and / or extending below the protein pore complex (e.g., extending below the transmembrane domain (e.g., barrel) on the trans side of the protein pore complex). In some embodiments, the accessory protein or fusion protein (or a portion of the accessory protein or fusion protein, such as the first portion or the second portion) is attached to a nanopore (e.g., a CsgG nanopore). In some embodiments, the accessory protein or fusion protein (or portion thereof) is covalently attached to a nanopore. In some embodiments, the accessory protein or fusion protein (or portion thereof) is non-covalently attached to a nanopore. In some embodiments, the first accessory protein and the second accessory protein are attached to each other (e.g., covalently attached, non-covalently attached, etc.). In some embodiments, the first portion of the fusion protein and the second portion of the fusion protein are attached to each other (e.g., covalently attached, non-covalently attached, etc.). In some embodiments, the accessory protein or fusion protein (or a portion of the accessory protein or fusion protein, such as the first portion or the second portion) is not attached to a nanopore (e.g., a CsgG nanopore). In some embodiments, the first accessory protein and the second accessory protein are not attached to each other.

[0079] In some embodiments, the accessory protein (e.g., the first accessory protein) is not CsgF or a CsgF peptide, or a functional homologue, fragment or modified form thereof. In some embodiments, a portion (e.g., the first portion and / or the second portion) of the fusion protein is not CsgF or a CsgF peptide, or a functional homologue, fragment or modified form thereof. In some embodiments, the accessory protein is not a CsgG nanopore, or a homologue, fragment or modified form thereof. In some embodiments, a portion (e.g., the first portion and / or the second portion) of the fusion protein is not a CsgG nanopore, or a homologue, fragment or modified form thereof.

[0080] In some embodiments, the auxiliary protein is not a polynucleotide binding protein. In some embodiments, the auxiliary protein is not a functional polynucleotide binding protein, for example, the auxiliary protein is not a polynucleotide binding protein with enzymatic activity. In some embodiments, the auxiliary protein can be a protein other than a nucleic acid processing enzyme, for example, an auxiliary protein that is not a helicase or a polymerase, or a protein derived from such an enzyme. In some embodiments, the auxiliary protein does not have enzymatic activity. In some embodiments, the auxiliary protein does not undergo conformational changes when the target analyte passes through the continuous channel formed in the protein pore complex.

[0081] In some embodiments, the accessory protein or fusion protein (e.g., part of a fusion protein) is a component of a nanopore system or a modified component of such a system, other than a component that forms a transmembrane pore. An example of such a component is CsgF or a truncated version of CsgF. In some embodiments, the accessory protein or fusion protein comprises a CsgF protein or a homologue or modified form thereof, such as a fragment. In some embodiments, the pore complex comprises a CsgF protein or peptide and a non-CsgG pore, a homologue or a modified form thereof, such as a fragment.

[0082] The term "CsgF protein" or "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 (e.g., Figure 3A), 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 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% complete sequence identity with wild-type E. coli CsgF. 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. Mature CsgF (e.g., Figure 3A The CsgF peptide (shown in Figure 2) 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 distinct from the contraction of the pore as described herein. The "head" region of the CsgF peptide may also be referred to as a "C-terminal head domain". The structure of CsgF is discussed in detail in WO 2019 / 002893 (incorporated herein by reference in its entirety).

[0083] In some embodiments, the CsgF peptide is 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 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 wild-type E. coli CsgF). In some embodiments, the CsgF peptide comprises a CsgG binding region and a region that forms a constriction in the lumen of the pore. The CsgG binding region typically comprises residues 1 to 11 and / or 29 to 32 of a CsgF protein (e.g., wild-type E. coli CsgF or a homolog from another species) and may include one or more modifications. The region that forms a constriction in the pore typically comprises residues 9 to 28 of a CsgF protein (e.g., wild-type E. coli CsgF or a homolog from another species) and may include one or more modifications. In some embodiments, residues 9 to 17 comprise the conserved motif N9PXFGGXXX 17, and form a turn region. In some embodiments, residues 9 to 28 form an α-helix. In some embodiments, the amino acid residue at position 17 of the CsgF peptide forms the apex of the constriction region, corresponding to the narrowest part of the CsgF constriction in the pore. In some embodiments, the CsgF constriction region also makes stabilizing contacts with the CsgG β-barrel primarily at residues 8, 9, 11, 12, 18, 21, and 22 of the CsgF peptide. In some embodiments, the CsgF peptide comprises or consists of the amino acid sequence GTMTFQFRNPNFGGNPNNGAFLLNSAQAQN (SEQ ID NO: 60), which corresponds to amino acid residues 1-30 of wild-type E. coli CsgF. In some embodiments, the CsgF peptide is a first auxiliary protein. In some embodiments, the CsgF peptide is a portion (e.g., a first portion or a second portion) of a fusion protein. In some embodiments, the CsgF peptide comprises or consists of amino acid residues 1-23 of wild-type E. coli CsgF. In some embodiments, the CsgF peptide comprises or consists of amino acid residues 1-23 of wild-type E. coli CsgF. In some embodiments, the CsgF peptide comprises or consists of amino acid residues 1-24 of wild-type E. coli CsgF. In some embodiments, the CsgF peptide comprises or consists of amino acid residues 1-24 of wild-type E. coli CsgF.

[0084] In some embodiments, the CsgF peptide has a length of 28 to 60 amino acids, such as 29 to 49, 30 to 45, or 32 to 40 amino acids. In some embodiments, the CsgF peptide comprises 29 to 35 amino acids or 29 to 45 amino acids. In some embodiments, the CsgF peptide comprises 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, 55, 56, 57, 58, 59, or 60 amino acids. In some embodiments, the CsgF peptide comprises all or part of FCP, which corresponds to residues 1 to 35 of wild-type E. coli CsgF (or the corresponding residues in a CsgF homolog). In some embodiments, where the CsgF peptide is shorter than FCP, it is preferably truncated at the C-terminus.

[0085] 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, M3, T4, F5, R8, N9, N11, F12, N17, A20, N24, A26, and Q29 of SEQ ID NO: 60. In some embodiments, the CsgF peptide is 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: 60: G1, T4, F5, R8, N9, N11, F12, N17, A20, N24, A26, Q27, and Q29. Any number and combination of such introductions may be made. Preferably, introduction is by substitution.

[0086] In some embodiments, the CsgF peptide comprises a modification at a position corresponding to one or more of the following positions in SEQ ID NO: 60: N15, N17, A20, N24, and A28. In some embodiments, the CsgF peptide comprises one or more of the following substitutions: N15 S / A / T / Q / G / L / V / I / F / Y / W / R / K / D / C / E; N17 S / A / T / Q / G / L / V / I / F / Y / W / R / K / D / C / E; A20 S / T / Q / N / G / L / V / I / F / Y / W / R / K / D / C / E; N24 S / T / Q / A / G / L / V / I / F / Y / W / R / K / D / C / E; or A28 S / T / Q / N / G / L / V / I / F / Y / W / R / K / D / C / E.

[0087] In some embodiments, the CsgF peptide is preferably a variant of any of the CsgF sequences discussed above (including SEQ ID NO: 60) comprising one or more modifications compared to the comparison sequence. Over the entire length of the amino acid sequence of SEQ ID NO: 60, the variant will preferably be at least 40% homologous to the sequence based on amino acid identity. 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% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 60 over the entire sequence. Over the entire length of the amino acid sequence of SEQ ID NO: 60, 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: 60 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 15 or more, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more consecutive amino acids ("hard homology"). These homology / identity levels apply equally to any of the other CsgF peptides described above.

[0088] Any number of CsgF peptides in a pore or pore complex, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, may contain one or more substitutions compared to SEQ ID NO: 60. In some embodiments, all six to ten monomers in a pore or pore complex preferably contain one or more substitutions compared to SEQ ID NO: 60. 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 present disclosure.

[0089] Aspects of the present disclosure relate to accessory proteins or fusion proteins comprising one or more alpha helices. In some embodiments, such proteins may be referred to as "helix-forming proteins". The present disclosure is based in part on the recognition that helix-forming proteins can be located in the lumen of certain nanopores (e.g., CsgG nanopores) to form one or more constrictions in the lumen of the nanopore, and that the presence of such one or more constrictions improves the signal-to-noise ratio (e.g., discrimination of polynucleotide bases) of the resulting protein pore complex. The term "helix" or "helical" generally refers to the coiled structural arrangement of a protein, which forms a helix and is generated by the formation of hydrogen bonds between the backbones of non-contiguous amino acid residues in a repeating pattern. In some embodiments, the helix is ​​an alpha-helix (also known as a 3.6 13 -helix), each helical turn of which contains about 3.6 amino acid residues, 13 of which participate in the loop formed by hydrogen bonds. In some embodiments, the helix is ​​3 10 The helix contains about three residues per turn and has 10 atoms in the loop formed by hydrogen bonding.

[0090] Helices in accessory proteins or fusion proteins (e.g., α-helices, 3 10 In some embodiments, the number of helices in the accessory protein (e.g., the first accessory protein, the second accessory protein, etc.) is in the range of about 0 to about 15, such as 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15. In some embodiments, the number of helices in the accessory protein (e.g., the first accessory protein, the second accessory protein, etc.) is greater than 15 (e.g., 20, 25, etc.). In some embodiments, the fusion protein (e.g., the first part of the fusion protein, the second part of the fusion protein, etc.) comprises between 0 and about 15 helices (e.g., α helices, 3 π helices, etc.). 10 helix, π-helix, etc.), for example 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 helices.

[0091] Helices (e.g., α-helices, 3 10 The number of turns in each helix (e.g., α-helix, π-helix, etc.) of the accessory protein or fusion protein may vary. 10A helix (e.g., an alpha helix, a π helix, etc.) may comprise from about 0 to about 15 helical turns, e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 helical turns. A helix (e.g., an alpha helix, a π helix, etc.) may comprise one or more half helices (e.g., half turns), e.g., 0.5, 1.5, 2.5, 3.5, 4.5, 5.5, 6.5, 7.5, 8.5, 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, etc. helical turns.

[0092] Helical formation (e.g., α-helix, 3 10 The number of amino acids in each helix (e.g., α-helix, π-helix, etc.) of the accessory protein or fusion protein may vary. 10 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, or 55 amino acid residues.

[0093] The angle of the helix of the accessory protein or fusion protein may vary. In some embodiments, the helix includes an angle within the range of about -45° to -90° (e.g., -45°, -46°, -47°, -48°, -49°, -50°, -51°, -52°, -53°, -54°, ​​-55°, -56°, -57°, -58°, -59°, -60°, -61°, -62°, -63°, -64°, -65°, -66°, -67°, -68°, -69°, -70°, -71°, -72°, -73°, -74°, -75°, -76°, -77°, -78°, -79°, -80°, -81°, -82°, -83°, -84°, -85°, -86°, -87°, -88°, -89°, -90°, -91°, -92°, -93°, -94°, -95°, -96°, -97°, -98°, -99°, -100°, -101°, -102°, -103°, -104°, -105°, -106°, -107°, -108°, -109° -84°, -85°, -86°, -87°, -88°, -89° or -90°). In some embodiments, the spiral comprises a range of about 0° to -70° (e.g., 0°, -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°, -40°, -41°, -42°, -43°, -44°, -45°, -46°, -47°, -48°, -49°, -50°, -51°, -52°, -53°, -54°, ​​-55°, -56°, -57°, -58°, -59°, -60°, -61°, -62°, -63°, -64°, -65°, -66°, -67°, -68°, -69°, -70°, -71°, -72°, -73°, -74°, -75°, -76°, -77°, -78°, -79°, -80°, -81°, -82°, -83°, -84° In some embodiments, each of the helices comprises 1 to 20 amino acid residues having a Phi angle in the range of about -45° to -90° and a Psi angle in the range of about 0° to -70°. In some embodiments, each of the helices comprises 1 to 30 amino acid residues having a Phi angle in the range of about -45° to -90° and a Psi angle in the range of about 0° to -70°.

[0094] In some embodiments, one or more spirals of the auxiliary protein or fusion protein include structural features that are conducive to stacking spirals together. The "stacking" of spirals generally refers to two or more spirals that are closely associated with each other due to covalent or non-covalent interactions between spirals, such as salt bridges, hydrogen bonds, disulfide bonds, and tight hydrophobic side chains and side chains, side chains and main chains, main chains and main chains, etc., as described in Walther and Argos, J Mol Biol. January 26, 1996; 255 (3): 536-53.doi: 10.1006 / jmbi.1996.0044. Methods for predicting spiral stacking are known, such as Eilers et al. Proc Natl Acad Sci USA. May 23, 2000; 97 (11): 5796–5801.

[0095] Aspects of the present disclosure relate to the recognition that cyclized fusion proteins improve discrimination of target analytes in protein pore complexes. A "cyclized" protein generally refers to a protein (e.g., a fusion protein) that contains one or more intramolecular interactions that result in the formation of one or more bonds arranged in a ring. Examples of cyclization include side chain to side chain cyclization (e.g., intramolecular disulfide bond formation), head to tail cyclization (e.g., amide bond formation between the N-terminal and C-terminal amino acids of a protein), tail to side chain cyclization, and head to side chain cyclization, such as described in Hayes et al. Org Biomol Chem. 2021 May 12; 19(18): 3983–4001. In some embodiments, the fusion protein contains one or more side chain to side chain cyclization bonds. In some embodiments, at least one of the side chain to side chain cyclization bonds is a disulfide bond. In some embodiments, one or more cyclization bonds result in cyclization between a first portion of the fusion protein and a second portion of the fusion protein (e.g., cyclization between a CsgF peptide and a helix-forming protein). In some embodiments, the accessory protein or fusion protein comprises a loop region (eg, a linker forming a loop region) containing one or more cyclization bonds. In some embodiments, the cyclization bonds are formed by chemical cross-linking agents and / or comprise disulfide bonds.

[0096] CsgG nanopore

[0097] Aspects of the present disclosure relate to protein pore complexes. In some embodiments, the protein pore complexes described herein comprise a nanopore (e.g., a CsgG nanopore). A nanopore is a hole or channel through a membrane that allows hydrated ions driven by an applied potential to flow through or within the membrane.

[0098] In certain embodiments, nanopore is a transmembrane protein hole. Transmembrane protein holes generally span the entire membrane, and may have a structure extending beyond the membrane on one or both sides. Transmembrane protein holes are single or multimeric proteins that allow hydrated ions to flow from one side of the membrane to the other side of the membrane. Transmembrane protein holes include passages that allow analytes (e.g., polynucleotides, such as DNA or RNA) to move or be moved into and / or through holes.

[0099] Transmembrane protein pores typically include a barrel or channel through which ions can flow. The subunits of the pore typically surround a central axis and contribute strands to a transmembrane β-barrel or channel or a transmembrane α-helical bundle or channel.

[0100] The barrel or channel of the transmembrane protein pore usually contains amino acids that promote interaction with polynucleotides. These amino acids are preferably located near the contraction of the barrel or channel (such as within 1, 2, 3, 4 or 5 nm). The transmembrane protein pore usually contains one or more polar or hydrophobic residues. These amino acids usually facilitate the interaction between the hole and nucleotides, polynucleotides or nucleic acids.

[0101] In some embodiments, the nanopore is a CsgG pore, such as, for example, CsgG from E. coli strain K-12 substrain MC4100, or a homolog or mutant thereof. The mutant CsgG pore may comprise one or more mutant monomers. The CsgG pore may be a homopolymer comprising the same monomer, or a heteropolymer comprising two or more different monomers. Suitable pores derived from CsgG are disclosed in WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317, WO 2017 / 149318, International Patent Application Nos. PCT / GB2018 / 051191 and PCT / GB2018 / 051858, and Chinese Patent Publication Nos. CN113773373, CN113896776, CN113912683, and CN113754743, each of which is incorporated herein by reference in its entirety. Other examples of CsgG pores include, but are not limited to, Uniprot references K4KIX7, A0A086D1N6, A0A1I1MNE8, A0A143HJG2, AoA090RS48, and A0A090SZM0.

[0102] A CsgG pore typically comprises one or more CsgG monomers. A CsgG pore monomer is a monomer capable of forming a CsgG pore. Such monomers are known in the art, in particular from WO 2019 / 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 CsgG pore formed by the monomer may have any structure, but preferably has or comprises the structure of a wild-type E. coli CsgG pore (e.g., as described by PDB accession number 4UV3). 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.

[0103] The CsgG pore can be of any size, but preferably has the dimensions of a wild-type E. coli CsgG pore (e.g., as described in PDB accession number 4UV3). These dimensions are as follows: Fig.19 In some embodiments, the CsgG pore has a diameter of about 100 to about 20 mm at its widest point. The outer diameter of the or about 115 to about In some embodiments, the CsgG pore has a width of about 100 nm at its widest point. In some embodiments, the CsgG pore has an outer diameter of about 80 to about The total length is, for example, about 90 to about or about 95 to about In some embodiments, the CsgG pore has a References to "total length" and "length" refer to the length of a pore or pore region when viewed from the side (see, for example, a cis-to-trans cross section of a pore in an insert membrane). This can be Fig.19 In some embodiments, the CsgG pore is formed by calculating the CsgG residues that are farthest apart on the outside of the pore. a To C a In some embodiments, the outer diameter is measured by calculating the distance of the van der Waals radii of the farthest amino acid residues on the exterior of the CsgG pore.

[0104] In some embodiments, the cap region has a thickness of about 20 to about The length of the or about 35 to about In some embodiments, the cap region has about In some embodiments, the channel defined by the cap region has a diameter of about 30 to about The opening, such as a diameter of about 40 to about or about 45 to about In some embodiments, the channel defined by the cap region has a diameter of about In some embodiments, the diameter of the channel defined by the cap region at its narrowest point is about 20 to about Such as having a diameter of about 30 to about 10 mm at its narrowest point. or about 32 to about In some embodiments, the diameter of the channel defined by the cap region at its narrowest point is preferably about In some embodiments, the CsgG pore is determined by calculating the CsgG residues that are closest together on the channel of the cap region of the pore. a To C a In some embodiments, the outer diameter is measured by calculating the distance of the van der Waals radii of the amino acid residues that are closest together on the channel of the cap region.

[0105] In some embodiments, the constriction formed by the CsgG pore (when present) has a diameter of about 5 to about The length of the or about 15 to about In some embodiments, the contraction region has a In some embodiments, the diameter of the channel defined by the constriction at its narrowest point is about 2 to about Such as having a diameter of about 5 to about 10 mm at its narrowest point. About 8 to about or about 10 to about In some embodiments, the diameter of the channel defined by the constriction is about In some embodiments, the diameter of the channel defined by the constriction is about In some embodiments, the contracted diameter is about 2 to about Such as a diameter of about 5 to about About 8 to about or about 10 to about In some embodiments, the contracted diameter is about In some embodiments, the C of the amino acid residue that extends furthest into the lumen of the pore and forms the constriction is calculated. a To C a In some embodiments, the outer diameter is measured by calculating the distance of the van der Waals radius of the amino acid residue that extends farthest into the lumen of the pore and forms the constriction.

[0106] In some embodiments, the transmembrane β barrel region has about 20 to about The length of the or about 35 to about In some embodiments, the transmembrane β barrel has about In some embodiments, the diameter of the channel defined by the transmembrane β barrel region at its narrowest point is about 20 to about Such as having a diameter of about 30 to about 10 mm at its narrowest point. or about 35 to about In some embodiments, the channel defined by the transmembrane β-barrel region has a diameter at its narrowest point of about

[0107] All of the above measurements are based on the backbone-to-backbone measurements of the amino acids that form the different regions (e.g. Fig.19 shown).

[0108] SEQ ID NO: 59 shows the sequence of wild-type E. coli CsgG as a mature protein. Residues 1 to 41 of SEQ ID NO: 59 form the cap region. Residues 64 to 131 of SEQ ID NO: 59 form the constriction region. Residues 156 to 180 and 212 to 262 of SEQ ID NO: 59 form the transmembrane β-barrel region.

[0109] In some embodiments, the CsgG pore monomer is a variant of SEQ ID NO:59 because it has a cysteine ​​at a position corresponding to position 153 or 133 of SEQ ID NO:59. In some embodiments, the variant CsgG monomer may also be referred to as a modified CsgG pore monomer or a mutant 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:59. 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 homologues and the CsgG structure can be found at http: / / pfam.xfam.org / / family / PF03783.

[0110] In some embodiments, the CsgG pore monomer is a variant of SEQ ID NO:59 comprising one or more modifications in addition to the cysteine ​​at the position corresponding to position 153 or 133 in SEQ ID NO:59. Variants will preferably be at least 40% homologous to the amino acid sequence of SEQ ID NO:59 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:59 over the entire sequence. Variants will preferably be at least 40% identical to the amino acid sequence of SEQ ID NO:59 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:59 over the entire sequence.

[0111] 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:59, 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:59. 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"). In some embodiments, 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) 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 1 to 41 of SEQ ID NO: 59. In some embodiments, the variant comprises a sequence that is at least 40% identical to residues 1 to 41 of SEQ ID NO: 59. In some embodiments, the variant comprises 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 of SEQ ID NO: 59.

[0112] In some embodiments, the CsgG pore monomer is a variant of SEQ ID NO:59 comprising a sequence that is at least 40% homologous to the constriction region (residues 64 to 131) of SEQ ID NO:59. In some embodiments, the variant comprises 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 64 to 131 of SEQ ID NO:59. In some embodiments, the variant comprises a sequence that is at least 40% identical to residues 64 to 131 of SEQ ID NO:59. In some embodiments, the variant comprises 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 64 to 131 of SEQ ID NO:59.

[0113] In some embodiments, the CsgG pore monomer is a variant of SEQ ID NO:59 comprising a sequence that is at least 40% homologous to the transmembrane β barrel region (residues 156-180 and 212-262) of SEQ ID NO:3. In some embodiments, the variant comprises 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 156-180 and 212-262 of SEQ ID NO:59. In some embodiments, the variant comprises a sequence that is at least 40% identical to residues 156-180 and 212-262 of SEQ ID NO:59. In some embodiments, the variant comprises 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 156-180 and 212-262 of SEQ ID NO:59.

[0114] 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: 59, equivalent positions for mutations in CsgG pore monomers other than SEQ ID NO: 59 can be determined.

[0115] Thus, reference to a mutant CsgG pore monomer comprising a variant of the sequence as shown in SEQ ID NO: 59 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, CN113912683 A and CN 113754743 A, or variants thereof.

[0116] 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 / ).

[0117] SEQ ID NO:59 is a wild-type CsgG pore monomer from E. coli strain K-12 substrain MC4100. Variants of SEQ ID NO:59 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 of the 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:59, including one or more

[0118] The CsgG pore monomer in the pore monomer conjugates of the present disclosure 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: 59. 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.

[0119] In addition to those discussed above, amino acid substitutions can also be made to the amino acid sequence of SEQ ID NO:59, such as up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side chain volumes. The introduced amino acids can have similar polarity, hydrophilicity, hydrophobicity, alkalinity, acidity, neutrality or charge as the amino acids they replace. Alternatively, conservative substitutions can introduce another aromatic or aliphatic amino acid to replace a pre-existing aromatic or aliphatic amino acid.

[0120] In some embodiments, the CsgG pore monomer is 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. Preferably, introduction is by substitution.

[0121] One or more amino acid residues of the amino acid sequence of SEQ ID NO: 59 may additionally be deleted from the above polypeptide. Up to 1, 2, 3, 4, 5, 10, 20 or 30 or more residues may be deleted.

[0122] Variants may include fragments of SEQ ID NO:59. 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 domain of SEQ ID NO:59, i.e. K135-Q153 and S183-S208.

[0123] 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:59 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 to the amino acid sequence. Other fusion proteins are discussed in more detail elsewhere in the present disclosure, for example, in the section entitled "auxiliary protein".

[0124] A variant of SEQ ID NO:59 is a polypeptide having an amino acid sequence that differs from the amino acid sequence of SEQ ID NO:59 and retains its ability to form a pore. The variant generally contains the region of SEQ ID NO:59 that is responsible for pore formation. The pore-forming ability of β-barrel-containing CsgG is provided by β-sheets in the transmembrane β-barrel region of each subunit monomer. A variant of SEQ ID NO:59 generally comprises the β-sheet-forming region of SEQ ID NO:59, i.e., K134-Q154 and S183-S208. One or more modifications may be made to the β-sheet-forming region of SEQ ID NO:3, as long as the resulting variant retains its ability to form a pore.

[0125] 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 contemplated to alter the number, size, shape, placement or orientation of constrictions within the channel from the pore monomer conjugates of the present disclosure. The CsgG pore monomer or variant of SEQ ID NO: 59 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).

[0126] Preferred modifications or substitutions in SEQ ID NO: 59 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:

[0127] (a) a substitution at position Y51, such as Y51I, Y51L, Y51A, Y51V, Y51T, Y51S, Y51Q or Y51N;

[0128] (b) a substitution at position N55, such as N55I, N55L, N55A, N55V, N55T, N55S or N55Q;

[0129] (c) a substitution at position F56, such as F56I, F56L, F56A, F56V, F56T, F56S, F56Q or F56N;

[0130] (d) a substitution at position L90, such as L90N, L90D, L90E, L90R or L90K;

[0131] (e) a substitution at position N91, such as N91D, N91E, N91R or N91K;

[0132] (f) a substitution at position K94, such as K94R, K94F, K94Y, K94Q, K94W, K94L, K94S or K94N;

[0133] (g) a substitution at position R192, such as R192Q, R192F, R192S, R192D or R192T; and

[0134] (i) a substitution at position C215, such as C215T, C215S, C215I, C215L, C215A, C215V or C215G.

[0135] 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.

[0136] Any number of CsgG pore monomers in a pore or pore complex, such as 6, 7, 8, 9 or 10, may be a variant of SEQ ID NO: 59. All six to ten monomers in a pore or pore complex are preferably variants of SEQ ID NO: 59. 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.

[0137] Connectors

[0138] In some embodiments, the protein pore complex is stabilized by attaching (e.g., covalently attaching) an auxiliary protein or fusion protein to the nanopore. Covalent attachment can be, for example, a disulfide bond or click chemistry. As a further example, cysteine ​​residues can be connected by a linker such as BMOE. The auxiliary protein or fusion protein and / or transmembrane protein nanopore can be modified to promote such covalent interactions. In some embodiments, the auxiliary protein or fusion protein is non-covalently attached to the nanopore. In some embodiments, the auxiliary protein or fusion protein is attached to the nanopore by one or more (e.g., 1, 2, 3, 4, 5 or more) linkers.

[0139] In some embodiments, the accessory protein or fusion protein is attached to the nanopore by hydrophobic interactions and / or by one or more disulfide bonds. One or more monomers in the pore, such as 2, 3, 4, 5, 6, 8, 9, for example all monomers, can be modified to enhance such interactions. This can be achieved in any suitable manner. Other suitable interactions include salt bridges, electrostatic interactions, hydrogen bond formation, peptide bond formation, and π-π interactions.

[0140] At least one cysteine ​​residue in the amino acid sequence of the transmembrane protein nanopore at the interface between the nanopore and the auxiliary protein (or fusion protein) can be disulfide bonded with at least one cysteine ​​residue in the amino acid sequence of the auxiliary protein at the interface between the nanopore and the auxiliary protein. In some embodiments, at least one cysteine ​​residue in the amino acid sequence of the first auxiliary protein is disulfide bonded with at least one cysteine ​​residue in the amino acid sequence of the second auxiliary protein. In some embodiments, at least one cysteine ​​residue in the amino acid sequence of the first part of the fusion protein is disulfide bonded with at least one cysteine ​​residue in the amino acid sequence of the second part of the fusion protein. The cysteine ​​residue in the nanopore and / or the cysteine ​​residue in the auxiliary protein or fusion protein can be a cysteine ​​residue that does not exist in the wild-type transmembrane protein pore monomer or the wild-type auxiliary protein. A plurality of disulfide bonds, such as 2, 3, 4, 5, 6, 7, 8 or 9 to 16, 18, 24, 27, 32, 36, 40, 45, 48, 54, 56 or 63, may be formed between the nanopore and the accessory protein (or fusion protein) in the pore complex. One or both of the nanopore and the accessory protein (or fusion protein) may comprise at least one monomer or subunit, such as up to 8, 9 or 10 monomers or subunits, comprising a cysteine ​​residue at the interface between the nanopore and the accessory protein (or fusion protein).

[0141] The nanopore and / or the auxiliary protein (or fusion protein) may comprise one or more hydrophobic amino acid residues at the interface between the nanopore and the auxiliary protein (or fusion protein), which are more hydrophobic than the residues present at the corresponding positions in the wild-type nanopore and / or the auxiliary protein (or fusion protein). At least one monomer or subunit in the nanopore and / or at least one monomer or subunit in the auxiliary protein (or fusion protein) may comprise at least one residue at the interface between the nanopore and the auxiliary protein (or fusion protein), which is more hydrophobic than the residues present at the corresponding positions in the wild-type hole or the auxiliary protein (or fusion protein). For example, 2 to 10, such as 3, 4, 5, 6, 7, 8 or 9 residues in the nanopore and / or the auxiliary protein (or fusion protein) may be more hydrophobic than the residues at the same positions in the corresponding wild-type nanopore and / or the auxiliary protein (or fusion protein). Such hydrophobic residues enhance the interaction between the nanopore and the auxiliary protein (or fusion protein) in the pore complex. In the case where the residue at the interface in the wild-type nanopore or accessory protein (or fusion protein) is R, Q, N or E, the hydrophobic residue is typically I, L, V, M, F, W, A or Y. In the case where the residue at the interface in the wild-type nanopore or accessory protein (or fusion protein) is I, the hydrophobic residue is typically L, V, M, F, W, A or Y. In the case where the residue at the interface in the wild-type nanopore or accessory protein (or fusion protein) is L, the hydrophobic residue is typically I, V, M, F, W, A or Y.

[0142] Molecular dynamics simulations can be performed to determine which residues in the accessory protein and the nanopore are in close proximity. This information can be used to design accessory protein and / or transmembrane protein nanopore mutants that can increase the stability of the complex. For example, simulations can be performed using the GROMACS software package version 4.6.5, with the GROMOS 53a6 force field and the SPC water model using the cryo-EM structure of the protein. The complex can be solvated and then the energy minimized using the steepest descent algorithm. Throughout the simulation, constraints can be imposed on the main chain of the protein, but the residue side chains can move freely. The system can be simulated in the NPT ensemble for 20ns using a Berendsen thermostat and Berendsen barostat to 300K. The contacts between the accessory protein and the nanopore can be analyzed using GROMACS analysis software and / or locally written code. If two residues are within 3 angstroms of each other, they can be defined as having contact.

[0143] For example, in the pore complex, the interaction between the CsgF peptide and the CsgG pore may be stabilized, for example, by hydrophobic interactions, electrostatic interactions, or covalent bonds at one or more of the following pairs of positions corresponding to SEQ ID NO: 60 and SEQ ID NO: 59, respectively: 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, 29 and 144, or 30 and 196. Residues at one or more of these positions in CsgF and / or CsgG may be modified to enhance the interaction between CsgG and CsgF in the pore.

[0144] Covalent attachment or binding is, for example, via cysteine ​​attachment, wherein the sulfhydryl side group of cysteine ​​is covalently attached to another amino acid residue or moiety and / or via interactions between non-natural (photo) reactive amino acids. (Photo) reactive amino acids refer to artificial analogs of natural amino acids that can be used for cross-linking protein complexes and can be incorporated into proteins and peptides in vivo or in vitro. Commonly used photoreactive amino acid analogs are photoreactive diaziridine analogs of leucine and methionine and p-benzoyl-phenyl-alanine, as well as azidohomoalanine, homopropargylglycine, homoisotopic glycine, p-acetyl-Phe, p-azido-Phe, p-propargyloxy-Phe and p-benzoyl-Phe (Wang et al. 2012; Chin et al. 2002). When exposed to ultraviolet light, they are activated and covalently bound to interacting proteins that are a few angstroms away from the photoreactive amino acid analogs.

[0145] Hole complexes can be prepared and disulfide bonds can be induced by using oxidants (e.g., copper-o-phenanthroline). Other interactions (e.g., hydrophobic interactions, charge-charge interactions / electrostatic interactions) can also be used for these positions instead of cysteine ​​interactions. In another embodiment, non-natural amino acids can also be incorporated into those positions. In this embodiment, covalent bonds are 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.

[0146] For example, the CsgG pore may comprise at least one, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 CsgG monomers modified to facilitate attachment to an accessory protein or fusion protein. For example, cysteine ​​residues may be introduced at one or more of the positions corresponding to 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:59 and / or at any position predicted to contact an accessory protein or fusion protein to facilitate covalent attachment to an accessory protein or fusion protein. As an alternative or in addition to covalent attachment via cysteine ​​residues, the pore may be stabilized by hydrophobic interactions or electrostatic interactions. To facilitate such interactions, a non-natural reactive or photoreactive amino acid is present at a position 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:59.

[0147] For example, the CsgF peptide may be modified to facilitate attachment to the CsgG pore. For example, a cysteine ​​residue may be introduced at one or more of the positions corresponding to positions 1, 4, 5, 8, 9, 11, 12, 26, or 29 of SEQ ID NO: 60 and / or at any position predicted to contact CsgG to facilitate covalent attachment to CsgG. As an alternative or in addition to covalent attachment via cysteine ​​residues, the pore may be stabilized by hydrophobic interactions or electrostatic interactions. To facilitate such interactions, a non-natural reactive or photoreactive amino acid at one or more of the positions corresponding to positions 1, 2, 3, 4, 5, 8, 9, 11, 12, 26, or 29 of SEQ ID NO: 60.

[0148] Such stabilizing mutations can be combined with any other modifications to the accessory protein or fusion protein, such as modifications to improve the interaction of the pore complex with the polynucleotide, or modifications to improve certain properties of the complex (e.g., discrimination of polymer units, such as nucleotides of a polynucleotide).

[0149] In some embodiments, the nanopore can be isolated, substantially isolated, purified, or substantially purified. If the pore is completely free of any other components, such as lipids or other pores, the pore is isolated or purified. If the pore is mixed with a carrier or diluent that does not interfere with its intended use, it is substantially isolated. For example, if the pore is 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), the pore is substantially isolated or substantially purified. Alternatively, the pore can be present in a membrane. Suitable membranes are discussed below.

[0150] The pore complex may exist in the membrane as a single or individual pore. Alternatively, the pore complex may exist in a homogenous or heterogenous population of two or more pores.

[0151] An accessory protein or fusion can be attached directly to the transmembrane protein nanopore, or two proteins (e.g., a first accessory protein and a second accessory protein; a first portion of a fusion protein and a second portion of a fusion protein, etc.) can be attached using a linker (such as a chemical cross-linker or a peptide linker).

[0152] Suitable chemical cross-linking agents are well known in the art. Examples of cross-linking agents include, but are not limited to, 2,5-dioxopyrrolidin-1-yl 3-(pyridine-2-yl disulfide) propionate, 2,5-dioxopyrrolidin-1-yl 4-(pyridine-2-yl disulfide) butyrate and 2,5-dioxopyrrolidin-1-yl 8-(pyridine-2-yl disulfide) octanoate. In certain embodiments, the cross-linking agent is 3-(2-pyridine disulfide) propionic acid succinimidyl ester (SPDP). Typically, before the molecule / cross-linking agent complex is covalently attached to the mutant monomer, the molecule is covalently attached to the bifunctional cross-linking agent, but the bifunctional cross-linking agent / monomer complex can also be covalently attached to the monomer before the molecule is attached. In certain embodiments, the joint is resistant to dithiothreitol (DTT). Other suitable joints include, but are not limited to, iodoacetamide-based and maleimide-based joints.

[0153] 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 accessory protein or fusion protein forms a constriction in the pore complex. 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.

[0154] 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).

[0155] 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.

[0156] 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 the accessory protein or fusion protein is covalently attached to the CsgG pore monomer, the linker is preferably resistant to dithiothreitol (DTT).

[0157] In some embodiments, the crosslinking agent is selected from the group consisting of 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, dimaleimide PEG 1k, dimaleimide PEG 3.4k, dimaleimide PEG 5k, dimaleimide 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)). In some embodiments, the cross-linker is maleimide-propyl-SRDFWRS-(1,2-diaminoethane)-propyl-maleimide.

[0158] The linked CsgG pore monomer and accessory protein or fusion protein may be coupled via 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.

[0159] 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. 32P), 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.

[0160] The 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.

[0161] Another preferred attachment method is via 4-azidophenylalanine (Faz) linkage. This can be mediated by a bifunctional chemical linker or by a polypeptide linker with a terminally present Faz residue.

[0162] In some embodiments, the linker is a bond formed by a sulfur (VI) fluoride exchange (SuFEx) reaction. In some embodiments, the accessory protein (e.g., CsgF or a portion of CsgF) can be functionalized with a sulfonyl fluoride group that can react with a nucleophilic amino acid (e.g., a nucleophilic amino acid of a CsgG pore monomer, a nucleophilic amino acid of another accessory protein, etc.) to form a sulfonyl bond (SuFEX) when in appropriate proximity.

[0163] The auxiliary protein or fusion protein can be genetically fused to the transmembrane protein nanopore. If the entire construct is expressed by a single polynucleotide coding sequence, the hole monomer and the auxiliary protein (or fusion protein) are genetically fused. The monomer or subunit, the auxiliary protein (or fusion protein) can be directly fused to the monomer or subunit of the transmembrane protein nanopore. Alternatively, the monomer or subunit, the auxiliary protein (or fusion protein) can be fused to the monomer or subunit of the transmembrane protein nanopore via one or more joints.

[0164] The distance between the CsgG pore monomer and the accessory protein or fusion protein in the CsgG pore monomer conjugate and / or the length of the linker is preferably less than about 2.00 nm, such as 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. The distance between the CsgG pore monomer and the accessory protein or fusion protein in the pore monomer conjugate and / or the length of the linker is preferably less than about 1.20 nm. This distance / length can be achieved using maleimidocaproic acid, as discussed in more detail below. The distance between the CsgG pore monomer and the accessory protein or fusion protein in the pore monomer conjugate and / or the length of the linker is preferably less than about 0.8 nm. This distance / length can be achieved using maleimidopropionic acid as discussed below.

[0165] The distance between the CsgG pore monomer and the accessory protein or fusion protein in the pore monomer conjugate and / or the length of the linker is preferably about 0.40 nm to about 2.0 nm, such as about 0.45 nm to about 1.90 nm, about 0.50 nm to about 1.80 nm, about 0.55 nm to about 1.7 nm, about 0.60 nm to about 1.6 nm, about 0.65 nm to about 1.5 nm, about 0.7 nm to about 1.4 nm, about 0.75 nm to about 1.3 nm, about 0.80 nm to about 1.2 nm, about 0.85 nm to about 1.1 nm, and about 0.90 nm to about 1.00 nm. The distance between the CsgG pore monomer and the accessory protein or fusion protein in the pore monomer conjugate and / or the length of the linker is preferably about 0.50 nm to about 1.50 nm. The distance between the CsgG pore monomer and the accessory protein or fusion protein in the pore monomer conjugate and / or the length of the linker is preferably about 0.60 nm to about 1.2 nm. This distance / length can be achieved using any of the specific maleimide-containing linkers discussed below.

[0166] The maleimide-containing linker can be any linker discussed below with reference to the constructs described herein. The maleimide-containing linker preferably comprises or consists of a maleimide group and a linear carbon chain of 2, 3, 4, 5, 6 or more carbon atoms. The linear carbon chain is usually attached to the nitrogen atom in the maleimide group. The linear carbon chain also preferably comprises a terminal carboxyl group. The carboxyl group can form an amide bond with an amino acid in an auxiliary protein or fusion protein. The linker is preferably maleimidoacetic acid, maleimidopropionic acid, maleimidobutyric acid, maleimidovaleric acid or maleimidocaproic acid. The linker is most preferably maleimidopropionic acid. The linker is preferably Fig.15 shown.

[0167] The present disclosure also provides a pore monomer conjugate comprising a CsgG pore monomer covalently attached to an auxiliary protein or a fusion protein, wherein the auxiliary protein or the fusion protein is covalently attached to a cysteine ​​residue in the CsgG pore monomer via a linker comprising a thiol-reactive group. The thiol-reactive group may be a maleimide group, a pyridyl disulfide group, a halogen group, a para-fluoro group, an alkene group, an alkyne group, a vinyl sulfone group or a thiosulfone group. These groups, for example Fig.16 As shown. The linker comprising a thiol-reactive group can be any linker discussed below with reference to the construct disclosed herein. The linker preferably comprises a thiol-reactive group and a straight carbon chain of 2, 3, 4, 5, 6 or more carbon atoms or consists of it. The straight carbon chain also preferably comprises a terminal carboxyl group. The carboxyl group can form an amide bond with an amino acid in an auxiliary protein or fusion protein. The linker can be any specific maleimide-containing linker discussed above, wherein the maleimide is replaced by different thiol-reactive groups. The linker containing a thiol-reactive group can be any length discussed above.

[0168] Appropriate linking groups can be designed using conventional modeling techniques. The linker is typically flexible enough to allow the monomers or subunits to assemble into their respective protein oligomers and align along their common axis of symmetry to create a continuous channel within the pore complex.

[0169] Identification and selection of accessory proteins

[0170] Aspects of the present disclosure relate to computer-based methods for designing and / or selecting accessory proteins and / or fusion proteins for inclusion in a protein pore complex (e.g., a protein pore complex comprising a CsgG nanopore). In some embodiments, the method comprises providing an amino acid sequence (e.g., a CsgF amino acid sequence) as input to software comprising code implementing a protein backbone sequence selection technique and processing the amino acid sequence to generate a backbone amino acid sequence as output. In some embodiments, the protein backbone selection technique may be MASTER (e.g., as described in Zhou and Grigoryan, Protein Sci. 2015 Apr;24(4):508–524, the entire contents of which are incorporated herein by reference). In some embodiments, the protein backbone selection technique comprises selecting a protein backbone structure having one or more target features (e.g., the ability to form one or more helical regions, the ability to stack with one or more helical regions of a protein pore, etc.) from known protein backbone structures (e.g., as described in the protein data bank PDB). In some embodiments, the backbone structure is provided as input to software comprising code implementing a protein sequence design and structure prediction technique and processing the backbone structure to generate one or more de novo designed peptide sequences. In some embodiments, the protein sequence design and structure prediction technology can be Rosetta (e.g., as described in Chapter 19 of Leaver-Fay et al. -Rosetta 3: An Object-Oriented Software Suite for the Simulation and Design of Macromolecules, Methods in Enzymology, Academic Press, Vol. 487, 2011, pp. 545-574, doi.org / 10.1016 / B978-0-12-381270-4.00019-6., the entire contents of which are incorporated herein by reference). In some embodiments, the peptide sequence designed from scratch comprises one or more target features that are the same as one or more desired features of the main chain amino acid sequence.

[0171] Methods of producing nanopore complexes

[0172] In one embodiment, a pore complex comprising an auxiliary protein or fusion protein and a transmembrane protein nanopore can be prepared via coexpression. In some embodiments, the method comprises the following steps: expressing both a pore monomer and an auxiliary protein or fusion protein, or an auxiliary protein or monomer in a suitable host cell, and allowing in vivo complex pore formation. In this embodiment, at least one gene encoding a pore monomer in one vector and a gene encoding an auxiliary protein or fusion protein, or at least one auxiliary protein subunit or monomer in a second vector can be transformed together to express protein in a transformed cell and produce a complex. This is preferably performed in vitro or in vitro. Alternatively, two genes encoding a pore monomer and an auxiliary protein (or fusion protein) or its subunit can be placed in one vector, under the control of a single promoter or under the control of two separate promoters, and these promoters can be the same or different.

[0173] Another method for producing a hole complex formed by an auxiliary protein or fusion protein and a transmembrane protein nanopore is the in vitro reconstruction of a protein to obtain a functional hole. In certain embodiments, the method includes contacting a monomer of a transmembrane protein nanopore with an auxiliary protein (or fusion protein), or an auxiliary protein subunit or a monomer in a suitable system to allow a step of complex formation. The system may be an "in vitro system", which refers to a system comprising at least components and an environment necessary for performing the method, and utilizing biomolecules, organisms, cells (or a part of a cell) outside their normal naturally occurring environment, allowing for more detailed, more convenient or more effective analysis than with a whole organism. The in vitro system may also include a suitable buffer composition provided in a test tube, to which the protein component forming the complex has been added. Those skilled in the art will appreciate the option of providing the system.

[0174] In this embodiment, nanopores can be produced by expressing monomers separately from auxiliary proteins or fusion proteins. The pore monomers or nanopores can be purified from cells transformed with a vector encoding at least one pore monomer or with more than one vector each expressing a pore monomer. The auxiliary protein or fusion protein can be purified from cells transformed with a vector encoding at least one auxiliary protein or fusion protein. The purified pore monomer / nanopore can then be incubated with the auxiliary protein or fusion protein to prepare a pore complex.

[0175] In another embodiment, the nanopore monomer and / or the accessory protein or fusion protein are produced separately by in vitro translation and transcription (IVTT).The nanopore monomer can then be incubated with the accessory protein or fusion protein to prepare the pore complex.

[0176] The above embodiments can be combined so that, for example, (i) the nanopore is produced in vivo and the accessory protein or fusion protein is produced in vivo; (ii) the nanopore is produced in vitro and the accessory protein or fusion protein is produced in vivo; (iii) the nanopore is produced in vivo and the accessory protein or fusion protein is produced in vitro; or (iv) the nanopore is produced in vitro and the accessory protein or fusion protein is produced in vitro.

[0177] One or both of the nanopore monomer and the accessory protein or fusion protein may be labeled to facilitate purification. Purification may also be performed when the nanopore monomer and / or the accessory protein or fusion protein 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 the components of the pore complex.

[0178] Any known tag can be used for either of the two proteins. In one embodiment, two tag purifications can be used to purify the pore complex from the components of the pore complex. For example, a Strep tag can be used in the nanopore and a His tag can be used in the accessory protein (or fusion protein), or vice versa. Similar end results can be obtained when the two proteins are purified separately and mixed together, followed by another round of Strep and His purification.

[0179] The pore complex can be prepared before the membrane is inserted or after the nanopore is inserted into the membrane. However, the nanopore can be inserted into the membrane, and then the auxiliary protein (or fusion protein) can be added so that the pore complex can be formed in situ. For example, in one embodiment, in a system where the trans side or cis side of the membrane is accessible (e.g., in a chip or chamber for electrophysiological measurements), the nanopore can be inserted into the membrane, and then the auxiliary protein (or fusion protein) can be added from the trans side or cis side of the membrane so that the complex can be formed in situ.

[0180] In one embodiment, the auxiliary protein can include a protease cleavage site (e.g., TEV, HRV 3 or any other protease cleavage site) and be cleaved before or after association with the nanopore. For example, a full-length auxiliary protein (or fusion protein) can be used to form a hole. The cleavage of amino acid residues that do not form a part of the channel construction and do not need to interact with the transmembrane pore can be cut from the auxiliary protein or fusion protein. In this embodiment, once the hole complex is formed, a protease is used to cut the auxiliary protein or fusion protein. Alternatively, a protease can be used to produce an auxiliary protein or fusion protein before the hole complex is assembled.

[0181] Some protease sites leave an additional tag (or a portion thereof, such as one or more amino acids of the tag) after cleavage. For example, the TEV protease cleavage sequence is ENLYFQS. TEV protease cleaves the protein between Q and S, leaving ENLYFQ intact at the C-terminus of the CsgF peptide. As another example, the HRV C3 cleavage site is LEVLFQGP, and the enzyme cleaves between Q and G, leaving LEVLFQ intact at the C-terminus of the CsgF peptide.

[0182] 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.

[0183] Proteins can be attached to polynucleotide binding proteins. This forms a modular sequencing system. 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 methods described above.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] system

[0188] On the other hand, the present disclosure relates to a system for characterizing a target polynucleotide, the system comprising a membrane and a pore complex; wherein the pore complex comprises: (i) a nanopore located in the membrane, and (ii) an auxiliary protein or a fusion protein attached to the nanopore; wherein the nanopore and the auxiliary protein or the fusion protein together form a continuous channel across the membrane, the channel comprising a first constriction region and a second constriction region.

[0189] The pore complex, nanopore and accessory protein or fusion protein may be any as described above.

[0190] 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 polynucleotide, the system may further comprise a target polynucleotide, wherein the target polynucleotide is transiently located in the continuous channel, and wherein one end of the target polynucleotide is located in the first chamber, and one end of the target polynucleotide is located in the second chamber.

[0191] In one embodiment, the system further includes a conductive solution in contact with the nanopore, an electrode providing a voltage potential across the membrane, and a measurement system for measuring the current through the nanopore. In one embodiment, the voltage applied across the membrane and the hole complex is +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 the increased applied potential, the distinction between different nucleotides can be increased by the hole. 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 an 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).

[0192] 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 the current to indicate the presence of nucleotides identified against a normal current fluctuation background.

[0193] 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 6.9.

[0194] The system can include an array of pore complexes present in a membrane. In a preferred embodiment, each membrane in the array comprises a pore complex. Due to the manner in which the array is formed, for example, the array can include one or more layers of membrane that do not include pore complexes, and / or one or more layers of membrane that include two or more pore complexes. The array can include about 2 to about 12,000 layers, such as about 10 to about 800 layers, about 20 to about 600 layers, about 30 to about 500 layers, about 250 to about 2000 layers, about 500 to about 4000 layers, about 1000 to about 5000 layers, about 2500 to about 10,000 layers, or about 5000 to about 12,000 layers of membrane. In some embodiments, the array includes more than 12,000 layers of membrane.

[0195] 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.

[0196] In one embodiment, the apparatus includes a sensor device capable of supporting a plurality of wells and a membrane and operable to perform analyte characterization using the wells and the membrane; and at least one port for delivering a material for performing the characterization.

[0197] In one embodiment, the apparatus includes a sensor device capable of supporting a plurality of wells and a membrane, the sensor device being operable to perform analyte characterization using the wells and the membrane; and at least one reservoir for containing a material for performing the characterization.

[0198] In one embodiment, the apparatus includes a sensor device capable of supporting a membrane and a plurality of pores and membranes and operable to perform analyte characterization using the pores and membranes; at least one reservoir for containing a material for characterization; a fluid system configured to controllably supply material from at least one reservoir to the sensor device; and one or more containers for receiving corresponding samples, the fluid system configured to selectively supply samples from the one or more containers to the sensor device.

[0199] The device may also comprise circuitry capable of applying an electrical potential and measuring an electrical signal across the membrane and pore complex.The device may be any of those described in WO 2008 / 102120, WO 2009 / 077734, WO 2010 / 122293, WO 2011 / 067559 or WO00 / 28312.

[0200] membrane

[0201] 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 (composed 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.

[0202] Archaeal bipolar tetraether lipids are naturally occurring lipids that are constructed so that lipids form monolayers. These lipids are generally found in extreme microorganisms, thermophilic microorganisms, halophilic microorganisms, and acidophilic microorganisms that survive in harsh biological environments. Its stability is believed to be derived from the fusogenic properties of the final double layer. It is simple to construct block copolymer materials that simulate these biological entities by producing triblock polymers with the general motif hydrophilicity-hydrophobicity-hydrophilicity. This material can form a monomeric membrane that behaves similarly to a lipid bilayer and covers a series of stage performances from vesicles to lamellar membranes. The membranes formed by these triblock copolymers maintain several advantages over biological lipid membranes. Because the triblock copolymers are synthesized, the accurate construction can be carefully controlled to provide the correct chain length and properties required to form a membrane and interact with holes and other proteins.

[0203] Block copolymers can also be composed of subunits that are not classified as lipid submaterials; for example, hydrophobic polymers can be made from siloxanes or other non-hydrocarbon monomers. The hydrophilic subsegments of the block copolymers can also possess low protein binding properties, which allows the production of membranes that are highly resistant when exposed to raw biological samples. This head group unit can also be derived from non-classical lipid head groups.

[0204] Compared to biological lipid membranes, triblock copolymer membranes also have increased mechanical and environmental stability, such as much higher operating temperature or pH range.The synthetic nature of block copolymers provides a platform to tailor polymer-based membranes for a variety of applications.

[0205] The membrane is most preferably one of the membranes disclosed in International Application No. WO2014 / 064443 or WO2014 / 064444.

[0206] The amphiphilic molecule can be chemically modified or functionalized to facilitate coupling of 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.

[0207] Amphiphilic membranes are usually mobile in nature and act essentially as two-dimensional fluids, with lipid diffusion rates of about 10 -8 cms -1 This means that the pore and the coupled polynucleotide can generally move within the amphiphilic membrane.

[0208] 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.

[0209] Methods for forming lipid bilayers are known in the art. Lipid bilayers are usually formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972; 69: 3561-3566), wherein the lipid monolayer is carried on the aqueous solution / air interface on either side of the perforation, and the perforation is perpendicular to the interface. Usually, lipids are added to the surface of the aqueous electrolyte solution by first dissolving the lipids in an organic solvent and then evaporating the solvent droplets on the surface of the aqueous solution on either side of the perforation. Once the organic solvent evaporates, the solution / air interface on either side of the perforation will physically move up and down through the perforation until a double layer is formed. A planar lipid bilayer can be formed across the perforation in the membrane or across the opening in the groove.

[0210] The method of Montal & Mueller is popular because it is a cost-effective and relatively simple method for forming good-quality lipid bilayers suitable for protein pore insertion. Other common methods for bilayer formation include tip dipping, smeared bilayers, and patch clamping of liposomal bilayers.

[0211] Tip dip bilayer formation requires contacting the surface of the aperture (e.g., pipette tip) to the surface of the test solution carrying the monolayer lipids. Similarly, lipid monolayers are first generated at the solution / air interface by allowing lipid droplets dissolved in an organic solvent to evaporate on the surface of the solution. Bilayers are then formed by the Langmuir-Schaefer method, and mechanical automation is required to move the aperture relative to the solution surface.

[0212] For smearing the bilayer, lipid droplets dissolved in an organic solvent are applied directly onto the opening, which is immersed in the aqueous test solution. The lipid solution is thinly painted onto the opening using a paintbrush or equivalent. The thinning of the solvent causes the formation of the lipid bilayer. However, complete removal of the solvent from the bilayer is difficult, and thus the bilayer formed by this method is less stable and more susceptible to noise during electrochemical measurements.

[0213] Patch clamping is commonly used in the study of biological cell membranes. The cell membrane is clamped to the end of a pipette by suction and the patch is attached to the opening. The method has been adapted to produce lipid bilayers by clamping liposomes, which then rupture, leaving the lipid bilayer sealed over the opening in the pipette. The method requires stable, large, unilamellar liposomes and small openings made in a material with a glass surface.

[0214] Liposomes can be formed by ultrasonic treatment, extrusion or Mozafari method (Colas et al. (2007) Micron 38: 841-847). In a preferred embodiment, the lipid bilayer is formed as described in International Application No. WO 2009 / 077734. Advantageously, in this method, the lipid bilayer is formed by dried lipids. In a most preferred embodiment, the lipid bilayer is formed across an opening, as described in WO 2009 / 077734.

[0215] The lipid bilayer is formed by two relative lipid layers. Two layers of lipids are arranged so that their hydrophobic tail groups face each other to form a hydrophobic interior. The hydrophilic head groups of lipids face the aqueous environment on each side of the bilayer outward. The bilayer can be present in many lipid phases, including but not limited to liquid disordered phase (fluid lamellar), liquid ordered phase, solid ordered phase (lamellar gel phase, interdigitated gel phase) and planar bilayer crystals (lamellar subgel phase, lamellar crystalline phase).

[0216] Any lipid composition that forms lipid bilayer can be used.Select lipid composition so that the lipid bilayer with desired properties (such as surface charge, ability to support membrane proteins, packing density or mechanical properties) is formed.Lipid composition can comprise one or more different lipids.For example, lipid composition can contain up to 100 lipids.Lipid composition preferably contains 1 to 10 lipids.Lipid composition can comprise naturally occurring lipids and / or artificial lipids.

[0217] Lipids generally comprise a head group, an interface portion, and two hydrophobic tail groups that may be the same or different. Suitable head groups include, but are not limited to, neutral head groups such as diacylglycerides (DG) and ceramides (CM); zwitterionic head groups such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM); negatively charged head groups such as phosphatidylglycerol (PG); phosphatidylserine (PS), phosphatidylinositol (PI), phosphoric acid (PA), and cardiolipin (CA); and positively charged head groups such as trimethylammonium propane (TAP). Suitable interface portions include, but are not limited to, naturally occurring interface portions such as glycerol-based or ceramide-based portions. Suitable hydrophobic tail groups include, but are not limited to, saturated hydrocarbon chains such as lauric acid (n-dodecanoic acid), myristic acid (n-tetradecanoic acid), palmitic acid (n-hexadecanoic acid), stearic acid (n-octadecanoic acid) and arachidic acid (n-eicosanoic acid); unsaturated hydrocarbon chains such as oleic acid (cis-9-octadecanoic acid); and branched hydrocarbon chains such as phytanoyl. The length of the chain in the unsaturated hydrocarbon chain and the position and number of double bonds can vary. The length of the chain in the branched hydrocarbon chain and the position and number of branches such as methyl groups can vary. The hydrophobic tail group can be connected to the interface portion as an ether or ester. The lipid can be a mycolic acid.

[0218] The lipids may also be chemically modified. The head group or tail group of the lipid may be chemically modified. Suitable lipids whose head groups have been chemically modified include, but are not limited to, PEG-modified lipids, such as 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]; functionalized PEG lipids, such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)2000]; and lipids modified for conjugation, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl). Suitable lipids whose tail groups have been chemically modified include, but are not limited to, polymerizable lipids such as 1,2-bis(10,12-trimethylbenzene)-sn-glycero-3-phosphocholine; fluorinated lipids such as 1-palmitoyl-2-(16-fluoropalmitoyl)-sn-glycero-3-phosphocholine; deuterated lipids such as 1,2-dipalmitoyl-D62-sn-glycero-3-phosphocholine; and ether-linked lipids such as 1,2-di-O-phytyl-sn-glycero-3-phosphocholine. The lipids may be chemically modified or functionalized to facilitate coupling of polynucleotides.

[0219] The amphiphilic layer, such as a lipid composition, typically comprises one or more additives that will affect the properties of the layer. Suitable additives include, but are not limited to, fatty acids such as palmitic acid, myristic acid, and oleic acid; fatty alcohols such as palmityl, myristyl alcohol, and oleyl alcohol; sterols such as cholesterol, ergosterol, lanosterol, sitosterol, and stigmasterol; lysophospholipids such as 1-acyl-2-hydroxy-sn-glycero-3-phosphocholine; and ceramides.

[0220] In another preferred embodiment, the membrane 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 WO 2009 / 035647. If the membrane comprises a solid layer, the pores are typically present in the amphiphilic membrane or layer contained in the solid layer, for example in a hole, hole, gap, channel, groove or crevice in the solid layer. A technician can prepare a suitable solid / amphiphilic hybrid system. Suitable systems are disclosed in WO 2009 / 020682 and WO2012 / 005857. Any of the amphiphilic membranes or layers discussed above can be used.

[0221] 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 methods of the present disclosure are typically performed in vitro.

[0222] Methods for characterizing analytes

[0223] On the other hand, a method for determining the presence, absence, or one or more characteristics of a target analyte is disclosed. The method includes contacting the target analyte with a membrane comprising a pore complex so that the target analyte moves relative to a continuous channel, such as entering or passing through the continuous channel, the continuous channel comprising at least two structures provided by a nanopore and an auxiliary protein or peptide in the pore complex, respectively, and performing one or more measurements as the analyte moves relative to the channel, and thereby determining the presence, absence, or one or more characteristics of the analyte. The analyte may contract through the nanopore and then contract through the auxiliary protein. In an alternative embodiment, the analyte may contract through the auxiliary protein and then contract through the nanopore, depending on the orientation of the pore complex in the membrane.

[0224] In one embodiment, the method is used to determine the presence, absence or one or more characteristics of a 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.

[0225] The binding of molecules in the channel of the pore complex or near any opening of the channel will have an effect on the open channel ion flow through the pore, which is the essence of the "molecular sensing" of the pore channel. 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 WO 2009 / 077734). The degree of reduction in ion flow measured by the reduction in current is related to the size of the obstacles in or near the pore. Therefore, the binding of molecules of interest (also referred to as "analytes") 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 has applications in personalized drug development, medicine, diagnostics, life science research, environmental monitoring, and the security and / or defense industries.

[0226] The target analyte can be 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, a polysaccharide, a dye, a bleach, a drug, a diagnostic agent, a recreational drug, an explosive, a toxic compound, or an environmental pollutant. The method can 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 can 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).

[0227] 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.

[0228] In one embodiment, the analyte is 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 as described above. It should be understood that the target analyte may be modified by any method available in the art.

[0229] In a preferred embodiment, the analyte is a polynucleotide, such as a nucleic acid. A polynucleotide is defined as a macromolecule comprising two or more nucleotides. 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. The change in ion flow can be recorded. Suitable electrical measurement techniques for recording changes in ion flow are described in, for example, WO 2000 / 28312 and D. Stoddart et al., Proc. Natl. Acad. Sci., 2010, 106, pp. 7702-7 (single-channel recording equipment); and, for example, in WO 2009 / 077734 (multi-channel recording technology). By 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 each nucleotide of the nucleic acid sequence of interest passes through the channel of the nanopore in turn, the open channel ion flow is reduced. 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 measured ion flow of known nucleotides through the channel, thereby generating a means for determining which nucleotide is passing through the channel, and therefore, when performed sequentially, providing a way to determine the nucleotide sequence of the nucleic acid passing through the nanopore. 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 a single nucleotide passing through the constriction structure (or "reading head"). It should be understood that the complete nucleic acid polymer that "spirals" through the hole via the action of, for example, a related polymerase or helicase can be sequenced. Alternatively, the sequence can be determined by the passage of nucleotide triphosphate bases sequentially removed from the target nucleic acid near the hole (see, for example, WO 2014 / 187924).

[0230] 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 monophosphates, diphosphates or triphosphates. 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 a 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: (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.

[0231] 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.

[0232] The nucleotide may have any identity (ii) and include, but are 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.

[0233] A pore complex comprising at least two constrictions is particularly useful in analyzing homopolymers. For example, the pore 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, the pore can be used to sequence a polynucleotide comprising polyA, polyT, polyG and / or polyC regions.

[0234] In some embodiments, the CsgG pore constriction consists of residues at positions 51, 55, and 56 of SEQ ID NO:59. As the 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. Although some CsgG pores (e.g., CsgG pores lacking one or more accessory proteins or fusion proteins as described herein) 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 some information. Because 5 bases dominate the signal for CsgG and its constriction mutants, homopolymers longer than 5 are difficult to discern without the use of additional residence time information. However, if the DNA is passing through a second constriction, more DNA bases will interact with the combined constriction, increasing the length of homopolymers that can be discerned.

[0235] Reagent test kit

[0236] On the other hand, the present disclosure also provides a kit for characterizing a target polynucleotide. The kit includes components of the disclosed pore complex and membrane. The membrane is preferably formed by these components. The pore complex is preferably present in the membrane, forming a transmembrane pore complex channel together. The kit may include components of any type of membrane (such as an amphiphilic layer or a triblock copolymer membrane). The kit may further include a polynucleotide binding protein, such as a nucleic acid processing enzyme, such as a polymerase or a helicase. The kit may further include one or more anchors, such as cholesterol, for coupling the polynucleotide to the membrane. The kit may further include one or more polynucleotide adapters, which may be attached to the target polynucleotide to facilitate the characterization of the polynucleotide. In one embodiment, an anchor, such as cholesterol, is attached to a polynucleotide adapter. The 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 items: a suitable buffer (aqueous solution), a tool for obtaining a sample from a subject (such as a container or instrument comprising a needle), a tool or voltage or patch clamp device for amplifying and / or expressing polynucleotides. The reagents may be present in the kit in a dry state so that the fluid sample resuspends the reagents. The kit may also optionally include instructions for enabling the kit to be used in the disclosed method or details about which organisms the method may be used for. Finally, the kit may also include additional components useful in polynucleotide characterization.

[0237] 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 disclosure, various changes or modifications may be made in form and detail without departing from the scope and spirit of the present disclosure. 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.

[0238] Examples

[0239] Example 1

[0240] To generate helical constrictions, de novo design is used to select small protein domains that fold well and project into the lumen of the nanopore to the desired extent. Many programs can be used for this purpose. This example describes a workflow that uses extent MASTER to facilitate backbone design and Rosetta with variable backbone geometry for sequence selection.

[0241] To create new domains that are projected into the lumen of the pore, programs such as RF diffusion, CHROMA or the program MASTER can be used. Here we used MASTER. The Protein Data Bank (PDB) was searched for structures that met the following criteria: 1) stability of the target region of CsgF (residues 16-30); 2) projection into the nanopore to create a diameter between 1.0 and 2.0 when all units were generated using a 9-fold symmetry operator. and 3) the new domain should not conflict with any atom in CsgG or with symmetry pairing from CsgF.

[0242] First, helices docking to the target region in CsgF and its symmetric neighbors were identified whose geometries were frequently observed in native proteins in the PDB and were therefore "designable". After clustering the output according to the RMSD of the target region plus the found helices, the best candidates were selected based on the number of closely related helix-helix pairs found in the database (Figure 1). In this way, the geometry of the helices that were closely packed relative to the target and its N-terminal four amino acids was selected. In addition, the database was searched for helices that made favorable helix-helix interactions with symmetry-related partners. The linker (e.g., loop structure) connecting the helices was selected using a database of helical backbones (Figure 1). The sequence of the resulting backbone was then designed using Rosetta. Representative sequences (e.g., SEQ ID NOs: 1-58) were generated.

[0243] Sequences for experimental validation were selected based on the lowest energy score and highest PackStat score, e.g. Figure 2To further prioritize sequences, aggregation propensity can also be tested using one of a variety of aggregation and amyloid prediction programs.

[0244] Example 2

[0245] Materials and methods

[0246] E. coli CsgG pore production

[0247] 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 on LB agar plates containing appropriate antibiotics for selection and incubated overnight at 37°C. A single colony from the agar plate was inoculated into LB medium containing appropriate antibiotics and grown overnight at 37°C with shaking. The culture was diluted into autoinduction medium supplemented with the necessary antibiotics and incubated at 18°C ​​with shaking for 68 hours. The cells were harvested by centrifugation and then lysed and extracted into a buffer containing 1x Bugbuster extraction reagent (Merck 70921) and 0.1% DDM. The lysate was centrifuged and the pores were purified from the soluble extract using affinity chromatography, heat treatment, and then size exclusion chromatography to select for oligomeric nanopores as judged by SDS-PAGE.

[0248] CsgG / CsgF or fusion protein complex formation protocol

[0249] CsgG-CsgF complexes were prepared from nanopores purified as described above and chemically synthesized de novo fusion proteins with or without maleimide modification. For cysteine-containing fusion proteins, cyclization of the fusion proteins was achieved by crosslinking thiols at the appropriate cysteines. The nanopores were buffer exchanged into pH 7.0 buffer without reducing agent and incubated with an 8-fold molar excess of peptide relative to CsgG monomer for 1 h at 25 °C. The samples were then heated at 60 °C for 15 min, subsequently centrifuged to remove any precipitate, and DTT was added to prevent any further reaction.

[0250] SDS-PAGE analysis

[0251] 1 μg of complex and CsgG only well control were added to each 0.5 mL ProteinLoBind 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.

[0252] For some analyses, 1 ug of complex and well controls of CsgG alone were added to individual PCR tubes and made up to a 10 μL volume with reaction buffer. A freshly prepared 1 M DTT stock solution was prepared and spiked into individual PCR tubes at a final concentration of 10 mM. The final volume was made up to 20 μL by adding 10 μL of 2x Laemmli buffer. Each sample was heated at 95°C for 2 minutes on a PCR thermal cycler. This was cooled for 5 minutes, and then all material from each sample was loaded onto a 4%-20% TGX gel (BioRad, 5671093) run with 1x TGS buffer (Sigma, T7777). This was run at 300 V for 21 minutes. To image the gel, Spyro Ruby (Merk, S4942) stain was used as per the manufacturer's instructions. This was then imaged on a GE Typhoon gel imager using a 450 nm laser.

[0253] Electrical Measurement

[0254] Electrical measurements were acquired from CsgG-only, CsgG / CsgF, or CsgG / fusion protein complexes inserted into MinION flow cells. After inserting a single pore 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.

[0255] The analyte used to assess the DNA profile was a 3.6 kilobase DNA segment from the 3' end of the lambda genome as shown in Figure 23. 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-LSK110 protocol.

[0256] Electrical measurements were obtained using a minION Mk1b from Oxford Nanopore Technologies. A standard sequencing script was run at -180 mV for 6 h with static flicking every 5 min to remove extended nanopore blocks. Raw data were collected in batch FAST5 files using MinKNOW software (Oxford Nanopore Technologies).

[0257] Discrimination analysis

[0258] A FAST5 containing DNA curve (eg, electrical measurements) was obtained for a 3.6 kilobase DNA segment from the 3' end of the lambda genome (3.6 Kb lambda). The DNA curve was trimmed using a custom Python script to remove any electrical signal measurements captured before DNA sequencing began.

[0259] The trimmed 3.6 Kb lambda curve and the genomic reference corresponding to this region are used to train the parameters of the neural network. The neural network contains 4 layers and models the sequences of the user-specified window length and the associated current levels of these sequences. The window length specified for these models allows regions of + / - 12 nucleotides to contribute to the current level at any one position.

[0260] The trained neural network was used to predict the current level that would correspond to a 3.6Kb lambda DNA reference sequence. It was also used to predict the current level based on all possible single base edits to that sequence.

[0261] Changing the base at a single position (L) in the sequence changes the current predicted when that base passes through the main constriction of the pore, but also changes the current before and after the base passes through the main constriction. The edited set of 3.6 Kb lambda sequences was analyzed for predicted current levels to calculate the range of predicted currents at position L+X (offset) when the base at position L was changed. Offsets between -16 and +16 were analyzed at each position. The median range of predicted currents at each offset was calculated to give the data in the figure. The model was centered so that the largest peak indicating CsgG constriction corresponds to position 0.

[0262] Example 3

[0263] De novo fusion protein sequences designed using Rosetta were analyzed and sequences were selected for experimental validation based on the lowest energy score and highest PackStat score ( Figure 2PSIPRED (e.g., as described in McGuffin LJ, Bryson, K, Jones D, Bioinformatics, 16, 404-405, 2000) analysis was performed to predict the secondary structure of the fusion protein. Residues are colored according to whether they are predicted to be strands, helices, and coils, respectively. The secondary structure analysis of the de novo designed fusion protein (e.g., extended CsgF protein) and the mature sequence of wild-type CsgF is shown in Figure 3A The structural analysis of the de novo designed fusion proteins ONT1 to ONT10, ONT11 to ONT20, and ONT21 to ONT25 is shown in Figures 3B to 3C middle.

[0264] The three-dimensional structures of alternative sequences of the de novo designed fusion proteins were also studied using a protein folding algorithm. The predicted 3-D structures of the de novo designed fusion proteins ONT1 to ONT10, ONT11 to ONT20, and ONT21 to ONT25 are shown in Figures 4A to 4C Middle. Structures are colored according to a confidence metric, the predicted local distance difference test (pLDDT).

[0265] SDS-PAGE gel analysis of CsgG-only wells and CsgG / fusion protein complexes containing CsgF-del(S31-F119) control or de novo designed fusion proteins with or without maleimide crosslinker ( Figure 5 ). Complexes containing the fusion protein showed a band shift, indicating that these samples are nanopore complexes. Note that the samples were not heated before loading on the gel. SDS-PAGE gel analysis was also performed on CsgG-only pores and CsgG / fusion protein complexes. These complexes contained CsgF-del(S31-F119) control or de novo designed fusion proteins with or without maleimide crosslinker ( Figure 6 ). The pores were resolved into their constituent monomer components upon boiling in the presence of DTT prior to loading onto the gel. Note that no band shift was observed in the absence of maleimide crosslinking, indicating that these bands consist of CsgG monomers only. Lane 7 shows a band shift compared to the CsgG only control, indicating that the fusion protein is covalently bound to the CsgG pore due to the presence of maleimide. Lanes 8 and 9 show further band shifts due to the increased mass of the fusion protein. This indicates that the fusion protein is covalently bound to the CsgG pore.

[0266] The ionic current (pA) versus time (s) was measured for single-stranded DNA translocation 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 observed pore opening current for the CsgG-only pore was approximately 180 pA. Table 1 below shows representative data for the median range, median noise, and median signal-to-noise ratio (SNR) of a protein pore complex as described in the present disclosure.

[0267] Table 1: Index table

[0268]

[0269] Figures 7 to 11 Representative ion current (pA) versus time (s) traces are shown as single-stranded DNA translocates through a pore of CsgG alone, CsgG containing the del(S31-F119)CsgF peptide, or CsgG containing a de novo designed fusion protein. 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 complete DNA current trace, while the bottom row shows a magnified view of the first portion of the current trace. The opening current of the CsgG-only pore was observed to be approximately 175 pA to 200 pA, and the median current of the DNA curve was approximately 75 pA. For the pore containing the CsgF peptide, the opening current was approximately 90 pA to 120 pA, and the median current was approximately 35 pA to 50 pA. Figure 7 The trace is shown as DNA translocates through a CsgG-only pore. Figure 8 Shown are representative ionic current (pA) versus time (s) traces as single-stranded DNA translocates through CsgG containing the del(S31-F119) CsgF peptide, with (right) or without (left) a maleimide cross-linker. Fig. 9 Shown are representative ionic current (pA) versus time (s) traces as single-stranded DNA translocates through CsgG containing the de novo designed fusion protein ONLP20623 in the absence of a maleimide cross-linker. Fig.10 Shown are representative ionic current (pA) versus time (s) traces as single-stranded DNA translocates through CsgG containing de novo designed fusion proteins ONLP20624 (without maleimide crosslinks) or ONLP20627 (with maleimide crosslinks). Fig.11 Representative ionic current (pA) versus time (s) traces are shown when single-stranded DNA is translocated through CsgG containing the de novo designed fusion proteins ONLP20628 (with maleimide crosslinker) or ONLP20625 (without maleimide crosslinker). In some embodiments, the fusion protein comprises a 37R residue as well as a cysteine ​​residue to form an internal disulfide bond within the peptide, i.e., a circularized fusion protein.

[0270] As the DNA molecule translocates through the pore, a curve is generated showing the position within the pore and its contribution to the overall change in ionic current levels ("discrimination"). Distance within the pore is measured in nucleotide steps relative to the main constriction. Negative values ​​correspond to positions below the main constriction, while positive values ​​correspond to positions above the main constriction (CsgG). The dotted box shows the region that will be affected by the introduction of the de novo designed fusion protein. Fig.12 Representative curves are shown as a DNA molecule translocates through a CsgG-only pore. The CsgG-only pore (plus / minus Q153C) shows one major discernible peak at position 0. Fig.13 Representative curves are shown as a DNA molecule translocates through a CsgG / CsgF pore. The dashed box shows the region that would be affected by the introduction of a de novo designed fusion protein. CsgG-CsgF-del(S31-F119) pores with (right) or without (left) a maleimide crosslinker show two discrimination peaks. As seen in the CsgG-only pore, the main discrimination peak is at position 0, and an additional discrimination peak is located 4 to 6 nucleotides below the main constriction (positions -4 to -6). This additional discrimination region has less impact on the ion current than the main discrimination peak at position 0. Fig.14 Representative curves are shown as a DNA molecule translocates through a CsgG / fusion protein (ONLP20641 or ONLP20644) pore. Complexes containing CsgG and a de novo designed fusion protein containing K37R, with (right) or without (left) a maleimide crosslinker; with cyclization, show three discernible peaks. As seen in the CsgG-only pore, the main discernible peak is at position 0, and additional peaks are at positions -6 and -9. The peak at position -9 corresponds to the expected contraction produced by the de novo designed fusion protein when folded in the correct orientation.

[0271] Example 3

[0272] The pore formed by the nine subunits shown in SEQ ID NO: 61 (with or without a maleimide cross-linker; both were not cyclized) was also tested as described in Example 2. Figures 17 to 18 shown.

[0273] Representative sequences

[0274] >(SEQ ID NO:1)CsgF-WT-del(S31-F119))-Ext(31-GGELAAKLWANGDETNALSLFQTIIQS)(ONLP20623)

[0275] <h2 style=";text-align:left;direction:ltr">GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNGGELAAKLWANGDETNALSLFQTIIQS>(SEQ ID NO:2)CsgF-WT-K37R-del(S31-F119)-Ext(31-GGELAAKLWANGDETNALSLFQTIIQS)(ONLP20624)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0276] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNGGELAARLWANGDETNALSLFQTIIQS>(SEQ ID NO:3)CsgF-WT-N24C / K37R-del(S31-F119)-Ext(31-GGELAAKLWANGDETNALSLFQTIIQSC)(ONLP20625)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0277] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLCSAQAQNGGELAARLWANGDETNLSLFQTIIQSC>(SEQ IDNO:4)Mat-CsgF-Eco-(WT-Del(S31-F119)-Ext(31-AGELAKKLWENGNVNQALSLFQTVIQS)(ONLZ19432,DGLONT76)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0278] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLWENGNVNQALSLFQTVIQS>(SEQ ID NO:5)Mat-CsgF-Eco-(WT-K36R / K37R-Del(S31-F119)-Ext(31-AGELAKKLWENGNVNQALSLFQTVIQS)(ONLZ19431)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0279] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELARRLWENGNVNQALSLFQTVIQS>(SEQ ID NO:6)Mat-CsgF-Eco-(WT-N24C / K36R / K37R-Del(S31-F119)-Ext(31-AGELARRLWENGNVNQALSLFQTVIQSC)(ONLZ19781)<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0280] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLCSAQAQNAGELARRLWENGNVNQALSLFQTVIQSC>(SEQ IDNO:7)ONT113_2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0281] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAAELAAKLWANADETNALSLFQTIIQS>(SEQ ID NO:8)ONT113_3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0282] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAAELAAKLWANADETNLSLFQTLIQS>(SEQ ID NO:9)ONT1<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0283] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLFKKGDLTNLSLFQTVIQS>(SEQ ID NO:10)ONT2<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0284] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELVEKLFKNGDWTNAISIFQTVIQS>(SEQ ID NO:11)ONT 3<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0285] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAEKLWRNGDETNALSLFQTVIQS>(SEQ ID NO:12)ONT 4<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0286] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAEKLWKNGDETNALSLFQTVIQS>(SEQ ID NO:13)ONT 5<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0287] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLWENGDETNALSLFQTVVQS>(SEQ ID NO:14)ONT 6<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0288] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAEKLWRNGNESDALSLFQTVIQS>(SEQ ID NO:15)ONT 7<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0289] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLFENGDKTNALSLFQTVIQS>(SEQ ID NO:16)ONT 8<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0290] <h2 style=";text-align:left;direction:ltr">GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLWENGDETNALSLFQTVIQS>(SEQ ID NO:17)ONT 9<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0291] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLWEKGNSEDALALFRTVVQS>(SEQ ID NO:18)ONT 10<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0292] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLFDNGDMENAMKLFQTVIAS>(SEQ ID NO:19)ONT 11<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0293] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAEKLWRNGDKDRALALFRTVIQS>(SEQ ID NO:20)ONT 12<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0294] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELADKLWKNGDKDRALSLFQTVIQS>(SEQ ID NO:21)ONT 13<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0295] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLFDNGDMDRALALFRTVIAS>(SEQ ID NO:22)ONT 14<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0296] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLFDNGNEEDALALFRTVVAS>(SEQ ID NO:23)ONT 15<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0297] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLWKKGDEENALKLFRTVVTS>(SEQ ID NO:24)ONT 16<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0298] <h2 style=";text-align:left;direction:ltr"> GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLFKNGNMEDALKLFRTVIAS>(SEQ ID NO:25)ONT 17<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0299] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGKVAAILWKNGNKSDALSLFQTVVTS>(SEQ ID NO:26)ONT 18

[0300] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLFKNGDLTNALSLFQTVVQS>(SEQ ID NO:27)ONT 19

[0301] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELGLKLLRKGDVETALTLFAQVISG>(SEQ ID NO:28)ONT 20

[0302] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELGLKLILKGDLETALKLFAIVIAG>(SEQ ID NO:29)ONT 21

[0303] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELGLKLLRKGDVETALKLFAIVIAG>(SEQ ID NO:30)ONT 22

[0304] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLYENGLIELALMLFALVIAS>(SEQ ID NO:31)ONT 23

[0305] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELYKKLWDNGEVDKALDLFAKIIAG>(SEQ ID NO:32)ONT 24

[0306] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELGKKLIEKGDLETALKLFAIVIAG>(SEQ ID NO:33)ONT 25

[0307] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGEIALRLLKNGKEEEALKTLLVTIAG>(SEQ ID NO:34)ONT26

[0308] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLWKKGDETNALSLFQTVVTS>(SEQ ID NO:35)ONT27

[0309] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGKVAAILWKNGNKSDALSLFQTVVTS>(SEQ ID NO:36)ONT28

[0310] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLWEKGDETNALSLFQTVVTS>(SEQ ID NO:37)ONT29

[0311] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGDLAAKLWKKGDETNALSLFQTVVTS>(SEQ ID NO:38)ONT30

[0312] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLWKNGNSSDALSLFQTVVTS>(SEQ ID NO:39)ONT31

[0313] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLWEKGDETNALSLFQTVVTS>(SEQ ID NO:40)ONT32

[0314] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLWEKGDSSNALSLFQTVVTS>(SEQ ID NO:41)ONT33

[0315] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGDLAAKLWKNGDETNALSLFQTVVTS>(SEQ ID NO:42)ONT34

[0316] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLFKNGDLTNALSLFQTVVQS>(SEQ ID NO:43)ONT35

[0317] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLWKKGDETNALSLFQTVVTS>(SEQ ID NO:44)ONT36

[0318] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLFNSGDLDRALALFRTVVTS>(SEQ ID NO:45)ONT37

[0319] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGKVAKELYDNGDEKWALLLFRTVVTS>(SEQ ID NO:46)ONT38

[0320] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGKVAAELYKNGDEKNALLLFRTVVAS>(SEQ ID NO:47)ONT39

[0321] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLFKNGDMENALALFRTVVTS>(SEQ ID NO:48)ONT40

[0322] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLWEKGNSEDALALFRTVVQS>(SEQ ID NO:49)ONT41

[0323] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLFNKGDEDRALALFRTVVQS>(SEQ ID NO:50)ONT42

[0324] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLWKNGDEENALALFRTVVTS>(SEQ ID NO:51)ONT43

[0325] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAEKLWRSGDADRALALFRTVVTS>(SEQ ID NO:52)ONT44

[0326] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLWKNGNEEDALALFRTVVTS>(SEQ ID NO:53)ONT45

[0327] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLFNNGDEDRALALFRTVVQS>(SEQ ID NO:54)ONT46

[0328] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLWKKGDEDRALALFRTVVTS>(SEQ ID NO:55)ONT47

[0329] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLFNSGDEDRALALFRTVVQS>(SEQ ID NO:56)ONT48

[0330] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLYNNGDLDRADATFRTVVQS>(SEQ ID NO:57)ONT49

[0331] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLWENGNEEDALALFRTVVTS>(SEQ ID NO:58)ONT50

[0332] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGEIAKQLWEKGDESSAITVATIVLSS

[0333] >(SEQ ID NO:59)Wild-type Escherichia coli CsgG protein monomer (without signal sequence)

[0334] CLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQFKPYPASNFSTAVP

[0335] QSATAMLVTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIM

[0336] VEGSIIGYESNVKSGGVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILS

[0337] YEVQAGVFRFIDYQRLLEGEVGYTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNK

[0338] AERQNDILVKYRHMSVPPES

[0339] (SEQ ID NO:60) Residues 1 - 30 of the CsgF peptide

[0340] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQN

[0341] (SEQ ID NO:61) CsgF - WT - del(S31 - F119) - Ext(31 - AGILAAQLWNNGDYDRALSLFIAVVQS - 57)

[0342] GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGILAAQLWNNGDYDRALSLFIAVVQS

Claims

1. A protein nanopore complex comprising: (a) a CsgG nanopore comprising an inner lumen; and (b) a fusion polypeptide comprising a first portion comprising a CsgF protein and a second portion comprising a helix formation accessory protein, wherein the fusion protein is attached to the nanopore.

2. The protein nanopore complex of claim 1, wherein the first portion of the fusion protein is attached to the CsgG nanopore.

3. The protein nanopore complex of claim 1 or 2, wherein the first portion of the fusion protein is located inside the lumen of the CsgG nanopore.

4. The protein nanopore complex of any one of claims 1 to 3, wherein the first portion of the fusion protein extends outside the lumen of the CsgG nanopore.

5. The protein nanopore complex of any one of claims 1 to 4, wherein the first portion forms a first constriction in the lumen of the CsgG nanopore. The protein nanopore complex according to claim 5 , wherein the second portion forms a second constriction.

7. The protein nanopore complex of any one of claims 1 to 6, wherein the CsgG nanopore further comprises a constriction.

8. The protein nanopore complex of any one of claims 1 to 7, wherein the second moiety is not attached to the CsgG nanopore.

9. The protein nanopore complex according to any one of claims 1 to 8, wherein the second portion comprises one or more α-helices.

10. The protein nanopore complex of any one of claims 1 to 9, wherein each of the α-helices comprises between 0 and 15 α-helical turns. 11 . The protein nanopore complex according to any one of claims 1 to 10 , wherein the second portion comprises a first α-helix comprising one to four α-helical turns and a second α-helix comprising three to six α-helical turns.

12. The protein nanopore complex of claim 11, wherein the second α-helix is ​​stacked relative to the first α-helix.

13. The protein nanopore complex according to any one of claims 9 to 12, wherein the second portion comprises between 1 and 55 amino acid residues.

14. The protein nanopore complex according to any one of claims 6 to 13, wherein the distance between the first constriction and the second constriction is about to about within the range.

15. The protein nanopore complex according to any one of claims 1 to 14, wherein the protein nanopore complex has a An axial length of about to about within the range.

16. The protein nanopore complex of any one of claims 1 to 15, wherein the fusion protein is attached to the nanopore via a linker.

17. The protein nanopore complex of claim 16, wherein the linker comprises a bond, a peptide linker, or a chemical linker.

18. The protein nanopore complex of claim 16 or 17, wherein the linker comprises a bond formed by a sulfur (VI) fluoride exchange (SuFEx) reaction.

19. The protein nanopore complex of claim 16 or 17, wherein the linker comprises one or more maleimide molecules.

20. The protein nanopore complex according to any one of claims 1 to 19, wherein the fusion protein is circularized.

21. The protein nanopore complex of claim 20, wherein the cyclization comprises one or more side chain to side chain cyclization bonds.

22. The protein nanopore complex of claim 21, wherein at least one of the side chain to side chain cyclization bonds is a disulfide bond.

23. A protein nanopore complex comprising: (a) a CsgG nanopore comprising a lumen and a first constriction formed within the lumen of the nanopore; and (b) a fusion protein comprising a first part comprising a CsgF protein and a second part comprising a helix formation accessory protein, wherein the fusion protein is attached to the nanopore.

24. The protein nanopore complex of claim 23, wherein the first portion of the fusion protein is attached to the CsgG nanopore.

25. The protein nanopore complex of claim 23 or 24, wherein the first portion of the fusion protein is located inside the lumen of the CsgG nanopore.

26. The protein nanopore complex of any one of claims 23 to 25, wherein the second portion of the fusion protein is located outside the lumen of the CsgG nanopore.

27. The protein nanopore complex of any one of claims 23 to 26, wherein the first portion forms a second constriction in the lumen of the CsgG nanopore.

28. The protein nanopore complex of claim 27, wherein the second portion forms a third constriction in the lumen of the CsgG nanopore.

29. The protein nanopore complex of any one of claims 23 to 28, wherein the second moiety is not attached to the CsgG nanopore.

30. The protein nanopore complex of any one of claims 23 to 29, wherein the second portion comprises one or more α-helices.

31. The protein nanopore complex of claim 30, wherein each of the α-helices comprises between 0 and 15 α-helical turns.

32. The protein nanopore complex of any one of claims 23 to 31, wherein the second portion comprises between 1 and 55 amino acid residues.

33. The protein nanopore complex of any one of claims 23 to 32, wherein the fusion protein is circularized.

34. The protein nanopore complex of claim 33, wherein the cyclization comprises one or more side chain to side chain cyclization bonds.

35. The protein nanopore complex of claim 34, wherein at least one of the side chain to side chain cyclization bonds is a disulfide bond.

36. A protein nanopore complex comprising: (a) A CsgG nanopore comprising an inner cavity and a first constriction formed within the inner cavity of the nanopore; (b) a first accessory protein that attaches to the CsgG nanopore and forms a second constriction in the lumen of the nanopore; and (c) a second accessory protein that attaches to the CsgG nanopore or the first accessory protein and forms a third constriction.

37. The protein nanopore complex of claim 36, wherein the first accessory protein is located inside the lumen of the CsgG nanopore.

38. The protein nanopore complex of claim 36 or 37, wherein the first accessory protein comprises a CsgF protein or peptide.

39. The protein nanopore complex of any one of claims 36 to 38, wherein the second accessory protein comprises one or more α-helices.

40. The protein nanopore complex of claim 39, wherein each of the one or more α-helices comprises between 0 and 15 α-helical turns.

41. The protein nanopore complex of claim 39 or 40, wherein the second accessory protein comprises two α-helices.

42. The protein nanopore complex of claim 41, wherein one of the α-helices comprises between 1 and 6 α-helical turns.

43. The protein nanopore complex of claim 41 or 42, wherein one of the α-helices comprises between 1 and 10 α-helical turns.

44. The protein nanopore complex of any one of claims 41 to 43, wherein one of the α-helices comprises three α-helical turns and the other α-helix comprises three or four α-helical turns.

45. The protein nanopore complex of any one of claims 36 to 44, wherein the second accessory protein comprises at least one alpha helix that is stacked relative to the alpha-helical stack of the first accessory protein.

46. ​​The protein nanopore complex of any one of claims 36 to 45, wherein the second accessory protein comprises between 1 and 55 amino acid residues.

47. The protein nanopore complex of any one of claims 36 to 46, wherein the distance between the first constriction and the second constriction is about to about within the range.

48. The protein nanopore complex of any one of claims 36 to 47, wherein the distance between the second constriction and the third constriction is about to about within the range.

49. The protein nanopore complex of any one of claims 36 to 48, wherein the protein nanopore complex has a An axial length of about to about within the range.

50. The protein nanopore complex of any one of claims 36 to 49, wherein the first accessory protein and the second accessory protein are attached via a linker.

51. The protein nanopore complex of claim 50, wherein the linker comprises a bond, a peptide linker, or a chemical linker.

52. The protein nanopore complex of claim 50 or 51, wherein the linker comprises a bond formed by a sulfur (VI) fluoride exchange (SuFEx) reaction.

53. The protein nanopore complex of claim 50 or 51, wherein the linker comprises one or more maleimide molecules.

54. The protein nanopore complex of any one of claims 36 to 53, wherein the first accessory protein and the second accessory protein comprise one or more side chain to side chain cyclization bonds.

55. The protein nanopore complex of claim 54, wherein at least one of the side chain to side chain cyclization bonds is a disulfide bond.

56. A system for characterizing a target analyte, the system comprising the protein nanopore complex according to any one of claims 1 to 55 inserted into a membrane.

57. The system of claim 56, further comprising: A conductive solution in contact with the protein nanopore complex, electrodes providing a voltage potential across the membrane, and a measurement system for measuring the current passing through the protein nanopore complex.

58. A method for characterizing a target analyte, the method comprising the steps of: (a) contacting the system according to claim 56 with the target analyte; (b) applying an electric potential across the membrane so that the target analyte moves relative to the lumen formed by the protein nanopore complex; and (c) taking one or more measurements as the target analyte moves relative to the lumen, thereby characterizing the target analyte.

59. The method of claim 58, wherein the target analyte comprises a target polynucleotide.

60. The method of claim 58 or 59, wherein step (c) comprises measuring a current passing through a continuous channel, wherein the current indicates the presence and / or one or more characteristics of the target analyte, and thereby detecting and / or characterizing the target analyte.

61. A method according to any one of claims 58 to 60, wherein the target analyte is a polynucleotide, and the nucleotides in the polynucleotide interact with a first contraction region, a second contraction region, and optionally a third contraction region within the inner cavity, and wherein each of the first contraction region, the second contraction region, and optionally the third contraction region is capable of discriminating different nucleotides, so that the total current passing through the inner cavity is affected by the interaction between each of the first contraction region, the second contraction region, and the third contraction region and the nucleotides located at each of the regions.

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