JP2025528144A - DE NOVO Pore - Google Patents

Abstract

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

Description

Two key elements of polymer characterization using nanopore sensing are (1) control of polymer transport through the pore and (2) discrimination of the constituent building blocks as the polymer passes through the pore. During nanopore sensing, the narrowest part of the pore forms the constriction, which is the most discriminatory part of the nanopore in terms of current signature as a function of the analyte passing through it. CsgG was identified as an ungated, nonselective protein secretion channel from Escherichia coli (Goyal et al., 2014) and has been used as a nanopore to detect and characterize analytes. Mutations to 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 by reference in their entirety).

When the analyte is a polynucleotide, nucleotide discrimination is achieved by passing it through such a mutant pore; however, the current signature was shown to be sequence-dependent, with multiple nucleotides contributing to the observed current; therefore, the height of the channel constriction and the degree of interaction surface with the analyte influence the relationship between the observed current and the polynucleotide sequence. While the current range for nucleotide discrimination has been improved through mutations in the CsgG pore, sequencing systems would have higher performance if the current difference between nucleotides could be further improved.

The present disclosure, in some aspects, relates to protein pore complexes and their use in the detection and characterization of analytes. The disclosure is based, in part, on nanopore complexes formed by a CsgG pore and one or more auxiliary proteins that form one or more channel constrictions within the nanopore complex. In some embodiments, the one or more auxiliary proteins are fusion proteins. As further described in the Examples, it has surprisingly been discovered that auxiliary proteins that impart specific desired characteristics to a CsgG protein nanopore (e.g., tuning the pore width, extending the pore lumen, forming one or more additional constrictions, etc.) can be de novo designed using computer-based structural analysis tools. In some embodiments, the de novo designed auxiliary proteins (e.g., fusion proteins) form one or more constrictions in the lumen of the CsgG nanopore, improving discrimination of polymer units as analytes translocate through the nanopore.

Some aspects of the present disclosure further relate to methods for designing auxiliary proteins and generating nanopore complexes, as well as for use in molecular sensing and nucleic acid sequencing applications.

In some aspects, the present disclosure provides a protein nanopore complex comprising a CsgG nanopore comprising a lumen, and a fusion polypeptide comprising a first portion comprising a CsgF protein and a second portion comprising a helix-forming assisting protein, wherein the fusion protein is bound to the nanopore.

In some aspects, the first portion of the fusion protein is bound to the CsgG nanopore. In some aspects, the first portion of the fusion protein is located within 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 region in the lumen of the CsgG nanopore.

In some embodiments, the second portion forms a second constriction region.

In some embodiments, the CsgG nanopore further comprises a constriction region.

In some embodiments, the second portion is not bound to the CsgG nanopore. In some embodiments, the second portion comprises one or more helices (e.g., alpha helices).

In some embodiments, each of the helices (e.g., alpha helices) of the second portion comprises between 0 and 15 alpha helical turns. In some embodiments, the second portion comprises a first alpha helix comprising between 1 and 4 alpha helical turns and a second alpha helix comprising between 3 and 6 alpha helical turns. In some embodiments, the second alpha helix is packed against the first alpha 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°.

In some embodiments, the distance (e.g., perpendicular distance) between the first constriction region and the second constriction region ranges from about 5 Å to about 80 Å (e.g., measured as the distance between the alpha carbons (C _a ) of the amino acid residues that extend furthest into the constricted lumen of the nanopore forming the first constriction and the amino acid residues that extend furthest into the lumen of the nanopore forming the second constriction). In some embodiments, the protein nanopore complex has an axial length of greater than 90 Å, optionally, the axial length ranges from about 95 Å to about 160 Å.

In some embodiments, the fusion protein is attached to the nanopore by 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.

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.

In some aspects, the present disclosure provides a protein nanopore complex comprising: a CsgG nanopore comprising an inner lumen and a first constriction region formed within the inner lumen of the nanopore; and a fusion protein comprising a first portion comprising a CsgF protein and a second portion comprising a helix-forming assisting protein, wherein the fusion protein is bound to the nanopore.

In some embodiments, the first portion of the fusion protein is bound to the CsgG nanopore. In some embodiments, the first portion of the fusion protein is located within the lumen of the CsgG nanopore.

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

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

In some embodiments, the second portion is not bound to the CsgG nanopore.

In some embodiments, the second portion comprises one or more helices (e.g., alpha helices). In some embodiments, each of the helices (e.g., alpha helices) comprises between 0 and 15 alpha 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°.

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-terminus) cyclization bonds. In some embodiments, at least one of the cyclization bonds is a disulfide bond.

In some aspects, the present disclosure provides a protein nanopore complex comprising: a CsgG nanopore having a lumen and a first constriction region formed within the lumen of the nanopore; a first auxiliary protein bound to the CsgG nanopore and forming a second constriction region in the lumen of the nanopore; and a second auxiliary protein bound to the CsgG nanopore or the first auxiliary protein and forming a third constriction region.

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

In some embodiments, the second auxiliary protein comprises one or more helices (e.g., alpha helices). In some embodiments, each of the one or more helices (e.g., alpha helices) comprises between 0 and 15 alpha helical turns. In some embodiments, the second auxiliary protein comprises two alpha helices.

In some embodiments, one of the alpha helices contains between 1 and 6 alpha helical turns. In some embodiments, one of the alpha helices contains between 1 and 10 alpha helical turns. In some embodiments, one of the alpha helices contains three alpha helical turns, and the other alpha helix contains three or four alpha helical turns. In some embodiments, each of the helices contains 1 to 36 amino acid residues with 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 contains 1 to 36 amino acid residues with 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, the second auxiliary protein comprises at least one alpha helix that packs against an alpha helix of the first auxiliary protein. In some embodiments, the second auxiliary protein comprises between 1 and 55 amino acid residues.

In some embodiments, the distance (e.g., perpendicular distance) between the first and second constrictions ranges from about 20 Å to about 80 Å (e.g., measured as the distance between the alpha carbons (C _a ) of the amino acid residues that extend furthest into the constriction lumen of the nanopore forming the first constriction and the amino acid residues that extend furthest into the lumen of the nanopore forming the second constriction). In some embodiments, the distance between the second and third constrictions ranges from about 5 Å to about 80 Å. In some embodiments, the protein nanopore complex has an axial length of greater than 90 Å, optionally, the axial length ranges from about 95 Å to about 160 Å.

In some embodiments, the first auxiliary protein and the second auxiliary protein are linked by 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. In some embodiments, the linker comprises one or more cyclization bonds (e.g., a first amino acid of a linker may be covalently or non-covalently bonded to a second amino acid of the linker, e.g., by a crosslinker).

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-terminus) cyclization bonds. In some embodiments, at least one of the cyclization bonds is a disulfide bond.

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

In some embodiments, the system further comprises a conductive solution in contact with the protein nanopore complex, electrodes that provide a voltage potential across the membrane, and a measurement system that measures the current through the protein nanopore complex.

In some aspects, the present disclosure provides a method for characterizing a target analyte, the method comprising contacting the target analyte with a system described herein, applying a potential across the membrane such that the target analyte enters the lumen formed by the protein nanopore complex, and performing one or more measurements as the target analyte migrates relative to the lumen, thereby characterizing the target analyte.

In some embodiments, the target analyte comprises a target polynucleotide.

In some embodiments, the step of taking one or more measurements includes measuring a current through a continuous channel, the current indicating the presence and/or one or more characteristics of the target analyte, thereby detecting and/or characterizing the target analyte.

In some embodiments, the target analyte is a polynucleotide, and nucleotides in the polynucleotide interact with the first and second (and optionally, third) constriction regions within the lumen, and each of the first, second (and optionally, third) constriction regions can discriminate between different nucleotides such that the overall current through the lumen is affected by interactions between each of the first, second, and third constriction regions and the nucleotides located in each of the regions.

In some aspects, the present disclosure provides a method of producing a protein nanopore complex, said protein nanopore complex comprising:
(a) a CsgG nanopore comprising a lumen;
(b) a fusion polypeptide comprising a first portion comprising a CsgF protein and a second portion comprising a helix-forming assisting protein, wherein the fusion protein is bound to the nanopore, and at least one domain of the fusion polypeptide is designed using a computer-generated algorithm.

Figure 1 shows the workflow for de novo design of fusion proteins. Figure 2 shows the design workflow using a CsgG nanopore. Wild-type CsgF (residues 1-35, left panel) is shown in orange. Residues 17-30 (red) of wild-type CsgF were selected as the target we explored, and we constructed geometrically consistent designable helices that packed the target and projected it onto the pore to create a new constriction (cyan) between 10 Å and 30 Å in diameter. Two helices were looped (yellow), and sequence design of the resulting backbone was performed via Rosetta. 1 shows the workflow for de novo design of fusion proteins. 2 shows helix-helix interactions with symmetry-related partners. Figure 1 shows the workflow for de novo design of fusion proteins. Figure 2 shows a top view of the nonameric CsgG-fusion protein complex demonstrating the additional constriction achieved by the de novo designed fusion protein. Representative data for sequence prioritization of de novo fusion proteins designed using Rosetta are shown. Sequences for experimental validation were selected based on lowest energy score and highest PackStat score. Figure 1 shows a secondary structure analysis of the PSIPRED protein based on the amino acid sequence of the de novo designed fusion protein. The secondary structure predictions of the fusion protein and the mature sequence of wild-type CsgF are shown. Residues are shaded according to whether they are predicted to be strands, helices, and coils, respectively. Figure 1 shows secondary structure analysis of the PSIPRED protein based on the amino acid sequence of the de novo designed fusion proteins. Figure 2 shows secondary structure analysis of the de novo designed fusion proteins ONT1 to ONT10. Residues are shaded according to whether they are predicted to be strands, helices, and coils, respectively. Figure 1 shows secondary structure analysis of the PSIPRED protein based on the amino acid sequence of the de novo designed fusion proteins. Figure 2 shows secondary structure analysis of the de novo designed fusion proteins ONT11 to ONT20. Residues are shaded according to whether they are predicted to be strands, helices, and coils, respectively. Secondary structure analysis of the PSIPRED protein based on the amino acid sequence of the de novo designed fusion proteins is shown. Residues are shaded according to whether they are predicted to be chains, helices, and coils, respectively. Secondary structure analysis of the de novo designed fusion proteins ONT21 to ONT25 is shown. 1 shows the predicted three-dimensional structures of alternative sequences of de novo designed fusion proteins. 2 shows the predicted structures of de novo designed fusion proteins ONT1 to ONT10. 1 shows the predicted three-dimensional structures of alternative sequences of de novo designed fusion proteins. 2 shows the predicted structures of de novo designed fusion proteins ONT11 to ONT20. 1 shows the predicted three-dimensional structures of alternative sequences of de novo designed fusion proteins. 2 shows the predicted structures of de novo designed fusion proteins ONT21 to ONT25. Representative SDS-PAGE gel analysis of CsgG-only pores and CsgG/fusion protein complexes containing either the CsgF-del(S31-F119) control or de novo designed fusion proteins, with or without the maleimide crosslinker, is shown. Complexes containing fusion proteins show a band shift indicating that these samples are pore complexes. Note that samples were not heated before loading onto the gel. Representative SDS-PAGE gel analysis of CsgG-only pores and CsgG/fusion protein complexes containing either the CsgF-del(S31-F119) control or the de novo designed fusion protein, with or without the maleimide crosslinker, is shown. The pores were disassembled into their constituent monomeric components by boiling in the presence of DTT before loading onto the gel. Representative ionic current (pA) versus time (s) traces are shown as single-stranded DNA translocates through a CsgG-only pore. The raw current trace is shown as a black line, and the event detection signal is shown as a red line. For each pore, the top row shows the full DNA current trace, and the bottom row shows a zoomed-in view of the first portion of the current trace. Representative ionic current (pA) versus time (s) traces are shown as single-stranded DNA translocates through CsgG containing the del(S31-F119) CsgF peptide, with or without a maleimide crosslinker. Representative ionic current (pA) versus time (s) traces are shown as single-stranded DNA translocates through a CsgG containing de novo designed fusion protein without a maleimide crosslinker. Representative ionic current (pA) versus time (s) traces are shown as single-stranded DNA translocates through a CsgG containing de novo designed fusion protein with and without maleimide cross-linking. Representative ionic current (pA) versus time (s) traces are shown as single-stranded DNA translocates through a CsgG containing de novo designed fusion protein with and without a maleimide crosslinker. The fusion protein contains a K37R mutation along with a cysteine residue to form an internal disulfide bond within the peptide, i.e., cyclize the fusion protein. Representative profiles are shown showing the position within the pore and their contribution to the overall change in ionic current level ("discrimination") as a DNA molecule translocates through the pore. CsgG-only pores (with or without Q153C) show one major discrimination peak at position 0. Representative profiles are shown showing the position within the pore and their contribution to the overall change in ion current level ("discrimination") as a DNA molecule translocates through the pore. The dashed box indicates the region affected by the introduction of the de novo designed fusion protein. The CsgG-CsgF-del(S31-F119) pore, with or without the maleimide crosslinker, shows two discrimination peaks. The major discrimination peak is at position 0, as seen in the CsgG-only pore, and an additional discrimination peak is 4 to 6 nucleotides below the major constriction (positions -4 to -6). This additional region of discrimination has a smaller effect on the ion current compared to the major discrimination peak at position 0. Representative profiles are shown showing the position within the pore and their contribution to the overall change in ionic current level ("discrimination") as a DNA molecule translocates through the pore. Distance within the pore is measured in nucleotide steps relative to the major constriction. Negative values correspond to positions below the major constriction, and positive values correspond to positions above the major constriction (CsgG). The dashed box indicates the region affected by the introduction of the de novo-designed fusion protein. A complex composed of CsgG and a de novo-designed fusion protein containing K37R (with or without a maleimide crosslinker, and with cyclization) shows three discrimination peaks. The major discrimination peak is at position 0, as seen in the CsgG-only pore, with additional peaks 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. An example of two proteins connected by a maleimidopropionic acid linker is shown. 1 provides examples of pore proteins and auxiliary proteins (eg, fusion proteins) functionalized with reactive modifiers, such as thiol modifiers. Representative ionic current (pA) versus time (s) traces are shown as single-stranded DNA translocates through CsgG containing a de novo designed fusion protein (SEQ ID NO: 61) in the presence (bottom two traces) or absence (top two traces) of a maleimide crosslinker. The raw current traces are shown as black lines, and the event detection signal is shown as a red line. For each pore, the top row shows the full DNA current trace, and the bottom row shows a zoomed-in view of the first portion of the current trace. Representative profiles are shown showing the position within the pore and their contribution to the overall change in ionic current level ("discrimination") as a DNA molecule translocates through the pore. Distance within the pore is measured in nucleotide steps relative to the major constriction. Negative values correspond to positions below the major constriction, and positive values correspond to positions above the major constriction (CsgG). The dashed box indicates the region affected by the introduction of the de novo designed fusion protein. A complex composed of CsgG and the de novo designed fusion protein (SEQ ID NO: 61) (with (lower profile) or without (upper profile) the maleimide crosslinker, both without cyclization) shows three discrimination peaks. The major discrimination peak is at position 0, as seen in the CsgG-only pore, with additional peaks 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. Figure 1 shows the structure and size of the wild-type CsgG pore from E. coli K12 (the databank access code for this structure is 4UV3). Distances shown are measured from backbone to backbone of the amino acids that form the pore structure. The CsgG pore is a tightly interconnected, symmetric nonameric pore resembling a crown. The overall height is 98 Å, and the maximum outer diameter is 120 Å. It defines a central channel and consists of three parts: (A) the cap region, (B) the constriction region, and (C) the transmembrane beta-barrel region. The axial length, or height, of the cap is 39 Å. The internal diameter is 43 Å, and the opening is 66 Å. The beta-barrel has 36 strands, an axial length of 39 Å, and an internal diameter of 55 Å. The transition between the pore cap and the beta-barrel is abrupt, with a constriction located between them at the level of the predicted lipid-aqueous interface. The constriction is approximately 18.5 Å in diameter and 20 Å in length along the axis of the channel.

Aspects of the present disclosure relate to compositions and methods for characterizing analytes using nanopore-based systems. The disclosure is based, in part, on a protein nanopore complex formed by a CsgG pore and one or more auxiliary proteins that form one or more channel constrictions within the nanopore complex. In some embodiments, the one or more auxiliary proteins are fusion proteins. As further described in the Examples, it has surprisingly been discovered that auxiliary proteins that impart specific desired characteristics to a CsgG nanopore (e.g., tuning the pore width, extending the pore lumen, forming one or more additional constrictions, etc.) can be designed de novo using computer-based structural analysis tools. In some embodiments, the de novo-designed auxiliary proteins (e.g., fusion proteins) form one or more additional constrictions in the lumen of the CsgG pore, improving discrimination of polymer units as analytes translocate through the nanopore.

Auxiliary Proteins Protein nanopore complexes (also referred to interchangeably as protein pore complexes) described by the present disclosure may comprise 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 a peptide bond. In some embodiments, a protein (also referred to as a polypeptide or peptide) comprises 2 to 2000 amino acids. In some embodiments, a protein comprises 2 to 10 amino acids, 2 to 25 amino acids, 2 to 50 amino acids, 2 to 100 amino acids, 2 to 500 amino acids, or 2 to 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, a protein comprises more than 2000 amino acids. In some embodiments, the peptide, polypeptide, or protein is synthetic (e.g., not occurring in nature, e.g., not naturally expressed in any organism). In some embodiments, the peptide, polypeptide, or protein is naturally occurring (e.g., naturally expressed in an organism that has not been genetically modified to express the peptide, polypeptide, or protein). In some embodiments, the peptide, polypeptide, or protein may 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 include one or more naturally occurring amino acids (e.g., L-amino acids, D-amino acids, etc.), one or more non-naturally occurring amino acids (e.g., radiolabeled amino acids, non-standard amino acids, unnatural amino acids, etc.), or a combination of one or more naturally occurring amino acids and one or more non-naturally occurring amino acids.

In some embodiments, the auxiliary protein is a fusion protein. The term "fusion protein" refers to a naturally occurring, synthetic, semi-synthetic, or recombinant single protein molecule that contains all or portions of two or more heterologous polypeptides (e.g., polypeptides that are heterologous to each other) linked by peptide bonds. In some embodiments, a fusion protein contains all or portions of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 heterologous polypeptides linked by peptide bonds. As used herein, a "portion of a peptide" refers to two or more amino acids of a peptide. In some embodiments, a portion of a peptide is 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, 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, 110, 111, 112, 113, 114, 115, 116, 117, 1 9, 98, 99, or 100 amino acids), or the full amino acid sequence of a peptide. The portions of the fusion protein may be arranged in any suitable manner (e.g., C-terminus to N-terminus, N-terminus to C-terminus, C-terminus to C-terminus, N-terminus to N-terminus, etc.). In some embodiments, the C-terminus of a first portion may be linked (e.g., connected) to the N-terminus of a second portion. The portions of a fusion protein may be directly linked (e.g., an amino acid of one portion may be directly linked to an amino acid of a second portion via a peptide bond between the terminal amino acids of the portions) or indirectly linked (e.g., an amino acid of one portion of a fusion protein may be linked, e.g., by a first peptide bond, to a linker that is linked to the second portion of the fusion protein by 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. Linking portions of fusion proteins using linkers is further described herein, e.g., in the section entitled "Linkers."

In some embodiments, the protein nanopore complex comprises multiple subunits or monomers (e.g., multiple CsgG monomers) arranged around a central cavity or opening (also referred to as the "lumen" of the nanopore). Formation of the protein nanopore is further described herein, e.g., 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 disposed within or with the lumen of the nanopore to form a continuous channel (e.g., a continuous lumen). In some embodiments, the protein nanopore complex comprises a ratio of pore monomer (e.g., CsgG pore monomer) to auxiliary protein 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 auxiliary proteins or one or more fusion proteins may have the same symmetry as the nanopore. For example, if the nanopore comprises eight monomers around a central axis, there will be eight auxiliary proteins (or eight fusion proteins), or if the nanopore comprises nine monomers around a central axis, there will be nine auxiliary proteins (or nine fusion proteins). In some embodiments, one or more auxiliary proteins (or one or more fusion proteins) may comprise more or fewer monomers than the nanopore, e.g., one more or one less.

The lumen of a nanopore or protein nanopore complex may have one or more constrictions. As used interchangeably herein, the terms "constriction," "opening," "constricted region," "channel constriction," or "constriction site" refer to an opening defined by the luminal surface of a pore or protein pore complex that acts to allow the passage of ions and target molecules (e.g., but not limited to, polynucleotides or individual nucleotides) but not other non-target molecules through the pore or protein pore complex channel. The constriction(s) are typically the narrowest opening(s) within the pore or protein pore complex or within the channel defined by the pore or pore complex. The constriction(s) may serve to limit the passage of molecules through the pore. The size of the constriction is typically a key factor determining the suitability of the pore or pore complex for analyte characterization. If the constriction is too small, the molecule to be characterized will not be able to pass. However, to achieve the greatest effect on ion flow through the channel, each constriction should not be too large. For example, each constriction should be no wider than the solvent-accessible lateral diameter of the target analyte. Ideally, each constriction should be as close as possible to the lateral diameter of the analyte passing through it.

The number of constrictions within a protein pore complex described by the present disclosure may vary. In some embodiments, the protein pore complex comprises at least one, two, three, four, five, or more constrictions. In some embodiments, the protein pore complex comprises two or three constrictions. In some embodiments, the protein pore complex comprises two constrictions. In some embodiments, the first constriction is formed by a first auxiliary protein and the second constriction is formed by a second auxiliary protein. In some embodiments, the first constriction is formed by a portion of the CsgG nanopore and the second constriction is formed by an auxiliary protein or fusion protein. In some embodiments, the protein pore complex comprises three constrictions. In some embodiments, the first constriction is formed by a portion of the CsgG nanopore, the second constriction is formed by a first auxiliary protein, and the third constriction is formed by a second auxiliary protein. In some embodiments, the first constriction is formed by a portion of the CsgG nanopore, and the second and third constrictions are formed by the fusion protein.

The narrowest point of the central cavity or opening typically forms a constriction in the continuous channel. In some embodiments, the diameter of the constriction is calculated by measuring the distance between the alpha carbons (C _a ) of the amino acid residues that extend furthest into the lumen of the nanopore to form the constriction. 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 furthest into the lumen of the nanopore to form the constriction. In some embodiments, the smallest diameter of a constriction (e.g., a constriction formed by a portion of a 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 nanometers (e.g., as measured by the distance between 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 nanometers, or about 0.5 to about 2.0 nanometers, preferably about 0.7 to about 1.8 nanometers, about 0.8 to about 1.7 nanometers, about 0.9 to about 1.6 nanometers, or about 1.0 to about 1.5 nanometers, e.g., about 1.1, 1.2, 1.3, or 1.4 nanometers. In some embodiments, the minimum diameter of the constriction is in the range of about 10 Å to about 30 Å, e.g., 10 Å, 11 Å, 12 Å, 13 Å, 14 Å, 15 Å, 16 Å, 17 Å, 18 Å, 19 Å, 20 Å, 21 Å, 22 Å, 23 Å, 24 Å, 25 Å, 26 Å, 27 Å, 28 Å, 29 Å, or 30 Å (e.g., as measured by _Ca - _Ca ). In some embodiments, the minimum diameter of the constriction ranges from about 10 Å to about 30 Å (e.g., as measured by C _a -C _a ). In some embodiments, the minimum diameter of the constriction ranges from about 15 Å to about 25 Å (e.g., as measured by C _a -C _a ).

The distance between one or more constrictions within the lumen of the protein pore complex may vary. In some embodiments, the distance between the first constriction region and the second constriction region ranges from about 5 Å to about 80 Å. In some embodiments, the distance between the first constriction region and the second constriction region ranges from about 5 Å, 6 Å, 7 Å, 8 Å, 9 Å, 10 Å, 11 Å, 12 Å, 13 Å, 14 Å, 15 Å, 16 Å, 17 Å, 18 Å, 19 Å, 20 Å, 21 Å, 22 Å, 23 Å, 24 Å, 25 Å, 26 Å, 27 Å, 28 Å, 29 Å, 30 Å, 31 Å, 32 Å, 33 Å, 34 Å, 35 Å, 36 Å, 37 Å, 38 Å, 39 Å, 40 Å, 41 Å, 42 Å, 43 Å, 44 Å, 45 Å, 46 Å, 47 Å, 48 Å, 49 Å, 50 Å, 51 Å, 52 Å, 53 Å, 54 Å, 55 Å, 56 Å, 57 Å, 58 Å, 59 Å, 60 Å, 61 Å, 62 Å, 63 Å, 64 Å, 65 Å, 66 Å, 67 Å, 68 Å, 69 Å, 70 Å, 71 Å, 72 Å, 73 Å, 74 Å, 75 Å, 76 Å, 7 9 Å, 40 Å, 41 Å, 42 Å, 43 Å, 44 Å, 45 Å, 46 Å, 47 Å, 48 Å, 49 Å, 50 Å, 51 Å, 52 Å, 53 Å, 54 Å, 55 Å, 56 Å, 57 Å, 58 Å, 59 Å, 60 Å, 61 Å, 62 Å, 63 Å, 64 Å, 65 Å, 66 Å, 67 Å, 68 Å, 69 Å, 70 Å, 71 Å, 72 Å, 73 Å, 74 Å, 75 Å, 76 Å, 77 Å, 78 Å, 79 Å, or 80 Å. In some embodiments, the distance between the first and second constriction regions is greater than 80 Å in length (e.g., 90 Å, 100 Å, etc.).

In some embodiments, the distance between the second and third constriction regions ranges from about 5 Å to about 80 Å. In some embodiments, the distance between the first and second constriction regions ranges from about 5 Å, 6 Å, 7 Å, 8 Å, 9 Å, 10 Å, 11 Å, 12 Å, 13 Å, 14 Å, 15 Å, 16 Å, 17 Å, 18 Å, 19 Å, 20 Å, 21 Å, 22 Å, 23 Å, 24 Å, 25 Å, 26 Å, 27 Å, 28 Å, 29 Å, 30 Å, 31 Å, 32 Å, 33 Å, 34 Å, 35 Å, 36 Å, 37 Å, 38 Å, 39 Å, 40 Å, 41 Å, 42 Å, 43 Å, 44 Å, 45 Å, 46 Å, 47 Å, 48 Å, 49 Å, 50 Å, 51 Å, 52 Å, 53 Å, 54 Å, 55 Å, 56 Å, 57 Å, 58 Å, 59 Å, 60 Å, 61 Å, 62 Å, 63 Å, 64 Å, 65 Å, 66 Å, 67 Å, 68 Å, 69 Å, 70 Å, 71 Å, 72 Å, 73 Å, 74 Å, 75 Å, 76 Å, 77 Å, 78 Å, 79 Å, 80 Å, 81 Å, 82 9 Å, 40 Å, 41 Å, 42 Å, 43 Å, 44 Å, 45 Å, 46 Å, 47 Å, 48 Å, 49 Å, 50 Å, 51 Å, 52 Å, 53 Å, 54 Å, 55 Å, 56 Å, 57 Å, 58 Å, 59 Å, 60 Å, 61 Å, 62 Å, 63 Å, 64 Å, 65 Å, 66 Å, 67 Å, 68 Å, 69 Å, 70 Å, 71 Å, 72 Å, 73 Å, 74 Å, 75 Å, 76 Å, 77 Å, 78 Å, 79 Å, or 80 Å. In some embodiments, the distance between the second and third constriction regions is greater than 80 Å in length (e.g., 90 Å, 100 Å, etc.).

In some embodiments, the distance between the first and third constriction regions ranges from about 10 Å to about 160 Å. In some embodiments, the distance between the first and second constriction regions ranges from about 10 Å, 11 Å, 12 Å, 13 Å, 14 Å, 15 Å, 16 Å, 17 Å, 18 Å, 19 Å, 20 Å, 21 Å, 22 Å, 23 Å, 24 Å, 25 Å, 26 Å, 27 Å, 28 Å, 29 Å, 30 Å, 31 Å, 32 Å, 33 Å, 34 Å, 35 Å, 36 Å, 37 Å, 38 Å, 39 Å, 40 Å, 41 Å, 42 Å, 43 Å, 44 Å, 45 Å, 46 Å, 47 Å, 48 Å, 49 Å, 50 Å, 51 Å, 52 Å, 53 Å, 54 Å, 55 Å, 56 Å, 57 Å, 58 Å, 59 Å, 60 Å, 61 Å, 62 Å, 63 Å, 64 Å, 65 Å, 66 Å, 67 Å, 68 Å, 69 Å, 70 Å, 71 Å, 72 Å, 73 Å, 74 Å, 75 Å, 76 Å, 77 Å, 78 Å, 79 Å, 80 Å, 81 Å, 82 Å, 83 Å, 84 Å, 85 Å, 86 Å, 45 Å, 46 Å, 47 Å, 48 Å, 49 Å, 50 Å, 51 Å, 52 Å, 53 Å, 54 Å, 55 Å, 56 Å, 57 Å, 58 Å, 59 Å, 60 Å, 61 Å, 62 Å, 63 Å, 64 Å, 65 Å, 66 Å, 67 Å, 68 Å, 69 Å, 70 Å, 71 Å, 72 Å, 73 Å, 74 Å, 75 Å, 76 Å, 77 Å, 78 Å, 79 Å, 80 Å, 81 Å, 82 Å, 83 Å, 84 Å, 85 Å, 86 Å, 87 Å, 88 Å , 89Å, 90Å, 91Å, 92Å, 93Å, 94Å, 95Å, 96Å, 97Å, 98Å, 99Å, 100Å, 101Å, 102Å, 103Å, 104Å, 105Å, 106Å, 107Å, 108 Å, 109 Å, 110 Å, 111 Å, 112 Å, 113 Å, 114 Å, 115 Å, 116 Å, 117 Å, 118 Å, 119 Å, 120 Å, 121 Å, 122 Å, 123 Å, 124 Å, 125 Å, 12 6 Å, 127 Å, 128 Å, 129 Å, 130 Å, 131 Å, 132 Å, 133 Å, 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, 139 Å, 140 Å, 141 Å, 142 Å, 143 Å, 144 Å, 145 Å, 146 Å, 147 Å, 148 Å, 149 Å, 150 Å, 151 Å, 152 Å, 153 Å, 154 Å, 155 Å, 156 Å, 157 Å, 158 Å, 159 Å, or 160 Å. In some embodiments, the distance between the first and third constriction regions is greater than 160 Å in length (e.g., 190 Å, 200 Å, etc.).

In some embodiments, auxiliary proteins (or fusion proteins) may be modified from their native state to provide a constriction with a desired minimum diameter. For example, auxiliary proteins may be modified to form a constriction with a minimum diameter within the above ranges, e.g., by introducing one or more bulky residues by targeted mutation. The maximum height of the auxiliary protein, in one embodiment, is from about 3 nm to about 20 nm, e.g., from about 4 nm to about 10 nm. In one embodiment, the length of the channel within the auxiliary protein is from about 3 nm to about 20 nm, e.g., from about 4 nm to about 10 nm. The height is the dimension of the auxiliary protein perpendicular to the membrane.

In some embodiments, an auxiliary protein (e.g., a first auxiliary protein or a second auxiliary protein) or a fusion protein (e.g., a first portion of a fusion protein or a second portion of a fusion protein) extends outside the lumen of a protein pore complex. The auxiliary protein or fusion protein may extend outside the cis or trans side of the lumen of a protein pore complex (e.g., when the protein pore complex is inserted into a membrane). In some embodiments, the distance that an auxiliary protein or fusion protein extends outside the lumen of a protein pore complex is calculated by measuring the Ca distance between the amino acid residue of the auxiliary protein or fusion protein that extends furthest outside the lumen and a reference amino acid of the protein pore (e.g., the CsgG pore), e.g., amino acid residue Phe144 or Tyr196 of the wild-type _CsgG monomer. In some embodiments, the auxiliary protein or fusion protein extends outside the lumen by about 0 Å to about 50 Å. In some embodiments, the auxiliary protein or fusion protein extends outside the lumen by about 5 Å to about 30 Å. In some embodiments, the auxiliary protein or fusion protein extends outside the lumen by about 10 Å to about 25 Å. In some embodiments, the auxiliary protein or fusion protein extends outside the lumen by about 1 Å, 2 Å, 3 Å, 4 Å, 5 Å, 6 Å, 7 Å, 8 Å, 9 Å, 10 Å, 11 Å, 12 Å, 13 Å, 14 Å, 15 Å, 16 Å, 17 Å, 18 Å, 19 Å, 20 Å, 21 Å, 22 Å, 23 Å, 24 Å, 25 Å, 26 Å, 27 Å, 28 Å, 29 Å, 30 Å, 31 Å, 32 Å, 33 Å, 34 Å, 35 Å, 36 Å, 37 Å, 38 Å, 39 Å, 40 Å, 41 Å, 42 Å, 43 Å, 44 Å, 45 Å, 46 Å, 47 Å, 48 Å, 49 Å, or about 50 Å.

The distance between the first and second constrictions of a protein pore complex typically 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 axial length of the protein pore complex is greater than 90 Å. In some embodiments, the axial length of the protein pore complex (e.g., a protein pore complex comprising one or more auxiliary proteins or one or more fusion proteins) is in the range of about 95 Å to about 160 Å, e.g., 95 Å, 96 Å, 97 Å, 98 Å, 99 Å, 100 Å, 101 Å, 102 Å, 103 Å, 104 Å, 105 Å, 106 Å, 107 Å, 108 Å, 109 Å, 110 Å, 111 Å, 112 Å, 113 Å, 114 Å, 115 Å, 116 Å, 117 Å, 118 Å, 119 Å, 120 Å, 121 Å, 122 Å, 123 Å, 124 Å, 125 Å, 126 Å, 127 Å, 128 Å, 129 Å, 130 Å, 131 Å, 132 Å, 133 Å, 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, 139 Å, 140 Å, 141 Å, 142 Å, 143 Å, 144 Å, 145 Å, 146 Å, 147 Å, 148 Å, 149 Å, 150 Å, 151 Å, 152 Å, 153 Å, 154 Å, 155 Å, 156 Å, 157 Å, 158 Å, 159 Å, 120 Å, 121 Å, 122 Å, 123 Å, 124 Å, 125 Å, 126 Å, 127 Å, 128 Å, 129 Å, 130 Å, 131 Å, 132 Å, 133 Å, 134 Å, 135 Å, 136 Å, 137 Å, 138 Å, 139 Å, 140 Å, 141 Å, 142 Å, 143 Å, 144 Å, 145 Å, 146 Å, 147 Å, 148 Å, 149 Å, 150 Å, 151 Å, 152 Å, 153 Å, 154 Å, 155 Å, 156 Å, 157 Å, 158 Å, 159 Å, or 160 Å.

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, located at or near the constriction formed by the auxiliary protein or fusion protein (e.g., within about 1, 2, 3, 4, or 5 nm of the constriction). In some embodiments, the auxiliary protein or fusion protein comprises one or more polar, negative, or hydrophobic amino acids located at or near the constriction formed by the auxiliary protein or fusion protein (e.g., within about 1, 2, 3, 4, or 5 nm of the constriction). In some embodiments, the one or more amino acids located at or near the constriction formed by the auxiliary protein or fusion protein (e.g., within about 1, 2, 3, 4, or 5 nm of the constriction) are asparagine, threonine, serine, or glutamic acid. These amino acids typically facilitate the interaction between the pore and the polynucleotide.

The location of one or more auxiliary proteins (or one or more fusion proteins) in a protein pore complex may vary. In some embodiments, the auxiliary protein (or fusion protein) is located entirely within the lumen of the protein pore complex. In some embodiments, the auxiliary protein or fusion protein includes a portion that extends beyond the lumen of the protein pore complex, e.g., extends above the lumen of the protein pore complex (e.g., extends above the cap region on the cis side of the protein pore complex) and/or extends below the protein pore complex (e.g., extends below the transmembrane domain (e.g., barrel) on the trans side of the protein pore complex). In some embodiments, the auxiliary protein or fusion protein (or a portion of the auxiliary protein or fusion protein, e.g., the first portion or the second portion) is bound to a nanopore (e.g., a CsgG nanopore). In some embodiments, the auxiliary protein or fusion protein (or a portion thereof) is covalently bound to the nanopore. In some embodiments, the auxiliary protein or fusion protein (or a portion thereof) is non-covalently bound to the nanopore. In some embodiments, the first auxiliary protein and the second auxiliary protein are bound to each other (e.g., covalently, non-covalently, etc.). In some embodiments, the first portion of the fusion protein and the second portion of the fusion protein are bound to each other (e.g., covalently, non-covalently, etc.). In some embodiments, the auxiliary protein or fusion protein (or a portion of the auxiliary protein or fusion protein, e.g., the first portion or the second portion) is not bound to the nanopore (e.g., the CsgG nanopore). In some embodiments, the first auxiliary protein and the second auxiliary protein are not bound to each other.

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

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, e.g., the auxiliary protein is not a polynucleotide binding protein with enzymatic activity. In some embodiments, the auxiliary protein may be a protein other than a nucleic acid processing enzyme, e.g., an auxiliary protein that is not a helicase or 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 change conformation when the target analyte passes through the continuous channel formed within the protein pore complex.

In some embodiments, the auxiliary 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 the component that forms the transmembrane pore. Examples of such components include CsgF or truncated forms of CsgF. In some embodiments, the auxiliary protein or fusion protein comprises a CsgF protein or a modified form thereof, such as a homolog or fragment thereof. In some embodiments, the pore complex comprises a CsgF protein or peptide and a modified form thereof, such as a non-CsgF pore, homolog, or fragment thereof.

The terms "CsgF protein" or "CsgF peptide" preferably define a CsgF peptide truncated from its C-terminus (i.e., an N-terminal fragment). The CsgF peptide may be a fragment of wild-type E. coli CsgF (e.g., as shown in FIG. 3A) or a fragment of a wild-type homolog of E. coli CsgF, such as a peptide comprising any one of the amino acid sequences set forth in WO 2019/002893 (incorporated herein by reference in its entirety). A CsgF homolog is a polypeptide that shares at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% complete sequence identity with wild-type E. coli CsgF. A CsgF homolog is also referred to as a polypeptide that contains the PFAM domain PF10614, which is characteristic of CsgF-like proteins. A list of currently known CsgF homologs and CsgF architectures can be found at http://pfam.xfam.org/family/PF10614. Mature CsgF (e.g., as shown in Figure 3A) can be divided into three main regions: the "CsgF constriction peptide" (FCP), the "neck" region, and the "head" region. The "head" region of the CsgF peptide is distinct from the pore constriction described herein. The "head" region of the CsgF peptide may also be referred to as the "C-terminal head domain." The structure of CsgF is discussed in detail in WO2019/002893, which is incorporated herein by reference in its entirety.

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 include only a portion of the neck domain of CsgF), or lacks the C-terminal head and the neck domain of CsgF. The CsgF peptide can lack a portion of the CsgF neck domain, for example, the CsgF peptide can include a portion of the neck domain from amino acid residue 36 at the N-terminus of the neck domain (e.g., residues 36-40, 36-41, 36-42, 36-43, 36-45, 36-46, or at most residues 36-50 or 36-60 of wild-type E. coli CsgF). In some embodiments, the CsgF peptide includes the CsgG binding region and the region that forms the constriction within the lumen of the pore. The CsgG-binding region typically comprises residues 1-11 and/or 29-32 of a CsgF protein (e.g., wild-type E. coli CsgF or a homologue from another species) and may include one or more modifications. The region that forms the constriction within the pore typically comprises residues 9-28 of a CsgF protein (wild-type E. coli CsgF or a homologue from another species) and may include one or more modifications. In some embodiments, residues 9-17 comprise the conserved motif _N9PXFGGXXX17 and form a turn region. In some embodiments, residues 9-28 form an alpha-helix. In some embodiments, the amino acid residue at position ₁₇ of the CsgF peptide forms the apex of the constriction region, which corresponds to the narrowest part of the CsgF constriction within the pore. In some embodiments, the CsgF constriction region also makes stabilizing contacts with the CsgG beta-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 accessory protein. In some embodiments, the CsgF peptide is part of a fusion protein (e.g., the first portion or the second portion). 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.

In some embodiments, the CsgF peptide has a length of 28 to 60 amino acids, e.g., 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 length. In some embodiments, the CsgF peptide comprises all or a portion of an FCP corresponding to residues 1 to 35 of wild-type E. coli CsgF (or the corresponding residues in a CsgF homologue). In some embodiments, when the CsgF peptide is shorter than FCP, the truncation is preferably at the C-terminus.

One or more residues in the CsgF peptide may be modified. For example, the CsgF peptide may include modifications in SEQ ID NO:6 at positions corresponding to one or more of 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 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, for example, at positions corresponding to one or more of G1, T4, F5, R8, N9, N11, F12, N17, A20, N24, A26, Q27, and Q29 of SEQ ID NO:60. Such introductions may be made in any number and combination. Introduction is preferably made by substitution.

In some embodiments, the CsgF peptide includes modifications at positions corresponding to one or more of positions N15, N17, A20, N24, and A28 in SEQ ID NO: 60. In some embodiments, the CsgF peptide includes one or more of the following substitutions: N15S/A/T/Q/G/L/V/I/F/Y/W/R/K/D/C/E, N17S/A/T/Q/G/L/V/I/F/Y/W/R/K/D/C/E, A20S/T/Q/N/G/L/V/I/F/Y/W/R/K/D/C/E, N24S/T/Q/A/G/L/V/I/F/Y/W/R/K/D/C/E, or A28S/T/Q/N/G/L/V/I/F/Y/W/R/K/D/C/E.

In some embodiments, the CsgF peptide is a variant of any of the above-described CsgF sequences, including SEQ ID NO: 60, preferably containing 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 is preferably at least 40% homologous to that 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%, more preferably at least 95%, 97%, or 99% homologous to the amino acid sequence of SEQ ID NO: 60 over the entire sequence based on amino acid identity. Over the entire length of the amino acid sequence of SEQ ID NO: 60, the variant is preferably at least 40% identical to that 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%, more preferably at least 95%, 97%, or 99% identical to SEQ ID NO: 60 over the entire sequence. There may also be at least 80%, e.g., at least 85%, 90%, or 95%, amino acid identity ("hard homology") over a stretch of 15 or more consecutive amino acids, e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more. These levels of homology/identity are similar for any of the other CsgF peptides described above.

Any number of CsgF peptides within 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 6 to 10 monomers within a pore or pore complex preferably contain one or more substitutions compared to SEQ ID NO: 60. The CsgF peptides within a pore complex may be the same or different. The CsgF peptides are preferably identical in each pore monomer conjugate within a pore complex of the present disclosure.

Aspects of the present disclosure relate to auxiliary 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 may be located within the lumen of certain nanopores (e.g., CsgG nanopores) to form one or more constrictions within the lumen of the nanopore, and that the presence of such one or more constrictions improves the signal-to-noise ratio (e.g., polynucleotide base discrimination) of the resulting protein-pore complex. The term "helix" or "helix" generally refers to a coiled structural arrangement of a protein that forms a spiral and results from 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 referred to as a 3.6 ₁₃ -helix) containing approximately 3.6 amino acid residues per helical turn, with 13 atoms participating in the ring formed by hydrogen bonds. In some embodiments, the helix is a 3 ₁₀ helix containing approximately three residues per turn and with 10 atoms in the ring formed by hydrogen bond formation.

The number of helices (e.g., alpha helices, 3 _' helices, pi helices, etc.) in an auxiliary protein or fusion protein can vary. In some embodiments, the number of helices in an auxiliary protein (e.g., a first auxiliary protein, a second auxiliary protein, etc.) ranges from about 0 to about 15, e.g., 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 an auxiliary protein (e.g., a first auxiliary protein, a second auxiliary protein, etc.) is greater than 15 (e.g., 20, 25, etc.). In some embodiments, the fusion protein (e.g., a first portion of a fusion protein, a second portion of a fusion protein, etc.) comprises from 0 to about 15 helices (e.g., alpha helices, 3 ₁₀ helices, pi helices, etc.), e.g., 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 helices.

The number of turns within a helix (e.g., an alpha helix, a _3:10 helix, a pi helix, etc.) can vary. In some embodiments, each helix (e.g., an alpha helix, a _3:10 helix, a pi helix, etc.) of an auxiliary protein or fusion protein contains 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 3:10 helix, a pi helix, etc.) can contain 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.

The number of turns within a helix (e.g., an alpha helix, a _3'10 helix, a pi helix, etc.) can vary. In some embodiments, each helix (e.g., an alpha helix, a _3'10 helix, a pi helix, etc.) of the auxiliary protein or fusion protein comprises 2 to 55 amino acid residues, e.g., 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, or 55 amino acid residues.

The angle of the helix of the auxiliary protein or fusion protein may vary. In some embodiments, the helix has a phi angle ranging from 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°, or -90°). In some embodiments, the helix has a psi angle ranging from 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°, 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 with 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, one or more helices of the auxiliary protein or fusion protein contain structural features that promote the packing of the helices together. "Packing" of helices typically refers to the close association of two or more helices through covalent or non-covalent bonds between the helices, such as salt bridges, hydrogen bonds, disulfide bonds, and hydrophobic side chain-side chain close contacts, side chain-main chain contacts, or main chain-main chain contacts, as described in Walther and Argos, J Mol Biol. 1996 Jan 26; 255(3):536-53. doi:10.1006/jmbi.1996.0044. Methods for predicting helical packing are described, for example, in Eilers et al. Proc Natl Acad Sci U S A. It is known as described in 2000 May 23;97(11):5796-5801.

Aspects of the present disclosure relate to the recognition that cyclized fusion proteins improve target analyte discrimination within protein pore complexes. A "cyclized" protein typically refers to a protein (e.g., a fusion protein) that includes one or more intramolecular interactions that result in the formation of one or more cyclic arrangements of bonds. Examples of cyclization include side chain-to-side chain cyclization (e.g., formation of an intramolecular disulfide bond), head-to-tail cyclization (e.g., formation of an amide bond between the N-terminal and C-terminal amino acids of a protein), and head-to-side chain cyclization, as described, for example, in Hayes et al. Org Biomol Chem. 2021 May 12;19(18):3983-4001. In some embodiments, the fusion protein includes 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, the one or more cyclization bonds result in cyclization between a first portion of a 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 auxiliary protein or fusion protein includes a loop region (e.g., a linker that forms a loop region) that includes one or more cyclization bonds. In some embodiments, the cyclization bonds are formed by a chemical crosslinker and/or include a disulfide bond.

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

In some embodiments, the nanopore is a transmembrane protein pore. Transmembrane protein pores typically span the entire membrane and may have a structure that extends beyond the membrane on one or both sides. Transmembrane protein pores are single or multimeric proteins that allow hydrated ions to flow from one side of the membrane to the other side. Transmembrane protein pores contain a channel that allows an analyte, e.g., a polynucleotide such as DNA or RNA, to move or be moved into and/or through the pore.

Transmembrane protein pores typically contain a barrel or channel through which ions may flow. The subunits of the pore typically surround a central axis and contribute strands to a transmembrane beta barrel or channel or a transmembrane alpha-helical bundle or channel.

The barrel or channel of a transmembrane protein pore typically contains amino acids that facilitate interaction with a polynucleotide. These amino acids are preferably located near (e.g., within 1, 2, 3, 4, or 5 nm) the constriction of the barrel or channel. A transmembrane protein pore typically contains one or more polar or hydrophobic residues. These amino acids typically facilitate interaction of the pore with a nucleotide, polynucleotide, or nucleic acid.

In some embodiments, the nanopore is a CsgG pore, such as, for example, CsgG from E. coli Str. K-12 substr. 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 identical monomers, or a heteropolymer comprising two or more different monomers. Suitable pores derived from CsgG are disclosed in WO2016/034591, WO2017/149316, WO2017/149317, WO2017/149318, International Patent Application Nos. PCT/GB2018/051191 and PCT/GB2018/051858, and Chinese Patent Publication Nos. CN113773373, CN113896776, CN113912683, and CN113754743, which are incorporated herein by reference in their entireties. Additional examples of CsgG pores include, but are not limited to, Uniprot reference numbers K4KIX7, A0A086D1N6, A0A1I1MNE8, A0A143HJG2, AoA090RS48, and A0A090SZM0.

The 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 (herein incorporated by reference in its entirety). The CsgG pore preferably comprises one or more of: (a) a cap region; (b) a constriction region; and (c) a transmembrane beta-barrel region, e.g., (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c). The CsgG pore monomer preferably comprises one or more of (a) a cap-forming region, (b) a constriction-forming region, and (c) a transmembrane beta-barrel-forming region, e.g., (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 the wild-type E. coli CsgG pore (e.g., as described by PDB accession number 4UV3). The protein structure of CsgG defines a channel or hole that allows the translocation of molecules and ions from one side of the membrane to the other.

The CsgG pore may be of any size, but preferably has the dimensions of the wild-type E. coli CsgG pore (e.g., as described by PDB accession number 4UV3). These dimensions are shown in Figure 19. In some embodiments, the CsgG pore has an outer diameter of about 100 to about 150 Å at its widest point, e.g., about 110 to about 140 Å or about 115 to about 125 Å at its widest point. In some embodiments, the CsgG pore has an outer diameter of about 120 Å at its widest point. In some embodiments, the CsgG pore has a total length of about 80 to about 120 Å, e.g., about 90 to about 110 Å or about 95 to about 105 Å. In some embodiments, the CsgG pore has a total length of about 98 Å. References to "total length" and "length" relate to the length of the pore or pore region when viewed from the side (e.g., referring to a cis-to-trans cross-section of the pore inserted into a membrane). This may be a side view of Figure 19. In some embodiments, the outer diameter is measured by calculating the _Ca - _Ca distance of the most distant amino acid residues on the exterior of the CsgG pore. In some embodiments, the outer diameter is measured by calculating the van der Waals radii distance of the most distant amino acid residues on the exterior of the CsgG pore.

In some embodiments, the cap region has a length of about 20 to about 60 Å, e.g., about 30 to about 50 Å or about 35 to about 45 Å. In some embodiments, the cap region has a length of about 39 Å. In some embodiments, the channel defined by the cap region has an opening with a diameter of about 30 to about 70 Å, e.g., about 40 to about 60 Å or about 45 to about 55 Å. In some embodiments, the channel defined by the cap region has an opening with a diameter of about 66 Å. In some embodiments, the channel defined by the cap region has a diameter of about 20 to about 66 Å at its narrowest point, e.g., about 30 to about 50 Å or about 32 to about 43 Å at its narrowest point. In some embodiments, the channel defined by the cap region preferably has a diameter of about 43 Å at its narrowest point. In some embodiments, the outer diameter is measured by calculating the _Ca - _Ca distance of the closest amino acid residues on the channel of the cap region of the CsgG pore. In some embodiments, the outer diameter is measured by calculating the distance between the van der Waals radii of the nearest amino acid residues on the channel of the cap region.

In some embodiments, the constriction region formed by the CsgG pore (if present) has a length of about 5 to about 40 Å, e.g., about 10 to about 30 Å or about 15 to about 25 Å. In some embodiments, the constriction region has a length of about 20 Å. In some embodiments, the channel defined by the constriction region has a diameter of about 2 to about 30 Å at its narrowest point, e.g., about 5 to about 25 Å, about 8 to about 20 Å, or about 10 to about 15 Å at its narrowest point. In some embodiments, the channel defined by the constriction region has a diameter of about 9 Å. In some embodiments, the channel defined by the constriction region has a diameter of about 18.5 Å. In some embodiments, the constriction has a diameter of about 2 to about 30 Å, e.g., about 5 to about 25 Å, about 8 to about 20 Å, or about 10 to about 15 Å. In some embodiments, the constriction has a diameter of about 12 Å. In some embodiments, the constriction region of the CsgG pore is measured by calculating the C _a -C _a distance of the amino acid residues that extend furthest into the lumen of the pore forming the constriction, hi some embodiments, the outer diameter is measured by calculating the distance of the van der Waals radii of the amino acid residues that extend furthest into the lumen of the pore forming the constriction.

In some embodiments, the transmembrane beta-barrel region has a length of about 20 to about 60 Å, e.g., about 30 to about 50 Å or about 35 to about 45 Å. In some embodiments, the transmembrane beta-barrel region has a length of about 39 Å. In some embodiments, the channel defined by the transmembrane beta-barrel region has a diameter at its narrowest point of about 20 to about 60 Å, e.g., about 30 to about 50 Å or about 35 to about 45 Å. In some embodiments, the channel defined by the transmembrane beta-barrel region has a diameter at its narrowest point of about 55 Å.

All of the above measurements are based on backbone-to-backbone measurements of the amino acids that make up the different regions (as shown in Figure 19).

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

In some embodiments, the CsgG pore monomer is a variant of SEQ ID NO:59, as 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. Modifications or mutations in the variant include, but are not limited to, any one or more of the modifications disclosed herein, or combinations of the modifications. The CsgG pore monomer may also be a CsgG homolog monomer. A CsgG homolog monomer is a polypeptide that has at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% complete sequence identity to the wild-type E. coli CsgG set forth in SEQ ID NO:59. A CsgG homolog is also referred to as a polypeptide that comprises the PFAM domain PF03783, which is characteristic of CsgG-like proteins. A list of currently known CsgG homologues and CsgG architectures can be found at http://pfam.xfam.org//family/PF03783.

In some embodiments, the CsgG pore monomer is a variant of SEQ ID NO:59 that includes one or more modifications in addition to a cysteine at a position corresponding to position 153 or 133 of SEQ ID NO:59. Over the entire length of the amino acid sequence of SEQ ID NO:59, the variant is preferably at least 40% homologous to that 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%, more preferably at least 95%, 97%, or 99% homologous to the amino acid sequence of SEQ ID NO:59 over the entire sequence based on amino acid identity. Over the entire length of the amino acid sequence of SEQ ID NO:59, the variant is preferably at least 40% homologous to that 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%, more preferably at least 95%, 97%, or 99% identical to SEQ ID NO:59 over the entire sequence.

Sequence identity can also relate to fragments or portions of the CsgG pore monomer. Thus, a sequence may have less than 40% overall sequence homology/identity with SEQ ID NO:59, but the sequence of a particular region, domain, or subunit can 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%, e.g., at least 85%, 90%, or 95% amino acid identity ("hard homology") over a stretch of 100 or more, e.g., 125, 150, 175, or 200 or more contiguous amino acids. 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 1-41) of SEQ ID NO:3. More preferably, a 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%, more preferably at least 95%, 97%, or 99% homologous based on amino acid identity to amino acid residues 1-41 of SEQ ID NO: 59. In some embodiments, a variant comprises a sequence that is at least 40% identical to residues 1-41 of SEQ ID NO: 59. More preferably, a 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%, more preferably at least 95%, 97%, or 99% identical to residues 1-41 of SEQ ID NO: 59.

In some embodiments, the CsgG pore monomer is a variant of SEQ ID NO: 59 comprising a sequence at least 40% homologous to the constriction region (residues 64-131) 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%, more preferably at least 95%, 97%, or 99% homologous based on amino acid identity to residues 64-131 of SEQ ID NO: 59. In some embodiments, the variant comprises a sequence that is at least 40% identical to residues 64-131 of SEQ ID NO: 59. More preferably, 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 64-131 of SEQ ID NO:59.

In some embodiments, the CsgG pore monomer is a variant of SEQ ID NO:59, comprising a sequence at least 40% homologous to the transmembrane beta-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%, 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. More preferably, 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%, more preferably at least 95%, 97% or 99% identical to residues 156-180 and 212-262 of SEQ ID NO:59.

The CsgG pore monomer is highly conserved (as can be readily seen from Figures 45-47 of WO 2017/149317). Furthermore, knowledge of the mutations relative to SEQ ID NO:59 makes it possible to determine the equivalent positions of mutations in the CsgG pore monomer other than the mutation in SEQ ID NO:59.

Thus, reference to a mutant CsgG pore monomer comprising a variant of the sequence set forth in SEQ ID NO:59 and the specific amino acid mutations thereof described in the claims and elsewhere in this specification also encompasses mutant CsgG pore monomers comprising any variant of the sequences set forth in SEQ ID NOs:68-88 of WO2019/002893 (which is incorporated herein by reference in its entirety) and their corresponding amino acid mutations. The CsgG pore monomer may also be any of the sequences set forth in CN113773373A, CN113896776A, CN113912683A, and CN113754743A, or a variant thereof.

Standard methods in the art can be used to determine homology. For example, the UWGCG package provides the BESTFIT program, which can be used to calculate homology, e.g., with its default settings (Devereux et al. (1984) Nucleic Acids Research 12, pp. 387-395). For example, the PILEUP and BLAST algorithms can be used to calculate homology or align sequences (identify equivalent residues or corresponding sequences, typically with their default settings), as described in Altschul S. F. (1993) J Mol Evol 36:290-300 and Altschul, S. F. et al. (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

SEQ ID NO:59 is the wild-type CsgG pore monomer from E. coli Str. K-12 substr. MC4100. Variants of SEQ ID NO:59 may include any of the substitutions present in other CsgG homologs. Preferred CsgG homologs are set forth in SEQ ID NOs:68-88 of WO 2019/002893 (incorporated herein by reference in their entirety). Variants may include one or more combinations of the substitutions present in SEQ ID NOs:68-88 of WO 2019/002893 (incorporated herein by reference in their entirety), including one or more, compared to SEQ ID NO:59.

The CsgG pore monomer in the pore monomer conjugates of the present disclosure typically retains the ability to form the same 3D structure as a wild-type CsgG pore monomer, for example, 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 by the mutations.

In addition to those described above, amino acid substitutions may be made to the amino acid sequence of SEQ ID NO:59, for example, up to 1, 2, 3, 4, 5, 10, 20, or 30 substitutions. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties, or similar side chain volume. The introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acids they replace. Alternatively, conservative substitutions may introduce another amino acid that is aromatic or aliphatic in place of an existing aromatic or aliphatic amino acid.

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. Such introductions may be made in any number and combination. Introduction is preferably made by substitution.

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

Variants may include fragments of SEQ ID NO:59. Such fragments retain pore-forming activity. Fragments may be at least 50, at least 100, at least 150, at least 200, or at least 250 amino acids in length. Such fragments may be used to produce pores. The fragment preferably includes the transmembrane domain of SEQ ID NO:59, designated K135-Q153 and S183-S208.

Alternatively, or in addition, one or more amino acids may be added to the above-described polypeptides. The extension may be provided at the amino or carboxy terminus of the amino acid sequence of SEQ ID NO: 59, or a polypeptide variant or fragment thereof. The extension may be very short, for example, 1 to 10 amino acids in length. Alternatively, the extension may be longer, for example, up to 50 or 100 amino acids. A carrier protein may be fused to the amino acid sequence. Other fusion proteins are discussed in more detail elsewhere in this disclosure, for example, in the section entitled "Accessory Proteins."

A variant of SEQ ID NO:59 is a polypeptide having an amino acid sequence different from that of SEQ ID NO:59 but which retains the ability to form a pore. The variant typically contains the region of SEQ ID NO:59 responsible for pore formation. The pore-forming ability of β-barrel-containing CsgG is provided by a β-sheet in the transmembrane beta-barrel of each subunit monomer. A variant of SEQ ID NO:59 typically includes the regions of SEQ ID NO:59 that form β-sheets, designated K134-Q154 and S183-S208. One or more modifications can be made to the regions of SEQ ID NO:3 that form β-sheets, as long as the resulting variant retains the ability to form a pore.

One or more modifications in the CsgG pore monomer preferably improve the ability of a pore complex containing the pore monomer to characterize an analyte. For example, modifications/mutations/substitutions are contemplated that alter the number, size, shape, location, or orientation of constrictions within a channel from a pore monomer conjugate of the present disclosure. A 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 by reference in their entirety).

Preferred modifications or substitutions in SEQ ID NO: 59 are:
(a) a substitution at position Y51, for example Y51I, Y51L, Y51A, Y51V, Y51T, Y51S, Y51Q or Y51N;
(b) a substitution at position N55, for example N55I, N55L, N55A, N55V, N55T, N55S or N55Q;
(c) a substitution at position F56, for example F56I, F56L, F56A, F56V, F56T, F56S, F56Q or F56N;
(d) a substitution at position L90, e.g., L90N, L90D, L90E, L90R or L90K;
(e) a substitution at position N91, e.g., N91D, N91E, N91R or N91K;
(f) a substitution at position K94, e.g., K94R, K94F, K94Y, K94Q, K94W, K94L, K94S, or K94N;
(g) a substitution at position R192, e.g., R192Q, R192F, R192S R192D, or R192T;
(i) substitutions at position C215, e.g., C215T, C215S, C215I, C215L, C215A, C215V, or C215G, including, but not limited to, one or more, e.g., two or more, three or more, four or more, five or more, six or more, seven or more, or all of the following:

Variant of SEQ ID NO: 3 may further include a deletion of one or more positions, for example, a deletion of T104 to N109, a deletion of F193 to L199, or a deletion of F195 to L199.

Any number of CsgG pore monomers within a pore or pore complex, such as 6, 7, 8, 9, or 10, may be a variant of SEQ ID NO: 59. Preferably, all 6 to 10 monomers within a pore or pore complex are variants of SEQ ID NO: 59. The variants within a pore complex may be the same or different. The variant is preferably the same in each pore monomer conjugate within the pore complex.

Linkers In some embodiments, the protein pore complex is stabilized by the attachment (e.g., covalent attachment) of an auxiliary protein or fusion protein to the nanopore. The covalent attachment can be, for example, a disulfide bond or click chemistry. As a further example, cysteine residues may be connected by a linker such as BMOE. The auxiliary protein or fusion protein and/or the transmembrane protein nanopore may be modified to facilitate 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.

In some embodiments, the auxiliary protein or fusion protein is bound to the nanopore by hydrophobic interactions and/or one or more disulfide bonds. One or more, e.g., 2, 3, 4, 5, 6, 8, 9, or even all, of the monomers within the pore may be modified to enhance such interactions. This may be achieved in any suitable manner. Further suitable interactions include salt bridges, electrostatic interactions, hydrogen bond formation, peptide bond formation, and Pi-Pi interactions.

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) may be disulfide bonded to 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 a first auxiliary protein is disulfide bonded to at least one cysteine residue in the amino acid sequence of a second auxiliary protein. In some embodiments, at least one cysteine residue in the amino acid sequence of a first portion of a fusion protein is disulfide bonded to at least one cysteine residue in the amino acid sequence of a second portion of the fusion protein. The cysteine residue in the nanopore and/or the auxiliary protein or fusion protein may be a cysteine residue not present in the wild-type transmembrane protein pore monomer or 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 auxiliary protein (or fusion protein) in the pore complex. One or both of the nanopore and the auxiliary protein (or fusion protein) may include at least one monomer or subunit, e.g., up to 8, 9, or 10 monomers or subunits, that includes a cysteine residue at the interface between the nanopore and the auxiliary protein (or fusion protein).

The nanopore and/or 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) that are more hydrophobic than the residues present at the corresponding position in the wild-type nanopore or 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 residue is more hydrophobic than the residues present at the corresponding position in the wild-type pore or auxiliary protein (or fusion protein). For example, 2 to 10, e.g., 3, 4, 5, 6, 7, 8, or 9, residues in the nanopore and/or auxiliary protein (or fusion protein) may be more hydrophobic than the residues present at the same position in the corresponding wild-type nanopore and/or 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. When a residue at the interface of the wild-type nanopore or auxiliary protein (or fusion protein) is R, Q, N, or E, the hydrophobic residue is typically I, L, V, M, F, W, A, or Y. When a residue at the interface of the wild-type nanopore or auxiliary protein (or fusion protein) is I, the hydrophobic residue is typically L, V, M, F, W, A, or Y. When a residue at the interface of the wild-type nanopore or auxiliary protein (or fusion protein) is L, the hydrophobic residue is typically I, V, M, F, W, A, or Y.

Molecular dynamics simulations can be performed to determine which residues in the auxiliary protein and nanopore are in close proximity. This information can be used to design auxiliary protein and/or transmembrane protein nanopore mutants that can enhance the stability of the complex. For example, simulations can be performed using the GROMACS package version 4.6.5 with the GROMOS 53a6 force field and an SPC water model using the protein's cryo-EM structure. The complex can be solvated and then energy minimized using a steepest descent algorithm. Constraints can be applied to the protein backbone throughout the simulation, while residue side chains are free to move. The system can be simulated for 20 ns in the NPT ensemble up to 300 K using a Berendsen thermostat and Berendsen barostat. Contacts between the auxiliary protein and nanopore can be analyzed using the GROMACS analysis software and/or locally written code. Two residues can be defined as in contact if they are within 3 Å of each other.

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

Covalent linkages or bonds can occur, for example, via cysteine bonds, where the sulfhydryl side group of cysteine is covalently linked to another amino acid residue or moiety, and/or through interactions between non-natural (photo)reactive amino acids. (Photo)reactive amino acids refer to artificial analogs of natural amino acids that can be used to crosslink protein complexes and may be incorporated into proteins and peptides in vivo or in vitro. Commonly used photoreactive amino acid analogs are photoreactive diazirine analogs of leucine and methionine, as well as parabenzoyl-phenyl-alanine, azidohomoalanine, homopropargylglycine, homoallelicglycine, p-acetyl-Phe, p-azido-Phe, p-propargyloxy-Phe, and p-benzoyl-Phe (Wang et al. 2012; Chin et al. 2002). Upon exposure to UV light, they become activated and covalently bond to interacting proteins within a few angstroms of the photoreactive amino acid analog.

Pore complexes can be prepared, and disulfide bond formation can be induced using an oxidizing agent (e.g., copper-orthophenanthroline). Instead of cysteine interactions, other interactions (e.g., hydrophobic interactions, charge-charge interactions/electrostatic interactions) can also be used at these positions. In another embodiment, unnatural amino acids can also be incorporated at these positions. In this embodiment, covalent bonds are created by click chemistry. For example, unnatural amino acids bearing azides or alkynes, or bearing dibenzocyclooctyne (DBCO) and/or bicyclo[6.1.0]nonyne (BCN) groups, can be introduced at one or more of these positions.

For example, the CsgG pore may comprise at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, CsgG monomers modified to facilitate binding to an auxiliary protein or fusion protein. For example, cysteine residues may be introduced at one or more of 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 auxiliary protein or fusion protein, to facilitate covalent binding to the auxiliary protein or fusion protein. Alternatively, or in addition to covalent binding via cysteine residues, the pore may be stabilized by hydrophobic or electrostatic interactions. To facilitate such interactions, non-naturally occurring reactive or photoreactive amino acids at positions 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.

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

Such stabilizing mutations can be combined with any other modifications to the auxiliary protein or fusion protein, such as modifications to improve the interaction of the pore complex with the polynucleotide or to improve a particular property of the complex (e.g., discrimination of polymer units such as nucleotides of the polynucleotide).

In some embodiments, the nanopore may be isolated, substantially isolated, purified, or substantially purified. A pore is isolated or purified if it is free of any other components, such as lipids or other nanopores. A pore is substantially isolated if it is mixed with a carrier or diluent that does not interfere with its intended use. For example, a pore is substantially isolated or substantially purified if it is present in a form that contains less than 10%, less than 5%, less than 2%, or less than 1% of other components, such as block copolymers, lipids, or other nanopores. Alternatively, the pore may be present in a membrane. Suitable membranes are discussed below.

A pore complex may exist as an individual pore or a single pore in the membrane. Alternatively, a pore complex may exist as a homogeneous or heterogeneous population of two or more pores.

The auxiliary protein or fusion protein may be directly attached to the transmembrane protein nanopore, or two proteins (e.g., a first auxiliary protein and a second auxiliary protein, a first portion of a fusion protein and a second portion of a fusion protein, etc.) may be attached using a linker, such as a chemical crosslinker or a peptide linker.

Suitable chemical crosslinkers are well known in the art. Examples of crosslinkers include, but are not limited to, 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldisulfanyl)propanoate, 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfanyl)butanoate, and 2,5-dioxopyrrolidin-1-yl 8-(pyridin-2-yldisulfanyl)octananoate. In some embodiments, the crosslinker is succinimidyl 3-(2-pyridyldithio)propionate (SPDP). Typically, the molecule is covalently attached to the bifunctional crosslinker before the molecule/crosslinker complex is covalently attached to the mutant monomer; however, it is also possible to covalently attach the bifunctional crosslinker to the monomer before the bifunctional crosslinker/monomer complex is attached to the molecule. In some embodiments, the linker is resistant to dithiothreitol (DTT). Additional suitable linkers include, but are not limited to, iodoacetamide-based and maleimide-based linkers.

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 typically designed to allow the auxiliary protein or fusion protein to form a constriction within the pore complex. Preferred flexible peptide linkers are stretches of 2 to 20, e.g., 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 , where S is serine and G is glycine. Preferred rigid linkers are stretches of 2 to 30, e.g., 4, 6, 8, 16, or 24, proline amino acids. More preferred rigid linkers include (P) ₁₂ , where P is proline.

Suitable chemical crosslinkers include, but are not limited to, those containing the following functional groups: maleimide, active ester, succinimide, azide, alkyne (such as dibenzocyclooctynol (DIBO or DBCO), difluorocycloalkyne, and linear alkyne), phosphines (such as those used in traceless and non-traceless Staudinger ligation), haloacetyl (such as iodoacetamide), phosgene-type reagents, sulfonyl chloride reagents, isothiocyanates, acyl halides, hydrazine, disulfide, vinyl sulfone, aziridine, and photoreactive reagents (such as aryl azides and diaziridine).

The reaction between the amino acid and the functional group can be spontaneous, such as cysteine/maleimide, or can require an external reagent, such as Cu(I) to link an azide and a linear alkyne.

Linkers can include any molecule that spans the required distance. Linkers can vary in length from one carbon (phosgene-type linkers) 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, and polyamides. These linkers can be inert or reactive; in particular, they may be chemically cleavable at defined positions or may themselves be modified with fluorophores or ligands. Linkers are preferably resistant to dithiothreitol (DTT) after covalent attachment of an auxiliary protein or fusion protein to the CsgG pore monomer.

In some embodiments, preferred crosslinkers are 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldisulfanyl)propanoate, 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfanyl)butanoate, and 2,5-dioxopyrrolidin-1-yl 8-(pyridin-2-yldisulfanyl)octananoate, di-maleimide PEG 1k, di-maleimide PEG 3.4k, di-maleimide PEG 5k, di-maleimide PEG 10k, bis(maleimido)ethane (BMOE), bis-maleimidohexane (BMH), 1,4-bis-maleimidobutane (BMB), 1,4 bis-maleimidyl-2,3-dihydroxybutane (BMDB), BM[PEO]2 (1,8-bis-maleimidodiethylene glycol), BM[PEO]3 (1,11-bis-maleimidotriethylene glycol), tris[2-maleimidoethyl]amine (TMEA), DTME dithiobismaleimidoethane, bis-maleimidoPEG3, bis-maleimidoPEG11, 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-S-S-PEG3-biotin, DBCO-S-S-PEG3-biotin, DBCO-S-S-PEG11-biotin, (succinimidyl 3-(2-pyridyldithio)propionate (SPDP), and maleimide-PEG(2 kDa)-maleimide (alpha, omega-bis-maleimide poly(ethylene glycol)). In some embodiments, the crosslinker is maleimide-propyl-SRDFWRS-(1,2-diaminoethane)-propyl-maleimide.

The linked CsgG pore monomer and auxiliary protein or fusion protein may be attached via the formation of a covalent bond between the groups. Any of the specific linkers disclosed in WO 2010/086602 (hereby incorporated by reference in its entirety) may be used.

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

A preferred method of connecting pore monomer conjugates is via a cysteine bond. This can be mediated by a bifunctional chemical crosslinker or by an amino acid linker with a terminally presented cysteine residue.

Another preferred method of attachment is via a 4-azidophenylalanine (Faz) linkage. This can be mediated by a bifunctional chemical linker or by a polypeptide linker bearing a terminally-disposed Faz residue.

In some embodiments, the linker is a bond formed by a sulfur(VI) fluoride exchange (SuFEx) reaction. In some embodiments, an auxiliary 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 acid of another auxiliary protein, etc.) when in appropriate proximity to form a sulfonyl bond (SuFEX).

The auxiliary protein or fusion protein may be genetically fused to the transmembrane protein nanopore. When the entire construct is expressed from a single polynucleotide coding sequence, the pore monomer and auxiliary protein (or fusion protein) are genetically fused. The monomer or subunit, auxiliary protein (or fusion protein) may be fused directly to the transmembrane protein nanopore monomer or subunit. Alternatively, the monomer or subunit, auxiliary protein (or fusion protein) may be fused to the transmembrane protein nanopore monomer or subunit via one or more linkers.

The distance between the CsgG pore monomer and the auxiliary protein or fusion protein in a CsgG pore monomer conjugate and/or the length of the linker is preferably less than about 2.00 nm, e.g., 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 auxiliary protein or fusion protein in a 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 maleimidohexanoic acid, as described in more detail below. The distance and/or linker length between the CsgG pore monomer and the auxiliary protein or fusion protein in the pore monomer conjugate is preferably less than about 0.8 nm. This distance/length can be achieved using maleimidopropionic acid, as described below.

The distance and/or linker length between the CsgG pore monomer and the auxiliary protein or fusion protein in the pore monomer conjugate is preferably about 0.40 nm to about 2.0 nm, e.g., 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 and/or linker length between the CsgG pore monomer and the auxiliary protein or fusion protein in the pore monomer conjugate is preferably about 0.50 nm to about 1.50 nm. The distance and/or linker length between the CsgG pore monomer and the auxiliary protein or fusion protein in the pore monomer conjugate 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 described below.

The maleimide-containing linker may be any of the linkers described 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 typically attached to a nitrogen atom in the maleimide group. The linear carbon chain also preferably comprises a terminal carboxyl group. This carboxyl group is capable of forming an amide bond with an amino acid in the auxiliary protein or fusion protein. The linker is preferably maleimidoacetic acid, maleimidopropionic acid, maleimidobutyric acid, maleimidopentanoic acid, or maleimidohexanoic acid. The linker is most preferably maleimidopropionic acid. This linker is shown in Figure 15.

The present disclosure also provides a pore monomer conjugate comprising a CsgG pore monomer covalently linked to an auxiliary protein or fusion protein, where the auxiliary protein or fusion protein is covalently linked to a cysteine residue in the CsgG pore monomer by a linker comprising a thiol-reactive group. The thiol-reactive group may be a maleimide group, a pyridyldithio group, a halogeno group, a parafluoro group, an ene group, an yne group, a vinylsulfone group, or a thiosulfone group. These groups are shown in Figure 16. The linker comprising a thiol-reactive group may be any of the linkers described below with reference to the constructs of the present disclosure. The linker preferably comprises or consists of a thiol-reactive group and a linear carbon chain of 2, 3, 4, 5, 6, or more carbon atoms. The linear carbon chain also preferably comprises a terminal carboxyl group that can form an amide bond with an amino acid in the auxiliary protein or fusion protein. The linker may be any of the specific maleimide-containing linkers described above, with the maleimide being replaced with a different thiol-reactive group. The linker containing the thiol-reactive group may be any of the lengths described above.

Suitable linking groups may be designed using conventional modeling techniques. The linker typically has sufficient flexibility to allow the monomers or subunits to assemble into their respective protein oligomers and align along their common axis of symmetry to generate continuous channels within the pore complex.

Aspects of the present disclosure relate to computer-based methods for designing and/or selecting auxiliary 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 that performs a protein backbone sequence selection technique and processes 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 involves selecting, from known protein backbone structures (e.g., as described in the Protein Data Bank, PDB), a protein backbone structure that has one or more target properties (e.g., the ability to form one or more helical regions, the ability to fill one or more helical regions of a protein pore, etc.). In some embodiments, the backbone structure is provided as input to software that performs protein sequence design and structure prediction techniques and includes code that processes the backbone structure to generate one or more de novo designed peptide sequences. In some embodiments, the protein sequence design and structure prediction technology may be Rosetta (e.g., as described in Leaver-Fay et al. Chapter nineteen-Rosetta 3: An Object-Oriented Software Suite for the Simulation and Design of Macromolecules, Methods in Enzymology, Academic Press, Volume 487, 2011, pages 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 de novo designed peptide sequence contains one or more target properties that are the same as one or more desired properties of the backbone amino acid sequence.

Methods for Producing Nanopore Complexes Pore complexes comprising an auxiliary protein or fusion protein and a transmembrane protein nanopore can be produced, in one embodiment, by co-expression. In some embodiments, the method involves expressing a pore monomer and an auxiliary protein or fusion protein, or both auxiliary proteins or monomers, in a suitable host cell and allowing in vivo formation of the complex pore. In this embodiment, at least one gene encoding the pore monomer in one vector and a gene encoding the auxiliary protein or fusion protein or at least one auxiliary protein subunit or monomer in a second vector may be co-transformed to express the proteins and produce the complex in the transformed cell. This is preferably performed ex vivo or in vitro. Alternatively, the two genes encoding the pore monomer and the auxiliary protein (or fusion protein) or subunit thereof can be placed in one vector under the control of a single promoter or under the control of two separate promoters, which may be the same or different.

Another method for generating a pore complex formed by an auxiliary protein or fusion protein and a transmembrane protein nanopore is in vitro reconstitution of the proteins to obtain a functional pore. In some embodiments, the method comprises contacting a monomer of the transmembrane protein nanopore with an auxiliary protein (or fusion protein), or an auxiliary protein subunit or monomer, in a suitable system to allow complex formation. The system may be an "in vitro system," which refers to a system that includes at least the components and environment necessary to carry out the method, utilizes biomolecules, organisms, or cells (or portions of cells) outside their normal, naturally occurring environment, and allows for more detailed, convenient, or efficient analysis than can be performed with whole organisms. An in vitro system may also include a suitable buffer composition provided in a test tube, to which the protein components for complex formation are added. Those skilled in the art will be aware of options for providing such a system.

In this embodiment, the nanopore may be generated by expressing the monomer(s) separately from the auxiliary protein or fusion protein. The pore monomer or nanopore may be purified from cells transformed with a vector encoding at least one pore monomer or two or more vectors expressing pore monomers, respectively. The auxiliary protein or fusion protein may be purified from cells transformed with a vector encoding at least one auxiliary protein or fusion protein. The purified pore monomer(s)/nanopore may then be incubated with the auxiliary protein or fusion protein to generate a pore complex.

In another embodiment, the nanopore monomer(s) and/or the auxiliary protein or fusion protein are produced separately by in vitro translation and transcription (IVTT). The nanopore monomer(s) may then be incubated with the auxiliary protein or fusion protein to produce the pore complex.

The above embodiments may be combined, for example, (i) the nanopore is generated in vivo and the auxiliary protein or fusion protein is generated in vivo; (ii) the nanopore is generated in vitro and the auxiliary protein or fusion protein is generated in vivo; (iii) the nanopore is generated in vivo and the auxiliary protein or fusion protein is generated in vitro; or (iv) the nanopore is generated in vitro and the auxiliary protein or fusion protein is generated in vitro.

One or both of the nanopore monomer and the auxiliary protein or fusion protein may be tagged to facilitate purification. Purification can also be performed when the nanopore monomer and/or the auxiliary protein or fusion protein are untagged. Methods known in the art (e.g., ion exchange, gel filtration, hydrophobic interaction column chromatography, etc.) can be used alone or in different combinations to purify the components of the pore complex.

Any known tag can be used on either of the two proteins. In one embodiment, two-tag purification can be used to purify the pore complex from its constituent parts. For example, a Strep tag can be used on the nanopore and a His tag on the auxiliary protein (or fusion protein), or vice versa. The two proteins can be purified separately, mixed together, and then Strep and His purified again to achieve the same end result.

The pore complex can be generated before insertion into the membrane or after insertion of the nanopore into the membrane. However, the nanopore can also be inserted into the membrane and then the auxiliary protein (or fusion protein) added so that the pore complex can form in situ. For example, in one embodiment, in systems where the trans 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 or cis side of the membrane so that the complex can form in situ.

In one embodiment, the auxiliary protein includes a protease cleavage site (e.g., TEV, HRV3, or any other protease cleavage site) and may be cleaved before or after association with the nanopore. For example, a full-length auxiliary protein (or fusion protein) may be used to form the pore. Amino acid residues that do not form part of the channel structure and are not required for interaction with the transmembrane pore may be cleaved from the auxiliary protein or fusion protein. In this embodiment, a protease is used to cleave the auxiliary protein or fusion protein once the pore complex is formed. Alternatively, a protease may be used to generate the auxiliary protein or fusion protein prior to assembly of the pore complex.

Some protease sites leave an additional tag (or a portion thereof, e.g., one or more amino acids of the tag) after cleavage. For example, the TEV protease cleavage sequence is ENLYFQS. The 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; the enzyme cleaves between Q and G, leaving LEVLFQ intact at the C-terminus of the CsgF peptide.

The protein may be chemically modified with a molecular adaptor that facilitates interaction between the pore containing the monomer and the target nucleotide or target polynucleotide sequence. Suitable adaptors, including cyclic molecules, cyclodextrins, hybridizable species, DNA binders or interchelators, peptides or peptide analogs, synthetic polymers, aromatic planar molecules, small positively charged molecules, or small molecules capable of hydrogen bonding, are described in WO 2019/002893, which is incorporated herein by reference in its entirety. The molecular adaptor may be attached using any of the methods and linkers described above.

The protein may be attached to a polynucleotide binding protein, forming a modular sequencing system. Polynucleotide binding proteins are discussed below. The protein may be covalently attached to the monomer using any method known in the art. The monomer and protein may be chemically fused or genetically fused. Genetic fusion of a monomer to a polynucleotide binding protein is discussed in WO 2010/004265, which is incorporated herein by reference in its entirety. The polynucleotide binding protein may be attached via a cysteine linkage using any of the methods described above.

The polynucleotide-binding protein may be directly attached to the protein via one or more linkers. The molecule may be attached to the CsgG pore monomer using a hybridization linker as described in WO 2010/086602 (incorporated herein by reference in its entirety). Alternatively, a peptide linker may be used. Suitable peptide linkers are described above.

Any of the proteins can be produced using standard methods known in the art. Polynucleotide sequences encoding the proteins can be obtained and replicated using standard methods in the art. Polynucleotide sequences encoding the proteins can be expressed in bacterial host cells using standard techniques in the art. Proteins can be produced intracellularly by expressing the polypeptide in situ from a recombinant expression vector. The expression vector optionally carries an inducible promoter to control expression of the polypeptide. 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.

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

Systems In another aspect, the present disclosure relates to a system for characterizing a target polynucleotide, the system comprising a membrane and a pore complex, the pore complex comprising (i) a nanopore located in the membrane, and (ii) an accessory protein or fusion protein bound to the nanopore, wherein the nanopore and the accessory protein or fusion protein together form a continuous channel across the membrane, the channel comprising a first constriction region and a second constriction region.

The pore complex, nanopore and auxiliary protein or fusion protein may be any of those described herein above.

In one embodiment, the system further comprises a first chamber and a second chamber, the first and second chambers being separated by a membrane. When used to characterize a target polynucleotide, the system may further comprise a target polynucleotide, the target polynucleotide being temporarily located within the continuous channel, with one end of the target polynucleotide located in the first chamber and one end of the target polynucleotide located in the second chamber.

In one embodiment, the system further includes a conductive solution in contact with the nanopore, electrodes that provide a voltage potential across the membrane, and a measurement system that measures the current through the nanopore. In one embodiment, the voltage applied to the membrane and pore complex is between +5 V and -5 V, e.g., between -600 mV and +600 mV or between -400 mV and +400 mV. The voltage used is preferably in the range of 100 mV and 240 mV, more preferably between 120 mV and 220 mV. Using an increased applied potential can increase the discrimination between different nucleotides per pore. Any suitable conductive solution may be used. For example, the solution may include a charge carrier such as a metal salt, e.g., an alkali metal salt; a halide salt, e.g., a chloride salt, e.g., an alkali metal chloride salt. The charge carrier may include an ionic liquid or an organic salt, e.g., tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride, or 1-ethyl-3-methylimidazolium chloride. In an exemplary system, a salt is present in the aqueous solution within the chamber. Potassium chloride (KCl), sodium chloride (NaCl), cesium chloride (CsCl), or a mixture of potassium ferrocyanide and potassium ferricyanide are typically used. KCl, NaCl, and a mixture of potassium ferrocyanide and potassium ferricyanide are preferred. Charge carriers can be asymmetric across the membrane. For example, the type and/or concentration of charge carriers can be different on each side of the membrane, e.g., within each chamber.

The salt concentration may be saturating. The salt concentration may be 3 M or less, 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 carried out using a salt concentration of at least 0.3 M, e.g., 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, allowing identification of currents indicative of the presence of nucleotides against a background of normal current fluctuations.

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.

The system may include an array of pore complexes present in a membrane. In preferred embodiments, each membrane in the array includes one pore complex. Depending on the manner in which the array is formed, for example, the array may include one or more membranes that do not include a pore complex and/or one or more membranes that include two or more pore complexes. The array may include from about 2 to about 12,000 membranes, e.g., from about 10 to about 800, from about 20 to about 600, from about 30 to about 500, from about 250 to about 2000, from about 500 to about 4000, from about 1000 to about 5000, from about 2500 to about 10,000, or from about 5000 to about 12,000 membranes. In some embodiments, the array includes more than 12,000 membranes.

The system may be included in a device. The device may be any conventional device for analyte analysis, such as an array or chip. The device is preferably configured to perform the disclosed method. For example, the device may include a chamber containing an aqueous solution and a barrier separating the chamber into two sections. The barrier typically has an opening through which a membrane containing pores is formed. Alternatively, the barrier forms a membrane in which the pores reside.

In one embodiment, the apparatus includes a sensor device operable to support a plurality of pores and membranes and perform characterization of an analyte using the pores and membranes, and at least one port for delivering material for performing the characterization.

In one embodiment, the apparatus includes a sensor device operable to support a plurality of pores and membranes and to perform characterization of an analyte using the pores and membranes, and at least one reservoir for holding material for performing the characterization.

In one embodiment, the apparatus includes a sensor device supporting a membrane and a plurality of pores and membranes and operable to characterize an analyte using the pores and membranes; at least one reservoir for holding material for characterization; a fluidics system configured to controllably deliver material from the at least one reservoir to the sensor device; and one or more containers for receiving individual samples, the fluidics system configured to selectively deliver sample from the one or more containers to the sensor device.

The device may also include an electrical circuit capable of applying an electrical potential and measuring an electrical signal between the membrane and the pore complex. The device may be any of those described in WO 2008/102120, WO 2009/077734, WO 2010/122293, WO 2011/067559, or WO 00/28312.

Membrane Any suitable membrane can be used in the system. The membrane is preferably an amphiphilic layer. An amphiphilic layer is a layer formed from amphiphilic molecules, such as phospholipids, that have both hydrophilic and lipophilic properties. The amphiphilic molecules can be synthetic or naturally occurring. Non-naturally occurring amphiphiles and amphiphiles that form monolayers are known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). Block copolymers are polymeric materials in which two or more monomer subunits are polymerized together to create a single polymer chain. Block copolymers typically have properties contributed by each monomer subunit. However, block copolymers may have unique properties not possessed by polymers formed from individual subunits. Block copolymers may be designed so that one of the monomer subunits is hydrophobic (i.e., lipophilic) and the other subunit(s) is/are hydrophilic in aqueous media. In this case, the block copolymer may have amphiphilic properties and form structures that mimic biological membranes. Block copolymers may be diblock (composed of two monomer subunits), but may also be composed of more than two monomer subunits to form more complex arrangements that function as amphiphiles. The copolymer may be a triblock, tetrablock, or pentablock copolymer. The membrane is preferably a triblock copolymer membrane.

Archaeal bipolar tetraether lipids are naturally occurring lipids that organize to form lipid monolayer membranes. These lipids are commonly found in extremophilic, thermophilic, halophilic, and acidophilic bacteria, which survive in harsh biological environments. Their stability is thought to derive from the fused nature of the final bilayer. It is straightforward to construct block copolymers that mimic these biological entities by creating triblock polymers with the general motif hydrophilic-hydrophobic-hydrophilic. This material forms monomeric membranes that behave similarly to lipid bilayers and can encompass a wide range of phase behaviors, from vesicles to lamellar membranes. Membranes formed from these triblock copolymers have several advantages over biological lipid membranes. Because the triblock copolymers are synthetic, their precise structure can be carefully controlled to provide the correct chain length and properties necessary to form membranes and interact with pores and other proteins.

Block copolymers may also be constructed from subunits that are not classified as lipid submaterials. For example, hydrophobic polymers may be formed from siloxane or other non-hydrocarbon-based monomers. The hydrophilic subsection of the block copolymer may also have low protein binding properties, allowing for the formation of films that are highly resistant when exposed to live biological samples. This head group unit may also be derived from a non-classified lipid head group.

Triblock copolymer membranes also have increased mechanical and environmental stability compared to biological lipid membranes, e.g., much higher operating temperature or pH ranges. The synthetic nature of block copolymers provides a platform for customizing polymer-based membranes for a wide range of applications.

The membrane is most preferably one of the membranes disclosed in WO2014/064443 or WO2014/064444.

The amphiphilic molecules may be chemically modified or functionalized to facilitate coupling of polynucleotides. The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic layer is typically planar. The amphiphilic layer may also be curved. The amphiphilic layer may also be supported.

Amphiphilic membranes are typically mobile in nature, essentially behaving as two-dimensional fluids with lipid diffusion rates of approximately 10 ⁻⁸ cm s ⁻¹ , which means that pores and coupled polynucleotides are typically able to move within the amphiphilic membrane.

The membrane can be a lipid bilayer. Lipid bilayers are models of cell membranes and serve as an excellent platform for a wide range of experimental studies. For example, lipid bilayers can be used for in vitro investigation of membrane proteins by single-channel recording. Alternatively, lipid bilayers can be used as biosensors to detect the presence of a wide 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.

Methods for forming lipid bilayers are known in the art. Lipid bilayers are typically formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA, 1972; 69:3561-3566), in which a lipid monolayer is placed on the aqueous solution/air interface through an opening on either side that is perpendicular to the interface. Lipids are typically added to the surface of an aqueous electrolyte solution by first dissolving them in an organic solvent and then evaporating a small amount of solvent onto the aqueous solution interface on either side of the opening. As the organic solvent evaporates, the solution/air interfaces on either side of the opening physically move up and down across the opening until a bilayer is formed. Planar lipid bilayers can form across the opening in a membrane or across the opening in a recess.

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

Tip-dipping bilayer formation involves contacting an aperture (e.g., a pipette tip) onto the surface of a test solution bearing a lipid monolayer. Again, the lipid monolayer is generated at the solution/air interface by first evaporating a small amount of lipid dissolved in an organic solvent at the solution surface. The bilayer is then formed by the Langmuir-Schaefer method, which requires mechanical automation to move the aperture relative to the solution surface.

For bilayer coating, a small amount of lipid dissolved in an organic solvent is applied directly to an aperture immersed in the test aqueous solution. The lipid solution is spread thinly across the aperture using a paintbrush or equivalent. Diluting the solvent results in the formation of a lipid bilayer. However, it is difficult to completely remove the solvent from the bilayer, and as a result, bilayers formed by this method are less stable and more likely to generate noise during electrochemical measurements.

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, causing a patch of membrane to adhere across the opening. This method has been adapted to produce lipid bilayers by clamping and then rupturing liposomes to seal the lipid bilayer across the pipette opening. This method requires the creation of stable, large, unilamellar liposomes and a small opening in a material with a glass surface.

Liposomes can be formed by sonication, extrusion, or the 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 from dry lipids. In a most preferred embodiment, the lipid bilayer is formed across an opening as described in WO 2009/077734.

A lipid bilayer is formed from two opposing layers of lipids. These two lipid layers are arranged so that their hydrophobic tail groups face each other, forming a hydrophobic interior. The hydrophilic head groups of the lipids face outward toward the aqueous environment on either side of the bilayer. Bilayers may exist in several lipid phases, including, but not limited to, liquid disordered phases (fluid lamellar), liquid ordered phases, solid ordered phases (lamellar gel phase, interdigitated gel phase), and planar bilayer crystals (lamellar subgel phase, lamellar crystalline phase).

Any lipid composition that forms a lipid bilayer may be used. The lipid composition is selected to form a lipid bilayer with the desired properties, such as surface charge, ability to support membrane proteins, packing density, or mechanical properties. The lipid composition can include one or more different lipids. For example, the lipid composition can include up to 100 lipids. The lipid composition preferably includes 1 to 10 lipids. The lipid composition can include naturally occurring lipids and/or artificial lipids.

Lipids typically comprise a head group, an interfacial moiety, and two hydrophobic tail groups, which may be the same or different. Suitable head groups include, but are not limited to, neutral head groups such as diacylglyceride (DG) and ceramide (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), phosphate (PA), and cardiolipin (CA), and positively charged head groups such as trimethylammonium propane (TAP). Suitable interfacial moieties include naturally occurring interfacial moieties, such as, but not limited to, glycerol-based moieties or ceramide-based moieties. Suitable hydrophobic tail groups include, but are not limited to, saturated hydrocarbon chains such as lauric acid (n-dodecanolic acid), myristic acid (n-tetradecononinoic 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 chain length and the position and number of double bonds in the unsaturated hydrocarbon chain may vary. The chain length and the position and number of branches, such as methyl groups in the branched hydrocarbon chain, may vary. The hydrophobic tail group may be linked to the interfacial moiety as an ether or ester. The lipid may be a mycolic acid.

Lipids can also be chemically modified. The head group or tail group of the lipid can be chemically modified. Suitable lipids with chemically modified head groups 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 with chemically modified tail groups include, but are not limited to, polymerizable lipids such as 1,2-bis(10,12-tricosadinoyl)-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-phytanyl-sn-glycero-3-phosphocholine. Lipids may be chemically modified or functionalized to facilitate coupling of polynucleotides.

The amphiphilic layer, e.g., lipid composition, typically contains 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 palmitic alcohol, myristic alcohol, and oleic alcohol; sterols such as cholesterol, ergosterol, lanosterol, sitosterol, and stigmasterol; lysophospholipids such as 1-acyl-2-hydroxy-sn-glycero-3-phosphocholine; and ceramides.

In another preferred embodiment, the membrane comprises a solid-state layer. The solid-state layer can be formed from both 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 polyamides, plastics such as Teflon®, or elastomers such as two-component addition-cured silicone rubber, and glass. The solid-state layer can be formed from graphene. Suitable graphene layers are disclosed in WO 2009/035647. When the membrane comprises a solid-state layer, the pores are typically present in the amphiphilic membrane or layer contained within the solid-state layer, for example, within holes, wells, gaps, channels, grooves, or slits within the solid-state layer. Those skilled in the art can prepare suitable solid-state/amphiphilic hybrid systems. Suitable systems are disclosed in WO 2009/020682 and WO 2012/005857. Any of the amphiphilic membranes or layers discussed above may be used.

The method is typically carried out using (i) an artificial amphiphile layer containing a pore, (ii) an isolated natural lipid bilayer containing a pore, or (iii) a cell into which a pore has been inserted. The method is typically carried out using an artificial amphiphile layer, such as an artificial triblock copolymer layer. In addition to the pore, the layer may contain other transmembrane and/or intramembrane proteins, as well as other molecules. Suitable equipment and conditions are discussed below. The method of the present disclosure is typically carried out in vitro.

Methods for Characterizing Analytes In a further aspect, methods for determining the presence, absence, or one or more properties of a target analyte are disclosed. The method includes contacting the target analyte with a membrane containing a pore complex such that the target analyte migrates relative to, e.g., in or through, a continuous channel comprising at least two structures provided by a nanopore and an auxiliary protein or peptide, respectively, within the pore complex, and performing one or more measurements as the analyte migrates relative to the channel, thereby determining the presence, absence, or one or more properties of the analyte. The analyte may pass through a constriction of the nanopore followed by a constriction of the auxiliary protein. In an alternative embodiment, the analyte may pass through a constriction of the auxiliary protein followed by a constriction of the nanopore, depending on the orientation of the pore complex within the membrane.

In one embodiment, the method is for determining the presence, absence, or one or more characteristics of a target analyte. The method may be for determining the presence, absence, or one or more characteristics of at least one analyte. The method may involve determining the presence, absence, or one or more characteristics of two or more analytes. The method may include determining the absence or one or more characteristics of any number of analytes, for example, 2, 5, 10, 15, 20, 30, 40, 50, 100, or more analytes. Any number of characteristics of one or more analytes may be determined, for example, 1, 2, 3, 4, 5, 10, or more characteristics.

Binding of a molecule within the channel of a pore complex or near any opening of the channel affects open-channel ionic flow through the pore, which is the essence of pore-channel "molecular sensing." In a manner similar to nucleic acid sequencing applications, fluctuations in open-channel ionic flow can be measured using suitable measurement techniques based on changes in electrical current (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 decrease in ionic flow, measured by a decrease in electrical current, is related to the size of the obstacle within or near the pore. Thus, binding of a molecule of interest, also referred to as an "analyte," within or near the pore provides a detectable and measurable event, thereby forming the basis of a "biological sensor." Molecules suitable for nanopore sensing include nucleic acids, proteins, peptides, polysaccharides, and small molecules (herein referring to organic or inorganic compounds with low molecular weight (e.g., <900 Da or <500 Da)), such as pharmaceuticals, toxins, cytokines, and pollutants. Detecting the presence of biomolecules finds applications in personalized drug development, medicine, diagnostics, life science research, environmental monitoring, and the security and/or defense industries.

The target analyte may be a metal ion, inorganic salt, polymer, amino acid, peptide, polypeptide, protein, nucleotide, oligonucleotide, polynucleotide, monosaccharide, polysaccharide, dye, bleach, pharmaceutical, diagnostic agent, recreational drug, explosive, toxic compound, or environmental pollutant. The method may involve determining the presence, absence, or one or more characteristics of two or more analytes of the same type, e.g., two or more proteins, two or more nucleotides, or two or more pharmaceuticals. Alternatively, the method may involve determining the presence, absence, or one or more characteristics of two or more different types of analytes, e.g., one or more proteins, one or more nucleotides, and one or more pharmaceuticals.

The target analyte may be secreted from the cell. Alternatively, the target analyte may be an analyte that is present intracellularly, and therefore the analyte must be extracted from the cell before the method may be performed.

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 therein. Several different types of modifications to amino acids are known in the art. Suitable amino acids and their modifications are described above. It should be understood that the target analyte may be modified by any method available in the art.

In a preferred embodiment, the analyte is a polynucleotide, such as a nucleic acid. A polynucleotide is defined as a polymer containing two or more nucleotides. Naturally occurring nucleic acid bases in DNA and RNA can be distinguished by their physical size. As a nucleic acid molecule or individual bases pass through the nanopore channel, the size differences between the bases result in a directly correlated reduction in 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, for example, in WO 2000/28312 and D. Stoddart et al., Proc. Natl. Acad. Sci., 2010, 106, pp. 7702-7 (single-channel recording instruments), and, for example, in WO 2009/077734 (multi-channel recording techniques). With appropriate calibration, the characteristic reduction in ion flow can be used to identify specific nucleotides and associated bases passing through the channel in real time. In typical nanopore nucleic acid sequencing, open-channel ion current decreases as individual nucleotides of a nucleotide sequence of interest pass sequentially through the nanopore channel due to partial blockage of the channel by the nucleotide. It is this reduction in ion current that is measured using the suitable recording techniques described above. The reduction in ion current can be calibrated to the reduction in ion current measured for known nucleotides passing through the channel, providing a means for determining which nucleotides pass through the channel, and thus, when performed sequentially, provides a method for determining the nucleotide sequence of a nucleic acid passing through the nanopore. To accurately determine individual nucleotides, it is typically necessary that the reduction in ion current through the channel directly correlate with the size of the individual nucleotides passing through the constriction (or "leading head"). It will be understood that sequencing may be performed on an intact nucleic acid polymer "threaded" through the pore, for example, via the action of an associated polymerase or helicase. Alternatively, the sequence may be determined by passing nucleotide triphosphate groups sequentially removed from the target nucleic acid adjacent to the pore (see, e.g., WO 2014/187924).

A polynucleotide or nucleic acid can contain any combination of any nucleotides. Nucleotides can be naturally occurring or artificial. One or more nucleotides in a polynucleotide can be oxidized or methylated. One or more nucleotides in a polynucleotide can be damaged. For example, a polynucleotide can contain a pyrimidine dimer. Such dimers are typically associated with UV damage and are a major cause of cutaneous melanoma. One or more nucleotides in a polynucleotide can be modified, for example, with a label or tag, suitable examples of which are known to those skilled in the art. A polynucleotide can contain one or more spacers. A nucleotide typically contains a nucleobase, a sugar, and at least one phosphate group. The nucleobase and sugar form a nucleoside. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines, more specifically, adenine (A), guanine (G), thymine (T), uracil (U), and cytosine (C). The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar is preferably deoxyribose. Polynucleotides preferably contain the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and/or thymidine (dT), deoxyguanosine (dG), and deoxycytidine (dC). Nucleotides are typically ribonucleotides or deoxyribonucleotides. Nucleotides typically contain monophosphate, diphosphate, or triphosphate. Nucleotides may contain more than three phosphates, for example, four or five phosphates. The phosphates may be attached to the 5' or 3' side of the nucleotide. Nucleotides in a polynucleotide can be attached to each other in any manner. Nucleotides are typically attached via their sugar and phosphate groups, similar to nucleic acids. Nucleotides may be connected via nucleobases, similar to pyrimidine dimers. Polynucleotides may be single-stranded or double-stranded. At least a portion of the polynucleotide is preferably double-stranded. The polynucleotide is most preferably ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In particular, the method alternatively using a polynucleotide as an analyte includes 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.

Polynucleotides can be of any length (i). For example, polynucleotides 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 in length. Polynucleotides can be 1,000 nucleotides or nucleotide pairs or more in length, 5,000 nucleotides or nucleotide pairs or more in length, or 100,000 nucleotides or nucleotide pairs or more in length. Any number of polynucleotides can be investigated. For example, the method can involve characterizing 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100, or more polynucleotides. When two or more polynucleotides are characterized, they can be different polynucleotides or two instances of the same polynucleotide. Polynucleotides can be naturally occurring or artificial. For example, the method can be used to verify the sequence of manufactured oligonucleotides. The method is typically performed in vitro.

The nucleotides may have identity (ii) and may 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 nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP, and dUMP. A nucleotide may be abasic (i.e., lacking a nucleobase). A nucleotide may also lack a nucleobase and a sugar (i.e., a C3 spacer). The sequence (iii) of a nucleotide is determined by the sequential identities of the following nucleotides, linked together in the 5' to 3' direction of the strand, throughout the polynucleotide strand:

Pore complexes containing at least two constrictions are particularly useful for analyzing homopolymers. For example, the pore can be used to sequence polynucleotides that contain two or more identical nucleotides, e.g., at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides. For example, the pore can be used to sequence polynucleotides that contain poly-A tracts, poly-T tracts, poly-G tracts, and/or poly-C tracts.

In some embodiments, the CsgG pore constriction is generated by residues at positions 51, 55, and 56 of SEQ ID NO: 59. As DNA passes through the constriction, the interaction of approximately five bases of DNA with the pore constriction at any given time dominates the current signal. Certain CsgG pores (e.g., CsgG pores lacking one or more auxiliary proteins or fusion proteins described herein) are very good at reading mixed-sequence regions of DNA (when A, T, G, and C are mixed), but when there are homopolymeric regions within the DNA (e.g., poly-T, poly-G, poly-A, poly-C), the signal becomes flat and lacks some information. Because five bases dominate the signal for CsgG and its constriction variants, it is difficult to discriminate between homopolymers longer than five without using additional dwell time information. However, when DNA passes through a second constriction, more DNA bases interact with the combined constrictions, increasing the length of homopolymers that can be discriminated.

Kits In a further aspect, the present disclosure also provides kits for characterizing target polynucleotides. The kits include the disclosed pore complex and membrane components. The membrane is preferably formed from the components. The pore complex is preferably present within the membrane, and together they form a transmembrane pore complex channel. The kits may include components of any type of membrane, such as an amphiphilic layer or a triblock copolymer membrane. The kits may further include a polynucleotide-binding protein, such as a nucleic acid processing enzyme, e.g., a polymerase or a helicase. The kits may further include one or more anchors, such as cholesterol, for coupling the polynucleotide to the membrane. The kits may further include one or more polynucleotide adaptors capable of binding to the target polynucleotide to facilitate characterization of the polynucleotide. In one embodiment, an anchor, such as cholesterol, is attached to the polynucleotide adaptor. The kits may further include one or more other reagents or equipment that enable any of the above embodiments to be performed. Such reagents or equipment may include one or more of the following: suitable buffer(s) (aqueous solution), means for obtaining a sample from a subject (such as an apparatus including a container or needle), means for amplifying and/or expressing polynucleotides, or voltage or patch clamp apparatus. The reagents may be present in the kit in a dry state so that a fluid sample resuspends the reagents. The kit may also optionally include instructions to enable the kit to be used in the methods of the disclosure, or details regarding organisms for which the methods may be used. Finally, the kit may also include additional components useful for characterizing polynucleotides.

While specific embodiments, particular configurations, and materials and/or molecules have been discussed herein for engineered cells and methods according to the present disclosure, it should be understood that various changes or modifications in form and detail may be made 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, which is limited only by the claims.

Example 1
To create the helical constriction, de novo design was used to select a small protein domain that was well-folded and protruded into the lumen of the nanopore to a desired extent. Several programs can be used for this purpose. This example describes a workflow using the programs MASTER, which facilitates backbone design, and Rosetta, which has variable backbone geometries for sequence selection.

To create a new domain to project into the pore lumen, programs such as RF-diffusion, CHROMA, or the program MASTER can be used. Here, the inventors used MASTER. They searched the Protein Data Bank (PDB) for structures that met the following criteria: 1) stabilization of the target region of CsgF (residues 16-30); 2) projection into the nanopore to create a new constriction (the Ca-Ca distance of the amino acid residues that extend furthest into the pore lumen) with a diameter between 10 Å and 30 Å when all units are generated using 9-fold symmetry operators; and 3) the new domain does not clash with any atoms in CsgG or with symmetry mates from CsgF.

First, helices docking to the target region in CsgF and its symmetrically adjacent regions were identified in geometries frequently observed in natural proteins in the PDB and therefore "designable." Top candidates were selected based on the number of closely related helix-helix pairs found in the database after clustering the output based on the RMSD of the target region and the discovered helices (Figure 1). In this way, helix geometries that pack well against the target amino acid and its four N-terminal amino acids were selected. Furthermore, the database was searched for helices that form favorable helix-helix interactions with symmetry-related partners. Linkers (e.g., loop structures) connecting the helices were selected using a database of helix backbones (Figure 1). The sequences of the resulting backbones were then designed using Rosetta. Representative sequences were generated (e.g., SEQ ID NOS: 1-58).

Sequences for experimental validation were selected based on lowest energy score and highest PackStat score, as shown in Figure 2. To further prioritize sequences, aggregation propensity may be tested using one of several aggregation and amyloid prediction programs.

Example 2
Materials and Methods: E. coli CsgG Pore Generation. A recombinant expression vector encoding a CsgG variant nanopore with a C-terminal Strep affinity tag and an ampicillin resistance gene was transformed into chemically competent E. coli cells. Cells were plated onto LB agar plates containing the appropriate antibiotic for selection and incubated overnight at 37°C. LB medium containing the appropriate antibiotic was inoculated with a single colony from the agar plate and grown overnight at 37°C with shaking. The culture was diluted with autoinduction medium and the required antibiotic and incubated for 68 hours at 18°C with shaking. Cells were harvested by centrifugation before lysis and extraction into a buffer containing 1x Bugbuster extraction reagent (Merck 70921) and 0.1% DDM. The lysate was spun down, 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.

CsgG/CsgF or Fusion Protein Complex Formation Protocol. CsgG-CsgF complexes were prepared from purified nanopores as described above, and de novo fusion proteins were chemically synthesized with or without maleimide modification. For cysteine-containing fusion proteins, cyclization of the fusion protein was achieved by crosslinking the thiol at the appropriate cysteine. The nanopores were buffer-exchanged into a pH 7.0 buffer containing no reducing agent and incubated with an 8-fold molar excess of peptide over CsgG monomer at 25°C for 1 hour. The samples were then heated at 60°C for 15 minutes, after which they were centrifuged to remove any precipitates, and DTT was added to prevent any further reaction.

SDS-PAGE analysis. 1 μg of conjugate and CsgG-only pore control were added to individual 0.5 mL ProteinLoBind Eppendorf tubes (Fisher, 10316752) and brought to a volume of 10 μL with reaction buffer. This was brought to a final volume of 20 μL by adding 10 μL of 2x Laemmli buffer. The entire sample was loaded onto a 4-20% TGX gel (BioRad, 5671093) run in 1x TGS buffer (Sigma, T7777). This was run at 300 V for 21 minutes. Spyro Ruby (Merck, S4942) stain was used to image the gel, according to the manufacturer's instructions. This was then imaged on a GE Typhoon gel imager using the 450 nm laser.

For some analyses, 1 μg of complex and a CsgG-only pore control were added to individual PCR tubes and brought to a volume of 10 μL with reaction buffer. A freshly prepared 1 M DTT stock was spiked into each PCR tube at a final concentration of 10 mM. This was brought to a final volume of 20 μL by adding 10 μL of 2x Laemmli buffer. Each sample was heated to 95°C for 2 minutes in a PCR thermocycler. This was allowed to cool for 5 minutes before the entire material from each sample was loaded onto a 4-20% TGX gel (BioRad, 5671093) run in 1x TGS buffer (Sigma, T7777). This was run at 300V for 21 minutes. To image the gel, Spyro Ruby (Merck, S4942) stain was used according to the manufacturer's instructions. This was then imaged on a GE Typhoon gel imager using a 450 nm laser.

Electrical measurements were taken from CsgG-only complexes, CsgG/CsgF complexes, or CsgG/fusion protein complexes inserted into MinION flow cells. After inserting a single pore into the block copolymer membrane, 1 mL of buffer containing 25 mM potassium phosphate, 150 mM potassium ferrocyanide(II), 150 mM potassium ferricyanide(III), pH 8.0 was flowed through the system to remove any excess nanopores.

As shown in Figure 23, the analyte used to assess DNA waviness was a 3.6 kilobase DNA fragment from the 3' end of the lambda genome. Analyte preparation, ligation of the analyte to a Y-adapter, SPRI-bead cleanup of the ligated analyte, and application to the minION flow cell were performed using the Oxford Nanopore Technologies Q-SQK-LSK110 protocol.

Electrical measurements were taken using a minION Mk1b instrument manufactured by Oxford Nanopore Technologies. A standard sequencing script was run at -180 mV for 6 hours, with static flicks every 5 minutes to remove any elongation nanopore blockages. Raw data were collected in bulk FAST5 files using MinKNOW software (Oxford Nanopore Technologies).

Differential Profiling: A FAST5 containing DNA squiggles (e.g., electrical measurements) of a 3.6 kilobase DNA segment from the 3' end of the lambda genome (3.6 Kb lambda) was acquired. The DNA squiggles were trimmed using a custom Python script to remove any electrical signal measurements captured before DNA sequencing began.

The trimmed 3.6 Kb lambda curve and the corresponding genomic reference for this region were used to train the parameters of a neural network. The neural network, containing four layers, modeled sequences in a user-specified window length and the current level associated with those sequences. The window length specified for these models allowed a region of +/- 12 nucleotides to contribute to the current level at any one position.

The trained neural network was used to predict the current level corresponding to a 3.6 Kb lambda DNA reference sequence. This was also used to predict the current level from all possible single-base edits of that sequence.

Changing a base at a single position (L) within a sequence not only changes the predicted current as this base passes through the main constriction of the pore, but also changes the current before and after the base passes through this main constriction. The predicted current levels for a set of edited 3.6 Kb lambda sequences were analyzed to calculate the range of predicted current at position L + X (offset) when the base at position L is changed. Offsets of -16 to +16 were analyzed at each position. The midpoint of the range of predicted current at each offset was calculated to obtain the data in the figure. The model was centered so that the largest peak, representing the CsgG constriction, corresponds to position 0.

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

The three-dimensional structures of alternative sequences of the de novo designed fusion proteins were also investigated using protein folding algorithms. The predicted 3D structures of the de novo designed fusion proteins ONT1-ONT10, ONT11-ONT20, and ONT21-ONT25 are shown in Figures 4A-4C. The structures are shaded according to a measure of confidence, the predicted local distance difference test (pLDDT).

SDS-PAGE gel analysis of CsgG-only pores and CsgG/fusion protein complexes was performed. The complexes contained either the CsgF-del(S31-F119) control or the de novo-designed fusion protein, with or without a maleimide crosslinker (Figure 5). Complexes containing the fusion protein exhibited a band shift, indicating that these samples were nanopore complexes. Note that the samples were not heated before loading onto the gel. SDS-PAGE gel analysis of CsgG-only pores and CsgG/fusion protein complexes was also performed. These complexes contained either the CsgF-del(S31-F119) control or the de novo-designed fusion protein, with or without a maleimide crosslinker (Figure 6). The pores were disassembled into their constituent monomeric components by boiling in the presence of DTT before loading onto the gel. Note that no band shift was observed in the absence of maleimide crosslinking, indicating that these bands consist solely of CsgG monomers. 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 a further band shift due to the increased mass of the fusion protein, indicating that the fusion protein is covalently bound to the CsgG pore.

Ion current (pA) versus time (s) was measured as single-stranded DNA translocated through the CsgG-only pore. Each individual graph corresponds to a single pore inserted into the minION flow cell. The open pore current observed for the CsgG-only pore was approximately 180 pA at an applied voltage of -180 mV. Table 1 below shows representative data for the mean range, mean noise, and mean signal-to-noise ratio (SNR) of the protein-pore complex as described in this disclosure.
Table 1: Metrics table

Figures 7-11 show representative ionic current (pA) versus time (s) traces as single-stranded DNA translocates through a CsgG-only pore, CsgG containing the del(S31-F119) CsgF peptide, or CsgG containing a de novo designed fusion protein. The raw current traces are shown as black lines, and the event detection signal is shown as a red line. For each pore, the top row shows the complete DNA current trace, and the bottom row shows a zoomed-in view of the first portion of the current trace. The open pore current for the CsgG-only pore is observed to be approximately 175-200 pA, with the mean current for the DNA squiggly line being approximately 75 pA. For the pore containing the CsgF peptide, the open pore current is approximately 90-120 pA, with the mean current being approximately 35-50 pA. Figure 7 shows the traces as DNA translocates through a CsgG-only pore. Figure 8 shows representative ion current (pA) versus time (s) traces when single-stranded DNA translocates through CsgG containing the del(S31-F119) CsgF peptide, with (right) or without (left) a maleimide crosslinker. Figure 9 shows representative ion current (pA) versus time (s) traces when single-stranded DNA translocates through CsgG containing the de novo designed fusion protein ONLP20623, without the maleimide crosslinker. Figure 10 shows representative ion current (pA) versus time (s) traces when single-stranded DNA translocates through CsgG containing the de novo designed fusion protein ONLP20624 (without the maleimide crosslinker) or ONLP20627 (with the maleimide crosslinker). Figure 11 shows representative ionic current (pA) versus time (s) traces as single-stranded DNA translocates through CsgG containing de novo designed fusion proteins ONLP20628 (with maleimide bridge) or ONLP20625 (without maleimide bridge). In some embodiments, the fusion protein contains a 37R residue along with a cysteine residue to form an internal disulfide bond within the peptide, i.e., cyclize the fusion protein.

As a DNA molecule translocates through the pore, a profile is generated showing its position within the pore and its contribution to the overall change in ionic current level ("discrimination"). Distance within the pore is measured in nucleotide steps relative to the major constriction. Negative values correspond to positions below the major constriction, and positive values correspond to positions above the major constriction (CsgG). The dashed box indicates the region affected by the introduction of the de novo designed fusion protein. Figure 12 shows a representative profile when a DNA molecule translocates through a CsgG-only pore. CsgG-only pores (with or without Q153C) show one major discrimination peak at position 0. Figure 13 shows a representative profile when a DNA molecule translocates through a CsgG/CsgF pore. The dashed box indicates the region affected by the introduction of the de novo designed fusion protein. The CsgG-CsgF-del (S31-F119) pore with (right) or without (left) the maleimide crosslinker shows two discrimination peaks. The major discrimination peak is at position 0, as seen in the CsgG-only pore, and an additional discrimination peak is 4 to 6 nucleotides below the major constriction (positions -4 to -6). This additional region of discrimination has a smaller effect on the ion current compared to the major discrimination peak at position 0. Figure 14 shows a representative profile of a DNA molecule translocating through a CsgG/fusion protein (ONLP20641 or ONLP20644) pore. Complexes containing CsgG and a de novo designed fusion protein containing K37R, with or without (left) the maleimide crosslinker, with or without cyclization, show three discrimination peaks. The major discriminatory peak is at position 0, as seen in the CsgG-only pore, with additional peaks 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.

Example 3
Pores formed from nine of the subunits set forth in SEQ ID NO:61 (with or without the maleimide crosslinker, both without cyclization) were also tested as described in Example 2. The results are shown in Figures 17-18.

Representative sequence: (SEQ ID NO: 1) CsgF-WT-del(S31-F119))-Ext(31-GGELAAKLWANGDETNALSLFQTIIQS) (ONLP20623)
GTMTFQFRNPNFGGNPNNGAFLLLNSAQAQNGGELAAKLWANGDETNALSLFQTIIQS
>(SEQ ID NO: 2) CsgF-WT-K37R-del(S31-F119)-Ext(31-GGELAAKLWANGDETNALSLFQTIIQS) (ONLP20624)
GTMTFQFRNPNFGGNPNNGAFLLLNSAQAQNGGELAARLWANGDETNALSLFQTIIQS
>(SEQ ID NO: 3) CsgF-WT-N24C/K37R-del(S31-F119)-Ext(31-GGELAAKLWANGDETNALSLFQTIIQSC) (ONLP20625)
GTMTFQFRNPNFGGNPNNGAFLLCSAQAQNGGELAARLWANGDETNALSLFQTIIQSC
>(SEQ ID NO: 4) Mat-CsgF-Eco-(WT-Del(S31-F119)-Ext(31-AGELAKKLWENGNVNQALSLFQTVIQS) (ONLZ19432, DGLONT76)
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAKKLWENGNVNQALSLFQTVIQS
>(SEQ ID NO: 5) Mat-CsgF-Eco-(WT-K36R/K37R-Del(S31-F119)-Ext(31-AGELAKKLWENGNVNQALSLFQTVIQS) (ONLZ19431)
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELARRLWENGNVNQALSLFQTVIQS
>(SEQ ID NO: 6) Mat-CsgF-Eco-(WT-N24C/K36R/K37R-Del(S31-F119)-Ext(31-AGELARRLWENGNVNQALSLFQTVIQSC) (ONLZ19781)
GTMTFQFRNPNFGGNPNNGAFLLCSAQAQNAGELARRLWENGNVNQALSLFQTVIQSC
>(SEQ ID NO: 7) ONT113_2
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAAELAAKLWANADETNALSLFQTIIQS
>(SEQ ID NO: 8) ONT113_3
GTMTFQFRNPNFGGNPNNGAFLLLNSAQAQNAAELAAKLWANADETNALSLFQTLIQS
>(SEQ ID NO: 9) ONT1
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAAKLFKKGDLTNALSLFQTVIQS
>(SEQ ID NO: 10) ONT2
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELVEKLFKNGDWTNAISIFQTVIQS
>(SEQ ID NO: 11) ONT3
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAEKLWRNGDETNALSLFQTVIQS
>(SEQ ID NO: 12) ONT4
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAEKLWKNGDETNALSLFQTVIQS
>(SEQ ID NO: 13) ONT5
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAKKLWENGDETNALSLFQTVVQS
>(SEQ ID NO: 14) ONT6
GTMTFQFRNPNFGGNPNNGAFLLLNSAQAQNAGELAEKLWRNGNESDALSLFQTVIQS
>(SEQ ID NO: 15) ONT7
GTMTFQFRNPNFGGNPNNGAFLLLNSAQAQNAGELAKKLFENGDKTNALSLFQTVIQS
>(SEQ ID NO: 16) ONT8
GTMTFQFRNPNFGGNPNNGAFLLLNSAQAQNAGELAKKLWENGDETNALSLFQTVIQS
>(SEQ ID NO: 17) ONT9
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAKKLWEKGNSEDALALFRTVVQS
>(SEQ ID NO: 18) ONT10
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAKKLFDNGDMENAMKLFQTVIAS
>(SEQ ID NO: 19) ONT11
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAEKLWRNGDKDRALALFRTVIQS
>(SEQ ID NO: 20) ONT12
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELADKLWKNGDKDRALSLFQTVIQS
>(SEQ ID NO: 21) ONT13
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAKKLFDNGDMDRALALFRTVIAS
>(SEQ ID NO: 22) ONT14
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAKKLFDNGNEEDALALFRTVVAS
>(SEQ ID NO: 23) ONT15
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAAKLWKKGDEENALKLFRTVVTS
>(SEQ ID NO: 24) ONT16
GTMTFQFRNPNFGGNPNNGAFLLLNSAQAQNAGELAAKLFKNGNMEDALKLFRTVIAS
>(SEQ ID NO: 25) ONT17
GTMTFQFRNPNFGGNPNNGAFLLLNSAQAQNAGKVAAILWKNGNKSDALSLFQTVVTS
>(SEQ ID NO: 26) ONT18
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAAKLFKNGDLTNALSLFQTVVQS
>(SEQ ID NO: 27) ONT19
GTMTFQFRNPNFGGNPNNGAFLLLNSAQAQNAGELGLKLLRKGDVETALTLFAQVISG
>(SEQ ID NO: 28) ONT20
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELGLKLILKGDLETALKLFAIVIAG
>(SEQ ID NO: 29) ONT21
GTMTFQFRNPNFGGNPNNGAFLLLNSAQAQNAGELGLKLLRKGDVETALKLFAIVIAG
>(SEQ ID NO: 30) ONT22
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAKKLYENGLIELALMLFALVIAS
>(SEQ ID NO: 31) ONT23
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELYKKLWDNGEVDKALDLFAKIIAG
>(SEQ ID NO: 32) ONT24
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELGKKLIEKGDLETALKLFAIVIAG
>(SEQ ID NO: 33) ONT25
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGEIALRLLKNGKEEEEALKTLLVTIAG
>(SEQ ID NO: 34) ONT26
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAAKLWKKGDETNALSLFQTVVTS
>(SEQ ID NO: 35) ONT27
GTMTFQFRNPNFGGNPNNGAFLLLNSAQAQNAGKVAAILWKNGNKSDALSLFQTVVTS
>(SEQ ID NO: 36) ONT28
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAKKLWEKGDETNALSLFQTVVTS
>(SEQ ID NO: 37) ONT29
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGDLAAKLWKKGDETNALSLFQTVVTS
>(SEQ ID NO: 38) ONT30
GTMTFQFRNPNFGGNPNNGAFLLLNSAQAQNAGELAAKLWKNGNSSDALSLFQTVVTS
>(SEQ ID NO: 39) ONT31
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAKKLWEKGDETNALSLFQTVVTS
>(SEQ ID NO: 40) ONT32
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAKKLWEKGDSSNALSLFQTVVTS
>(SEQ ID NO: 41) ONT33
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGDLAAKLWKNGDETNALSLFQTVVTS
>(SEQ ID NO: 42) ONT34
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAAKLFKNGDLTNALSLFQTVVQS
>(SEQ ID NO: 43) ONT35
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAAKLWKKGDETNALSLFQTVVTS
>(SEQ ID NO: 44) ONT36
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAAKLFNSGDLDRALALFRTVVTS
>(SEQ ID NO: 45) ONT37
GTMTFQFRNPNFGGNPNNGAFLLLNSAQAQNAGKVAKELYDNGDEKWALLFRTVVTS
>(SEQ ID NO: 46) ONT38
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGKVAAELYKNGDEKNALLFRTVAS
>(SEQ ID NO: 47) ONT39
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAAKLFKNGDMENALALFRTVVTS
>(SEQ ID NO: 48) ONT40
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAKKLWEKGNSEDALALFRTVVQS
>(SEQ ID NO: 49) ONT41
GTMTFQFRNPNFGGNPNNGAFLLLNSAQAQNAGELAAKLFNKGDEDRALALFRTVVQS
>(SEQ ID NO: 50) ONT42
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAAKLWKNGDEENALALFRTVVTS
>(SEQ ID NO: 51) ONT43
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAEKLWRSGDADRALALFRTVVTS
>(SEQ ID NO: 52) ONT44
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAAKLWKNGNEEDALALFRTVVTS
>(SEQ ID NO: 53) ONT45
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAAKLFNNGDEDRALALFRTVVQS
>(SEQ ID NO: 54) ONT46
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAAKLWKKGDEDRALALFRTVVTS
>(SEQ ID NO: 55) ONT47
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAAKLFNSGDEDRALALFRTVVQS
>(SEQ ID NO: 56) ONT48
GTMTFQFRNPNFGGNPNNGAFLLLNSAQAQNAGELAAKLYNNGDLDRADATFRTVVQS
>(SEQ ID NO: 57) ONT49
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGELAKKLWENGNEEDALALFRTVVTS
>(SEQ ID NO: 58) ONT50
GTMTFQFRNPNFGGNPNNNGAFLLNSAQAQNAGEIAKQLWEKGDESSAITVATIVLSS
(SEQ ID NO: 59) Wild-type E. coli CsgG protein monomer (without signal sequence)
CLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQFKPYPASNFSTAVPQSAT AMLVTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIMVEGSIIGYE SNVKSGGVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSSVNTSKTILSYEVQAGVFRFIDY QRLLEGEVGYTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPES
(SEQ ID NO: 60) Residues 1-30 of the CsgF peptide
GTMTFQFRNPNFGGNPNNGAFLLLNSAQAQN
>(SEQ ID NO: 61) CsgF-WT-del(S31-F119)-Ext(31-AGILAAQLWNNGDYDRALSLFIAVVQS-57)GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGILAAQLWNNGDYDRALSLFIAVVQS

Claims (61)

1. A protein nanopore complex comprising:
(a) a CsgG nanopore comprising a lumen;
(b) a fusion polypeptide comprising a first portion comprising a CsgF protein and a second portion comprising a helix-forming assisting protein, wherein the fusion protein is bound to the nanopore. The protein nanopore complex of claim 1, wherein the first portion of the fusion protein is bound to the CsgG nanopore. The protein nanopore complex of claim 1 or 2, wherein the first portion of the fusion protein is located within the lumen of the CsgG nanopore. 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. The protein nanopore complex of any one of claims 1 to 4, wherein the first portion forms a first constriction region in the lumen of the CsgG nanopore. The protein nanopore complex of claim 5, wherein the second portion forms a second constriction region. The protein nanopore complex of any one of claims 1 to 6, wherein the CsgG nanopore further comprises a constriction region. The protein nanopore complex of any one of claims 1 to 7, wherein the second portion is not bound to the CsgG nanopore. The protein nanopore complex of any one of claims 1 to 8, wherein the second portion comprises one or more alpha helices. The protein nanopore complex of any one of claims 1 to 9, wherein each of the alpha helices contains between 0 and 15 alpha helix turns. The protein nanopore complex of any one of claims 1 to 10, wherein the second portion comprises a first alpha helix comprising one to four alpha helix turns and a second alpha helix comprising three to six alpha helix turns. The protein nanopore complex of claim 11, wherein the second alpha helix is packed against the first alpha helix. The protein nanopore complex described in any one of claims 9 to 12, wherein the second portion comprises between 1 and 55 amino acid residues. The protein nanopore complex described in any one of claims 6 to 13, wherein the distance between the first constriction region and the second constriction region is in the range of about 5 Å to about 80 Å. The protein nanopore complex of any one of claims 1 to 14, wherein the protein nanopore complex has an axial length of greater than 90 Å, and optionally, the axial length is in the range of about 95 Å to about 160 Å. The protein nanopore complex described in any one of claims 1 to 15, wherein the fusion protein is bound to the nanopore by a linker. The protein nanopore complex of claim 16, wherein the linker comprises a bond, a peptide linker, or a chemical linker. The protein nanopore complex of claim 16 or 17, wherein the linker comprises a bond formed by a sulfur(VI) fluoride exchange (SuFEx) reaction. The protein nanopore complex of claim 16 or 17, wherein the linker comprises one or more maleimide molecules. The protein nanopore complex described in any one of claims 1 to 19, wherein the fusion protein is cyclized. The protein nanopore complex of claim 20, wherein the cyclization comprises one or more side chain-to-side chain cyclization bonds. The protein nanopore complex of claim 21, wherein at least one of the side chain-to-side chain cyclization bonds is a disulfide bond. 1. A protein nanopore complex comprising:
(a) a CsgG nanopore comprising a lumen and a first constriction region formed within the lumen of the nanopore;
(b) a fusion protein comprising a first portion comprising a CsgF protein and a second portion comprising a helix-forming assisting protein, wherein the fusion protein is bound to the nanopore. The protein nanopore complex of claim 23, wherein the first portion of the fusion protein is bound to the CsgG nanopore. The protein nanopore complex of claim 23 or 24, wherein the first portion of the fusion protein is located within the lumen of the CsgG nanopore. 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. The protein nanopore complex of any one of claims 23 to 26, wherein the first portion forms a second constriction region in the lumen of the CsgG nanopore. The protein nanopore complex of claim 27, wherein the second portion forms a third constriction region in the lumen of the CsgG nanopore. The protein nanopore complex of any one of claims 23 to 28, wherein the second portion is not bound to the CsgG nanopore. The protein nanopore complex of any one of claims 23 to 29, wherein the second portion comprises one or more alpha helices. The protein nanopore complex of claim 30, wherein each of the alpha helices contains between 0 and 15 alpha helix turns. The protein nanopore complex described in any one of claims 23 to 31, wherein the second portion comprises between 1 and 55 amino acid residues. The protein nanopore complex described in any one of claims 23 to 32, wherein the fusion protein is cyclized. The protein nanopore complex of claim 33, wherein the cyclization comprises one or more side chain-to-side chain cyclization bonds. The protein nanopore complex of claim 34, wherein at least one of the side chain-to-side chain cyclization bonds is a disulfide bond. 1. A protein nanopore complex comprising:
(a) a CsgG nanopore comprising a lumen and a first constriction region formed within the lumen of the nanopore;
(b) a first accessory protein bound to the CsgG nanopore and forming a second constriction region in the lumen of the nanopore;
(c) a second accessory protein bound to the CsgG nanopore or the first accessory protein and forming a third constriction region. The protein nanopore complex of claim 36, wherein the first auxiliary protein is located within the lumen of the CsgG nanopore. The protein nanopore complex of claim 36 or 37, wherein the first auxiliary protein comprises a CsgF protein or a CsgF peptide. The protein nanopore complex described in any one of claims 36 to 38, wherein the second auxiliary protein comprises one or more alpha helices. The protein nanopore complex of claim 39, wherein each of the one or more alpha helices contains between 0 and 15 alpha helix turns. The protein nanopore complex of claim 39 or 40, wherein the second auxiliary protein comprises two alpha helices. The protein nanopore complex of claim 41, wherein one of the alpha helices contains between 1 and 6 alpha helix turns. The protein nanopore complex of claim 41 or 42, wherein one of the alpha helices contains between 1 and 10 alpha helix turns. A protein nanopore complex according to any one of claims 41 to 43, wherein one of the alpha helices contains three alpha helix turns and the other alpha helix contains three or four alpha helix turns. The protein nanopore complex of any one of claims 36 to 44, wherein the second auxiliary protein comprises at least one alpha helix that packs against an alpha helix of the first auxiliary protein. The protein nanopore complex described in any one of claims 36 to 45, wherein the second auxiliary protein contains between 1 and 55 amino acid residues. The protein nanopore complex described in any one of claims 36 to 46, wherein the distance between the first constriction region and the second constriction region is in the range of about 10 Å to about 80 Å. The protein nanopore complex described in any one of claims 36 to 47, wherein the distance between the second constriction region and the third constriction region is in the range of about 5 Å to about 80 Å. The protein nanopore complex of any one of claims 36 to 48, wherein the protein nanopore complex has an axial length of greater than 90 Å, and optionally, the axial length is in the range of about 95 Å to about 160 Å. The protein nanopore complex described in any one of claims 36 to 49, wherein the first auxiliary protein and the second auxiliary protein are linked by a linker. The protein nanopore complex of claim 50, wherein the linker comprises a bond, a peptide linker, or a chemical linker. The protein nanopore complex of claim 50 or 51, wherein the linker comprises a bond formed by a sulfur(VI) fluoride exchange (SuFEx) reaction. The protein nanopore complex of claim 50 or 51, wherein the linker comprises one or more maleimide molecules. The protein nanopore complex described in any one of claims 36 to 53, wherein the first auxiliary protein and the second auxiliary protein contain one or more side chain-to-side chain cyclization bonds. The protein nanopore complex of claim 54, wherein at least one of the side chain-to-side chain cyclization bonds is a disulfide bond. A system for characterizing a target analyte, the system comprising a protein nanopore complex described in any one of claims 1 to 55 inserted into a membrane. The system of claim 56, further comprising a conductive solution in contact with the protein nanopore complex, electrodes that provide a voltage potential across the membrane, and a measurement system that measures the current through the protein nanopore complex. A method for characterizing a target analyte, the method comprising: (a) contacting the target analyte with the system described in claim 56; (b) applying a potential across the membrane such that the target analyte moves relative to the lumen formed by the protein nanopore complex; and (c) performing one or more measurements as the target analyte moves relative to the lumen, thereby characterizing the target analyte. The method of claim 58, wherein the target analyte comprises a target polynucleotide. The method of claim 58 or 59, wherein step (c) comprises measuring a current through the continuous channel, the current indicating the presence and/or one or more properties of the target analyte, thereby detecting and/or characterizing the target analyte. The method of any one of claims 58 to 60, wherein the target analyte is a polynucleotide, nucleotides in the polynucleotide interact with the first, second, and optionally, third constriction regions in the lumen, and each of the first, second, and optionally, third constriction regions can distinguish between different nucleotides such that the overall current through the lumen is affected by interactions between each of the first, second, and third constriction regions and the nucleotides located in each of the regions.