KR20250048551A - New pore - Google Patents

Abstract

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

Description

New pore

Two important components of polymer characterization using nanopore sensing are (1) control of polymer movement through the pore and (2) discrimination of the constituent building blocks as the polymer moves through the pore. During nanopore sensing, the narrowest portion of the pore forms the constriction, which is the most discriminative portion of the nanopore with respect to the current signature as a function of the analyte passing through it. CsgG was identified as a non-gated, non-selective protein secretion channel from Escherichia coli (Goyal et al., 2014) and has been used as a nanopore to detect and characterize analytes. In this context, mutations in the wild-type CsgG pore that improve the properties of the pore have also been disclosed (WO2016/034591, WO2017/149316, WO2017/149317 and WO2017/149318, PCT/GB2018/051191, all of which are herein incorporated by reference in their entirety).

For analytes that are polynucleotides, nucleotide discrimination is achieved via passage through these mutant pores, but the current signature appears to be sequence dependent, with multiple nucleotides contributing to the observed current, such that the height of the channel constriction and the extent of the interaction surface with the analyte influence the relationship between the observed current and the polynucleotide sequence. While mutations in the CsgG pore improve the current range for nucleotide discrimination, the sequencing system would perform better if the current differential between nucleotides could be further improved.

In some aspects, the present disclosure relates to protein pore complexes and their use in analyte detection and characterization. The present disclosure is based in part on a nanopore complex formed by a CsgG pore and one or more accessory proteins, which form one or more channel constrictions in the nanopore complex. In some embodiments, the one or more accessory proteins are fusion proteins. As further described in the Examples, it has surprisingly been discovered that, using computational structural analysis tools, accessory proteins can be de novo designed that impart certain desirable features to a CsgG protein nanopore (e.g., tuning the pore width, elongating the pore lumen, forming one or more additional constrictions, etc.). In some embodiments, the de novo designed accessory proteins (e.g., fusion proteins) form one or more constrictions in the lumen of the CsgG nanopore and improve discrimination of polymer units as an analyte moves through the nanopore.

Certain aspects of the present disclosure further relate to methods of designing accessory proteins and forming nanopore complexes and their use in molecular sensing and nucleic acid sequencing applications.

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

In some embodiments, the first portion of the fusion protein is attached to a CsgG nanopore. In some embodiments, the first portion of the fusion protein is positioned 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 additionally comprises a constriction region.

In some embodiments, the second moiety is not attached to a CsgG nanopore. In some embodiments, the second moiety comprises one or more helices (e.g., an alpha helix, etc.).

In some embodiments, each helix (e.g., an alpha helix, etc.) of the second portion comprises 0 to 15 alpha helical turns. In some embodiments, the second portion comprises a first alpha helix comprising 1 to 4 alpha helical turns and a second alpha helix comprising 3 to 6 alpha helical turns. In some embodiments, the second alpha helix is packed into the first alpha helix. In some embodiments, the second portion comprises 1 to 55 amino acid residues. In some embodiments, each helix 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 helix 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., vertical distance) between the first constriction region and the second constriction region is in the range of about 5 (e.g., as measured as the distance between the alpha carbon (C _a ) of the amino acid residue extending furthest into the lumen of the nanopore forming the first constriction and the amino acid residue extending furthest into the lumen of the nanopore forming the second constriction). About 80 In some embodiments, the protein nanopore complex is 90 It has an excess shaft length, optionally with a shaft length range of about 95 About 160 am.

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 embodiments, the present disclosure provides a protein nanopore complex comprising a CsgG nanopore comprising a lumen and a first constriction region formed within the lumen of the nanopore; and a fusion protein comprising a first portion comprising a CsgF protein and a second portion comprising a helix-forming accessory protein, wherein the fusion protein is attached to the nanopore.

In some embodiments, the first portion of the fusion protein is attached to a CsgG nanopore. In some embodiments, the first portion of the fusion protein is positioned 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 attached to the CsgG nanopore.

In some embodiments, the second portion comprises one or more helices (e.g., alpha helices, etc.). In some embodiments, each helix (e.g., alpha helices) comprises 0 to 15 alpha helical turns. In some embodiments, the second portion comprises 1 to 54 amino acid residues. In some embodiments, each helix 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 helix 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-terminal) cyclization bonds. In some embodiments, at least one of the cyclization bonds is a disulfide bond.

In some embodiments, the present disclosure provides a protein nanopore complex comprising a CsgG nanopore comprising a lumen and a first constriction region formed within the lumen of the nanopore; a first accessory protein attached to the CsgG nanopore to form a second constriction region within the lumen of the nanopore; and a second accessory protein attached to either the CsgG nanopore or the first accessory protein to form 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 peptide.

In some embodiments, the second accessory protein comprises one or more helices (e.g., alpha helices, etc.). In some embodiments, each of the one or more helices (e.g., alpha helices) comprises from 0 to 15 alpha helical turns. In some embodiments, the second accessory protein comprises two alpha helices.

In some embodiments, one of the alpha helices comprises 1 to 6 alpha helical turns. In some embodiments, one of the alpha helices comprises 1 to 10 alpha helical turns. In some embodiments, one of the alpha helices comprises 3 alpha helical turns and the other alpha helices comprise 3 or 4 alpha helical turns. In some embodiments, each helix 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 helix 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 second accessory protein comprises at least one alpha helix that is packed into an alpha helix of the first accessory protein. In some embodiments, the second accessory protein comprises 1 to 55 amino acid residues.

In some embodiments, the distance (e.g., vertical distance) between the first constriction and the second constriction is in the range of about 20 (e.g., as measured as the distance between the alpha carbon (C _a ) of the amino acid residue extending furthest into the lumen of the nanopore forming the first constriction and the amino acid residue extending furthest into the lumen of the nanopore forming the second constriction). About 80 In some embodiments, the distance between the second constriction and the third constriction is in the range of about 5 About 80 In some embodiments, the protein nanopore complex is 90 It has an excess shaft length, optionally with a shaft length range of about 95 About 160 am.

In some embodiments, the first accessory protein and the second accessory protein are attached 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 the linker can be covalently or noncovalently attached to a second amino acid of the linker, e.g., by a cross-linking agent).

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

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

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

In some embodiments, the present disclosure provides a method for characterizing a target analyte, comprising: contacting a system as described herein with the target analyte; applying a potential across a membrane to cause the target analyte to enter a lumen formed by a protein nanopore complex; and performing one or more measurements as the target analyte moves about the lumen, thereby characterizing the target analyte.

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

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

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

In some aspects, the present disclosure provides a method of preparing a protein nanopore complex, wherein the protein nanopore complex comprises:

(a) CsgG nanopores comprising an inner lumen; and

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

Figures 1a-1c illustrate the workflow for the de novo design of fusion proteins. Figure 1a illustrates the design workflow using the CsgG nanopore. Wild-type CsgF (residues 1-35; left panel) is shown in orange. Residues 17-30 of wild-type CsgF (red) are packed and projected onto the pore to obtain a diameter of 10 30 inland Geometrically aligned and designable helices that generate novel constrictions (cyan) were selected as targets for searching. Two helices were looped (yellow) and sequence design of the generated backbone was performed via Rosetta. Figure 1b shows helix-helix interactions with symmetry-related partners. Figure 1c shows a planar view of the nine-membered CsgG-fusion protein complex demonstrating the additional constriction achieved by the newly designed fusion protein.
Figure 2 shows representative data for prioritization of novel fusion protein sequences designed using Rosetta. Sequences for experimental validation were selected based on the lowest energy score and highest PackStat score.
Figures 3a to 3d show PSIPRED protein secondary structure analysis based on amino acid sequences for the newly designed fusion proteins. Residues are shaded according to whether they are predicted as strands, helices, and coils, respectively. Figure 3a shows the secondary structure prediction of the mature sequence of the fusion proteins and wild-type CsgF. Figure 3b shows the secondary structure analysis for the newly designed fusion proteins ONT1 to ONT10. Figure 3c shows the secondary structure analysis for the newly designed fusion proteins ONT11 to ONT20. Figure 3d shows the protein secondary structure analysis for the newly designed fusion proteins ONT21 to ONT25.
Figures 4a to 4c show predicted three-dimensional structures of alternative sequences for the newly designed fusion proteins. Figure 4a shows the predicted structure for the newly designed fusion proteins ONT1 to ONT10. Figure 4b shows the predicted structure for the newly designed fusion proteins ONT11 to ONT20. Figure 4c shows the predicted structure for the newly designed fusion proteins ONT21 to ONT25.
Figure 5 shows representative SDS-PAGE gel analyses of CsgG-only pore and CsgG/fusion protein complexes, wherein the complexes include CsgF-del(S31-F119) control or the novel designed fusion proteins, with or without maleimide cross-linker. Complexes including the fusion proteins show a band shift, indicating that these samples are pore complexes. Samples were not heated prior to loading onto the gel.
Figure 6 shows representative SDS-PAGE gel analyses of CsgG-only pore and CsgG/fusion protein complexes, wherein the complexes contain CsgF-del(S31-F119) control or the newly designed fusion proteins, with or without maleimide cross-linker. The pore was immediately disassembled into its constituent monomeric components upon boiling in the presence of DTT prior to loading onto the gel.
Figure 7 shows representative ionic current (pA) versus time (s) traces 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 entire DNA current trace, and the bottom row shows an enlarged view of the first section of the current trace.
Figure 8 shows representative ion current (pA) versus time (s) traces when single-stranded DNA is translocated through CsgG containing the del(S31-F119) CsgF peptide, with or without a maleimide crosslinker.
Figure 9 shows representative ion current (pA) versus time (s) traces when single-stranded DNA is translocated through CsgG containing the novel designed fusion protein in the absence of a maleimide cross-linker.
Figure 10 shows representative ion current (pA) versus time (s) traces when single-stranded DNA is translocated through CsgG containing the novel designed fusion protein +/- maleimide crosslinks.
Figure 11 shows representative ion current (pA) versus time (s) traces when single-stranded DNA is translocated through CsgG containing the novel designed fusion proteins, with or without a maleimide crosslinker. The fusion proteins include a K37R mutation with a cysteine residue, which forms an internal disulfide bond within the peptide, i.e., cyclization of the fusion protein.
Figure 12 shows representative profiles demonstrating their contribution to the overall change in position and ionic current level within the pore (“discrimination”) as DNA molecules translocate through the pore. The CsgG-only pore (+/- Q153C) shows one major discrimination peak at position 0.
Figure 13 shows representative profiles demonstrating their contribution to the overall change in ion current level (“discrimination”) and position within the pore as a DNA molecule translocates through the pore. Dashed boxes indicate regions affected by introduction of the novel designed fusion proteins. CsgG-CsgF-del(S31-F119) pores, with or without the maleimide crosslinker, exhibit two discriminant peaks: a major discriminant peak at position 0 as seen in the CsgG-only pore, and an additional discriminant peak 4-6 nucleotides below the major constriction (positions -4 to -6). This additional discriminant region has less of an effect on ion current than the major discriminant peak at position 0.
Figure 14 shows representative profiles demonstrating their contribution to the overall change in ion current level ("discrimination") and position within the pore 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 is a novel Indicates the region affected by introduction of the designed fusion protein. Complexes consisting of the newly designed fusion protein containing CsgG and K37R (with or without maleimide crosslinker; with cyclization) exhibit three discriminant peaks. As seen in the CsgG-only pore, the main discriminant peak is at position 0, with additional peaks at positions -6 and -9. The peak at position -9 corresponds to the expected constriction created by the newly designed fusion protein when folded in the correct orientation.
Figure 15 shows an example of two proteins linked by a maleimidopropionic acid linker.
Figure 16 shows an example of a pore protein and an adjuvant (e.g., a fusion protein) functionalized with a reactive modifier, such as a thiol modifier.
Figure 17 shows representative ionic current (pA) versus time (s) traces when single-stranded DNA is translocated through CsgG comprising the novel designed fusion protein (SEQ ID NO: 61) with or without (bottom two traces) a maleimide crosslinker (top two traces). The raw current traces are shown as black lines, and the event detection signals are shown as red lines. For each pore, the top row shows the entire DNA current trace, and the bottom row shows an enlarged view of the first section of the current trace.
Figure 18 shows representative profiles demonstrating their contribution to the overall change in ion current level (“discrimination”) and positions within the pore 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 introduction of the novel designed fusion protein. Three discriminant peaks are shown for complexes consisting of CsgG and the novel designed fusion protein (SEQ ID NO: 61) with (bottom profile) or without (top profile) a maleimide crosslinker; both without cyclization. As seen in the CsgG-only pore, the major discriminant peak is at position 0 with additional peaks at positions -5 and -11. The peak at position -11 corresponds to the expected constriction created by the novel designed fusion protein when folded in the correct orientation.
Figure 19 shows the structure and dimensions of the wild-type CsgG pore of E. coli strain K12 (the databank accession code for this structure is 4UV3). The distances shown are measured from backbone to backbone of the amino acids forming the pore structure. The CsgG pore is a tightly interconnected symmetrical nine-membered pore resembling a crown. The overall height is 98 , the maximum outer diameter is 120 It defines the central channel and consists of three parts: (A) the cap region, (B) the constriction region, and (C) the transmembrane beta-barrel region. The cap axis length or height is 39 It is. The inner diameter is 43 And the entrance is 66 The beta barrel has 36 strands and the axis length is 39 , inner diameter is 55 The transition between the fore cap and the beta barrel is abrupt, with a constriction located between them at the predicted lipid-aqueous interface level. The constriction has a diameter of about 18.5 and along the channel axis 20 Indicates the length of .

Aspects of the present disclosure relate to compositions and methods for characterizing analytes using nanopore-based systems. The present disclosure is based in part on a protein nanopore complex formed by a CsgG pore and one or more accessory proteins, which form one or more channel constrictions in the nanopore complex. In some embodiments, the one or more accessory proteins are fusion proteins. As further described in the Examples, it has surprisingly been discovered that, using computational structural analysis tools, accessory proteins can be de novo designed that impart certain desirable features to a CsgG nanopore (e.g., tuning the pore width, elongating the pore lumen, forming one or more additional constrictions, etc.). In some embodiments, the de novo designed accessory proteins (e.g., fusion proteins) form one or more additional constrictions in the lumen of the CsgG nanopore and improve discrimination of polymer units as an analyte moves through the nanopore.

Auxiliary protein

The protein nanopore complexes (also referred to interchangeably as protein pore complexes) described herein may comprise one or more accessory proteins. The terms "peptide,""polypeptide," or "protein," as used herein, are used interchangeably herein and refer to two or more amino acids joined together by peptide bonds. In some embodiments, the protein (also referred to as a polypeptide or peptide) comprises from 2 to 2000 amino acids. In some embodiments, the 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, the protein comprises more than 2000 amino acids. In some embodiments, the peptide, polypeptide or protein is synthetic in origin (e.g., not existing in nature, e.g., not naturally expressed in any living organism). In some embodiments, the peptide, polypeptide, or protein occurs naturally (e.g., is expressed naturally in a living organism that has not been genetically modified to express the peptide, polypeptide, or protein). In some embodiments, the peptide, polypeptide, or protein can be naturally expressed by the organism. In some embodiments, the peptide, polypeptide, or protein is heterologously expressed by the 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, or the like). The peptide, polypeptide, or protein can comprise 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 one or more naturally occurring amino acids and one or more non-naturally occurring amino acids.

In some embodiments, the accessory protein is a fusion protein. The term "fusion protein" refers to a naturally occurring synthetic, semi-synthetic, or recombinant single protein molecule comprising all or portions of two or more heterologous polypeptides (e.g., polypeptides that are heterologous to one another) joined by peptide bonds. In some embodiments, the fusion protein comprises all or portions of at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 heterologous polypeptides joined by peptide bonds. As used herein, "portion of a peptide" refers to two or more amino acids of a peptide. In some embodiments, a portion of a peptide comprises the complete amino acid sequence of the peptide or at least 5, 10, 20, 30, 50 or 100 amino acids, contiguous or including gaps, of the entire amino acid sequence of the peptide (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 or 100 amino acids). The portions of the fusion protein can 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 the first portion can be linked (e.g., connected) to the N-terminus of the second portion. The portions of the fusion protein can be directly linked (e.g., an amino acid of one portion can be directly linked to an amino acid of the second portion, e.g., via a peptide bond between the terminal amino acids of the portions) or indirectly linked (e.g., an amino acid of one portion can 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 accessory protein is the first portion of the fusion protein and the second accessory protein is the second portion of the fusion protein. Linking of fusion protein portions 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 perforation (also referred to as the "lumen" of the nanopore). Formation of protein nanopores 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 arranged 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 monomers (e.g., CsgG pore monomers) to accessory 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 accessory proteins or the one or more fusion proteins can have the same symmetry as the nanopore. For example, if the nanopore comprises eight monomers around a central axis, then eight accessory proteins (or eight fusion proteins) are present, if the nanopore comprises nine monomers around a central axis, then nine accessory proteins (or nine fusion proteins) are present, and so on. In some embodiments, the one or more accessory proteins (or the one or more fusion proteins) may comprise more or fewer, for example, one more or one less, monomers than the nanopore.

The lumen of a nanopore or protein nanopore complex may have one or more constrictions. The terms "constriction," "pore," "constriction region," "channel constriction," or "constriction region," as used interchangeably herein, refer to an opening defined by the lumenal surface of a pore or protein pore complex that acts to allow passage of ions and target molecules (e.g., but not limited to, polynucleotides or individual nucleotides) but prevent other nontarget molecules from passing through the pore or protein pore complex channel. A constriction is typically the narrowest opening within a pore or protein pore complex or the narrowest hole within a channel defined by a pore or pore complex. A constriction may serve to restrict passage of molecules through the pore. The size of the constriction is typically a key factor in determining the suitability of a pore or pore complex for analyte characterization. If the constriction is too small, the molecules to be characterized will not be able to pass through. However, to achieve maximum effect on ion flow through the channel, each constriction should not be too large. For example, each constriction should not be wider than the solvent-accessible transverse diameter of the target analyte. Ideally, each constriction should be as close as possible to the transverse diameter of the analyte passing through it.

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

The narrowest point of the central cavity or perforation 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 minimum diameter of a constriction (e.g., a constriction formed by a portion of the CsgG protein, a constriction formed by an accessory protein, a constriction formed by a fusion protein, etc.) is in the range of about 0.5 nm to about 4.0 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, for example 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 (e.g., as measured by C _a to C _a ). About 30 , for example 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 or 30 In some embodiments, the minimum diameter of the constriction is in the range of about 10 (e.g., as measured by C _a to C _a ). About 30 In some embodiments, the minimum diameter of the stenosis is in the range of about 15 (e.g., as measured by C _a to C _a ). About 25 am.

The distance between one or more constrictions in the lumen of the protein pore complex can vary. In some embodiments, the distance between the first constriction region and the second constriction region is in the range of about 5 About 80 In some embodiments, the distance between the first constriction region and the second constriction region is about 5 in length. , 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 or 80 In some embodiments, the distance between the first constriction region and the second constriction region is 80 in length. Excess (e.g. 90 , 100 etc.)

In some embodiments, the distance between the second stenosis region and the third stenosis region is in the range of about 5 About 80 In some embodiments, the distance between the first constriction region and the second constriction region is about 5 in length. , 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 or 80 In some embodiments, the distance between the second constriction region and the third constriction region is 80 in length. Excess (e.g. 90 , 100 etc.)

In some embodiments, the distance between the first stenosis region and the third stenosis region is in the range of about 10 About 160 In some embodiments, the distance between the first constriction region and the second constriction region is about 10 in length. , 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 , 118 , 119 , 120 , 121 , 122 , 123 , 124 , 125 , 126 , 127 , 128 , 129 , 130 , 131 , 132 , 133 , 134 , 135 , 136 , 137 , 138 , 139 , 140 , 141 , 142 , 143 , 144 , 145 , 146 , 147 , 148 , 149 , 150 , 151 , 152 , 153 , 154 , 155 , 156 , 157 , 158 , 159 or 160 In some embodiments, the distance between the first stenosis region and the third stenosis region is 160 in length. Excess (e.g. 190 , 200 etc.)

The accessory protein (or fusion protein) can, in some embodiments, be modified from its native state to provide a constriction having a desired minimum diameter. For example, the accessory protein can be modified by introducing one or more bulky residues by targeted mutation to create a constriction having a minimum diameter within the ranges specified above. The maximum height of the accessory protein is, in one embodiment, from about 3 nm to about 20 nm, such as from about 4 nm to about 10 nm. In one embodiment, the length of the channel in the accessory protein is from about 3 nm to about 20 nm, such as from about 4 nm to about 10 nm. The height is the size of the accessory protein in the direction perpendicular to the membrane.

In some embodiments, the accessory protein (e.g., the first accessory protein or the second accessory protein) or the fusion protein (e.g., the first portion of the fusion protein or the second portion of the fusion protein) extends outside the lumen of the protein pore complex. The accessory protein or fusion protein can extend outside the lumen of the protein pore complex, either cis-side or trans-side (e.g., when the protein pore complex is inserted into a membrane). In some embodiments, the distance that the accessory protein or fusion protein extends outside the lumen of the protein pore complex is calculated by measuring the distance between the C a of the amino acid residue of the accessory protein or fusion protein that extends furthest outside the lumen and a reference amino acid of the protein pore (e.g., a CsgG pore), e.g., amino acid residue Phe144 or Tyr196 of a wild-type CsgG monomer. In some embodiments, the accessory protein or fusion protein has a C _a of about 0 About 50 extends outside the lumen by about 5. In some embodiments, the accessory protein or fusion protein is about About 30 extends outside the lumen by about 10. In some embodiments, the accessory protein or fusion protein is About 25 extends about 1 degree outside the lumen. In some embodiments, the accessory protein or fusion protein extends about 1 degree outside the lumen. , 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 or about 50 It is extended by that much.

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

In some embodiments, the accessory protein or fusion protein comprises one or more positively charged amino acids, such as arginine, lysine, or histidine, or an aromatic amino acid, such as tyrosine or tryptophan, positioned at or near a constriction formed by the accessory protein or fusion protein (e.g., within about 1, 2, 3, 4, or 5 nm of the constriction). In some embodiments, the accessory protein or fusion protein comprises one or more polar amino acids, negative amino acids, or hydrophobic amino acids positioned at or near a constriction formed by the accessory 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 positioned at or near a constriction formed by the accessory protein or fusion protein (e.g., within about 1, 2, 3, 4, or 5 nm of the constriction) are asparagine, threonine, serine, or glutamate. Such amino acids typically facilitate interactions between the pore and the polynucleotide.

The location of one or more of the accessory proteins (or one or more fusion proteins) of the protein pore complex can vary. In some embodiments, the accessory protein (or fusion protein) is positioned entirely within the lumen of the protein pore complex. In some embodiments, the accessory protein or fusion protein comprises a portion that extends beyond the lumen of the protein pore complex, e.g., a portion that extends above the lumen of the protein pore complex (e.g., a portion that extends above the cap region on the cis side of the protein pore complex) and/or a portion that extends below the protein pore complex (e.g., a portion that extends below a transmembrane domain (e.g., barrel) on the trans side of the protein pore complex). In some embodiments, the accessory protein or fusion protein (or a portion of the accessory protein or fusion protein, such as the first portion or the second portion) is attached to a nanopore (e.g., a CsgG nanopore). In some embodiments, the accessory protein or fusion protein (or a portion thereof) is covalently attached to the nanopore. In some embodiments, the assistant protein or fusion protein (or portion thereof) is noncovalently attached to the nanopore. In some embodiments, the first assistant protein and the second assistant protein are attached to each other (e.g., covalently attached, noncovalently attached, etc.). In some embodiments, the first portion of the fusion protein and the second portion of the fusion protein are attached to each other (e.g., covalently attached, noncovalently attached, etc.). In some embodiments, the assistant protein or fusion protein (or portion of the assistant protein or fusion protein, e.g., the first portion or the second portion) is not attached to the nanopore (e.g., a CsgG nanopore). In some embodiments, the first assistant protein and the second assistant protein are not attached to each other.

In some embodiments, the accessory protein (e.g., the first accessory protein) is not CsgF or a CsgF peptide or a functional homolog, fragment or modified version thereof. In some embodiments, neither portion (e.g., the first portion and/or the second portion) of the fusion protein is CsgF or a CsgF peptide or a functional homolog, fragment or modified version thereof. In some embodiments, the accessory protein is not a CsgG nanopore, or a homolog, fragment or modified version thereof. In some embodiments, neither portion (e.g., the first portion and/or the second portion) of the fusion protein is a CsgG nanopore or a homolog, fragment or modified version thereof.

In some embodiments, the accessory protein is not a polynucleotide binding protein. In some embodiments, the accessory protein is not a functional polynucleotide binding protein. For example, the accessory protein is not a polynucleotide binding protein having enzymatic activity. In some embodiments, the accessory protein can be a protein other than a nucleic acid processing enzyme, for example, an accessory protein other than a helicase or a polymerase, or a protein derived from such an enzyme. In some embodiments, the accessory protein does not have enzymatic activity. In some embodiments, the accessory protein does not undergo a structural change when a target analyte passes through the continuous channel formed in the protein pore complex.

In some embodiments, the accessory protein or fusion protein (e.g., portion of a fusion protein) is a component of the nanopore system other than a component forming a transmembrane pore, or a modified component of such a system. An example of such a component is CsgF or a truncated version of CsgF. In some embodiments, the accessory protein or fusion protein comprises a CsgF protein or a homolog or modified version thereof, such as a fragment. In some embodiments, the pore complex comprises a CsgF protein or peptide and a non-CsgG pore, a homolog or modified version thereof, such as a fragment.

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

In some embodiments, the CsgF peptide is a truncated CsgF peptide lacking the C-terminal head; a truncated CsgF peptide lacking the C-terminal head and a portion of the neck domain of CsgF (e.g., the truncated CsgF peptide can comprise only a portion of the neck domain of CsgF); or a truncated CsgF peptide lacking 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 comprise a portion of the neck domain, for example, from amino acid residues 36 at the N-terminal end of the neck domain (e.g., residues 36-40, 36-41, 36-42, 36-43, 36-45, 36-46 to 36-50 or 36-60 of wild-type E. coli CsgF). In some embodiments, the CsgF peptide comprises a CsgG binding region and a region that forms a constriction in the lumen of the pore. The CsgG binding region typically comprises residues 1 to 11 and/or 29 to 32 of the CsgF protein (e.g., wild-type E. coli CsgF or a homolog of another species) and may comprise one or more modifications. The region that forms a constriction in the pore typically comprises residues 9 to 28 of the CsgF protein (e.g., wild-type E. coli CsgF or a homolog of another species) and may comprise one or more modifications. In some embodiments, residues 9 to 17 comprise a conserved motif, N ₉ PXFGGXXX _{17 .} forming a turn region. In some embodiments, residues 9 to 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 in 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 a portion (e.g., the first portion or the second portion) of the fusion protein. In some embodiments, the CsgF peptide comprises or consists of amino acid residues 1-23 of wild-type E. coli CsgF. In some embodiments, the CsgF peptide comprises or consists of amino acid residues 1-23 of wild-type E. coli CsgF. In some embodiments, the CsgF peptide comprises or consists of amino acid residues 1-24 of wild-type E. coli CsgF. In some embodiments, the CsgF peptide comprises or consists of amino acid residues 1-24 of wild-type E. coli CsgF.

In some embodiments, the CsgF peptide has a length of 28 to 60 amino acids, such as 29 to 49, 30 to 45, or 32 to 40 amino acids. In some embodiments, the CsgF peptide comprises 29 to 35 amino acids or 29 to 45 amino acids. In some embodiments, the CsgF peptide comprises a length of 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59 or 60 amino acids. 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 homolog). In some embodiments, when the CsgF peptide is shorter than FCP, the cleavage preferably occurs at the C-terminal end.

One or more residues in the CsgF peptide can be modified. For example, the CsgF peptide can comprise a modification at a position corresponding to one or more of the following positions in SEQ ID NO: 6: G1, M3, T4, F5, R8, N9, N11, F12, N17, A20, N24, A26, and Q29 of SEQ ID NO: 60. In some embodiments, the CsgF peptide is modified to introduce 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 a position corresponding to one or more of the following positions: G1, T4, F5, R8, N9, N11, F12, N17, A20, N24, A26, Q27, and Q29 of SEQ ID NO: 60. Such introductions can be made in any number and combination. The introductions are preferably by substitution.

In some embodiments, the CsgF peptide comprises a modification at one or more of the following positions: N15, N17, A20, N24, and A28 in SEQ ID NO: 60. In some embodiments, the CsgF peptide comprises 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 CsgF sequences discussed above, including SEQ ID NO: 60, preferably comprising one or more modifications compared to the comparable sequence. Over the entire length of the amino acid sequence of SEQ ID NO: 60, the variant will preferably be at least 40% homologous to that sequence, based on amino acid identity. More preferably, the variant can be at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous to the amino acid sequence of SEQ ID NO: 60, over the entire sequence. Over the entire length of the amino acid sequence of SEQ ID NO: 60, the variant will preferably be at least 40% identical to 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% and more preferably at least 95%, 97% or 99% identical to SEQ ID NO: 60 over the entire sequence. There may be at least 80%, for example at least 85%, 90% or 95% amino acid identity over a stretch of 15 or more, for example 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more contiguous amino acids (“strong homology”). These levels of homology/identity apply equally to any other CsgF peptide described above.

Any number of the CsgF peptides in a pore or pore complex, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, may contain one or more substitutions compared to SEQ ID NO: 60. In some embodiments, all 6 to 10 monomers in a pore or pore complex preferably contain one or more substitutions compared to SEQ ID NO: 60. The CsgF peptides in a pore complex can be identical or different. The CsgF peptides are preferably identical in each pore monomer conjugate of the pore complex of the present disclosure.

Aspects of the present disclosure relate to accessory proteins or fusion proteins comprising one or more alpha helices. In some embodiments, such proteins may be referred to as "helix-forming proteins." The present disclosure is based in part on the recognition that helix-forming proteins can be positioned in the lumen of a particular nanopore (e.g., a CsgG nanopore) to form one or more constrictions in the lumen of the nanopore, and that the presence of such one or more constrictions improves the signal-to-noise ratio (e.g., discrimination of polynucleotide bases) of the resulting protein-pore complex. The terms "helix" or "helical" generally refer to a coiled-coil structural arrangement of a protein that forms a helix and is formed by hydrogen bonding between 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), comprising about 3.6 amino acid residues per helical turn, with 13 atoms in the loop formed by hydrogen bonding. In some embodiments, the helix is a 3 ₁₀ helix, containing about 3 residues per turn and having 10 atoms in a loop formed by hydrogen bonding.

The helix number of the accessory protein or fusion protein can vary (e.g., alpha helix, 3 ₁₀ helix, π helix, etc.). In some embodiments, the helix number of the accessory protein (e.g., the first accessory protein, the second accessory protein, etc.) is in the range of about 0 to about 15, for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15. In some embodiments, the helix number of the accessory protein (e.g., the first accessory protein, the second accessory protein, etc.) is greater than 15 (e.g., 20, 25, etc.). In some embodiments, the fusion protein (e.g., the first portion of the fusion protein, the second portion of the fusion protein, etc.) comprises from 0 to about 15 helices (e.g., alpha helices, _3-10 helices, π helices, etc.), for example, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 helices.

The number of turns of the helix (e.g., alpha helix, 3 ₁₀ helix, π helix, etc.) can vary. In some embodiments, each helix (e.g., alpha helix, 3 ₁₀ helix, π helix, etc.) of the accessory protein or fusion protein comprises from about 0 to about 15 helical turns, for example, 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 310 helix, a π helix, etc.) can contain one or more half-helices (e.g., half-turns), for example, 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 amino acids that form a helix (e.g., alpha helix, _3–10 helix, π helix, etc.) can vary. In some embodiments, each helix (e.g., an alpha helix, a _3-10 helix, a π helix, etc.) of the accessory protein or fusion protein comprises from 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, Contains 53, 54 or 55 amino acid residues.

The angle of the helix of an accessory protein or fusion protein can vary. In some embodiments, the spiral is 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°, Includes Phi angles in the range of -89° or -90°. In some embodiments, the spiral has an angle of about 0° to -70° (e.g., 0°, -1°, -2°, -3°, -4°, -5°, -6°, -7°, -8°, -9°, -10°, -11°, -12°, -13°, -14°, -15°, -16°, -17°, -18°, -19°, -20°, -21°, -22°, -23°, -24°, -25°, -26°, -27°, -28°, -29°, -30°, -31°, -32°, -33°, -34°, -35°, -36°, -37°, -38°, -39°, -40°, -41°, -42°, -43°, -44°, -45°, -46°, -47°, -48°, -49°, -50°, -51°, -52°, -53°, -54°, -55°, -56°, -57°, -58°, -59°, -60°, -61°, -62°, -63°, -64°, -65°, -66°, -67°, -68°, -69° or -70°. In some embodiments, each helix 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 helix 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 accessory protein or fusion protein comprises structural features that facilitate packing of one or more helices with respect to one another. "Packing" of a helix typically refers to close association of two or more helices with respect to one another due to covalent or non-covalent interactions between the helices, such as salt bridges, hydrogen bonds, disulfide bonds, and close hydrophobic side-chain-to-side-chain contacts, side-chain-to-backbone contacts, backbone-to-backbone contacts, etc., as described in the literature [Walther and Argos, J Mol Biol . 26 Jan 1996;255(3):536-53. doi: 10.1006/jmbi.1996.0044]. Methods for predicting helix packing are described, for example, in the literature [Eilers et al. Proc Natl Acad Sci USA . 23 May 2000;97(11):5796-5801].

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

CsgG nanopore

Aspects of the present disclosure relate to protein pore complexes. In some embodiments, the protein pore complexes described by the present 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 within the membrane, driven by an applied potential.

The nanopore, in some embodiments, is a transmembrane protein pore. A transmembrane protein pore typically spans the entire membrane and can have a structure that extends beyond the membrane on one or both sides. A transmembrane protein pore is a single or multimeric protein that allows hydrated ions to flow from one side of the membrane to the other side of the membrane. A transmembrane protein pore includes a channel that allows an analyte, such as a polynucleotide such as DNA or RNA, to move into and/or through the pore.

A transmembrane protein pore typically contains a barrel or channel through which ions can flow. The subunits of the pore typically surround a central axis and provide strands to a transmembrane β barrel or channel or a transmembrane α helix bundle or channel.

The barrel or channel of the transmembrane protein pore comprises amino acids that facilitate interaction with a typical polynucleotide. Such amino acids are preferably located near the narrow portion of the barrel or channel (e.g., within 1, 2, 3, 4 or 5 nm). The transmembrane protein pore typically comprises one or more polar or hydrophobic residues. Such amino acids typically facilitate interaction between the pore and a nucleotide, polynucleotide or nucleic acid.

In some embodiments, the nanopore is a CsgG pore, e.g., CsgG of E. coli strain K-12 substrain MC4100 or a homolog or mutant thereof. The mutant CsgG pore can comprise one or more mutant monomers. The CsgG pore can be a homopolymer comprising identical monomers or a heteropolymer comprising two or more different monomers. Suitable pores derived from CsgG are disclosed in WO 2016/034591, WO2017/149316, WO2017/149317, WO2017/149318, International Patent Application Nos. PCT/GB2018/051191 and PCT/GB2018/051858 and Chinese Patent Application Nos. CN113773373, CN113896776, CN113912683 and CN113754743, which are each 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.

A CsgG pore typically comprises one or more CsgG monomers. A CsgG pore monomer is a monomer capable of forming a CsgG pore. Such monomers are known in the art, particularly from WO 2019/002893 (which is incorporated herein 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, such as (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 can have any structure, but preferably has or comprises the structure of a wild-type E. coli CsgG pore (e.g., as described by PDB Accession No. 4UV3). The protein structure of CsgG defines a channel or hole that allows translocation of molecules and ions from one side of the membrane to the other.

The CsgG pore can be of any size, but preferably has the size of a wild-type E. coli CsgG pore (e.g., as described by PDB Accession No. 4UV3). This size is shown in FIG. 19. In some embodiments, the CsgG pore is about 100 to about 150 at its widest point. , for example, about 110 to about 140 at its widest point. or about 115 to about 125 has an outer diameter of about 120 at its widest point. In some embodiments, the CsgG pore is has an outer diameter of about 80 to about 120. In some embodiments, the CsgG pore has an outer diameter of about 80 to about 120 , for example, about 90 to about 110 or about 95 to about 105 has a total length of about 98. In some embodiments, the CsgG pore has a total length of about 98 has a total length of . References to "total length" and "length" relate to the length of the pore or pore region when viewed from the side (e.g., see a cis-to-trans cross-section of a pore inserted into a membrane). This may be the side view of FIG. 19 . In some embodiments, the outer diameter is measured by calculating the C _a to C _a distance of the most distant amino acid residues outside the CsgG pore. In some embodiments, the outer diameter is measured by calculating the distance of the van der Waals radii of the most distant amino acid residues outside the CsgG pore.

In some embodiments, the cap area is about 20 to about 60 , for example, about 30 to about 50 or about 35 to about 45 has a length of about 39. In some embodiments, the cap region is has a length of about 30 to about 70. In some embodiments, the channel defined by the cap region has a diameter of about 30 to about 70. , for example, a diameter of about 40 to about 60 or about 45 to about 55 has an opening. In some embodiments, the channel defined by the cap region has a diameter of about 66 has a diameter of from about 20 to about 66 at its narrowest point. In some embodiments, the channel defined by the cap region has a diameter of from about 20 to about 66 at its narrowest point. , and has a diameter of about 30 to about 50 at its narrowest point. or about 32 to about 43 In some embodiments, the channel defined by the cap region preferably has a diameter of about 43 at its narrowest point. In some embodiments, the outer diameter is measured by calculating the C _a to _C a distance between amino acid residues on the channel of the cap region of the CsgG pore that are closest to each other. In some embodiments, the outer diameter is measured by calculating the distance between the van der Waals radii of amino acid residues on the channel of the cap region that are closest to each other.

In some embodiments, the constriction region formed by the CsgG pore (if present) is about 5 to about 40 , for example, about 10 to about 30 or about 15 to about 25 has a length of about 20. In some embodiments, the stenotic region is about 20 has a length of about 2 to about 30. In some embodiments, the channel defined by the constriction region has a diameter at its narrowest point of about 2 to about 30. , for example, from about 5 to about 25 in diameter at its narrowest point. , about 8 to about 20 or about 10 to about 15 In some embodiments, the channel defined by the constricted region has a diameter of about 9 In some embodiments, the channel defined by the constricted region has a diameter of about 18.5 In some embodiments, the diameter of the constriction is from about 2 to about 30 , for example, the diameter is about 5 to about 25 , about 8 to about 20 or about 10 to about 15 In some embodiments, the constriction has a diameter of about 12 In some embodiments, the constriction area of a CsgG pore is measured by calculating the distance between C _a and C _a of the amino acid residues that extend furthest into the lumen of the pore to form the constriction. In some embodiments, the outer diameter is measured by calculating the distance between the van der Waals radii of the amino acid residues that extend furthest into the lumen of the pore to form the constriction.

In some embodiments, the transmembrane beta barrel region is about 20 to about 60 , for example, about 30 to about 50 or about 35 to about 45 has a length of about 39. In some embodiments, the transverse beta barrel has a length of about 39. has a length of about 20 to about 60 cm. In some embodiments, the channel defined by the transverse beta-barrel region has a diameter at its narrowest point of about 20 to about 60 cm. , and for example, the diameter at the narrowest point is about 30 to about 50 or about 35 to about 45 In some embodiments, the channel defined by the transverse beta barrel region has a diameter of about 55 at its narrowest point. am.

All of the above measurements are based on measurements from backbone to backbone of amino acids forming different domains (as shown in Figure 19).

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

In some embodiments, the CsgG pore monomer is a variant of SEQ ID NO: 59 because it has a cysteine at a position corresponding to position 153 or 133 of SEQ ID NO: 59. In some embodiments, the variant CsgG monomer may also be referred to as a modified CsgG pore monomer or a mutant CsgG pore monomer. The modifications or mutations in the variant include, but are not limited to, any one or more of the modifications disclosed herein, or a combination of such modifications. The CsgG pore monomer can be a CsgG homolog monomer. A CsgG homolog monomer is a polypeptide having at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% complete sequence identity to the wild-type E. coli CsgG as set forth in SEQ ID NO: 59. CsgG homologues are also referred to as polypeptides containing 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 comprising 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 will preferably be at least 40% homologous, based on amino acid identity, to that sequence. More preferably, the variant can be at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous, based on amino acid identity, to the amino acid sequence of SEQ ID NO: 59 over the entire sequence. Over the entire length of the amino acid sequence of SEQ ID NO: 59, the variant will preferably be 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% and more preferably at least 95%, 97% or 99% identical to SEQ ID NO: 59 over the entire sequence.

The sequence identity can also relate to a fragment or portion of a CsgG pore monomer. Thus, while a sequence may have less than 40% overall sequence homology/identity with SEQ ID NO: 59, the sequence of a particular region, domain or subunit may share at least 80%, 90%, or at most 99% sequence homology/identity with a corresponding region of SEQ ID NO: 59. There may be at least 80%, for example, at least 85%, 90% or 95% amino acid identity over a stretch of 100 or more, for example, 125, 150, 175 or 200 or more contiguous amino acids (“strong homology”). In some embodiments, the CsgG pore monomer is a variant of SEQ ID NO: 3, preferably comprising a sequence that is at least 40% homologous to the cap region (residues 1 to 41) of SEQ ID NO: 3. More preferably, the variant comprises a sequence which is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous to residues 1 to 41 of SEQ ID NO: 59. In some embodiments, the variant comprises a sequence which is at least 40% identical to residues 1 to 41 of SEQ ID NO: 59. In some embodiments, the variant comprises a sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% identical to residues 1 to 41 of SEQ ID NO: 59.

In some embodiments, the CsgG pore monomer is a variant of SEQ ID NO: 59 comprising a sequence that is at least 40% homologous to the stricture region (residues 64 to 131) of SEQ ID NO: 59. In some embodiments, the variant comprises a sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, and more preferably at least 95%, 97% or 99% homologous based on amino acid identity to residues 64 to 131 of SEQ ID NO: 59. In some embodiments, the variant comprises a sequence that is at least 40% identical to residues 64 to 131 of SEQ ID NO: 59. In some embodiments, the variant comprises a sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% identical to residues 64 to 131 of SEQ ID NO: 59.

In some embodiments, the CsgG pore monomer is a variant of SEQ ID NO: 59 comprising a sequence that is at least 40% homologous to the transmembrane 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%, and more preferably at least 95%, 97% or 99% homologous based on amino acid identity to residues 156-180 and 212-262 of SEQ ID NO: 59. In some embodiments, the variant comprises a sequence that is at least 40% identical to residues 156-180 and 212-262 of SEQ ID NO: 59. In some embodiments, the variant comprises a sequence which is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% identical to residues 156-180 and 212-262 of SEQ ID NO: 59.

The CsgG pore monomer is highly conserved (as readily apparent in FIGS. 45 to 47 of WO 2017/149317). Furthermore, from knowledge of the mutations associated with SEQ ID NO: 59, it is possible to determine equivalent positions for mutations in the CsgG pore monomer other than those of SEQ ID NO: 59.

Thus, a reference to a mutant CsgG pore monomer comprising a variant of the sequence set forth in SEQ ID NO: 59 and certain amino acid mutations specified in the claims and elsewhere in the specification also encompasses mutant CsgG pore monomers comprising a variant of any one of the sequences set forth in SEQ ID NO: 68 to 88 of WO 2019/002893 (which is herein incorporated by reference in its entirety) and the corresponding amino acid mutations thereof. The CsgG pore monomer may also be any one of the sequences set forth in CN 113773373 A, CN 113896776 A, CN 113912683 A and CN 113754743 A or variants 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, for example, with default settings (Devereux et al. (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or to align sequences (e.g., to identify equivalent residues or corresponding sequences (typically with their default settings)), as described, for example, in Altschul S. F. (1993) J Mol Evol 36:290-300; Altschul, S. F et al. (1990) J Mol Biol 215:403-10). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).

SEQ ID NO: 59 is a wild-type CsgG pore monomer of E. coli strain K-12 substrain MC4100. Variants of SEQ ID NO: 59 may comprise any substitution present in other CsgG homologues. Preferred CsgG homologues are set forth in SEQ ID NOs: 68 to 88 of WO 2019/002893 (which is incorporated herein by reference in its entirety). Variants may comprise a combination of one or more substitutions present in SEQ ID NOs: 68 to 88 of WO 2019/002893 (which is incorporated herein by reference in its entirety) compared to SEQ ID NO: 59.

The CsgG pore monomer in the pore monomer conjugate of the present disclosure typically retains the ability to form the same 3D structure as a wild-type CsgG pore monomer, e.g., 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. Any number of mutations may be made in the wild-type CsgG sequence in addition to the mutations described herein, provided that the CsgG pore monomer retains the improved properties conferred by the mutation.

Amino acid substitutions may be made, for example, up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions, for the amino acid sequence of SEQ ID NO: 59, in addition to those discussed above. A conservative substitution replaces an amino acid with another amino acid having a similar chemical structure, similar chemical properties or similar side chain volume. The introduced amino acid may have a polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge similar to the amino acid it replaces. Alternatively, a conservative substitution may introduce another amino acid which is aromatic or aliphatic instead of the 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 can be made in any number and combination. The introductions are preferably by substitution.

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

The variant may comprise a fragment of SEQ ID NO: 59. This fragment retains pore-forming activity. The fragment may be at least 50, at least 100, at least 150, at least 200 or at least 250 amino acids in length. This fragment may be used to generate a pore. The fragment preferably comprises the membrane spanning domain of SEQ ID NO: 59, i.e. K135-Q153 and S183-S208.

One or more amino acids may alternatively or additionally be added to the polypeptide described above. An extension may be provided at the amino terminus 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, from 1 to 10 amino acids in length. Alternatively, the extension may be longer, for example, up to 50 or 100 amino acids. A carrier protein may be fused to 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 that differs from the amino acid sequence of SEQ ID NO: 59 and retains the ability to form a pore. The variant typically comprises the region of SEQ ID NO: 59 that is responsible for pore formation. The pore-forming ability of CsgG containing a β-barrel is provided by a β-sheet in the transmembrane β-barrel region of each subunit monomer. A variant of SEQ ID NO: 59 typically comprises the region of SEQ ID NO: 59 that forms a β-sheet, namely K134-Q154 and S183-S208. One or more modifications may be made in the region of SEQ ID NO: 3 that forms a β-sheet, as long as the resulting variant retains the ability to form a pore.

One or more modifications of the CsgG pore monomer preferably improve the ability of the pore complex comprising the pore monomer to characterize an analyte. For example, the modifications/mutations/substitutions are contemplated to alter the number, size, shape, arrangement or orientation of constrictions within the channel from the pore monomer conjugates of the present disclosure. The CsgG pore monomer or variants of SEQ ID NO: 59 may have specific modifications or substitutions disclosed in WO 2016/034591, WO 2017/149316, WO 2017/149317, WO 2017/149318, WO 2018/211241 and WO 2019/002893 (all of which are incorporated herein by reference in their entireties).

Preferred modifications or substitutions of SEQ ID NO: 59 include, but are not limited to, one or more of the following, for example, two or more, three or more, four or more, five or more, six or more, seven or more, or all:

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

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

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

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

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

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

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

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

The variant of SEQ ID NO: 3 may additionally comprise a deletion of one or more positions, for example a deletion of T104-N109, a deletion of F193-L199 or a deletion of F195-L199.

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

Linker

In some embodiments, the protein pore complex is stabilized by attaching (e.g., covalently attaching) an auxiliary protein or fusion protein to the nanopore. The covalent linkage can be, for example, a disulfide bond or click chemistry. As a further example, the cysteine residues can be linked by a linker, such as BMOE. The auxiliary protein or fusion protein and/or the transmembrane protein nanopore can be modified to facilitate such covalent interactions. In some embodiments, the auxiliary protein or fusion protein is noncovalently 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 accessory protein or fusion protein is attached to the nanopore by hydrophobic interactions and/or one or more disulfide bonds. One or more of the monomers in the pore, such as 2, 3, 4, 5, 6, 8, 9, for example all, may be modified to enhance these interactions. This may be accomplished in any suitable manner. Additional 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 accessory protein (or fusion protein) can be disulfide bonded with at least one cysteine residue in the amino acid sequence of the accessory protein at the interface between the nanopore and the accessory protein. In some embodiments, at least one cysteine residue in the amino acid sequence of the first accessory protein is disulfide bonded to at least one cysteine residue in the amino acid sequence of the second accessory protein. In some embodiments, at least one cysteine residue in the amino acid sequence of the first portion of the fusion protein is disulfide bonded to at least one cysteine residue in the amino acid sequence of the second portion of the fusion protein. The cysteine residue in the nanopore and/or the cysteine residue in the accessory protein or fusion protein can be a cysteine residue that is not present in the wild-type transmembrane protein pore monomer or in the wild-type accessory protein. Multiple 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, can 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) can comprise at least one monomer or subunit comprising a cysteine residue at the interface between the nanopore and the auxiliary protein (or fusion protein), for example up to 8, 9 or 10 monomers or subunits.

The nanopore and/or the accessory protein (or fusion protein) can comprise one or more hydrophobic amino acid residues at the interface between the nanopore and the accessory protein (or fusion protein) that are more hydrophobic than the residues present at the corresponding positions in the wild-type nanopore or the accessory protein (or fusion protein). At least one of the monomers or subunits in the nanopore and/or at least one of the monomers or subunits in the accessory protein (or fusion protein) can comprise at least one residue at the interface between the nanopore and the accessory protein (or fusion protein), which residue is more hydrophobic than the residues present at the corresponding positions in the wild-type pore or the accessory protein (or fusion protein). For example, from two to ten, i.e., three, four, five, six, seven, eight or nine, residues in the nanopore and/or the accessory protein (or fusion protein) can be more hydrophobic than the residues present at the same positions in the corresponding wild-type nanopore and/or the accessory protein (or fusion protein). These hydrophobic residues enhance the interaction between the nanopore and the accessory protein (or fusion protein) in the pore complex. When the residue at the interface of the wild-type nanopore or the accessory protein (or fusion protein) is R, Q, N or E, the hydrophobic residues are typically I, L, V, M, F, W, A or Y. When the residue at the interface of the wild-type nanopore or the accessory protein (or fusion protein) is I, the hydrophobic residues are typically L, V, M, F, W, A or Y. When the residue at the interface of the wild-type nanopore or the accessory protein (or fusion protein) is L, the hydrophobic residues are typically I, V, M, F, W, A or Y.

Molecular dynamics simulations can be performed to establish which residues of the auxiliary protein and the nanopore are in close proximity. This information can be used to design auxiliary protein and/or transmembrane protein nanopore mutants that may increase the stability of the complex. For example, simulations can be performed using the GROMOS 53a6 force field and the SPC water model using the cryo-EM structure of the protein using the GROMACS package version 4.6.5. The complex can be solvated and energy minimized using the steepest descent algorithm. The protein backbone can be restrained throughout the simulation, but the residue side chains can move freely. The system can be simulated for 20 ns in the NPT ensemble up to 300 K using the Berendsen thermostat and Berendsen pressure regulator. Contacts between the auxiliary protein and the nanopore can be analyzed using the GROMACS analysis software and/or locally written code. Contacts are defined as two residues within 3 angstroms of each other.

For example, the interaction between a CsgF peptide and a CsgG pore in the pore complex can be stabilized by hydrophobic interactions, electrostatic interactions or covalent bonds at positions corresponding to, for example, one or more of the following position pairs of SEQ ID NO: 60 and SEQ ID NO: 59: 1 and 153, 4 and 133, 5 and 136, 8 and 187, 8 and 203, 9 and 203, 11 and 142, 11 and 201, 12 and 149, 12 and 203, 26 and 191, 29 and 144 or 30 and 196. Residues of CsgF and/or CsgG at one or more of these positions can be modified to enhance the interaction between CsgG and CsgF in the pore.

Covalent linkages or bonds are formed, for example, via cysteine linkages, wherein the sulfhydryl side group of the cysteine is covalently linked to another amino acid residue or moiety and/or via interactions between non-natural (photo)reactive amino acids. (Photo)reactive amino acids refer to artificial analogues of natural amino acids that can be used for cross-linking protein complexes, which can be incorporated into proteins and peptides in vivo or in vitro . Commonly used photoreactive amino acid analogues are leucine and methionine and para-benzoyl-phenyl-alanine, as well as photoreactive diazirine analogues for azidohomoalanine, homopropargylglycine, homoallelglycine, p-acetyl-Phe, p-azido-Phe, p-propargyloxy-Phe and p-benzoyl-Phe (Wang et al. 2012; Chin et al. 2002). When exposed to ultraviolet light, it becomes activated and covalently binds to interacting proteins within a few angstroms of the photoreactive amino acid analogue.

An oxidizing agent (e.g., copper-orthophenanthroline) can be used to form a pore complex and induce disulfide bond formation. Other interactions (e.g., hydrophobic interactions, charge-charge interactions/electrostatic interactions) can also be used at this position in place of the cysteine interaction. In other embodiments, a non-natural amino acid can also be incorporated at this position. In such embodiments, the covalent bond is formed via click chemistry. For example, a non-natural amino acid having an azide or alkyne or dibenzocyclooctyne (DBCO) group and/or a bicyclo[6.1.0]nonyne (BCN) group can be introduced at one or more of these positions.

For example, the CsgG pore can comprise at least one, e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10 CsgG monomers modified to facilitate attachment to an accessory protein or fusion protein. For example, a cysteine residue can 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 accessory protein or fusion protein to facilitate covalent attachment to an accessory protein or fusion protein. As an alternative or addition to covalent attachment via a cysteine residue, the pore may be stabilized by hydrophobic interactions or electrostatic interactions. To facilitate such interactions, a non-naturally reactive or photoreactive amino acid is positioned at a position corresponding to one or more of positions 132, 133, 136, 138, 140, 142, 144, 145, 147, 149, 151, 153, 155, 183, 185, 187, 189, 191, 201, 203, 205, 207 and 209 of SEQ ID NO: 59.

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

These stabilizing mutations may be combined with any other modifications to the accessory protein or fusion protein, for example, modifications that improve the interaction of the polynucleotide with the pore complex or that improve certain properties of the complex (for example, the discrimination of polymeric units such as nucleotides in the polynucleotide).

In some embodiments, the nanopores can be isolated, substantially isolated, purified, or substantially purified. The pores are isolated or purified when they are completely free of any other components, such as lipids or other pores. The pores are substantially isolated when they are mixed with a carrier or diluent that does not interfere with the intended use. For example, the pores are substantially isolated or substantially purified when they are present in a form that includes less than 10%, less than 5%, less than 2%, or less than 1% of other components, such as block copolymers, lipids, or other pores. Alternatively, the pores can be present in a membrane. Suitable membranes are discussed below.

Pore complexes may exist in the membrane as individual or single pores. Alternatively, pore complexes may exist as homogeneous or heterogeneous populations of two or more pores.

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

Suitable chemical cross-linkers are well known in the art. Examples of cross-linkers 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)octanoate. In some embodiments, the cross-linker is succinimidyl 3-(2-pyridyldithio)propionate (SPDP). Typically, the molecule is covalently attached to the bifunctional cross-linker before the molecule/cross-linker complex is covalently attached to the mutant monomer, but it is also possible to covalently attach the bifunctional cross-linker to the monomer before the bifunctional cross-linker/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 accessory protein or fusion protein to form a constriction in 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 are (SG) ₁ , (SG) ₂ , (SG) ₃ , (SG) ₄ , (SG) ₅ , (SG) ₈ , (SG) ₁₀ , (SG) ₁₅ or (SG) ₂₀ , wherein S is serine and G is glycine. Preferred rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or 24, proline amino acids. A more preferred rigid linker comprises (P) ₁₂ , wherein P is proline.

Suitable chemical cross-linkers include, but are not limited to, those containing the following functional groups: maleimides, activated esters, succinimides, azides, alkynes (e.g., dibenzocyclooctynols (DIBO or DBCO), difluoro cycloalkynes, and linear alkynes), phosphines (e.g., those used in trace-free and trace Staudinger ligations), haloacetyls (e.g., iodoacetamide), phosgene type reagents, sulfonyl chloride reagents, isothiocyanates, acyl halides, hydrazines, disulfides, vinyl sulfones, aziridines, and photoreactive reagents (e.g., aryl azides, diaziridines).

Reactions between amino acids and functional groups can be spontaneous, such as cysteine/maleimide, or may require external reagents, such as Cu(I) to link azides and linear alkynes.

The linker can comprise any molecule that extends over the required distance. The length of the linker can range from one carbon (phosgene type linker) to several angstroms. Examples of linker molecules include, but are not limited to, polyethylene glycol (PEG), polypeptides, polysaccharides, deoxyribonucleic acid (DNA), peptide nucleic acid (PNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), saturated and unsaturated hydrocarbons, polyamides, and the like. Such linkers can be inert or reactive, and in particular can be chemically cleavable at defined positions or can themselves be modified with fluorophores or ligands. The linker is preferably resistant to dithiothreitol (DTT) following covalent attachment of the accessory protein or fusion protein to the CsgG pore monomer.

In some embodiments, the crosslinker is selected from: 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)octanoate, 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-maleimide PEG3, bis-maleimide PEG11, DBCO-maleimide, DBCO-PEG4-maleimide, DBCO-PEG4-NH2, DBCO-PEG4-NHS, DBCO-NHS, DBCO-PEG-DBCO 2.8 kDa, 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(2kDa)-maleimide(alpha,omega-bis-maleimido poly(ethylene glycol)). In some embodiments, the cross-linker is maleimide-propyl-SRDFWRS-(1,2-diaminoethane)-propyl-maleimide.

The linked CsgG pore monomer and the accessory protein or fusion protein can be coupled via covalent bond formation between the groups. Any one of the specific linkers disclosed in WO 2010/086602 (which is incorporated herein by reference in its entirety) can be used.

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

A preferred method of linking the pore monomer conjugates is via a cysteine linkage. This can be mediated by a bifunctional chemical cross-linker or an amino acid linker having cysteine residues presented at the terminal end.

Another preferred method of attachment is via a 4-azidophenylalanine (Faz) linkage. This can be mediated by a bifunctional chemical linker or a polypeptide linker having Faz residues presented at the termini.

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

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

The distance between the CsgG pore monomer and the accessory protein or fusion protein in the CsgG pore monomer conjugate and/or the length of the linker is preferably less than about 2.00 nm, such as less than about 1.90 nm, less than about 1.80 nm, less than about 1.70 nm, less than about 1.60 nm, less than about 1.50 nm, less than about 1.40 nm, less than about 1.30 nm, less than about 1.20 nm, less than about 1.10 nm, less than about 1.00 nm, less than about 0.90 nm, less than about 0.80 nm, less than about 0.70 nm, less than about 0.60 nm, less than about 0.50 nm or less than about 0.40 nm. The distance between the CsgG pore monomer and the accessory protein or fusion protein in the pore monomer conjugate and/or the length of the linker is preferably less than about 1.20 nm. This distance/length can be achieved using maleimidohexanoic acid as discussed in more detail below. The distance and/or length of the linker between the CsgG pore monomer and the accessory 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 discussed below.

The distance between the CsgG pore monomer and the pore accessory protein or fusion protein in the monomer conjugate and/or the length of the linker is preferably about 0.40 nm to about 2.0 nm, for example about 0.45 nm to about 1.90 nm, about 0.50 nm to about 1.80 nm, about 0.55 nm to about 1.7 nm, about 0.60 nm to about 1.6 nm, about 0.65 nm to about 1.5 nm, about 0.7 nm to about 1.4 nm, about 0.75 nm to about 1.3 nm, about 0.80 nm to about 1.2 nm, about 0.85 nm to about 1.1 nm and about 0.90 nm to about 1.00 nm. The distance between the CsgG pore monomer and the accessory protein or fusion protein in the pore monomer conjugate and/or the length of the linker is preferably from about 0.50 nm to about 1.50 nm. The distance between the CsgG pore monomer and the accessory protein or fusion protein in the pore monomer conjugate and/or the length of the linker is preferably from about 0.60 nm to about 1.2 nm. Such distances/lengths can be achieved using certain maleimide-containing linkers discussed below.

The maleimide-containing linker can be any one of the linkers discussed below in connection with the constructs described herein. The maleimide-containing linker preferably comprises or consists of a linear carbon chain consisting of a maleimide group and 2, 3, 4, 5, 6 or more carbon atoms. The linear carbon chain is typically attached to the nitrogen atom of 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 of the accessory 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. Such linkers are illustrated in FIG. 15 .

The present disclosure also provides a pore monomer conjugate comprising a CsgG pore monomer covalently attached to an accessory protein or fusion protein, wherein the accessory protein or fusion protein is covalently attached to a cysteine residue of the CsgG pore monomer by a linker comprising a thiol reactive group. The thiol reactive group can be a maleimide group, a pyridyldithio group, a halogeno group, a parafluoro group, an ene group, a phosphorus group, a vinylsulfone group or a thiosulfone group. Such groups are illustrated in FIG. 16 . The linker comprising a thiol reactive group can be any of the linkers discussed below in connection with the constructs of the present disclosure. The linker preferably comprises or consists of a linear carbon chain of 2, 3, 4, 5, 6 or more carbon atoms with the thiol reactive group. The linear carbon chain preferably also comprises a terminal carboxyl group. Such carboxyl group is capable of forming an amide bond with an amino acid of the accessory protein or fusion protein. The linker can be one of the specific maleimide-containing linkers discussed above, wherein the maleimide is replaced with a different thiol reactive group. The linker containing the thiol reactive group can be any one of the lengths discussed above.

Suitable linkage groups can be designed using existing modeling techniques. The linker is typically flexible enough to allow the monomers or subunits to assemble into individual protein oligomers and align along a common axis of symmetry to create a continuous channel within the pore complex.

Identification and selection of auxiliary proteins

Aspects of the present disclosure relate to computer-based methods for designing and/or selecting accessory proteins and/or fusion proteins for inclusion in a protein pore complex (e.g., a protein pore complex comprising a CsgG nanopore). In some embodiments, the method comprises providing an amino acid sequence (e.g., a CsgF amino acid sequence) as input to software comprising code that implements 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 can be MASTER (e.g., as described in Zhou and Grigoryan, Protein Sci . Apr. 2015; 24(4): 508-524, the entire contents of which are incorporated herein by reference). In some embodiments, the protein backbone selection technique comprises selecting a protein backbone structure having one or more target properties (e.g., the ability to form one or more helical regions, the ability to pack into one or more helical regions of a protein pore, etc.) from known protein backbone structures (e.g., as described in the Protein Data Bank, PDB). In some embodiments, the backbone structures are provided as input to software comprising code that implements protein sequence design and structural prediction techniques and processes the backbone structures to generate one or more novel designed peptide sequences. In some embodiments, the protein sequence design and structure prediction technique can be Rosetta (see, e.g., Leaver-Fay et al., Chapter 19 - Rosetta3: An Object-Oriented Software Suite for the Simulation and Design of Macromolecules, Methods in Enzymology, Academic Press, Vol. 487, 2011, pp. 545-574, doi.org/10.1016/B978-0-12-381270-4.00019-6., the entire contents of which are incorporated herein by reference). In some embodiments, the de novo designed peptide sequence comprises one or more target features that are identical to one or more desirable features of the backbone amino acid sequence.

Method for producing nanopore complexes

A pore complex comprising an accessory protein or fusion protein and a transmembrane protein nanopore can, in one embodiment, be produced by co-expression. In some embodiments, the method comprises expressing in a suitable host cell both a pore monomer and an accessory protein or fusion protein, or an accessory protein or monomer, and allowing formation of a complex pore in vivo . In such embodiments, at least one gene encoding a pore monomer in one vector and a gene encoding an accessory protein or fusion protein or at least one accessory protein subunit or monomer in a second vector are co-transformed to express the proteins and form a complex within the transformed cell. This is preferably done ex vivo or in vitro . Alternatively, the two genes encoding the pore monomer and the accessory 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 can be identical or different.

Another method for generating a pore complex formed by an accessory protein or fusion protein and a transmembrane protein nanopore is to reconstitute the protein in vitro to obtain a functional pore. In some embodiments, the method comprises contacting a monomer of the transmembrane protein nanopore with an accessory protein (or fusion protein) or an accessory protein subunit or monomer in a suitable system to allow for complex formation. The system may be an " in vitro system," which refers to a system comprising at least the components and environment necessary to perform the method, and which utilizes a biological molecule, organism, cell (or part of a cell) outside of its normal, naturally occurring environment, thereby allowing for more detailed, more convenient, or more efficient analysis than would be possible with a whole organism. The in vitro system may also comprise a suitable buffer composition provided in a test tube, to which the protein component for forming the complex has been added. Those skilled in the art are aware of options for providing such a system.

In such embodiments, the nanopore can be generated by expressing the monomer separately from the accessory protein or fusion protein. The pore monomer or nanopore can be purified from a cell transformed with a vector encoding at least one pore monomer or from a cell transformed with two or more vectors each expressing a pore monomer. The accessory protein or fusion protein can be purified from a cell transformed with a vector encoding at least one accessory protein or fusion protein. The purified pore monomer/nanopore can then be incubated with the accessory protein or fusion protein to form a pore complex.

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

The above embodiments can 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 .

Either or both of the nanopore monomer and the accessory protein or fusion protein may be tagged to facilitate purification. Purification may also be performed when the nanopore monomer and/or the accessory protein or fusion protein is not tagged. Components of the pore complex may be purified using methods known in the art, such as ion exchange, gel filtration, hydrophobic interaction column chromatography, and the like, alone or in different combinations.

Any known tag may be used on either protein. In one embodiment, a two-tag purification may be used to purify the pore complex from its component parts. For example, a Strep tag may be used on the nanopore and a His tag may be used on the accessory protein (or fusion protein), or vice versa. Similar end results may be obtained by purifying the two proteins separately and mixing them together followed by another Strep and His purification.

The pore complex can be formed prior to membrane insertion or after the nanopore is inserted into the membrane. However, the pore complex can also be formed in situ by adding an accessory protein (or fusion protein) after the nanopore is inserted into the membrane. For example, in one embodiment, in a system 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 the accessory protein (or fusion protein) can then be added from the trans or cis side of the membrane to form the complex in situ.

In one embodiment, the accessory protein can include a protease cleavable site (e.g., TEV, HRV 3, or any other protease cleavable site) and can be cleaved prior to or after associating with the nanopore. For example, a full-length accessory protein (or a fusion protein) can be used to form the pore. Cleavage of amino acid residues that do not form part of the channel configuration and are not required for interaction with the transmembrane pore can be cleaved from the accessory protein or fusion protein. In such embodiments, once the pore complex has been formed, a protease is used to cleave the accessory protein or fusion protein. Alternatively, the protease can be used to generate the accessory protein or fusion protein prior to pore complex assembly.

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

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 binding agents or intercalators, peptides or peptide analogs, synthetic polymers, aromatic planar molecules, positively charged small 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 discussed above.

The protein can be attached to the polynucleotide binding protein. This forms a modular sequencing system. The polynucleotide binding protein is discussed below. The protein can be covalently attached to the monomer using any method known in the art. The monomer and protein can 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 can be attached via a cysteine linkage using any of the methods described above.

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

Any protein can be produced using standard methods known in the art. A polynucleotide sequence encoding the protein can be derived and cloned using standard methods known in the art. A polynucleotide sequence encoding the protein can be expressed in a bacterial host cell using standard techniques known in the art. The protein can be produced in the cell by in situ expression of the polypeptide from a recombinant expression vector. The expression vector optionally carries an inducible promoter to control expression of the polypeptide. Such methods are described in Sambrook, J. and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

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

System

In another aspect, the present disclosure relates to a system for characterizing a target polynucleotide, the system comprising a membrane and a pore complex; wherein the pore complex comprises (i) a nanopore positioned in the membrane and (ii) an accessory protein or fusion protein attached 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 accessory 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, wherein the first chamber and the second chamber are separated by a membrane. When used to characterize a target polynucleotide, the system can further comprise a target polynucleotide, wherein the target polynucleotide is transiently positioned within the continuous channel, wherein one end of the target polynucleotide is positioned in the first chamber and one end of the target polynucleotide is positioned in the second chamber.

In some embodiments, the system further comprises an electrically conductive solution in contact with the nanopore, an electrode providing a voltage potential across the membrane, and a measurement system for measuring the current passing through the nanopore. In one embodiment, the voltage applied across the membrane and the pore complex is from +5 V to -5 V, such as from -600 mV to +600 mV or from -400 mV to +400 mV. The voltage used is preferably in the range of 100 mV to 240 mV, more preferably in the range of 120 mV to 220 mV. By using an increased applied potential, it is possible to increase the discrimination between different nucleotides by the pore. Any suitable electrically conductive solution can be used. For example, the solution can comprise a charge carrier, such as a metal salt, such as an alkali metal salt, a halide salt, such as a chloride salt, such as an alkali metal chloride salt. The charge carrier can include an ionic liquid or an organic salt, such as tetramethyl ammonium chloride, trimethylphenyl ammonium chloride, phenyltrimethyl ammonium chloride, or 1-ethyl-3-methyl imidazolium chloride. In an exemplary system, the salt is present in an aqueous solution within the chamber. Potassium chloride (KCl), sodium chloride (NaCl), cesium chloride (CsCl), or mixtures of potassium ferrocyanide and potassium ferricyanide are typically used. KCl, NaCl, and mixtures of potassium ferrocyanide and potassium ferricyanide are preferred. The charge carrier can be asymmetric across the membrane. For example, the type and/or concentration of the charge carrier can be different on each side of the membrane, e.g., in each chamber.

The salt concentration can be saturating. The salt concentration can be up to 3 M, and is typically from 0.1 to 2.5 M, from 0.3 to 1.9 M, from 0.5 to 1.8 M, from 0.7 to 1.7 M, from 0.9 to 1.6 M, or from 1 M to 1.4 M. The salt concentration is preferably from 150 mM to 1 M. The method is preferably performed using a salt concentration of at least 0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M or at least 3.0 M. High salt concentrations provide a high signal-to-noise ratio, allowing currents indicating the presence of nucleotides to be discerned against a background of normal current fluctuations.

The electrically conductive solution may contain a buffer. Typically, the buffer is a phosphate buffer. Other suitable buffers are HEPES and Tris-HCl buffer. The pH of the electrically conductive solution may be from 4.0 to 12.0, from 4.5 to 10.0, from 5.0 to 9.0, from 5.5 to 8.8, from 6.0 to 8.7, or from 7.0 to 8.8, or from 7.5 to 8.5. The pH used is preferably about 6.9.

The system can include an array of pore complexes present in the membrane. In a preferred embodiment, each membrane of the array includes a pore complex. Due to the manner in which the array is formed, for example, the array can 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 can include from about 2 to about 12,000 membranes, for example, 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 incorporated into a device. The device may be any conventional device for analyte analysis, such as an array or a 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 perforations through which a membrane containing pores is formed. Alternatively, the barrier forms a membrane through which pores are present.

In one embodiment, the device comprises a sensor device capable of supporting a plurality of pores and membranes and operable to perform characterization of an analyte using the pores and membranes; and at least one port for transferring data for performing the characterization.

In one embodiment, the device comprises 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 reservoir for holding a material for performing characterization.

In one embodiment, the device comprises a membrane and a plurality of pores and a sensor device operable to support the membrane and perform characterization of an analyte using the pores and the membrane; at least one reservoir for holding a substance for performing the characterization; a fluidics system configured to controllably supply a substance from the at least one reservoir to the sensor device; and one or more vessels for receiving each sample, wherein the fluidics system is configured to selectively supply a sample from the one or more vessels to the sensor device.

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

membrane

Any suitable membrane may 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, which have both hydrophilic and lipophilic properties. The amphiphilic molecules may be synthetic or naturally occurring. Non-naturally occurring amphiphiles and monolayer-forming amphiphiles 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 sub-units are polymerized together to form a single polymer chain. Block copolymers typically have properties contributed by each of the monomer sub-units. However, block copolymers may have unique properties that are not possessed by polymers formed from the individual sub-units. Block copolymers can be engineered so that one of the monomer sub-units is hydrophobic (i.e., lipophilic) while the other sub-unit(s) is hydrophilic while in an aqueous medium. In this case, the block copolymer can possess amphiphilic properties and can form structures that mimic biological membranes. The block copolymers can be diblocks (consisting of two monomer sub-units), but can also be constructed from more than two monomer sub-units to form more complex arrangements that behave like amphiphiles. The copolymers can be triblock, tetrablock, or pentablock copolymers. The membrane is preferably a triblock copolymer membrane.

Archaeal amphiphilic tetraether lipids are naturally occurring lipids engineered to form monolayer membranes. These lipids are commonly found in extremophiles, thermophiles, halophiles, and acidophiles that survive in harsh biological environments. Their stability is thought to derive from the fused nature of the final bilayer. It is straightforward to engineer block copolymer materials that mimic these biological entities by producing triblock polymers with a general motif of hydrophilic-hydrophobic-hydrophilic. These materials behave similarly to lipid bilayers and can form monomeric membranes with a variety of phase behaviors, from vesicles to laminar membranes. Membranes formed from these triblock copolymers have several advantages over biological lipid membranes. Since triblock copolymers are synthetic, their precise construction can be carefully controlled to provide the precise chain lengths and properties necessary to form membranes and interact with pores and other proteins.

Block copolymers may also be composed of sub-units that are not classified as lipid sub-substances. For example, the hydrophobic polymer may be made of siloxanes or other non-hydrocarbon based monomers. The hydrophilic sub-section of the block copolymer may also possess low protein binding properties, which allows for the production of highly resistant membranes when exposed to unprocessed biological samples. The head group unit may also be derived from non-classical lipid head-groups.

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

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

The amphiphilic molecule may be chemically modified or functionalized to facilitate coupling of the polynucleotides. The amphiphilic layer may be a single layer or a double layer. The amphiphilic layer is typically planar. The amphiphilic layer may be curved. The amphiphilic layer may be supported.

Amphiphilic membranes are typically mobile in nature, essentially behaving as two-dimensional fluids with lipid diffusion rates of the order of 10 ^-8 cm s ^-1 . This means that pores and coupled polynucleotides can typically move within an amphiphilic membrane.

The membrane may be a lipid bilayer. Lipid bilayers are models of cell membranes and serve as excellent platforms for a variety 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 various substances. The lipid bilayer may 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), wherein the lipid monolayer is transported across the aqueous/air interface across one side of the perforation perpendicular to that interface. The lipid is usually added to the surface of an aqueous electrolyte solution by first dissolving it in an organic solvent and then allowing droplets of the solvent to evaporate from the surface of the aqueous solution on one side of the perforation. As the organic solvent evaporates, the solution/air interface on the side of one of the perforations physically moves up and down through the perforation until a bilayer is formed. Planar lipid bilayers can be formed across perforations in the membrane or across openings that enter a recess.

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

Tip-immersion bilayer formation involves touching a perforated surface (e.g., a pipette tip) to the surface of a test solution carrying a lipid monolayer. In other words, the lipid monolayer is first created at the solution/air interface by allowing a droplet of lipid dissolved in an organic solvent to evaporate from the solution surface. The bilayer is then formed by the Langmuir-Schaefer process, requiring mechanical automation to move the perforation relative to the solution surface.

For painted bilayers, a drop of lipid dissolved in an organic solvent is applied directly to the perforation, which is then immersed in the test solution. The lipid solution is spread thinly across the perforation using a paintbrush or equivalent. Thinning of the solvent results in the formation of a lipid bilayer. However, complete removal of the solvent from the bilayer is difficult, and consequently, bilayers formed by this method are less stable and more susceptible to noise during electrochemical measurements.

Patch-clamping is commonly used in the study of biological membranes. The membrane is clamped to the end of a pipette by suction, and a patch of membrane is attached over the perforation. This method has been applied to produce lipid bilayers by clamping liposomes, which are then ruptured, leaving a lipid bilayer seal over the perforation of the pipette. This method requires the fabrication of stable, large unilamellar liposomes and small perforations in materials with a glassy 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 dried lipids. In a most preferred embodiment, the lipid bilayer is formed across the opening as described in WO2009/077734.

Lipid bilayers are formed of two opposing lipid layers. The two lipid layers are arranged so that the hydrophobic tail groups point toward each other to form a hydrophobic interior. The hydrophilic head groups of the lipids point outward toward the aqueous environment on each side of the bilayer. Bilayers can exist in a number of lipid phases, including but not limited to a liquid disordered phase (fluid lamellar), a liquid ordered phase, a solid ordered phase (lamellar gel phase, interdigitated gel phase), and a planar bilayer crystal (lamellar sub-gel phase, lamellar crystalline phase).

Any lipid composition that forms a lipid bilayer can be used. The lipid composition is selected such that a lipid bilayer having the desired properties, such as surface charge, ability to support membrane proteins, packing density, or mechanical properties, is formed. The lipid composition can comprise one or more different lipids. For example, the lipid composition can contain up to 100 lipids. The lipid composition preferably contains 1 to 10 lipids. The lipid composition can comprise 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 acylglycerides (DG) and ceramides (CM); zwitterionic head groups, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM); negatively charged head groups, such as phosphatidylglycerol (PG); phosphatidylserine (PS), phosphatidylinositol (PI), phosphate (PA), and cardiolipin (CA); and positively charged head groups, such as trimethylammonium-propane (TAP). Suitable interfacial moieties include, but are not limited to, naturally occurring interfacial moieties, such as glycerol-based 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 -tetradecanoic acid), palmitic acid ( n- hexadecanoic acid), stearic acid ( n- octadecanoic acid), and arachidic acid ( n- eicosanoic acid); unsaturated hydrocarbon chains, such as oleic acid ( cis -9-octadecanoic acid); and branched hydrocarbon chains, such as phytanoyl. The length of the chain and the position and number of double bonds in the unsaturated hydrocarbon chain can vary. The length of the chain and the position and number of branches, such as methyl groups, can vary. The hydrophobic tail group can be linked to the interfacial moiety as an ether or ester. The lipid can be mycolic acid.

Lipids can also be chemically modified. The head group or tail group of the lipid can be chemically modified. Suitable lipids having a chemically modified head group 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 having chemically modified tail groups include polymerizable lipids, such as 1,2-bis(10,12-tricosadiinoyl)-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. The lipids can be chemically modified or functionalized to facilitate coupling of the polynucleotides.

The amphiphilic layer, e.g., the lipid composition, typically includes 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 of both organic and inorganic materials, including but not limited to microelectronic materials, insulating materials such as Si ₃ N ₄ , Al ₂ O ₃ and SiO, organic and inorganic polymers such as polyamides, plastics such as Teflon® or elastomers such as two-component addition-cured silicone rubbers, and glasses. The solid state layer can be formed of graphene. Suitable graphene layers are disclosed in WO 2009/035647. When the membrane comprises a solid state layer, the pores are typically present within an amphiphilic membrane or layer contained within the solid state layer, for example, holes, wells, gaps, channels, trenches 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 performed using (i) an artificial amphiphilic layer comprising a pore, (ii) an isolated naturally occurring lipid bilayer comprising a pore, or (iii) a cell having a pore inserted therein. The method is typically performed using an artificial amphiphilic layer, such as an artificial triblock copolymer layer. The layer may comprise other molecules in addition to the pore, as well as other transmembrane and/or intramembrane proteins. Suitable apparatus and conditions are discussed below. The methods of the present disclosure are typically performed in vitro .

How to characterize analytes

In a further aspect, a method of determining the presence, absence, or one or more characteristics of a target analyte is disclosed. The method comprises the steps of contacting a target analyte with a membrane comprising a pore complex, allowing the target analyte to migrate across a continuous channel comprising a nanopore within the pore complex and at least two structures provided by an auxiliary protein or peptide, for example, into or through the continuous channel, and performing one or more measurements as the analyte migrates across the channel, thereby determining the presence, absence, or one or more characteristics of the analyte. The analyte can pass through the nanopore constriction and then through the auxiliary protein constriction. In an alternative embodiment, the analyte can pass through the nanopore constriction and then through the auxiliary protein constriction depending on the orientation of the pore complex in the membrane.

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

Binding of a molecule within or near the opening of a pore complex will affect the open-channel ion flux through the pore, which is the essence of "molecular sensing" of the pore channel. In a manner analogous to nucleic acid sequencing applications, the variation in the open-channel ion flux can be measured using a suitable measurement technique by changes in current (see, e.g., WO 2000/28312 and D. Stoddart et al., Proc. Natl. Acad. Sci., 2010, 106, 7702-7 or WO 2009/077734). The extent of the decrease in ion flux, as measured by a decrease in current, is related to the size of the obstruction 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, forming the basis of a "biological sensor". Molecules suitable for nanopore sensing include nucleic acids; proteins; peptides; Polysaccharides and small molecules (herein referred to as low molecular weight (e.g., <900 Da or <500 Da) organic or inorganic compounds), such as pharmaceuticals, toxins, cytokines and pollutants. Detecting the presence of biological molecules has applications in personalized drug development, medicine, diagnostics, life science research, environmental monitoring, and in the security and/or defense industries.

The target analyte can be a metal ion, an inorganic salt, a polymer, an amino acid, a peptide, a polypeptide, a protein, a nucleotide, an oligonucleotide, a polynucleotide, a monosaccharide, a polysaccharide, a dye, a bleach, a pharmaceutical, a diagnostic agent, a recreational drug, an explosive, a toxic compound, or an environmental pollutant. The method can involve determining the presence, absence, or one or more characteristics of two or more analytes of the same type, such as two or more proteins, two or more nucleotides, or two or more pharmaceuticals. Alternatively, the method can involve determining the presence, absence, or one or more characteristics of two or more analytes of different types, such as 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 inside the cell, and thus the analyte must be extracted from the cell before the method is performed.

In one embodiment, the analyte is an amino acid, a peptide, a polypeptide, or a 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 listed 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 macromolecule comprising two or more nucleotides. Naturally occurring nucleic acid bases in DNA and RNA can be distinguished by their physical size. When a nucleic acid molecule or individual base passes through the channel of a nanopore, the size difference between the bases causes a directly correlated decrease in ion flux through the channel. The variation in ion flux can be recorded. Suitable electrical measurement techniques for recording the variation in ion flux are, for example, WO 2000/28312 and [D. Stoddart et al., Proc. Natl. Acad. Sci., 2010, 106, pp 7702-7] (single channel recording apparatus); and, for example, WO 2009/077734 (multi-channel recording technique). With suitable compensation, the characteristic decrease in ion flux can be used to identify specific nucleotides and associated bases traversing the channel in real time. In typical nanopore nucleic acid sequencing, the open-channel ion flux is reduced as individual nucleotides of a nucleic acid sequence of interest pass sequentially through the channel of the nanopore due to partial blockage of the channel by nucleotides. It is this reduction in ion flux that is measured using the appropriate recording techniques described above. The reduction in ion flux can be compensated for by a measured reduction in ion flux for a known nucleotide through the channel, resulting in a means of determining which nucleotides pass through the channel, and thus, when performed sequentially, the nucleotide sequence of the nucleic acid passing through the nanopore. For accurate determination of individual nucleotides, it has typically been desired that the reduction in ion flux through the channel be directly correlated to the size of the individual nucleotides passing through the constriction (or "read head"). It will be appreciated that sequencing can be performed on intact nucleic acid polymers that are "threaded" through the pore, for example, by the action of an associated polymerase or helicase. Alternatively, the sequence can be determined by passage of nucleotide triphosphate bases sequentially removed from the target nucleic acid in proximity to the pore (see, e.g., WO 2014/187924).

The polynucleotide or nucleic acid can comprise any combination of any nucleotides. The nucleotides can be naturally occurring or artificial. One or more of the nucleotides in the polynucleotide can be oxidized or methylated. One or more of the nucleotides in the polynucleotide can be damaged. For example, the polynucleotide can comprise a pyrimidine dimer. Such dimers are typically associated with damage caused by ultraviolet radiation and are a major cause of skin melanoma. One or more of the nucleotides in the polynucleotide can be modified, for example, using a label or tag, suitable examples of which are known to those skilled in the art. The polynucleotide can comprise one or more spacers. The nucleotides typically contain a nucleobase, a sugar, and at least one phosphate group. The nucleobase and the 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. The polynucleotide preferably comprises the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU), and/or thymidine (dT), deoxyguanosine (dG), and deoxycytidine (dC). The nucleotides are typically ribonucleotides or deoxyribonucleotides. The nucleotides typically contain a monophosphate, a diphosphate, or a triphosphate. The nucleotides can contain more than three phosphates, such as four or five phosphates. The phosphates can be attached to the 5' or 3' side of the nucleotide. The nucleotides in the polynucleotide can be attached to each other in any manner. The nucleotides are typically attached by sugar and phosphate groups, as in nucleic acids. The nucleotides can be linked via nucleobases, as in pyrimidine dimers. The polynucleotide can 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 using a polynucleotide as an analyte alternatively comprises the step of 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.

The polynucleotide (i) can be of any length. For example, the polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotides or nucleotide pairs in length. The polynucleotide can be at least 1000 nucleotides or nucleotide pairs in length, at least 5000 nucleotides or nucleotide pairs in length, or at least 100000 nucleotides or nucleotide pairs in length. Any number of polynucleotides can be examined. For example, the method can relate to 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 may be different polynucleotides or two instances of the same polynucleotide. The polynucleotides may be naturally occurring or artificial. For example, the method may be used to determine the sequence of a manufactured oligonucleotide. The method is typically performed in vitro .

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

A pore complex comprising at least two constrictions is particularly useful for analyzing homopolymers. For example, the pore can be used to determine the sequence of a polynucleotide comprising two or more identical, for example, at least 3, 4, 5, 6, 7, 8, 9 or 10 consecutive nucleotides. For example, the pore can be used to sequence a polynucleotide comprising a polyA, a polyT, a polyG and/or a polyC region.

In some embodiments, the CsgG pore constriction is comprised of residues at positions 51, 55, and 56 of SEQ ID NO: 59. As DNA passes through the constriction, at any given time, the interaction of the pore's constriction with approximately 5 bases of DNA dominates the current signal. Certain CsgG pores (e.g., CsgG pores lacking one or more accessory proteins or fusion proteins as described herein) are very good at reading mixed sequence regions of DNA (where As, Ts, Gs, and Cs are mixed), but the signal flattens and lacks some information when there are homopolymer regions within the DNA (e.g., poly-T, poly-G, poly-A, poly-C). Because the 5 bases dominate the signal of CsgG and its constriction mutants, it is difficult to discern homopolymers longer than 5 bases without using additional residence time information. However, as the DNA passes through the second constriction, more DNA bases interact with the combined constriction, increasing the length of the recognizable homopolymer.

Kit

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

Although specific embodiments, specific configurations, as well as materials and/or molecules, for the engineered cells and methods according to the present disclosure have been discussed herein, 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 are not to be construed as limiting the application. The application is limited only by the claims.

Example

Example 1

To generate the helical constriction, a novel design was used to select small protein domains that are well folded and project into the lumen of the nanopore to a desired extent. Several programs are available for this purpose. This example describes a workflow using the MASTER program to facilitate backbone design and Rosetta to perform sequence selection using variable backbone geometry.

Programs such as RF diffusion, CHROMA, or MASTER can be used to project new domains into the pore lumen. Here, MASTER was used. The Protein Data Bank (PDB) was searched for structures matching the following criteria: 1) stabilization of the target region of CsgF (residues 16–30); 2) diameter (Ca-to-Ca distance of the amino acid residues extending furthest into the pore lumen) of 10 when all units were generated using the ninefold symmetry operator. and 30 3) The new domain must not collide with atoms of CsgG or its symmetric counterpart in CsgF;

First, helices that are frequently observed in natural proteins in the PDB and dock to the target region of CsgF and its symmetric neighbors in a “designable” geometry were identified. After clustering the output based on the RMSD of the target region and the discovered helices, the top candidates were selected based on the number of closely related helix-helix pairs found in the database (Fig. 1). In this way, well-packed helical geometries were selected for the target and the four amino acids at the N-terminus of the target. In addition, helices that participate in favorable helix-helix interactions with symmetry-related partners were searched in the database. Linkers (e.g., loop structures) that connect the helices were selected using the helix backbone database (Fig. 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 the lowest energy score and highest PackStat score, as shown in Figure 2. To further prioritize sequences, their aggregation propensity can also be tested using one of several aggregation and amyloid prediction programs.

Example 2

Materials and Methods

Escherichia coli CsgG pore generation

A recombinant expression vector encoding a CsgG mutant nanopore with a C-terminal Strep affinity tag and an ampicillin resistance gene was transformed into chemocompetent E. coli cells. Cells were streaked on LB agar plates containing the appropriate antibiotics for selection and grown overnight at 37°C. A single colony from the agar plate was inoculated into LB medium containing the appropriate antibiotics and grown overnight at 37°C with shaking. The culture was diluted with autoinduction medium and the appropriate antibiotics and grown at 18°C with shaking for 68 h. Cells were harvested by centrifugation, lysed, and extracted with 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 size exclusion chromatography, and oligomeric nanopores were selected for by SDS-PAGE.

Protocol for formation of CsgG/CsgF or fusion protein complexes

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

SDS-PAGE analysis

1 μg of complex and CsgG-only pore control were added to individual 0.5 mL ProteinLoBind Eppendorf tubes (Fisher, 10316752) and made up to 10 μL with reaction buffer. Final volume was made up to 20 μL by adding 10 uL of 2x Laemmli buffer. Each sample was loaded in its entirety onto a 4-20% TGX gel (BioRad, 5671093) run with 1x TGS buffer (Sigma, T7777). It was run at 300 V for 21 minutes. To image the gel, Spyro Ruby (Merk, S4942) dye was used according to the manufacturer's instructions. It was then imaged on a GE Typhoon gel imager using a 450 nm laser.

For some assays, 1 ug of complex and CsgG-only pore control were added to individual PCR tubes and made up to 10 μL volume with reaction buffer. Freshly prepared 1 M DTT stock was prepared and added to individual PCR tubes to a final concentration of 10 mM. 10 μL of 2x Laemmli buffer was added to make up to a final volume of 20 μL. Each sample was heated at 95 °C for 2 minutes in a PCR thermocycler. After cooling for 5 minutes, material from each sample was loaded in its entirety onto a 4-20% TGX gel (BioRad, 5671093) run using 1x TGS buffer (Sigma, T7777). This was run at 300 V for 21 minutes. Spyro Ruby (Merk, S4942) dye was used to image the gel according to the manufacturer's instructions. Subsequently, images were obtained on a GE Typhoon gel imager using a 450 nm laser.

Electrical Measurement

Electrical measurements were obtained from CsgG-only, CsgG/CsgF, or CsgG/fusion protein complexes inserted into the MinION flow cell. 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.

The analyte used to evaluate DNA waveforms was a 3.6-kilobase DNA section from the 3' end of the lambda genome as described in Figure 23. Preparation of the analyte, ligation of the analyte to the Y-adapter, SPRI-bead clean-up of the ligated analyte, and addition to the minION flow cell were performed using the Oxford Nanopore Technologies Q-SQK-LSK110 protocol.

Electrical measurements were acquired using a minION Mk1b from Oxford Nanopore Technologies. A standard sequencing script was run for 6 h at -180 mV with static flicks every 5 min to remove extended nanopore block. Raw data were collected as bulk FAST5 files using MinKNOW software (Oxford Nanopore Technologies).

Discriminant profiling

FAST5 containing DNA waveforms (i.e., electrical measurements) for a 3.6-kilobase DNA section from the 3' end of the lambda genome (3.6 Kb lambda) was acquired. DNA waveforms were trimmed using a custom Python script to remove any electrical signal measurements captured before DNA sequencing was initiated.

The parameters of the neural network were trained using the trimmed 3.6 Kb lambda-waveline and the corresponding genome references for these regions. The neural network, which consists of four layers, contains modeled sequences of user-specified span lengths and their associated current levels. The span length specified for these models allowed +/- 12 nucleotide regions to contribute to the current level at any one location.

The trained neural network was used to predict current levels corresponding to a 3.6 Kb lambda DNA reference sequence, and also to predict current levels for all possible single-base edits of that sequence.

Changing bases at a single position (L) in the sequence changes the predicted current when that base passes through the major constriction of the pore, but also changes the current before and after the base passes through this major constriction. The predicted current levels were analyzed for the edited 3.6 Kb lambda sequence set to calculate the range of predicted current at positions L+X (offsets) when the base at position L is changed. Offsets between -16 and +16 were analyzed for each position. The median range of predicted current at each offset was calculated to provide data for the diagrams. The model was centered so that the largest peak representing the CsgG constriction corresponds to position 0.

Example 3

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

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

SDS-PAGE gel analysis of CsgG-only pore and CsgG/fusion protein complexes was performed. The complexes were composed of CsgF-del(S31-F119) control or newly designed fusion proteins with or without maleimide crosslinker (Fig. 5). The complexes containing the fusion proteins showed a band shift, indicating that these samples were nanopore complexes. The samples were not heated prior to loading onto the gel. SDS-PAGE gel analysis of CsgG-only pore and CsgG/fusion protein complexes was also performed. The complexes were composed of CsgF-del(S31-F119) control or newly designed fusion proteins with or without maleimide crosslinker (Fig. 6). The pores were immediately decomposed into their constituent monomer components upon boiling in the presence of DTT prior to loading onto the gel. No band shift was observed in the absence of maleimide crosslinker, indicating that these bands were composed solely of CsgG monomers. Lane 7 showed 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 showed additional band shifts due to the increase in mass of the fusion protein, indicating that the fusion protein is covalently bound to the CsgG pore.

Ionic current (pA) versus time (s) was measured as single-stranded DNA translocated through a 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 presents representative data for median range, median noise, and median signal-to-noise ratio (SNR) of protein pore complexes as described by the present disclosure.

Matrix table Pore monomer (conjugate) Median range (pA) Median noise (pA) Median SNR CsgG-F56Q 25.01 3.84 6.49 CsgG-F56Q / del(S31-F119) 12.64 1.77 7.11 CsgG-F56Q/del(S31-F119)-EXT1 12.63 1.78 6.99 CsgG-F56Q/K37R-del(S31-F119)-EXT1 12.46 1.74 7.04 CsgG-F56Q/N24C-K37R-del(S31-F119)-EXT2 15.72 2.59 5.71 CsgG-F56Q-Q153C 25.62 3.53 7.26 CsgG-F56Q-Q153C / Mal-Del(S31-F119) 13.17 1.75 7.48 CsgG-F56Q-Q153C / Mal-K37R-Del(S31-F119)-EXT1 12.85 1.77 7.25 CsgG-F56Q-Q153C / Mal-N24C/K37R-Del(S31-F119)-EXT2 15.81 2.15 7.36

Figures 7-11 show representative ion current (pA) versus time (s) traces as single-stranded DNA translocates through a CsgG-only pore, CsgG comprising the del(S31-F119) CsgF peptide, or CsgG comprising the novel designed fusion proteins. The raw current traces are shown as black lines, and the event detection signals are shown as red lines. For each pore, the top row shows the entire DNA current trace, and the bottom row shows an enlarged view of the first section of the current trace. The open pore current for the CsgG-only pore was observed to be approximately 175-200 pA, with a median current of the DNA waveform being approximately 75 pA. For the pore comprising the CsgF peptide, the open pore current was approximately 90-120 pA, with a median current of approximately 35-50 pA. Figure 7 shows traces as DNA translocates through a CsgG-only pore. Figure 8 shows representative ion current (pA) versus time (s) traces when single-stranded DNA is translocated through CsgG comprising del(S31-F119) CsgF peptide with (right) and without (left) a maleimide crosslinker. Figure 9 shows representative ion current (pA) versus time (s) traces when single-stranded DNA is translocated through CsgG comprising the novel designed fusion protein, ONLP20623, in the absence of a maleimide crosslinker. Figure 10 shows representative ion current (pA) versus time (s) traces when single-stranded DNA is translocated through CsgG comprising the novel designed fusion protein, ONLP20624 (without maleimide crosslink) or ONLP20627 (with a maleimide crosslink). Figure 11 shows representative ion current (pA) versus time (s) traces when single-stranded DNA is translocated through CsgG comprising the novel designed fusion proteins, ONLP20628 (with maleimide cross-linker) or ONLP20625 (without maleimide cross-linker). In some embodiments, the fusion protein comprises a 37R residue together with a cysteine residue to form an internal disulfide bond within the peptide, i.e., cyclization of the fusion protein.

Profiles were generated demonstrating their contribution to the overall change in ionic current level ("discrimination") and position within the pore when a DNA molecule is translocated 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). Dashed boxes indicate regions affected by introduction of the novel designed fusion proteins. Figure 12 shows representative profiles when a DNA molecule is translocated through a CsgG-only pore. The CsgG-only pore (+/- Q153C) exhibits one major discriminant peak at position 0. Figure 13 shows representative profiles when a DNA molecule is translocated through a CsgG/CsgF pore. Dashed boxes indicate regions affected by introduction of the novel designed fusion proteins. CsgG-CsgF-del(S31-F119) pores with (right) or without (left) the maleimide crosslinker exhibit two discriminant peaks: a major discriminant peak at position 0 as seen in the CsgG-only pore and an additional discriminant peak 4-6 nucleotides below the major constriction (positions -4 to -6). This additional discriminant region has less influence on the ion current than the major discriminant peak at position 0. Figure 14 shows representative profiles when DNA molecules are translocated through a CsgG/fusion protein (ONLP20641 or ONLP20644) pore. Complexes composed of CsgG and the newly designed fusion proteins containing K37R with cyclization with (right) or without (left) the maleimide crosslinker exhibit three discriminant peaks. As seen in the CsgG-only pore, the main discriminant peak is at position 0, with additional peaks at positions -6 and -9. The peak at position -9 corresponds to the expected constriction created by the newly designed fusion protein when folded in the correct orientation.

Example 3

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

Representative sequence

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

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNGGELAAKLWANGDETNALSLFQTIIQS

> (SEQ ID NO: 2) CsgF-WT-K37R-del(S31-F119)-Ext(31-GGELAAKLWANGDETNALSLFQTIIQS) (ONLP20624)

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNGGELAARLWANGDETNALSLFQTIIQS

> (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)

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLWENGNVNQALSLFQTVIQS

> (SEQ ID NO: 5) Mat-CsgF-Eco-(WT-K36R/K37R-Del(S31-F119)-Ext(31-AGELAKKLWENGNVNQALSLFQTVIQS) (ONLZ19431)

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELARRLWENGNVNQALSLFQTVIQS

> (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

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAAELAAKLWANADETNALSLFQTIIQS

>(SEQ ID NO: 8) ONT113_3

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAAELAAKLWANADETNALSLFQTLIQS

> (SEQ ID NO: 9) ONT1

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLFKKGDLTNALSLFQTVIQS

> (SEQ ID NO: 10) ONT2

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELVEKLFKNGDWTNAISIFQTVIQS

> (SEQ ID NO: 11) ONT 3

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAEKLWRNGDETNALSLFQTVIQS

> (SEQ ID NO: 12) ONT 4

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAEKLWKNGDETNALSLFQTVIQS

> (SEQ ID NO: 13) ONT 5

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLWENGDETNALSLFQTVVQS

> (SEQ ID NO: 14) ONT 6

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAEKLWRNGNESDALSLFQTVIQS

> (SEQ ID NO: 15) ONT 7

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLFENGDKTNALSLFQTVIQS

> (SEQ ID NO: 16) ONT 8

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLWENGDETNALSLFQTVIQS

> (SEQ ID NO: 17) ONT 9

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLWEKGNSEDALALFRTVVQS

> (SEQ ID NO: 18) ONT 10

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLFDNGDMENAMKLFQTVIAS

> (SEQ ID NO: 19) ONT 11

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAEKLWRNGDKDRALALFRTVIQS

> (SEQ ID NO: 20) ONT 12

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELADKLWKNGDKDRALSLFQTVIQS

> (SEQ ID NO: 21) ONT 13

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLFDNGDMDRALALFRTVIAS

> (SEQ ID NO: 22) ONT 14

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLFDNGNEEDALALFRTVVAS

> (SEQ ID NO: 23) ONT 15

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLWKKGDEENALKLFRTVVTS

> (SEQ ID NO: 24) ONT 16

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLFKNGNMEDALKLFRTVIAS

> (SEQ ID NO: 25) ONT 17

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGKVAAILWKNGNKSDALSLFQTVVTS

> (SEQ ID NO: 26) ONT 18

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLFKNGDLTNALSLFQTVVQS

> (SEQ ID NO: 27) ONT 19

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELGLKLLRKGDVETALTLFAQVISG

> (SEQ ID NO: 28) ONT 20

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELGLKLILKGDLETALKLFAIVIAG

> (SEQ ID NO: 29) ONT 21

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELGLKLLRKGDVETALKLFAIVIAG

> (SEQ ID NO: 30) ONT 22

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLYENGLIELALMLFALVIAS

> (SEQ ID NO: 31) ONT 23

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELYKKLWDNGEVDKALDLFAKIIAG

> (SEQ ID NO: 32) ONT 24

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELGKKLIEKGDLETALKLFAIVIAG

> (SEQ ID NO: 33) ONT 25

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGEIALRLLKNGKEEEALKTLLVTIAG

> (SEQ ID NO: 34) ONT26

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLWKKGDETNALSLFQTVVTS

> (SEQ ID NO: 35) ONT27

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGKVAAILWKNGNKSDALSLFQTVVTS

> (SEQ ID NO: 36) ONT28

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLWEKGDETNALSLFQTVVTS

> (SEQ ID NO: 37) ONT29

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGDLAAKLWKKGDETNALSLFQTVVTS

> (SEQ ID NO: 38) ONT30

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLWKNGNSSDALSLFQTVVTS

> (SEQ ID NO: 39) ONT31

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLWEKGDETNALSLFQTVVTS

> (SEQ ID NO: 40) ONT32

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLWEKGDSSNALSLFQTVVTS

> (SEQ ID NO: 41) ONT33

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGDLAAKLWKNGDETNALSLFQTVVTS

> (SEQ ID NO: 42) ONT34

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLFKNGDLTNALSLFQTVVQS

> (SEQ ID NO: 43) ONT35

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLWKKGDETNALSLFQTVVTS

> (SEQ ID NO: 44) ONT36

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLFNSGDLDRALALFRTVVTS

> (SEQ ID NO: 45) ONT37

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGKVAKELYDNGDEKWALLLFRTVVTS

> (SEQ ID NO: 46) ONT38

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGKVAAELYKNGDEKNALLLFRTVVAS

>(SEQ ID NO: 47) ONT39

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLFKNGDMENALALFRTVVTS

>(SEQ ID NO: 48) ONT40

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLWEKGNSEDALALFRTVVQS

> (SEQ ID NO: 49) ONT41

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLFNKGDEDRALALFRTVVQS

> (SEQ ID NO: 50) ONT42

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLWKNGDEENALALFRTVVTS

> (SEQ ID NO: 51) ONT43

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAEKLWRSGDADRALALFRTVVTS

> (SEQ ID NO: 52) ONT44

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLWKNGNEEDALALFRTVVTS

> (SEQ ID NO: 53) ONT45

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLFNNGDEDRALALFRTVVQS

> (SEQ ID NO: 54) ONT46

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLWKKGDEDRALALFRTVVTS

> (SEQ ID NO: 55) ONT47

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLFNSGDEDRALALFRTVVQS

> (SEQ ID NO: 56) ONT48

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAAKLYNNGDLDRADATFRTVVQS

> (SEQ ID NO: 57) ONT49

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGELAKKLWENGNEEDALALFRTVVTS

> (SEQ ID NO: 58) ONT50

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGEIAKQLWEKGDESSAITVATIVLSS

>(SEQ ID NO: 59) Wild-type E. coli CsgG protein monomer (without signal sequence)

CLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQFKPYPASNFSTAVPQSATAMLVTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIMVEGSIIGYE SNVKSGGVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGEVGYTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPES

>(SEQ ID NO: 60) Residues 1-30 of CsgF peptide

GTMTFQFRNPNFGGNPNNGAFLLNSAQAQN

>(SEQ ID NO: 61) CsgF-WT-del(S31-F119)-Ext(31-AGILAAQLWNNGDYDRALSLFIAVVQS-57) GTMTFQFRNPNFGGNPNNGAFLLNSAQAQNAGILAAQLWNNGDYDRALSLFIAVVQS

Attach electronic file of sequence list

Claims (61)

As a protein nanopore complex,
(a) CsgG nanopores comprising an inner lumen; and
(b) a protein nanopore complex comprising a fusion polypeptide comprising a first portion comprising a CsgF protein and a second portion comprising a helix-forming accessory protein, wherein the fusion protein is attached to the nanopore. In the first aspect, the first portion of the fusion protein is a protein nanopore complex attached to the CsgG nanopore. A protein nanopore complex according to claim 1 or 2, wherein the first portion of the fusion protein is positioned within the lumen of the CsgG nanopore. A protein nanopore complex according to any one of claims 1 to 3, wherein the first portion of the fusion protein extends outside the lumen of the CsgG nanopore. A protein nanopore complex according to any one of claims 1 to 4, wherein the first portion forms a first constricted region in the lumen of the CsgG nanopore. In the fifth paragraph, the second portion is a protein nanopore complex forming a second constricted region. A protein nanopore complex according to any one of claims 1 to 6, wherein the CsgG nanopore further comprises a constriction region. A protein nanopore complex according to any one of claims 1 to 7, wherein the second portion is not attached to the CsgG nanopore. A protein nanopore complex according to any one of claims 1 to 8, wherein the second portion comprises at least one alpha helix. A protein nanopore complex according to any one of claims 1 to 9, wherein each of said alpha helices comprises 0 to 15 alpha helical turns. A protein nanopore complex according to any one of claims 1 to 10, wherein the second portion comprises a first alpha helix comprising 1 to 4 alpha helix turns and a second alpha helix comprising 3 to 6 alpha helix turns. In claim 11, a protein nanopore complex wherein the second alpha helix is packed into the first alpha helix. A protein nanopore complex according to any one of claims 9 to 12, wherein the second portion comprises 1 to 55 amino acid residues. In any one of claims 6 to 13, the distance between the first constriction region and the second constriction region is in a range of about 5 About 80 Human protein nanopore complex. In any one of claims 1 to 14, the protein nanopore complex is 90 having an excess shaft length, optionally the shaft length being in the range of about 95 About 160 Human protein nanopore complex. A protein nanopore complex according to any one of claims 1 to 15, wherein the fusion protein is attached to the nanopore by a linker. In claim 16, the linker is a protein nanopore complex comprising a bond, a peptide linker or a chemical linker. A protein nanopore complex according to claim 16 or 17, wherein the linker comprises a bond formed by a sulfur (VI) fluoride exchange (SuFEx) reaction. A protein nanopore complex according to claim 16 or 17, wherein the linker comprises one or more maleimide molecules. A protein nanopore complex according to any one of claims 1 to 19, wherein the fusion protein is a cyclized protein. In claim 20, the cyclization is a protein nanopore complex comprising one or more side chain-to-side chain cyclization bonds. A protein nanopore complex in claim 21, wherein at least one of the side chain-to-side chain cyclization bonds is a disulfide bond. As a protein nanopore complex,
(a) a CsgG nanopore comprising a lumen and a first constricted region formed within the lumen of the nanopore; and
(b) a protein nanopore complex comprising a fusion protein comprising a first portion comprising a CsgF protein and a second portion comprising a helix-forming accessory protein, wherein the fusion protein is attached to the nanopore. In claim 23, a protein nanopore complex wherein the first portion of the fusion protein is attached to the CsgG nanopore. A protein nanopore complex according to claim 23 or 24, wherein the first portion of the fusion protein is positioned within the lumen of the CsgG nanopore. A protein nanopore complex according to any one of claims 23 to 25, wherein the second portion of the fusion protein is positioned outside the lumen of the CsgG nanopore. A protein nanopore complex according to any one of claims 23 to 26, wherein the first portion forms a second constriction region in the lumen of the CsgG nanopore. In claim 27, the second portion is a protein nanopore complex forming a third constriction region in the lumen of the CsgG nanopore. A protein nanopore complex according to any one of claims 23 to 28, wherein the second portion is not attached to the CsgG nanopore. A protein nanopore complex according to any one of claims 23 to 29, wherein the second portion comprises at least one alpha helix. A protein nanopore complex in claim 30, wherein each of said alpha helices comprises 0 to 15 alpha helical turns. A protein nanopore complex according to any one of claims 23 to 31, wherein the second portion comprises 1 to 55 amino acid residues. A protein nanopore complex according to any one of claims 23 to 32, wherein the fusion protein is a cyclized protein. In claim 33, the cyclization is a protein nanopore complex comprising one or more side chain-to-side chain cyclization bonds. A protein nanopore complex in claim 34, wherein at least one of the side chain-to-side chain cyclization bonds is a disulfide bond. As a protein nanopore complex,
(a) a CsgG nanopore comprising a lumen and a first constricted region formed within the lumen of the nanopore;
(b) a first accessory protein that attaches to the CsgG nanopore and forms a second constriction region within the lumen of the nanopore; and
(c) a protein nanopore complex comprising a second accessory protein attached to the CsgG nanopore or the first accessory protein to form a third constriction region. In claim 36, the first auxiliary protein is a protein nanopore complex positioned within the lumen of the CsgG nanopore. A protein nanopore complex according to claim 36 or 37, wherein the first auxiliary protein comprises a CsgF protein or peptide. A protein nanopore complex according to any one of claims 36 to 38, wherein the second auxiliary protein comprises at least one alpha helix. A protein nanopore complex in claim 39, wherein each of said one or more alpha helices comprises 0 to 15 alpha helical turns. A protein nanopore complex according to claim 39 or 40, wherein the second auxiliary protein comprises two alpha helices. A protein nanopore complex in claim 41, wherein one of the alpha helices comprises 1 to 6 alpha helical turns. A protein nanopore complex according to claim 41 or 42, wherein one of the alpha helices comprises 1 to 10 alpha helical turns. A protein nanopore complex according to any one of claims 41 to 43, wherein one of the alpha helices comprises three alpha helical turns and the other alpha helix comprises three or four alpha helical turns. A protein nanopore complex according to any one of claims 36 to 44, wherein the second auxiliary protein comprises at least one alpha helix packed into an alpha helix of the first auxiliary protein. A protein nanopore complex according to any one of claims 36 to 45, wherein the second auxiliary protein comprises 1 to 55 amino acid residues. In any one of claims 36 to 46, the distance between the first constriction and the second constriction is in a range of about 10 About 80 Human protein nanopore complex. In any one of claims 36 to 47, the distance between the second constriction and the third constriction is in the range of about 5 About 80 Human protein nanopore complex. In any one of claims 36 to 48, the protein nanopore complex is 90 having an excess shaft length, optionally the shaft length being in the range of about 95 About 160 Human protein nanopore complex. A protein nanopore complex according to any one of claims 36 to 49, wherein the first auxiliary protein and the second auxiliary protein are attached by a linker. In claim 50, the linker is a protein nanopore complex comprising a bond, a peptide linker or a chemical linker. A protein nanopore complex in claim 50 or 51, wherein the linker comprises a bond formed by a sulfur (VI) fluoride exchange (SuFEx) reaction. A protein nanopore complex according to claim 50 or 51, wherein the linker comprises one or more maleimide molecules. A protein nanopore complex according to any one of claims 36 to 53, wherein the first auxiliary protein and the second auxiliary protein comprise one or more side chain-to-side chain cyclization bonds. A protein nanopore complex in 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, comprising a protein nanopore complex of any one of claims 1 to 55 inserted into a membrane. A system further comprising: an electrically conductive solution in contact with the protein nanopore complex, an electrode providing a voltage potential across the membrane; and a measurement system for measuring current passing through the protein nanopore complex, in claim 56. A method for characterizing a target analyte, comprising: (a) contacting a system according to claim 56 with the target analyte; (b) applying a potential across the membrane so that the target analyte moves about the lumen formed by the protein nanopore complex; and (c) performing one or more measurements as the target analyte moves about the lumen, thereby characterizing the target analyte. In claim 58, a method wherein the target analyte comprises a target polynucleotide. A method according to claim 58 or 59, wherein step (c) comprises measuring a current passing through the continuous channel, wherein the current is indicative of the presence and/or one or more characteristics of the target analyte, thereby detecting and/or characterizing the target analyte. A method according to any one of claims 58 to 60, wherein the target analyte is a polynucleotide, wherein nucleotides in the polynucleotide interact with the first constriction region, the second constriction region, and optionally the third constriction region within the lumen, and wherein each of the first constriction region, the second constriction region, and optionally the third constriction region is capable of discriminating a different nucleotide, such that the total current passing through the lumen is affected by the interaction between each of the first constriction region, the second constriction region, and the third constriction region and the nucleotides located in each of the regions.