Methods and kits for coupling a polypeptide to a membrane

Abstract

The invention relates to methods, kits and other products for coupling polypeptides to membranes. The methods and kits are particularly suited to determining the presence, absence or one or more characteristics of polypeptide analytes, especially using nanopores. The invention also provides a method of determining the presence, absence or one or more characteristics of polypeptide analytes.

Description

[0001] METHODS AND KITS

[0002] TECHNICAL FIELD

[0003] The invention relates to methods, kits and other products for coupling polypeptides to membranes. The methods and kits are particularly suited to determining the presence, absence or one or more characteristics of polypeptide analytes, especially using nanopores. The invention also provides a method of determining the presence, absence or one or more characteristics of polypeptide analytes.

[0004] INTRODUCTION

[0005] Biological pores (and other nanopores) have great potential as direct, electrical biosensors for polymers and a variety of small molecules. In particular, recent focus has been given to nanopores as technology for sequencing polymeric analytes, such as polynucleotides or polypeptides. When a potential is applied across a nanopore, there is a change in the current flow as the monomeric components of the polymeric analyte, such as a nucleotide or an amino acid, reside transiently in the barrel for a certain period of time. Nanopore detection of the monomer gives a current change of known signature and duration. In the strand sequencing method, a single polymer strand is passed through the pore and the identities of the monomers are derived. Strand sequencing can involve the use of a molecular brake to control the movement of the polymer through the pore.

[0006] When a potential is applied across a nanopore, there is a drop in the current flow when an analyte resides transiently in the barrel for a certain period of time. Nanopore detection of the analyte gives a current blockade of known signature and duration. The concentration of an analyte can then be determined by the number of blockade events per unit time to a single pore. For nanopore applications, efficient capture of analytes from solution is required. For instance, the number of interactions between the analyte and the nanopore needs to be maximal. Therefore, it is preferred to have the analyte at as high a concentration as is possible. This becomes a particular problem as the concentration of analyte in some samples can be limiting. Methods for increasing the concentration of the analyte in the proximity of the nanopore by coupling the analyte to the membrane containing the nanopore are known in the art (e.g., WO 2012 / 164270 incorporated by reference in its entirety). Improved methods of coupling are needed in the art.

[0007] SUMMARY OF THE INVENTION

[0008] The inventors have developed novel methods and kits for coupling polypeptides to a membrane. The inventors have surprisingly demonstrated increased polypeptide delivery by coupling the polypeptide to a membrane in which the relevant detector is present using specifically designed adaptors comprising at least one coupling polypeptide. This lowers by several orders of magnitude the amount of polypeptide required in order to be detected. The different adaptors used in the invention are typically designed so they bind to each other rather than themselves. This results in an efficient method of coupling the polypeptide to the membrane. It ensures the polypeptide is coupled to the membrane and polypeptides are not coupled together.

[0009] In addition, coupling the polypeptide to the membrane using the adaptors of the invention has added advantages for various applications. For instance, coupling the polypeptide to the membrane in accordance with the invention can reduce the any permanent or temporary blocking of the detector. It can also reduce the capture of other polypeptides or proteins, such as unfoldases, polynucleotide binding proteins or ATP regeneration enzymes, by the detector. Coupling the polypeptide to the membrane can orient the intended end for capture by the detector and improve its movement with respect to, such as through, the detector. It can also be used to set an upper limit of polypeptide concentration. For instance, a specific concentration of polypeptide can be coupled to the membrane and the excess can be removed from or flushed out of the system.

[0010] The invention provides a method for coupling a polypeptide to a membrane, comprising coupling the polypeptide to the membrane using (1) one or more first coupling adaptors each comprising one or more first amphipathic helices, and (2) one or more second coupling adaptors each comprising one or more second amphipathic helices which specifically bind to the one or more first amphipathic helices.

[0011] The invention also provides:

[0012] - a kit for coupling a polypeptide to a membrane, comprising (1) one or more first coupling adaptors each comprising one or more first amphipathic helices, and (2) one or more second coupling adaptors comprising one or more second amphipathic helices which specifically bind to the one or more first amphipathic helices; a method for determining the presence, absence or one or more characteristics of a polypeptide analyte, comprising (a) coupling the polypeptide analyte to a membrane using a kit of the invention or method of the invention and (b) allowing the coupled polypeptide analyte to interact with a detector present in the membrane and thereby determining the presence, absence or one or more characteristics of the polypeptide analyte; use of a kit of the invention for coupling a polypeptide or a plurality of polypeptides to a membrane;

[0013] - a membrane comprising a polypeptide or a plurality of polypeptides coupled to it using a kit of the invention or a method of the invention; an array comprising a plurality of membranes of the invention; a system comprising (a) a membrane of the invention or an array of the invention, (b) means for applying a potential across the membrane(s) and (c) means for detecting electrical or optical signals across the membrane(s); and

[0014] - an apparatus (a) comprising a polypeptide or a plurality of polypeptides coupled to an in vitro membrane using a kit of the invention or a method of the invention or (b) produced by a method comprising coupling a polypeptide or a plurality of polypeptides to an in vitro membrane using a kit of the invention or a method of the invention.

[0015] DESCRIPTION OF THE FIGURES

[0016] It is to be understood that Figures are for the purpose of illustrating particular embodiments of the invention only and are not intended to be limiting.

[0017] Figure 1 : Panel A shows a construct consisting of a portion of a polypeptide analyte featuring a leader (1) and leucine zipper (2). The leucine zipper (2) forms a non-covalent interaction with another leucine zipper (3) that is covalently attached to an oligonucleotide

[0018] (4). Panel B shows the sequencing analyte bound to a surface via a oligonucleotide tether

[0019] (5) to bring it in proximity to a detector (protein nanopore; 6).

[0020] Figure 2: Panel A shows the average number of captures per nanopore per hour at 20, 200, and 2000 pM of analyte without the leucine zipper monomer-DNA conjugate shown in designs 1 and 2 ( / .e., without coupling to the membrane). Panel B shows the average number of captures per nanopore per hour at 0.2, 2, 20, 200, and 2000 pM using the same analyte in panel A with the addition of leucine zipper monomer-DNA conjugate based on design 1. Panel C shows the average number of captures per nanopore per hour at 0.2, 2, 20, 200, and 2000 pM using the same analyte in panel A with the addition of leucine zipper monomer-DNA conjugate based on design 2.

[0021] Figure 3: Panel A shows the normalised increase in number of reads from an experiment including the unfoldase CIpX. Panel B shows reads taken from the two experiments with either no basic leucine zipper monomer, i.e., no coupling to the membrane, (top) or with the 3 heptad basic leucine zipper monomer present, i.e., with coupling to the membrane (bottom).

[0022] Figures 4-7: Schematics of preferred coupling methods and kits of the invention. The cylinders may be any of the one or more amphipathic helices described below.

[0023] DESCRIPTION OF THE SEQUENCE LISTING

[0024] SEQ ID NOs: 1-253 are shown and described below.

[0025] DETAILED DESCRIPTION It is to be understood that different applications of the disclosed products and methods may be tailored to the specific needs in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention only and is not intended to be limiting.

[0026] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0027] The invention, both as to organization and method of operation, together with features and advantages thereof, may best be understood by reference to the following detailed description when read in conjunction with the accompanying Figures. The aspects and advantages of the invention will be apparent from and elucidated with reference to the embodiment(s) described hereinafter. Reference throughout this specification to "embodiment(s)" means that a particular feature, structure, or characteristic described in connection with the embodiment(s) is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment", "in another embodiment" or "in a preferred embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment, but may do so. Similarly, it should be appreciated that in the description of exemplary embodiments of the invention, various features of the invention are sometimes grouped together in a single embodiment, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, inventive aspects lie in less than all features of a single foregoing disclosed embodiment.

[0028] Definitions

[0029] Where an indefinite or definite article is used when referring to a singular noun e.g., "a” or "an", "the", this includes a plural of that noun unless something else is specifically stated. Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps. The term "comprising" is interchangeable with "consisting of" or "consisting essentially of". Furthermore, the terms first, second, third and the like in the description and in the claims, are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0030] The following terms or definitions are provided solely to aid in the understanding of the invention. Unless specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the invention. Practitioners are particularly directed to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016), for definitions and terms of the art. The definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

[0031] "About" as used herein when referring to a measurable value such as an amount, percentage, a temporal duration, and the like, is meant to encompass variations of ± 20 % or ± 10 %, more preferably ± 5 %, even more preferably ± 1 %, and still more preferably ± 0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods. Any embodiment containing the term "about" includes the same feature without the term. For example, about 40% includes 40%.

[0032] "Polynucleotide" as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule. Thus, this term includes double- and single-stranded DNA, and RNA. The term "polynucleotide" as used herein, is a single or double stranded covalently linked sequence of nucleotides in which the 3' and 5' ends on each nucleotide are joined by phosphodiester bonds. The polynucleotide may be made up of deoxyribonucleotide bases or ribonucleotide bases. Polynucleotides may be manufactured synthetically in vitro or isolated from natural sources. Polynucleotides may further include modified DNA or RNA, for example DNA or RNA that has been methylated, or RNA that has been subject to post- translational modification, for example 5'-capping with 7-methylguanosine, 3'-processing such as cleavage and polyadenylation, and splicing. Polynucleotides may also include synthetic nucleic acids (XNA), such as hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), locked nucleic acid (LNA), peptide nucleic acid (PNA) and (poly)ADPribose modified DNA. Sizes of polynucleotides are typically expressed as the number of base pairs (bp) or nucleotide pairs for double stranded polynucleotides, or in the case of single stranded polynucleotides as the number of nucleotides (nt). One thousand bp or nt equal a kilobase (kb). Polynucleotides of less than around 40 nucleotides in length are typically called "oligonucleotides" and may comprise primers for use in manipulation of DNA such as via polymerase chain reaction (PCR). The term "polynucleotide" is interchangeable with "polynucleotide sequence", "nucleotide sequence", "DNA sequence", "nucleic acid", or "nucleic acid molecule(s)".

[0033] The term "amino acid" in the context of the present disclosure is used in its broadest sense and is meant to include organic compounds containing amine (NH2) and carboxyl (COOH) functional groups, along with a side chain (e.g., a R group) specific to each amino acid. The amino acids typically refer to naturally occurring L o-amino acids or residues. The commonly used one and three letter abbreviations for naturally occurring amino acids are used herein: A=Ala; C=Cys; D=Asp; E=Glu; F=Phe; G=Gly; H=His; I=Ile; K=Lys; L=Leu; M = Met; N=Asn; P=Pro; Q=Gln; R=Arg; S=Ser; T=Thr; V=Val; W=Trp; and Y=Tyr (Lehninger, A. L., (1975) Biochemistry, 2d ed., pp. 71-92, Worth Publishers, New York). The general term "amino acid" further includes D-amino acids, retro-inverso amino acids as well as chemically modified amino acids such as amino acid analogues, naturally occurring amino acids that are not usually incorporated into proteins such as norleucine, and chemically synthesised compounds having properties known in the art to be characteristic of an amino acid, such as P-amino acids. For example, analogues or mimetics of phenylalanine or proline, which allow the same conformational restriction of the peptide compounds as do natural Phe or Pro, are included within the definition of amino acid. Such analogues and mimetics are referred to herein as "functional equivalents" of the respective amino acid. Other examples of amino acids are listed by Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, eds., Vol. 5 p. 341, Academic Press, Inc., N.Y. 1983, which is incorporated herein by reference.

[0034] The terms "polypeptide", and "peptide" are interchangeably used herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides can also undergo maturation or post-translational modification processes that may include, but are not limited to glycosylation, proteolytic cleavage, lipidization, signal peptide cleavage, propeptide cleavage, phosphorylation, and such like. A peptide can be made using recombinant techniques, e.g., through the expression of a recombinant or synthetic polynucleotide. A recombinantly produced peptide it typically substantially free of culture medium, e.g., culture medium represents less than about 20 %, more preferably less than about 10 %, and most preferably less than about 5 % of the volume of the protein preparation.

[0035] The term "protein" is used to describe a folded polypeptide having a secondary or tertiary structure. The protein may be composed of a single polypeptide or may comprise multiple polypepties that are assembled to form a multimer. The multimer may be a homooligomer, or a heterooligmer. The protein may be a naturally occurring or wild type protein, or a modified, or non-naturally, occurring protein. The protein may, for example, differ from a wild type protein by the addition, substitution, or deletion of one or more amino acids.

[0036] The term "polypeptide" when being used in the context of what is being coupled to the membrane in accordance with the invention is interchangeable with "protein". Similarly, the term "polypeptide analyte" is interchangeable with "protein analyte".

[0037] A "variant" of a polypeptide or protein encompasses peptides, oligopeptides, polypeptides, proteins, and enzymes having structural similarity with the unmodified or wild-type polypeptide or protein. Structural variants are defined below with reference to root mean square deviation (RMSD). Standard methods in the art can be used to determine structural similarity, including RMSD. Suitable methods include, but are not limited to, AlphaFold, PSIPRED, TM-Align, US-Align or FATCAT. In all instances herein, RMSD is preferably measured over all alpha carbon atoms (Co atoms). RMSD may be measured over all heavy atoms.

[0038] A "variant" of a polypeptide or protein encompasses peptides, oligopeptides, polypeptides, proteins, and enzymes having amino acid substitutions, deletions and / or insertions relative to the unmodified or wild-type polypeptide or protein in question and having similar biological and functional activity as the unmodified polypeptide or protein from which they are derived. The term "amino acid identity" as used herein refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity" is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Vai, Leu, He, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison ( / .e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.

[0039] Standard methods in the art may be used to determine identity and / or homology. For example, the UWGCG Package provides the BESTFIT program which can be used to calculate homology, for example used on its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387-395). The PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described 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 / ). In all instances herein, identity or homology is typically measured over the entire length of the reference sequence. The term "wild-type" refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal" or "wild-type" form of the gene. In contrast, the term "modified", "mutant" or "variant" refers to a gene or gene product that displays modifications in sequence (e.g., substitutions, truncations, or insertions), post-translational modifications and / or functional properties (e.g., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product. Methods for introducing or substituting naturally occurring amino acids are well known in the art. For instance, methionine (M) may be substituted with arginine (R) by replacing the codon for methionine (ATG) with a codon for arginine (CGT) at the relevant position in a polynucleotide encoding the mutant monomer. Methods for introducing or substituting non-naturally occurring amino acids are also well known in the art. For instance, non-naturally occurring amino acids may be introduced by including synthetic aminoacyl-tRNAs in the IVTT system used to express the mutant monomer. Alternatively, they may be introduced by expressing the mutant monomer in E. coli that are auxotrophic for specific amino acids in the presence of synthetic ( / .e., non-naturally occurring) analogues of those specific amino acids. They may also be produced by naked ligation if the mutant monomer is produced using partial peptide synthesis. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties, or similar side-chain volume. The amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acids they replace. Alternatively, the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid. Conservative amino acid changes are well- known in the art.

[0040] A mutant or variant protein can also be chemically modified in any way and at any site. A mutant or variant protein is preferably chemically modified by attachment of a molecule to one or more cysteines (cysteine linkage), attachment of a molecule to one or more lysines, attachment of a molecule to one or more non-natural amino acids, enzyme modification of an epitope or modification of a terminus. Suitable methods for carrying out such modifications are well-known in the art. The mutant or variant protein may be chemically modified by the attachment of any molecule. For instance, the mutant or variant protein may be chemically modified by attachment of a dye or a fluorophore.

[0041] In the context of the invention, the terms "couple", "coupling" or the like are interchangeable with "tether", "tethering" or the like. In the context of the invention, the term "one or more" includes the singular. For instance, one or more first coupling adaptors each comprising one or more amphipathic helices includes a first coupling adaptor comprising an amphipathic helix.

[0042] Coupling methods of the invention

[0043] The invention provides a method for coupling a polypeptide to a membrane. This method may also be called the coupling method of the invention.

[0044] Membranes are defined in more detail below. The method comprises coupling the polypeptide to the membrane using (1) one or more first coupling adaptors and (2) one or more second coupling adaptors.

[0045] Coupling the polypeptide to the membrane containing a detector lowers by several orders of magnitude the amount of polypeptide required. The method is of course advantageous for detecting polypeptides that are present at low concentrations. The method preferably allows the presence, absence or characteristics of the polypeptide to be determined when the polypeptide (also called the polypeptide analyte below) is present at a concentration of from about O.OOlpM to about InM, such as less than O.OlpM, less than O.lpM, less than IpM, less than lOpM or less than lOOpM.

[0046] Coupling one end of the polypeptide to the membrane, even temporarily, also means the end is prevented from interfering with any detector in the membrane or any detector- based process. Other advantages of the method of the invention are discussed above.

[0047] The coupling may be permanent or stable. In other words, the coupling may be such that the polypeptide remains coupled to the membrane during the method. The coupling may be transient. In other words, the coupling may be such that the polypeptide decouples from the membrane during the method. For certain applications, especially the detection or characterisation methods discussed below, the transient nature of the coupling is preferred. If the coupling is permanent or stable, then some polypeptide data may be lost as the detector cannot continue to the end of the polypeptide. If the coupling is transient, then when the coupled end of the polypeptide randomly becomes free of the membrane, then the polypeptide can be processed to completion. Chemical groups that form permanent / stable or transient links with the membrane are discussed in more detail below. The polypeptide may be transiently coupled to the membrane using cholesterol or a fatty acyl chain. Any fatty acyl chain having a length of from about 6 to 30 about carbon atoms, such as hexadecanoic acid, may be used.

[0048] Polypeptide

[0049] The polypeptide can be any polypeptide. The polypeptide may be a polypeptide that is secreted from cells. Alternatively, the polypeptide can be a polypeptide that is present inside cells such that the polypeptide must be extracted from the cells before the invention can be carried out. The polypeptide may be present in blood or an extract thereof.

[0050] The polypeptide can be naturally-occurring or non-naturally-occurring. The polypeptide can include within it synthetic or modified amino acids. A number of different types of modification to amino acids are known in the art. For the purposes of the invention, it is to be understood that the polypeptide can be modified by any method available in the art.

[0051] The polypeptide may comprise the products of cellular expression of a plasmid, e.g., a plasmid used in cloning of proteins in accordance with the methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016).

[0052] The polypeptide can be provided as an impure mixture of one or more polypeptides and one or more impurities. Impurities may comprise truncated forms of the target polypeptide which are distinct from the "polypeptide analyte" or "target polypeptide analyte" for characterisation. For example, the polypeptide may be a full-length protein and impurities may comprise fractions of the protein. Impurities may also comprise proteins other than the target protein, e.g., which may be co-purified from a cell culture or obtained from a sample.

[0053] A polypeptide may comprise any combination of any amino acids, amino acid analogs and modified amino acids ( / .e., amino acid derivatives). Amino acids (and derivatives, analogs etc) in the polypeptide can be distinguished by their physical size and charge. The amino acids / derivatives / analogs can be naturally occurring or artificial. The polypeptide may comprise any naturally occurring amino acid.

[0054] The polypeptide may be modified. The polypeptide may be modified for detection using the detection or characterisation method of the invention. The detection or characterisation method may be for characterising modifications in the target polypeptide.

[0055] One or more of the amino acids / derivatives / analogs in the polypeptide may be modified. One or more of the amino acids / derivatives / analogs in the polypeptide may be post- translationally modified. As such, the detection or characterisation method of the invention can be used to detect the presence, absence, number of positions of post-translational modifications in a polypeptide. The method can be used to characterise the extent to which a polypeptide has been post-translationally modified.

[0056] Any one or more post-translational modifications may be present in the polypeptide. Typical post-translational modifications include modification with a hydrophobic group, modification with a cofactor, addition of a chemical group, glycation (the non-enzymatic attachment of a sugar), biotinylation and pegylation. Post-translational modifications can also be non- natural, such that they are chemical modifications done in the laboratory for biotechnological or biomedical purposes. This can allow monitoring the levels of the laboratory made peptide, polypeptide, or protein in contrast to the natural counterparts.

[0057] Examples of post-translational modification with a hydrophobic group include myristoylation, attachment of myristate, a Ci4saturated acid; palmitoylation, attachment of palmitate, a Ci6saturated acid; isoprenylation or prenylation, the attachment of an isoprenoid group; farnesylation, the attachment of a farnesol group; geranylgeranylation, the attachment of a geranylgeraniol group; and glypiation, and glycosylphosphatidylinositol (GPI) anchor formation via an amide bond.

[0058] Examples of post-translational modification with a cofactor include lipoylation, attachment of a lipoate (C8) functional group; flavination, attachment of a flavin moiety (e.g. flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD)); attachment of heme C, for instance via a thioether bond with cysteine; phosphopantetheinylation, the attachment of a 4'-phosphopantetheinyl group; and retinylidene Schiff base formation.

[0059] Examples of post-translational modification by addition of a chemical group include acylation, e.g. O-acylation (esters), N-acylation (amides) or S-acylation (thioesters); acetylation, the attachment of an acetyl group for instance to the N-terminus or to lysine; formylation; alkylation, the addition of an alkyl group, such as methyl or ethyl; methylation, the addition of a methyl group for instance to lysine or arginine; amidation; butyrylation; gamma-carboxylation; glycosylation, the enzymatic attachment of a glycosyl group for instance to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine or tryptophan; polysialylation, the attachment of polysialic acid; malonylation; hydroxylation; iodination; bromination; citrulination; nucleotide addition, the attachment of any nucleotide such as any of those discussed above, ADP ribosylation; oxidation; phosphorylation, the attachment of a phosphate group for instance to serine, threonine or tyrosine (O-linked) or histidine (N-linked); adenylylation, the attachment of an adenylyl moiety for instance to tyrosine (O-linked) or to histidine or lysine (N-linked); propionylation; pyroglutamate formation; S-glutathionylation; Sumoylation; S-nitrosylation; succinylation, the attachment of a succinyl group for instance to lysine; selenoylation, the incorporation of selenium; and ubiquitinilation, the addition of ubiquitin subunits (N-linked).

[0060] The polypeptide may be labelled with a molecular label. A molecular label may be a modification to the polypeptide which promotes the detection of the polypeptide in the detection or characterisation method of the invention. For example, the label may be a modification to the polypeptide which alters the signal obtained as conjugate is characterised. For example, the label may interfere with a flux of ions through the nanopore. In such a manner, the label may improve the sensitivity of the method. The polypeptide may contain one or more cross-linked sections, e.g., C-C bridges. The polypeptide may not be cross-linked prior to being characterised using the method.

[0061] The polypeptide may comprise sulphide-containing amino acids and thus has the potential to form disulphide bonds. Typically, in such embodiments, the polypeptide is reduced using a reagent such as DTT (Dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine) prior to being characterised using the method.

[0062] The polypeptide may be a full-length protein or naturally occurring polypeptide. The protein or naturally occurring polypeptide may be fragmented prior to conjugation to the polynucleotide. The protein or polypeptide may be chemically or enzymatically fragmented. The polypeptides or polypeptide fragments can be conjugated to form a longer target polypeptide.

[0063] The polypeptide can be any suitable length. The polypeptide preferably has a length of from about 2 to about 10,000 amino acids. The polypeptide may have a length of from about 3 to about 5,000, from about 5 to about 1,000, from about 10 to about 500 amino acids, from about 20 to about 100 amino acids. The polypeptide may have a length of at least about 10 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, at least about 50 amino acids, at least about 100 amino acids, at least about 200 amino acids, at least about 500 amino acids, at least about 1,000 amino acids, at least about 2,000 amino acids, at least about 5,000 amino acids or at least about 10,000 amino acids.

[0064] Any number of polypeptides may be coupled to the membrane in accordance with the invention. From about 2 to about 1 x 1013, from about 5 to about 1 x 1012, from about 10 to about 1 x 1011, from about 20 to about 1 x 1010, from about 50 to 1 x 1010, from about 100 to 1 x 109, from about 100 to 1 x 108, from about 500 to 1 x 107, from about 1,000 to 1 x 108, from about 5,000 to 1 x 107or from about 10,000 to 1 x 105polypeptides may be coupled to the membrane. At least about 10, at least about 50, at least about 100, at least about 500, at least about 1,000, at least about 5,000, at least about 10,000, at least about 50,000, at least about 1 x 105, at least about 1 x 106, at least about 1 x 107, at least about 1 x 108, at least about 1 x 109, at least about 1 x 1010, at least about 1 x 1011, at least about 1 x 1012or at least about 1 x 1013or more polypeptides can be coupled to the membrane. In the context of the invention, multiple instances of a polypeptide are called a plurality of polypeptides. The plurality may comprise any of the numbers of polypeptides in this paragraph. The polypeptides may be the same. The polypeptides may be a homologous plurality of polypeptides. The polypeptides may be different. The polypeptides may be a heterologous plurality of different polypeptides. The invention provides a coupling a plurality of polypeptides to a membrane, comprising coupling the plurality of polypeptides to the membrane using (1) one or more first coupling adaptors each comprising one or more coupling polypeptides, and (2) one or more one or more second coupling adaptors each comprising one or more coupling domains which specifically bind to the one or more coupling polypeptides.

[0065] The polypeptide can be any polypeptide or protein. The polypeptide can be an enzyme, antibody, hormone, structural protein, storage protein, transport protein, contractile protein, a toxin, a drug. The protein may be eukaryotic, prokaryotic, viral, an artificially designed protein or an engineered protein. The polypeptide may be a bacterial protein, fungal protein, virus protein or parasite-derived protein. Before it is contacted with the detector, the polypeptide may be unfolded to form a linear polypeptide chain. The plurality of polypeptides may comprise any of these polypeptides.

[0066] The polypeptide or the plurality of polypeptides is typically present in or derived from any suitable sample. The invention is typically carried out on a sample that is known to contain or suspected to contain the polypeptide or the plurality of polypeptides.

[0067] The sample may be a biological sample. The invention may be carried out in vitro on a sample obtained from or extracted from any organism or microorganism. The organism or microorganism is typically archaeal, prokaryotic or eukaryotic and typically belongs to one of the five kingdoms: plantae, animalia, fungi, monera and protista.

[0068] The sample is preferably a fluid sample. The sample typically comprises a body fluid of the patient. The sample may be urine, lymph, saliva, mucus or amniotic fluid but is preferably blood, plasma or serum. Typically, the sample is human in origin, but alternatively it may be from another mammal animal such as from commercially farmed animals such as horses, cattle, sheep or pigs or may alternatively be pets such as cats or dogs. Alternatively a sample of plant origin is typically obtained from a commercial crop, such as a cereal, legume, fruit or vegetable, for example wheat, barley, oats, canola, maize, soya, rice, bananas, apples, tomatoes, potatoes, grapes, tobacco, beans, lentils, sugar cane, cocoa or cotton.

[0069] The polypeptide or the plurality of polypeptides may be derived from one or more cells. The cells may be any type of cells. The cells may be a prokaryotic cells. The cells may be bacterial or archaeal. The cells are typically eukaryotic cells. The cells may be a protozoan, algal, fungal, plant or animal cells. The animal cells may be derived from the ectoderm, endoderm, or mesoderm. The cells may be stem cells, such as embryonic stem cells, induced pluripotent stem cells or mesenchymal stem cells, bone cells, such as osteoclasts, osteoblasts or osteocytes, tendon cells, such as tenoblasts or tenocytes, chondrocytes, synovial cells, vascular cells, blood cells, such as red blood cells, immune cells, platelet, neutrophils or basophils, muscle cells, such as skeletal muscle cells, cardiac muscle cells or smooth muscle cells, reproductive cells, such as sperm, oocytes, duct cells or epididymal cells, secretory cells, adipocytes, liver lipocytes, epithelial cells, odontoblasts, cementoblasts, hormone-secreting cells, barrier cells, exocrine secretory epithelial cells, nerve cells, astrocytes, oligodendrocytes, or neurons.

[0070] The immune cells may be neutrophil granulocyte and precursors, such as myeloblasts, promyelocytes, myelocytes, or metamyelocytes, eosinophil granulocyte and precursors, basophil granulocyte and precursors, mast cells, leukocytes, lymphocytes, helper T cells, regulatory T cells, cytotoxic T cells, natural killer T cells, B cells, macrophages, dendritic cells, plasma cells, neutrophils, or monocytes.

[0071] The cells may be wild-type or naturally occurring. The cells may be genetically modified or genetically engineered. For instance, the immune cells may be genetically engineered to express a recombinant chimeric antigen receptor (CAR) or T cell receptor (TCR). The cells may be genetically modified or genetically engineered using transduction or transfection or any other common techniques known to those skilled in the art.

[0072] The cells may be healthy cells or obtained from a healthy donor or source. The cells may be diseased or damaged, associated with a disease or damage or obtained from a diseased or damaged donor or source.

[0073] The sample may be a non-biological sample. The non-biological sample is preferably a fluid sample. Examples of a non-biological sample include surgical fluids, water such as drinking water, sea water or river water, and reagents for laboratory tests.

[0074] The sample is typically processed prior to being assayed, for example by centrifugation or by passage through a membrane that filters out unwanted molecules or cells, such as red blood cells. The sample may be measured immediately upon being taken. The sample may also be typically stored prior to assay, preferably below -70°C. The polypeptide or the plurality of polypeptides is typically extracted from the sample before it is used in the coupling method of the invention. Polypeptide and protein extraction kits are commercially available from, for instance, Thermo Fischer Scientific® and QIAGEN®.

[0075] Membrane

[0076] Any suitable membrane may be used in the coupling method of the invention. 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 amphiphiles which form a monolayer are known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). Block copolymers are polymeric materials in which two or more monomer sub-units that are polymerized together to create a single polymer chain. Block copolymers typically have properties that are contributed by each monomer sub-unit. However, a block copolymer may have unique properties that polymers formed from the individual sub-units do not possess. Block copolymers can be engineered such that one of the monomer subunits is hydrophobic ( / .e., lipophilic), whilst the other sub-unit(s) are hydrophilic whilst in aqueous media. In this case, the block copolymer may possess amphiphilic properties and may form a structure that mimics a biological membrane. The block copolymer may be a diblock (consisting of two monomer sub-units) but may also be constructed from more than two monomer sub-units to form more complex arrangements that behave as amphipiles. The copolymer may be a triblock, tetrablock or pentablock copolymer. The membrane may be a triblock copolymer membrane.

[0077] Archaebacterial bipolar tetraether lipids are naturally occurring lipids that are constructed such that the lipid forms a monolayer membrane. These lipids are generally found in extremophiles that survive in harsh biological environments, thermophiles, halophiles and acidophiles. Their stability is believed to derive from the fused nature of the final bilayer. It is straightforward to construct block copolymer materials that mimic these biological entities by creating a triblock polymer that has the general motif hydrophilic-hydrophobic- hydrophilic. This material may form monomeric membranes that behave similarly to lipid bilayers and encompass a range of phase behaviours from vesicles through to laminar membranes. Membranes formed from these triblock copolymers hold several advantages over biological lipid membranes. Because the triblock copolymer is synthesised, the exact construction can be carefully controlled to provide the correct chain lengths and properties required to form membranes and to interact with pores and other proteins.

[0078] Block copolymers may also be constructed from sub-units that are not classed as lipid submaterials; for example, a hydrophobic polymer may be made from siloxane or other non- hydrocarbon-based monomers. The hydrophilic sub-section of block copolymer can also possess low protein binding properties, which allows the creation of a membrane that is highly resistant when exposed to raw biological samples. This head group unit may also be derived from non-classical lipid head-groups.

[0079] Triblock copolymer membranes also have increased mechanical and environmental stability compared with biological lipid membranes, for example a much higher operational temperature or pH range. The synthetic nature of the block copolymers provides a platform to customise polymer-based membranes for a wide range of applications.

[0080] The membrane may be one of the membranes disclosed in WO 2014 / 064443 or WO 2014 / 064444 (both of which are incorporated herein by reference in their entireties). The amphiphilic molecules may be chemically modified or functionalised to facilitate coupling of the polynucleotide. The amphiphilic layer may be a monolayer or a bilayer. The amphiphilic layer is typically planar. The amphiphilic layer may be curved. The amphiphilic layer may be supported.

[0081] Amphiphilic membranes are typically naturally mobile, essentially acting as two-dimensional fluids with lipid diffusion rates of approximately IO-8cm s4. This means that the pore and coupled polynucleotide can typically move within an amphiphilic membrane.

[0082] The membrane may be a lipid bilayer. Lipid bilayers are models of cell membranes and serve as excellent platforms for a range of experimental studies. For example, lipid bilayers can be used for in vitro investigation of membrane proteins by single-channel recording. Alternatively, lipid bilayers can be used as biosensors to detect the presence of a range of substances. The lipid bilayer may be any lipid bilayer. Suitable lipid bilayers include, but are not limited to, a planar lipid bilayer, a supported bilayer, or a liposome. 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 (incorporated herein by reference in their entireties).

[0083] Methods for forming lipid bilayers are known in the art. Lipid bilayers are commonly formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972; 69: 3561-3566).

[0084] A lipid bilayer may be formed as described in WO 2009 / 077734 (incorporated herein by reference in its entirety). In this method, the lipid bilayer is formed from dried lipids. A lipid bilayer may be formed across an opening as described in W02009 / 077734.

[0085] The membrane may comprise a solid-state layer. Solid state layers can be formed from both organic and inorganic materials including, but not limited to, microelectronic materials, insulating materials such as Si3N4, A12O3, and SiO, organic and inorganic polymers such as polyamide, plastics such as Teflon® or elastomers such as two-component addition-cure silicone rubber, and glasses. The solid-state layer may be formed from graphene. Suitable graphene layers are disclosed in WO 2009 / 035647 (incorporated herein by reference in its entirety). If the membrane comprises a solid-state layer, the pore is typically present in an amphiphilic membrane or layer contained within the solid-state layer, for instance within a hole, well, gap, channel, trench or slit within the solid-state layer. The skilled person can prepare suitable solid state / amphiphilic hybrid systems. Suitable systems are disclosed in WO 2009 / 020682 and WO 2012 / 005857 (incorporated herein by reference in their entireties). Any of the amphiphilic membranes or layers discussed above may be used.

[0086] The methods disclosed herein are typically carried out 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 methods are typically carried out using an artificial amphiphilic layer, such as an artificial triblock copolymer layer. The layer may comprise other transmembrane and / or intramembrane proteins as well as other molecules in addition to the pore. Suitable apparatus and conditions are discussed below. The coupling method of the invention and / or the detection or characterisation method of the invention is typically carried out in vitro.

[0087] The polypeptide or the plurality of polypeptides may be coupled to the membrane via the adaptors using any known method including those described in WO 2012 / 164270 (incorporated by reference in its entirety). If the membrane is an amphiphilic layer, such as a lipid bilayer or any of the copolymer membranes discussed above, the polypeptide plurality of polypeptides is preferably coupled to the membrane via a protein present in the membrane (also known as a membrane protein) or one or more hydrophobic anchors present in the membrane. These may be form part of the one or more second adaptors or one or more third coupling adaptors as discussed in more detail below. Any number of one or more hydrophobic anchors may be used, such as 1, 2, 3, 4, 5 or more hydrophobic anchors. One or two hydrophobic anchors are preferably used. The one or more hydrophobic anchors are preferably selected from a lipid, fatty acid, sterol, carbon nanotube, polypeptide, protein or amino acid. The one or more hydrophobic anchors may be cholesterol, palmitate or tocopherol. The one or more hydrophobic anchors are preferably one or more cholesterols. In preferred embodiments, the polypeptide is not coupled to the membrane via the detector.

[0088] The hydrophobic modification may for example comprise a phosphorothioate such as a charge-neutralized alkyl-phosphorothioate (PPT) as described in Jones et al, J. Am. Chem. Soc. 2021, 143, 22, 8305 (incorporated by reference in its entirety). Suitable alkyl groups include for example C1-C10 alkyl groups such as C2-C6 alkyl groups, e.g., methyl, ethyl, propyl, butyl, pentyl and hexyl groups. Incorporation of the charge-neutralized alkyl- phosphorothioate into an adaptor allows the adaptor to couple to a hydrophobic region such the membrane.

[0089] The components of the membrane, such as the copolymer monomers, amphiphilic molecules or lipids, may be chemically-modified or functionalised to facilitate coupling of the polypeptide or the plurality of polypeptides to the membrane via the adaptors. Examples of suitable chemical modifications and suitable ways of functionalising the components of the membrane are discussed in more detail below. Any proportion of the membrane components may be functionalized, for example at least 0.01%, at least 0.1%, at least 1%, at least 10%, at least 25%, at least 50% or 100%.

[0090] Coupling of molecules to synthetic lipid bilayers has been carried out previously with various different tethering strategies. These are summarised in Table 1 below. Any of these may be used to couple the polypeptide or the plurality of polypeptides to the membrane via the adaptors

[0091] Table 1 - Coupling chemistries Any of the adaptors discussed below may be functionalised using a modified phosphoramidite in the synthesis reaction, which is easily compatible for the direct addition of suitable coupling moieties, such as cholesterol, tocopherol or palmitate, as well as for reactive groups, such as thiol, cholesterol, lipid and biotin groups. These different attachment chemistries give a suite of options for attachment to adaptors. Each different modification group tethers the adaptors in a slightly different way and coupling is not always permanent so giving different dwell times for the polypeptide or the plurality of polypeptides to the membrane. The advantages of transient coupling are discussed above.

[0092] Coupling to a membrane or functionalised membrane can also be achieved by a number of other means provided that a complementary reactive group or a tether can be added to the relevant adaptor. The addition of reactive groups to either end of polynucleotide adaptors has been reported previously. A thiol group can be added to the 5' of ssDNA or dsDNA using T4 polynucleotide kinase and ATPyS (Grant, G. P. and P. Z. Qin (2007). "A facile method for attaching nitroxide spin labels at the 5' terminus of nucleic acids." Nucleic Acids Res 35(10): e77). An azide group could be added to the 5'-phosphate of ssDNA or dsDNA using T4 polynucleotide kinase and y-[2-Azidoethyl]-ATP or y-[6-Azidohexyl]-ATP. Using thiol or Click chemistry a tether, containing either a thiol, iodoacetamide OPSS or maleimide group (reactive to thiols) or a DIBO (dibenzocyclooxtyne) or alkyne group (reactive to azides), can be covalently attached to the analyte. A more diverse selection of chemical groups, such as biotin, thiols and fluorophores, can be added using terminal transferase to incorporate modified oligonucleotides to the 3' of ssDNA (Kumar, A., P. Tchen, et al. (1988). "Nonradioactive labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase." Anal Biochem 169(2): 376-82). Streptavidin / biotin coupling may be used. It may also be possible that tethers could be directly added to polynucleotide adaptors using terminal transferase with suitably modified nucleotides (e.g. cholesterol or palmitate).

[0093] The reactive group may be ligated to a single strand or double stranded polynucleotide adaptor. Ligation of short pieces of ssDNA have been reported using T4 RNA ligase I (Troutt, A. B., M. G. McHeyzer-Williams, et al. (1992). "Ligation-anchored PCR: a simple amplification technique with single-sided specificity." Proc Natl Acad Sci U S A 89(20): 9823-5).

[0094] Adenylated nucleic acids (AppDNA) are intermediates in ligation reactions, where an adenosine-monophostate is attached to the 5'-phosphate of the nucleic acid. Various kits are available for generation of this intermediate, such as the 5' DNA Adenylation Kit from NEB. By substituting ATP in the reaction for a modified nucleotide triphosphate, then addition of reactive groups (such as thiols, amines, biotin, azides, etc) to the 5' of polynucleotide adaptors should be possible. It may also be possible that tethers could be directly added to polynucleotide adaptors using a 5' DNA adenylation kit with suitably modified nucleotides (e.g. cholesterol or palmitate).

[0095] A common technique for the amplification of sections of genomic DNA is using polymerase chain reaction (PCR). Here, using two synthetic oligonucleotide primers, a number of copies of the same section of DNA can be generated, where for each copy the 5' of each strand in the duplex will be a synthetic polynucleotide. By using an antisense primer single or multiple nucleotides can be added to 3' end of single or double stranded DNA by employing a polymerase. Examples of polymerases which could be used include, but are not limited to, Terminal Transferase, Klenow and E. coli Poly(A) polymerase). By substituting ATP in the reaction for a modified nucleotide triphosphate then reactive groups, such as a cholesterol, thiol, amine, azide, biotin or lipid, can be incorporated into the polynucleotide adaptors. Therefore, each copy of the amplified polynucleotide adaptors will contain a reactive group for coupling. Coupling polynucleotide adaptors to a membrane can also be achieved by anchoring a binding group, such as a polynucleotide binding protein or a chemical group, to the membrane and allowing the binding group to interact with the polynucleotide adaptors or by functionalizing the membrane. The binding group may be coupled to the membrane by any of the methods described herein. In particular, the binding group may be coupled to the membrane using one or more linkers, such as maleimide functionalised linkers.

[0096] The binding group can be any group that interacts with single or double stranded nucleic acids, specific nucleotide sequences within the polynucleotide adaptors or patterns of modified nucleotides within the polynucleotide adaptors, or any other ligand that is present on the polynucleotide.

[0097] Suitable binding proteins include E. coli single stranded binding protein, P5 single stranded binding protein, T4 gp32 single stranded binding protein, the TOPO V dsDNA binding region, human histone proteins, E. coli HU DNA binding protein and other archaeal, prokaryotic or eukaryotic single- or double-stranded nucleic acid binding proteins, including those listed below.

[0098] The specific nucleotide sequences in the polynucleotide adaptors could be sequences recognised by transcription factors, ribosomes, endonucleases, topoisomerases or replication initiation factors. The patterns of modified nucleotides could be patterns of methylation or damage.

[0099] The chemical group can be any group which intercalates with or interacts with a polynucleotide adaptor. The group may intercalate or interact with the polynucleotide adaptor via electrostatic, hydrogen bonding or Van der Waals interactions. Such groups include a lysine monomer, poly-lysine (which will interact with ssDNA or dsDNA), ethidium bromide (which will intercalate with dsDNA), universal bases or universal nucleotides (which can hybridise with any polynucleotide analyte) and osmium complexes (which can react to methylated bases). A polynucleotide adaptor may therefore be coupled to the membrane using one or more universal nucleotides attached to the membrane. Each universal nucleotide residue may be attached to the membrane using one or more linkers. A universal nucleotide is preferably one which will hybridise or bind to some degree to nucleotides comprising the nucleosides adenosine (A), thymine (T), uracil (U), guanine (G) and cytosine (C). Universal nucleotides may hybridise or bind more strongly to some nucleotides than to others. For instance, a universal nucleotide (I) comprising the nucleoside, 2'-deoxyinosine, will show a preferential order of pairing of I-C>I-A>I-G approximately=I-T. The polymerase will replace a nucleotide species with a universal nucleotide if the universal nucleotide takes the place of the nucleotide species in the population. For instance, the polymerase will replace dGMP with a universal nucleotide, if it is contacted with a population of free dAMP, dTMP, dCMP and the universal nucleotide. The universal nucleotide preferably comprises one of the following nucleobases: hypoxanthine, 4-nitroindole, 5-nitroindole, 6-nitroindole, formylindole, 3-nitropyrrole, nitroimidazole, 4-nitropyrazole, 4-nitrobenzimidazole, 5-nitroindazole, 4- aminobenzimidazole or phenyl (C6-aromatic ring). The universal nucleotide more preferably comprises one of the following nucleosides: 2'-deoxyinosine, inosine, 7-deaza-2'- deoxyinosine, 7-deaza-inosine, 2-aza-deoxyinosine, 2-aza-inosine, 2-O'-methylinosine, 4- nitroindole 2'-deoxyribonucleoside, 4-nitroindole ribonucleoside, 5-nitroindole 2'- deoxyribonucleoside, 5-nitroindole ribonucleoside, 6-nitroindole 2'-deoxyribonucleoside, 6- nitroindole ribonucleoside, 3-nitropyrrole 2'-deoxyribonucleoside, 3-nitropyrrole ribonucleoside, an acyclic sugar analogue of hypoxanthine, nitroimidazole 2'- deoxyribonucleoside, nitroimidazole ribonucleoside, 4-nitropyrazole 2'-deoxyribonucleoside, 4-nitropyrazole ribonucleoside, 4-nitrobenzimidazole 2'-deoxyribonucleoside, 4- nitrobenzimidazole ribonucleoside, 5-nitroindazole 2'-deoxyribonucleoside, 5-nitroindazole ribonucleoside, 4-aminobenzimidazole 2'-deoxyribonucleoside, 4-aminobenzimidazole ribonucleoside, phenyl C-ribonucleoside, phenyl C-2'-deoxyribosyl nucleoside, 2'- deoxynebularine, 2'-deoxyisoguanosine, K-2'-deoxyribose, P-2'-deoxyribose and pyrrolidine. The universal nucleotide more preferably comprises 2'-deoxyinosine. The universal nucleotide is more preferably IMP or dIMP. The universal nucleotide is most preferably dPMP (2'-Deoxy-P-nucleoside monophosphate) or dKMP (N6-methoxy-2, 6- diaminopurine monophosphate).

[0100] Where the binding group is a protein, it may be able to anchor directly into the membrane without further functionalisation, for example if it already has an external hydrophobic region which is compatible with the membrane. Examples of such proteins include transmembrane proteins. Alternatively the protein may be expressed with a genetically fused hydrophobic region which is compatible with the membrane. Such hydrophobic protein regions are known in the art.

[0101] Examples of methods of coupling adaptors to membranes are disclosed in WO 2012 / 164270 and WO 2015 / 150786 (incorporated herein by reference in their entireties).

[0102] Any of the adaptors may be coupled to the membrane using a tethering complex comprising one or more hydrophilic components connected by a hydrophobic linker as described in WO 2021 / 111139 (incorporated herein by reference in its entirety). The tethering complex represents a hydrophobic anchor as described herein.

[0103] First coupling a dap tor (s)

[0104] The method comprises coupling the polypeptide or the plurality of polypeptides to the membrane using (1) one or more first coupling adaptors each comprising one or more coupling polypeptides. Coupling polypeptides are discussed in more detail below. Any number of one or more first coupling adaptors may be used. The method may comprise using 1, 2, 3, 4, 5 or more first coupling adaptors. The method preferably comprises using 1 or 2 first coupling adaptors. Any of these may be referred to as "types of" first coupling adaptors to distinguish them from multiple instances of the one or more first coupling adaptors in the populations described below. The one or more first coupling adaptors or one or more types of first coupling adaptors may differ based on one or more of their (a) length, (b), structure, (c) number and / or identity of coupling polypeptides and (d) presence or absence of a blocking moiety. The one or more first coupling adaptors or one or more types of first coupling adaptors may differ in terms of (a); (b); (c); (d); (a) and (b); (a) and (c); (a) and (d); (b) and (c); (b) and (d); (c) and (d); (a), (b) and (c); (a), (b) and (d); (a), (c) and (d); (b), (c) and (d); or (a), (b), (c) and (d). Item (c) may be the number of coupling polypeptides, the identity of the coupling polypeptides or the number and identity of the coupling polypeptides. The embodiments in this paragraph apply to any of the first coupling adaptors discussed below.

[0105] The method preferably comprises using a first adaptor comprising one or more coupling polypeptides. The method preferably comprises using a first adaptor comprising one or more amphipathic helices. The one or more amphipathic helices may be any of those described in more detail below.

[0106] The one or more first coupling adaptors may be one or more polypeptide adaptors. The one or more polypeptide adaptors may be modified in any of the ways discussed above with reference to the polypeptide being coupled to the membrane. One or more of the amino acids / derivatives / analogs in the one or more first coupling adaptors may be modified. Any one or more post-translational modifications may be present in the one or more first coupling adaptors. The one or more first coupling adaptors may be labelled with a molecular label. The one or more first coupling adaptors may contain one or more cross-linked sections, e.g., C-C bridges. The one or more first coupling adaptors may comprise sulphide- containing amino acids and thus have the potential to form disulphide bonds.

[0107] The one or more first coupling adaptors can be any suitable length. The one or more first coupling adaptors preferably comprise one or more polypeptide adaptors having a length of from about 2 to about 500 amino acids. The one or more polypeptide adaptors preferably have a length of from about 5 to about 450 amino acids, from about 10 to about 400 amino acids, from about 20 to about 300 amino acids, from about 50 to about 200 amino acids or from about 60 to about 100 amino acids. The one or more polypeptide adaptors may have a length of at least about 10 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, at least about 50 amino acids, at least about 60 amino acids, at least about 70 amino acids, at least about 80 amino acids, at least about 90 amino acids, at least about 100 amino acids, at least about 150 amino acids, at least about 200 amino acids, at least about 300 amino acids, at least about 400 amino acids or at least about 500 amino acids.

[0108] The one or more first coupling adaptors may comprise a polynucleotide and a polypeptide. The one or more first coupling adaptors may be one or more polynucleotide-polypeptide conjugates. The one or more conjugates preferably comprise a polynucleotide conjugated to a polypeptide. The polypeptide section typically comprises or consists of the one or more coupling polypeptides. The polypeptide section may be any of the polypeptides discussed above for polypeptide adaptors.

[0109] The polypeptide can be conjugated to the polynucleotide at any suitable position. For example, the polypeptide can be conjugated to the polynucleotide at the N-terminus or the C-terminus of the polypeptide. The polypeptide can be conjugated to the polynucleotide via a side chain group of a residue (e.g., an amino acid residue) in the polypeptide. The polypeptide may have a naturally occurring reactive functional group which can be used to facilitate conjugation to the polynucleotide. For example, a cysteine residue can be used to form a disulphide bond to the polynucleotide or to a modified group thereon.

[0110] The polypeptide may be modified in order to facilitate its conjugation to the polynucleotide. For example, the polypeptide may be modified by attaching a moiety comprising a reactive functional group for attaching to the polynucleotide. For example, the polypeptide can be extended at the N-terminus or the C-terminus by one or more residues (e.g., amino acid residues) comprising one or more reactive functional groups for reacting with a corresponding reactive functional group on the polynucleotide. For example, the polypeptide can be extended at the N-terminus and / or the C-terminus by one or more cysteine residues. Such residues can be used for attachment to the polynucleotide portion of the conjugate, e.g., by maleimide chemistry (e.g., by reaction of cysteine with an azido-maleimide compound such as azido-[Pol]-maleimide wherein [Pol] is typically a short chain polymer such as PEG, e.g., PEG2, PEG3, or PEG4; followed by coupling to appropriately functionalised polynucleotide e.g., polynucleotide carrying a BCN group for reaction with the azide). Such chemistry is described in Example 2. For avoidance of doubt, when the polypeptide comprises an appropriate naturally occurring residue at the N- and / or C- terminus (e.g., a naturally occurring cysteine residue at the N- and / or C-terminus) then such residue(s) can be used for attachment to the polynucleotide.

[0111] A residue in the polypeptide may be modified to facilitate attachment of the polypeptide to the polynucleotide. A residue (e.g., an amino acid residue) in the polypeptide may be chemically modified for attachment to the polynucleotide. A residue (e.g., an amino acid residue) in the polypeptide may be enzymatically modified for attachment to the polynucleotide. The conjugation chemistry between the polynucleotide and the polypeptide in the conjugate is not particularly limited. Any suitable combination of reactive functional groups can be used. Many suitable reactive groups and their chemical targets are known in the art. Some exemplary reactive groups and their corresponding targets include aryl azides which may react with amine, carbodiimides which may react with amines and carboxyl groups, hydrazides which may react with carbohydrates, hydroxmethyl phosphines which may react with amines, imidoesters which may react with amines, isocyanates which may react with hydroxyl groups, carbonyls which may react with hydrazines, maleimides which may react with sulfhydryl groups, NHS-esters which may react with amines, PFP-esters which may react with amines, psoralens which may react with thymine, pyridyl disulfides which may react with sulfhydryl groups, vinyl sulfones which may react with sulfhydryl amines and hydroxyl groups, vinylsulfonamides, and the like. Other suitable chemistry for conjugating the polypeptide to the polynucleotide includes click chemistry. Many suitable click chemistry reagents are known in the art. Suitable examples of click chemistry include, but are not limited to, the following: copper(I)-catalyzed azide-alkyne cycloadditions (azide alkyne Huisgen cycloadditions); strain-promoted azide-alkyne cycloadditions; including alkene and azide [3+2] cycloadditions; alkene and tetrazine inverse-demand Diels-Alder reactions; and alkene and tetrazole photoclick reactions; copper-free variant of the 1,3 dipolar cycloaddition reaction, where an azide reacts with an alkyne under strain, for example in a cyclooctane ring such as in bicycle[6.1.0]nonyne (BCN); the reaction of an oxygen nucleophile on one linker with an epoxide or aziridine reactive moiety on the other; and the Staudinger ligation, where the alkyne moiety can be replaced by an aryl phosphine, resulting in a specific reaction with the azide to give an amide bond.

[0112] Any reactive group may be used to form the conjugate. Some suitable reactive groups include [1, 4-Bis[3-(2-pyridyldithio)propionamido]butane; 1,1 1-bis- maleimidotriethyleneglycol; 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide ester); ethylene glycol-bis(succinic acid N-hydroxysuccinimide ester); 4,4'-diisothiocyanatostilbene- 2,2'-disulfonic acid disodium salt; Bis[2-(4-azidosalicylamido)ethyl] disulphide; 3-(2- pyridyldithio)propionic acid N-hydroxysuccinimide ester; 4-maleimidobutyric acid N- hydroxysuccinimide ester; lodoacetic acid N-hydroxysuccinimide ester; S-acetylthioglycolic acid N-hydroxysuccinimide ester; azide-PEG-maleimide; and alkyne-PEG-maleimide. The reactive group may be any of those disclosed in WO 2010 / 086602, particularly in Table 3 of that application. The reactive functional group may be comprised in the polynucleotide and the target functional group may be comprised in the polypeptide prior to the conjugation step. The reactive functional group may be comprised in the polypeptide and the target functional group may be comprised in the polynucleotide prior to the conjugation step. The reactive functional group may be attached directly to the polypeptide. The reactive functional group may be attached to the polypeptide via a spacer. Any suitable spacer can be used. Suitable spacers include for example alkyl diamines such as ethyl diamine, etc.

[0113] Any polynucleotide, such as a nucleic acid, in the one or more first coupling adaptors is a macromolecule comprising two or more nucleotides. The polynucleotide or nucleic acid may comprise any combination of any nucleotides. The nucleotides can be naturally occurring or artificial. One or more nucleotides in the polynucleotide can be oxidized or methylated. One or more nucleotides in the polynucleotide may be modified, for instance with a label or a tag, for which suitable examples are known by a skilled person. The polynucleotide may comprise one or more spacers. A nucleotide typically contains a nucleobase, a sugar and at least one phosphate group. The nucleobase and sugar form a nucleoside. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and 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 a deoxyribose. The polynucleotide preferably comprises the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and / or thymidine (dT), deoxyguanosine (dG) and deoxycytidine (dC). The nucleotide is typically a ribonucleotide or deoxyribonucleotide. The nucleotide typically contains a monophosphate, diphosphate, or triphosphate. The nucleotide may comprise more than three phosphates, such as 4 or 5 phosphates. Phosphates may be attached on the 5' or 3' side of a nucleotide. The nucleotides in the polynucleotide may be attached to each other in any manner. The nucleotides are typically attached by their sugar and phosphate groups as in nucleic acids. The nucleotides may be connected via their nucleobases as in pyrimidine dimers. The polynucleotide may be single stranded or double stranded. At least a portion of the polynucleotide is preferably double stranded. The polynucleotide is most preferably ribonucleic nucleic acid (RNA) or deoxyribonucleic acid (DNA).

[0114] The polynucleotide in the one or more first coupling adaptors can be any length. For example, the polynucleotide can have a length of from about 5 to about 500 nucleotides or nucleotide pairs, from about 10 to about 200 nucleotides or nucleotide pairs, from about 30 to about 150 nucleotides or nucleotide pairs, from about 50 to about 100 nucleotides or nucleotide pairs or from about 60 to about 90 nucleotides or nucleotide pairs. The polynucleotide can be at least about 10, at least about 20, at least about 25, at least about 50, at least about 100, at least about 150, at least about 200, at least about 250, at least about 300, at least about 400 or at least about 500 nucleotides or nucleotide pairs in length.

[0115] Suitable nucleotides include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), 5-methylcytidine monophosphate, 5-hydroxymethylcytidine monophosphate, cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP), deoxycytidine monophosphate (dCMP) and deoxymethylcytidine monophosphate. The nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP and dUMP. A nucleotide may be abasic (i.e., lack a nucleobase). A nucleotide may also lack a nucleobase and a sugar (i.e., is a C3 spacer).

[0116] The one or more first coupling adaptors typically couple to the polypeptide or the plurality of polypeptides. The one or more second coupling adaptors typically couple to the membrane either directly or via one or more third coupling adaptors. The specific binding of the one or more second coupling adaptors to the one or more first coupling adaptors typically couples the polypeptide or the plurality of polypeptides to the membrane.

[0117] Coupling polypeptide(s)

[0118] The one or more first coupling adaptors each comprises one or more coupling polypeptides. Polypeptides are defined above. The one or more coupling polypeptides may be any polypeptide(s) that specifically bind the one or more coupling domains in the one or more second coupling adaptors. Specific binding is discussed in more detail below in the context of the one or more second coupling adaptors.

[0119] Any number of one or more coupling polypeptides may be used. Each first coupling adaptor may comprise 1, 2, 3, 4, 5, 6, 7, 8 or more coupling polypeptides. Each first coupling adaptor preferably comprises 1,2, 3 or 4 coupling polypeptides. Each first coupling adaptor preferably comprises 1 or 2 coupling polypeptides. Any of these may be referred to as "types of" coupling polypeptides to distinguish them from multiple instances of the one or more coupling polypeptides in the populations described below. The one or more coupling polypeptides or one or more types of coupling polypeptide may differ based on one or more of their (a) type, (b), sequence and (c) length. The one or more coupling polypeptides or one or more types of coupling polypeptide may differ in terms of (a); (b); (c); (a) and (b); (a) and (c); (b) and (c); or (a), (b) and (c). The embodiments in this paragraph apply to any of the one or more coupling polypeptides discussed below, including the one or more amphipathic helices, one or more amphipathic helix oligomers, one or more helical hairpins, one or more coiled-coil monomers, one or more coiled-coil oligomers, one or more coiled- coil hairpins, one or more leucine zipper monomers, one or more leucine zipper oligomers and one or more leucine zipper hairpins.

[0120] The method preferably comprises using a first adaptor comprising one or two coupling polypeptides. The method preferably comprises using a first adaptor comprising one or two amphipathic helices. The one or two amphipathic helices may be any of those described in more detail below.

[0121] The one or more coupling polypeptides may be any of the lengths discussed above with reference to the polypeptide in the one or more first coupling adaptors. The one or more coupling polypeptides, especially the one or more amphipathic helices, coiled-coil monomers and one or more leucine zipper monomers discussed below, may be from about 7 to about 56 amino acids in length. The one or more coupling polypeptides are preferably from about 10 to about 50, from about 20 to about 40 or from about 25 to about 30 amino acids in length. The one or more coupling polypeptides may be at least about 7, at least about 14, at least about 21, at least about 28, at least at least about 35, at least about 42 or at least about 56 amino acids in length.

[0122] As explained in more detail below, various monomer coupling polypeptides may be used to form oligomeric coupling polypeptides. The one or more coupling polypeptides, especially the one or more oligomers or hairpins discussed below, may be from about 7 to about 500 amino acids in length. The one or more coupling polypeptides are preferably from about 10 to about 450, from about 20 to about 400, from about 25 to about 300 amino acids or from about 50 to about 200 amino acids in length. The one or more coupling polypeptides may be at least about 7, at least about 10, at least about 20, at least about 50, at least at least about 100, at least about 150, at least about 200, at least about 300, at least about 400 or at least about 500 amino acids in length.

[0123] The one or more coupling polypeptides preferably comprise one or more amphipathic helices, one or more polyhistidine-tags, one or more transcription factors, one or more antigens, one or more antibodies or one or more fragments thereof, one or more enzymes, one or more peptide nucleic acids (PNAs), one or more gamma PNAs, one or more polypeptide analytes, one or more split proteins, one or more affimers or one or more combinations thereof. Preferred coupling domains which specifically bind to these polypeptides are discussed below.

[0124] Antibodies and fragments are discussed below.

[0125] Split proteins are known in the art. They are formed from the splitting and engineering of proteins which naturally form an intramolecular bond. Suitable split proteins include, but are not limited to, a split section of a beta-barrel protein, a split section of GFP and SpyTag or SpyCatcher from the SpyTag / SpyCatcher system.

[0126] The one or more coupling polypeptides preferably comprise one or more, such as 1, 2, 3, 4, 5, 6, 7 or 8 or more, amphipathic helices. Amphipathic helices are structures comprising both hydrophobic and polar ( / .e., hydrophilic) amino acid residues arranged in such a way as to create two faces on opposite sides of the helix, one face being hydrophobic.

[0127] Preferred one or more amphipathic helices include one or more helical bundle monomers and one or more coiled-coil monomers.

[0128] The one or more amphipathic helices may be one or more monomer units of amphipathic helices. The one or more coupling polypeptides may comprise two or more, such as 2, 3, 4, 5, 6, 7 or 8, amphipathic helices. The one or more amphipathic helices may be one or more amphipathic helix dimers, one or more amphipathic helix trimers, one or more amphipathic helix tetramers, one or more amphipathic helix pentamers, one or more amphipathic helix hexamers, one or more amphipathic helix heptamers or one or more one or more amphipathic helix octamers. These one or more oligomers may be homomers or heteromers. The one or more oligomers are preferably heteromers. The one or more oligomers can be parallel or antiparallel.

[0129] The monomer units in the oligomers may be linked by polypeptide loops. Amphipathic helices linked by polypeptide loops can be called helical hairpins. The one or more coupling polypeptides may comprise one or more helical hairpins. The helical hairpins can be parallel or antiparallel. The one or more helical hairpins may comprise any of the numbers of amphipathic helices discussed above, such as 1, 2, 3, 4, 5, 6, 7, 8 or more. Preferred one or more helical hairpins include one or more helical hairpins dimers (helix-loop-helix) and one or more helical hairpin trimers (helix-loop-helix-loop-helix). Examples of helical hairpins include coiled-coil hairpins and these are discussed in more detail below. The one or more helical hairpins can be homomers or heteromers. The one or more helical hairpins are preferably heteromers. The one or more helical hairpins can be parallel or antiparallel.

[0130] The one or more amphipathic helices may be oligomers formed from both amphipathic helix monomer units and helical hairpins. Examples include, but are not limited to, one or more tetramers each comprising two helical hairpins dimers (helix-loop-helix), one or more tetramers each comprising two amphipathic helix monomers and a helical hairpin dimer (helix-loop-helix), one or more tetramers each comprising an amphipathic helix monomer and a helical hairpin trimer (helix-loop-helix-loop-helix), and one or more helical hairpin trimers each comprising an amphipathic helix monomer and a helical hairpin dimer (helixloop-helix). The one or more oligomers can be homomers or heteromers. The one or more oligomers are preferably heteromers. The one or more oligomers can be parallel or antiparallel. The skilled person is capable of designing other suitable oligomers for use in the invention.

[0131] The one or more coupling polypeptides preferably comprise one or more, such as 1, 2, 3, 4, 5, 6, 7, 8 or more, coiled-coil monomers. Coiled-coil monomers have a general heptad pattern of HPPHPPP where H = hydrophobic amino acid residue and P = polar amino acid residue. They are typically presented as ABCDEFG where A and D are generally hydrophobic amino acid residues. The hydrophobic amino acid residues may be selected from alanine (A), methionine (M), tyrosine (Y), tryptophan (W), leucine (L), isoleucine (I), valine (V) and phenylalanine (F). The hydrophobic amino acid residues are preferably selected from leucine (L), isoleucine (I), valine (V) and phenylalanine (F). Patterning these residues can control the oligomeric state of the monomer. Substituting the hydrophobic amino acid residues for other residues, particularly asparagine (N) and arginine (R), can be used to control affinity, specificity and stability. The number of heptads can also be tuned to control affinity, specificity and stability. The one or more coiled-coil monomers may comprise any number of heptads. The one or more coiled-coil monomers may comprise 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5 or 8 heptads. The one or more coiled-coil monomers may comprise 1, 1.5, 2, 2.5, 3, 3.5, or 4, 4.5 heptads. These lengths also apply to the zipper monomers discussed below, including the leucine zipper monomers.

[0132] The vast majority of coiled-coil monomers form dimers (antiparallel and parallel). Coiled-coil monomers may form a trimer, a tetramer or a pentamer.

[0133] Preferred one or more coiled-coil monomers include one or more alacoil monomers, one or more leucine zipper monomers, one or more alanine zipper monomers, one or more isoleucine zipper monomers and one or more phenylalanine zipper monomers. Leucine zipper monomers are discussed in more detail below.

[0134] The one or more coiled-coil monomers may be one or more monomer units of coiled-coil monomers. The one or more coupling polypeptides may comprise two or more, such as 2, 3, 4, 5, 6, 7 or 8, coiled-coil monomers. The one or more coiled-coil monomers may be one or more coiled-coil dimers, one or more coiled-coil trimers, one or more coiled-coil tetramers, one or more coiled-coil pentamers, one or more coiled-coil hexamers, one or more coiled-coil heptamers or one or more one or more coiled-coil octamers. These one or more oligomers may be homomers or heteromers. The one or more oligomers are preferably heteromers. The one or more oligomers can be parallel or antiparallel.

[0135] The monomer units in the oligomers may be linked by polypeptide loops. Coiled-coil monomers linked by polypeptide loops can be called coiled-coil hairpins. The one or more coupling polypeptides may comprise one or more coiled-coil hairpins. The coiled-coil hairpins can be parallel or antiparallel. The one or more coiled-coil hairpins may comprise any of the numbers of coiled-coil monomers discussed above, such as 1, 2, 3, 4, 5, 6, 7, 8 or more. Preferred one or more coiled-coil hairpins include one or more coiled-coil hairpins dimers (CC-loop-CC; where CC = coiled-coil monomer) and one or more coiled-coil hairpin trimers (CC-loop-CC-loop-CC; where CC= coiled-coil monomer). The one or more coiled-coil hairpins can be homomers or heteromers. The one or more coiled-coil hairpins are preferably heteromers. The one or more coiled-coil hairpins can be parallel or antiparallel.

[0136] The one or more coiled-coil monomers may be oligomers formed from both coiled-coil monomer units and coiled-coil hairpins. Examples include, but are not limited to, one or more tetramers each comprising two coiled-coil hairpins dimers (CC-loop-CC), one or more tetramers each comprising two coiled-coil monomers and a coiled-coil hairpin dimer (CC- loop-CC), one or more tetramers each comprising a coiled-coil monomer and a coiled-coil hairpin trimer (CC-loop-CC-loop-CC), and one or more helical coiled-coil trimers each comprising a coiled-coil monomer and a coiled-coil hairpin dimer (CC-loop-CC). The one or more oligomers can be homomers or heteromers. The one or more oligomers are preferably heteromers. The one or more oligomers can be parallel or antiparallel. The skilled person is capable of designing other suitable oligomers for use in the invention.

[0137] The one or more coiled-coil monomers may be any of those described in Erin K O'Shea et al., Current Biology, Volume 3, Issue 10, 1993, Pages 658-667; Franziska Thomas et al. Journal of the American Chemical Society 2013 135 (13), 5161-5166; Jennifer R. Litowski et al., Journal of Biological Chemistry, Volume 277, Issue 40, 2002, Pages 37272-37279; Moll et al., Protein Science, Volume 10, Issue 3, March 2001, Pages 649-655; Christopher Negron and Amy E. Keating, Journal of the American Chemical Society 2014 136 (47), 16544-16556; and Rhys et al. Chem Biol 18, 999-1004 (2022) (incorporated herein by reference in their entireties).

[0138] Suitable coiled-coil hairpins for use in the invention are disclosed in Abigail et al., ACS Synthetic Biology 2023 12 (6), 1845-1858 and Chen, Z., Boyken, S.E., Jia, M. et al. Programmable design of orthogonal protein heterodimers. Nature 565, 106-111 (2019) (incorporated herein by reference in their entireties).

[0139] The one or more coiled-coil monomers may be modified by substitution of one or more of the leucine amino acid residues in their sequence. Any number of leucine amino acid residues may be substituted. For instance, 1, 2, 3, 4, 5, 6 or more leucine residues may be substituted. The one or more leucine residues may be substituted with any other amino acid residue(s). The one or more leucine residues may be substituted with isoleucine (I) and / or valine (V).

[0140] Preferred one or more coiled-coil monomers are shown in SEQ ID NOs: 60-253. The one or more coiled-coil monomers preferably comprise one or more structural variants having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with a protein having the sequence shown in any one of SEQ ID NOs: 60-253.

[0141] The one or more coiled-coil monomers preferably comprise one or more variant sequences having at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in any one of SEQ ID NOs: 60-253. The one or more coiled-coil monomers preferably comprises one or more variant sequences having at least about 80% homology and / or identity to the sequence shown in any one of SEQ ID NOs: 60-253. The one or more coiled- coil monomers preferably comprise a sequence having at least about 90% homology and / or identity to the sequence shown in any one of SEQ ID NOs: 60-253. The one or more coiled- coil monomers preferably comprises a sequence having 100% homology and / or identity to the sequence shown in any one of SEQ ID NOs: 60-253. Homology and / or identity is typically measured over the entire length of the reference sequence.

[0142] Sequence homology and / or identity can also relate to a fragment or portion of the reference sequence. Hence, a sequence may have less than 40% overall sequence homology and / or identity with the sequence shown in any one of SEQ ID NOs: 60-253, but the sequence of a particular region, domain or subunit could share at least about 80%, at least about 90%, or as much as about 99% sequence homology and / or identity with the corresponding region of the sequence. There may be at least about 80%, at least 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% homology and / or identity over a stretch of about 10 or more, for example about 12, about 15, about 17, about 18, about 20, about 25 or more, contiguous amino acids ("hard homology").

[0143] The one or more coiled-coil monomers preferably comprise one or more coiled-coil oligomers and / or one or more coiled-coil hairpins comprising any of SEQ ID NOs: 60-253 or structural variants and / or variant sequences thereof. Coiled-coil oligomers and hairpins are described above.

[0144] Preferred one or more coiled-coil oligomers for use in the invention comprise one or more of:

[0145] - the sequence shown in SEQ ID NO: 236 or a structural variant and / or variant sequence thereof and the sequence shown in SEQ ID NO: 237 or a structural variant and / or variant sequence thereof; - the sequence shown in SEQ ID NO: 238 or a structural variant and / or variant sequence thereof and the sequence shown in SEQ ID NO: 239 or a structural variant and / or variant sequence thereof;

[0146] - the sequence shown in SEQ ID NO: 240 or a structural variant and / or variant sequence thereof and the sequence shown in SEQ ID NO: 241 or a structural variant and / or variant sequence thereof;

[0147] - the sequence shown in SEQ ID NO: 242 or a structural variant and / or variant sequence thereof and the sequence shown in SEQ ID NO: 243 or a structural variant and / or variant sequence thereof;

[0148] - the sequence shown in SEQ ID NO: 244 or a structural variant and / or variant sequence thereof and the sequence shown in SEQ ID NO: 245 or a structural variant and / or variant sequence thereof;

[0149] - the sequence shown in SEQ ID NO: 246 or a structural variant and / or variant sequence thereof and the sequence shown in SEQ ID NO: 247 or a structural variant and / or variant sequence thereof;

[0150] - the sequence shown in SEQ ID NO: 248 or a structural variant and / or variant sequence thereof and the sequence shown in SEQ ID NO: 249 or a structural variant and / or variant sequence thereof;

[0151] - the sequence shown in SEQ ID NO: 250 or a structural variant and / or variant sequence thereof and the sequence shown in SEQ ID NO: 251 or a structural variant and / or variant sequence thereof; and / or

[0152] - the sequence shown in SEQ ID NO: 252 or a structural variant and / or variant sequence thereof and the sequence shown in SEQ ID NO: 253 or a structural variant and / or variant sequence thereof.

[0153] The one or more coupling polypeptides preferably comprise one or more, such as 1, 2, 3, 4, 5, 6, 7, 8 or more, leucine zipper monomers. In all instances herein, the term "zipper" is interchangeable with "scissor".

[0154] Leucine zippers are coiled-coil motifs made up of two alpha helices. The leucine residues are typically repeated every seventh position along the helices, placing them on one side. The side chains of the leucine residues extend from one alpha helix and interdigitate with those of a similar alpha helix in a second monomer. This allows the monomers to form a parallel or anti-parallel coiled-coil. Suitable leucine zippers for use in the invention are known in the art. Preferred leucine zipper monomers are discussed in more detail below. The one or more leucine zipper monomers may be one or more monomer units of leucine zipper monomers. The one or more coupling polypeptides may comprise two or more, such as 2, 3, 4, 5, 6, 7 or 8, leucine zipper monomers. The one or more leucine zipper monomers may be one or more leucine zipper dimers, one or more leucine zipper trimers, one or more leucine zipper tetramers, one or more leucine zipper pentamers, one or more leucine zipper hexamers, one or more leucine zipper heptamers or one or more one or more leucine zipper octamers. These one or more oligomers may be homomers or heteromers. The one or more oligomers are preferably heteromers. The one or more oligomers can be parallel or anti parallel.

[0155] The monomer units in the oligomers may be linked by polypeptide loops. Leucine zipper monomers linked by polypeptide loops can be called leucine zipper hairpins. The one or more coupling polypeptides may comprise one or more leucine zipper hairpins. The leucine zipper hairpins can be parallel or antiparallel. The one or more leucine zipper hairpins may comprise any of the numbers of leucine zipper monomers discussed above, such as 1, 2, 3, 4, 5, 6, 7, 8 or more. Preferred one or more leucine zipper hairpins include one or more leucine zipper hairpins dimers (LZ-loop-LZ; where LZ = leucine zipper monomer) and one or more leucine zipper hairpin trimers (LZ-loop-LZ-loop-LZ; where LZ= leucine zipper monomer). The one or more leucine zipper hairpins can be homomers or heteromers. The one or more leucine zipper hairpins are preferably heteromers. The one or more leucine zipper hairpins can be parallel or antiparallel.

[0156] The one or more leucine zipper monomers may be oligomers formed from both leucine zipper monomer units and leucine zipper hairpins. Examples include, but are not limited to, one or more tetramers each comprising two leucine zipper hairpins dimers (LZ-loop-LZ), one or more tetramers each comprising two leucine zipper monomers and a leucine zipper hairpin dimer (LZ-loop-LZ), one or more tetramers each comprising a leucine zipper monomer and a leucine zipper hairpin trimer (LZ-loop-LZ-loop-LZ), and one or more helical leucine zipper trimers each comprising a leucine zipper monomer and a leucine zipper hairpin dimer (LZ-loop-LZ). The one or more oligomers can be homomers or heteromers. The one or more oligomers are preferably heteromers. The one or more oligomers can be parallel or antiparallel. The skilled person is capable of designing other suitable oligomers for use in the invention.

[0157] The one or more leucine zipper monomers may be modified by substitution of one or more of the leucine amino acid residues in their sequence. Any number of leucine amino acid residues may be substituted. For instance, 1, 2, 3, 4, 5, 6 or more leucine residues may be substituted. The one or more leucine residues may be substituted with any other amino acid residue(s). The one or more leucine residues may be substituted with isoleucine (I) and / or valine (V). Preferred one or more leucine zipper monomers are shown in SEQ ID NOs: 9, 10, 3-7 and 11-59. SEQ ID NOs: 9, 10, 3-7 and 11-51 are leucine zipper monomers. SEQ ID NOs: 52-59 are leucine zipper hairpins. All references herein to SEQ ID NOs: 11-59 explicitly include SEQ ID NOs: 11-51. The leucine zipper shown in SEQ ID NO: 9 specifically binds to the leucine zipper shown in SEQ ID NO: 3 or 4. The leucine zipper shown in SEQ ID NO: 10 specifically binds to the leucine zipper shown in SEQ ID NO: 5, 6 or 7.

[0158] The one or more leucine zipper monomers are preferably one or more structural variants having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with a protein having the sequence shown in any one of SEQ ID NOs: 9, 10, 3-7 and 11-59, preferably SEQ ID NOs: 9, 10 and 3-7.

[0159] The one or more leucine zipper monomers preferably comprise one or more variant sequences having at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in any one of SEQ ID NOs: 9, 10, 3-7 and 11-59, preferably SEQ ID NOs:

[0160] 9, 10 and 3-7. The one or more leucine zipper monomers preferably comprise one or more variant sequences having at least about 80% homology and / or identity to the sequence shown in any one of SEQ ID NOs: 9, 10, 3-7 and 11-59, preferably SEQ ID NOs: 9, 10 and 3-7. The one or more monomers preferably comprise one or more sequences having at least about 90% homology and / or identity to the sequence shown in any one of SEQ ID NOs: 9,

[0161] 10, 3-7 and 11-59, preferably SEQ ID NOs: 9, 10 and 3-7. The one or more monomers preferably comprise one or more sequences having 100% homology and / or identity to the sequence shown in any one of SEQ ID NOs: 9, 10, 3-7 and 11-59, preferably SEQ ID NOs: 9, 10 and 3-7. Homology and / or identity is typically measured over the entire length of the reference sequence.

[0162] Sequence homology and / or identity can also relate to a fragment or portion of the reference sequence. Hence, a sequence may have less than 40% overall sequence homology and / or identity with the sequence shown in any one of SEQ ID NOs: 9, 10, 3-7 and 11-59, preferably SEQ ID NOs: 9, 10 and 3-7, but the sequence of a particular region, domain or subunit could share at least about 80%, at least about 90%, or as much as about 99% sequence homology and / or identity with the corresponding region of the sequence. There may be at least about 80%, at least 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% homology and / or identity over a stretch of about 10 or more, for example about 12, about 15, about 17, about 18, about 20, about 25 or more, contiguous amino acids ("hard homology"). Any of SEQ ID NOs: 9, 10, and 3-7 and 11-253 may be modified by substitution of one or more of their leucine amino acid residues. Any of the leucine substitution embodiments discussed above equally apply to SEQ ID NOs: 9, 10, and 3-7 and 11-253. Any of SEQ ID NOs: 9, 10, and 3-7 and 11-253 or structural variants and / or variant sequences thereof may be used to create coiled-coil oligomers, coiled-coil hairpins, leucine zipper oligomers or leucine zipper hairpins as discussed above.

[0163] In some embodiments, the one or more coupling domains in the one or more second coupling adaptors are one or more second coupling polypeptides. In those instance, the one or more coupling polypeptides in the one or more first coupling adaptors may be called one or more first coupling polypeptides.

[0164] Leader sequence

[0165] The one or more first coupling adaptors may further comprise a leader. Any suitable leader may be used. The leader may be a charged polymer, e.g., a negatively charged polymer. The leader may comprise a polymer such as PEG or a polysaccharide. The leader may be a polypeptide. The leader may be a polynucleotide. The leader may be a polypeptidepolynucleotide conjugate. The leader may be any of the polypeptides, polynucleotides, or polypeptide-polynucleotide conjugates discussed above.

[0166] The leader may be from about 10 to about 150 monomer units (e.g., ethylene glycol or saccharide units, amino acids, nucleotides or a combination thereof) in length. The leader may be from about 12 to about 120, from about 15 to about 100, from about 18 to about 80 or about 20 to about 60 monomer units in length.

[0167] Blocking moiety

[0168] The one or more first coupling adaptors may further comprise a blocking moiety. The blocking moiety may be any of those described in WO 2021 / 111125 (incorporated by reference in its entirety). The blocking moiety may be located between the one or more coupling polypeptides and the polypeptide. The blocking moiety may be located at any of the positions shown in Figures 4-7.

[0169] Second coupling adaptor(s)

[0170] The method comprises coupling the polypeptide to the membrane using (2) one or more second coupling adaptors each comprising one or more coupling domains.

[0171] Any number of one or more second coupling adaptors may be used. The method may comprise using 1, 2, 3, 4, 5 or more second coupling adaptors. The method preferably comprises using 1 or 2 second coupling adaptors. Any of these may be referred to as "types of" second coupling adaptors to distinguish them from multiple instances of the one or more second coupling adaptors in the populations described below. The one or more second coupling adaptors or one or more types of second coupling adaptors may differ based on one or more of their (a) length, (b), structure, (c) number of coupling domains and (d) identity of coupling domains. The one or more second coupling adaptors or one or more types of second coupling adaptors may differ in terms of (a); (b); (c); (d); (a) and (b); (a) and (c); (a) and (d); (b) and (c); (b) and (d); (c) and (d); (a), (b) and (c); (a), (b) and (d); (a), (c) and (d); (b), (c) and (d); or (a), (b), (c) and (d). The embodiments in this paragraph apply to any of the second coupling adaptors discussed below.

[0172] The method preferably comprises using one or two second adaptors each comprising one or more coupling domains. The method preferably comprises using one or two second adaptors each comprising one or more amphipathic helices. The amphipathic helices may be any of those described in more detail below.

[0173] The one or more second coupling adaptors may comprise a polypeptide. The one or more second coupling adaptors may be one or more polypeptide adaptors. The polypeptide(s) may be any of the types and / or lengths discussed above for the one or more first coupling adaptors. The one or more second coupling adaptors preferably comprise one or more second coupling polypeptides which specifically bind to the one or more first coupling polypeptides in the one or more first coupling adaptors. Preferred combinations of first and second coupling polypeptides are discussed in more detail below.

[0174] The one or more second coupling adaptors may comprise a polynucleotide. The one or more second coupling adaptors may be one or more polynucleotide adaptors. The polynucleotide(s) may be any of the types and / or lengths discussed above for the one or more first coupling adaptors.

[0175] The one or more second coupling adaptors may comprise a polynucleotide and a polypeptide. The one or more second coupling adaptors may be one or more polynucleotidepolypeptide conjugates. The one or more conjugate preferably comprise a polynucleotide conjugated to a polypeptide. The one or more second coupling adaptors may be any of the types of polynucleotide-polypeptide conjugates and / or and have any of the lengths discussed above for the one or more first coupling adaptors. The one or more second coupling adaptors preferably comprises one or more second coupling polypeptides which specifically bind to the one or more first coupling polypeptides in the one or more first coupling adaptors. Preferred combinations of first and second coupling polypeptides are discussed in more detail below. The one or more second coupling adaptors preferably comprise a first oligonucleotide which hybridizes to a second oligonucleotide in one or more third coupling adaptors. The one or more second coupling adaptors preferably comprise one or more second coupling polypeptides which specifically bind to one or more first coupling polypeptides in the one or more first coupling adaptors and a first oligonucleotide which hybridizes to a second oligonucleotide in one or more third coupling adaptors.

[0176] The one or more second coupling adaptors are preferably functionalised to couple to the membrane. The one or more second coupling adaptors may be functionalised in any of the ways discussed above. The one or more second coupling adaptors preferably comprise a membrane protein or one or more hydrophobic anchors. The one or more second coupling adaptors may comprise a membrane protein or one or more hydrophobic anchors even if one or more third coupling adaptors are used.

[0177] Coupling domain(s)

[0178] The one or more second coupling adaptors each comprise one or more coupling domains. The one or more coupling domains specifically bind to the one or more coupling polypeptides. The one or more coupling polypeptides are in the one or more first coupling adaptors. In all instances herein, the one or more coupling domains specifically binding to the one or more coupling polypeptides is interchangeable with the one or more coupling polypeptides specifically binding to the one or more coupling domains.

[0179] Any number of one or more coupling domains may be used. Each second coupling adaptor may comprise 1, 2, 3, 4, 5, 6, 7 or 8 or more coupling domains. Each second coupling adaptor preferably comprises 1, 2, 3 or 4 coupling domains. Each second coupling adaptor preferably comprises 1 or 2 coupling domains. Any of these may be referred to as "types of" coupling domains to distinguish them from multiple instances of the one or more coupling domains in the populations described below. The one or more coupling domains or one or more types of coupling domain may differ based on one or more of their (a) type, (b), sequence and (c) length. The one or more coupling domains or one or more types of coupling domain may differ in terms of (a); (b); (c); (a) and (b); (a) and (c); (b) and (c); or (a), (b) and (c). The embodiments in this paragraph apply to any of the one or more coupling domains discussed above and below, including the one or more amphipathic helices, one or more amphipathic helix oligomers, one or more helical hairpins, one or more coiled-coil monomers, one or more coiled-coil oligomers, one or more coiled-coil hairpins, one or more leucine zipper monomers, one or more leucine zipper oligomers and one or more leucine zipper hairpins.

[0180] The method preferably comprises using a second adaptor comprising two coupling domains. The method preferably comprises using a second adaptor comprising two amphipathic helices. The method preferably comprises using two second adaptors each comprising one coupling domain. The method preferably comprises using two second adaptors each comprising one amphipathic helix. The amphipathic helices may be any of those described in more detail below. The one or more coupling domains can be any molecules as long as they specifically bind the one or more coupling polypeptides. Suitable molecules are discussed in more detail below.

[0181] The term "specifically binds to" means binding that is measurably different from a nonspecific or non-selective interaction (e.g., with a non-target molecule). Specific binding can be measured, for example, by measuring binding of one or more coupling domains to one or more coupling polypeptides (or vice versa) and comparing it to binding to a non-target molecule. Specific binding can also be determined by competition with a control molecule that mimics the epitope recognized on the target molecule. Such methods are routine in the art. All instances herein of the term "specifically binds to" is interchangeable with "specifically interacts with," "specific for," "selectively binds to" "selectively interacts with" and "selective for".

[0182] The one or more coupling domains specifically bind to the one or more coupling polypeptides with preferential or high affinity, but do not bind or bind with only low affinity to other or different molecules, such as other or different polypeptides, other or different polypeptides or proteins or other or different polynucleotides. Preferably, the one or more coupling domains bind to the one or more coupling polypeptides with an affinity that is at least about 10 times, such as at least about 50 times, at least about 100 times, at least about 200 times, at least about 300 times, at least about 400times, at least about 500 times, at least about 1000 times or at least about 10,000 times, greater than its / their affinity for other molecules.

[0183] The term "Kassoc" or "Kon", as used herein, is intended to refer to the association rate of a particular binding molecule-target, whereas the term "Kdis" or "Koff," as used herein, is intended to refer to the dissociation rate of a particular binding molecule-target interaction. The term "Kd", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kon to Koff (i.e. Kon / Koff) and is expressed as a molar concentration (M).

[0184] The one or more coupling domains have affinity for the one or more coupling polypeptides. The one or more coupling domains preferably haves high affinity for the one or more coupling polypeptides. The one or more coupling domains have high affinity if it / they bind(s) to the one or more coupling polypeptides with a Kd of about 1 x IO-6M or less, such as about 1 x IO-7M or less, about 5 x IO-8M or less, about 1 x IO-8M or less, or about 5 x IO-9M or less. The one or more coupling domains preferably have a Kd for the one or more coupling polypeptides of about 100 nM or less, such as about 50 nM or less, about 20 nM or less, about 10 nM or less, about 1 nM or less, about 100 pM or less, about 50 pM or less, about 20 pM or less or about 10 pM or less. A molecule or group binds with low affinity if it binds with a Kd of about 1 x IO-6M or more, about 1 x IO-5M or more, about 1 x IO-4M or more, about 1 x IO-3M or more, or about 1 x IO-2M or more.

[0185] Affinity can be measured using known binding assays, such as those that make use of fluorescence and radioisotopes. Competitive binding assays are also known in the art. The strength of binding between peptides or proteins and polynucleotides can be measured using nanopore force spectroscopy as described in Hornblower et al., Nature Methods. 4: 315-317. (2007) or Isothermal Titration Calorimetry (ITC), which is a label-free quantification technique used in studies of a wide variety of biomolecular interactions. ITC works by directly measuring the heat that is either released or absorbed during a biomolecular binding event. Kd values for antibodies and other binding domains can be determined using surface plasmon resonance, such as a Biacore® system, or solution equilibrium titration (SET) (see Friguet, et al., (1985) J. Immunol. Methods, 77(2):305-319, and Hanel et al., (2005) Anal. Biochem., 339(1): 182-184).

[0186] The one or more coupling domains may specifically bind to at least a part of the one or more coupling polypeptides and vice versa. The one or more coupling domains may specifically bind to at least about 5% of the one or more coupling polypeptides, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90% or about 100% of the one or more coupling polypeptides and vice versa. %s are usually calculated on the basis on the number of amino acids to which the one or more coupling domains bind. The one or more coupling domains may specifically bind to at least about 3 amino acids in the one or more coupling polypeptides and vice versa. The one or more coupling domains may specifically bind to at least about 4 amino acids, at least about 5 amino acids, at least about 10 amino acids, at least about 15 amino acids, at least about 20 amino acids, at least about 30 amino acids, at least about 40 amino acids, at least about

[0187] 50 amino acids, at least about 60 amino acids, at least about 70 amino acids, at least about

[0188] 80 amino acids, at least about 90 amino acids, at least about 100 amino acids, at least about 110 amino acids, at least about 120 amino acids, at least about 130 amino acids, at least about 140 amino acids or at least about 150 amino acids in the one or more coupling polypeptides and vice versa.

[0189] The one or more coupling domains specifically bind the one or more coupling polypeptides. The one or more coupling polypeptides specifically bind the one or more coupling domains. The one or more coupling domains do not specifically bind itself / themselves. The one or more coupling domains specifically bind to the one or more coupling polypeptides with preferential or high affinity, but do not bind or bind with only low affinity to itself / themselves. The one or more coupling polypeptides does not specifically bind itself / themselves. The one or more coupling polypeptides specifically bind to the one or more coupling domains with preferential or high affinity, but do not bind or bind with only low affinity to itself / themselves.

[0190] The one or more coupling domains may be one or more peptides, one or more polypeptides, one or more proteins, one or more nucleotides, one or more oligonucleotide, one or more polynucleotides, one or more polynucleotide-polypeptide conjugates, one or more monosaccharides, one or more oligosaccharides, one or more polysaccharides, one or more polyethyleneglycols (PEGs), one or more saturated and unsaturated hydrocarbons or one or more polyamides. The one or more polypeptides, one or more polynucleotides or one or more polynucleotide-polypeptide conjugates may be any of the types discussed above with reference to the one or more first coupling adaptors.

[0191] The one or more coupling domains may be one or more second coupling polypeptides which specifically bind the one or more first coupling polypeptides in the one or more first coupling adaptors. The one or more second coupling polypeptides may be any of the lengths discussed above with reference to the one or more first coupling polypeptides in the one or more first coupling adaptors. The one or more second coupling polypeptides may be from about 8 to about 60 amino acids in length. The one or more second coupling polypeptides are preferably from about 10 to about 50, from about 20 to about 40 or from about 25 to about 30 amino acids in length. The one or more second coupling polypeptides may be at least about 10, at least about 20, at least about 25, at least about 30, at least at least about 40, at least about 50 or at least about 60 amino acids in length.

[0192] The one or more coupling domains preferably comprise one or more amphipathic helices, one or more derivatives of nitrilotriacetic acid (NTA), one or more promoter sequences, one or more antibodies or one or more fragments thereof, one or more antigens, one or more polynucleotides, one or more analytes, one or more enzymes, one or more aptamers, one or more split proteins, one or more affimers or one or more combinations thereof.

[0193] Antibodies and fragments are discussed below. The one or more split proteins may be any of those discussed above with reference to the coupling polypeptide.

[0194] The one or more amphipathic helices may be any of those discussed above with reference to the one or more coupling polypeptides, including one or more amphipathic helices, one or more amphipathic helix oligomers, one or more helical hairpins, one or more coiled-coil monomers, one or more coiled-coil oligomers, one or more coiled-coil hairpins, one or more leucine zipper monomers, one or more leucine zipper oligomers, one or more leucine zipper hairpins.

[0195] Table 2 below summarises preferred combinations of coupling polypeptides and coupling domains. The combination from any row may be used in the coupling method of the invention.

[0196] The one or more amphipathic helices, one or more amphipathic helix oligomers and one or more helical hairpins may be any of those discussed above with reference to the one or more coupling polypeptides. The one or more coiled-coil monomers, one or more coiled-coil oligomers and one or more coiled-coil hairpins may be any of those discussed above with reference to the one or more coupling polypeptides. The one or more leucine zipper monomers, one or more leucine zipper oligomers and one or more leucine zipper hairpins may be any of those discussed above with reference to the one or more coupling polypeptides. The skilled person is capable of designing combinations of amphipathic helices, coiled-coiled monomers and leucine zipper monomers so they specifically bind each other and couple the one or more first coupling adaptors and the one or more second coupling adaptors.

[0197] The term "antibody" as referred to herein includes whole antibodies. Naturally occurring antibodies typically comprise a tetramer which is usually composed of at least two heavy (H) chains and at least two light (L) chains. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, usually comprised of three domains (CHI, CH2 and CH3). Heavy chains can be of any isotype, including IgG (IgGl, IgG2, IgG3 and IgG4 subtypes), IgA (IgAl and IgA2 subtypes), IgM and IgE. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL). Light chain includes kappa (K) chains and lambda (A) chains. The heavy and light chain variable region is typically responsible for antigen recognition, whilst the heavy and light chain constant region may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g. effector cells) and the first component (Clq) of the classical complement system. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.

[0198] The term "fragment thereof" with reference to an antibody refers to any fragment of an intact antibody. Such fragments include, but are not limited to, an antibody heavy chain, the variable region of an antibody heavy chain (VH), an antibody light chain and the variable region of an antibody light chain (VL). The fragment retains the ability to specifically bind to a target antigen, such as CD38, although in some instances an accessory protein is required for specific binding. For instance, if the fragment is an antibody heavy chain or the variable region of an antibody heavy chain (VH), an antibody light chain or the variable region of an antibody light chain (VL) is needed for specific binding. The antibody light chain or the variable region of an antibody light chain (VL) need not form part of the fusion protein of the invention and can be supplied or expressed separately so that the fusion protein forms an antibody or functional fragment thereof capable of specific binding to a target antigen. The fragment is preferably a functional fragment. The term "functional fragment" with reference to an antibody, including a "functional antibody fragment", refers to a fragment of an intact antibody that retains the ability to specifically bind to a target antigen. Such fragments include, but are not limited to, Fab fragments, Fab' fragments, monovalent fragments consisting of the VL, VH, CL and CHI domains; F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragment consisting of the VH and CHI domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g. Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term "functional fragment" of an antibody. These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0199] The antibody, fragment thereof or functional fragment thereof typically comprises one or more, such as 6 or 12, complementarity determining regions (CDRs). CDRs are defined according to the Kabat definition unless specified that the CDR are defined according to another definition. The precise amino acid sequence boundaries of a given CDR can be determined using any of a number of well-known schemes, including those described by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5thEd. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme), Al- Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme) and ImMunoGenTics (IMGT) numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc, M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003) ("IMGT" numbering scheme). For example, for classic formats, under Kabat, the CDR amino acid residues in the heavy chain variable domain (VH) are numbered 31-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3); and the CDR amino acid residues in the light chain variable domain (VL) are numbered 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3). Under Chothia the CDR amino acids in the VH are numbered 26-32 (HCDR1), 52-56 (HCDR2), and 95-102 (HCDR3); and the amino acid residues in VL are numbered 26-32 (LCDR1), 50-52 (LCDR2), and 91-96 (LCDR3). By combining the CDR definitions of both Kabat and Chothia, the CDRs consist of amino acid residues 26-35 (HCDR1), 50-65 (HCDR2), and 95-102 (HCDR3) in human VH and amino acid residues 24-34 (LCDR1), 50-56 (LCDR2), and 89-97 (LCDR3) in human VL. Under IMGT the CDR amino acid residues in the VH are numbered approximately 26-35 (CDR1), 51-57 (CDR2) and 93-102 (CDR3), and the CDR amino acid residues in the VL are numbered approximately 27-32 (CDR1), 50-52 (CDR2), and 89-97 (CDR.3) (numbering according to "Kabat"). Under IMGT, the CDR regions of an antibody can be determined using the program IMGT / DomainGap Align.

[0200] By convention, the CDR regions in the heavy chain are typically referred to as HCDR1, HCDR2 and HCDR3 and in the light chain as LCDR1, LCDR2 and LCDR3. They are numbered sequentially in the direction from the amino terminus to the carboxy terminus.

[0201] The antibody may be a monoclonal antibody, a fragment thereof or a functional fragment thereof. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0202] The antibody or fragment thereof may be human

[0203] An antibody can be prepared using an antibody having one or more of the VH and / or VL sequences shown herein as starting material to engineer a modified antibody, which modified antibody may have altered properties from the starting antibody. An antibody can be engineered by modifying one or more residues within one or both variable regions (i.e., VH and / or VL), for example within one or more CDR regions and / or within one or more framework regions. Additionally or alternatively, an antibody can be engineered by modifying residues within the constant region(s), for example to alter the effector function(s) of the antibody. Antibody engineering is well known in the art.

[0204] Antibody proteins obtained from members of the camel and dromedary {Camelus bactrianus and Camelus dromaderius) family including new world members such as llama species {Lama paccos, Lama glama and Lama vicugna) have been characterized with respect to size, structural complexity and antigenicity for human subjects. The antibody or fragment thereof may be derived from any of these species.

[0205] A region of the camelid antibody which is the small single variable domain identified as VHH can be obtained by genetic engineering to yield a small protein having high affinity for a target, resulting in a low molecular weight antibody-derived protein known as a "camelid nanobody". See US5,759,808; see also Stijlemans, B. et al., 2004 J Biol Chem 279: 1256- 1261; Dumoulin, M. et al., 2003 Nature 424: 783-788; Pleschberger, M. et al. 2003 Bioconjugate Chem 14: 440-448; Cortez-Retamozo, V. et al. 2002 Int J Cancer 89: 456-62; and Lauwereys, M. et al. 1998 EMBO J 17: 3512-3520. Engineered libraries of camelid antibodies and antibody fragments are commercially available, for example, from Ablynx, Ghent, Belgium. As with other antibodies of non-human origin, an amino acid sequence of a camelid antibody can be altered recombinantly to obtain a sequence that more closely resembles a human sequence, i.e., the nanobody can be "humanized". Thus the natural low antigenicity of camelid antibodies to humans can be further reduced. The camelid nanobody has a molecular weight approximately one-tenth that of a human IgG molecule, and the protein has a physical diameter of only a few nanometers. One consequence of the small size is the ability of camelid nanobodies to bind to antigenic sites that are functionally invisible to larger antibody proteins, i.e., camelid nanobodies are useful as reagents detect antigens that are otherwise cryptic using classical immunological techniques, and as possible therapeutic agents. Thus yet another consequence of small size is that a camelid nanobody can inhibit as a result of binding to a specific site in a groove or narrow cleft of a target protein, and hence can serve in a capacity that more closely resembles the function of a classical low molecular weight drug than that of a classical antibody.

[0206] The low molecular weight and compact size further result in camelid nanobodies being extremely thermostable, stable to extreme pH and to proteolytic digestion, and poorly antigenic. Another consequence is that camelid nanobodies readily move from the circulatory system into tissues, and even cross the blood-brain barrier and can treat disorders that affect nervous tissue. Nanobodies can further facilitate drug transport across the blood brain barrier, see US2004 / 0161738. These features combined with the low antigenicity to humans indicate great therapeutic potential. Further, these molecules can be fully expressed in prokaryotic cells such as E. coli and are expressed as fusion proteins with bacteriophage and are functional.

[0207] The one or more antibodies or one or more fragments thereof can be one or more camelid antibodies or one or more nanobodies, one or more fragments thereof or one or more functional fragments thereof. In one embodiment, the camelid antibody, nanobody, fragment thereof or a functional fragment thereof is obtained by grafting the CDRs sequences of the heavy or light chain of a human antibody into nanobody or single domain antibody framework sequences, as described for example in WO 94 / 04678 (incorporated herein by reference in its entirety).

[0208] The antibody or fragment thereof may comprise non-immunoglobulin frameworks. Known non-immunoglobulin frameworks or scaffolds include, but are not limited to, Adnectins (fibronectin) (Compound Therapeutics, Inc., Waltham, MA), ankyrin (Molecular Partners AG, Zurich, Switzerland), domain antibodies (Domantis, Ltd (Cambridge, MA), now part of GSK) and Ablynx nv (Zwijnaarde, Belgium)), lipocalin (Anticalin) (Pieris Proteolab AG, Freising, Germany), small modular immuno-pharmaceuticals (Trubion Pharmaceuticals Inc., Seattle, WA, now Emergent BioSolutions), maxybodies (Avidia, Inc. (Mountain View, CA)), Protein A (Affibody AB, Sweden) and affilin (gamma-crystallin or ubiquitin) (Scil Proteins GmbH, Halle, Germany), protein epitope mimetics (Polyphor Ltd, Allschwil, Switzerland).

[0209] The antibody or fragment thereof may be modified using any method known in the art. Numbers of adaptors, polypeptides and domains

[0210] The method uses (1) one or more first coupling adaptors comprising one or more coupling polypeptides, and (2) one or more second coupling adaptors comprising one or more coupling domains which specifically bind to the one or more coupling polypeptides. Each row of the Table 3 below summarises preferred combinations of numbers of (the types of) one or more first coupling adaptors, one or more coupling polypeptides, one or more second coupling adaptors and one or more coupling domains. The one or more first coupling adaptors, one or more coupling polypeptides, one or more second coupling adaptors and one or more coupling domains may be any of those described above. The one or more coupling polypeptides and / or the one or more coupling domains may be any of the amphipathic helices, amphipathic helix oligomers, helical hairpins, coiled-coil monomers, coiled-coil oligomers, coiled-coil hairpins, leucine zipper monomers, leucine zipper oligomers, and leucine zipper hairpins discussed above. The last column relates to the optional number of third coupling adaptors. The means no third coupling adaptors are used. The third coupling adaptors may be any of those described above. The method may use any of the numbers of these components shown in Figures 4-7.

[0211] Table 3 - Preferred numbers of adaptors, coupling polypeptides and coupling domains

[0212] Preferred coupling method of the invention The coupling method of the invention preferably uses combinations of one or more amphipathic helices which specifically bind to each other.

[0213] The one or more coupling polypeptides preferably comprises one or more amphipathic helices and the one or more coupling domains preferably comprises one or more amphipathic helices which specifically bind to the one or more amphipathic helices in the one or more first coupling adaptors.

[0214] The one or more first coupling polypeptides preferably comprise one or more first amphipathic helices and the one or more second coupling polypeptides preferably comprise one or more second amphipathic helices. The one or more first amphipathic helices specifically bind the one or more second amphipathic helices. The one or more second amphipathic helices specifically bind the one or more first amphipathic helices.

[0215] The one or more first coupling adaptors preferably comprise one or more first amphipathic helices and the one or more second coupling adaptors preferably comprises one or more second amphipathic helices which specifically bind to the one or more first amphipathic helices.

[0216] The one or more amphipathic helices in first adaptors and / or the second adaptors may be any of the amphipathic helices, amphipathic helix oligomers and helical hairpins discussed above. The one or more first amphipathic helices and / or the one or more second amphipathic helices may be any of the amphipathic helices, amphipathic helix oligomers and helical hairpins discussed above.

[0217] The one or more first amphipathic helices are preferably acidic and the one or more second amphipathic helices are preferably basic. The one or more first amphipathic helices are preferably basic and the one or more second amphipathic helices are preferably acidic.

[0218] In all embodiments herein, the one or more amphipathic helices, one or more coiled-coil monomers or one or more leucine zipper monomers, including one or more oligomers and one or more hairpins formed thereof, are acidic if they have an overall or net negative charge. In all instances herein, the term "are acidic" is interchangeable with "have an overall negative charge" or "have a net negative charge". This can be achieved if the one or more amphipathic helices, one or more coiled-coil monomers or one or more leucine zipper monomers, including one or more oligomers and one or more hairpins formed thereof, comprise one or more negatively charged amino acids residues, such as aspartic acid (D) or glutamic acid (E). Even though the one or more monomers, oligomers or hairpins are acidic, they may comprise one or more sections that are basic. Even though the one or more monomers, oligomers or hairpins have an overall negative charge, they may comprise one or more sections that are positively charged. This allows the specificity of the one or more monomers, oligomers or hairpins to be fine-tuned. In all embodiments herein, the one or more amphipathic helices, one or more coiled-coil monomers or one or more leucine zipper monomers, including one or more oligomers and one or more hairpins formed thereof, are basic if they have an overall or net positive charge. In all instances herein, the term "are basic" is interchangeable with "have an overall positive charge" or "have a net positive charge". This can be achieved if the one or more amphipathic helices, one or more coiled-coil monomers or one or more leucine zipper monomers, including one or more oligomers and one or more hairpins formed thereof, comprise one or more positively charged amino acids residues, such as such as arginine (R) or lysine (K). Even though the one or more monomers, oligomers or hairpins are basic, they may comprise one or more sections that are acidic. Even though the one or more monomers, oligomers or hairpins have an overall positive charge, they may comprise one or more sections that are negatively charged. This allows the specificity of the one or more monomers, oligomers or hairpins to be fine-tuned.

[0219] The one or more coupling polypeptides preferably comprises one or more coiled-coil monomers and the one or more coupling domains preferably comprises one or more coiled- coil monomers which specifically bind to the one or more coiled-coil monomers in the one or more first coupling adaptors.

[0220] The one or more first coupling polypeptides preferably comprise one or more first coiled-coil monomers and the one or more second coupling polypeptides preferably comprise one or more second coiled-coil monomers. The one or more first coiled-coil monomers specifically bind the one or more second coiled-coil monomers. The one or more second coiled-coil monomers specifically bind the one or more first coiled-coil monomers.

[0221] The one or more first coupling adaptors preferably comprise one or more first coiled-coil monomers and the one or more second coupling adaptors preferably comprises one or more second coiled-coil monomers which specifically bind to the one or more first coiled-coil monomers.

[0222] The one or more coiled-coil monomers in first adaptors and / or the second adaptors may be any of the coiled-coil monomers, coiled-coil oligomers and coiled-coil hairpins discussed above. The one or more first coiled-coil monomers and / or the one or more second coiled- coil monomers may be any of the coiled-coil monomers, coiled-coil oligomers and coiled-coil hairpins discussed above.

[0223] The one or more first coiled-coil monomers are preferably acidic and the one or more second coiled-coil monomers are preferably basic. The one or more first coiled-coil monomers are preferably basic and the one or more second coiled-coil monomers are preferably acidic. These terms are defined above and any of those embodiments equally apply to the one or more coiled-coil monomers. The one or more first coiled-coil monomers may be any of the proteins discussed above with reference to SEQ ID NOs: 60-253, including structural variants and variant sequences thereof and coiled-coil oligomers and coiled-coil hairpins comprising such sequences.

[0224] The one or more second coiled-coil monomers preferably comprise one or more structural variants having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with a protein having the sequence shown in any one of SEQ ID NOs: 60-253.

[0225] The one or more second coiled-coil monomers preferably comprise one or more variant sequences having at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in any one of SEQ ID NOs: 60-253. The one or more second coiled-coil monomers preferably comprises one or more variant sequences having at least about 80% homology and / or identity to the sequence shown in any one of SEQ ID NOs: 60-253. The one or more second coiled-coil monomers preferably comprise a sequence having at least about 90% homology and / or identity to the sequence shown in any one of SEQ ID NOs: 60- 253. The one or more second coiled-coil monomers preferably comprises a sequence having 100% homology and / or identity to the sequence shown in any one of SEQ ID NOs: 60-253. Homology and / or identity is typically measured over the entire length of the reference sequence.

[0226] Sequence homology and / or identity can also relate to a fragment or portion of the reference sequence. Hence, a sequence may have less than 40% overall sequence homology and / or identity with the sequence shown in any one of SEQ ID NOs: 60-253, but the sequence of a particular region, domain or subunit could share at least about 80%, at least about 90%, or as much as about 99% sequence homology and / or identity with the corresponding region of the sequence. There may be at least about 80%, at least 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% homology and / or identity over a stretch of about 10 or more, for example about 12, about 15, about 17, about 18, about 20, about 25 or more, contiguous amino acids ("hard homology").

[0227] The one or more second coiled-coil monomers preferably comprise one or more coiled-coil oligomers and / or one or more coiled-coil hairpins comprising any of SEQ ID NOs: 60-253 or structural variants and / or variant sequences thereof. Coiled-coil oligomers and hairpins are described above.

[0228] Preferred one or more coiled-coil oligomers comprise one or more of: - the sequence shown in SEQ ID NO: 236 or a structural variant and / or variant sequence thereof and the sequence shown in SEQ ID NO: 237 or a structural variant and / or variant sequence thereof;

[0229] - the sequence shown in SEQ ID NO: 238 or a structural variant and / or variant sequence thereof and the sequence shown in SEQ ID NO: 239 or a structural variant and / or variant sequence thereof;

[0230] - the sequence shown in SEQ ID NO: 240 or a structural variant and / or variant sequence thereof and the sequence shown in SEQ ID NO: 241 or a structural variant and / or variant sequence thereof;

[0231] - the sequence shown in SEQ ID NO: 242 or a structural variant and / or variant sequence thereof and the sequence shown in SEQ ID NO: 243 or a structural variant and / or variant sequence thereof;

[0232] - the sequence shown in SEQ ID NO: 244 or a structural variant and / or variant sequence thereof and the sequence shown in SEQ ID NO: 245 or a structural variant and / or variant sequence thereof;

[0233] - the sequence shown in SEQ ID NO: 246 or a structural variant and / or variant sequence thereof and the sequence shown in SEQ ID NO: 247 or a structural variant and / or variant sequence thereof;

[0234] - the sequence shown in SEQ ID NO: 248 or a structural variant and / or variant sequence thereof and the sequence shown in SEQ ID NO: 249 or a structural variant and / or variant sequence thereof;

[0235] - the sequence shown in SEQ ID NO: 250 or a structural variant and / or variant sequence thereof and the sequence shown in SEQ ID NO: 251 or a structural variant and / or variant sequence thereof; and / or

[0236] - the sequence shown in SEQ ID NO: 252 or a structural variant and / or variant sequence thereof and the sequence shown in SEQ ID NO: 253 or a structural variant and / or variant sequence thereof.

[0237] The one or more first coiled-coil monomers preferably comprise one or more leucine zipper monomers. The one or more second coiled-coil monomers preferably comprise one or more leucine zipper monomers. The one or more first coiled-coil monomers preferably comprises one or more first leucine zipper monomers and the one or more second coiled-coil monomers preferably comprises one or more second leucine zipper monomers. The one or more first leucine zipper monomers specifically bind the one or more second leucine zipper monomers. The one or more second leucine zipper monomers specifically bind the one or more first leucine zipper monomers.

[0238] The one or more leucine zipper monomers in first adaptors and / or the second adaptors may be any of the leucine zipper monomers, leucine zipper oligomers and leucine zipper hairpins discussed above. The one or more first leucine zipper monomers and / or the one or more second leucine zipper monomers may be any of the leucine zipper monomers, leucine zipper oligomers and leucine zipper hairpins discussed above.

[0239] The one or more first leucine zipper monomers are preferably acidic and the one or more second leucine zipper monomers are preferably basic. The one or more first leucine zipper monomers are preferably basic and the one or more second leucine zipper monomers are preferably acidic. These terms are defined above and any of those embodiments equally apply to the one or more leucine zipper monomers.

[0240] The one or more first leucine zipper monomers may be any of the proteins discussed above with reference to SEQ ID NOs: 9, 10, 3-7 and 11-59, including structural variants and variant sequences thereof and leucine zipper oligomers and leucine zipper hairpins comprising such sequences.

[0241] The one or more second leucine zipper monomers preferably comprise one or more structural variants having a root mean square deviation (RMSD) of less than about 4.0 Angstroms (A), such as less than about 3.5 A, less than about 3.0 A, less than about 2.5 A, less than about 2.0 A, less than about 1.5 A, less than about 1.0 A or less than about 0.5 A, when compared with a protein having the sequence shown in any one of SEQ ID NOs: 9, 10, 3-7 and 11-59, preferably SEQ ID NOs: 9, 10 and 3-7.

[0242] The one or more second leucine zipper monomers preferably comprise one or more variant sequences having at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% homology and / or identity to the sequence shown in any one of SEQ ID NOs: 9, 10, 3-7 and 11-59, preferably SEQ ID NOs: 9, 10 and 3-7. The one or more second leucine zipper monomers preferably comprises one or more variant sequences having at least about 80% homology and / or identity to the sequence shown in any one of SEQ ID NOs: 9, 10, 3-7 and 11-59, preferably SEQ ID NOs: 9, 10 and 3-7. The one or more second leucine zipper monomers preferably comprise a sequence having at least about 90% homology and / or identity to the sequence shown in any one of SEQ ID NOs: 9, 10, 3-7 and 11-59, preferably SEQ ID NOs: 9, 10 and 3-7. The one or more second leucine zipper monomers preferably comprises a sequence having 100% homology and / or identity to the sequence shown in any one of SEQ ID NOs: 9, 10, 3- 7 and 11-59, preferably SEQ ID NOs: 9, 10 and 3-7. Homology and / or identity is typically measured over the entire length of the reference sequence.

[0243] Sequence homology and / or identity can also relate to a fragment or portion of the reference sequence. Hence, a sequence may have less than 40% overall sequence homology and / or identity with the sequence shown in any one of SEQ ID NOs: 9, 10, 3-7 and 11-59, preferably SEQ ID NOs: 9, 10 and 3-7, but the sequence of a particular region, domain or subunit could share at least about 80%, at least about 90%, or as much as about 99% sequence homology and / or identity with the corresponding region of the sequence. There may be at least about 80%, at least 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98% or at least about 99% homology and / or identity over a stretch of about 10 or more, for example about 12, about 15, about 17, about 18, about 20, about 25 or more, contiguous amino acids ("hard homology").

[0244] The one or more second leucine zipper monomers preferably comprise one or more leucine zipper oligomers and / or one or more leucine zipper hairpins comprising any of SEQ ID NOs: 9, 10, 3-7 and 11-59 or structural variants and / or variant sequences thereof. Leucine zipper oligomers and hairpins are described above.

[0245] Any of SEQ ID NOs: 9, 10, and 3-7 and 11-253 may be modified by substitution of one or more of their leucine amino acid residues. Any of the leucine substitution embodiments discussed above equally apply to SEQ ID NOs: 9, 10, and 3-7 and 11-253. Any of SEQ ID NOs: 9, 10, and 3-7 and 11-253 or structural variants and / or variant sequences thereof may be used to create coiled-coil oligomers, coiled-coil hairpins, leucine zipper oligomers or leucine zipper hairpins as discussed above.

[0246] Table 4 below also summarises preferred combinations of leucine zipper monomers for use in the coupling method of the invention. For any row, the one or more first leucine zipper monomers may comprise one or more of the the leucine zipper monomer in the left column and the one or more second leucine zipper may comprise one or more of the leucine zipper monomer in the right column. The structural variants and / or variant sequences may be any of those defined above. The structural variants include all of the leucine zipper oligomers and leucine zipper hairpins discussed above.

[0247] Table 4 - Preferred combination of first and second leucine zipper monomers

[0248] Third coupling adaptor(s)

[0249] The method preferably further comprises using (3) one or more third coupling adaptors. The one or more third coupling adaptors may comprise a polypeptide. The polypeptide may be any of the types and / or lengths discussed above for the one or more first coupling adaptors.

[0250] The one or more third coupling adaptors may comprise a polynucleotide and a polypeptide. The one or more third coupling adaptors may be one or more polynucleotide-polypeptide conjugates. The one or more conjugates preferably comprise a polynucleotide conjugated to a polypeptide. The one or more third coupling adaptors may be any of the types of a polynucleotide-polypeptide conjugates and / or and have any of the lengths discussed above for the one or more first coupling adaptors. The one or more third coupling adaptors preferably comprise a polynucleotide. The polynucleotide may be any of the types and / or lengths discussed above with reference to the one or more first coupling adaptors.

[0251] The polynucleotide is preferably at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 11 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 27 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, or at least about 50 nucleotides in length.

[0252] When one or more third coupling adaptors are used, the one or more second coupling adaptors may further comprise a first oligonucleotide and the one or more third coupling adaptors may further comprises a second oligonucleotide which hybridizes to the first oligonucleotide. The first and / or second oligonucleotide may be any length. The first and / or second oligonucleotide is preferably at least about 5 nucleotides, at least about 6 nucleotides, at least about 7 nucleotides, at least about 8 nucleotides, at least about 9 nucleotides, at least about 10 nucleotides, at least about 11 nucleotides, at least about 12 nucleotides, at least about 13 nucleotides, at least about 14 nucleotides, at least about 15 nucleotides, at least about 20 nucleotides, at least about 25 nucleotides, at least about 27 nucleotides, at least about 30 nucleotides, at least about 35 nucleotides, at least about 40 nucleotides, at least about 45 nucleotides, or at least about 50 nucleotides in length.

[0253] The first oligonucleotide preferably specifically hybridises to the second oligonucleotide. Oligonucleotides "specifically hybridise" when they hybridise with preferential or high affinity each other but do not substantially hybridise, does not hybridise, or hybridises with only low affinity to other polynucleotide sequences, especially other polynucleotide sequences used in the invention. Conditions that permit the hybridisation are well-known in the art (for example, Sambrook et al., 2001, Molecular Cloning: a laboratory manual, 3rd edition, Cold Spring Harbour Laboratory Press; and Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishing and Wiley-lnterscience, New York (1995)). Hybridisation can be carried out under low stringency conditions, for example in the presence of a buffered solution of 30 to 35% formamide, 1 M NaCI and 1 % SDS (sodium dodecyl sulfate) at 37 °C followed by a 20 wash in from IX (0.1650 M Na+) to 2X (0.33 M Na+) SSC (standard sodium citrate) at 50 °C. Hybridisation can be carried out under moderate stringency conditions, for example in the presence of a buffer solution of 40 to 45% formamide, 1 M NaCI, and 1 % SDS at 37 °C, followed by a wash in from 0.5X (0.0825 M Na+) to IX (0.1650 M Na+) SSC at 55 °C. Hybridisation can be carried out under high stringency conditions, for example in the presence of a buffered solution of 50% formamide, 1 M NaCI, 1% SDS at 37 °C, followed by a wash in 0.1X (0.0165 M Na+) SSC at 60 °C. The oligonucleotides "specifically hybridise" if they hybridise with a melting temperature (Tm) that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C or at least 10 °C, greater than its Tm for other polynucleotide sequences. More preferably, the oligonucleotides hybridise with a Tm that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C, at least 10 °C, at least 20 °C, at least 30 °C or at least 40 °C, greater than its Tm for other polynucleotide sequences. Preferably, the oligonucleotides hybridise with a Tm that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C, at least 10 °C, at least 20 °C, at least 30 °C or at least 40 °C, greater than its Tm for a polynucleotide which differs from the oligonucleotides by one or more nucleotides, such as by 1, 2, 3, 4 or 5 or more nucleotides. The strands or parts thereof typically hybridise with a Tm of at least 90 °C, such as at least 92 °C or at least 95 °C. Tm can be measured experimentally using known techniques, including the use of DNA microarrays, or can be calculated using publicly available Tm calculators, such as those available over the internet.

[0254] The one or more third coupling adaptors are preferably functionalised to couple to the membrane. The one or more third coupling adaptors may be functionalised in any of the ways discussed above. The one or more third coupling adaptors preferably comprise a membrane protein or one or more hydrophobic anchors.

[0255] Linkers

[0256] Any of the coupling adaptors may comprise one or more linkers such as 2, 3, 4 or more linkers. The (a) one or more first coupling adaptors, (b) the one or more second coupling adaptors and / or (c) the one or more third coupling adaptors, such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c) or (a), (b) and (c), may comprise one or more linkers.

[0257] Preferred linkers include, but are not limited to, polymers, such as polynucleotides, polyethylene glycols (PEGs), polysaccharides and polypeptides. These linkers may be linear, branched, or circular. For instance, the linker may be a circular polynucleotide. The adaptor(s) may hybridise to a complementary sequence on a circular polynucleotide linker. The one or more linkers may comprise a component that can be cut or broken down, such as a restriction site or a photolabile group. The one or more linkers may be functionalised with maleimide groups to attach to cysteine residues in proteins. Suitable linkers are described in WO 2010 / 086602 (incorporated herein by reference in its entirety).

[0258] Adaptor synthesis

[0259] The adaptors of the invention are typically synthetic or semi-synthetic. For example, DNA or RNA may be purely synthetic, synthesised by conventional DNA synthesis methods such as phosphoramidite based chemistries. Synthetic polynucleotides subunits may be joined together by known means, such as ligation or chemical linkage, to produce longer strands. Internal self-forming structures (e.g., hairpins, quadruplexes) can be designed into the substrate, e.g., by ligating appropriate sequences. Synthetic polynucleotides can be copied and scaled up for production by means known in the art, including PCR, incorporation into bacterial factories, and the like. Synthetic polypeptides may be produced using any of the methods described herein. The production of polynucleotide-polypeptide conjugates is discussed above.

[0260] Spacers

[0261] Any of the coupling adaptors may comprise one or more spacers. The (a) one or more first coupling adaptors, (b) the one or more second coupling adaptors and / or (c) the one or more third coupling adaptors, such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c) or (a), (b) and (c), may comprise one or more spacers.

[0262] Any of the adaptors may comprise from about one to about 10 spacers, e.g., from about 1 to about 5 spacers, e.g., about 1, 2, 3, 4 or 5 spacers.

[0263] One or more spacers are typically included in the adaptors to provide a distinctive signal when they pass through or across a nanopore. One or more spacers may be used to define or separate one or more regions of an adaptor, e.g., to separate an adaptor from the target polypeptide.

[0264] A spacer may comprise a linear molecule, such as a polymer, e.g., a polypeptide or a polyethylene glycol (PEG). Typically, such a spacer has a different structure from the target polynucleotide. For instance, if the target polynucleotide is DNA, the or each spacer typically does not comprise DNA. In particular, if the target polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), the or each spacer preferably comprises peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA) or a synthetic polymer with nucleotide side chains. A spacer may comprise one or more nitroindoles, one or more inosines, one or more acridines, one or more 2- aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuridines, one or more inverted thymidines (inverted dTs), one or more inverted dideoxy-thymidines (ddTs), one or more dideoxy-cytidines (ddCs), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2'-O-Methyl RNA bases, one or more Isodeoxycytidines (Iso-dCs), one or more Iso-deoxyguanosines (Iso-dGs), one or more C3 (OC3H6OPO3) groups, one or more photo-cleavable (PC) [OC3H6-C(O)NHCH2-C6H3NO2- CH(CH3)OPO3] groups, one or more hexandiol groups, one or more spacer 9 (iSp9) [(OCH2CH2)3OPO3] groups, or one or more spacer 18 (iSplS) [(OCH2CH2)6OPO3] groups; or one or more thiol connections. A spacer may comprise any combination of these groups. Many of these groups are commercially available from IDT® (Integrated DNA Technologies®). For example, C3, iSp9 and iSpl8 spacers are all available from IDT®. A spacer may comprise any number of the above groups as spacer units.

[0265] A spacer may comprise one or more chemical groups, e.g., one or more pendant chemical groups. The one or more chemical groups may be attached to one or more nucleobases in an adaptor. The one or more chemical groups may be attached to the backbone of an adaptor. Any number of appropriate chemical groups may be present, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more. Suitable groups include, but are not limited to, fluorophores, streptavidin and / or biotin, cholesterol, methylene blue, dinitrophenols (DNPs), digoxigenin and / or anti-digoxigenin and dibenzylcyclooctyne groups.

[0266] A spacer may comprise one or more abasic nucleotides ( / .e., nucleotides lacking a nucleobase), such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more abasic nucleotides. The nucleobase can be replaced by -H (idSp) or -OH in the abasic nucleotide. Abasic spacers can be inserted into target polynucleotides by removing the nucleobases from one or more adjacent nucleotides. For instance, polynucleotides may be modified to include 3- methyladenine, 7-methylguanine, l,N6-ethenoadenine inosine or hypoxanthine and the nucleobases may be removed from these nucleotides using Human Alkyladenine DNA Glycosylase (hAAG). Alternatively, polynucleotides may be modified to include uracil and the nucleobases removed with Uracil-DNA Glycosylase (UDG). The one or more spacers preferably do not comprise any abasic nucleotides.

[0267] Suitable spacers can be designed or selected depending on the nature of the adaptor, the polynucleotide binding protein, and the conditions under which the method is to be carried out.

[0268] Tags

[0269] Any of the adaptors used in the invention may comprise a tag. The tag may comprise or be an oligonucleotide (e.g., DNA, RNA, LNA, BNA, PNA, or morpholino). The oligonucleotide (e.g., DNA, RNA, LNA, BNA, PNA, or morpholino) can have about 10-30 nucleotides in length or about 10-20 nucleotides in length. The oligonucleotide (e.g., DNA, RNA, LNA, BNA, PNA, or morpholino) for use in the tag can have at least one end (e.g., 3'- or 5'-end) modified for conjugation to other modifications or to a solid substrate surface including, e.g., a bead. The end modifiers may add a reactive functional group which can be used for conjugation. Examples of functional groups that can be added include, but are not limited to amino, carboxyl, thiol, maleimide, aminooxy, and any combinations thereof. The functional groups can be combined with different length of spacers (e.g., C3, C9, C12, Spacer 9 and 18) to add physical distance of the functional group from the end of the oligonucleotide sequence. The tag may comprise or be a morpholino oligonucleotide. The morpholino oligonucleotide can have about 10-30 nucleotides in length or about 10-20 nucleotides in length. The morpholino oligonucleotides can be modified or unmodified. For example, the morpholino oligonucleotide can be modified on the 3' and / or 5' ends of the oligonucleotides. Examples of modifications on the 3' and / or 5' end of the morpholino oligonucleotides include, but are not limited to 3' affinity tag and functional groups for chemical linkage (including, e.g., 3'- biotin, 3'-primary amine, 3'-disulfide amide, 3'-pyridyl dithio, and any combinations thereof); 5' end modifications (including, e.g., 5'-primary ammine, and / or 5'-dabcyl), modifications for click chemistry (including, e.g., 3'-azide, 3'-alkyne, 5'-azide, 5'-alkyne), and any combinations thereof.

[0270] The tag may further comprise a polymeric linker, e.g., to facilitate coupling to a detector e.g., a nanopore. An exemplary polymeric linker includes, but is not limited to, polyethylene glycol (PEG). The polymeric linker may have a molecular weight of about 500 Da to about 10 kDa (inclusive), or about 1 kDa to about 5 kDa (inclusive). The polymeric linker (e.g., PEG) can be functionalized with different functional groups including, e.g., but not limited to maleimide, NHS ester, di benzocyclooctyne (DBCO), azide, biotin, amine, alkyne, aldehyde, and any combinations thereof. The tag may further comprise a 1 kDa PEG with a 5'- maleimide group and a 3'-DBCO group. The tag may further comprise a 2 kDa PEG with a 5'-maleimide group and a 3'-DBCO group. The tag may further comprise a 3 kDa PEG with a 5'-maleimide group and a 3'-DBCO group. The tag may further comprise a 5 kDa PEG with a 5'-maleimide group and a 3'-DBCO group.

[0271] The tag may be a peptide, polypeptide or protein. The tag may be a CsgA polypeptide. The CsgA polypeptide may be any of those described in WO 2024 / 100270 (incorporated herein in its entirety).

[0272] Other examples of a tag include, but are not limited to His tags, biotin or streptavidin, antibodies that bind to analytes, aptamers that bind to analytes, analyte binding domains such as DNA binding domains (including, e.g., peptide zippers such as leucine zippers, single-stranded DNA binding proteins (SSB)), and any combinations thereof.

[0273] The tag may be attached to the external surface of a nanopore, e.g., on the cis side of a membrane, using any methods known in the art. For example, one or more tags can be attached to the nanopore via one or more cysteines (cysteine linkage), one or more primary amines such as lysines, one or more non-natural amino acids, one or more histidines (His tags), one or more biotin or streptavidin, one or more antibody-based tags, one or more enzyme modification of an epitope (including, e.g., acetyl transferase), and any combinations thereof. Suitable methods for carrying out such modifications are well-known in the art. Suitable non-natural amino acids include, but are not limited to, 4-azido-L- phenylalanine (Faz) and any one of the amino acids numbered 1-71 in Figure 1 of Liu C. C. and Schultz P. G., Annu. Rev. Biochem., 2010, 79, 413-444.

[0274] Where one or more tags are attached to a nanopore via cysteine linkage(s), the one or more cysteines can be introduced to one or more monomers that form the nanopore by substitution. The nanopore may be chemically modified by attachment of (i) Maleimides including diabromomaleimides such as: 4-phenylazomaleinanil, l.N-(2- Hydroxyethyl)maleimide, N-Cyclohexylmaleimide, 1.3-Maleimidopropionic Acid, 1.1-4- Aminophenyl-lH-pyrrole,2,5,dione, l.l-4-Hydroxyphenyl-lH-pyrrole,2,5,dione, N- Ethylmaleimide, N-Methoxycarbonylmaleimide, N-tert-Butylmaleimide, N-(2- Aminoethyl)maleimide , 3-Maleimido-PROXYL , N-(4-Chlorophenyl)maleimide, l-[4- (dimethylamino)-3,5-dinitrophenyl]-lH-pyrrole-2, 5-dione, N-[4-(2- Benzimidazolyl)phenyl]maleimide, N-[4-(2-benzoxazolyl)phenyl]maleimide, N-(l-naphthyl)- maleimide, N-(2,4-xylyl)maleimide, N-(2,4-difluorophenyl)maleimide , N-(3-chloro-para- tolyl)-maleimide, l-(2-amino-ethyl)-pyrrole-2, 5-dione hydrochloride, l-cyclopentyl-3- methyl-2,5-dihydro-lH-pyrrole-2, 5-dione, l-(3-aminopropyl)-2,5-dihydro-lH-pyrrole-2,5- dione hydrochloride, 3-methyl-l-[2-oxo-2-(piperazin-l-yl)ethyl]-2,5-dihydro-lH-pyrrole- 2, 5-dione hydrochloride, l-benzyl-2,5-dihydro-lH-pyrrole-2, 5-dione, 3-methyl-l-(3,3,3- trifluropropyl)-2,5-dihydro-lH-pyrrole-2, 5-dione, l-[4-(methylamino)cyclohexyl]-2,5- dihydro-lH-pyrrole-2, 5-dione trifluroacetic acid, SMILES O=C1C=CC(=O)N1CC=2C=CN=CC2, SMILES O=C1C=CC(=O)N1CN2CCNCC2, l-benzyl-3- methyl-2,5-dihydro-lH-pyrrole-2, 5-dione, l-(2-fluorophenyl)-3-methyl-2,5-dihydro 1H- pyrrole-2, 5-dione, N-(4-phenoxyphenyl)maleimide , N-(4-nitrophenyl)maleimide (ii) lodocetamides such as :3-(2-Iodoacetamido)-proxyl, N-(cyclopropylmethyl)-2- iodoacetamide, 2-iodo-N-(2-phenylethyl)acetamide, 2-iodo-N-(2,2,2- trifluoroethyl)acetamide, N-(4-acetylphenyl)-2-iodoacetamide, N-(4- (aminosulfonyl)phenyl)-2-iodoacetamide, N-(l,3-benzothiazol-2-yl)-2-iodoacetamide, N- (2,6-diethylphenyl)-2-iodoacetamide, N-(2-benzoyl-4-chlorophenyl)-2-iodoacetamide, (iii) Bromoacetamides: such as N-(4-(acetylamino)phenyl)-2-bromoacetamide , N-(2- acetylphenyl)-2-bromoacetamide , 2-bromo-n-(2-cyanophenyl)acetamide, 2-bromo-N-(3- (trifluoromethyl)phenyl)acetamide, N-(2-benzoylphenyl)-2-bromoacetamide , 2-bromo-N- (4-fluorophenyl)-3-methylbutanamide, N-Benzyl-2-bromo-N-phenylpropionamide, N-(2- bromo-butyryl)-4-chloro-benzenesulfonamide, 2-Bromo-N-methyl-N-phenylacetamide, 2- bromo-N-phenethyl-acetamide,2-adamantan-l-yl-2-bromo-N-cyclohexyl-acetamide, 2- bromo-N-(2-methylphenyl)butanamide, Monobromoacetanilide, (iv) Disulphides such as: aldrithiol-2 , aldrithiol-4 , isopropyl disulfide, l-(Isobutyldisulfanyl)-2-methylpropane, Dibenzyl disulfide, 4-aminophenyl disulfide, 3-(2-Pyridyldithio)propionic acid, 3-(2- Pyridyldithio)propionic acid hydrazide, 3-(2-Pyridyldithio)propionic acid N-succinimidyl ester, am6amPDPl-[3CD and (v) Thiols such as: 4-Phenylthiazole-2-thiol, Purpald, 5, 6, 7, 8- tetrahydro-quinazoline-2-thiol. The tag may be attached directly to a nanopore or via one or more linkers. The tag may be attached to the nanopore using the hybridization linkers described in WO 2010 / 086602 (incorporated herein by reference in its entirety). Alternatively, peptide linkers may be used. Peptide linkers are amino acid sequences. The length, flexibility and hydrophilicity of the peptide linker are typically designed such that it does not to disturb the functions of the monomer and pore. Preferred flexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10 or 16, serine and / or glycine amino acids. More preferred flexible linkers include (SG)i, (SG)2, (SG)3, (SG)4, (SG)5and (SG)8wherein S is serine and G is glycine. Preferred rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or 24, proline amino acids. More preferred rigid linkers include (P)i2wherein P is proline.

[0275] Suitable pore tags are also described in WO 2018 / 100370, which describes non-hairpin methods for characterising double-stranded polynucleotides and is herein incorporated by reference in its entirety.

[0276] Biotin enrichment

[0277] Any of the adaptors may comprise biotin. The biotin may be used to isolate the adaptors. Suitable methods for biotin-based enrichment are known in the art. For instance, a surface, such as a bead, comprising avidin / or streptavidin may be used to barcoded constructs comprising biotin.

[0278] Method steps

[0279] The coupling method may comprise (a) attaching the one or more first coupling adaptors to the polypeptide or the plurality of polypeptides. The one or more first coupling adaptors may be any of those discussed above. The one or more first coupling adaptors may be attached to the polypeptide or the plurality of polypeptides in any way. The one or more first coupling adaptors are typically covalently attached to the polypeptide or the plurality of polypeptides in any way. The method may comprise (a) ligating the one or more first coupling adaptor to the polypeptide or the plurality of polypeptides. Suitable methods are known in the art.

[0280] The method may also comprise (b) coupling the one or more first coupling adaptors to the membrane using the one or more second coupling adaptors. The one or more second coupling adaptors may be any of those described above. The one or more second coupling adaptors are preferably functionalised to couple to the membrane. The one or more second coupling adaptors may be functionalised in any of the ways discussed above. The one or more second coupling adaptors preferably comprise a membrane protein or one or more hydrophobic anchors. Step (b) typically comprises contacting the polypeptide-first coupling adaptor conjugate or the plurality of polypeptide-first coupling adaptor conjugates from step (a) with the one or more second coupling adaptors under conditions which allow the one or more coupling polypeptides to specifically bind to the one or more coupling domains and allow the one or more second coupling adaptors to couple to the membrane.

[0281] The method may also comprise (b) coupling the one or more first coupling adaptors to the membrane using the one or more second coupling adaptors and the one or more third coupling adaptors. The one or more second coupling adaptors may be any of those described above. The one or more third coupling adaptors may be any of those described above. The one or more third coupling adaptors are preferably functionalised to couple to the membrane. The one or more third coupling adaptors may be functionalised in any of the ways discussed above. The one or more third coupling adaptors preferably comprises a membrane protein or one or more hydrophobic anchors. The one or more second coupling adaptors preferably further comprises a first oligonucleotide and the one or more third coupling adaptors preferably further comprises a second oligonucleotide which hybridizes to the first oligonucleotide. Step (b) typically comprises contacting the polypeptide-first coupling adaptor conjugate or the plurality of polypeptide-first coupling adaptor conjugates from step (a) with the one or more second coupling adaptors and the one or more third coupling adaptors under conditions which allow the one or more coupling polypeptides to specifically bind to the one or more coupling domains, allow the first oligonucleotide to specifically hybridise to the second oligonucleotide and allow the one or more third coupling adaptors to couple to the membrane.

[0282] The method may also comprise in step (a) attaching the blocking moiety to the polypeptide or the plurality of polypeptides. Blocking moieties are discussed above. The blocking moiety may be attached to the opposite side of the polypeptide from the one or more first adaptors. The blocking moiety may be attached at any of the positions shown in Figures 4- 7.

[0283] The method may comprise using a population of one or more first coupling adaptors and / or a population of one or more second coupling adaptors. The one or more first coupling adaptors in the population are typically all the same. The one or more second coupling adaptors in the population are typically the same. The one or more first coupling adaptors in the population may be any of the adaptors of invention defined above. The one or more second coupling adaptors in the population may be any of the adaptors of invention defined above.

[0284] The population(s) may contain any number of one or more first coupling adaptors and / or one or more second coupling adaptors. For instance, the method may use a (1) a population of at least about 2, at least about 5, at least about 10, at least about 50, at least about 100, at least about 500, at least about 1,000, at least about 5,000, at least about 10,000, at least about 50,000, at least about 1 x 105, at least about 1 x 106, at least about 1 x 107, at least about 1 x 108, at least about 1 x 109, at least about 1 x 1010, at least about 1 x 1011, at least about 1 x 1012or at least about 1 x 1013one or more first coupling adaptors and / or (2) a population of at least about 2, at least about 5, at least about 10, at least about 50, at least about 100, at least about 500, at least about 1,000, at least about 5,000, at least about 10,000, at least about 50,000, at least about 1 x 105, at least about 1 x 106, at least about 1 x 107, at least about 1 x 108, at least about 1 x 109, at least about 1 x 1010, at least about 1 x 1011, at least about 1 x 1012or at least about 1 x 1013one or more second coupling adaptors. The number of adaptors in the populations are typically the same as or greater than the number of polypeptides in the plurality. The number of polypeptides in the plurality may not be known.

[0285] The coupling method may comprise (a) attaching a population of one or more first coupling adaptors to the plurality of polypeptides. The one or more first coupling adaptors may be attached in any of the ways discussed above.

[0286] The method may also comprise (b) coupling the one or more first coupling adaptors to the membrane using a population of one or more second coupling adaptors. The one or more second coupling adaptors may be coupled to the membrane in any of the ways discussed above.

[0287] The method may also comprise (b) coupling the one or more first coupling adaptors to the membrane using a population of one or more second coupling adaptors and a population of one or more third coupling adaptors. The one or more third coupling adaptors may be any of those described above. The population of one or more third coupling adaptors may comprise any of the numbers of adaptors discussed above for the first and / or second coupling adaptors. The one or more third coupling adaptors are preferably functionalised to couple to the membrane. The one or more second coupling adaptors and the one or more third coupling adaptors may be used in any of the ways discussed above.

[0288] Kits

[0289] The invention provides a kit for coupling a polypeptide or a plurality of polypeptides to a membrane. The kit comprises (1) one or more first coupling adaptors each comprising one or more coupling polypeptides. The kit also comprises (2) one or more second coupling adaptors comprising one or more coupling domains which specifically bind to the one or more coupling polypeptides. The one or more first coupling adaptors may be any of those discussed above with reference to the coupling method of the invention. The one or more second coupling adaptors may be any of those discussed above with reference to the coupling method of the invention. The one or more second coupling adaptors may comprise a membrane protein or one or more hydrophobic anchors. The kit preferably further comprises (3) one or more third coupling adaptors comprising a membrane protein or one or more hydrophobic anchors. The invention also provides a kit for coupling a polypeptide or a plurality of polypeptides to a membrane. The kit comprises (1) one or more first coupling adaptors each comprising one or more first amphipathic helices. The kit also comprises (2) one or more second coupling adaptors each comprising one or more second amphipathic helices which specifically bind to the one or more first amphipathic helices. The one or more first coupling adaptors may be any of those discussed above with reference to the preferred coupling method of the invention. The one or more first amphipathic helices may be any of those discussed above with reference to the preferred coupling method of the invention. The one or more second coupling adaptors may be any of those discussed above with reference to the preferred coupling method of the invention. The one or more second amphipathic helices may be any of those discussed above with reference to the preferred coupling method of the invention. The one or more second coupling adaptors may comprise a membrane protein or one ore more hydrophobic anchors. The kit preferably further comprises (3) one or more third coupling adaptors comprising a membrane protein or one or more hydrophobic anchors.

[0290] The one or more first amphipathic helices and / or the one or more second amphipathic helices may be any of the amphipathic helices, amphipathic helix oligomers and helical hairpins discussed above.

[0291] The invention also provides a kit for coupling a polypeptide or a plurality of polypeptides to a membrane. The kit comprises (1) one or more first coupling adaptors each comprising one or more first coiled-coil monomers. The kit also comprises (2) one or more second coupling adaptors each comprising one or more second coiled-coil monomers which specifically bind to the one or more first coiled-coil monomers. The one or more first coupling adaptors may be any of those discussed above with reference to the preferred coupling method of the invention. The one or more first coiled-coil monomers may be any of those discussed above with reference to the preferred coupling method of the invention, including one or more monomers defined with reference to SEQ ID NOs: 60-253 and including the first leucine zipper monomer embodiments and one or more monomers defined with reference to SEQ ID NOs: 9, 10, 3-7 and 11-59. The one or more second coupling adaptors may be any of those discussed above with reference to the preferred coupling method of the invention. The one or more second coiled-coil monomers may be any of those discussed above with reference to the preferred coupling method of the invention, including one or more monomers defined with reference to SEQ ID NOs: 60-253 and including the second leucine zipper monomer embodiments and one or more monomers defined with reference to SEQ ID NOs: 9, 10, 3-7 and 11-59. The one or more second coupling adaptors may comprise a membrane protein or one or more hydrophobic anchors. The kit preferably further comprises (3) one or more third coupling adaptors comprising a membrane protein or one or more hydrophobic anchors. The one or more first coiled-coil monomers and / or the one or more second coiled-coil monomers may be any of the coiled-coil monomers, coiled-coil oligomers, coiled-coil hairpins, leucine zipper monomer, leucine zipper oligomers and leucine zipper hairpins discussed above.

[0292] The kit typically comprises (1) a population of one or more first coupling adaptors as defined above and (2) a population of one or more second coupling adaptors as defined above. The populations of adaptors can be used to couple a plurality of polypeptides to a membrane in accordance with the invention. The one or more first coupling adaptors in the population are typically all the same. The one or more second coupling adaptors in the population are typically the same.

[0293] The one or more first coupling adaptors in the population may be any of the adaptors of invention defined above. The one or more second coupling adaptors in the population may be any of the adaptors of invention defined above.

[0294] The kit may comprise any numbers of (the types of) one or more first coupling adaptors, one or more coupling polypeptides, one or more second coupling adaptors, one or more coupling domains and optionally one or more third coupling adaptors discussed above with reference to the method of the invention. The kit may comprise any of the preferred numbers of (the types of) one or more first coupling adaptors, one or more coupling polypeptides, one or more second coupling adaptors, one or more coupling domains and optionally one or more third coupling adaptors shown in Table 3 above.

[0295] The population(s) may contain any number of one or more first coupling adaptors and / or one or more second coupling adaptors. For instance, the kit may comprise (1) a population of at least about 2, at least about 5, at least about 10, at least about 20, at least about 50, at least about 100, at least about 500, at least about 1000, at least about 5000, at least about 10000, at least about 50000, at least about 100000 one or more first coupling adaptors and / or (2) a population of at least about 2, at least about 5, at least about 10, at least about 20, at least about 50, at least about 100, at least about 500, at least about 1000, at least about 5000, at least about 10000, at least about 50000, at least about 100000 one or more second coupling adaptors.

[0296] The number of adaptors in the populations are typically the same as or greater than the number of polypeptides in the plurality. The number of polypeptides in the plurality may not be known.

[0297] The invention provides a kit for coupling a polypeptide or a plurality of polypeptides to a membrane. The kit comprises (1) one or more polynucleotides encoding one or more first coupling adaptors each comprising one or more first coupling polypeptides. The kit also comprises (2) one or more polynucleotide encoding one or more second coupling adaptors each comprising one or more second coupling polypeptides which specifically bind to the one or more first coupling polypeptides. The one or more first coupling adaptors may be any of those discussed above with reference to the coupling method of the invention. The one or more second coupling adaptors may be any of those discussed above with reference to the coupling method of the invention. The one or more second coupling adaptors may comprise a membrane protein or one or more hydrophobic anchors. The kit preferably further comprises (3) one or more third coupling adaptors comprising a membrane protein or one or more hydrophobic anchors. The invention also provides a kit for coupling a polypeptide or a plurality of polypeptides to a membrane. The kit comprises (1) one or more polynucleotides encoding one or more first coupling adaptors comprising one or more first amphipathic helices. The kit also comprises (2) one or more polynucleotides encoding one or more second coupling adaptors comprising one or more second amphipathic helices which specifically bind to the one or more first amphipathic helices. The one or more first coupling adaptors may be any of those discussed above with reference to the preferred coupling method of the invention. The one or more first amphipathic helices may be any of those discussed above with reference to the preferred coupling method of the invention, including one or more monomers defined with reference to SEQ ID NOs: 60-253 and including the first leucine zipper monomer embodiments and one or more monomers defined with reference to SEQ ID NOs: 9, 10, 3-7 and 11-59. The one or more second coupling adaptors may be any of those discussed above with reference to the preferred coupling method of the invention. The one or more second amphipathic helices may be any of those discussed above with reference to the preferred coupling method of the invention, including one or more monomers defined with reference to SEQ ID NOs: 60-253 and including the second leucine zipper monomer embodiments and one or more monomers defined with reference to SEQ ID NOs: 9, 10, 3-7 and 11-59. The one or more first amphipathic helices and / or one or more second amphipathic helices may be any of the one or more amphipathic helices, one or more amphipathic helix oligomers, helical hairpins, coiled-coil monomers, coiled-coil oligomers, coiled-coil hairpins, leucine zipper monomers, leucine zipper oligomers and leucine zipper hairpins discussed above.

[0298] The one or more second coupling adaptors may comprise a membrane protein or one or more hydrophobic anchors. The kit preferably further comprises (3) one or more third coupling adaptors comprising a membrane protein or one or more hydrophobic anchors.

[0299] The one or more polynucleotides in the kit may be any of those discussed above. The kit may comprise any number of one or more polynucleotides, such as 1, 2, 3, 4, 5 or more polynucleotides. The kit may encode any of the numbers of (the types of) one or more first coupling adaptors, one or more coupling polypeptides, one or more second coupling adaptors, one or more coupling domains and optional one or more third coupling adaptors discussed above with reference to the method of the invention. The kit may encode any of the preferred numbers of (the types of) one or more first coupling adaptors, one or more coupling polypeptides, one or more second coupling adaptors, one or more coupling domains and optional one or more third coupling adaptors shown in Table 3 above.

[0300] The invention also provides one or more expression vectors comprising a kit of the invention. The polynucleotides in the kit defined in (1) and (2) above may be in the same or different expression vectors. The invention also provides a host cell comprising a kit of the invention or one or more expression vectors of the invention. Suitable vectors and host cells are known in the art.

[0301] The kit may additionally comprise one or more other reagents or instruments which enable any of the embodiments mentioned above or below to be carried out. Such reagents or instruments include one or more of the following: suitable buffer(s) (aqueous solutions), means to obtain a sample from a subject (such as a vessel or an instrument comprising a needle), means to amplify and / or express polynucleotides, a membrane as defined below or voltage or patch clamp apparatus. Reagents may be present in the kit in a dry state such that a fluid sample is used to resuspend the reagents. The kit may also, optionally, comprise instructions to enable the kit to be used in the methods described herein or details regarding for which organism the method may be used. The kit may comprise a magnet or an electromagnet. The kit may, optionally, comprise nucleotides.

[0302] Characterisation methods

[0303] The invention provides a method for determining the presence, absence or one or more characteristics of a polypeptide analyte. This method may also be known as the detection or characterisation method of the invention.

[0304] The polypeptide analyte may be any of the polypeptides defined above with reference to the coupling method of the invention.

[0305] The invention may be for determining the presence, absence or one or more characteristics of a plurality of polypeptide analytes. There may be any number of polypeptide analytes in the plurality. For instance, there may be at least about 2, at least about 5, at least about 10, at least about 20, at least about 50, at least about 100, at least about 500, at least about 1000, at least about 5000, at least about 10000, at least about 50000, at least about 100000 or more polypeptides analytes in the plurality.

[0306] The polypeptide analyte or the plurality of polypeptide analytes may be called the target polypeptide analyte or the plurality of target polypeptide analytes.

[0307] The method comprises (a) coupling the polypeptide analyte or the plurality of polypeptide analytes to a membrane using a kit of the invention or a coupling method of the invention. The kit may be any of the kits of the invention described above. The method may be any of the coupling methods of the invention described above.

[0308] The kit preferably comprises (1) one or more first coupling adaptors each comprising one or more first amphipathic helices, and (2) one or more second coupling adaptors each comprising one or more second amphipathic helices which specifically bind to the one or more first amphipathic helices. The one or more first coupling adaptors may be any of those discussed above with reference to the preferred coupling method of the invention. The one or more first amphipathic helices may be any of those discussed above with reference to the preferred coupling method of the invention, including one or more monomers defined with reference to SEQ ID NOs: 60-253 and including the first leucine zipper monomer embodiments and one or more monomers defined with reference to SEQ ID NOs: 9, 10, 3-7 and 11-59. The one or more second coupling adaptors may be any of those discussed above with reference to the preferred coupling method of the invention. The one or more second amphipathic helices may be any of those discussed above with reference to the preferred coupling method of the invention, including one or more monomers defined with reference to SEQ ID NOs: 60-253 and including the second leucine zipper monomer embodiments and one or more monomers defined with reference to SEQ ID NOs: 9, 10, 3-7 and 11-59. The one or more first amphipathic helices and / or one or more second amphipathic helices may be any of the one or more amphipathic helices, one or more amphipathic helix oligomers, helical hairpins, coiled-coil monomers, coiled-coil oligomers, coiled-coil hairpins, leucine zipper monomers, leucine zipper oligomers and leucine zipper hairpins discussed above. The one or more second coupling adaptors may comprise a membrane protein or one or more hydrophobic anchors. The kit preferably further comprises (3) one or more third coupling adaptors comprising a membrane protein or one or more hydrophobic anchors. The kit may comprise (1) a population of one or more first coupling adaptors as defined in this paragraph and (2) a population of one or more second coupling adaptors as defined in this paragraph.

[0309] In the coupling method of the invention, the one or more first coupling adaptors preferably comprise one or more first amphipathic helices, and the one or more second coupling adaptors preferably comprise one or more second amphipathic helices which specifically bind to the one or more first amphipathic helices. In the coupling method of the invention, the one or more first coupling adaptors preferably comprise one or more first coiled-coil monomers, and the one or more second coupling adaptors preferably comprise one or more second coiled-coil monomers which specifically bind to the one or more first coiled-coil monomers. In the coupling method of the invention, the one or more first coupling adaptors preferably comprise one or more first leucine zipper monomers, and the one or more second coupling adaptors preferably comprise one or more second leucine zipper monomers which specifically bind to the one or more first leucine zipper monomers. The one or more first coupling adaptors may be any of those discussed above with reference to the preferred method of the invention. The one or more second coupling adaptors may be any of those discussed above with reference to the preferred method of the invention. The one or more first coupling adaptors and the one or more second coupling adaptors used in the coupling method may be any of those defined in the previous paragraph. The coupling method may use (1) a population of one or more first coupling adaptors as defined in this paragraph or the previous paragraph and (2) a population of one or more second coupling adaptors as defined in this paragraph or the previous paragraph.

[0310] The kit may comprise or the method may use any of the numbers of (the types of) one or more first coupling adaptors, one or more coupling polypeptides, one or more second coupling adaptors, one or more coupling domains and optional one or more third coupling adaptors discussed above with reference to the method of the invention. The kit may comprise or the method may use any of the preferred numbers of (the types of) one or more first coupling adaptors, one or more coupling polypeptides, one or more second coupling adaptors, one or more coupling domains and optional one or more third coupling adaptors shown in Table 3 above.

[0311] The method also comprises (b) allowing the coupled polypeptide analyte or the plurality of coupled polypeptide analytes to interact with a detector present in the membrane and thereby determining the presence, absence or one or more characteristics of the polypeptide analyte or the plurality of polypeptide analytes.

[0312] The invention also provides a method for detecting analyte polypeptides, comprising (a) providing a membrane in which is present a nanopore that provides a channel through the membrane; (b) contacting the membrane, in an ionic solution, with analyte polypeptides, wherein following contact with the membrane the analytes polypeptides are coupled or tethered to the membrane using a kit of the invention or a coupling method of the invention; (c) applying a potential difference across the membrane and detecting a first analyte polypeptide using the nanopore, from among the analytes polypeptides coupled or tethered to the membrane; and (d)detecting a second analyte polypeptide, from among the analytes polypeptides coupled or tethered to the membrane, using the same nanopore, wherein the second polypeptide is not the first polypeptide. The kit may be any of the kits of the invention described above. The method may be any of the coupling methods of the invention described above.

[0313] Any method of characterisation may be used. The method preferably uses next generation sequencing (NGS).

[0314] The coupled polypeptide analyte or the plurality of coupled polypeptide analytes are allowed to interact with a detector present in the membrane. The coupled polypeptide analyte or the plurality of coupled polypeptide analytes are preferably moved with respect to the detector in the membrane. Any suitable measurements can be taken using the detector as the polypeptide analyte or the plurality of polypeptide analytes move with respect to the detector. The detector preferably detects the polypeptide analyte or the plurality of polypeptide analytes via electrical or optical means. The detector may be selected from (i) a zero-mode waveguide, (ii) a field-effect transistor, optionally a nanowire field-effect transistor; (iii) an AFM tip; (iv) a nanotube, optionally a carbon nanotube; and (v) a nanopore. Preferably, the detector comprises a nanopore.

[0315] The coupled polypeptide analyte or the plurality of coupled polypeptide analytes may be characterised in the detection or characterisation method of the invention in any suitable manner. The coupled polypeptide analyte or the plurality of coupled polypeptide analytes are preferably characterised by detecting an ionic current or optical signal as they move with respect to a nanopore. This is described in more detail herein. The method is amenable to these and other methods of characterising analytes.

[0316] Nanopore characterisation

[0317] The coupled polypeptide or the coupled polypeptides are preferably characterised using a nanopore. In step (b), the method preferably comprises (i) allowing the coupled polypeptide analyte or the plurality of coupled polypeptide analytes to interact with, such as move through, the nanopore and (ii) measuring the current passing through the pore during the interaction and thereby determining the presence, absence or one or more characteristics of the polypeptide analyte or the plurality of polypeptide analytes. The coupled polypeptide analyte or the plurality of coupled polypeptide analytes typically become(s) uncoupled from the membrane as they interact with, such as move through, the nanopore.

[0318] The one or more characteristics are preferably selected from (i) the length(s) of the polypeptide analyte or the plurality of polypeptide analytes, (ii) the identity / identities of the polypeptide analyte or the plurality of polypeptide analytes, (iii) the sequence(s) of the polypeptide analyte or the plurality of polypeptide analytes, (iv) the secondary structure(s) of the polypeptide analyte or the plurality of polypeptide analytes and (v) whether or not the polypeptide analyte or the plurality of polypeptide analytes is / are modified. The polypeptide analyte or the plurality of polypeptide analytes may be modified in any of the ways discussed above. The one or more characteristics of the polypeptide analyte or the plurality of polypeptide analytes are preferably measured by electrical measurement and / or optical measurement. The electrical measurement is preferably a current measurement, an impedance measurement, a tunnelling measurement, or a field effect transistor (FET) measurement. The method more preferably comprises (i) contacting the coupled polypeptide analyte or the plurality of coupled polypeptide analytes with a nanopore such that the polypeptide analyte or the plurality of polypeptide analytes move(s) through the pore and (ii) measuring the current moving through the pore as the polypeptide analyte or the plurality of polypeptide analytes move(s) through the pore wherein the current is indicative of one or more characteristics of the polypeptide analyte or the plurality of polypeptide analytes and thereby characterising the polypeptide analyte or the plurality of polypeptide analytes. The one or more characteristics may be any of those defined above.

[0319] The polypeptide analyte or the plurality of polypeptide analytes may be in a relaxed form. The polypeptide analyte or the plurality of polypeptide analytes may be held in a linearized form. Holding polypeptides in a linearized form can facilitate their characterisation on a residue-by-residue basis as "bunching up" of the polypeptides within the transmembrane protein pore is prevented. The polypeptide analyte or the plurality of polypeptide analytes can be held in a linearized form using any suitable means. For example, if the polypeptide analyte or the plurality of polypeptide analytes is / are charged, the polypeptide or the plurality of polypeptide analytes can be held in a linearized form by applying a voltage.

[0320] If the polypeptide analyte or the plurality of polypeptide analytes is / are not charged or is / are only weakly charged then the charge can be altered or controlled by adjusting the pH. For example, the polypeptide analyte or the plurality of polypeptide analytes can be held in a linearized form by using high pH to increase the relative negative charge of the polypeptide(s). Increasing the negative charge of the polypeptide(s) allows it / them to be held in a linearized form under, e.g., a positive voltage. Alternatively, the polypeptide analyte or the plurality of polypeptide analytes can be held in a linearized form by using low pH to increase the relative positive charge of the polypeptide(s). Increasing the positive charge of the polypeptide analyte or the plurality of polypeptide analytes allows it / them to be held in a linearized form under, e.g., a negative voltage. In the disclosed methods below, a polynucleotide-handling protein is used to control the movement of a polynucleotide with respect to a nanopore. As a polynucleotide is typically negatively charged it is generally most suitable to increase the linearization of the polypeptide analyte or the plurality of polypeptide analytes by increasing the pH thus making the polypeptide(s) more negatively charged, in common with the polynucleotide. In this way, the conjugate retains an overall negative charge and thus can readily move, e.g., under an applied voltage.

[0321] The polypeptide analyte or the plurality of polypeptide analytes can be held in a linearized form by using suitable denaturing conditions. Suitable denaturing conditions include, for example, the presence of appropriate concentrations of denaturants such as guanidine HCI and / or urea. The concentration of such denaturants to use in the disclosed methods is dependent on the polypeptide analyte(s) to be characterised in the methods and can be readily selected by those of skill in the art. If denaturing conditions are used, the conditions preferably do not affect or interfere with specific binding between the coupling polypeptide and coupling domain.

[0322] The polypeptide analyte or the plurality of polypeptide analytes can be held in a linearized form by using suitable detergents. Suitable detergents for use in the disclosed methods include SDS (sodium dodecyl sulfate). The polypeptide(s) can be held in a linearized form by carrying out the disclosed methods at an elevated temperature. Increasing the temperature overcomes intra-strand bonding and allows the polypeptide to adopt a linearized form.

[0323] The polypeptide analyte or the plurality of polypeptide analytes can be held in a linearized form by carrying out the method under strong electro-osmotic forces. Such forces can be provided by using asymmetric salt conditions and / or providing suitable charge in the channel of the nanopore. The charge in the channel of a nanopore can be altered, e.g., by mutagenesis. Altering the charge of a pore is well within the capacity of those skilled in the art. Altering the charge of a pore generates strong electro-osmotic forces from the unbalanced flow of cations and anions through the nanopore when a voltage potential is applied across the nanopore.

[0324] The polypeptide analyte or the plurality of polypeptide analytes can be held in a linearized form by passing it / then through a structure such an array of nanopillars, through a nanoslit or across a nanogap. The physical constraints of such structures can force the polypeptide(s) to adopt a linearized form.

[0325] Any suitable nanopore can be used. The nanopore is preferably a transmembrane pore. A transmembrane pore is a structure that crosses the membrane to some degree. It permits hydrated ions driven by an applied potential to flow across or within the membrane. The transmembrane pore typically crosses the entire membrane so that hydrated ions may flow from one side of the membrane to the other side of the membrane. However, the transmembrane pore does not have to cross the membrane. It may be closed at one end. For instance, the pore may be a well, gap, channel, trench or slit in the membrane along which or into which hydrated ions may flow.

[0326] The nanopore or transmembrane pore typically has a first opening and a second opening. The first opening is typically the cis opening and the second opening is typically the trans opening. However, the first opening may be the trans opening and the second opening may be the cis opening. Any polynucleotide binding protein used in the detection or characterisation method of the invention is typically provided at the first opening of the nanopore and thus controls the movement of the target polynucleotide in the direction from the second opening of the nanopore towards the first opening of the nanopore. Any transmembrane pore may be used in the detection or characterisation method of the invention. The pore may be biological or artificial. Suitable pores include, but are not limited to, protein pores, polynucleotide pores and solid-state pores. The pore may be a DNA origami pore (Langecker et al., Science, 2012; 338: 932-936). Suitable DNA origami pores are disclosed in WO2013 / 083983.

[0327] The nanopore may be a transmembrane protein pore which is a monomer or an oligomer. The pore is preferably made up of several repeating subunits, such as at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, or at least about 16 subunits. The pore is preferably a hexameric, heptameric, octameric or nonameric pore. The pore may be a homo-oligomer or a hetero-oligomer.

[0328] The transmembrane protein pore may comprise a barrel or channel through which the ions may flow. The subunits of the pore typically surround a central axis and contribute strands to a transmembrane [3-barrel or channel or a transmembrane a-helix bundle or channel.

[0329] Typically, the barrel or channel of the transmembrane protein pore comprises amino acids that facilitate interaction with an analyte, such as a target polynucleotide (as described herein). These amino acids are preferably located near a constriction of the barrel or channel. The transmembrane protein pore typically comprises one or more positively charged amino acids, such as arginine, lysine or histidine, or aromatic amino acids, such as tyrosine or tryptophan. These amino acids typically facilitate the interaction between the pore and nucleotides, polynucleotides, or nucleic acids.

[0330] The transmembrane protein pore may be from or derived from Wza, Iota toxin, Anthrax protective antigen, Vibrio cholerae cytolysin, Cytotoxin K (CytK), CELIII, CsgG, CsgF, CsgG- CsgF, Aerolysin, alpha hemolysin, MspA, MspB, MspC, PorARr, PorBRr, PorARc, PilQ, necrotic enteritis B-like toxin (NetB), FraC, portal proteins including G20c, P23_45, T4, SPP1, P22 and Phi29, gamma hemolysin, Monalysin, Lysenin, ClyA, an actinoporin, Clostridium perfringens beta toxin, parasporin-2, epsilon toxin, lectin from the parasitic mushroom Laetiporus sulphureus (LSL), volvatoxin, Cry toxins, CytlAa, Cyt2Aa, Complement component 9 (C9), Perfringolysin O, Pleurotolysin, Listeriolysin, Perforin-2, Gasdermin-A3, L-, P- and M-ring protein, Type II secretion system protein D, GspD, InvG, VirB7, SpoIIIAG, Cag8, Cag3, Cag or other proteins in the Type IV secretion system apparatus protein CagY, WzzB, Pentraxin, Afp2, Major vault protein, Thioredoxin-dependent peroxidase reductase, Arf-GAP, Respiratory syncytial virus ribonucleoprotein, Chikungunya virus nonstructural protein 1, PRC, YaxA, XaxA, HfaB, NfpAB, leukocidin or PrgH.

[0331] Suitable transmembrane protein pores for use in the invention include those described in WO 2016 / 034591, WO 2017 / 149316, WO 2017 / 149317, WO 2017 / 149318, WO 2018 / 211241, WO 2019 / 002893, WO 2023 / 118404, WO 2023 / 198911, WO 2024 / 033421, WO 2024 / 033422, WO 2024 / 033443, and WO 2024 / 089270 (all incorporated by reference herein in their entirety).

[0332] The transmembrane protein pore may also be any of the CsgG pores described in WO 2023 / 060420, WO 2023 / 60418, WO 2023 / 60422, WO 2023 / 060421, WO 2023 / 019470, CN114957412, WO 2023 / 019471, W02023 / 060419 and WO 2023 / 050031 (all incorporated herein by reference in their entireties) or a variant thereof.

[0333] The transmembrane protein pore may be any of the pores described in WO 2023 / 123370, WO 2024 / 138470, WO 2024 / 138472, WO 2024 / 138424, WO 2024 / 138425, WO 2024 / 138512 and WO 2024 / 138565 (all incorporated herein by reference in their entireties) or a variant thereof.

[0334] The transmembrane pore may be formed from a chimeric pore monomer comprising two or more regions, wherein at least two of the two or more regions are from at least two different pores. The chimeric pore monomer may comprise any number of regions, such as three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more regions, from different pores. The chimeric pore monomer may comprise two or three regions. The regions are preferably selected from a cap region, a constriction region, and a transmembrane region. The regions may be a cap region and a constriction region. The regions may be a cap region, a constriction region, and a transmembrane region. The at least two different pores are typically at least two different pores that appear in nature. The at least two different pores are typically at least two different wild-type or naturally occurring pores. The at least two different pores are preferably different before any artificial or synthetic modifications, such as additions, deletions and / or substitutions, are made to them. The at least two different pores are preferably homologues, for example structural homologues. A structural homologue refers to a protein or molecule that shares a similar three-dimensional structure with another protein or molecule. This can be determined using standard methods in the art (e.g., AlphaFold or PSIPRED). Structural homologues typically have similar sequences. Structural homologues are normally identified in similar species. The at least two different pores may be selected from any of the pores listed above. The at least two different pores may be two different PorARc pores or three different PorARc pores. The at least two different pores may be two different CsgG pores or three different CsgG pores. The chimeric pore monomer may be any of those described in WO 2024 / 089270; PCT / EP2023 / 080135 (incorporated by reference herein in its entirety).

[0335] The transmembrane pore may be formed from a pore monomer comprising (a) a CsgG monomer and (b) a fusion polypeptide comprising a first portion comprising a CsgF peptide and a second portion comprising a helix-forming auxiliary protein, wherein the fusion protein is attached to the pore monomer. The pore monomer may be derived from a protein transmembrane pore complex comprising (a) a CsgG transmembrane pore comprising a lumen and (b) a fusion polypeptide comprising a first portion comprising a CsgF protein and a second portion comprising a helix-forming auxiliary protein, wherein the fusion protein is attached to the transmembrane pore. The auxiliary protein can be designed de novo using computer-based structural analysis tools to confer certain desirable features to the CsgG monomer (e.g., modulation of pore width, lengthening of pore lumen, formation of one or more additional constrictions, etc.). The de novo designed auxiliary protein may form one or more additional constrictions in the lumen of a CsgG pore formed from the monomer, and improve discrimination of polymer units as an analyte moves through the pore. The pore monomer may be any of the pore monomers described in WO 2024 / 033447 (incorporated by reference herein in its entirety).

[0336] Protein translocase

[0337] The movement of the polypeptide analyte or the plurality of polypeptide analytes with respect to, such as through, the detector, nanopore or transmembrane is preferably controlled using a protein translocase. The protein translocase is typically a protein unfoldase, such as CIpX. Suitable methods for doing this are described in WO 2013 / 123379 and WO 2024 / 091124 (incorporated by reference in their entireties).

[0338] Polynucleotide binding protein

[0339] The movement of the polypeptide analyte or the plurality of polypeptide analytes with respect to, such as through, the detector, nanopore or transmembrane is preferably controlled using a polynucleotide binding protein. This can be achieved by conjugating the polypeptide analyte or the plurality of polypeptide analytes to a polynucleotide or to polynucleotides to form a polynucleotide-polypeptide conjugate or polynucleotidepolypeptide conjugates. This is disclosed in WO 2021 / 111125 (incorporated by reference in its entirety). The polynucleotide may be any of those discussed above. Polynucleotidepolypeptide conjugates and methods of making them are discussed in more detail above.

[0340] As those skilled in the art will appreciate, any suitable polynucleotide binding protein can be used in the invention. The polynucleotide binding protein may be any protein that is capable of binding to a polynucleotide and controlling its movement with respect to a detector, e.g., a nanopore.

[0341] In more detail, polynucleotide binding proteins such as helicases can typically control the movement of polynucleotides in at least two active modes of operation (when is provided with all the necessary components to facilitate movement e.g., ATP and Mg2+) and one inactive mode of operation (when not provided with the necessary components to facilitate movement; or when the polynucleotide binding protein is modified in order to prevent the active mode).

[0342] When provided with all the necessary components to facilitate movement, a polynucleotide binding protein may move along a polynucleotide in either a 5'-3' direction or a 3'-5' direction. Many polynucleotide binding proteins process polynucleotides in a 5'-3' direction. Polynucleotide binding proteins which control the movement of polynucleotides in this manner are typically suitable for use in the detection or characterisation method of the invention.

[0343] However, when a polynucleotide binding protein is not provided with the necessary components to facilitate movement or is modified in order to prevent it from actively controlling the movement of the polynucleotide with respect to the nanopore, it can still passively control the movement of the polynucleotide with respect to the nanopore. For example, the polynucleotide binding protein can bind to the polynucleotide and act as a brake slowing the movement of the polynucleotide when it is pulled into the pore by an applied field (e.g., by the first force in the detection or characterisation method of the invention). In the "inactive" mode it typically does not matter whether the polynucleotide is captured either 3' or 5' down ( / .e., moves through the nanopore in a 5'-3' direction or in a 3'-5' direction), as the applied force provides the impetus to move the polynucleotide through the nanopore. However, in such embodiments, the polynucleotide binding protein may still control the movement of the polynucleotide with respect to the nanopore e.g., by acting as a brake. When in the inactive mode the movement control of a polynucleotide by a polynucleotide binding protein can be described in a number of ways including ratcheting, sliding, and braking. Typically the detection or characterisation method of the invention do not comprise the use of a polynucleotide binding protein operating in the passive mode. However, when a polynucleotide binding protein the polynucleotide binding protein is used, it may be a polynucleotide binding protein operating in the passive mode.

[0344] Some methods of the invention may comprise use of a polynucleotide binding protein as a pausing moiety to impede the movement of the polynucleotide strand through the nanopore. The polynucleotide binding protein may be a protein which binds to polynucleotides but which does not have polynucleotide processing capacity, i.e., it is not a polynucleotide binding protein.

[0345] A polynucleotide-handling enzyme is a polypeptide that is capable of interacting with a polynucleotide. The enzyme may modify the polynucleotide by cleaving it to form individual nucleotides or shorter chains of nucleotides, such as di- or trinucleotides. The enzyme may modify the polynucleotide by orienting it or moving it to a specific position. A polynucleotide binding protein as used herein may be, or may be derived from a polynucleotide handling enzyme. A polynucleotide binding protein may be, or may be derived from a polynucleotide- handling enzyme.

[0346] The polynucleotide binding protein may be derived from a member of any of the Enzyme Classification (EC) groups 3.1.11, 3.1.13, 3.1.14, 3.1.15, 3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30 and 3.1.31.

[0347] Typically, the polynucleotide binding protein is a helicase, a polymerase, an exonuclease, a topoisomerase, or a variant thereof.

[0348] The polynucleotide binding protein may be modified to prevent the polynucleotide binding protein disengaging from the polynucleotide. Thus, the target polynucleotide preferably does not disengage from the polynucleotide binding protein.

[0349] As used herein, the term "disengaging" refers to the dissociation of the polynucleotide binding protein from the target polynucleotide. Thus, a polynucleotide binding protein may be modified to prevent it from dissociating from the target polynucleotide, e.g., into the reaction medium. It is important to distinguish potential "disengagement" of a polynucleotide binding protein from "unbinding" of a polynucleotide binding protein from a target polynucleotide. As used herein, "unbinding" refers to the transient release of the target polynucleotide the active site of the polynucleotide binding protein (described in more detail herein) but does not imply disengagement. Thus, for example, a polynucleotide binding protein may be modified to prevent the polynucleotide binding protein from disengaging from a polynucleotide, but without preventing the polynucleotide binding protein from unbinding from the polynucleotide. When unbound, the polynucleotide binding protein remains engaged with the target polynucleotide. For example, the polynucleotide binding protein may remain engaged with the target polynucleotide ( / .e., it may be prevented from disengaging from the target polynucleotide) because it is topologically closed around the target polynucleotide. The polynucleotide binding site may remain free to bind or unbind the target polynucleotide such that the polynucleotide binding protein may bind or unbind to the target polynucleotide, whilst the polynucleotide binding protein remains engaged with the target polynucleotide. When the polynucleotide binding protein is unbound from the target polynucleotide it may be able to move on (e.g., along) the target polynucleotide under an applied force and may be capable of re-binding to the target polynucleotide. When engaged on the target polynucleotide but unbound from the target polynucleotide, the polynucleotide binding protein is not capable of dissociating from the target polynucleotide.

[0350] The polynucleotide binding protein can be adapted to prevent disengagement in any suitable way. For example, the polynucleotide binding protein can be loaded on the polynucleotide and then modified in order to prevent it from disengaging from the polynucleotide. Alternatively, the polynucleotide binding protein can be modified to prevent it from disengaging from the polynucleotide before it is loaded onto the polynucleotide. Modification of a polynucleotide binding protein and / or a polynucleotide binding protein in order to prevent it from disengaging from a polynucleotide can be achieved using methods known in the art, such as those discussed in WO 2014 / 013260, which is hereby incorporated by reference in its entirety, and with particular reference to passages describing the modification of polynucleotide binding proteins such as helicases in order to prevent them from disengaging with polynucleotide strands. For example, a polynucleotide binding protein can be modified by treating with tetramethylazodicarboxamide (TMAD). Various other closing moieties are described in WO 2021 / 255476 (incorporated herein by reference in its entirety).

[0351] For example, a polynucleotide binding protein and / or a polynucleotide binding protein may have a polynucleotide-unbinding opening, e.g., a cavity, cleft or void through which a polynucleotide strand may pass when the polynucleotide binding protein disengages from the strand. The polynucleotide-unbinding opening may be the opening through which a polynucleotide may pass when the polynucleotide binding protein disengages from the polynucleotide. The polynucleotide-unbinding opening for a given polynucleotide binding protein can be determined by reference to its structure, e.g., by reference to its X-ray crystal structure. The X-ray crystal structure may be obtained in the presence and / or the absence of a polynucleotide substrate. The location of a polynucleotide-unbinding opening in a given polynucleotide binding protein may be deduced or confirmed by molecular modelling using standard packages known in the art. The polynucleotide-unbinding opening may be transiently produced by movement of one or more parts e.g., one or more domains of the polynucleotide binding protein.

[0352] The polynucleotide binding protein may be modified by closing the polynucleotide-unbinding opening. The polynucleotide-unbinding opening may be closed with a closing moiety.

[0353] Closing the polynucleotide-unbinding opening may therefore prevent the polynucleotide binding protein from disengaging from the polynucleotide. For example, the polynucleotide binding protein may be modified by covalently closing the polynucleotide-unbinding opening. However, as explained above closing the polynucleotide-unbinding opening does not necessarily prevent the target polynucleotide from unbinding from the polynucleotide binding site of the polynucleotide binding protein. A preferred protein for addressing in this way is a helicase.

[0354] The polynucleotide binding protein may be modified with a closing moiety for (i) topologically closing the polynucleotide binding site of the polynucleotide binding protein around the target polynucleotide and (ii) promoting unbinding of the target polynucleotide from the polynucleotide binding site of the polynucleotide binding protein and / or retarding re-binding of the target polynucleotide to the polynucleotide binding site of the polynucleotide binding protein. The polynucleotide binding protein may be modified in any suitable manner to facilitate attachment of such a closing moiety.

[0355] A closing moiety may comprise a bifunctional cross-linking moiety. The closing moiety may comprise a bifunctional cross-linker. The bifunctional crosslinker may attach at two points on the polynucleotide binding protein and close the polynucleotide-unbinding opening of the polynucleotide binding protein thereby preventing disengagement of the polynucleotide from the polynucleotide binding protein whilst allowing unbinding of the polynucleotide from the polynucleotide-binding site of the polynucleotide binding protein.

[0356] The closing moiety may attach at any suitable positions on the polynucleotide binding protein. For example, the closing moiety may crosslink two amino acid residues of the polynucleotide binding protein. Typically, at least one amino acid crosslinked by the closing moiety is a cysteine or a non-natural amino acid. The cysteine or non-natural amino acid may be introduced into the polynucleotide binding protein by substitution or modification of a naturally occurring amino acid residue of the polynucleotide binding protein. Methods for introducing non-natural amino acids are well known in the art and include for example native chemical ligation with synthetic polypeptide strands comprising such non-natural amino acids. Methods for introducing cysteines into a polynucleotide binding protein are likewise within the capability of one of skill in the art, for example using techniques disclosed in references such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 4thed., Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016).

[0357] The closing moiety may have a length of from about 1 A to about 100 A. The length of the closing moiety may be calculated according to static bond lengths or more preferably using molecular dynamics simulations. The length may for example be from about 2 A to about 80 A, such as from about 5 A to about 50 A, e.g., from about 8 to about 30 A such as from about 10 to about 25 A or about 20 A, e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 A.

[0358] Polynucleotide binding proteins suitable for being closed using a closing moiety as described above are discussed in more detail herein. The polynucleotide binding protein is preferably a helicase.

[0359] The polynucleotide binding protein may be or may be derived from an exonuclease. Suitable enzymes include, but are not limited to, exonuclease I from E. coli, exonuclease III enzyme from E. coli, RecJ from T. thermophilus and bacteriophage lambda exonuclease, TatD exonuclease and variants thereof.

[0360] The polynucleotide binding protein may be a polymerase. The polymerase may be PyroPhage® 3173 DNA Polymerase (which is commercially available from Lucigen® Corporation), SD Polymerase (commercially available from Bioron®), Klenow from NEB or variants thereof. In one embodiment, the enzyme is Phi29 DNA polymerase or a variant thereof. Modified versions of Phi29 polymerase that may be used in the invention are disclosed in US Patent No. 5,576,204.

[0361] The polynucleotide binding protein may be a topoisomerase. In one embodiment, the topoisomerase is a member of any of the Moiety Classification (EC) groups 5.99.1.2 and 5.99.1.3. The topoisomerase may be a reverse transcriptase, which are enzymes capable of catalysing the formation of cDNA from a RNA template. They are commercially available from, for instance, New England Biolabs® and Invitrogen®.

[0362] The polynucleotide binding protein is preferably a helicase. Any suitable helicase can be used in accordance with the detection or characterisation method of the invention. The helicase is preferably a member of superfamily 1 or superfamily 2. The helicase is more preferably a member of one of the following families: Pifl-like, Upfl-like, UvrD / Rep, Skilike, Rad3 / XPD, NS3 / NPH-II, DEAD, DEAH / RHA, RecG-like, REcQ-like, TIR-like, Swi / Snf-like and Rig-I-like. The first three of those families are in superfamily 1 and the second ten families are in superfamily 2. The helicase is more preferably a member of one of the following subfamilies: RecD, Upfl , PcrA, Rep, UvrD, Hel308, Mtr4, XPD, NS3, Mssll6, Prp43, RecG, RecQ, T1R, RapA and Hef. The first five of those subfamilies are in superfamily 1 and the second eleven subfamilies are in superfamily 2. Members of the Upfl, Mtr4, NS3, Mssll6, Prp43 and Hef subfamilies are RNA helicases. Members of the remaining subfamilies are DNA helicases.

[0363] The polynucleotide binding protein is preferably a NS3 helicase or a modified NS3 helicase. The NS3 helicase or modified NS3 helicase may be any of those described in WO 2024 / 235919; PCT / EP2024 / 063118 (incorporated herein by reference in its entirety).

[0364] For example, the or each enzyme used in accordance with the present disclosure may be independently selected from a Hel308 helicase, a RecD helicase, a Tral helicase, a TrwC helicase, an XPD helicase, and a Dda helicase, or a variant thereof. Monomeric helicases may comprise several domains attached together. For instance, Tral helicases and Tral subgroup helicases may contain two RecD helicase domains, a relaxase domain and a C- terminal domain. The domains typically form a monomeric helicase that is capable of functioning without forming oligomers. Particular examples of suitable helicases include Hel308, NS3, Dda, UvrD, Rep, PcrA, Pifl and Tral. These helicases typically work on single stranded DNA. Examples of helicases that can move along both strands of a double stranded DNA include FtfK and hexameric enzyme complexes, or multisubunit complexes such as RecBCD. The polynucleotide binding protein may be a Dda (DNA-dependent ATPase) helicase. Hel308 helicases are described in publications such as WO 2013 / 057495, the entire contents of which are incorporated by reference. RecD helicases are described in publications such as WO 2013 / 098562, the entire contents of which are incorporated by reference. XPD helicases are described in publications such as WO 2013 / 098561, the entire contents of which are incorporated by reference. Dda helicases are described in publications such as WO 2015 / 055981 and WO 2016 / 055777, the entire contents of each of which are incorporated by reference.

[0365] The helicase may be Trwc Cba or a variant thereof, Hel308 Mbu or a variant thereof or Dda or a variant thereof. Variants may differ from the native sequences in any of the ways discussed herein.

[0366] General methods

[0367] As mentioned above, the detection or characterisation method of the invention may be operated using any suitable detector, and as such any suitable apparatus for detecting polypeptides can be used.

[0368] The detection or characterisation method of the invention may be carried out using any apparatus that is suitable for nanopore sensing. For example, the apparatus may comprise a chamber comprising an aqueous solution and a barrier that separates the chamber into two sections. The barrier may have an aperture in which a membrane containing a transmembrane pore is formed. Transmembrane pores are described herein.

[0369] The methods may be carried out using the apparatus described in WO 2008 / 102120, WO 2010 / 122293, or WO 00 / 28312 (incorporated herein by reference in their entireties). In brief, the binding of the polypeptide analyte or the plurality of polypeptide analytes in the channel of a pore will have an effect on the open-channel ion flow through the pore, which is the essence of "molecular sensing" of pore channels. Variation in the open-channel ion flow can be measured using suitable measurement techniques by the change in electrical current. The degree of reduction in ion flow, as measured by the reduction in electrical current, is related to the size of the obstruction within, or in the vicinity of, the pore. Binding of the polypeptide analyte or the plurality of polypeptide analytes in or near the pore therefore provides a detectable and measurable event, thereby forming the basis of a "biological sensor".

[0370] When used to characterize the polypeptide analyte or the plurality of polypeptide analytes, the presence, absence or one or more characteristics of the polypeptide analyte or the plurality of polypeptide analytes are determined. The methods may be for determining the presence, absence or one or more characteristics of the polypeptide analyte or the plurality of polypeptide analytes. The methods may concern determining the presence, absence or one or more characteristics of two or more the plurality of polypeptide analytes. The methods may comprise determining the presence, absence or one or more characteristics of any number of polypeptide analytes, such as 2, 5, 10, 15, 20, 30, 40, 50, 100 or more polypeptide analytes. Any number of characteristics of the one or more polypeptide analytes may be determined, such as 1, 2, 3, 4, 5, 10 or more characteristics. Characteristics amenable to being detected in the methods provide herein include the identity or sequence of the polypeptide analyte or the plurality of polypeptide analytes, the length of the polypeptide analyte or the plurality of polypeptide analytes, whether or not the polypeptide analyte or the plurality of polypeptide analytes is / are modified, etc. In some embodiments, the detection or characterisation method of the invention is a method of sequencing the polypeptide analyte or the plurality of polypeptide analytes. In some embodiments the sequences of the polypeptide analyte or the plurality of polypeptide analytes may be determined in real-time by aligning real-time signal or amino calling to known references. Exemplary methods of determining a polynucleotide sequence are described in WO 2016 / 059427 (incorporated herein by reference in its entirety).

[0371] The method may involve measuring the ion current flow through the pore, typically by measurement of a current. Alternatively, the ion flow through the pore may be measured optically, such as disclosed by Heron et al: J. Am. Chem. Soc. 9 Vol. 131, No. 5, 2009. Therefore, the apparatus may also comprise an electrical circuit capable of applying a potential and measuring an electrical signal across the membrane and pore. The characterisation methods may be carried out using a patch clamp or a voltage clamp. The characterisation methods preferably involve the use of a voltage clamp.

[0372] The method may involve measuring an optical signal as described in Chen et al, Nature Communications (2018)9: 1733, the entire contents of which are hereby incorporated by reference. For example, a nanopore such as an optically engineered nanopore structure (e.g., a plasmonic nanoslit) may be used to locally enable single-molecule surface enhanced Raman spectroscopy (SERS) to allow the characterisation of the polynucleotide through direct Raman spectroscopic detection.

[0373] The method may be carried out on a silicon-based array of wells where each array comprises 128, 256, 512, 1024, 2000, 3000, 4000, 6000, 10000, 12000, 15000 or more wells.

[0374] The method may involve the measuring of a current flowing through the pore. The method is typically carried out with a voltage applied across the membrane and pore. The voltage used is typically from +2 V to -2 V, typically -400 mV to +400mV. The voltage used is preferably in a range having a lower limit selected from -400 mV, -300 mV, -200 mV, -150 mV, -100 mV, -50 mV, -20mV and 0 mV and an upper limit independently selected from + 10 mV, + 20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV. The voltage used is more preferably in the range 100 mV to 240mV and most preferably in the range of 120 mV to 220 mV. It is possible to increase discrimination between different nucleotides by a pore by using an increased applied potential.

[0375] The methods are typically carried out in the presence of any charge carriers, such as metal salts, for example alkali metal salts, halide salts, for example chloride salts, such as alkali metal chloride salt. Charge carriers may include ionic liquids or organic salts, for example tetramethyl ammonium chloride, trimethylphenyl ammonium chloride, phenyltrimethyl ammonium chloride, or l-ethyl-3-methyl imidazolium chloride. In the exemplary apparatus discussed above, the salt is present in the aqueous solution in the chamber. Potassium glutamate (KC5H8NO4), potassium chloride (KCI), sodium chloride (NaCI) or caesium chloride (CsCI) is typically used. KCI is preferred. The salt may be an alkaline earth metal salt such as calcium chloride (CaCI2). The salt concentration may be at saturation. The salt concentration may be 3M or lower 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 carried out using a salt concentration of at least 0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M or at least 3.0 M. High salt concentrations provide a high signal to noise ratio and allow for currents indicative of binding / no binding to be identified against the background of normal current fluctuations.

[0376] The methods are typically carried out in the presence of a buffer. In the exemplary apparatus discussed above, the buffer is present in the aqueous solution in the chamber. Any suitable buffer may be used. Typically, the buffer is HEPES. Another suitable buffer is Tris-HCI buffer. The methods are typically carried out at a pH of 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 7.5 to 8.5. The pH used is preferably about 7.5.

[0377] The methods may be carried out at from 0 °C to 100 °C, from 15 °C to 95 °C, from 16 °C to 90 °C, from 17 °C to 85 °C, from 18 °C to 80 °C, 19 °C to 70 °C, or from 20 °C to 60 °C. The methods are typically carried out at room temperature. The methods are optionally carried out at a temperature that supports enzyme function, such as about 37 °C.

[0378] Any of the proteins described herein, such as the protein pores, may be made synthetically or by recombinant means. For example, the pore may be synthesised by in vitro translation and transcription (IVTT). The amino acid sequence of the pore may be modified to include non-naturally occurring amino acids or to increase the stability of the protein. When a protein is produced by synthetic means, such amino acids may be introduced during production. The pore may also be altered following either synthetic or recombinant production. Any of the proteins described herein, such as the protein pores, can be produced using standard methods known in the art. Polynucleotide sequences encoding a pore or construct may be derived and replicated using standard methods in the art. Polynucleotide sequences encoding a pore or construct may be expressed in a bacterial host cell using standard techniques in the art. The pore may be produced in a cell by in situ expression of the polypeptide from a recombinant expression vector. The expression vector optionally carries an inducible promoter to control the expression of the polypeptide. These methods are described in Sambrook, J. and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[0379] The protein may be produced in large scale following purification by any protein liquid chromatography system from protein producing organisms or after recombinant expression. Typical protein liquid chromatography systems include FPLC, AKTA systems, the Bio-Cad system, the Bio-Rad BioLogic system, and the Gilson HPLC system.

[0380] Methods for introducing or substituting non-naturally occurring amino acids in proteins are also well known in the art and described in WO 2019 / 002893 (incorporated by reference herein in its entirety). The proteins may be modified to assist their identification or purification, for example by the addition of a streptavidin tag or by the addition of a signal sequence to promote their secretion from a cell where the monomer does not naturally contain such a sequence. The proteins may also be produced using D-amino acids or a mixture of L-amino acids and D-amino acids. This is conventional in the art for producing such proteins or peptides.

[0381] Any protein of the invention may be chemically modified. The protein can be chemically modified in any way and at any site. The protein may be chemically modified by attachment of a molecule to one or more cysteines (cysteine linkage), attachment of a molecule to one or more lysines, attachment of a molecule to one or more non-natural amino acids, enzyme modification of an epitope or modification of a terminus. Suitable methods for carrying out such modifications are well-known in the art. The protein may be chemically modified by the attachment of any molecule, such as a dye or a fluorophore.

[0382] The protein may be chemically modified with a molecular adaptor that facilitates the interaction between a pore comprising the monomer and a target nucleotide or target polynucleotide sequence. Suitable adaptors, including a cyclic molecule, a cyclodextrin, a species that is capable of hybridization, a DNA binder or interchelator, a peptide or peptide analogue, a synthetic polymer, an aromatic planar molecule, a small positively charged molecule or a small molecule capable of hydrogen-bonding, are described in WO 2019 / 002893 (incorporated by reference herein in its entirety). The molecular adaptor may be attached using any of the methods and linkers discussed above. Any of the proteins may be modified to assist their identification or purification, for example by the addition of histidine residues (a his tag), aspartic acid residues (an asp tag), a streptavidin tag, a flag tag, a SUMO tag, a GST tag or a MBP tag, or by the addition of a signal sequence to promote their secretion from a cell where the polypeptide does not naturally contain such a sequence. An alternative to introducing a genetic tag is to chemically react a tag onto a native or engineered position on the protein. An example of this would be to react a gel-shift reagent to a cysteine engineered on the outside of the protein. This has been demonstrated as a method for separating hemolysin heterooligomers (Chem Biol. 1997 Jul;4(7):497-505).

[0383] Any of the proteins may be labelled with a revealing label. The revealing label may be any suitable label which allows the protein to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g., 1251, 35S, enzymes, antibodies, antigens, polynucleotides, and ligands such as biotin.

[0384] The protein may also contain other non-specific modifications as long as they do not interfere with the function of the protein. A number of non-specific side chain modifications are known in the art and may be made to the side chains of the protein(s). Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH4, amidation with methylacetimidate or acylation with acetic anhydride.

[0385] Membrane

[0386] The invention also provides a membrane comprising a polypeptide or a plurality of polypeptides coupled to it using a kit of the invention or a coupling method of the invention. The membrane may be any of those discussed above. The polypeptide or the plurality of polypeptides may be any of those discussed above. The kit or the coupling method may be any of those discussed above, especially with reference to the preferred kits or preferred coupling methods. The polypeptide or the plurality of polypeptides may be coupled to the membrane in any of the ways discussed above. The membrane preferably further comprises a detector, a nanopore or a transmembrane pore.

[0387] Arrays

[0388] The invention also provides an array comprising a plurality of membranes of the invention. Any of the embodiments discussed above with respect to the membranes of the invention, kits of the invention and coupling methods of the invention equally apply the array of the invention. The array may be set up to perform any of the methods described herein.

[0389] In a preferred embodiment, each membrane in the array comprises one detector, nanopore or transmembrane pore. Due to the manner in which the array is formed, for example, the array may comprise one or more membranes that do not comprise a detector, nanopore or transmembrane pore, and / or one or more membranes that comprise two or more detectors, nanopores or transmembrane pores. The array may comprise from about 2 to about 1000, such as from about 10 to about 800, from about 20 to about 600 or from about 30 to about 500 membranes.

[0390] Systems

[0391] The invention provides a system comprising (a) a membrane of the invention or an array of the invention, (b) means for applying a potential across the membrane(s) and (c) means for detecting electrical or optical signals across the membrane(s). The membranes may be any as described herein.

[0392] In one embodiment, the system further comprises a first chamber and a second chamber, wherein the first and second chambers are separated by the membrane(s). When used to characterise a polypeptide analyte or the plurality of polypeptide analytes, the system may further comprise a polypeptide analyte, wherein the polypeptide analyte is transiently located within the continuous channel and wherein one end of the polypeptide analyte is located in the first chamber and one end of the polypeptide analyte is located in the second chamber.

[0393] In one embodiment, the system further comprises an electrically conductive solution in contact with the oligomeric pore construct(s), electrodes providing a voltage potential across the membrane(s), and a measurement system for measuring the current through the oligomeric pore construct (s). The voltage applied across the membranes and construct is preferably from +5 V to -5 V, such as -600 mV to +600mV or -400 mV to +400 mV. The voltage used is preferably in the range 100 mV to 240 mV and more preferably in the range of 120 mV to 220 mV. It is possible to increase discrimination between different amino acids or nucleotides by the oligomeric pore construct by using an increased applied potential. Any suitable electrically conductive solution may be used. For example, the solution may comprise charge carriers, such as metal salts, for example alkali metal salt, halide salts, for example chloride salts, such as alkali metal chloride salt. Charge carriers may include ionic liquids or organic salts, for example tetramethyl ammonium chloride, trimethylphenyl ammonium chloride, phenyltrimethyl ammonium chloride, or l-ethyl-3-methyl imidazolium chloride. In an exemplary system, salt is present in the aqueous solution in the chamber. Potassium chloride (KCI), sodium chloride (NaCI), caesium chloride (CsCI) or a mixture of potassium ferrocyanide and potassium ferricyanide is typically used. KCI, NaCI and a mixture of potassium ferrocyanide and potassium ferricyanide are preferred. The charge carriers may be asymmetric across the membrane. For instance, the type and / or concentration of the charge carriers may be different on each side of the membrane, e.g., in each chamber. The salt concentration may be at saturation. The salt concentration may be 3 M or lower 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 carried out using a salt concentration of at least 0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M or at least 3.0 M. High salt concentrations provide a high signal to noise ratio and allow for currents indicative of the presence of an amino acid or nucleotide to be identified against the background of normal current fluctuations.

[0394] A buffer may be present in the electrically conductive solution. Typically, the buffer is phosphate buffer. Other suitable buffers are HEPES and Tris-HCI 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 7.5 to 8.5. The pH used is preferably about 7.5.

[0395] The system may be comprised in an apparatus. The apparatus may be any conventional apparatus for analyte analysis, such as an array or a chip. The apparatus is preferably set up to carry out the disclosed method. For example, the apparatus may comprise a chamber comprising an aqueous solution and a barrier that separates the chamber into two sections. The barrier typically has an aperture in which the membrane(s) containing the oligomeric pore construct (s) are formed. Alternatively, the barrier forms the membrane in which the oligomeric pore construct is present.

[0396] The apparatus may also comprise an electrical circuit capable of applying a potential and measuring an electrical signal across the membrane and pore.

[0397] The apparatus may be any of those described in WO 2008 / 102120, WO 2009 / 077734, WO 2010 / 122293, WO 2011 / 067559, or WO 00 / 28312 (all incorporated herein by reference in their entirety).

[0398] Apparatus

[0399] The invention also provides an apparatus comprising a polypeptide or a plurality of polypeptides coupled to an in vitro membrane using a kit of the invention or a coupling method of the invention.

[0400] The invention also provides an apparatus produced by a method comprising coupling a polypeptide or a plurality of polypeptides to an in vitro membrane using a kit of the invention or a coupling method of the invention.

[0401] The membrane preferably comprises a detector, a nanopore or a transmembrane pore. Any of the specific embodiments discussed above are equally applicable to the apparatuses of the invention.

[0402] SEQUENCE LISTING

[0403] Table 5 - Sequences used in the Examples

[0404] SEQ ID NO: 9 (underlined sequence in SEQ ID NO: 1)

[0405] EDLEIEAAFLEQENTALETEVAELEQEVQRLENIVSQYETRYGPLGGG

[0406] SEQ ID NO: 10 (underlined sequence in SEQ ID NO: 2)

[0407] EIAALEQEIAALEKENAALEWEIAALEQGG Table 6 - Preferred monomers, oligomers and hairpins for use in the invention

[0408] The following Examples illustrate the invention. It is to be understood that although particular embodiments, specific configurations as well as materials and / or molecules, have been discussed herein for methods according to the invention, various changes or modifications in form and detail may be made without departing from the scope and spirit of this invention. The following examples are provided to better illustrate particular embodiments, and they should not be considered limiting the application. The application is limited only by the claims.

[0409] EXAMPLES

[0410] Example 1

[0411] This Example describes implementation of a coupling system utilising leucine zippers. The system enables an increase in sensitivity and helps to control the orientation of a construct to improve capture into a nanopore.

[0412] Expression and purification of polypeptide analytes

[0413] Polypeptide analytes were expressed as an N-terminal extension to a blocker domain with an N-terminal leader (SEQ ID NOs: 1-2) containing an acidic leucine zipper monomer (underlined) in E. coli BL21(DE3). Proteins were purified by immobilised metal affinity chromatography and exchanged into 50 mM Tris-HCI pH 8.0, 300 mM NaCI.

[0414] Assembly of leucine zipper-DNA conjugates

[0415] Basic leucine zipper peptides (SEQ ID NOs: 3-7) with an N-terminal azide moiety were produced by solid phase synthesis using well established methods in the art. The leucine zipper peptides were attached to DNA oligonucleotide (SEQ ID NO: 8) using click chemistry via the 5' BCN group. Peptides (SEQ ID NOs: 3-4) were purified by SPRI. Peptides (SEQ ID NOs: 5-7) were purified by HPLC using anion exchange chromatography.

[0416] Electrophysiology

[0417] Electrical data were collected using RP4 MinlON flow cells (Oxford Nanopore Technologies pic). Sequencing libraries were prepared in 75 pL Sequencing Buffer (25 mM HEPES-KOH (pH 7.6), 450 mM K-glutamate, 10 mM ATP, 20 mM MgCI2). Flow cells were first flushed with 1 mL of Sequencing Buffer with addition of FCT taken from an SQK-LSK114 sequencing kit (Oxford Nanopore Technologies pic). Sample (75 pL) was then introduced into the flow cell via the SpotON port. Electrical data were acquired with a sample rate of 4 kHz at 30°C and applied potential of 140 mV. Results and discussion

[0418] Electrophysiology measurements were collected using a sequencing library consisting of various concentrations of polypeptide analyte with the leader sequence (SEQ ID NO: 1) either in the absence of leucine zipper monomer-DNA (Figure 1A), or with 4nM of leucine zipper monomer-DNA of design 1 (Figure IB, SEQ ID NO: 3) or design 2 (Figure 2B, SEQ ID NO: 4). Capture events were recorded and normalised per nanopore per hour (Figure 1). It was found that capture frequency was increased for all input concentrations with the addition of the basic leucine zipper.

[0419] Next electrophysiology measurements were collected using a sequencing library consisting of 2nM polypeptide analyte with the leader sequence (SEQ ID NO: 2) and optionally including 4 nM of leucine zipper-DNA of either design 3 (SEQ ID NO: 5), 4 (SEQ ID NO: 6), or 5 (SEQ ID NO: 7) which possess 3, 3.5, 4 heptad repeats respectively and the unfoldase CIpX at 50 nM. CIpX reads were recorded for each separate experiment and were normalised by the no basic zipper control (Figure 2). All leucine zipper designs showed an increase in throughout of reads compared to the no leucine zipper control, with the 3 and 3.5 heptad designs showing the highest read throughput.

Claims

CLAIMS1. A method for coupling a polypeptide to a membrane, comprising coupling the polypeptide to the membrane using (1) one or more first coupling adaptors each comprising one or more coupling polypeptides, and (2) one or more second coupling adaptors each comprising one or more coupling domains which specifically bind to the one or more coupling polypeptides.

2. A method according to claim 1, wherein the one or more coupling polypeptides comprise one or more amphipathic helices, one or more polyhistidine-tags, one or more transcription factors, one or more antigens, one or more antibodies or one or more fragments thereof, one or more enzymes, one or more peptide nucleic acids (PNAs), one or more gamma PNAs, one or more polypeptide analytes, one or more split proteins, one or more affimers or one or more combinations thereof.

3. A method according to claim 1 or 2, wherein the one or more coupling domains comprise one or more amphipathic helices, one or more derivatives of nitrilotriacetic acid (NTA), one or more promoter sequences, one or more antibodies or one or more fragments thereof, one or more antigens, one or more polynucleotides, one or more analytes, one or more enzymes, one or more aptamers, one or more split proteins, one or more affimers or one or more combinations thereof.

4. A method according to any one of the preceding claims, wherein the one or more coupling polypeptides comprise one or more coiled-coil monomers and the one or more coupling domains comprise one or more coiled-coil monomers or wherein the one or more coupling polypeptides comprise one or more leucine zipper monomers and the one or more coupling domains comprise one or more leucine zipper monomers.

5. A method according to any one of the preceding claims, wherein the one or more coupling polypeptides comprise one or more acidic coiled-coil monomers and the one or more coupling domains comprise one or more basic coiled-coil monomers or vice versa or wherein the one or more coupling polypeptides comprise one or more acidic leucine zipper monomers and the one or more coupling domains comprise one or more basic leucine zipper monomers or vice versa.

6. A method according to any one of the preceding claims, wherein the one or more first coupling adaptors comprise a polypeptide leader.

7. A method according to any one of the preceding claims, wherein the one or more second coupling adaptors comprise a membrane protein or one or more hydrophobic anchors.

8. A method according to any one of claims 1-6, wherein the method further comprises using (3) one or more third coupling adaptors comprising a membrane protein or one or more hydrophobic anchors.

9. A method according to claim 8, wherein the one or more second coupling adaptors further comprises a first oligonucleotide and the one or more third coupling adaptors further comprises a second oligonucleotide which hybridizes to the first oligonucleotide.

10. A method according to any one of the preceding claims, wherein the one or more first coupling adaptors further comprise a blocking moiety.

11. A method according to any one of the preceding claims, wherein the method comprises (a) attaching the one or more first coupling adaptors to the polypeptide, and (b) coupling the one or more first coupling adaptors to the membrane using the one or more second coupling adaptors or the one or more second coupling adaptors and the one or more third coupling adaptors.

12. A method according to claim 11, wherein the method also comprises in step (a) attaching the blocking moiety to the polypeptide.

13. A method according to any one of the preceding claims, wherein the membrane is an amphiphilic layer or a solid state layer.

14. A method according to any one of the preceding claims, wherein the polypeptide is coupled transiently or permanently to the membrane.

15. A kit for coupling a polypeptide to a membrane, comprising (1) one or more first coupling adaptors each comprising one or more coupling polypeptides, and (2) one or more second coupling adaptors each comprising one or more coupling domains which specifically bind to the one or more coupling polypeptides.

16. A kit according to claim 15, wherein the one or more first coupling adaptors and / or the one or more second coupling adaptors are as defined in any one of claims 2-7.

17. A kit for coupling a polypeptide to a membrane, comprising (1) one or more first coupling adaptors each comprising one or more first coiled-coil monomers or one or more first leucine zipper monomers, and (2) one or more second coupling adaptors each comprising one or more second coiled-coil monomers or one or more second leucine zipper monomers which specifically binds to the one or more first coiled-coil monomers or one or more first leucine zipper monomers in the one or more first coupling adaptors.

18. A kit according to any one of claims 15-17, wherein the kit further comprises (3) one or more third coupling adaptors comprising a membrane protein or one or more hydrophobic anchors.

19. A method for determining the presence, absence or one or more characteristics of a polypeptide analyte, comprising (a) coupling the polypeptide analyte to a membrane using a kit according to any of claims 15-18 or method according to any one of claims 1-14 and (b) allowing the coupled polypeptide analyte to interact with a detector present in the membrane and thereby determining the presence, absence or one or more characteristics of the polypeptide analyte.

20. A method according to claim 19, wherein the detector detects the analyte via electrical or optical means.

21. A method according to claim 19 or 20, wherein the detector comprises a transmembrane pore.

22. A method according to claim 21, wherein step (b) comprises (i) allowing the polypeptide analyte to interact with the transmembrane pore and (ii) measuring the current passing through the pore during the interaction and thereby determining the presence, absence or one or more characteristics of the polypeptide analyte.

23. Use of a kit according to any one of claims 15-18 for coupling a polypeptide or a plurality of polypeptides to a membrane.

24. A membrane comprising a polypeptide or a plurality of polypeptides coupled to it using a kit according to any one of claims 15-18 or a method according to any one of claims 1-14.

25. A membrane according to claim 24, wherein the membrane further comprises a detector.

26. An array comprising a plurality of membranes according to claim 24 or 25.

27. A system comprising (a) a membrane according to claim 24 or 25 or an array according to claim 24, (b) means for applying a potential across the membrane(s) and (c) means for detecting electrical or optical signals across the membrane(s).

28. An apparatus comprising a polypeptide or a plurality of polypeptides coupled to an in vitro membrane using a kit according to any one of claims 15-18 or a method according to any one of claims 1-14.10429. An apparatus produced by a method comprising coupling a polypeptide or a plurality of polypeptides to an in vitro membrane using a kit according to any one of claims 15-18 or a method according to any one of claims 1-14.

30. An apparatus according to claim 28 or 29, wherein the membrane comprises a detector.105

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