Modification of nanopore-forming protein oligomers

Abstract

Provided herein are methods for chemically modifying polypeptide or polynucleotide monomers. The monomers can be monomers of protein pores, such as monomeric or oligomeric protein pores. Related monomers and oligomers, as well as methods for using the disclosed pores for characterizing analytes, are also disclosed.

Description

[Technical Field]

[0001] This invention relates to a method for chemically modifying proteins and polynucleotides, as well as the modified proteins and polynucleotides themselves. This method is particularly useful for generating chemically modified protein nanopores. [Background technology]

[0002] Nanopore sensing is a technique for analyte detection and characterization that relies on observing individual binding or interaction events between analyte molecules and ion conduction channels. Nanopore sensors can be created by placing a single nanometer-sized pore in an electrical insulating film and measuring the voltage-driven ion current through the pore in the presence of analyte molecules. The presence of analyte inside or near the nanopore alters the ion flow through the pore, resulting in a change in the ion current or current measured on the channel. The identity of the analyte is revealed through its distinctive current signature, particularly the duration and degree of current interruption, and any discrepancies in current levels during its interaction with the pore.

[0003] Polynucleotides are important analytes for sensing in this manner. Nanopore sensing of polynucleotide analytes can reveal identity and perform single-molecule counting of the sensed analytes, as well as provide information about their composition, such as their nucleotide sequences, and the presence of features such as base modifications, oxidation, reduction, decarboxylation, and deamination. Nanopore sensing has the potential to enable rapid and inexpensive polynucleotide sequencing, providing single-molecule sequences of polynucleotides ranging from tens to tens of thousands of base pairs in length.

[0004] Known methods of nanopore sensing utilize the use of biological protein nanopores. Such nanopores are typically multimeric proteins composed of multiple monomer subunits. For example, the known pore-forming toxin α-hemolidine assembles into heptameric pores. Other pore stoichiometry for different pore-forming proteins are known.

[0005] In many cases, it is desirable to modify protein nanopores to produce effects that are not seen in unmodified pores, or are seen to a lesser extent. A wide variety of modifications have been disclosed to achieve diverse and different results. For example, nanopores are known to be chemically modified to include binding sites for molecular adapters such as cyclodextrins, and such adapters have found specific applications in sensing small analytes such as individual nucleotides. Nanopores can also be modified to introduce specific modifications that alter the properties of natural or artificial constrictions within the pore channel. Such constrictions, sometimes known as "read heads," typically interact with the analyte when the analyte interacts with the pore, altering the properties of the read head and thereby altering the analyte detection signal accordingly. In yet another example, nanopores can be modified to include membrane anchors such as cholesterol. Such anchors can interact with the bilayer to promote the stable localization of nanopores within the membrane.

[0006] All of these modification strategies may involve chemical modification of nanopores. However, current methods for modifying nanopores and related proteins, as discussed below, have problems.

[0007] One known method for modifying proteins, such as protein monomers in protein nanopores, relies on modifications that can be generated by the cells expressing the protein. For example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 thUsing conventional molecular biology techniques, such as those discussed in *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)*, amino acids in proteins can be altered, introduced, or deleted. Once expressed, the modified protein can be purified for use. However, the range of chemical modifications that can be performed in this manner is extremely limited, as it depends on the range of chemical species that can be processed by the expressing cell and the existence of cellular mechanisms for processing such species. Such techniques are insufficient for modifying proteins across a wide range of chemical parts. Furthermore, such techniques are not universally applicable: typically, different proteins require different cloning and expression strategies, and the expression of a first protein in a given system may not be successful for a second protein.

[0008] A second known method for modifying proteins, such as protein monomers in protein nanopores, is to express an unmodified protein in the usual manner and then attempt to modify the expressed protein. Essentially, there are two alternative approaches that can be taken. In the first approach, the expressed protein is modified before purification, and then an attempt is made to purify the modified protein. In the second approach, the expressed protein is purified, and then the purified protein is modified. However, both of these approaches are fraught with difficulties. As will be explained below, these difficulties stem from the fact that chemical modification of expressed proteins is rarely efficient, and typically, even the most efficient modification strategies result in only about 95% modification, leaving more than 5% of the protein molecule unmodified.

[0009] In the first method, the target protein is chemically modified before purification. As described above, even with modification strategies generally considered efficient, typically less than 95% of the available protein molecules are modified, with more than 5% remaining unmodified. Attempts may then be made to purify the modified protein molecules from the unmodified molecules (and other impurities). Unfortunately, isolating only modified protein molecules from unmodified analogs is typically difficult or impossible. Modifications may typically have little effect on the overall mass of the protein molecule, meaning that separation techniques that rely on distinguishing proteins by mass may not be effective. Modifications may not change the charge of the protein, and therefore techniques that rely on distinguishing proteins based on charge interactions may similarly fail. As a result, in many cases, purification strategies fail to separate modified proteins from their unmodified counterparts, and the resulting product from the purification process contains a mixture of modified and unmodified protein molecules in proportions determined by the efficiency of the modification chemistry used.

[0010] In the second method, the target protein is chemically modified after purification. The purification protocol used may be efficient in separating the unmodified protein from impurities present in the expression medium. However, problems arise again when modification chemistry is performed on purified protein. Since modification chemistry is not 100% efficient, the result of the modification process is again a mixture of modified and unmodified protein molecules in proportions determined by the efficiency of the modification chemistry used.

[0011] The presence of relatively low levels (around 5%) of unmodified proteins in a population of proteins modified in other ways can be problematic. This is especially true when the protein is a monomer of oligomeric protein pores. For example, the presence of some unmodified monomers in a population of modified monomers typically means that a certain distribution of aggregated pores will occur. For instance, in heptameric pores such as aggregated α-hemolidine from a mixture of both modified and unmodified monomers, the aggregated pores may contain only modified monomers, only unmodified monomers, or a mixture of both. While such pore distributions can be statistically calculated, characterizing individual pores aggregated into a membrane is by no means straightforward, for example, for use in nanopore sensing. Furthermore, this presents problems when interpreting data obtained from pores in functional devices, as the signal must be interpreted based on the inherently heterogeneous nature of the population of pores used to generate the device. In some cases, this difficulty can result in the abandonment of functional pores, data loss or convolution, the introduction of artifacts in the resulting data by the pores, and / or a loss of reproducibility when a given homogeneous sample is analyzed using a sample with heterogeneous pores.

[0012] Therefore, there is an urgent need for new strategies for chemically modifying proteins, such as monomers of protein nanopores. The methods of this disclosure overcome some or all of the aforementioned problems. [Overview of the project]

[0013] This disclosure relates to a method for chemically modifying polypeptides or polynucleotide monomers, typically monomers of oligomeric protein nanopores. The monomer is brought into contact with a polyfunctional molecule comprising (i) a reactive group for reacting with the monomer, (ii) a chemical modification group for providing the chemical modification, and (iii) a cleavable purification tag for purifying the monomer. The reactive group of the polyfunctional molecule is reacted with the monomer, thereby binding the chemical modification group and the cleavable purification tag to the monomer to form a chemically modified tagged monomer. The chemically modified monomer is brought into contact with a support such as a chromatography matrix or magnetic beads, and the purification tag is bound to the support. Binding of the purification tag to the support results in binding of the chemically modified tagged monomer to the support. The purification tag is then cleaved by any preferred means. The chemically modified monomer is released from the support by cleaving of the purification tag. A key advantage of this method is the combined modification and purification strategy, which avoids or minimizes problems associated with inefficiencies in any of the steps described above.

[0014] Therefore, a method for chemically modifying polypeptides or polynucleotide monomers (e.g., monomers of oligomeric protein nanopores), a) Contacting a monomer with a polyfunctional molecule, wherein the polyfunctional molecule includes (i) a reactive group, (ii) a chemically modifying group, and (iii) a cleavable purified tag. b) Reacting the reactive groups of a polyfunctional molecule with a monomer, thereby attaching chemically modified groups and cleavable purified tags to the monomer to form chemically modified tagged monomers, c) Contacting the chemically modified tagged monomer formed in step (b) with the support, d) Attaching a purified tag to a support, thereby attaching a chemically modified tagged monomer to the support, e) A method is provided herein that includes cleaving a purified tag, thereby releasing a chemically modified monomer from a support.

[0015] In some embodiments, the multifunctional molecule is of formula (I) or formula (II),

[0016]

Chemical formula

[0017] In some embodiments, the monomer contains a reactive functional group, and step (b) includes reacting the reactive group of the multifunctional molecule with the reactive functional group of the monomer.

[0018] In some embodiments, the reactive group of the multifunctional molecule is an amine-reactive group; a carboxyl-reactive group; a sulfhydryl-reactive group or a carbonyl-reactive group; preferably, it contains a cysteine-reactive group. In some embodiments, the reactive group contains maleimide, azide, thiol, alkyne, NHS ester, or haloacetamide.

[0019] In some embodiments, the chemical modification group introduces hydrophilic, hydrophobic, positive charge, negative charge, hydrogen bonding, supramolecular assembly, or zwitterionic properties to the protein monomer. In some embodiments, the chemical modification group contains an amino acid, a nucleotide, a polymer, a hydrogen bonding group, a membrane anchor, a sugar, a pigment, a chromophore, a fluorophore, or a molecular adapter. In some embodiments, the chemical modification group contains a natural or non-natural amino acid, a polypeptide, a nucleotide or a nucleotide analog, an oligonucleotide or an oligonucleotide analog, a polysaccharide, a lipid, polyethylene glycol, cyclodextrin, a DNA intercalator, an aptamer, or an analyte binding domain.

[0020] In some embodiments, the support comprises a chromatography matrix, preferably an agarose or sepharose resin; one or more beads, preferably magnetic beads; or a solid surface, preferably a glass, silica, polymer, or ceramic surface. In some embodiments, the support is functionalized for binding to a purification tag. In some embodiments, the purification tag comprises a biotin group and the support comprises streptavidin, neutravidin, or avidin, preferably streptavidin.

[0021] In some embodiments, the cleavable linker is cleaved by physical or chemical means. In some embodiments, the cleavable linker comprises a nitro-benzyl moiety that is cleavable by UV light.

[0022] In some embodiments, in step (e) of the method, cleaving the purification tag comprises exposing the support and / or the tagged monomer to light, preferably UV light. In some embodiments, in step (e), cleaving the purification tag comprises exposing the support and / or the tagged monomer to a change in pH. In some embodiments, in step (e), cleaving the purification tag comprises exposing the support and / or the tagged monomer to a chemical reagent, preferably a reducing reagent. In some embodiments, in step (e), cleaving the purification tag comprises exposing the support and / or the tagged monomer to an enzyme, preferably a protease.

[0023] In some embodiments, the monomer is a polypeptide having a mass of about 10 kDa to about 1 MDa. In some embodiments, the monomer is an oligomeric protein pore, preferably a lysenin pore, a γ-hemolysin pore, an α-hemolysin pore; a NetB pore; a CytK pore, or a leukocidin pore; or a monomer of a homolog or paralog thereof. In some embodiments, the oligomeric protein pore is a multi-component pore.

[0024] In some embodiments of the disclosure, the monomer is a polynucleotide, preferably a monomer of a DNA origami pore.

[0025] In some embodiments, the monomer is the monomer of the oligomeric pore, and when the monomer is oligomerized to form a pore, the polyfunctional molecule reacts with the reactive functional group located on the monomer at a surface-exposed position. In some embodiments, the surface-exposed position is located on the surface of the channel through the pore, or on the outer surface of the pore. In some embodiments, when the monomer is oligomerized to form a pore, the polyfunctional molecule reacts with the reactive functional group located on the monomer at a position located at or near the constriction of the channel through the pore.

[0026] In some embodiments, the method includes, prior to step (a), (i) expressing the monomer in a cell expression system or a cell-free expression system, and (ii) isolating and / or purifying the monomer.

[0027] In some embodiments, step (d) of the method further includes removing unmodified monomers and / or unreacted polyfunctional molecules from the support, if present.

[0028] In some embodiments, this method f) Further comprising the step of oligomerizing a chemically modified monomer to form a chemically modified oligomer, for example, forming a chemically modified oligomer protein nanopore. In some embodiments, the monomer is a protein monomer, and step (f) includes forming an oligomeric protein. In some embodiments, step (f) includes oligomerizing two or more chemically modified monomers to form a homooligomer, for example, to form an oligomeric protein nanopore. In some embodiments, the monomer is a protein monomer, and the homooligomer is a homooligomeric protein pore.

[0029] In some embodiments, step (f) includes oligomerizing one or more chemically modified monomers with one or more unmodified or differently modified monomers to form a heterooligomer, for example, an oligomeric protein nanopore. In some embodiments, the monomers are protein monomers, and the heterooligomers are heterooligomeric protein pores.

[0030] In some embodiments, step (f) comprises oligomerizing one or more chemically modified first monomers with one or more chemically modified second monomers to form a heterooligomer, for example, to form an oligomeric protein nanopore, wherein the chemical modifications performed on the first monomer are the same as or different from the chemical modifications performed on the second monomer. In some embodiments, the monomers are protein monomers, the first monomer has a different amino acid sequence from the second monomer, and the heterooligomer is a heterooligomeric protein nanopore.

[0031] A method for generating homo-oligomeric proteins, for example, homo-oligomeric protein nanopores, i) The method described herein produces multiple chemically modified protein monomers, ii) A method is also provided which comprises oligomerizing two or more of the chemically modified protein monomers obtained in step (i) to form a homo-oligomeric protein. In some embodiments, the homo-oligomeric protein is a homo-oligomeric protein pore.

[0032] A method for generating hetero-oligomeric proteins, for example, hetero-oligomeric protein nanopores, i) To produce one or more chemically modified first protein monomers by the method described herein, ii) To produce one or more chemically modified second protein monomers by the method described herein, iii) A method is also provided which comprises oligomerizing one or more first monomers and one or more second monomers to form a hetero-oligomeric protein. In some embodiments, the hetero-oligomeric protein is a hetero-oligomeric protein pore.

[0033] A method for generating oligomeric proteins, for example, oligomeric protein nanopores, i) To produce one or more chemically modified first protein monomers by the method described herein, ii) To provide one or more unmodified second protein monomers, iii) A method is also provided which comprises oligomerizing one or more first monomers and one or more second monomers to form a hetero-oligomeric protein. In some embodiments, the hetero-oligomeric protein is a hetero-oligomeric protein pore.

[0034] Chemically modified monomers, such as chemically modified monomers of oligomeric protein nanopores, which can be obtained by performing the methods described herein are also provided.

[0035] Also provided is a homogeneous population comprising multiple chemically modified monomers, wherein at least 95% of the monomers in the population are chemically modified with a chemical modification group, for example, the monomers may be monomers of one or more oligomeric protein nanopores (e.g., one or more types of protein nanopores). In some embodiments, the chemically modified monomers are as described in more detail herein.

[0036] Chemically modified oligomers, such as oligomeric protein nanopores, which can be obtained by performing the methods described herein are also provided.

[0037] Also provided is a homogeneous population comprising multiple chemically modified oligomers, for example, oligomeric protein nanopores, wherein at least 95% of the oligomers in the population comprise a predetermined number of monomers chemically modified with chemical modification groups. In some embodiments, the chemically modified monomers are as described in more detail herein.

[0038] A method for characterizing an analyte, i) To generate chemically modified oligomeric pores by the method described herein, or to provide chemically modified oligomeric pores as described herein, ii) A method is also provided which includes obtaining one or more measurements as the analyte moves into the pore, wherein the one or more measurements represent one or more features of the analyte, thereby characterizing the analyte as it moves into the pore. In some embodiments, the analyte is a polynucleotide, polypeptide, or polysaccharide.

[0039] Related systems, methods, and uses are also provided. [Brief explanation of the drawing]

[0040] [Figure 1] A schematic diagram illustrating how the disclosed method may be used to introduce multiple modifications to the barrel of a two-component pore-forming toxin such as gamma-hemolyzin. Specific residues are shown for illustrative purposes only and are not limiting. [Figure 2-1] UV cleavage products of photocleavable polyfunctional molecules, as discussed in the examples (see, for example, Example 1). [Figure 2-2] UV cleavage products of photocleavable polyfunctional molecules, as discussed in the examples (see, for example, Example 1). [Figure 3] A schematic diagram illustrating the chemical modification of monomer cysteine ​​variants using polyfunctional molecules, as discussed in the examples. (See Example 2). [Figure 4]A gel demonstrating the successful modification of the lysenin mutant Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / S98C / C272A / C283A) according to the method disclosed in Example 2. Lane 1 - Unmodified protein monomer (Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / S98C / C272A / C283A)); Lane 2 - Crude reaction mixture - Maleimide-isoleucine adduct containing Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / S98C / C272A / C283A); Lane 3 - Flow-through after equilibration of modified protein monomer on StrepTactin beads (the flow-through is bound to the StrepTactin beads). Lane 4 / 5 - Lane 4 shows unmodified protein monomers; Lane 5 shows further washing of StrepTactin beads bound with modified monomers to remove unmodified protein monomers (the gel shows that unmodified monomers do not elute during further washing); Lane 6 shows modified Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / S98C-maleimide-icoleucine / C272A / C283A) eluted from StrepTactin beads after UV cleavage of the linker. [Figure 5]Gel demonstrating the successful modification of the lysenin mutant Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / T63C / C272A / C283A) according to the method disclosed in Example 2. Lane 1 - Unmodified protein monomer (Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / T63C / C272A / C283A)) Lane 2 - Crude reaction mixture - Maleimide-isoleucine adduct containing Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / T63C / C272A / C283A); Lane 3 - Flow-through after equilibration of modified protein monomers on StrepTactin beads (flow-through shows unmodified protein monomers not bound to StrepTactin beads); Lane 4 / 5 - Unmodified tan Further washing of StrepTactin beads bound with modified monomers to remove protein monomers (the gel shows that unmodified monomers do not elute during further washing); modified Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / T63C-maleimido-icoleucine / C272A / C283A) eluted from StrepTactin beads after UV cleavage of the lane 6-linker. [Figure 6]A gel demonstrating successful modification of the F component mutant of gamma-hemolyzin pore LukF-T142C-Del(E1-K15) according to the method disclosed in Example 2. Lane 1 - Unmodified protein monomer, LukF-T142C-Del(E1-K15); Lane 2 - Crude reaction mixture - Maleimide-isoleucine adduct containing LukF-T142C-Del(E1-K15); Lane 3 - Flow-through after equilibration of modified protein monomer on StrepTactin beads (flow-through shows unmodified protein monomer not bound to StrepTactin beads); Lane 4 / 5 - Further washing of StrepTactin beads bound with modified monomer to remove unmodified protein monomer (gel shows no elution of unmodified monomer during further washing); Lane 6 - Modified LukF-T142C-maleimide-alanine-Del(E1-K15) eluted from StrepTactin beads after UV cleavage of the linker. [Figure 7] A gel demonstrating successful modification of a variant of the S component of gamma-hemolyzin pore Hlg2-G114C-Del(E1-G10) according to the method disclosed in Example 2. Lane 1 - Unmodified protein monomer, Hlg2-G114C-Del(E1-G10); Lane 2 - Crude reaction mixture - Maleimide-isoleucine adduct containing Hlg2-G114C-Del(E1-G10); Lane 3 - Flow-through after equilibration of modified protein monomer on StrepTactin beads (flow-through shows unmodified protein monomer not bound to StrepTactin beads); Lane 4 / 5 - Further washing of StrepTactin beads bound with modified monomer to remove unmodified protein monomer (gel shows no elution of unmodified monomer during further washing); Lane 6 - Modified Hlg2-G114C-maleimide-isoleucine-Del(E1-G10) eluted from StrepTactin beads after UV cleavage of the linker. [Figure 8]A gel demonstrating successful oligomerization of modified lysenin-[Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / S98C-maleimide-icoleucine / C272A / C283A)] for the formation of nonameric pores. The results are discussed in Example 3. Lane 1 - Modified protein monomer, Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / S98C-maleimido-icoleucine / C272A / C283A); Lane 2 - 1:1 crude reaction mixture of Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / S98C-maleimido-icoleucine / C272A / C283A) and sphingomyelin after overnight storage. The reactant mixture shows oligomeric pore formation (upper band) and unreacted modified monomer Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / S98C-maleimido-icoleucine / C272A / C283A); lane 3 - pores - supernatant after centrifugation to separate sphingomyelin pellet. Several pores are also visible in the supernatant; lane 4 - modified [Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / S98C-maleimido-icoleucine / C272A / C283A) 9 pores; [Figure 9]A gel demonstrating successful oligomerization of lysenin-[Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / T63C-maleimide-PNA(thymine) / C272A / C283A)] for the formation of nonomer pores. The results are discussed in Example 3. Lane 1 - Modified protein monomer, Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / T63C-maleimide-PNA(thymine)C272A / C283A); Lane 2 - 1:1 crude reaction mixture of Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / T63C-maleimide-PNA(thymine)C272A / C283A) and sphingomyelin after overnight storage. The reactant mixture shows oligomeric pore formation (top band) and unreacted modified monomer Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / S98C-maleimide-icoleucine / C272A / C283A); lane 3 - pores - supernatant after centrifugation to separate sphingomyelin pellet. Several pores are also visible in the supernatant; lane 4 - modified [Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / T63C-maleimide-PNA(thymine) / C272A / C283A)] pores. [Figure 10]A gel demonstrating the successful oligomerization of various gamma-hemolysin variants described in Example 4. Lane 1 - Unmodified (LukF-T142C-Del(E1-K15)) monomer - A guide to see where the monomer appears on the gel; Lane 2 - Unmodified (Hlg2-G114C-Del(E1-G10)) monomer - A guide to see where the monomer appears on the gel; Lane 3 - Modified pore oligomer [(LukF-T142C-maleimide-isoleucine-Del(E1-K15))4(Hlg2-G114C-maleimide-alanine-Del(E1-G10))4] - Band on the gel. Bands of modified monomers that have not been oligomerized can also be seen. ;Lane 4 - Modified pore oligomer [(LukF-T142C-maleimide-isoleucine-Del(E1-K15))4(Hlg2-G114C-maleimide-isoleucine-Del(E1-G10))4] - band above. You can also see the band of the modified monomer that has not been oligomerized. ;Lane 5 - Modified pore oligomer [(LukF-T142C-maleimide-alanine-Del(E1-K15))4(Hlg2-G114C-maleimide-alanine-Del(E1-G10))4] - band above. You can also see the band of the modified monomer that has not been oligomerized. ;Lane 6-Modified porous oligomer [(LukF-T142C-maleimide-isoleucine-Del(E1-K15))4(Hlg2-G114C-maleimide-alanine-Del(E1-G10))4]-Band above. Also, bands of non-oligomerized, modified monomers can be seen. [Figure 11]Characterization data for the polynucleotide analyte of Sequence ID No. 20, when characterized using a panel of chemically modified lysenin pores generated according to the method of disclosure. Two lysenin pores: Lys-(E84Q / E85S / E92Q / E94D / E97S / S98C / T106K / D126G / C272A / C283A) and Lys-(T63C / E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / C272A / C283A) were modified with five different chemical molecules, and the effect of the modification was analyzed by comparing the open pore current level of the modified pores with that of the unmodified pores as lambda DNA passed through each pore. The highest level within each channel represents the open pore current level. The lowest level within each channel represents the current level observed as DNA passes through the pore. [Figure 12] Signal changes in 3.6Kb lambda DNA translocated through lysenin mutant pores, correlated with modifying factor molecules. Panel A shows data for unmodified Lys-(E84Q / E85S / E92Q / E94D / E97S / S98C / T106K / D126G / C272A / C283A), and Panel B shows data for modified pore Lys-(E84Q / E85S / E92Q / E94D / E97S / S98C-maleimide-isoleucine / T106K / D126G / C272A / C283A). The left panel shows events for the entire 3.6Kb of DNA passing through the pore. The right panel shows a magnified version of the irregular curve over 0.4 seconds. [Figure 13]Changes in the GGAA region of 3.6Kb lambda DNA translocated through lysenin mutant pores, correlated with modifying factor molecules. Panel A shows data for unmodified Lys-(E84Q / E85S / E92Q / E94D / E97S / S98C / T106K / D126G / C272A / C283A). Panel B shows data for modified pore Lys-(E84Q / E85S / E92Q / E94D / E97S / S98C-maleimide-isoleucine / T106K / D126G / C272A / C283A), and Panel C shows data for modified pore Lys-(E84Q / E85S / E92Q / E94D / E97S / S98C-maleimide-PNA(thymine) / T106K / D126G / C272A / C283A). [Figure 14] Gel demonstrating successful modification of a variant of component F of gamma-hemolyzin pore LukF-T142C-Del(E1-K15) according to the method disclosed in Example 2. A: Modification with maleimide-asparagine. B: Modification with maleimide-CH2-NH2. C: Modification with maleimide-arginine. D: Modification with maleimide-isoleucine. E: Modification with maleimide-aspartic acid. For each of A to E, lane 1 = unmodified protein monomer, LukF-T142C-Del(E1-K15); lane 2 = monomer modified with a cleavable complex; lane 3 = flow-through after equilibration of the modified protein monomer on StrepTactin beads (flow-through shows unmodified protein monomer not bound to StrepTactin beads); lane 4 = modified LukF-T142C-Del(E1-K15)-maleimide-[modified] eluted from StrepTactin beads after UV cleavage of the linker. [Figure 15]Gel demonstrating successful modification of a variant of the S component of gamma-hemolyzin pore Hlg2-N130C-Del(E1-G10) according to the method disclosed in Example 2. A: Modification with maleimide-asparagine. B: Modification with maleimide-CH2-NH2. C: Modification with maleimide-arginine. D: Modification with maleimide-isoleucine. E: Modification with maleimide-aspartic acid. For each of A to E, lane 1 = unmodified protein monomer, Hlg2-N130C-Del(E1-G10); lane 2 = monomer modified with a cleavable complex; lane 3 = flow-through after equilibration of the modified protein monomer on StrepTactin beads (flow-through shows unmodified protein monomer not bound to StrepTactin beads); lane 4 = modified Hlg2-N130C-Del(E1-G10)-maleimide-[modified] eluted from StrepTactin beads after UV cleavage of the linker. [Figure 16] Gel demonstrating successful oligomerization of (LukF-T142C-maleimide-isoleucine-Del(E1-K15)) by wild-type (WT) Hlg2 monomer. Lane 1 - Modified (LukF-T142C-maleimide-isoleucine-Del(E1-K15)) monomer; Lane 2 - Unmodified wild-type (WT) Hlg2 monomer. Lane 3 - Modified pore oligomer [(LukF-T142C-maleimide-isoleucine-Del(E1-K15))4(Hlg2-WT)4] - Band indicated by arrow. [Figure 17]Characterization data for the polynucleotide analyte of SEQ ID NO: 20 when characterized using a gamma-hemolidine pore modified according to the method of disclosure. A: Representative characterization data for the translocation of SEQ ID NO: 20 when characterized using (left) an unmodified gamma-hemolidine-[(LukF-T142C-Del(E1-K15))4(Hlg2-WT)4] pore generated and oligomerized as discussed herein, compared with the corresponding modified gamma-hemolidine-[(LukF-T142C-Del(E1-K15))4(Hlg2-WT)4] pore generated and oligomerized as discussed herein. B: Changes when the GGAA region of 3.6Kb lambda DNA (residues 38-41 and 52-55 of SEQ ID NO: 20) translocates through the pore. [Figure 18]A gel demonstrating the successful modification of alpha-hemolysin (a-HL) monomers and the successful oligomerization of the modified monomers by the disclosed method. A: Successful modification of a-HL M113C monomer (Lane 1 - Unmodified protein monomer, a-HL-M113C; Lane 2 - Crude reaction mixture - Maleimide-Isoleucine (Mal-Ile) adduct containing a-HL-M113C; Lane 3 - Flow-through after equilibration of the modified protein monomers on StrepTactin beads (flow-through shows unmodified protein monomers not bound to StrepTactin beads); Lane 4 / 5 - Further washing of StrepTactin beads bound to modified monomers to remove unmodified protein monomers (the gel is (Lane 6 - showing that the unmodified monomer does not elute during further washing); a-HL-M113C modified with maleimide-isoleucine (Mal-Ile). B: Successful oligomerization of the modified a-HL monomer. (Lane 1 - modified protein monomer, a-HL-M113C-Mal-Ile; Lane 2 - 1:1 crude reaction mixture of a-HL-M113C-Mal-Ile and sphingomyelin after overnight storage. Lane 3 - pores - supernatant after centrifugation to separate the sphingomyelin pellet. Several pores are also visible in the supernatant; Lane 4 - 7 pores of modified (a-HL-M113C-Mal-Ile). [Figure 19-1] Characterization data for the polynucleotide analyte of SEQ ID NO: 20 when characterized using alpha-hemolidine pores modified according to the method of disclosure. A: Representative characterization data for the translocation of SEQ ID NO: 20 when characterized using (left) unmodified a-HL M113C pores generated and oligomerized as described above, compared with the corresponding modified a-HL[a-HL-M113C-Mal-Ile)7] pores generated and oligomerized as described above. B: Change in translocation (irregular curve) data from (A). C: Change in the irregular curve of the translocation for the first 0.3 seconds following the characteristic sp18 signal of the polynucleotide analyte. [Figure 19-2]Characterization data for the polynucleotide analyte of SEQ ID NO: 20 when characterized using alpha-hemolidine pores modified according to the method of disclosure. A: Representative characterization data for the translocation of SEQ ID NO: 20 when characterized using (left) unmodified a-HL M113C pores generated and oligomerized as described above, compared with the corresponding modified a-HL[a-HL-M113C-Mal-Ile)7] pores generated and oligomerized as described above. B: Change in translocation (irregular curve) data from (A). C: Change in the irregular curve of the translocation for the first 0.3 seconds following the characteristic sp18 signal of the polynucleotide analyte. [Figure 20]A gel demonstrating the successful modification of cytotoxin-K (Cyt-K) monomers and the successful oligomerization of the modified monomers by the disclosed method. A: Successful modification of Cyt-K WT-Q123S / K129C / E140S / Q146S monomer (Lane 1 - Unmodified protein monomer, Cyt-K (WT-Q123S / K129C / E140S / Q146S); Lane 2 - Crude reaction mixture - Maleimide-isoleucine (Mal-Ile) adduct containing Cyt-K (WT-Q123S / K129C / E140S / Q146S); Lane 3 - Flowthrough after equilibration of the modified protein monomers on StrepTactin beads) (Flow-through shows unmodified protein monomers not bound to StrepTactin beads); Lane 4 / 5 - Further washing of StrepTactin beads bound to modified monomers to remove unmodified protein monomers (gel shows that unmodified monomers do not elute during further washing); Lane 6 - Cyt-K (WT-Q123S / K129C / E140S / Q146S) modified with maleimido-isoleucine (Mal-Ile). B: Successful oligomerization of modified Cyt-K monomers. (Lane 1 - Modified protein monomer, CytK-(WT-Q123S / K129C-Mal-Ile / E140S / Q146S; Lane 2 - Crude 1:1 reaction mixture of CytK-(WT-Q123S / K129C-Mal-Ile / E140S / Q146S) and sphingomyelin after overnight storage. Lane 3 - Pores - Supernatant after centrifugation to separate sphingomyelin pellet. Several pores are visible in the supernatant; Lane 4 - Modified (CytK-(WT-Q123S / K129C-Mal-Ile / E140S / Q146S) 7 pores.) [Figure 21-1]Characterization data for the polynucleotide analyte of SEQ ID NO: 20 when characterized using Cyt-K pores modified according to the method of disclosure. A: Representative characterization data for the translocation of SEQ ID NO: 20 when characterized using (left) unmodified Cyt-K WT-Q123S / K129S / E140S / Q146S pores generated and oligomerized as described above, compared with the corresponding modified Cyt-K WT-Q123S / K129C-maleimido-isoleucine / E140S / Q146S pores generated and oligomerized as described above. B: Change in translocation (irregular curve) data from (A). C: Representative change in the irregular curve of the translocation for the first 0.3 seconds following the characteristic sp18 signal of the polynucleotide analyte. [Figure 21-2] Characterization data for the polynucleotide analyte of SEQ ID NO: 20 when characterized using Cyt-K pores modified according to the method of disclosure. A: Representative characterization data for the translocation of SEQ ID NO: 20 when characterized using (left) unmodified Cyt-K WT-Q123S / K129S / E140S / Q146S pores generated and oligomerized as described above, compared with the corresponding modified Cyt-K WT-Q123S / K129C-maleimido-isoleucine / E140S / Q146S pores generated and oligomerized as described above. B: Change in translocation (irregular curve) data from (A). C: Representative change in the irregular curve of the translocation for the first 0.3 seconds following the characteristic sp18 signal of the polynucleotide analyte. [Figure 22] A gel comparing the modification method of the present invention with conventional methods for modifying proteins. Results obtained for lysenin modified with maleimide-isoleucine (Lys-( / E84Q / E85K / E92Q / E94D / E97S / S98C / T106K / D126G / C272A / C283A)) using both methods: one without a cleavable purification tag (conventional method, lane 2) and one according to the disclosed method (lanes 3 and 6). The results are described in Example 9. [Modes for carrying out the invention]

[0041] While the present invention is described with reference to certain drawings in relation to specific embodiments, the invention is not limited thereto and is limited only by the claims. No reference numerals in the claims should be construed as limiting the scope. Of course, it should be understood that not all aspects or advantages can necessarily be achieved according to any particular embodiment of the present invention. Therefore, for example, a person skilled in the art will recognize that the present invention may be embodied or practiced in a manner that achieves or optimizes one or more advantages taught herein, rather than necessarily achieving other aspects or advantages that can be taught or suggested herein.

[0042] The present invention, when viewed in conjunction with the accompanying drawings, will be best understood by referring to the following detailed description, both in terms of its configuration and operation, along with its features and advantages. The aspects and advantages of the present invention will become apparent from and be illustrated by the embodiments described below. Throughout this specification, any reference to “one embodiment” or “an embodiment” means that a particular feature, structure, or characteristic described in conjunction with the embodiment is included in at least one embodiment of the present invention. Therefore, where the phrase “in one embodiment” or “in an embodiment” appears in various places throughout this specification, it may, though not, refer to the same embodiment. Similarly, in the description of exemplary embodiments of the present invention, it should be understood that various features of the invention may be grouped together in a single embodiment, figure, or description for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various aspects of the present invention. However, this method of disclosure should not be construed as reflecting an intention that the claimed invention requires more features than those explicitly described in each claim. Rather, as reflected in the following claims, aspects of the present invention are fewer in scope than all the features of a single, aforementioned embodiment of disclosure.

[0043] The “embodiments” of this disclosure should be understood to be specifically combined unless otherwise indicated by the context. Any particular combination of all the embodiments of the disclosure (unless otherwise implied by the context) constitutes a further disclosed embodiment of the claimed invention.

[0044] In addition, as used herein and in the appended claims, the singular forms "a," "an," and "the" include multiple referents unless the context clearly indicates otherwise. Thus, for example, a reference to "polynucleotide" includes two or more polynucleotides, a reference to "motor protein" includes two or more such proteins, a reference to "helicase" includes two or more helicases, a reference to "monomer" refers to two or more monomers, and a reference to "pore" includes two or more pores.

[0045] All publications, patents, and patent applications cited herein, whether in whole or in part, are incorporated herein by reference.

[0046] definition Where an indefinite or definite article, e.g., “a” or “an” or “the,” is used to refer to a singular noun, this includes the plural form of that noun unless otherwise specifically stated. Where the term “comprising” is used herein and in the claims, it does not exclude other elements or steps. Furthermore, terms such as first, second, third, etc., in the description and claims are used to distinguish similar elements and are not necessarily used to describe a sequential or chronological order. Terms used in this way are interchangeable under appropriate circumstances, and it should be understood that the embodiments of the invention described herein can be operated in an order other than those described or illustrated herein. The following terms or definitions are provided solely to aid in understanding the invention. Unless otherwise specifically defined herein, all terms used herein have the same meaning as they would to a person skilled in the art of the invention. For definitions and terms of the art, the practitioner should refer in particular to Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 th This document follows ed., 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). The definitions provided herein should not be construed as having a smaller scope than that understood by those skilled in the art.

[0047] As used herein, "about" when referring to a measurable value such as quantity or duration means that it includes a variation of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from a given value, such variation is appropriate for carrying out the method of disclosure.

[0048] As used herein, “nucleotide sequence,” “DNA sequence,” or “nucleic acid molecule” refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of a molecule. Therefore, this term includes double-stranded and single-stranded DNA, as well as RNA. As used herein, the term “nucleic acid” is a single-stranded or double-stranded covalent nucleotide sequence in which the 3' and 5' ends of each nucleotide are linked by phosphodiester bonds. Polynucleotides may consist of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acids can be produced in vitro by synthesis or isolated from natural sources. Further examples of nucleic acids include modified DNA or RNA, such as methylated DNA or RNA, or RNA that has undergone post-translational modifications, such as 3'-treatments including 5'-capping, cleavage, and polyadenylation with 7-methylguanosine, and spliced ​​RNA. Other examples of nucleic acids include synthetic nucleic acids (XNAs) such as hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), locked nucleic acid (LNA), and peptide nucleic acid (PNA). Furthermore, the size of nucleic acids, also referred to herein as "polynucleotides," is typically expressed as the number of base pairs (bp) for double-stranded polynucleotides, or as the number of nucleotides (nt) for single-stranded polynucleotides. 1000 bp or nt is equal to kilobase (kb). Polynucleotides less than approximately 40 nucleotides in length are typically called "oligonucleotides" and may include primers used for DNA manipulation via polymerase chain reaction (PCR), etc.

[0049] In the context of this disclosure, the term “amino acid” is used in its broadest sense and means including organic compounds containing an amine (NH2) functional group and a carboxyl (COOH) functional group, along with a side chain specific to each amino acid (e.g., an R group). In some embodiments, an amino acid refers to a natural Lα-amino acid or residue. The one- and three-letter abbreviations commonly used for natural 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, AL, (1975) Biochemistry, 2nd ed., pp. 71-92, Worth Publishers, New York). The general term “amino acid” further includes chemically modified amino acids such as D-amino acids, retro-inversoamino acids, and amino acid analogs, natural amino acids not typically incorporated into proteins such as norleucine, and chemically synthesized compounds having properties known in the art to be characteristic of amino acids, such as β-amino acids. For example, analogs or mimetic compounds of phenylalanine or proline that allow for the same conformational limitation of peptide compounds as natural Phe or Pro are included within the definition of an amino acid. Such analogs and mimetic compounds are referred to herein as “functional equivalents” of their respective amino acids. Other examples of amino acids are enumerated by reference in Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, eds., Vol. 5 p. 341, Academic Press, Inc., NY 1983, which is incorporated herein by reference.

[0050] The terms “polypeptide” and “peptide” are used interchangeably herein to refer to polymers of amino acid residues, as well as their variants and synthetic analogs. Therefore, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic non-natural amino acids, such as chemical analogs of corresponding natural amino acids, as well as to natural amino acid polymers. Polypeptides may also undergo maturation or post-translational modification processes, including, but not limited to, glycosylation, proteolytic cleavage, lipidation, signal peptide cleavage, propeptide cleavage, and phosphorylation. Peptides may be prepared using recombinant techniques, for example, through the expression of recombinant or synthetic polynucleotides. Recombinant peptides typically contain substantially no culture medium, for example, less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.

[0051] The term "protein" is used to describe folded polypeptides having a secondary or tertiary structure. A protein may consist of a single polypeptide or may contain multiple polypeptides that assemble to form a multimer. The multimer may be a homooligomer or a heterooligomer. A protein may be a native protein or a wild-type protein, or a modified protein or a non-native protein. A protein may differ from a wild-type protein, for example, by the addition, substitution, or deletion of one or more amino acids.

[0052] Protein “variants” include peptides, oligopeptides, polypeptides, proteins, and enzymes that have amino acid substitutions, deletions, and / or insertions compared to the unmodified or wild-type protein in question, and that possess similar biological and functional activity to the unmodified protein from which they are derived. As used herein, the term “amino acid identity” refers to the degree to which sequences are identical amino acid-wise across a comparison frame. Thus, the “percentage of sequence identity” is calculated by comparing two optimally aligned sequences across a comparison frame, determining the number of positions in which identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) appear in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison frame (i.e., frame size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0053] In all aspects and embodiments of the present invention, the "variant" has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% complete sequence identity with the amino acid sequence of the corresponding wild-type protein. Sequence identity can also be to a full-length polynucleotide or a fragment or portion of a polypeptide. Thus, a sequence may have only 50% overall sequence identity with the full-length reference sequence, while the sequence of a particular region, domain, or subunit may share as much as 80%, 90%, or 99% sequence identity with the reference sequence.

[0054] The term "wild-type" refers to a gene or gene product isolated from a natural source. Wild-type genes are the most frequently observed genes in a population and are therefore arbitrarily designed as the "normal" or "wild-type" form of the gene. In contrast, the terms "modified," "mutant," or "variant" refer to a gene or gene product that exhibits sequence modifications (e.g., substitutions, cleavage, or insertions), post-translational modifications, and / or functional characteristics (e.g., altered features) compared to a wild-type gene or gene product. It should be noted that these may be identified by isolating natural mutants and, compared to a wild-type gene or gene product, by the fact that they possess altered features. Methods for introducing or substituting natural amino acids are well known in the art. For example, methionine (M) can be substituted with arginine (R) by replacing the methionine codon (ATG) with the arginine codon (CGT) at the relevant position in the polynucleotide encoding the mutant monomer. Methods for introducing or substituting non-natural amino acids are also well known in the art. For example, non-natural amino acids can be introduced by including synthetic aminoacyl-tRNA in the IVTT system used to express mutant monomers. Alternatively, they can be introduced by expressing mutant monomers in E. coli that are nutrient-dependent to a particular amino acid in the presence of a synthetic (i.e., non-natural) analogue of that particular amino acid. They can also be produced by naked ligation when the mutant monomers are produced using partial peptide synthesis. Conservative substitutions replace an amino acid with another amino acid having a similar chemical structure, similar chemical properties, or similar side-chain volume. The introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino acid they replace. Alternatively, a conservative substitution may introduce another amino acid that is aromatic or aliphatic in place of an existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and can be selected according to the properties of 20 major amino acids defined in Table 1 below.If amino acids have similar polarity, this can also be determined by referring to the hydrophobic scale for amino acid side chains in Table 2.

[0055] [Table 1]

[0056] [Table 2]

[0057] As described in more detail herein, mutant or modified proteins, monomers, or peptides can be chemically modified in any manner and at any site. Mutant or modified monomers or peptides are preferably chemically modified by the attachment of molecules to one or more cysteine ​​molecules (cysteine ​​bond), the attachment of molecules to one or more lysines, the attachment of molecules to one or more non-natural amino acids, enzymatic modification of epitopes, or terminal modification. Suitable methods for carrying out such modifications are well known in the art. Modified protein, monomer, or peptide mutants can be chemically modified by the attachment of any molecule. For example, modified protein, monomer, or peptide mutants can be chemically modified by the attachment of dyes or fluorophores.

[0058] Chemically modified monomers This disclosure relates to a method for modifying monomers, such as monomers of protein nanopores. As will be described in more detail below, this method has the advantage of improving the production of modified proteins, such as nanopores. The resulting population of modified nanopores typically has improved properties compared to the population of nanopores produced by prior arts.

[0059] In nanopore sensing, it is particularly important that the population of nanopores used in any sensing application is homogeneous. If the population is heterogeneous, it becomes problematic to know whether any discrepancies in the signal obtained when a sample is analyzed originate from the nanopores used to detect the analyte or from the analyte itself. This can reduce the efficiency of characterizing the analyte due to the increased need for data processing, or, in the worst case, result in the discarding of good data. Therefore, it is necessary that the population of nanopores used in a sensing device has known homogeneity, and that any deviations in the reproducibility of the nanopores themselves are minimized.

[0060] This problem is particularly serious when chemically modified nanopores are used. There are many reasons why chemical modification of nanopores may be necessary: ​​for example, to alter the characteristics of the signal obtained when the analyte interacts with the pore, to change the pore stability, or to change the physical interaction between the analyte and the pore (e.g., by changing the rate at which the analyte interacts with the pore). Chemical modification of protein nanopores can be carried out by known pathways. However, as described above, the problem is that the modification methods used are typically not highly efficient, and a considerable percentage of protein molecules are not modified. Therefore, in many cases, obtaining a homogeneous population of chemically modified nanopores is difficult or impossible.

[0061] A further challenge lies in separating chemically modified proteins, such as those with chemically modified nanopores, from their unmodified analogues. When modifications result in small changes to the overall mass, charge, or three-dimensional structure of a protein, it may not be possible to efficiently separate the modified protein from the unmodified protein.

[0062] These difficulties, combined, prevent the formation of homogeneous populations of proteins, such as protein nanopores. When modifications are performed before purification, limitations in purification techniques often mean that homogeneous populations cannot be formed. When purification is performed before modification, inefficiencies in modification chemistry again often mean that homogeneous populations cannot be formed.

[0063] The method of this disclosure addresses these problems. By combining the modification step and the purification step together, the inefficiencies in these processes are "offset". The method involves modifying a monomer of interest with a portion that combines both the desired chemical modification and the purification tag. Only monomers that are successfully modified have the purification tag, and therefore only these monomers bind to a purified substrate such as a chromatography matrix. Upon binding, the cleavable linker between the chemical modification and the purification tag is cleaved, and therefore the modified monomer is released from the purified substrate. Since only modified monomers have the tag necessary to bind to the purified substrate, and only monomers that are successfully cleaved are released from the purified substrate, the yield of the modified monomer can be very pure, typically yielding a monomer population with a purity of over 95%, and often up to 100%. Therefore, the method of this disclosure has significant advantages compared to known methods in which the modification strategy and the purification strategy are separated.

[0064] Therefore, a method for chemically modifying a polypeptide or polynucleotide monomer, wherein the method is a) Contacting a monomer with a polyfunctional molecule, wherein the polyfunctional molecule includes (i) a reactive group, (ii) a chemically modifying group, and (iii) a cleavable purified tag. b) Reacting the reactive groups of a polyfunctional molecule with a monomer, thereby attaching chemically modified groups and cleavable purified tags to the monomer to form chemically modified tagged monomers, c) Contacting the chemically modified tagged monomer formed in step (b) with the support, d) Attaching a purified tag to a support, thereby attaching a chemically modified tagged monomer to the support, e) A method is provided herein that includes cleaving a purified tag, thereby releasing a chemically modified monomer from a support. Typically, monomers are monomers in oligomeric protein nanopores.

[0065] In some embodiments, the monomer is a polynucleotide monomer. For example, the monomer may be a monomer of DNA origami pores, or origami pores formed from DNA analogs such as PNA. In some embodiments, the monomer is a polypeptide, such as a protein. For example, the monomer may be a monomer of protein nanopores, such as a monomer of monomeric protein nanopores, or more typically, a monomer of oligomeric protein nanopores. Monomers of nanopores particularly suitable for modification according to the claimed method are discussed below.

[0066] This method involves contacting a monomer with a polyfunctional molecule. As used herein, the polyfunctional molecule is a molecule comprising at least three components: (i) a reactive group for reacting with a suitable site on the monomer, such as a reactive functional group on the monomer; (ii) a chemically modifying group corresponding to a chemical modification performed on the monomer; and (iii) a cleavable purification tag. The cleavable purification tag typically comprises (i) a cleavable linker and (ii) the purification tag. The purification tag is suitable for binding to a purification support, such as a chromatography matrix, and thus enables the purification of the modified monomer. The cleavable linker allows the modified monomer to be released from the purification support. This is described in more detail below.

[0067] Therefore, this method involves reacting a reactive group on a polyfunctional molecule with a monomer. Typical reactive groups suitable for use in this method are described in more detail below.

[0068] Once the reactive group reacts with the monomer, and the monomer is thus modified with the polyfunctional molecule, the modified monomer is brought into contact with a support, typically a purified support. The purified tag on the polyfunctional molecule binds to the support, thereby binding the modified monomer to the support. The bound monomer can then be washed or subjected to purification techniques to remove impurities, including the unmodified monomer and / or unreacted polyfunctional molecule.

[0069] This method further includes cleaving the purified tag. As described herein, this releases the chemically modified monomer from the support. Methods for cleaving the cleavable linker are discussed in more detail herein.

[0070] polyfunctional molecules The method provided involves the use of the polyfunctional molecules described above.

[0071] In some embodiments, polyfunctional molecules may be represented by the following formula (I) or formula (II):

[0072] [ka] During the ceremony, A is a reactive group, B is a chemically modified group, DC forms a detachable refined tag.

[0073] In some embodiments, D comprises a cleavable linker as discussed herein. In some embodiments, C comprises a support-binding group as discussed herein.

[0074] The polyfunctional molecule according to formula (I) offers an in-line design. In this scheme, the chemically modifying group is "hidden" by a cleavable linker and a reactive group, and is exposed by cleavage of the cleavable linker. Such a design may be useful, for example, when the chemically modifying group is reactive under the conditions of binding the polyfunctional molecule to a purified support.

[0075] The polyfunctional molecule according to formula (II) offers a “branched” design. The modifying group is exogenous and typically available throughout the steps of the claimed method. Such a design may be useful, for example, when the desired chemical modification is incompatible with the chemistry of the cleavable linker.

[0076] The cleavage of a cleavable linker may, in some cases, leave residues from the linker. In some embodiments, the residues (if any) remaining after the linker has been cleaved correspond to the chemically modifying groups used herein. This is particularly true when the polyfunctional molecule is of formula (I). In other embodiments, any residues from the cleavable linker are separate from the chemically modifying groups. This is particularly true when the polyfunctional molecule is of formula (II).

[0077] Therefore, in some embodiments, the reactive group includes a chemically modified group. For example, in some embodiments, the polyfunctional molecule is of the following formula (III):

[0078] [ka] During the ceremony, A is a reactive group containing a chemically modified group, DC forms a detachable refined tag.

[0079] Reactive group The reactive group (A) of the polyfunctional molecule is suitable for reacting with the monomer subjected to the method disclosed herein. Any suitable reactive group may be used. For example, the reactive group may be an amine reactive group; a carboxyl reactive group; a sulfhydryl reactive group or a carbonyl reactive group. In some embodiments, the reactive group of the polyfunctional molecule includes a cysteine ​​reactive group. In some embodiments, the reactive group includes maleimide, azide, thiol, alkyne, NHS ester, or haloacetamide.

[0080] In some embodiments, the reactive group may be a group capable of reacting with a non-natural amino acid such as 4-azido-L-phenylalanine (Faz), and any one of the amino acids numbered 1 to 71 in Figure 1 of Liu CC and Schultz PG, Annu. Rev. Biochem., 2010, 79, 413-444. Such a group is particularly useful when the corresponding non-natural amino acid is contained in the monomer.

[0081] In some embodiments, the reactive group is a click chemistry group. Click chemistry is a term first introduced in 2001 by Kolb et al. to describe an extended set of strong, selective modular building blocks that reliably function in both small-scale and large-scale applications (Kolb HC, Finn, MG, Sharpless KB, Click chemistry: diverse chemical function from a few good reactions, Angew. Chem. Int. Ed. 40 (2001) 2004-2021). They defined a set of strict criteria for click chemistry as follows: "The reaction must be modular, broad in scope, yield very high yields, produce only harmless by-products that can be removed by non-chromatographic methods, and be stereospecific (but not necessarily enantioselective). Required process features include simple reaction conditions (ideally, the process should be insensitive to oxygen and water), readily available starting materials and reagents, no solvents used, or solvents that are harmless (e.g., water) or easily removed, and simple product isolation. Purification, if necessary, must be by non-chromatographic methods such as crystallization or distillation, and the product must be stable under physiological conditions."

[0082] Suitable examples of click chemistry include, but are not limited to, the following: (a) For example, a copper-free variant of a 1,3-dipolar cycloaddition reaction in which an azide is reacted with an alkyne under strain in a cyclooctane ring, (b) Reaction of an oxygen nucleophile on one linker with an epoxide or aziridine reactive moiety on the other linker, (c) Staudinger ligation, in which the alkyne moiety can be replaced by an arylphosphine, resulting in a specific reaction with the azide to give an amide bond.

[0083] In this method, any reactive group may be used. Therefore, reactive groups may be suitable for click chemistry, particularly when complementary groups are present on the monomer. The reactive groups may be any of those disclosed in International Publication 2010 / 086602, in particular in Table 4 of that application.

[0084] In some embodiments, the reactive group is a haloacetamide, such as iodoacetamide, bromoacetamide, or chloroacetamide.

[0085] In some embodiments, the reactive group is selected from vinyl groups, TCOs, tetrazines and strained alkynes; DBCOs; activated acids, such as acid chlorides, as well as piperazines and reactive amines.

[0086] In some embodiments, the reactive group is a polynucleotide or polynucleotide analog, e.g., PNA. In some embodiments, the reactive group of the polyfunctional molecule includes a nucleotide analog such as a PNA base or PNA polymer, which can interact non-covalently with a monomer, e.g., a monomer containing a complementary PNA base or polymer, via supramolecular association and / or hydrogen bonding.

[0087] In some embodiments, a polynucleotide or polynucleotide analog is bonded to a complementary polynucleotide or polynucleotide analog on a monomer.

[0088] Host-guest chemistry can also be used to provide reactions between reactive groups and monomers. For example, in some embodiments, the monomer includes a ligand for binding to a metal complex, and the reactive group includes a metal complex. Thus, in some embodiments, the reactive group of a polyfunctional molecule includes a metal complex that can interact non-covalently with a monomer containing a site that can act as a ligand to form a complex with a modifying factor molecule by forming a stable association, via chelation or supramolecular association.

[0089] The reactive group may be one of those disclosed in Sakamoto and Hamachi, "Recent progress in chemical modification of proteins," Anal. Sci 2019 (35) 5-27, or McKay and Finn, "Click chemistry in complex mixtures: biorthogonal bioconjugation," Chem. Biol. 2014, 21 (9) 1075-1101, both of which are incorporated herein by reference in their entirety.

[0090] The methods disclosed herein are particularly suitable for the use of polyfunctional molecules and include thiol or maleimide groups for reaction with cysteine ​​residues on monomers; NHS-ester groups for reaction with amine groups on monomers; or azides or alkynes for click chemistry reactions with corresponding groups on monomers.

[0091] Therefore, the reactive group can react with the reactive functional group on the monomer. In other words, in some embodiments, the monomer contains a reactive functional group, and step (b) includes reacting the reactive group of a polyfunctional molecule with the reactive functional group of the monomer.

[0092] Reactive functional groups may be naturally present in the monomer or may be introduced, for example, by genetic engineering or chemical modification of the monomer. Reactive functional groups may also originate from unnatural amino acids incorporated into the monomer during its synthesis or expression, for example, during cell-free expression, or, for example, via in vitro transcription / translation.

[0093] chemical modification group The chemical modification groups of polyfunctional molecules provide the desired chemical modification. Any suitable chemical modification group can be used in the provided method.

[0094] In some embodiments, the chemically modified group has a molecular weight of at most 500 Da, for example, at most 400 Da, at most 300 Da, for example, at most 200 Da, or at most kDa. Typical amino acids have an approximate mass of about 110 Da and are exemplary chemically modified groups for use in the methods disclosed herein, but the methods disclosed herein are not limited to such groups.

[0095] In some embodiments, the chemically modified group is an aliphatic group such as an alkane or alkene. The chemically modified group may be, for example, a linear or branched alkane containing 1 to 20 carbon atoms, for example, 2 to 10 carbon atoms. The chemically modified group may be, for example, a linear or branched alkene containing 1 to 20 carbon atoms, for example, 2 to 10 carbon atoms. The chemically modified group may be an alkyl, alkenyl; alkynyl; or alkoxy group. The chemically modified group may be a cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl group; or a cyclic group such as piperazine, piperidine, morpholine, 1,3-oxazinane, pyrrolidine, imidazolidine, and oxazolidine.

[0096] In some embodiments, the chemically modified group is C6-C 10 Aromatic groups such as aromatic rings (e.g., benzene / phenyl); or 5-10 membered heteroaromatic groups, such as pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyridine, pyridazine, pyrimidine, and pyrazine.

[0097] In some embodiments, the chemically modified group includes amino acids, nucleotides; polymers; hydrogen-bonding groups; membrane anchors; sugars, dyes, chromophores, fluorophores, or molecular adapters. In some embodiments, the chemically modified group includes natural or unnatural amino acids; polypeptides; nucleotides or nucleotide analogs; oligonucleotides or oligonucleotide analogs; polysaccharides; lipids; polyethylene glycol; cyclodextrins; DNA intercalators; aptamers, or analyte-binding domains.

[0098] In some embodiments, the chemically modified group introduces hydrophilic, hydrophobic, positive, negative charge, hydrogen bonding, supramolecular association, or zwitterionic properties to the protein monomer.

[0099] In some embodiments, the chemical modification group is or contains an amino acid. The amino acid may be a natural amino acid or a non-natural amino acid. Multiple amino acids may be included in the chemical modification group; for example, the chemical modification group may contain a peptide.

[0100] In some embodiments, the chemical modification group is a nucleotide or polynucleotide, or includes them. Analogues of natural nucleotides / polynucleotides are also included. For example, the chemical modification group may include RNA, PNA, LNA, or BNA.

[0101] In some embodiments, the chemically modified group may include sugars or polysaccharides such as dextrose, maltose, and glucose.

[0102] In some embodiments, the chemically modifying group may include dyes such as anthraquinoine or phthalocyanine. In some embodiments, the chemically modifying group may include fluorophores such as hydroxycoumarin, aminocoumarin, methoxycoumarin, fluorescein, X-rhodamine, Texas Red, Cy5, and Cy7.

[0103] In some embodiments, the chemically modifying group is an adapter, which is a compound that, when monomers assemble into the pore, affects the physical or chemical properties of the nanopore. Typically, the adapter improves the interaction between the pore and an analyte such as a nucleotide or polynucleotide. The adapter may alter the charge of the pore barrel or channel, or may specifically interact with or bind to a nucleotide or polynucleotide, thereby facilitating that interaction with the pore.

[0104] The adapter may be a cyclic molecule. The adapter may contain one or more chemical groups capable of interacting with a nucleotide or polynucleotide, for example, by hydrophobic interactions, hydrogen bonding, van der Waals forces, π-cation interactions, and / or electrostatic forces.

[0105] The adapter may contain one or more amino groups. The amino groups may be bonded to primary, secondary, or tertiary carbon atoms. The adapter may contain a ring of amino groups, such as a ring of 6, 7, 8, or 9 amino groups. Alternatively, the adapter may contain one or more hydroxyl groups. The hydroxyl groups may be bonded to primary, secondary, or tertiary carbon atoms. The hydroxyl groups may form hydrogen bonds with the uncharged amino acids in the pores.

[0106] Suitable chemical modification groups include, but are not limited to, cyclodextrins, cyclic peptides, and cucurbituryls. The chemical modification group may be any of those disclosed in Eliseev, AV, and Schneider, HJ. (1994) J.Am. Chem. Soc. 116, 6081-6088. The adapter may be heptakis-6-amino-β-cyclodextrin (am7-βCD), 6-monodeoxy-6-monoamino-β-cyclodextrin (am1-βCD), or heptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu7-βCD). The guanidino group of gu7-βCD has a much higher pKa than the primary amine of am7-βCD, and is therefore more positively charged. The adapter may also be a γ-cyclodextrin.

[0107] In some embodiments, the chemically modified group is (i) 4-phenylazomareinanyl, 1.N-(2-hydroxyethyl)maleimide, N-cyclohexylmaleimide, 1.3-maleimidopropionic acid, 1.1-4-aminophenyl-1H-pyrrole,2,5,dione, 1.1-4-hydroxyphenyl-1H-pyrrole,2,5,dione, N-ethylmaleimide, N-methoxycarbonylmaleimide, N-tert-butyl Maleimide, N-(2-aminoethyl)maleimide, 3-maleimide-PROXYL, N-(4-chlorophenyl)maleimide, 1-[4-(dimethylamino)-3,5-dinitrophenyl]-1H-pyrrole-2,5-dione, N-[4-(2-benzimidazolyl)phenyl]maleimide, N-[4-(2-benzoxazolyl)phenyl]maleimide, N-(1-naphthyl)-maleimide, N-(2,4-xylyl)maleimide Imide, N-(2,4-difluorophenyl)maleimide, N-(3-chloro-para-tolyl)-maleimide, 1-(2-amino-ethyl)-pyrrole-2,5-dione hydrochloride, 1-cyclopentyl-3-methyl-2,5-dihydro-1H-pyrrole-2,5-dione, 1-(3-aminopropyl)-2,5-dihydro-1H-pyrrole-2,5-dione hydrochloride, 3-methyl-1-[2-oxo-2-(pipe [Razin-1-yl)ethyl]-2,5-dihydro-1H-pyrrole-2,5-dione hydrochloride, 1-benzyl-2,5-dihydro-1H-pyrrole-2,5-dione, 3-methyl-1-(3,3,3-trifluoropropyl)-2,5-dihydro-1H-pyrrole-2,5-dione, 1-[4-(methylamino)cyclohexyl]-2,5-dihydro-1H-pyrrole-2,5-dione trifluoroacetic acid, SMILES O=C1C=CC(=O)N1CC=2C=CN=CC2, SMILES O=C1C=CC(=O)N1CN2CCNCC2, 1-benzyl-3-methyl-2,5-dihydro-1H-pyrrole-2,5-dione, 1-(2-fluorophenyl)-3-methyl-2,5-dihydro-1H-pyrrole-2,Maleimides including diabromomaleimides such as 5-dione, N-(4-phenoxyphenyl)maleimide, and N-(4-nitrophenyl)maleimide; (ii) 3-(2-iodoacetamide)-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-io Iodocetamides such as (iii) N-(4-(acetylamino)phenyl)-2-bromoacetamide, N-(2-acetylphenyl)-2-bromoacetamide, 2-bromo-n-(2-cyanophenyl)acetamide, and 2-bromo-N-(3-(trifluoromethyl)phenyl)acetamide. Cetoamide, N-(2-benzoylphenyl)-2-bromoacetamide, 2-bromo-N-(4-fluorophenyl)-3-methylbutanamide, N-benzyl-2-bromo-N-phenylpropionamide, N-(2-bromo-butyryl)-4-chlorobenzenesulfonamide, 2-bromo-N-methyl-N-phenylacetamide, 2-bromo-N-phenethylacetamide, 2-adamantan-1-yl-2-bromo-N-cyclohexylacetamide, 2-bromo-N-(2-methylphenyl)butanamide, monobromoacetanili Bromoacetamide such as (iv) aldrithiol-2, aldrithiol-4, isopropyl disulfide, 1-(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, am6amPDP1-βCD and other disulfides, as well as (v) 4-phenylthiazole-2-thiol, Purpald, 5,6,7,Selected from thiols such as 8-tetrahydroquinazoline-2-thiol.

[0108] Cuttable refined tags The polyfunctional molecule includes a cleavable purification tag for binding chemically modified monomers to a purified support during the claimed method. Typically, the cleavable purification tag includes a cleavable linker and the purification tag itself.

[0109] In the disclosed method, any suitable cleavable linker may be used. The linker may include, for example, a short-chain oligopeptide or oligonucleotide containing about 1 to about 20 amino acids or nucleotides. The linker may include a polymer such as polyethylene glycol or sugar containing about 1 to about 20 repeating units. For example, the linker may include PEG2, PEG3, or PEG4.

[0110] In some embodiments, the severable linker is cut by physical or chemical means. Any suitable means may be used.

[0111] The cleavable portion of a cleavable linker may be, for example, a pH-sensitive group; a redox-sensitive group; a photosensitive group; or a temperature-sensitive or chemical-sensitive group that is susceptible to cleavage by the reaction of a group containing a specific chemical.

[0112] In some embodiments, the cleavable linker is cleavable by exposure to light, i.e., photocleavable. Therefore, in some embodiments of the method disclosed, cleaving the purified tag in step (e) includes exposing the support and / or tagged monomer to light, preferably UV light. The photocleavable linker comprises a nitrobenzyl moiety. Such a group is cleavable under UV irradiation.

[0113] In some embodiments, the cleavable linker is cleavable by exposure to a change in pH. Therefore, in some embodiments of the method disclosed, cleaving the purified tag in step (e) includes exposing the support and / or tagged monomer to a change in pH. Examples of pH-sensitive cleavable linkers include hydrazones and cis-aconityl. An example of a cleavable hydrazone linker is shown below.

[0114] [ka] In the formulas, the wavy lines represent bonding points to the rest of the polyfunctional molecule. Those skilled in the art will understand that the PEG linkers shown above may be replaced with other linkers, including those discussed herein, and similarly, the bond chemistry shown may be replaced with other suitable bond chemistry (for example, the amide bond shown may be replaced with an ester bond).

[0115] In some embodiments, the cleavable linker is cleavable by exposure to a chemical reagent. Therefore, in some embodiments of the method disclosed, cleaving the purified tag in step (e) involves exposing the support and / or tagged monomer to a chemical reagent, preferably a reducing reagent. Chemically sensitive cleavable linkers include disulfides. Disulfide bonds are readily cleaved by the addition of reducing agents such as DTT and beta-mercaptoethanol. Examples of cleavable disulfide linkers are shown below:

[0116] [ka] In the formula, the wavy line represents a bond point to the rest of the polyfunctional molecule. Those skilled in the art will understand that the PEG linker shown above may be replaced with other linkers, including those discussed herein, and similarly, the bond chemistry shown may be replaced with other suitable bond chemistry (for example, the ester bond shown may be replaced with an amide bond).

[0117] In some embodiments, the cleavable linker can be cleaved by exposure to an enzyme such as a protease or nuclease. Thus, in some embodiments of the method disclosed, cleaving the purified tag in step (e) includes exposing the support and / or tagged monomer to an enzyme, preferably a protease.

[0118] Enzyme-sensitive linkers include protease-sensitive peptide linkers containing recognition sequences for one or more endo and / or exoproteases. Examples include the sequences DDDDK (SEQ ID NO: 24; cleaved by enteropeptidases from E. coli and S. cerevisiae); LVPRGS (SEQ ID NO: 25; cleaved by thrombin and factor Xa); ENLYFQG (SEQ ID NO: 26; cleaved by TEV protease); and LEVLFQGP (SEQ ID NO: 27; cleaved by rhinovirus 3C protease). β-glucuronide linkers can be cleaved by lysosomal β-glucuronidases.

[0119] Examples of linkers that can be enzymatically cleaved are shown below.

[0120] [ka]

[0121] In some embodiments, the cleavable linker is in the form of Cl-Lk, Lk-Cl, or Lk-Cl-Lk, where Cl is the cleavable portion and Lk is the linker.

[0122] In some embodiments, the cleavable linker comprises a polymer and a photocleavable portion. In some embodiments, the cleavable linker comprises a PEG linker and a nitrobenzyl portion. In some embodiments, the cleavable linker comprises the following forms of structure:

[0123] [ka] The wavy lines represent the binding points to the purified tag and chemically modified group (either directly or via a linker).

[0124] In a cleavable purification tag contained within a polyfunctional molecule, any suitable purification tag may be used. For example, the purification tag may contain or consist of biotin. Biotin is particularly suitable for use in the disclosed method because it forms strong non-covalent bonds with streptavidin and related proteins (such as neutraavidin and avidin).

[0125] Other purification tags include peptide purification tags suitable for IMAC (Immigrated Metal Affinity Chromatography) chemistry. For example, purification tags may include poly-His tags (e.g., HHHH, HHHHHH, or HHHHHHHH; SEQ ID NOs. 28-30). Such tags are suitable for binding to purification supports containing metals such as nickel or cobalt. Other examples of purification tags include peptide tags such as Strep (WSHPQFEK; SEQ ID NOs. 31), FLAG (DYKDDDDK; SEQ ID NOs. 32), human influenza hemagglutinin (HA) (YPYDVPDYA; SEQ ID NOs. 33), Myc (EQKLISEED; SEQ ID NOs. 34), and V5 (GKPIPNPLLGLDST; SEQ ID NOs. 35).

[0126] Other suitable purification tags include biotin-carboxy carrier protein (BCCP); calmodulin binding peptide (CBP); chitin binding domain (CBD); histidine affinity tag (HAT); polyarginine (Arg-tag); polyaspartate (Asp-tag); polylysine (Lys-tag); polyphenylalanine (Phe-tag); streptavidin-binding peptide (SBP); tetrazine tag; TCO tag; azide tag; and DBCO / alkyne tag.

[0127] In some embodiments, the cleavable purified tag comprises a polymer and a cleavable linker including a photocleavable moiety, as well as biotin. In some embodiments, the cleavable purified tag comprises a PEG linker, a nitrobenzyl moiety, and biotin. In some embodiments, the cleavable purified tag comprises the following forms of structure:

[0128] [ka] In the formula, the wavy line indicates a bond point to a chemically modified group (either directly or via a linker).

[0129] support The disclosed method includes binding a monomer functionalized with a polyfunctional molecule to a support for purification.

[0130] Any suitable support can be used.

[0131] In some embodiments, the support comprises a chromatographic matrix, preferably an agarose or Sepharose resin. Such resins are commercially available from suppliers such as Sigma Aldrich.

[0132] In some embodiments, the support comprises beads (i.e., one or more beads). Magnetic beads are preferred because they allow for easy purification, for example, by washing with a buffer solution. Functionalized magnetic beads with a variety of functionalizations are commercially available from suppliers such as Sigma Aldrich and Bio-Rad.

[0133] In some embodiments, the support includes a solid surface. Any suitable material can be used. Suitable materials include glass, silica, polymers such as polyester, and ceramics such as hydroxyapatite.

[0134] In some embodiments, the support is functionalized for binding to a purified tag. Those skilled in the art will understand that the support may be functionalized in accordance with the purified tag contained in the polyfunctional molecule used. Alternatively, the purified tag may be selected in accordance with the support material used. Thus, the selection of the purified tag and the support material are operational parameters that can be determined by the user of the method disclosed.

[0135] In some embodiments, the support comprises streptavidin, neutraavidin, or avidin, or derivatives of streptavidin, neutraavidin, or avidin, such as traptabidine. Such a support is particularly useful when the polyfunctional molecule contains a purified tag containing biotin.

[0136] In some embodiments, the support comprises a metal such as nickel or cobalt. The metal ion may be provided with a suitable chelating agent such as nitriloacetic acid (NTA) or iminodiacetic acid (IDA). For example, the support may comprise Ni-NTA. Such a support is particularly useful when the polyfunctional molecule contains a purification tag including a His tag.

[0137] In some embodiments, the support comprises streptactin. Such a support is particularly useful when the polyfunctional molecule contains a purified tag that includes a Strep tag.

[0138] In some embodiments, the support comprises an antibody against the sequence FLAG, HA, Myc, or V5 as described above.

[0139] In some specific embodiments of the disclosed method, the purified tag comprises a biotin group, and the support comprises streptavidin, neutraavidin, or avidin, preferably streptavidin.

[0140] In some embodiments, the severable purification tag includes the following structure:

[0141] [ka] In the formula, the wavy line represents a bond point to a chemically modified group (either directly or via a linker), and the support comprises streptavidin. For example, the support may comprise an agarose or Sepharose resin containing streptavidin, or magnetic beads coated with streptavidin.

[0142] monomer The methods provided involve monomer modification. The discussion herein primarily focuses on the modification of protein monomers in protein nanopores, particularly in multicomponent protein nanopores. However, the methods disclosed are not limited to such monomers and are useful for the chemical modification of monomer proteins, polynucleotides, components of polynucleotide origami structures such as origami pores (e.g., DNA origami), enzymes (including motor proteins discussed herein), and more.

[0143] In some embodiments, the monomer is a polypeptide having a mass of about 10 kDa to about 1 MDa.

[0144] In embodiments of the present invention relating to nanopore monomers, any suitable nanopore may be used. In one embodiment, the nanopore is a transmembrane pore.

[0145] Transmembrane pores are structures that, to some extent, cross the membrane. They allow hydrated ions, driven by an applied potential, to flow across or within the membrane. Transmembrane pores typically cross the entire membrane so that hydrated ions can flow from one side of the membrane to the other. However, transmembrane pores do not necessarily have to cross the membrane. They may be closed at one end. For example, pores can be wells, gaps, channels, trenches, or slits within the membrane, through which hydrated ions can flow or into which they can flow.

[0146] The monomers may be monomers for biological or artificial nanopores. Suitable pores include, but are not limited to, protein pores and polynucleotide pores.

[0147] In one embodiment, the monomer is a monomer of a polynucleotide pore. For example, the polynucleotide pore may be a DNA origami pore (Langecker et al., Science, 2012;338:932-936). A suitable DNA origami pore is disclosed in International Publication No. 2013 / 083983. The monomers of the polynucleotide origami pore are typically polynucleotides of 50 nt to 1000 kb, such as 100 nt to 100 kb, e.g., 1000 nt (1 kb) to 10 kb. The monomers assemble into a structure that enables ion transport from one chamber to another. One or more monomers may assemble into such a structure. Typically, multiple monomers assemble into that structure, and the origami pore is typically an oligomer. The monomers that aggregate into the origami pore may be of the same type (i.e., the pore may be a homo-oligomer) or they may be of two or more different types (i.e., the pore may be a multi-component hetero-oligomer pore).

[0148] More often, monomers are monomers of transmembrane protein pores. Transmembrane protein pores are polypeptides or aggregates of polypeptides, such as polynucleotides, that allow hydrated ions to flow from one side of a membrane to the other. In the methods provided herein, transmembrane protein pores are typically capable of forming pores that allow hydrated ions, driven by an applied potential, to flow from one side of a membrane to the other. Transmembrane protein pores preferably allow polynucleotides to flow from one side of a membrane to the other, such as a triblock copolymer membrane. Transmembrane protein pores allow polynucleotides to move through the pores.

[0149] In one embodiment, the monomer is a monomer of monomer nanopores, i.e., the monomer forms transmembrane protein pores. In one embodiment, the monomer is a monomer of oligomeric pores. The monomer may be a monomer of pores composed of several repeating subunits, such as at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 subunits. In some embodiments, the monomer is a monomer of pores containing at least 20 subunits, at least 30 subunits, at least 40 subunits, or at least 50 subunits.

[0150] For example, members of the MACPF superfamily form large transmembrane pores and pore complexes. For instance, pleurotolysin (PlyAB) from Pleurotus ostreatus consists of two distinct monomer components: pleurotolysin A (PlyA) and pleurotolysin B (PlyB). PlyA binds to the membrane and acts as a scaffold for recruiting the second component, PlyB, to form a transmembrane channel across the membrane. To introduce different chemical groups into the channel, the PlyB monomer can be modified in the manner described. Thus, in some embodiments, the monomer is either a monomer of PlyA or a monomer of PlyB. The use of PlyAB in protein detection is discussed in its entirety in Huang et al, "Electro-osmotic vortices promote the capture of folded proteins by PlyAB nanopores," Nano Letters 2020, 20(5), 3819-3827, the entirety of which is incorporated herein by reference.

[0151] The monomer may be a monomer with hexamer, heptamer, octamer, or nonamer pores. The pores may be homooligomers or heterooligomers.

[0152] Transmembrane protein pores typically contain barrels or channels through which ions can flow. The pore subunits typically surround a central axis and give rise to transmembrane β-barrels or channels, or transmembrane α-helix bundles or chains leading to channels.

[0153] Typically, the barrel or channel of a transmembrane protein pore contains amino acids that facilitate interaction with an analyte, such as a target polynucleotide (as described herein). These amino acids are preferably located near the constriction of the barrel or channel. The transmembrane protein pore typically contains 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 interaction between the pore and a nucleotide, polynucleotide, or nucleic acid.

[0154] In one embodiment, the monomer is a monomer of a transmembrane protein pore derived from a β-barrel pore or an α-helix bundle pore. A β-barrel pore includes a barrel or channel formed from a β-chain. Suitable β-barrel pores include, but are not limited to, β-toxins such as α-hemolysin, anthrax toxin, and leucosidine, outer membrane phospholipase A, and other pores such as lysenin, NetB, and CytK. An α-helix bundle pore includes a barrel or channel formed from an α-helix. Suitable α-helix bundle pores include, but are not limited to, inner membrane proteins and α-outer membrane proteins.

[0155] In one embodiment, the monomer is a transmembrane pore monomer derived from or based on α-hemolidine (α-HL), lysenin, or the hemolytic protein fragaceatoxin C (FraC).

[0156] In one embodiment, the monomer is an actinporin monomer. Examples of actinporins include equinatoxin II (EqtII) from Actinia equina and fragaceatoxin C (FraC) from Actinia fragacea.

[0157] In one embodiment, the monomer is a monomer of any one homolog or paralog of any of the protein pores discussed herein.

[0158] In one embodiment, the monomer is a transmembrane pore monomer derived from lysenin, or its paralog or homolog. Examples of suitable pores derived from lysenin are disclosed in International Publication No. 2013 / 153359.

[0159] In one embodiment, the monomer is a transmembrane pore monomer derived from or based on α-hemolidine (α-HL), or its paralog or homolog. The wild-type α-hemolidine pore is formed from seven identical monomers or subunits (i.e., it is a heptamer). The α-hemolidine pore may be α-hemolidine-NN or a variant thereof. The variant preferably contains N residues at positions E111 and K147.

[0160] In one embodiment, the monomer is a transmembrane pore monomer derived from or based on NetB, or its paralog or homolog. NetB is a pore-forming toxin produced by Clostridium perfringens.

[0161] In one embodiment, the monomer is a transmembrane pore monomer derived from or based on CytK, or its paralog or homolog. CytK is a pore-forming toxin produced by Bacillus cerius.

[0162] In one embodiment, the monomer is a gamma-hemolyzin pore, or a monomer of its paralog or homolog.

[0163] In some embodiments, the monomer is a monomer of a multicomponent pore. As used herein, a multicomponent pore is a pore containing two or more different monomers. For example, gamma-hemolyzin is a multicomponent pore containing two different types of monomers, in other words, a binary pore. The disclosed method is particularly suitable for modifying multicomponent pores because it can be used to introduce different modifications to different components of the pore. Thus, a first modification can be performed on a first component, a second modification on a second component, and so on. By performing different modifications on different components of a multicomponent pore, the functional groups of the chemical modifications can be precisely located at multiple positions (e.g., two or more positions) within the pore, for example, within the channels of the pore. For example, modifications can be performed to introduce or modify two recognition sites within the channels of a multifunctional pore, which can improve the characterization of analytes such as polynucleotides. An example of this strategy is shown in Figure 1, which illustrates how the disclosed method can be used to introduce multiple modifications to a binary pore such as gamma-hemolyzin.

[0164] As described above, the monomer may be any of the nanopore monomers described above. The method provided herein also relates to a method for generating chemically modified nanopores. The nanopores used in such a method may be any of the nanopores discussed in the context of the monomers above.

[0165] Chemical modifications by the disclosed method can be performed at specific locations within a monomer. These specific locations can be determined based on the structure of the free monomer, or, if the monomer forms an oligomer (if applicable), the structure adopted by the monomer. The structure used to determine the modification location of the oligomerizing monomer is typically the three-dimensional structure of the oligomer formed from the monomer. For example, the structure could be the 3D structure of an oligomeric protein pore. The 3D X-ray crystal structures of many oligomeric pores are known and / or can be determined by computer modeling.

[0166] In some embodiments, modifications may be made to position chemically modifying groups at specific locations within the assembled pores. In some embodiments, modifications are made such that they are located in a position accessible to a solvent on the surface of the pore. In some embodiments, modifications are located on the outer surface of the pore. In some embodiments, modifications are positioned to interact with the membrane once the pores are assembled within the membrane, for example, to improve the fixation of the pores to the membrane. In some embodiments, modifications are located at or near the opening of the pore, for example, at or near the cis or trans opening of a channel extending through the pore. In some embodiments, modifications are located within the channels of the pore. In some embodiments, modifications are located on inward-facing residues within the channels or barrels of the pore. In some embodiments, modifications are located at or near the constrictions within the channels of the pore. In some embodiments, modifications are positioned to increase the constrictions within the channels of the pore. In some embodiments, modifications are positioned to introduce constrictions into the channels of the pore. In some embodiments, modifications alter the properties of the channels passing through the pore, for example, by introducing chemically functional groups into the channels.

[0167] In some embodiments, the monomer may be modified to ensure that there are a sufficient number of modification sites for reaction with the reactive groups of a polyfunctional molecule. In some embodiments, the monomer may be modified to introduce one or more modification sites. In some embodiments, the monomer may be modified to remove or delete one or more modification sites. In some embodiments, the monomer may be modified to delete one or more sites that would react with the reactive groups of a polyfunctional molecule, and to introduce one or more modification sites for reaction with the reactive groups of a polyfunctional molecule; that is, the monomer may be modified to replace one or more modification sites with one or more different modification sites.

[0168] One or more modification sites can be introduced into a monomer by any preferred means. For example, in some embodiments, the monomer is a polypeptide monomer of an oligomeric protein pore, and one or more modification sites are introduced by mutations in the native amino acid sequence of the polypeptide monomer. In some embodiments, one or more modification sites are introduced by insertion of one or more residues into the native sequence. In some embodiments, one or more modification sites are introduced by substitution of one or more residues into the native sequence.

[0169] For example, one or more cysteine ​​residues can be introduced into the native sequence of a monomer. Cysteine ​​residues can be used, for example, when a polyfunctional molecule contains a sulfhydryl reactive group as a reactive group, or when a polyfunctional molecule contains a thiol or maleimide group as a reactive group. One or more cysteine ​​residues can be introduced, for example, by substitution of one or more non-cysteine ​​residues in the monomer. One or more cysteine ​​residues can be introduced, for example, by insertion of one or more amino acids into the native amino acid sequence of the monomer.

[0170] For example, one or more amino acid-containing residues, such as lysine, can be introduced into the native sequence of a monomer. Amino acid-containing residues can be used, for example, when a polyfunctional molecule contains an amino-reactive group as a reactive group, for example, when a polyfunctional molecule contains an NHS-ester group as a reactive group. One or more amino acid-containing residues, such as lysine, can be introduced, for example, by substitution of one or more residues in the monomer. One or more amino acid-containing residues, such as lysine, can be introduced, for example, by inserting one or more amino acids into the native amino acid sequence of the monomer.

[0171] Amino acid insertions, deletions, and substitutions are, for example, described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 4. thThis can be done to the natural amino acid sequences of monomers using techniques known in the field, such as those described in ed., 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).

[0172] One or more reactive non-natural amino acids can be introduced into the natural sequence of a monomer. Reactive non-natural amino acids can be used, for example, when a polyfunctional molecule contains a reactive group for reacting with such non-natural amino acids, such as a click reagent. One or more non-natural amino acids can be introduced, as described herein, for example, by including synthetic aminoacyl-tRNA in an IVTT system used to express the mutant monomer, or by expressing the mutant monomer in a suitable bacterial expression system (e.g., E. coli) containing bacteria that are nutrient-dependent on a particular amino acid in the presence of a synthetic (i.e., non-natural) analog of such a particular amino acid. They can also be produced by naked ligation when the mutant monomer is produced using partial peptide synthesis.

[0173] In some embodiments, the monomer is modified to include one or more modification sites. In some embodiments, the monomer is modified to include one, two, three, four, or five modification sites. In some embodiments, the monomer is modified to include exactly one or two modification sites, such as exactly one modification site.

[0174] Therefore, in some embodiments of the disclosed method, the monomer is a monomer of an oligomeric pore, and when the monomer is oligomerized to form a pore, the polyfunctional molecule reacts with reactive functional groups located on the monomer at a surface-exposed position. In some embodiments, the surface-exposed position is located on the surface of the channel through the pore. In some embodiments, the surface-exposed position is located on the outer surface of the pore.

[0175] In some embodiments, when a monomer is oligomerized to form a pore, the polyfunctional molecule reacts with the reactive functional group located on the monomer at a position within or near the constriction of the channel through the pore (e.g., within 10 nm, within 5 nm such as within 3 nm, within 2 nm, within 1 nm).

[0176] In some embodiments, the monomer is an erolysine-like pore monomer, such as erolysine, lysenin, epsilon toxin (ε-toxin) type B, parasporin-2, LSL, monalisin, enterotoxin, or hemolytic lectin, or their paralogs or homologs. In some embodiments, the monomer is a leucosidine-like pore monomer, such as gamma-hemolidine (HlGAB or HlgCB), LukAB / HG, LukED, Panton-Valentine leucosidine (LukSF-PV / PVL), or LukMF', or their paralogs or homologs. In some embodiments, the monomer is a hemolidine-like pore monomer, such as VCC, CytK, NetB, or alpha-hemolidine, or their paralogs or homologs. In some embodiments, the monomer is an AB toxin-like pore monomer, such as anthrax toxin protective antigen, or their paralogs or homologs.

[0177] In some embodiments, the monomer is the LukF subunit of gamma-hemolidine (SEQ ID NO: 10), or a monomer of its variant, paralog, or homolog. The structure of gamma-hemolidine is deposited in the Protein Data Bank (PDB) under accession code 3B07. For example, in some embodiments, the monomer is a variant having at least 90%, for example, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or at least 99.9%, of SEQ ID NO: 10, for example, at least 70%, for example, at least 80%, for example, at least 85%, typically at least 95%.

[0178] In some embodiments, the monomer is modified at one or more of the positions corresponding to K146, N144, T142, S140, A138, N136, N134, G132, S130, G128, S126, S124, D122, G120, T118, G116, T114, Q112, Q110, or E108 of SEQ ID NO: 10. These positions (when assembled) correspond to positions within the barrel of the gamma-hemolidine pore and, in some embodiments, may be modified to alter, for example, improve, the interaction between the pore and an analyte such as a polynucleotide.

[0179] In some embodiments, the monomer is modified at one or more of the positions corresponding to K43, D44, K45, S46, Y47, D48, or K49 in SEQ ID NO: 10. These positions (when assembled) correspond to the cap domain positions of the gamma-hemolidine pore and, in some embodiments, can be modified to alter, for example, improve, the interaction between the pore and an analyte such as a polynucleotide.

[0180] In some embodiments, the monomer is modified at one or more of the positions corresponding to Y145, I143, E141, F139, T137, G135, L133, G131, L129, N127, I125, I123, G121, F119, Y117, L115, N113, V111, or F109 of SEQ ID NO: 10. These positions (when combined) correspond to positions outside the barrel of the gamma-hemolidine pore, and in some embodiments, they may be modified to alter, for example, improve pore fixation by altering or improving the interaction between the membrane and the pore.

[0181] In some embodiments, the monomer is modified at one or more of the positions corresponding to A196, G197, R198, Q199, S200, S201, A202, Y203, W257, N258, G259, F260, Y261, or W262 of SEQ ID NO: 10. These positions (when combined) correspond to the positions of the edge domains of the gamma-hemolidine pore, and in some embodiments, they may be modified to alter, for example, improve pore fixation by altering or improving the membrane-pore interaction.

[0182] Those skilled in the art will understand that the corresponding positions of other monomers (e.g., other monomers disclosed herein) can be modified according to the methods disclosed. The corresponding positions can be determined, for example, by sequence alignment and / or structural modeling.

[0183] In some embodiments, the monomer is the Hlg2 subunit of gamma-hemolyzin (SEQ ID NO: 11), or a monomer of its variant, paralog, or homolog. For example, in some embodiments, the monomer is a variant having at least 90%, for example, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or at least 99.9%, of SEQ ID NO: 11, such as at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, typically at least 95%. In some embodiments, the monomer is modified at one or more of the positions corresponding to N138, S136, T134, S132, N130, S128, S126, G124, S122, P121, Q118, S119, N116, G114, N112, G110, K108, S106, D104, or S102 of SEQ ID NO: 11. These positions (when assembled) correspond to positions within the barrel of the gamma-hemolidine pore and, in some embodiments, can be modified to alter, for example, improve, the interaction between the pore and an analyte such as a polynucleotide.

[0184] In some embodiments, the monomer is modified at one or more of the positions corresponding to K37, D38, K37, K39, K40, Y41, N42, K43, or D44 of SEQ ID NO: 11. These positions (when assembled) correspond to the cap domain positions of the gamma-hemolidine pore and, in some embodiments, can be modified to alter, for example, improve, the interaction between the pore and an analyte such as a polynucleotide.

[0185] In some embodiments, the monomer is modified at one or more of the positions corresponding to Q139, Y137, I135, K133, Y131, F129, G127, G125, I123, P121, S119, F117, G115, I113, Y111, L109, Q107, V105, or A103 of SEQ ID NO: 11. These positions (when combined) correspond to positions outside the barrel of the gamma-hemolidine pore, and in some embodiments, they may be modified to alter, for example, improve pore fixation by altering or improving the interaction between the membrane and the pore.

[0186] In some embodiments, the monomer is modified at one or more of the positions corresponding to Q180, D181, P182, T183, G184, P185, T241, R242, H243, or R244 in SEQ ID NO: 11. These positions (when combined) correspond to the positions of the edge domains of the gamma-hemolidine pore, and in some embodiments, they may be modified to alter, for example, improve pore fixation by altering or improving the membrane-pore interaction.

[0187] Those skilled in the art will understand that the corresponding positions of other monomers (e.g., other monomers disclosed herein) can be modified according to the methods disclosed. The corresponding positions can be determined, for example, by sequence alignment and / or structural modeling.

[0188] In some embodiments, the monomer is lysenin (SEQ ID NO: 12), or a monomer of its variant, paralog, or homolog. The structure of lysenin is deposited in the Protein Databank (PDB) under depositary code 5EC5. For example, in some embodiments, the monomer is a variant having at least 90%, for example, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or at least 99.9%, sequence identity or sequence homology with SEQ ID NO: 12, such as at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, typically at least 95%. In some embodiments, the monomer is modified at one or more positions corresponding to D35, K37, T39, T41, G43, K45, V47, S49, T51, T53, T55, T57, S59, G61, T63, S65, G67, A69, I72, S74, E76, S78, S80, S82, Q84, S86, V88, M90, Q92, D94, Y96, S98, V100, E102, T104, or K106 of SEQ ID NO: 12.

[0189] In some embodiments, the monomer is a monomer of cytotoxin K (CytK) (SEQ ID NO: 13) from Bacillus cereus, or a variant, paralog, or homolog thereof. For example, in some embodiments, the monomer is a variant having at least 90%, for example, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or at least 99.9%, sequence identity or sequence homology with SEQ ID NO: 13, such as at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, typically at least 95%. In some embodiments, the monomer is modified at one or more of the positions corresponding to E113, T115, T117, S119, S121, Q123, G125, S127, K129, S131, T133, G136, S138, E140, G142, T144, Q146, T148, S150, S152, S154, or K156 of SEQ ID NO: 13.

[0190] In some embodiments, the monomer is erollysine (SEQ ID NO: 14), or a monomer of its variant, paralog, or homolog. The structure of erollysine is deposited in the Protein Databank (PDB) under the deposit code 5JZT. For example, in some embodiments, the monomer is a variant having at least 90%, for example, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or at least 99.9%, sequence identity or sequence homology with SEQ ID NO: 14, such as at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, typically at least 95%. In some embodiments, the monomer is modified at one or more positions corresponding to G214, D216, T218, R220, D222, A224, N226, S228, T230, T232, G234, S236, K238, T240, T242, K244, K246, P248, V250, E252, E254, S256, E258, A260, N262, S264, A266, Q268, G270, S272, S274, S276, S278, S280, R282, or T284 of SEQ ID NO: 14.

[0191] In some embodiments, the monomer is a monomer of NetB (SEQ ID NO: 15), or a variant, paralog, or homolog thereof. The structure of NetB is deposited in the Protein Databank (PDB) under depositary code 4H56. For example, in some embodiments, the monomer is a variant having at least 90%, for example, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or at least 99.9%, sequence identity or sequence homology with SEQ ID NO: 15, such as at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, typically at least 95%. In some embodiments, the monomer is modified at one or more of the positions corresponding to I112, K114, D116, S118, S120, G122, S124, G126, N128, S130, E132, T135, G137, G139, N141, S143, N145, Q147, T149, E151, S153, or P155 of SEQ ID NO: 15.

[0192] In some embodiments, the monomer is alpha-hemolidine (SEQ ID NO: 16), or a monomer of its variant, paralog, or homolog. The structure of alpha-hemolidine is deposited in the Protein Databank (PDB) under depositary code 7AHL. For example, in some embodiments, the monomer is a variant having at least 90%, for example, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or at least 99.9%, sequence identity or sequence homology with SEQ ID NO: 16, such as at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, typically at least 95%. In some embodiments, the monomer is modified at one or more of the positions corresponding to E111, M113, T115, T117, G119, N121, N123, T125, D127, D128, T129, G130, K131, G133, L135, G137, N139, S141, G143, T145, or K147 of SEQ ID NO: 16.

[0193] In some embodiments, the monomer is VCC (SEQ ID NO: 17), or a monomer of its variant, paralog, or homolog. The structure of VCC is deposited in the Protein Databank (PDB) under depositary code 3O44. For example, in some embodiments, the monomer is a variant having at least 90%, for example, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or at least 99.9%, sequence identity or sequence homology with SEQ ID NO: 17, such as at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, typically at least 95%. In some embodiments, the monomer is modified at one or more of the positions corresponding to E281, K283, V285, G287, E289, G291, T293, G295, E297, S299, K304, K306, E308, R310, S312, T314, S316, W318, T320, or N322 of Sequence ID No. 17.

[0194] In some embodiments, the monomer is a monomer of Bacillus anthrax PA (SEQ ID NO: 18), or a variant, paralog, or homolog thereof. The structure of the Bacillus anthrax protective antigen is deposited in the Protein Databank (PDB) under accession code 3J9C. For example, in some embodiments, the monomer is a variant having at least 90%, for example, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or at least 99.9%, sequence identity or sequence homology with SEQ ID NO: 18, such as at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, typically at least 95%. In some embodiments, the monomer is modified at one or more of the positions corresponding to D276, S278, Q280, T282, S284, T286, T288, S290, N292, S294, S296, T298, T300, E302, H304, N306, E308, H310, S312, D315, G317, S319, S321, G323, S325, S327, S329, T331, A333, D335, S337, S339, A341, E343, T345, A347, T349, or G351 of SEQ ID NO: 18.

[0195] In some embodiments, the monomer is a monomer of ε-toxin (SEQ ID NO: 19), or a variant, paralog, or homolog thereof. The structure of ε-toxin type B is deposited in the Protein Databank (PDB) under deposit code 6RB9. For example, in some embodiments, the monomer is a variant having at least 90%, for example, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or at least 99.9%, sequence identity or sequence homology with SEQ ID NO: 19, such as at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, typically at least 95%. In some embodiments, the monomer is modified at one or more positions corresponding to S90, T92, K94, T96, T98, T100, T102, T104, T106, G108, S110, Q112, 114, K116, T118, E123, G125, S127, S131, S133, A135, T137, T139, T141, S143, E145, T147, or P151 of SEQ ID NO: 19.

[0196] In some embodiments, the monomer is a monomer of fragaceatoxin C (FraC) (SEQ ID NO: 21) from Actinia fragacea, or a variant, paralog, or homolog thereof. The structure of FraC from Actinia fragacea is deposited in the Protein Databank (PDB) under the deposit code 3W9P. For example, in some embodiments, the monomer is a variant having at least 90%, for example, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or at least 99.9%, of SEQ ID NO: 21, such as at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, typically at least 95%.

[0197] As described above, in some embodiments, the monomer is PlyA (SEQ ID NO: 22) or PlyB (SEQ ID NO: 23) from Pleurotus ostreatus, or a monomer of a variant, paralog, or homolog thereof. The structure of PlyAB is deposited in the Protein Databank (PDB) under the deposit code 4V2T. For example, in some embodiments, the monomer is a variant having at least 90%, for example, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or at least 99.9%, sequence identity or sequence homology with SEQ ID NO: 22, such as at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, typically at least 95%. In some embodiments, the monomer is a variant having at least 90%, for example, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5%, or at least 99.9%, sequence identity or sequence homology with SEQ ID NO: 23, for example, at least 60%, for example, at least 70%, for example, at least 80%, for example, at least 85%, typically at least 95%.

[0198] In some embodiments, the monomers are monomers of insecticidal proteins of Bacillus thuringiensis, such as LukF / HlgB (PDB code 1LKF), the S component of Panton-Valentine leucocidine (PDB code 1T5R), and the F component of Panton-Valentine leucocidine (PDB code 1PVL); AB toxins such as anthrax toxin protective antigens; erolysine-like pores such as parasporin-2 (PDB code 2ZTB), Clostridium perfringens epsilon toxin (PDB code 6RB9), and hemolytic lectin (PDB code 1W3A); and Cry and Cyt proteins.

[0199] In some embodiments, the monomer is one of six known leucosidines from S. aureus: gamma hemolyzins HlGAB and HlgCB; LukAB / HG; LukED; Panton-Valentine leucosidine (LukSF-PV / PVL); and LukMF'.

[0200] As described above, in some embodiments, the monomer is a monomer of an enzyme such as an oligomeric enzyme. In one embodiment, the monomer is a monomer of an oligomeric helicase. These and other enzymes suitable for modification according to the present invention may, in some embodiments, be motor proteins, which are described in more detail herein.

[0201] Further steps Those skilled in the art will understand that the method of disclosure may include additional steps.

[0202] For example, in some embodiments, the method includes, prior to step (a), (i) expressing the monomer in a cell expression system or a cell-free expression system, and (ii) isolating and / or purifying the monomer. The monomer can be expressed in any suitable expression system. Suitable expression systems include bacterial expression systems, such as those using an E. coli strain to express the monomer from a suitable vector. Other suitable expression systems include expression in insect cells or yeast. For expression methods, see Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 thAs discussed in references such as ed., 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), this is well known to those skilled in the art. Cell-free expression systems (e.g., in vitro transcription / translation, IVTT) are well known in the art and are commercially available from suppliers such as Promega. IVTT is used to prepare monomers for oligomeric protein pores.

[0203] In some embodiments, step (d) of the method may further include removing unmodified monomers and / or unreacted polyfunctional molecules from the support, if present. Any preferred technique may be used. For example, the bound modified monomers may be washed with an aqueous or non-aqueous dissolved solution, such as a washing buffer, to remove unbound or weakly bound impurities from the support.

[0204] Method for generating oligomers In some embodiments, the methods disclosed herein are used to generate oligomers.

[0205] In some embodiments, the methods disclosed herein are f) Further comprising the step of oligomerizing a chemically modified monomer to form a chemically modified oligomer. In some embodiments, the monomer is a protein monomer, and step (f) includes forming an oligomeric protein. The monomer may be a monomer of an oligomeric protein nanopore, and step (f) may include forming an oligomeric protein nanopore.

[0206] In some embodiments, step (f) includes oligomerizing two or more chemically modified monomers to form a homooligomer. In such cases, the two or more monomers are identical, i.e., two or more monomers have the same monomeric structure and are chemically modified in the same manner. In some embodiments, the two or more monomers are protein monomers, and the homooligomer is a homooligomeric protein pore.

[0207] In some embodiments, step (f) includes oligomerizing one or more chemically modified monomers with one or more unmodified or differently modified monomers to form a heterooligomer. In some embodiments, two or more monomers are protein monomers, and the homooligomer is a homooligomeric protein pore.

[0208] One or more different monomers may be unmodified monomers, being of the same type (e.g., the same sequence) as a chemically modified monomer. One or more different monomers may be unmodified monomers, being of a different type (e.g., a different sequence) than a chemically modified monomer. One or more different monomers may be chemically modified monomers, being of the same type (e.g., the same sequence) as a first chemically modified monomer but modified with a different chemical modification. One or more different monomers may be modified monomers, being of a different type (e.g., a different sequence) than a first chemically modified monomer.

[0209] One or more first monomers may comprise two or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 first monomers. One or more second monomers may comprise two or more, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10 second monomers.

[0210] Therefore, this disclosure also relates to a method for producing homooligomeric proteins, i) The method described herein produces multiple chemically modified protein monomers, The present invention provides a method comprising: ii) oligomerizing two or more of the chemically modified protein monomers obtained in step (i) to form a homo-oligomeric protein. In some embodiments, the homooligomeric protein is a homooligomeric protein pore, such as the protein pores described herein.

[0211] This disclosure also relates to a method for generating heterooligomeric proteins, i) To produce one or more chemically modified first protein monomers by the method described herein, ii) To produce one or more chemically modified second protein monomers by the method described herein, iii) A method is provided which comprises oligomerizing one or more first monomers and one or more second monomers to form a hetero-oligomeric protein. In some embodiments, the hetero-oligomeric protein is a hetero-oligomeric protein pore.

[0212] This disclosure relates to an oligomeric protein, i) To produce one or more chemically modified first protein monomers by the method described herein, ii) To provide one or more unmodified second protein monomers, iii) The present invention provides an oligomeric protein comprising oligomerizing one or more first monomers and one or more second monomers to form a hetero-oligomeric protein. In some embodiments, the hetero-oligomeric protein is a hetero-oligomeric protein pore.

[0213] Monomers and oligomers provided in this disclosure This disclosure also provides products of the claimed method.

[0214] Accordingly, this disclosure provides chemically modified monomers that can be obtained by performing the methods described herein. In some embodiments, the monomer is a monomer described herein. In some embodiments, the monomer is chemically modified by one or more modifications described herein. In some embodiments, the monomer is a protein nanopore monomer described herein.

[0215] This disclosure also provides chemically modified oligomers that can be obtained by performing the methods described herein. In some embodiments, the oligomer is an oligomer comprising two or more monomers described herein. In some embodiments, the oligomer is chemically modified by one or more modifications described herein. In some embodiments, the oligomer is an oligomeric protein nanopore described herein.

[0216] This disclosure also provides a population of chemically modified monomers. Such a population of monomers is characterized, among other things, by its homogeneity. Thus, in some embodiments, this disclosure provides a homogeneous population comprising a plurality of chemically modified monomers, wherein at least 95% of the monomers in the population are chemically modified with a chemical modification group. In some embodiments, at least 96%, e.g., at least 98%, e.g., at least 99%, e.g., at least 99%, at least 99.9%, or at least 99.99%, e.g., at least 99.5%, e.g., at least 97% of the monomers in the population are chemically modified with a chemical modification group. In some embodiments, 100% of the monomers in the population are modified with a chemical modification group. As described above, modification chemistry is typically not 100% efficient, and modified monomers are typically not easily separated or purified from impurities, so previously known methods are typically not capable of providing such a homogeneous population. In some embodiments, the population is a population of monomers of protein pores, e.g., protein pores as described herein.

[0217] This disclosure also provides a population of chemically modified oligomers, such as oligomeric protein pores. Such a population of oligomers is characterized, among other things, by their homogeneity. Thus, in some embodiments, this disclosure provides a homogeneous population comprising a plurality of chemically modified oligomers, wherein at least 95% of the oligomers in the population are chemically modified with a chemical modification group. In some embodiments, at least 96%, e.g., at least 98%, e.g., at least 99%, e.g., at least 99%, at least 99.9%, or at least 99.99%, e.g., at least 99.5%, e.g., at least 97% of the oligomers in the population are chemically modified with a chemical modification group. In some embodiments, 100% of the oligomers in the population are modified with a chemical modification group. As described above, modification chemistry is typically not 100% efficient, and modified monomers are typically not easily separated or purified from impurities in order to provide a population of highly homogeneous oligomers; therefore, previously known methods are typically not capable of providing such a homogeneous population. In some embodiments, the population is an oligomeric protein pore, for example, a population of protein pores as described herein.

[0218] Methods using monomers and oligomers provided herein The chemically modified monomers and oligomers provided herein are useful in a variety of ways.

[0219] Chemically modified proteins have found common utility in the fields of biotechnology and medicine. For example, modified enzymes may have improved or altered functionality compared to their natural counterparts.

[0220] In some embodiments, the monomer is a monomer of nanopores and / or the oligomer is an oligomeric nanopore. Such nanopores can be used for the detection and characterization of analytes such as polynucleotides.

[0221] Therefore, a method for characterizing an analyte, i) To generate chemically modified oligomeric pores by the method described herein, ii) A method is provided herein that includes obtaining one or more measurements as the analyte moves into a pore, wherein the one or more measurements represent one or more features of the analyte, thereby characterizing the analyte as it moves into the pore.

[0222] A method for characterizing an analyte, i) To provide the chemically modified oligomeric pores described herein, ii) A method is also provided which includes obtaining one or more measurements as the analyte moves into the pore, wherein the one or more measurements represent one or more features of the analyte, thereby characterizing the analyte as it moves into the pore. In some embodiments, the analyte is a polynucleotide, which is described in more detail herein.

[0223] Characterization of the analyte The disclosed feature evaluation method can be performed using any apparatus suitable for investigating membrane / pore systems in which pores are inserted into a membrane. Membranes are described in more detail herein.

[0224] The feature evaluation method can be carried out using any apparatus suitable for transmembrane pore sensing. For example, the apparatus may comprise a chamber containing an aqueous solution and a barrier separating the chamber into two compartments. The barrier may have openings, through which a membrane containing transmembrane pores, such as the chemically modified transmembrane pores described herein, is formed. Transmembrane pores are described herein.

[0225] The feature evaluation method may be carried out using the apparatus described in International Publication No. 2008 / 102120, No. 2010 / 122293, or No. 00 / 28312.

[0226] The binding of molecules (e.g., target polynucleotides) in the pore channels will affect the open-channel ion flow through the pores, which is essential to the "molecular sensing" of the pore channels. Therefore, characterization methods may typically involve measuring the flow of ionic current through the pores by measuring current (e.g., International Publication 2000 / 28312, and D. Stoddart et al., Proc. Natl. Acad. Sci., 2010, 106, 7702-7, or International Publication 2009 / 077734). Alternatively, the ion flow through the pores may be measured optically, as disclosed by Heron et al: J. Am. Chem. Soc. 9 Vol. 131, No. 5, 2009, etc. Thus, the apparatus may also include an electrical circuit capable of applying a potential and measuring electrical signals across the membrane and pores. Characterization methods may be performed using patch clamps or voltage clamps. Characterization methods preferably involve the use of voltage clamps.

[0227] For precise determination of individual nucleotides, it is advantageous that a reduction in ion flow through the channel correlates with the size of individual nucleotides passing through the constriction (or "reading head"). Therefore, modification of the nanopore reading head by the claimed method may be useful in tuning this interaction and improving the characterization signal that can be obtained.

[0228] The feature evaluation method can be performed on a silicon array with wells, where each array contains 128, 256, 512, 1024, 2000, 3000, 4000, 6000, 10000, 12000, 15000 or more wells.

[0229] The feature evaluation method may include measuring the current flowing through the pores. This method is typically performed using a voltage applied across the membrane and pores. The voltage used is typically +2V to -2V, and typically -400mV to +400mV. The voltage used is preferably in the range having a lower limit selected from -400mV, -300mV, -200mV, -150mV, -100mV, -50mV, -20mV, and 0mV, and an upper limit independently selected from +10mV, +20mV, +50mV, +100mV, +150mV, +200mV, +300mV, and +400mV. The voltage used is more preferably in the range of 100mV to 240mV, and most preferably in the range of 120mV to 220mV. By using an increased applied potential, it is possible to increase the distinction between different nucleotides by the pores.

[0230] The characterization method is typically carried out in the presence of any charge carrier, such as a metal salt, such as an alkali metal salt or a halide salt, or a chloride salt, such as an alkali metal chloride salt. Examples of charge carriers include ionic liquids or organic salts, such as tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride, or 1-ethyl-3-methylimidazolium chloride. In the exemplary apparatus described above, the salt is present in an aqueous solution within the chamber. Typically, potassium chloride (KCl), sodium chloride (NaCl), or cesium chloride (CsCl) are used. KCl is preferred. The salt may be an alkaline earth metal salt, such as calcium chloride (CaCl2). The salt concentration may be saturated. The salt concentration may be 3M or less, and is typically 0.1-2.5M, 0.3-1.9M, 0.5-1.8M, 0.7-1.7M, 0.9-1.6M, or 1M-1.4M. The salt concentration is preferably 150 mM to 1 M. The characteristic evaluation method is preferably performed using a salt concentration of at least 0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M, or at least 3.0 M. Higher salt concentrations provide a high signal-to-noise ratio, allowing coupled / uncoupled currents to be identified against a background of normal current fluctuations.

[0231] The characterization method is typically performed in the presence of a buffer. In the exemplary apparatus described above, the buffer is present in an aqueous solution within the chamber. Any suitable buffer can be used. Typically, the buffer is HEPES. Another suitable buffer is Tris-HCl buffer. The method is typically performed at pH values ​​of 4.0–12.0, 4.5–10.0, 5.0–9.0, 5.5–8.8, 6.0–8.7, or 7.0–8.8, or 7.5–8.5. The pH used is preferably about 7.5.

[0232] The characterization method may be performed at temperatures of 0°C to 100°C, 15°C to 95°C, 16°C to 90°C, 17°C to 85°C, 18°C ​​to 80°C, 19°C to 70°C, or 20°C to 60°C. Typically, the characterization method is performed at room temperature. Optionally, the characterization method may be performed at temperatures supporting enzyme function, such as approximately 37°C.

[0233] film In embodiments of the present invention involving the use of transmembrane nanopores, the transmembrane nanopores are typically located within the membrane. Any suitable membrane can be used in the system.

[0234] The membrane is preferably an amphiphilic layer. The amphiphilic layer is a layer formed from amphiphilic molecules, such as phospholipids, that possess both hydrophilic and lipophilic properties. The amphiphilic molecules may be synthetic or natural. Unnatural amphiphilic substances and amphiphilic substances forming monolayers are known in the art, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). A block copolymer is a polymer material in which two or more monomer subunits polymerize together to form a single polymer chain. Block copolymers typically possess properties contributed by each monomer subunit. However, block copolymers may possess unique properties not found in polymers formed from individual subunits. Block copolymers can be manipulated in an aqueous medium such that one of the monomer subunits is hydrophobic (i.e., lipophilic) and the other subunits are hydrophilic. In this case, the block copolymer may possess amphiphilic properties and form structures that mimic biological membranes. Block copolymers can be diblocks (consisting of two monomer subunits), but they can also be constructed from more than two monomer subunits to form more complex arrangements that behave as amphiphilic materials. Copolymers can be triblocks, tetrablocks, or pentablock copolymers. The membrane is preferably a triblock copolymer membrane.

[0235] Archaeal bipolar tetraether lipids are natural lipids that are constructed such that the lipids form a monolayer membrane. These lipids are generally found in extremophilic microorganisms, thermophiles, halophiles, and acidophiles that survive in harsh biological environments. Their stability is thought to be due to the fused nature of the resulting bilayer. It is straightforward to construct block copolymer materials that mimic these biological entities by creating triblock polymers having a hydrophilic-hydrophobic-hydrophilic general motif. This material behaves like a lipid bilayer and can form monomeric membranes that encompass a range of phase behaviors from vesicles to lamellar membranes. The membranes formed from these triblock copolymers retain several advantages over biological lipid membranes. Since the triblock copolymers are synthesized, the exact structure can be carefully controlled to provide the correct chain lengths and properties necessary to form membranes and interact with pores and other proteins.

[0236] Block copolymers can be constructed from subunits that are not classified as lipid submaterials; for example, hydrophobic polymers can be made from siloxanes or other non-hydrocarbon-based monomers. The hydrophilic subdomain of the block copolymer can also have low protein-binding properties, which enables the fabrication of highly resistant membranes when exposed to untreated biological samples. This head group unit can be derived from non-classical lipid head groups.

[0237] Triblock copolymer membranes also have increased mechanical and environmental stability compared to biological lipid membranes, for example, having much higher operating temperatures or pH ranges. The synthetic nature of the block copolymers provides a platform for customizing polymer-based membranes for a wide range of applications.

[0238] In some embodiments, the membrane is one of the membranes disclosed in International Publication No. WO 2014 / 064443 or WO 2014 / 064444.

[0239] Amphiphilic molecules can be chemically modified or functionalized to facilitate the binding of polynucleotides. The amphiphilic layer can be a monolayer or a bilayer. The amphiphilic layer is typically planar. The amphiphilic layer may be curved. The amphiphilic layer may be supported.

[0240] The amphiphilic membrane is typically naturally mobile and essentially acts as a two-dimensional fluid with a lipid diffusion rate of approximately 10 -8 cm s -1 . This means that pores and bound polynucleotides can typically move within the amphiphilic membrane.

[0241] The membrane can be a lipid bilayer. The lipid bilayer is a model of the cell membrane and serves as an excellent platform for a range of experimental studies. For example, the lipid bilayer can be used for in vitro investigation of membrane proteins by single-channel recording. Alternatively, the lipid bilayer can be used as a biosensor for detecting the presence of a range of substances. The lipid bilayer can be any lipid bilayer. Suitable lipid bilayers include, but are not limited to, planar lipid bilayers, supported bilayers or liposomes. The lipid bilayer is preferably a planar lipid bilayer. Suitable lipid bilayers are disclosed in WO 2008 / 102121, WO 2009 / 077734, and WO 2006 / 100484.

[0242] Methods for forming lipid bilayers are known in the art. Lipid bilayers are generally formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972; 69: 3561-3566), in which a lipid monolayer is supported on the aqueous solution / air interface, passing through one side of the opening perpendicular to the interface. The lipid is usually added to the surface of the electrolyte aqueous solution by first dissolving it in an organic solvent and then evaporating a droplet of solvent on the surface of the aqueous solution on one side of the opening. As the organic solvent evaporates, the solution / air interface on one side of the opening physically moves up and down through the opening until the bilayer is formed. Planar lipid bilayers can be formed across the opening of the membrane or across the opening into a recess.

[0243] The Montal & Mueller method is common because it is cost-effective and relatively simple for forming a good quality lipid bilayer suitable for protein pore insertion. Other common methods for bilayer formation include tip dipping, bilayer coating, and liposome bilayer patch clamping.

[0244] Tip dipping bilayer formation involves bringing the opening surface (e.g., pipette tip) into contact with the surface of the test solution supporting a lipid monolayer. Again, the lipid monolayer is initially formed at the solution / air interface by evaporating droplets of lipid dissolved in an organic solvent on the solution surface. Subsequently, the bilayer is formed by the Langmuir-Schaefer process, which requires mechanical automation to move the opening relative to the solution surface.

[0245] In the coated bilayer, droplets of lipid dissolved in an organic solvent are applied directly to the opening and immersed in an aqueous test solution. A paintbrush or equivalent is used to spread the lipid solution thinly over the opening. By thinning the solvent, the lipid bilayer is formed. However, it is difficult to completely remove the solvent from the bilayer, and as a result, the bilayer formed by this method has low stability and is prone to generating noise during electrochemical measurements.

[0246] Patch clamping is commonly used in the study of biological cell membranes. The cell membrane is fixed to the end of a pipette by aspiration, and a patch of the membrane is attached over the opening. This method is adapted to generate a lipid bilayer by fixing liposomes, which then rupture, leaving a lipid bilayer that covers and seals the pipette opening. The method requires the creation of stable, large unilamellar liposomes and smaller openings in materials with glass surfaces.

[0247] Liposomes can be formed by sonication, extrusion, or the Mozafari method (Colas et al. (2007) Micron 38:841-847).

[0248] In some embodiments, the lipid bilayer is formed as described in International Publication No. 2009 / 077734. Advantageously, in this method, the lipid bilayer is formed from dry lipids. In the most preferred embodiment, the lipid bilayer is formed across the opening as described in International Publication No. 2009 / 077734.

[0249] A lipid bilayer is formed from two opposing layers of lipids. The two lipid layers are arranged such that their hydrophobic tail groups face each other, forming a hydrophobic interior. The hydrophilic head groups of the lipids face outward toward the aqueous environment on each side of the bilayer. Bilayers can exist in a variety of lipid phases, including, but not limited to, liquid disordered phases (fluid lamellae), liquid ordered phases, solid ordered phases (lamellar gel phases, interlocking gel phases), and planar bilayer crystals (lamellar subgel phases, lamellar crystal phases).

[0250] Any lipid composition that forms a lipid bilayer may be used. The lipid composition is selected so as to form a lipid bilayer having the desired properties, such as surface charge, ability to support membrane proteins, packing density, or mechanical properties. The lipid composition may contain one or more different lipids. For example, the lipid composition may contain up to 100 lipids. The lipid composition preferably contains 1 to 10 lipids. The lipid composition may contain natural lipids and / or artificial lipids.

[0251] Lipids typically consist of a head group, an interface, and two hydrophobic tail groups, which may be the same or different. Suitable head groups include, but are not limited to, neutral head groups such as diacylglyceride (DG) and ceramide (CM); zwitterionic head groups such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM); negatively charged head groups such as phosphatidylglycerol (PG); positively charged head groups such as phosphatidylserine (PS), phosphatidylinositol (PI), phosphatic acid (PA), and cardiolipin (CA); and trimethylammonium-propane (TAP). Suitable interface portions include, but are not limited to, natural interface portions such as glycerol-based or ceramide-based portions. Suitable hydrophobic tail groups include, but are not limited to, saturated hydrocarbon chains such as lauric acid (n-dodecanoic acid), myristic acid (n-tetradecanoic acid), palmitic acid (n-hexadecanoic acid), stearic acid (n-octadecanoic acid), and arachidic acid (n-eicosanoic acid); unsaturated hydrocarbon chains such as oleic acid (cis-9-octadecanoic acid); and branched hydrocarbon chains such as phytanoyl. The length of the unsaturated hydrocarbon chain and the position and number of double bonds may vary. The length of the branched hydrocarbon chain and the position and number of branches such as methyl groups may vary. The hydrophobic tail groups can be linked to the interface portion as ethers or esters. The lipid may be mycolic acid.

[0252] Lipids can also be chemically modified. The head group or tail group of a lipid can be chemically modified. Suitable lipids with chemically modified head groups include, but are not limited to, PEG-modified lipids such as 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]; functionalized PEG lipids such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)2000]; and lipids modified for conjugates such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl). Suitable lipids with chemically modified tail groups include, but are not limited to, polymerizable lipids such as 1,2-bis(10,12-tricosadiinoyl)-sn-glycero-3-phosphocholine; fluorinated lipids such as 1-palmitoyl-2-(16-fluoropalmitoyl)-sn-glycero-3-phosphocholine; deuterated lipids such as 1,2-dipalmitoyl-D62-sn-glycero-3-phosphocholine; and ether-binding lipids such as 1,2-di-O-phytanyl-sn-glycero-3-phosphocholine. Lipids can be chemically modified or functionalized to facilitate the binding of polynucleotides.

[0253] The amphiphilic layer, for example, a lipid composition, typically contains one or more additives that will affect the properties of the layer. Suitable additives include, but are not limited to, fatty acids such as palmitic acid, myristic acid, and oleic acid; fatty alcohols such as palmitic alcohol, myristic alcohol, and oleic alcohol; sterols such as cholesterol, ergosterol, lanosterol, sitosterol, and stigmasterol; lysophospholipids such as 1-acyl-2-hydroxy-sn-glycero-3-phosphocholine; and ceramides.

[0254] In another embodiment, the film includes a solid state layer. The solid state layer may 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 polyamides, plastics such as Teflon®, or elastomers such as two-component addition-cured silicone rubber, and glass. The solid state layer may be formed from graphene. A suitable graphene layer is disclosed in International Publication No. 2009 / 035647. When the film includes a solid state layer, pores are typically present in the amphiphilic film or layer contained within the solid state layer, for example, in holes, wells, gaps, channels, trenches, or slits within the solid state layer. Those skilled in the art can prepare suitable solid state / amphiphilic hybrid systems. Suitable systems are disclosed in International Publication Nos. 2009 / 020682 and 2012 / 005857. Any of the amphiphilic films or layers described above may be used.

[0255] Motor protein In a method of disclosure that includes characterizing an analyte as it moves into nanopores, such movement can be controlled using a motor protein. In addition, the methods of chemically modifying monomers disclosed herein are also applicable to the chemical modification of such motor proteins (or their monomers), whether used in the disclosed method or any other method.

[0256] As used herein, a motor protein is any protein that binds to a polynucleotide and is capable of controlling its movement toward, for example, through a nanopore.

[0257] In one embodiment, the motor protein is, or is derived from, a polynucleotide handling enzyme. A polynucleotide handling enzyme is a polypeptide that interacts with a polynucleotide and is capable of modifying at least one property of the polynucleotide. The enzyme can modify the polynucleotide by cleaving the polynucleotide to form individual nucleotides or shorter nucleotide chains such as dinucleotides or trinucleotides. The enzyme can modify the polynucleotide by orienting or moving the polynucleotide to a specific location.

[0258] In one embodiment, the motor protein is derived from any member of 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.

[0259] Typically, the motor protein is a helicase, polymerase, exonuclease, topoisomerase, or a variant thereof.

[0260] In some embodiments, the motor protein can be modified to prevent the motor protein from detaching from the polynucleotide or polynucleotide adapter. For example, the modification of the motor protein to prevent detachment from a spacer on a polynucleotide adapter is discussed in WO 2014 / 013260, which is hereby incorporated by reference in its entirety, and in particular, see the section describing the modification of motor proteins such as helicases to prevent detachment from a polynucleotide chain. For example, the motor protein can be modified by treatment with tetramethylazodicarboxamide.

[0261] For example, a motor protein may have a polynucleotide-unbound opening, such as a cavity, crack, or void, through which the polynucleotide chain can pass when the motor protein detaches from the chain. A motor protein can be modified by closing the polynucleotide-unbound opening. Therefore, detachment of the motor protein can be prevented by closing the polynucleotide-unbound opening. For example, a motor protein can be modified by covalently closing the polynucleotide-unbound opening. In some embodiments, a preferred motor protein for this purpose is a helicase.

[0262] In one embodiment, the motor protein is an exonuclease. Preferred enzymes include, but are not limited to, exonuclease I from E. coli (SEQ ID NO: 1), exonuclease III from E. coli (SEQ ID NO: 2), RecJ from T. thermophilus (SEQ ID NO: 3), and bacteriophage lambda exonuclease (SEQ ID NO: 4), TatD exonuclease, and their variants. Three subunits, including the sequence shown in SEQ ID NO: 3 or its variants, interact to form a trimer exonuclease.

[0263] In one embodiment, the motor protein is a polymerase. The polymerase may be PyroPhage® 3173 DNA polymerase (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 (SEQ ID NO: 5) or a variant thereof. Modified versions of Phi29 polymerase that may be used in the present invention are disclosed in U.S. Patent No. 5,576,204.

[0264] In one embodiment, the motor protein is a topoisomerase. In one embodiment, the topoisomerase is a member of either subcategory (EC) group 5.99.1.2 or 5.99.1.3. The topoisomerase may be a reverse transcriptase, an enzyme capable of catalyzing the formation of cDNA from an RNA template. These are commercially available, for example, from New England Biolabs® and Invitrogen®.

[0265] In one embodiment, the motor protein is a helicase. Any suitable helicase may be used according to the methods provided herein. For example, the motor protein used according to this disclosure, or each motor protein, may be independently selected from Hel308 helicase, RecD helicase, TraI helicase, TrwC helicase, XPD helicase, and Dda helicase, or variants thereof. Monomer helicases may contain several domains that are bound together. For example, TraI helicase and TraI subgroup helicases may contain two RecD helicase domains, one relaxase domain, and one C-terminal domain. The domains typically form a monomer helicase that is capable of functioning without forming an oligomer. Specific examples of suitable helicases include Hel308, NS3, Dda, UvrD, Rep, PcrA, Pif1, and TraI. These helicases typically act on single-stranded DNA. Examples of helicases that can move along both strands of double-stranded DNA include the FtfK and hexamer enzyme complex, or multi-subunit complexes such as RecBCD.

[0266] The Hel308 helicase is described in its entirety in publications such as International Publication No. 2013 / 057495, which are incorporated by reference. The RecD helicase is described in its entirety in publications such as International Publication No. 2013 / 098562, which are incorporated by reference. The XPD helicase is described in its entirety in publications such as International Publication No. 2013 / 098561, which are incorporated by reference. The Dda helicase is described in publications such as International Publication No. 2015 / 055981 and International Publication No. 2016 / 055777, which are incorporated by reference.

[0267] In one embodiment, the helicase includes the sequence shown in SEQ ID NO: 6 (Trwc Cba) or a variant thereof, the sequence shown in SEQ ID NO: 7 (Hel308Mbu) or a variant thereof, or the sequence shown in SEQ ID NO: 8 (Dda) or a variant thereof. The variant may differ from the natural sequence in any of the ways discussed herein. An exemplary variant of SEQ ID NO: 8 includes E94C / A360C. Further exemplary variants of SEQ ID NO: 8 include E94C / A360C followed by (ΔM1)G1G2 (i.e., deletion of M1 followed by addition of G1 and G2).

[0268] In some embodiments, the motor protein (e.g., helicase) has at least two active modes of operation (the motor protein has all the components necessary to facilitate movement, e.g., fuel and ATP and Mg 2+ The movement of polynucleotides can be controlled (when provided with cofactors such as), and in one inactive mode of operation (when the motor protein is not provided with the components necessary to facilitate movement).

[0269] When provided with all the components necessary to facilitate movement (i.e., in active mode), a motor protein (e.g., a helicase) moves along the polynucleotide in a 5' to 3' or 3' to 5' direction (depending on the motor protein). In embodiments where a motor protein is used to control the movement of a polynucleotide chain into a nanopore, the motor protein can be used to move the polynucleotide away from the pore (e.g., outwards) (e.g., relative to an applied field) or toward the pore (e.g., into the pore) (e.g., relative to an applied field). For example, if the end of the polynucleotide to which the motor protein is moving is trapped by the pore, the motor protein acts in the direction of the field resulting from the applied potential, pulling the threaded polynucleotide away from the pore (e.g., into the cis chamber). However, if the end to which the motor protein is moving away is trapped within the pore, the motor protein acts in the direction of the field resulting from the applied potential, pushing the threaded polynucleotide into the pore (e.g., into the trans chamber).

[0270] If a motor protein (e.g., a helicase) is not provided with the necessary components to facilitate movement (i.e., in inactive mode), the motor protein can bind to a polynucleotide and act as a brake, slowing down the movement of the polynucleotide as it moves into a nanopore, for example, by being drawn into the pore by a field resulting from an applied potential. In inactive mode, it is irrelevant which end of the polynucleotide is captured; it is the applied field that determines the movement of the polynucleotide into the pore, and the motor protein acts as a brake. In inactive mode, the control of polynucleotide movement by the motor protein can be described in several ways, including ratcheting, sliding, and braking.

[0271] In active mode, motor proteins typically consume fuel molecules. These fuel molecules are typically free nucleotides or free nucleotide analogs. Free nucleotides are not limited to adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), and cytidine triphosphate (cytidine deoxyadenosine monophosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyadenosine diphosphate (dADP), deoxyadenosine triphosphate (dATP), deoxyguanosine monophosphate (dGMP), deoxyguanosine diphosphate (deoxyguanosineIt can be one or more of diphosphate, dGDP, deoxyguanosine triphosphate (dGTP), deoxythymidine monophosphate (dTMP), deoxythymidine diphosphate (dTDP), deoxythymidine triphosphate (dTTP), deoxyuridine monophosphate (dUMP), deoxyuridine diphosphate (dUDP), deoxyuridine triphosphate (dUTP), deoxycytidine monophosphate (dCMP), deoxycytidine diphosphate (dCDP), and deoxycytidine triphosphate (dCTP). The free nucleotide is usually selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, or dCMP. The free nucleotide is typically adenosine triphosphate (ATP).

[0272] The cofactor of the motor protein is a factor that enables the motor protein to function. The cofactor is preferably a divalent metal cation. The divalent metal cation is preferably Mg 2+ , Mn 2+ , Ca 2+ or Co 2+ is. The cofactor is most preferably Mg 2+ is.

[0273] Polynucleotide analyte In embodiments of the disclosed method that include detecting or characterizing an analyte, the analyte is typically a polynucleotide.

[0274] Polynucleotides, such as nucleic acids, are macromolecules containing two or more nucleotides. Polynucleotides can be single-stranded or double-stranded. Double-stranded polynucleotides are made from two single-stranded polynucleotides that have hybridized together. Target polynucleotides can be single-stranded or double-stranded polynucleotides.

[0275] Polynucleotides can contain any combination of any nucleotides. Nucleotides can be natural or artificial.

[0276] Nucleotides typically contain a nucleic acid base, a sugar, and at least one phosphate group. The nucleic acid base and sugar form a nucleoside.

[0277] Nucleic acid bases are typically heterocyclic. Examples of nucleic acid bases include, but are not limited to, purines and pyrimidines, more specifically adenine (A), guanine (G), thymine (T), uracil (U), and cytosine (C).

[0278] The sugar is typically a pentose sugar. Examples of nucleotide sugars, though not limited to them, include ribose and deoxyribose. The sugar is preferably deoxyribose. The polynucleotide preferably comprises the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU), and / or thymidine (dT), deoxyguanosine (dG), and deoxycytidine (dC).

[0279] Nucleotides are typically ribonucleotides or deoxyribonucleotides. Nucleotides typically contain monophosphate, diphosphate, or triphosphate. Nucleotides may contain four or more phosphates, such as four or five phosphates. Phosphates may be attached to the 5' or 3' end of the nucleotide. Examples of nucleotides, but not limited to, include 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.

[0280] Nucleotides can be debased (i.e., lacking a nucleic acid base). Nucleotides can also lack both a nucleic acid base and a sugar (i.e., they are C3 spacers).

[0281] The nucleotides of a polynucleotide can be linked to each other in any manner. Typically, nucleotides are linked by their sugar and phosphate groups, as in nucleic acids. Nucleotides can also be linked via their nucleic acid bases, as in pyrimidine dimers.

[0282] Polynucleotides can be nucleic acids such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). A polynucleotide may contain one strand of RNA hybridized to one strand of DNA. Polynucleotides can be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), roq nucleic acid (LNA), bridged nucleic acid (BNA), or other synthetic polymers having nucleotide side chains. The PNA backbone consists of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The GNA backbone consists of repeating glycol units linked by phosphodiester bonds. The TNA backbone consists of repeating threose sugars linked together by phosphodiester bonds. LNA is formed from the above-mentioned ribonucleotides having an excess of crosslinks connecting the 2' oxygen and 4' carbon of the ribose moiety.

[0283] The polynucleotide is preferably DNA, RNA, or a DNA or RNA hybrid, most preferably DNA. The DNA / RNA hybrid may contain both DNA and RNA on the same strand. Preferably, the DNA / RNA hybrid contains one DNA strand hybridized to an RNA strand.

[0284] The polynucleotide backbone can be modified to reduce the likelihood of strand breaks. For example, DNA is known to be more stable than RNA under many conditions. The polynucleotide chain backbone can be modified to avoid damage caused by harsh chemicals, such as free radicals.

[0285] DNA or RNA containing non-native or modified bases can be produced by amplifying native DNA or RNA polynucleotides in the presence of modified NTPs using an appropriate polymerase.

[0286] The nucleotides of a polynucleotide can be modified. Nucleotides can be oxidized or methylated. One or more nucleotides of a polynucleotide can be damaged. For example, a polynucleotide may contain pyrimidine dimers. Such dimers are typically associated with UV damage and are a major cause of cutaneous melanoma. One or more nucleotides of a polynucleotide can be modified with labels or tags.

[0287] Single-stranded polynucleotides may contain regions with strong secondary structures, such as hairpin, quadruple-stranded, or triple-stranded DNA. These types of structures can be used to control the movement of polynucleotides into nanopores. For example, secondary structures can be used to temporarily halt the movement of polynucleotides through nanopores, as described in more detail herein. Each continuous secondary structure along the strand pauses the movement of the strand into the nanopore when it unravels and translocates. After moving through the nanopores, the polynucleotides can reform the secondary structures. Such secondary structures can be used to prevent the polynucleotides from returning through the nanopores under low or absent negative voltages (applied to the trans side of the nanopores), and thus to assist in controlling the movement of polynucleotides, so that the movement of polynucleotides occurs only in a controlled manner in the relevant steps of the methods provided herein.

[0288] As used herein, a double-stranded polynucleotide may include a single-stranded region as well as regions having other structures such as hairpin loops, triple-stranded, and / or quadruple-stranded regions. Such secondary structures may be useful in the context of a single-stranded polynucleotide as described above.

[0289] The two strands of a double-stranded molecule can be covalently bonded at the ends of the molecule, for example, by linking the 5' end of one strand to the 3' end of the other in a hairpin structure.

[0290] The target polynucleotide can be of any length. For example, the target polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotides or nucleotide pairs. The target polynucleotide can be 1,000 or more nucleotides or nucleotide pairs, 5,000 or more nucleotides or nucleotide pairs, 100,000 or more nucleotides or nucleotide pairs, 500,000 or more nucleotides or nucleotide pairs, 1,000,000 or more nucleotides or nucleotide pairs, 10,000,000 or more nucleotides or nucleotide pairs, 100,000,000 or more nucleotides or nucleotide pairs, 200,000,000 or more nucleotides or nucleotide pairs, or the entire length of the chromosome.

[0291] The target polynucleotide may be an oligonucleotide. An oligonucleotide is typically a short nucleotide polymer having 50 or fewer nucleotides, such as 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer, or 5 or fewer nucleotides. The target oligonucleotide is preferably about 15 to about 30 nucleotides long, such as about 20 to about 25 nucleotides long. For example, the oligonucleotide may be about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, or about 30 nucleotides long.

[0292] The target polynucleotide may be a longer polynucleotide fragment. In this embodiment, the longer polynucleotide is typically fragmented into two or more shorter polynucleotides.

[0293] The target polynucleotides may include products of PCR reactions, genomic DNA, products of endonuclease digestion, and / or DNA libraries.

[0294] The target polynucleotide may be naturally occurring. The target polynucleotide may be secreted from cells. Alternatively, since the target analyte may be an analyte present inside cells, the analyte must be extracted from cells before this method can be performed.

[0295] Target polynucleotides can be sourced from common organisms such as viruses, bacteria, archaea, plants, or animals. Such organisms may be selected or modified to modulate the sequence of the target polynucleotide, for example, by altering the base composition or removing undesirable sequence elements. The selection and modification of organisms to achieve desired polynucleotide characteristics is commonplace for those skilled in the art.

[0296] The source organism for the target polynucleotide can be selected based on the desired characteristics of the sequence. Desired characteristics include the ratio of single-stranded to double-stranded polynucleotides produced by the organism; the complexity of the sequence of the polynucleotide produced by the organism; the composition of the polynucleotide produced by the organism (e.g., GC composition); or the length of the continuous polynucleotide chain produced by the organism. For example, if a continuous polynucleotide chain of approximately 50 kb is required, lambda phage DNA can be used. If a longer continuous chain is needed, other organisms can be used to produce the polynucleotide; for example, E. coli produces approximately 4.5 Mb of continuous dsDNA.

[0297] Target polynucleotides are often obtained from humans or animals, for example, from urine, lymph, saliva, mucus, semen, or amniotic fluid, or from whole blood, plasma, or serum. Target polynucleotides can also be obtained from plants, for example, cereals, legumes, fruits, or vegetables. Target polynucleotides may include genomic DNA. Genomic DNA can be fragmented. DNA can be fragmented by any preferred method. For example, methods for fragmenting DNA are known in the art, and such methods may use transposases such as MuA transposases. In many cases, genomic DNA is not fragmented.

[0298] In some embodiments, polynucleotides are synthetic or semi-synthetic. For example, DNA or RNA may be purely synthetic and can be synthesized by conventional DNA synthesis methods such as phosphoramidite-based chemistry. Synthetic polynucleotide subunits can be linked together by known means such as ligation or chemical bonding to produce longer chains. In some embodiments, internally self-forming structures (e.g., hairpins, quadruples) can be designed in the substrate, for example, by ligating a suitable sequence. Synthetic polynucleotides can be copied and scaled up for production by means known in the art, including PCR and integration into bacterial factories.

[0299] In some embodiments, polynucleotides may have a simplified nucleotide composition. In some embodiments, polynucleotides have a repeating pattern of the same subunit. For example, the repeating unit may be (AmGn)q, where m, n, and q are positive integers. For example, m is often 1 to 10, 1 to 20 such as 1 to 5, e.g., 1, 2, 3, 4, or 5. n is often 1 to 10, 1 to 20 such as 1 to 5, e.g., 1, 2, 3, 4, or 5. m and n may be the same or different. q is often 1 to about 100,000. A typical repeating unit may be, for example, (AAAAAAGGGGGG)q (SEQ ID NO: 36). Repeating polynucleotides can be prepared by many means known in the art, for example, by linking together synthetic subunits having sticky ends that allow ligation. Thus, in some embodiments, polynucleotides may be linked polynucleotides. The method for linking polynucleotides is described in PCT / GB2017 / 051493.

[0300] Polynucleotide adapter Target polynucleotides evaluated by the methods provided herein may include polynucleotide adapters. Polynucleotide adapters can be used to control the movement of polynucleotides into nanopores, for example, to load motor proteins onto polynucleotides. For example, International Publication 2015 / 110813 describes the loading of motor proteins onto target polynucleotides such as adapters, and is incorporated herein by reference in its entirety.

[0301] The adapter typically comprises a polynucleotide chain capable of binding to the ends of a target polynucleotide. The polynucleotide adapter may be attached to both ends of the target polynucleotide, or different adapters may be attached to two ends of the target polynucleotide. The adapter may also be attached to only one end of the target polynucleotide. Methods for attaching adapters to polynucleotides are known in the art. The adapter may be attached to the polynucleotide, for example, by ligation, click chemistry, tagmentation, topoisomerization, or any other suitable method.

[0302] The adapter may be synthetic or artificial. Typically, the adapter comprises the polymers described herein. In some embodiments, the adapter comprises polynucleotides. In some embodiments, the adapter may comprise single-stranded polynucleotide chains. In some embodiments, the adapter may comprise double-stranded polynucleotides. Polynucleotide adapters may comprise DNA, RNA, modified DNA (such as basic DNA), RNA, PNA, LNA, BNA, and / or PEG. Typically, the adapter comprises single-stranded and / or double-stranded DNA or RNA.

[0303] The adapter may be a Y-adapter. A Y-adapter is typically double-stranded and includes (a) a region at one end where the two strands are hybridized together, and (b) a region at the other end where the two strands are not complementary. The non-complementary portion of the strands forms an overhang. The hybridized stem of the adapter typically binds to the 5' end of the first strand of the double-stranded polynucleotide and the 3' end of the second strand of the double-stranded polynucleotide; or to the 3' end of the first strand of the double-stranded polynucleotide and the 5' end of the second strand of the double-stranded polynucleotide. Unlike the double-stranded portion, the two strands typically do not hybridize to each other, so the presence of a non-complementary region in the Y-adapter gives the adapter its Y shape. A motor protein or polynucleotide-binding protein may bind to the overhang of an adapter such as a Y-adapter. In another embodiment, a motor protein or polynucleotide-binding protein may bind to the double-stranded region. In other embodiments, a motor protein or polynucleotide-binding protein may bind to the single-stranded and / or double-stranded regions of the adapter. In other embodiments, a first motor protein or polynucleotide-binding protein may bind to the single-stranded region of such an adapter, and a second motor protein or polynucleotide-binding protein may bind to the double-stranded region of the adapter.

[0304] In some embodiments, one of the non-complementary strands of a polynucleotide adapter, such as a Y adapter, may contain a leader sequence that, upon contact with a transmembrane pore, can pass into the nanopore. The leader sequence typically includes polynucleotides, e.g., DNA or RNA, modified polynucleotides (such as debasalized DNA), PNA, LNA, polyethylene glycol (PEG), or polymers such as polypeptides. In some embodiments, the leader sequence contains a single strand of DNA, such as a polydT compartment. The leader sequence can be of any length, but is typically 10 to 150 nucleotides long, such as 20 to 120, 30 to 100, 40 to 80, or 50 to 70 nucleotides long.

[0305] In one embodiment, the polynucleotide adapter is a hairpin loop adapter. The hairpin loop adapter is an adapter comprising a single polynucleotide chain, wherein the ends of the polynucleotide chains are capable of hybridizing with each other, or are hybridized with each other, and the central compartments of the polynucleotides form a loop. A suitable hairpin loop adapter can be designed using methods known in the art. Typically, the 3' end of the hairpin loop adapter is bound to the 5' end of the first strand of a double-stranded polynucleotide and the 5' end of the hairpin loop adapter is bound to the 3' end of the second strand of a double-stranded polynucleotide, or the 5' end of the hairpin loop adapter is bound to the 3' end of the first strand of a double-stranded polynucleotide and the 3' end of the hairpin loop adapter is bound to the 5' end of the second strand of a double-stranded polynucleotide.

[0306] A polynucleotide or polynucleotide adapter may include one or more spacers, e.g., 1 to about 10 spacers, e.g., 1 to about 5 spacers, e.g., 1, 2, 3, 4, or 5 spacers. The spacer may include any suitable number of spacer units. The spacer typically provides an energy barrier that hinders the movement of the polynucleotide-binding protein. For example, the spacer may hinder the movement of a motor protein or polynucleotide-binding protein by reducing the traction of the protein, for example, by using a debase spacer. The spacer may physically block the movement of the protein, for example, by introducing a bulky chemical group to physically hinder the movement of the polynucleotide-binding protein.

[0307] In some embodiments, one or more spacers that provide a specific signal when passing through or across nanopores are included in the polynucleotide or polynucleotide adapter. One or more spacers may be used to delineate or isolate one or more regions of the polynucleotide, for example, to isolate the adapter from the target polynucleotide.

[0308] In some embodiments, the spacer may comprise a polymer, such as a polypeptide or a linear molecule such as polyethylene glycol (PEG). Typically, such a spacer has a structure different from the target polynucleotide. For example, if the target polynucleotide is DNA, the spacer or each spacer typically does not contain DNA. In particular, if the target polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), the spacer or each spacer preferably comprises peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), roq nucleic acid (LNA), or a synthetic polymer having nucleotide side chains. In some embodiments, the spacer is 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-bromodeoxyuridines, one or more inverted thymidines (inverted dTs), one or more inverted dideoxy-thymidines (dideoxy-thymidines, ddTs), one or more dideoxy-cytidines (dideoxy-cytidines, ddCs), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2'-O-methylRNA bases, one or more isodeoxycytidines The spacers may include Iso-deoxycytidines (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 hexanediol groups, one or more spacer 9 (iSp9) [(OCH2CH2)3OPO3] groups, or one or more spacer 18 (iSp18) [(OCH2CH2)6OPO3] groups; or one or more thiol bonds. The spacers may include any combination of these groups. Many of these groups are commercially available from IDT® (Integrated DNA Technologies®). For example, C3, iSp9, and iSp18 spacers are all available from IDT®.A spacer can contain any number of upper bases as a spacer unit.

[0309] In some embodiments, the spacer may include one or more chemical groups, for example, one or more pendant chemical groups. One or more chemical groups may be bound to one or more nucleic acid bases of the polynucleotide adapter. One or more chemical groups may be bound to the backbone of the polynucleotide adapter. Any number of suitable chemical groups may exist, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more. Suitable groups, but not limited to, include fluorophores, streptavidin and / or biotin, cholesterol, methylene blue, dinitrophenols (DNPs), digoxigenin and / or anti-digoxigenin, and dibenzylcyclooctin groups.

[0310] In some embodiments, the spacer may contain one or more debasalized nucleotides (i.e., nucleotides lacking nucleic acid bases), such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 or more debasalized nucleotides. The nucleic acid bases may be replaced by -H(idSp) or -OH in the debasalized nucleotides. The debasalized spacer may be inserted into a target polynucleotide by removing nucleic acid bases from one or more adjacent nucleotides. For example, the polynucleotide may be modified to contain 3-methyladenine, 7-methylguanine, 1,N6-ethenoadenine inosine, or hypoxanthine, and the nucleic acid bases may be removed from these nucleotides using human alkyladenine DNA glycosylase (hAAG). Alternatively, the polynucleotide may be modified to contain uracil and nucleic acid bases removed with uracil-DNA glycosylase (UDG). In one embodiment, one or more spacers do not contain any debased nucleotides.

[0311] A suitable spacer may be designed or selected depending on the properties of the polynucleotide or polynucleotide adapter, the motor protein, and the conditions under which the method is performed.

[0312] tag In some embodiments, the polynucleotide or polynucleotide adapter may include a tag or tether. For example, the polynucleotide may be bound to a tag on a nanopore via its adapter and released at some point during the characterization of the polynucleotide by the nanopore.

[0313] The tag or tether does not need to be charged. This ensures that the tag or tether is not drawn into the nanopore under the influence of a potential difference.

[0314] One or more molecules that attract or bind a polynucleotide or adapter may be linked to a nanopore, for example, the nanopore described herein. Any molecule that hybridizes to the adapter and / or target polynucleotide may be used. The molecules linked to the pore may be selected from PNA tags, PEG linkers, short oligonucleotides, positively charged amino acids, and aptamers. Pores linked to such molecules are known in the art. For example, pores linked to short oligonucleotides are disclosed in Howarka et al (2001) Nature Biotech. 19:636-639 and International Publication No. 2010 / 086620, and pores containing PEG bound within the lumen of the pore are disclosed in Howarka et al (2000) J.Am.Chem.Soc. 122(11):2411-2416.

[0315] Short oligonucleotides bound to the pores, containing a sequence complementary to a sequence in the leader sequence or another single-stranded sequence in the adapter, may be used in the methods described herein to improve the capture of the target polynucleotide.

[0316] The tag or tether may comprise, or may contain, an oligonucleotide described herein, for example, 10 to 30 nucleotides in length or about 10 to 20 nucleotides in length. Such oligonucleotides may have at least one modified end (e.g., a 3' end or a 5' end) for conjugation to other modifications or, for example, to the surface of a solid substrate including beads. End modification factors can add reactive functional groups that 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 combination thereof. Functional groups can be added physically away from the ends of the oligonucleotide sequence by combining them with spacers of different lengths (e.g., C3, C9, C12, spacers 9 and 18).

[0317] In some embodiments, the tag or tether may or may not contain a morpholino oligonucleotide. The morpholino oligonucleotide may have a length of about 10 to 30 nucleotides or about 10 to 20 nucleotides. The morpholino oligonucleotide may be modified or unmodified. For example, in some embodiments, the morpholino oligonucleotide may be modified at the 3' and / or 5' ends of the oligonucleotide. Examples of modifications at the 3' and / or 5' ends of the morpholino oligonucleotide include, but are not limited to, functional groups for 3' affinity and chemical bonding (e.g., 3'-biotin, 3'-primary amine, 3'-disulfideamide, 3'-pyridyldithio, and any combination thereof); 5' end modifications (e.g., 5'-primary ammine, and / or 5'-dabsyl); modifications for click chemistry (e.g., 3'-azide, 3'-alkyne, 5'-azide, 5'-alkyne); and any combination thereof.

[0318] The tag or tether may further include a polymer linker to facilitate binding to, for example, a detector, such as a nanopore. Examples of polymer linkers include, but are not limited to, polyethylene glycol (PEG). The polymer linker may have a molecular weight of about 500 Da to about 10 kDa (inclusive of both ends), or about 1 kDa to about 5 kDa (inclusive of both ends). The polymer linker (e.g., PEG) may be functionalized with different functional groups, including, but are not limited to, maleimide, NHS esters, dibenzocyclooctyne (DBCO), azides, biotin, amines, alkynes, aldehydes, and any combination thereof. In some embodiments, the tag or tether may further include 1 kDa PEG having a 5'-maleimide group and a 3'-DBCO group. In some embodiments, the tag or tether may further include 2 kDa PEG having a 5'-maleimide group and a 3'-DBCO group. In some embodiments, the tag or tether may further comprise a 3 kDa PEG having a 5'-maleimide group and a 3'-DBCO group.

[0319] The tags may be incorporated on the nanopores using the modification methods disclosed. In some embodiments, the tags or tethers may be bound to the nanopores directly or via one or more linkers. The tags or tethers may be bound to the nanopores using hybridization linkers described in International Publication No. 2010 / 086602. Alternatively, peptide linkers may be used. Peptide linkers are amino acid sequences. The length, flexibility, and hydrophilicity of the peptide linkers are typically designed so as not to interfere with the function of the monomers and pores. Preferred flexible peptide linkers are a series of 2 to 20 serine and / or glycine amino acids, such as 4, 6, 8, 10, or 16. More preferred flexible linkers include (SG)1, (SG)2, (SG)3, (SG)4, (SG)5, and (SG)8, where S is serine and G is glycine. A preferred rigid linker is a series of 2 to 30 proline amino acids, such as 4, 6, 8, 16, or 24. A more preferred rigid linker is (P) 12 The following is given, and in the formula, P is proline.

[0320] anchor In one embodiment, the polynucleotide or polynucleotide adapter may include a membrane anchor or a transmembrane pore anchor. In one embodiment, the anchor assists in the characterization of a target polynucleotide according to the method disclosed herein. For example, the membrane anchor or transmembrane pore anchor may facilitate localization to a selected polynucleotide around a nanopore.

[0321] The anchor may be a polypeptide anchor and / or a hydrophobic anchor that can be inserted into the membrane. In one embodiment, the hydrophobic anchor is a lipid, fatty acid, sterol, carbon nanotube, polypeptide, protein, or amino acid, such as cholesterol, palmitate, or tocopherol. The anchor may also include thiols, biotin, or surfactants.

[0322] In one embodiment, the anchor may be biotin (for binding to streptavidin), amylose (for binding to maltose-binding proteins or fusion proteins), Ni-NTA (for binding to polyhistidine or polyhistidine-tagged proteins), or a peptide (such as an antigen).

[0323] In one embodiment, the anchor comprises one linker, or two, three, four or more linkers. Preferred linkers include, but are not limited to, polymers such as polynucleotides, polyethylene glycol (PEG), polysaccharides, and polypeptides. These linkers may be linear, branched, or cyclic. For example, the linker may be a cyclic polynucleotide. The adapter can hybridize to a complementary sequence on the cyclic polynucleotide linker. One or more anchors or one or more linkers may contain components that can be cleaved or degraded, such as limiting sites or photodissociable groups. The linkers may be functionalized with maleimide groups to bind to cysteine ​​residues in the protein. Preferred linkers are described in International Publication No. 2010 / 086602.

[0324] In one embodiment, the anchor is cholesterol or a fatty acyl chain. For example, any fatty acyl chain having a length of 6 to 30 carbon atoms, such as hexadecanoic acid, can be used. Examples of suitable anchors and methods for bonding the anchor to the adapter are disclosed in International Publication Nos. 2012 / 164270 and 2015 / 150786.

[0325] Further Embodiments Polyfunctional molecules suitable for use in the manner of disclosure are also provided. The polyfunctional molecules provided are typically those described in more detail herein.

[0326] kit Kits for modifying monomers are also provided. In one embodiment, the kit comprises a polyfunctional molecule disclosed herein and a support for binding the polyfunctional molecule to a purification tag. In one embodiment, the kit further comprises a buffer for performing binding and / or an optional washing step. Those skilled in the art will understand that any polyfunctional molecule and support described herein may be used in the kits of disclosure.

[0327] system A system for characterization comprising the chemically modified monomer or oligomer described herein is also provided. In one embodiment, the system comprises the chemically modified monomer or oligomer described herein, along with a motor protein for controlling the movement of an analyte, such as a polynucleotide analyte, into a pore formed from the monomer or oligomer.

[0328] In some embodiments, the system comprises a homogeneous collection of multiple monomers, typically a homogeneous collection of chemically modified monomers as described herein. In some embodiments, the system comprises a homogeneous collection of multiple oligomers, typically a homogeneous collection of chemically modified monomers as described herein. In some embodiments, the system comprises a homogeneous collection of multiple nanopores, typically a homogeneous collection of chemically modified nanopores as described herein. In some embodiments, the collection is 100% homogeneous, i.e., all monomers / oligomers / pores in the collection are chemically modified. In some embodiments, the multiple monomers / oligomers / pores are present on a chip for analyzing (e.g., characterizing) an analyte.

[0329] In some embodiments, the system further includes analytes such as polynucleotide analytes and / or other components such as membranes; membrane anchors; tethers as described herein, etc.

[0330] Further characteristics The following are numbered embodiments of the present invention. 1. A method for chemically modifying a polypeptide or polynucleotide monomer. a) Contacting a monomer with a polyfunctional molecule, wherein the polyfunctional molecule includes (i) a reactive group, (ii) a chemically modifying group, and (iii) a cleavable purified tag. b) Reacting the reactive groups of a polyfunctional molecule with a monomer, thereby attaching chemically modified groups and cleavable purified tags to the monomer to form chemically modified tagged monomers, c) Contacting the chemically modified tagged monomer formed in step (b) with the support, d) Attaching a purified tag to a support, thereby attaching a chemically modified tagged monomer to the support, e) A method comprising cleaving a purified tag and thereby releasing a chemically modified monomer from a support. 2. The polyfunctional molecule is of formula (I) or formula (II),

[0331] [ka] During the ceremony, A is a reactive group, B is a chemically modified group, DC forms a detachable refined tag, Preferably, the method according to embodiment 1, wherein D comprises a cleavable linker and C comprises a support-binding group. 3. The method according to embodiment 1, wherein the reactive group includes a chemically modified group. 4. The method according to any one of embodiments 1 to 3, wherein the monomer comprises a reactive functional group, and step (b) comprises reacting a reactive group of a polyfunctional molecule with a reactive functional group of the monomer. 5. The reactive group of the polyfunctional molecule includes an amine-reactive group; a carboxyl-reactive group; a sulfhydryl-reactive group or a carbonyl-reactive group. Preferably, the method according to any one of embodiments 1 to 4, wherein the reactive group of the polyfunctional molecule includes a cysteine-reactive group. 6. The method according to any one of embodiments 1 to 5, wherein the reactive group comprises maleimide, azide, thiol, alkyne, NHS ester, or haloacetamide. 7. Chemical modification groups introduce hydrophilic, hydrophobic, positively charged, negatively charged, hydrogen-bonded, supramolecular association, or zwitterionic properties to protein monomers. Preferably, the method according to any one of embodiments 1 and 3 to 6, wherein the chemically modifying group comprises (i) an amino acid, nucleotide, polymer, hydrogen bonding group, membrane anchor, sugar, dye, chromophore, fluorophore, or molecular adapter; or (ii) a natural or unnatural amino acid, polypeptide, nucleotide or nucleotide analog, oligonucleotide or oligonucleotide analog, polysaccharide, lipid, polyethylene glycol, cyclodextrin, DNA intercalator, aptamer, or analyte-binding domain. 8. The method according to any one of embodiments 1 to 7, wherein the support comprises a chromatography matrix, preferably agarose or Sepharose resin; one or more beads, preferably magnetic beads; or a solid surface, preferably glass, silica, polymer, or ceramic surface. 9. The method according to any one of embodiments 1 to 8, wherein the support is functionalized for binding to a purified tag. 10. The method according to any one of embodiments 1 to 9, wherein the purified tag contains a biotin group, and the support contains streptavidin, neutraavidin, or avidin, preferably streptavidin. 11. A severable linker is cut by physical or chemical means. Preferably, the method according to any one of embodiments 1 to 10, wherein the severable linker includes a UV-cuttable nito-benzyl portion. 12. The method according to any one of embodiments 1 to 11, wherein step (e) comprises cleaving the purified tag, which involves exposing the support and / or tagged monomer to light, preferably UV light. 13. The method according to any one of embodiments 1 to 12, wherein step (e) comprises cleaving the purified tag, which involves exposing the support and / or tagged monomer to a change in pH. 14. The method according to any one of embodiments 1 to 13, wherein step (e) comprises cleaving the purified tag, exposing the support and / or tagged monomer to a chemical reagent, preferably a reducing reagent. 15. The method according to any one of embodiments 1 to 14, wherein step (e) comprises cleaving the purified tag, which involves exposing the support and / or tagged monomer to an enzyme, preferably a protease. 16. The method according to any one of embodiments 1 to 15, wherein the monomer is a peptide having a mass of about 10 kDa to about 1 MDa. 17. The method according to any one of embodiments 1 to 16, wherein the monomer is a monomer of an oligomeric protein pore, preferably a lysenin pore, a γ-hemolidine pore, an α-hemolidine pore; a NetB pore; a CytK pore, or a leucosidine pore; or a homolog or paralog thereof. 18. The method according to embodiment 16, wherein the oligomeric protein pore is a multicomponent pore. 19. The method according to any one of embodiments 1 to 15, wherein the monomer is a polynucleotide, and preferably the monomer is a monomer of DNA origami pores. 20. When a monomer is a monomer of an oligomeric pore, and the monomer is oligomerized to form a pore, the polyfunctional molecule reacts with the reactive functional group located on the monomer at the surface-exposed position. Preferably, the method according to any one of embodiments 1 to 19, wherein the surface exposure position is located on the surface of the channel passing through the pore, or on the outer surface of the pore. 21. The method according to embodiment 20, wherein when a monomer is oligomerized to form a pore, a polyfunctional molecule reacts with a reactive functional group located on the monomer at a position located at or near the constriction of a channel passing through the pore. 22. The method according to any one of embodiments 1 to 21, wherein the method comprises, prior to step (a), (i) expressing a monomer in a cell expression system or a cell-free expression system, and (ii) isolating and / or purifying the monomer. 23. The method according to any one of embodiments 1 to 22, wherein step (d) further comprises removing unmodified monomers and / or unreacted polyfunctional molecules (if present) from the support. twenty four. f) Further comprising the step of oligomerizing a chemically modified monomer to form a chemically modified oligomer, Preferably, the method according to any one of embodiments 1 to 23, wherein the monomer is a protein monomer, and step (f) comprises forming an oligomeric protein. 25. Step (f) comprises oligomerizing two or more chemically modified monomers to form a homooligomer, Preferably, the method according to embodiment 24, wherein the monomer is a protein monomer and the homooligomer is a homooligomeric protein pore. 26. Step (f) comprises oligomerizing one or more chemically modified monomers with one or more unmodified or differently modified monomers to form a heterooligomer, Preferably, the method according to embodiment 24, wherein the monomer is a protein monomer and the heterooligomer is a heterooligomeric protein pore. 27. Step (f) comprises oligomerizing one or more chemically modified first monomers with one or more chemically modified second monomers to form a heterooligomer, wherein the chemical modification performed on the first monomer is the same as or different from the chemical modification performed on the second monomer. Preferably, the monomer is a protein monomer, the first monomer has a different amino acid sequence from the second monomer, and the heterooligomer is a heterooligomeric protein pore, according to embodiment 24. 28. A method for producing homooligomeric proteins, i) A method according to any one of embodiments 1 to 23, which produces a plurality of chemically modified protein monomers, ii) oligomerizing two or more of the chemically modified protein monomers obtained in step (i) to form a homo-oligomeric protein, A method wherein the homo-oligomeric protein is preferably a homo-oligomeric protein pore. 29. A method for producing heterooligomeric proteins, i) Producing one or more chemically modified first protein monomers by the method described in any one of embodiments 1 to 23, ii) Producing one or more chemically modified second protein monomers by the method described in any one of embodiments 1 to 23, iii) oligomerizing one or more first monomers and one or more second monomers to form a hetero-oligomeric protein, A method wherein the hetero-oligomeric protein is preferably a hetero-oligomeric protein pore. 30. A method for producing oligomeric proteins, i) Producing one or more chemically modified first protein monomers by the method described in any one of embodiments 1 to 23, ii) To provide one or more unmodified second protein monomers, iii) oligomerizing one or more first monomers and one or more second monomers to form a hetero-oligomeric protein, A method wherein the hetero-oligomeric protein is preferably a hetero-oligomeric protein pore. 31. A chemically modified monomer that can be obtained by performing the method described in any one of embodiments 1 to 23. 32. A homogeneous population comprising multiple chemically modified monomers, wherein at least 95% of the monomers in the population are chemically modified with a chemical modification group. A homogeneous group of chemically modified monomers, preferably as defined in any one of embodiments 5 to 7 or 16 to 21. 33. A chemically modified oligomer that can be obtained by performing the method described in any one of embodiments 24 to 30. 34. A homogeneous population comprising multiple chemically modified oligomers, wherein at least 95% of the oligomers in the population contain a specified number of monomers chemically modified with chemically modifying groups. A homogeneous group of chemically modified monomers, preferably as defined in any one of embodiments 5 to 7 or 16 to 21. 35. A method for characterizing an analyte, i) To generate chemically modified oligomer pores by the method described in any one of embodiments 24 to 30, or to provide chemically modified oligomer pores as described in embodiment 33, ii) Obtaining one or more measurements as the analyte moves into the pore, wherein one or more measurements indicate one or more characteristics of the analyte, thereby characterizing the analyte as it moves into the pore, Preferably, the method wherein the analyte is a polynucleotide, polypeptide, or polysaccharide. While specific embodiments, configurations, and materials and / or molecules of the methods according to the present invention have been discussed herein, it is understood that various changes or modifications in form and detail can be made without departing from the scope and spirit of the invention. The following examples are provided to better illustrate specific embodiments and should not be considered as limiting this application. This application is limited only by the claims. [Examples]

[0332] These examples describe the modification of polypeptide monomers by the claimed method and the subsequent use of oligomeric pores generated from such monomers in the characterization of analytes.

[0333] Example 1 General synthesis of modification factor molecules containing UV-cleavable linker sites and amino acids as modification B)

[0334] [ka] 2-ethylaminomaleimide was reacted with the -COOH terminus of a preferred amino acid to form a maleimide-amino acid adduct. This was purchased from Cambridge Research Biochemicals as a custom-synthesized molecule. A biotin tag containing a photocleavable linker was purchased from Broadpharm USA (catalog number BP-24161 / PC biotin-PEG3-NHS carbonate). 0.04 mmol of the maleimide-amino acid adduct was dissolved in 1 ml of DMF, and 1 equivalent (mol / mol) of biotin photocleavable linker was added. 1.5 equivalents (mol / mol) of TEA were added to the mixture, and the mixture was stirred overnight. Molecular formation was confirmed by mass spectrometry and used for protein / peptide modification without further purification.

[0335] 1. Isoleucine as a chemically modified group Maleimide-isoleucine adduct (MW-253.2 g / mol) was purchased from Cambridge Research Biochemicals as a custom-synthesized molecule. A biotin tag containing a photocleavable linker was purchased from Broadpharm USA (catalog number BP-24161 / PC Biotin-PEG3-NHS carbonate MW-840.9 g / mol). 0.04 mmol of maleimide-isoleucine adduct was dissolved in 1 ml of DMF, and 1 equivalent (mol / mol) of biotin photocleavable linker was added. 1.5 equivalents (mol / mol) of TEA were added to the mixture, and the mixture was stirred overnight. Molecular formation was confirmed by mass spectrometry, and the mixture was used for protein / peptide modification without further purification.

[0336] 2-Alanine as a chemically modified group Maleimide-alanine adduct (MW-211.2 g / mol) was purchased from Cambridge Research Biochemicals as a custom-synthesized molecule. A biotin tag containing a photocleavable linker was purchased from Broadpharm USA (catalog number BP-24161 / PC Biotin-PEG3-NHS carbonate MW-840.9 g / mol). 0.04 mmol of maleimide-alanine adduct was dissolved in 1 ml of DMF, and 1 equivalent (mol / mol) of biotin photocleavable linker was added. 1.5 equivalents (mol / mol) of TEA were added to the mixture, and the mixture was stirred overnight. Molecular formation was confirmed by mass spectrometry, and the mixture was used for protein / peptide modification without further purification.

[0337] 3. Arginine as a chemically modified group Maleimide-arginine adduct (MW-296.3 g / mol) was purchased from Cambridge Research Biochemicals as a custom-synthesized molecule. A biotin tag containing a photocleavable linker was purchased from Broadpharm USA (catalog number BP-24161 / PC Biotin-PEG3-NHS carbonate MW-840.9 g / mol). 10 mg (0.04 mmol) of the maleimide-arginine adduct was dissolved in 1 ml of DMF, and 1 equivalent (mol / mol) of the biotin photocleavable linker was added. 1.5 equivalents (mol / mol) of TEA were added to the mixture, and the mixture was stirred overnight. Molecular formation was confirmed by mass spectrometry, and the mixture was used for protein / peptide modification without further purification.

[0338] 4. Aspartic acid as a chemically modifying group Maleimide-aspartate adduct (MW-255.2 g / mol) was purchased from Cambridge Research Biochemicals as a custom-synthesized molecule. A biotin tag containing a photocleavable linker was purchased from Broadpharm USA (catalog number BP-24161 / PC Biotin-PEG3-NHS carbonate MW-840.9 g / mol). 10 mg (0.04 mmol) of maleimide-aspartate adduct was dissolved in 1 ml of DMF, and 1 equivalent (mol / mol) of biotin photocleavable linker was added. 1.5 equivalents (mol / mol) of TEA were added to the mixture, and the mixture was stirred overnight. Molecular formation was confirmed by mass spectrometry, and the mixture was used for protein / peptide modification without further purification.

[0339] 5. Asparagine as a chemically modified group Maleimide-asparagine adduct (MW-254.2 g / mol) was purchased from Cambridge Research Biochemicals as a custom-synthesized molecule. A biotin tag containing a photocleavable linker was purchased from Broadpharm USA (catalog number BP-24161 / PC Biotin-PEG3-NHS carbonate MW-840.9 g / mol). 10 mg (0.04 mmol) of maleimide-asparagine adduct was dissolved in 1 ml of DMF, and 1 equivalent (mol / mol) of biotin photocleavable linker was added. 1.5 equivalents (mol / mol) of TEA were added to the mixture, and the mixture was stirred overnight. Molecular formation was confirmed by mass spectrometry, and the mixture was used for protein / peptide modification without further purification.

[0340] 6. Glutamine as a chemically modified group Maleimide-glutamine adduct (MW-266.2 g / mol) was purchased from Cambridge Research Biochemicals as a custom-synthesized molecule. A biotin tag containing a photocleavable linker was purchased from Broadpharm USA (catalog number BP-24161 / PC Biotin-PEG3-NHS carbonate MW-840.9 g / mol). 10 mg (0.04 mmol) of maleimide-glutamine adduct was dissolved in 1 ml of DMF, and 1 equivalent (mol / mol) of biotin photocleavable linker was added. 1.5 equivalents (mol / mol) of TEA were added to the mixture, and the mixture was stirred overnight. Molecular formation was confirmed by mass spectrometry, and the mixture was used for protein / peptide modification without further purification.

[0341] 7. Phenylalanine as a chemically modified group Maleimide-phenylalanine adduct (MW-287.3 g / mol) was purchased from Cambridge Research Biochemicals as a custom-synthesized molecule. A biotin tag containing a photocleavable linker was purchased from Broadpharm USA (catalog number BP-24161 / PC Biotin-PEG3-NHS carbonate MW-840.9 g / mol). 0.04 mmol of maleimide-phenylalanine adduct was dissolved in 1 ml of DMF, and 1 equivalent (mol / mol) of biotin photocleavable linker was added. 1.5 equivalents (mol / mol) of TEA were added to the mixture, and the mixture was stirred overnight. Molecular formation was confirmed by mass spectrometry, and the mixture was used for protein / peptide modification without further purification.

[0342] 8. Lysine as a chemical modification group The maleimide-lysine adduct (MW-268.3 g / mol) was purchased from Cambridge Research Biochemicals as a custom-synthesized molecule. The biotin tag containing a photocleavable linker was purchased from Broadpharm USA (catalog number BP-24161 / PC biotin-PEG3-NHS carbonate MW-840.9 g / mol). 0.04 mmol of the maleimide-lysine adduct was dissolved in 1 ml of DMF, and 1 equivalent (mol / mol) of the biotin photocleavable linker was added. 1.5 equivalents (mol / mol) of TEA were added to the mixture, and the mixture was stirred overnight. Molecular formation was confirmed by mass spectrometry, and the mixture was used for protein / peptide modification without further purification.

[0343] 9. PNA (thymine) as a chemical modification group Maleimide-PNA(thymine) adduct (MW-406.3 g / mol) was purchased from Cambridge Research Biochemicals as a custom-synthesized molecule. A biotin tag containing a photocleavable linker was purchased from Broadpharm USA (catalog number BP-24161 / PC Biotin-PEG3-NHS carbonate MW-840.9 g / mol). 10 mg (0.04 mmol) of maleimide-PNA(thymine) adduct was dissolved in 1 ml of DMF, and 1 equivalent (mol / mol) of biotin photocleavable linker was added. 1.5 equivalents (mol / mol) of TEA was added to the mixture, and the mixture was stirred overnight. Molecular formation was confirmed by mass spectrometry, and the mixture was used for protein / peptide modification without further purification.

[0344] 10. CH2-NH2 as a chemically modified group Maleimide-CH2-NH2 was conjugated to a biotin tag containing a photocleavable linker according to the examples described above and used for the modification of the described proteins / peptides.

[0345] Figure 2 shows the cleavage of the polyfunctional molecules described in Examples 1 to 10.

[0346] Example 2 General modification of protein monomers by modifying factor molecules Chemical modification of cysteine ​​variants (generally - e.g., leucocidine and lysenin) To avoid disulfide bond formation, Cys variants of protein monomers were stored with a reducing agent (e.g., -DTT-dithiol-threitol). The reducing agent was removed from the purified monomer solution by buffer exchange on a 7K MWCO desalting column using 50 mM Tris-HCl, 150 mM NaCl, pH 7. The selected linker was added to each sample and allowed to stand at room temperature (RT) for 16 hours to allow the monomers to be bound to the chemical modifiers via maleimide-cysteine ​​(thiol) chemistry. Unreacted linker molecules were removed from the solution by passing through a 7K MWCO desalting column and centrifugation at 1500 rcf for 2 minutes. The flow-through was added to equilibrated StrepTactin® Sepharose High Performance resin (GE Healthcare Life Sciences, catalog no. 28935600) and allowed to stand on a rotating apparatus at 23°C for 2 hours to allow the modified monomers to be bound to beads via their biotin tags. Unbound / unmodified protein monomers were removed by centrifugation at 1500 rcf for 2 minutes in three washing steps using buffer. After washing, the same buffer was added to StrepTactin® resin, which was then subjected to two UV light exposures at 30-second intervals to cleave the photocleavable linker, releasing the modified monomers from the beads, which were collected in a flow-through.

[0347] This general modification is shown in Figure 3.

[0348] Chemical modifications of 1-lysenin mutants Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / S98C / C272A / C283A) Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / S98C / C272A / C283A)TEV, cleaved to remove the thioredoxin-strep portion, was used as the starting material. The monomer was buffered once with 50 mM Tris-HCl, 150 mM NaCl, pH 7, and the DTT solution was removed. 3 μl of maleimide-isoleucine adduct at 10 mg / mL in DMF was added to 120 μL of Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / S98C / C272A / C283A) that had been buffered with 0.6 mg / mL. The solution was left at RT for 16 hours. Unreacted linker molecules were removed from the solution by passing the mixture through a 7K MWCO desalting column and centrifuging at 1500 rcf for 2 minutes. The flow-through was added to equilibrated StrepTactin® Sepharose High Performance resin (GE Healthcare Life Sciences, catalog no. 28935600) and left on a rotating apparatus at 23°C for 2 hours to allow the modified monomers to bind to the beads via their biotin tags. Unbound / unmodified protein monomers were removed by centrifugation at 1500 rcf for 2 minutes in three washing steps using buffer. After washing, the same buffer solution was added to the StrepTactin® resin, and it was exposed to UV light twice at 30-second intervals to cleave the photocleavable linker, releasing the modified monomer from the beads. This was collected in a flow-through as the modified monomer -Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / S98C-maleimide-isoleucine / C272A / C283A). The success of the modification was confirmed by SDS-PAGE electrophoresis on 4-20% gels (Figure 4).

[0349] Chemical modifications of 2-lysenin mutants Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / T63C / C272A / C283A) Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / T63C / C272A / C283A)TEV, cleaved to remove the thioredoxin-strep portion, was used as the starting material. The monomer was buffered once with 50 mM Tris-HCl, 150 mM NaCl, pH 7, and the DTT solution was removed. 3 μl of maleimide-isoleucine adduct at 10 mg / mL in DMF was added to 120 μL of Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / T63C / C272A / C283A) that had been buffered with 0.6 mg / mL. The solution was left at RT for 16 hours. Unreacted linker molecules were removed from the solution by passing the mixture through a 7K MWCO desalting column and centrifuging at 1500 rcf for 2 minutes. The flow-through was added to equilibrated StrepTactin® Sepharose High Performance resin (GE Healthcare Life Sciences, catalog no. 28935600) and left on a rotating apparatus at 23°C for 2 hours to allow the modified monomers to bind to the beads via their biotin tags. Unbound / unmodified protein monomers were removed by centrifugation at 1500 rcf for 2 minutes in three washing steps using buffer. After washing, the same buffer solution was added to the StrepTactin® resin, and it was exposed to UV light twice at 30-second intervals to cleave the photocleavable linker, releasing the modified monomer from the beads. This was collected in a flow-through as the modified monomer Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / T63C-maleimide-PNA (thymine) / C272A / C283A). The success of the modification was confirmed by SDS-PAGE electrophoresis on 4-20% gels. (Figure 5)

[0350] Chemical modification of the 3-gamma-hemolyzin mutant, LukF-T142C-Del(E1-K15) The DTT solution was removed by changing the buffer once with LukF-T142C-Del(E1-K15)TEV monomer to 50 mM Tris-HCl, 150 mM NaCl, pH 7. 3 µl of maleimide-alanine adduct at 10 mg / mL in DMF was added to 120 µl of LukF-T142C-Del(E1-K15) that had been buffered with 0.6 mg / mL. The solution was left to stand at RT for 16 hours. Unreacted linker molecules were removed from the solution by passing it through a 7K MWCO desalting column and centrifugation at 1500 rcf for 2 minutes. The flow-through was added to equilibrated StrepTactin® Sepharose High Performance resin (GE Healthcare Life Sciences, catalog no. 28935600) and left on a rotating apparatus at 23°C for 2 hours to allow the modified monomers to bind to beads via their biotin tags. Unbound / unmodified protein monomers were removed by centrifugation at 1500 rcf for 2 minutes in three washing steps using buffer. After washing, the same buffer was added to StrepTactin® resin, and the photocleavable linker was cleaved by two exposures of UV light at 30-second intervals, releasing the modified monomer from the beads. This was collected in a flow-through as the modified monomer-LukF-T142C-maleimide-alanine-Del(E1-K15). The success of the modification was confirmed by SDS-PAGE electrophoresis on 4-20% gels. (Figure 6)

[0351] Chemical modification of the 4-gamma-hemolyzin mutant, Hlg2-G114C-Del(E1-G10) The Hlg2-G114C-Del(E1-G10) monomer was buffered once with 50 mM Tris-HCl, 150 mM NaCl, pH 7 to remove the DTT solution. 3 μl of maleimide-isoleucine adduct at 10 mg / mL in DMF was added to 120 μL of 0.6 mg / mL buffered Hlg2-G114C-Del(E1-G10). The solution was left to stand at RT for 16 hours. Unreacted linker molecules were removed from the solution by passing it through a 7K MWCO desalting column and centrifugation at 1500 rcf for 2 minutes. The flow-through was added to equilibrated StrepTactin® Sepharose High Performance resin (GE Healthcare Life Sciences, catalog no. 28935600) and left on a rotating apparatus at 23°C for 2 hours to allow the modified monomers to bind to beads via their biotin tags. Unbound / unmodified protein monomers were removed by centrifugation at 1500 rcf for 2 minutes in three washing steps using buffer. After washing, the same buffer was added to StrepTactin® resin, and the photocleavable linker was cleaved by two exposures of UV light at 30-second intervals, releasing the modified monomer from the beads. This was collected in a flow-through as the modified monomer-Hlg2-G114C-maleimide-isoleucine-Del(E1-G10). The success of the modification was confirmed by SDS-PAGE electrophoresis on 4-20% gels. (Figure 7)

[0352] Further chemical modifications of 5-gamma-hemolyzin mutants, LukF-T142C-Del(E1-K15) and Hlg2-N130C-Del(E1-G10). Monomers of the LukF and Hlg2 mutant subunits of gamma-hemolidine were modified with maleimide-asparagine, maleimide-CH2-NH2, maleimide-arginine, maleimide-isoleucine, and maleimide-aspartic acid according to the procedure described above. The success of the modifications was confirmed by SDS-PAGE electrophoresis (Figures 14 and 15).

[0353] Example 3 Oligomerization of modified lysenin monomers to form oligomeric pores 1-Lysenin-[Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / S98C-maleimide-icoleucine / C272A / C283A)]9 A custom 5-lipid mixture designed to replicate rabbit blood cells was ordered from Encapsula Nanosciences with the following composition: phosphatidylserine (0.325 mg / ml), POPE (0.55 mg / ml), Egg PC (0.9 mg / ml), Sphingomyelin (0.275 mg / ml), Cholesterol (0.45 mg / ml). The modified monomer, Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / S98C-maleimide-icoleucine / C272A / C283A) solution and five lipid mixtures were combined in a 1:1 ratio, thoroughly mixed, and left at 23°C for 16 hours. The mixture was centrifuged at 21,000 rcf for 10 minutes to pelletize the sphingomyelin / pore complex. The supernatant was removed, and the pellet was resuspended in buffer (50 mM Tris, 150 mM NaCl, 2% SDS, pH 9). The mixture was heated at 60°C for 2 hours to release the formed pores from the lipid-protein complex. After the tube was cooled to room temperature, it was centrifuged at 21,000 rcf for 10 minutes. The supernatant was collected and diluted 10-fold in 50 mM Tris, 150 mM NaCl, pH 9 to obtain a final SDS concentration of 0.2% in the final pore solution. The success of oligomerization was confirmed by SDS-PAGE electrophoresis on a 4-20% gel. (Figure 8)

[0354] 2-Lysenine-[Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / T63C-maleimide-PNA(thymine) / C272A / C283A)]9 A custom 5-lipid mixture designed to replicate rabbit blood cells was ordered from Encapsula Nanosciences with the following composition: phosphatidylserine (0.325 mg / ml), POPE (0.55 mg / ml), Egg PC (0.9 mg / ml), sphingomyelin (0.275 mg / ml), and cholesterol (0.45 mg / ml). The modified monomer, Lys-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / S98C-maleimide-icoleucine / C272A / C283A) solution and the 5-lipid mixture were combined in a 1:1 ratio, thoroughly mixed, and left at 23°C for 16 hours. The mixture was centrifuged at 21,000 rcf for 10 minutes to pelletize the sphingomyelin / pore complex. The supernatant was removed, and the pellet was resuspended in buffer (50 mM Tris, 150 mM NaCl, 2% SDS, pH 9). The mixture was heated at 60°C for 2 hours to release the formed pores from the lipid-protein complex. After cooling the tube to room temperature, it was centrifuged at 21,000 rcf for 10 minutes. The supernatant was collected and diluted 10-fold in 50 mM Tris, 150 mM NaCl, pH 9 to obtain a final SDS concentration of 0.2% in the final pore solution. The success of oligomerization was confirmed by SDS-PAGE electrophoresis on 4-20% gels. (Figure 9)

[0355] Example 4 Oligomerization of modified gamma-hemolidine monomers to form oligomeric pores General oligomerization protocols for modified gamma-hemolyzin pores The monomer solutions were concentrated to 0.5 mg / ml each, and the LukF and Hlg2 components were combined in a 1:1 ratio. "Leukocyte" liposomes were added (final concentration 17%), and the solution was incubated at 37°C for 16 hours to induce oligomerization. SDS was added to the oligomer sample (final concentration 1.7%), and the sample was sonicated in a bath for 10 minutes. The success of oligomerization was confirmed by SDS-PAGE electrophoresis on 4-20% gels.

[0356] "Leukocyte" preparation To prepare liposomes with the lipid composition of human leukocytes, 965 μl of 5 mg / ml phosphatidylcholine (38.6%), 197 μl of 25 mg / ml phosphatydulethanoamine (33.4%), 18.75 μl of 100 mg / ml phosphatidylserine (15%), 13.1 μl of 100 mg / ml sphingomyelin (10.5%), and 6.5 μl of 25 mg / ml cardiolipid (1.3%) (all from Avanti Polar Lipids) were combined in chloroform in a glass vial and sonicated in a bath for 15 minutes. The organic solvent was evaporated using a nitrogen stream, and 1 ml of buffer (50 mM HEPES, 30 mM NaCl, pH 7.5) was added to the vial to solubilize the lipids. The mixture was vortexed and then sonicated in a bath for a further 15 minutes. It was then passed through a 0.4 μm filter and extruded. The liposomes were stored at 4°C.

[0357] 1-Gamma-hemolyzin-[(LukF-T142C-maleimide-isoleucine-Del(E1-K15))4(Hlg2-G114C-maleimide-alanine-Del(E1-G10))4] Modified monomer solutions, (LukF-T142C-maleimide-isoleucine-Del(E1-K15)) and (Hlg2-G114C-maleimide-alanine-Del(E1-G10)), were each concentrated to 0.5 mg / ml, and the LukF and Hlg2 components were combined in a 1:1 ratio. "Leukocyte" liposomes were added (final concentration 17%), and the solution was incubated at 37°C for 16 hours to induce oligomerization. SDS was added to the oligomer sample (final concentration 1.7%), and the sample was sonicated in a bath for 10 minutes. The success of oligomerization was confirmed by SDS-PAGE electrophoresis on a 4-20% gel (Rubi stained) - Figure 10 (lane 3).

[0358] 2-Gamma-hemolyzin-[(LukF-T142C-maleimide-isoleucine-Del(E1-K15))4(Hlg2-G114C-maleimide-isoleucine-Del(E1-G10))4] Modified monomer solutions, (LukF-T142C-maleimide-isoleucine-Del(E1-K15)) and (Hlg2-G114C-maleimide-isoleucine-Del(E1-G10)), were each concentrated to 0.5 mg / ml, and the LukF and Hlg2 components were combined in a 1:1 ratio. "Leukocyte" liposomes were added (final concentration 17%), and the solution was incubated at 37°C for 16 hours to perform oligomerization. SDS was added to the oligomer sample (final concentration 1.7%), and the sample was sonicated in a bath for 10 minutes. The success of oligomerization was confirmed by SDS-PAGE electrophoresis on a 4-20% gel (Rubi stained) - Figure 10 (lane 4).

[0359] 3-Gamma-hemolyzin-[(LukF-T142C-maleimide-alanine-Del(E1-K15))4(Hlg2-G114C-maleimide-alanine-Del(E1-G10))4] Modified monomer solutions, (LukF-T142C-maleimide-alanine-Del(E1-K15)) and (Hlg2-G114C-maleimide-alanine-Del(E1-G10)), were each concentrated to 0.5 mg / ml, and the LukF and Hlg2 components were combined in a 1:1 ratio. "Leukocyte" liposomes were added (final concentration 17%), and the solution was incubated at 37°C for 16 hours to perform oligomerization. SDS was added to the oligomer sample (final concentration 1.7%), and the sample was sonicated in a bath for 10 minutes. The success of oligomerization was confirmed by SDS-PAGE electrophoresis on a 4-20% gel (Rubi stained) - Figure 10 (lane 5).

[0360] 4-Gamma-hemolyzin-[(LukF-T142C-maleimide-alanine-Del(E1-K15))4(Hlg2-G114C-maleimide-isoleucine-Del(E1-G10))4] Modified monomer solutions, (LukF-T142C-maleimide-alanine-Del(E1-K15)) and (Hlg2-G114C-maleimide-isoleucine-Del(E1-G10)), were each concentrated to 0.5 mg / ml, and the LukF and Hlg2 components were combined in a 1:1 ratio. "Leukocyte" liposomes were added (final concentration 17%), and the solution was incubated at 37°C for 16 hours to induce oligomerization. SDS was added to the oligomer sample (final concentration 1.7%), and the sample was sonicated in a bath for 10 minutes. The success of oligomerization was confirmed by SDS-PAGE electrophoresis on a 4-20% gel (Rubi stained) - Figure 10 (lane 6).

[0361] 5-Gamma-hemolyzin-[(LukF-T142C-maleimide-isoleucine-Del(E1-K15))4(Hlg2-WT)4] The modified monomer solution (LukF-T142C-maleimide-isoleucine-Del(E1-K15)) and the wild-type (WT) Hlg2 component were combined and oligomerized as described above. The success of the oligomerization was confirmed by SDS-PAGE as shown above (Figure 16, lane 3).

[0362] Example 5 General methods, protein synthesis and purification DNA synthesis (leucocidine and lysenin) All constructs were cloned into pT7 expression vectors and validated by Sanger Sequencing (Source Bioscience). Genes encoding point mutations were generated by PCR mutagenesis using restriction endonucleases (NdeI / HindIII). A set of cloning primers was designed to introduce the desired mutations onto the DNA template. The DNA template was digested at specific recognition sites using restriction enzymes (NdeI / EconI-HindIII / BamHI). Mutagenic primers were then added to the digested DNA template, and PCR was performed using Q5HotStart DNA polymerase (NEB, catalog number M0494S). Genes encoding point mutations were generated by in vivo recombination using XL10 Gold Ultracompetent Cells (Agilent, catalog number 200315) and ampicillin agar plates. One colony was inoculated into LB medium and grown at 37°C for 16 hours. DNA plasmid preparations were made using the Qiagen Plasmid Plus Midi Kit (Qiagen, catalog number 12945).

[0363] Protein expression and purification (leucocidine) Hlg2, LukF, and their variants were overexpressed and separately purified as soluble monomer proteins. Transformed Escherichia coli strain Lemo21(DE3) (NEB, catalog number C2528J) containing expression vectors encoding the desired LukF or Hlg2 monomer was grown at 37°C in LB medium supplemented with 41 ug / uL chloramphenicol and 100 ug / uL carbenicillin until the logarithmic growth phase was achieved. Expression of the desired protein was induced using isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM. The temperature was reduced to 25°C and expression was continued for 18 hours. Cells were collected by centrifugation at 6000×g at 4°C for 20 minutes. The pellet was resuspended in buffer (50mM HEPES, 300mM NaCl, 2mM EDTA, 0.1% DDM, 1×bugbuster, benzonase nuclease, protease inhibitor tablets, pH 8), then sonicated to disrupt the cells, and dissolved in a magnetic stirrer for 4 hours. The lysate was clarified at 39,000×g at 4°C for 35 minutes. The supernatant was diluted 10-fold with 50mM HEPES and packed into an HS50 cation exchange column (Poros Media by Applied BioSciences), and used with a gradually increasing NaCl gradient for elution. The fraction containing the desired protein was further purified using a Superdex75 10 / 300GL size exclusion column (GE Healthcare Life Sciences).

[0364] Protein expression and purification (lysenin) Transformed Escherichia coli strain BL21(DE3) (NEB, catalog number C2527H) containing an expression vector encoding a desired lysenin monomer including the thioredoxin-strep moiety was grown at 37°C in TB medium supplemented with 0.1 mg / ml ampicillin until the logarithmic growth phase was achieved (OD600 0.8). Expression of the desired protein was induced using isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM. The temperature was reduced to 18°C ​​and expression was continued for 16 hours. Cells were collected by centrifugation at 6000×g at 4°C for 20 minutes. The pellet was resuspended in buffer (50 mM Tris-HCl, 0.5 M NaCl, 2 mM DTT, benzonase nuclease, protease inhibitor, 2 mM EDTA, pH 8.0) and left in a magnetic stirrer for 3 hours, then disrupted by sonication. The lysate was clarified at 45,000 × g, 4°C for 60 minutes. The supernatant was packed into a 5 ml StrepTrap® High Performance column (GE Healthcare Life Sciences, catalog no. 28907548), and the protein was eluted with 2 mg / ml desthiobiotin. The fraction containing the desired protein was further purified using a Superdex200 increase10 / 300GL size exclusion column (GE Healthcare Life Sciences, catalog no. 28990944) and analyzed by SDS-PAGE. The peak fraction was pooled and cleaved with TEV-strep protease to remove the thioredoxin-strep portion. The mixture was returned to a StrepTrap® High Performance column and packed to obtain untagged lysenin monomers in flow-through. The final eluate was concentrated using an Amicon® Ultra-15 Centrifugal Filter Unit (Merck Millipore, catalog no. UFC901024).

[0365] Example 6 Oligomerized and chemically modified pores, generated according to the disclosed method, were assembled into a nanopore detection device and used to characterize polynucleotide analytes.

[0366] Electrical methods Electrical measurements were obtained from various lysenine nanopores (chemically modified and unmodified according to the method of disclosure) inserted into block copolymer membranes of a MinION flow cell (Oxford Nanopore Technologies). Lysenine pore samples in 0.2% SDS (V / V) were incubated with Brij58 (final concentration 0.1% (V / V)) at room temperature for 10 minutes. For pore insertion, pore samples (0.05 ug / mL) were diluted in MinION flow cell buffer (25 mM potassium phosphate, 150 mM potassium ferrocyanide, 150 mM potassium ferricyanide, pH 8.0). All pore experiments were performed using a MinION Mk1b device (Oxford Nanopore Technologies, ONT). Scripts were controlled throughout all experiments using MinKNOW core version 1.11.5 software developed and provided by ONT.

[0367] Pore ​​insertion For pore insertion, 300 μL of diluted pore sample was packed into the priming port of the flow cell. Using the MinKNOW pore insertion script, voltage was applied starting at -100 mV and increasing by 10 mV every 15 seconds up to -450 mV. Any excess pores were removed by perfusing 1 mL of flow cell buffer through the priming port. The groups and locations with single pores were evaluated using a standard flow cell check protocol with MinKNOW.

[0368] 3.6kb library / analyte preparation A 3.6kb double-stranded DNA analyte (SEQ ID NO: 20) was prepared using specific primers and PCR. The PCR product was subjected to NEBNext end repair and the NEBNext dA-tailing module (New England Biolabs (NEB)) to generate a 3'A overhang.

[0369] Ligation of the Y adapter to the analyte A 1 μg portion of 3.6 kb analyte was ligated in 100 μL volume to AMX from the Oxford Nanopore Technologies sequencing kit (LSK-SQK109) using LNB and T4 DNA ligase (NEB) from (LSK-SQK109). The sample was purified using Agencourt AMPure XP (Beckman Coulter) beads and washed twice with LFB / SFB from the Oxford Nanopore Technologies sequencing kit (LSK-SQK109). The ligated substrate was eluted in EB from (LSK-SQK109) according to all manufacturer guidelines. This is also referred to as the "3.6 kb lambda DNA library".

[0370] Flow cell filling For flow cell packing, 800 μL of FLB from the Oxford Nanopore Technologies sequencing kit (SQK-LSK109), prepared using FLT (SQK-LSK109), was flowed through the system, waited for 5 minutes, and then 200 μL of FLB+FT was flowed through the system with the SpotON port open. 37.5 μL of SQB from the Oxford Nanopore Technologies sequencing kit (SQK-LSK109), 12 μL of recovered beads, a purified 3.6 kb lambda DNA library (the preparation described below), and 25.5 μL of LLB (SQK-LSK109) were mixed. 75 μL of the 3.6 kb sequencing mixture was added to the MinION flow cell using the SpotOn flow cell port. 75 μL of the sequencing mixture was added to the MinION flow cell using the SpotOn flow cell port. Using MinKNOW software (Oxford Nanopore Technologies), raw data was collected at -180mV (acquisition frequency of 4000kHz) to monitor helicase-controlled DNA movement.

[0371] Figure 11 shows the feature data for the polynucleotide analyte of SEQ ID NO: 20, when featured using a panel of chemically modified lysenine pores generated according to the method of disclosure. Two lysenine pores: Lys-(E84Q / E85S / E92Q / E94D / E97S / S98C / T106K / D126G / C272A / C283A) and Lys-(T63C / E84Q / E85S / E92Q / E94D / E97S / T106K / D126G / C272A / C283A) The lysenin pores were modified with five different chemical molecules, and the effects of the modifications were analyzed by comparing the open-pore current levels of the modified pores with those of the unmodified pores as lambda DNA passed through each pore. The modifications made to the lysenin pores covered aromatic, aliphatic, hydrophobic, hydrophilic, positively charged, and H-binding residues, including DNA base-binding PNA (thymine).

[0372] Compared to unmodified pores, the open pore current level of modified pores changes significantly as a result of changes in diameter and chemical environment within the barrel.

[0373] Figure 12 shows enhanced signal detail observed when a 3.6Kb lambda DNA translocation occurs through a modified lysenin mutant pore, correlated with the modifying factor molecule. Figure 12(A) shows the unmodified pore. The data obtained from Lys-(E84Q / E85S / E92Q / E94D / E97S / S98C / T106K / D126G / C272A / C283A) is shown. Figure 12(B) shows the corresponding data from the modified pore Lys-(E84Q / E85S / E92Q / E94D / E97S / S98C-maleimide-isoleucine / T106K / D126G / C272A / C283A). The left panels of Figures 12(A) and 12(B) respectively show the event of an entire 3.6Kb DNA passing through a pore. As can be seen, as the DNA passes through the pore, the open pore current level (240 pA in A and 250 pA in B) decreases to approximately 160 pA. The current fluctuation at 160 pA indicates different base compositions passing through the narrowest region (constriction) of the pore. The right panel shows a magnified version of the current-time data "irregular curve" over 0.4 seconds. The shape and range (current fluctuations within the irregular curve level) vary significantly between unmodified and modified pores.

[0374] Figure 13 shows the changes in the GGAA region of 3.6Kb lambda DNA (residues 38-41 and 52-55 of SEQ ID NO: 20) correlated with modifying factor molecules during translocation through lysenin mutant pores. The DNA analyte was translocated through unmodified lysenin pores (Lys-(E84Q / E85S / E92Q / E94D / E97S / S98C / T106K / D126G / C272A / C283A); panel A), and the resulting signal was compared to pores modified according to the method described herein: The signals were compared with those of (Lys-(E84Q / E85S / E92Q / E94D / E97S / S98C-maleimide-isoleucine / T106K / D126G / C272A / C283A); panel B and Lys-(E84Q / E85S / E92Q / E94D / E97S / S98C-maleimide-PNA(thymine) / T106K / D126G / C272A / C283A; panel C). The GGAA signal area is circled in each trace. As can be seen, the G and A levels of the signals obtained from the modified pores differ in the modified pores compared to the unmodified pores, with a particularly significant change in the signals obtained from the Lys-(E84Q / E85S / E92Q / E94D / E97S / S98C-maleimide-PNA(thymine) / T106K / D126G / C272A / C283A pores (Panel C).

[0375] Figure 17 shows representative feature data for the polynucleotide analyte of Sequence ID No. 20 when featured using (left) the unmodified gamma-hemolidine-[(LukF-T142C-Del(E1-K15))4(Hlg2-WT)4] pore, which is generated and oligomerized as discussed herein, compared with the corresponding modified gamma-hemolidine-[(LukF-T142C-Del(E1-K15))4(Hlg2-WT)4] pore, which is generated and oligomerized as described above. Panel A shows the unmagnified trace. Compared to the unmodified pore, the open pore current levels and translocation features of the polynucleotide analyte vary significantly between the unmodified and modified pores as a result of the changes in barrel diameter and chemical environment resulting from the modification. Panel B shows the changes that occur when the GGAA region (residues 38-41 and 52-55 of SEQ ID NO: 20) of a 3.6Kb lambda DNA translocation through the pore. The GGAA signal compartment is circled in each trace. As can be seen, the G and A levels of the signal obtained from the modified pore differ in the modified pore compared to the unmodified pore.

[0376] Example 7 Modification, oligomerization, and analyte characterization of alpha-hemolidine (α-HL) monomers. Modification of the alpha-hemolidine (a-HL) monomer (M113C) with maleimide-isoleucine was carried out according to the general modification method described in the previous paragraph. Once the modified monomers were obtained, they were combined with five lipid mixtures in a 1:1 ratio and left to stand at 23°C for 16 hours. The mixtures were centrifuged at 21,000 rcf for 10 minutes to pelletize the sphingomyelin / pore complex. The supernatant was removed, and the pellet was resuspended in buffer (50 mM Tris, 150 mM NaCl, pH 8). The mixtures were heated at 60°C for 2 hours to release the formed pores from the lipid-protein complex. After cooling the tubes to room temperature, they were centrifuged at 21,000 rcf for 10 minutes. The supernatant was collected and diluted 10-fold in 50 mM Tris, 150 mM NaCl, pH 8 to obtain a final SDS concentration of 0.2% in the final pore solution. The success of oligomerization was confirmed by SDS-PAGE electrophoresis on 4-20% gels.

[0377] Figure 18A shows the modification of the a-HL M113C monomer (Lane 1 - Unmodified protein monomer, a-HL-M113C; Lane 2 - Crude reaction mixture - Maleimide-isoleucine (Mal-Ile) adduct containing a-HL-M113C; Lane 3 - Flow-through after equilibration of the modified protein monomer on StrepTactin beads (flow-through shows unmodified protein monomer not bound to StrepTactin beads); Lane 4 / 5 - Further washing of StrepTactin beads bound with modified monomer to remove unmodified protein monomer (gel shows that unmodified monomer does not elute during further washing); Lane 6 - a-HL-M113C modified with maleimido-isoleucine (Mal-Ile). Figure 18B shows the oligomerization of modified a-HL-113C-maleimido-isoleucine to form homooligomeric heptameric pores (Lane 1 - Modified protein monomer, a-HL-M113C-Mal-Ile; Lane 2 - 1:1 crude reaction mixture of a-HL-M113C-Mal-Ile and sphingomyelin after overnight storage; Lane 3 - Pores - Supernatant after centrifugation to separate sphingomyelin pellet. Several pores are visible in the supernatant; Lane 4 - Modified (HL-M113C-Mal-Ile) heptameric oligomerized pores).

[0378] The above DNA analytes were characterized using modified α-HL heptamer pores, and the characteristic data were compared with the corresponding data from unmodified pores. Representative data are shown in Figure 19.

[0379] Figure 19A shows electrophysiological data for unmodified pores ((a-HL-M113C)7, left panel) and modified pores ((a-HL-M113C-Mal-Ile)7, right panel). The open-pore current of the unmodified pores is higher than that of the modified pores, which (though not theoretically bound) is thought to be due to a reduction in the inner diameter of the pore after modification. The drop in current (delta) when DNA is translocating through the pore is higher in the unmodified pores (delta approximately 35 pA) compared to the modified pores (delta approximately 25 pA). Open-pore noise is also higher in the unmodified pores compared to the modified pores. These indicate that the modified pores altered the characteristics of the irregular curve of the DNA compared to the unmodified version. Figure 19B shows an enlarged region of the data from Figure 19A. Figure 19C shows the signal trace, concentrated in the first 0.3 seconds after the sp18 signal from the analyte. The difference in signals resulting from the modification by the method described herein can be clearly detected.

[0380] Example 8 Modification, oligomerization, and characterization of cytotoxin-K (Cyt-K) monomers. Modification of the cytotoxin-K (Cyt-K WT-Q123S / K129C / E140S / Q146S-H6(C)) monomer with maleimide-isoleucine was carried out as described under the general modification method described in the previous paragraph. Once the modified monomers were obtained (Figure 1 - modified with maleimide-isoleucine group-Mal-Ile), they were combined with five lipid mixtures in a 1:1 ratio and left at 23°C for 16 hours. The mixtures were centrifuged at 21,000 rcf for 10 minutes to pelletize the sphingomyelin / pore complex. The supernatant was removed, and the pellets were resuspended in buffer (50 mM Tris, 150 mM NaCl, pH 8), and the mixtures were heated at 60°C for 2 hours to release the formed pores from the lipid-protein complex. After the tubes were cooled to room temperature, they were centrifuged at 21,000 rcf for 10 minutes. The supernatant was collected and diluted 10-fold in 50 mM Tris, 150 mM NaCl, pH 8 to obtain a final SDS concentration of 0.2% in the final pore solution. The success of oligomerization was confirmed by SDS-PAGE electrophoresis on 4-20% gels.

[0381] Figure 20A shows the modification of the Cyt-K monomer (Lane 1 - Unmodified protein monomer, Cyt-K (WT-Q123S / K129C / E140S / Q146S); Lane 2 - Crude reaction mixture - Maleimide-isoleucine (Mal-Ile) adduct containing Cyt-K (WT-Q123S / K129C / E140S / Q146S); Lane 3 - Flow-through after equilibration of the modified protein monomer on StrepTactin beads (Flow-through) Lane 4 / 5 - Lane 4 shows unmodified protein monomers not bound to StrepTactin beads; Lane 5 - Lane 6 - Lane 6 shows Cyt-K (WT-Q123S / K129C / E140S / Q146S) modified with maleimide-isoleucine (Mal-Ile). Figure 20B shows the oligomerization of modified Cyt-K WT-Q123S / K129C-maleimide-isoleucine / E140S / Q146S to form homooligomeric pores (Lane 1 - Modified protein monomer, CytK-(WT-Q123S / K129C-Mal-Ile / E140S / Q146S; Lane 2 - 1:1 crude reaction mixture of CytK-(WT-Q123S / K129C-Mal-Ile / E140S / Q146S) and sphingomyelin after overnight storage. Lane 3 - Pores - Supernatant after centrifugation to separate the sphingomyelin pellet. Several pores are also visible in the supernatant; Lane 4 - Modified (CytK-(WT-Q123S / K129C-Mal-Ile / E140S / Q146S) 7 pores).

[0382] The above DNA analytes were characterized using modified Cyt-K pores, and the characteristic data were compared with the corresponding data from unmodified pores. Representative data are shown in Figure 21.

[0383] Figure 21A shows electrophysiological data for unmodified CytK-(WT-Q123S / K129S / E140S / Q146S)7 and modified CytK-(WT-Q123S / K129C-Mal-Ile / E140S / Q146S)7 pores. The open pore current and sequencing current (i.e., the level of irregular curves) of the unmodified pores are higher than those of the modified pores, which (though not theoretically bound) is thought to result from a reduction in the inner diameter of the pores after modification. This data indicates that the modified pores altered the characteristics of the irregular curves of the DNA compared to the unmodified version. Figure 21B shows characteristic data from poly-T, GGAA regions in a 3.6Kb asymmetric library, magnified to the first 0.3 seconds of the trace following the sp18 signal from the analyte. Both the first and second poly-T regions are clearly visible in the unmodified pore (underlined; poly-T1 and 2), but in the unmodified pore, the first poly-T region is indistinguishable, and the signal from the second poly-T region is more dominant. This data demonstrates that the modified pore altered the characteristics of the irregular curve of the DNA compared to the unmodified version.

[0384] Example 9 The advantages of the method provided herein compared to other modification methods are illustrated in this embodiment.

[0385] As discussed herein, achieving 100% reaction efficiency in a reaction is typically impossible. When modification is performed on purified protein monomers of oligomeric pores, heterogeneity arises in the resulting pore population, which contains a mixture of modified and unmodified proteins.

[0386] This is illustrated in Figure 22, which shows the results of modifying the lysenine monomer (Lys-( / E84Q / E85K / E92Q / E94D / E97S / S98C / T106K / D126G / C272A / C283A)) with maleimide-isoleucine containing a cleavable purified tag. Maleimide-isoleucine reacts at position S98C. Lane 1 shows the unmodified starting material. Lane 2 shows the crude incomplete reaction reaction containing both modified and unmodified protein monomers. Oligomerization with this monomer mixture will result in a heterogeneous pore population. The effect of modification on electrophysiological data obtained from oligomerized protein pores is illustrated above using the significant difference resulting from modified pores compared to unmodified pores. If the monomers of the crude reaction mixture in lane 2 were used to generate pores in electrophysiological experiments, the data obtained would depend on the position and number of modifications within the pores and would therefore be difficult or impossible to interpret. For example, chromatography cannot be used to purify the crude reaction mixture before oligomerization. The difference in molecular weight between modified and unmodified pore monomers is small and can hinder efficient separation, meaning that homogeneous pores cannot be efficiently and easily produced. Even if such purification is achieved in some cases, the development of thorough methods tailored to the functionalities of the monomers and modifying factors will be necessary.

[0387] In contrast, in the method disclosed herein, all unmodified monomers can be removed, for example, by washing or elution, whereas the modified monomers bind to the support material, thus forming a homogeneous population. This is illustrated in lane 3 of Figure 22, showing the elution of unmodified monomers. This method is applicable to modifying and purifying modified porous monomers independently of the substrate without requiring the development of individual methods.

[0388] Protein monomers modified according to the method disclosed herein can be cleanly released from the support material by cleaving the purification tag. As shown in lane 6 of Figure 22, the modified monomers are obtained without contamination from the unmodified monomers. Furthermore, the method disclosed herein is suitable for a wide range of proteins and does not require the development of a specific purification strategy for each monomer of interest.

[0389] Array enumeration description Sequence ID 1 shows the amino acid sequence of (hexa-histidine tagged) exonuclease I (EcoExo I) from E. coli. Sequence ID 2 shows the amino acid sequence of the exonuclease III enzyme from E. coli. Sequence ID 3 shows the amino acid sequence of the RecJ enzyme (TthRecJ-cd) from T. thermophilus. Sequence ID 4 shows the amino acid sequence of bacteriophage lambda exonuclease. The sequence is one of three identical subunits that assemble into a trimer. (http: / / www.neb.com / nebecomm / products / productM0262.asp) Sequence ID 5 shows the amino acid sequence of Phi29 DNA polymerase from the Bacillus subtilis phage Phi29. Sequence ID 6 shows the amino acids of Trwc Cba (Citromicrobium bathyomarinum) helicase. Sequence ID 7 shows the amino acid sequence of Hel308 Mbu (Methanococcoides burtonii) helicase. Sequence ID 8 shows the amino acid sequence of Dda helicase 1993 from the intestinal bacterium phage T4. Sequence ID 10 shows the amino acid sequence of the LukF subunit (Del1-15) of gamma-hemolyzin. Sequence ID 11 shows the amino acid sequence of the Hlg2 subunit (Del1-10) of gamma-hemolysin. Sequence ID 12 shows the amino acid sequence of the monomer lysenin-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G). Sequence ID 13 shows the amino acid sequence of the monomer for cytotoxin K (CytK) from Bacillus cereus. Sequence ID 14 shows the amino acid sequence of the erolidine monomer. Sequence ID 15 shows the amino acid sequence of the monomer for necrotizing enteritis toxin B (NetB) from Clostridium perfringens. Sequence ID 16 shows the amino acid sequence of the alpha-hemolyzin (α-HL) monomer. Sequence ID 17 shows the amino acid sequence of the monomer for Vibrio cholerae cytolysin (HlyA) / VCC. Sequence ID 18 shows the amino acid sequence of the monomer of the anthrax toxin protective antigen (Anthrax PA). Sequence ID 19 shows the amino acid sequence of the monomer of epsilon toxin (ε-toxin). Sequence ID 20 shows the polynucleotide sequence of the 3.6 kb lambda DNA analyte used in the example. Sequence ID 21 shows the amino acid sequence of the monomer for fragaceatoxin C (FraC) from Actinia fragacea. Sequence ID 22 shows the amino acids of the PlyA monomer of plurotrycin PlyAB from Pleurotus ostreatus. Sequence ID 23 shows the amino acid sequence of the PlyB monomer of plurotrycin PlyAB from Pleurotus ostreatus. Sequence IDs 24-27 show the amino acid sequences of various protease-sensitive peptide linkers mentioned herein. Sequence IDs 28-35 show the amino acid sequences of various peptide tags referred to herein. Sequence ID 36 shows the amino acid sequence of the polynucleotide repeating unit referred to herein.

[0390] Sequence List Exonuclease I from sequence number 1-E.coli MMNDGKQQSTFLFHDYETFGTHPALDRPAQFAAIRTDSEFNVIGEPEVFYCKPADDYLPQ PGAVLITGITPQEARAKGENEAAFAARIHSLFTVPKTCILGYNNVRFDDEVTRNIFYRNF YDPYAWSWQHDNSRWDLLLDVMRACYALRPEGINWPENDDGLPSFRLEHLTKANGIEHSNA HDAMADVYATIAMAKLVKTRQPRLFDYLFTHRNKHKLMALIDVPQMKPLVHVSGMFGAWR GNTSWVAPLAWHPENRNAVIMVDLAGDISPLLELDSDTLRERLYTAKTDLGDNAAVPVKL VHINKCPVLAQANTLRPEDADRLGINRQHCLDNLKILRENPQVREKVVAIFAEAEPFTPS DNVDAQLYNGFFSDADRAAMKIVLETEPRNLPALDITFVDKRIEKLLFNYRARNFPGTLD YAEQQRWLEHRRQVFTPEFLQGYADELQMLVQQYADDKEKVALLKALWQYAEEIVSGSGH HHHHH

[0391] Exonuclease III enzyme from Sequence ID No. 2-E. coli MKFVSFNINGLRARPHQLEAIVEKHQPDVIGLQETKVHDDMFPLEEVAKLGYNVFYHGQK GHYGVALLTKETPIAVRRGFPGDDEEAQRRIIMAEIPSLLGNVTVINGYFPQGESRDHPI KFPAKAQFYQNLQNYLETELKRDNPVLIMGDMNISPTDLDIGIGEENRKRWLRTGKCSFL PEEREWMDRLMSWGLVDTFRHANPQTADRFSWFDYRSKGFDDNRGLRIDLLLASQPLAEC CVETGIDYEIRSMEKPSDHAPVWATFRR

[0392] RecJ enzyme from T. thermophilus, sequence number 3. MFRRKEDLDPPLALLPLKGLREAAALLEEALRQGKRIRVHGDYDADGLTGTAILVRGLAA LGADVHPFIPHRLEEGYGVLMERVPEHLEASDLFLTVDCGITNHAELRELLENGVEVIVT DHHTPGKTPPPGLVVHPALTPDLKEKPTGAGVAFLLLWALHERLGLPPPLEYADLAAVGT IADVAPLWGWNRALVKEGLARIPASSWVGLRLLAEAVGYTGKAVEVAFRIAPRINAASRL GEAEKALRLLLTDDAAEAQALVGELHRLNARRQTLEEAMLRKLLPQADPEAKAIVLLDPE GHPGVMGIVASRILEATLRPVFLVAQGKGTVRSLAPISAVEALRSAEDLLLRYGGHKEAA GFAMDEALFPAFKARVEAYAARFPDPVREVALLLDLLPEPGLLPQVFRELALLEPYGEGNP EPLFL

[0393] Sequence ID 4-bacteriophagelambdaexonuclease MTPDIILQRTGIDVRAVEQGDDAWHKLRLGVITASEVHNVIAKPRSGKKWPDMKMSYFHT LLAEVCTGVAPEVNAKALAWGKQYENDARTLFEFTSGVNVTESPIIYRDESMRTACSPDG LCSDGNGLELKCPFTSRDFMKFRLGGFEAIKSAYMAQVQYSMWVTRKNAWYFANYDPRMK REGLHYVVIERDEKYMASFDEIVPEFIEKMDEALAEIGFVFGEQWR

[0394] Sequence ID 5-Phi29 DNA polymerase MKHMPRKMYSCAFETTTKVEDCRVWAYGYMNIEDHSEYKIGNSLDEFMAWVLKVQADLYF HNLKFDGAFIINWLERNGFKWSADGLPNTYNTIISRMGQWYMIDICLGYKGKRKIHTVIY DSLKKLPFPVKKIAKDFKLTVLKGDIDYHKERPVGYKITPEEYAYIKNDIQIIAEALLIQ FKQGLDRMTAGSDSLKGFKDIITTKKFKKVFPTLSLGLLDKEVRYAYRGGFTWLNDRFKEK EIGEGMVFDVNSLYPAQMYSRLLPYGEPIVFEGKYVWDEDYPLHIQHIRCEFELKEGYIP TIQIKRSRFYKGNEYLKSSGGEIADLWLSNVDLELMKEHYDLYNVEYISGLKFKATTGLF KDFIDKWTYIKTTSEGAIKQLAKLMLNSLYGKFASNPDVTGKVPYLKENGALGFRLGEEE TKDPVYTPMGVFITAWARYTTITAAQACYDRIIYCDTDSIHLTGTEIPDVIKDIVDPKKL GYWAHESTFKRAKYLRQKTYIQDIYMKEVDGKLVEGSPDDYTDIKFSVKCAGMTDKIKKE VTFENFKVGFSRKMKPKPVQVPGGVVLVDDTFTIKSGGSAWSHPQFEKGGGSGGGSGGSA WSHPQFEK

[0395] Sequence ID 6-Trwc Cba helicase MLSVANVRSPSAAASYFASDNYYASADADRSGQWIGDGAKRLGLEGKVEARAFDALLRGE LPDGSSVGNPGQAHRPGTDLTFSVPKSWSLLALVGKDERIIAAYREAVVEALHWAEKNAA ETRVVEKGMVVTQATGNLAIGLFQHDTNRNQEPNLHFHAVIANVTQGKDGKWRTLKNDRL WQLNTTLNSIAMARFRVAVEKLGYEPGPVLKHGNFEARGISREQVMAFSTRRKEVLEARR GPGLDAGRIAALDTRASKEGIEDRATLSKQWSEAAQSIGLDLKPLVDRARTKALGQGMEA TRIGSLVERGRAWLSRFAAHVRGDPADPLVPPSVLKQDRQTIAAAQAVASAVRHLSQREA AFERTALYKAALDFGLPTTIADVEKRTRALVRSGDLIAGKGEGHKGWLASRDAVVTEQRIL SEVAAGKGDSSPAITPQKAAASVQAAALTGQGFRLNEGQLAARLILISKDRTIAVQGIA GAGCSSVLKPVAEVLRDEGHPVIGLAIQNTLVQMLERDTGIGSQTLARFLGGWNKLLDDP GNVALRAEAQASLKDHVLVLDEASMVSNEDKEKLVRLANLAGVHRLVLIGDRKQLGAVDA GKPFALLQRAGIARAEMATNLRARDPVVREAQAAAQAGDVRKALRHLKSHTVEARGDGAQ VAAETWLALDKETRARTSIYASGRAIRSAVNAAVQQGLLASREIGPAKMKLEVLDRVNTT REELRHLPAYRAGRVLEVSRKQQALGLFIGEYRVIGQDRKGKLVEVEDKRGKRFRFDPAR IRAGKGDDNLTLLEPRKLEIHEGDRIRWTRNDHRRGLFNADQARVVEIANGKVTFETSKG DLVELKKDDPMLKRIDLAYALNVHMAQGLTSDRGIAVMDSRERNLSNQKTFLVTVTRLRD HLTLVVDSADKLGAAVARNKGEKASAIEVTGSVKPTATKGSGVDQPKSVEANKAEKELTR SKSKTLDFGI

[0396] SEQ ID NO:7 - Hel308 Mbu Helicase MMIRELDIPRDIIGFYEDSGIKELYPPQAEAIEMGLLEKKNLLAAIPTASGKTLLAELAM IKAIREGGKALYIVPLRALASEKFERFKELAPFGIKVGISTGDLDSRADWLGVNDIIVAT SEKTDSLLRNGTSWMDEITTVVVDEIHLLDSKNRGPTLEVTITKLMRLNPDVQVVALSAT VGNAREMADWLGAALVLSEWRPTDLHEGVLFGDAINFPGSQKKIDRLEKDDAVNLVLDTI KAEGQCLVFESSRRNCAGFAKTASSKVAKILDNDIMIKLAGIAEEVESTGETDTAIVLAN CIRKGVAFHHAGLNSNHRKLVENGFRQNLIKVISSTPTLAAGLNLPARRVIIRSYRRFDS NFGMQPIPVLEYKQMAGRAGRPHLDPYGESVLLAKTYDEFAQLMENYVEADAEDIWSKLG TENALRTHVLSTIVNGFASTRQELFDFFGATFFAYQQDKWMLEEVINDCLEFLIDKAMVS ETEDIEDASKLFLRGTRLGSLVSMLYIDPLSGSKIVDGFKDIGKSTGGNMGSLEDDKGDD ITVTDMTLLHLVCSTPDMRQLYLRNTDYTIVNEYIVAHSDEFHEIPDKLKETDYEWFMGE VKTAMLLEEWVTEVSAEDITRHFNVGEGDIHALADTSEWLMHAAAKLAELLGVEYSSHAY SLEKRIRYGSGLDLMELVGIRGVGRVRARKLYNAGFVSVAKLKGADISVLSKLVGPKVAY NILSGIGVRVNDKHFNSAPISSNTLDTLLDKNQKTFNDFQ

[0397] Sequence ID 8-Dda helicase MTFDDLTEGQKNAFNIVMKAIKEKKHHVTINGPAGTGKTTLTKFIIEALISTGETGIILA APTHAAKKILSKLSGKEASTIHSILKINPVTYEENVLFEQKEVPDLAKCRVLICDEVSMY DRKLFKILLSTIPPWCTIIGIGDNKQIRPVDPGENTAYISPFFTHKDFYQCELTEVKRSN APIIDVATDVRNGKWIYDKVVDGHGVRGFTGDTALRDFMVNYFSIVKSLDDLFENRVMAF TNKSVDKLNSIIRKKIFETDKDFIVGEIIVMQEPLFKTYKIDGKPVSEIIFNNGQLVRII EAEYTSTFVKARGVPGEYLIRHWDLTVETYGDDEYYREKIKIISSDEELYKFNLFLGKTA ETYKNWNKGGKAPWSDFWDAKSQFSKVKALPASTFHKAQGMSVDRAFIYTPCIHYADVEL AQQLLYVGVTRGRYDVFYV

[0398] Sequence ID 10-LukF (gamma-hemolyzin). AEGKITPVSVKKVDDKVTLYKTTATADSDKFKISQILTFNFIKDKSYDKDTLVLKATGNI NSGFVKPNPNDYDFSKLYWGAKYNVSISSQSNDSVNVVDYAPKNQNEEFQVQNTLGYTFG GDISISNGLSGGLNGNTAFSETINYKQESYRTTLSRNTNYKNVGWGVEAHKIMNNGWGPY GRDSFHPTYGNELFLAGRQSSAYAGQNFIAQHQMPLLSRSNFNPEFLSVLSHRQDGAKKS KITVTYQREMDLYQIRWNGFYWAGANYKNFKTRTFKSTYEIDWENHKVKLLDTKETENNK

[0399] Sequence ID 11-Hlg2 (gamma-hemolyzin). ENKIEDIGQGAEIIKRTQDITSKRLAITQNIQFDFVKDKKYNKDALVVKMQGFISSRTTY SDLKKYPYIKRMIWPFQYNISLKTKDSNVDLINYLPKNKIDSADVSQKLGYNIGGNFQSA PSIGGSGSFNYSKTISYNQKNYVTEVESQNSKGVKWGVKANSFVTPNGQVSAYDQYLFAQ DPTGPAARDYFVPDNQLPPLIQSGFNPSFITTLSHERGKGDKSEFEITYGRNMDATYAYV TRHRLAVDRKHDAFKNRNVTVKYEVNWKTHEVKIKSITPK

[0400] Sequence ID 12-Lysenine-(E84Q / E85S / E92Q / E94D / E97S / T106K / D126G). MSAKAAEGYEQIEVDVVAVWKEGYVYENRGSTSVDQKITITKGMKNVNSETRTVTATHSIGSTISTGDAFEIGSVEVSYSHSHQKSQVSMTQTDVYSSKVIEHTIKIPPTSKFTRWQLNADVGGAGIEYMYLIDEVTPIGGTQSIPQV ITSRAKIIVGRQIILGKTEIRIKHAERKEYMTVVSRKSWPAATLGHSKLFKFVLYEDWGGFRIKTLNTMYSGYEYAYSSDQGGIYFDQGTDNPKQRWAINKSLPLRHGDVVTFMNKYFTRSGLCYDDGPATNVYCLDKREDKWILEVVG

[0401] Sequence ID 13-CytK (cytotoxin K from Bacillus cereus). MQTTSQVVTDIGQNAKTHTSYNTFNNEQADNMTMSLKVTFIDDPSADKQIAVINTTGSFM KANPTLSDAPVDGYPIPGASVTLRYPSQYDIAMNLQDNTSRFFHVAPTNAVEETTVTSSV SYQLGGSIKASVTPSGPSGESGATGQVTWSDSVSYKQTSYKTNLIDQTNKHVKWNVFFNG YNNQNWGIYTRDSYHALYGNQLFMYSRTYPHETDARGNLVPMNDLPALTNSGFSPGMIAV VISEKDTEQSSIQVAYTKHADDYTLRPGFTFGTGNWVGNNIKDVDQKTFNKSFVLDWKNK KLVEKK

[0402] Sequence ID 14-Erolyzine. AEPVYPDQLRLFSLGQGVCGDKYRPVNREEAQSVKSNIVGMMGQWQISGLANGWVIMGPGYNGEIKPGTASNTWCYPTNPVTGEIPTLSALDIPDGDEVDVQWRLV HDSANFIKPTSYLAHYLGYAWVGGNHSQYVGEDMDVTRDGDGGWVIRGNNDGGCDGYRCGDKTAIKVSNFAYNLDPDSFKHGDVTQSDRQLVKTVVGWAVNDSDTPQ SGYDVTLRYDTATNWSKTNTYGLSEKVTTKNKFKWPLVGETELSIEIAANQSWASQNGGSTTTSLSQSVRPTVPARSKIPVKIELYKADISYPYEFKADVSYDLTL SGFLRWGGNAWYTHPDNRPNWNHTFVIGPYKDKASSIRYQWDKRYIPGEVKWWDWNWTIQQNGLSTMQNNLARVLRPVRAGITGDFSAESQFAGNIEIGAPVPLAA

[0403] Sequence ID 15 - NetB from Clostridium perfringens. SELNDINKIELKNLSGEIIKENGKEAIKYTSSDTASHKGWKATLSGTFIEDPHSDKKTAL LNLEGFIPSDKQIFGSKYYGKMKWPETYRINVKSADVNNNIKIANSIPKNTIDKKDVSNS IGYSIGGNISVEGKTAGAGINASYNVQNTISYEQPDFRTIQRKDDANLASWDIKFVETKD GYNIDSYHAIYGNQLFMKSRLYNNGDKNFTDDRDLSTLISGGFSPNMALALTAPKNAKES VIIVEYQRFNDDYILNWETTQWRGTNKLSSTSEYNEFMFKINWQDHKIEYYL

[0404] Sequence ID 16-alpha-hemolyzin. ADSDINIKTGTTDIGSNTTVKTGDLVTYDKENGMHKKVFYSFIDDKNHNKKLLVIRTKGTIAGQYRVYSEEGANKSGLAWPSAFKVQLQLPDNEVAQISDYYPRNSIDTKEYMSTLTYGFNGNVTGDDTGKIGGLIGANVSIGHTL KYVQPDFKTILESPTDKKVGWKVIFNNMVNQNWGPYDRDSWNPVYGNQLFMKTRNGSMKAADNFLDPNKASSLLSSGFSPDFATVITMDRKASKQQTNIDVIYERVRDDYQLHWTSTNWKGTNTKDKWTDRSSERYKIDWEKEEMTN

[0405] Sequence ID No. 17 - Vibrio cholerae cytolysin (HlyA) / VCC. NINEPSGEAADIISQVADSHAIKYYNAADWQAEDNALPSLAELRDLVINQQKRVLVDFSQISDAEGQAEMQAQFRKAYGVGFANQFIVITEHKGELLFTPFDQAEEVDPQLLEAPRTARLLARSGFASPAPANSETNTLPHVAFYISVNRAISDEECTFNNSWLWKNEKGSRPFCKDAN ISLIYRVNLERSLQYGIVGSATPDAKIVRISLDDDSTGAGIHLNDQLGYRQFGASYTTLDAYFREWSTDAIAQDYRFVFNASNNKAQILKTFPVDNINEKFERKEVSGFELGVTGGVEVSGDPGKAKLEARASYTQSRWLTYNTQDYRIERNAKNAQAVSFTWNRQQYATAESLLNRST DALWVNTYPVDVNRISPLSYASFVPKMDVIYKASATETGSTDFIIDSSVNIRPIYNGAYKHYYVVGAHQSYHGFEDTPRRRITKSASFTVDWDHPVFTGGRPVNLQLASFNNRCIQVDAQGRLAANTCDSQQSAQSFIYDQLGRYVSASNTKLCLDGEALDALQPCNQNLTQRWEWRKG TDELTNVYSGESLGHDKQTGELGLYASSNDAVSLRTITAYTDVFNAQESSPILGYTQGKMNQQRVGQDHRLYVRAGAAIDALGSASDLLVGGNGGSLSSVDLSGVKSITATSGDFQYGGQQLVALTFTYQDGRQQTVGSKAYVTNAHEDRFDLPAAAKITQLKIWSDDWLVKGVQFDLN

[0406] Sequence ID 18 - Anthrax toxin protective antigen. EVKQENRLLNESESSSQGLLGYYFSDLNFQAPMVVTSSTTGDLSIPSSELENIPSENQYFQSAIWSGFIKVKKSDEYTFATSADNHVTMWVDDQEVINKASNSNKIRLEKGRLYQIKIQYQRENPTEKGLDFKLYWTDSQNKKEVISSDNLQLPELKQKSSNSRKKRSTSAGPTVPDRDNDGIPDSLEVEGYTVDVKNKRTFLSPWISNIHEKKGLTKYKSSPEKWSTASDPYSDFEKVTGRIDKNVSPEARHPLVAAYPIVHVDMENIILSKNEDQSTQNTDSQTRTISKNTSTSRTHTSEVHGNAEVHASFFDIGGSVSAGFSNSNSSTVAIDHSLSLAGERTWAETMGLNTADTARLNANIRYVNTGTAPIYNVLPTTSLVLGKNQTLATIKAKENQLSQILAPNNYYPSKNLAPIALNAQDDFSSTPITMNYNQFLELEKTKQLRLDTDQVYGNIATYNFENGRVRVDTGSNWSEVLPQIQETTARIIFNGKDLNLVERRIAAVNPSDPLETTKPDMTLKEALKIAFGFNEPNGNLQYQGKDITEFDFNFDQQTSQNIKNQLAELNATNIYTVLDKIKLNAKMNILIRDKRFHYDRNNIAVGADESVVKEAHREVINSSTEGLLLNIDKDIRKILSGYIVEIEDTEGLKEVINDRYDMLNISSLRQDGKTFIDFKKYNDKLPLYISNPNYKVNVYAVTKENTIINPSENGDTSTNGIKKILIFSKKGYEIG

[0407] Sequence number 19 - ε - toxin. KASYDNVDTLIEKGRYNTKYNYLKRMEKYYPNAMAYFDKVTINPQGNDFYINNPKVELDGEPSMNYLEDVYVGKALLTNDTQQEQKLKSQSFTCKNTDTVTATTTHTVGTSIQATAKFTVPFNETGVSLTTSYSFANTNTN TNSKEITHNVPSQDILVPANTTVEVIAYLKKVNVKGNVKLVGQVSGSEWGEIPSYLAFPRDGYKFSLSDTVNKSDLNEDGTININGKGNYSAVMGDELIVKVRNLNTNNVQEYVIPVDKKEKSNDSNIVKYRSLYIKAPGIK

[0408] Lambda DNA sequence number 20-3.6kb

[0409] Sequence ID 21 - FraC from Actinia fragacea SADVAGAVIDGAGLGFDVLKTVLEALGNVKRKIAVGIDNESGKTWTAMNTYFRSGTSDIV LPHKVAHGKALLYNGQKNRGPVATGVVGVIAYSMSDGNTLAVLFSVPYDYNWYSNWWNVR VYKGQKRADQRMYEELYYHRSPFRGDNGWHSRGLGYGLKSRGFMNSSGHAILEIHVTKA

[0410] Sequence ID 22 - PlyA from Pleurotus ostreatus MAYAQWVIIIIHNVGSKDVKIKNLKPSWGKLHADGDKDTEVSASKYEGTVIKPDEKLQIN ACGRSDAAEGTTGTFDLVDPADGDKQVRHFYWDCPWGSKTNTWTVSGSNTKWMIEYSGQN LDSGALGTITVDTLKKGN

[0411] Sequence ID 23 - PlyB from Pleurotus ostreatus MEAVLSRQAATAEAIGRFQDSSTSVGLVAGSPSTRIRRQADNVVLKSTSQAGDTLNDVIQ DPTRRNKLINDNNLLKGIIMGRDGPVPSSRELIVRPDTLRAIINNRATIETTTMEAEFTE TLMESNYNSASVKVSAPFITANSEYSESSSFKNTETEKSMYTSSRYLFPQGRIDFTTPDS GFDDVIKLSPQFTSGVQAALAKATGTEKREALQNLFQEYGHVFRTKVHIGGVLSAHTMET FSRSENETEVKQDVKAGLEGAVKGWGGGATAGHGNTQGTITTSQNRKLNVKYIVNGGDYT KIQNTEEWVASTNQSEHWRVIEVTEVTAVADLLPQPIRGQVKDLLKPLLGKWVDVEKVPG LESLPVSVYRPKGAIPAGWFWLGDTADASKALLVKPTLPARSGRNPALTSLHQGSGMTEQ PFVDLPQYQYLSTYFGFSFAHDTPPGSTLRGLRPDHVLPGRYEMHGDTISTAVYVTRPVDV PFPEDECFDLKSLVRVKLPGSGNPPKPRSALKSMVLFDSGEK

Claims

1. A method for chemically modifying monomers in oligomeric protein nanopores, a) Contacting the monomer with a polyfunctional molecule, wherein the polyfunctional molecule includes (i) a reactive group, (ii) a chemically modifying group, and (iii) a cleavable purified tag. b) Reacting the reactive group of the polyfunctional molecule with the monomer, thereby bonding the chemically modified group and the cleavable purified tag to the monomer to form a chemically modified tagged monomer, c) Contacting the chemically modified tagged monomer formed in step (b) with a support, d) Attaching the purified tag to the support, thereby attaching the chemically modified tagged monomer to the support, e) A method comprising cutting the purified tag, thereby releasing the chemically modified monomer from the support.

2. The polyfunctional molecule is of formula (I) or formula (II), 【Chemistry 1】 During the ceremony, A is a reactive group, and B is a chemically modifying group. D-C forms a severable purified tag, Preferably, the method according to claim 1, wherein D comprises a cleavable linker and C comprises a support-binding group.

3. The method according to claim 1, wherein the reactive group includes the chemically modifying group.

4. The method according to any one of claims 1 to 3, wherein the monomer comprises a reactive functional group, and step (b) comprises reacting the reactive group of the polyfunctional molecule with the reactive functional group of the monomer.

5. The reactive group of the polyfunctional molecule comprises an amine-reactive group; a carboxyl-reactive group; a sulfhydryl-reactive group or a carbonyl-reactive group. Preferably, the method according to any one of claims 1 to 4, wherein the reactive group of the polyfunctional molecule comprises a cysteine-reactive group.

6. The method according to any one of claims 1 to 5, wherein the reactive group comprises maleimide, azide, thiol, alkyne, NHS ester, or haloacetamide.

7. The aforementioned chemical modification group introduces hydrophilic, hydrophobic, positive charge, negative charge, hydrogen bonding, supramolecular association, or zwitterionic properties to the protein monomer. Preferably, the method according to any one of claims 1 and 3 to 6, wherein the chemically modifying group comprises (i) an amino acid, nucleotide, polymer, hydrogen bonding group, membrane anchor, sugar, dye, chromophore, fluorophore, or molecular adapter; or (ii) a natural or unnatural amino acid, polypeptide, nucleotide or nucleotide analog, oligonucleotide or oligonucleotide analog, polysaccharide, lipid, polyethylene glycol, cyclodextrin, DNA intercalator, aptamer, or analyte-binding domain.

8. The method according to any one of claims 1 to 7, wherein the support comprises a chromatography matrix, preferably an agarose or Sepharose resin; one or more beads, preferably magnetic beads; or a solid surface, preferably glass, silica, polymer, or ceramic surface.

9. The method according to any one of claims 1 to 8, wherein the support is functionalized for binding to the purified tag.

10. The method according to any one of claims 1 to 9, wherein the purified tag comprises a biotin group, and the support comprises streptavidin, neutraavidin, or avidin, preferably streptavidin.

11. The severable linker is cut by physical or chemical means, Preferably, the method according to any one of claims 1 to 10, wherein the severable linker includes a UV-cuttable nitrobenzyl portion.

12. The method according to any one of claims 1 to 11, wherein step (e) involves severing the purified tag, which includes exposing the support and / or the tagged monomer to light, preferably UV light.

13. The method according to any one of claims 1 to 12, wherein step (e) involves cleaving the purified tag, which in turn involves exposing the support and / or the tagged monomer to a change in pH.

14. The method according to any one of claims 1 to 13, wherein step (e) involves cleaving the purified tag, which includes exposing the support and / or the tagged monomer to a chemical reagent, preferably a reducing reagent.

15. The method according to any one of claims 1 to 14, wherein step (e) involves cleaving the purified tag, which includes exposing the support and / or the tagged monomer to an enzyme, preferably a protease.

16. The method according to any one of claims 1 to 15, wherein the monomer has a mass of about 10 kDa to about 1 MDa.

17. The method according to any one of claims 1 to 16, wherein the monomer is a monomer of a lysenin pore, a γ-hemolidine pore, an α-hemolidine pore; a Net B pore; a CytK pore, or a leucosidine pore; or a homolog or paralog thereof.

18. The method according to any one of claims 1 to 17, wherein the oligomeric protein pore is a multicomponent pore.

19. When the monomer is oligomerized to form the pore, the polyfunctional molecule reacts with the reactive functional group located on the monomer at the surface-exposed position. Preferably, the surface exposure position is located on the surface of the channel through the pore or on the outer surface of the pore, according to any one of claims 1 to 18.

20. The method according to claim 19, wherein, when the monomer is oligomerized to form the pore, the polyfunctional molecule reacts with a reactive functional group located on the monomer at a position located at or near the constricted portion of the channel passing through the pore.

21. The method according to any one of claims 1 to 20, wherein the method comprises, prior to step (a), (i) expressing the monomer in a cell expression system or a cell-free expression system, and (ii) isolating and / or purifying the monomer.

22. The method according to any one of claims 1 to 21, further comprising step (d) removing unmodified monomers and / or unreacted polyfunctional molecules (if present) from the support.

23. f) The method according to any one of claims 1 to 22, further comprising the step of oligomerizing the chemically modified monomer to form chemically modified oligomeric protein nanopores.

24. The method according to claim 23, wherein step (f) comprises oligomerizing two or more chemically modified monomers to form homo-oligomeric protein nanopores.

25. The method according to claim 23, wherein step (f) comprises oligomerizing one or more chemically modified monomers with one or more unmodified or differently modified monomers to form hetero-oligomeric protein nanopores.

26. The method according to claim 23, wherein step (f) comprises oligomerizing one or more chemically modified first monomers with one or more chemically modified second monomers to form heterooligomeric protein nanopores, wherein the chemical modifications performed on the first monomers are the same as or different from the chemical modifications performed on the second monomers, and the first monomers have a different amino acid sequence from the second monomers.

27. A method for generating homo-oligomeric protein nanopores, i) The method according to any one of claims 1 to 22, which produces a plurality of chemically modified protein monomers, ii) oligomerizing two or more of the chemically modified protein monomers obtained in step (i) to form homo-oligomeric protein nanopores, ,method.

28. A method for generating hetero-oligomeric protein nanopores, i) Producing one or more chemically modified first protein monomers by the method according to any one of claims 1 to 22, ii) The method according to any one of claims 1 to 22, wherein one or more chemically modified second protein monomers are produced, iii) A method comprising oligomerizing one or more first monomers and one or more second monomers to form hetero-oligomeric protein nanopores.

29. A method for generating oligomeric protein nanopores, i) Producing one or more chemically modified first protein monomers by the method according to any one of claims 1 to 22, ii) To provide one or more unmodified second protein monomers, iii) A method comprising oligomerizing one or more first monomers and one or more second monomers to form hetero-oligomeric protein nanopores.

30. A method can be obtained by performing the method described in any one of claims 1 to 22. Chemically modified monomers of oligomeric protein nanopores.

31. A homogeneous group comprising multiple chemically modified monomers, wherein at least 95% of the monomers in the group are chemically modified with a chemical modification group. A homogeneous group wherein the chemically modified monomers are as defined in any one of claims 5 to 7 or 16 to 20.

32. Chemically modified oligomeric protein nanopores that can be obtained by performing the method according to any one of claims 23 to 29.

33. A homogeneous population comprising multiple chemically modified oligomeric protein nanopores, wherein at least 95% of the oligomeric protein nanopores in the population contain a specified number of monomers chemically modified with chemically modifying groups. A homogeneous group wherein the chemically modified monomers are as defined in any one of claims 5 to 7 or 16 to 20.

34. A method for characterizing an analyte, i) To generate chemically modified oligomeric nanopores by the method of any one of claims 23 to 29, or to provide chemically modified oligomeric nanopores as described in claim 32, ii) Obtaining one or more measurements as the analyte moves into the nanopores, wherein the one or more measurements indicate one or more characteristics of the analyte, thereby characterizing the analyte as it moves into the nanopores, Preferably, the method wherein the analyte is a polynucleotide, polypeptide, or polysaccharide.

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