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WO2022074397A1 - Modification d'un oligomère protéique formant un nanopore - Google Patents

Modification d'un oligomère protéique formant un nanopore Download PDF

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Publication number
WO2022074397A1
WO2022074397A1 PCT/GB2021/052609 GB2021052609W WO2022074397A1 WO 2022074397 A1 WO2022074397 A1 WO 2022074397A1 GB 2021052609 W GB2021052609 W GB 2021052609W WO 2022074397 A1 WO2022074397 A1 WO 2022074397A1
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Prior art keywords
monomer
pore
protein
monomers
modified
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Inventor
Lakmal Nishantha JAYASINGHE
Elizabeth Jayne Wallace
Ranga Prabhath MALAVIARACHCHIGE RABEL
John Joseph KILGOUR
Kimberley Emma BLACKWELL
Pratik Raj SINGH
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Oxford Nanopore Technologies PLC
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Oxford Nanopore Technologies PLC
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Priority to CA3193980A priority Critical patent/CA3193980A1/fr
Priority to JP2023521511A priority patent/JP2023545759A/ja
Priority to EP21794929.6A priority patent/EP4225768A1/fr
Priority to AU2021356235A priority patent/AU2021356235A1/en
Priority to CN202180069111.6A priority patent/CN116323634A/zh
Publication of WO2022074397A1 publication Critical patent/WO2022074397A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/22—Affinity chromatography or related techniques based upon selective absorption processes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48721—Investigating individual macromolecules, e.g. by translocation through nanopores
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N2015/0038—Investigating nanoparticles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1024—Counting particles by non-optical means

Definitions

  • the present invention relates to methods of chemically modifying proteins and polynucleotides, and to the modified proteins and polynucleotides themselves.
  • the methods are particularly useful for producing chemically modified protein nanopores.
  • Nanopore sensing is an approach to analyte detection and characterization that relies on the observation of individual binding or interaction events between the analyte molecules and an ion conducting channel.
  • Nanopore sensors can be created by placing a single pore of nanometre dimensions in an electrically insulating membrane and measuring voltage-driven ion currents through the pore in the presence of analyte molecules. The presence of an analyte inside or near the nanopore will alter the ionic flow through the pore, resulting in altered ionic or electric currents being measured over the channel. The identity of an analyte is revealed through its distinctive current signature, notably the duration and extent of current blocks and the variance of current levels during its interaction time with the pore.
  • Nanopore sensing of polynucleotide analytes can reveal the identity and perform single molecule counting of the sensed analytes, but can also provide information on their composition such as their nucleotide sequence, as well as the presence of characteristics such as base modifications, oxidation, reduction, decarboxylation, deamination and more. Nanopore sensing has the potential to allow rapid and cheap polynucleotide sequencing, providing single molecule sequence reads of polynucleotides of tens to tens of thousands bases length.
  • Nanopore sensing exploit the use of biological protein nanopores.
  • Such nanopores are typically multimeric proteins made up of multiple monomeric subunits.
  • the known pore-forming toxin a-hemolysin assembles into a heptameric pore.
  • Other pore stoichiometries are known for different pore-forming proteins. It is often desirable to modify protein nanopores to lead to effects which are not seen, or are seen to a lesser extent, in unmodified pores.
  • Various modifications have been disclosed to achieve a wide variety of different outcomes.
  • a nanopore can be chemically modified to comprise attachment points for a molecular adapter such as a cyclodextrin, with such adapters finding particular use in sensing small analytes such as individual nucleotides.
  • Nanopores can also be modified to introduce specific modifications to alter the properties of naturally or artificially occurring constrictions within the pore channel. Such constrictions, which are sometimes known as "reading heads", typically interact with analytes as the analyte interacts with the pore and by altering the properties of the reading head the analyte detection signal can be altered accordingly.
  • nanopores can be modified to include membrane anchors such as cholesterol. Such anchors can interact with bilayers in order to promote the stable localisation of the nanopore in the membrane.
  • One known method of modifying a protein such as a protein monomer of a protein nanopore is to rely on modifications that can be generated by a cell expressing the protein.
  • routine molecular biology techniques such as those discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 th 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), can be used to alter, introduce or delete amino acids in a protein. Once expressed, the modified protein can be purified for use.
  • a second known method of modifying a protein such as a protein monomer of a protein nanopore is to express the unmodified protein in a routine manner, and then to seek to modify the expressed protein.
  • the expressed protein is modified prior to being purified, and attempts are then made to purify the modified protein.
  • the expressed protein is purified, and then the purified protein is modified. Both of these approaches, however, are associated with difficulties. As explained below, the origin of these difficulties is that chemical modification of expressed proteins is rarely efficient, and typically even the most efficient modification strategies result in only around 95% modification, with ⁇ 5% or more protein molecules remaining unmodified.
  • the protein of interest is subjected to chemical modification prior to being purified.
  • modification strategies generally considered efficient typically result in no more than 95% of the available protein molecules being modified, with 5% or more remaining unmodified. Attempts can then be made to purify the modified protein molecules from the unmodified molecules (and other impurities).
  • Modifications may typically have a small impact on the overall mass of the protein molecule, meaning that separation techniques that depend on distinguishing proteins by mass may not be effective. Modifications may not alter the charge of the protein and so techniques relying on distinguishing proteins based on charge interactions may similarly fail. The consequence is that in many cases purification strategies fail to separate modified proteins from their unmodified counterparts, and the resulting output from the purification process contains a mixture of modified and unmodified protein molecules in proportions determined by the efficiency of the modification chemistry used.
  • the protein of interest is subjected to chemical modification after being purified.
  • the purification protocol used may be efficient in separating the unmodified protein of interest from impurities present from the expression medium.
  • the presence of even relatively low levels ( ⁇ 5%) unmodified protein in a population of otherwise modified protein can be problematic. This is particularly the case when the protein is a monomer of an oligomeric protein pore.
  • the presence of some unmodified monomers in a population of otherwise modified monomers means that a distribution of assembled pores typically arises.
  • assembled pores may comprise only modified monomers, only unmodified monomers, or a mixture of modified and unmodified monomers. Whilst the distribution of such pores can be statistically calculated, characterisation of an individual pore assembled into a membrane for use e.g.
  • the disclosure relates to a method of chemically modifying a polypeptide or polynucleotide monomer, typically a monomer of an oligomeric protein nanopore.
  • the monomer is contacted with a multifunctional molecule comprising (i) a reactive group for reacting with the monomer; (ii) a chemical modifying group for providing the chemical modification; and (iii) a cleavable purification tag for purifying the monomer.
  • the reactive group of the multi-functional molecule is allowed to react with the monomer, thereby attaching the chemical modifying group and cleavable purification tag to the monomer to form a chemically modified tagged monomer.
  • the chemically modified monomer is contacted with a support such as a chromatography matrix or magnetic beads, and the purification tag is allowed to bind to the support.
  • the binding of the purification tag to the support results in binding the chemically modified tagged monomer to the support.
  • the purification tag is then cleaved by any suitable means. Cleavage of the purification tag releases the chemically modified monomer from the support.
  • a method of chemically modifying a polypeptide or polynucleotide monomer comprising: a) contacting the monomer with a multifunctional molecule, wherein the multi- functional molecule comprises (i) a reactive group; (ii) a chemical modifying group and (iii) a cleavable purification tag; b) allowing the reactive group of the multi-functional molecule to react with the monomer thereby attaching the chemical modifying group and cleavable purification 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) allowing the purification tag to bind to the support thereby binding the chemically modified tagged monomer to the support; and e) cleaving the purification tag thereby releasing the chemically modified monomer from the support.
  • a polypeptide or polynucleotide monomer e.g. a monomer of an oligomeric protein
  • the multifunctional molecule is of Formula (I) or Formula (II):
  • A is a reactive group
  • B is a chemical modifying group
  • D-C forms a cleavable purification tag; preferably wherein D comprises a cleavable linker and C comprises a support- binding group.
  • the wherein the reactive group comprises the chemical modifying group.
  • the multifunctional molecule is of Formula (III) as defined herein.
  • the monomer comprises a reactive functional group and step (b) comprises allowing the reactive group of the multifunctional molecule to react with the reactive functional group of the monomer.
  • the reactive group of the multifunctional molecule comprises an amine-reactive group; a carboxyl -reactive group; a sulfhydryl -reactive group or a carbonyl -reactive group; preferably a cysteine-reactive group.
  • the reactive group comprises a maleimide, an azide, a thiol, an alkyne, an NHS ester or a haloacetamide.
  • the chemical modifying group introduces hydrophilic, hydrophobic, positively charged, negatively charged, hydrogen-bonding, supramolecular associations or zwitterionic properties to the protein monomer.
  • the chemical modifying group comprises an amino acid, a nucleotide, a polymer, a hydrogen- bonding group, a membrane anchor, a sugar, a dye, a chromophore, a fluorophore or a molecular adapter.
  • the chemical modifying group comprises a natural or unnatural amino acid, a polypeptide, a nucleotide or nucleotide analog, an oligonucleotide or oligonucleotide analog, a polysaccharide, a lipid, a polyethylene glycol, a cyclodextrin, a DNA intercalator, an aptamer or an analyte binding domain.
  • 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.
  • the support is functionalised for binding to the purification tag.
  • the purification tag comprises a biotin group and the support comprises streptavidin, neutravidin or avidin, preferably streptavidin.
  • the cleavable linker is cleaved by physical or chemical means. In some embodiments the cleavable linker comprises a UV photocleavable nitro- benzyl moiety.
  • 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 a enzyme; preferably a protease.
  • the monomer is a polypeptide having a mass of from about 10 kDa to about 1 MDa.
  • the monomer is a monomer of an oligomeric protein pore, preferably a lysenin pore, a ⁇ -hemolysin pore, an a-hemolysin pore; a NetB pore; a CytK pore or a leukocidin pore; or a homolog or paralog thereof.
  • the oligomeric protein pore is a multi-component pore.
  • the monomer is a polynucleotide; preferably a monomer of a DNA origami pore.
  • the monomer is a monomer of an oligomeric pore; and the multifunctional molecule reacts with a reactive functional group located on the monomer at a surface-exposed position when the monomer is oligomerised to form the pore.
  • the surface-exposed position is located at the surface of the channel through the pore; or on the exterior surface of the pore.
  • the multifunctional molecule reacts with a reactive functional group located on the monomer at a position located at or near a constriction of the channel through the pore when the monomer is oligomerised to form the pore.
  • the method comprises, prior to step (a), the steps of (i) expressing the monomer in a cellular expression system or a cell-free expression system; and (ii) isolating and/or purifying the monomer.
  • step (d) of the method further comprises the step of removing unmodified monomer(s) and/or unreacted multifunctional molecule(s), if present, from the support.
  • the method further comprises the step of: f) oligomerising the chemically modified monomer to form a chemically modified oligomer, e.g. to form a chemically modified oligomeric protein nanopore.
  • the monomer is a protein monomer and step (f) comprises forming an oligomeric protein.
  • step (f) comprises oligomerising two or more chemically modified monomers to form a homooligomer, e.g. to form an oligomeric protein nanopore.
  • said monomers are protein monomers and said homooligomer is a homooligomeric protein pore.
  • step (f) comprises oligomerising one or more chemically modified monomers with one or more unmodified or differently-modified monomers to form a heterooligomer, e.g. to form an oligomeric protein nanopore.
  • said monomers are protein monomers and said heterooligomer is a heterooligomeric protein pore.
  • step (f) comprises oligomerising one or more chemically modified first monomers with one or more chemically modified second monomers to form a heterooligomer, e.g. to form an oligomeric protein nanopore; wherein the chemical modification made to the first monomer is the same or different to the chemical modification made to the second monomer.
  • said monomers are protein monomers, said first monomer has a different amino acid sequence to said second monomer, and the heterooligomer is a heterooligomeric protein pore.
  • a method of producing a homooligomeric protein e.g.
  • a homooligomeric protein nanopore comprising i) producing a plurality of chemically modified protein monomers in a method as described herein; and ii) oligomerising two or more of the chemically modified protein monomers obtained in step (i) to form a homooligomeric protein.
  • said homooligomeric protein is a homooligomeric protein pore.
  • a method of producing a heterooligomeric protein comprising i) producing one or more chemically modified first protein monomers in a method as described herein; and ii) producing one or more chemically modified second protein monomers in a method as described herein; and iii) oligomerising said one or more first monomers and said one or more second monomers to form a hetero-oligomeric protein.
  • said heterooligomeric protein is a heterooligomeric protein pore.
  • an oligomeric protein e.g. an oligomeric protein nanopore
  • a method of producing an oligomeric protein comprising i) producing one or more chemically modified first protein monomers in a method as described herein; and ii) providing one or more unmodified second protein monomers; and iii) oligomerising said one or more first monomers and said one or more second monomers to form a hetero-oligomeric protein.
  • said heterooligomeric protein is a heterooligomeric protein pore.
  • a chemically modified monomer e.g. a chemically modified monomer of an oligomeric protein nanopore, obtainable by carrying out a method as described herein.
  • 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 modifying group; e.g. the monomers may be monomers of one or more oligomeric protein nanopores (e.g. of one or more types of protein nanopore).
  • the chemically modified monomers are as described in more detail herein.
  • a chemically modified oligomer e.g. an oligomeric protein nanopore, obtainable by carrying out a method as described herein.
  • a homogeneous population comprising a plurality of chemically modified oligomers, e.g. of oligomeric protein nanopores; wherein at least 95% of the oligomers in the population comprise a defined number of monomers chemically modified with a chemical modifying group.
  • the chemically modified monomers are as described in more detail herein.
  • Also provided is a method of characterising an analyte comprising: i) producing a chemically modified oligomeric pore in a method as described herein; or providing a chemically modified oligomeric pore as described herein; and ii) taking one or more measurements as the analyte moves with respect to the pore, wherein the one or more measurements are indicative of one or more characteristics of the analyte, and thereby characterising the analyte as it moves with respect to the pore.
  • the analyte is a polynucleotide, a polypeptide or a polysaccharide. Also provided are related systems, methods and uses.
  • Figure 1 A schematic showing how the disclosed methods can be used to introduce multiple modifications into the barrel of a bi-component pore-forming toxin such as gamma-hemolysin. Specific residues are depicted for illustration only and are non- limiting.
  • Figure 4 Gel showing successful modification of Lysenin mutant Lys- (E84Q/E85S/E92Q/E94D/E97S/T106K/D126G/S98C/C272A/C283A) in accordance with the disclosed methods, as discussed 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 with Lys- (E84Q/E85S/E92Q/E94D/E97S/T106K/D126G/S98C/C272A/C283A); Lane 3 - Flow through after equilibrating the modified protein monomers on StrepTactin beads (flow through indicates unmodified protein monomers unbound to StrepTactin beads); Lane 4/5
  • FIG. Gel showing successful modification of Lysenin mutant Lys- (E84Q/E85S/E92Q/E94D/E97S/T106K/D126G/T63C/C272A/C283A) in accordance with the disclosed methods, as discussed 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 with Lys- (E84Q/E85S/E92Q/E94D/E97S/T106K/D126G/T63C/C272A/C283A); Lane 3 - Flow through after equilibrating the modified protein monomers on StrepTactin beads (flow through indicates unmodified protein monomers unbound to StrepTactin beads); Lane 4/5
  • Figure 6 Gel showing successful modification of a mutant of the F component of the Gamma-Hemolysin pore, LukF-T142C-Del(E1-K15) in accordance with the disclosed methods, as discussed in Example 2.
  • Lane 1 Unmodified protein monomer, LukF- T142C-Del(E1-K15); Lane 2 - Crude reaction mixture - Maleimide-Isoleucine adduct with LukF-T142C-Del(E1-K15); Lane 3 - Flow through after equilibrating the modified protein monomers on StrepTactin beads (flow through indicates unmodified protein monomers unbound to StrepTactin beads); Lane 4/5 - Further washings of the modified monomer bound StrepTactin beads to remove unmodified protein monomer (gel indicates no unmodified monomer eluting in further washes); Lane 6 - Modified LukF-T142C- Maleimide-Alanine-Del(E1-K15)
  • Figure 7 Gel showing successful modification of a mutant of the S component of the Gamma-Hemolysin pore, Hlg2-Gl 14C-Del(E1-G10) in accordance with the disclosed methods, as discussed in Example 2.
  • Lane 1 Unmodified protein monomer, Hlg2- G114C-Del(E1-G10); Lane 2 - Crude reaction mixture - Maleimide-Isoleucine adduct with Hlg2-Gl 14C-Del(E1-G10); Lane 3 - Flow through after equilibrating the modified protein monomers on StrepTactin beads (flow through indicates unmodified protein monomers unbound to StrepTactin beads); Lane 4/5 - Further washings of the modified monomer bound StrepTactin beads to remove unmodified protein monomer (gel indicates no unmodified monomer eluting in further washes); Lane 6 - Modified Hlg2-Gl 14C- Maleimide-Isoleucine-Del(E1-G
  • Figure 8 Gel showing successful oligomerisation of modified Lysenin - [Lys- (E84Q/E85 S/E92Q/E94D/E97S/T 106K/D 126G/S98C-Maleimide- Icoleucine/C272A/C283A)] to form a nonameric pore. Results discussed in Example 3.
  • Lane 1 Modified protein monomer, Lys- (E84Q/E85 S/E92Q/E94D/E97S/T 106K/D 126G/S98C-Maleimide- Icoleucine/C272A/C283A); Lane 2 - Crude reaction mixture of Lys- (E84Q/E85 S/E92Q/E94D/E97S/T 106K/D 126G/S98C-Maleimide- Icoleucine/C272A/C283A) with 1 : 1 Sphingomyelin after overnight storage.
  • Reaction mixture shows oligomeric pore formation (upper band) and unreacted modified monomer Lys-(E84Q/E85S/E92Q/E94D/E97S/T106K/D126G/S98C-Maleimide- Icoleucine/C272A/C283 A); Lane 3 - Supernatant after centrifuging to separate pore- sphingomyelin pellet. Some pores are seen in supernatant as well.; Lane 4 - Modified [Lys-(E84Q/E85S/E92Q/E94D/E97S/T106K/D126G/S98C-Maleimide- Icoleucine/C272A/C283A)]9 pore;
  • Figure 9 Gel showing successful oligomerisation of Lysenin - [Lys- (E84Q/E85 S/E92Q/E94D/E97S/T 106K/D 126G/T63 C-Maleimide- PNA(Thymine)/C272A/C283 A)] to form a nonameric pore. Results discussed in Example 3. Lane 1 - Modified protein monomer, Lys- (E84Q/E85 S/E92Q/E94D/E97S/T 106K/D 126G/T63C-Maleimide-PNA(Thymine) /C272A/C283A); Lane 2 - Crude reaction mixture of Lys-
  • Figure 10 Gel showing successful oligomerisation of various gamma-hemolysin mutants described in Example 4.
  • Lane 1 Unmodified (LukF-T142C-Del(E1-K15)) monomer - guide to see where the monomer would appear on the gel;
  • Lane 2 Unmodified (Hlg2- G114C-Del(E1-G10)) monomer - guide to see where the monomer would appear on the gel;
  • Lane 3 modified pore oligomer [(LukF-T142C-Maleimide-Isoleucine-Del(E1- K15))4 (Hl g2-G114C-Mal eimide- Alanine -Del(E1-G10))4] - top band.
  • Lane 4 - modified pore oligomer [(LukF-T142C-Maleimide-Isoleucine-Del(E1-K15))4 (Hlg2-Gl 14C-Maleimide- Isoleucine-Del(E1-G10))4] - top band.
  • Lane 5 - modified pore oligomer [(LukF-T 142C-Mal eimide- Alanine- Del(E1-K15))4 (Hlg2-Gl 14C-Mal eimide- Alanine -Del(E1-G10))4] - top band.
  • Lane 6 - modified pore oligomer [(LukF-T 142C-Maleimide-Isoleucine-Del(El -KI 5))4 (Hlg2-Gl 14C-Mal eimide- Alanine- Del(E1-G10))4] - top band. Also, can be seen the non-oligomerised modified monomer bands.
  • Lys-(E84Q/E85 S/E92Q/E94D/E97S/S98C/T 106K/D 126G/C272A/C283A) and Ly s-(T63 C/E84Q/E85 S/E92Q/E94D/E97 S/T 106K/D 126G/C272 A/C283A) were modified with 5 different chemical molecules and the effect of the modification was analysed by comparing the open pore current level of the modified pore against the unmodified pore when the 3.6 lambda DNA is passing through each pore. Top level within each channel represents the open pore current level. Bottom level within each channel represents the current level observed when the DNA is passing through the pore.
  • Figure 12 Changes in the signal of the 3.6Kb Lambda DNA translocating through lysenin mutant pores as a function of the modifier molecule.
  • Panel A shows data for unmodified Lys-(E84Q/E85S/E92Q/E94D/E97S/S98C/T106K/D126G/C272A/C283A) and
  • panel B shows data for the modified pore Lys-(E84Q/E85S/E92Q/E94D/E97S/S98C-maleimide- Isoleucine/T 106K/D 126G/C272A/C283 A).
  • the left panel shows an event of the entire 3.6Kb DNA passing through the pore.
  • the right panel shows the expanded version of the squiggle for 0.4 seconds.
  • FIG. 13 Changes in the GGAA region of the 3.6Kb Lambda DNA translocating through lysenin mutant pores as a function of the modifier molecule.
  • Panel A shows data for unmodified Lys-(E84Q/E85S/E92Q/E94D/E97S/S98C/T106K/D126G/C272A/C283A).
  • Panel B shows data for the modified pore Lys-(E84Q/E85S/E92Q/E94D/E97S/S98C-maleimide- Isoleucine/T106K/D126G/C272A/C283A); and Panel C shows data for the modified pore Lys-(E84Q/E85S/E92Q/E94D/E97S/S98C-maleimide- PNA(Thymine)/T106K/D126G/C272A/C283A).
  • Figure 14 Gel showing successful modification of a mutant of the F component of the Gamma-Hemolysin pore, LukF-T142C-Del(E1-K15) in accordance with the disclosed methods, as discussed in Example 2.
  • A Modification with maleimide-asparagine.
  • B Modification with maleimide-CH 2 -NH 2 .
  • C Modification with maleimide-arginine.
  • D Modification with maleimide-isoleucine.
  • E Modification with maleimide-asparatic acid.
  • Figure 15 Modified LukF- T142C-Del(E1-K15)-Maleimide-[modification] eluted from StrepTactin beads after UV cleavage of the linker.
  • Figure 16 Gel showing successful oligomerisation of (LukF-T142C-Maleimide- Isoleucine-Del(E1-K15)) with 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 with arrow.
  • FIG. 17 Characterisation data for the polynucleotide analyte of SEQ ID NO: 20 when characterised using gamma-hemolysin pores modified in accordance with the disclosed methods.
  • A representative characterisation data for the translocation of SEQ ID NO: 20 when characterised using (left) unmodified Gamma-Hemolysin - [(LukF-T142C-Del(E1- K15))4 (Hlg2-WT)4] pores produced and oligomerised as discussed herein, as compared to corresponding modified Gamma-Hemolysin - [(LukF-T142C-Maleimide-Aspartate- Del(E1-K15))4 (Hlg2-WT)4] pores produced and oligomerised as discussed herein.
  • B Changes in the GGAA region of the 3.6Kb Lambda DNA (residues 38-41 and 52-55 of SEQ ID NO: 20) as it translocates
  • FIG. 18 Gel showing successful modification of an alpha-hemolysin (a-HL) monomer and the successful oligomerisation of the modified monomer, in accordance with the disclosed methods.
  • FIG. 19 Characterisation data for the polynucleotide analyte of SEQ ID NO: 20 when characterised using alpha-hemolysin pores modified in accordance with the disclosed methods.
  • A representative characterisation data for the translocation of SEQ ID NO: 20 when characterised using (left) unmodified a-HL Ml 13C pores produced and oligomerised as discussed above, as compared to corresponding modified a-HL [a-HL-Ml 13C-Mal- He)7] pores produced and oligomerised as discussed above.
  • B Changes in the translocation (squiggle) data from (A).
  • C Changes in the translocation squiggle in the first 0.3 s following the characteristic spl8 signal of the polynucleotide analyte.
  • FIG. 20 Gel showing successful modification of cytotoxin-K (Cyt-K) monomers and the successful oligomerisation of the modified monomers, in accordance with the disclosed methods.
  • FIG. 21 Characterisation data for the polynucleotide analyte of SEQ ID NO: 20 when characterised using Cyt-K pores modified in accordance with the disclosed methods.
  • A representative characterisation data for the translocation of SEQ ID NO: 20 when characterised using (left) unmodified Cyt-K WT-Q123S/K129S/E140S/Q146S pores produced and oligomerised as discussed above, as compared to corresponding modified Cyt-K WT-Q123S/K129C-Maleimide-isoleucine/E140S/Q146S pores produced and oligomerised as discussed above.
  • B Changes in the translocation (squiggle) data from (A).
  • C Representative changes in the translocation squiggle in the first 0.3 s following the characteristic spl8 signal of the polynucleotide analyte.
  • Figure 22 Gel comparing the modification methods of the invention with conventional methods for modifying proteins.
  • "Nucleotide sequence”, “DNA sequence” or “nucleic acid molecule(s)” as used herein refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. This term refers only to the primary structure of the molecule.
  • nucleic acid is a single or double stranded covalently-linked sequence of nucleotides in which the 3' and 5' ends on each nucleotide are joined by phosphodiester bonds.
  • the polynucleotide may be made up of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acids may be manufactured synthetically in vitro or isolated from natural sources.
  • Nucleic acids may further include modified DNA or RNA, for example DNA or RNA that has been methylated, or RNA that has been subject to post-translational modification, for example 5'-capping with 7-methylguanosine, 3' -processing such as cleavage and polyadenylation, and splicing.
  • Nucleic acids may also include synthetic nucleic acids (XNA), such as hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), locked nucleic acid (LNA) and peptide nucleic acid (PNA).
  • HNA hexitol nucleic acid
  • CeNA cyclohexene nucleic acid
  • TAA threose nucleic acid
  • GNA glycerol nucleic acid
  • LNA locked nucleic acid
  • PNA peptide nucle
  • nucleic acids also referred to herein as “polynucleotides” are typically expressed as the number of base pairs (bp) for double stranded polynucleotides, or in the case of single stranded polynucleotides as the number of nucleotides (nt). One thousand bp or nt equal a kilobase (kb). Polynucleotides of less than around 40 nucleotides in length are typically called “oligonucleotides” and may comprise primers for use in manipulation of DNA such as via polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • amino acid in the context of the present disclosure is used in its broadest sense and is meant to include organic compounds containing amine (NH2) and carboxyl (COOH) functional groups, along with a side chain (e.g., a R group) specific to each amino acid.
  • NH2 amine
  • COOH carboxyl
  • side chain e.g., a R group
  • the amino acids refer to naturally occurring L a- amino acids or residues.
  • amino acid further includes D- amino acids, retro-inverso amino acids as well as chemically modified amino acids such as amino acid analogues, naturally occurring amino acids that are not usually incorporated into proteins such as norleucine, and chemically synthesised compounds having properties known in the art to be characteristic of an amino acid, such as ⁇ -amino acids.
  • amino acid analogues naturally occurring amino acids that are not usually incorporated into proteins such as norleucine
  • chemically synthesised compounds having properties known in the art to be characteristic of an amino acid, such as ⁇ -amino acids such as ⁇ -amino acids.
  • analogues or mimetics of phenylalanine or proline which allow the same conformational restriction of the peptide compounds as do natural Phe or Pro, are included within the definition of amino acid.
  • Such analogues and mimetics are referred to herein as "functional equivalents" of the respective amino acid.
  • polypeptide and “peptide” are interchangeably used herein to refer to a polymer of amino acid residues and to variants and synthetic analogues of the same. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid, such as a chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally-occurring amino acid polymers.
  • Polypeptides can also undergo maturation or post-translational modification processes that may include, but are not limited to: glycosylation, proteolytic cleavage, lipidization, signal peptide cleavage, propeptide cleavage, phosphorylation, and such like.
  • a peptide can be made using recombinant techniques, e.g., through the expression of a recombinant or synthetic polynucleotide.
  • a recombinantly produced peptide it typically substantially free of culture medium, e.g., culture medium represents less than about 20 %, more preferably less than about 10 %, and most preferably less than about 5 % of the volume of the protein preparation.
  • the term "protein” is used to describe a folded polypeptide having a secondary or tertiary structure.
  • the protein may be composed of a single polypeptide, or may comprise multiple polypepties that are assembled to form a multimer.
  • the multimer may be a homooligomer, or a heterooligmer.
  • the protein may be a naturally occurring, or wild type protein, or a modified, or non-naturally, occurring protein.
  • the protein may, for example, differ from a wild type protein by the addition, substitution or deletion of one or more amino acids.
  • a "variant" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified or wild-type protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.
  • amino acid identity refers to the extent that sequences are identical on an amino acid- by-amino acid basis over a window of comparison.
  • a "percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gin, Cys and Met
  • a "variant" has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% complete sequence identity to the amino acid sequence of the corresponding wild-type protein. Sequence identity can also be to a fragment or portion of the full length polynucleotide or polypeptide. Hence, a sequence may have only 50 % overall sequence identity with a full length reference sequence, but a sequence of a particular region, domain or subunit could share 80 %, 90 %, or as much as 99 % sequence identity with the reference sequence.
  • wild-type refers to a gene or gene product isolated from a naturally occurring source.
  • a wild-type gene is that which is most frequently observed in a population and is thus arbitrarily designed the "normal” or “wild-type” form of the gene.
  • modified refers to a gene or gene product that displays modifications in sequence (e.g., substitutions, truncations, or insertions), post- translational modifications and/or functional properties (e.g., altered characteristics) when compared to the wild-type gene or gene product. It is noted that naturally occurring mutants can be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product.
  • methionine (M) may be substituted with arginine (R) by replacing the codon for methionine (ATG) with a codon for arginine (CGT) at the relevant position in a polynucleotide encoding the mutant monomer.
  • Methods for introducing or substituting non-naturally-occurring amino acids are also well known in the art.
  • non- naturally-occurring amino acids may be introduced by including synthetic aminoacyl- tRNAs in the IVTT system used to express the mutant monomer. Alternatively, they may be introduced by expressing the mutant monomer in E.
  • coli that are auxotrophic for specific amino acids in the presence of synthetic (i.e. non-naturally-occurring) analogues of those specific amino acids. They may also be produced by naked ligation if the mutant monomer is produced using partial peptide synthesis. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side-chain volume.
  • the amino acids introduced may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge to the amino acids they replace.
  • the conservative substitution may introduce another amino acid that is aromatic or aliphatic in the place of a pre-existing aromatic or aliphatic amino acid.
  • Conservative amino acid changes are well-known in the art and may be selected in accordance with the properties of the 20 main amino acids as defined in Table 1 below. Where amino acids have similar polarity, this can also be determined by reference to the hydropathy scale for amino acid side chains in Table 2.
  • a mutant or modified protein, monomer or peptide can be chemically modified in any way and at any site.
  • a mutant or modified monomer or peptide is preferably chemically modified by attachment of a molecule to one or more cysteines (cysteine linkage), attachment of a molecule to one or more lysines, attachment of a molecule to one or more non-natural amino acids, enzyme modification of an epitope or modification of a terminus. Suitable methods for carrying out such modifications are well-known in the art.
  • the mutant of modified protein, monomer or peptide may be chemically modified by the attachment of any molecule.
  • the mutant of modified protein, monomer or peptide may be chemically modified by attachment of a dye or a fluorophore.
  • the disclosure relates to a method of modifying a monomer such a monomer of a protein nanopore.
  • the method has the advantage of improving the production of modified proteins such as nanopores.
  • Populations of modified nanopores thus produced typically have improved properties compared to populations of nanopores produced by prior techniques.
  • nanopore sensing it is particularly important that the population of nanopores used in any sensing application is homogeneous. If the population is non-homogeneous then it is problematic to know if any variance in the signal obtained when a sample is analysed derives from the nanopore that is used to detect the analyte, or the analyte itself. This can reduce the efficiency at which an analyte is characterised due to increased need for data processing, or at a worst case can result in otherwise good data being discarded. Accordingly, it is necessary that the population of nanopores used in sensing apparatuses is of known homogeneity and that any deviation in the reproducibility at which the nanopores themselves are produced is minimised.
  • the problem is particularly acute when chemically modified nanopores are used.
  • chemical modification of nanopores may be required: for example, in order to modify the properties of the signal obtained when an analyte interacts with the pore, to alter the pore stability, or to alter the physical interaction of the analyte with the pore (e.g. by altering the kinetics at which the analyte interacts with the pore).
  • chemical modification can be effected by known routes.
  • a problem is that the modification methods used are typically not highly efficient, with a significant percentage of protein molecules not being modified. Accordingly, it is often difficult or impossible to obtain a homogeneous population of chemically modified nanopores.
  • a further difficulty lies in the separation of chemically modified proteins such as chemically modified nanopores from analogs which have not been chemically modified. If the modification results in a small change in the overall mass, charge or conformation of the protein it may not be possible to separate modified from unmodified protein in an efficient manner.
  • the methods of the present disclosure address these issues. By combining the modification and purification steps together the inefficiencies in these processes are “cancelled out”.
  • the methods involve modifying the monomer of interest with a moiety which combines both the desired chemical modification and a purification tag. Only those monomers which are successfully modified have the purification tag, and so only these monomers bind to a purification substrate such as a chromatography matrix. Once bound, a cleavable linker between the chemical modification and the purification tag is cleaved, thus releasing the modified monomer from the purification substrate.
  • modified monomers Because only modified monomers have the required tag to bind to the purification substrate, and because only monomers which are successfully cleaved are released from the purification substrate, the yield of modified monomer can be very pure, with monomer populations obtained typically greater than 95% pure, and often up to 100% pure.
  • the methods of the present disclosure thus have significant advantages compared to known methods in which modification and purification strategies are decoupled.
  • a method of chemically modifying a polypeptide or polynucleotide monomer comprising: a) contacting the monomer with a multifunctional molecule, wherein the multi- functional molecule comprises (i) a reactive group; (ii) a chemical modifying group and (iii) a cleavable purification tag; b) allowing the reactive group of the multi-functional molecule to react with the monomer thereby attaching the chemical modifying group and cleavable purification 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) allowing the purification tag to bind to the support thereby binding the chemically modified tagged monomer to the support; and e) cleaving the purification tag thereby releasing the chemically modified monomer from the support.
  • the monomer is a monomer of an oligomeric protein nanopore.
  • the monomer is a polynucleotide monomer.
  • the monomer may be a monomer of a DNA origami pore, or an origami pore formed from an analog of DNA such as PNA.
  • the monomer is a polypeptide such as a protein.
  • the monomer may be a monomer of a protein nanopore, such as a monomer of a monomeric protein nanopore or, more typically, the monomer of an oligomeric protein nanopore.
  • Monomers of nanopores which are particularly amenable to being modified in accordance with the claimed methods are discussed below.
  • a multifunctional molecule is a molecule comprising at least three components: (i) a reactive group for reacting with an appropriate site on the monomer, such as reactive functional group on the monomer; (ii) a chemical modifying group which corresponds to the chemical modification being made to the monomer; and (iii) a cleavable purification tag.
  • the cleavable purification tag typically comprises (i) a cleavable linker; and (ii) a purification tag.
  • the purification tag is suitable for binding to a purification support such as a chromatography matrix, thus allowing the modified monomer to be purified.
  • the cleavable linker allows the modified monomer to be released from the purification support. This is described in more detail below.
  • the method thus comprises allowing the reactive group on the multifunctional molecule to react with the monomer.
  • Typical reactive groups suitable for use in this way are described in more detail below.
  • the modified monomer is contacted with a support, typically a purification support.
  • a support typically a purification support.
  • the purification tag on the multifunctional molecule binds to the support thereby binding the modified monomer to the support.
  • the bound monomer can then be washed or otherwise subjected to purification techniques to eliminate impurities including unmodified monomer and/or unreacted multifunctional molecules.
  • the method further comprises cleaving the purification tag. As explained herein, this releases the chemically modified monomer from the support. Methods for cleaving cleavable linkers are discussed in more detail herein. Multifunctional Molecule
  • the provided methods comprise the use of a multifunctional molecule as discussed above.
  • the multifunctional molecule may be in some embodiments represented by Formula (I) or Formula (II) below:
  • A is a reactive group
  • B is a chemical modifying group
  • D-C forms a cleavable purification tag.
  • D comprises a cleavable linker as discussed herein.
  • C comprises a support-binding group as discussed herein.
  • a multifunctional molecule according to Formula (I) provides an in-line design.
  • the chemical modifying group is "hidden” by the cleavable linker and the reactive group and is exposed by the cleaving of the cleavable linker.
  • Such designs can be useful when the chemical modifying group is reactive under the conditions of the binding of the multifunctional molecule to the purification support, for example.
  • a multifunctional molecule according to Formula (II) provides a "branched" design.
  • the modification group is extrinsic and is typically accessible throughout the steps of the claimed method. Such designs can be useful when the desired chemical modification is incompatible with the chemistry of the cleavable linker, for example.
  • Cleavage of the cleavable linker can in some cases leave a residue from the linker.
  • the residue if present once the linker has been cleaved corresponds to a chemical modifying group as used herein. This is particularly the case when the multifunctional molecule is according to Formula (I).
  • any residue from the cleavable linker is separate to the chemical modifying group. This is particularly the case when the multifunctional molecule is according to Formula (II).
  • the reactive group comprises the chemical modifying group.
  • the multifunctional molecule is of Formula (III) below:
  • A is a reactive group comprising a chemical modifying group
  • D-C forms a cleavable purification tag.
  • the reactive group (A) of the multifunctional molecule is suitable for reacting with the monomer being subjected to the methods disclosed herein.
  • Any suitable reactive group can be used.
  • the reactive group may be an amine-reactive group; a carboxyl- reactive group; a sulfhydryl-reactive group or a carbonyl-reactive group.
  • the reactive group of the multi-functional molecule comprises a cysteine- reactive group.
  • the reactive group comprises a maleimide, an azide, a thiol, an alkyne, an NHS ester or a haloacetamide.
  • 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 1-71 in Figure 1 of Liu C. C. and Schultz P. G., Annu. Rev. Biochem., 2010, 79, 413-444.
  • a non-natural amino acid such as 4-azido-L-phenylalanine (Faz) and any one of the amino acids numbered 1-71 in Figure 1 of Liu C. C. and Schultz P. G., Annu. Rev. Biochem., 2010, 79, 413-444.
  • Such groups are particularly useful when corresponding non-natural amino acids are comprised in the monomer.
  • the reactive group is a click chemistry group.
  • Click chemistry is a term first introduced by Kolb et al. in 2001 to describe an expanding set of powerful, selective, and modular building blocks that work reliably in both small- 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 have defined the set of stringent criteria for click chemistry as follows: "The reaction must be modular, wide in scope, give very high yields, generate only inoffensive by-products that can be removed by non-chromatographic methods, and be stereospecific (but not necessarily enantioselective).
  • the required process characteristics include simple reaction conditions (ideally, the process should be insensitive to oxygen and water), readily available starting materials and reagents, the use of no solvent or a solvent that is benign (such as water) or easily removed, and simple product isolation. Purification if required must be by non-chromatographic methods, such as crystallization or distillation, and the product must be stable under physiological conditions".
  • Suitable examples of click chemistry include, but are not limited to, the following:
  • the reactive group may thus be one that is suitable for click chemistry, particularly when a complementary group is present on the monomer.
  • the reactive group may be any of those disclosed in WO 2010/086602, particularly in Table 4 of that application.
  • the reactive group is a haloacetamide, for example, iodoacetamide, bromoacetemide or chloroacetamide.
  • the reactive group is selected from a vinyl group, TCO, tetrazine and a strained alkyne; DBCO; an activated acid e.g. an acid chloride; and piperazine and reactive amines.
  • the reactive group is a polynucleotide or polynucleotide analog, e.g. PNA.
  • the reactive group of the multifunctional molecule comprises a nucleotide analog such as a PNA base or PNA polymer which can interact non-covalently via supramolecular associations and/or hydrogen bonding with the monomer, e.g. with a monomer containing the complementary PNA base or polymer.
  • polynucleotide or polynucleotide analog binds to a complementary polynucleotide or polynucleotide analog on the monomer.
  • Host -guest chemistry can also be used to provide the reaction between the reactive group and the monomer.
  • the monomer comprises a ligand for binding to a metal complex
  • the reactive group comprises a metal complex.
  • the reactive group of the multifunctional molecule comprises a metal complex which can interact non-covalently via chelation or supramolecular association with the monomer containing a site that can act as a ligand to complex with the modifier molecule by forming a stable association.
  • the reactive group may be any of those disclosed in Sakamoto and Hamachi, “Recent progress in chemical modification of proteins” , Anal.
  • the reactive group may therefore react with a reactive functional group on the monomer.
  • the monomer comprises a reactive functional group and step (b) comprises allowing the reactive group of the multifunctional molecule to react with the reactive functional group of the monomer.
  • the reactive functional group may be present naturally in the monomer or may be introduced, e.g. by genetic manipulation or by chemical modification of the monomer.
  • the reactive functional group may originate from a non-natural amino acid incorporated into the monomer during its synthesis or expression, e.g. during cell-free expression, e.g. via in vitro transcription/translation.
  • the chemical modifying group of the multifunctional molecule provides the chemical modification desired. Any suitable chemical modifying group can be used in the provided methods.
  • the chemical modifying group has a molecular mass of at most kDa, such as at most 500 Da, e.g at most 400 Da, such as at most 300 Da, e.g. at most 200 Da.
  • Typical amino acids have an approximate mass of around 110 Da and are exemplary chemical modifying groups for use in the methods disclosed herein, although the methods disclosed herein are not limited to such groups.
  • the chemical modifying group is an aliphatic group such as an alkane or alkene.
  • the chemical modifying group may be a linear or branched alkane e.g. comprising from 1 to 20 carbon atoms, e.g. from 2 to 10 carbon atoms.
  • the chemical modifying group may be a linear or branched alkene e.g. comprising from 1 to 20 carbon atoms, e.g. from 2 to 10 carbon atoms.
  • the chemical modifying group may be an alkyl, alkenyl; alkynyl; or alkoxy group.
  • the chemical modifying group may be a cyclic group such as cyclopropyl, cyclobutyl, cyclopentyl and cyclohexyl groups; piperazine, piperidine, morpholine, 1,3-oxazinane, pyrrolidine, imidazolidine, and oxazolidine.
  • the chemical modifying group is an aromatic group such as a C 6 to C 10 aromatic ring (e.g. benzene/phenyl); or a 5- to 10-membered heteroaromatic group, e.g. pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyridine, pyridazine, pyrimidine, and pyrazine.
  • aromatic group such as a C 6 to C 10 aromatic ring (e.g. benzene/phenyl); or a 5- to 10-membered heteroaromatic group, e.g. pyrrole, furan, thiophene, imidazole, oxazole, thiazole, pyridine, pyridazine, pyrimidine, and pyrazine.
  • the chemical modifying group comprises an amino acid, a nucleotide; a polymer; a hydrogen-bonding group; a membrane anchor; a sugar, a dye, a chromophore, a fluorophore or a molecular adapter.
  • the chemical modifying group comprises a natural or unnatural amino acid; a polypeptide; a nucleotide or nucleotide analog; an oligonucleotide or oligonucleotide analog; a polysaccharide; a lipid; a polyethylene glycol; a cyclodextrin; a DNA intercalator; an aptamer or an analyte binding domain.
  • the chemical modifying group introduces hydrophilic, hydrophobic, positively charged, negatively charged, hydrogen-bonding, supramolecular associations or zwitterionic properties to the protein monomer.
  • the chemical modifying group is or comprises an amino acid.
  • the amino acid may be a natural or unnatural amino acid.
  • a plurality of amino acids may be comprised in the chemical modifying group; e.g. the chemical modifying group may comprise a peptide.
  • the chemical modifying group is or comprises a nucleotide or polynucleotide. Analogs of naturally occurring nucleotides/polynucleotides are also included.
  • the chemical modifying group may comprise RNA, PNA, LNA, or BNA.
  • the chemical modifying group may comprise a saccharide or polysaccharide such as dextrose, maltose, glucose, etc.
  • the chemical modifying group may comprise a dye such as an anthraquinoine or phthalocyanine.
  • the chemical modifying group may comprise a fluorophore such as hydroxycoumarin, aminocoumarin, methoxycoumarin, fluorescein, X-Rhodamine, Texas Red, Cy5, Cy7 etc.
  • the chemical modifying group is an adaptor which is a compound which has an effect on the physical or chemical properties of a nanopore once the monomer has assembled into the pore.
  • an adapter improves the interaction of the pore with an analyte such as a nucleotide or polynucleotide.
  • the adaptor may alter the charge of the barrel or channel of the pore or specifically interact with or bind to the nucleotide or polynucleotide thereby facilitating its interaction with the pore.
  • An adapter may be a cyclic molecule.
  • An adaptor may comprise one or more chemical groups that are capable of interacting with a nucleotide or polynucleotide e.g. by hydrophobic interactions, hydrogen bonding, Van der Waal' s forces, ⁇ -cation interactions and/or electrostatic forces.
  • An adapter may comprise one or more amino groups.
  • the amino groups can be attached to primary, secondary or tertiary carbon atoms.
  • the adaptor may comprise a ring of amino groups, such as a ring of 6, 7, 8 or 9 amino groups.
  • an adapter may comprise one or more hydroxyl groups.
  • the hydroxyl groups can be attached to primary, secondary or tertiary carbon atoms.
  • the hydroxyl groups may form hydrogen bonds with uncharged amino acids in the pore.
  • Suitable chemical modifying groups include, but are not limited to, cyclodextrins, cyclic peptides and cucurbiturils.
  • the chemical modifying group may be any of those disclosed in Eliseev, A. V., and Schneider, H-J. (1994) J. Am. Chem. Soc. 116, 6081-6088.
  • the adaptor may be heptakis-6-amino-P-cyclodextrin (arm- ⁇ CD), 6-monodeoxy-6- monoamino-P-cyclodextrin (ami-pCD) or heptakis-(6-deoxy-6-guanidino)-cyclodextrin (gU7-pCD).
  • the guanidino group in gu 7 -pCD has a much higher pKa than the primary amines in am?-pCD and so it more positively charged.
  • the adapter may be a y- cyclodextrins.
  • the chemical modifying group is selected from (i) Maleimides including diabromomaleimides such as: 4-phenylazomaleinanil, l.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-Butylmaleimide, N-(2- Aminoethyl)maleimide , 3-Maleimido-PROXYL , N-(4-Chlorophenyl)maleimide, 1-[4- (dimethylamino)-3,5-dinitrophenyl]-1H-pyrrole-2, 5-dione, N-[4-(2- Benzyl)
  • the multifunctional molecule comprises a cleavable purification tag for binding the chemically modified monomer to a purification support during the claimed methods.
  • the cleavable purification tag comprises a cleavable linker and a purification tag.
  • the linker may comprise a short chain oligopeptide or oligonucleotide containing e.g. from about 1 to about 20 amino acids or nucleotides.
  • the linker may comprise a polymer such as a polyethylene glycol or a saccharide containing from about 1 to about 20 repeat units.
  • the linker may comprise PEG2, PEG3 or PEG 4.
  • the cleavable linker is cleaved by physical or chemical means. Any suitable means can be used.
  • the cleavable moiety of the cleavable linker may be e.g. a pH sensitive group; a redox sensitive group; a light-sensitive group; a temperature sensitive group or a chemical- sensitive group which is sensitive to cleavage by reaction of the group with a specific chemical.
  • cleavable linker is cleavable by exposure to light; i.e. it is photocleavable.
  • cleaving the purification tag comprises exposing the support and/or the tagged monomer to light; preferably UV light.
  • Photocleavable linkers include nitobenzyl moieties. Such groups are cleavable under UV irradiation.
  • cleavable linker is cleavable by exposure to a change in pH.
  • cleavable linkers include hydrazones and cis-aconityl.
  • An example of a cleavable hydrazone linker is shown below: wherein the wave lines represent the points of attachment to the rest of the multifunctional molecule.
  • cleavable linker is cleavable by exposure to a chemical reagent.
  • cleaving the purification tag comprises exposing the support and/or the tagged monomer to a chemical reagent; preferably a reducing reagent.
  • Chemical-sensitive cleavable linkers include disulphides. Disulphide bonds are susceptible to cleavage by addition of a reducing agent such as DTT and beta-mercaptoethanol.
  • a cleavable disulphide linker is shown below: wherein the wave lines represent the points of attachment to the rest of the multifunctional molecule.
  • cleavable linker is cleavable by exposure to an enzyme such as a protease or nuclease.
  • an enzyme such as a protease or nuclease.
  • cleaving the purification tag comprises exposing the support and/or the tagged monomer to an enzyme; preferably a protease.
  • Enzyme-sensitive linkers include protease-sensitive peptide linkers comprising recognition sequences for one or more endo- and/or exo-proteases. Examples include the sequences DDDDK (SEQ ID NO: 24; cleaved by enteropeptidase 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).
  • B-glucuronide linkers can be cleaved by lysosomal ⁇ - glucuronidase.
  • the cleavable linker is of the form Cl-Lk, Lk-Cl or Lk-Cl-Lk wherein Cl is a cleavable moiety and Lk is a linker.
  • the cleavable linker comprises a polymer and a photocleavable moiety. In some embodiments the cleavable linker comprises a PEG linker and a nitobenzyl moiety. In some embodiments the cleavable linker comprises a structure of the form: wherein the wavy lines represent the points of attachment to the purification tag and chemical modifying group (either directly or via a linker), respectively.
  • any suitable purification tag can be used in the cleavable purification tag comprised in the multifunctional molecule.
  • the purification tag may comprise or consist of biotin. Biotin is particularly suitable for use in the disclosed methods as it forms a strong non-covalent attachment with streptavidin and related proteins (neutravidin, avidin, etc).
  • purification tags include peptide purification tags suitable for IMAC (immobilised metal affinity chromatography) chemistry.
  • the purification tag may comprise a poly-His tag (e g. HHHH, HHHHHH or HHHHHHHH; SEQ ID NOs: 28- 30).
  • poly-His tag e g. HHHH, HHHHHH or HHHHHHHH; SEQ ID NOs: 28- 30.
  • Such tags are suitable for binding to a purification support comprising a metal such as nickel or cobalt.
  • Still other purification tags include peptide tags such as Strep (WSHPQFEK; SEQ ID NO: 31), FLAG (DYKDDDDK; SEQ ID NO: 32), Human influenza hemagglutinin (HA) (YPYDVPDYA; SEQ ID NO: 33), Myc (EQKLISEED; SEQ ID NO: 34), and V5 (GKPIPNPLLGLDST; SEQ ID NO: 35), etc.
  • BCCP Biotin-carboxy carrier protein
  • CBP Calmodulin binding peptide
  • CBD Chitin binding domain
  • HAT Histidine affinity tag
  • Polyarginine Arg-tag
  • Polyaspartate Asp-tag
  • Polylysine Lys-tag
  • Polyphenylalanine Phe-tag
  • Streptavadin-binding peptide SBP
  • Tetrazine tag TCO tag
  • Azide tag Azide tag
  • DBCO / Alkyne tag Suitable purification tags
  • the cleavable purification tag comprises a cleavable linker comprising a polymer and a photocleavable moiety; and biotin.
  • the cleavable purification tag comprises a PEG linker and a nitobenzyl moiety and biotin.
  • the cleavable purification tag comprises a structure of the form: wherein the wavy line represents the point of attachment to the chemical modifying group (either directly or via a linker).
  • the disclosed methods comprise allowing the monomer, once functionalised with the multifunctional molecule, to bind to a support for purification.
  • Any suitable support can be used.
  • the support comprises a chromatography matrix, preferably an agarose or sepharose resin.
  • a chromatography matrix preferably an agarose or sepharose resin.
  • Such resins are commercially available from suppliers such as Sigma Aldrich.
  • the support comprises beads (i.e. one or more beads).
  • Magnetic beads are preferred as such beads allow for facile purification e.g. using washing with buffer.
  • Functionalised magnetic beads are commercially available with a variety of functionalisations from suppliers such as Sigma Aldrich and Bio-Rad.
  • the support comprises a solid surface.
  • Any suitable material can be used. Suitable materials include glass, silica, polymers such as polyester, and ceramics such as hydroxyapatite.
  • the support is functionalised for binding to the purification tag.
  • the support can be functionalised depending on the purification tag comprised in the multifunctional molecule that is used.
  • the purification tag can be chosen depending on the support material to be used.
  • the choice of purification tag and support material is an operational parameter which can be determined by the user of the disclosed methods.
  • the support comprises streptavidin, neutravidin or avidin, or a derivative of streptavidin, neutravidin or avidin such as traptavidin.
  • Such supports are particularly useful when the multifunctional molecule comprises a purification tag comprising biotin.
  • the support comprises a metal such as nickel or cobalt.
  • the metal ion may be provided with a suitable chelator such as nitriloacetic acid (NTA) or iminodiacetic acid (IDA)
  • NTA nitriloacetic acid
  • IDA iminodiacetic acid
  • the support may comprise Ni-NTA.
  • Such supports are particularly useful when the multifunctional molecule comprises a purification tag comprising a His tag.
  • the support comprises streptactin.
  • Such supports are particularly useful when the multifunctional molecule comprises a purification tag comprising a Strep tag.
  • the support comprises an antibody for a sequence such as FLAG, HA, Myc or V5 as discussed above.
  • the purification tag comprises a biotin group and the support comprises streptavidin, neutravidin or avidin, preferably streptavidin.
  • the cleavable purification tag comprises a structure of the wherein the wavy line represents the point of attachment to the chemical modifying group (either directly or via a linker) and the support comprises streptavidin; e.g. the support may comprise an agarose or sepharose resin comprising streptavidin or streptavidin-coated magnetic beads.
  • the provided methods comprise the modification of a monomer.
  • the discussion herein focusses primarily on the modification of protein monomers of protein nanopores, and particularly the modification of monomers of multicomponent protein nanopores.
  • the disclosed methods are not limited to such monomers and are useful for the chemical modification of monomeric proteins, polynucleotides, components of polynucleotide origami (e.g. DNA origami) structures such as origami pores, enzymes (including motor proteins as discussed herein), and the like.
  • the monomer is a polypeptide having a mass of from about 10 kDa to about 1 MDa.
  • any suitable nanopore can be used.
  • a nanopore is a transmembrane pore.
  • a transmembrane pore is a structure that crosses the membrane to some degree. It permits hydrated ions driven by an applied potential to flow across or within the membrane.
  • the transmembrane pore typically crosses the entire membrane so that hydrated ions may flow from one side of the membrane to the other side of the membrane.
  • the transmembrane pore does not have to cross the membrane. It may be closed at one end.
  • the pore may be a well, gap, channel, trench or slit in the membrane along which or into which hydrated ions may flow.
  • the monomer may be a monomer of a biological or artificial nanopore.
  • Suitable pores include, but are not limited to, protein pores and polynucleotide pores.
  • the monomer is a monomer of a polynucleotide pore.
  • a polynucleotide pore may be a DNA origami pore (Langecker et al., Science, 2012; 338: 932-936). Suitable DNA origami pores are disclosed in WO2013/083983.
  • a monomer of a polynucleotide origami pore is typically a polynucleotide of between 50 nt and 1000 kb; such as between 100 nt and 100 kb, e.g. between 1000 nt (1 kb) and 10 kb. The monomer assembles into a structure permitting ion transport from one chamber to another.
  • One or more monomers may assemble into such a structure.
  • an origami pore is typically oligomeric.
  • the monomers that assemble into an origami pore may be of the same type (i.e. the pore may be homooligomeric) or may be of two or more different types (i.e. the pore may be a multicomponent heterooligomeric pore).
  • the monomer is a monomer of a transmembrane protein pore.
  • a transmembrane protein pore is a polypeptide or a collection of polypeptides that permits hydrated ions, such as polynucleotide, to flow from one side of a membrane to the other side of the membrane.
  • the transmembrane protein pore typically is capable of forming a pore that permits hydrated ions driven by an applied potential to flow from one side of the membrane to the other.
  • the transmembrane protein pore preferably permits polynucleotides to flow from one side of the membrane, such as a triblock copolymer membrane, to the other.
  • the transmembrane protein pore allows a polynucleotide to be moved through the pore.
  • the monomer is a monomer of a monomeric nanopore; i.e. the monomer forms a transmembrane protein pore.
  • the monomer is a monomer of an oligomeric pore.
  • the monomer may be a monomer of a pore made up 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.
  • the monomer is a monomer of a pore comprising at least 20 subunits, at least 30 subunits, at least 40 subunits or at least 50 subunits.
  • pleurotolysin from Pleurotus ostreatus consists of two distinct monomeric components.
  • Pleurotolysin A Ply A
  • Pleurotolysin B PlyB
  • PlyA binds on the membrane and acts as a scaffold to recruit the second component PlyB which spans the membrane to form the transmembrane channel.
  • PlyB monomers can be modified with the said method to introduce different chemical groups within the channel. Accordingly, in some embodiments the monomer is a monomer of PlyA or Ply B.
  • the monomer may be a monomer of a hexameric, heptameric, octameric or nonameric pore.
  • the pore may be a homo-oligomer or a hetero-oligomer.
  • a transmembrane protein pore typically comprises a barrel or channel through which the ions may flow.
  • the subunits of the pore typically surround a central axis and contribute strands to a transmembrane P-barrel or channel or a transmembrane a-helix bundle or channel.
  • the barrel or channel of a transmembrane protein pore comprises amino acids that facilitate interaction with an analyte, such as a target polynucleotide (as described herein). These amino acids are preferably located near a constriction of the barrel or channel.
  • a transmembrane protein pore typically comprises one or more positively charged amino acids, such as arginine, lysine or histidine, or aromatic amino acids, such as tyrosine or tryptophan. These amino acids typically facilitate the interaction between the pore and nucleotides, polynucleotides or nucleic acids.
  • the monomer is a monomer of a transmembrane protein pore derived from ⁇ -barrel pores or a-helix bundle pores.
  • P-barrel pores comprise a barrel or channel that is formed from P-strands.
  • Suitable P-barrel pores include, but are not limited to, P-toxins, such as a-hemolysin, anthrax toxin and leukocidins, outer membrane phospholipase A and other pores, such as lysenin, NetB and CytK.
  • a-helix bundle pores comprise a barrel or channel that is formed from a -helices.
  • Suitable a-helix bundle pores include, but are not limited to, inner membrane proteins and a outer membrane proteins.
  • the monomer is a monomer of a transmembrane pore derived from or based on a-hemolysin (a-HL), lysenin, or haemolytic protein fragaceatoxin C (FraC).
  • a-HL a-hemolysin
  • lysenin lysenin
  • FraC haemolytic protein fragaceatoxin C
  • the monomer is a monomer of an actinporin.
  • Actinporins include Equinatoxin II (Eqtll) from Actinia equina and Fragaceatoxin C (FraC) from Actinia fragacea.
  • the monomer is a monomer of a homolog or paralog of any one of the protein pores discussed herein.
  • the monomer is a monomer of a transmembrane pore derived from lysenin, or a paralog or homolog thereof.
  • suitable pores derived from lysenin are disclosed in WO 2013/153359.
  • the monomer is a monomer of a transmembrane pore derived from or based on a-hemolysin (a-HL), or a paralog or homolog thereof.
  • a-HL a-hemolysin
  • the wild type a- hemolysin pore is formed of 7 identical monomers or sub-units (i.e., it is heptameric).
  • An a-hemolysin pore may be a-hemolysin-NN or a variant thereof.
  • the variant preferably comprises N residues at positions El 11 and K147.
  • the monomer is a monomer of a transmembrane pore derived from or based on NetB, or a paralog or homolog thereof.
  • NetB is a pore-forming toxin produced by Clostridium perfringens.
  • the monomer is a monomer of a transmembrane pore derived from or based on CytK, or a paralog or homolog thereof.
  • CytK is a pore-forming toxin produced by Bacillus cerius.
  • the monomer is a monomer of a gamma-hemolysin pore, or a paralog or homolog thereof.
  • the monomer is a monomer of a multi-component pore.
  • a multi-component pore is a pore which comprises two or more different monomers.
  • gamma-hemolysin is a multicomponent pore comprising two different types of monomer: in other words it is a bicomponent pore.
  • the disclosed method is particularly suitable for modifying multicomponent pores because it can be used to inctroduce different modifications into the different components of the pore. Thus, a first modification can be made in a first component, a second modification can be made in a second component, etc.
  • the functionality of the chemical modification can be precisely located at multiple locations (e.g at two or more locations) within the pore, e.g. within the channel of the pore.
  • modifications can be made to introduce or modify two recognition sites within the channel of a multifunctional pore allowing for improved characterisation of analytes such as polynucleotides.
  • Figure 1 depicts how the disclosed methods could be used to introduce multiple modifications into a bicomponent pore such as gamma-hemolysin.
  • the monomer may be a monomer of any of the nanopores discussed above.
  • the methods provided herein also relate to methods of producing a chemically modified nanopore.
  • the nanopore used in such methods may be any of the nanopores discussed in the context of monomers above.
  • the chemical modification in accordance with the disclosed methods can be made at a specific position within the monomer.
  • the specific position can be determined based on the structure of the free monomer or the structure adopted by the monomer when the monomer forms an oligomer (if applicable).
  • the structure used to determine the positioning of the modification is typically the three- dimensional structure of the oligomer formed from the monomer.
  • the structure may be the 3D structure of an oligomeric protein pore. 3D X-ray crystal structures are known for many oligomeric pores, and/or can determined by computational modelling.
  • the modification can be made to place the chemical modifying group at a set position in the assembled pore.
  • the modification is such that the modification is positioned at a solvent-accessible position on a surface of the pore.
  • the modification is positioned on an external surface of the pore.
  • the modification is positioned to interact with a membrane when the pore is assembled in the membrane, e.g. to improve anchoring of the pore to the membrane.
  • the modification is positioned at or near an opening of a pore; e.g. at or near the cis or trans opening of a channel running through the pore.
  • the modification is positioned within the channel of the pore.
  • the modification is at an internal-facing residue within the channel or barrel of the pore. In some embodiments the modification is positioned at or near a constriction within the channel of the pore. In some embodiments the modification is positioned to increase a constriction within the channel of the pore. In some embodiments the modification is positioned to introduce a constriction within the channel of the pore. In some embodiments the modification alters the properties of a channel through the pore, e.g. by introducing chemical functionality to the channel.
  • the monomer can be modified to ensure the presence of an appropriate number of modification sites for reaction with the reactive group of the multifunctional molecule. In some embodiments the monomer can be modified to introduce one or more modification sites. In some embodiments the monomer can be modified to remove or delete one or more modification sites. In some embodiments the monomer can be modified to delete one or more sites which would otherwise react with the reactive group of the multifunctional molecule and also to introduce one or more modification sites for reacting with the reactive group of the multifunctional molecule; i.e. the monomer can be modified to replace one or more modification sites with one or more different modification sites.
  • One or more modification sites can be introduced into the monomer by any suitable means.
  • the monomer is a polypeptide monomer of an oligomeric protein pore and one or more modification sites are introduced by mutation of the native amino acid sequence of the polypeptide monomer.
  • the one or more modification sites are introduced by insertion of one or more residues into the native sequence.
  • the one or more modification sites are introduced by substitution of one or more residues of the native sequence.
  • one or more cysteine residues may be introduced into the native sequence of the monomer.
  • Cysteine residues may be used e.g. when the multifunctional molecule comprises a sulfhydryl-reactive group as the reactive group, e.g. when the multifunctional molecule comprises a thiol or maleimide group as the reactive group.
  • One or more cysteine residues may be introduced e.g. by substitution of one or more non- cysteine residues in the monomer.
  • One or more cysteine residues may be introduced e.g. by insertion of one or more amino acids into the native amino acid sequence of the monomer.
  • one or more amino-containing residues may be introduced into the native sequence of the monomer.
  • Amino-containing residues may be used e.g. when the multifunctional molecule comprises a amino-reactive group as the reactive group, e.g. when the multifunctional molecule comprises an NHS-ester group as the reactive group.
  • One or more amino-containing residues, e.g. lysines may be introduced e.g. by substitution of one or more residues in the monomer.
  • One or more amino-containing residues, e.g. lysines may be introduced e.g. by insertion of one or more amino acids into the native amino acid sequence of the monomer.
  • Amino acid insertions, deletions and substitutions may be made in the native amino acid sequence of the monomer using techniques known in the art; e.g. those described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 th 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).
  • One or more reactive non-natural amino acids may be introduced into the native sequence of the monomer.
  • Reactive non-natural amino acids may be used e.g. when the multifunctional molecule comprises a reactive group for reacting with such non-natural amino acids, such as a click reagent.
  • One or more non-natural amino acids may be introduced as described herein e.g. by including synthetic aminoacyl-tRNAs in the IVTT system used to express the mutant monomer or by expressing the mutant monomer in a suitable bacterial expression system (e.g. E. coll) comprising bacteria that are auxotrophic for specific amino acids in the presence of synthetic (i.e. non-naturally-occurring) analogues of those specific amino acids. They may also be produced by naked ligation if the mutant monomer is produced using partial peptide synthesis.
  • the monomer is modified to comprise one or more modification sites. In some embodiments the monomer is modified to comprise 1, 2, 3, 4 or 5 modification sites. In some embodiments the monomer is modified to comprise exactly one or two modification sites, such as exactly one modification site.
  • the monomer is a monomer of an oligomeric pore; and the multifunctional molecule reacts with a reactive functional group located on the monomer at a surface-exposed position when the monomer is oligomerised to form the pore.
  • the surface-exposed position is located at the surface of the channel through the pore.
  • the surface- exposed position is located on the exterior surface of the pore.
  • the multifunctional molecule reacts with a reactive functional group located on the monomer at a position located at or near (e.g. within 10 nm, e.g. within 5 nm, such as within 3 nm, e.g. within 2 nm, e.g. within 1 nm) a constriction of the channel through the pore when the monomer is oligomerised to form the pore.
  • a reactive functional group located on the monomer at a position located at or near (e.g. within 10 nm, e.g. within 5 nm, such as within 3 nm, e.g. within 2 nm, e.g. within 1 nm) a constriction of the channel through the pore when the monomer is oligomerised to form the pore.
  • the monomer is a monomer of an aerolysin-like pore, such as aerolysin, lysenin, epsilon toxin ( ⁇ -Toxin) type B, parasporin-2, LSL, monalysin, enterotoxin or haemolytic lectin, or a paralog or homolog thereof.
  • aerolysin-like pore such as aerolysin, lysenin, epsilon toxin ( ⁇ -Toxin) type B, parasporin-2, LSL, monalysin, enterotoxin or haemolytic lectin, or a paralog or homolog thereof.
  • the monomer is a monomer of a leukocidin-like pore, such as gamma-hemolysin, (HlgAB or HlgCB), LukAB/HG, LukED, Panton-Valentine leukocidin (LukSF-PV/PVL), or LukMF', or a paralog or homolog thereof.
  • the monomer is a monomer of a hemolysin-like pore, such as VCC, CytK, NetB or alpha-hemolysin, or a paralog or homolog thereof.
  • the monomer is a monomer of an AB toxin-like pore, such as anthrax toxin protective antigen, or a paralog or homolog thereof.
  • the monomer is a monomer of the LukF subunit of gamma- hemolysin (SEQ ID NO: 10) or a variant, paralog or homolog thereof.
  • the structure of gamma-hemolysin has been deposited at the protein data bank (PDB) under accession code 3B07.
  • the monomer is a variant having at least 60%, e.g. at least 70%, e.g. at least 80%, for example at least 85%, typically at least 90%, such as at least 95%, e.g. 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 to SEQ ID NO: 10.
  • 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 correspond to positions within the barrel of the gamma-hemolysin pore (once assembled) and may in some embodiments be modified in order to alter, e.g. to improve, interaction of an analyte such as a polynucleotide with the pore.
  • the monomer is modified at one or more of the positions corresponding to K43, D44, K45, S46, Y47, D48 or K49 of SEQ ID NO: 10. These positions correspond to positions in the cap domain of the gamma-hemolysin pore (once assembled) and may in some embodiments be modified in order to alter, e.g. to improve, interaction of an analyte such as a polynucleotide with the pore.
  • the monomer is modified at one or more of the positions corresponding to Y145, 1143, E141, F139, T137, G135, L133, G131, L129, N127, 1125, 1123, G121, F119, Y117, L115, N113, V111 or F109 of SEQ ID NO: 10.
  • These positions correspond to positions outside of the barrel of the gamma-hemolysin pore (once assembled) and may in some embodiments be modified in order to alter, e.g. to improve anchoring of the pore, e.g. by altering or improving interaction of the pore with a membrane.
  • 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 correspond to positions in the rim domain of the gamma-hemolysin pore (once assembled) and may in some embodiments be modified in order to alter, e.g. to improve anchoring of the pore, e.g. by altering or improving interaction of the pore with a membrane.
  • the monomer is a monomer of the Hlg2 subunit of gamma- hemolysin (SEQ ID NO: 11) or a variant, paralog or homolog thereof.
  • the monomer is a variant having at least 60%, e.g. at least 70%, e.g. at least 80%, for example at least 85%, typically at least 90%, such as at least 95%, e.g. 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 to SEQ ID NO: 11.
  • 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 correspond to positions within the barrel of the gamma-hemolysin pore (once assembled) and may in some embodiments be modified in order to alter, e.g. to improve, interaction of an analyte such as a polynucleotide with the pore.
  • 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 correspond to positions in the cap domain of the gamma-hemolysin pore (once assembled) and may in some embodiments be modified in order to alter, e.g. to improve, interaction of an analyte such as a polynucleotide with the pore.
  • the monomer is modified at one or more of the positions corresponding to Q139, Y137, 1135, K133, Y131, F129, G127, G125, 1123, P121, S119, Fl 17, G115, 1113, Y111, L109, Q107, V105 or A103 of SEQ ID NO: 11.
  • These positions correspond to positions outside of the barrel of the gamma-hemolysin pore (once assembled) and may in some embodiments be modified in order to alter, e.g. to improve anchoring of the pore, e.g. by altering or improving interaction of the pore with a membrane.
  • the monomer is modified at one or more of the positions corresponding to Q180, D181, P182, T183, G184, P185, T241, R242, H243 or R244 of SEQ ID NO: 11. These positions correspond to positions in the rim domain of the gamma- hemolysin pore (once assembled) and may in some embodiments be modified in order to alter, e.g. to improve anchoring of the pore, e.g. by altering or improving interaction of the pore with a membrane.
  • the monomer is a monomer of lysenin (SEQ ID NO: 12) or a variant, paralog or homolog thereof.
  • the structure of lysenin has been deposited at the protein data bank (PDB) under accession code 5EC5.
  • the monomer is a variant having at least 60%, e.g. at least 70%, e.g. at least 80%, for example at least 85%, typically at least 90%, such as at least 95%, e.g. 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 to SEQ ID NO: 12.
  • the monomer is modified at one or more of the positions corresponding to D35, K37, T39, T41, G43, K45, V47, S49, T51, T53, T55, T57, S59, G61, T63, S65, G67, A69, 172, S74, E76, S78, S80, S82, Q84, S86, V88, M90, Q92, D94, Y96, S98, V100, E102, T104 or K106 of SEQ ID NO: 12.
  • the monomer is a monomer of Cytotoxin K from Bacillus cereus (CytK) (SEQ ID NO: 13) or a variant, paralog or homolog thereof.
  • the monomer is a variant having at least 60%, e.g. at least 70%, e.g. at least 80%, for example at least 85%, typically at least 90%, such as at least 95%, e.g. 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 to SEQ ID NO: 13.
  • the monomer is modified at one or more of the positions corresponding to E113, T115, T117, SI 19, S121, Q123, G125, S127, K129, S131, T133, G136, S138, E140, G142, T144, Q146, T148, S150, S152, S154, or K156 of SEQ ID NO: 13.
  • the monomer is a monomer of aerolysin (SEQ ID NO: 14) or a variant, paralog or homolog thereof.
  • the structure of aerolysin has been deposited at the protein data bank (PDB) under accession code 5JZT.
  • the monomer is a variant having at least 60%, e.g. at least 70%, e.g. at least 80%, for example at least 85%, typically at least 90%, such as at least 95%, e.g. 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 to SEQ ID NO: 14.
  • the monomer is modified at one or more of the 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.
  • the monomer is a monomer of NetB (SEQ ID NO: 15) or a variant, paralog or homolog thereof.
  • the structure of NetB has been deposited at the protein data bank (PDB) under accession code 4H56.
  • the monomer is a variant having at least 60%, e.g. at least 70%, e.g. at least 80%, for example at least 85%, typically at least 90%, such as at least 95%, e.g. 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 to SEQ ID NO: 15.
  • 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.
  • the monomer is a monomer of alpha-hemolysin (SEQ ID NO: 16) or a variant, paralog or homolog thereof.
  • the structure of alpha-hemolysin has been deposited at the protein data bank (PDB) under accession code 7AHL.
  • the monomer is a variant having at least 60%, e.g. at least 70%, e.g. at least 80%, for example at least 85%, typically at least 90%, such as at least 95%, e.g. 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 to SEQ ID NO: 16.
  • 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.
  • the monomer is a monomer of VCC (SEQ ID NO: 17) or a variant, paralog or homolog thereof.
  • the structure of VCC has been deposited at the protein data bank (PDB) under accession code 3044.
  • the monomer is a variant having at least 60%, e.g. at least 70%, e.g. at least 80%, for example at least 85%, typically at least 90%, such as at least 95%, e.g. 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 to SEQ ID NO: 17.
  • 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 SEQ ID NO: 17.
  • the monomer is a monomer of Anthrax PA (SEQ ID NO: 18) or a variant, paralog or homolog thereof.
  • the structure of Anthrax protective antigen has been deposited at the protein data bank (PDB) under accession code 3 J9C.
  • the monomer is a variant having at least 60%, e.g. at least 70%, e.g. at least 80%, for example at least 85%, typically at least 90%, such as at least 95%, e.g. 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 to SEQ ID NO: 18.
  • 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.
  • the monomer is a monomer of ⁇ -Toxin (SEQ ID NO: 19) or a variant, paralog or homolog thereof.
  • the structure of ⁇ -Toxin type B has been deposited at the protein data bank (PDB) under accession code 6RB9.
  • the monomer is a variant having at least 60%, e.g. at least 70%, e.g. at least 80%, for example at least 85%, typically at least 90%, such as at least 95%, e.g. 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 to SEQ ID NO: 19.
  • the monomer is modified at one or more of the positions corresponding to S90, T92, K94, T96, T98, T100, T102, T104, T106, G108, SI 10, Q112, 114, K116, T118, E123, G125, S127, S131, S133, A135, T137, T139, T141, S143, E145, T147 or P151 of SEQ ID NO: 19.
  • the monomer is a monomer of Fragaceatoxin C (FraC) from Actinia fragacea (SEQ ID NO: 21) or a variant, paralog or homolog thereof.
  • the structure of FraC from Actinia fragacea has been deposited at the protein data bank (PDB) under accession code 3W9P.
  • the monomer is a variant having at least 60%, e.g. at least 70%, e.g. at least 80%, for example at least 85%, typically at least 90%, such as at least 95%, e.g. 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 to SEQ ID NO: 21.
  • the monomer is a monomer of PlyA (SEQ ID NO: 22) or PlyB (SEQ ID NO: 23) from Pleurotus ostreatus or a variant, paralog or homolog thereof.
  • the structure of Ply AB has been deposited at the protein data bank (PDB) under accession code 4V2T.
  • the monomer is a variant having at least 60%, e.g. at least 70%, e.g. at least 80%, for example at least 85%, typically at least 90%, such as at least 95%, e.g.
  • the monomer is a variant having at least 60%, e.g. at least 70%, e.g. at least 80%, for example at least 85%, typically at least 90%, such as at least 95%, e.g. 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 to SEQ ID NO: 23.
  • the monomer is a monomer of a leukocidin pore such as LukF/HlgB (PDB code 1LKF), the S component of Panton-Valentine leukocidin (PDB code 1T5R) and the F component of Panton-Valentine leukocidin (PDB code 1PVL); an AB toxin such as Anthrax toxin protective antigen; an Aerolysin-like pore such as Parasporin-2 (PDB code 2ZTB), Clostridium perfringens epsilon toxin (PDB code 6RB9), Hemolytic Lectin (PDB code 1W3 A); and pesticidal proteins of Bacillus thuringiensis such as Cry and Cyt proteins.
  • a leukocidin pore such as LukF/HlgB (PDB code 1LKF), the S component of Panton-Valentine leukocidin (PDB code 1T5R) and the F component of Panton-
  • the monomer is a monomer of one of the six known leukocidins of S. aureus: gamma hemolysins HlgAB and HlgCB; LukAB/HG; LukED; Panton-Valentine leukocidin (LukSF-PV/PVL) and LukMF'.
  • the monomer is a monomer of an enzyme, such as an oligomeric enzyme.
  • the monomer is a monomer of an oligomeric helicase.
  • enzymes suitable for modification in accordance with the invention can, in some embodiments, be motor proteins as described in more detail herein.
  • the method comprises, prior to step (a), the steps of (i) expressing the monomer in a cellular expression system or a cell-free expression system; and (ii) isolating and/or purifying the monomer.
  • the monomer may be expressed in any suitable expression system. Suitable expression systems include bacterial expression systems such as those which use strains of E. coli to express the monomer from a suitable vector. Other suitable expression systems include expression in insect cells or yeast.
  • step (d) of the method can further comprise the step of removing unmodified monomer(s) and/or unreacted multifunctional molecule(s), if present, from the support.
  • Any suitable technique can be used.
  • the bound modified monomer can be washed using an aqueous or non-aqueous solved, e.g. a wash buffer, in order to remove unbound or weakly bound impurities from the support.
  • the methods of the present disclosure are used to produce oligomers.
  • the method disclosed herein further comprises the step of: f) oligomerising the chemically modified monomer to form a chemically modified oligomer.
  • the monomer is a protein monomer and step (f) comprises forming an oligomeric protein.
  • the monomer may be a monomer of an oligomeric protein nanopore and step (f) may comprise forming an oligomeric protein nanopore.
  • step (f) comprises oligomerising two or more chemically modified monomers to form a homooligomer.
  • the two or more monomers are the same; i.e. the two or monomers are of the same monomeric structure and have been chemically modified in the same way.
  • the two or more monomers are protein monomers and said homooligomer is a homooligomeric protein pore.
  • step (f) comprises oligomerising one or more chemically modified monomers with one or more unmodified or differently-modified monomers to form a heterooligomer.
  • the two or more monomers are protein monomers and said homooligomer is a homooligomeric protein pore.
  • the one or more different monomers can be unmodified monomers which are of the same type (e.g. the same sequence) as the chemically modified monomer but have not been modified.
  • the one or more different monomers can be unmodified monomers which are of a different type (e.g. are of a different sequence) as the chemically modified monomer and have not been modified.
  • the one or more different monomers can be chemically modified monomers which are of the same type (e.g. the same sequence) as the first chemically modified monomer but have been modified with a different chemical modification.
  • the one or more different monomers can be modified monomers which are of a different type (e.g. are of a different sequence) as the first chemically modified monomer and have not been modified.
  • the one or more first monomers can comprise two or more, e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 first monomers.
  • the one or more second monomers can comprise two or more, e.g. 2, 3, 4, 5, 6, 7, 8, 9 or 10 second monomers.
  • the disclosure also provides a method of producing a homooligomeric protein; comprising i) producing a plurality of chemically modified protein monomers in a method as described herein; and ii) oligomerising two or more of the chemically modified protein monomers obtained in step (i) to form a homooligomeric protein.
  • the homooligomeric protein is a homooligomeric protein pore, such as a protein pore described herein.
  • the disclosure also provides a method of producing a heterooligomeric protein; comprising i) producing one or more chemically modified first protein monomers in a method as described herein; and ii) producing one or more chemically modified second protein monomers in a method as described herein; and iii) oligomerising said one or more first monomers and said one or more second monomers to form a hetero-oligomeric protein.
  • the heterooligomeric protein is a heterooligomeric protein pore.
  • the disclosure further provides an oligomeric protein; comprising i) producing one or more chemically modified first protein monomers in a method as described herein; and ii) providing one or more unmodified second protein monomers; and iii) oligomerising said one or more first monomers and said one or more second monomers to form a hetero-oligomeric protein.
  • the heterooligomeric protein is a heterooligomeric protein pore.
  • the present disclosure also provides the products of the claimed methods.
  • the disclosure provides a chemically modified monomer obtainable by carrying out a method as described herein.
  • the monomer is a monomer as described herein.
  • the monomer is chemically modified by one or more modifications as described herein.
  • the monomer is a monomer of a protein nanopore as described herein.
  • the disclosure also provides a chemically modified oligomer obtainable by carrying out a method as described herein.
  • the oligomer is an oligomer comprising two or more monomers as described herein.
  • the oligomer is chemically modified by one or more modifications as described herein.
  • the oligomer is an oligomeric protein nanopore as described herein.
  • the disclosure also provides populations of chemically modified monomers.
  • the populations of such monomers are characterised inter alia by their homogeneity.
  • the disclosure thus 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 modifying group.
  • at least 96%, such as at least 97%, e.g. at least 98%, for example at least 99%, e.g. at least 99.5% such as at least 99.9% or at least 99.99% of the monomers in the population are chemically modified with a chemical modifying group.
  • 100% of the monomers in the population are modified with a chemical modifying group.
  • the population is a population of monomers of a protein pore, e.g. a protein pore as described herein.
  • the disclosure also provides populations of chemically modified oligomers, such as oligomeric protein pores.
  • the populations of such oligomers are characterised inter alia by their homogeneity.
  • the disclosure thus 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 modifying group.
  • at least 96%, such as at least 97%, e.g. at least 98%, for example at least 99%, e.g. at least 99.5% such as at least 99.9% or at least 99.99% of the oligomers in the population are chemically modified with a chemical modifying group.
  • 100% of the oligomers in the population are modified with a chemical modifying group.
  • a chemical modifying group typically not capable of providing such homogeneous populations as modification chemistry is typically not 100% efficient, and modified monomers typically cannot be readily separated or purified from impurities in order to provide highly homogeneous populations of oligomers.
  • the population is a population of oligomeric protein pores, e.g. protein pores as described herein.
  • modified proteins find general utility in the fields of biotechnology and medicine.
  • modified enzymes can have enhanced or altered functionality compared to their native counterparts.
  • the monomers are monomers of a nanopore; and/or the oligomer is an oligomeric nanopore.
  • Such nanopores can be used in the detection and characterisation of analytes such as polynucleotides.
  • analyte comprising: i) producing a chemically modified oligomeric pore in a method as described herein; and ii) taking one or more measurements as the analyte moves with respect to the pore, wherein the one or more measurements are indicative of one or more characteristics of the analyte, and thereby characterising the analyte as it moves with respect to the pore.
  • Also provided is a method of characterising an analyte comprising: i) providing a chemically modified oligomeric pore as described herein; and ii) taking one or more measurements as the analyte moves with respect to the pore, wherein the one or more measurements are indicative of one or more characteristics of the analyte, and thereby characterising the analyte as it moves with respect to the pore.
  • the analyte is a polynucleotide as described in more detail herein.
  • the disclosed characterisation methods may be carried out using any apparatus that is suitable for investigating a membrane/pore system in which a pore is inserted into a membrane.
  • Membranes are described in more detail herein.
  • the characterisation method may be carried out using any apparatus that is suitable for transmembrane pore sensing.
  • the apparatus may comprise a chamber comprising an aqueous solution and a barrier that separates the chamber into two sections.
  • the barrier may have an aperture in which a membrane containing a transmembrane pore, e.g. a chemically modified transmembrane pore as described herein, is formed. Transmembrane pores are described herein.
  • the characterisation methods may be carried out using the apparatus described in WO 2008/102120, WO 2010/122293 or WO 00/28312.
  • the binding of a molecule (e.g. a target polynucleotide) in the channel of the pore will have an effect on the open-channel ion flow through the pore, which is the essence of "molecular sensing" of pore channels.
  • the characterisation methods may thus involve measuring the ion current flow through the pore, typically by measurement of a current (for example, WO 2000/28312 and D. Stoddart et al., Proc. Natl. Acad. Sci., 2010, 106, 7702-7 or WO 2009/077734).
  • the ion flow through the pore may be measured optically, such as disclosed by Heron et al: J. Am. Chem. Soc. 9 Vol. 131, No. 5, 2009. Therefore the apparatus may also comprise an electrical circuit capable of applying a potential and measuring an electrical signal across the membrane and pore.
  • the characterisation methods may be carried out using a patch clamp or a voltage clamp.
  • the characterisation methods preferably involve the use of
  • the reduction in ion flow through the channel is advantageously correlated to the size of the individual nucleotide passing through the constriction (or "reading head"). Accordingly, modification of the reading head of a nanopore in accordance with the claimed methods can be useful in tuning this interaction and improving the characterisation signal that can be obtained.
  • the characterisation methods may be carried out on a silicon-based array of wells where each array comprises 128, 256, 512, 1024, 2000, 3000, 4000, 6000, 10000, 12000, 15000 or more wells.
  • the characterisation methods may involve the measuring of a current flowing through the pore.
  • the method is typically carried out with a voltage applied across the membrane and pore.
  • the voltage used is typically from +2 V to -2 V, typically -400 mV to +400mV.
  • the voltage used is preferably in a range having a lower limit selected from -400 mV, -300 mV, -200 mV, -150 mV, -100 mV, -50 mV, -20m V and 0 mV and an upper limit independently selected from +10 mV, + 20 mV, +50 mV, +100 mV, +150 mV, +200 mV, +300 mV and +400 mV.
  • the voltage used is more preferably in the range 100 mV to 240mV and most preferably in the range of 120 mV to 220 mV. It is possible to increase discrimination between different nucleotides by a pore by using an increased applied potential.
  • the characterisation methods are typically carried out in the presence of any charge carriers, such as metal salts, for example alkali metal salts, halide salts, for example chloride salts, such as alkali metal chloride salt.
  • Charge carriers may include ionic liquids or organic salts, for example tetramethyl ammonium chloride, trimethylphenyl ammonium chloride, phenyltrimethyl ammonium chloride, or l-ethyl-3 -methyl imidazolium chloride.
  • the salt is present in the aqueous solution in the chamber. Potassium chloride (KC1), sodium chloride (NaCl) or caesium chloride (CsCl) is typically used. KC1 is preferred.
  • the salt may be an alkaline earth metal salt such as calcium chloride (CaC12).
  • the salt concentration may be at saturation.
  • the salt concentration may be 3M or lower and is typically from 0.1 to 2.5 M, from 0.3 to 1.9 M, from 0.5 to 1.8 M, from 0.7 to 1.7 M, from 0.9 to 1.6 M or from 1 M to 1.4 M.
  • the salt concentration is preferably from 150 mM to 1 M.
  • the characterisation method is preferably carried out using a salt concentration of at least 0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M or at least 3.0 M.
  • High salt concentrations provide a high signal to noise ratio and allow for currents indicative of binding/no binding to be identified against the background of normal current fluctuations.
  • the characterisation methods are typically carried out in the presence of a buffer.
  • the buffer is present in the aqueous solution in the chamber. Any suitable buffer may be used.
  • the buffer is HEPES.
  • Another suitable buffer is Tris-HCl buffer.
  • the methods are typically carried out at a pH of from 4.0 to 12.0, from 4.5 to 10.0, from 5.0 to 9.0, from 5.5 to 8.8, from 6.0 to 8.7 or from 7.0 to 8.8 or 7.5 to 8.5.
  • the pH used is preferably about 7.5.
  • the characterisation methods may be carried out at from 0 °C to 100 °C, from 15 °C to 95 °C, from 16 °C to 90 °C, from 17 °C to 85 °C, from 18 °C to 80 °C, 19 °C to 70 °C, or from 20 °C to 60 °C.
  • the characterisation methods are typically carried out at room temperature.
  • the characterisation methods are optionally carried out at a temperature that supports enzyme function, such as about 37 °C.
  • the transmembrane nanopore is typically present in a membrane. Any suitable membrane may be used in the system.
  • the membrane is preferably an amphiphilic layer.
  • An amphiphilic layer is a layer formed from amphiphilic molecules, such as phospholipids, which have both hydrophilic and lipophilic properties.
  • the amphiphilic molecules may be synthetic or naturally occurring.
  • Non-naturally occurring amphiphiles and amphiphiles which form a monolayer are known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450).
  • Block copolymers are polymeric materials in which two or more monomer sub-units that are polymerized together to create a single polymer chain. Block copolymers typically have properties that are contributed by each monomer sub-unit.
  • a block copolymer may have unique properties that polymers formed from the individual sub-units do not possess.
  • Block copolymers can be engineered such that one of the monomer sub-units is hydrophobic (i.e. lipophilic), whilst the other sub- units) are hydrophilic whilst in aqueous media.
  • the block copolymer may possess amphiphilic properties and may form a structure that mimics a biological membrane.
  • the block copolymer may be a diblock (consisting of two monomer sub- units), but may also be constructed from more than two monomer sub-units to form more complex arrangements that behave as amphipiles.
  • the copolymer may be a triblock, tetrablock or pentablock copolymer.
  • the membrane is preferably a triblock copolymer membrane.
  • Archaebacterial bipolar tetraether lipids are naturally occurring lipids that are constructed such that the lipid forms a monolayer membrane. These lipids are generally found in extremophiles that survive in harsh biological environments, thermophiles, halophiles and acidophiles. Their stability is believed to derive from the fused nature of the final bilayer. It is straightforward to construct block copolymer materials that mimic these biological entities by creating a triblock polymer that has the general motif hydrophilic-hydrophobic-hydrophilic.
  • This material may form monomeric membranes that behave similarly to lipid bilayers and encompass a range of phase behaviours from vesicles through to laminar membranes.
  • Membranes formed from these triblock copolymers hold several advantages over biological lipid membranes. Because the triblock copolymer is synthesised, the exact construction can be carefully controlled to provide the correct chain lengths and properties required to form membranes and to interact with pores and other proteins.
  • Block copolymers may also be constructed from sub-units that are not classed as lipid sub-materials; for example a hydrophobic polymer may be made from siloxane or other non-hydrocarbon based monomers.
  • the hydrophilic sub-section of block copolymer can also possess low protein binding properties, which allows the creation of a membrane that is highly resistant when exposed to raw biological samples.
  • This head group unit may also be derived from non-classical lipid head-groups.
  • Triblock copolymer membranes also have increased mechanical and environmental stability compared with biological lipid membranes, for example a much higher operational temperature or pH range.
  • the synthetic nature of the block copolymers provides a platform to customise polymer based membranes for a wide range of applications.
  • the membrane is one of the membranes disclosed in International Application No. WO2014/064443 or WO2014/064444.
  • the amphiphilic molecules may be chemically-modified or functionalised to facilitate coupling of the polynucleotide.
  • the amphiphilic layer may be a monolayer or a bilayer.
  • the amphiphilic layer is typically planar.
  • the amphiphilic layer may be curved.
  • the amphiphilic layer may be supported.
  • Amphiphilic membranes are typically naturally mobile, essentially acting as two dimensional fluids with lipid diffusion rates of approximately 10 -8 cm s' 1 . This means that the pore and coupled polynucleotide can typically move within an amphiphilic membrane.
  • the membrane may be a lipid bilayer. Lipid bilayers are models of cell membranes and serve as excellent platforms for a range of experimental studies. For example, lipid bilayers can be used for in vitro investigation of membrane proteins by single-channel recording. Alternatively, lipid bilayers can be used as biosensors to detect the presence of a range of substances.
  • the lipid bilayer may be any lipid bilayer.
  • Suitable lipid bilayers include, but are not limited to, a planar lipid bilayer, a supported bilayer or a liposome.
  • the lipid bilayer is preferably a planar lipid bilayer.
  • Suitable lipid bilayers are disclosed in WO 2008/102121, WO 2009/077734 and WO 2006/100484.
  • Lipid bilayers are commonly formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972; 69: 3561-3566), in which a lipid monolayer is carried on aqueous solution/air interface past either side of an aperture which is perpendicular to that interface.
  • the lipid is normally added to the surface of an aqueous electrolyte solution by first dissolving it in an organic solvent and then allowing a drop of the solvent to evaporate on the surface of the aqueous solution on either side of the aperture. Once the organic solvent has evaporated, the solution/air interfaces on either side of the aperture are physically moved up and down past the aperture until a bilayer is formed.
  • Planar lipid bilayers may be formed across an aperture in a membrane or across an opening into a recess.
  • Montal & Mueller The method of Montal & Mueller is popular because it is a cost-effective and relatively straightforward method of forming good quality lipid bilayers that are suitable for protein pore insertion.
  • Other common methods of bilayer formation include tip- dipping, painting bilayers and patch-clamping of liposome bilayers.
  • Tip-dipping bilayer formation entails touching the aperture surface (for example, a pipette tip) onto the surface of a test solution that is carrying a monolayer of lipid. Again, the lipid monolayer is first generated at the solution/air interface by allowing a drop of lipid dissolved in organic solvent to evaporate at the solution surface. The bilayer is then formed by the Langmuir-Schaefer process and requires mechanical automation to move the aperture relative to the solution surface.
  • the aperture surface for example, a pipette tip
  • lipid dissolved in organic solvent is applied directly to the aperture, which is submerged in an aqueous test solution.
  • the lipid solution is spread thinly over the aperture using a paintbrush or an equivalent. Thinning of the solvent results in formation of a lipid bilayer.
  • complete removal of the solvent from the bilayer is difficult and consequently the bilayer formed by this method is less stable and more prone to noise during electrochemical measurement.
  • Patch-clamping is commonly used in the study of biological cell membranes. The cell membrane is clamped to the end of a pipette by suction and a patch of the membrane becomes attached over the aperture.
  • the method has been adapted for producing lipid bilayers by clamping liposomes which then burst to leave a lipid bilayer sealing over the aperture of the pipette.
  • the method requires stable, giant and unilamellar liposomes and the fabrication of small apertures in materials having a glass surface.
  • Liposomes can be formed by sonication, extrusion or the Mozafari method (Colas et al. (2007) Micron 38:841-847).
  • a lipid bilayer is formed as described in International Application No. WO 2009/077734.
  • the lipid bilayer is formed from dried lipids.
  • the lipid bilayer is formed across an opening as described in W02009/077734.
  • a lipid bilayer is formed from two opposing layers of lipids.
  • the two layers of lipids are arranged such that their hydrophobic tail groups face towards each other to form a hydrophobic interior.
  • the hydrophilic head groups of the lipids face outwards towards the aqueous environment on each side of the bilayer.
  • the bilayer may be present in a number of lipid phases including, but not limited to, the liquid disordered phase (fluid lamellar), liquid ordered phase, solid ordered phase (lamellar gel phase, interdigitated gel phase) and planar bilayer crystals (lamellar sub-gel phase, lamellar crystalline phase).
  • lipid composition that forms a lipid bilayer may be used.
  • the lipid composition is chosen such that a lipid bilayer having the required properties, such surface charge, ability to support membrane proteins, packing density or mechanical properties, is formed.
  • the lipid composition can comprise one or more different lipids.
  • the lipid composition can contain up to 100 lipids.
  • the lipid composition preferably contains 1 to 10 lipids.
  • the lipid composition may comprise naturally-occurring lipids and/or artificial lipids.
  • the lipids typically comprise a head group, an interfacial moiety and two hydrophobic tail groups which may be the same or different.
  • Suitable head groups include, but are not limited to, neutral head groups, such as diacylglycerides (DG) and ceramides (CM); zwitterionic head groups, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin (SM); negatively charged head groups, such as phosphatidylglycerol (PG); phosphatidylserine (PS), phosphatidylinositol (PI), phosphatic acid (PA) and cardiolipin (CA); and positively charged headgroups, such as trimethylammonium-Propane (TAP).
  • neutral head groups such as diacylglycerides (DG) and ceramides (CM)
  • zwitterionic head groups such as phosphatidylcholine (PC), phosphatidylethanolamine (PE
  • Suitable interfacial moieties include, but are not limited to, naturally-occurring interfacial moieties, such as glycerol-based or ceramide- based moieties.
  • Suitable hydrophobic tail groups include, but are not limited to, saturated hydrocarbon chains, such as lauric acid (n-Dodecanolic acid), myristic acid (n- Tetradecononic acid), palmitic acid (n-Hexadecanoic acid), stearic acid (n-Octadecanoic) and arachidic (n-Eicosanoic); unsaturated hydrocarbon chains, such as oleic acid (cis-9- Octadecanoic); and branched hydrocarbon chains, such as phytanoyl.
  • the length of the chain and the position and number of the double bonds in the unsaturated hydrocarbon chains can vary.
  • the length of the chains and the position and number of the branches, such as methyl groups, in the branched hydrocarbon chains can vary.
  • the hydrophobic tail groups can be linked to the interfacial moiety as an ether or an ester.
  • the lipids may be mycolic acid.
  • the lipids can also be chemically-modified.
  • the head group or the tail group of the lipids may be chemically-modified.
  • Suitable lipids whose head groups have been chemically-modified include, but are not limited to, PEG-modified lipids, such as 1,2- Diacyl-sn-Glycero-3-Phosphoethanolamine-N -[Methoxy(Polyethylene glycol)-2000]; functionalised PEG Lipids, such as 1,2-Distearoyl-sn-Glycero-3 Phosphoethanolamine-N- [Biotinyl(Polyethylene Glycol)2000]; and lipids modified for conjugation, such as 1,2- Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(succinyl) and 1,2-Dipalmitoyl-sn- Glycero-3-Phosphoethanolamine-N-(Biotinyl).
  • Suitable lipids whose tail groups have been chemically-modified include, but are not limited to, polymerisable lipids, such as 1,2- bis(10,12-tricosadiynoyl)-sn-Glycero-3-Phosphocholine; fluorinated lipids, such as 1- Palmitoyl-2-(16-Fluoropalmitoyl)-sn-Glycero-3 -Phosphocholine; deuterated lipids, such as 1,2-Dipalmitoyl-D62-sn-Glycero-3 -Phosphocholine; and ether linked lipids, such as 1,2- Di-O-phytanyl-sn-Glycero-3 -Phosphocholine.
  • the lipids may be chemically-modified or functionalised to facilitate coupling of the polynucleotide.
  • the amphiphilic layer typically comprises one or more additives that will affect the properties of the layer.
  • Suitable additives include, but are not limited to, fatty acids, such as palmitic acid, myristic acid and oleic acid; fatty alcohols, such as 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.
  • the membrane comprises a solid state layer.
  • Solid state layers can be formed from both organic and inorganic materials including, but not limited to, microelectronic materials, insulating materials such as Si 3 N 4 , A1 2 O 3 , and SiO, organic and inorganic polymers such as polyamide, plastics such as Teflon® or elastomers such as two-component addition-cure silicone rubber, and glasses.
  • the solid state layer may be formed from graphene. Suitable graphene layers are disclosed in WO 2009/035647. If the membrane comprises a solid state layer, the pore is typically present in an amphiphilic membrane or layer contained within the solid state layer, for instance within a hole, well, gap, channel, trench or slit within the solid state layer.
  • suitable solid state/amphiphilic hybrid systems Suitable systems are disclosed in WO 2009/020682 and WO 2012/005857. Any of the amphiphilic membranes or layers discussed above may be used.
  • a motor protein can be used to control said movement.
  • the methods of chemically modifying a monomer as disclosed herein are also applicable to the chemical modification of such a motor protein (or a monomer thereof) whether for use in a disclosed method or in any other method.
  • a motor protein is any protein that is capable of binding to a polynucleotide and controlling its movement with respect to a nanopore, e.g. through the pore.
  • a motor protein is or is derived from a polynucleotide handling enzyme.
  • a polynucleotide handling enzyme is a polypeptide that is capable of interacting with and modifying at least one property of a polynucleotide.
  • the enzyme may modify the polynucleotide by cleaving it to form individual nucleotides or shorter chains of nucleotides, such as di- or trinucleotides.
  • the enzyme may modify the polynucleotide by orienting it or moving it to a specific position.
  • the motor protein is derived from a member of any of the Enzyme Classification (EC) groups 3.1.11, 3.1.13, 3.1.14, 3.1.15, 3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30 and 3.1.31.
  • EC Enzyme Classification
  • the motor protein is a helicase, a polymerase, an exonuclease, a topoisomerase, or a variant thereof.
  • a motor protein can be modified to prevent the motor protein disengaging from a polynucleotide or polynucleotide adapter.
  • modification of a motor protein in order to prevent it from disengaging from a spacer on a polynucleotide adapter is discussed in WO 2014/013260, which is hereby incorporated by reference in its entirety, and with particular reference to passages describing the modification of motor proteins such as helicases in order to prevent them from disengaging from polynucleotide strands.
  • a motor protein can be modified by treating with tetramethyl azodi carb oxami de .
  • the motor protein may have a polynucleotide-unbinding opening; e.g. a cavity, cleft or void through which a polynucleotide strand may pass when the motor protein disengages from the strand.
  • the motor protein may be modified by closing the polynucleotide-unbinding opening. Closing the polynucleotide-unbinding opening may therefore prevent the motor protein from disengaging.
  • the motor protein may be modified by covalently closing the polynucleotide-unbinding opening.
  • a preferred motor protein for addressing in this way is a helicase.
  • a motor protein is an exonuclease.
  • Suitable enzymes include, but are not limited to, exonuclease I from E. coli (SEQ ID NO: 1), exonuclease III enzyme from E. coli (SEQ ID NO: 2), Red from T. thermophilus (SEQ ID NO: 3) and bacteriophage lambda exonuclease (SEQ ID NO: 4), TatD exonuclease and variants thereof.
  • Three subunits comprising the sequence shown in SEQ ID NO: 3 or a variant thereof interact to form a trimer exonuclease.
  • a motor protein is a polymerase.
  • the polymerase may be PyroPhage® 3173 DNA Polymerase (which is commercially available from Lucigen® Corporation), SD Polymerase (commercially available from Bioron®), Klenow from NEB or variants thereof.
  • 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 invention are disclosed in US Patent No. 5,576,204.
  • a motor protein is a topoisomerase.
  • the topoisomerase is a member of any of the Moiety Classification (EC) groups 5.99.1.2 and 5.99.1.3.
  • the topoisomerase may be a reverse transcriptase, which are enzymes capable of catalysing the formation of cDNA from a RNA template. They are commercially available from, for instance, New England Biolabs® and Invitrogen®.
  • a motor protein is a helicase.
  • Any suitable helicase can be used in accordance with the methods provided herein.
  • the or each motor protein used in accordance with the present disclosure may be independently selected from a Hel308 helicase, a RecD helicase, a Tral helicase, a TrwC helicase, an XPD helicase, and a Dda helicase, or a variant thereof.
  • Monomeric helicases may comprise several domains attached together. For instance, Tral helicases and Tral subgroup helicases may contain two RecD helicase domains, a relaxase domain and a C-terminal domain.
  • the domains typically form a monomeric helicase that is capable of functioning without forming oligomers.
  • suitable helicases include Hel308, NS3, Dda, UvrD, Rep, PcrA, Pifl and Tral. These helicases typically work on single stranded DNA.
  • Hel308 helicases are described in publications such as WO 2013/057495, the entire contents of which are incorporated by reference.
  • RecD helicases are described in publications such as WO 2013/098562, the entire contents of which are incorporated by reference.
  • XPD helicases are described in publications such as WO 2013/098561, the entire contents of which are incorporated by reference.
  • Dda helicases are described in publications such as WO 2015/055981 and WO 2016/055777, the entire contents of each of which are incorporated by reference.
  • a helicase comprises the sequence shown in SEQ ID NO: 6 (Trwc Cba) or a variant thereof, the sequence shown in SEQ ID NO: 7 (Hel308 Mbu) or a variant thereof or the sequence shown in SEQ ID NO: 8 (Dda) or a variant thereof.
  • Variants may differ from the native sequences in any of the ways discussed herein.
  • An example variant of SEQ ID NO: 8 comprises E94C/A360C.
  • a further example variant of SEQ ID NO: 8 comprises E94C/A360C and then (AM1)G1G2 (i.e. deletion of Ml and then addition of G1 and G2).
  • a motor protein e.g. a helicase
  • the motor protein moves along the polynucleotide in a 5' to 3' or a 3' to 5' direction (depending on the motor protein).
  • the motor protein can be used to either move the polynucleotide away from (e.g. out of) a pore (e.g. against an applied field) or the polynucleotide towards (e.g. into) a pore (e.g. with an applied field).
  • the motor protein works against the direction of the field resulting from the applied potential and pulls the threaded polynucleotide out of the pore (e.g. into the cis chamber).
  • the motor protein works with the direction of the field resulting from the applied potential and pushes the threaded polynucleotide into the pore (e.g. into the trans chamber).
  • the motor protein e.g. helicase
  • the motor protein can bind to the polynucleotide and act as a brake slowing the movement of the polynucleotide when it is moved with respect to a nanopore, e.g. by being pulled into the pore by a field resulting from an applied potential.
  • the inactive mode it does not matter which end of the polynucleotide is captured, it is the applied field which determines the movement of the polynucleotide with respect to the pore, and the motor protein acts as a brake.
  • the movement control of the polynucleotide by the motor protein can be described in a number of ways including ratcheting, sliding and braking.
  • Fuel is typically free nucleotides or free nucleotide analogues.
  • the free nucleotides may be one or more of, but 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), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cAMP), cyclic aden
  • the free nucleotides are usually selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP.
  • the free nucleotides are typically adenosine triphosphate (ATP).
  • a cofactor for a motor protein is a factor that allows 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+ .
  • the cofactor is most preferably Mg 2+ .
  • the analyte is typically a polynucleotide.
  • a polynucleotide such as a nucleic acid, is a macromolecule comprising two or more nucleotides.
  • a polynucleotide can be single-stranded or double-stranded.
  • a double- stranded polynucleotide is made of two single stranded polynucleotides hybridised together.
  • the target polynucleotide can be a single-stranded polynucleotide or a double- stranded polynucleotide.
  • a polynucleotide may comprise any combination of any nucleotides.
  • the nucleotides can be naturally occurring or artificial.
  • a nucleotide typically contains a nucleobase, a sugar and at least one phosphate group.
  • the nucleobase and sugar form a nucleoside.
  • the nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C).
  • A adenine
  • G guanine
  • T thymine
  • U uracil
  • C cytosine
  • the sugar is typically a pentose sugar.
  • Nucleotide sugars include, but are not limited to, ribose and deoxyribose.
  • the sugar is preferably a deoxyribose.
  • the polynucleotide preferably comprises the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and/or thymidine (dT), deoxyguanosine (dG) and deoxycytidine (dC).
  • the nucleotide is typically a ribonucleotide or deoxyribonucleotide.
  • the nucleotide typically contains a monophosphate, diphosphate or triphosphate.
  • the nucleotide may comprise more than three phosphates, such as 4 or 5 phosphates. Phosphates may be attached on the 5' or 3' side of a nucleotide.
  • Nucleotides include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), 5-methylcytidine monophosphate, 5-hydroxymethylcytidine monophosphate, cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP), deoxycytidine monophosphate (dCMP) and deoxymethylcytidine monophosphate.
  • the nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP and dUMP.
  • a nucleotide may be abasic (i.e. lack a nucleobase).
  • a nucleotide may also lack a nucleobase and a sugar (i.e. is a C3 spacer).
  • the nucleotides in the polynucleotide may be attached to each other in any manner.
  • the nucleotides are typically attached by their sugar and phosphate groups as in nucleic acids.
  • the nucleotides may be connected via their nucleobases as in pyrimidine dimers.
  • the polynucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the polynucleotide can comprise one strand of RNA hybridized to one strand of DNA.
  • the polynucleotide may be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), bridged nucleic acid (BNA) or other synthetic polymers with nucleotide side chains.
  • PNA peptide nucleic acid
  • GNA glycerol nucleic acid
  • TAA threose nucleic acid
  • LNA locked nucleic acid
  • BNA bridged nucleic acid
  • the PNA backbone is composed of repeating N-(2- aminoethyl)-glycine units linked by peptide bonds
  • the GNA backbone is composed of repeating glycol units linked by phosphodi ester bonds.
  • the TNA backbone is composed of repeating threose sugars linked together by phosphodiester bonds.
  • LNA is formed from ribonucleotides as discussed above having an extra bridge connecting the 2' oxygen and 4' carbon in the ribose moiety.
  • the polynucleotide is preferably DNA, RNA or a DNA or RNA hybrid, most preferably DNA.
  • a DNA/RNA hybrid may comprise DNA and RNA on the same strand.
  • the DNA/RNA hybrid comprises one DNA strand hybridized to a RNA strand.
  • the backbone of the polynucleotide can be altered to reduce the possibility of strand scission.
  • DNA is known to be more stable than RNA under many conditions.
  • the backbone of the polynucleotide strand can be modified to avoid damage caused by e.g. harsh chemicals such as free radicals.
  • DNA or RNA that contains unnatural or modified bases can be produced by amplifying natural DNA or RNA polynucleotides in the presence of modified NTPs using an appropriate polymerase.
  • the nucleotides in the polynucleotide may be modified.
  • the nucleotides may be oxidized or methylated.
  • One or more nucleotides in the polynucleotide may be damaged.
  • the polynucleotide may comprise a pyrimidine dimer. Such dimers are typically associated with damage by ultraviolet light and are the primary cause of skin melanomas.
  • One or more nucleotides in the polynucleotide may be modified with a label or a tag.
  • a single-stranded polynucleotide may contain regions with strong secondary structures, such as hairpins, quadruplexes, or triplex DNA. Structures of these types can be used to control the movement of the polynucleotide with respect to the nanopore. For example, secondary structures can be used to pause the movement of the polynucleotide through a nanopore, as described in more detail herein. Each successive secondary structure along the strand pauses the movement of the strand with respect to the nanopore as it is unwound and translocated. The polynucleotide may reform secondary structures after it has translocated through the nanopore.
  • secondary structures can be used to pause the movement of the polynucleotide through a nanopore, as described in more detail herein. Each successive secondary structure along the strand pauses the movement of the strand with respect to the nanopore as it is unwound and translocated.
  • the polynucleotide may reform secondary structures after it has translocated through the nano
  • Such secondary structures can be used to prevent the polynucleotide from moving back through the nanopore under low or no applied negative voltages (applied to the trans side of the nanopore) and therefore assist in controlling the movement of the polynucleotide so it only occurs in a controlled manner in the relevant steps of the methods provided herein.
  • a double stranded polynucleotide may comprise single stranded regions and regions with other structures, such as hairpin loops, triplexes and/or quadruplexes.
  • Such secondary structures can be useful as described above in the context of single-stranded polynucleotides.
  • the two strands of a double-stranded molecule may be covalently linked, for example at the ends of the molecules by joining the 5' end of one strand to the 3' end of the other with a hairpin structure.
  • a target polynucleotide can be any length.
  • 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 in length.
  • the target polynucleotide can be 1000 or more nucleotides or nucleotide pairs, 5000 or more nucleotides or nucleotide pairs in length or 100000 or more nucleotides or nucleotide pairs in length or 500,000 or more nucleotides or nucleotide pairs in length, or 1,000,000 or more nucleotides or nucleotide pairs in length, 10, 000,000 or more nucleotides or nucleotide pairs in length, or 100,000,000 or more nucleotides or nucleotide pairs in length, or 200,000,000 or more nucleotides or nucleotide pairs in length, or the entire length of a chromosome.
  • a target polynucleotide may be an oligonucleotide.
  • Oligonucleotides are short nucleotide polymers which typically have 50 or fewer nucleotides, such 40 or fewer, 30 or fewer, 20 or fewer, 10 or fewer or 5 or fewer nucleotides.
  • the target oligonucleotide is preferably from about 15 to about 30 nucleotides in length, such as from about 20 to about 25 nucleotides in length.
  • the oligonucleotide can 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 in length.
  • the target polynucleotide may be a fragment of a longer polynucleotide.
  • the longer polynucleotide is typically fragmented into multiple, such as two or more, shorter polynucleotides.
  • the target polynucleotide may comprise the products of a PCR reaction, genomic DNA, the products of an endonuclease digestion and/or a DNA library.
  • the target polynucleotide may be naturally occurring.
  • the target polynucleotide may be secreted from cells.
  • the target analyte can be an analyte that is present inside cells such that the analyte must be extracted from the cells before the method can be carried out.
  • the target polynucleotide may be sourced from common organisms such as viruses, bacteria, archaea, plants or animals. Such organisms may be selected or altered to adjust the sequence of the target polynucleotide, for example by adjusting the base composition, removing unwanted sequence elements, and the like. The selection and alteration of organisms in order to arrive at desired polynucleotide characteristics is routine for one of ordinary skill in the art.
  • the source organism for the target polynucleotide may be chosen based on desired characteristics of the sequence. Desired characteristics include the ratio of single-stranded vs double-stranded polynucleotides produced by the organism; the complexity of the sequences of polynucleotides produced by the organism, the composition of the polynucleotides produced by the organism (such as the GC composition), or the length of contiguous polynucleotide strands produced by the organism. For example, when a contiguous polynucleotide strand of around 50 kb is required, lambda phage DNA can be used. If longer contiguous strands are required, other organisms can be used to produce the polynucleotide; for example A. coli produces around 4.5 Mb of contiguous dsDNA.
  • the target polynucleotide is often obtained from a human or animal, e.g. from urine, lymph, saliva, mucus, seminal fluid or amniotic fluid, or from whole blood, plasma or serum.
  • the target polynucleotide may be obtained from a plant e.g. a cereal, legume, fruit or vegetable.
  • the target polynucleotide may comprise genomic DNA.
  • the genomic DNA may be fragmented.
  • the DNA may be fragmented by any suitable method. For example, methods of fragmenting DNA are known in the art, Such methods may use a transposase, such as a MuA transposase. Often the genomic DNA is not fragmented.
  • the polynucleotide is synthetic or semi-synthetic.
  • DNA or RNA may be purely synthetic, synthesised by conventional DNA synthesis methods such as phosphoramidite based chemistries.
  • Synthetic polynucleotides subunits may be joined together by known means, such as ligation or chemical linkage, to produce longer strands.
  • internal self-forming structures e.g. hairpins, quadruplexes
  • Synthetic polynucleotides can be copied and scaled up for production by means known in the art, including PCR, incorporation into bacterial factories, and the like.
  • the polynucleotide may have a simplified nucleotide composition.
  • the polynucleotide has a repeating pattern of the same subunit.
  • a repeating unit may be (AmGn)q, wherein m, n and q are positive integers.
  • m is often from 1 to 20, such as from 1 to 10 e.g. from 1 to 5, e.g. 1, 2, 3, 4 or 5.
  • n is often from 1 to 20, such as from 1 to 10 e.g. from 1 to 5, e.g. 1, 2, 3, 4 or 5.
  • m and n may be the same or different, q is often from 1 to about 100,000.
  • a typical repeating unit may be for example (AAAAAAGGGGGG)q (SEQ ID NO: 36).
  • Repeating polynucleotides can be made by many means known in the art, for example by concatenating together synthetic subunits with sticky ends that enable ligation.
  • the polynucleotide may therefore be a concatenated polynucleotide. Methods of concatenating polynucleotides are described in PCT/GB2017/051493.
  • a target polynucleotide assessed in a method provided herein may comprise a polynucleotide adapter.
  • a polynucleotide adapter can be used to load e.g. a motor protein onto a polynucleotide in order to control the movement of the polynucleotide with respect to a nanopore.
  • WO 2015/110813 describes the loading of motor proteins onto a target polynucleotide such as an adapter, and is hereby incorporated by reference in its entirety.
  • An adapter typically comprises a polynucleotide strand capable of being attached to the end of a target polynucleotide.
  • a polynucleotide adapter may be added to both ends of the target polynucleotide.
  • different adapters may be added to the two ends of the target polynucleotide.
  • An adapter may be added to just one end of the target polynucleotide. Methods of adding adapters to polynucleotides are known in the art.
  • Adapters may be attached to polynucleotides, for example, by ligation, by click chemistry, by tagmentation, by topoisomerisation or by any other suitable method.
  • an adapter may be synthetic or artificial. Typically, an adapter comprises a polymer as described herein. In some embodiments, the adapter comprises a polynucleotide. In some embodiments an adapter may comprise a single-stranded polynucleotide strand. In some embodiments an adapter may comprise a double-stranded polynucleotide.
  • a polynucleotide adapter may comprise DNA, RNA, modified DNA (such as a basic DNA), RNA, PNA, LNA, BNA and/or PEG. Usually, the adapter comprises single stranded and/or double stranded DNA or RNA.
  • An adapter may be a Y adapter.
  • a Y adapter is typically double stranded and comprises (a) at one end, a region where the two strands are hybridised together and (b), at the other end, a region where the two strands are not complementary. The non- complementary parts of the strands form overhangs.
  • the hybridised stem of the adapter typically attaches to the 5' end of a first strand of a double-stranded polynucleotide and the 3' end of a second strand of a double-stranded polynucleotide; or to the 3' end of a first strand of a double-stranded polynucleotide and the 5' end of a second strand of a double- stranded polynucleotide.
  • the presence of a non-complementary region in the Y adapter gives the adapter its Y shape since the two strands typically do not hybridise to each other unlike the double stranded portion.
  • a motor protein or polynucleotide binding protein may bind to an overhang of an adapter such as a Y adapter.
  • a motor protein or polynucleotide binding protein may bind to the double stranded region.
  • a motor protein or polynucleotide binding protein may bind to a single- stranded and/or a double-stranded region of the adapter.
  • 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.
  • one of the non-complementary strands of a polynucleotide adapter such as a Y adapter may comprise a leader sequence, which when contacted with a transmembrane pore is capable of threading into a nanopore.
  • the leader sequence typically comprises a polymer such as a polynucleotide, for instance DNA or RNA, a modified polynucleotide (such as abasic DNA), PNA, LNA, polyethylene glycol (PEG) or a polypeptide.
  • the leader sequence comprises a single strand of DNA, such as a poly dT section.
  • the leader sequence can be any length, but is typically 10 to 150 nucleotides in length, such as from 20 to 120, 30 to 100, 40 to 80 or 50 to 70 nucleotides in length.
  • a polynucleotide adapter is a hairpin loop adapter.
  • a hairpin loop adapter is an adapter comprising a single polynucleotide strand, wherein the ends of the polynucleotide strand are capable of hybridising to each other, or are hybridized to each other, and wherein the middle section of the polynucleotide forms a loop.
  • Suitable hairpin loop adapters can be designed using methods known in the art.
  • the 3' end of a hairpin loop adapter attaches to the 5' end of a first strand of a double-stranded polynucleotide and the 5' end of the hairpin loop adapter attaches to the 3' end of a second strand of a double-stranded polynucleotide; or the 5' end of a hairpin loop adapter attaches to the 3' end of a first strand of a double-stranded polynucleotide and the 3' end of the hairpin loop adapter attaches to the 5' end of a second strand of a double-stranded polynucleotide.
  • a polynucleotide or polynucleotide adapter may comprise one or more spacers, e.g. from one to about 10 spacers, e.g. from 1 to about 5 spacers, e.g. 1, 2, 3, 4 or 5 spacers.
  • the spacer may comprise any suitable number of spacer units.
  • a spacer typically provides an energy barrier which impedes movement of a polynucleotide binding protein.
  • a spacer may impede movement of a motor protein or polynucleotide binding protein by reducing the traction of the protein, e.g. using an abasic spacer.
  • a spacer may physically block movement of the protein, for instance by introducing a bulky chemical group to physically impede the movement of the polynucleotide binding protein.
  • one or more spacers are included in the polynucleotide or in a polynucleotide adapter to provide a distinctive signal when they pass through or across a nanopore.
  • One or more spacers may be used to define or separate one or more regions of a polynucleotide; e.g. to separate an adapter from the target polynucleotide.
  • a spacer may comprise a linear molecule, such as a polymer, e.g. a polypeptide or a polyethylene glycol (PEG).
  • a spacer has a different structure from the target polynucleotide. For instance, if the target polynucleotide is DNA, the or each spacer typically does not comprise DNA.
  • the or each spacer preferably comprises peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA) or a synthetic polymer with nucleotide side chains.
  • PNA peptide nucleic acid
  • GNA glycerol nucleic acid
  • TAA threose nucleic acid
  • LNA locked nucleic acid
  • a spacer may comprise one or more nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuridines, one or more inverted thymidines (inverted dTs), one or more inverted dideoxy-thymidines (ddTs), one or more dideoxy-cytidines (ddCs), one or more 5-methylcytidines, one or more 5- hydroxym ethyl cytidines, one or more 2'-O-Methyl RNA bases, one or more Iso- deoxycytidines (Iso-dCs), one or more Iso-deoxyguanosines (Iso-dGs), one or more C3 (OC 3 H 6 OPO 3 ) groups, one or more photo-cleavable (PC) [OC 3 H
  • a spacer may comprise any combination of these groups. Many of these groups are commercially available from IDT® (Integrated DNA Technologies®). For example, C3, iSp9 and iSp 18 spacers are all available from IDT®. A spacer may comprise any number of the above groups as spacer units.
  • a spacer may comprise one or more chemical groups, e.g. one or more pendant chemical groups.
  • the one or more chemical groups may be attached to one or more nucleobases in a polynucleotide adapter.
  • the one or more chemical groups may be attached to the backbone of a polynucleotide adapter. Any number of appropriate chemical groups may be present, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more.
  • Suitable groups include, but are not limited to, fluorophores, streptavidin and/or biotin, cholesterol, methylene blue, dinitrophenols (DNPs), digoxigenin and/or anti-digoxigenin and dibenzyl cyclooctyne groups.
  • a spacer may comprise one or more abasic nucleotides (i.e. nucleotides lacking a nucleobase), such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more abasic nucleotides.
  • the nucleobase can be replaced by -H (idSp) or -OH in the abasic nucleotide.
  • Abasic spacers can be inserted into target polynucleotides by removing the nucleobases from one or more adjacent nucleotides.
  • polynucleotides may be modified to include 3 -methyladenine, 7-methylguanine, l,N6-ethenoadenine inosine or hypoxanthine and the nucleobases may be removed from these nucleotides using Human Alkyladenine DNA Glycosylase (hAAG).
  • polynucleotides may be modified to include uracil and the nucleobases removed with Uracil-DNA Glycosylase (UDG).
  • the one or more spacers do not comprise any abasic nucleotides.
  • Suitable spacers can be designed or selected depending on the nature of the polynucleotide or polynucleotide adapter, the motor protein and the conditions under which the method is to be carried out.
  • a polynucleotide or polynucleotide adapter may comprise a tag or tether.
  • a polynucleotide can bind to a tag on a nanopore, e.g., via its adaptor, and release at some point, e.g., during characterization of the polynucleotide by the nanopore.
  • a tag or tether may be uncharged. This can ensure that the tags or tethers are not drawn into the nanopore under the influence of a potential difference.
  • One or more molecules that attract or bind a polynucleotide or adaptor may be linked to a nanopore, e.g. a nanopore as described herein. Any molecule that hybridizes to the adaptor and/or target polynucleotide may be used.
  • the molecule attached to the pore may be selected from a PNA tag, a PEG linker, a short oligonucleotide, a positively charged amino acid and an aptamer. Pores having such molecules linked to them are known in the art. For example, pores having short oligonucleotides attached thereto are disclosed in Howarka et al (2001) Nature Biotech. 19: 636-639 and WO 2010/086620, and pores comprising PEG attached within the lumen of the pore are disclosed in Howarka et al (2000) J. Am. Chem. Soc. 122(11): 2411-2416.
  • a short oligonucleotide attached to the pore comprising a sequence complementary to a sequence in the leader sequence or another single stranded sequence in an adaptor may be used to enhance capture of the target polynucleotide in the methods described herein.
  • a tag or tether may comprise or be an oligonucleotide as described herein, e.g. of length 10-30 nucleotides or about 10-20 nucleotides.
  • Such an oligonucleotide can have at least one end (e.g, 3'- or 5'-end) modified for conjugation to other modifications or to a solid substrate surface including, e.g, a bead.
  • the end modifiers may add a reactive functional group which can be used for conjugation. Examples of functional groups that can be added include, but are not limited to amino, carboxyl, thiol, mal eimide, aminooxy, and any combinations thereof.
  • the functional groups can be combined with different length of spacers (e.g., C3, C9, C12, Spacer 9 and 18) to add physical distance of the functional group from the end of the oligonucleotide sequence.
  • the tag or tether may comprise or be a morpholino oligonucleotide.
  • the morpholino oligonucleotide can have about 10-30 nucleotides in length or about 10-20 nucleotides in length.
  • the morpholino oligonucleotides can be modified or unmodified.
  • the morpholino oligonucleotide can be modified on the 3' and/or 5' ends of the oligonucleotides.
  • modifications on the 3' and/or 5' end of the morpholino oligonucleotides include, but are not limited to 3' affinity tag and functional groups for chemical linkage (including, e.g., 3'- biotin, 3'-primary amine, 3'-disulfide amide, 3 '-pyridyl dithio, and any combinations thereof); 5' end modifications (including, e.g., 5'-primary ammine, and/or 5'-dabcyl), modifications for click chemistry (including, e.g., 3'-azide, 3'-alkyne, 5'-azide, 5'-alkyne), and any combinations thereof.
  • 3' affinity tag and functional groups for chemical linkage including, e.g., 3'- biotin, 3'-primary amine, 3'-disulfide amide, 3 '-pyridyl dithio, and any combinations thereof
  • 5' end modifications including, e.
  • a tag or tether may further comprise a polymeric linker, e.g., to facilitate coupling to a detector e.g. a nanopore.
  • An exemplary polymeric linker includes, but is not limited to polyethylene glycol (PEG).
  • the polymeric linker may have a molecular weight of about 500 Da to about 10 kDa (inclusive), or about 1 kDa to about 5 kDa (inclusive).
  • the polymeric linker e.g, PEG
  • the tag or tether may further comprise a 1 kDa PEG with a 5'-maleimide group and a 3 '-DBCO group. In some embodiments, the tag or tether may further comprise a 2 kDa PEG with a 5'-mal eimide group and a 3 '-DBCO group. In some embodiments, the tag or tether may further comprise a 3 kDa PEG with a 5'-mal eimide group and a 3'-DBCO group. In some embodiments, the tag or tether may further comprise a 5 kDa PEG with a 5'-maleimide group and a 3'-DBCO group.
  • a tag can be included on a nanopore using the disclosed modification methods.
  • the tag or tether may be attached directly to a nanopore or via one or more linkers.
  • the tag or tether may be attached to the nanopore using the hybridization linkers described in WO 2010/086602.
  • peptide linkers may be used.
  • Peptide linkers are amino acid sequences. The length, flexibility and hydrophilicity of the peptide linker are typically designed such that it does not to disturb the functions of the monomer and pore.
  • Preferred flexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10 or 16, serine and/or glycine amino acids.
  • More preferred flexible linkers include (SG) 1 , (SG) 2 , (SG) 3 , (SG) 4 , (SG) 5 and (SG) 8 wherein S is serine and G is glycine.
  • Preferred rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or 24, proline amino acids. More preferred rigid linkers include (P) 12 wherein P is proline.
  • a polynucleotide or polynucleotide adapter may comprise a membrane anchor or a transmembrane pore anchor.
  • the anchor assists in the characterisation of a target polynucleotide in accordance with the methods disclosed herein.
  • a membrane anchor or transmembrane pore anchor may promote localisation of the selected polynucleotides around a nanopore.
  • the anchor may be a polypeptide anchor and/or a hydrophobic anchor that can be inserted into the membrane.
  • the hydrophobic anchor is a lipid, fatty acid, sterol, carbon nanotube, polypeptide, protein or amino acid, for example cholesterol, palmitate or tocopherol.
  • the anchor may comprise thiol, biotin or a surfactant.
  • the anchor may be biotin (for binding to streptavidin), amylose (for binding to maltose binding protein or a fusion protein), Ni-NTA (for binding to poly-histidine or poly-histidine tagged proteins) or peptides (such as an antigen).
  • the anchor comprises a linker, or 2, 3, 4 or more linkers.
  • Preferred linkers include, but are not limited to, polymers, such as polynucleotides, polyethylene glycols (PEGs), polysaccharides and polypeptides. These linkers may be linear, branched or circular. For instance, the linker may be a circular polynucleotide. The adapter may hybridise to a complementary sequence on a circular polynucleotide linker.
  • the one or more anchors or one or more linkers may comprise a component that can be cut or broken down, such as a restriction site or a photolabile group.
  • the linker may be functionalised with maleimide groups to attach to cysteine residues in proteins. Suitable linkers are described in WO 2010/086602.
  • the anchor is cholesterol or a fatty acyl chain.
  • any fatty acyl chain having a length of from 6 to 30 carbon atom, such as hexadecanoic acid may be used.
  • suitable anchors and methods of attaching anchors to adapters are disclosed in WO 2012/164270 and WO 2015/150786.
  • a provided multifunctional molecule is typically a multifunctional molecule as described in more detail herein.
  • kits for modifying a monomer comprising a multifunctional molecule as disclosed herein and a support for binding the purification tag of the multifunctional molecule.
  • the kit further comprises buffer solutions for carrying out the binding and/or any washing steps.
  • systems for characterising comprising a chemically-modified monomer or chemically modified oligomer as described herein.
  • the system comprises a chemically modified monomer or oligomer as described herein, together with a motor protein for controlling the movement of a analyte, such as a polynucleotide analyte, with respect to a pore formed from the monomer or oligomer.
  • the system comprises a plurality of monomers, typically a homogeneous population of chemically modified monomers as described herein.
  • the system comprises a plurality of oligomers, typically a homogeneous population of chemically modified monomers as described herein.
  • the system comprises a plurality of nanopores, typically a homogeneous population of chemically modified nanopores as described herein.
  • the population is 100% homogeneous: ie all of the monomers/oligomers/pores in the population are chemically modified.
  • the plurality of monomers/oligomers/pores are present on a chip for analysing (e.g. characterising) an analyte.
  • system further comprises an analyte such as a polynucleotide analyte and/or other components such as a membrane; membrane anchor; tether, etc as described herein.
  • analyte such as a polynucleotide analyte and/or other components such as a membrane; membrane anchor; tether, etc as described herein.
  • a method of chemically modifying a polypeptide or polynucleotide monomer comprising: a) contacting the monomer with a multifunctional molecule, wherein the multi- functional molecule comprises (i) a reactive group; (ii) a chemical modifying group and (iii) a cleavable purification tag; b) allowing the reactive group of the multi-functional molecule to react with the monomer thereby attaching the chemical modifying group and cleavable purification 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) allowing the purification tag to bind to the support thereby binding the chemically modified tagged monomer to the support; and e) cleaving the purification tag thereby releasing the chemically modified monomer from the support.
  • A is a reactive group
  • B is a chemical modifying group
  • D-C forms a cleavable purification tag; preferably wherein D comprises a cleavable linker and C comprises a support-binding group.
  • step (b) comprises allowing the reactive group of the multifunctional molecule to react with the reactive functional group of the monomer.
  • the reactive group of the multifunctional molecule comprises an amine-reactive group; a carboxyl- reactive group; a sulfhydryl-reactive group or a carbonyl-reactive group; preferably wherein the reactive group of the multi-functional molecule comprises a cysteine-reactive group.
  • the reactive group comprises a maleimide, an azide, a thiol, an alkyne, an NHS ester or a haloacetamide.
  • the chemical modifying group introduces hydrophilic, hydrophobic, positively charged, negatively charged, hydrogen-bonding, supramolecular associations or zwitterionic properties to the protein monomer; preferably wherein the chemical modifying group comprises (i) an amino acid, a nucleotide, a polymer, a hydrogen-bonding group, a membrane anchor, a sugar, a dye, a chromophore, a fluorophore or a molecular adapter; or (ii) a natural or unnatural amino acid, a polypeptide, a nucleotide or nucleotide analog, an oligonucleotide or oligonucleotide analog, a polysaccharide, a lipid, a polyethylene glycol, a cyclodextrin, a DNA intercalator, an aptamer or an analyte binding domain.
  • the chemical modifying group comprises (i) an amino acid, a nucleotide, a polymer
  • 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.
  • the purification tag comprises a biotin group and the support comprises streptavidin, neutravidin or avidin, preferably streptavidin.
  • the cleavable linker is cleaved by physical or chemical means; preferably wherein the cleavable linker comprises a UV photocleavable nito-benzyl moiety.
  • step (e) cleaving the purification tag comprises exposing the support and/or the tagged monomer to light; preferably UV light.
  • step (e) cleaving the purification tag comprises exposing the support and/or the tagged monomer to a change in pH.
  • step (e) cleaving the purification tag comprises exposing the support and/or the tagged monomer to a chemical reagent; preferably a reducing reagent.
  • step (e) cleaving the purification tag comprises exposing the support and/or the tagged monomer to an enzyme; preferably a protease.
  • the monomer is a polypeptide having a mass of from about 10 kDa to about 1 MDa.
  • the monomer is a monomer of an oligomeric protein pore, preferably a lysenin pore, a y-hemolysin pore, an a-hemolysin pore; a NetB pore; a CytK pore or a leukocidin pore; or a homolog or paralog thereof.
  • the monomer is a monomer of an oligomeric pore; and wherein the multifunctional molecule reacts with a reactive functional group located on the monomer at a surface-exposed position when the monomer is oligomerised to form the pore; preferably wherein the surface-exposed position is located at the surface of the channel through the pore; or on the exterior surface of the pore.
  • step (a) comprises, prior to step (a), the steps of (i) expressing the monomer in a cellular expression system or a cell-free expression system; and (ii) isolating and/or purifying the monomer.
  • step (d) further comprises the step of removing unmodified monomer(s) and/or unreacted multifunctional molecule(s), if present, from the support.
  • a method according to any one of the preceding aspects further comprising the step of: f) oligomerising the chemically modified monomer to form a chemically modified oligomer; wherein preferably the monomer is a protein monomer and step (f) comprises forming an oligomeric protein.
  • step (f) comprises oligomerising two or more chemically modified monomers to form a homooligomer, wherein preferably said monomers are protein monomers and said homooligomer is a homooligomeric protein pore.
  • step (f) comprises oligomerising one or more chemically modified monomers with one or more unmodified or differently-modified monomers to form a heterooligomer, wherein preferably said monomers are protein monomers and said heterooligomer is a heterooligomeric protein pore.
  • step (f) comprises oligomerising one or more chemically modified first monomers with one or more chemically modified second monomers to form a heterooligomer; wherein the chemical modification made to the first monomer is the same or different to the chemical modification made to the second monomer; wherein preferably said monomers are protein monomers, said first monomer has a different amino acid sequence to said second monomer, and the heterooligomer is a heterooligomeric protein pore.
  • a method of producing a homooligomeric protein comprising i) producing a plurality of chemically modified protein monomers in a method according to any one of aspects 1 to 23; and ii) oligomerising two or more of the chemically modified protein monomers obtained in step (i) to form a homooligomeric protein; wherein said homooligomeric protein is preferably a homooligomeric protein pore.
  • a method of producing a heterooligomeric protein comprising i) producing one or more chemically modified first protein monomers in a method according to any one of aspects 1 to 23; and ii) producing one or more chemically modified second protein monomers in a method according to any one of aspects 1 to 23; and iii) oligomerising said one or more first monomers and said one or more second monomers to form a hetero-oligomeric protein; wherein said heterooligomeric protein is preferably a heterooligomeric protein pore.
  • a method of producing an oligomeric protein comprising i) producing one or more chemically modified first protein monomers in a method according to any one of aspects 1 to 23; and ii) providing one or more unmodified second protein monomers; and iii) oligomerising said one or more first monomers and said one or more second monomers to form a hetero-oligomeric protein; wherein said heterooligomeric protein is preferably a heterooligomeric protein pore.
  • a chemically modified monomer obtainable by carrying out a method according to any one of aspects 1 to 23.
  • 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 modifying group; wherein the chemically modified monomers are preferably as defined in any one of aspects 5 to 7 or 16 to 21.
  • a chemically modified oligomer obtainable by carrying out a method according to any one of aspects 24 to 30.
  • a homogeneous population comprising a plurality of chemically modified oligomers; wherein at least 95% of the oligomers in the population comprise a defined number of monomers chemically modified with a chemical modifying group; wherein the chemically modified monomers are preferably as defined in any one of aspects 5 to 7 or 16 to 21.
  • a method of characterising an analyte comprising: i) producing a chemically modified oligomeric pore in a method according to any one of aspects 24 to 30; or providing a chemically modified oligomeric pore according to aspect 33; and ii) taking one or more measurements as the analyte moves with respect to the pore, wherein the one or more measurements are indicative of one or more characteristics of the analyte, and thereby characterising the analyte as it moves with respect to the pore; wherein preferably the analyte is a polynucleotide, a polypeptide or a polysaccharide.
  • 2-Ethylamino maleimide was reacted with the -COOH end of the preferred amino acid to form the maleimide - amino acid adduct.
  • This was purchased as a custom synthesised molecule from Cambridge Research Biochemicals.
  • Biotin tag with photocleavable linker was purchased from Broadpharm USA (cat number - BP-24161 / PC Biotin-PEG3-NHS carbonate ester).
  • 0.04 mmol of maleimide-amino acid adduct was dissolved in 1 ml of DMF and was added 1 eq. (mol/mol) of the Biotin photocleavable linker. 1.5 eq. (mol/mol) TEA to the mixture and the mixture was stirred overnight. Formation of the molecule was confirmed by mass spectrometry and was used in the modification of the protein/ peptides without further purification.
  • Maleimide-Isoleucine adduct (M.W. - 253.2 g/mol) was purchased as a custom synthesised molecule from Cambridge Research Biochemicals. Biotin tag with photocleavable linker was purchased from Broadpharm USA (Cat No. - BP-24161 / PC Biotin-PEG3-NHS carbonate ester M.W. - 840.9 g/mol). 0.04 mmol of maleimide- Isoleucine adduct was dissolved in 1 ml of DMF and was added 1 eq. (mol/mol) of the Biotin photocleavable linker. 1.5 eq. (mol/mol) TEA to the mixture and the mixture was stirred overnight. Formation of the molecule was confirmed by mass spectrometry and was used in the modification of the protein/ peptides without further purification.
  • Maleimide- Alanine adduct (M.W. - 211.2 g/mol) was purchased as a custom synthesised molecule from Cambridge Research Biochemicals. Biotin tag with photocleavable linker was purchased from Broadpharm USA (Cat No. - BP-24161 / PC Biotin-PEG3-NHS carbonate ester M.W. - 840.9 g/mol). 0.04 mmol of maleimide-alanineadduct was dissolved in 1 ml of DMF and was added 1 eq. (mol/mol) of the Biotin photocleavable linker. 1.5 eq. (mol/mol) TEA to the mixture and the mixture was stirred overnight. Formation of the molecule was confirmed by mass spectrometry and was used in the modification of the protein/ peptides without further purification.
  • Maleimide-Arginine adduct (M.W. - 296.3 g/mol) was purchased as a custom synthesised molecule from Cambridge Research Biochemicals. Biotin tag with photocleavable linker was purchased from Broadpharm USA (Cat No. - BP-24161 / PC Biotin-PEG3-NHS carbonate ester M.W. - 840.9 g/mol). 10 mg (0.04 mmol) of maleimide-arginine adduct was dissolved in 1 ml of DMF and was added 1 eq. (mol/mol) of the Biotin photocleavable linker. 1.5 eq. (mol/mol) TEA to the mixture and the mixture was stirred overnight. Formation of the molecule was confirmed by mass spectrometry and was used in the modification of the protein/ peptides without further purification. 4 -Aspartic acid as the chemical modifying group
  • Maleimide-Aspartic acid adduct (M.W. - 255.2 g/mol) was purchased as a custom synthesised molecule from Cambridge Research Biochemicals. Biotin tag with photocleavable linker was purchased from Broadpharm USA (Cat No. - BP-24161 / PC Biotin-PEG3-NHS carbonate ester M.W. - 840.9 g/mol). 10 mg (0.04 mmol) of maleimide- aspartic acid adduct was dissolved in 1 ml of DMF and was added 1 eq. (mol/mol) of the Biotin photocleavable linker. 1.5 eq. (mol/mol) TEA to the mixture and the mixture was stirred overnight. Formation of the molecule was confirmed by mass spectrometry and was used in the modification of the protein/ peptides without further purification.
  • Maleimide-Asparagine adduct (M.W. - 254.2 g/mol) was purchased as a custom synthesised molecule from Cambridge Research Biochemicals. Biotin tag with photocleavable linker was purchased from Broadpharm USA (Cat No. - BP-24161 / PC Biotin-PEG3-NHS carbonate ester M.W. - 840.9 g/mol). 10 mg (0.04 mmol) of maleimide- asparagine adduct was dissolved in 1 ml of DMF and was added 1 eq. (mol/mol) of the Biotin photocleavable linker. 1.5 eq. (mol/mol) TEA to the mixture and the mixture was stirred overnight. Formation of the molecule was confirmed by mass spectrometry and was used in the modification of the protein/ peptides without further purification.
  • Maleimide-Glutamine adduct (M.W. - 266.2 g/mol) was purchased as a custom synthesised molecule from Cambridge Research Biochemicals. Biotin tag with photocleavable linker was purchased from Broadpharm USA (Cat No. - BP -24161 / PC Biotin-PEG3-NHS carbonate ester M.W. - 840.9 g/mol). 10 mg (0.04 mmol) of maleimide- glutamine adduct was dissolved in 1 ml of DMF and was added 1 eq. (mol/mol) of the Biotin photocleavable linker. 1.5 eq. (mol/mol) TEA to the mixture and the mixture was stirred overnight. Formation of the molecule was confirmed by mass spectrometry and was used in the modification of the protein/ peptides without further purification.
  • Maleimide-Phenylalanine adduct (M.W. - 287.3 g/mol) was purchased as a custom synthesised molecule from Cambridge Research Biochemicals. Biotin tag with photocl eavable linker was purchased from Broadpharm USA (Cat No. - BP-24161 / PC Biotin-PEG3-NHS carbonate ester M.W. - 840.9 g/mol). 0.04 mmol of maleimide- phenylalanine adduct was dissolved in 1 ml of DMF and was added 1 eq. (mol/mol) of the Biotin photocleavable linker. 1.5 eq. (mol/mol) TEA to the mixture and the mixture was stirred overnight. Formation of the molecule was confirmed by mass spectrometry and was used in the modification of the protein/ peptides without further purification.
  • Maleimide-Lysine adduct (M.W. - 268.3 g/mol) was purchased as a custom synthesised molecule from Cambridge Research Biochemicals. Biotin tag with photocleavable linker was purchased from Broadpharm USA (Cat No. - BP-24161 / PC Biotin-PEG3-NHS carbonate ester M.W. - 840.9 g/mol). 0.04 mmol of maleimide-lysine adduct was dissolved in 1 ml of DMF and was added 1 eq. (mol/mol) of the Biotin photocleavable linker. 1.5 eq. (mol/mol) TEA to the mixture and the mixture was stirred overnight. Formation of the molecule was confirmed by mass spectrometry and was used in the modification of the protein/ peptides without further purification.
  • Maleimide-PNA(Thymine) adduct (M.W. - 406.3 g/mol) was purchased as a custom synthesised molecule from Cambridge Research Biochemicals. Biotin tag with photocleavable linker was purchased from Broadpharm USA (Cat No. - BP-24161 / PC Biotin-PEG3-NHS carbonate ester M.W. - 840.9 g/mol). 10 mg (0.04 mmol) of maleimide- PNA(thymine) adduct was dissolved in 1 ml of DMF and was added 1 eq. (mol/mol) of the Biotin photocleavable linker. 1.5 eq. (mol/mol) TEA to the mixture and the mixture was stirred overnight. Formation of the molecule was confirmed by mass spectrometry and was used in the modification of the protein/ peptides without further purification.
  • Maleimide-CH 2 -NH 2 was conjugated to a biotin tag with a photocleavable linker in accordance with the preceding examples and used in the modification of the protein/ peptides as described.
  • Example 2 The cleavage of the multifunctional molecules described in Examples 1 to 10 is shown in Figure 2.
  • Example 2 The cleavage of the multifunctional molecules described in Examples 1 to 10 is shown in Figure 2.
  • Cys mutants of the protein monomers were stored with a reducing agent (e.g. - DTT - Dithiolthreitol) to avoid di-sulfide bond formation.
  • Reducing agent was removed from the purified monomer solutions by buffer exchange in a 7K MWCO desalting column using 50mM Tris-HCl, 150mM NaCl, pH7.
  • a linker of choice was added to each sample and left at room temperature (RT) for 16h to allow the monomers to bind to the chemical modifiers via maleimide-cysteine (thiol) chemistry.
  • Unreacted linker molecules were removed from the solution by centrifugation at 1500rcf for 2 minutes through a 7K MWCO desalting column. The flow through was added to equilibrated StrepTactinTM Sepharose High Performance resin (GE Healthcare Life Sciences, Cat No. 28935600) and left on a rotator for 2h at 23°C, allowing modified monomers to bind to the beads via their biotin tag. Unbound/unmodified protein monomers were removed via centrifugation at 1500rcf for 2 minutes in three wash steps using buffer.
  • Lys- (E84Q/E85S/E92Q/E94D/E97S/T106K/D126G/S98C/C272A/C283A)
  • TEV cleaved to remove thioredoxin-strep moiety was used as the starting material.
  • the monomer was buffered exchanged once to 50mM Tris-HCl, 150mM NaCl, pH7 to remove DTT solution.
  • TEV cleaved to remove thioredoxin-strep moiety was used as the starting material.
  • the monomer was buffered exchanged once to 50mM Tris-HCl, 150mM NaCl, pH7 to remove DTT solution.
  • LukF-T142C-Del(E1-K15) TEV monomer was buffered exchanged once to 50mM Tris- HC1, 150mM NaCl, pH7 to remove DTT solution. 3ul of lOmg/mL mal eimide- Alanine adduct in DMF was added to 120 uL of 0.6 mg/ml buffer exchanged LukF-T142C-Del(E1- K15). The solution was left at RT for 16h. Unreacted linker molecules were removed from the solution by centrifugation at 1500rcf for 2 minutes through a 7K MWCO desalting column.
  • Hlg2-G114C-Del(E1-G10) Hlg2-Gl 14C-Del(E1-G10) monomer was buffered exchanged once to 50mM Tris-HCl, 150mM NaCl, pH7 to remove DTT solution. 3ul of lOmg/mL maleimide-Isoleucine adduct in DMF was added to 120 uL of 0.6 mg/ml buffer exchanged Hlg2-Gl 14C-Del(E1-G10). The solution was left at RT for 16h.
  • Unreacted linker molecules were removed from the solution by centrifugation at 1500rcf for 2 minutes through a 7K MWCO desalting column. The flow through was added to equilibrated StrepTactinTM Sepharose High Performance resin (GE Healthcare Life Sciences, Cat No. 28935600) and left on a rotator for 2h at 23 °C, allowing modified monomers to bind to the beads via their biotin tag. Unbound/unmodified protein monomers were removed via centrifugation at 1500rcf for 2 minutes in three wash steps using buffer.
  • Monomers of LukF and Hlg2 mutant subunits of gamma-hemolysin were modified with maleimide-asparagine, maleimide-CH 2 -NH 2 , maleimide-arginine, maleimide-isoleucine and maleimide-aspartic acid in accordance with the procedures set out above. Successful modification was confirmed by SDS-PAGE electrophoresis ( Figures 14 and 15).
  • a custom 5-lipid mix designed to replicate rabbit blood cells was ordered from Encapsula Nanosciences with the following composition: Phosphatidylserine (0.325mg/ml),
  • Icoleucine/C272A/C283A solution and five lipid mix were combined in a 1 : 1 ratio, mixed well and left at 23°C for 16h.
  • the mixture was spun in a centrifuge at 21,000rcf for 10 minutes to pellet the sphingomyelin/pore complex.
  • the supernatant was removed, the pellet resuspended in buffer (50mM Tris, 150mM NaCl, 2% SDS, pH9) and the mixture heated for 2h at 60°C to release the formed pore from the lipid-protein complex.
  • the tube was allowed to cool down to room temperature before centrifugation at 21,000rcf for 10 minutes.
  • a custom 5-lipid mix designed to replicate rabbit blood cells was ordered from Encapsula Nanosciences with the following composition: Phosphatidylserine (0.325mg/ml),
  • Icoleucine/C272A/C283A solution and five-lipid mix were combined in a 1 : 1 ratio, mixed well and left at 23°C for 16h.
  • the mixture was spun in a centrifuge at 21,000rcf for 10 minutes to pellet the sphingomyelin/pore complex.
  • the supernatant was removed, the pellet resuspended in buffer (50mM Tris, 150mM NaCl, 2% SDS, pH9) and the mixture heated for 2h at 60°C to release the formed pore from the lipid-protein complex.
  • the tube was allowed to cool down to room temperature before centrifugation at 21,000rcf for 10 minutes.
  • Monomer solutions were each concentrated to 0.5mg/ml and LukF and Hlg2 components were combined in a 1 : 1 ratio.
  • 'Leukocyte' liposomes were added (17% final concentration) and the solutions were incubated at 37°C for 16h to allow oligomerisation to take place.
  • SDS was added to the oligomer samples (1.7 % final concentration) and were bath sonicated for 10 minutes. Successful oligomerisation was checked by SDS-PAGE electrophoresis on a 4-20% gel.
  • liposomes with the lipid composition of human leukocytes 965 pl of 5mg/ml Phosphatidyl Choline (38.6%), 197pl of 25mg/ml Phosphatydulethanoamine (33.4%), 18.75pl of 100mg/ml Phosphatidylserine (15%), 13.1 ⁇ l of 100mg/ml Sphingomyelin (10.5%) and 6.5 ⁇ l of 25mg/ml Cardiolipid (1.3%) (all from Avanti Polar Lipids) in chloroform were combined in a glass vial and bath sonicated for 15 minutes.
  • Modified monomer solutions (LukF-T142C-Maleimide-Isoleucine-Del(E1-K15)) and (Hlg2-Gl 14C-Maleimide-Alanine-Del(E1-G10) were each concentrated to 0.5mg/ml and LukF and Hlg2 components were combined in a 1 : 1 ratio. ‘Leukocyte' liposomes were added (17% final concentration) and the solutions were incubated at 37°C for 16h to allow oligomerisation to take place. SDS was added to the oligomer samples (1.7 % final concentration) and were bath sonicated for 10 minutes. Successful oligomerisation was checked by SDS-PAGE electrophoresis on a 4-20% gel (Rubi staining) - Figure 10 (lane
  • Modified monomer solutions (LukF-T142C-Maleimide-Isoleucine-Del(E1-K15)) and (Hlg2-Gl 14C-Mal eimide- Isol eucine-Del(El -G10) were each concentrated to 0.5mg/ml and LukF and Hlg2 components were combined in a 1 : 1 ratio. ‘Leukocyte' liposomes were added (17% final concentration) and the solutions were incubated at 37°C for 16h to allow oligomerisation to take place. SDS was added to the oligomer samples (1.7 % final concentration) and were bath sonicated for 10 minutes. Successful oligomerisation was checked by SDS-PAGE electrophoresis on a 4-20% gel (Rubi staining) - Figure 10 (lane
  • Modified monomer solutions (LukF-T142C-Maleimide- Alanine -Del(E1-K15)) and (Hlg2-Gl 14C-Maleimide-Alanine-Del(E1-G10) were each concentrated to 0.5mg/ml and LukF and Hlg2 components were combined in a 1 : 1 ratio. ‘Leukocyte' liposomes were added (17% final concentration) and the solutions were incubated at 37°C for 16h to allow oligomerisation to take place. SDS was added to the oligomer samples (1.7 % final concentration) and were bath sonicated for 10 minutes. Successful oligomerisation was checked by SDS-PAGE electrophoresis on a 4-20% gel (Rubi staining) - Figure 10 (lane
  • Modified monomer solutions (LukF-T142C-Maleimide- Alanine-Del(E1-K15)) and (Hlg2-G114C-Maleimide-Isoleucine-Del(E1-G10) were each concentrated to 0.5mg/ml and LukF and Hlg2 components were combined in a 1 : 1 ratio. ‘Leukocyte' liposomes were added (17% final concentration) and the solutions were incubated at 37°C for 16h to allow oligomerisation to take place. SDS was added to the oligomer samples (1.7 % final concentration) and were bath sonicated for 10 minutes. Successful oligomerisation was checked by SDS-PAGE electrophoresis on a 4-20% gel (Rubi staining) - Figure 10 (lane
  • Hlg2, LukF and their mutants were overexpressed and purified separately as soluble monomeric proteins.
  • Transformed Escherichia coli strain Lemo21(DE3) (NEB, Cat No. C2528J) harbouring the expression vector encoding the desired LukF or Hlg2 monomer was grown at 37°C in LB media supplemented with 41ug/uL chloramphenicol and lOOug/uL carbenicillin until logarithmic growth phase was achieved.
  • Expression of the desired protein was induced using Isopropyl-P-D-thiogalactoside (IPTG) to a final concentration of 0.5mM. The temperature was reduced to 25°C and allowed to express for 18h.
  • IPTG Isopropyl-P-D-thiogalactoside
  • Cells were harvested by centrifugation at 6000xg for 20 minutes at 4°C and pellets resuspended in buffer (50mM HEPES, 300mM NaCl, 2mM EDTA, 0.1% DDM, Ixbugbuster, benzonase nuclease, protease inhibitor tablets, pH8) before being disrupted by sonication and left to lyse for 4h on a magnetic stirrer. Lysate was clarified at 39,000xg for 35 minutes at 4°C. The supernatant was diluted 10-fold with 50mM HEPES and loaded onto a HS50 cation exchange column (Poros Media by Applied BioSciences) where an increasing NaCl gradient was used for elution. Fractions containing the desired protein were further purified on an Superdex 75 10/300 GL size exclusion column (GE Healthcare Life Sciences).
  • buffer 50mM HEPES, 300mM NaCl, 2mM EDTA, 0.1% DDM, Ix
  • Transformed Escherichia coli strain BL21(DE3) (NEB, Cat No. C2527H) harbouring an expression vector encoding the desired lysenin monomer with an thioredoxin-strep moiety was grown at 37°C in TB media supplemented with O. lmg/ml ampicillin until logarithmic growth phase was achieved (OD600 0.8).
  • Expression of the desired protein was induced using Isopropyl-P-D-thiogalactoside (IPTG) to a final concentration of 0.5mM. The temperature was reduced to 18°C and allowed to express for 16h. Cells were harvested by centrifugation at 6000xg for 20 minutes at 4°C.
  • Pellets were resuspended in buffer (50mM Tris-HCl, 0.5M NaCl, 2mM DTT, benzonase nuclease, protease inhibitors, 2mM EDTA, pH 8.0) and left on a magnetic stirrer for 3h before being disrupted by sonication. Lysate was clarified at 45,000xg for 60 minutes at 4°C. The supernatant was loaded onto 5ml StrepTrapTM High Performance columns (GE Healthcare Life Sciences, Cat No. 28907548) and proteins were eluted with 2mg/ml desthiobiotin.
  • buffer 50mM Tris-HCl, 0.5M NaCl, 2mM DTT, benzonase nuclease, protease inhibitors, 2mM EDTA, pH 8.0
  • Fractions containing the desired protein were further purified on an Superdex200 increase 10/300 GL size exclusion column (GE Healthcare Life Sciences, Cat No. 28990944) and analysed by SDS-PAGE. Peak fractions were pooled and cleaved with TEV-strep protease to remove the thioredoxin-strep moiety. The mixture was loaded back onto a StrepTrapTM High Performance column to gain untagged lysenin monomers in the flow through. The final elution was concentrated using an Amicon® Ultra- 15 Centrifugal Filter Unit (Merck Millipore, Cat No. UFC901024).
  • Oligomerised chemically modified pores produced in accordance with the disclosed methods were assembled into nanopore detection devices and used to characterise a polynucleotide analyte.
  • a double stranded 3.6 kb DNA analyte (SEQ ID NO: 20) was prepared using specific primers and PCR.
  • the PCR product was subjected to NEBNext end repair, NEBNext dA- tailing modules (New England Biolabs (NEB)), to generate a 3' A overhangs.
  • Ly s-(T63 C/E84Q/E85 S/E92Q/E94D/E97 S/T 106K/D 126G/C272 A/C283 A) were modified with 5 different chemical molecules and the effect of the modification was analysed by comparing the open pore current level of the modified pore against the unmodified pore when the 3.6 lambda DNA is passing through each pore.
  • the modifications made to the lysenin pores covered aromatic, aliphatic, hydrophobic, hydrophilic, positive charge and H-bonding residues including DNA base attachment - PNA(Thymine).
  • the open pore current levels of the modified pores change significantly as a result of the change in diameter and change in chemical environment within the barrels.
  • Figure 12 shows enhanced details of the signal observed as the 3.6Kb Lambda DNA translocates through the modified lysenin mutant pores, as a function of the modifier molecule.
  • Figure 12(A) shows data obtained from the unmodified pore Lys-(E84Q/E85S/E92Q/E94D/E97S/S98C/T106K/D126G/C272A/C283A).
  • Figure 12(B) shows corresponding data from the modified pore Lys-(E84Q/E85S/E92Q/E94D/E97S/S98C-maleimide- isoleucine/T106K/D126G/C272A/C283A).
  • the left panel shows an event of the entire 3.6Kb DNA passing through the pore.
  • the open pore current level (240pA in A and 250pA in B) drops down to about 160pA when the DNA is passing through the pore. Fluctuations of the current at 160pA indicate different base compositions passing through the narrowest region (constriction) of the pore.
  • the right panel shows the expanded version of the current-time data "squiggle" for 0.4 seconds. The shape and range (current variation within the squiggle level) vary significantly between the unmodified and modified pores.
  • Figure 13 shows changes in the GGAA region of the 3.6Kb Lambda DNA (residues 38-41 and 52-55 of SEQ ID NO: 20) as it translocates through lysenin mutant pores as a function of the modifier molecule.
  • the DNA analyte was translocated through an unmodified lysenin pore (Lys-(E84Q/E85S/E92Q/E94D/E97S/S98C/T106K/D126G/C272A/C283A); panel A) and the signal obtained compared to that for pores modified in accordance with the methods described herein:
  • the G and A levels of the signal obtained from the modified pore is different in the modified pores compared to the unmodified pore, with a particularly significant alteration in the signal obtained from the Lys-(E84Q/E85S/E92Q/E94D/E97S/S98C-maleimide- PNA(Thymine)/T106K/D126G/C272A/C283A pore (panel C).
  • Figure 17 shows representative characterisation data for the polynucleotide analyte of SEQ ID NO: 20 when characterised using (left) unmodified Gamma-Hemolysin - [(LukF- T142C-Del(E1-K15))4 (Hlg2-WT)4] pores produced and oligomerised as discussed above, as compared to corresponding modified Gamma-Hemolysin - [(LukF-T142C-Maleimide- Aspartate-Del(E1-K15))4 (Hlg2-WT)4] pores produced and oligomerised as discussed above.
  • Panel A shows the unexpanded trace.
  • the open pore current levels and translocation characteristics of the polynucleotide analyte vary significantly between the unmodified pore and the modified pore, as a result of the change in diameter and change in chemical environment within the barrels resulting from the modification.
  • Panel B shows changes in the GGAA region of the 3.6Kb Lambda DNA (residues 38-41 and 52-55 of SEQ ID NO: 20) as it translocates through the pores.
  • the GGAA signal section is circled in each of the traces. As can be seen, the G and A levels of the signal obtained from the modified pore is different in the modified pores compared to the unmodified pore.
  • alpha hemolysin (a-HL) monomers Modification of alpha hemolysin (a-HL) monomers, oligomerisation, and analyte characterisation
  • alpha hemolysin (a-HL) monomers Ml 13C
  • maleimide-isoleucine maleimide-isoleucine
  • the tube was allowed to cool down to room temperature before centrifugation at 21,000rcf for 10 minutes. The supernatant was collected and diluted 10-fold in 50mM Tris, 150mM NaCl, pH8 to give a final SDS concentration of 0.2% in the final pore solution. Successful oligomerisation was checked by SDS-PAGE electrophoresis on a 4-20% gel.
  • Figure 18A shows modification of a-HL Ml 13C monomers (Lane 1 - Unmodified protein monomer, a-HL-Ml 13C; Lane 2 - Crude reaction mixture - Maleimide-isoleucine (Mal- He) adduct with a-HL-Ml 13C; Lane 3 - Flow through after equilibrating the modified protein monomers on StrepTactin beads (flow through indicates unmodified protein monomers unbound to StrepTactin beads); Lane 4/5 - Further washings of the modified monomer bound StrepTactin beads to remove unmodified protein monomer (gel indicates no unmodified monomer eluting in further washes); Lane 6 - Modified a-HL-M113C with Maleimide-isoleucine (Mal-Ile)).
  • Figure 18B shows the oligomerisation of modified a- HL-113C-maleimide-isoleucine to form a homooligomeric heptameric pore
  • Anae 1 - modified protein monomer a-HL-M113C-Mal-Ile
  • Lane 2 Crude reaction mixture of a-HL-M113C-Mal-Ile with 1 : 1 Sphingomyelin after overnight storage.
  • Lane 3 Supernatant after centrifuging to separate pore-sphingomyelin pellet. Some pores are seen in supernatant as well.
  • Lane 4 Modified (HL-M113C-Mal-Ile)7 heptameric oligomerised pore.).
  • the modified a-HL heptameric pores were used to characterise a DNA analyte as described above and the characterisation data compared with corresponding data from unmodified pores. Representative data is shown in Figure 19.
  • Figure 19A shows electrophysiology data for unmodified ((a-HL-M113C)7, left panel) and modified ((a-HL-Ml 13C-Mal-Ile)7, right panel) pores.
  • Open pore current of the unmodified pores is higher than the modified pores, which (without being bound by theory) is considered to arise as the internal diameter of the pore is reduced after modification.
  • the drop in current (delta) when DNA is translocating through the pore is higher for the unmodified pore (delta ⁇ 35 pA) compared to the modified pore (delta ⁇ 25 pA).
  • Open pore noise is also higher for the unmodified pore compared to the modified pore.
  • Figure 19B shows a zoomed-in region of the data in Figure 19A.
  • Figure 19C shows traces of the signal focussed on the first 0.3 seconds after the spl8 signal from the analyte. Differences in the signal can be clearly detected arising from the modification in accordance with the methods herein.
  • Cyt-K cytotoxin-K
  • the supernatant was removed, the pellet resuspended in buffer (50mM Tris, 150mM NaCl, pH8) and the mixture heated for 2h at 60°C to release the formed pore from the lipid- protein complex.
  • the tube was allowed to cool down to room temperature before centrifugation at 21,000rcf for 10 minutes.
  • the supernatant was collected and diluted 10- fold in 50mM Tris, 150mM NaCl, pH8 to give a final SDS concentration of 0.2% in the final pore solution.
  • Successful oligomerisation was checked by SDS-PAGE electrophoresis on a 4-20% gel.
  • Figure 20A shows modification of Cyt-K monomers (Lane 1 - Unmodified protein monomer, Cyt-K(WT-Q123S/K129C/E140S/Q146S); Lane 2 - Crude reaction mixture - Maleimide-Isoleucine (Mal-Ile) adduct with Cyt-K(WT-Q123S/K129C/E140S/Q146S); Lane 3 - Flow through after equilibrating the modified protein monomers on StrepTactin beads (flow through indicates unmodified protein monomers unbound to StrepTactin beads); Lane 4/5 - Further washings of the modified monomer bound StrepTactin beads to remove unmodified protein monomer (gel indicates no unmodified monomer eluting in further washes); Lane 6 - Modified Cyt-K(WT-Q123S/K129C/E140S/Q146S) with Maleimide-Isoleucine (Mal-Ile)).
  • Figure 20B shows the oligomerisation of modified Cyt- K WT-Q123S/K129C-maleimide-isoleucine/E140S/Q146S to form a homooligomeric pore
  • Lane 1 Modified protein monomer, CytK-(WT-Q123S/K129C-Mal-Ile/E140S/Q146S
  • Lane 2 Crude reaction mixture of CytK-(WT-Q123S/K129C-Mal-Ile/E140S/Q146S with 1 : 1 Sphingomyelin after overnight storage.
  • Lane 3 Supernatant after centrifuging to separate pore-sphingomyelin pellet. Some pores are seen in supernatant as well.
  • Lane 4 Modified (CytK-(WT-Q123S/K129C-Mal-Ile/E140S/Q146S)7 pore.).
  • the modified Cyt-K pores were used to characterise a DNA analyte as described above and the characterisation data compared with corresponding data from unmodified pores. Representative data is shown in Figure 21.
  • Figure 21 A shows electrophysiology data for unmodified CytK-(WT- Q123S/K129S/E140S/Q146S)7 and modified CytK-(WT-Q123S/K129C-Mal- Ile/E140S/Q146S)7 pores.
  • Both open pore current and sequencing current (i.e. squiggle level) of the unmodified pores is higher than the modified pores, which (without being bound by theory) is considered to arise as the internal diameter of the pore is reduced after modification.
  • This data indicates the modified pore has changed the characteristics of the DNA squiggle compared to the unmodified version.
  • Figure 21B shows characteristic data from the poly-T,GGAA region in the 3.6 Kb asymmetric library, zoomed into the first 0.3 s of the trace following the spl8 signal from the analyte.
  • Both first and second poly-T regions can be clearly seen in the unmodified pore (underlined; poly-T 1 and 2), whereas in the unmodified pore the first poly-T region cannot be distinguished and the signal from the second poly-T region is more dominant.
  • This data confirms that the modified pore has changed the characteristics of the DNA squiggle compared to the unmodified version.
  • Figure 22 shows the results of modifying a lysenin monomer (Lys-(/E84Q/E85K/E92Q/E94D/E97S/S98C/T 106K/D 126G/C272A/C283 A)) with maleimide-isoleucine with a cleavable purification tag.
  • the maleimide-isoleucine reacts at position S98C.
  • Lane 1 shows the unmodified starting material.
  • Lane 2 shows crude incomplete reaction mixture containing both modified and unmodified protein monomers. Oligomerising with this monomer mixture would lead to heterogeneous pore populations.
  • the methods disclosed herein produce a homogeneous population, as all unmodified monomers can be eliminated, e.g. by washing or elution whereas modified monomers bind to a support material. This is illustrated in lane 3 of Figure 22 which shows that non-modified monomers are eluted. This method is applicable to modify and purify the modified pore monomers independent of the substrate without the requirement for individual method development.
  • Protein monomers modified in accordance with the methods disclosed herein can be released from the support material cleanly by cleaving the purification tag. As shown in Lane 6 of Figure 22, the modified monomers are obtained without contamination from unmodified monomers. Furthermore, the methods disclosed herein are amenable to a wide variety of proteins without requiring specific purification strategies to be developed for each monomer of interest.
  • SEQ ID NO: 1 shows the amino acid sequence of (hexa-histidine tagged) exonuclease I (EcoExo I) from E. coli.
  • SEQ ID NO: 2 shows the amino acid sequence of the exonuclease III enzyme from E. coli.
  • SEQ ID NO: 3 shows the amino acid sequence of the Red enzyme from T. thermophilus (TthRecJ-cd).
  • SEQ ID NO: 4 shows the amino acid sequence of bacteriophage lambda exonuclease. The sequence is one of three identical subunits that assemble into a trimer.
  • SEQ ID NO: 5 shows the amino acid sequence of Phi29 DNA polymerase from Bacillus subtilis phage Phi29.
  • SEQ ID NO: 6 shows the amino acid sequence of Trwc Cba (Citromicrobium bathyomarinum) helicase.
  • SEQ ID NO: 7 shows the amino acid sequence of Hel308 Mbu (Methanococcoides burtonii) helicase .
  • SEQ ID NO: 8 shows the amino acid sequence of the Dda helicase 1993 from Enterobacteria phage T4.
  • SEQ ID NO: 10 shows the amino acid sequence of the LukF subunit of gamma-hemolysin (Del 1-15).
  • SEQ ID NO: 11 shows the amino acid sequence of the Hlg2 subunit of gamma-hemolysin (Del 1-10).
  • SEQ ID NO: 12 shows the amino acid sequence of a monomer of lysenin- (E84Q/E85 S/E92Q/E94D/E97S/T 106K/D 126G).
  • SEQ ID NO: 13 shows the amino acid sequence of a monomer of cytotoxin K from Bacillus cereus (CytK).
  • SEQ ID NO: 14 shows the amino acid sequence of a monomer of aerolysin.
  • SEQ ID NO: 15 shows the amino acid sequence of a monomer of Necrotic enteritis toxin B (NetB) from Clostridium perfringens .
  • ID NO: 16 shows the amino acid sequence of a monomer of alpha-hemolysin (a-HL).
  • SEQ ID NO: 17 shows the amino acid sequence of a monomer of Vibrio cholera cytolysin (HlyA) / VCC.
  • SEQ ID NO: 18 shows the amino acid sequence of a monomer of Anthrax toxin protective antigen (Anthrax PA).
  • SEQ ID NO: 19 shows the amino acid sequence of a monomer of epsilon toxin ( ⁇ -Toxin).
  • SEQ ID NO: 20 shows the polynucleotide sequence of a 3.6 kb lambda DNA analyte used in the Examples.
  • SEQ ID NO: 21 shows the amino acid sequence of a monomer of Fragaceatoxin C (FraC) from Actinia fragacea.
  • SEQ ID NO: 22 shows the amino acid sequence of the PlyA monomer of the pleurotolysin PlyAB from Pleurotus ostreatus.
  • SEQ ID NO: 23 shows the amino acid sequence of the PlyB monomer of the pleurotolysin PlyAB from Pleurotus ostreatus.
  • SEQ ID NOs: 24-27 show the amino acid sequences of various protease-sensitive peptide linkers referred to herein.
  • SEQ ID NOs: 28-35 show the amino acid sequences of various peptide tags referred to herein.
  • SEQ ID NO: 36 shows the amino acid sequence of a polynucleotide repeating unit referred to herein.

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Abstract

L'invention concerne un procédé de modification chimique d'un polypeptide ou d'un monomère polynucléotidique. Le monomère peut être un monomère d'un pore protéique tel qu'un pore protéique monomère ou oligomère. L'invention concerne également des monomères et des oligomères associés et des procédés d'utilisation des pores décrits dans la caractérisation d'analytes.
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WO2023026056A1 (fr) * 2021-08-26 2023-03-02 Oxford Nanopore Technologies Plc Nanopore
WO2024138472A1 (fr) * 2022-12-28 2024-07-04 青岛华大基因研究院 Monomère de porine, porine, mutant correspondant et utilisation associée
WO2025129045A1 (fr) * 2023-12-15 2025-06-19 Ginkgo Bioworks, Inc. Gènes pesticides et procédés d'utilisation

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WO2025138260A1 (fr) * 2023-12-29 2025-07-03 深圳华大生命科学研究院 Polypeptide et procédé de séquençage de protéines, kit et utilisation

Citations (26)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5576204A (en) 1989-03-24 1996-11-19 Consejo Superior Investigaciones Cientificas φ29 DNA polymerase
WO2000028312A1 (fr) 1998-11-06 2000-05-18 The Regents Of The University Of California Support miniature pour films minces contenant des canaux uniques ou des nanopores et procedes d'utilisation de ces derniers
WO2006100484A2 (fr) 2005-03-23 2006-09-28 Isis Innovation Limited Administration de molecules dans une bicouche lipidique
WO2008102121A1 (fr) 2007-02-20 2008-08-28 Oxford Nanopore Technologies Limited Formation de bicouches lipidiques
WO2009020682A2 (fr) 2007-05-08 2009-02-12 The Trustees Of Boston University Fonctionnalisation chimique d'ensembles de nanopores et de nanopores à semi-conducteurs, et leurs applications
WO2009035647A1 (fr) 2007-09-12 2009-03-19 President And Fellows Of Harvard College Capteur moléculaire haute résolution en feuille de carbone avec ouverture dans la couche de feuille de carbone
WO2009077734A2 (fr) 2007-12-19 2009-06-25 Oxford Nanopore Technologies Limited Formation de couches de molécules amphiphiles
WO2010086620A1 (fr) 2009-02-02 2010-08-05 Itis Holdings Plc Appareil et procédés de fourniture d'informations de voyage
WO2010086602A1 (fr) 2009-01-30 2010-08-05 Oxford Nanopore Technologies Limited Lieurs d'hybridation
WO2010122293A1 (fr) 2009-04-20 2010-10-28 Oxford Nanopore Technologies Limited Réseau de capteurs de bicouche lipidique
WO2012005857A1 (fr) 2010-06-08 2012-01-12 President And Fellows Of Harvard College Dispositif nanoporeux à membrane lipidique artificielle sur support de graphène
WO2012164270A1 (fr) 2011-05-27 2012-12-06 Oxford Nanopore Technologies Limited Procédé de couplage
WO2013057495A2 (fr) 2011-10-21 2013-04-25 Oxford Nanopore Technologies Limited Procédé enzymatique
WO2013083983A1 (fr) 2011-12-06 2013-06-13 Cambridge Enterprise Limited Contrôle de la fonctionnalité de nanopore
WO2013098562A2 (fr) 2011-12-29 2013-07-04 Oxford Nanopore Technologies Limited Procédé enzymatique
WO2013098561A1 (fr) 2011-12-29 2013-07-04 Oxford Nanopore Technologies Limited Procédé de caractérisation d'un polynucléotide au moyen d'une hélicase xpd
WO2013153359A1 (fr) 2012-04-10 2013-10-17 Oxford Nanopore Technologies Limited Pores formés de lysenine mutante
WO2014013260A1 (fr) 2012-07-19 2014-01-23 Oxford Nanopore Technologies Limited Hélicases modifiées
WO2014064444A1 (fr) 2012-10-26 2014-05-01 Oxford Nanopore Technologies Limited Interfaces de gouttelettes
WO2014064443A2 (fr) 2012-10-26 2014-05-01 Oxford Nanopore Technologies Limited Formation de groupement de membranes et appareil pour celle-ci
WO2015055981A2 (fr) 2013-10-18 2015-04-23 Oxford Nanopore Technologies Limited Enzymes modifiées
WO2015110813A1 (fr) 2014-01-22 2015-07-30 Oxford Nanopore Technologies Limited Procédé de fixation d'une ou plusieurs protéines de liaison de polynucléotides dans un polynucléotide cible
WO2015150786A1 (fr) 2014-04-04 2015-10-08 Oxford Nanopore Technologies Limited Méthode de caractérisation d'un acide nucléique double brin au moyen d'un nano-pore et de molécules d'ancrage aux deux extrémités dudit acide nucléique
WO2016055777A2 (fr) 2014-10-07 2016-04-14 Oxford Nanopore Technologies Limited Enzymes modifiées
WO2017162828A1 (fr) * 2016-03-24 2017-09-28 Genia Technologies, Inc. Procédés et compositions de bioconjugaison spécifique d'un site utiles pour des systèmes nanoporeux
WO2017174990A1 (fr) * 2016-04-06 2017-10-12 Oxford Nanopore Technologies Limited Pore mutant

Patent Citations (27)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5576204A (en) 1989-03-24 1996-11-19 Consejo Superior Investigaciones Cientificas φ29 DNA polymerase
WO2000028312A1 (fr) 1998-11-06 2000-05-18 The Regents Of The University Of California Support miniature pour films minces contenant des canaux uniques ou des nanopores et procedes d'utilisation de ces derniers
WO2006100484A2 (fr) 2005-03-23 2006-09-28 Isis Innovation Limited Administration de molecules dans une bicouche lipidique
WO2008102121A1 (fr) 2007-02-20 2008-08-28 Oxford Nanopore Technologies Limited Formation de bicouches lipidiques
WO2008102120A1 (fr) 2007-02-20 2008-08-28 Oxford Nanopore Technologies Limited Système de capteur bicouche lipidique
WO2009020682A2 (fr) 2007-05-08 2009-02-12 The Trustees Of Boston University Fonctionnalisation chimique d'ensembles de nanopores et de nanopores à semi-conducteurs, et leurs applications
WO2009035647A1 (fr) 2007-09-12 2009-03-19 President And Fellows Of Harvard College Capteur moléculaire haute résolution en feuille de carbone avec ouverture dans la couche de feuille de carbone
WO2009077734A2 (fr) 2007-12-19 2009-06-25 Oxford Nanopore Technologies Limited Formation de couches de molécules amphiphiles
WO2010086602A1 (fr) 2009-01-30 2010-08-05 Oxford Nanopore Technologies Limited Lieurs d'hybridation
WO2010086620A1 (fr) 2009-02-02 2010-08-05 Itis Holdings Plc Appareil et procédés de fourniture d'informations de voyage
WO2010122293A1 (fr) 2009-04-20 2010-10-28 Oxford Nanopore Technologies Limited Réseau de capteurs de bicouche lipidique
WO2012005857A1 (fr) 2010-06-08 2012-01-12 President And Fellows Of Harvard College Dispositif nanoporeux à membrane lipidique artificielle sur support de graphène
WO2012164270A1 (fr) 2011-05-27 2012-12-06 Oxford Nanopore Technologies Limited Procédé de couplage
WO2013057495A2 (fr) 2011-10-21 2013-04-25 Oxford Nanopore Technologies Limited Procédé enzymatique
WO2013083983A1 (fr) 2011-12-06 2013-06-13 Cambridge Enterprise Limited Contrôle de la fonctionnalité de nanopore
WO2013098562A2 (fr) 2011-12-29 2013-07-04 Oxford Nanopore Technologies Limited Procédé enzymatique
WO2013098561A1 (fr) 2011-12-29 2013-07-04 Oxford Nanopore Technologies Limited Procédé de caractérisation d'un polynucléotide au moyen d'une hélicase xpd
WO2013153359A1 (fr) 2012-04-10 2013-10-17 Oxford Nanopore Technologies Limited Pores formés de lysenine mutante
WO2014013260A1 (fr) 2012-07-19 2014-01-23 Oxford Nanopore Technologies Limited Hélicases modifiées
WO2014064444A1 (fr) 2012-10-26 2014-05-01 Oxford Nanopore Technologies Limited Interfaces de gouttelettes
WO2014064443A2 (fr) 2012-10-26 2014-05-01 Oxford Nanopore Technologies Limited Formation de groupement de membranes et appareil pour celle-ci
WO2015055981A2 (fr) 2013-10-18 2015-04-23 Oxford Nanopore Technologies Limited Enzymes modifiées
WO2015110813A1 (fr) 2014-01-22 2015-07-30 Oxford Nanopore Technologies Limited Procédé de fixation d'une ou plusieurs protéines de liaison de polynucléotides dans un polynucléotide cible
WO2015150786A1 (fr) 2014-04-04 2015-10-08 Oxford Nanopore Technologies Limited Méthode de caractérisation d'un acide nucléique double brin au moyen d'un nano-pore et de molécules d'ancrage aux deux extrémités dudit acide nucléique
WO2016055777A2 (fr) 2014-10-07 2016-04-14 Oxford Nanopore Technologies Limited Enzymes modifiées
WO2017162828A1 (fr) * 2016-03-24 2017-09-28 Genia Technologies, Inc. Procédés et compositions de bioconjugaison spécifique d'un site utiles pour des systèmes nanoporeux
WO2017174990A1 (fr) * 2016-04-06 2017-10-12 Oxford Nanopore Technologies Limited Pore mutant

Non-Patent Citations (19)

* Cited by examiner, † Cited by third party
Title
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 2016, JOHN WILEY & SONS
CHANG TSUNG-CHE ET AL: "A photo-cleavable biotin affinity tag for the facile release of a photo-crosslinked carbohydrate-binding protein", BIOORGANIC, ELSEVIER, AMSTERDAM, NL, vol. 24, no. 6, 27 January 2016 (2016-01-27), pages 1216 - 1224, XP029439441, ISSN: 0968-0896, DOI: 10.1016/J.BMC.2016.01.050 *
COLAS ET AL., MICRON, vol. 38, 2007, pages 841 - 847
D. STODDART ET AL., PROC. NATL. ACAD. SCI., vol. 106, 2010, pages 7702 - 444
ELISEEV, A. V.SCHNEIDER, H-J., J. AM. CHEM. SOC., vol. 116, 1994, pages 6081 - 6088
GEOFFRAY LERICHE ET AL: "Cleavable linkers in chemical biology", BIOORGANIC, vol. 20, no. 2, 15 January 2012 (2012-01-15), pages 571 - 582, XP028442289, ISSN: 0968-0896, [retrieved on 20110730], DOI: 10.1016/J.BMC.2011.07.048 *
GONZALEZ-PEREZ ET AL., LANGMUIR, vol. 25, 2009, pages 10447 - 10450
HERON ET AL., J. AM. CHEM. SOC. 9, vol. 131, 2009
HOWARKA ET AL., J. AM. CHEM. SOC., vol. 122, no. 11, 2000, pages 2411 - 2416
HOWARKA ET AL., NATURE BIOTECH, vol. 19, 2001, pages 636 - 639
HUANG ET AL.: "Electro-osmotic vortices promote the capture of folded proteins by PlyAB nanopores''", NANO LETTERS, vol. 20, no. 5, 2020, pages 3819 - 3827
KOLB HCFINN, MGSHARPLESS KB: "Click chemistry: diverse chemical function from a few good reactions", ANGEW. CHEM. INT. ED., vol. 40, 2001, pages 2004 - 2021, XP055718455, DOI: 10.1002/1521-3773(20010601)40:11<2004::AID-ANIE2004>3.0.CO;2-5
LANGECKER ET AL., SCIENCE, vol. 338, 2012, pages 932 - 936
LEHNINGER, A. L, BIOCHEMISTRY, 1975, pages 71 - 92
MCKAYFINN: "Click chemistry in complex mixtures: bioorthogonal bioconjugation", CHEM. BIOL., vol. 21, no. 9, 2014, pages 1075 - 1101, XP055398778, DOI: 10.1016/j.chembiol.2014.09.002
MONTALMUELLER, PROC. NATL. ACAD. SCI. USA., vol. 69, 1972, pages 3561 - 3566
ROBERTSON JOSEPH W F ET AL: "Nanopore sensing: A physical-chemical approach", BBA - BIOMEMBRANES ACTA, ELSEVIER, AMSTERDAM, NL, vol. 1863, no. 9, 11 May 2021 (2021-05-11), XP086620274, ISSN: 0005-2736, [retrieved on 20210511], DOI: 10.1016/J.BBAMEM.2021.183644 *
ROBERTSVELLACCIO: "The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer", vol. 5, 1983, ACADEMIC PRESS, INC., pages: 341
SAKAMOTOHAMACHI: "Recent progress in chemical modification of proteins", ANAL. SCI, no. 35, 2019, pages 5 - 27

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WO2024138472A1 (fr) * 2022-12-28 2024-07-04 青岛华大基因研究院 Monomère de porine, porine, mutant correspondant et utilisation associée
WO2025129045A1 (fr) * 2023-12-15 2025-06-19 Ginkgo Bioworks, Inc. Gènes pesticides et procédés d'utilisation

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