WO2024100270A1 - Nouveaux monomères à pores et pores - Google Patents

Nouveaux monomères à pores et pores Download PDF

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Publication number
WO2024100270A1
WO2024100270A1 PCT/EP2023/081472 EP2023081472W WO2024100270A1 WO 2024100270 A1 WO2024100270 A1 WO 2024100270A1 EP 2023081472 W EP2023081472 W EP 2023081472W WO 2024100270 A1 WO2024100270 A1 WO 2024100270A1
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pore
polypeptide
monomer
polynucleotide
seq
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PCT/EP2023/081472
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English (en)
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Elizabeth Jayne Wallace
Ranga Prabhath MALAVIARACHCHIGE RABEL
Paul Richard Moody
Mark John BRUCE
Richard George HAMBLEY
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Oxford Nanopore Technologies Plc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/24—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • C07K14/245—Escherichia (G)

Definitions

  • the present invention relates to novel pore monomer conjugates comprising pore monomers and functionalised partner molecules, pore complexes formed from the conjugates and their uses in analyte detection and characterisation.
  • 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.
  • Two of the essential components of analyte characterization using nanopore sensing are (1) the control of analyte movement through the pore and (2) the discrimination of the composing building blocks as the analyte is moved through the pore.
  • the narrowest part of the pore forms the most discriminating part of the nanopore with respect to the current signatures as a function of the passing analyte.
  • CsgG was identified as an ungated, non-selective protein secretion channel from Escherichia coli (Goyal et al., 2014) and has been used as a nanopore for detecting and characterising analytes. Mutations to the wild-type CsgG pore that improve the properties of the pore in this context have also been disclosed (WO 2016/034591, WO 2017/149316, WO 2017/149317, WO 2017/149318, WO 2018/211241, and WO 2019/002893, all incorporated by reference herein in their entirety).
  • Methods for sequencing double-stranded polynucleotides have been developed, e.g., involving translocation of both the template and complement strands connected by a hairpin. Measurement of both strands in this way is advantageous as information from the two complementary linked strands can be combined and used to provide higher confidence observations than may be achieved from measurement of template strands only.
  • preparation of such a hairpin linked polynucleotide can increase sample preparation time and result in a loss of valuable analyte.
  • translocation of a hairpin linked template and complement polynucleotide strands through a nanopore can give rise to rehybridization of the strands on the other (trans) side of the nanopore.
  • partner molecules can be used to functionalise pore monomers and pores complexes formed from these functionalised pore monomers have improved abilities to determine the presence, absence or one or more characteristics of target analytes.
  • a CsgA partner polypeptide can be used to functionalise a CsgG pore with a pore tether and increase the percentage total reads of double stranded polynucleotides by the pore.
  • the pore monomers and pores of the invention can be used in connection with a variety of different applications as described in more detail below.
  • the invention therefore provides a pore monomer conjugate comprising a pore monomer, a partner molecule, and a functional binding moiety, wherein the partner molecule has affinity for the pore monomer and wherein the functional binding moiety is attached to the pore monomer via the partner molecule.
  • the invention also provides:
  • a construct comprising two or more covalently attached pore monomer conjugates of the invention; a pore complex comprising at least one pore monomer conjugate of the invention or at least one construct of the invention; a pore multimer comprising two or more pores, wherein at least one of the pores is a pore complex of the invention; a membrane comprising a pore complex of the invention or a pore multimer of the invention; a method for determining the presence, absence or one or more characteristics of a target analyte, comprising the steps of:
  • kits for characterising a target analyte comprising (a) a pore complex of the invention or a pore multimer of the invention and (b) the components of a membrane;
  • a kit for characterising a target polynucleotide or a target polypeptide comprising (a) a pore complex of the invention or a pore multimer of the invention and (b) a polynucleotide binding protein or a polypeptide binding protein;
  • an apparatus for characterising a target polynucleotide or a target polypeptide in a sample comprising (a) a plurality of pore complexes of the invention or a plurality of pore multimers of the invention and (b) a plurality of polynucleotide binding proteins or a plurality of polypeptide binding proteins; an array comprising a plurality of membranes of the invention; a system comprising (a) a membrane of the invention or an array of the invention, (b) means for applying a potential across the membrane(s) and (c) means for detecting electrical or optical signals across the membrane(s); an apparatus comprising a pore complex of the invention or a pore multimer of the invention inserted into an in vitro membrane;
  • an apparatus produced by a method comprising (i) obtaining a pore complex of the invention or a pore multimer of the invention and (ii) contacting the pore complex or a pore multimer with an in vitro membrane such that the pore complex or the pore multimer is inserted in the in vitro membrane; a pore monomer conjugate comprising a CsgG pore monomer, a partner molecule, and a functional binding moiety, wherein the CsgG pore monomer comprises a sequence which is at least about 40% homologous or identical to the amino acid sequence of SEQ ID NO: 3 over the entire sequence, wherein the partner molecule comprises SEQ ID NO: 21, 23, 25, 27, 29, 31, 63, 64, 65, 66, 67, or 68, wherein the K at position 7 of SEQ ID NO: 21, 23, 25, 27, 29 or 31 is covalently attached to the CsgG pore monomer by a sulfonyl group or the K at position 8 of SEQ ID NO: 63, 64
  • Figure 1 SDS-PAGE gel analysis of the CsgG-only pore control (CsgG-F56Q) and CsgG- CsgA-morpholino complexes when broken down to their constituent monomer components upon boiling in the presence of DTT.
  • Lanes 2 - 12 correspond to CsgA polypeptide variants whereby the position of the sulphonyl fluoride is moved along the CsgA polypeptide.
  • Lanes 2-12 demonstrate a band shift compared with the pore-only sample in lane 1. This implies that CsgG monomers are covalently modified with a CsgA-morpholino strand.
  • Lanes 8-11 demonstrate a high reaction efficiency, with the majority of CsgG monomer being modified with a CsgA-morpholino strand.
  • FIG. 2 SDS-PAGE gel analysis of CsgG/CsgF complex-only control and CsgG/CsgF-CsgA- N3 complexes with polypeptides ranging between 14 and 19 residues in length and where each complex was clicked with a BCN-morpholino strand.
  • Lanes 2, 4, 6, 8 and 10 each show a small band shift relative to the pore-only sample in lane 1.
  • Lanes 3, 5, 7, 9 and 11 each show a band shift to approximately 40kDa corresponding to the pore-CsgA-morpholino conjugate which is absent in the pore-only sample in lane 1. This implies that each of the CsgA polypeptides covalently attach to the CsgG monomer.
  • Figure 3a Ionic current (pA) vs time (s) traces as a duplex pair of DNA strands translocate through a CsgG-CsgA-morpholino modified pore inserted into a minion flow cell.
  • the raw current trace is shown in black lines and the event detected signal is shown in red lines.
  • Shorter time scale segments of the template and complement strands of the duplex pair are indicated. Aligned basecalls of this pair are shown, indicating alignment to the forward and reverse of the same genomic position in the reference.
  • Figure 3b A time-segmented bar chart of sequencing data where the percentage of sequenced DNA bases within that time segment are assigned to being part of a duplex pair (duplex template in dark grey, duplex complement in black) or a ID strand (light grey). Data from three minion flow cells is shown where two flow cells of CsgG-CsgA-morpholino modified pore are compared to an unmodified pore control. The total amount of duplex pair data is uplifted in the modified pore and maintained over the course of the sequencing experiment.
  • Figure 3c A bar chart showing the percentage of sequenced DNA bases within 3 separate minlON sequencing experiments. Sequenced bases are assigned to being part of a duplex pair (duplex template in dark grey, duplex complement in black) or a ID strand (light grey). Data from three minion flow cells is shown where two flow cells of CsgG-CsgA-morpholino modified pore are compared to an unmodified control. The total amount of duplex pair data is uplifted in the modified pore.
  • Figure 3d A bar chart showing the total amount of sequenced DNA bases within 8 separate minlON sequencing experiments.
  • the first 3 bars show the same data as in Figure 3c.
  • Bars 4-8 show data from modified pores in which the CsgA polypeptides (SEQ ID NOs: 43-47 in Table 3 in order from left to right) were longer than the CsgA polypeptide (SEQ ID NO: 40 in Table 1) used in Figure 3c.
  • Sequenced bases are assigned to being part of a duplex pair (duplex template in dark grey, duplex complement in black) or a ID strand (light grey). The total amount of duplex pair data is uplifted in all tested linker contexts compared to the unmodified pore control.
  • Figure 3e A bar chart showing the total amount of sequenced DNA bases within 8 separate minlON sequencing experiments.
  • the data relate to the CsgA polypeptides shown in Table 5.
  • the first two columns (left to right) relate to SEQ ID NO: 48.
  • the next two columns relate to SEQ ID NO: 40.
  • the next two columns relate to SEQ ID NO: 49.
  • the final two columns (left to right) relate to SEQ ID NO: 50.
  • Sequenced bases are assigned to being part of a duplex pair (duplex template in dark grey, duplex complement in black) or a ID strand (light grey).
  • the total amount of duplex pair data is uplifted in all tested linker contexts compared to the unmodified pore control.
  • FIG. 4 Native PAGE gel analysis of CsgG pore control and CsgG pore complexed with two linked CsgA polypeptides (SEQ ID NOs: 69 and 70), where each CsgA polypeptide contained an SO 2 F group.
  • the C-terminus of one CsgA polypeptide (SEQ ID NO: 70) was attached to the R group of the K at the C-terminus of another CsgA polypeptide (SEQ ID NO: 69). This provided a linked construct with one CsgA polypeptide N to C linked to another C to N. The N-terminus of each CsgA polypeptide was then available to attach to a CsgG pore.
  • CsgG single pore is the band shown between 242 kDa and 480 kDa, while the dimer band sits between 480 and 720 kDa.
  • CsgG pore control we observed a small overall reduction in the single pore band intensity at higher temperatures, presumably due to breakdown of pore oligomers to single polypeptide chains.
  • CsgG pore complexed with the linked CsgA polypeptides no significant difference was observed in the ratio of dimer to single pore over the temperature range tested.
  • We observed some overall reduction in band intensity at higher temperatures presumably due to breakdown of pore oligomers to single polypeptide chains.
  • Substantially more dimer pore was seen than compared with the control sample, indicating the CsgA peptide stabilises the pore dimer.
  • Figure 5 A bar chart showing the total amount of sequenced DNA bases within 3 separate minlON sequencing experiments.
  • the data relate to CsgG-CsgF nanopore complexes variably modified with CsgA-morpholino as indicated. The presence or absence of a competitor to the morpholino sequence is also indicated. Sequenced bases are assigned to being part of a duplex pair (duplex template in dark grey, duplex complement in black) or a ID strand (light grey).
  • the total amount of duplex pair data is uplifted when the nanopore is modified and a competitor is not present, the presence of competitor returns the total amount of duplex pair data to that of the control unmodified pore.
  • FIG. 6 The structure and size of the wild-type CsgG pore from Escherichia coli strain K12 (the databank accession code for this structure is 4UV3). The distances shown are measured from backbone to backbone of the amino acids forming the pore structure.
  • the CsgG pore is a tightly interconnected symmetrical nonameric pore that resembles a crown.
  • the overall height is 98 A, and the largest outer diameter is 120 A. It defines a central channel and consists of three parts: (A) the cap region, (B) the constriction region and (C) the transmembrane beta barrel region.
  • Cap axial length, or height, is 39 A. It has an inner diameter of 43 A and a 66 A mouth.
  • the beta barrel has 36 strands, an axial length of 39 A and inner diameter of 55 A. Transition between pore cap and beta barrel is sharp, being the constriction located among them, at the level of the predicted lipid-aqueous interface.
  • the constriction is approximately 18.5 A in diameter and exhibits a length of 20A along the axis of the channel.
  • Figure 7 Compared to pore alone, high polynucleotide binding protein to pore ratios result in an upwards shift of the pore band, indicating that the polynucleotide binding protein and pore are forming a complex.
  • the dashed white line indicates the pore-only band, confirming that the polynucleotide binding protein: pore band is shifted compared to the pore-only band.
  • the polypeptide binding protein is labelled "motor".
  • Figure 8 The proportion of polynucleotide binding protein-driven strand capture events which initialise within the first 40 nucleotides of the strand, as determined by mapping the signal to the reference using an in-house hidden Markov model (HMM)-based algorithm. This proportion is larger when the polynucleotide binding protein is attached to the nanopore than when the polynucleotide binding protein is un-modified, demonstrating that the events originate from polynucleotide binding proteins which are attached to the nanopore.
  • HMM hidden Markov model
  • Figure 9 The total number of polynucleotide binding protein-driven DNA capture events obtained during the experiment. Compared to the experiment using control un-modified polynucleotide binding protein, the experiment using tethered polynucleotide binding protein shows an increased rate of capture of polynucleotide binding protein-driven DNA strands.
  • SEQ ID NO: 1 shows the polynucleotide sequence of wild-type E. coli CsgG from strain K12, including signal sequence (Gene ID: 945619).
  • SEQ ID NO: 2 shows the amino acid sequence of wild-type E. coli CsgG including signal sequence (Uniprot accession number P0AEA2).
  • SEQ ID NO: 3 shows the amino acid sequence of wild-type E. coli CsgG as a mature protein (Uniprot accession number P0AEA2).
  • SEQ ID NO: 4 shows the polynucleotide sequence of wild-type E. coli CsgA from strain K12, including signal sequence.
  • SEQ ID NO: 5 shows the amino acid sequence of wild-type E. coli CsgA including signal sequence.
  • SEQ ID NO: 6 shows the amino acid sequence of wild-type E. coli CsgA as a mature protein.
  • SEQ ID NO: 7 shows the sequence of a fragment of CsgA, GVVPQYGGGG.
  • SEQ ID NO: 8 shows the sequence of a CsgA polypeptide for use in the invention, GVVPQYKGGG.
  • SEQ ID NO: 9 shows the sequence of a fragment of CsgA, GVVPQYGGGGN.
  • SEQ ID NO: 10 shows the sequence of a CsgA polypeptide for use in the invention, GVVPQYKGGGN.
  • SEQ ID NO: 11 shows the sequence of a fragment of CsgA, GVVPQYGGGGNH.
  • SEQ ID NO: 12 shows the sequence of a CsgA polypeptide for use in the invention
  • SEQ ID NO: 13 shows the sequence of a fragment of CsgA, GVVPQYGGGG NHG.
  • SEQ ID NO: 14 shows the sequence of a CsgA polypeptide for use in the invention
  • SEQ ID NO: 15 shows the sequence of a fragment of CsgA, GVVPQYGGGGNHGG.
  • SEQ ID NO: 16 shows the sequence of a CsgA polypeptide for use in the invention
  • SEQ ID NO: 17 shows the sequence of a fragment of CsgA, GVVPQYGGGGNHGGG.
  • SEQ ID NO: 18 shows the sequence of a CsgA polypeptide for use in the invention
  • SEQ ID NO: 19 shows the sequence of a linker, SGS.
  • SEQ ID NO: 20 shows the sequence of a CsgA polypeptide for use in the invention
  • SEQ ID NO: 21 shows the sequence of the CsgA polypeptide used in Example 1, GVVPQYKGGGSGSK.
  • SEQ ID NO: 22 shows the sequence of a CsgA polypeptide for use in the invention, GVVPQYGGGGNSGSK.
  • SEQ ID NO: 23 shows the sequence of the CsgA polypeptide used in Example 1, GVVPQYKGGGNSGSK.
  • SEQ ID NO: 24 shows the sequence of a CsgA polypeptide for use in the invention, GVVPQYGGGGN HSGSK.
  • SEQ ID NO: 25 shows the sequence of the CsgA polypeptide used in Example 1, GVVPQYKGGGNHSGSK.
  • SEQ ID NO: 26 shows the sequence of a CsgA polypeptide for use in the invention, GVVPQYGGGGN HGSGSK.
  • SEQ ID NO: 27 shows the sequence of the CsgA polypeptide used in Example 1, GVVPQYKGGGNHGSGSK.
  • SEQ ID NO: 28 shows the sequence of a CsgA polypeptide for use in the invention, GVVPQYGGGGN HGGSGSK.
  • SEQ ID NO: 29 shows the sequence of the CsgA polypeptide used in Example 1, GVVPQYKGGGNHGGSGSK.
  • SEQ ID NO: 30 shows the sequence of a CsgA polypeptide for use in the invention, GVVPQYGGGGN HGGGSGSK.
  • SEQ ID NO: 31 shows the sequence of the CsgA polypeptide used in Example 1, GVVPQYKGGGNHGGGSGSK.
  • SEQ ID Nos: 32-42 are the modified CsgA polypeptides in Table 1 in Example 1.
  • SEQ ID Nos: 43-47 are the modified CsgA polypeptides in Table 3 in Example 1.
  • SEQ ID Nos: 48-50 are the modified CsgA polypeptides in Table 5 in Example 1.
  • SEQ ID NOs: 51-56 are the oligonucleotides used in Example 2.
  • SEQ ID NO: 57 shows the sequence of a CsgA polypeptide for use in the invention, GVVPQYGKGG.
  • SEQ ID NO: 58 shows the sequence of a CsgA polypeptide for use in the invention, GVVPQYGKGGN.
  • SEQ ID NO: 59 shows the sequence of a CsgA polypeptide for use in the invention, GVVPQYGKGGNH.
  • SEQ ID NO: 60 shows the sequence of a CsgA polypeptide for use in the invention, GVVPQYGKGGNHG.
  • SEQ ID NO: 61 shows the sequence of a CsgA polypeptide for use in the invention, GVVPQYGKGGNHGG.
  • SEQ ID NO: 62 shows the sequence of a CsgA polypeptide for use in the invention, GVVPQYGKGGNHGGG.
  • SEQ ID NO: 63 shows the sequence of a CsgA polypeptide for use in the invention, GVVPQYGKGGSGSK.
  • SEQ ID NO: 64 shows the sequence of a CsgA polypeptide for use in the invention, GVVPQYGKGGNSGSK.
  • SEQ ID NO: 65 shows the sequence of a CsgA polypeptide for use in the invention, GVVPQYGKGGNHSGSK.
  • SEQ ID NO: 66 shows the sequence of a CsgA polypeptide for use in the invention, GVVPQYGKGGNHGSGSK.
  • SEQ ID NO: 67 shows the sequence of a CsgA polypeptide for use in the invention, GVVPQYGKGGNHGGSGSK.
  • SEQ ID NO: 68 shows the sequence of a CsgA polypeptide for use in the invention, GVVPQYGKGGNHGGGSGSK.
  • SEQ ID NO: 69 is a sequence used in Example 1, GVVPQY(KSO2F)GPPGGPPK.
  • SEQ ID NO: 70 is a sequence used in Example 1, GVVPQY(KSO2F)PPGGPPK.
  • a polynucleotide includes two or more polynucleotides
  • reference to “a polynucleotide binding protein” includes two or more such proteins
  • reference to “a helicase” includes two or more helicases
  • reference to “a monomer” refers to two or more monomers
  • reference to “a pore” includes two or more pores and the like.
  • Standard substitution notation is also used, i.e., Q42R means that Q at position 42 is replaced with R.
  • the I symbol means "or".
  • Q87R/K means Q87R or Q87K.
  • the I symbol means "and” such that Y51/N55 is Y51 and N55.
  • the invention provides pore monomer conjugates comprising a pore monomer, a partner molecule, and a functional binding moiety.
  • the partner molecule has affinity for the pore monomer.
  • the partner molecule typically binds to or is attached to the pore monomer.
  • the attachment may comprise covalent, non-covalent, supramolecular and/or native interactions.
  • the attachment may comprise non-covalent, supramolecular and/or native interactions.
  • the partner molecule is preferably covalently attached to the pore monomer.
  • the functional binding moiety is attached, preferably covalently attached, to the partner molecule.
  • the functional binding moiety is attached to the pore monomer via the partner molecule.
  • the pore monomer is bound or attached, preferably covalently attached, to the partner molecule, which is then attached, preferably covalently attached, to the functional binding moiety.
  • the functional binding moiety is indirectly attached to the pore monomer through the partner molecule.
  • the pore monomer is not directly attached to the functional binding moiety.
  • the functional binding moiety does not typically function to attach the partner molecule to the pore monomer.
  • the pore monomer may be from or derived from any pore.
  • the pore is typically a protein pore.
  • Pores that are suitable for characterising target analytes are known in the art. For instance, examples are disclosed in WO 2010/004265, WO2012/107778, WO 2013/153359, WO 2015/166275, WO 2016/055778, WO 2015/166276, WO 2016/132123, WO 2017/174990, WO2018/146491, WO 2020/095052, WO 2020/208357, PCT/GB2022/052196, PCT/EP2022/077537, WO 2016/034591, WO 2017/149316, WO 2017/149317, WO 2017/149318, WO 2018/211241, and WO 2019/002893 (all incorporated by reference herein in their entirety).
  • the pore monomer is preferably from or derived from Wza, Iota toxin, Anthrax protective antigen, Vibrio cholerae cytolysin, Cytotoxin K (CytK), CELIII, CsgG, Aerolysin, alpha hemolysin, InvG, GspD, MspA, MspB, MspC, PorARr, PorBRr, PorARc, PilQ, necrotic enteritis B-like toxin (NetB), FraC, portal proteins including G20c, P23_45, T4, SPP1, P22 and Phi29, gamma hemolysin, Monalysin, Lysenin, ClyA, and Clostridium perfringens beta toxin.
  • the pore monomer may be constructed from two or more different pore monomers from or derived from any of these pores.
  • the pore monomer may be a chimeric pore monomer comprising two or more regions, such as 3, 4, 5, 6, 7 or more regions, wherein at least two, such as at least 3, 4, 5, 6 or 7, of the two or more regions are from at least two different pores, such as from at least 3, 4, 5, 6, or 7 different pores.
  • a pore monomer is "from” or “derived from” a pore if it shares significant homology/identity with the sequence of the wild-type or naturally occurring pore monomer from the pore.
  • the pore monomer preferably comprises a sequence having at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or more preferably at least about 95%, at least about 97%, at least about 98% or at least about 99% homology to the sequence of the wild-type or naturally occurring pore monomer.
  • the pore monomer preferably comprises a sequence having at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90% or more preferably at least about 95%, at least about 97%, at least about 98% or at least about 99% identity to the sequence of the wild-type or naturally occurring pore monomer. Homology and/or identity is typically measured over the entire length of the region. Methods for measuring homology and/or identity are discussed in more detail below. Sequences for the pore listed above are publicly available through GenBank and various references supra and infra.
  • the pore monomer may be "from” or "derived from” a pore if it shares significant homology/identity with a fragment or portion of the sequence of the wild-type or naturally occurring pore monomer. Hence, a sequence may have less than 40% overall sequence homology/identity with the overall sequence of the pore monomer, but the sequence of a particular region, domain or subunit could share at least about 80%, at least about 90%, or as much as at least about 99% sequence homology or identity with the corresponding region of the pore monomer.
  • the pore is preferably a CsgG pore monomer. Such monomers are discussed in more detail below.
  • the partner molecule may be any molecule.
  • the partner molecule may be a molecule or a modified version of a molecule that interacts with the pore monomer in the course of their natural functions.
  • the partner molecule may be a molecule or a modified version of a molecule which forms a complex with the pore monomer, for instance in nature.
  • the partner molecule preferably comprises or consists of a polymer, an amino acid, a peptide, a polypeptide, a protein, a nucleotide, an oligonucleotide, a polynucleotide, a polynucleotide-polypeptide conjugate, a monosaccharide, an oligosaccharide, or a polysaccharide.
  • the partner molecule preferably comprises or consists of an oligonucleotide or a polynucleotide, such as a nucleic acid.
  • An oligonucleotide or a polynucleotide is defined as a macromolecule comprising two or more nucleotides.
  • the oligonucleotide, polynucleotide or nucleic acid may comprise any combination of any nucleotides.
  • the nucleotides can be naturally occurring or artificial.
  • One or more nucleotides in the oligonucleotide or polynucleotide can be oxidized or methylated.
  • One or more nucleotides in the oligonucleotide or polynucleotide may be damaged.
  • the oligonucleotide or 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 may be modified, for instance with a label or a tag, for which suitable examples are known by a skilled person.
  • the oligonucleotide or polynucleotide may comprise one or more spacers.
  • a nucleotide typically contains a nucleobase, a sugar and at least one phosphate group. The nucleobase and sugar form a nucleoside.
  • the nucleobase is typically heterocyclic.
  • Nucleobases include, but are not limited to, purines and pyrimidines and more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C).
  • the sugar is typically a pentose sugar.
  • Nucleotide sugars include, but are not limited to, ribose and deoxyribose.
  • the sugar is preferably a deoxyribose.
  • the polynucleotide preferably comprises the following nucleosides: deoxyadenosine (dA), deoxyuridine (dll) and/or thymidine (dT), deoxyguanosine (dG) and deoxycytidine (dC).
  • the nucleotide is typically a ribonucleotide or deoxyribonucleotide.
  • the nucleotide typically contains a monophosphate, diphosphate, or triphosphate.
  • the nucleotide may comprise more than three phosphates, such as 4 or 5 phosphates. Phosphates may be attached on the 5' or 3' side of a nucleotide.
  • the nucleotides 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 oligonucleotide or polynucleotide may be single stranded or double stranded. At least a portion of the polynucleotide may be double stranded.
  • the polynucleotide may be ribonucleic nucleic acid (RNA) or deoxyribonucleic acid (DNA).
  • the oligonucleotide or polynucleotide can be any length.
  • the partner oligonucleotide or polynucleotide can be at least 10, at least 25, at least 30, at least 40, 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 oligonucleotide or 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.
  • 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.
  • AMP adenosine monophosphate
  • GFP guanosine monophosphate
  • TMP thymidine monophosphate
  • UMP uridine monophosphate
  • CMP
  • 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 partner molecule preferably comprises or is an amino acid, a peptide, a polypeptide, or protein.
  • the amino acid, peptide, polypeptide, or protein can be naturally occurring or non- naturally occurring.
  • the polypeptide or protein can include within them synthetic or modified amino acids. Several different types of modification to amino acids are known in the art. It is to be understood that the partner molecule can be modified by any method available in the art.
  • a polypeptide may comprise any combination of any amino acids, amino acid analogs and modified amino acids (i.e., amino acid derivatives).
  • the amino acids/derivatives/analogs can be naturally occurring or artificial.
  • the polypeptide may comprise any naturally occurring amino acid.
  • One or more of the amino acids/derivatives/analogs in the polypeptide may be post- translationally modified. Suitable post-translational modifications are discussed in more detail below with reference to target analytes.
  • the polypeptide may be labelled with a molecular label.
  • the polypeptide may contain one or more cross-linked sections, e.g., C-C bridges.
  • the polypeptide may comprise sulphide- containing amino acids and thus has the potential to form disulphide bonds. All of these embodiments are discussed in more detail below with reference to target analytes. Any of the polypeptides discussed below with reference to target analytes may be used as a partner molecule.
  • the partner polypeptide can be any suitable length.
  • the polypeptide preferably has a length of from about 2 to about 300 peptide units or amino acids.
  • the polypeptide has a length of from about 2 to about 100 peptide units, for example from about 2 to about 50 peptide units, e.g., from about 3 to about 50 peptide units, such as from about 5 to about 25 peptide units, e.g., from about 7 to about 16 peptide units, such as from about 9 to about 13 peptide units.
  • “Peptide unit” is interchangeable with "amino acid”.
  • the partner polypeptide may be a polypeptide or a modified version of the polypeptide that interacts with the pore monomer in the course of their natural functions.
  • the partner polypeptide may be from or derived from a polypeptide which forms a complex with the pore monomer, for instance in nature.
  • a partner polypeptide is "from” or “derived from” a polypeptide which forms a complex with the pore monomer if it shares significant homology or identity with the sequence of the polypeptide which forms a complex with the pore monomer.
  • the partner polypeptide preferably comprises a sequence having at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% or more preferably at least about 95%, at least about 97%, at least about 98% or at least about 99% homology to the sequence of the polypeptide which forms a complex with the pore monomer.
  • the pore monomer preferably comprises a sequence having at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90% or more preferably at least about 95%, at least about 97%, at least about 98% or at least about 99% identity to the sequence of polypeptide which forms a complex with the pore monomer. Homology and/or identity is typically measured over the entire length of the region. Methods for measuring homology and/or identity are discussed in more detail below. Sequences for the pore listed above are publicly available through GenBank and various references supra and infra.
  • the partner polypeptide may be "from” or "derived from” a pore if it shares significant homology or identity with a fragment or portion of the sequence of the wild-type or naturally occurring polypeptide which forms a complex with the pore monomer.
  • a sequence may have less than 40% overall sequence homology or identity with the overall sequence of the polypeptide which forms a complex with the pore monomer, but the sequence of a particular region, domain or subunit could share at least about 80%, at least about 90%, or as much as at least about 99% sequence homology or identity with the corresponding region of the polypeptide which forms a complex with the pore monomer.
  • the partner polypeptide may comprise or consists of a fragment or portion of the polypeptide which forms a complex with the pore monomer. Such fragments or portions typically share 100% homology or identity with the corresponding fragment or portion of the polypeptide which forms a complex with the pore monomer.
  • the partner polypeptide may comprise or consists at least 5 consecutive or contiguous amino acids from the polypeptide which forms a complex with the pore monomer, such as at least 4, at least 5, 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, at least 16, at least 17, at least 18, at least 19, at least 20, at least 30, at least 40, at least 50, at least 100 or at least 150 consecutive or contiguous amino acids from the polypeptide which forms a complex with the pore monomer.
  • the partner molecule is preferably a CsgA polypeptide as discussed in more detail below.
  • the partner molecule may comprise a polynucleotide and a polypeptide.
  • the partner molecule may comprise or be a polynucleotide-polypeptide conjugate.
  • the conjugate preferably comprises a polynucleotide conjugated to a polypeptide.
  • Polynucleotidepolypeptide conjugates are discussed in more detail below with reference to target analytes. Any of those polynucleotide-polypeptide conjugates may be used as a partner molecule.
  • the partner molecule has affinity for the pore monomer.
  • the partner molecule preferably has high affinity for the pore monomer.
  • the partner molecule has high affinity for the pore monomer if it binds with a Kd of 1 x 10' 6 M or less, more preferably 1 x 10' 7 M or less, 5 x 10' 8 M or less, more preferably 1 x 10' 8 M or less or more preferably 5 x 10' 9 M or less.
  • a molecule or group binds with low affinity if it binds with a Kd of 1 x 10' 6 M or more, more preferably 1 x 10' 5 M or more, more preferably 1 x 10' 4 M or more, more preferably 1 x 10' 3 M or more, even more preferably 1 x 10' 2 M or more.
  • the partner molecules such as the partner polynucleotide or partner polypeptide, preferably has affinity for the pore monomer without any modification, such as the use of one or more reactive groups discussed below.
  • the partner molecules, such as the partner polynucleotide or partner polypeptide preferably has natural affinity for the pore monomer, i.e., affinity based on its wild-type or naturally occurring structure. An example of this is a CsgA polypeptide having affinity for a CsgG pore monomer.
  • the partner molecule typically binds to the pore monomer.
  • the partner molecule preferably specifically binds to the pore monomer.
  • the partner molecule specifically binds to the pore monomer if it binds to the pore monomer with preferential or high affinity, but does not bind or binds with only low affinity to other or different molecules, such as other or different pore monomers, other or different polypeptides and/or polynucleotides.
  • the partner molecule binds to the pore monomer with an affinity that is at least 10 times, such as at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 1000 or at least 10,000 times, greater than its affinity for other polynucleotides.
  • Affinity can be measured using known binding assays, such as those that make use of fluorescence and radioisotopes.
  • Competitive binding assays are also known in the art.
  • the strength of binding between peptides or proteins and polynucleotides can be measured using nanopore force spectroscopy as described in Hornblower et al., Nature Methods. 4: 315-317. (2007) or Isothermal Titration Calorimetry (ITC), which is a label-free quantification technique used in studies of a wide variety of biomolecular interactions. ITC works by directly measuring the heat that is either released or absorbed during a biomolecular binding event.
  • the partner molecule is typically attached to the pore monomer.
  • the partner molecule is preferably covalently attached to the pore monomer.
  • the partner molecule is preferably attached to the pore monomer by one or more reactive groups.
  • the attachment preferably comprises one or more reactive groups.
  • the attachment preferably comprises a reaction between a position, nucleotide, residue, or linker in the partner molecule with the pore monomer.
  • Suitable reactive groups for use in the invention include, but are not limited to, amine-reactive groups, oxygen-reactive groups and a fluroacetamide groups.
  • the aminereactive group is preferably a thioester, a NHS-ester, a pentafluorophenyl ester, a benzylic halide, a sulfonyl fluoride, a fluorosulfate, or a sulfonyl triazole.
  • the oxygen-reactive group is preferably an alkyl halide, a sulfonyl fluoride, a fluorosulfate, or a sulfonyl triazoles.
  • the partner molecule is preferably attached to the pore monomer by a sulfonyl fluoride reaction.
  • a reactive group on the pore monomer such as the R group of a threonine, cysteine, tyrosine, lysine, serine, or histidine, preferably displaces the fluoride group in the sulphonyl fluoride group on the partner molecule to covalently link the pore monomer and the partner molecule.
  • the pore monomer is preferably covalently attached to the partner molecule by a sulfonyl group.
  • the attachment preferably comprises one or more reactive groups which react with lysine, cysteine, tyrosine, serine, threonine, proline, tryptophan, arginine, histidine, methionine, or phenylalanine in the pore monomer.
  • the attachment preferably comprises a reaction between a position, nucleotide, residue, or linker in the partner molecule with lysine, cysteine, tyrosine, serine, threonine, proline, tryptophan, arginine, histidine, methionine, or phenylalanine in the pore monomer.
  • the lysine, cysteine, tyrosine, serine, threonine, proline, tryptophan, arginine, histidine, methionine, or phenylalanine may be native to the pore monomer.
  • the lysine, cysteine, tyrosine, serine, threonine, proline, tryptophan, arginine, histidine, methionine, or phenylalanine may be introduced into the pore monomer, preferably by substitution or addition.
  • Reactive groups which react with lysine include, but are not limited to, maleimide, activated esters, anhydrides, carbonates, isocyanates, isothiocyanates, a range of other acylating and alkylating agents, oxidative coupling O-aminophenols, aldehydes, activated carbodiimides, ketenes, sulfonyl halides, fluorosulfates, and sulfonyl triazoles.
  • Positions, residues, or linkers may also be attached to lysine using periodate oxidation, reductive amination, transamination, aniline/arylamine conjugation via oxidative coupling, azaelectrocyclization, iminoboronate formation, or conjugation of arene diazonium salts.
  • Reactive groups which react with cysteine include, but are not limited to, haloacetamides and other alpha-halocarbonyls, maleimides, acrylates, vinyl sulfones, vinylpyridines, epoxides, oxanorbornadienes, methylsulfonyl functioanlised heteroaromatic, allenes, allyl selenosulfate salts, perfluoroaromatic, thiol-ene and thiol-yne click chemistry, pyridyl dithiol, vinylsulfones, sulfonyl halides, fluorosulfates, and sulfonyl triazoles.
  • Positions, residues, or linkers may also be attached to cysteine using strain-release alkylation, nickel(II)-catalyzed oxidative coupling, oxidative coupling with aminophenols, conjugation with allenes (in the presence of gold catalyst, or allyl selenosulfate salts), native chemical ligation, Pd-catalysed arylation/alkynylation, or allylation followed by cross-metathesis.
  • Reactive groups which react with tyrosine include, but are not limited to, sulfonyl halides, fluorosulfates, and sulfonyl triazoles. Positions, residues, or linkers may also be attached to tyrosine using oxidative conjugation of tyrosines including O-alkylation, hydrazone and oxime condensations, addition reactions with electron deficient alkynes such as alkynones, alkynoate, amide or esters, cyclic diazodicarboxamides, Pd catalysed alkylation, diazonium salts, or Mannich reaction with imines formed from aldehydes, cyclic diazodicarboxamides, modification with Rhodium carbenoids.
  • Reactive groups which react with serine or threonine include, but are not limited to, sulfonyl halides, fluorosulfates, and sulfonyl triazoles. Positions, residues, or linkers may also be attached to serine or threonine using periodate oxidation and subsequent transimination reactions of ketones/aldehydes with hydrazides/alkoxyamines. Resultant aldehydes/ketones may also modified through aldol ligation.
  • Positions, residues, or linkers may be attached to proline using oxidative coupling with O- aminophenols at N-terminus.
  • Reactive groups which react with tryptophan include, but are not limited to, aldehydes, ketones, and tetrazoles. Positions, residues, or linkers may be attached to tryptophan using a condensation reaction, modification with Rhodium carbenoids, conjugation with N/O centred radicals, and N-terminal Trp modification using Pictet-Spengler reaction.
  • Positions, residues, or linkers may be attached to arginine using condensation with o,p- dicarbonyl compounds.
  • Reactive groups which react with histidine include, but are not limited to, vinylsulfones, sulfonyl halides, fluorosulfates, and sulfonyl triazoles. Positions, residues, or linkers may be attached to hisitidine using C2 alkylation and N3 alkylation/thiophosphorylation.
  • Positions, residues, or linkers may be attached to methionine using S-alkylation/imidation.
  • Positions, residues, or linkers may be attached to phenylalanine using modification with Rhodium carbenoids.
  • the attachment such as covalent attachment, preferably comprises one or more reactive groups which react with any amino acid in the pore monomer.
  • Reactive groups which react with any amino acid include, but are not limited to, activated esters, anhydrides, carbonates, isocyanates, isothiocyanates, and a range of other acylating and alkylating agents, oxidative coupling O-aminophenols, aldehydes, activated carbodiimides, ketenes, transamination, and vinylboronic acids.
  • the attachment such as covalent attachment, preferably comprises reacting a position, nucleotide, residue, or linker in the partner molecule with any amino acid in the pore monomer. Positions, residues, or linkers may be attached to any amino acid using periodate oxidation, or reductive amination.
  • the attachment such as covalent attachment, preferably comprises one or more reactive groups which undergo click chemistry.
  • Suitable click chemistries include, but are not limited to, CuAAC Azide/alkyne, staudinger ligation, strain-promoted azide-alkyne cycloaddition, inverse-electron demand Diels-Alder reaction between 1,2,4,5-tetrazines and strained alkenes.
  • the pore monomer preferably binds to the partner molecule by a linker.
  • the pore monomer is preferably attached, such as covalently attached, to the partner molecule by a linker.
  • the pore monomer preferably binds to the partner molecule or is preferably attached, such as covalently attached, to the partner molecule by two or more linkers, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more linkers. If two or more linkers are used, they may be the same. If two or more linkers are used, they may be different.
  • the skilled person is capable of designing one or more linkers for use in the invention.
  • the linker or one or more linkers may be any of those discussed below.
  • the linker or one or more linkers preferably comprise any of the reactive groups discussed above for attaching the pore monomer to the partner molecule.
  • the linker(s) preferably comprise(s) or consist(s) of a linear carbon chain of 2, 3, 4, 5, 6 or more carbon atoms and/or cyclic groups containing 3, 5 or 6 carbon atoms.
  • the distance between the pore monomer and the partner molecule in the pore monomer conjugate and/or the length of the linker is preferably less than about 2.00 nm, such as less than about 1.90 nm, less than about 1.80 nm, less than about 1.70 nm, less than about 1.60 nm, less than about 1.50 nm, less than about 1.40 nm, less than about 1.30 nm, less than about 1.20 nm, less than about 1.10 nm, less than about 1.00 nm, less than about 0.90 nm, less than about 0.80 nm, less than about 0.70 nm, less than about 0.60 nm, less than about 0.50 nm, or less than about 0.40 nm.
  • the distance between the pore monomer and the partner molecule in the pore monomer conjugate and/or the length of the linker is preferably from about 0.40 nm to about 2.0 nm, such as about 0.45 nm to about 1.90 nm, from about 0.50 nm to about 1.80 nm, from about 0.55 nm to about 1.7 nm, from about 0.60 nm to about 1.6 nm, from about 0.65 nm to about 1.5 nm, from about 0.7 nm to about 1.4 nm, from about 0.75 nm to about 1.3 nm, from about 0.80 nm to about 1.2 nm, from about 0.85 nm to about 1.1 nm and from about 0.90 nm to about 1.00 nm.
  • the pore monomer conjugate may comprise any number of partner molecules, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more partner molecules for each pore monomer.
  • the partner molecules may be the same.
  • the partner molecules may be different.
  • the partner molecules may be attached to the pore monomer using any of the reactive groups and/or linker discussed above.
  • Combinations of pore monomers and partner molecules include, but are not limited to, (a) a CsgG pore monomer and a CsgA polypeptide, (b) a CsgG pore monomer and a CsgB polypeptide, (c) a CsgG pore monomer and a CsgC polypeptide, (d) a CsgG pore monomer and a CsgD polypeptide, or (e) a CsgG pore monomer and a CsgE polypeptide.
  • the pore monomer and partner molecule are preferably selected from (i) a CsgG pore monomer and a CsgA polypeptide, (ii) a CsgG pore monomer and a CsgB polypeptide, (iii) a CsgG pore monomer and a CsgC polypeptide, or (iv) a CsgG pore monomer and a CsgE polypeptide.
  • the pore monomer is preferably a CsgG pore monomer and the partner molecule is preferably selected from (a) a CsgA polypeptide, (b) a CsgB polypeptide and (c) a CsgE polypeptide, such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c) or (a), (b) and (c).
  • the pore monomer is preferably a CsgG pore monomer and the partner molecule is preferably a CsgA polypeptide. CsgG pore monomers and CsgA polypeptides are discussed in more detail below.
  • the functional binding moiety has affinity for another molecule.
  • the functional binding moiety is capable of binding to or attaching to another molecule. Affinity and measuring binding is discussed above.
  • the functional binding moiety is capable of binding to or attaching to a target analyte.
  • the target analyte preferably comprises or consists of a metal ion, an inorganic salt, a polymer, an amino acid, a peptide, a polypeptide, a protein, a nucleotide, an oligonucleotide, a polynucleotide, a polynucleotide-polypeptide conjugate, a monosaccharide, an oligosaccharide, a polysaccharide, a dye, a bleach, a pharmaceutical, a diagnostic agent, a recreational drug, an explosive, a toxic compound, or an environmental pollutant.
  • the target analyte preferably comprises or consists of a polypeptide, a protein, an oligonucleotide, a polynucleotide, a polynucleotide-polypeptide conjugate, an oligosaccharide, or a polysaccharide.
  • the functional binding moiety comprises a region that is capable of binding to or attaching to the molecule or target analyte.
  • the functional binding moiety preferably comprises an oligonucleotide, a polynucleotide, a polynucleotide analog, a morpholino, a peptide nucleic acid, a polypeptide, a ligand, a cyclodextrin, a monosaccharide, an oligosaccharide, a polysaccharide, a boronic acid, an enzyme, a peptide, a cyclic peptide, an antibody or fragment thereof, or an aptamer.
  • Oligonucleotides and polynucleotides are defined above and below. Any polynucleotide analog may be used. Polynucleotide analogs typically contain nucleobases connected by modified backbones. Suitable polynucleotide analogs include, but are not limited to, peptide nucleic acid (PNA), threose nucleic acid (TNA), and glycerol nucleic acid (GNA). The skilled person can identify other suitable polynucleotide analogs for use in the invention.
  • PNA peptide nucleic acid
  • TAA threose nucleic acid
  • GNA glycerol nucleic acid
  • Morpholinos contain nucleobases connected using methylenemorpholine rings linked through phosphorodiamidate groups. Morpholinos are also known as morpholino oligomers or a phosphorodiamidate morpholino oligomers (PMOs).
  • the functional binding moiety may comprise or consist of any of these oligonucleotides, polynucleotides, polynucleotide analogs or morpholinos.
  • the functional binding moiety preferably comprises an oligonucleotide, polynucleotide, polynucleotide analog or morpholino which is capable of hybridizing, preferably specifically hybridizing, to a target polynucleotide.
  • An oligonucleotide, polynucleotide, polynucleotide analog or morpholino specifically hybridizes to a target polynucleotide when it hybridizes with preferential or high affinity to the target polynucleotide but does not substantially hybridize, does not hybridize or hybridizes with only low affinity to other polynucleotides.
  • An oligonucleotide, polynucleotide, polynucleotide analog or morpholino specifically hybridizes if it hybridizes to the target polynucleotide with a melting temperature (Tm) that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C or at least 10 °C, greater than its Tm for other sequences.
  • Tm melting temperature
  • the oligonucleotide, polynucleotide, polynucleotide analog or morpholino hybridizes to the target polynucleotide with a Tm that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C, at least 10 °C, at least 20 °C, at least 30 °C or at least 40 °C, greater than its Tm for other nucleic acids.
  • the oligonucleotide, polynucleotide, polynucleotide analog or morpholino hybridizes to the target polynucleotide with a Tm that is at least 2 °C, such as at least 3 °C, at least 4 °C, at least 5 °C, at least 6 °C, at least 7 °C, at least 8 °C, at least 9 °C, at least 10 °C, at least 20 °C, at least 30 °C or at least 40 °C, greater than its Tm for a sequence which differs from the target polynucleotide by one or more nucleotides, such as by 1, 2, 3, 4 or 5 or more nucleotides.
  • the oligonucleotide, polynucleotide, polynucleotide analog or morpholino typically hybridizes to the target polynucleotide with a Tm of at least 90 °C, such as at least 92 °C or at least 95 °C.
  • Tm can be measured experimentally using known techniques, including the use of DNA microarrays, or can be calculated using publicly available Tm calculators, such as those available over the internet.
  • Hybridization can be carried out under low stringency conditions, for example in the presence of a buffered solution of 30 to 35% formamide, 1 M NaCI and 1 % SDS (sodium dodecyl sulfate) at 37 °C followed by a 20 wash in from IX (0.1650 M Na+) to 2X (0.33 M Na+) SSC (standard sodium citrate) at 50 °C.
  • Hybridization can be carried out under moderate stringency conditions, for example in the presence of a buffer solution of 40 to 45% formamide, 1 M NaCI, and 1 % SDS at 37 °C, followed by a wash in from 0.5X (0.0825 M Na+) to IX (0.1650 M Na+) SSC at 55 °C.
  • Hybridization can be carried out under high stringency conditions, for example in the presence of a buffered solution of 50% formamide, 1 M NaCI, 1% SDS at 37 °C, followed by a wash in 0.1X (0.0165 M Na+) SSC at 60 °C.
  • the oligonucleotide, polynucleotide, polynucleotide analog or morpholino may be any length as described above with reference to oligonucleotides and polynucleotides (where references to nucleotides are replaced with nucleobases for polynucleotide analogs and morpholinos).
  • the oligonucleotide, polynucleotide, polynucleotide analog or morpholino preferably comprises a portion or region which is substantially complementary, or complementary to, to a portion or region of the target polynucleotide. The portion or region in the target polynucleotide may also be known as a binding region.
  • the portion or region in the oligonucleotide, polynucleotide, polynucleotide analog or morpholino and/or in the target polynucleotide is typically at least 10 nucleotides or nucleobases in length, such at least 15 nucleotides or nucleobases, at least 20 nucleotides or nucleobases, at least 25 nucleotides or nucleobases, at least 30 nucleotides or nucleobases, at least 40 nucleotides or nucleobases or at least 50 nucleotides or nucleobases in length.
  • the region or portion of the oligonucleotide, polynucleotide, polynucleotide analog or morpholino may therefore have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mismatches across a region of 5, 10, 15, 20, 21, 22, 30, 40 or 50 nucleotides or nucleobases compared with the portion or region in the target polynucleotide.
  • a portion or region is typically 50 nucleotides/nucleobases or fewer in length, such as 40 nucleotides/nucleobases or fewer, 30 nucleotides/nucleobases or fewer, 20 nucleotides/nucleobases or fewer, 10 nucleotides/nucleobases or fewer or 5 nucleotides/nucleobases or fewer in length.
  • the portion or region may be from 5 to 50 nucleotides/nucleobases in length, such as from 10 to 40 nucleotides/nucleobases in length or from 20 to 30 nucleotides/nucleobases.
  • the portion or region is preferably 25 nucleotides/nucleobases in length.
  • antibody includes whole antibodies. Naturally occurring antibodies typically comprise a tetramer which is usually composed of at least two heavy (H) chains and at least two light (L) chains. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region, usually comprised of three domains (CHI, CH2 ad CH3). Heavy chains can be of any isotype, including IgG (IgGl, IgG2, IgG3 and IgG4 subtypes), IgA (IgAl and IgA2 subtypes), IgM and IgE. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region (CL).
  • VH heavy chain variable region
  • CL light chain constant region
  • Light chain includes kappa (K) chains and lambda (A) chains.
  • the heavy and light chain variable region is typically responsible for antigen recognition, whilst the heavy and light chain constant region may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the term "functional fragment” refers to a fragment of an intact antibody that retains the ability to specifically bind to a given molecule or target analyte.
  • Such fragments include Fab fragments, Fab' fragments, monovalent fragments consisting of the VL, VH, CL and CHI domains; F(ab')2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragment consisting of the VH and CHI domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; a dAb fragment (Ward et al., 1989 Nature 341 :544-546), which consists of a VH domain; and an isolated complementarity determining region (CDR).
  • CDR complementarity determining region
  • the two domains of the Fv fragment, VL and VH are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); see e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988 Proc. Natl. Acad. Sci. 85:5879-5883).
  • single chain Fv single chain Fv
  • Such single chain antibodies are also intended to be encompassed within the term "functional fragment" of an antibody.
  • These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.
  • Aptamers are small molecules that bind to one or more molecules or target analytes. Aptamers can be produced using SELEX (Stoltenburg, R. et al., (2007), Biomolecular Engineering 24, p381 403; Tuerk, C. et al., Science 249, p505-510; Bock, L. C. et al., (1992), Nature 355, p564-566) or NON-SELEX (Berezovski, M. et al. (2006), Journal of the American Chemical Society 128, pl410-1411).
  • the aptamer may be capable of binding to or attaching to two or more molecules or target analytes. Aptamers that bind to more than one analyte member can be produced using Toggle SELEX (White, R. et al., (2001), Molecular Therapy 4, p567-573).
  • the aptamer is preferably a peptide aptamer or an oligonucleotide aptamer.
  • the peptide aptamer may comprise any amino acids.
  • the amino acids may be any of those discussed above.
  • the oligonucleotide aptamer may comprise any nucleotides.
  • the nucleotides may be any of those discussed above.
  • the aptamer can be any length.
  • the aptamer is typically at least 15 amino acids or nucleotides in length, such as at least 20, at least 25, at least 30 or at least 35 amino acids or nucleotides in length.
  • the aptamer is preferably from about 15 to about 50, from about 20 to about 40 or from about 25 to about 30 amino acids or nucleotides in length.
  • the functional binding moiety is preferably capable of binding to or attaching to a polynucleotide binding protein, a pore monomer, an aptamer, a ring-shaped protein, or a DNA origami structure. Attaching these structures to pore monomers or pores in accordance with the invention improve their abilities to characterise target analytes as discussed in more detail below.
  • the functional binding moiety is preferably capable of binding to or attaching to a polynucleotide binding protein, a polypeptide binding protein, a pore monomer, an aptamer, a ring-shaped protein, or a DNA origami structure. Attaching these structures to pore monomers or pores in accordance with the invention improve their abilities to characterise target analytes as discussed in more detail below.
  • the functional binding moiety may be capable of binding or attaching to a polynucleotide binding protein.
  • Polynucleotide binding proteins are polymerases, exonucleases, helicases, and topoisomerases, such as gyrases.
  • Suitable polynucleotide binding proteins include, but are not limited to, exonuclease I from E. coli, exonuclease III enzyme from E. coli, RecJ from T. thermophilus and bacteriophage lambda exonuclease, TatD exonuclease and variants thereof.
  • thermophilus or a variant thereof interact to form a trimer exonuclease.
  • the polymerase may be PyroPhage® 3173 DNA Polymerase (which is commercially available from Lucigen® Corporation), SD Polymerase (commercially available from Bioron®) or variants thereof.
  • the polynucleotide binding protein may be Phi29 DNA polymerase or a variant thereof.
  • the topoisomerase is preferably a member of any of the Moiety Classification (EC) groups 5.99.1.2 and 5.99.1.3.
  • the polynucleotide binding proteins is most preferably derived from a helicase, such as Hel308 Mbu, Hel308 Csy, Hel308 Tga, Hel308 Mhu, Tral Eco, XPD Mbu or a variant thereof.
  • a helicase such as Hel308 Mbu, Hel308 Csy, Hel308 Tga, Hel308 Mhu, Tral Eco, XPD Mbu or a variant thereof.
  • Any helicase may be used in the invention.
  • the helicase may be or be derived from a Hel308 helicase, a RecD helicase, such as Tral helicase or a TrwC helicase, a XPD helicase or a Dda helicase.
  • the helicase may be any of the helicases, modified helicases or helicase constructs disclosed in WO 2013/057495; WO 2013/098562; WO 2013098561; WO 2014/013260; WO 2014/013259; WO 2014/013262 and WO 2015/055981. All of these are incorporated by reference herein in their entirety.
  • the functional binding moiety may be capable of binding or attaching to a polypeptide binding protein.
  • Polypeptide binding proteins are known in the art.
  • the polypeptide binding protein may be a nucleoside triphosphate (NTP) driven unfoldase.
  • NTP nucleoside triphosphate
  • the unfoldase may be driven by any NTP. Suitable nucleosides which can form the basis of NTPs are discussed below with reference to polynucleotides.
  • the NTP unfoldase may be an ATPase Associated with diverse cellular Activities (AAA+) enzyme.
  • the AAA+ enzyme may be CIpX, CIpAP, CIpXP, CIpCP, HsIVU or Lon.
  • the polypeptide binding protein is preferably CIpX.
  • the proteins listed in this paragraph are defined in WO 2013/123379, incorporated by reference in its entirety. Aptamers are discussed above.
  • ring-shaped proteins include, but are not limited to, pentraxin, GroES, SP1, any pore, MspA, aHL, CsgG, lysenin, InvG, GspD, leukocidin, FraC, aerolysin, NetB and any of those discussed above, and a hexameric enzyme, such as a hexameric helicase or a hexameric unfoldase.
  • the pore monomer may be any of the pore monomers discussed above.
  • the functional binding moiety may bind the pore monomer in the pore monomer conjugate. This is capable of stabilising cis loops in the pore monomer and reducing the signal-to-noise ratio (SNR) of the pore complexes formed from the pore monomer.
  • the pore monomer is not attached to the partner molecule only by the functional binding moiety.
  • the functional binding moiety is not the only link between the pore monomer and the partner molecule.
  • the partner molecule is preferably not a CsgF peptide.
  • the DNA origami structure is preferably a DNA origami pore.
  • Such pores are known in the art, e.g., Langecker et al., Science, 2012; 338: 932-936).
  • the partner molecule is attached, preferably covalently attached, to the functional binding moiety.
  • the partner molecule may be attached, preferably covalently attached, to the functional binding moiety using any known method, including using any of the reactive groups discussed above or alternative attachment methods discussed below.
  • the partner molecule is preferably attached or covalently attached to the functional binding moiety by a linker.
  • the linker may be any of the linkers discussed above or below.
  • the linker is preferably a polypeptide linker or a polynucleotide linker. Polypeptides and polynucleotides are defined above.
  • the partner molecule is a polypeptide, such as a CsgA polypeptide, it may be genetically fused to the polynucleotide binding protein, a pore monomer, a ring-shaped protein, optionally by a linker.
  • the partner polypeptide and polynucleotide binding protein, a pore monomer, or a ring-shaped protein with or without a linker may be expressed as a single polypeptide or protein construct.
  • the partner molecule is a polypeptide, such as a CsgA polypeptide, it may be genetically fused to the polynucleotide binding protein, the polypeptide binding protein, a pore monomer, a ring-shaped protein, optionally by a linker.
  • the partner polypeptide and polynucleotide binding protein, the polypeptide binding protein, a pore monomer, or a ring-shaped protein with or without a linker may be expressed as a single polypeptide or protein construct. Any of the linkers discussed above may be used.
  • the linker is preferably an amino acid or peptide linker. Suitable amino acid linkers, such as peptide linkers, are known in the art. Flexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10 or 16, serine and/or glycine amino acids. More flexible linkers include (SG)i, (SG) 2 , (SG) 3 , (SG) 4 , (SG) 5 , (SG) 8 , (SG)IQ, (SG)i 5 or (SG) 2 o wherein S is serine and G is glycine. Rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or 24, proline amino acids. More rigid linkers include (P) i2 wherein P is proline. The linker is preferably SGS (SEQ ID NO: 19) wherein S is serine and G is glycine. The linker is preferably SEQ ID NO: 19 further comprising a K or K(N3) at its C-terminus.
  • the pore monomer conjugates of the invention are capable of forming a pore or a pore complex. This can be measured using routine methods, including any of those described in WO 2016/034591, WO 2017/149316, WO 2017/149317, WO 2017/149318, WO 2018/211241, and WO 2019/002893 (all incorporated by reference herein in their entirety) and in the Examples.
  • the pore monomer is preferably a CsgG pore monomer.
  • a CsgG pore monomer is a monomer that is capable of forming a CsgG pore. Such monomers are known in the art, especially from WO 2019/002893 (incorporated by reference herein in its entirety).
  • the CsgG pore preferably comprises one or more of (a) a cap region, (b) a constriction region, and (c) a transmembrane beta barrel region, such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c).
  • the CsgG pore monomer preferably comprises one or more of (a) a cap forming region, (b) a constriction forming region, and (c) a transmembrane beta barrel forming region, such as (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b) and (c).
  • the residues of SEQ ID NO: 3 which form these regions are defined below.
  • the CsgG pore formed by the monomer may have any structure but preferably has or comprises the structure of the wild-type CsgG pore ( Figure 6).
  • the protein structure of CsgG defines a channel or hole that allows the translocation of molecules and ions from one side of the membrane to the other.
  • constriction refers to an aperture defined by a luminal surface of a pore or pore complex, which acts to allow the passage of ions and target analytes (e.g., but not limited to polynucleotides or individual nucleotides) but not other non-target analytes through the pore or pore complex channel.
  • target analytes e.g., but not limited to polynucleotides or individual nucleotides
  • the constriction(s) are typically the narrowest aperture(s) within a pore or pore complex or within the channel defined by the pore or pore complex.
  • the constriction(s) may serve to limit the passage of molecules through the pore.
  • the size of the constriction is typically a key factor in determining suitability of a pore or pore complex for analyte characterisation. If the constriction is too small, the molecule to be characterised will not be able to pass through. However, to achieve a maximal effect on ion flow through the channel, the constriction should not be too large. For example, the constriction should not be wider than the solvent-accessible transverse diameter of a target analyte. Ideally, any constriction should be as close as possible in diameter to the transverse diameter of the analyte passing through.
  • the CsgG pore may be any size but preferably has the dimensions of the wild-type CsgG pore ( Figure 6).
  • the CsgG pore preferably has an external diameter of from about 100 to about 150 A at its widest point, such as from about 110 to about 140 A or from about 115 to about 125 A at its widest point.
  • the CsgG pore preferably has an external diameter of about 120 A at its widest point.
  • the CsgG pore preferably has a total length of from about 80 to about 120 A, such as from about 90 to about 110 A or from about 95 to about 105 A.
  • the CsgG pore preferably has a total length of about 98 A. References to "total length” and “length” relate to the length of the pore or pore region when viewed from the side (see, e.g., the side view in Figure 6).
  • the cap region preferably has a length of from about 20 to about 60 A, such as from about 30 to about 50 A or from about 35 to about 45 A.
  • the cap region preferably has a length of about 39 A.
  • the channel defined by the cap region preferably has an opening of from about 45 to about 85 A in diameter, such as from about 55 to about 75 A or from about 60 to about 70 A in diameter.
  • the channel defined by the cap region preferably has an opening of about 66 A in diameter.
  • the channel defined by the cap region is preferably from about 30 to about 70 A in diameter at its narrowest point, such as from about 35 to about 60 A or from about 40 to about 50 A in diameter at its narrowest point.
  • the channel defined by the cap region is preferably about 43 A in diameter at its narrowest point.
  • the constriction region preferably has a length of from about 5 to about 40 A, such as from about 10 to about 30 A or from about 15 to about 25 A.
  • the constriction region preferably has a length of about 20 A.
  • the channel defined by the constriction region is preferably from about 2 to about 40 A in diameter at its narrowest point, such as from about 5 to about 35 A, from about 8 to about 25 A or from about 10 to about 20 A in diameter at its narrowest point.
  • the channel defined by the constriction region is preferably about 9 A or 12 A in diameter.
  • the channel defined by the constriction region is preferably about 18.5 A in diameter.
  • the constriction is preferably from about 2 to about 40 A in diameter, such as from about 5 to about 35 A, from about 8 to about 25 A or from about 10 to about 20 A in diameter.
  • the constriction is preferably about 9 A or 12 A in diameter.
  • the constriction is preferably about 12 A in diameter.
  • the transmembrane beta barrel region preferably has a length of from about 20 to about 60 A, such as from about 30 to about 50 A or from about 35 to about 45 A.
  • the transmembrane beta barrel preferably has a length of about 39 A.
  • the channel defined by the transmembrane beta barrel region is preferably from about 35 to about 75 A in diameter at its narrowest point, such as from about 45 to about 65 A or from about 50 to about 60 A in diameter at its narrowest point.
  • the channel defined by the transmembrane beta barrel region is preferably about 55 A in diameter at its narrowest point.
  • SEQ ID NO: 3 shows the sequence of wild-type E. coli CsgG as a mature protein. Residues 1 to 41, 64 to 131, 156 to 180 and 212 to 262 of SEQ ID NO: 3 form the cap region. Residues 42 to 63 of SEQ ID NO: 3 form the constriction region. Residues 132 to 155 and 181 to 211 of SEQ ID NO: 3 form the transmembrane beta barrel region.
  • the CsgG pore monomer is preferably a variant of SEQ ID NO: 3.
  • the variant CsgG momomer may also be referred to as a modified CsgG pore monomer or a mutant CsgG pore monomer.
  • the modifications, or mutations, in the variant include but are not limited to any one or more of the modifications disclosed herein, or combinations of said modifications.
  • the CsgG pore monomer may be a CsgG homologue monomer.
  • a CsgG homologue monomer is a polypeptide that has at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% complete sequence identity to wildtype E.
  • a CsgG homologue is also referred to as a polypeptide that contains the PFAM domain PF03783, which is characteristic for CsgG-like proteins.
  • PFAM domain PF03783 A list of presently known CsgG homologues and CsgG architectures can be found at httD://Dfam.xfam.oro//familv/PF03783.
  • a variant will preferably be at least about 40% homologous to that sequence based on amino acid identity. More preferably, the variant may be at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of SEQ ID NO: 3 over the entire sequence. Over the entire length of the amino acid sequence of SEQ ID NO: 3, a variant will preferably be at least about 40% identical to that sequence.
  • the variant may be at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% identical to SEQ ID NO: 3 over the entire sequence.
  • Sequence identity can also relate to a fragment or portion of the CsgG pore monomer.
  • a sequence may have less than 40% overall sequence homology or identity with SEQ ID NO: 3, but the sequence of a particular region, domain or subunit could share at least about 80%, 90%, or as much as 99% sequence homology or identity with the corresponding region of SEQ ID NO: 3.
  • the CsgG pore monomer is preferably a variant of SEQ ID NO: 3 comprising a sequence that is at least about 40% homologous to the cap region of SEQ ID NO: 3 (residues 1 to 41, 64 to 131, 156 to 180 and 212 to 262).
  • the variant may comprise a sequence that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% homologous based on amino acid identity to residues 1 to 41, 64 to 131, 156 to 180 and 212 to 262 of SEQ ID NO: 3.
  • the variant preferably comprises a sequence that is at least about 40% identical to residues 1 to 41, 64 to 131, 156 to 180 and 212 to 262 of SEQ ID NO: 3.
  • the variant may comprise a sequence that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% identical to residues of 1 to 41, 64 to 131, 156 to 180 and 212 to 262 of SEQ ID NO: 3. Homology and/or identity is typically measured over the entire length of the cap region.
  • the CsgG pore monomer is preferably a variant of SEQ ID NO: 3 comprising a sequence that is at least about 40% homologous to the constriction region of SEQ ID NO: 3 (residues 42 to 63). More preferably, the variant may comprise a sequence that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% homologous based on amino acid identity to residues 42 to 63 of SEQ ID NO: 3.
  • the variant preferably comprises a sequence that is at least about 40% identical to residues 42 to 63 of SEQ ID NO: 3. More preferably, the variant may comprise a sequence that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% identical to residues of 42 to 63 of SEQ ID NO: 3. Homology and/or identity is typically measured over the entire length of the constriction region.
  • the CsgG pore monomer is preferably a variant of SEQ ID NO: 3 comprising a sequence that is at least about 40% homologous to the transmembrane beta barrel region of SEQ ID NO: 3 (residues 132 to 155 and 181 to 211). More preferably, the variant may comprise a sequence that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% homologous based on amino acid identity to residues 132 to 155 and 181 to 211 of SEQ ID NO: 3.
  • the variant preferably comprises a sequence that is at least about 40% identical to residues 132 to 155 and 181 to 211 of SEQ ID NO: 3. More preferably, the variant may comprise a sequence that is at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% identical to residues of 132 to 155 and 181 to 211 of SEQ ID NO: 3. Homology and/or identity is typically measured over the entire length of the transmembrane beta barrel region.
  • CsgG pore monomers are highly conserved (as can be readily appreciated from Figures 45 to 47 of WO 2017/149317). Furthermore, from knowledge of the mutations in relation to SEQ ID NO: 3 it is possible to determine the equivalent positions for mutations of CsgG pore monomers other than that of SEQ ID NO: 3.
  • mutant CsgG pore monomer comprising a variant of the sequence as shown in SEQ ID NO: 3 and specific amino-acid mutations thereof as set out in the claims and elsewhere in the specification also encompasses a mutant CsgG pore monomer comprising a variant of any of the sequences shown in SEQ ID NOs: 68 to 88 of WO 2019/002893 (incorporated by reference herein in its entirety) and corresponding aminoacid mutations thereof.
  • the CsgG pore monomer may also be any of the sequences shown in CN 113773373 A, CN 113896776 A, CN 113912683 A, and CN 113754743 A or a variant thereof. It will further be appreciated that the invention extends to other variant CsgG pore monomers not expressly identified in the specification that show highly conserved regions.
  • Standard methods in the art may be used to determine homology.
  • the UWGCG Package provides the BESTFIT program which can be used to calculate homology, for example used on its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387-395).
  • the PILEUP and BLAST algorithms can be used to calculate homology or line up sequences (such as identifying equivalent residues or corresponding sequences (typically on their default settings)), for example as described in Altschul S. F. (1993) J Mol Evol 36:290- 300; Altschul, S.F et al (1990) J Mol Biol 215:403-10.
  • SEQ ID NO: 3 is the wild-type CsgG pore monomer from Escherichia coli Str. K-12 substr. MC4100.
  • a variant of SEQ ID NO: 3 may comprise any of the substitutions present in another CsgG homologue.
  • CsgG homologues are shown in SEQ ID NOs: 68 to 88 of WO 2019/002893 (incorporated by reference herein in its entirety).
  • the variant may comprise combinations of one or more of the substitutions present in SEQ ID NOs: 68 to 88 WO 2019/002893 (incorporated by reference herein in its entirety) compared with SEQ ID NO: 3, including one or more substitutions, one or more conservative mutations, one or more deletions or one or more insertion mutations, such as deletion or insertion of 1 to 10 amino acids, such as of 2 to 8 or 3 to 6 amino acids.
  • the CsgG pore monomer in the pore monomer conjugate of the invention typically retains the ability to form the same 3D structure as the wild-type CsgG pore monomer, such as the same 3D structure as a CsgG pore monomer having the sequence of SEQ ID NO: 3.
  • the 3D structure of CsgG is known in the art and is disclosed, for example, in Goyal et al (2014) Nature 516(7530):250-3. Any number of mutations may be made in the wild-type CsgG sequence in addition to the mutations described herein provided that the CsgG pore monomer retains the improved properties imparted on it by the mutations of the present invention.
  • the CsgG pore monomer will retain the ability to form a structure comprising five alpha-helices and five beta-strands. Therefore, it is envisaged that further mutations may be made in any of these regions in any CsgG pore monomer without affecting the ability of the monomer to form a pore that can translocate polynucleotides. It is also expected that deletions of one or more amino acids can be made in any of the loop regions linking the alpha helices and beta-strands and/or in the N-terminal and/or C-terminal regions of the CsgG pore monomer without affecting the ability of the monomer to form a pore that can translocate polynucleotides.
  • Amino acid substitutions may be made to the amino acid sequence of SEQ ID NO: 3 in addition to those discussed above, for example up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions.
  • Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties, or similar side-chain volume.
  • the 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.
  • the CsgG pore monomer may be modified to introduce one or more cysteines, one or more hydrophobic amino acids, one or more charged amino acids, one or more non-native amino acids, one or more polar amino acids, or one or more photoreactive amino acids. Any number and combination of such introductions may be made. The introduction is preferably by substitution or addition.
  • One or more amino acid residues of the amino acid sequence of SEQ ID NO: 3 may additionally be deleted from the polypeptides described above. Up to 1, 2, 3, 4, 5, 10, 20 or 30 or more residues may be deleted.
  • Variants may include fragments of SEQ ID NO: 3. Such fragments retain pore forming activity. Fragments may be at least 50, at least 100, at least 150, at least 200 or at least 250 amino acids in length. Such fragments may be used to produce the pores.
  • a fragment preferably comprises the transmembrane beta barrel region of SEQ ID NO: 3, namely residues 132 to 155 and 181 to 211, or a variant thereof as discussed above.
  • One or more amino acids may be alternatively or additionally added to the polypeptides described above.
  • An extension may be provided at the amino terminal or carboxy terminal of the amino acid sequence of SEQ ID NO: 3 or polypeptide variant or fragment thereof.
  • the extension may be quite short, for example from 1 to 10 amino acids in length. Alternatively, the extension may be longer, for example up to 50 or 100 amino acids.
  • a carrier protein may be fused to an amino acid sequence according to the invention. Other fusion proteins are discussed in more detail below.
  • a variant of SEQ ID NO: 3 is a polypeptide that has an amino acid sequence which varies from that of SEQ ID NO: 3 and which retains its ability to form a pore.
  • a variant typically contains the regions of SEQ ID NO: 3 that are responsible for pore formation. The pore forming ability of CsgG, which contains a p-barrel, is provided by p-strands in the transmembrane beta barrel region of each monomer.
  • a variant of SEQ ID NO: 3 typically comprises the region in SEQ ID NO: 3 that forms p-strands, namely residues 132 to 155 and 181 to 211, or a variant thereof as discussed above. One or more modifications can be made to the region of SEQ ID NO: 3 that form p-strands as long as the resulting variant retains its ability to form a pore.
  • the one or more modifications in the CsgG pore monomer preferably improve the ability of a pore complex comprising the pore monomer to characterise an analyte.
  • modifications/mutations/substitutions are contemplated to alter the number, size, shape, placement or orientation of the constriction within a channel from the pore monomer conjugate of the invention.
  • the CsgG pore monomer or the variant of SEQ ID NO: 3 may have any of the particular modifications or substitutions disclosed in WO 2016/034591, WO 2017/149316, WO 2017/149317, WO 2017/149318, WO 2018/211241, and WO 2019/002893 (all incorporated by reference herein in their entirety).
  • Modifications or substitutions in SEQ ID NO: 3 include, but are not limited to, one or more of, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more or all of:
  • a substitution at position Y51 such as Y51I, Y51L, Y51A, Y51V, Y51T, Y51S, Y51Q or Y51N;
  • N55 a substitution at position N55, such as N55I, N55L, N55A, N55V, N55T, N55S or N55Q;
  • N91 a substitution at position N91, such as N91D, N91E, N91R or N91K;
  • a substitution at position C215 such as C215T, C215S, C215I, C215L, C215A, C215V, or C215G.
  • the variant of SEQ ID NO: 3 preferably comprises F56Q.
  • the variant of SEQ ID NO: 3 may further comprise a deletion of one or more positions, such as a deletion of T104-N109, a deletion of F193-L199 or a deletion of F195-L199.
  • any number of the CsgG pore monomers in the pore or pore complex of the invention may be a variant of SEQ ID NO: 3. All six to ten monomers in the pore or pore complex are preferably variants of SEQ ID NO: 3.
  • the variants in the pore complex may be the same or different.
  • the variants are preferably identical in each pore monomer conjugate in the pore complex of the invention.
  • the partner molecule may be covalently attached to the pore monomer.
  • the partner molecule is preferably attached to one or more residues in the CsgG pore monomer corresponding to one or more of positions 1 to 9 in SEQ ID NO: 3. Any of the amino acids at these positions may be modified as discussed above or replaced, for instance by addition or substitution, with amino acids which attach to reactive groups as discussed above.
  • the pore monomer conjugate comprising a CsgG pore monomer preferably further comprises a CsgF peptide.
  • Such peptides are described in WO 2016/034591, WO 2017/149316, WO 2017/149317, WO 2019/002893, WO 2017/149318, WO 2018/211241, and WO 2019/002893 (herein all incorporated by reference in their entirety).
  • the partner molecule preferably comprises a CsgA polypeptide.
  • CsgA has natural affinity for CsgG pore monomers and it is straightforward to design a CsgA polypeptide which binds to any of the CsgG pore monomers discussed above.
  • Wild-type CsgA has a Kd of 23.8 x 10' 6 M for wild-type CsgG (Yan, Z., Yin, M., Chen, J. et al. Assembly and substrate recognition of curli biogenesis system. Nat Commun 11, 241 (2020)).
  • the pore monomer is a CsgG pore monomer and the partner molecule is a CsgA polypeptide
  • the CsgA polypeptide is preferably bound or attached to, preferably covalently attached, to the cap region of the CsgG pore monomer.
  • the cap region of CsgG is defined above.
  • a CsgA polypeptide is a polypeptide from or derived from CsgA.
  • the wild-type E. coli CsgA sequence is shown in SEQ ID NO: 5 and the same sequence without the signal peptide is shown in SEQ ID NO: 6.
  • the CsgA polypeptide may have any length.
  • the CsgA polypeptide may have a length of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55 amino acids.
  • the CsgA polypeptide may be from 5 to 150 amino acids, such from 7 to 100 amino acids, from 10 to 75 amino acids, or about 10 to 60 amino acids.
  • the CsgA polypeptide is preferably a fragment of SEQ ID NO: 5 or SEQ ID NO: 6 which has affinity for, binds to or attaches to the pore monomer, preferably a CsgG pore monomer.
  • the CsgA polypeptide preferably comprises or consists of at least 5 consecutive or contiguous amino acids from SEQ ID NO: 5 or SEQ ID NO: 6, such as at least 4, at least 5, 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, at least 16, at least 17, at least 18, at least 19, at least 20, at least
  • the fragment preferably comprises amino acids 21 to 30, 21 to
  • the CsgA polypeptide preferably comprises or consists of SEQ ID NO: 7, 9, 11, 13, 15 or 17.
  • the CsgA polypeptide is preferably a variant of any of the CsgA sequences discussed above, including any of the fragments and SEQ ID NO: 7, 9, 11, 13, 15 or 17. Over the entire length of the amino acid sequence of the CsgA fragment or SEQ ID NO: 7, 9, 11, 13, 15 or 17, a variant will preferably be at least about 40% homologous to that sequence based on amino acid identity.
  • the variant may be at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of the CsgA fragment or SEQ ID NO: 7, 9, 11, 13, 15 or 17 over the entire sequence. Over the entire length of the amino acid sequence of the CsgA fragment or SEQ ID NO: 7, 9, 11, 13, 15 or 17, a variant will preferably be at least about 40% identical to that sequence.
  • the variant may be at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% identical to the CsgA fragment or SEQ ID NO: 7, 9, 11, 13, 15 or 17 over the entire sequence.
  • the amino acid at any position in SEQ ID NO: 7, 9, 11, 13, 15 or 17 is preferably substituted with a K and/or modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • SEQ ID NO: 7, 9, 11, 13, 15 or 17 preferably further comprises a K at its N-terminus (/.e., before the G at position 1) modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the reactive group in any of these modified versions of SEQ ID NOs: 7, 9, 11, 13, 15 or 17 may be any of those discussed above.
  • the reactive group is preferably a sulfonyl group.
  • the amino acid at any of positions 1 to 10 of SEQ ID NO: 7, 9, 11, 13, 15 or 17, such as the G at position 1, the V at position 2, the V at position 3, the P at position 4, the Q at position 5, the Y at position 6, the G at position 7, the G at position 8, the G at position 9 or the G at position 10, is more preferably substituted with a K and/or modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • amino acid at any of positions 1 to 8 of SEQ ID NO: 7, 9, 11, 13, 15 or 17, such as the G at position 1, the V at position 2, the V at position 3, the P at position 4, the Q at position 5, the Y at position 6, the G at position 7, or the G at position 8, is more preferably substituted with a K and/or modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the amino acid at position 6, 7 or 8 of SEQ ID NO: 7, 9, 11, 13, 15 or 17, such as the Y at position 6, the G at position 7 or the G at position 8, is more preferably substituted with a K and/or modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the amino acid at position 8 of SEQ ID NO: 7, 9, 11, 13, 15 or 17 is more preferably substituted with a K and/or modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the reactive group may be any of those discussed above.
  • the reactive group is preferably a sulfonyl group.
  • amino acid at any of positions 1 to 7 of SEQ ID NO: 7, 9, 11, 13, 15 or 17, such as the G at position 1, the V at position 2, the V at position 3, the P at position 4, the Q at position 5, the Y at position 6, or the G at position 7, is more preferably substituted with a K and/or modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the amino acid at position 6 or 7 of SEQ ID NO: 7, 9, 11, 13, 15 or 17, such as the Y at position 6 or the G at position 7, is more preferably substituted with a K and/or modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the amino acid at position 7 of SEQ ID NO: 7, 9, 11, 13, 15 or 17 is more preferably substituted with a K and/or modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the reactive group may be any of those discussed above.
  • the reactive group is preferably a sulfonyl group.
  • the CsgA polypeptide preferably comprises or consists of SEQ ID NO: 7, 9, 11, 13, 15 or 17 and comprises a substitution of the G at position 7 with K (G7K).
  • the CsgA polypeptide preferably comprises or consists of SEQ ID NO: 8, 10, 12, 14, 16 or 18.
  • the G at position 7 of SEQ ID NO: 7, 9, 11, 13, 15 or 17 or the K at position 7 of SEQ ID NO: 8, 10, 12, 14, 16 or 18 is preferably modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the reactive group may be any of those discussed above.
  • the reactive group is preferably a sulfonyl group.
  • the CsgA polypeptide comprising or consisting of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 is preferably attached, preferably covalently attached, to the pore monomer, preferably CsgG pore monomer, by the reaction of a sulfonyl fluoride group at position 7 of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 with the pore monomer.
  • the CsgA polypeptide preferably comprises or consists of SEQ ID NO: 7, 9, 11, 13, 15 or 17 and comprises a substitution of the G at position 8 with K (G8K).
  • the CsgA polypeptide preferably comprises or consists of SEQ ID NO: 57, 58, 59, 60, 61, or 62.
  • the G at position 8 of SEQ ID NO: 7, 9, 11, 13, 15 or 17 or the K at position 8 of SEQ ID NO: 57, 58, 59, 60, 61, or 62 is preferably modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the reactive group may be any of those discussed above.
  • the reactive group is preferably a sulfonyl group.
  • the CsgA polypeptide comprising or consisting of SEQ ID NO: 7, 9, 11, 13, 15, 17, 57, 58, 59, 60, 61, or 62 is preferably attached, preferably covalently attached, to the pore monomer, preferably CsgG pore monomer, by the reaction of a sulfonyl fluoride group at position 8 of SEQ ID NO: 7, 9, 11, 13, 15, 17, 57, 58, 59, 60, 61, or 62 with the pore monomer.
  • any of the CsgA polypeptides discussed above preferably further comprises a linker.
  • the linker is preferably attached to the C-terminus of the CsgA polypeptide, such as any of the fragments discussed above or SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18.
  • the linker is preferably attached to the C-terminus of the CsgA polypeptide, such as SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 57, 58, 59, 60, 61, or 62.
  • the linker is preferably an amino acid or peptide linker. Suitable amino acid linkers, such as peptide linkers, are known in the art and discussed above.
  • the length, flexibility and hydrophilicity of the amino acid or peptide linker are typically designed to control the positioning of the functional binding moiety in relation to the CsgA polypeptide.
  • Flexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10 or 16, serine and/or glycine amino acids. More flexible linkers include (SG) X , (SG) 2 , (SG) 3 , (SG) 4 , (SG) 5 , (SG) 8 , (SG)i 0 , (SG)i 5 or (SG) 20 wherein S is serine and G is glycine.
  • Rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or 24, proline amino acids.
  • More rigid linkers include (P) i2 wherein P is proline.
  • the linker is preferably SGS (SEQ ID NO: 19) wherein S is serine and G is glycine.
  • the linker is preferably SEQ ID NO: 19 further comprising a K or K with an azide group, such as K(N3), at its C-terminus.
  • the CsgA polypeptide is preferably attached or covalently attached to the functional binding moiety by the linker.
  • the linker may be any of the linkers discussed above or below.
  • the CsgA polypeptide preferably comprises or consists of SEQ ID NO: 20, 22, 24, 26, 28 or 30. These sequences are SEQ ID NOs: 7, 9, 11, 13, 15 and 17 with a preferred linker attached to their C-termini.
  • the amino acid at any position in SEQ ID NO: 20, 22, 24, 26, 28 or 30 is preferably substituted with a K and/or modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • SEQ ID NO: 20, 22, 24, 26, 28 or 30 preferably further comprises a K at its N-terminus (/.e., before the G at position 1) modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the reactive group in any of these modified versions of SEQ ID NOs: 20, 22, 24, 26, 28 and 30 may be any of those discussed above.
  • the reactive group is preferably a sulfonyl group.
  • the amino acid at any of positions 1 to 10 of SEQ ID NO: 20, 22, 24, 26, 28 or 30, such as the G at position 1, the V at position 2, the V at position 3, the P at position 4, the Q at position 5, the Y at position 6, the G at position 7, the G at position 8, the G at position 9 or the G at position 10, is more preferably substituted with a K and/or modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the amino acid at any of positions 1 to 8 of SEQ ID NO: 20, 22, 24, 26, 28 or 30, such as the G at position 1, the V at position 2, the V at position 3, the P at position 4, the Q at position 5, the Y at position 6, the G at position 7, or the G at position 8, is more preferably substituted with a K and/or modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the amino acid at position 6, 7 or 8 of SEQ ID NO: 20, 22, 24, 26, 28 or 30, is more preferably substituted with a K and/or modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the amino acid at position 8 of SEQ ID NO: 20, 22, 24, 26, 28 or 30 is more preferably substituted with a K and/or modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the reactive group may be any of those discussed above.
  • the reactive group is preferably a sulfonyl group.
  • amino acid at any of positions 1 to 7 of SEQ ID NO: 20, 22, 24, 26, 28 or 30, such as the G at position 1, the V at position 2, the V at position 3, the P at position 4, the Q at position 5, the Y at position 6, or the G at position 7, is more preferably substituted with a K and/or modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the amino acid at position 6 or 7 of SEQ ID NO: 20, 22, 24, 26, 28 or 30, such as the Y at position 6 or the G at position 7, is more preferably substituted with a K and/or modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the reactive group may be any of those discussed above.
  • the reactive group is preferably a sulfonyl group.
  • the CsgA polypeptide comprising or consisting of SEQ ID NO: 20, 22, 24, 26, 27, 28 or 30 or any of the modified versions discussed above is preferably attached, preferably covalently attached, to the pore monomer, preferably CsgG pore monomer, by the reaction of a sulfonyl fluoride group with the pore monomer to form a sulfonyl group.
  • the K at the C- terminus of SEQ ID NO: 20, 22, 24, 26, 27, 28 or 30 or any of the modified versions discussed above is preferably modified with an azide group, such as N3.
  • the CsgA polypeptide preferably comprises or consists of SEQ ID NO: 21, 23, 25, 27, 29 or 31. These are SEQ ID NOs: 8, 10, 12, 14, 16 or 18 with a preferred linker attached to their C-termini.
  • the K at position 7 of SEQ ID NO: 21, 23, 25, 27, 29 or 31 is preferably modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the reactive group may be any of those discussed above.
  • the reactive group is preferably a sulfonyl group.
  • the CsgA polypeptide comprising or consisting of SEQ ID NO: 21, 23, 25, 27, 29 or 31 is preferably attached, preferably covalently attached, to the pore monomer, preferably CsgG pore monomer, by the reaction of a sulfonyl fluoride group at position 7 of SEQ ID NO: 21, 23, 25, 27, 29 or 31 with the pore monomer.
  • the K at the C-terminus of SEQ ID NO: 21, 23, 25, 27, 29 or 31 is preferably modified with an azide group, such as N3.
  • the CsgA polypeptide preferably comprises or consists of SEQ ID NO: 21, 23, 25, 27, 29, 31, 63, 64, 65, 66, 67 or 68. These are SEQ ID NOs: 8, 10, 12, 14, 16, 18, 57, 58, 59, 60, 61 or 62 with a preferred linker attached to their C-termini.
  • the K at position 7 of SEQ ID NO: 21, 23, 25, 27, 29 or 31 or the K at position 8 of SEQ ID NO: 63, 64, 65, 66, 67 or 68 is preferably modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the reactive group may be any of those discussed above.
  • the reactive group is preferably a sulfonyl group.
  • the CsgA polypeptide comprising or consisting of SEQ ID NO: 21, 23, 25, 27, 29, 31, 63, 64, 65, 66, 67 or 68 is preferably attached, preferably covalently attached, to the pore monomer, preferably CsgG pore monomer, by the reaction of a sulfonyl fluoride group at position 7 of SEQ ID NO: 21, 23, 25, 27, 29 or 31 or of a sulfonyl fluoride group at position 8 of SEQ ID NO: 63, 64, 65, 66, 67 or 68 with the pore monomer.
  • the K at the C-terminus of SEQ ID NO: 21, 23, 25, 27, 29, 31, 63, 64, 65, 66, 67 or 68 is preferably modified with an azide group, such as N3.
  • the CsgA polypeptide preferably comprises or consists of SEQ ID NO: 21. This is SEQ ID NO: 8 with a preferred linker attached to its C-terminus.
  • the K at position 7 of SEQ ID NO: 21 is preferably modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the reactive group may be any of those discussed above.
  • the reactive group is preferably a sulfonyl group.
  • the CsgA polypeptide comprising or consisting of SEQ ID NO: 21 is preferably attached, preferably covalently attached, to the pore monomer, preferably CsgG pore monomer, by the reaction of a sulfonyl fluoride group at position 7 of SEQ ID NO: 21 with the pore monomer.
  • the K at the C-terminus of SEQ ID NO: 21 is preferably modified with an azide group, such as N3.
  • the CsgA polypeptide preferably comprises or consists of SEQ ID NO: 63.
  • This is SEQ ID NO: 57 with a preferred linker attached to its C-terminus.
  • the K at position 8 of SEQ ID NO: 63 is preferably modified to include a reactive group which attaches the CsgA polypeptide to the pore monomer, preferably the CsgG pore monomer.
  • the reactive group may be any of those discussed above.
  • the reactive group is preferably a sulfonyl group.
  • the CsgA polypeptide comprising or consisting of SEQ ID NO: 63 is preferably attached, preferably covalently attached, to the pore monomer, preferably CsgG pore monomer, by the reaction of a sulfonyl fluoride group at position 8 of SEQ ID NO: 63 with the pore monomer.
  • the K at the C-terminus of SEQ ID NO: 63 is preferably modified with an azide group, such as N3.
  • Any of the CsgA polypeptides discussed above, especially those include linkers at the C- terminus, may further comprise a modified C-terminal group.
  • the CsgA polypeptide may comprise a -CONH2 group at its C-terminus.
  • the CsgA polypeptide preferably comprises or consists of SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47. These sequences are shown in Table 1 or 3 where SO2F is a sulfonyl fluoride group and N3 is an azide.
  • the CsgA polypeptide preferably comprises or consists of SEQ ID NO: 38, 39, 40, 41, 43, 44, 45, 46 or 47.
  • the CsgA polypeptide comprising or consisting of SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47 is preferably attached, preferably covalently attached, to the pore monomer, preferably CsgG pore monomer, by the reaction of the sulfonyl fluoride group with the pore monomer to form a sulfonyl group.
  • the CsgA polypeptide preferably comprises or consists of SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48. These sequences are shown in Table 1, 3 and 5 where SO2F is a sulfonyl fluoride group and N3 is an azide. SEQ ID NO: 48 also has a -CONH2 group at its C-terminus.
  • the CsgA polypeptide preferably comprises or consists of SEQ ID NO: 38, 39, 40, 41, 43, 44, 45, 46, 47 or 48.
  • the CsgA polypeptide comprising or consisting of SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47 or 48 is preferably attached, preferably covalently attached, to the pore monomer, preferably CsgG pore monomer, by the reaction of the sulfonyl fluoride group with the pore monomer to form a sulfonyl group.
  • the CsgA polypeptide preferably comprises or consists of SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50. These sequences are shown in Table 1, 3 and 5 where SO2F is a sulfonyl fluoride group and N3 is an azide. SEQ ID NOs: 48 and 49 also have a -CONH2 group at their C-terminii.
  • the CsgA polypeptide preferably comprises or consists of SEQ ID NO: 38, 39, 40, 41, 43, 44, 45, 46, 47, 48, 49 or 50.
  • the CsgA polypeptide comprising or consisting of SEQ ID NO: 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 is preferably attached, preferably covalently attached, to the pore monomer, preferably CsgG pore monomer, by the reaction of the sulfonyl fluoride group with the pore monomer to form a sulfonyl group.
  • the CsgA polypeptide preferably comprises or consists of SEQ ID NO: 49 or 50. These sequences are shown in Table 5 where SO2F is a sulfonyl fluoride group and N3 is an azide. SEQ ID NO: 49 also has a -CONH2 group at its C-terminus.
  • the CsgA polypeptide comprising or consisting of SEQ ID NO: 49 or 50 is preferably attached, preferably covalently attached, to the pore monomer, preferably CsgG pore monomer, by the reaction of the sulfonyl fluoride group with the pore monomer to form a sulfonyl group.
  • the CsgA polypeptide preferably comprises or consists of SEQ ID NO: 69 or 70. These sequences are shown in the description of the sequence listing (where SO2F is a sulfonyl fluoride group) and are used in Example 1 and Figure 4 to create a linked multimer.
  • the CsgA polypeptide comprising or consisting of SEQ ID NO: 69 and/or 70 is preferably attached, preferably covalently attached, to the pore monomer, preferably CsgG pore monomer, by the reaction of the sulfonyl fluoride group with the pore monomer to form a sulfonyl group.
  • the CsgA polypeptide is preferably a variant of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47.
  • a variant will preferably be at least about 40% homologous to that sequence based on amino acid identity.
  • the variant may be at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of the CsgA fragment or SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47 over the entire sequence. Over the entire length of the amino acid sequence of the CsgA fragment or SEQ ID NO: 8, 10, 12, 14,
  • a variant will preferably be at least about 40% identical to that sequence. More preferably, the variant may be at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% identical to the CsgA fragment or SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47 over the entire sequence.
  • the CsgA polypeptide is preferably a variant of SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16,
  • ID NO: 8 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 57, 58,
  • a variant will preferably be at least about
  • the variant may be at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of the CsgA fragment or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
  • a variant will preferably be at least about 40% identical to that sequence. More preferably, the variant may be at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% identical to the CsgA fragment or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 57, 58, 59, 60,
  • the G at position 1 may be substituted or deleted.
  • the G at position 1 may be substituted or deleted.
  • the CsgA polypeptides above including those based on SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28,
  • the Y at position 6 or 7 may be substituted, preferably with W.
  • the G at position 1 may be substituted or deleted.
  • the Y at position 6 or 7 may be substituted, preferably with W.
  • the CsgA polypeptides above including those based on SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 39, 40, 42, 43, 44, 45, 46, 47, 48, 49, 50, 57, 58, 59, 60, 61,
  • the Y at position 6 may be substituted, preferably with W.
  • the pore monomer is preferably attached or covalently attached to the positions discussed above in the CsgA polypeptide.
  • the pore monomer is typically attached or covalently attached to any of positions 1 to 10 in the CsgA polypeptide.
  • the pore monomer is typically attached or covalently attached to any of positions 1 to 8 in the CsgA polypeptide.
  • the pore monomer is typically attached or covalently attached to any of positions 1 to 7 in the CsgA polypeptide.
  • the pore monomer is typically attached or covalently attached to any of positions 6, 7 and 8 in the CsgA polypeptide.
  • the pore monomer is typically attached or covalently attached to position 7 or 8 in the CsgA polypeptide.
  • the pore monomer is typically attached or covalently attached to position 7 in the CsgA polypeptide.
  • the pore monomer is typically attached or covalently attached to position 8 in the CsgA polypeptide.
  • the functional binding moiety is typically attached or covalently attached the C-terminus of the CsgA polypeptide, or any linker attached thereto.
  • the CsgA polypeptide may be a CsgA multimer comprising two or more linked CsgA polypeptides.
  • the two or more linked CsgA polypeptides may be selected from any of those discussed above, including from SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 and 70 and variants thereof.
  • the CsgA multimer may comprise any number of two or more linked CsgA polypeptides, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more CsgA polypeptides.
  • the two or more CsgA polypeptides may be the same CsgA polypeptide.
  • the two or more CsgA polypeptides may be different CsgA polypeptides.
  • the two or more CsgA polypeptides may be linked in any manner.
  • the two or more CsgA polypeptides are preferably covalently linked.
  • the two or more CsgA polypeptides are preferably covalently linked via the K at the C-terminus of one or more of the CsgA polypeptides.
  • the two or more CsgA polypeptides may be covalently linked directly or via any of the linkers disclosed herein.
  • the skilled person is capable of linking two or more CsgA polypeptides, for instance using peptide bonds, via the R groups of any of the positions in the CsgA polypeptides or using the reactive groups discussed above.
  • the two or more CsgA polypeptides may be linked in any orientation.
  • the two or more CsgA polypeptides may be linked N-terminus to C-terminus or C-terminus to N-terminus.
  • the two or more CsgA polypeptides may be linked N-terminus to N-terminus.
  • the two or more CsgA polypeptides may be linked C-terminus to C-terminus.
  • One or more CsgA polypeptides may branch off one or more positions within the sequence of one or more CsgA polypeptides.
  • the multimer preferably comprises two CsgA polypeptides in which the C-terminus of one CsgA polypeptide is linked to the K at the C-terminus of the other CsgA polypeptide. This provides a linked multimer with one CsgA polypeptide N to C linked to another C to N. The N-terminus of each CsgA polypeptide is then available to attach to a pore monomer, such as a CsgG pore monomer.
  • the two CsgA polypeptides may be selected from SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 and 70 and variants thereof.
  • the two CsgA polypeptides are preferably SEQ ID NO: 69 and SEQ ID NO: 70.
  • the C-terminus of SEQ ID NO: 70 is preferably linked to the K at the C-terminus of SEQ ID NO: 69.
  • a preferred pore monomer conjugate of the invention is a pore monomer conjugate comprising a CsgG pore monomer, a partner molecule, and a functional binding moiety, wherein the CsgG pore monomer comprises a sequence which is at least about 40% homologous or identical to the amino acid sequence of SEQ ID NO: 3 over the entire sequence, wherein the partner molecule comprises SEQ ID NO: 21, 23, 25, 27, 29, 31, 63, 64,
  • the K at position 7 of SEQ ID NO: 21, 23, 25, 27, 29 or 31 is covalently attached to the CsgG pore monomer by a sulfonyl group or the K at position 8 of SEQ ID NO: 63, 64, 65, 66, 67, or 68 is covalently attached to the CsgG pore monomer by a sulfonyl group
  • the functional binding moiety comprises an oligonucleotide, polynucleotide, polynucleotide analog or morpholino which is capable of specifically hybridizing to a target polynucleotide analyte, and wherein the functional binding moiety is covalently attached to C-terminal lysine (K) of SEQ ID NO: 21, 23, 25, 27, 29, 31, 63, 64, 65, 66, 67, or 68.
  • the C-terminal K is at position 14 in SEQ ID NO: 21, position 15 in SEQ ID NO: 23, position 16 in SEQ ID NO: 25, position 17 in SEQ ID NO: 27, position 18 in SEQ ID NO: 29, position 19 in SEQ ID NO: 31, position 14 in SEQ ID NO: 63, position 15 in SEQ ID NO: 64, position 16 in SEQ ID NO: 65, position 17 in SEQ ID NO: 66, position 18 in SEQ ID NO: 67 and position 19 in SEQ ID NO:
  • Another preferred pore monomer conjugate of the invention is a pore monomer conjugate comprising a CsgG pore monomer, a partner molecule, and a functional binding moiety, wherein the CsgG pore monomer comprises a sequence which is at least about 40% homologous or identical to the amino acid sequence of SEQ ID NO: 3 over the entire sequence, wherein the partner molecule comprises SEQ ID NO: 21, 23, 25, 27, 29 or 31, wherein the K at position 7 of SEQ ID NO: 21, 23, 25, 27, 29 or 31 is covalently attached to the CsgG pore monomer by a sulfonyl group, wherein the functional binding moiety comprises an oligonucleotide, polynucleotide, polynucleotide analog or morpholino which is capable of specifically hybridizing to a target polynucleotide analyte, and wherein the functional binding moiety is covalently attached to position 14 of SEQ ID NO: 21, 23, 25, 27, 29 or 31.
  • the CsgG pore monomer may have any of the percentages of homology or identity to SEQ ID NO: 3 set out above.
  • the CsgG pore monomer sequence preferably comprises F56Q.
  • the K at the C-terminus of SEQ ID NO: 21, 23, 25, 27, 29 or 31 is preferably modified with an azide group, such as N3.
  • the K at the C-terminus of SEQ ID NO: 21, 23, 25, 27, 29, 31, 63, 64, 65, 66, 67, or 68 is preferably modified with an azide group, such as N3.
  • the partner molecule preferably comprises or consists of SEQ ID NO: 21 or 63.
  • the partner molecule preferably comprises or consists of SEQ ID NO: 21.
  • the partner molecule preferably comprises or consists of SEQ ID NO: 63.
  • the partner molecule preferably comprises or consists of SEQ ID NO: 40, 43, 44, 45, 46, 47, 48, 49 or 50.
  • the partner molecule preferably comprises or consists of SEQ ID NO: 40, 43, 44, 45, 46 or 47.
  • the partner molecule preferably comprises or consists of SEQ ID NO: 40.
  • the partner molecule preferably comprises a -CONH2 group at its C-terminus.
  • the functional binding moiety preferably comprises a morpholino.
  • the invention also provides a construct comprising two or more covalently attached pore monomer conjugates of the invention.
  • the construct may comprise 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more pore monomer conjugates of the invention.
  • the construct may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9 or at least 10 pore monomer conjugates of the invention.
  • the two or more pore monomer conjugates may be the same or different.
  • the two or more pore monomer conjugates may differ based on one or more of
  • the pore monomer conjugates may differ based on (a);
  • the two or more pore monomer conjugates are preferably the same (i.e., identical).
  • the construct preferably comprises two pore monomer conjugates.
  • the two or more pore monomer conjugates may be the same or different.
  • the two or more pore monomer conjugates are preferably the same (i.e., identical).
  • the pore monomer conjugates may be genetically fused, optionally via a linker, or chemically fused, for instance via a chemical crosslinker. Methods for covalently attaching monomers are disclosed in WO 2017/149316, WO 2017/149317, and WO 2017/149318 (incorporated herein by reference in their entirety).
  • the linker is preferably an amino acid sequence and/or a chemical crosslinker.
  • Suitable amino acid linkers such as peptide linkers, are known in the art.
  • the length, flexibility and hydrophilicity of the amino acid or peptide linker are typically designed such that the pore monomer conjugates in the construct are in the correct orientation to form a pore complex.
  • Flexible and rigid linkers that are useful in the constructs are discussed above.
  • Suitable chemical crosslinkers are well-known in the art. Suitable chemical crosslinkers include, but are not limited to, those including the following functional groups: maleimide, active esters, succinimide, azide, alkyne (such as dibenzocyclooctynol (DIBO or DBCO), difluoro cycloalkynes and linear alkynes), phosphine (such as those used in traceless and non-traceless Staudinger ligations), haloacetyl (such as iodoacetamide), phosgene type reagents, sulfonyl chloride reagents, isothiocyanates, acyl halides, hydrazines, disulfides, vinyl sulfones, aziridines and photoreactive reagents (such as aryl azides, diaziridines).
  • alkyne such as dibenzocyclooctynol (DIBO or DBCO), di
  • Reactions between amino acids and functional groups may be spontaneous, such as cysteine/maleimide, or may require external reagents, such as Cu(I) for linking azide and linear alkynes.
  • Linkers can comprise any molecule that stretches across the distance required. Linkers can vary in length from one carbon (phosgene-type linkers) to many Angstroms. Examples of linker molecules, include but are not limited to, are polyethyleneglycols (PEGs), polypeptides, polysaccharides, deoxyribonucleic acid (DNA), peptide nucleic acid (PNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), saturated and unsaturated hydrocarbons, polyamides. These linkers may be inert or reactive, in particular they may be chemically cleavable at a defined position, or may be themselves modified with a fluorophore or ligand. The linker is preferably resistant to reducing agents, such as dithiothreitol (DTT), following the covalent attachment.
  • DTT dithiothreitol
  • Crosslinkers include 2,5-dioxopyrrolidin-l-yl 3-(pyridin-2-yldisulfanyl)propanoate, 2,5- dioxopyrrolidin-l-yl 4-(pyridin-2-yldisulfanyl)butanoate and 2,5-dioxopyrrolidin-l-yl 8- (pyridin-2-yldisulfanyl)octananoate, di-maleimide PEG Ik, di-maleimide PEG 3.4k, di- maleimide PEG 5k, di-maleimide PEG 10k, bis(maleimido)ethane (BMOE), bis- maleimidohexane (BMH), 1,4-bis-maleimidobutane (BMB), 1,4 bis-maleimidyl-2,3- di hydroxybutane (BMDB), BM[PEO]2 (1,8-bis-maleimidodiethyleneglycol),
  • the linker is preferably resistant to dithiothreitol (DTT).
  • Suitable linkers include, but are not limited to, iodoacetamide-based and maleimide-based linkers.
  • the pore monomer conjugates may be connected using two or more linkers each comprising a hybridizable region and a group capable of forming a covalent bond.
  • the hybridizable regions in the linkers hybridize and link the pore monomer conjugates.
  • the linked CsgG pore monomer conjugates are then coupled via the formation of covalent bonds between the groups.
  • Any of the specific linkers disclosed in WO 2010/086602 (incorporated herein by reference in its entirety) may be used in accordance with the invention.
  • the linkers may be labelled. Suitable labels include, but are not limited to, fluorescent molecules (such as Cy3 or AlexaFluor®555), radioisotopes, e.g. 125 I, 35 S, 32 P, enzymes, antibodies, antigens, polynucleotides and ligands such as biotin. Such labels allow the amount of linker to be quantified.
  • the label could also be a cleavable purification tag, such as biotin, or a specific sequence to show up in an identification method, such as a peptide that is not present in the protein itself, but that is released by trypsin digestion.
  • a method of connecting the pore monomer conjugates is via cysteine linkage. This can be mediated by a bi-functional chemical crosslinker or by an amino acid linker with a terminal presented cysteine residue.
  • Another method of attachment via 4-azidophenylalanine or Faz linkage can be mediated by a bi-functional chemical linker or by a polypeptide linker with a terminal presented 4-azidophenylalanine or Faz residue. Additional suitable linkers are discussed in more detail below.
  • pore complex refers to an oligomeric pore complex comprising at least one pore monomer conjugate of the invention (including, e.g., one or more pore monomer conjugates such as two or more pore monomer conjugates, three or more pore monomer conjugates etc.).
  • the pore complex of the invention has the features of a biological pore, i.e., it has a typical protein structure and defines a channel. When the pore complex is provided in an environment having membrane components, membranes, cells, or an insulating layer, the pore complex will insert in the membrane or the insulating layer and form a "transmembrane pore complex".
  • the CsgG part of the pore complex of the invention i.e., the part formed from the at least one CsgG pore monomer in the at least one conjugate of the invention
  • the CsgG constriction in the pore complex of the invention preferably has or comprises any of the constriction diameters described above.
  • the partner molecule functionalises the pore complex by attaching or tethering a functional binding moiety to one or more of the pore monomers in the pore complex.
  • Functionalised pores can be used in any of the applications discussed below with reference to the methods of the invention.
  • the pore complex or transmembrane pore complex of the invention includes a pore complex with two constrictions, i.e., two channel constrictions positioned in such a way that one constriction does not interfere in the accuracy of the other constriction.
  • the pore monomers are CsgG pore monomers
  • the pore complex preferably comprises one or more CsgF peptides.
  • the pore complex preferably comprises a CsgF peptide attached, preferably covalently attached, to each CsgG pore monomer in the pore complex.
  • Said pore complexes may include any of the mutations, CsgG pore monomers or CsgF peptides are described in WO 2016/034591, WO 2017/149316, WO 2017/149317, WO 2019/002893, WO 2017/149318, WO 2018/211241, and WO 2019/002893 (herein all incorporated by reference in their entirety).
  • the pore complex or transmembrane pore complex of the invention includes a pore complex with one constriction.
  • the invention provides a pore complex comprising at least one pore monomer conjugate of the invention.
  • the pore complex typically comprises at least 6, 7, 8, 9 or 10 pore monomer conjugates of the invention.
  • the pore complex preferably comprises 8 or 9 pore monomer conjugates of the invention.
  • the pore monomer conjugates are typically the same (i.e., identical).
  • the pore complex is preferably a homooligomer comprising 6 to 10, such as 6, 7, 8, 9 or 10, pore monomer conjugates of the invention.
  • the pore monomer conjugates are typically identical.
  • the pore complex preferably comprises 8 or 9 identical pore monomer conjugates of the invention.
  • the pore monomer conjugates may be any of those discussed above.
  • the invention provides a pore complex comprising at least one construct of the invention.
  • the pore complex typically comprises at least 1, 2, 3, 4 or 5 constructs of the invention.
  • the pore complex comprises sufficient pore monomers to form a pore.
  • an octameric pore may comprise (a) four constructs each comprising two pore monomer conjugates, (b) two constructs each comprising four pore monomer conjugates, (c) one construct comprising two pore monomer conjugates and six pore monomer conjugates that do not form part of a construct, (d) three constructs comprising two pore monomer conjugates and two pore monomer conjugates that do not form part of a construct, and (e) combinations thereof. Same and additional possibilities are provided for a nonameric pore for instance.
  • constructs of the invention may be used to form a pore complex for characterising, such as sequencing, polynucleotides.
  • the pore complex preferably comprises 4 constructs of the invention each of which comprises two pore monomer conjugates.
  • the constructs are typically the same (i.e., identical).
  • the pore complex is preferably a homooligomer comprising 1-5, such as 1, 2, 3, 4, 5, constructs of the invention.
  • the constructs are typically the same (i.e., identical).
  • the pore complex preferably comprises 4 identical constructs of the invention each of which comprises two pore monomer conjugate.
  • the constructs may be any of those discussed above.
  • the pore monomers in the pore complex are preferably all approximately the same length or are the same length.
  • the barrels of the pore monomers of the invention in the pore are preferably approximately the same length or are the same length. Length may be measured in number of amino acids and/or units of length.
  • the pore complex of the invention may be isolated, substantially isolated, purified or substantially purified.
  • a pore complex of the invention is isolated or purified if it is completely free of any other components, such as lipids or other pores.
  • a pore complex is substantially isolated if it is mixed with carriers or diluents which will not interfere with its intended use.
  • a pore complex is substantially isolated or substantially purified if it is present in a form that comprises less than 10%, less than 5%, less than 2% or less than 1% of other components, such as block copolymers, lipids or other pores.
  • a pore complex of the invention may be present in a membrane. Suitable membranes are discussed below.
  • a pore complex of the invention may be present as an individual or single pore complex.
  • a pore complex of the invention may be present in a homologous or heterologous population of two or more pore complexes or pores.
  • Other formats involving the pore complexes of the invention are discussed in more detail below.
  • the invention also provides a pore multimer comprising two or more pores, wherein at least one of the pores is a pore complex of the invention.
  • the multimer may comprise any number of pores, such as 3, 4, 5, 6, 7 or 8 or more pores. Any number of the pores in the multimer, including all of them, may be a pore complex of the invention.
  • the pore multimer may be a double pore complex comprising a first pore complex of the invention and a second pore or complex.
  • the second pore or complex is typically derived from the same type of pore, such as CsgG.
  • the second pore complex may be a complex of the invention. Both the first pore complex and the second pore complex are preferably pore complexes of the invention.
  • the first pore complex may be attached to the second pore (complex) by hydrophobic interactions and/or by one or more disulfide bonds.
  • One or more, such as 2, 3, 4, 5, 6, 8, 9, for example all, of the monomers in the first pore complex and/or the second pore (complex) may be modified to enhance such interactions. This may be achieved in any suitable way. Particular methods of forming double pores from CsgG-derived pores are described in WO 2019/002893 (incorporated by reference herein in its entirety).
  • the pore multimer of the invention may be isolated, substantially isolated, purified or substantially purified. Such terms are defined above with reference to the pore complexes of the invention.
  • the invention also provides a pore complex of the invention or a pore multimer of the invention which is comprised in a membrane.
  • the invention also provides a membrane comprising a pore complex of the invention or a pore multimer of the invention.
  • proteins may be modified to assist their identification or purification, for example by the addition of a streptavidin tag or by the addition of a signal sequence to promote their secretion from a cell where the monomer does not naturally contain such a sequence.
  • the proteins may also be produced using D- amino acids or a mixture of L-amino acids and D-amino acids. This is conventional in the art for producing such proteins or peptides.
  • the pore monomer, the partner polypeptide or protein, the polypeptide or protein functional binding moiety, the pore monomer conjugate, the construct, the pore complex, or the pore multimer may be chemically modified.
  • the protein can be chemically modified in any way and at any site.
  • the protein may be chemically modified by attachment of a molecule to one or more cysteines (cysteine linkage), attachment of a molecule to one or more lysines, attachment of a molecule to one or more non-natural amino acids, enzyme modification of an epitope or modification of a terminus. Suitable methods for carrying out such modifications are well-known in the art.
  • the protein may be chemically modified by the attachment of any molecule, such as a dye or a fluorophore.
  • the protein may be chemically modified with a molecular adaptor that facilitates the interaction between a pore comprising the monomer and a target nucleotide or target polynucleotide sequence.
  • Suitable adaptors including a cyclic molecule, a cyclodextrin, a species that is capable of hybridization, a DNA binder or interchelator, a peptide or peptide analogue, a synthetic polymer, an aromatic planar molecule, a small positively charged molecule or a small molecule capable of hydrogen-bonding, are described in WO 2019/002893 (incorporated by reference herein in its entirety).
  • the molecular adaptor may be attached using any of the methods and linkers discussed above.
  • the protein may be attached to a polynucleotide binding protein.
  • the polynucleotide binding protein may be attached using the functional binding moiety or through any other method. Polynucleotide binding proteins are discussed above.
  • the protein can be covalently attached to the monomer using any method known in the art.
  • the monomer and protein may be chemically fused or genetically fused. Genetic fusion of a monomer to a polynucleotide binding protein is discussed in WO 2010/004265 (incorporated herein by reference in its entirety).
  • the polynucleotide binding protein may be attached via cysteine linkage using any method described above. This equally applies to polypeptide binding proteins.
  • the polynucleotide binding protein may be attached directly to the protein via one or more linkers.
  • the polynucleotide binding protein or the polypeptide binding protein may be attached directly to the protein via one or more linkers.
  • the molecule may be attached to the pore monomer using the hybridization linkers described in as WO 2010/086602 (incorporated herein by reference in its entirety).
  • peptide linkers may be used. Suitable peptide linkers are discussed above.
  • any of the proteins may be modified to assist their identification or purification, for example by the addition of histidine residues (a his tag), aspartic acid residues (an asp tag), a streptavidin tag, a flag tag, a SUMO tag, a GST tag or a MBP tag, or by the addition of a signal sequence to promote their secretion from a cell where the polypeptide does not naturally contain such a sequence.
  • An alternative to introducing a genetic tag is to chemically react a tag onto a native or engineered position on the protein. An example of this would be to react a gel-shift reagent to a cysteine engineered on the outside of the protein. This has been demonstrated as a method for separating hemolysin heterooligomers (Chem Biol. 1997 Jul;4(7):497-505).
  • any of the proteins may be labelled with a revealing label.
  • the revealing label may be any suitable label which allows the protein to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes, e.g., 1251, 35S, enzymes, antibodies, antigens, polynucleotides, and ligands such as biotin.
  • the protein may also contain other non-specific modifications as long as they do not interfere with the function of the protein.
  • a number of non-specific side chain modifications are known in the art and may be made to the side chains of the protein(s). Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH4, amidation with methylacetimidate or acylation with acetic anhydride.
  • Polynucleotide sequences encoding a protein may be derived and replicated using standard methods in the art. Polynucleotide sequences encoding a protein may be expressed in a bacterial host cell using standard techniques in the art. The protein may be produced in a cell by in situ expression of the polypeptide from a recombinant expression vector. The expression vector optionally carries an inducible promoter to control the expression of the polypeptide. These methods are described in Sambrook, J. and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
  • Proteins may be produced in large scale following purification by any protein liquid chromatography system from protein producing organisms or after recombinant expression.
  • Typical protein liquid chromatography systems include FPLC, AKTA systems, the Bio-Cad system, the Bio-Rad BioLogic system and the Gilson HPLC system.
  • the invention provides methods for producing a pore monomer conjugate of the invention.
  • the method comprises combining or contacting the pore monomer, the partner molecule, and the functional binding moiety under conditions which attach, preferably covalently attach, the functional binding moiety to the pore monomer via the partner molecules. Such conditions are well known to a person skilled in the art and are discussed in the Examples.
  • the method is typically carried out in vitro as defined below.
  • the functional binding moiety is preferably covalently attached to the partner molecule when they are combined with the pore monomer.
  • the method preferably comprises attaching, preferably covalently attaching, the pore monomer to the partner molecule. Any of the linkers and reactive groups discussed above may be used.
  • the method preferably comprises attaching, preferably covalently attaching, the functional binding moiety to the partner molecule and then attaching, preferably covalently attaching, the partner molecule to the pore monomer.
  • the invention provides methods for attaching a functional binding moiety to a pore monomer.
  • the invention provides methods for functionalising a pore monomer.
  • the method comprises using a partner molecule which has affinity for the pore monomer as a linker.
  • the functional binding moiety is attached, preferably covalently attached, to the pore monomer via the partner molecule.
  • the invention also provides methods for producing a pore complex of the invention or a pore multimer of the invention.
  • the method may involve expressing the pore complex in a host cell.
  • the method may comprise expressing at least one pore monomer conjugate of the invention or a construct of the invention and sufficient pore monomers or constructs to form the pore complex or the pore multimer in a host cell and allowing the pore complex or pore multimer to form in the host cell.
  • the sufficient pore monomers or constructs are preferably sufficient pore monomer conjugates of the invention or sufficient constructs of the invention.
  • the numbers of pore monomers, pore monomer conjugates or constructs needed to form the pore complexes of the invention or pore multimers of the invention are discussed above. Suitable host cells and expression systems are known in the art and are discussed in the Examples.
  • the method may involve forming the pore complex in a non-cellular or in vitro context.
  • the method may comprise contacting at least one pore monomer conjugate of the invention or a construct of the invention with sufficient pore monomers or constructs in vitro and allowing the formation of the pore complex or pore multimer.
  • the pore monomer conjugate or the construct may be produced separately by in vitro translation and transcription (IVTT) and then incubated with the sufficient pore monomers or constructs.
  • the sufficient pore monomers or constructs are preferably sufficient pore monomer conjugates of the invention or sufficient constructs of the invention. The numbers of pore monomers, pore monomer conjugates or constructs needed to form the pore complexes of the invention or pore multimers of the invention are discussed above.
  • the method may be conducted in an "in vitro system", which refers to a system comprising at least the necessary components and environment to execute said method, and makes use of biological molecules, organisms, a cell (or part of a cell) outside of their normal naturally occurring environment, permitting a more detailed, more convenient, or more efficient analysis than can be done with whole organisms.
  • An in vitro system may also comprise a suitable buffer composition provided in a test tube, wherein said protein components to form the complex have been added.
  • Some or all of the components of the pore complex or pore multimer may be tagged to facilitate purification. Purification can also be performed when the components are untagged. Methods known in the art (e.g., ion exchange, gel filtration, hydrophobic interaction column chromatography etc.) can be used alone or in different combinations to purify the components of the pore.
  • the pore complex or pore multimer can be made prior to insertion into a membrane or after insertion of the components into a membrane.
  • the invention provides a method of determining the presence, absence or one or more characteristics of a target analyte.
  • the method involves contacting the target analyte with a pore complex of the invention or pore multimer of the invention such that the target analyte moves with respect to, such as into or through, the pore complex or pore multimer and taking one or more measurements as the target analyte moves with respect to the pore complex or pore multimer and thereby determining the presence, absence or one or more characteristics of the target analyte.
  • the target analyte may also be called the template analyte or the analyte of interest.
  • the pore complex of the invention or the pore multimer of the invention may be any of those discussed above.
  • the method is for determining the presence, absence or one or more characteristics of a target analyte.
  • the method may be for determining the presence, absence or one or more characteristics of at least one target analyte.
  • the method may concern determining the presence, absence or one or more characteristics of two or more target analytes.
  • the method may comprise determining the presence, absence or one or more characteristics of any number of target analytes, such as 2, 5, 10, 15, 20, 30, 40, 50, 100 or more analytes. Any number of characteristics of the one or more target analytes may be determined, such as 1, 2, 3, 4, 5, 10 or more characteristics.
  • the degree of reduction in ion flow is related to the size of the obstruction within, or in the vicinity of, the pore. Binding of a molecule of interest, also referred to as an "analyte", in or near the pore therefore provides a detectable and measurable event, thereby forming the basis of a "biological sensor".
  • Suitable molecules for nanopore sensing include nucleic acids; proteins; peptides; polysaccharides and small molecules (refers here to a low molecular weight (e.g., ⁇ 900Da or ⁇ 500Da) organic or inorganic compound) such as pharmaceuticals, toxins, cytokines, and pollutants. Detecting the presence of biological molecules finds application in personalised drug development, medicine, diagnostics, life science research, environmental monitoring and in the security and/or the defence industry.
  • the pore complex or pore multimer may serve as a molecular or biological sensor.
  • the target analyte molecule that is to be detected may bind to either face of the channel, or within the lumen of the channel itself. The position of binding may be determined by the size of the molecule to be sensed.
  • the target analyte preferably comprises or consists of a metal ion, an inorganic salt, a polymer, an amino acid, a peptide, a polypeptide, a protein, a nucleotide, an oligonucleotide, a polynucleotide, a polynucleotide-polypeptide conjugate, a monosaccharide, an oligosaccharide, a polysaccharide, a dye, a bleach, a pharmaceutical, a diagnostic agent, a recreational drug, an explosive, a toxic compound, or an environmental pollutant.
  • the target analyte preferably comprises or consists of a polypeptide, a protein, an oligonucleotide, a polynucleotide, a polynucleotide-polypeptide conjugate, an oligosaccharide, or a polysaccharide.
  • the target analyte may comprise two or more different molecules, such as a peptide and a polypeptide.
  • the target analyte may be a polynucleotide-polypeptide conjugate.
  • the method may concern determining the presence, absence or one or more characteristics of two or more target analytes of the same type, such as two or more proteins, two or more nucleotides or two or more pharmaceuticals. Alternatively, the method may concern determining the presence, absence or one or more characteristics of two or more target analytes of different types, such as one or more proteins, one or more nucleotides and one or more pharmaceuticals.
  • the target analyte can be secreted from cells.
  • the target analyte can be an analyte that is present inside cells such that the target analyte must be extracted from the cells before the method can be carried out.
  • the target analyte may be obtained from or extracted from any organism or microorganism.
  • the target analyte may be 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 analyte may be obtained from a plant e.g., a cereal, legume, fruit, or vegetable.
  • the pore complex or pore multimer may be modified via recombinant or chemical methods to increase the strength of binding, the position of binding, or the specificity of binding of the molecule to be sensed. Typical modifications include addition of a specific binding moiety complimentary to the structure of the molecule to be sensed.
  • this binding moiety may comprise a cyclodextrin or an oligonucleotide; for small molecules this may be a known complimentary binding region, for example the antigen binding portion of an antibody or of a non-antibody molecule, including a single chain variable fragment (scFv) region or an antigen recognition domain from a T- cell receptor (TCR); or for proteins, it may be a known ligand of the target protein.
  • scFv single chain variable fragment
  • TCR T- cell receptor
  • the pore complex or pore multimer may be rendered capable of acting as a molecular sensor for detecting presence in a sample of suitable antigens (including epitopes) that may include cell surface antigens, including receptors, markers of solid tumours or haematologic cancer cells (e.g. lymphoma or leukaemia), viral antigens, bacterial antigens, protozoal antigens, allergens, allergy related molecules, albumin (e.g. human, rodent, or bovine), fluorescent molecules (including fluorescein), blood group antigens, small molecules, drugs, enzymes, catalytic sites of enzymes or enzyme substrates, and transition state analogues of enzyme substrates.
  • suitable antigens including epitopes
  • suitable antigens including epitopes
  • suitable antigens including epitopes
  • suitable antigens including epitopes
  • suitable antigens including epitopes
  • suitable antigens including epitopes
  • suitable antigens including epitopes
  • modifications may be achieved using known genetic engineering and recombinant DNA techniques.
  • the positioning of any adaptation would be dependent on the nature of the molecule to be sensed, for example, the size, three-dimensional structure, and its biochemical nature.
  • the choice of adapted structure may make use of computational structural design. Determination and optimization of protein-protein interactions or protein-small molecule interactions can be investigated using technologies such as a BIAcore® which detects molecular interactions using surface plasmon resonance (BIAcore, Inc., Piscataway, NJ; see also www.biacore.com).
  • the target analyte preferably comprises or consists of an amino acid, a peptide, a polypeptides, or protein.
  • the amino acid, peptide, polypeptide, or protein can be naturally occurring or non-naturally occurring.
  • the polypeptide or protein can include within them synthetic or modified amino acids.
  • Suitable amino acids and modifications thereof are above. It is to be understood that the target analyte can be modified by any method available in the art.
  • the target analyte preferably comprises a polypeptide.
  • Any suitable polypeptide can be characterised.
  • the polypeptide may be an unmodified protein or a portion thereof, or a naturally occurring polypeptide or a portion thereof.
  • the target polypeptide may be secreted from cells.
  • the target polypeptide can be produced inside cells such that it must be extracted from cells for characterisation.
  • the polypeptide may comprise the products of cellular expression of a plasmid, e.g., a plasmid used in cloning of proteins in accordance with the methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 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).
  • a plasmid e.g., a plasmid used in cloning of proteins in accordance with the methods described in Sambrook et al., Molecular Cloning: A Laboratory Manual, 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).
  • the polypeptide can be provided as an impure mixture of one or more polypeptides and one or more impurities.
  • Impurities may comprise truncated forms of the target polypeptide which are distinct from the "target polypeptides" for characterisation.
  • the target polypeptide may be a full-length protein and impurities may comprise fractions of the protein.
  • Impurities may also comprise proteins other than the target protein, e.g., which may be co-purified from a cell culture or obtained from a sample.
  • a polypeptide may comprise any combination of any amino acids, amino acid analogs and modified amino acids (/.e., amino acid derivatives).
  • Amino acids (and derivatives, analogs etc) in the polypeptide can be distinguished by their physical size and charge.
  • the amino acids/derivatives/analogs can be naturally occurring or artificial.
  • the polypeptide may comprise any naturally occurring amino acid.
  • the polypeptide may be modified.
  • the polypeptide may be modified for detection using the method of the invention.
  • the method may be for characterising modifications in the target polypeptide.
  • One or more of the amino acids/derivatives/analogs in the polypeptide may be modified.
  • One or more of the amino acids/derivatives/analogs in the polypeptide may be post- translationally modified.
  • the method of the invention can be used to detect the presence, absence, number of positions of post-translational modifications in a polypeptide.
  • the method can be used to characterise the extent to which a polypeptide has been post- translationally modified. Any one or more post-translational modifications may be present in the polypeptide.
  • Post-translational modifications include modification with a hydrophobic group, modification with a cofactor, addition of a chemical group, glycation (the non-enzymatic attachment of a sugar), biotinylation and pegylation.
  • Post-translational modifications can also be nonnatural, such that they are chemical modifications done in the laboratory for biotechnological or biomedical purposes. This can allow monitoring the levels of the laboratory made peptide, polypeptide, or protein in contrast to the natural counterparts.
  • Examples of post-translational modification with a hydrophobic group include myristoylation, attachment of myristate, a C i4 saturated acid; palmitoylation, attachment of palmitate, a Ci 6 saturated acid; isoprenylation or prenylation, the attachment of an isoprenoid group; farnesylation, the attachment of a farnesol group; geranylgeranylation, the attachment of a geranylgeraniol group; and glypiation, and glycosylphosphatidylinositol (GPI) anchor formation via an amide bond.
  • GPI glycosylphosphatidylinositol
  • post-translational modification with a cofactor examples include lipoylation, attachment of a lipoate (C 8 ) functional group; flavination, attachment of a flavin moiety (e.g. flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD)); attachment of heme C, for instance via a thioether bond with cysteine; phosphopantetheinylation, the attachment of a 4'-phosphopantetheinyl group; and retinylidene Schiff base formation.
  • flavin mononucleotide FMN
  • flavin adenine dinucleotide flavin adenine dinucleotide
  • attachment of heme C for instance via a thioether bond with cysteine
  • phosphopantetheinylation the attachment of a 4'-phosphopantetheinyl group
  • retinylidene Schiff base formation examples include lipoylation, attachment of a lipoate
  • Examples of post-translational modification by addition of a chemical group include acylation, e.g. O-acylation (esters), N-acylation (amides) or S-acylation (thioesters); acetylation, the attachment of an acetyl group for instance to the N-terminus or to lysine; formylation; alkylation, the addition of an alkyl group, such as methyl or ethyl; methylation, the addition of a methyl group for instance to lysine or arginine; amidation; butyrylation; gamma-carboxylation; glycosylation, the enzymatic attachment of a glycosyl group for instance to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine or tryptophan; polysialylation, the attachment of polysialic acid; malonylation; hydroxylation; iodination; bromination; citrulin
  • the polypeptide may be labelled with a molecular label.
  • a molecular label may be a modification to the polypeptide which promotes the detection of the polypeptide in the method of the invention.
  • the label may be a modification to the polypeptide which alters the signal obtained as conjugate is characterised.
  • the label may interfere with a flux of ions through the nanopore. In such a manner, the label may improve the sensitivity of the method.
  • the polypeptide may contain one or more cross-linked sections, e.g., C-C bridges.
  • the polypeptide may not be cross-linked prior to being characterised using the method.
  • the polypeptide may comprise sulphide-containing amino acids and thus has the potential to form disulphide bonds.
  • the polypeptide is reduced using a reagent such as DTT (Dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine) prior to being characterised using the method.
  • DTT Dithiothreitol
  • TCEP tris(2-carboxyethyl)phosphine
  • the polypeptide may be a full-length protein or naturally occurring polypeptide.
  • the protein or naturally occurring polypeptide may be fragmented prior to conjugation to the polynucleotide.
  • the protein or polypeptide may be chemically or enzymatically fragmented.
  • the polypeptides or polypeptide fragments can be conjugated to form a longer target polypeptide.
  • the polypeptide can be any suitable length.
  • the polypeptide preferably has a length of from about 2 to about 300 peptide units or amino acids.
  • the polypeptide has a length of from about 2 to about 100 peptide units, for example from about 2 to about 50 peptide units, e.g., from about 3 to about 50 peptide units, such as from about 5 to about 25 peptide units, e.g., from about 7 to about 16 peptide units, such as from about 9 to about 12 peptide units.
  • “Peptide unit” is interchangeable with "amino acid”.
  • the one or more characteristics of the polypeptide are preferably selected from (i) the length of the polypeptide, (ii) the identity of the polypeptide, (iii) the sequence of the polypeptide, (iv) the secondary structure of the polypeptide and (v) whether or not the polypeptide is modified.
  • the one or more characteristics may be the sequence of the polypeptide or whether or not the polypeptide is modified, e.g., by one or more post- translational modifications.
  • the one or more characteristics are preferably the sequence of the polypeptide.
  • the polypeptide may be in a relaxed form.
  • the polypeptide may be held in a linearized form. Holding the polypeptide in a linearized form can facilitate the characterisation of the polypeptide on a residue-by-residue basis as "bunching up" of the polypeptide within the nanopore is prevented.
  • the polypeptide can be held in a linearized form using any suitable means. For example, if the polypeptide is charged, the polypeptide can be held in a linearized form by applying a voltage. If the polypeptide is not charged or is only weakly charged then the charge can be altered or controlled by adjusting the pH. For example, the polypeptide can be held in a linearized form by using high pH to increase the relative negative charge of the polypeptide.
  • Increasing the negative charge of the polypeptide allows it to be held in a linearized form under, e.g., a positive voltage.
  • the polypeptide can be held in a linearized form by using low pH to increase the relative positive charge of the polypeptide.
  • Increasing the positive charge of the polypeptide allows it to be held in a linearized form under, e.g., a negative voltage.
  • a polynucleotide-handling protein is used to control the movement of a polynucleotide with respect to a nanopore.
  • a polynucleotide As a polynucleotide is typically negatively charged it is generally most suitable to increase the linearization of the polypeptide by increasing the pH thus making the polypeptide more negatively charged, in common with the polynucleotide. In this way, the conjugate retains an overall negative charge and thus can readily move, e.g., under an applied voltage.
  • the polypeptide can be held in a linearized form by using suitable denaturing conditions.
  • suitable denaturing conditions include, for example, the presence of appropriate concentrations of denaturants such as guanidine HCI and/or urea.
  • concentration of such denaturants to use in the disclosed methods is dependent on the target polypeptide to be characterised in the methods and can be readily selected by those of skill in the art.
  • the polypeptide can be held in a linearized form by using suitable detergents.
  • suitable detergents for use in the disclosed methods include SDS (sodium dodecyl sulfate).
  • SDS sodium dodecyl sulfate
  • the polypeptide can be held in a linearized form by carrying out the disclosed methods at an elevated temperature. Increasing the temperature overcomes intra-strand bonding and allows the polypeptide to adopt a linearized form.
  • the polypeptide can be held in a linearized form by carrying out the method under strong electro-osmotic forces.
  • Such forces can be provided by using asymmetric salt conditions and/or providing suitable charge in the channel of the nanopore.
  • the charge in the channel of a pore can be altered, e.g., by mutagenesis. Altering the charge of a pore is well within the capacity of those skilled in the art. Altering the charge of a pore generates strong electro-osmotic forces from the unbalanced flow of cations and anions through the nanopore when a voltage potential is applied across the nanopore.
  • the polypeptide can be held in a linearized form by passing it through a structure such an array of nanopillars, through a nanoslit or across a nanogap.
  • a structure such an array of nanopillars, through a nanoslit or across a nanogap.
  • the physical constraints of such structures can force the polypeptide to adopt a linearized form.
  • the movement of the polypeptide with respect to the pore, such as through the pore, is preferably controlled using a polypeptide binding protein. Suitable proteins are discussed in more detail above.
  • the invention provides a method for determining the presence, absence or one or more characteristics of a target polypeptide, comprising the steps of:
  • the target analyte is preferably a polynucleotide, such as a nucleic acid, which is defined as a macromolecule comprising two or more nucleotides.
  • Nucleic acids are particularly suitable for nanopore sequencing.
  • the naturally occurring nucleic acid bases in DNA and RNA may be distinguished by their physical size.
  • the variation in ion flow may be recorded. Suitable electrical measurement techniques for recording ion flow variations are discussed above. Through suitable calibration, the characteristic reduction in ion flow can be used to identify the particular nucleotide and associated base traversing the channel in realtime.
  • the open-channel ion flow is reduced as the individual nucleotides of the nucleic sequence of interest sequentially pass through the channel of the nanopore due to the partial blockage of the channel by the nucleotide. It is this reduction in ion flow that is measured using the suitable recording techniques described above.
  • the reduction in ion flow may be calibrated to the reduction in measured ion flow for known nucleotides through the channel resulting in a means for determining which nucleotide is passing through the channel, and therefore, when done sequentially, a way of determining the nucleotide sequence of the nucleic acid passing through the nanopore.
  • sequencing may be performed upon an intact nucleic acid polymer that is 'threaded' through the pore via the action of an associated polymerase, for example.
  • sequences may be determined by passage of nucleotide triphosphate bases that have been sequentially removed from a target nucleic acid in proximity to the pore (see for example WO 2014/187924 incorporated herein by reference in its entirety).
  • the polynucleotide or nucleic acid may comprise any combination of any nucleotides.
  • the nucleotides can be any of those discussed above with reference to the pore monomer conjugates of the invention.
  • said method using a polynucleotide as an analyte alternatively comprises determining one or more characteristics selected from (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide and (v) whether or not the polynucleotide is modified.
  • the polynucleotide can be any length (i).
  • the polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400 or at least 500 nucleotides or nucleotide pairs in length.
  • the polynucleotide can be 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. Any number of polynucleotides can be investigated. For instance, the method may concern characterising 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more polynucleotides.
  • polynucleotides may be different polynucleotides or two instances of the same polynucleotide.
  • the polynucleotide can be naturally occurring or artificial.
  • the method may be used to verify the sequence of a manufactured oligonucleotide. The method is typically carried out in vitro.
  • Nucleotides can have any identity (ii). Possible nucleotides are defined above with reference to the pore monomer conjugates of the invention. The sequence of the nucleotides (iii) is determined by the consecutive identity of following nucleotides attached to each other throughout the polynucleotide strain, in the 5' to 3' direction of the strand.
  • the pore complexes and pore multimers of the invention are particularly useful in analysing homopolymers. For example, they may be used to determine the sequence of a polynucleotide comprising two or more, such as at least 3, 4, 5, 6, 7, 8, 9 or 10, consecutive nucleotides that are identical. For example, they may be used to sequence a polynucleotide comprising a polyA, polyT, polyG and/or polyC region.
  • the CsgG pore constriction is made of the residues at the 51, 55 and 56 positions of SEQ ID NO: 3.
  • the constriction of CsgG and its constriction mutants are generally sharp.
  • interactions of approximately 5 bases of DNA with the constriction of the pore at any given time dominate the current signal.
  • these sharper constrictions are very good in reading mixed sequence regions of DNA (when A, T, G and C are mixed)
  • the signal becomes flat and lack information when there is a homopolymeric region within the DNA (eg: polyT, polyG, polyA, polyC). Because 5 bases dominate the signal of the CsgG and its constriction mutants, it's difficult to discriminate photopolymers longer than 5 without using additional dwell time information.
  • the movement of the polynucleotide with respect to the pore, such as through the pore, is preferably controlled using a polynucleotide binding protein. Suitable proteins are discussed in more detail above.
  • the invention provides a method for determining the presence, absence or one or more characteristics of a target polynucleotide, comprising the steps of:
  • the one or more characteristics of the target analyte are preferably measured by electrical measurement and/or optical measurement.
  • the electrical measurement is a current measurement, an impedance measurement, a tunnelling measurement, or a field effect transistor (FET) measurement.
  • FET field effect transistor
  • the method preferably comprises measuring the current flowing through the pore complex or the pore multimer as the target analyte moves with respect to, such as through, the pore.
  • the partner molecule functionalises the pore monomers and pore complexes of the invention by attaching a functional binding protein.
  • the functionalised pore complexes and pore multimers of the invention can be used in a variety of different specific applications of the method of the invention.
  • the functional binding moiety preferably interacts with or binds to the target analyte and facilitates its movement with respect to the pore complex or multimer.
  • “facilitates” is synonymous with “improves”.
  • the invention therefore provides a method of determining the presence, absence or one or more characteristics of a target analyte, comprising the steps of (a) contacting the target analyte with a pore complex of the invention or pore multimer of the invention such that the functional binding moiety interacts with or binds to the target analyte and facilitates its movement with respect to, such as into or through, the pore complex or multimer and (b) taking one or more measurements as the target analyte moves with respect to the pore complex or pore multimer and thereby determining the presence, absence or one or more characteristics of the target analyte.
  • the target analyte is preferably a polynucleotide and the functional binding moiety is preferably comprises an oligonucleotide, polynucleotide, polynucleotide analog or morpholino which is capable of hybridising to the target analyte.
  • the invention therefore provides a method of determining the presence, absence or one or more characteristics of a target polynucleotide, comprising the steps of (a) contacting the target polynucleotide with a pore complex of the invention or pore multimer of the invention wherein the functional binding moiety comprises an oligonucleotide, polynucleotide, polynucleotide analog or morpholino which is capable of hybridising to the target polynucleotide such that the functional binding moiety interacts with or binds to the target polynucleotide and facilitates its movement with respect to, such as into or through, the pore complex or pore multimer and (b) taking one or more measurements as the target polynucleotide moves with respect to the pore complex or pore multimer and thereby determining the presence, absence or one or more characteristics of the target polynucleotide.
  • the functional binding moiety preferably hybridises to the target polynucleotide.
  • the functional binding moiety preferably facilitates capture of the target polynucleotide by the pore complex or pore multimer.
  • the functional binding moiety preferably comprises a morpholino.
  • the target polynucleotide is preferably double stranded.
  • the double stranded polynucleotide typically comprises template and complement strands.
  • the movement of the target polynucleotide with respect to, such as through or into, the pore complex or pore multimer typically separates the two strands.
  • the movement of the template strand with respect to, such as through or into, the pore complex or pore multimer typically separates the two strands.
  • the movement of the target polynucleotide with respect to, such as through or into, the pore complex or pore multimer is preferably controlled by a polynucleotide binding protein, such as a helicase.
  • the polynucleotide binding protein preferably separates the two strands of the double stranded polynucleotide as the double stranded polynucleotide or template strand moves with respect to, such as through or into, the pore complex or pore multimer.
  • the double stranded polynucleotide preferably the complement strand, preferably comprises a portion or region on one of the strands to which the functional binding moiety is capable of hybridising.
  • the double stranded polynucleotide, preferably the complement strand preferably comprises a binding region that is capable of hybridising to the functional binding moiety
  • the separation of the strands of the double stranded polynucleotide preferably reveals the portion or region of one of the strands to which the functional binding moiety is capable of hybridising.
  • the separation of the strands of the double stranded polynucleotide preferably reveals the binding region that is capable of hybridising to the functional binding moiety.
  • the functional binding moiety preferably comprises a portion or region which is substantially complementary or complementary to the portion or region or binding region revealed as the two strands separate.
  • the functional binding protein facilitates or improves the movement of the strand with respect to the pore complex or pore multimer by bringing it into closer proximity to the pore complex or pore multimer or by facilitating its capturing.
  • the functional binding moiety which is tethered to the pore, hybridises to the portion or region or binding region it facilitates or improves the capture of the strand, preferably the complement strand, by the pore complex or pore multimer.
  • An example of improved capture in accordance with the invention is shown in Figures 3a, 3b, 3c and 3d.
  • sequencing adaptors such as the adaptors described in WO 2016/034591 and WO 2018/100370 (both incorporated herein by reference in their entirety), to attach a suitable portion or region to a double stranded polynucleotide.
  • These adaptors also comprise suitable binding sites for polynucleotide binding proteins.
  • the skilled person is also capable of designing a functional binding moiety comprising a portion or region that is capable of hybridising to the revealed portion or region.
  • the method preferably comprises (i) separating the two strands of the double stranded polynucleotide such that the template strand moves with respect to, such as into or through, the pore complex or pore multimer and such that a portion or region on the complement strand to which the functional binding moiety is capable of hybridising is revealed, (ii) allowing the functional binding moiety to hybridise to the portion or region on the complement strand and facilitate the movement of the complement strand with respect to, such as into or through, the pore complex or pore multimer and (iii) taking one or more measurements as the template strand and the complement strand of the double stranded target polynucleotide move with respect to the pore complex or pore multimer and thereby determining the presence, absence or one or more characteristics of the target double stranded polynucleotide.
  • the separating in step (i) is preferably using a polynucleotide binding protein.
  • the movement of the complement strand in step (ii) is also preferably controlled using a polynucleotide binding protein.
  • the method preferably comprises (i) contacting the double stranded polynucleotide with the pore complex or pore multimer such that the two stands are separated as the template strand moves with respect to, such as into or through, the pore complex or pore multimer and such that the binding region on the complement strand is revealed wherein the functional binding moiety hybridises to the binding region in order to facilitate the capture of the complement strand by the pore complex or pore multimer and (iii) taking one or more measurements as the template strand and the complement strand move with respect to the pore complex or pore multimer wherein the measurements of both the template and complement strands are used to determine the presence, absence or one or more characteristics of the target double stranded polynucleotide.
  • the invention therefore provides a method of determining the presence, absence or one or more characteristics of a target double stranded polynucleotide, comprising the steps of (a) contacting the target double stranded polynucleotide with a pore complex of the invention or pore multimer of the invention wherein the functional binding moiety comprises an oligonucleotide, polynucleotide, polynucleotide analog or morpholino which is capable of hybridising to a portion or region on the complement strand of the target double stranded polynucleotide, (b) separating the two strands of the double stranded polynucleotide such that the template strand moves with respect to, such as into or through, the pore complex or pore multimer and such that the portion or region on the complement strand is revealed, (c) allowing the functional binding moiety to hybridise to the portion or region on the complement strand and facilitate the movement of the complement strand with respect to, such as into
  • the separating in step (a) is preferably using a polynucleotide binding protein.
  • the movement of the complement strand in step (b) is also preferably controlled using a polynucleotide binding protein.
  • the functional binding moiety preferably comprises a morpholino.
  • the method preferably comprises before step (a) modifying the target double stranded polynucleotide with a double stranded sequencing adaptor which comprises on one strand a portion or region to which the functional binding moiety is capable of hybridising.
  • the adaptor is typically ligated to the target double stranded polynucleotide.
  • the portion or region is typically on strand of the adaptor which is ligated to the complement strand of the target double stranded polynucleotide.
  • the adaptor preferably further comprises one or more binding sites for polynucleotide binding proteins and/or a leader sequence.
  • the invention also provides a method of determining the presence, absence or one or more characteristics of a target polynucleotide, comprising the steps of (a) contacting a double stranded polynucleotide comprising template and complement strands with a pore complex or pore multimer comprising at least one pore monomer conjugate of the invention, wherein the complement strand comprises a binding region that is capable of hybridising to the functional binding moiety, such that the two strands are separated as the template strand moves through the pore complex or pore multimer to reveal the binding region on the complement strand wherein the functional binding moiety hybridises to the binding region in order to facilitate the capture of the complement strand by said pore complex or pore multimer and (b) taking one or more measurements as the template strand and the complement strand move with respect to the pore complex or pore multimer wherein the measurements of both the template and complement strands are used to determine the presence, absence or one or more characteristics of the target polynucleotide.
  • the method preferably comprises before step (a) modifying a double stranded polynucleotide with a double stranded sequencing adaptor which comprises on one strand a binding region that is capable of hybridising to the functional binding moiety.
  • the adaptor is typically ligated to the target polynucleotide.
  • the portion or region is typically on strand of the adaptor which is ligated to the complement strand of the target polynucleotide.
  • the adaptor preferably further comprises one or more binding sites for polynucleotide binding proteins and/or a leader sequence.
  • the pore complex or pore multimer may be used in any of the embodiments disclosed in WO 2018/100370 (incorporated herein by reference in its entirety).
  • the partner molecule functions as a pore tether in that document.
  • the most preferred pore monomer conjugate is preferably used.
  • the target analyte is preferably a polynucleotide and the functional binding moiety is preferably attached to a polynucleotide binding protein which controls the movement of the target polynucleotide with respect to the pore complex or pore multimer.
  • the invention therefore provides a method of determining the presence, absence or one or more characteristics of a target polynucleotide, comprising the steps of (a) contacting the target polynucleotide with a pore complex of the invention or pore multimer of the invention wherein the functional binding moiety is attached to a polynucleotide binding protein which controls the movement of the target polynucleotide with respect to, such as into or through, the pore complex or multimer and (b) taking one or more measurements as the target polynucleotide moves with respect to the pore complex or pore multimer and thereby determining the presence, absence or one or more characteristics of the target polynucleotide.
  • Polynucleotide binding proteins are discussed in more detail above.
  • the target analyte is preferably a ligand for an enzyme and the functional binding moiety is preferably attached to the enzyme.
  • the invention therefore provides a method of determining the presence, absence or one or more characteristics of a target ligand, comprising the steps of (a) contacting the target ligand with a pore complex of the invention or pore multimer of the invention wherein the functional binding moiety is attached to an enzyme which binds to the target ligand and facilitates its movement with respect to, such as into or through, the pore complex or multimer and (b) taking one or more measurements as the target ligand moves with respect to the pore complex or pore multimer and thereby determining the presence, absence or one or more characteristics of the target ligand.
  • the skilled person is capable of identifying suitable ligand and enzyme pairs for use in the invention.
  • the functional binding moiety is preferably an antibody or a functional fragment thereof or an aptamer which binds to the target analyte.
  • the invention therefore provides a method of determining the presence, absence or one or more characteristics of a target analyte, comprising the steps of (a) contacting the target analyte with a pore complex of the invention or pore multimer of the invention wherein the functional binding moiety is an antibody or a functional fragment thereof or an aptamer which binds to the target analyte and facilitates its movement with respect to, such as into or through, the pore complex or multimer and (b) taking one or more measurements as the target analyte moves with respect to the pore complex or pore multimer and thereby determining the presence, absence or one or more characteristics of the target analyte.
  • Antibodies, functional fragments and aptamers are discussed above.
  • the target analyte is preferably a target polypeptide.
  • the pore complexes and pore multimers of the invention can also be used in a variety of other contexts.
  • the functional binding moiety is preferably attached to a pore, a ringshaped protein or a DNA origami structure to increase the distance between a polynucleotide binding protein and the pore complex or pore multimer or to provide one or more additional constrictions.
  • the invention therefore provides a method of determining the presence, absence or one or more characteristics of a target analyte, comprising the steps of (a) contacting the target analyte with a pore complex of the invention or pore multimer of the invention wherein the functional binding moiety is attached to a pore, a ring-shaped protein or a DNA origami structure to increase the distance between a polynucleotide binding protein and the pore complex or pore multimer, (b) using the polynucleotide binding protein to control the movement of the target analyte with respect to, such as into or through, the pore complex or multimer and (c) taking one or more measurements as the target analyte moves with respect to the pore complex or pore multimer and thereby determining the presence, absence or one or more characteristics of the target analyte.
  • the invention also provides a method of determining the presence, absence or one or more characteristics of a target analyte, comprising the steps of (a) contacting the target analyte with a pore complex of the invention or pore multimer of the invention wherein the functional binding moiety is attached to a pore, a ring-shaped protein or a DNA origami to provide one or more additional constrictions such that target analyte moves with respect to, such as into or through, the pore complex or multimer and (b) taking one or more measurements as the target analyte moves with respect to the pore complex or pore multimer and thereby determining the presence, absence or one or more characteristics of the target analyte.
  • the functional binding moiety preferably binds to the pore complex or pore multimer to stabilise cis loops and reduce the signal-to-noise ratio of the pore complex or pore multimer.
  • the invention therefore provides a method of determining the presence, absence or one or more characteristics of a target analyte, comprising the steps of (a) contacting the target analyte with a pore complex of the invention or pore multimer of the invention wherein the functional binding moiety binds to the pore complex or pore multimer to stabilise cis loops such that target analyte moves with respect to, such as into or through, the pore complex or multimer and (b) taking one or more measurements as the target analyte moves with respect to the pore complex or pore multimer and thereby determining the presence, absence or one or more characteristics of the target analyte.
  • the functional binding moiety preferably attaches the pore complex or pore multimer to a second pore or a second pore multimer.
  • An example of this is shown in Figure 4.
  • the CsgA multimers of the invention may be used to link two or more pores or two or more pore multimers.
  • the invention therefore provides a method of determining the presence, absence or one or more characteristics of a target analyte, comprising the steps of (a) contacting the target analyte with a pore complex of the invention or pore multimer of the invention wherein the functional binding moiety attaches the pore complex or pore multimer to a second pore or a second pore multimer such that target analyte moves with respect to, such as into or through, the pore complex, pore multimer, second pore or second multimer and (b) taking one or more measurements as the target analyte moves with respect to the pore complex, pore multimer, second pore or second multimer and thereby determining the presence, absence or one or more characteristics of the target analyte.
  • the pore and second pore and/or the pore multimer and the second multimer may be the same or different.
  • the second pore or the second pore multimer may be a pore complex or a pore multimer of the invention.
  • the second pore may be a pore complex of the invention.
  • the functional binding moiety- functional binding moiety is preferably a peptide link (or genetic fusion) between the two partner molecules, especially if they are partner polypeptides.
  • the two partner polypeptides may be linked or fused using a linker, such has a polypeptide linker.
  • the method comprises attaching two CsgG pores
  • the C-terminus of one CsgA polypeptide is preferably attached to the R group of the K at the C-terminus of another CsgA polypeptide.
  • This provides a linked construct with one CsgA polypeptide N to C linked to another C to N.
  • the N-terminus of each CsgA polypeptide is then available to attach to a CsgG pore thereby linking two CsgA pores.
  • the functional binding moiety-functional binding moiety is a peptide link (or genetic fusion) between the two CsgA polypeptides.
  • the CsgA polypeptides may be any of those discussed above.
  • the CsgA polypeptides may be the same or different.
  • the second pore multimer could be a pore multimer of the invention.
  • the movement of the target polypeptide with respect to, such as through or into, the pore complex or pore multimer is preferably controlled by a polypeptide binding protein.
  • a polypeptide binding protein Such proteins are discussed above.
  • the polypeptide binding protein preferably unfolds the target polypeptide as it moves with respect to, such as through or into, the pore complex or pore multimer. Unfoldases are also discussed above.
  • the target analyte is preferably a polypeptide and the functional binding moiety is preferably attached to a polypeptide binding protein which controls the movement of the target polypeptide with respect to the pore complex or pore multimer.
  • the invention therefore provides a method of determining the presence, absence or one or more characteristics of a target polypeptide, comprising the steps of (a) contacting the target polypeptide with a pore complex of the invention or pore multimer of the invention wherein the functional binding moiety is attached to a polypeptide binding protein which controls the movement of the target polypeptide with respect to, such as into or through, the pore complex or multimer and (b) taking one or more measurements as the target polypeptide moves with respect to the pore complex or pore multimer and thereby determining the presence, absence or one or more characteristics of the target polypeptide.
  • Polypeptide binding proteins are discussed in more detail above.
  • the method preferably comprises before step (a) modifying the target polypeptide with a sequencing adaptor which comprises a portion or region to which the functional binding moiety is capable of hybridising.
  • the sequencing adaptor preferably further comprises one or more binding sites for polypeptide binding proteins and/or a leader sequence.
  • the invention also provides a method of determining the presence, absence or one or more characteristics of a target polypeptide, comprising the steps of (a) modifying the target polypeptide with a sequencing adaptor which comprises a portion or region to which the functional binding moiety is capable of hybridising, (b) contacting the modified target polypeptide with a pore complex or pore multimer comprising at least one pore monomer conjugate of the invention, wherein the functional binding moiety hybridises to the sequencing adaptor in order to facilitate the capture of the target polypeptide by said pore complex or pore multimer and (c) taking one or more measurements as the target polypeptide moves with respect to the pore complex or pore multimer wherein the measurements are used to determine the presence, absence or one or more characteristics of the target polypeptide.
  • the functional binding moiety is preferably an antibody or a functional fragment thereof or an aptamer which binds to the target polypeptide.
  • the invention therefore provides a method of determining the presence, absence or one or more characteristics of a target polypeptide, comprising the steps of (a) contacting the target polypeptide with a pore complex of the invention or pore multimer of the invention wherein the functional binding moiety is an antibody or a functional fragment thereof or an aptamer which binds to the target polypeptide and facilitates its movement with respect to, such as into or through, the pore complex or multimer and (b) taking one or more measurements as the target polypeptide moves with respect to the pore complex or pore multimer and thereby determining the presence, absence or one or more characteristics of the target polypeptide.
  • Antibodies, functional fragments, and aptamers are discussed above.
  • the movement of the target polypeptide with respect to, such as through or into, the pore complex or pore multimer is preferably controlled by a polypeptide binding protein.
  • the invention provides a method of determining the presence, absence or one or more characteristics of a target double stranded polynucleotide, comprising the steps of (a) contacting the target double stranded polynucleotide with a pore complex or pore multimer, (b) separating the two strands of the double stranded polynucleotide such that the template strand moves with respect to, such as into or through, the pore complex or pore multimer and such that a portion or region on the complement strand is revealed, (c) allowing the functional binding moiety to hybridise to the portion or region on the complement strand and facilitate the movement of the complement strand with respect to, such as into or through, the pore complex or pore multimer and (d) taking one or more measurements as the template strand and the complement strand of the double stranded target polynucleotide move with respect to the pore complex or pore multimer and thereby determining the presence, absence or one or more characteristics of the target double
  • the CsgG pore monomer sequence preferably comprises F56Q.
  • the K at the C-terminus of SEQ ID NO: 21, 23, 25, 27, 29 or 31 is preferably modified with an azide group, such as N3.
  • the partner molecule preferably comprises or consists of SEQ ID NO: 21.
  • the partner molecule preferably comprises or consists of SEQ ID NO: 40, 43, 44, 45, 46 or 47.
  • the partner molecule preferably comprises or consists of SEQ ID NO: 40.
  • the functional binding moiety preferably comprises a morpholino.
  • the pore monomer conjugate comprises a CsgG pore monomer, a partner molecule, and a functional binding moiety
  • the CsgG pore monomer comprises a sequence which is at least about 40% homologous or identical to the amino acid sequence of SEQ ID NO: 3 over the entire sequence
  • the partner molecule comprises SEQ ID NO: 21, 23, 25, 27, 29, 31, 63, 64, 65, 66, 67, or 68, wherein the K at position 7 of SEQ ID NO: 21, 23, 25, 27, 29 or 31 is covalently attached to the CsgG pore monomer by a sulfonyl group or the K at position 8 of SEQ ID NO: 63, 64, 65, 66, 67, or 68 is covalently attached to the CsgG pore monomer by a sulfonyl group
  • the functional binding moiety comprises an oligonucleotide, polynucleotide, polynucleotide analog or morph
  • the CsgG pore monomer may have any of the percentages of homology or identity to SEQ ID NO: 3 set out above.
  • the CsgG pore monomer sequence preferably comprises F56Q.
  • the K at the C-terminus of SEQ ID NO: 21, 23, 25, 27, 29, 31, 63, 64, 65, 66, 67, or 68 is preferably modified with an azide group, such as N3.
  • the partner molecule preferably comprises or consists of SEQ ID NO: 21 or 63.
  • the partner molecule preferably comprises or consists of SEQ ID NO: 40, 43, 44, 45, 46, 47, 48, 49 or 50.
  • the partner molecule preferably comprises or consists of SEQ ID NO: 40.
  • the partner molecule preferably comprises a -CONH2 group at its C-terminus.
  • the functional binding moiety preferably comprises a morpholino.
  • the invention provides a method of determining the presence, absence or one or more characteristics of a target polynucleotide, comprising the steps of (a) contacting a double stranded polynucleotide comprising template and complement strands with a pore complex or pore multimer, wherein the complement strand comprises a binding region that is capable of hybridising to the functional binding moiety, such that the two strands are separated as the template strand moves through the pore complex or pore multimer to reveal the binding region on the complement strand wherein the functional binding moiety hybridises to the binding region in order to facilitate the capture of the complement strand by said pore complex or pore multimer and (b) taking one or more measurements as the template strand and the complement strand move with respect to the pore complex or pore multimer wherein the measurements of both the template and complement strands are used to determine the presence, absence or one or more characteristics of the target polynucleotide, wherein the pore complex or pore multimer comprises at least one pore
  • the CsgG pore monomer sequence preferably comprises F56Q.
  • the K at the C-terminus of SEQ ID NO: 21, 23, 25, 27, 29 or 31 is preferably modified with an azide group, such as N3.
  • the partner molecule preferably comprises or consists of SEQ ID NO: 21.
  • the partner molecule preferably comprises or consists of SEQ ID NO: 40, 43, 44, 45, 46 or 47.
  • the partner molecule preferably comprises or consists of SEQ ID NO: 40.
  • the functional binding moiety preferably comprises a morpholino.
  • the pore monomer conjugate comprises a CsgG pore monomer, a partner molecule, and a functional binding moiety
  • the CsgG pore monomer comprises a sequence which is at least about 40% homologous or identical to the amino acid sequence of SEQ ID NO: 3 over the entire sequence
  • the partner molecule comprises SEQ ID NO: 21, 23, 25, 27, 29, 31, 63, 64, 65, 66, 67, or 68, wherein the K at position 7 of SEQ ID NO: 21, 23, 25, 27, 29 or 31 is covalently attached to the CsgG pore monomer by a sulfonyl group or the K at position 8 of SEQ ID NO: 63, 64, 65, 66, 67, or 68 is covalently attached to the CsgG pore monomer by a sulfonyl group
  • the functional binding moiety comprises an oligonucleotide, polynucleotide, polynucleotide analog or morph
  • the CsgG pore monomer may have any of the percentages of homology or identity to SEQ ID NO: 3 set out above.
  • the CsgG pore monomer sequence preferably comprises F56Q.
  • the K at the C-terminus of SEQ ID NO: 21, 23, 25, 27, 29, 31, 63, 64, 65, 66, 67, or 68 is preferably modified with an azide group, such as N3.
  • the partner molecule preferably comprises or consists of SEQ ID NO: 21 or 63.
  • the partner molecule preferably comprises or consists of SEQ ID NO: 40, 43, 44, 45, 46, 47, 48, 49 or 50.
  • the partner molecule preferably comprises or consists of SEQ ID NO: 40.
  • the partner molecule preferably comprises a -CONH2 group at its C-terminus.
  • the functional binding moiety preferably comprises a morpholino.
  • a pore complex comprises at least one pore monomer conjugate as defined.
  • the pore complex is preferably a homooligomer comprising 6 to 10, such as 6, 7, 8, 9 or 10, of the pore monomer conjugates.
  • the pore monomer conjugates are typically identical.
  • the pore complex preferably comprises 8 or 9 identical pore monomer conjugates.
  • the CsgG pore monomer may have any of the percentages of homology or identity to SEQ ID NO: 3 set out above.
  • the method preferably uses a polynucleotide binding protein to control the movement of one or both strands of the double stranded polynucleotide.
  • the movement of the template and/or complement strands is preferably controlled using a polynucleotide binding protein.
  • the method preferably comprises before step (a) modifying the target double stranded polynucleotide with a double stranded sequencing adaptor which comprises on one strand a portion or region to which the oligonucleotide, polynucleotide, polynucleotide analog or morpholino in the functional binding moiety is capable of hybridising.
  • the method preferably comprises before step (a) modifying a double stranded polynucleotide with a double stranded sequencing adaptor which comprises on one strand a binding region that is capable of hybridising to the functional binding moiety.
  • the adaptor is typically ligated to the target polynucleotide.
  • the portion or region or binding region is typically on strand of the adaptor which is ligated to the complement strand of the target polynucleotide.
  • the adaptor preferably further comprises one or more binding sites for polynucleotide binding proteins and/or a leader sequence.
  • the invention also provides a polypeptide consisting of the sequence of SEQ ID NO: 7, 9, 11, 13, 15 or 17.
  • the invention also provides a polypeptide comprising or consisting of SEQ ID NO: 8, 10, 12, 14, 16, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47.
  • the invention also provides a polypeptide comprising or consisting of SEQ ID NO: 8, 10, 12,
  • the invention also provides a polypeptide comprising or consisting of a variant of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47. Over the entire length of the amino acid sequence of the CsgA fragment or SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14,
  • a variant will preferably be at least about 40% homologous to that sequence based on amino acid identity.
  • the variant may be at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of the CsgA fragment or SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27,
  • a variant will preferably be at least about 40% identical to that sequence. More preferably, the variant may be at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% identical to the CsgA fragment or SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 or 47 over the entire sequence.
  • the invention also provides a polypeptide comprising or consisting of a variant of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 57, 58, 59,
  • a variant will preferably be at least about 40% homologous to that sequence based on amino acid identity. More preferably, the variant may be at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% homologous based on amino acid identity to the amino acid sequence of the CsgA fragment or SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14,
  • a variant will preferably be at least about 40% identical to that sequence.
  • the variant may be at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90% and more preferably at least about 95%, 97% or 99% identical to the CsgA fragment or SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 over the entire sequence.
  • the amino acid at any of positions 1 to 7 of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31, such as the G at position 1, the V at position 2, the V at position 3, the P at position 4, the Q at position 5, the Y at position 6, or the G or K at position 7, is preferably modified to include a reactive group.
  • the amino acid at position 6 or 7 of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31, such as the Y at position 6 or the G or K at position 7, is preferably modified to include a reactive group.
  • the reactive group may be any of those discussed above.
  • the reactive group is preferably a sulfonyl fluoride.
  • the K at the C- terminus of SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 is preferably modified with an azide group, such as N3.
  • the reactive group may be any of those discussed above.
  • the reactive group is preferably a sulfonyl fluoride.
  • the K at the C-terminus of SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 is preferably modified with an azide group, such as N3.
  • the G or K at position 7 of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 is preferably modified to include a reactive group.
  • the G or K at position 8 of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 is preferably modified to include a reactive group.
  • the reactive group may be any of those discussed above.
  • the reactive group is preferably a sulfonyl fluoride.
  • the K at the C-terminus of SEQ ID NO: 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 31 is preferably modified with an azide group, such as N3.
  • the K at the C-terminus of SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 or 70 is preferably modified with an azide group, such as N3.
  • the G at position 1 may be substituted or deleted.
  • the G at position 1 may be substituted or deleted.
  • Any of the CsgA polypeptides of the invention may comprise a -CONH2 group at its C- terminus.
  • the invention also provides a CsgA multimer comprising two or more linked CsgA polypeptides.
  • the two or more linked CsgA polypeptides may be selected from any of those discussed above, including from SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 and 70 and variants thereof.
  • the CsgA multimer may comprise any number of two or more linked CsgA polypeptides, such as 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more or 10 or more CsgA polypeptides.
  • the two or more CsgA polypeptides may be the same CsgA polypeptide.
  • the two or more CsgA polypeptides may be different CsgA polypeptides.
  • the two or more CsgA polypeptides may be linked in any manner.
  • the two or more CsgA polypeptides are preferably covalently linked.
  • the two or more CsgA polypeptides are preferably covalently linked via the K at the C-terminus of one or more of the CsgA polypeptides.
  • the two or more CsgA polypeptides may be covalently linked directly or via any of the linkers disclosed herein.
  • the skilled person is capable of linking two or more CsgA polypeptides, for instance using peptide bonds, via the R groups of any of the positions in the CsgA polypeptides or using the reactive groups discussed above.
  • the two or more CsgA polypeptides may be linked in any orientation.
  • the two or more CsgA polypeptides may be linked N-terminus to C-terminus or C-terminus to N-terminus.
  • the two or more CsgA polypeptides may be linked N-terminus to N-terminus.
  • the two or more CsgA polypeptides may be linked C-terminus to C-terminus.
  • One or more CsgA polypeptides may branch off one or more positions within the sequence of one or more CsgA polypeptides.
  • the multimer preferably comprises two CsgA polypeptides in which the C-terminus of one CsgA polypeptide is linked to the K at the C-terminus of the other CsgA polypeptide. This provides a linked multimer with one CsgA polypeptide N to C linked to another C to N. The N-terminus of each CsgA polypeptide is then available to attach to a pore monomer, such as a CsgG pore monomer.
  • the two CsgA polypeptides may be selected from SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69 and 70 and variants thereof.
  • the two CsgA polypeptides are preferably SEQ ID NO: 69 and SEQ ID NO: 70.
  • the C-terminus of SEQ ID NO: 70 is preferably linked to the K at the C-terminus of SEQ ID NO: 69.
  • the invention also provides a polynucleotide which encodes any of these polypeptides.
  • the polynucleotide may be any of those discussed above.
  • the invention also provides an expression vector comprising a polynucleotide of the invention.
  • the invention also provides a host cell comprising a polynucleotide of the invention or a host cell of the invention. Suitable vectors and host cells are known in the art.
  • kits for characterising a target analyte or for producing pore monomer conjugates of the invention comprises (a) a pore monomer and (b) a partner molecule which has affinity for the pore monomer and which is attached to a functional binding moiety. Any of the embodiments discussed above with reference to the pore monomer conjugates of the invention equally apply here.
  • the invention also provides kits for characterising a target analyte.
  • the kit comprises (a) a pore complex of the invention or a pore multimer of the invention and (b) the components of a membrane. Suitable membranes and components are discussed below.
  • the invention also provides kits for characterising a target polynucleotide.
  • the kit comprises (a) a pore complex of the invention or a pore multimer of the invention and (b) a polynucleotide binding protein.
  • the invention also provides kits for characterising a target polynucleotide or a target polypeptide.
  • the kit comprises (a) a pore complex of the invention or a pore multimer of the invention and (b) a polynucleotide binding protein or a polypeptide binding protein.
  • the kit preferably further comprises the components of a membrane.
  • the kit may comprise components of any type of membranes, such as an amphiphilic layer, such as a triblock copolymer membrane.
  • the kit may further comprise one or more anchors, such as cholesterol, for coupling the target analyte to the membrane.
  • the kit may further comprise one or more polynucleotide adaptors that can be attached to a target polynucleotide to facilitate characterisation of the polynucleotide.
  • the anchor such as cholesterol, is preferably attached to the polynucleotide adaptor.
  • the kit may additionally comprise one or more other reagents or instruments which enable any of the embodiments mentioned above to be carried out.
  • reagents or instruments include one or more of the following: suitable buffer(s) (aqueous solutions), means to obtain a sample from a subject (such as a vessel or an instrument comprising a needle), means to amplify and/or express polynucleotides or voltage or patch clamp apparatus.
  • Reagents may be present in the kit in a dry state such that a fluid sample resuspends the reagents.
  • the kit may also, optionally, comprise instructions to enable the kit to be used in the method of the invention or details regarding for which organism the method may be used.
  • the kit may also comprise additional components useful in analyte characterization.
  • the invention also provides an apparatus for characterising target analytes in a sample, comprising (a) a plurality of pore complexes of the invention or a plurality of pore multimers of the invention and (b) a plurality of polynucleotide binding proteins.
  • the invention also provides an apparatus for characterising target analytes in a sample, comprising (a) a plurality of pore complexes of the invention or a plurality of pore multimers of the invention and (b) a plurality of polynucleotide binding proteins or a plurality of polypeptide binding proteins.
  • the invention also provides an apparatus for characterising a target polynucleotide or a target polypeptide in a sample, comprising (a) a plurality of pore complexes of the invention or a plurality of pore multimers of the invention and (b) a plurality of polynucleotide binding proteins or a plurality of polypeptide binding proteins.
  • the plurality of pore complexes or plurality of pore multimers may be any of those discussed above.
  • the invention also provides an apparatus comprising a pore complex of the invention or a pore multimer of the invention inserted into an in vitro membrane.
  • the invention also provides an apparatus produced by a method comprising: (i) obtaining a pore complex of the invention or a pore multimer of the invention and (ii) contacting the pore complex or pore multimer with an in vitro membrane such that the pore complex or pore multimer is inserted in the in vitro membrane.
  • the invention also provides an array comprising a plurality of membranes of the invention. Any of the embodiments discussed above with respect to the membranes of the invention equally apply the array of the invention.
  • the array may be set up to perform any of the methods described below.
  • each membrane in the array comprises one pore complex or pore multimer. Due to the manner in which the array is formed, for example, the array may comprise one or more membranes that do not comprise a pore complex or pore multimer, and/or one or more membranes that comprise two or more pores complexes or multimers.
  • the array may comprise from about 2 to about 1000, such as from about 10 to about 800, from about 20 to about 600 or from about 30 to about 500 membranes.
  • the invention provides a system comprising (a) a membrane of the invention or an array of the invention, (b) means for applying a potential across the membrane(s) and (c) means for detecting electrical or optical signals across the membrane(s).
  • the electrical signal may be a measurement of ion flow through the nanopore such as the measurement of a current or voltage over time.
  • the pores and membranes may be any as described above and below.
  • the system further comprises a first chamber and a second chamber, wherein the first and second chambers are separated by the membrane(s).
  • the system may further comprise a target analyte, wherein the target analyte is transiently located within the continuous channel and wherein one end of the target analyte is located in the first chamber and one end of the target analyte is located in the second chamber.
  • the target analyte is preferably a target polypeptide or a target polynucleotide.
  • the system further comprises an electrically conductive solution in contact with the pore(s), electrodes providing a voltage potential across the membrane(s), and a measurement system for measuring the current through the pore(s).
  • the voltage applied across the membranes and pore is preferably from +5 V to -5 V, such as -600 mV to +600mV or -400 mV to +400 mV.
  • the voltage used is preferably in the range 100 mV to 240 mV and more preferably in the range of 120 mV to 220 mV. It is possible to increase discrimination between different amino acids or nucleotides by a pore by using an increased applied potential. Any suitable electrically conductive solution may be used.
  • the solution may comprise charge carriers, such as metal salts, for example alkali metal salt, halide salts, for example chloride salts, such as alkali metal chloride salt.
  • Charge carriers may include ionic liquids or organic salts, for example tetramethyl ammonium chloride, trimethylphenyl ammonium chloride, phenyltrimethyl ammonium chloride, or 1- ethyl-3-methyl imidazolium chloride.
  • salt is present in the aqueous solution in the chamber. Potassium chloride (KCI), sodium chloride (NaCI), caesium chloride (CsCI) or a mixture of potassium ferrocyanide and potassium ferricyanide is typically used.
  • KCI, NaCI and a mixture of potassium ferrocyanide and potassium ferricyanide are preferred.
  • the charge carriers may be asymmetric across the membrane. For instance, the type and/or concentration of the charge carriers may be different on each side of the membrane, e.g., in each chamber.
  • the salt concentration may be at saturation.
  • the salt concentration may be 3 M or lower and is typically from 0.1 to 2.5 M, from 0.3 to 1.9 M, from 0.5 to 1.8 M, from 0.7 to 1.7 M, from 0.9 to 1.6 M or from 1 M to 1.4 M.
  • the salt concentration is preferably from 150 mM to 1 M.
  • the method is preferably carried out using a salt concentration of at least 0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M or at least 3.0 M.
  • High salt concentrations provide a high signal to noise ratio and allow for currents indicative of the presence of an amino acid or nucleotide to be identified against the background of normal current fluctuations.
  • a buffer may be present in the electrically conductive solution.
  • the buffer is phosphate buffer.
  • Other suitable buffers are HEPES and Tris-HCI buffer.
  • the pH of the electrically conductive solution may be from 4.0 to 12.0, from 4.5 to 10.0, from 5.0 to 9.0, from 5.5 to 8.8, from 6.0 to 8.7 or from 7.0 to 8.8 or 7.5 to 8.5.
  • the pH used is preferably about 7.5.
  • the system may be comprised in an apparatus.
  • the apparatus may be any conventional apparatus for analyte analysis, such as an array or a chip.
  • the apparatus is preferably set up to carry out the disclosed method.
  • the apparatus may comprise a chamber comprising an aqueous solution and a barrier that separates the chamber into two sections.
  • the barrier typically has an aperture in which the membrane(s) containing the pore(s) are formed.
  • the barrier forms the membrane in which the pore is present.
  • the apparatus may also comprise an electrical circuit capable of applying a potential and measuring an electrical signal across the membrane and pore.
  • the apparatus may be any of those described in WO 2008/102120, WO 2009/077734, WO 2010/122293, WO 2011/067559, WO 2014/06442, or WO2020/183172 (all incorporated herein by reference in their entirety).
  • the method for determining the presence, absence or one or more characteristics of a target polymer analyte may comprise estimating or determining the sequence of polymer units.
  • the signal measured during movement of the polymer, such as a polynucleotide, with respect to the nanopore may be dependent at any one time upon multiple polymer units such as nucleotides.
  • the presence of multiple nucleotides in the lumen of the nanopore and potentially nucleotides outside of the nanopore can influence the ion flow and therefore current or voltage signal.
  • the polynucleotide may also contain modified bases which can affect the measurement signal and as such the estimation or determination of the sequence may be non-trivial.
  • the method according to an aspect of the invention may comprise the measurement of the template and complement strands of a target polynucleotide or a polynucleotide-polypeptide conjugate wherein measurements of both strands can be used to estimate or determine an overall sequence.
  • Various known methods may be used.
  • the sequence of template and complete strands may be initially determined from the series of measurements taken during the movement of the polynucleotides with respect to the nanopore and the results combined to provide an overall sequence. More preferably the series of measurements of both the template and complement may be treated by a probabilistic or machine learning technique as plural series of measurements in plural dimensions wherein an overall sequence determination is made without the initial determination of the sequences of the template and complement strands.
  • a non-limiting example of a method suitable for use in the invention is disclosed in WO2015140535 (incorporated by reference in its entirety).
  • the membrane is preferably an amphiphilic layer.
  • An amphiphilic layer is a layer formed from amphiphilic molecules, such as phospholipids, which have both hydrophilic and lipophilic properties.
  • the amphiphilic molecules may be synthetic or naturally occurring.
  • Non-naturally occurring amphiphiles and amphiphiles which form a monolayer are known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450).
  • Block copolymers are polymeric materials in which two or more monomer sub-units that are polymerized together to create a single polymer chain.
  • Block copolymers typically have properties that are contributed by each monomer sub-unit. However, a block copolymer may have unique properties that polymers formed from the individual sub-units do not possess. Block copolymers can be engineered such that one of the monomer sub-units is hydrophobic (i.e., lipophilic), whilst the other sub-unit(s) are hydrophilic whilst in aqueous media. In this case, the block copolymer may possess amphiphilic properties and may form a structure that mimics a biological membrane. The block copolymer may be a diblock (consisting of two monomer sub-units) but may also be constructed from more than two monomer sub-units to form more complex arrangements that behave as amphiphiles. The amphiphilic layer may comprise a diblock, triblock, tetrablock or pentablock copolymer.
  • the membrane may comprise one of the membranes disclosed in International Application No. WO 2014/064443 or WO 2014/064444 (both incorporated herein by reference in their entirety).
  • 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.
  • lipid bilayers can be used for in vitro investigation of membrane proteins by single-channel recording.
  • 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 (all incorporated herein by reference in their entirety).
  • the membrane may comprise a solid-state layer.
  • Solid state layers can be formed from both organic and inorganic materials including, but not limited to, microelectronic materials, insulating materials such as 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 (incorporated herein by reference in its entirety).
  • the pore is typically present in an amphiphilic membrane or layer contained within the solid-state layer, for instance within a hole, well, gap, channel, trench or slit within the solid-state layer.
  • the skilled person can prepare suitable solid state/amphiphilic hybrid systems. Suitable systems are disclosed in WO 2009/020682 and WO 2012/005857 (both incorporated herein by reference in their entirety). Any of the amphiphilic membranes or layers discussed above may be used.
  • the method is typically carried out using (i) an artificial amphiphilic layer comprising a pore, (ii) an isolated, naturally occurring lipid bilayer comprising a pore, or (iii) a cell having a pore inserted therein.
  • the method is typically carried out using an artificial amphiphilic layer, such as a di- or tri-block copolymer layer.
  • the layer may comprise other transmembrane and/or intramembrane proteins as well as other molecules in addition to the pore. Suitable apparatus and conditions are discussed below.
  • the method of the invention is typically carried out in vitro.
  • SEQ ID NO: 2 (>P0AEA2 (1 :277); WT Pro-CsgG from E. coli K12)
  • SEQ ID Nos: 32-42 are shown in Table 1 below.
  • SEQ ID Nos: 33-47 are shown in Table 3 below.
  • SEQ ID Nos: 48-50 are shown in Table 5 below.
  • SEQ ID Nos: 51-52 are shown in Example 2 below.
  • SEQ ID NOs: 57-70 are shown above in the description of the sequence listing.
  • Recombinant expression vectors encoding the CsgG variant nanopores with a C-terminal Strep affinity tag and ampicillin resistance gene were transformed into chemically competent E. coli cells.
  • the cells were plated onto an LB Agar plate containing appropriate antibiotics for selection. A single colony from the agar plate was inoculated in LB Media with antibiotics and grown overnight.
  • the culture was diluted into autoinduction media plus necessary antibiotics and incubated at 18°C for 68 hours.
  • the cells were harvested through centrifugation before being lysed and extracted into lx Bugbuster extraction reagent (Merck 70921) and 0.1% DDM.
  • the pore was purified from the supernatant using affinity chromatography, heat treatment and then size exclusion chromatography, selecting for oligomeric nanopores as judged by SDS-PAGE.
  • CsgG-CsgF complexes were prepared from nanopores purified as above and chemically synthesised CsgF peptides. Nanopores were buffer exchanged into a pH 7.0 buffer and incubated in a 8x molar excess of peptide to CsgG monomer for Jackpot at 25°C. Reactions were stopped with heating at 60°C for 15 mins followed by centrifugation to remove any precipitate.
  • CsgG-CsgA complexes were prepared from nanopores purified as above and chemically synthesised CsgA peptides with or without a sulphonyl fluoride modification. Nanopores were buffer exchanged into a pH 7.4 buffer free of reducing agents and incubated with 250x molar equivalents of peptide to CsgG monomer. For the addition of the morpholino strand to the CsgG-CsgA complexes, the complex was buffer exchanged to remove any unbound CsgA peptide and then 20x molar equivalents of morpholino to CsgG monomer was added. Each morpholino strand contained a 5' BCN and was clicked to the C-terminal azide on the CsgA peptide.
  • FIG. 3 CsgA-Morpholino modified CsgG-CsgF nanopore complexes (including CsgG-F56Q) were prepared by first incubating CsgA polypeptide containing sulphonylflouride and azide reactive moieties (SEQ ID NO: 40 in Table 1) in a large excess with CsgG-CsgF nanopore complexes in appropriate buffer conditions. Excess reactants were removed using buffer exchange. A second reaction was completed by incubating CsgA modified pore with morpholino-DNA strand containing BCN reactive moiety. The results are shown in Figures 3a, 3b and 3c.
  • the analyte being used to assess the DNA squiggle was from a randomly fragmented library of the ZymoBIOMICS Microbial Community standard. Preparation of the analyte, ligating the analyte to the Y-adapter, SPRI-bead clean-up of the ligated analyte and addition to a minlON flow cell was carried out using the Oxford Nanopore Technologies SQK-LSK114 protocol.
  • CsgG pore (0.15 mg/ml, 0.4 nmol, in PBS buffer with 0.1% SDS, pH 7.4) was added the linked CsgA polypeptides (SEQ ID NOs: 69 and 70; see Figure legend; 20 mg/ml in DMSO, lOOx molar excess).
  • the sample was incubated with shaking (25 °C, 600 rpm) for 14h. Following this, 12 x 5 ul aliquots were taken and heated on a PCRmax Alpha Cycler 4 (Stone, UK) across a temperature gradient ranging from 62.0 to 87.8 °C for 30 minutes.
  • each 5 ul sample was mixed with 10 ul Native Sample Buffer (Bio-Rad, Hercules, USA).
  • the samples were analysed by blue native PAGE gels (4-20% TGX precast gels, Bio-Rad) in lx Tris-glycine buffer (Sigma-Aldrich, Burlington, USA). Initially, the gels were run for 30 minutes at 180 V with lx NativePageTM Cathode Buffer Additive (Invitrogen, Waltham, USA) present in the cathode compartment. Subsequently, this was replaced with lx TG buffer, and the gel run for a further 60 minutes at 180 V.
  • a CsgA-CsgG-CsgF variant nanopore complex was prepared with SEQ ID NO: 40. Electrical measurements were acquired from unmodified CsgG-CsgF nanopore complexes or CsgG-CsgF-CsgA-Morpholino complexes that were inserted into MinlON flow cells. After a single pore complex inserted into the block co-polymer membrane, 1 mL of a buffer comprising 25 mM Potassium Phosphate, 150 mM Potassium Ferrocyanide (II), 150 mM Potassium Ferricyanide (III), pH 8.0 were flowed through the system to remove any excess nanopores.
  • a buffer comprising 25 mM Potassium Phosphate, 150 mM Potassium Ferrocyanide (II), 150 mM Potassium Ferricyanide (III), pH 8.0 were flowed through the system to remove any excess nanopores.
  • the analyte being used to assess the DNA squiggle was from a randomly fragmented library of the ZymoBIOMICS Microbial Community standard. Preparation of the analyte, ligating the analyte to the Y-adapter, SPRI-bead clean-up of the ligated analyte and addition to a minlON flow cell was carried out using the Oxford Nanopore Technologies SQK-LSK114 protocol.
  • the gel in Figure 1 shows the results for the following CsgA polypeptides (Table 1; SO2F is a sulfonyl fluoride group and N3 is an azide group)
  • the gel in Figure 2 shows the results for the following CsgA polypeptides (Table 3; SO2F is a sulfonyl fluoride group and N3 is an azide group)
  • This example demonstrates the attachment of a polynucleotide binding protein to a nanopore via a CsgA peptide, which enabled the polynucleotide binding protein-controlled translocation of DNA through the nanopore.
  • Functionalised CsgG nanopores were prepared by reacting the nanopore protein with excess modified CsgA peptide.
  • This peptide has two modifications: an internal sulfonyl fluoride group which reacts to form a covalent bond with the nanopore; and an azide group, which is reacted with a BCN-modified morpholino oligo 5'-GCAATACGTAACTGAACGAAGTACA-3' (SEQ ID NO: 51).
  • a tethered polynucleotide binding protein was prepared by functionalising the N-terminus of a Hel308 helicase with an azide group, then attaching a BCN-modified morpholino oligo 5'- TGTACTTCGTTCAGTTACGTATTGC-3' (SEQ ID NO: 52) via click reaction of the BCN with the azide group.
  • This morpholino oligo has a sequence which is complementary to the sequence of the morpholino oligo in the functionalised nanopore.
  • the tethered polynucleotide binding protein was confirmed to be able to attach to the functionalised nanopore by blue native electrophoresis. Tethered polynucleotide binding protein was mixed with functionalised in different excess concentrations of tethered polynucleotide binding protein and incubated at room temperature for 15 minutes. The resulting products were loaded in a 4-20% CriterionTM TGXTM Precast Midi Protein Gel (BioRad), after being mixed with Native Sample Buffer for Protein Gels (Bio-Rad).
  • the gel was run with NativePAGETM Cathode Buffer Additive (20X) diluted to lx in lx Tris-Glycine Buffer (Sigma-Aldrich) as the cathode buffer and lx Tris-Glycine Buffer (Sigma-Aldrich) as the anode buffer for 30 minutes at 180V in a CriterionTM Cell (Bio-Rad). After this period, the cathode buffer was replaced by lx Tris-Glycine Buffer (Sigma-Aldrich). Electrophoresis proceeded for 60 minutes further at 180V. The gel was stained with Quick Coomasie Stain (Neo-Biotech). The results are shown in Figure 7.
  • a 3' overhang adapter was prepared by annealing DNA oligonucleotides SEQ ID NO: 53 and SEQ ID NO: 54.
  • a Y-adapter was prepared by annealing DNA oligonucleotides SEQ ID NO: 53 and SEQ ID NO: 55.
  • a 3.6-kilobase DNA analyte was obtained via PCR amplification from bacteriophage Lambda, then end-repaired and dA-tailed using an Ultra II end-repair and dA-tailing kit (New England Biolabs), to generate 3' dA overhangs at both ends of each fragment.
  • the sample was ligated to the T overhang of either the 3' overhang adapter or the Y-adapter using LNB (ONT) and T4 DNA Ligase.
  • the adapter-ligated samples were then purified using Agencourt AMPure XP (Beckman Coulter) beads, to yield the 3' overhang- adapted DNA library and the Y-adapter-adapted DNA library.
  • a control mix was prepared by combining 75pL of SB (ONT), 45pL of nuclease free water and 30pL of unmodified polynucleotide binding protein (800nM final). A total 150pL were flowed into each flow cell and left to incubate for 30 minutes. 500pL of FCF (ONT) were flowed into each flow cell to displace polynucleotide binding protein that did not pair with the complementary morpholino on the functionalised nanopore. After 5 minutes a further 500pL were flowed into each flow cell with the spot-on port open.
  • a first sequencing mix was prepared by combining 37.5pL of SB (ONT), 11.6pL nuclease free water, 15.0pL of buffer consisting of 50mM HEPES pH7, 384mM NaCI and 5% Glycerol, and 10.9pL 3' overhang-adapted DNA library (3.56nM final). 75 pL of this sequencing mix was added to each flow cell through the spot-on port. Current measurements were obtained using the MinKNOW (ONT) software.
  • a second sequencing mix was prepared by combining 37.5pL SB (ONT), 11.8pL LS (ONT), 15.0pL buffer consisting of 50mM HEPES pH7, 384mM NaCI and 5%
  • a pore monomer conjugate comprising a pore monomer, a partner molecule, and a functional binding moiety, wherein the partner molecule has affinity for the pore monomer and wherein the functional binding moiety is attached to the pore monomer via the partner molecule.
  • a pore monomer conjugate according to embodiment 7, wherein the functional binding moiety comprises an oligonucleotide, a polynucleotide, a polynucleotide analog, a morpholino, a peptide nucleic acid, a polypeptide, a ligand, a cyclodextrin, a monosaccharide, an oligosaccharide, a polysaccharide, a boronic acid, an enzyme, a peptide, a cyclic peptide as the functional, an antibody or fragment thereof, or an aptamer.
  • the partner molecule is covalently attached to the pore monomer, optionally by a linker.
  • a construct comprising two or more covalently attached pore monomer conjugates according to any one of embodiments 1-10.
  • a pore complex comprising at least one pore monomer conjugate according to any one of the embodiments 1-10 or at least one construct according to embodiment 11.
  • a pore multimer comprising two or more pores, wherein at least one of the pores is a pore complex according to embodiment 12.
  • a membrane comprising a pore complex according to embodiment 12 or a pore multimer according to embodiment 13.
  • a method for determining the presence, absence or one or more characteristics of a target analyte comprising the steps of:
  • the target analyte is a polynucleotide and the functional binding moiety comprises an oligonucleotide, polynucleotide, polynucleotide analog or morpholino which is capable of hybridising to the target polynucleotide
  • the target analyte is a polynucleotide and the functional binding moiety is attached to a polynucleotide binding protein which controls the movement of the target analyte with respect to the pore complex or pore multimer
  • the target analyte is a ligand for an enzyme and the functional binding moiety is attached to the enzyme
  • the functional binding moiety is an antibody or functional fragment thereof or an aptamer which binds to the target analyte.
  • a kit for characterising a target analyte comprising (a) a pore complex according to embodiment 12 or a pore multimer according to embodiment 13 and (b) the components of a membrane.
  • kit according to embodiment 24, wherein the kit further comprises an enzyme, a pore, a pore multimer, an aptamer, a ring-shaped protein, or a DNA origami structure.
  • a kit for characterising a target polynucleotide or target polypeptide comprising (a) a pore complex according to embodiment 12 or a pore multimer according to embodiment 13 and (b) a polynucleotide binding protein or a polypeptide binding protein.
  • An apparatus for characterising a target polynucleotide or a target polypeptide in a sample comprising (a) a plurality of pore complexes according to embodiment 12 or a plurality of pore multimers according to embodiment 13 and (b) a plurality of polynucleotide binding proteins or a plurality of polypeptide binding proteins.
  • 29. An array comprising a plurality of membranes according to embodiment 15.
  • a system comprising (a) a membrane according to embodiment 15 or an array according to embodiment 29, (b) means for applying a potential across the membrane(s) and (c) means for detecting electrical or optical signals across the membrane(s).
  • An apparatus comprising a pore complex according to embodiment 12 or a pore multimer according to embodiment 13 inserted into an in vitro membrane.
  • An apparatus produced by a method comprising (i) obtaining a pore complex according to embodiment 12 or a pore multimer according to embodiment 13 and (ii) contacting the pore complex or a pore multimer with an in vitro membrane such that the pore complex or the pore multimer is inserted in the in vitro membrane.
  • a method of determining the presence, absence or one or more characteristics of a target polynucleotide comprising the steps of (a) contacting a double stranded polynucleotide comprising template and complement strands with a pore complex or pore multimer comprising at least one pore monomer conjugate according to any one of embodiments 1-10, wherein the complement strand comprises a binding region that is capable of hybridising to the functional binding moiety, such that the two strands are separated as the template strand moves through the pore complex or pore multimer to reveal the binding region on the complement strand wherein the functional binding moiety hybridises to the binding region in order to facilitate the capture of the complement strand by said pore complex or pore multimer and (b) taking one or more measurements as the template strand and the complement strand move with respect to the pore complex or pore multimer wherein the measurements of both the template and complement strands are used to determine the presence, absence or one or more characteristics of the target polynucleotide.

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Abstract

La présente invention concerne de nouveaux conjugués de monomères à pores comprenant des monomères à pores et des molécules partenaires fonctionnalisées, des complexes de pores formés à partir des conjugués et leurs utilisations dans la détection et la caractérisation d'analytes.
PCT/EP2023/081472 2022-11-11 2023-11-10 Nouveaux monomères à pores et pores WO2024100270A1 (fr)

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Citations (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
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
WO2010004265A1 (fr) 2008-07-07 2010-01-14 Oxford Nanopore Technologies Limited Constructions enzyme-pore
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
WO2011067559A1 (fr) 2009-12-01 2011-06-09 Oxford Nanopore Technologies Limited Instrument d'analyse biochimique
WO2012005857A1 (fr) 2010-06-08 2012-01-12 President And Fellows Of Harvard College Dispositif nanoporeux à membrane lipidique artificielle sur support de graphène
WO2012107778A2 (fr) 2011-02-11 2012-08-16 Oxford Nanopore Technologies Limited Pores mutants
WO2013041878A1 (fr) 2011-09-23 2013-03-28 Oxford Nanopore Technologies Limited Analyse d'un polymère comprenant des unités de polymère
WO2013057495A2 (fr) 2011-10-21 2013-04-25 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
WO2013098562A2 (fr) 2011-12-29 2013-07-04 Oxford Nanopore Technologies Limited Procédé enzymatique
WO2013121224A1 (fr) 2012-02-16 2013-08-22 Oxford Nanopore Technologies Limited Analyse de mesures d'un polymère
WO2013123379A2 (fr) 2012-02-16 2013-08-22 The Regents Of The University Of California Détecteur de nanopore pour la translocation de protéine à médiation par enzyme
WO2013153359A1 (fr) 2012-04-10 2013-10-17 Oxford Nanopore Technologies Limited Pores formés de lysenine mutante
WO2014006442A1 (fr) 2012-07-02 2014-01-09 Freescale Semiconductor, Inc. Dispositif de circuit intégré, circuit de sécurité, système essentiel à la sécurité et procédé de fabrication d'un dispositif de circuit intégré
WO2014013262A1 (fr) 2012-07-19 2014-01-23 Oxford Nanopore Technologies Limited Construction d'enzyme
WO2014013260A1 (fr) 2012-07-19 2014-01-23 Oxford Nanopore Technologies Limited Hélicases modifiées
WO2014013259A1 (fr) 2012-07-19 2014-01-23 Oxford Nanopore Technologies Limited Procédé ssb
WO2014064443A2 (fr) 2012-10-26 2014-05-01 Oxford Nanopore Technologies Limited Formation de groupement de membranes et appareil pour celle-ci
WO2014064444A1 (fr) 2012-10-26 2014-05-01 Oxford Nanopore Technologies Limited Interfaces de gouttelettes
WO2014187924A1 (fr) 2013-05-24 2014-11-27 Illumina Cambridge Limited Sequençage pyrophosphorolytique
WO2015055981A2 (fr) 2013-10-18 2015-04-23 Oxford Nanopore Technologies Limited Enzymes modifiées
WO2015140535A1 (fr) 2014-03-21 2015-09-24 Oxford Nanopore Technologies Limited Analyse d'un polymère à partir de mesures multi-dimensionnelles
WO2015166276A1 (fr) 2014-05-02 2015-11-05 Oxford Nanopore Technologies Limited Procédé pour améliorer le mouvement d'un polynucléotide cible par rapport à un pore transmembranaire
WO2016034591A2 (fr) 2014-09-01 2016-03-10 Vib Vzw Pores mutants
WO2016055778A1 (fr) 2014-10-07 2016-04-14 Oxford Nanopore Technologies Limited Pores mutants
WO2016132123A1 (fr) 2015-02-19 2016-08-25 Oxford Nanopore Technologies Limited Hétéro-pores
WO2016164422A2 (fr) * 2015-04-06 2016-10-13 President And Fellows Of Harvard College Matériaux biosynthétiques à base de substance amyloïde présentant des séquences de protéines fonctionnelles
WO2017149318A1 (fr) 2016-03-02 2017-09-08 Oxford Nanopore Technologies Limited Pores mutants
WO2017174990A1 (fr) 2016-04-06 2017-10-12 Oxford Nanopore Technologies Limited Pore mutant
WO2018100370A1 (fr) 2016-12-01 2018-06-07 Oxford Nanopore Technologies Limited Procédés et systèmes de caractérisation d'analytes à l'aide de nanopores
WO2018146491A1 (fr) 2017-02-10 2018-08-16 Oxford Nanopore Technologies Limited Nanopores modifiés, compositions les comprenant et leurs utilisations
WO2018203084A1 (fr) 2017-05-04 2018-11-08 Oxford Nanopore Technologies Limited Analyse d'apprentissage automatique de mesures de nanopores
WO2018211241A1 (fr) 2017-05-04 2018-11-22 Oxford Nanopore Technologies Limited Pore transmembranaire constitué de deux pores csgg
WO2019002893A1 (fr) 2017-06-30 2019-01-03 Vib Vzw Nouveaux pores protéiques
WO2020095052A1 (fr) 2018-11-08 2020-05-14 Oxford Nanopore Technologies Limited Pore
WO2020183172A1 (fr) 2019-03-12 2020-09-17 Oxford Nanopore Technologies Inc. Dispositif de détection à nanopores, composants et procédé de fonctionnement
WO2020208357A1 (fr) 2019-04-09 2020-10-15 Oxford Nanopore Technologies Limited Pore
CN113754743A (zh) 2021-10-12 2021-12-07 成都齐碳科技有限公司 孔蛋白单体的突变体、蛋白孔及其应用
CN113773373A (zh) 2021-10-12 2021-12-10 成都齐碳科技有限公司 孔蛋白单体的突变体、蛋白孔及其应用
CN113896776A (zh) 2021-10-12 2022-01-07 成都齐碳科技有限公司 孔蛋白单体的突变体、蛋白孔及其应用
CN113912683A (zh) 2021-10-12 2022-01-11 成都齐碳科技有限公司 孔蛋白单体的突变体、蛋白孔及其应用

Patent Citations (51)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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
WO2008102120A1 (fr) 2007-02-20 2008-08-28 Oxford Nanopore Technologies Limited Système de capteur 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
WO2010004265A1 (fr) 2008-07-07 2010-01-14 Oxford Nanopore Technologies Limited Constructions enzyme-pore
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
WO2011067559A1 (fr) 2009-12-01 2011-06-09 Oxford Nanopore Technologies Limited Instrument d'analyse biochimique
WO2012005857A1 (fr) 2010-06-08 2012-01-12 President And Fellows Of Harvard College Dispositif nanoporeux à membrane lipidique artificielle sur support de graphène
WO2012107778A2 (fr) 2011-02-11 2012-08-16 Oxford Nanopore Technologies Limited Pores mutants
WO2013041878A1 (fr) 2011-09-23 2013-03-28 Oxford Nanopore Technologies Limited Analyse d'un polymère comprenant des unités de polymère
WO2013057495A2 (fr) 2011-10-21 2013-04-25 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
WO2013098562A2 (fr) 2011-12-29 2013-07-04 Oxford Nanopore Technologies Limited Procédé enzymatique
WO2013121224A1 (fr) 2012-02-16 2013-08-22 Oxford Nanopore Technologies Limited Analyse de mesures d'un polymère
WO2013123379A2 (fr) 2012-02-16 2013-08-22 The Regents Of The University Of California Détecteur de nanopore pour la translocation de protéine à médiation par enzyme
WO2013153359A1 (fr) 2012-04-10 2013-10-17 Oxford Nanopore Technologies Limited Pores formés de lysenine mutante
WO2014006442A1 (fr) 2012-07-02 2014-01-09 Freescale Semiconductor, Inc. Dispositif de circuit intégré, circuit de sécurité, système essentiel à la sécurité et procédé de fabrication d'un dispositif de circuit intégré
WO2014013262A1 (fr) 2012-07-19 2014-01-23 Oxford Nanopore Technologies Limited Construction d'enzyme
WO2014013260A1 (fr) 2012-07-19 2014-01-23 Oxford Nanopore Technologies Limited Hélicases modifiées
WO2014013259A1 (fr) 2012-07-19 2014-01-23 Oxford Nanopore Technologies Limited Procédé ssb
WO2014064443A2 (fr) 2012-10-26 2014-05-01 Oxford Nanopore Technologies Limited Formation de groupement de membranes et appareil pour celle-ci
WO2014064444A1 (fr) 2012-10-26 2014-05-01 Oxford Nanopore Technologies Limited Interfaces de gouttelettes
WO2014187924A1 (fr) 2013-05-24 2014-11-27 Illumina Cambridge Limited Sequençage pyrophosphorolytique
WO2015055981A2 (fr) 2013-10-18 2015-04-23 Oxford Nanopore Technologies Limited Enzymes modifiées
WO2015140535A1 (fr) 2014-03-21 2015-09-24 Oxford Nanopore Technologies Limited Analyse d'un polymère à partir de mesures multi-dimensionnelles
WO2015166276A1 (fr) 2014-05-02 2015-11-05 Oxford Nanopore Technologies Limited Procédé pour améliorer le mouvement d'un polynucléotide cible par rapport à un pore transmembranaire
WO2015166275A1 (fr) 2014-05-02 2015-11-05 Oxford Nanopore Technologies Limited Pores mutants
WO2016034591A2 (fr) 2014-09-01 2016-03-10 Vib Vzw Pores mutants
WO2016055778A1 (fr) 2014-10-07 2016-04-14 Oxford Nanopore Technologies Limited Pores mutants
WO2016132123A1 (fr) 2015-02-19 2016-08-25 Oxford Nanopore Technologies Limited Hétéro-pores
WO2016164422A2 (fr) * 2015-04-06 2016-10-13 President And Fellows Of Harvard College Matériaux biosynthétiques à base de substance amyloïde présentant des séquences de protéines fonctionnelles
WO2017149318A1 (fr) 2016-03-02 2017-09-08 Oxford Nanopore Technologies Limited Pores mutants
WO2017149316A1 (fr) 2016-03-02 2017-09-08 Oxford Nanopore Technologies Limited Pore mutant
WO2017149317A1 (fr) 2016-03-02 2017-09-08 Oxford Nanopore Technologies Limited Pore mutant
WO2017174990A1 (fr) 2016-04-06 2017-10-12 Oxford Nanopore Technologies Limited Pore mutant
WO2018100370A1 (fr) 2016-12-01 2018-06-07 Oxford Nanopore Technologies Limited Procédés et systèmes de caractérisation d'analytes à l'aide de nanopores
WO2018146491A1 (fr) 2017-02-10 2018-08-16 Oxford Nanopore Technologies Limited Nanopores modifiés, compositions les comprenant et leurs utilisations
WO2018203084A1 (fr) 2017-05-04 2018-11-08 Oxford Nanopore Technologies Limited Analyse d'apprentissage automatique de mesures de nanopores
WO2018211241A1 (fr) 2017-05-04 2018-11-22 Oxford Nanopore Technologies Limited Pore transmembranaire constitué de deux pores csgg
WO2019002893A1 (fr) 2017-06-30 2019-01-03 Vib Vzw Nouveaux pores protéiques
WO2020095052A1 (fr) 2018-11-08 2020-05-14 Oxford Nanopore Technologies Limited Pore
WO2020183172A1 (fr) 2019-03-12 2020-09-17 Oxford Nanopore Technologies Inc. Dispositif de détection à nanopores, composants et procédé de fonctionnement
WO2020208357A1 (fr) 2019-04-09 2020-10-15 Oxford Nanopore Technologies Limited Pore
CN113754743A (zh) 2021-10-12 2021-12-07 成都齐碳科技有限公司 孔蛋白单体的突变体、蛋白孔及其应用
CN113773373A (zh) 2021-10-12 2021-12-10 成都齐碳科技有限公司 孔蛋白单体的突变体、蛋白孔及其应用
CN113896776A (zh) 2021-10-12 2022-01-07 成都齐碳科技有限公司 孔蛋白单体的突变体、蛋白孔及其应用
CN113912683A (zh) 2021-10-12 2022-01-11 成都齐碳科技有限公司 孔蛋白单体的突变体、蛋白孔及其应用

Non-Patent Citations (27)

* Cited by examiner, † Cited by third party
Title
"Gene", Database accession no. 945619
"Uniprot", Database accession no. POAEA2
ALTSCHUL S. F, J MOL EVOL, vol. 36, 1993, pages 290 - 300
ALTSCHUL, S.F ET AL., J MOL BIOL, vol. 215, 1990, pages 403 - 10
AUSUBEL ET AL.: "Current Protocols in Molecular Biology", 2016, GREENE PUBLISHING AND WILEY-INTERSCIENCE
BEREZOVSKI, M. ET AL., JOURNAL OF THE AMERICAN CHEMICAL SOCIETY, vol. 128, 2006, pages l410 - 1411
BIRD ET AL., SCIENCE, vol. 242, 1988, pages 423 - 426
BOCK, L. C. ET AL., NATURE, vol. 355, 1992, pages 564 - 566
CELL CHEMICAL BIOLOGY, vol. 27, 2020, pages 970 - 985
CHEM BIOL., vol. 4, no. 7, pages 497 - 505
CURRENT OPINION IN CHEMICAL BIOLOGY, 2015, pages 18 - 26
D. STODDART ET AL., PROC. NATL. ACAD. SCI., vol. 106, 2010, pages 7702 - 7
DEVEREUX ET AL., NUCLEIC ACIDS RESEARCH, vol. 12, 1984, pages 387 - 395
GONZALEZ-PEREZ ET AL., LANGMUIR, vol. 25, 2009, pages 10447 - 10450
GOYAL ET AL., NATURE, vol. 516, no. 7530, 2014, pages 250 - 3
HORNBLOWER ET AL., NATURE METHODS., vol. 4, 2007, pages 315 - 317
HUSTON ET AL., PROC. NATL. ACAD. SCI., vol. 85, 1988, pages 5879 - 5883
J. AM. CHEM. SOC., vol. 141, no. 7, 2019, pages 2782 - 2799
LANGECKER ET AL., SCIENCE, vol. 338, 2012, pages 932 - 936
NATURE CHEMISTRY, vol. 13, 2021, pages 1081 - 1092
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory", 2012, COLD SPRING HARBOR PRESS
SAMBROOK, J.RUSSELL, D.: "Molecular Cloning: A Laboratory Manual", 2001, COLD SPRING HARBOR LABORATORY PRESS
STOLTENBURG, R. ET AL., BIOMOLECULAR ENGINEERING, vol. 24, 2007, pages 381 403
WARD ET AL., NATURE, vol. 341, 1989, pages 544 - 546
WHITE, R. ET AL., MOLECULAR THERAPY, vol. 4, 2001, pages 567 - 573
YAN, Z.YIN, M.CHEN, J. ET AL.: "Assembly and substrate recognition of curli biogenesis system", NAT COMMUN, vol. 11, 2020, pages 241
ZHANG ET AL.: "A Guide to Signal Processing Algorithms for Nanopore Sensors", ACS SENS., vol. 6, no. 10, 2021, pages 3536 - 3555, XP055861844, DOI: 10.1021/acssensors.1c01618

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