pore

Abstract

The present invention relates to novel Rhodococcus porin monomers, and pores comprising the monomers, and methods of using the pores to characterize analytes such as polypeptides and polynucleotides.

Description

[Technical field]

[0001] The present invention relates to novel Rhodococcus porin monomers, pores containing the monomers, and methods of using the pores to characterize analytes such as polynucleotides and polypeptides.

[0002] Introduction Nanopore sensing is a technique for analyte detection and characterization that relies on the observation of individual binding or interaction events between analyte molecules and ion-conducting channels. Nanopore sensors can be made by placing a single pore of nanometer dimensions in an electrically insulating membrane and measuring the voltage-driven ionic current through the pore in the presence of analyte molecules. The presence of an analyte in or near the nanopore alters the ionic flow through the pore, resulting in a change in the ionic or electrical current measured on the channel. The identity of the analyte is revealed through its unique current signature, in particular the duration and extent of the current blockade, and the variation in the current level during its interaction time with the pore. Analytes can be organic and inorganic small molecules, as well as a variety of biological or synthetic macromolecules and polymers, including polynucleotides, polypeptides, and polysaccharides. Nanopore sensing can reveal the identity of the sensed analyte and perform single molecule counts of the sensed analyte, but can also provide information about the composition of the analyte, such as nucleotide, amino acid, or glycan sequence, and the presence of base, amino acid, or glycan modifications, such as methylation and acylation, phosphorylation, hydroxylation, oxidation, reduction, glycosylation, decarboxylation, deamination, etc. Nanopore sensing has the potential to enable rapid and inexpensive polynucleotide sequencing, providing single molecule sequence reads for polynucleotides that are tens to tens of thousands of bases long.

[0003] Two of the key elements of polymer characterization using nanopore sensing are (1) control of the transport of the polymer through the pore and (2) discrimination of the constituent building blocks as the polymer passes through the pore. During nanopore sensing, the narrowest part of the pore forms the reader head, which is the most discriminating part of the nanopore in terms of the current signature as a function of the analyte passing through it.

[0004] In the case of polynucleotide analytes, nucleotide discrimination is achieved by passing through such mutant pores, but the current signature was shown to be sequence-dependent, with multiple nucleotides contributing to the observed current, and the height of the channel constriction and the degree of interaction surface with the analyte affecting the relationship between the observed current and the polynucleotide sequence. Although the current range for nucleotide discrimination has been improved through mutations, if the current difference between nucleotides could be further improved, sequencing systems would have higher performance. Thus, there is a need to identify novel methods to improve nanopore sensing characteristics.

[0005] The cell wall channel of Rhodococcus corynebacteroides was purified according to established procedures (Riess and Benz, 2000, Biochim Biophys Acta, 20;1509(1-2):485-495). The protein was then subjected to Edman degradation and sequencing. The resulting partial sequence was then used as input for a BLAST search to identify a protein designated PorARc. A BLAST search for homologues to the partial sequence of the Rhodococcus corynebacteroides cell wall channel PorARc identified two primary polypeptide sequences from Rhodococcus ruber, PorARr and PorBRr. Summary of the Invention

[0006] The present invention relates to modified Rhodococcus porin monomers, and pores containing the monomers, which have been modified to facilitate the characterization of target analytes, particularly target polynucleotides and polypeptides.

[0007] The present inventors have surprisingly shown that novel variants of PorARr, PorBRr and PorARc show improved properties for estimating analyte characteristics, such as polynucleotide or polypeptide sequences.The variants surprisingly show improved polynucleotide capture and nucleotide discrimination.In particular, the variants surprisingly show an increase in current range, which makes it easier to distinguish between different nucleotides, and a reduction in state variation (referred to as "median sd"), which increases signal-to-noise ratio (referred to as "SNR").

[0008] In particular, the present invention provides the following: - a mutant Rhodococcus pore comprising a monomer which is a variant of the sequence shown in SEQ ID NO:2, wherein the variant is located at the following position: (a) D11, G12, G13, G14, N15, T52, G53, P54, D55, A56, E57, D58, F59, S60, G61, T62, T64, Y67, Q68, V69, Y71, P119, G120, I121, E122, T123, V124, E125, V126, S128, A130, A131, S132, G133, A134, H135, L143, H144, T146, T148, K149, Y159, Q161, V163, S164, D165, N166, G167, D168, V169, and T171, (b) S74, G76, E78, T80, D82, T84, Q108, G110, G112, T114, E116, and P117, and / or (c) one or more mutations in one or more of G87, E89, G91, S93, S94, A95, G96, A97, S99, T101, S103, and D104; A mutant Rhodococcus pore, in which one or more mutations alter the ability of the monomer to interact with a target analyte; a mutant Rhodococcus pore comprising a monomer which is a variant of the sequence shown in SEQ ID NO: 6, wherein the variant comprises one or more mutations between positions 9 and 200 of SEQ ID NO: 6, wherein the one or more mutations alter the ability of the pore to interact with a target analyte; A construct comprising two or more covalently linked Rhodococcus porin monomers. a polynucleotide encoding the mutant pore of the invention or the construct of the invention, a pore comprising at least one construct of the invention, - a kit for characterizing a target polynucleotide or a target polypeptide, comprising (a) a pore of the invention and (b) a polynucleotide binding protein or a polypeptide handling enzyme, - a device for characterizing a target polynucleotide or a target polypeptide in a sample, the device comprising: (a) a plurality of pores of the invention; and (b) a plurality of polynucleotide binding proteins or a plurality of polypeptide handling enzymes, a membrane comprising the pores of the invention, an array comprising a plurality of membranes of the invention; - a system comprising (a) a membrane of the invention or an array of the invention, (b) means for applying an electrical potential across the membrane(s), and (c) means for detecting an electrical or optical signal across the membrane(s), - a method for determining the presence, absence, or one or more characteristics of a target analyte, comprising: (a) contacting the target analyte with a pore of the invention; and (b) performing one or more measurements as the target analyte migrates relative to the pore, thereby determining the presence, absence, or one or more characteristics of the target analyte, - a method for characterizing a target polynucleotide, the method comprising: (a) contacting a target polynucleotide with a pore of the invention; and (b) performing one or more measurements as the target polynucleotide moves relative to the pore, thereby characterizing the target polynucleotide, - the use of a pore of the invention for determining the presence, absence or one or more characteristics of a target analyte or a target polynucleotide, - a device comprising a transmembrane protein pore inserted into an in vitro membrane, the transmembrane protein pore being a variant of a Rhodococcus porin monomer and located at the following position: (a) D11, G12, G13, G14, N15, T52, G53, P54, D55, A56, E57, D58, F59, S60, G61, T62, T64, Y67, Q68, V69, Y71, P119, G120, I121, E122, T123, V124, E125, V126, S128, A130, A131, S132, G133, A134, H135, L143, H144, T146, T148, K149, Y159, Q161, V163, S164, D165, N166, G167, D168, V169, and T171, (b) S74, G76, E78, T80, D82, T84, Q108, G110, G112, T114, E116, and P117, and / or (c) at least one monomer containing a mutation in one or more of G87, E89, G91, S93, S94, A95, G96, A97, S99, T101, S103, and D104, corresponding to the sequence set forth in SEQ ID NO:2; - an apparatus comprising: (i) obtaining a transmembrane protein pore comprising at least one Rhodococcus porin monomer, the monomer being located at position (a) D11, G12, G13, G14, N15, T52, G53, P54, D55, A56, E57, D58, F59, S60, G61, T62, T64, Y67, Q68, V69, Y71, P119, G120, I121, E122, T123, V124, E125, V126, S128, A130, A131, S132, G133, A134, H135, L143, H144, T146, T148, K149, Y159, Q161, V163, S164, D165, N166, G167, D168, V169, and T171, (b) S74, G76, E78, T80, D82, T84, Q108, G110, G112, T114, E116, and P117, and / or (c) obtaining a polypeptide comprising one or more amino acid mutations: G87, E89, G91, S93, S94, A95, G96, A97, S99, T101, S103, and D104, corresponding to the sequence set forth in SEQ ID NO:2; (ii) contacting the pore with an in vitro membrane such that the pore is inserted into the in vitro membrane. [Brief description of the drawings]

[0009] [Figure 1] Data is shown for PorARc wild type (WT) and PorARc mutants E78S / D82S / E89N, and E78S / D82S / E89N / E116T / E125A / D165S. [Diagram 2] Data is shown for PorARc mutants E78S / D82S / E89R, E78R / D82S / E89R, and E78R / D82S / E89R / E116T / E125A / D165S. [Diagram 3] Data is shown for PorARc mutants E57R / E78R / D82S / E89R / E116T / E125A / D165S, D55S / E57S / D58S / E78R / D82S / E89R / E116T / E125A / D165S, and E78R / D82S / E89R / D104S / E116T / E122S / E125A / D165S. [Figure 4]Data is shown for PorARc mutants D55S / E57S / D58S / E78R / D82S / E89R / D104S / E116T / E122S / E125A / D165S, and E57R / E78R / D82S / E89R / D104S / E116T / E122S / E125A / D165S. [Diagram 5] Data is shown for PorARc mutants E78R / D82S / E89G / E116T / E125A / D165S, E78R / D82S / E89A / E116T / E125A / D165S, and E78R / D82S / E89V / E116T / E125A / D165S. [Figure 6] Data is shown for PorARc mutants E78R / D82S / E89L / E116T / E125A / D165S, E78R / D82S / E89I / E116T / E125A / D165S, and E78R / D82S / E89F / E116T / E125A / D165S. [Figure 7] Data is shown for PorARc mutants E78R / D82S / E89S / E116T / E125A / D165S, E78R / D82S / E89T / E116T / E125A / D165S, and E78R / D82S / E89Q / E116T / E125A / D165S. [Figure 8] Data is shown for the PorARc mutant E78R / D82S / E89K / E116T / E125A / D165S. [Figure 9] Data is shown for PorARc mutants E78R / D82S / E89R / T101V / E116T / E125A / D165S, E78R / D82S / E89R / T101S / E116T / E125A / D165S, and E78R / D82S / E89R / T101N / E116T / E125A / D165S. [Figure 10] Data is shown for PorARc mutants E78R / D82S / E89R / T101Q / E116T / E125A / D165S and E78R / D82S / E89R / T101R / E116T / E125A / D165S. [Figure 11]Data is shown for PorARc mutants E78R / D82S / E89N / T101V / E116T / E125A / D165S, E78R / D82S / E89N / T101S / E116T / E125A / D165S, and E78R / D82S / E89N / T101N / E116T / E125A / D165S. [Figure 12] Data is shown for PorARc mutants E78R / D82S / E89N / T101Q / E116T / E125A / D165S and E78R / D82S / E89N / T101R / E116T / E125A / D165S. [Figure 13] Data is shown for PorARc mutants E78R / D82S / G87S / E89R / E116T / E125A / D165S, E78R / D82S / E89R / G91S / E116T / E125A / D165S, and E78R / D82S / E89R / G91N / E116T / E125A / D165S. [Figure 14] Data is shown for PorARc mutants E78R / D82S / E89R / G91N / I92N / E116T / E125A / D165S, E78R / D82S / E89R / I92N / E116T / E125A / D165S, and E78R / D82S / E89R / S99N / E116T / E125A / D165S. [Figure 15] Data is shown for PorARc mutants E78R / D82S / E89R / S103N / E116T / E125A / D165S, E78R / D82S / E89R / D104S / E116T / E125A / D165S, and E78R / D82S / E89R / E116T / E122S / E125A / D165S. [Figure 16] Data is shown for PorARc mutants E78G / D82S / E89L / G91N / I92N / E116G / E125A / D165S and E78G / D82S / E89L / E116G / E125A / D165S. [Figure 17] Data is shown for PorARr mutants E182R / E184R, E89N / E91N, and E89N / E91N / E168R / E182R / E184R. [Figure 18] Data is shown for PorARr mutants E89N / E91N / E96R / E99N / E113N / E123N / D130N / E182R / E184R and E89N / E91N / E96R / E99R / E113K / E123K / D130K / E182R / E184R. [Figure 19] Data is shown for PorBRr wild type (WT) and PorBRr mutants E90N and E92N. [Figure 20] Data are shown for PorBRr mutants E90N / E92N, E90N / E92N / E115R / E131R, and E90N / E92N / E113R / E115R / E129R / E131R.

[0010] Each of FIGS. 1 to 20 illustrates the following. a) Snapshots of ionic current versus time as DNA translocates through the pore, the x-axis spanning the entire experimental run time (typically 6 hours). b) Histogram of the current from each channel in the flow cell when an applied current of -180 mV was applied prior to the addition of analyte. c) Zoomed-in snapshots of ionic current versus time as DNA translocates through the pore, with the y-axis ranging over 150 pA to facilitate comparisons between pore variants, and the x-axis typically spanning over 2 sec. d) Zoomed-in snapshots of ionic current versus time as DNA translocates through the pore, with the y-axis ranging over 150 pA to facilitate comparisons between pore variants, and the x-axis typically spanning less than 2 s. [Figure 21] 4 shows strand mean from PorARr, PorBRr and PorARc wild type pores and their mutants as DNA translocates through the pore. SNR=current range divided by noise of current events, range=current range, median sd=noise of current events, and median current=median current as DNA translocates through the pore. [Figure 22]4 shows strand fiducials from PorBRr mutants as DNA translocates through the pore. SNR = current range divided by noise of current events, range = current range, median sd = noise of current events, and normalized MAD current = median absolute deviation of the current level in the signal as DNA translocates through the pore, normalized by dividing by the current range of the strand as DNA translocates through the pore.

[0011] Sequence Listing SEQ ID NO:1 shows the polynucleotide sequence encoding PorARc. SEQ ID NO: 2 shows the amino acid sequence of PorARc. SEQ ID NO: 3 shows the polynucleotide sequence encoding PorARr. SEQ ID NO: 4 shows the amino acid sequence of PorARr. SEQ ID NO:5 shows the polynucleotide sequence encoding PorBRr. SEQ ID NO: 6 shows the amino acid sequence of PorBRr. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention has been described with respect to certain embodiments and with reference to certain drawings, but the present invention is not limited thereto, but only by the claims. Any reference signs in the claims should not be interpreted as limiting the scope. Of course, it should be understood that not necessarily all aspects or advantages can be achieved according to any particular embodiment of the present invention. Thus, for example, a person skilled in the art will recognize that the present invention can be embodied or performed in a manner that achieves or optimizes one advantage or group of advantages taught herein, but does not necessarily achieve other aspects or advantages that may be taught or suggested herein.

[0013] The present invention, both with respect to its method of construction and operation, together with its features and advantages, may be best understood by reference to the following detailed description when read in conjunction with the accompanying drawings. Aspects and advantages of the present invention will become apparent from and be elucidated with reference to the embodiment(s) described below. References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with an embodiment is included in at least one embodiment of the present invention. Thus, although the phrases "in one embodiment" or "in an embodiment" appear in various places throughout this specification, they do not necessarily all refer to the same embodiment. Similarly, in describing exemplary embodiments of the present invention, it should be understood that various features of the invention may be grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. However, this method of disclosure is not to be interpreted as reflecting an intention that the claimed invention requires more features than are expressly recited in each claim. Rather, as the following claims reflect, aspects of the present invention lie in less than all of the features of a single foregoing disclosed embodiment.

[0014] Additionally, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "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, etc.

[0015] In all discussions herein, standard single letter codes for amino acids are used, which are as follows: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V). Standard substitution notation is also used, i.e., E78S means that E at position 78 is replaced with S.

[0016] In sections herein where different amino acids at a particular position are separated by a / symbol, the / symbol means "or", e.g., E89A / V means E89A or E89V.

[0017] In paragraphs herein where different positions or substitutions are separated by a / symbol, the / symbol means "and", so that E89A / E116T is E89A and E116T.

[0018] All amino acid substitutions, deletions, and / or additions disclosed herein relate to mutant monomers, including variants of the sequences shown in SEQ ID NOs: 2, 4 or 6, unless otherwise stated.

[0019] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. All publications, patents, and patent applications mentioned herein are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that the publications and patents or patent applications incorporated by reference conflict with the disclosure contained herein, the present specification is intended to supersede and / or take precedence over any such conflicting material.

[0020] definition When an indefinite or definite article is used when referring to a singular noun, e.g., "a" or "an" or "the," this includes the plural of that noun, unless otherwise specified. When the term "comprising" is used in the present specification and claims, it does not exclude other elements or steps. Furthermore, the terms first, second, third, etc. in the present specification and claims are used to distinguish between similar elements and are not necessarily used to describe a sequential or chronological order. It is to be understood that the terms used in this manner are interchangeable under appropriate circumstances and that the embodiments of the invention described herein can operate in sequences other than those described or illustrated herein. The following terms or definitions are provided solely to aid in the understanding of the present invention. Unless otherwise specifically defined herein, all terms used herein have the same meaning as they would to one skilled in the art of the present invention. Skilled artisans are particularly advised to refer to 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 definitions provided herein should not be construed to have a scope less than understood by a person of ordinary skill in the art.

[0021] As used herein, "about" when referring to a measurable value, such as an amount, duration, and the like, is meant to encompass variations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and even more preferably ±0.1% from the particular value, such variations being appropriate for carrying out the methods of the present disclosure.

[0022] As used herein, "polynucleotide", "nucleotide sequence", "DNA sequence" or "nucleic acid molecule(s)" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. The term refers only to the primary structure of the molecule. Thus, the term includes double- and single-stranded DNA, as well as RNA. As used herein, the term "nucleic acid" is a single- or double-stranded covalently linked nucleotide sequence in which the 3' and 5' ends on each nucleotide are linked by a phosphodiester bond. A polynucleotide can be composed of deoxyribonucleotide or ribonucleotide bases. Nucleic acids can be produced synthetically in vitro or isolated from natural sources. Nucleic acids can further include modified DNA or RNA, such as methylated DNA or RNA, or RNA that has undergone post-translational modifications, such as 5'-capping with 7-methylguanosine, 3'-processing such as truncation and polyadenylation, and splicing. Nucleic acids can also include synthetic nucleic acids (XNA), such as hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), locked nucleic acid (LNA) and peptide nucleic acid (PNA). The size of a nucleic acid, also referred to herein as a "polynucleotide", is typically expressed as the number of base pairs (bp) for double-stranded polynucleotides or the number of nucleotides (nt) for single-stranded polynucleotides. 1000 bp or nt is equivalent to a kilobase (kb). Polynucleotides less than about 40 nucleotides in length are typically referred to as "oligonucleotides" and can include primers for use in manipulating DNA, such as via polymerase chain reaction (PCR).

[0023] As used herein, "gene" includes both the promoter region and coding sequence of a gene. It refers to both the genomic sequence (including possible introns) and the cDNA derived spliced ​​messenger that is operably linked to the promoter sequence.

[0024] A "coding sequence" is a nucleotide sequence that is transcribed into mRNA and / or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5'-terminus and a translation stop codon at the 3'-terminus. A coding sequence can include, but is not limited to, mRNA, cDNA, recombinant nucleotide sequences, or genomic DNA, and can also contain introns under certain circumstances.

[0025] The term "amino acid" in the context of this disclosure is used in its broadest sense and is meant to include organic compounds containing an amine (NH2) functional group and a carboxyl (COOH) functional group, along with a side chain (e.g., R group) specific to each amino acid. In some embodiments, amino acid refers to a naturally occurring Lα-amino acid or residue. Commonly used one-letter and three-letter abbreviations for naturally occurring amino acids are used herein: A=Ala, C=Cys, D=Asp, E=Glu, F=Phe, G=Gly, H=His, I=Ile, K=Lys, L=Leu, M=Met, N=Asn, P=Pro, Q=Gln, R=Arg, S=Ser, T=Thr, V=Val, W=Trp, and Y=Tyr (Lehninger, AL, (1975) Biochemistry, 2d ed., pp. 71-92, Worth Publishers, New York). The general term "amino acid" further includes D-amino acids, retro-inverso amino acids, and chemically modified amino acids such as amino acid analogs, naturally occurring amino acids that are not normally incorporated into proteins, such as norleucine, and chemically synthesized compounds that have properties known in the art to be characteristic of amino acids, such as β-amino acids. For example, analogs or mimetics of phenylalanine or proline that allow the same conformational restriction of peptide compounds as natural Phe or Pro are included within the definition of amino acids. Such analogs and mimetics are referred to herein as "functional equivalents" of the respective amino acids. Other examples of amino acids are listed by Roberts and Vellaccio, The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, eds., Vol. 5 p. 341, Academic Press, Inc., NY 1983, which is incorporated herein by reference.

[0026] The terms "polypeptide" and "peptide" are further used interchangeably herein to refer to a polymer of amino acid residues, as well as variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues are synthetic, non-naturally occurring amino acids, such as chemical analogs of the corresponding naturally occurring amino acids, as well as to naturally occurring amino acid polymers. Polypeptides may also undergo maturation or post-translational modification processes, including, but not limited to, glycosylation, proteolytic cleavage, lipidation, signal peptide cleavage, propeptide cleavage, phosphorylation, and the like. "Recombinant polypeptide" refers to a polypeptide made using recombinant techniques, e.g., via expression of a recombinant or synthetic polynucleotide. When a chimeric polypeptide or a biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, e.g., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the amount of a protein preparation. "Isolated" refers to a material that is substantially or essentially free from components that normally accompany it in its non-denatured state. For example, an "isolated polypeptide" as used herein refers to a polypeptide purified from a Rhodococcus porin monomer that has been removed from molecules adjacent to it in its naturally occurring state, e.g., molecules present in a production host adjacent to said polypeptide. An isolated peptide may be produced by amino acid chemical synthesis or by recombinant production. An isolated pore may be produced by in vitro reconstitution following purification of the components of the pore, or may be produced by recombinant co-expression.

[0027] The term "protein" is used to describe a folded polypeptide having secondary or tertiary structure. A protein may be composed of a single polypeptide or may contain multiple polypeptides that assemble to form a multimer. A multimer may be a homo- or hetero-oligomer. A protein may be a naturally occurring or wild-type protein, or a modified or non-naturally occurring protein. A protein may differ from a wild-type protein, for example, by the addition, substitution, or deletion of one or more amino acids.

[0028] "Ortholog" and "paralog" encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogs are genes within the same species that arose by duplication of an ancestral gene, while orthologs are genes from different organisms that arose by speciation, also derived from a common ancestral gene.

[0029] Protein "variants," "homologues," and "homologues" include peptides, oligopeptides, polypeptides, proteins, and enzymes that have amino acid substitutions, deletions, and / or insertions compared to the unmodified or wild-type protein in question and that have similar biological and functional activity as the unmodified protein from which they are derived. As used herein, the term "amino acid identity" refers to the degree to which sequences are identical on an amino acid-by-amino acid basis over a comparison window. Thus, "percentage of sequence identity" is calculated by comparing two optimally aligned sequences across the comparison window, determining the number of positions at which an identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) appears in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to obtain the percentage of sequence identity.

[0030] The term "transmembrane protein pore" defines a pore comprising multiple pore monomers. Each monomer may be a wild type monomer or a variant thereof. A variant monomer may also be referred to as a modified or mutant monomer. Modifications or mutations in a variant include, but are not limited to, any one or more of the modifications disclosed herein, or a combination of said modifications.

[0031] The term "Rhodococcus pore" defines a pore comprising a plurality of PorARr, PorBRr or PorARc monomers. Each PorARr, PorBRr or PorARc monomer can be a wild-type monomer (SEQ ID NO: 2, 4 or 6), a wild-type homologue of PorARr, PorBRr or PorARc, or any variant thereof (e.g., any one of the variants of SEQ ID NO: 2, 4 or 6). A variant PorARr, PorBRr or PorARc monomer may also be referred to as a modified PorARr, PorBRr or PorARc monomer or a mutant PorARr, PorBRr or PorARc monomer. Modifications or mutations in the variant include, but are not limited to, any one or more of the modifications disclosed herein, or a combination of the modifications.

[0032] For all aspects and embodiments of the invention, a homologue refers to a polypeptide having at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% complete sequence identity to PorARr, PorBRr, or PorARc, e.g., the sequence shown in SEQ ID NO: 2, 4, or 6. Similarly, a homologous polynucleotide may include a polynucleotide having at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% complete sequence identity to a nucleic acid sequence encoding a wild-type protein. For example, a PorARr, PorBRr, or PorARc homologous polynucleotide may include a polynucleotide having at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% complete sequence identity to PorARr, PorBRr, or PorARc, as shown in SEQ ID NO: 1, 3, or 5.

[0033] Sequence identity may also be for a fragment or part of a full-length polynucleotide or polypeptide. Thus, a sequence may have only 50% overall sequence identity with a full-length reference sequence, but the sequence of a particular region, domain, or subunit may share 80%, 90%, or even 99% sequence identity with the reference sequence. Homology to the nucleic acid sequence of SEQ ID NO: 1, 3, or 5 for PorARr, PorBRr, or PorARc homologs, respectively, is not limited to just sequence identity. Many nucleic acid sequences may show significant biological homology to each other, despite apparently low sequence identity. Homologous nucleic acid sequences are considered to hybridize to each other under low stringency conditions (MR Green, J. Sambrook, 2012, Molecular Cloning: A Laboratory Manual, Fourth Edition, Books 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0034] The term "wild type" refers to a gene or gene product isolated from a natural source. A wild type gene is the gene most frequently observed in a population, and is therefore arbitrarily designated the "normal" or "wild type" form of the gene. In contrast, the terms "modified," "mutant," or "variant" refer to a gene or gene product that exhibits modifications in sequence (e.g., substitutions, truncations, or insertions), post-translational modifications, and / or functional properties (e.g., altered characteristics) when compared to the wild type gene or gene product. It should be noted that naturally occurring mutants can be isolated, which are identified by the fact that they have altered characteristics when compared to the wild type gene or gene product. Methods for introducing or substituting naturally occurring amino acids are well known in the art. For example, methionine (M) can be substituted with arginine (R) by replacing the codon for methionine (ATG) with the codon for arginine (CGT) at the relevant position in the polynucleotide encoding the mutant monomer. Methods for introducing or substituting non-naturally occurring amino acids are also well known in the art. For example, non-naturally occurring amino acids can be introduced by including synthetic aminoacyl-tRNA in the IVTT system used to express the mutant monomers. Alternatively, they can be introduced by expressing mutant monomers in E. coli that are auxotrophic for specific amino acids in the presence of synthetic (i.e., non-naturally occurring) analogs of those specific amino acids. They can also be generated by exposed ligation when the mutant monomers are generated using partial peptide synthesis. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties, or similar side chain volume. The introduced amino acids can have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge as the amino acids they replace. Alternatively, conservative substitutions may introduce another amino acid that is aromatic or aliphatic in place of an existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and can be selected according to the properties of the 20 major amino acids defined in Table 1 below.If the amino acids have a similar polarity, this can also be determined by reference to the hydrophobicity scale for the amino acid side chains in Table 2. [Table 1] [Table 2]

[0035] The mutant or modified protein, monomer, or peptide can also be chemically modified in any manner and at any site. The mutant or modified monomer or peptide is preferably chemically modified by binding a molecule to one or more cysteines (cysteine ​​binding), binding a molecule to one or more lysines, binding a molecule to one or more unnatural amino acids, enzymatic modification of an epitope, or modification of a terminal. Suitable methods for carrying out such modifications are well known in the art. The modified protein, monomer, or peptide variant can be chemically modified by binding any molecule. For example, the modified protein, monomer, or peptide variant can be chemically modified by binding a dye or fluorophore.

[0036] The protein may also be a fusion protein, which refers specifically to a genetic fusion produced, for example, by recombinant DNA technology. The protein may also be conjugated, or "conjugated to," as used herein, which refers specifically to chemical and / or enzymatic conjugation resulting in a stable covalent bond. For example, two or more or all of the polypeptide subunits of a multimeric auxiliary protein and / or nanopore may be fused, and / or a polypeptide subunit of an auxiliary protein may be fused to a monomer of a nanopore.

[0037] When several polypeptides or protein monomers bind or interact with each other, the proteins may form a protein complex. By "binding" is meant any interaction, whether direct or indirect. Direct interaction implies contact between binding partners, for example, via covalent bonds or couplings. Indirect interaction means any interaction where the interacting partners interact in a complex of three or more compounds. The interaction may be fully indirect with the aid of one or more bridging molecules, or may be partially indirect, where there is still a direct contact between the partners that is stabilized by the additional interaction of one or more compounds. A "complex" as referred to in this disclosure is defined as a group of two or more related proteins that may have different functions. The association between the different polypeptides of a protein complex may be through non-covalent interactions, such as hydrophobic or ionic forces, or may be covalent bonds or couplings, such as disulfide bridges or peptide bonds. Covalent "bonding" or "coupling" are used interchangeably herein and may also involve "cysteine ​​coupling" or "reactive or photoreactive amino acid coupling", respectively, referring to bioconjugation between cysteines or (photo)reactive amino acids, which are covalent chemical bonds to form stable complexes. Examples of photoreactive amino acids include azidohomoalanine, homopropargylglycine, homoallergylglycine, p-acetylPhe, p-azido-Phe, p-propargyloxy-Phe, and p-benzoyl-Phe (Wang et al. 2012, in Protein Engineering, DOI: 10.5772 / 28719; Chin et al. 2002, Proc. Nat. Acad. Sci. USA 99(17); 11020-24).

[0038] A "transmembrane protein pore" or "biological pore" is a transmembrane protein structure that defines a channel or hole that allows the translocation of molecules and ions from one side of a membrane to the other. Translocation of ionic species through the pore can be driven by a potential difference applied to either side of the pore. A "nanopore" is a pore in which the minimum diameter of the channel through which molecules or ions pass is on the order of nanometers (10 -9 The minimum diameter is the diameter at the narrowest point of the constriction. Transmembrane protein pores can be monomeric or oligomeric in nature. Typically, the pore comprises multiple polypeptide subunits arranged around a central axis, thereby forming a protein-lined channel that extends substantially perpendicular to the membrane in which the nanopore resides. The number of polypeptide subunits is not limited. Typically, the number of subunits is from 5 to 30, preferably the number of subunits is from 6 to 10. Alternatively, the number of subunits is not defined as in the case of perfringolysin or related large membrane pores. The portion of the protein subunits within the nanopore that forms the protein-lined channel typically comprises secondary structural motifs that may include one or more transmembrane β-barrel and / or α-helical segments.

[0039] The monomers, pores, or transmembrane protein pores of the present disclosure are suitable for characterization of analytes, particularly for characterization of polypeptides or polynucleotides. In some embodiments, the monomers, pores, or transmembrane protein pores described herein may be used to sequence polynucleotide sequences, for example, because they can distinguish different nucleotides with high sensitivity. In some embodiments, the monomers, pores, or transmembrane protein pores described herein may be used to sequence polypeptide sequences, for example, because they can distinguish different amino acids with high sensitivity. The monomers, pores, or transmembrane protein pores of the present disclosure may be isolated, substantially isolated, purified, or substantially purified. The monomers, pores, or transmembrane protein pores of the present disclosure are "isolated" or purified when they are completely free of any other components, such as lipids and / or other pores, or other proteins with which they are normally associated in their native state, or when they are sufficiently concentrated from the membrane compartment. The monomers, pores, or transmembrane protein pores are substantially isolated when they are mixed with a carrier or diluent that does not interfere with its intended use. For example, a monomer, pore, or transmembrane protein pore is substantially isolated or substantially purified if it is present in a form that contains less than 10%, less than 5%, less than 2%, or less than 1% of other components, such as triblock copolymers, lipids, or other pores. Alternatively, a monomer, pore, or transmembrane protein pore of the present disclosure can be a transmembrane pore when present in a membrane.

[0040] As used interchangeably herein, "constriction," "opening," "constriction region," "channel constriction," "constriction site," or "reader head" refer to an opening defined by the luminal surface of a pore that acts to allow the passage of ions and target molecules (e.g., but not limited to amino acids, polypeptides, polynucleotides, or individual nucleotides), but not other non-target molecules through the pore channel or continuous channel formed by the pore and auxiliary proteins or peptides. In some embodiments, the constriction(s) are the narrowest opening(s) in the pore. In this embodiment, the constriction(s) may serve to limit the passage of molecules through the pore. The size of the constriction is typically a critical factor in determining the suitability of a nanopore for nucleic acid sequencing applications. If the constriction is too small, the molecules to be sequenced will not be able to pass. However, to achieve the greatest effect on ion flow through the channel, the constriction should not be too large. For example, the constriction should preferably not be wider than the solvent-accessible lateral diameter of the target analyte. Ideally, any constriction should be as close as possible to the lateral diameter of the analyte to be passed. For nucleic acid and nucleic acid base sequencing, suitable constriction diameters are in the nanometer range (10 -9 The diameter is preferably in the range of 0.5 to 2.0 nm, or 0.5 to 4.0 nm, typically the diameter is in the range of 0.7 to 1.2 nm, e.g. 0.9 nm (9 Å). Such diameters may be particularly suitable for sequencing single-stranded nucleic acids. Larger diameters such as about 1.2 nm to about 4 nm, e.g. about 2 to about 4 nm or about 3 nm to about 4 nm, may be particularly suitable for sequencing double-stranded nucleic acids.

[0041] When two or more constrictions are present and spaced apart, each constriction can simultaneously interact with or "read" a separate nucleotide in a nucleic acid strand or a separate nucleotide in a polypeptide. In this situation, the reduction in ion flow through the channel results from the combined flow restriction of all constrictions containing nucleotides or amino acids. Thus, in some cases, double constrictions can lead to a composite current signal. In certain circumstances, the current readings of one constriction, or "read head", may not be individually determinable when two such read heads are present. Suitable distances between constriction regions are discussed below. The pore or transmembrane pore of the present invention includes a pore or transmembrane pore with two or more reader heads, meaning two or more channel constrictions positioned to provide suitable separate reader heads without interfering with the accuracy of the other constriction channel reader head.

[0042] The constriction region or site may be formed by one or more specific amino acid residues within the protein sequence of the transmembrane protein nanopore and / or the auxiliary protein or peptide.

[0043] In wild-type PorARr, PorBRr and PorARc, the constriction is located towards the bottom of the pore, which is depicted as part (c) below.

[0044] PorARc Pores and Monomers of the Invention The invention provides a mutant Rhodococcus pore comprising a monomer that is a variant of the sequence shown in SEQ ID NO:2, the variant comprising one or more mutations between positions 9 and 200 of SEQ ID NO:2, the one or more mutations altering the ability of the pore to interact with a target analyte. Any of the specific embodiments discussed below apply to this embodiment of the invention. The variant preferably comprises one or more mutations between positions 10 and 190, or between 11 and 180 of SEQ ID NO:2. The variant preferably comprises one or more mutations between positions 87 and 104.

[0045] The present invention also provides a mutant Rhodococcus pore comprising a monomer which is a variant of the sequence shown in SEQ ID NO:2, wherein the variant comprises one or more mutations at one or more of the following positions (a), (b) and / or (c), wherein the one or more mutations alter the ability of the pore to interact with a target analyte:

[0046] The target analyte may be any of those discussed below. The target analyte is preferably an amino acid, a polypeptide, a nucleotide, or a polynucleotide. The ability of the pore to interact with the target analyte may be measured using known methods, including those described in the Examples. The pore may include any number of monomers, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 monomers. Any number of monomers in the pore may be variants as defined above. The pore may be a homo-oligomer or a hetero-oligomer as defined above.

[0047] The invention also provides a mutant Rhodococcus porin monomer comprising a variant of the sequence shown in SEQ ID NO:2, the variant comprising one or more mutations between positions 9 and 200 of SEQ ID NO:2, the one or more mutations altering the ability of the monomer to interact with a target analyte such that the monomer can form a pore. Any of the specific embodiments discussed below apply to this embodiment of the invention. The variant preferably comprises one or more mutations between positions 10 and 190, or between 11 and 180 of SEQ ID NO:2. The variant preferably comprises one or more mutations between positions 87 and 104.

[0048] The invention also provides mutant Rhodococcus porin monomers comprising a variant of the sequence shown in SEQ ID NO: 2. The variants comprise one or more mutations at one or more of the following positions (a), (b) and / or (c), which alter the ability of the monomer to interact with a target analyte, such that the monomer can form a pore:

[0049] The target analyte is preferably an amino acid, a polypeptide, a nucleotide, or a polynucleotide. The ability of the monomer to interact with the target analyte can be measured using known methods, including those described in the Examples. The ability of the monomer to form a pore can be determined using known methods, including those in the Examples.

[0050] The mutant monomers have improved target analyte reading properties, i.e., they show improved analyte capture and discrimination. In particular, pores constructed from mutant monomers capture amino acids, polypeptides, nucleotides, and polynucleotides more easily than wild type. In addition, pores constructed from mutant monomers show increased current ranges, which make it easier to discriminate between different amino acids or nucleotides, and reduced fluctuations in conditions, which increases the signal-to-noise ratio. In addition, as a nucleic acid moves through a pore constructed from the mutant, the number of amino acids or nucleotides that contribute to the current decreases. This makes it easier to identify a direct relationship between the current observed as a polypeptide or polynucleotide moves through the pore and the polypeptide or polynucleotide sequence. The improved amino acid or nucleotide reading properties of the variants are achieved by changes in five main mechanisms: conformation (increasing or decreasing the size of the amino acid residues), charge (e.g., introducing a +ve charge to interact with the nucleic acid sequence), hydrogen bonding (e.g., introducing an amino acid that can hydrogen bond to base pairs), π-stacking (e.g., introducing an amino acid that interacts via a delocalized electron π-system), and / or changes in the structure of the pore (e.g., introducing an amino acid that increases the size of the anterior chamber and / or constriction).

[0051] Any one or more of these five mechanisms may be responsible for the improved properties of the pores of the invention, for example, the pores of the invention may exhibit improved amino acid or nucleotide reading properties as a result of conformational changes, hydrogen bonding changes, and structural changes.

[0052] Introduction of bulky residues such as phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H) increases the conformation of the pore. Introduction of aromatic residues such as phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H) also increases pi-stacking in the pore. Introduction of bulky or aromatic residues also changes the structure of the pore, for example by opening the pore and increasing the size of the antechamber and / or constriction. This is explained in more detail below.

[0053] The one or more positions occur in three regions of the monomer: the cap, the middle, or the bottom.

[0054] In cap (a), the positions are D11, G12, G13, G14, N15, T52, G53, P54, D55, A56, E57, D58, F59, S60, G61, T62, T64, Y67, Q68, V69, Y71, P119, G120, I121, E122, T123, V124, E125, V126, S128, A130, A131, S132, G133, A134, H135, L143, H144, T146, T148, K149, Y159, Q161, V163, S164, D165, N166, G167, D168, V169, and T171.

[0055] In center (b), the positions are S74, G76, E78, T80, D82, T84, Q108, G110, G112, T114, E116, and P117.

[0056] At the bottom (c), the positions are G87, E89, G91, S93, S94, A95, G96, A97, S99, T101, S103, and D104.

[0057] The variants may contain one or more mutations at any combination of positions in the cap, middle and bottom, i.e., at one or more of (a), (b), (c), (a) and (b), (b) and (c), (a) and (c), or (a), (b) and (c).

[0058] The variant may include any number of one or more mutations, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more mutations.

[0059] The variant preferably comprises at least the following position of SEQ ID NO:2: E78 / D82 / E89, E78 / D82 / E89 / E116 / E125 / D165, E57 / E78 / D82 / E89 / E116 / E125 / D165, D55 / E57 / D58 / E78 / D82 / E89 / E116 / E125 / D165, E78 / D82 / E89 / D104 / E116 / E122 / E125 / D165, D55 / E57 / D58 / E78 / D82 / E89 / D104 / E116 / E122 / E125 / D165, E57 / E78 / D82 / E89 / D104 / E116 / E122 / E125 / D165, E78 / D82 / E89 / T101 / E116 / E125 / D165, E78 / D82 / G87 / E89 / E116 / E125 / D165, E78 / D82 / E89 / G91 / I92 / E116 / E125 / D165, E78 / D82 / E89 / I92 / E116 / E125 / D165, E78 / D82 / E89 / S99 / E116 / E125 / D165, E78 / D82 / E89 / S103 / E116 / E125 / D165, E78 / D82 / E89 / D104 / E116 / E125 / D165, E78 / D82 / E89 / E116 / E122 / E125 / D165, E78 / D82 / E89 / G91 / I92 / E116 / E125 / D165, or Contains one or more mutations at E78 / D82 / E89 / E116 / E125 / D165.

[0060] The variants preferably comprise or consist of mutations at the positions set out immediately above SEQ ID NO:2.

[0061] The one or more mutations each independently (a) change the size of the amino acid residue at the modified position, (b) change the net charge of the amino acid residue at the modified position, (c) change the hydrogen bonding characteristics of the amino acid residue at the modified position, (d) introduce one or more chemical groups that interact through a delocalized electron π system into the amino acid residue at the modified position, or remove one or more chemical groups that interact through a delocalized electron π system from the amino acid residue at the modified position, and / or (e) change the structure of the amino acid residue at the modified position. The one or more mutations are one or more substitutions. Those skilled in the art can make suitable substitutions based on Tables 1 and 2 above.

[0062] In cap (a), mutations are preferably made to include positive, polar, or hydrophobic amino acids to either reduce negativity (so as to facilitate capture of negatively charged analytes) or improve / stabilize interactions with enzymes. The amino acid at position (a) may be substituted with R, K, S, T, N, Q, G, A, V, L, I, P, C, F, Y, or W.

[0063] In the middle (b), mutations are preferably made to include positive, polar, or hydrophobic amino acids to reduce negativity (so as to facilitate capture of negatively charged analytes). The amino acid at position (b) may be substituted with R, K, S, T, N, Q, G, A, V, L, I, P, C, F, Y, or W.

[0064] At the bottom (c), mutations are made, preferably including polar, hydrophobic, positive, or negative, to alter discrimination (central / cap residues may also affect discrimination, but likely to a lesser extent). The amino acid at position (c) may be substituted with S, T, N, Q, G, A, V, L, I, P, C, R, K, D, E, F, Y, or W.

[0065] Mutate to polar, hydrophobic, positive, or negative to alter discrimination (center / cap residues may also affect discrimination, but likely to a lesser extent)

[0066] The one or more mutations are preferably selected from the following specific substitutions: D to Q, S or Y, more preferably D to S; G to S, N, Q, T or A, more preferably G to S or N; N to S, Q, L, Y, M, I, V or G, T to V, S, N, Q, R, F or I, more preferably T to V, S, N, Q or R; From P to S, A to R, V, D, N, S or T, E to S, N, T, A, R, G, V, L, I, F, Q or K, F to R, K, S, T, N, Q, G, A, V, L, I, P, C, Y or W, S to N or C, more preferably S to N; Y to R, K, S, T, N, Q, G, A, V, L, I, P, C, D, E, F, or W, From Q to R, From V to I, I to G, L, S, F, V, A, P, W, Q, N, C, S, Y, K or T, more preferably I to N, E to R, H to F, N, Q or A, L through N, S, Y, V, Q, T, R, A, G, C, F or D, and K to R, S, T, N, Q, G, A, V, L, I, P, C, D, E, F, Y or W.

[0067] The variant preferably contains at least the following mutations in SEQ ID NO:2: E78S / D82S / E89N, E78S / D82S / E89N / E116T / E125A / D165S, E78S / D82S / E89R, E78R / D82S / E89R, E78R / D82S / E89R / E116T / E125A / D165S、 E57R / E78R / D82S / E89R / E116T / E125A / D165S、 D55S / E57S / D58S / E78R / D82S / E89R / E116T / E125A / D165S、 E78R / D82S / E89R / D104S / E116T / E122S / E125A / D165S、 D55S / E57S / D58S / E78R / D82S / E89R / D104S / E116T / E122S / E125A / D165S、 E57R / E78R / D82S / E89R / D104S / E116T / E122S / E125A / D165S、 E78R / D82S / E89G / E116T / E125A / D165S、 E78R / D82S / E89A / E116T / E125A / D165S、 E78R / D82S / E89V / E116T / E125A / D165S、 E78R / D82S / E89L / E116T / E125A / D165S、 E78R / D82S / E89I / E116T / E125A / D165S、 E78R / D82S / E89F / E116T / E125A / D165S、 E78R / D82S / E89S / E116T / E125A / D165S、 E78R / D82S / E89T / E116T / E125A / D165S、 E78R / D82S / E89Q / E116T / E125A / D165S、 E78R / D82S / E89K / E116T / E125A / D165S、 E78R / D82S / E89R / T101V / E116T / E125A / D165S、 E78R / D82S / E89R / T101S / E116T / E125A / D165S、 E78R / D82S / E89R / T101N / E116T / E125A / D165S、 E78R / D82S / E89R / T101Q / E116T / E125A / D165S, E78R / D82S / E89R / T101R / E116T / E125A / D165S, E78R / D82S / E89N / T101V / E116T / E125A / D165S, E78R / D82S / E89N / T101S / E116T / E125A / D165S, E78R / D82S / E89N / T101N / E116T / E125A / D165S, E78R / D82S / E89N / T101Q / E116T / E125A / D165S, E78R / D82S / E89N / T101R / E116T / E125A / D165S, E78R / D82S / G87S / E89R / E116T / E125A / D165S, E78R / D82S / E89R / G91S / E116T / E125A / D165S, E78R / D82S / E89R / G91N / E116T / E125A / D165S, E78R / D82S / E89R / G91N / I92N / E116T / E125A / D165S, E78R / D82S / E89R / I92N / E116T / E125A / D165S, E78R / D82S / E89R / S99N / E116T / E125A / D165S, E78R / D82S / E89R / S103N / E116T / E125A / D165S, E78R / D82S / E89R / D104S / E116T / E125A / D165S, E78R / D82S / E89R / E116T / E122S / E125A / D165S, E78G / D82S / E89L / G91N / I92N / E116G / E125A / D165S, or E78G / D82S / E89L / E116G / E125A / D165S.

[0068] The variant preferably comprises or consists of the mutations set out immediately above for SEQ ID NO: 2. The variant may be any of the mutations set out in Table 3.

[0069] A variant of SEQ ID NO:2 is preferably not identical to SEQ ID NO:4 or 6 (ie, does not share 100% amino acid identity with SEQ ID NO:4 or 6).

[0070] PorARr Pores and Monomers of the Invention The invention also provides a mutant Rhodococcus pore comprising a monomer that is a variant of the sequence shown in SEQ ID NO:4, the variant comprising one or more mutations between positions 9 and 250 of SEQ ID NO:4, the one or more mutations altering the ability of the pore to interact with a target analyte. Any of the specific embodiments discussed below apply to this embodiment of the invention. The variant preferably comprises one or more mutations between positions 10 and 200, or between 11 and 180 of SEQ ID NO:4. The variant preferably comprises one or more mutations between positions 87 and 158.

[0071] The present invention also provides a mutant Rhodococcus pore comprising a monomer which is a variant of the sequence shown in SEQ ID NO:4, wherein the variant comprises one or more mutations at one or more of the following positions (a), (b) and / or (c), wherein the one or more mutations alter the ability of the pore to interact with a target analyte:

[0072] The target analyte may be any of those discussed below. The target analyte is preferably an amino acid, a polypeptide, a nucleotide, or a polynucleotide. The ability of the pore to interact with the target analyte may be measured using known methods, including those described in the Examples. The pore may include any number of monomers, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 monomers. Any number of monomers in the pore may be variants as defined above. The pore may be a homo-oligomer or a hetero-oligomer as defined above.

[0073] The present invention provides a mutant Rhodococcus porin monomer comprising a variant of the sequence shown in SEQ ID NO:4, wherein the variant comprises one or more mutations between positions 9 and 250 of SEQ ID NO:4, wherein the one or more mutations alter the ability of the monomer to interact with a target analyte such that the monomer can form a pore. Any of the specific embodiments discussed below apply to this embodiment of the invention. The variant preferably comprises one or more mutations between positions 10 and 200, or between 11 and 180 of SEQ ID NO:4. The variant preferably comprises one or more mutations between positions 87 and 158.

[0074] The invention also provides a mutant Rhodococcus porin monomer comprising a variant of the sequence shown in SEQ ID NO: 4. The variant comprises one or more mutations at one or more of the following positions (a), (b) and / or (c), wherein the one or more mutations alter the ability of the monomer to interact with a nucleotide such that the monomer can form a pore.

[0075] The target analyte is preferably an amino acid, a polypeptide, a nucleotide, or a polynucleotide. The ability of the monomer to interact with the target analyte can be measured using known methods, including those described in the Examples. The ability of the monomer to form a pore can be determined using known methods, including those in the Examples.

[0076] Mutant monomers based on SEQ ID NO:4 have improved target analyte reading properties for the same reasons discussed above for SEQ ID NO:2.

[0077] The one or more positions occur in three regions of the monomer: the cap, the middle, or the bottom.

[0078] In cap (a), the positions are S11, D12, G13, H14, V51, G52, P53, D54, A55, A56, D57, F58, E59, G60, S61, Q64, Y67, Q68, F69, W71, P173, G174, I175, E176, E177, L178, V17 9, V180, E182, G183, E184, F185, D186, G187, D188, F189, V197, H198, A200, T202, G203, F213, R215, I217, T218, A219, N220, G221, D222, N223, and T225.

[0079] In center (b), the positions are S74, D76, S78, G80, A82, S84, Q162, T164, G166, E168, T170, and P171.

[0080] At the bottom (c), the positions are: Q87, E89, E91, V92, A93, P94, G95, E96, V97, Y98, E99, P100, A101, T102, E103, T104, K105, T106, V107, D108, G109, K110, E111, T112, E113, V114, P115, V116, T117, A118, P119, V120, L121, N122, E123 , D124, G125, S126, P127, I128, L129, D130, A131, D132, G133, K134, P135, V136, T137, E138, Q139, L140, Y141, R142, I143, K144, P145, D146, A147, T148, A149, T150, E151, N152, T153, T155, V156, G157, and G158.

[0081] The variants may contain one or more mutations at any combination of positions in the cap, middle and bottom, i.e., at one or more of (a), (b), (c), (a) and (b), (b) and (c), (a) and (c), or (a), (b) and (c).

[0082] The variant may include any number of one or more mutations, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more mutations.

[0083] The variant preferably comprises at least the following position of SEQ ID NO:4: E182 / E184, E89 / E91, E89 / E91 / E168 / E182 / E184, or Contains one or more mutations at E89 / E91 / E96 / E99 / E113 / E123 / D130 / E182 / E184.

[0084] The variants preferably comprise or consist of mutations at the positions shown immediately above for SEQ ID NO:4.

[0085] The one or more mutations each independently (a) change the size of the amino acid residue at the modified position, (b) change the net charge of the amino acid residue at the modified position, (c) change the hydrogen bonding characteristics of the amino acid residue at the modified position, (d) introduce one or more chemical groups that interact through a delocalized electron π system into the amino acid residue at the modified position, or remove one or more chemical groups that interact through a delocalized electron π system from the amino acid residue at the modified position, and / or (e) change the structure of the amino acid residue at the modified position. The one or more mutations are one or more substitutions. Those skilled in the art can make suitable substitutions based on Tables 1 and 2 above.

[0086] The one or more mutations are preferably selected from the preferred or specific substitutions set out above for SEQ ID NO:2.

[0087] The variant preferably contains at least the following mutations in SEQ ID NO:4: E182R / E184R, E89N / E91N, E89N / E91N / E168R / E182R / E184R, E89N / E91N / E96R / E99N / E113N / E123N / D130N / E182R / E184R, or E89N / E91N / E96R / E99R / E113K / E123K / D130K / E182R / E184R.

[0088] The variant preferably comprises or consists of the mutations shown immediately above for SEQ ID NO: 4. The variant may be any of the mutations shown in Table 3.

[0089] A variant of SEQ ID NO:4 is preferably not identical to SEQ ID NO:2 or 6 (ie, does not share 100% amino acid identity with SEQ ID NO:2 or 6).

[0090] PorBRr Pores and Monomers of the Invention The invention also provides a mutant Rhodococcus pore comprising a monomer that is a variant of the sequence shown in SEQ ID NO:6, the variant comprising one or more mutations between positions 9 and 200 of SEQ ID NO:6, the one or more mutations altering the ability of the pore to interact with a target analyte. Any of the specific embodiments discussed below apply to this embodiment of the invention. The variant preferably comprises one or more mutations between positions 10 and 190, or between 11 and 180 of SEQ ID NO:6. The variant preferably comprises one or more mutations between positions 88 and 105.

[0091] The present invention also provides a mutant Rhodococcus pore comprising a monomer which is a variant of the sequence shown in SEQ ID NO:6, wherein the variant comprises one or more mutations at one or more of the following positions (a), (b) and / or (c), wherein the one or more mutations alter the ability of the pore to interact with a target analyte:

[0092] The target analyte may be any of those discussed below. The target analyte is preferably an amino acid, a polypeptide, a nucleotide, or a polynucleotide. The ability of the pore to interact with the target analyte may be measured using known methods, including those described in the Examples. The pore may include any number of monomers, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 monomers. Any number of monomers in the pore may be variants as defined above. The pore may be a homo-oligomer or a hetero-oligomer as defined above.

[0093] The invention provides a mutant Rhodococcus porin monomer comprising a variant of the sequence shown in SEQ ID NO:6, wherein the variant comprises one or more mutations between positions 9 and 200 of SEQ ID NO:6, wherein the one or more mutations alter the ability of the monomer to interact with nucleotides such that the monomer can form a pore. Any of the specific embodiments discussed below apply to this embodiment of the invention. The variant preferably comprises one or more mutations between positions 10 and 190, or between 11 and 180 of SEQ ID NO:6. The variant preferably comprises one or more mutations between positions 88 and 105.

[0094] The invention also provides a mutant Rhodococcus porin monomer comprising a variant of the sequence shown in SEQ ID NO: 6. The variant comprises one or more mutations at one or more of the following positions (a), (b) and / or (c), wherein the one or more mutations alter the ability of the monomer to interact with a nucleotide such that the monomer can form a pore.

[0095] The target analyte is preferably an amino acid, a polypeptide, a nucleotide, or a polynucleotide. The ability of the monomer to interact with the target analyte can be measured using known methods, including those described in the Examples. The ability of the monomer to form a pore can be determined using known methods, including those in the Examples.

[0096] Mutant monomers based on SEQ ID NO:6 have improved target analyte reading properties for the same reasons discussed above for SEQ ID NO:2.

[0097] The one or more positions occur in three regions of the monomer: the cap, the middle, or the bottom.

[0098] In cap (a), the positions are S11, T12, D13, G14, Y15, V52, G53, A54, D55, A56, A57, D58, A59, E60, G61, T62, Q65, Y68, Q69, F70, W72, P120, G121, I122, E123, E124, L125, V 126, V127, E129, G130, E131, F132, D133, G134, D135, F136, V144, H145, T147, S149, G150, F160, R162, I164, T165, A166, N167, G168, D169, N170, and T172.

[0099] In center (b), the positions are S75, D77, A79, G81, T83, D85, Q109, Y111, E113, E115, T117, and P118.

[0100] At the bottom (c), the positions are G88, E90, E92, L93, S94, A95, K96, D97, G98, P99, T100, T102, V103, T104, and D105.

[0101] The variants may contain one or more mutations at any combination of positions in the cap, middle and bottom, i.e., at one or more of (a), (b), (c), (a) and (b), (b) and (c), (a) and (c), or (a), (b) and (c).

[0102] The variant may include any number of one or more mutations, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more mutations.

[0103] The variant preferably comprises at least the following position of SEQ ID NO:6: E90, E92, E90 / E92, E90 / E92 / E115 / E131, or Contains one or more mutations at E90 / E92 / E113 / E115 / E129 / E131.

[0104] The variant preferably comprises at least the following position of SEQ ID NO:6: E90 / E92 / E115 / E131, E90 / E92 / T100 / E115 / E131, E90 / E92 / T102 / E115 / E131, E90 / E92 / T104 / E115 / E131, E90 / E92 / D105 / E115 / E131, E90 / E92 / T102 / T104 / E115 / E131, or Contains one or more mutations at E90 / E92 / E113 / E115 / E129 / E131.

[0105] The variant preferably comprises at least the following position of SEQ ID NO:6: E90, E92, E90 / E92, E90 / E92 / E115 / E131, E90 / E92 / T100 / E115 / E131, E90 / E92 / T102 / E115 / E131, E90 / E92 / T104 / E115 / E131, E90 / E92 / D105 / E115 / E131, E90 / E92 / T102 / T104 / E115 / E131, or Contains one or more mutations at E90 / E92 / E113 / E115 / E129 / E131.

[0106] The variants preferably comprise or consist of mutations at the positions shown immediately above for SEQ ID NO:6.

[0107] The one or more mutations each independently (a) change the size of the amino acid residue at the modified position, (b) change the net charge of the amino acid residue at the modified position, (c) change the hydrogen bonding characteristics of the amino acid residue at the modified position, (d) introduce one or more chemical groups that interact through a delocalized electron π system into the amino acid residue at the modified position, or remove one or more chemical groups that interact through a delocalized electron π system from the amino acid residue at the modified position, and / or (e) change the structure of the amino acid residue at the modified position. The one or more mutations are one or more substitutions. Those skilled in the art can make suitable substitutions based on Tables 1 and 2 above.

[0108] The one or more mutations are preferably selected from the preferred or specific substitutions indicated above for SEQ ID NO:2. E at position 90 is preferably substituted with G, A, V, L, I, F, Y, S, T, Q, N, C or P. E at position 90 is preferably substituted with N, G, V, L or I. E at position 90 is preferably substituted with N (E90N). E at position 92 is preferably substituted with N, G, A, V, L, I, F, Y, S, T, Q, C or P. E at position 92 is preferably substituted with N, A, V, L or I. E at position 92 is preferably substituted with N (E92N). T at position 100 is preferably substituted with A, V, N or Q. T at position 100 is preferably substituted with N (T100N). T at position 102 is preferably substituted with A, V, N or Q. T at position 102 is preferably substituted with V or N. T at position 104 is preferably substituted with A, V, N or Q. T at position 104 is preferably substituted with A or N. D at position 105 is preferably substituted with A. E at position 113 is preferably substituted with R (E113R) or K (E113K). E at position 113 is preferably substituted with R (E113R). E at position 115 is preferably substituted with R (E115R) or K (E115K). E at position 115 is preferably substituted with R (E115R). E at position 131 is preferably substituted with R (E131R) or K (E131K). E at position 131 is preferably substituted with R (E131R).

[0109] The variant preferably contains at least the following mutations in SEQ ID NO:6: E90N, E92N, E90N / E92N, E90N / E92N / E115R / E131R, or E90N / E92N / E113R / E115R / E129R / E131R.

[0110] The variant preferably contains at least the following mutations in SEQ ID NO:6: E90C, E90P, E92C, E92P、 E90C / E92C、 E90P / E92P、 E90C / E92P、 E90P / E92C、 E90N / E92C、 E90N / E92P、 E90C / E92N、 E90P / E92N、 E90C / E92C / E115R / E131R、 E90C / E92P / E115R / E131R、 E90P / E92C / E115R / E131R、 E90C / E92C / E115R / E131R、 E90N / E92C / E115R / E131R、 E90N / E92P / E115R / E131R、 E90P / E92N / E115R / E131R、 E90C / E92N / E115R / E131R、 E90C / E92C / E113R / E115R / E129R / E131R、 E90C / E92P / E113R / E115R / E129R / E131R、 E90P / E92C / E113R / E115R / E129R / E131R、 E90P / E92P / E113R / E115R / E129R / E131R、 E90C / E92N / E113R / E115R / E129R / E131R、 E90P / E92N / E113R / E115R / E129R / E131R、 E90N / E92C / E113R / E115R / E129R / E131R、 E90N / E92P / E113R / E115R / E129R / E131R、 E90C、 E90P、 E92C、 E92P、 E90C / E92C、 E90P / E92P、 E90C / E92P、 E90P / E92C、 E90N / E92C、 E90N / E92P、 E90C / E92N、 E90P / E92N、 E90C / E92C / E115K / E131K、 E90C / E92P / E115K / E131K、 E90P / E92C / E115K / E131K、 E90C / E92C / E115K / E131K、 E90N / E92C / E115K / E131K、 E90N / E92P / E115K / E131K、 E90P / E92N / E115K / E131K、 E90C / E92N / E115K / E131K、 E90C / E92C / E113K / E115K / E129K / E131K、 E90C / E92P / E113K / E115K / E129K / E131K、 E90P / E92C / E113K / E115K / E129K / E131K、 E90P / E92P / E113K / E115K / E129K / E131K、 E90C / E92N / E113K / E115K / E129K / E131K、 E90P / E92N / E113K / E115K / E129K / E131K、 E90N / E92C / E113K / E115K / E129K / E131K、 E90N / E92P / E113K / E115K / E129K / E131K、 E90G / E92N / E115R / E131R、 E90A / E92N / E115R / E131R、 E90V / E92N / E115R / E131R、 E90L / E92N / E115R / E131R、 E90I / E92N / E115R / E131R、 E90F / E92N / E115R / E131R、 E90Y / E92N / E115R / E131R、 E90S / E92N / E115R / E131R、 E90T / E92N / E115R / E131R、 E90Q / E92N / E115R / E131R、 E90C / E92N / E115R / E131R、 E90P / E92N / E115R / E131R、 E90N / E92G / E115R / E131R、 E90N / E92A / E115R / E131R、 E90N / E92V / E115R / E131R、 E90N / E92L / E115R / E131R、 E90N / E92I / E115R / E131R、 E90N / E92F / E115R / E131R、 E90N / E92Y / E115R / E131R、 E90N / E92S / E115R / E131R、 E90N / E92T / E115R / E131R、 E90N / E92Q / E115R / E131R、 E90N / E92N / E115R / E131R、 E90N / E92C / E115R / E131R、 E90N / E92P / E115R / E131R、 E90C / E92C / E115R / E131R、 E90P / E92P / E115R / E131R、 E90C / E92P / E115R / E131R、 E90P / E92C / E115R / E131R、 E90N / E92N / T100N / E115R / E131R、 E90N / E92N / T102A / E115R / E131R、 E90N / E92N / T102V / E115R / E131R、 E90N / E92N / T102N / E115R / E131R、 E90N / E92N / T102Q / E115R / E131R、 E90N / E92N / T104A / E115R / E131R、 E90N / E92N / T104N / E115R / E131R、 E90N / E92N / D105A / E115R / E131R、 E90N / E92N / T102V / T104N / E115R / E131R、 E90N / E92N / T102N / T104N / E115R / E131R、 E90N / E92N / E113R / E115R / E129R / E131R、 E90C / E92N / T100N / E115R / E131R、 E90C / E92N / T102A / E115R / E131R、 E90C / E92N / T102V / E115R / E131R、 E90C / E92N / T102N / E115R / E131R、 E90C / E92N / T102Q / E115R / E131R、 E90C / E92N / T104A / E115R / E131R、 E90C / E92N / T104N / E115R / E131R、 E90C / E92N / D105A / E115R / E131R、 E90C / E92N / T102V / T104N / E115R / E131R、 E90C / E92N / T102N / T104N / E115R / E131R、 E90C / E92N / E113R / E115R / E129R / E131R、 E90N / E92C / T100N / E115R / E131R、 E90N / E92C / T102A / E115R / E131R、 E90N / E92C / T102V / E115R / E131R、 E90N / E92C / T102N / E115R / E131R、 E90N / E92C / T102Q / E115R / E131R、 E90N / E92C / T104A / E115R / E131R、 E90N / E92C / T104N / E115R / E131R、 E90N / E92C / D105A / E115R / E131R、 E90N / E92C / T102V / T104N / E115R / E131R、 E90N / E92C / T102N / T104N / E115R / E131R、 E90N / E92C / E113R / E115R / E129R / E131R、 E90C / E92C / T100N / E115R / E131R、 E90C / E92C / T102A / E115R / E131R、 E90C / E92C / T102V / E115R / E131R、 E90C / E92C / T102N / E115R / E131R、 E90C / E92C / T102Q / E115R / E131R、 E90C / E92C / T104A / E115R / E131R、 E90C / E92C / T104N / E115R / E131R、 E90C / E92C / D105A / E115R / E131R、 E90C / E92C / T102V / T104N / E115R / E131R、 E90C / E92C / T102N / T104N / E115R / E131R、 E90C / E92C / E113R / E115R / E129R / E131R、 E90C / E92P / T100N / E115R / E131R、 E90C / E92P / T102A / E115R / E131R、 E90C / E92P / T102V / E115R / E131R、 E90C / E92P / T102N / E115R / E131R、 E90C / E92P / T102Q / E115R / E131R、 E90C / E92P / T104A / E115R / E131R、 E90C / E92P / T104N / E115R / E131R、 E90C / E92P / D105A / E115R / E131R、 E90C / E92P / T102V / T104N / E115R / E131R、 E90C / E92P / T102N / T104N / E115R / E131R、 E90C / E92P / E113R / E115R / E129R / E131R、 E90P / E92C / T100N / E115R / E131R、 E90P / E92C / T102A / E115R / E131R、 E90P / E92C / T102V / E115R / E131R、 E90P / E92C / T102N / E115R / E131R、 E90P / E92C / T102Q / E115R / E131R、 E90P / E92C / T104A / E115R / E131R、 E90P / E92C / T104N / E115R / E131R、 E90P / E92C / D105A / E115R / E131R、 E90P / E92C / T102V / T104N / E115R / E131R、 E90P / E92C / T102N / T104N / E115R / E131R、 E90P / E92C / E113R / E115R / E129R / E131R、 E90G / E92N / E115K / E131K、 E90A / E92N / E115K / E131K、 E90V / E92N / E115K / E131K、 E90L / E92N / E115K / E131K、 E90I / E92N / E115K / E131K、 E90F / E92N / E115K / E131K、 E90Y / E92N / E115K / E131K、 E90S / E92N / E115K / E131K、 E90T / E92N / E115K / E131K、 E90Q / E92N / E115K / E131K、 E90C / E92N / E115K / E131K、 E90P / E92N / E115K / E131K、 E90N / E92G / E115K / E131K、 E90N / E92A / E115K / E131K、 E90N / E92V / E115K / E131K、 E90N / E92L / E115K / E131K、 E90N / E92I / E115K / E131K、 E90N / E92F / E115K / E131K、 E90N / E92Y / E115K / E131K、 E90N / E92S / E115K / E131K、 E90N / E92T / E115K / E131K、 E90N / E92Q / E115K / E131K、 E90N / E92N / E115K / E131K、 E90N / E92C / E115K / E131K、 E90N / E92P / E115K / E131K、 E90C / E92C / E115K / E131K、 E90P / E92P / E115K / E131K、 E90C / E92P / E115K / E131K、 E90P / E92C / E115K / E131K、 E90N / E92N / T100N / E115K / E131K、 E90N / E92N / T102A / E115K / E131K、 E90N / E92N / T102V / E115K / E131K、 E90N / E92N / T102N / E115K / E131K、 E90N / E92N / T102Q / E115K / E131K、 E90N / E92N / T104A / E115K / E131K、 E90N / E92N / T104N / E115K / E131K、 E90N / E92N / D105A / E115K / E131K、 E90N / E92N / T102V / T104N / E115K / E131K、 E90N / E92N / T102N / T104N / E115K / E131K、 E90N / E92N / E113K / E115K / E129K / E131K、 E90C / E92N / T100N / E115K / E131K、 E90C / E92N / T102A / E115K / E131K、 E90C / E92N / T102V / E115K / E131K、 E90C / E92N / T102N / E115K / E131K、 E90C / E92N / T102Q / E115K / E131K、 E90C / E92N / T104A / E115K / E131K、 E90C / E92N / T104N / E115K / E131K、 E90C / E92N / D105A / E115K / E131K、 E90C / E92N / T102V / T104N / E115K / E131K、 E90C / E92N / T102N / T104N / E115K / E131K、 E90C / E92N / E113K / E115K / E129K / E131K、 E90N / E92C / T100N / E115K / E131K、 E90N / E92C / T102A / E115K / E131K、 E90N / E92C / T102V / E115K / E131K、 E90N / E92C / T102N / E115K / E131K、 E90N / E92C / T102Q / E115K / E131K、 E90N / E92C / T104A / E115K / E131K、 E90N / E92C / T104N / E115K / E131K、 E90N / E92C / D105A / E115K / E131K、 E90N / E92C / T102V / T104N / E115K / E131K、 E90N / E92C / T102N / T104N / E115K / E131K、 E90N / E92C / E113K / E115K / E129K / E131K、 E90C / E92C / T100N / E115K / E131K、 E90C / E92C / T102A / E115K / E131K、 E90C / E92C / T102V / E115K / E131K、 E90C / E92C / T102N / E115K / E131K、 E90C / E92C / T102Q / E115K / E131K、 E90C / E92C / T104A / E115K / E131K、 E90C / E92C / T104N / E115K / E131K、 E90C / E92C / D105A / E115K / E131K、 E90C / E92C / T102V / T104N / E115K / E131K、 E90C / E92C / T102N / T104N / E115K / E131K、 E90C / E92C / E113K / E115K / E129K / E131K、 E90C / E92P / T100N / E115K / E131K、 E90C / E92P / T102A / E115K / E131K、 E90C / E92P / T102V / E115K / E131K、 E90C / E92P / T102N / E115K / E131K、 E90C / E92P / T102Q / E115K / E131K、 E90C / E92P / T104A / E115K / E131K、 E90C / E92P / T104N / E115K / E131K、 E90C / E92P / D105A / E115K / E131K、 E90C / E92P / T102V / T104N / E115K / E131K、 E90C / E92P / T102N / T104N / E115K / E131K、 E90C / E92P / E113K / E115K / E129K / E131K, E90P / E92C / T100N / E115K / E131K, E90P / E92C / T102A / E115K / E131K, E90P / E92C / T102V / E115K / E131K, E90P / E92C / T102N / E115K / E131K, E90P / E92C / T102Q / E115K / E131K, E90P / E92C / T104A / E115K / E131K, E90P / E92C / T104N / E115K / E131K, E90P / E92C / D105A / E115K / E131K, E90P / E92C / T102V / T104N / E115K / E131K, E90P / E92C / T102N / T104N / E115K / E131K, or E90P / E92C / E113K / E115K / E129K / E131K.

[0111] The variant preferably contains at least the following mutations in SEQ ID NO:6: E90N, E92N, E90N / E92N, E90N / E92N / E115R / E131R, E90N / E92N / E113R / E115R / E129R / E131R, E90C, E90P, E92C, E92P, E90C / E92C, E90P / E92P, E90C / E92P, E90P / E92C, E90N / E92C, E90N / E92P, E90C / E92N, E90P / E92N, E90C / E92C / E115R / E131R、 E90C / E92P / E115R / E131R、 E90P / E92C / E115R / E131R、 E90C / E92C / E115R / E131R、 E90N / E92C / E115R / E131R、 E90N / E92P / E115R / E131R、 E90P / E92N / E115R / E131R、 E90C / E92N / E115R / E131R、 E90C / E92C / E113R / E115R / E129R / E131R、 E90C / E92P / E113R / E115R / E129R / E131R、 E90P / E92C / E113R / E115R / E129R / E131R、 E90P / E92P / E113R / E115R / E129R / E131R、 E90C / E92N / E113R / E115R / E129R / E131R、 E90P / E92N / E113R / E115R / E129R / E131R、 E90N / E92C / E113R / E115R / E129R / E131R、 E90N / E92P / E113R / E115R / E129R / E131R、 E90C、 E90P、 E92C、 E92P、 E90C / E92C、 E90P / E92P、 E90C / E92P、 E90P / E92C、 E90N / E92C、 E90N / E92P、 E90C / E92N、 E90P / E92N、 E90C / E92C / E115K / E131K、 E90C / E92P / E115K / E131K、 E90P / E92C / E115K / E131K、 E90C / E92C / E115K / E131K、 E90N / E92C / E115K / E131K、 E90N / E92P / E115K / E131K、 E90P / E92N / E115K / E131K、 E90C / E92N / E115K / E131K、 E90C / E92C / E113K / E115K / E129K / E131K、 E90C / E92P / E113K / E115K / E129K / E131K、 E90P / E92C / E113K / E115K / E129K / E131K、 E90P / E92P / E113K / E115K / E129K / E131K、 E90C / E92N / E113K / E115K / E129K / E131K、 E90P / E92N / E113K / E115K / E129K / E131K、 E90N / E92C / E113K / E115K / E129K / E131K、 E90N / E92P / E113K / E115K / E129K / E131K、 E90G / E92N / E115R / E131R、 E90A / E92N / E115R / E131R、 E90V / E92N / E115R / E131R、 E90L / E92N / E115R / E131R、 E90I / E92N / E115R / E131R、 E90F / E92N / E115R / E131R、 E90Y / E92N / E115R / E131R、 E90S / E92N / E115R / E131R、 E90T / E92N / E115R / E131R、 E90Q / E92N / E115R / E131R、 E90C / E92N / E115R / E131R、 E90P / E92N / E115R / E131R、 E90N / E92G / E115R / E131R、 E90N / E92A / E115R / E131R、 E90N / E92V / E115R / E131R、 E90N / E92L / E115R / E131R、 E90N / E92I / E115R / E131R、 E90N / E92F / E115R / E131R、 E90N / E92Y / E115R / E131R、 E90N / E92S / E115R / E131R、 E90N / E92T / E115R / E131R、 E90N / E92Q / E115R / E131R、 E90N / E92C / E115R / E131R、 E90N / E92P / E115R / E131R、 E90C / E92C / E115R / E131R、 E90P / E92P / E115R / E131R、 E90C / E92P / E115R / E131R、 E90P / E92C / E115R / E131R、 E90N / E92N / T100N / E115R / E131R、 E90N / E92N / T102A / E115R / E131R、 E90N / E92N / T102V / E115R / E131R、 E90N / E92N / T102N / E115R / E131R、 E90N / E92N / T102Q / E115R / E131R、 E90N / E92N / T104A / E115R / E131R、 E90N / E92N / T104N / E115R / E131R、 E90N / E92N / D105A / E115R / E131R、 E90N / E92N / T102V / T104N / E115R / E131R、 E90N / E92N / T102N / T104N / E115R / E131R、 E90C / E92N / T100N / E115R / E131R、 E90C / E92N / T102A / E115R / E131R、 E90C / E92N / T102V / E115R / E131R、 E90C / E92N / T102N / E115R / E131R、 E90C / E92N / T102Q / E115R / E131R、 E90C / E92N / T104A / E115R / E131R、 E90C / E92N / T104N / E115R / E131R、 E90C / E92N / D105A / E115R / E131R、 E90C / E92N / T102V / T104N / E115R / E131R、 E90C / E92N / T102N / T104N / E115R / E131R、 E90C / E92N / E113R / E115R / E129R / E131R、 E90N / E92C / T100N / E115R / E131R、 E90N / E92C / T102A / E115R / E131R、 E90N / E92C / T102V / E115R / E131R、 E90N / E92C / T102N / E115R / E131R、 E90N / E92C / T102Q / E115R / E131R、 E90N / E92C / T104A / E115R / E131R、 E90N / E92C / T104N / E115R / E131R、 E90N / E92C / D105A / E115R / E131R、 E90N / E92C / T102V / T104N / E115R / E131R、 E90N / E92C / T102N / T104N / E115R / E131R、 E90N / E92C / E113R / E115R / E129R / E131R、 E90C / E92C / T100N / E115R / E131R、 E90C / E92C / T102A / E115R / E131R、 E90C / E92C / T102V / E115R / E131R、 E90C / E92C / T102N / E115R / E131R、 E90C / E92C / T102Q / E115R / E131R、 E90C / E92C / T104A / E115R / E131R、 E90C / E92C / T104N / E115R / E131R、 E90C / E92C / D105A / E115R / E131R、 E90C / E92C / T102V / T104N / E115R / E131R、 E90C / E92C / T102N / T104N / E115R / E131R、 E90C / E92C / E113R / E115R / E129R / E131R、 E90C / E92P / T100N / E115R / E131R、 E90C / E92P / T102A / E115R / E131R、 E90C / E92P / T102V / E115R / E131R、 E90C / E92P / T102N / E115R / E131R、 E90C / E92P / T102Q / E115R / E131R、 E90C / E92P / T104A / E115R / E131R、 E90C / E92P / T104N / E115R / E131R、 E90C / E92P / D105A / E115R / E131R、 E90C / E92P / T102V / T104N / E115R / E131R、 E90C / E92P / T102N / T104N / E115R / E131R、 E90C / E92P / E113R / E115R / E129R / E131R、 E90P / E92C / T100N / E115R / E131R、 E90P / E92C / T102A / E115R / E131R、 E90P / E92C / T102V / E115R / E131R、 E90P / E92C / T102N / E115R / E131R、 E90P / E92C / T102Q / E115R / E131R、 E90P / E92C / T104A / E115R / E131R、 E90P / E92C / T104N / E115R / E131R、 E90P / E92C / D105A / E115R / E131R、 E90P / E92C / T102V / T104N / E115R / E131R、 E90P / E92C / T102N / T104N / E115R / E131R、 E90P / E92C / E113R / E115R / E129R / E131R、 E90G / E92N / E115K / E131K、 E90A / E92N / E115K / E131K、 E90V / E92N / E115K / E131K、 E90L / E92N / E115K / E131K、 E90I / E92N / E115K / E131K、 E90F / E92N / E115K / E131K、 E90Y / E92N / E115K / E131K、 E90S / E92N / E115K / E131K、 E90T / E92N / E115K / E131K、 E90Q / E92N / E115K / E131K、 E90C / E92N / E115K / E131K、 E90P / E92N / E115K / E131K、 E90N / E92G / E115K / E131K、 E90N / E92A / E115K / E131K、 E90N / E92V / E115K / E131K、 E90N / E92L / E115K / E131K、 E90N / E92I / E115K / E131K、 E90N / E92F / E115K / E131K、 E90N / E92Y / E115K / E131K、 E90N / E92S / E115K / E131K、 E90N / E92T / E115K / E131K、 E90N / E92Q / E115K / E131K、 E90N / E92N / E115K / E131K、 E90N / E92C / E115K / E131K、 E90N / E92P / E115K / E131K、 E90C / E92C / E115K / E131K、 E90P / E92P / E115K / E131K、 E90C / E92P / E115K / E131K、 E90P / E92C / E115K / E131K、 E90N / E92N / T100N / E115K / E131K、 E90N / E92N / T102A / E115K / E131K、 E90N / E92N / T102V / E115K / E131K、 E90N / E92N / T102N / E115K / E131K、 E90N / E92N / T102Q / E115K / E131K、 E90N / E92N / T104A / E115K / E131K、 E90N / E92N / T104N / E115K / E131K、 E90N / E92N / D105A / E115K / E131K、 E90N / E92N / T102V / T104N / E115K / E131K、 E90N / E92N / T102N / T104N / E115K / E131K、 E90N / E92N / E113K / E115K / E129K / E131K、 E90C / E92N / T100N / E115K / E131K、 E90C / E92N / T102A / E115K / E131K、 E90C / E92N / T102V / E115K / E131K、 E90C / E92N / T102N / E115K / E131K、 E90C / E92N / T102Q / E115K / E131K、 E90C / E92N / T104A / E115K / E131K、 E90C / E92N / T104N / E115K / E131K、 E90C / E92N / D105A / E115K / E131K、 E90C / E92N / T102V / T104N / E115K / E131K、 E90C / E92N / T102N / T104N / E115K / E131K、 E90C / E92N / E113K / E115K / E129K / E131K、 E90N / E92C / T100N / E115K / E131K、 E90N / E92C / T102A / E115K / E131K、 E90N / E92C / T102V / E115K / E131K、 E90N / E92C / T102N / E115K / E131K、 E90N / E92C / T102Q / E115K / E131K、 E90N / E92C / T104A / E115K / E131K、 E90N / E92C / T104N / E115K / E131K、 E90N / E92C / D105A / E115K / E131K、 E90N / E92C / T102V / T104N / E115K / E131K、 E90N / E92C / T102N / T104N / E115K / E131K、 E90N / E92C / E113K / E115K / E129K / E131K、 E90C / E92C / T100N / E115K / E131K、 E90C / E92C / T102A / E115K / E131K、 E90C / E92C / T102V / E115K / E131K、 E90C / E92C / T102N / E115K / E131K、 E90C / E92C / T102Q / E115K / E131K、 E90C / E92C / T104A / E115K / E131K、 E90C / E92C / T104N / E115K / E131K、 E90C / E92C / D105A / E115K / E131K、 E90C / E92C / T102V / T104N / E115K / E131K、 E90C / E92C / T102N / T104N / E115K / E131K、 E90C / E92C / E113K / E115K / E129K / E131K、 E90C / E92P / T100N / E115K / E131K、 E90C / E92P / T102A / E115K / E131K、 E90C / E92P / T102V / E115K / E131K、 E90C / E92P / T102N / E115K / E131K、 E90C / E92P / T102Q / E115K / E131K、 E90C / E92P / T104A / E115K / E131K、 E90C / E92P / T104N / E115K / E131K、 E90C / E92P / D105A / E115K / E131K、 E90C / E92P / T102V / T104N / E115K / E131K、 E90C / E92P / T102N / T104N / E115K / E131K、 E90C / E92P / E113K / E115K / E129K / E131K、 E90P / E92C / T100N / E115K / E131K、 E90P / E92C / T102A / E115K / E131K、 E90P / E92C / T102V / E115K / E131K、 E90P / E92C / T102N / E115K / E131K、 E90P / E92C / T102Q / E115K / E131K、 E90P / E92C / T104A / E115K / E131K, E90P / E92C / T104N / E115K / E131K, E90P / E92C / D105A / E115K / E131K, E90P / E92C / T102V / T104N / E115K / E131K, E90P / E92C / T102N / T104N / E115K / E131K, or E90P / E92C / E113K / E115K / E129K / E131K.

[0112] The variant preferably comprises or consists of the mutations set out immediately above for SEQ ID NO: 6. The variant may be any of the mutations set out in Table 3 or Table 4.

[0113] A variant of SEQ ID NO:6 is preferably not identical to SEQ ID NO:2 or 4 (ie, does not share 100% amino acid identity with SEQ ID NO:2 or 4).

[0114] variant In addition to the specific mutations discussed above, the variant may include other mutations. Over the entire length of the amino acid sequence of SEQ ID NO: 2, 4 or 6, the variant is preferably at least 70% homologous to the sequence based on amino acid identity. More preferably, the variant may be at least 75%, at least 80%, at least 85%, at least 90%, more preferably at least 95%, 97%, 98% or 99% homologous to the amino acid sequence of SEQ ID NO: 2, 4 or 6 over the entire sequence based on amino acid identity. Over the entire length of the amino acid sequence of SEQ ID NO: 2, 4 or 6, the variant will preferably be at least 75%, at least 80%, at least 85%, at least 90%, more preferably at least 95%, 97%, 98% or 99% identical to the amino acid sequence of SEQ ID NO: 2, 4 or 6 over the entire sequence. Reference to homology or identity over the entire sequence may be referred to as "global" homology or identity.

[0115] There may be at least 80%, such as at least 85%, 90% or 95% amino acid identity over a stretch of 100 or more, such as 125, 150, 175 or 200 or more contiguous amino acids ("hard homology").

[0116] The package provides the BESTFIT program, which can be used to calculate homology, for example, using its default settings (Devereux et al (1984) Nucleic Acids Research 12, p387-395). For example, the PILEUP and BLAST algorithms can be used to calculate homology or align sequences (identify equivalent residues or corresponding sequences, typically at their default settings), as described in Altschul SF (1993) J Mol Evol 36:290-300, Altschul, SF et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0117] Amino acid substitutions may be made to the amino acid sequence of SEQ ID NO: 2, 4, or 6 in addition to those discussed above (e.g., 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 as the amino acids they replace. Alternatively, conservative substitutions may introduce another amino acid that is aromatic or aliphatic in place of an existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and may be selected according to the properties of the 20 main amino acids defined in Table 1 above. If the amino acids have similar polarity, this may also be determined by referring to the hydrophobicity scale for amino acid side chains in Table 2.

[0118] One or more amino acid residues of the amino acid sequence of SEQ ID NO: 2, 4 or 6 may further be deleted from the above-mentioned polypeptides. Up to 1, 2, 3, 4, 5, 10, 20, 30 or more residues may be deleted.

[0119] Variants may include fragments of SEQ ID NO: 2, 4, or 6. 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 long. Such fragments may be used to generate pores of the invention. The fragments preferably include the pore-forming domain of SEQ ID NO: 2, 4, or 6. The fragments preferably include amino acids 9-200 of SEQ ID NO: 2, 4, or 6.

[0120] One or more amino acids may alternatively or additionally be added to the above mentioned polypeptides. The extension may be provided at the amino or carboxy terminus of the amino acid sequence of SEQ ID NO: 2 or a polypeptide variant or fragment thereof. The extension may be very short, for example 1-10 amino acids long. Alternatively, the extension may be longer, for example up to 50 or 100 amino acids. A carrier protein may be fused to the amino acid sequence according to the invention. Other fusion proteins are discussed in more detail below.

[0121] As discussed above, a variant is a polypeptide having an amino acid sequence different from SEQ ID NO: 2, 4 or 6 that retains the ability to form a pore. A variant typically contains a region of SEQ ID NO: 2, 4 or 6 that is involved in pore formation. A variant of SEQ ID NO: 2, 4 or 6 typically includes a region that forms a β-strand of SEQ ID NO: 2, 4 or 6. One or more modifications can be made to the region that forms a β-strand of SEQ ID NO: 2, 4 or 6, as long as the resulting variant retains the ability to form a pore. A variant of SEQ ID NO: 2, 4 or 6 preferably includes one or more modifications, such as substitutions, additions or deletions, in its α-helical and / or loop regions.

[0122] Constructs The present invention also provides a construct comprising two or more covalently linked monomers derived from Rhodococcus. The construct of the present invention retains the ability to form pores. One or more constructs of the present invention can be used to form pores for characterization of target analytes, such as sequencing of polypeptides or polynucleotides. The construct can comprise 2, 3, 4, 5, 6, 7, 8, 9 or 10 monomers. The two or more monomers can be the same or different.

[0123] The monomers do not have to be mutant monomers of the invention. For example, all of the monomers may comprise the sequence shown in SEQ ID NO: 2, 4 or 6. For example, at least one monomer may comprise the sequence shown in SEQ ID NO: 2, 4 or 6. Alternatively, at least one monomer may comprise a variant of SEQ ID NO: 2, 4 or 6 that is at least 50% homologous to SEQ ID NO: 2, 4 or 6 over its entire sequence based on amino acid identity, but does not comprise any of the specific mutations required by the mutant monomers of the invention. More preferably, the variant may be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, more preferably at least 95%, 97%, or 99% homologous to the amino acid sequence of SEQ ID NO: 2, 4 or 6 over its entire sequence based on amino acid identity. In a preferred embodiment, at least one monomer in the construct is a mutant monomer of the invention. All of the monomers in the construct may be mutant monomers of the invention. The mutant monomers may be the same or different. In a more preferred embodiment, the construct comprises two monomers, at least one of which is a mutant monomer of the invention.

[0124] The monomers are preferably genetically fused. The monomers are genetically fused if the entire construct is expressed from a single polynucleotide sequence. The coding sequences of the monomers can be combined in any manner to form a single polynucleotide sequence that encodes the construct.

[0125] Monomers may be genetically fused in any configuration. Monomers may be fused via their terminal amino acids. For example, the amino terminus of one monomer may be fused to the carboxy terminus of another monomer. When a construct is formed from the genetic fusion of two or more monomers, each containing the sequence shown in SEQ ID NO: 2, 4 or 6, or a variant thereof, the second and subsequent monomers (amino to carboxy direction) in the construct may contain methionines at their amino termini (each of which is fused to the carboxy terminus of the previous monomer). For example, when M is a monomer containing the sequence shown in SEQ ID NO: 2, 4 or 6, or a variant (not containing an amino terminal methionine), and mM is a monomer containing the sequence shown in SEQ ID NO: 2, 4 or 6, or a variant containing an amino terminal methionine, the construct may contain the sequence M-mM, M-mM-mM, or M-mM-mM-mM. The presence of these methionines typically results from the expression of a start codon (i.e., ATG) at the 5' end of the polynucleotide encoding the second or subsequent monomer within the polynucleotide encoding the entire construct. The first monomer (amino to carboxy direction) within the construct may also contain a methionine (e.g., mM-mM, mM-mM-mM, or mM-mM-mM-mM).

[0126] Two or more monomers can be genetically fused together directly. The monomers are preferably genetically fused using a linker. The linker can be designed to limit the mobility of the monomers. A preferred linker is an amino acid sequence (i.e., a peptide linker). Any of the peptide linkers discussed above can be used.

[0127] In another preferred embodiment, the monomer is chemically fused. The subunit is chemically fused to the enzyme when the two parts of the subunit and the enzyme are chemically linked, for example, via a chemical crosslinker. Any of the chemical crosslinkers discussed above can be used. The linker can be attached to one or more cysteine ​​residues introduced into the mutant monomer of the present invention. Alternatively, the linker can be attached to the end of one of the monomers in the construct.

[0128] When the construct contains different monomers, crosslinking of the monomer to itself can be prevented by keeping the concentration of the linker at a large amount of monomer. Alternatively, a "lock and key" configuration can be used in which two linkers are used. Only one end of each linker may react together to form a longer linker, and the other end of the linker reacts with a different monomer, respectively. Such linkers are described in International Application No. PCT / GB10 / 000132 (published as WO2010 / 086602).

[0129] Polynucleotides The present invention also provides a polynucleotide sequence encoding a mutant pore of the invention or a mutant monomer of the invention. The mutant pore or mutant monomer may be any of those discussed above. The polynucleotide sequence preferably comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, or 95% homologous based on nucleotide identity to the sequence of SEQ ID NO: 1, 3 or 5 over the entire sequence. The polynucleotide sequence preferably comprises a sequence that is at least 50%, 60%, 70%, 80%, 90%, or 95% identical to the sequence of SEQ ID NO: 1, 3 or 5 over the entire sequence. Reference to homology or identity over the entire sequence may be referred to as "global" homology or identity.

[0130] There may be at least 80%, e.g., at least 85%, 90%, or 95% nucleotide identity ("hard homology") over a stretch of 300 or more, e.g., 375, 450, 525, or 600 or more contiguous nucleotides. Homology may be calculated as described above. A polynucleotide sequence may include sequences that differ from SEQ ID NO: 1, 3, or 5 based on the degeneracy of the genetic code.

[0131] The present invention also provides polynucleotide sequences encoding any of the genetically fused constructs of the present invention. The polynucleotide preferably comprises two or more of the sequences shown in SEQ ID NO: 1, 3 or 5 or variants thereof as described above.

[0132] Polynucleotide sequences can be obtained and replicated using standard methods in the art. Chromosomal DNA encoding wild-type Msp can be extracted from pore-producing organisms such as Rhodococcus corynebacteroides. Genes encoding pore subunits can be amplified using PCR with specific primers. The amplified sequences can then be subjected to site-directed mutagenesis. Suitable methods for site-directed mutagenesis are known in the art, including, for example, combinatorial chain reaction. Polynucleotides encoding constructs of the invention can be made using well-known techniques, such as those described in Sambrook, J. and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[0133] The resulting polynucleotide sequence can then be incorporated into a recombinant replicable vector, such as a cloning vector. The vector can be used to replicate the polynucleotide in a compatible host cell. Thus, a polynucleotide sequence can be produced by introducing the polynucleotide into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions that allow replication of the vector. The vector can be recovered from the host cell. Host cells suitable for cloning polynucleotides are known in the art and are described in more detail below.

[0134] The polynucleotide sequence can be cloned into a suitable expression vector. In the expression vector, the polynucleotide sequence is typically operably linked to a control sequence that can provide the expression of the coding sequence by the host cell. Such an expression vector can be used to express the pore subunit.

[0135] The term "operably linked" refers to a juxtaposition where the components described are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is ligated in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequences. Multiple copies of the same or different polynucleotide sequences can be introduced into the vector.

[0136] The expression vector can then be introduced into a suitable host cell. Thus, the mutant monomer or construct of the present invention can be produced by inserting a polynucleotide sequence into an expression vector, introducing the vector into a compatible bacterial host cell, and growing the host cell under conditions that result in expression of the polynucleotide sequence. The recombinantly expressed monomer or construct can self-assemble into a pore in the host cell membrane. Alternatively, the recombinant pore produced in this manner can be removed from the host cell and inserted into another membrane. When producing a pore that includes at least two different subunits, the different subunits can be expressed separately in different host cells as described above, removed from the host cell, and assembled into a pore in a separate membrane, such as a rabbit cell membrane.

[0137] The vector may be, for example, a plasmid, virus or phage vector provided with an origin of replication, optionally a promoter for the expression of the polynucleotide sequence, and optionally a regulator of the promoter. The vector may contain one or more selectable marker genes, for example the tetracycline resistance gene. Promoters and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed. Typically, T7, trc, lac, ara or lambda L A promoter is used.

[0138] The host cell typically expresses the pore subunit at high levels. The host cell transformed with the polynucleotide sequence is selected to be compatible with the expression vector used to transform the cell. The host cell is typically a bacterium, preferably Escherichia coli. Any cell with a λ DE3 lysogen, such as C41(DE3), BL21(DE3), JM109(DE3), B834(DE3), TUNER, Origami and Origami B, can express vectors containing a T7 promoter. In addition to the conditions listed above, any of the methods cited in Proc Natl Acad Sci USA.2008 Dec.30;105(52):20647-52 may be used to express the protein.

[0139] pore The present invention also provides various pores, including those described above. The pores of the present invention are ideal for characterization of analytes, such as sequencing of polypeptides or polynucleotides, because the pores of the present invention can distinguish different amino acids or nucleotides with high sensitivity. The pores can surprisingly distinguish between four nucleotides in DNA and RNA. The pores of the present invention can even distinguish between methylated and unmethylated nucleotides. The base resolution of the pores of the present invention is surprisingly high. The pores show almost complete separation of all four DNA nucleotides. The pores further distinguish between deoxycytidine monophosphate (dCMP) and methyl-dCMP based on the residence time in the pore and the current flowing through the pore.

[0140] The pores of the present invention can also distinguish different amino acids or nucleotides under various conditions. In particular, the pores distinguish amino acids or nucleotides under conditions favorable for characterization of analytes, such as sequencing of polypeptides or polynucleotides. The degree to which the pores of the present invention can distinguish different amino acids or nucleotides can be controlled by varying the applied potential, salt concentration, buffer, temperature, and the presence of additives such as urea, betaine, and DTT. This allows the function of the pore to be fine-tuned, especially when sequencing. This is discussed in more detail below. The pores of the present invention can also be used to identify polypeptides or polynucleotides from interactions with one or more monomers, rather than amino acid or nucleotide units.

[0141] The pore of the present invention can be isolated, substantially isolated, purified, or substantially purified. The pore of the present invention is isolated or purified when it does not contain any other components such as lipids or other pores. The pore is substantially isolated when it is mixed with a carrier or diluent that does not interfere with its intended use. For example, the pore is substantially isolated or substantially purified when it exists in a form that contains less than 10%, less than 5%, less than 2%, or less than 1% of other components, such as lipids or other pores. Alternatively, the pore of the present invention can exist in a lipid bilayer.

[0142] The pores of the present invention may exist as individual or single pores. Alternatively, the pores of the present invention may exist in a homogeneous or heterogeneous population of two or more pores.

[0143] Homo-oligomer pores The present invention also provides homo-oligomeric pores comprising the same mutant monomers of the present invention. The homo-oligomeric pores of the present invention are ideal for characterization of analytes, for example sequencing of polypeptides or polynucleotides. The homo-oligomeric pores of the present invention may have any of the advantages discussed above.

[0144] The homo-oligomeric pore may contain any number of mutant monomers. The pore typically contains 2, 3, 4, 5, 6, 7, 8, 9 or 10 identical mutant monomers. The pore preferably contains 7 or 8 identical mutant monomers. One or more of the mutant monomers, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10, are preferably chemically modified as discussed above.

[0145] Methods for creating pores are discussed in more detail below.

[0146] Hetero-oligomeric pores The present invention also provides a hetero-oligomeric pore comprising at least one mutant monomer of the present invention, at least one of the monomers being different from the other monomers. The hetero-oligomeric pore of the present invention is ideal for characterization of analytes, for example, sequencing of polypeptides or polynucleotides. The hetero-oligomeric pore can be made using methods known in the art (e.g., Protein Sci. 2002 Jul.; 11(7): 1813-24).

[0147] Hetero-oligomeric pores contain enough monomers to form a pore. The monomers can be of any type. The pores typically contain 2, 3, 4, 5, 6, 7, 8, 9 or 10 monomers. The pores preferably contain 7 or 8 monomers.

[0148] The pore may comprise at least one monomer comprising (a) a sequence as shown in sequence 2, 4 or 6, or (b) a variant thereof that does not have the mutation required by the mutant monomer of the invention. Suitable variants are discussed above. In this embodiment, the remaining monomers are preferably mutant monomers of the invention. Thus, the pore may comprise 9, 8, 7, 6, 5, 4, 3, 2, or 1 mutant monomer of the invention.

[0149] In a preferred embodiment, the pore comprises (a) one mutant of the invention and (b) seven identical monomers, where the mutant monomer of (a) is different from the identical monomer of (b). The identical monomers of (b) preferably comprise (i) a sequence as shown in sequence 2, 4 or 6, or (ii) a variant thereof that does not have the mutation present in the mutant monomer of the invention.

[0150] In another preferred embodiment, all of the monomers (i.e. 10, 9, 8, 7, 6, 5, 4, 3 or 2 of the monomers) are mutant monomers of the invention, at least one of which is different from the other monomers. In a more preferred embodiment, the pore comprises 8 mutant monomers of the invention, at least one of which is different from the other monomers.

[0151] In all embodiments discussed above, one or more of the mutant monomers, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10, are preferably chemically modified as discussed above. The preferred pores (a)-(c) above are preferably chemically modified by attachment of a molecule to one or more of the introduced cysteines.

[0152] Methods for creating pores are discussed in more detail below.

[0153] Pore ​​containing construct The present invention also provides a pore comprising at least one construct of the present invention. The construct of the present invention comprises two or more covalently linked Rhodococcus porin monomers. In other words, the construct must contain more than one monomer. The pore contains enough constructs, and optionally monomers, to form the pore. For example, an octameric pore may contain (a) two constructs, each containing four monomers, or (b) one construct containing two monomers and six monomers that do not form part of the construct. At least two of the monomers in the pore are in the form of a construct of the present invention. The monomers can be of any type. The pore typically contains a total of 2, 3, 4, 5, 6, 7, 8, 9, or 10 monomers (at least two of which must be in the construct). The pore preferably contains 7 or 8 monomers (at least two of which must be in the construct).

[0154] The pore typically contains (a) one construct comprising two monomers, and (b) 1, 2, 3, 4, 5, 6, 7 or 8 monomers. The constructs may be any of those discussed above. The monomers may be any of those discussed above, including mutant monomers of the invention.

[0155] Another exemplary pore comprises more than one construct of the invention, such as two, three, or four constructs of the invention. Such pores further comprise sufficient monomers to form the pore. The monomers can be any of those discussed above. Further pores of the invention comprise only constructs comprising two monomers, for example, the pore may comprise 1, 2, 3, 4, 5, 6, 7, or 8 constructs comprising two monomers. A particular pore according to the invention comprises four constructs, each comprising two monomers. The constructs may oligomerize into a pore having a structure such that only one monomer from the construct contributes to the barrel or anterior chamber of the pore. Typically, the other monomers of the construct are outside the barrel or anterior chamber of the pore. For example, the pore of the invention may comprise 1, 2, 3, 4, 5, 6, 7, or 8 constructs comprising two monomers, and the barrel or anterior chamber comprises 8 monomers.

[0156] As described above, mutations can be introduced into the construct. The mutations can be alternating, i.e., the mutations are different for each monomer in the two monomer construct, and the construct is assembled as a homo-oligomer resulting in alternating modifications. In other words, monomers containing MutA and MutB are fused and assembled to form an AB:AB:AB:AB pore. Alternatively, the mutations can be adjacent, i.e., the same mutation is introduced into two monomers in the construct, which are then oligomerized with different mutant monomers. In other words, a monomer containing MutA is fused and subsequently oligomerized with a MutB-containing monomer to form AA:B:B:B:B:B:B.

[0157] One or more of the monomers of the invention within the pore containing the construct may be chemically modified as discussed below.

[0158] Methods for Producing Modified Proteins Methods for introducing or substituting non-naturally occurring amino acids are also well known in the art. For example, non-naturally occurring amino acids can be introduced by including synthetic aminoacyl-tRNA in the IVTT system used to express mutant monomers. Alternatively, they can be introduced by expressing mutant monomers in E. coli that are auxotrophic for a particular amino acid in the presence of a synthetic (i.e., non-naturally occurring) analog of the particular amino acid. They can also be generated by exposed ligation when mutant monomers are generated using partial peptide synthesis.

[0159] Any of the proteins described herein, particularly the mutant monomers of the invention, can be modified to aid in their identification or purification, for example, by the addition of histidine residues (his tag), aspartic acid residues (asp tag), streptavidin tag, flag tag, SUMO tag, GST tag, or MBP tag, or by the addition of signal sequences to facilitate secretion of the monomers or subunits from cells that do not naturally contain such sequences. An alternative to introducing genetic tags is to chemically react the tag onto a native or engineered location on the protein. One example of this would be to react a gel shift reagent to a cysteine ​​engineered onto the outside of the protein.

[0160] The protein or monomer may be labeled with a revealing label. The revealing label may be any suitable label that allows the monomer or subunit to be detected. Suitable labels include fluorescent molecules, radioisotopes, e.g. 125 I, 35 These include, but are not limited to, S, enzymes, antibodies, antigens, polynucleotides, and ligands such as biotin.

[0161] The protein or monomer may in one embodiment be produced using D-amino acids. For example, the protein or monomer may contain a mixture of L- and D-amino acids. This is conventional in the art for producing such proteins or peptides.

[0162] The protein or monomer may contain one or more specific modifications to facilitate the discrimination of amino acids or nucleotides. The protein or monomer may also contain other non-specific modifications, so long as they do not interfere with pore formation. Many non-specific side chain modifications are known in the art and can be made to the side chains of amino acids in the transmembrane protein nanopore and / or auxiliary proteins. Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH4, amidination with methylacetimidate, or acylation with acetic anhydride.

[0163] The proteins or monomers can be produced using standard methods known in the art. The proteins or monomers can be made synthetically or by recombinant means. For example, the proteins can be synthesized by in vitro translation and transcription (IVTT). The amino acid sequence of the protein can be modified to include non-naturally occurring amino acids or to increase the stability of the protein. If the protein is produced by synthetic means, such amino acids may be introduced during production. The protein can also be modified following either synthetic or recombinant production. Suitable methods for producing transmembrane protein nanopores are discussed in International Application Nos. 2010 / 004273, 2010 / 004265, or 2010 / 086603. Methods are known for inserting pores into membranes.

[0164] Polynucleotide sequences encoding proteins can be obtained and replicated using standard methods in the art. Polynucleotide sequences encoding proteins can be expressed in bacterial host cells using standard methods in the art. Proteins can be produced intracellularly by expressing the polypeptide in situ 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.

[0165] Proteins can be produced on a large scale from protein producing organisms following purification by any protein liquid chromatography system or following recombinant expression.Typical protein liquid chromatography systems include FPLC, AKTA systems, Bio-Cad systems, Bio-Rad BioLogic systems, and Gilson HPLC systems.

[0166] Two or more monomers may be covalently linked to one another. For example, 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 monomers or subunits may be covalently linked. The covalently linked monomers or subunits may be the same or different.

[0167] The monomers or subunits may be genetically fused, optionally via a linker, or chemically fused, for example via a chemical crosslinker. Methods for covalently linking monomers or subunits are disclosed in WO2017 / 149316, WO2017 / 149317, and WO2017 / 149318.

[0168] chemical modification In some embodiments, the mutant monomer is chemically modified. The mutant monomer may be chemically modified in any manner and at any site. The mutant monomer may be chemically modified, for example, by binding of a molecule to one or more cysteines (cysteine ​​binding), binding of a molecule to one or more lysines, binding of a molecule to one or more unnatural amino acids, enzymatic modification of an epitope, or modification of a terminal end. Suitable methods for carrying out such modifications are well known in the art. The mutant monomer may be chemically modified by binding of any molecule. For example, the mutant monomer may be chemically modified by binding of a dye or fluorophore.

[0169] In some embodiments, the mutant monomer is chemically modified with a molecular adaptor that facilitates interaction between a pore containing the monomer and a target analyte, such as a target amino acid, a target polypeptide, a target nucleotide, or a target polynucleotide sequence. The presence of the adaptor improves the host-guest chemistry of the pore and the target analyte, thereby improving the sequencing ability of the pore formed from the mutant monomer. The principles of host-guest chemistry are well known in the art. The adaptor affects the physical or chemical properties of the pore that improves the interaction with a target analyte, such as a target amino acid, a polypeptide, a nucleotide, or a polynucleotide. The adaptor may change the charge of the barrel or channel of the pore, or may specifically interact or bind with the target analyte, thereby facilitating its interaction with the pore.

[0170] The molecular adaptor is preferably a circular molecule, a cyclodextrin, a hybridizable species, a DNA binding or interchelating agent, a peptide or peptide analog, a synthetic polymer, an aromatic planar molecule, a small positively charged molecule, or a small molecule capable of hydrogen bonding.

[0171] The adaptor may be annular. The annular adaptor preferably has the same symmetry as the pore. Since a pore typically has 2-10 subunits about a central axis, the adaptor preferably has 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, or 10-fold symmetry. This is discussed in more detail above.

[0172] The adaptor typically interacts with the target analyte, e.g., an amino acid, a polypeptide, a nucleotide, or a polynucleotide, via host-guest chemistry. The adaptor typically can interact with the target analyte. The adaptor comprises one or more chemical groups capable of interacting with the target analyte. The one or more chemical groups preferably interact with the target analyte by non-covalent interactions, such as hydrophobic interactions, hydrogen bonds, van der Waals forces, π-cation interactions, and / or electrostatic forces. The one or more chemical groups capable of interacting with the target analyte are preferably positively charged. The one or more chemical groups capable of interacting with the target analyte more preferably comprise an amino group. The amino group may be attached to a primary, secondary, or tertiary carbon atom. The adaptor even more preferably comprises a ring of amino groups, such as a ring of 6, 7, or 8 amino groups. The adaptor most preferably comprises a ring of 8 amino groups. The protonated ring of amino groups may interact with a negatively charged phosphate group in the target analyte.

[0173] The correct positioning of the adaptor in the pore can be facilitated by host-guest chemistry between the adaptor and the pore containing the mutant monomer. The adaptor preferably comprises one or more chemical groups capable of interacting with one or more amino acids in the pore. The adaptor more preferably comprises one or more chemical groups capable of interacting with one or more amino acids in the pore via non-covalent interactions such as hydrophobic interactions, hydrogen bonds, van der Waals forces, π-cation interactions, and / or electrostatic forces. The chemical groups capable of interacting with one or more amino acids in the pore are typically hydroxyl or amine. The hydroxyl group may be attached to a primary, secondary, or tertiary carbon atom. The hydroxyl group may form hydrogen bonds with an uncharged amino acid of the pore. Any adaptor that facilitates the interaction between the pore and the target analyte, such as an amino acid, a polypeptide, a nucleotide, or a polynucleotide, may be used.

[0174] Suitable adaptors include, but are not limited to, cyclodextrin, cyclic peptides, and cucurbituril. The adaptor is preferably a cyclodextrin or a derivative thereof. The cyclodextrin or a derivative thereof can be any of those disclosed in Eliseev, AV, and Schneider, HJ. (1994) J. Am. Chem. Soc. 116, 6081-6088. The adaptor is more preferably heptakis-6-amino-β-cyclodextrin (am7-βCD), 6-monodeoxy-6-monoamino-β-cyclodextrin (am1-βCD), or heptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu7-βCD). The guanidino group of gu7-βCD has a much higher pKa than the primary amine of am7-βCD, and is therefore more positively charged. This gu7-βCD adaptor can be used to increase the residence time of amino acids or nucleotides within the pore, increasing the precision of the measured residual current and increasing the rate of base detection at high temperatures or low data acquisition rates.

[0175] When succinimidyl 3-(2-pyridyldithio)propionate (SPDP) crosslinker is used as discussed in more detail below, the adapter is preferably heptakis(6-deoxy-6-amino)-6-N-mono(2-pyridyl)dithiopropanoyl-β-cyclodextrin (am6amPDP1-βCD).

[0176] More suitable adaptors include γ-cyclodextrins containing 2, 3, 4, 5, 6, 7, 8, 9, or 10 sugar units. The γ-cyclodextrins may contain linker molecules or may be modified to include all or many of the modified sugar units used in the β-cyclodextrin example discussed above.

[0177] The molecular adaptor can be covalently linked to the mutant monomer. The adaptor can be covalently linked to the pore using any method known in the art. The adaptor is typically linked via chemical bonds. When the molecular adaptor is linked via a cysteine ​​bond, one or more cysteines are preferably introduced into the mutant (e.g., into the barrel) by substitution. The mutant monomer can be chemically modified by binding the molecular adaptor to one or more cysteines introduced into the mutant monomer, for example, by substitution.

[0178] The reactivity of cysteine ​​residues can be enhanced by modification of adjacent residues. For example, the basic group of an adjacent arginine, histidine, or lysine residue changes the pKa of the cysteine ​​thiol group to that of the more reactive S-group. The reactivity of cysteine ​​residues can be protected by thiol protecting groups such as dTNB. These can be reacted with one or more cysteine ​​residues of the mutant monomer before the linker is attached.

[0179] The molecule may be attached directly to the mutant monomer. The molecule is preferably attached to the mutant monomer using a linker, such as a chemical crosslinker or a peptide linker.

[0180] Suitable chemical cross-linkers are well known in the art. Preferred cross-linkers include 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldisulfanyl)propanoate, 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldisulfanyl)butanoate, and 2,5-dioxopyrrolidin-1-yl 8-(pyridin-2-yldisulfanyl)octananoate. The most preferred cross-linker is succinimidyl 3-(2-pyridyldithio)propionate (SPDP). Typically, the molecule is covalently attached to the bifunctional cross-linker before the molecule / cross-linker complex is covalently attached to the mutant monomer, although it is also possible to covalently attach the bifunctional cross-linker to the monomer before the bifunctional cross-linker / monomer complex is attached to the molecule. Suitable examples of peptide linkers are defined above.

[0181] The linker is preferably resistant to dithiothreitol (DTT). Suitable linkers include, but are not limited to, iodoacetamide-based and maleimide-based linkers.

[0182] In other embodiments, the mutant monomers may be attached to a polynucleotide binding protein, forming a modular sequencing system that may be used in the sequencing methods of the invention. The polynucleotide binding protein may be a nucleic acid handling enzyme, such as a translocase, polymerase, or helicase.

[0183] The polynucleotide binding protein is preferably covalently linked to the mutant monomer. The protein can be covalently linked to the monomer using any method known in the art. The monomer and the protein can be chemically fused or genetically fused. If the entire construct is expressed from a single polynucleotide sequence, the monomer and the protein are genetically fused. Genetic fusion of monomers to polynucleotide binding proteins is discussed in WO2010 / 004265.

[0184] When the polynucleotide binding protein is bound via a cysteine ​​bond, one or more cysteines are preferably introduced into the mutant by substitution. One or more cysteines are preferably introduced into loop regions that have low conservation among homologues, indicating that mutations or insertions can be tolerated. Thus, they are suitable for binding the polynucleotide binding protein. The reactivity of cysteine ​​residues can be enhanced by the above-mentioned modifications.

[0185] The polynucleotide binding protein may be attached to the mutant monomer directly or via one or more linkers. The molecule may be attached to the mutant monomer using a hybridization linker as described in WO2010 / 086602. Alternatively, a peptide linker may be used. A peptide linker is an amino acid sequence. The length, flexibility, and hydrophilicity of the peptide linker are typically designed so that it does not interfere with the function of the monomer and the molecule. A preferred flexible peptide linker is a stretch of 2-20 serine and / or glycine amino acids, such as 4, 6, 8, 10, or 16. More preferred flexible linkers include (SG)1, (SG)2, (SG)3, (SG)4, (SG)5, and (SG)8, where S is serine and G is glycine. A preferred rigid linker is a stretch of 2-30 proline amino acids, such as 4, 6, 8, 16, or 24. More preferred rigid linkers include (P) 12 wherein P is proline.

[0186] The mutant monomers can be chemically modified with molecular adaptors and polynucleotide binding proteins.

[0187] Method for producing pores The present invention also provides methods for producing the pores of the invention in vivo and in vitro. One embodiment provides a method for producing the pores of the invention via co-expression. The method comprises expressing the necessary monomers in a suitable host cell to allow pore formation in vivo.

[0188] Another method for producing the pores of the present invention relates to the in vitro reconstitution of the above-mentioned monomers to obtain functional pores. The method includes a step of contacting the necessary monomers in a suitable system to allow complex formation. The system may be an "in vitro system", which refers to a system that includes at least the necessary components and environment to carry out the method, utilizes biomolecules, organisms, cells (or parts of cells) outside their normal naturally occurring environment, and allows for more detailed, more convenient, or more efficient analysis than can be performed with whole organisms. The in vitro system may also include a suitable buffer composition provided in a test tube, to which the protein components for forming the complex are added. The skilled person is aware of the options for providing the system.

[0189] Some or all of the monomers can be tagged to facilitate purification. Purification can also be performed when the monomers are not tagged. 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.

[0190] kit The present invention also provides a kit for characterizing a target polynucleotide or target polypeptide, the kit comprising a Rhodococcus pore, preferably an isolated Rhodococcus pore, and a polynucleotide-binding protein or a polypeptide-handling enzyme.

[0191] The Rhodococcus pore may be a wild-type pore. The pore may be formed from monomers comprising the sequence shown in SEQ ID NO: 2, 4 or 6. The pore may comprise 2, 3, 4, 5, 6, 7, 8, 9 or 10 monomers, preferably 2, 3, 4, 5, 6, 7, 8, 9 or 10 identical monomers comprising the sequence shown in SEQ ID NO: 2, 4 or 6. The pore is preferably a pore of the invention and may be any of the pores described above, including mutant pores, hetero-oligomeric pores, homo-oligomeric pores and construct-containing pores.

[0192] The polynucleotide binding protein may be any of those discussed above. The polynucleotide binding protein or polypeptide handling enzyme may be bound to the pore as discussed above.

[0193] The kit preferably further comprises a membrane component. The kit may comprise any type of membrane, such as an amphiphilic layer or a triblock copolymer membrane component. The kit may further comprise one or more anchors, such as cholesterol, for coupling a target polynucleotide to the membrane. The kit may further comprise one or more polynucleotide adaptors that can bind to a target polynucleotide to facilitate characterization of the polynucleotide. In one embodiment, an anchor, such as cholesterol, is bound to the polynucleotide adaptor.

[0194] The kit may further comprise one or more other reagents or equipment that allows for carrying out any of the above embodiments. Such reagents or equipment include one or more of the following: suitable buffer(s) (aqueous solution), means for obtaining a sample from a subject (such as an equipment including a container or needle), means for amplifying and / or expressing a polynucleotide, or voltage or patch clamp apparatus. The reagents may be present in the kit in a dry state, such that a fluid sample resuspends the reagents. The kit may also optionally comprise instructions for use to enable the kit to be used in the method of the invention, or details regarding the organisms for which the method may be used. Finally, the kit may also comprise additional components useful for characterization of the analyte.

[0195] Device The present invention also provides an apparatus for characterizing a target polynucleotide or target polypeptide in a sample. The apparatus comprises a plurality of Rhodococcus pores, preferably a plurality of isolated Rhodococcus pores, and a plurality of polynucleotide-binding proteins or a plurality of polypeptide-handling enzymes. The Rhodococcus pores can be any of those discussed above with reference to the kit of the present invention, including wild-type pores. The Rhodococcus pores are preferably pores of the present invention and can be any of the pores described above, including mutant pores, hetero-oligomeric pores, homo-oligomeric pores, and construct-containing pores. The polynucleotide-binding proteins can be any of those discussed above. The polynucleotide-binding proteins or polypeptide-handling enzymes can be bound to the pores as discussed above.

[0196] The invention also provides a device comprising a transmembrane protein pore inserted into an in vitro membrane, the transmembrane protein pore comprising at least one monomer which is a variant of a Rhodococcus porin monomer and which comprises a mutation at one or more of positions (a), (b) and / or (c) corresponding to the sequence shown in SEQ ID NO: 2. Positions (a), (b) and / or (c) with respect to SEQ ID NO: 2 are discussed above with reference to the pores and monomers of the invention.

[0197] The invention also provides a device produced by a method comprising: (i) obtaining a transmembrane protein pore comprising at least one Rhodococcus porin monomer, the monomer comprising an amino acid mutation at one or more of positions (a), (b) and / or (c) corresponding to the sequence shown in SEQ ID NO: 2; and (ii) contacting the pore with an in vitro membrane such that the pore is inserted into the in vitro membrane. Positions (a), (b) and / or (c) with respect to SEQ ID NO: 2 are discussed above with reference to the pores and monomers of the invention.

[0198] The invention also provides a device comprising a transmembrane protein pore inserted into an in vitro membrane, the transmembrane protein pore comprising at least one monomer which is a variant of a Rhodococcus porin monomer and which comprises a mutation at one or more of positions (a), (b) and / or (c) corresponding to the sequence shown in SEQ ID NO: 4. Positions (a), (b) and / or (c) with respect to SEQ ID NO: 4 are discussed above with reference to the pores and monomers of the invention.

[0199] The invention also provides a device produced by a method comprising: (i) obtaining a transmembrane protein pore comprising at least one Rhodococcus porin monomer, the monomer comprising an amino acid mutation at one or more of positions (a), (b) and / or (c) corresponding to the sequence shown in SEQ ID NO: 4; and (ii) contacting the pore with an in vitro membrane such that the pore is inserted into the in vitro membrane. Positions (a), (b) and / or (c) with respect to SEQ ID NO: 4 are discussed above with reference to the pores and monomers of the invention.

[0200] The invention also provides a device comprising a transmembrane protein pore inserted into an in vitro membrane, the transmembrane protein pore comprising at least one monomer which is a variant of a Rhodococcus porin monomer and which comprises a mutation at one or more of positions (a), (b) and / or (c) corresponding to the sequence shown in SEQ ID NO: 6. Positions (a), (b) and / or (c) with respect to SEQ ID NO: 6 are discussed above with reference to the pores and monomers of the invention.

[0201] The invention also provides a device produced by a method comprising: (i) obtaining a transmembrane protein pore comprising at least one Rhodococcus porin monomer, the monomer comprising an amino acid mutation at one or more of positions (a), (b) and / or (c) corresponding to the sequence shown in SEQ ID NO: 6; and (ii) contacting the pore with an in vitro membrane such that the pore is inserted into the in vitro membrane. Positions (a), (b) and / or (c) with respect to SEQ ID NO: 6 are discussed above with reference to the pores and monomers of the invention.

[0202] Any of the specific embodiments discussed above, particularly with respect to the pores and monomers of the present invention, are equally applicable to the devices of the present invention.

[0203] Pore-containing membrane The present invention also provides a membrane comprising a Rhodococcus pore, preferably an isolated Rhodococcus pore. The pores are preferably present in the membrane and together form a transmembrane pore. The membrane may comprise any type of membrane, such as an amphiphilic layer or a triblock copolymer membrane component. The membrane may further comprise a polynucleotide binding protein or a polypeptide handling enzyme bound to the pore. The membrane may further comprise one or more anchors for coupling a polynucleotide or a polypeptide to the membrane. The Rhodococcus pore may be any of those discussed above with reference to the kit of the present invention, including wild-type pores. The Rhodococcus pore is preferably a pore of the present invention and may be any of the pores described above, including mutant pores, hetero-oligomeric pores, homo-oligomeric pores, and construct-containing pores.

[0204] array The invention also provides an array comprising a plurality of the membranes of the invention. Any of the embodiments discussed above with respect to the membranes of the invention apply equally to the arrays of the invention. The arrays may be configured to carry out any of the methods described below.

[0205] In a preferred embodiment, each membrane in the array contains one pore. Depending on the manner in which the array is formed, for example, the array may contain one or more membranes that contain no pores and / or one or more membranes that contain two or more pores. The array may contain from about 2 to about 1000, e.g., from about 10 to about 800, from about 20 to about 600, or from about 30 to about 500 membranes.

[0206] system The invention provides a system comprising: (a) a membrane of the invention, or an array of the invention; (b) means for applying an electrical potential across the membrane(s); and (c) means for detecting an electrical or optical signal across the membrane(s).

[0207] The present invention also provides a system comprising: (a) a membrane or membranes comprising a Rhodococcus pore; (b) means for applying an electrical potential across the membrane(s); and (c) means for detecting an electrical or optical signal across the membrane(s). The Rhodococcus pore may be any of those discussed above with reference to the kit of the present invention, including wild-type pores. The Rhodococcus pore is preferably a pore of the present invention, and may be any of the pores described above, including mutant pores, hetero-oligomeric pores, homo-oligomeric pores, and construct-containing pores.

[0208] The pores and membranes can be any of those described herein above.

[0209] In one embodiment, the system further comprises a first chamber and a second chamber, the first and second chambers being separated by a membrane(s). When used to characterize a target analyte, the system may further comprise a target analyte, the target analyte being temporarily located in the continuous channel, one end of the target analyte being located in the first chamber and one end of the target analyte being located in the second chamber. The target analyte is preferably a target polypeptide or a target polynucleotide.

[0210] In one embodiment, the system further comprises a conductive solution in contact with the pore(s), electrodes providing a potential across the membrane(s), and a measurement system for measuring the current through the pore(s). In one embodiment, the voltage applied to the membrane and pore is +5V to -5V, for example -600mV to +600mV or -400mV to +400mV. The voltage used is preferably in the range of 100mV to 240mV, more preferably in the range of 120mV to 220mV. By using an increased applied potential, it is possible to increase the discrimination between different amino acids or nucleotides by the pore. Any suitable conductive solution can be used. For example, the solution may contain charge carriers such as metal salts, for example alkali metal salts, halide salts, for example chloride salts, for example alkali metal chloride salts. The charge carriers may include ionic liquids or organic salts, such as tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride, or 1-ethyl-3-methylimidazolium chloride. In an exemplary system, the salt is present in an aqueous solution in the chamber. Potassium chloride (KCl), sodium chloride (NaCl), cesium chloride (CsCl), or a mixture of potassium ferrocyanide and potassium ferricyanide are typically used. KCl, NaCl, and a mixture of potassium ferrocyanide and potassium ferricyanide are preferred. The charge carriers may be asymmetric across the membrane. For example, the type and / or concentration of the charge carriers may be different on each side of the membrane, for example in each chamber.

[0211] The salt concentration may be saturating. The salt concentration may be 3M or less, typically 0.1-2.5M, 0.3-1.9M, 0.5-1.8M, 0.7-1.7M, 0.9-1.6M, or 1M-1.4M. The salt concentration is preferably 150mM-1M. The method is preferably carried out using a salt concentration of at least 0.3M, such as at least 0.4M, at least 0.5M, at least 0.6M, at least 0.8M, at least 1.0M, at least 1.5M, at least 2.0M, at least 2.5M, or at least 3.0M. A high salt concentration provides a high signal-to-noise ratio, allowing identification of a current indicative of the presence of an amino acid or nucleotide against a background of normal current fluctuations.

[0212] A buffer may be present in the conductive solution. Typically, the buffer is a phosphate buffer. Other suitable buffers are HEPES and Tris-HCl buffers. The pH of the conductive solution may be 4.0-12.0, 4.5-10.0, 5.0-9.0, 5.5-8.8, 6.0-8.7, or 7.0-8.8, or 7.5-8.5. The pH used is preferably about 7.5.

[0213] The system may be included in a device. The device may be any conventional device for analyte analysis, such as an array or chip. The device is preferably configured to perform the disclosed method. For example, the device may comprise a chamber containing an aqueous solution and a barrier dividing the chamber into two compartments. The barrier typically has an opening in which a membrane(s) containing pore(s) is formed. Alternatively, the barrier forms a membrane in which the pores are present.

[0214] The device may also include electrical circuitry capable of applying an electrical potential and measuring the electrical signal across the membrane and pore.

[0215] The device may be any of those described in WO2008 / 102120, WO2009 / 077734, WO2010 / 122293, WO2011 / 067559, or WO00 / 28312.

[0216] film Any suitable membrane can be used in the system. The membrane is preferably an amphiphilic layer. An amphiphilic layer is a layer formed from amphiphilic molecules, such as phospholipids, that have both hydrophilic and lipophilic properties. The amphiphilic molecules can be synthetic or naturally occurring. Non-naturally occurring amphiphiles and amphiphiles that form monolayers are known in the art, including, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). Block copolymers are polymeric materials in which two or more monomer subunits polymerized together make a single polymer chain. Block copolymers typically have properties contributed by each monomer subunit. However, block copolymers can have unique properties that polymers formed from individual subunits do not have. Block copolymers can be engineered such that in aqueous media, one of the monomer subunits is hydrophobic (i.e., lipophilic) and the other subunit(s) is hydrophilic. In this case, the block copolymer may have amphiphilic properties and may form structures that mimic biological membranes. The block copolymer may be diblock (consisting of two monomer subunits), but may also be constructed from more than two monomer subunits to form more complex arrangements that behave as amphiphiles. The copolymer may be a triblock, tetrablock, or pentablock copolymer. The membrane is preferably a triblock copolymer membrane.

[0217] Archaeal bipolar tetraether lipids are naturally occurring lipids that are constructed such that the lipids form monolayer membranes. These lipids are commonly found in extremophiles, thermophiles, halophiles, and acidophiles that survive in harsh biological environments. Their stability is believed to derive from the fused nature of the final bilayer. It is straightforward to construct block copolymer materials that mimic these biological entities by creating triblock polymers with a general motif of hydrophilic-hydrophobic-hydrophilic. This material can form monomeric membranes that behave similarly to lipid bilayers and encompass a range of phase behaviors from vesicles to lamellar membranes. Membranes formed from these triblock copolymers hold several advantages over biological lipid membranes. As the triblock copolymers are synthesized, the exact structure can be carefully controlled to provide the correct chain length and properties required to form membranes and interact with pores and other proteins.

[0218] Block copolymers may also be constructed from subunits that are not classified as lipid submaterials, for example, hydrophobic polymers may be made from siloxanes or other non-hydrocarbon monomers. The hydrophilic subcompartments of block copolymers may also have low protein binding properties, allowing for the creation of membranes that are highly resistant when exposed to untreated biological samples. The head group units may be derived from non-classical lipid head groups.

[0219] Triblock copolymer membranes also have increased mechanical and environmental stability, e.g., much higher operating temperature or pH ranges, compared to biological lipid membranes. The synthetic nature of block copolymers provides a platform for customizing polymer-based membranes for a wide range of applications.

[0220] The membrane is most preferably one of the membranes disclosed in WO 2014 / 064443 or WO 2014 / 064444.

[0221] The amphiphilic molecules may be chemically modified or functionalized to facilitate coupling of polynucleotides. 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.

[0222] Amphiphilic membranes are typically mobile in nature, with a molecular weight of approximately 10 -8 cm s -1 The membrane essentially behaves as a two-dimensional fluid with a lipid diffusion rate of 100 s, which means that pores and attached polynucleotides can typically move within the amphiphilic membrane.

[0223] The membrane may be a lipid bilayer. Lipid bilayers are models of cell membranes and serve as an excellent platform for a range of experimental studies. For example, lipid bilayers can be used for in vitro investigation of membrane proteins by single channel recording. Alternatively, lipid bilayers can be used as biosensors to detect the presence of a range of substances. The lipid bilayer may be any lipid bilayer. Suitable lipid bilayers include, but are not limited to, planar lipid bilayers, supported bilayers or liposomes. The lipid bilayer is preferably a planar lipid bilayer. Suitable lipid bilayers are disclosed in WO2008 / 102121, WO2009 / 077734, and WO2006 / 100484.

[0224] Methods for forming lipid bilayers are known in the art. Lipid bilayers are usually formed by the method of Montal and Mueller (Proc. Natl. Acad. Sci. USA., 1972; 69: 3561-3566), in which a lipid monolayer is carried on the aqueous solution / air interface through either side of an opening that is perpendicular to the interface. Lipids are usually first dissolved in an organic solvent, then added to the surface of an aqueous electrolyte solution by evaporating a drop of the solvent on the surface of the aqueous solution on either side of the opening. As the organic solvent evaporates, the solution / air interface on either side of the opening physically moves up and down through the opening until a bilayer is formed. Planar lipid bilayers can be formed across the opening of the membrane, or across the opening into a recess.

[0225] The Montal & Mueller method is popular because it is a cost-effective and relatively simple method for forming good quality lipid bilayers suitable for protein pore insertion. Other common methods of bilayer formation include tip dipping, painting of bilayers, and patch clamping of liposomal bilayers.

[0226] Tip-dipping bilayer formation involves contacting an aperture surface (e.g., a pipette tip) onto the surface of a test solution carrying a lipid monolayer. Again, a lipid monolayer is first generated at the solution / air interface by evaporating a droplet of lipid dissolved in an organic solvent at the solution surface. The bilayer is then formed by the Langmuir-Schaefer process, which requires mechanical automation to move the aperture relative to the solution surface.

[0227] In coated bilayers, a drop of lipid dissolved in an organic solvent is applied directly to the aperture, which is then immersed in an aqueous test solution. A paintbrush or equivalent is used to spread the lipid solution thinly over the aperture. Thinning the solvent results in the formation of a lipid bilayer. However, it is difficult to completely remove the solvent from the bilayer, and as a result, bilayers formed by this method are less stable and more prone to noise during electrochemical measurements.

[0228] Patch clamping is commonly used in the study of biological cell membranes. The cell membrane is clamped to the end of a pipette by suction, and a patch of membrane is attached over the opening. The method has been adapted to generate lipid bilayers by clamping liposomes, which are then ruptured, leaving behind a lipid bilayer that covers and seals the opening of the pipette. The method requires the fabrication of stable giant unilamellar liposomes and a small opening in a material with a glass surface.

[0229] Liposomes can be formed by sonication, extrusion, or the Mozafari method (Colas et al. (2007) Micron 38:841-847).

[0230] In a preferred embodiment, the lipid bilayer is formed as described in WO2009 / 077734. Advantageously, in this method, the lipid bilayer is formed from dry lipids. In a most preferred embodiment, the lipid bilayer is formed across the opening as described in WO2009 / 077734.

[0231] A lipid bilayer is formed from two opposing layers of lipids. The two layers of lipids are arranged so that their hydrophobic tail groups face each other to form a hydrophobic interior. The hydrophilic head groups of the lipids face outward toward the aqueous environment on each side of the bilayer. Bilayers can exist in a number of lipid phases, including but not limited to liquid disordered phases (fluid lamella), liquid ordered phases, solid ordered phases (lamellar gel phase, interdigitated gel phase), and planar bilayer crystals (lamellar subgel phase, lamellar crystal phase).

[0232] Any lipid composition that forms a lipid bilayer may be used. The lipid composition is selected so that a lipid bilayer is formed with the required properties, such as surface charge, ability to support membrane proteins, packing density, or mechanical properties. The lipid composition may contain one or more different lipids. For example, the lipid composition may contain up to 100 lipids. The lipid composition preferably contains 1 to 10 lipids. The lipid composition may contain naturally occurring lipids and / or artificial lipids.

[0233] A lipid typically comprises a head group, an interface moiety, and two hydrophobic tail groups, which may be the same or different. Suitable head groups include, but are not limited to, neutral head groups, such as diacylglyceride (DG) and ceramide (CM), zwitterionic head groups, such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), and sphingomyelin (SM), negatively charged head groups, such as phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidic acid (PA), and cardiolipin (CA), and positively charged head groups, such as trimethylammonium-propane (TAP). Suitable interface moieties include, but are not limited to, naturally occurring interface moieties, such as glycerol-based moieties or ceramide-based moieties. Suitable hydrophobic tail groups include, but are not limited to, saturated hydrocarbon chains such as lauric acid (n-Dodecanolic acid), myristic acid (n-Tetradecononic acid), palmitic acid (n-Hexadecanoic acid), stearic acid (n-Octadecanoic acid), and arachidic acid (n-Eicosanoic acid), unsaturated hydrocarbon chains such as oleic acid (cis-9-Octadecanoic acid), and branched hydrocarbon chains such as phytanoyl. The chain length and the position and number of double bonds of the unsaturated hydrocarbon chains can vary. The chain length and the position and number of branches such as methyl groups of the branched hydrocarbon chains can vary. The hydrophobic tail group can be linked to the interface moiety as an ether or ester. The lipid can be a mycolic acid.

[0234] Lipids can also be chemically modified.The head group or tail group of lipids can be chemically modified.Suitable lipids with chemically modified head groups include, but are not limited to, PEG-modified lipids, such as 1,2-diacyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], functionalized PEG lipids, such as 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethylene glycol)2000], and lipids modified for conjugation, such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(succinyl) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(biotinyl). Suitable lipids with chemically modified tail groups include, but are not limited to, polymerizable lipids such as 1,2-bis(10,12-tricosadiynoyl)-sn-glycero-3-phosphocholine, fluorinated lipids such as 1-palmitoyl-2-(16-fluoropalmitoyl)-sn-glycero-3-phosphocholine, deuterated lipids such as 1,2-dipalmitoyl-D62-sn-glycero-3-phosphocholine, and ether-linked lipids such as 1,2-di-O-phytanyl-sn-glycero-3-phosphocholine. Lipids can be chemically modified or functionalized to facilitate coupling of polynucleotides.

[0235] Amphiphilic layer, for example, lipid composition, typically contains one or more additives that will affect the properties of the layer.Suitable additives include, but are not limited to, fatty acid, for example, palmitic acid, myristic acid, and oleic acid, fatty alcohol, for example, palmitic alcohol, myristic alcohol, and oleic alcohol, sterol, for example, cholesterol, ergosterol, lanosterol, sitosterol, and stigmasterol, lysophospholipid, for example, 1-acyl-2-hydroxy-sn-glycero-3-phosphocholine, and ceramide.

[0236] In another preferred embodiment, the membrane comprises a solid-state layer. The solid-state layer can be formed from both organic and inorganic materials, including but not limited to microelectronic materials, insulating materials such as Si3N4, A12O3, and SiO, organic and inorganic polymers such as polyamides, plastics such as Teflon® or elastomers such as two-component addition cured silicone rubber, and glass. The solid-state layer may be formed from graphene. A suitable graphene layer is disclosed in WO2009 / 035647. When the membrane comprises a solid-state layer, the pores are typically present in an amphiphilic membrane or layer contained within the solid-state layer, for example, in holes, wells, gaps, channels, trenches, or slits within the solid-state layer. Those skilled in the art can prepare suitable solid-state / amphiphilic hybrid systems. Suitable systems are disclosed in WO2009 / 020682 and WO2012 / 005857. Any of the amphiphilic membranes or layers discussed above may be used.

[0237] The method is typically carried out using (i) an artificial amphiphilic layer containing a pore, (ii) an isolated naturally occurring lipid bilayer containing a pore, or (iii) a cell with a pore inserted therein. The method is typically carried out using an artificial amphiphilic layer, such as an artificial triblock copolymer layer. In addition to the pore, the layer may contain other transmembrane and / or intramembrane proteins, as well as other molecules. Suitable equipment and conditions are discussed below. The method of the present invention is typically carried out in vitro.

[0238] Methods for characterizing an analyte The present invention also provides a method for determining the presence, absence, or one or more characteristics of a target analyte. The method includes contacting a target analyte with a Rhodococcus pore such that the target analyte moves relative to the pore, for example, into or through the pore, respectively, and performing one or more measurements as the analyte moves relative to the pore, thereby determining the presence, absence, or one or more characteristics of the analyte. The Rhodococcus pore can be any of those discussed above with reference to the kit of the present invention, including wild-type pores. The Rhodococcus pore is preferably a pore of the present invention and can be any of the pores described above, including mutant pores, hetero-oligomeric pores, homo-oligomeric pores, and construct-containing pores. The analyte may pass through the pore constriction. The pore may be in a membrane.

[0239] In one embodiment, 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 analyte. The method may relate to determining the presence, absence or one or more characteristics of two or more analytes. The method may include determining the absence or one or more characteristics of any number of analytes, for example 2, 5, 10, 15, 20, 30, 40, 50, 100 or more analytes. Any number of characteristics of one or more analytes may be determined, for example 1, 2, 3, 4, 5, 10 or more characteristics.

[0240] The binding of a molecule in the channel of the pore or near any opening of the channel affects the open channel ion flow through the pore, which is the essence of the "molecular sensing" of the pore channel. In a manner similar to nucleic acid sequencing applications, the variation of the open channel ion flow can be measured using a suitable measurement technique by changing the current (e.g., WO2000 / 28312 and D. Stoddart et al., Proc. Natl. Acad. Sci., 2010, 106, 7702-7 or WO2009 / 077734). The degree of the reduction in the ion flow, as measured by the reduction in the current, is related to the size of the obstacle in or near the pore. Thus, the binding of a molecule of interest, also called "analyte", in or near the pore provides a detectable and measurable event, thereby forming the basis of a "biological sensor". Molecules suitable for nanopore sensing include nucleic acids, proteins, peptides, polysaccharides, and small molecules (herein referring to organic or inorganic compounds of low molecular weight (e.g., <900 Da or <500 Da)), such as pharmaceuticals, toxins, cytokines, and pollutants. Detecting the presence of biomolecules finds applications in personalized drug development, medicine, diagnostics, life science research, environmental monitoring, and the security and / or defense industries.

[0241] The target analyte may be a metal ion, an inorganic salt, a polymer, an amino acid, a peptide, a polypeptide, a protein, a nucleotide, an oligonucleotide, a polynucleotide, a 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 is preferably or comprises a polypeptide, a polynucleotide, or a polysaccharide. The target analyte may be a metabolite.

[0242] The method may relate to determining the presence, absence, or one or more characteristics of two or more analytes of the same type, such as two or more proteins, two or more nucleotides, or two or more pharmaceuticals. Alternatively, the method may relate to determining the presence, absence, or one or more characteristics of two or more different types of analytes, such as one or more proteins, one or more nucleotides, and one or more pharmaceuticals.

[0243] The target analyte may be secreted from the cell. Alternatively, the target analyte may be an analyte that is present inside the cell, whereby the analyte must be extracted from the cell before the method can be performed.

[0244] In one embodiment, the analyte is an amino acid, a peptide, a polypeptide, or a protein. The amino acid, peptide, polypeptide, or protein may be naturally occurring or non-naturally occurring. The polypeptide or protein may include synthetic or modified amino acids therein. Several different types of modifications to amino acids are known in the art. Suitable amino acids and their modifications are described above. It should be understood that the target analyte may be modified by any method available in the art. The method preferably includes: (i) contacting the polypeptide with a polypeptide handling enzyme capable of controlling the movement of the polypeptide relative to the pore; and (ii) performing one or more measurements characteristic of the polypeptide as the polypeptide moves relative to the pore.

[0245] In a preferred embodiment, the analyte is a polynucleotide, such as a nucleic acid. The method preferably comprises: (i) contacting the polynucleotide with a polynucleotide-binding protein capable of controlling the movement of the polynucleotide relative to the pore; and (ii) performing one or more measurements characteristic of the polynucleotide as the polynucleotide moves relative to the pore. A polynucleotide is defined as a macromolecule that comprises two or more nucleotides. Naturally occurring nucleic acid bases in DNA and RNA may be distinguished by their physical size. When a nucleic acid molecule or individual bases pass through the channel of a nanopore, the difference in size between the bases directly correlates to a reduction in the ion flow through the channel. The fluctuations in the ion flow can be recorded. Suitable electrical measurement techniques for recording the fluctuations in the ion flow are described, for example, in WO2000 / 28312 and D.Stoddart et al., Proc.Natl.Acad.Sci.,2010,106,pp7702-7 (single channel recording device), and, for example, in WO2009 / 077734 (multi-channel recording technique). With suitable calibration, the characteristic reduction in ion current can be used to identify in real time the specific nucleotide and associated base passing through the channel. In a typical nanopore nucleic acid sequencing, the open channel ion current is reduced as the individual nucleotides of the nucleotide sequence of interest pass through the nanopore channel in succession due to partial blocking of the channel by the nucleotide. It is this reduction in ion current that is measured using the suitable recording technique described above. The reduction in ion current can be calibrated to the reduction in ion current measured for known nucleotides passing through the channel, which provides a means to determine which nucleotide passes through the channel, and thus, when performed sequentially, provides a way to determine the nucleotide sequence of the nucleic acid passing through the nanopore. In order to accurately determine individual nucleotides, it is typically necessary that the reduction in ion current through the channel is directly correlated to the size of the individual nucleotide passing through the constriction (or "leading head").It will be appreciated that sequencing can be performed on an intact nucleic acid polymer that has been "threaded" into the pore, for example, via the action of an associated polymerase or helicase. Alternatively, the sequence can be determined by the passage of nucleotide triphosphates successively removed from the target nucleic acid adjacent the pore (see, for example, WO2014 / 187924).

[0246] A polynucleotide or nucleic acid may contain any combination of any nucleotides. Nucleotides may be naturally occurring or artificial. One or more nucleotides in a polynucleotide may be oxidized or methylated. One or more nucleotides in a polynucleotide may be damaged. For example, a polynucleotide may contain pyrimidine dimers. Such dimers are typically associated with UV damage and are the main cause of skin melanoma. One or more nucleotides in a polynucleotide may be modified, for example by a label or tag, suitable examples of which are known to those skilled in the art. A polynucleotide may contain one or more spacers. A nucleotide typically contains a nucleobase, a sugar, and at least one phosphate group. The nucleobase and the sugar form a nucleoside. The nucleobase is typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines, more specifically adenine (A), guanine (G), thymine (T), uracil (U), and cytosine (C). The sugar is typically a pentose sugar. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar is preferably deoxyribose. The polynucleotide preferably comprises the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and / or thymidine (dT), deoxyguanosine (dG), and deoxycytidine (dC). The nucleotide is typically a ribonucleotide or a deoxyribonucleotide. The nucleotide typically contains a monophosphate, a diphosphate, or a triphosphate. The nucleotide may contain four or more phosphates, such as four or five phosphates. The phosphates may be attached to the 5' or 3' side of the nucleotide. The nucleotides of the polynucleotide may be attached to each other in any manner. The nucleotides are typically attached by their sugar and phosphate groups, as in nucleic acids. The nucleotides may be connected through their nucleobases, as in pyrimidine dimers. The polynucleotide may be single-stranded or double-stranded.At least a portion of the polynucleotide is preferably double-stranded. The polynucleotide is most preferably ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In particular, the method of alternatively using the polynucleotide as an analyte includes determining one or more characteristics selected from (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide, and (v) whether the polynucleotide is modified.

[0247] The polynucleotide can be of any length (i). For example, the polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotides or nucleotide pairs long. The polynucleotide can be 1000 nucleotides or nucleotide pairs long or more, 5000 nucleotides or nucleotide pairs long or more, or 100000 nucleotides or nucleotide pairs long or more. Any number of polynucleotides can be investigated. For example, the method can concern the characterization of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more polynucleotides. When two or more polynucleotides are characterized, they can be different polynucleotides or two instances of the same polynucleotide. The polynucleotide can be naturally occurring or artificial. For example, the method can be used to verify the sequence of a manufactured oligonucleotide. The method is typically carried out in vitro.

[0248] The nucleotide may have identity (ii) and may include, but is not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), 5-methylcytidine monophosphate, 5-hydroxymethylcytidine monophosphate, cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP), deoxycytidine monophosphate (dCMP), and deoxymethylcytidine monophosphate. The nucleotide is preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP, and dUMP. The nucleotides may be abasic (i.e., lack a nucleobase). The nucleotides may also lack a nucleobase and a sugar (i.e., are C3 spacers). The sequence (iii) of the nucleotides is determined by the consecutive identities of the following nucleotides linked together in the 5' to 3' direction of the strand throughout the polynucleotide strand:

[0249] The pores described herein are particularly useful for the analysis of homopolymers.For example, the pores can be used to determine the sequence of polynucleotides that contain two or more identical, for example at least 3, 4, 5, 6, 7, 8, 9, or 10 consecutive nucleotides.For example, the pores can be used to determine the sequence of polynucleotides that contain polyA, polyT, polyG, and / or polyC regions.

[0250] In a preferred embodiment, the target analyte comprises a polynucleotide-polypeptide conjugate, and the method comprises (i) contacting the conjugate with a polynucleotide binding protein capable of controlling movement of the polynucleotide of the conjugate relative to the pore, and (ii) performing one or more measurements characteristic of the polypeptide as the conjugate moves relative to the pore.

[0251] Although specific embodiments, specific configurations, and materials and / or molecules have been discussed herein for the engineered cells and methods according to the present invention, it should be understood that various changes or modifications in form and detail can be made without departing from the scope and spirit of the present invention. The following examples are provided to better illustrate specific embodiments, and they should not be considered as limiting applications. This application is limited only by the claims.

[0252] The following examples illustrate the invention. EXAMPLES

[0253] Example 1 method E. coli pore formation DNA encoding the mature forms of PorARr, PorBRr and PorARc proteins was synthesized by GENEWIZ Germany GmbH and Twist Bioscience plc.USA and cloned into pT7 vector containing ampicillin resistance gene. DNA concentration was adjusted to 400ng / μL.

[0254] Plasmid DNA was thawed at room temperature and mixed by gently pipetting up and down. Chemically competent Lemo21(DE3) E.coli cells were thawed on ice. 1 μl of DNA at 400 ng / μl was added to the cells and mixed by flicking and vortexing the tube. This was then left on ice for 30 minutes before the cells were heat shocked at 42°C for 10 seconds. The cells were then left on ice for 5 minutes. 250 μl of SOC (Sigma, S1797) medium pre-warmed to 37°C was added to the cells and left at 37°C for 1 hour with shaking at 250 rpm. 75ul of the grown cell culture was then plated on a large LB agar plate containing 100 μg / ml ampicillin and 41 μg / ml chloramphenicol and then incubated at 37°C overnight.

[0255] A single colony of transformed Lemo21(DE3) cells was picked and inoculated into 100 ml of LB medium with 100 μg / ml ampicillin and 41 μg / ml chloramphenicol. This starter culture was incubated overnight at 37° C. and 250 rpm in a 500 ml flask. A main medium of 500 ml of LB medium containing 100 μg / ml ampicillin and 41 μg / ml chloramphenicol was set up in a 2.5 1 flask. This was then spiked with 4 ml of the starter culture (diluted 1:125) and the cells were allowed to split at 37° C. and 250 rpm until an OD of 0.6 was reached. Once an OD of 0.6 was reached, the incubator temperature was reduced to 18° C. and the cells were induced with 0.2 mM IPTG (final concentration in the medium). The cells were incubated overnight at 18° C. and 250 rpm to allow overexpression of the protein of interest. Finally, the cells were harvested by spinning at 6000 g for 20 min at 4°C.

[0256] The cell paste was weighed to calculate the appropriate volume of functional lysis buffer to be prepared (cells are resuspended in 100 ml of lysis buffer per 10 g of paste). The required amount of functional lysis buffer was prepared by adding Benzonase (10 μl / 100 ml, Sigma), a tablet of EDTA-free protease inhibitor cocktail (1 tablet / 100 ml, Sigma) and 10x BugBuster Protein Extraction Reagent (10 ml / 100 ml, Sigma) to a buffer containing 50 mM Tris / HCl, 150 mM NaCl, 2 mM EDTA, 0.1% DDM. The pH of the resulting functional lysis buffer was then adjusted to pH 8 at room temperature. The cells were resuspended in the functional lysis buffer and mixed with a magnetic stirrer for 1 hour at room temperature to obtain a homogenous mixture. The cells were then lysed by sonication (Soniprep150, 10 cycles of 10 sec on / 20 sec off, 10 μm amplitude) on ice and stirred at room temperature for 3.5 h. The cell extract was then transferred to a 100 ml Beckman tube and centrifuged at 40,000 g for 35 min at room temperature. The resulting supernatant was then filtered using a sterile 0.2 μm high vacuum filter (Nalgene Rapid-Flow, Sigma).

[0257] Affinity chromatography The filtered supernatant was loaded onto a 5 mL StrepTrap HP column (Fisher, 10571680). The column was washed with 20 column volumes (CV) of mobile phase A (25 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.1% DDM, pH 8.0). The column was then washed with 7 CV of 25 mM Tris, 2 M NaCl, 2 mM EDTA, 0.1% DDM, pH 8.0 buffer before being returned to mobile phase A. Elution was performed by running 7 CV of mobile phase B buffer (25 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.1% DDM, 10 mM desthiobiotin pH 8.0) through the column.

[0258] Anion exchange chromatography The resulting fractions containing the affinity chromatography eluate were pooled together and diluted 5-fold with AEX mobile phase A (25 mM Tris, 0.1% DDM, pH 9.0) to reduce the salt concentration in the sample. The resulting Strep-purified sample was then subsequently loaded onto a POROS HQ10 2 ml column (ThermoFisher). The column was then washed with 20 CV of AEX mobile phase A before being eluted with a 0-50% gradient over 35 CV with 2 M NaCl, where AEX mobile phase B consisted of 25 mM Tris, 2 M NaCl, 0.1% DDM, pH 9.0.

[0259] Fractions of interest were screened using non-denaturing Native-PAGE. Fractions containing the protein of interest in the correct oligomeric state were pooled together and heated to 90°C for 15 min to denature misfolded proteins. The heated samples were then centrifuged at 21,130g for 10 min at room temperature to pellet the denatured proteins. The supernatant was retained and used for electrophysiological recordings.

[0260] Electrophysiology Data Electrical measurements were taken from PorARr, PorBRr, and PorARc wild-type and mutant nanopores inserted into a MinION flow cell. After inserting a single nanopore into the block copolymer membrane, 1 mL of buffer containing 25 mM calcium phosphate, 150 mM potassium ferrocyanide(II), 150 mM potassium ferricyanide(III), pH 8.0 was flowed through the system to remove any excess protein nanopores.

[0261] The analyte used to assess DNA irregular curvature was a 3.6 kilobase ssDNA segment from the 3' end of the lambda genome. Analyte preparation, ligation of the analyte to the Y-adapter, SPRI-bead cleanup of the ligated analyte, and loading onto the MinION flow cell were performed using the Oxford Nanopore Technologies Q-SQK-LSK109 protocol.

[0262] Electrical measurements were taken using a MinION Mk1b from Oxford Nanopore Technologies. Standard sequencing scripts were run at -180 mV for 1–6 h with a static flick (voltage back to +100 mV) every 5 min to remove elongation nanopore blocks. Raw data were collected into bulk FAST5 files using MinKNOW software (Oxford Nanopore Technologies).

[0263] result Wild-type PorARr, PorBRr and PorARc pores were tested with the following results: PorARr Some pores were observed Some of the pores observed appeared to be stable. No DNA capture PorBRr Somewhat more pores were observed than in PorARr, but still not many. -Many pore gates No DNA capture PorARc Many pores were observed The pore has several gatings Two populations of pores were observed No DNA capture

[0264] The results obtained from pores containing mutated PorARr, PorBRr and PorARc monomers are shown in Table 3. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7]

[0265] Example 2 - Additional PorBr Data method E. coli pore production DNA encoding the mature form of the PorBRr protein (SEQ ID NO: 6 with the substitutions shown in Table 4 below) was synthesized by GENEWIZ Germany GmbH and Twist Bioscience plc.USA and cloned into a pT7 vector containing an ampicillin resistance gene. The DNA concentration was adjusted to 400 ng / μL.

[0266] Plasmid DNA was thawed at room temperature and mixed by gently pipetting up and down. Chemically competent Lemo21(DE3) E.coli cells were thawed on ice. 1 μl of DNA at 400ng / μl was added to the cells and mixed by flicking and vortexing the tube. This was then left on ice for 30 minutes before the cells were heat shocked at 42°C for 10 seconds. The cells were then left on ice for 5 minutes. 150 μl of SOC (Sigma, S1797) medium pre-warmed to 37°C was added to the cells and left at 37°C for 1 hour with shaking at 250 rpm. 75ul of the grown cell culture was then plated on a large LB agar plate containing 100 μg / ml ampicillin, 41 μg / ml chloramphenicol and 0.2% glucose and then incubated at 37°C overnight.

[0267] A single colony of transformed Lemo21(DE3) cells was picked and inoculated into 100 ml of LB medium with 100 μg / ml ampicillin, 41 μg / ml chloramphenicol and 0.2% glucose. This starter culture was incubated overnight at 37° C. and 250 rpm in a 500 ml flask. A main medium of 500 ml of LB medium containing 100 μg / ml ampicillin, 41 μg / ml chloramphenicol and 0.2% glucose was set up in a 2.5 1 flask. This was then spiked with 4 ml of the starter culture (diluted 1:125) and the cells were split at 37° C. and 250 rpm until an OD of 0.6 was reached. Once OD of 0.6 was reached, the incubator temperature was reduced to 18° C. and the cells were induced with 0.2 mM IPTG (final concentration in the medium). The cells were incubated overnight at 18° C. and 250 rpm to allow overexpression of the protein of interest. Finally, the cells were harvested by spinning at 6000 g for 20 min at 4° C.

[0268] The cell paste was weighed to calculate the appropriate volume of functional lysis buffer to be prepared (cells are resuspended in 100 ml of lysis buffer per 10 g of paste). The required amount of functional lysis buffer was prepared by adding Benzonase (10 μl / 100 ml, Sigma), a tablet of EDTA-free protease inhibitor cocktail (1 tablet / 100 ml, Sigma) and 10x BugBuster Protein Extraction Reagent (10 ml / 100 ml, Sigma) to a buffer containing 50 mM Tris / HCl, 150 mM NaCl, 2 mM EDTA, 0.1% DDM. The pH of the resulting functional lysis buffer was then adjusted to pH 8 at room temperature. The cells were resuspended in the functional lysis buffer and mixed with a magnetic stirrer for 1 hour at room temperature to obtain a homogenous mixture. Cells were then lysed by sonication on ice (Soniprep150, 10 cycles of 10 sec on / 20 sec off, 10 μm amplitude) and agitation at room temperature for 3.5 h. The cell extract was then transferred to a 100 ml Beckman tube and centrifuged at 40,000 g for 35 min at room temperature. The resulting supernatant was then filtered using a sterile 0.2 μm high vacuum filter (Nalgene Rapid-Flow, Sigma).

[0269] Affinity chromatography The filtered supernatant was loaded onto a 5 mL StrepTrap HP column (Fisher, 10571680). The column was washed with 20 column volumes (CV) of mobile phase A (25 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.1% DDM, pH 8.0). The column was then washed with 7 CV of 25 mM Tris, 2 M NaCl, 2 mM EDTA, 0.1% DDM, pH 8.0 buffer before being returned to mobile phase A. Elution was performed by running 7 CV of mobile phase B buffer (25 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.1% DDM, 10 mM desthiobiotin pH 8.0) through the column.

[0270] Anion exchange chromatography The resulting fractions containing the affinity chromatography eluate were pooled together and diluted 5-fold with AEX mobile phase A (25 mM Tris, 0.1% DDM, pH 9.0) to reduce the salt concentration in the sample. The resulting Strep-purified sample was then subsequently loaded onto a POROS HQ10 2 ml column (ThermoFisher). The column was then washed with 20 CV of AEX mobile phase A before being eluted with a 0-50% gradient over 35 CV with 2 M NaCl, where AEX mobile phase B consisted of 25 mM Tris, 2 M NaCl, 0.1% DDM, pH 9.0.

[0271] Fractions of interest were screened using non-denaturing Native-PAGE. Fractions containing the protein of interest in the correct oligomeric state were pooled together and used for electrophysiological recordings.

[0272] Electrophysiology Data Electrical measurements were taken from PorBRr mutant nanopores inserted into a MinION flow cell. After inserting a single pore into the block copolymer membrane, 1 mL of buffer containing 25 mM calcium phosphate, 150 mM potassium ferrocyanide(II), 150 mM potassium ferricyanide(III), pH 8.0 was flowed through the system to remove any excess protein nanopores.

[0273] The analyte used to assess DNA irregular curvature was a 3.6 kilobase ssDNA segment from the 3' end of the lambda genome. Analyte preparation, ligation of the analyte to the Y-adapter, SPRI-bead cleanup of the ligated analyte, and loading onto the MinION flow cell were performed using the Oxford Nanopore Technologies Q-SQK-LSK109 protocol.

[0274] Electrical measurements were taken using a MinION Mk1b from Oxford Nanopore Technologies. Standard sequencing scripts were run at -180 mV for 1-6 hours, with a static flick (voltage back to +100 mV) every 5 minutes to remove extension nanopore blocks. Raw data was collected in bulk FAST5 files using MinKNOW software (Oxford Nanopore Technologies). Collected data was filtered for a small population of protein channels, taking into account that the signal criteria were better than those from other pore populations present. The median number of chains was 6000 across all pore variants. The results are shown in Figure 22 and Table 4 below. In both Figure 22 and Table 4, PorBRr-WT-E90N-E92N-E115R-E131R_ONLZ16955 (bold in Table 4) can be considered to be the baseline. All of the variants in Table 4 are improved in terms of one or more of SNR, range, median SD and normalized MAD current compared to PorBRr-WT-E90N-E92N-E115R-E131R_ONLZ16955. [Table 4-1] [Table 4-2] [Table 4-3] Sequence Listing SEQ ID NO:1 catatggcggttgatgatagcaacagcgttgtggatggtggcggtaacacgatcaccgtgagtcagagcgacaccttcatcaacagcgtgtttccgctggatggtagcccactgacccgcgaatggtttcataacggtcgcgccatcgttgatgtt accggtccggatgccgaagatttcagcggcaccgtgaccatcggctaccaagttggctatccagccagtctgggtggcgaactgacctttgactacaccaccccgggtctggaactgggtatcagcagcgcgggtgccgttagtggcacgctgagc gatgttctgccacaagccggcgttggcgttacgctggaaccgggcccgggtatcgaaaccgttgaagttgcgagtggtgcggccagtggcgcgcataccgaaatccagatcgccaatctgcacggcaccgcgacgaaaatcgccggcaatgttagc gtgcgcccatacgtgcaagttgttagcgataatggcgatgtggcgaccacctttggtcagccttggcggttcaatggtagtggtggtgagaatctgtattttcagggttctggtagtggttctgcgtggtcacatcctcaattcgagaaataatga கார்க்கை MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGELTFDYTTPGLEGISSAGAVSGTLSDVLPQAGVGVTLEPGPGIETVEVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSDNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK அக்க்கியுக்க்கு3 catatggtggtagatgatcaaaatcgtattgtctcagacggccatgaagtaatcgtaactcaagaagatacttttatccagggagttccgccaattggtggatcacctctttcgcgcgagtggttccataacggacgtgggatcgtaatattgtagggccggatgccgcagatttcgagggatcgacgttcca atttgggtatcaatttgcctggagcggcagtttagatgggagcattggggtcgcatggtcttctccgcaggccgaacttgaagttgctccgggggaagtctatgaacccgccaccgagactaagacagtagacgggaaagagacggaggtccccgtcacagctccggttttgaatgaggatggctcgcctattt tggacgccgacgggaaaccagttactgagcagctttatcgtatcaagccggacgcaacggcgacggagaatacgttgacggttggaggtttattaccacaacttacagctggcattgagcttactcccgctcccggcattgaggaacttgtggtggctgagggcgaatttgacggcgatttcaaatctgttcaa atcgccaatgtacacggagcggccacaggtgtcattggagctgtgagtgtccgcccctttgtgcgtgtcattactgccaacggggataatgtaactacttatggtaaggtttggacgctgggctctggggagagaatctttattccaaggctccggcagcggttccgcttggtcacatcctcagtttgagaaa sequence number 4 MVVDDQNRIVSDGHEVIVTQEDTFIQGVPPIGGSPLSREWFHNGRGIANIVGPDAADFEGSTFQFGYQFAWSGSLDGSIGVAWSSPQAELEVAPGEVYEPATETKTVDGKETEVPVTAPVLNEDGSPILDADGKPVTEQLYRIKPDATATENTLTVGGLLPQLTAGIELTPAPGIELVVAEGEFDGDFKSVQIANVHGAATGVIGAVSVRPFVRVITANGDNVTTYGKVWTLGSGGENLYFQGSGSGSHPQFEK sequence number 5 catatggctgtggacgaccagaatcgcatcgtgagcaccgatggttacgaggtggtggttacccaagaagatacgttcatccaaggcgttccagcgctgggtggtagtccgttcaaccgcgagttcttccacaatggtcgcggcaccgccaatct gttggtgccgatgccgcggatgccgaaggtaccacctttcagttcggctaccagtttgcgtgggcgggtagcattgatggcgcgatcggcgttacctatagcaccccgggtctggaactggaactgagcgccaaagatggcccgaccgcgaccgtt accgatattctgccacaagcctatgcggaactcgaactgaccccagcgccgggcatcgaagaactggtggttgcggagggtgaattcgacggcgatttcaagagcgtgcagttcagcaacgtgcatggtaccgccagcggtgttctgggtgcggtt caagttcgcccatttgtgcgcgcgatcaccgcgaatggcgacaacgttacaacctatggcaaaccttggaccgttggtagtggtggtgagaatctgtattttcagggttctggtagtggttctgcgtggtcacatcctcaattcgagaaataatga அக்க்கை MAVDDQNRIVSTDGYEVVVTQEDTFIQGVPALGGSPFNREFFHNGRGTANLVGADAADAEGTFQFGYQFAWAGSIDGAIGVTYSTPGLELELSAKDGPTATVTDILPQAYAELELTPAPGIEELVVAEGEFDGDFKSVQFSNVHGTASGVLGAVQVRPFVRAITANGDNVTTYGKPWTVGSGGENLYFQGSGSGSAWSHPQFEK

Claims

1. 2. A mutant Rhodococcus pore comprising a monomer which is a variant of the sequence shown in SEQ ID NO:2, said variant being located at the following position: (a) D11, G12, G13, G14, N15, T52, G53, P54, D55, A56, E57, D58, F59, S60, G61, T62, T64, Y67, Q68, V69, Y71, P119, G120, I121, E122, T123, V124, E125, V126, S128, A130, A131, S132, G133, A134, H135, L143, H144, T146, T148, K149, Y159, Q161, V163, S164, D165, N166, G167, D168, V169, and T171, (b) S74, G76, E78, T80, D82, T84, Q108, G110, G112, T114, E116, and P117, and / or (c) one or more mutations in one or more of G87, E89, G91, S93, S94, A95, G96, A97, S99, T101, S103, and D104; A mutant Rhodococcus pore, wherein said one or more mutations alter the ability of said monomer to interact with a target analyte.

2. 2. The mutant pore of claim 1, wherein the variant comprises one or more mutations at one or more of the positions: (a), (b), (c), (a) and (b), (b) and (c), (a) and (c), or (a), (b) and (c).

3. The variants are at least at the following positions: E78 / D82 / E89, E78 / D82 / E89 / E116 / E125 / D165, E57 / E78 / D82 / E89 / E116 / E125 / D165, D55 / E57 / D58 / E78 / D82 / E89 / E116 / E125 / D165, E78 / D82 / E89 / D104 / E116 / E122 / E125 / D165, D55 / E57 / D58 / E78 / D82 / E89 / D104 / E116 / E122 / E125 / D165, E57 / E78 / D82 / E89 / D104 / E116 / E122 / E125 / D165, E78 / D82 / E89 / T101 / E116 / E125 / D165, E78 / D82 / G87 / E89 / E116 / E125 / D165, E78 / D82 / E89 / G91 / I92 / E116 / E125 / D165, E78 / D82 / E89 / I92 / E116 / E125 / D165, E78 / D82 / E89 / S99 / E116 / E125 / D165, E78 / D82 / E89 / S103 / E116 / E125 / D165, E78 / D82 / E89 / D104 / E116 / E125 / D165, E78 / D82 / E89 / E116 / E122 / E125 / D165, E78 / D82 / E89 / G91 / I92 / E116 / E125 / D165, or 3. The mutant pore of claim 1 or 2, comprising one or more mutations at E78 / D82 / E89 / E116 / E125 / D165.

4. 2. The mutant pore of any one of the preceding claims, wherein the one or more mutations each independently (a) change the size of the amino acid residue at the modified position; (b) change the net charge of the amino acid residue at the modified position; (c) change the hydrogen bonding characteristics of the amino acid residue at the modified position; (d) introduce one or more chemical groups that interact via a delocalized electronic π-system to the amino acid residue at the modified position or remove one or more chemical groups that interact via a delocalized electronic π-system from the amino acid residue at the modified position; and / or (e) change the structure of the amino acid residue at the modified position.

5. 2. The mutant pore according to any one of the preceding claims, wherein the one or more mutations are one or more substitutions.

6. The one or more mutations are D to Q, S or Y, more preferably D to S, G to S, N, Q, T or A, more preferably G to S or N; N to S, Q, L, Y, M, I, V or G, T to V, S, N, Q, R, F or I, more preferably T to V, S, N, Q or R, From P to S, A to R, V, D, N, S or T, E to S, N, T, A, R, G, V, L, I, F, Q or K, F to R, K, S, T, N, Q, G, A, V, L, I, P, C, Y or W, S to N or C, more preferably S to N; Y to R, K, S, T, N, Q, G, A, V, L, I, P, C, D, E, F, or W; From Q to R, From V to I, I to G, L, S, F, V, A, P, W, Q, N, C, S, Y, K or T, more preferably I to N, E to R, H to F, N, Q or A, L through N, S, Y, V, Q, T, R, A, G, C, F, or D, and 2. The mutant pore of any one of the preceding claims, wherein K is selected from R, S, T, N, Q, G, A, V, L, I, P, C, D, E, F, Y or W.

7. The variant has at least the following mutations: E78S / D82S / E89N, E78S / D82S / E89N / E116T / E125A / D165S, E78S / D82S / E89R, E78R / D82S / E89R, E78R / D82S / E89R / E116T / E125A / D165S, E57R / E78R / D82S / E89R / E116T / E125A / D165S, D55S / E57S / D58S / E78R / D82S / E89R / E116T / E125A / D165S, E78R / D82S / E89R / D104S / E116T / E122S / E125A / D165S, D55S / E57S / D58S / E78R / D82S / E89R / D104S / E116T / E122S / E125A / D165S, E57R / E78R / D82S / E89R / D104S / E116T / E122S / E125A / D165S, E78R / D82S / E89G / E116T / E125A / D165S, E78R / D82S / E89A / E116T / E125A / D165S, E78R / D82S / E89V / E116T / E125A / D165S, E78R / D82S / E89L / E116T / E125A / D165S, E78R / D82S / E89I / E116T / E125A / D165S, E78R / D82S / E89F / E116T / E125A / D165S, E78R / D82S / E89S / E116T / E125A / D165S, E78R / D82S / E89T / E116T / E125A / D165S, E78R / D82S / E89Q / E116T / E125A / D165S, E78R / D82S / E89K / E116T / E125A / D165S, E78R / D82S / E89R / T101V / E116T / E125A / D165S, E78R / D82S / E89R / T101S / E116T / E125A / D165S, E78R / D82S / E89R / T101N / E116T / E125A / D165S, E78R / D82S / E89R / T101Q / E116T / E125A / D165S, E78R / D82S / E89R / T101R / E116T / E125A / D165S, E78R / D82S / E89N / T101V / E116T / E125A / D165S, E78R / D82S / E89N / T101S / E116T / E125A / D165S, E78R / D82S / E89N / T101N / E116T / E125A / D165S, E78R / D82S / E89N / T101Q / E116T / E125A / D165S, E78R / D82S / E89N / T101R / E116T / E125A / D165S, E78R / D82S / G87S / E89R / E116T / E125A / D165S, E78R / D82S / E89R / G91S / E116T / E125A / D165S, E78R / D82S / E89R / G91N / E116T / E125A / D165S, E78R / D82S / E89R / G91N / I92N / E116T / E125A / D165S, E78R / D82S / E89R / I92N / E116T / E125A / D165S, E78R / D82S / E89R / S99N / E116T / E125A / D165S, E78R / D82S / E89R / S103N / E116T / E125A / D165S, E78R / D82S / E89R / D104S / E116T / E125A / D165S, E78R / D82S / E89R / E116T / E122S / E125A / D165S, E78G / D82S / E89L / G91N / I92N / E116G / E125A / D165S, or 2. A mutant pore according to any one of the preceding claims, comprising E78G / D82S / E89L / E116G / E125A / D165S.

8. A mutant Rhodococcus pore comprising a monomer which is a variant of the sequence shown in SEQ ID NO:6, said variant comprising one or more mutations between positions 9 and 200 of SEQ ID NO:6, said one or more mutations altering the ability of said pore to interact with a target analyte.

9. 2. The mutant pore of any one of the preceding claims, wherein the variant has at least 70% identity with the sequence shown in SEQ ID NO:

2.

10. 2. The mutant pore of any one of the preceding claims, wherein the mutant is chemically modified.

11. The mutant pore of claim 10, wherein the mutant is chemically modified by attachment of a molecule to one or more cysteines, attachment of a molecule to one or more lysines, attachment of a molecule to one or more unnatural amino acids, enzymatic modification of an epitope, or terminal modification.

12. The mutant pore of claim 11 , wherein the one or more cysteines are introduced into the mutant by substitution.

13. 13. The mutant pore of claim 11 or 12, wherein the molecule is (a) a molecular adaptor that facilitates an interaction between the monomer and a target analyte, or (b) a polynucleotide binding protein.

14. A construct comprising two or more covalently linked Rhodococcus porin monomers.

15. The construct of claim 14, wherein at least one of the monomers is a variant as defined in any one of claims 1 to 13.

16. 16. The construct of claim 14 or 15, wherein the monomers are genetically fused and / or linked via a linker.

17. A polynucleotide encoding the mutant pore of any one of claims 1 to 13 or the construct of any one of claims 14 to 16.

18. A pore comprising at least one construct according to any one of claims 14 to 16.

19. 20. A kit for characterizing a target polynucleotide or a target polypeptide, comprising: (a) a pore according to any one of claims 1 to 13 and 18; and (b) a polynucleotide binding protein or a polypeptide handling enzyme.

20. 19. An apparatus for characterizing a target polynucleotide or a target polypeptide in a sample, the apparatus comprising: (a) a plurality of pores according to any one of claims 1 to 13 and 18; and (b) a plurality of polynucleotide binding proteins or a plurality of polypeptide handling enzymes.

21. A membrane comprising a pore according to any one of claims 1 to 13 and 18.

22. 22. An array comprising a plurality of the membranes of claim 21.

23. 23. A system comprising: (a) the membrane of claim 21 or the array of claim 22; (b) means for applying an electrical potential across the membrane(s); and (c) means for detecting an electrical or optical signal across the membrane(s).

24. 19. A method for determining the presence, absence, or one or more characteristics of a target analyte, comprising: (a) contacting the target analyte with a pore according to any one of claims 1 to 13 and 18; and (b) taking one or more measurements as the target analyte moves relative to the pore, thereby determining the presence, absence, or one or more characteristics of the target analyte.

25. 25. The method of claim 24, wherein the target analyte is or comprises a polypeptide, a polynucleotide, or a polysaccharide.

26. 26. The method of claim 24 or 25, wherein the target analyte comprises a polynucleotide, the method comprising: (i) contacting the polynucleotide with a polynucleotide binding protein capable of controlling movement of the polynucleotide relative to the pore; and (ii) performing one or more measurements characteristic of the polynucleotide as it moves relative to the pore.

27. 26. The method of claim 24 or 25, wherein the target analyte comprises a polypeptide, the method comprising: (i) contacting the polypeptide with a polypeptide handling enzyme capable of controlling the movement of the polypeptide relative to the pore; and (ii) taking one or more measurements characteristic of the polypeptide as it moves relative to the pore.

28. 26. The method of claim 24 or 25, wherein the target analyte comprises a polynucleotide-polypeptide conjugate, the method comprising: (i) contacting the conjugate with a polynucleotide binding protein capable of controlling movement of the polynucleotide of the conjugate relative to the pore; and (ii) performing one or more measurements characteristic of the polypeptide as the conjugate moves relative to the pore.

29. 19. A method for characterizing a target polynucleotide, the method comprising: (a) contacting the target polynucleotide with a pore according to any one of claims 1 to 13 and 18; and (b) taking one or more measurements as the target polynucleotide moves relative to the pore, thereby characterizing the target polynucleotide.

30. Use of a pore according to any one of claims 1 to 13 and 18 for determining the presence, absence or one or more characteristics of a target analyte or target polynucleotide.

31. 1. An apparatus comprising a transmembrane protein pore inserted into an in vitro membrane, said transmembrane protein pore being a variant of a Rhodococcus porin monomer, comprising the following positions: (a) D11, G12, G13, G14, N15, T52, G53, P54, D55, A56, E57, D58, F59, S60, G61, T62, T64, Y67, Q68, V69, Y71, P119, G120, I121, E122, T123, V124, E125, V126, S128, A130, A131, S132, G133, A134, H135, L143, H144, T146, T148, K149, Y159, Q161, V163, S164, D165, N166, G167, D168, V169, and T171, (b) S74, G76, E78, T80, D82, T84, Q108, G110, G112, T114, E116, and P117, and / or (c) at least one monomer comprising a mutation in one or more of G87, E89, G91, S93, S94, A95, G96, A97, S99, T101, S103, and D104, corresponding to the sequence set forth in SEQ ID NO:

2.

32. 1. An apparatus comprising: (i) obtaining a transmembrane protein pore comprising at least one Rhodococcus porin monomer, said monomer being located at position (a) D11, G12, G13, G14, N15, T52, G53, P54, D55, A56, E57, D58, F59, S60, G61, T62, T64, Y67, Q68, V69, Y71, P119, G120, I121, E122, T123, V124, E125, V126, S128, A130, A131, S132, G133, A134, H135, L143, H144, T146, T148, K149, Y159, Q161, V163, S164, D165, N166, G167, D168, V169, and T171, (b) S74, G76, E78, T80, D82, T84, Q108, G110, G112, T114, E116, and P117, and / or (c) obtaining a polypeptide comprising one or more amino acid mutations at G87, E89, G91, S93, S94, A95, G96, A97, S99, T101, S103, and D104, which correspond to the sequence set forth in SEQ ID NO:2; (ii) contacting the pore with an in vitro membrane such that the pore is inserted into the in vitro membrane.

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