Pore ​​monomers and pores

Abstract

This invention relates to novel pore monomers, pores formed from pore monomers, and their use in the detection and characterization of analytes.

Description

[Technical Field]

[0001] This invention relates to novel pore monomers, pores formed from pore monomers, and their use in the detection and characterization of analytes. [Background technology]

[0002] Nanopore sensing is an approach to analytes detection and characterization that relies on observing individual binding or interaction events between analyte molecules and ion conduction channels. Two key elements of analyte characterization using nanopore sensing are (1) controlling the movement of analytes through the pores, and (2) distinguishing constituent building blocks as the analyte passes through the pores. In nanopore sensing, the narrowest part of the pore forms the most distinguishing part of the nanopore with respect to the current signature as a function of the passing analyte.

[0003] For polynucleotide analytes, nucleotide differentiation is achieved by measuring the current as the polynucleotide passes through the pore. Since multiple nucleotides contribute to the observed current, the height of the channel constriction and the degree of interaction with the polynucleotide affect the relationship between the observed current and the polynucleotide sequence. While the current range and signal-to-noise ratio for nucleotide differentiation have been improved through protein pore mutations, sequencing systems would perform better if the current difference between nucleotides could be further improved. Therefore, it is necessary to identify novel methods for improving nanopore sensing properties.

[0004] Ghanem et al. (2022), FEBS J, 289:3505-3520, disclose chimeric mutants of alpha-hemolysin and gamma-hemolysin. However, these chimeric mutants have not been used for nanopore sensing. [Overview of the Initiative]

[0005] The inventors have shown, surprisingly, that chimeric pores formed from at least two different pores exhibit increased signal-to-noise ratio (SNR), increased current range, decreased noise, and increased normalized central absolute deviation (nMAD) during analyte characterization compared to the different pores from which the chimera originates. The increased SNR, increased current range, decreased noise, and increased nMAD improve the pore's ability to distinguish analytes as they pass through the pores. Accordingly, the present invention provides a chimeric pore monomer comprising two or more regions, where at least two of the two or more regions are sourced from at least two different pores, and the at least two different pores do not contain alpha-hemolysin and gamma-hemolysin.

[0006] The present invention also provides the following: - A chimeric construct comprising two or more covalently attached chimeric pore monomers according to the present invention; - Chimeric pores comprising at least one chimeric pore monomer or at least one construct of the present invention; - A chimeric pore multimer comprising two or more pores, wherein at least one of the pores is the chimeric pore of the present invention; - The chimeric pores or chimeric pore multimers of the present invention contained in the membrane; - A membrane containing the chimeric pores of the present invention or the chimeric pore multimer of the present invention; - A method for producing chimeric pore monomers according to the present invention, comprising attaching at least two regions from at least two different pores; - A method for determining the presence, absence, or one or more characteristics of a target analyte, (i) A step of bringing a target analyte into contact with (a) a chimeric pore comprising two or more regions, wherein at least two of the two or more regions are supplied by at least two different pores, or (b) a chimeric pore multimer comprising two or more pores, wherein at least one pore is a chimeric pore as defined in (a), (ii) A method comprising the step of obtaining one or more measurements as the target analyte moves toward the pore or pore multimer, thereby determining the presence, absence, or one or more characteristics of the target analyte; - A method for characterizing a target analyte using (a) a chimeric pore comprising two or more regions, wherein at least two of the two or more regions are supplied by at least two different pores, or (b) a chimeric pore multimer comprising two or more pores, wherein at least one pore is a chimeric pore as defined in (a); - To determine the presence or absence of a target analyte, or one or more characteristics, use (a) a chimeric pore comprising two or more regions, wherein at least two of the two or more regions are supplied by at least two different pores, or (b) a chimeric pore multimer comprising two or more pores, wherein at least one pore is a chimeric pore as defined in (a); - A kit for characterizing target polynucleotides, -(a) a chimeric pore comprising two or more regions, wherein at least two of the two or more regions are supplied by at least two different pores, or (b) a chimeric pore multimer comprising two or more pores, wherein at least one pore is a chimeric pore as defined in (a), - A kit containing polynucleotide-binding proteins; - An apparatus for characterizing target polynucleotides in a sample, -(a) a chimeric pore comprising two or more regions, wherein at least two of the two or more regions are supplied by at least two different pores, or (b) a chimeric pore multimer comprising two or more pores, wherein at least one pore is a chimeric pore as defined in (a), - A device containing multiple polynucleotide-binding proteins; -Polynucleotides encoding the chimeric pore monomer or chimeric construct of the present invention; - A kit for characterizing a target analyte, comprising (a) the chimeric pore or chimeric pore multimer of the present invention, and (b) a membrane component; - An array comprising multiple films of the present invention; - A system comprising (a) a film or array of the present invention, (b) means for applying a potential across the film(s), and (c) means for detecting an electrical or optical signal across the film(s); - Apparatus comprising the chimeric pores of the present invention or the chimeric pore multimers of the present invention inserted into an in vitro membrane; and - An apparatus manufactured by a method comprising: (i) obtaining a chimeric pore or a chimeric pore multimer of the present invention; and (ii) contacting the chimeric pore or the pore multimer with an in vitro membrane so that the chimeric pore or pore multimer is inserted into the in vitro membrane.

[0007] The inventors have also shown, surprisingly, that PorARc pores from various species can perform nanopore sensing with a high signal-to-noise ratio (SNR), a good current range, minimal noise, and a good normalized central absolute deviation (nMAD). Accordingly, the present invention provides PorARc pore monomers comprising a sequence having at least 88% identity to the sequence shown in SEQ ID NO: 2, or a sequence having at least about 20% identity to the sequences shown in SEQ ID NOs: 50, 51, 52, 53, 54, or 55.

[0008] The present invention also provides the following: - A PorARc construct comprising two or more covalently attached PorARc pore monomers according to the present invention; - A PorARc pore comprising at least one PorARc pore monomer of the present invention, or at least one construct of the present invention; - A PorARc pore multimer comprising two or more pores, wherein at least one of the pores is a PorARc pore of the present invention; - The PorARc pores or PorARc pore multimers of the present invention contained in the membrane; - A membrane containing the PorARc pores of the present invention or the PorARc pore multimer of the present invention; - A method for determining the presence, absence, or one or more characteristics of a target analyte, (i) A step of contacting a target analyte with the PorARc pore or PorARc pore multimer of the present invention, (ii) A method comprising the step of obtaining one or more measurements as the target analyte moves toward the pore or pore multimer, thereby determining the presence, absence, or one or more characteristics of the target analyte; - A method for characterizing a target analyte using the PorARc pores or PorARc pore multimers of the present invention; - Use of the PorARc pores or PorARc pore multimers of the present invention to determine the presence, absence, or one or more characteristics of a target analyte; -(a) a PorARc pore of the present invention or a PorARc pore multimer according to the claims of the present invention, and (b) a polynucleotide-binding protein, comprising a kit for characterizing a target polynucleotide; - An apparatus for characterizing a target polynucleotide in a sample, comprising (a) a plurality of PorARc pores of the present invention or a plurality of PorARc pore multimers of the present invention, and (b) a plurality of polynucleotide-binding proteins; - A polynucleotide encoding the PorARc pore monomer or the PorARc construct of the present invention; - A kit for characterizing a target analyte, the kit comprising: (a) the PorARc pore or the PorARc pore multimer of the present invention; and (b) a membrane component; - An array comprising a plurality of membranes of the present invention; - A system comprising: (a) the membrane of the present invention or the array of the present invention; (b) means for applying a potential across the membrane(s); and (c) means for detecting an electrical or optical signal across the membrane(s); - An apparatus comprising the PorARc pore or the PorARc pore multimer of the present invention, the apparatus being inserted into an in vitro membrane; and - An apparatus produced by a method comprising: (i) obtaining the PorARc pore or the PorARc pore multimer of the present invention; and (ii) contacting the chimeric pore or the pore multimer with the in vitro membrane such that the chimeric pore or the pore multimer is inserted into the in vitro membrane.

Brief Description of the Drawings

[0009] [Figure 1] A schematic diagram showing a chimeric pore formed from the cap region (also known as the scaffold) of one pore (A) and the constricted region of another pore (B). [Figure 2] An alignment of the sequences of the constricted pore monomer chimeras of the present invention. The dark shading indicates the consistency of the cap region (or scaffold) between the chimeras, and the unshaded portions indicate the differences between the constricted regions. [Figure 3] A snapshot of a run report showing the relationship of ionic current (pA) versus time (seconds) when a single-stranded DNA or peptide-DNA conjugate (lower irregular curve) translocates through PorARc (PorARc_Rco) from Rhodococcus corynebacteroides (for comparison purposes). [Figure 4]A snapshot of a run report showing the relationship between ion current (pA) and time (seconds) when a single-stranded DNA or peptide-DNA conjugate (irregular curve at the bottom) translocates through the PorARc pore (PorARc_Mph) from Mycolicibacterium phlei. [Figure 5] A snapshot of a run report showing the relationship between ion current (pA) and time (seconds) when a single-stranded DNA or peptide-DNA conjugate (irregular curve at the bottom) translocates through PorARc_Rco_Mel_ONLZ18401_ONLP19805 (SEQ ID NO: 18). [Figure 6] A snapshot of a run report showing the relationship between ion current (pA) and time (seconds) when a single-stranded DNA or peptide-DNA conjugate (irregular curve at the bottom) translocates through PorARc_Aku_Mph_ONLZ19310_ONLP20864 (SEQ ID NO: 40). [Figure 7] SNR, current range (pA), and noise (pA) for all chimeras tested in Example 1. The comparison line is for the PorARc pore (PorARc_Mph) from Mycolicibacterium phlei containing the D91N / D92N substitution (SEQ ID NO: 2). [Figure 8] nMAD for all chimeras tested in Example 1. The comparison line is for the PorARc pore (PorARc_Mph) from Mycolicibacterium phlei containing the D91N / D92N substitution (SEQ ID NO: 2). [Figure 9]This is a trace of the typical ion current (pA) versus time (seconds) relationship when single-stranded DNA displaces through a CsgG chimeric nanopore (CsgG-Eco-Vmae in Table 7) in Example 2. The raw current trace is shown by the black line, and the event detection signal is shown by the red line. A shows a series of DNA displacement events through a single nanopore, B shows individual DNA displacement events, C shows a magnified x-axis view of the first section of the current trace, and D shows magnified x-axis and y-axis views of the first section of the current trace. [Figure 10] The signal-to-noise ratio (SNR), current range (pA), and noise (pA) for the tested CsgG chimeras are shown. The results are presented in Table 7, from left to right, in the order the chimeras appear. The comparison line is for CsgG wild-type pores derived from E. coli (CsgG-Eco-WT). [Figure 11] This shows the structure and size of a wild-type CsgG pore from E. coli strain K12 (the databank access code for this structure is 4UV3). The distances shown are measured from backbone to backbone of the amino acids forming the pore structure. The CsgG pore is a tightly interconnected, symmetrical necaper pore resembling a crown. The total height is 98 Å and the maximum outer diameter is 120 Å. It consists of three parts that define the central channel: (A) the cap region, (B) the constriction region, and (C) the transmembrane beta-barrel region. The cap axial length, i.e., the height, is 39 Å. The inner diameter is 43 Å and the opening is 66 Å. The beta-barrel has 36 strands, with an axial length of 39 Å and an inner diameter of 55 Å. The transition between the pore cap and the beta-barrel is abrupt, and the constriction is located between them at the level of the predicted lipid-aqueous interface. The constricted portion has a diameter of approximately 18.5 Å and a length of 20 Å along the channel axis. [Figure 12]The structure and dimensions of PorARc are shown (cryo-electron microscope structure). Distances are measured from backbone to backbone of the amino acids forming the pore structure. The PorARc pore is a symmetrical octameric pore. The total height is 90.4 Å and the maximum outer diameter is 90.7 Å. The PorARc pore consists of a cap region (A) and a transmembrane beta-barrel region (B), both of which form a cap region (or scaffold) (C), and a constricted region (D). The height of the cap region (or scaffold) is 73.6 Å, corresponding to the height of the cap region (A) of 44.7 Å and the height of the transmembrane beta-barrel region (B) of 26.2 Å. The height of the constricted region (C) is 19.7 Å. The PorARc pore is generally funnel-shaped, with an entrance width of 49 Å, which narrows to 41.5 Å at the bottom of the cap region (A) and further to 39.8 Å in the transmembrane beta-barrel region (B). The constricted region (C) narrows rapidly to 27.4 Å, and then widens to 36.1 Å at the base of the pore structure. [Figure 13] This is a trace of the typical relationship between ionic current (pA) and time (seconds) when single-stranded DNA undergoes rearrangement through CsgG chimeric nanopores (CsgG-Eco-Vfu in Table 10) in Example 3. A-D are the same as in Figure 9. [Figure 14] The SNR, current range (pA), and noise (pA) for the tested CsgG chimeras are shown. The results are presented in Tables 7 and 12, in the order the chimeras appear from left to right. The data for the first nine chimeras from the left are from the chimera of Example 2 (and these data are identical to the data in Figure 10). The data for the last three chimeras from left to right are from the chimera of Example 3. The comparison line is for CsgG wild-type pores derived from E. coli (CsgG-Eco-WT).

[0010] Explanation of the sequence list Sequence ID 1 shows the amino acid sequence of PorARc (PorARc_Rco) from Rhodococcus corynebacteroides, including the substitutions E78R / D82S / E116T / E125A / D165S.

[0011] Sequence ID 2 shows the amino acid sequence of a PorARc pore (PorARc_Mph) from Mycolicibacterium phlei containing the D91N / D92N substitution.

[0012] Table 1 - Explanation of Sequence IDs 3-49 [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4]

[0013] Sequence ID 50 shows the amino acid sequence of a PorARc pore (PorARc_Msp) from the genus Mycobacterium, including the substitution D91N / D92N.

[0014] Sequence ID 51 shows the amino acid sequence of PorARc pore (PorARc_Mrh) from Mycolicibacterium rhodesiae, including the D91N / D92N substitution.

[0015] Sequence ID 52 is the amino acid sequence of the PorARc pore (PorARc_Mel) of Mycolicibacterium elephantis, containing the D91N / E101Q substitution.

[0016] Sequence ID 53 shows the amino acid sequence of a PorARc pore (PorARc_Mco) from Mycolicibacterium cosmeticum, containing the D91N / D92N substitution.

[0017] Sequence ID 54 shows the amino acid sequence of a PorARc pore (WP_056447532.1;PorARc_Rsp) from an unclassified Rhodococcus species, including the substitution E89Q / D91N / D93N / D100N.

[0018] Sequence ID 55 shows the amino acid sequence of the PorARc pore (WP_206003768.1; PorARc_Rsp) from Rhodococcus sp. PSBB049, including the D90N / D95N / D103N substitution.

[0019] Sequence IDs 56-64 and 73-75 show the amino acid sequences of the CsgG pores in Table 4 below. Signal peptides in each sequence are underlined. All the following considerations regarding the numbering of specific positions in Sequence ID 56 or Sequence IDs 57-64 and 73-75 (e.g., Q100) exclude signal peptides.

[0020] Sequence IDs 65-72 show the amino acid sequences of the CsgG constriction chimeras in Table 7 below. The signal peptides in each sequence are underlined.

[0021] Sequence IDs 76-78 show the amino acid sequences of the CsgG constriction chimeras in Table 12 below. The signal peptides in each sequence are underlined. [Modes for carrying out the invention]

[0022] All publications, patents, and patent applications cited herein, whether above or below, are incorporated herein by reference in their entirety. All publications, patents, and patent applications referenced herein are incorporated herein by reference in the same manner as individual publications, patents, or patent applications are specifically and individually indicated to be incorporated herein by reference. With respect to any extent that a publication or patent or patent application incorporated herein by reference conflicts with the Invention as contained herein, this Specified Version is intended to supersede and / or take precedence over any such conflicting material.

[0023] The present invention is described with respect to specific embodiments and with reference to certain drawings, but the present invention is not limited thereto and is limited only by the claims. None of the reference numerals in the claims should be construed as limiting the scope. Needless to say, it should be understood that not all aspects or advantages can necessarily be achieved according to any particular embodiment of the present invention. Accordingly, 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 or a group of advantages taught herein without necessarily achieving other aspects or advantages that can be taught or suggested herein.

[0024] In addition, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context otherwise clearly indicates. Thus, for example, a reference to "polynucleotide" includes two or more polynucleotides, a reference to "polynucleotide-binding protein" includes two or more such proteins, a reference to "helicase" includes two or more helicases, a reference to "monomer" refers to two or more monomers, a reference to "pore" includes two or more pores, and so on.

[0025] In all the examples presented here, the terms "comprises" or "comprising" encompass and can be replaced by "consists of" or "consisting of."

[0026] In all discussions herein, the standard single-letter codes for amino acids are used. These are: 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, namely D91N, which means that D at position 91 is replaced by N.

[0027] In paragraphs of this specification where different amino acids at specific positions are separated by the " / " symbol, the " / " symbol means "or". For example, D91N / Q means D91N or D91Q. Where different positions are separated by the " / " symbol, the " / " symbol means "and", so D91 / D92 means D91 and D92.

[0028] In the context of the present invention, “pore” is a transmembrane protein structure that defines a channel or hole that allows the rearrangement of molecules and ions from one side of a membrane to the other. The rearrangement of ionic species through a pore may be driven by a potential difference applied to either side of the pore. “Nanopore” is a biological pore in which the minimum diameter of the channel through which molecules or ions pass is on the order of nanometers (10 to 9 nanometers). In some embodiments, the pore may be a transmembrane protein pore. The transmembrane protein structure of a biological pore may be essentially monomers or oligomers. Typically, the pore comprises a plurality of polypeptide monomers or polypeptide subunits arranged around a central axis, thereby forming a protein-backed channel that extends substantially perpendicular to the membrane in which the pore resides. The number of polypeptide monomers or polypeptide subunits is not limited. Typically, the number of monomers or subunits is 5 to a maximum of 30, and preferably 6 to 10. The portion of a protein monomer or subunit within a pore that forms a protein-backed channel typically contains a secondary structural motif that may include one or more transmembrane beta barrels and / or alphalic sections.

[0029] The chimeric pore monomer of the present invention is formed from at least two different pores, i.e., from at least two monomers from at least two different pores. This means that the chimeric pore monomer is formed from pores or pore monomers that are pores as defined above in their natural state. The various pores are defined below.

[0030] The general definitions in WO2019 / 002893 are incorporated herein by reference in their entirety.

[0031] Chimeric pore monomer The present invention provides chimeric pore monomers, which are typically proteins or polypeptides. Chimeric pore monomers can form pores. This can be measured using any of the methods described in WO2016 / 034591, WO2017 / 149316, WO2017 / 149317, WO2017 / 149318, WO2018 / 211241, WO2019 / 002893, PCT / EP2023 / 059821, PCT / EP2023 / 072113, PCT / EP2023 / 072065, PCT / EP2023 / 072106, and PCT / EP2023 / 072068 (all incorporated herein in their entirety by reference) and the examples.

[0032] A chimeric pore monomer contains two or more regions. A chimeric pore monomer may contain any number of regions, such as three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more. A chimeric pore monomer may contain three, four, five, six, seven, eight, nine, or ten regions. A chimeric pore monomer preferably contains two regions. A chimeric pore monomer preferably contains three regions. A chimeric pore monomer preferably contains five regions.

[0033] Specific regions within pores, such as capped or constricted regions, may be identified using standard methods. Regions in proteins of unknown structure can be defined by aligning the protein sequence with a homologous protein of known structure. If the sequence identity or similarity between sequences is sufficiently high, the region boundaries are reliable. The reliability of region boundaries can be further enhanced by using tools that predict tertiary structure (such as AlphaFold) or secondary structure elements (such as PSIPRED). In the latter case, the reliability of the region boundary is higher if both the protein of unknown structure and the protein of known structure are flanked by the same secondary structure prediction elements.

[0034] At least two regions preferably include a capping region and a constricting region. The capping region typically forms the structural core of the protein pore and may be partially membrane-bound. When the capping region forms the structural core and is partially membrane-bound, it is also known as a scaffold. The constricting region typically includes at least one constriction. A “constriction” refers to an opening defined by the luminal surface of the pore, which acts to allow the passage of ions and target molecules (e.g., polynucleotides, polypeptides, or individual nucleotides) but prevents the passage of other non-target molecules through the pore channel. As will be described in more detail below, pores formed from the chimeric pore monomers of the present invention may include two or more constrictions. A constriction is typically the narrowest opening within the pore or within the channel defined by the pore. Constrictions can help restrict the passage of molecules through the pore. The size of the constriction is typically a key factor in determining the suitability of the pore for characterizing an analyte. If the constriction is too small, the molecule being characterized will not be able to pass through. However, to achieve the greatest effect on ion flow through the channel, the constriction must not be too large. For example, the constriction should not be wider than the lateral diameter through which the solvent of the target analyte can access. Ideally, any constriction should be as close as possible to the lateral diameter of the analyte passing through it.

[0035] The narrowest part of the constricted region preferably forms a constriction with a diameter of at least 5 Å, for example, at least about 10 Å, at least about 15 Å, at least about 18 Å, at least about 20 Å, at least about 25 Å, or at least about 27 Å.

[0036] The constricted region may include any number of constrictions, such as at least two, at least three, at least four, or at least five. The constricted region is typically located within the membrane. The constrictions are preferably transmembrane. Those skilled in the art can distinguish between the pore capping region (or scaffold) and the constricted region. Specific examples of capping and constricting regions are shown below.

[0037] The cap region (or scaffold) in the chimeric pore monomer may be longer, shorter, or the same length as the cap region (or scaffold) in the pore from which the constricted region in the chimeric pore monomer is sourced or derived. The constricted region in the chimeric pore monomer may be longer, shorter, or the same length as the constricted region in the pore from which the cap region (or scaffold) in the chimeric pore monomer is sourced or derived. Preferably, the constricted region in the chimeric pore monomer is shorter than the constricted region in the pore from which the cap region (or scaffold) in the chimeric pore monomer is sourced or derived. The length may be measured by the number of amino acids and / or the length along the sagittal plane (or longitudinal section) of the pore.

[0038] Preferably, at least two regions include a capping region, a constricting region, and a transmembrane region. The transmembrane region may be a transmembrane beta-barrel region or a transmembrane alpha-helical region. The transmembrane region is preferably a transmembrane beta-barrel region. CsgG pores typically include these three regions, which will be discussed in more detail below in relation to CsgG pores. In these embodiments having a capping region, a constricting region, and a transmembrane region, the capping region and the transmembrane region combined are also known as a scaffold.

[0039] The cap region may further include two sub-regions, namely a landing platform region and a carboxy-terminal (C-terminal) region. Preferably, at least two regions include the cap region, landing platform region, C-terminal region, constriction region, and transmembrane region. The transmembrane region may be a transmembrane beta-barrel region or a transmembrane alpha-helical region. The transmembrane region is preferably a transmembrane beta-barrel region. A CsgG pore typically includes these five regions, which will be discussed in more detail below in relation to the CsgG pore. In these embodiments, the cap region, landing platform region, C-terminal region, and transmembrane region together are also known as the scaffold.

[0040] The cap region in a chimeric pore monomer may be longer, shorter, or the same length as the cap region in the pore(s) from which the constricted region and / or transmembrane region of the chimeric pore monomer originates. The constricted region in a chimeric pore monomer may be longer, shorter, or the same length as the constricted region in the pore(s) from which the cap region and / or transmembrane region of the chimeric pore monomer originates. The transmembrane region in a chimeric pore monomer may be longer, shorter, or the same length as the transmembrane region in the pore(s) from which the cap region and / or constricted region of the chimeric pore monomer originates. The constricted region in a chimeric pore monomer may be shorter than the constricted region in the pore(s) from which the cap region and / or transmembrane region of the chimeric pore monomer originates. The length may be measured by the number of amino acids and / or the length along the sagittal plane (or longitudinal section) of the pore.

[0041] Chimeric pore monomers preferably comprise two regions, which are preferably a capped region (or scaffold) and a constricted region. In such cases, the capped region (or scaffold) and the constricted region typically originate from different pores. An example of this is shown in Figure 1. The constricted graft and capped graft (the latter also known as a scaffolded graft) tested in Example 1 are also examples of chimeric pore monomers formed from two different pores. In the context of the present invention, graft refers to cases where a chimeric pore monomer is created by transplanting all or part of a region of one pore monomer to a monomer of a different pore. For example, transplantation of a constricted region involves transplanting all or part of a constricted region from pore monomer A to pore monomer B.

[0042] Chimeric pore monomers preferably comprise three regions. These three regions are preferably a capping region, a constricting region, and a transmembrane region. As described above, the capping region and the transmembrane region together may also be known as a scaffold. In such cases, the constricting region may be supplied from one pore, and the capping region and the transmembrane region (collectively also known as a scaffold) may be supplied from different pores, i.e., the chimeric pore monomer is formed from / derived from two different pores. These are also constricting implants, such as the chimeric pores produced in Example 2. Alternatively, the capping region, the constricting region, and the transmembrane region may each be supplied from different pores, i.e., the chimeric pore monomer is formed from / derived from three different pores. The transmembrane region may be a transmembrane beta-barrel region or a transmembrane alpha-helical region. The transmembrane region is preferably a transmembrane beta-barrel region. Such regions are found in CsgG pores.

[0043] The chimeric pore monomer preferably comprises five regions. These five regions are preferably a cap region, a landing platform region, a C-terminal region, a constriction region, and a transmembrane region. As described above, the cap region, landing platform region, C-terminal region, and transmembrane region together may also be known as a scaffold. In such a case, the constriction region may be supplied from one pore, and the cap region, landing platform region, C-terminal region, and transmembrane region (collectively also known as the scaffold) may be supplied from different pores, i.e., the chimeric pore monomer is formed / derived from two different pores. These are also constriction implants, such as the chimeric pore produced in Example 2. Alternatively, the cap region, landing platform region, C-terminal region, constriction region, and transmembrane region may each be supplied from different pores, i.e., the chimeric pore monomer is formed / derived from five different pores. The transmembrane region may be a transmembrane beta-barrel region or a transmembrane alpha-helical region. The transmembrane region is preferably a transmembrane beta-barrel region. Such regions are found in CsgG pores.

[0044] The constricted region in the chimeric pore monomer may be formed from the constricted regions of two different pores. In other words, the constricted region may be a hybrid constricted region. For example, a portion of the constricted region in one pore (e.g., pore A) may be replaced with a portion or a corresponding portion of the constricted region of a different pore (e.g., pore B). In this example, the chimeric pore monomer includes a capped region (or scaffold) from pore A and constricted regions from pores A and B. In this sense, these two regions are supplied from two different pores. Any amount or portion of the constricted region of one pore may be replaced with any amount or portion of the constricted region of a different pore. For example, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, and at least about 99% of the constricted area in a certain pore may be replaced with at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, and at least about 99% of the constricted area in a different pore. At least about five amino acids in a constricted region of a pore, such as at least about 10, 15, 20, 25, 26, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 110 amino acids, may be replaced with at least about five amino acids from a constricted region of a different pore, such as at least about 10, 15, 20, 25, 26, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, or 110 amino acids. Specific examples will be discussed later with reference to CsgG chimeras. The entire constricted region of one pore may be replaced with the entire constricted region of a different pore. 100% of the constricted region of one pore may be replaced with 100% of the constricted region of a different pore. All amino acids in the constricted region of one pore may be replaced with all amino acids in the constricted region of a different pore.

[0045] At least two of the two or more regions are supplied with, or preferably derived from, at least two different pores. The pores may include any number of regions from any number of different pores, as long as at least two of the regions are supplied with or derived from two different pores. The chimeric pore monomer may include two or more different regions from at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten different pores. The number of two or more regions is typically the same as the number of at least two different pores, each region being supplied with or derived from a different pore. The chimeric pore monomer preferably includes two regions supplied with or derived from two different pores. The chimeric pore monomer preferably includes three regions supplied with or derived from three different pores. The chimeric pore monomer preferably includes five regions supplied with or derived from five different pores.

[0046] Regions from at least two different pores (or from two different pores) are typically attached to each other, preferably covalently, to form a chimeric pore monomer. The regions may be directly bonded or attached using one or more linkers, preferably one or more peptide linkers. Suitable linkers are described later with reference to the constructs of the present invention. The chimeric pore monomers of the present invention are typically produced by gene fusion of two or more regions. A polynucleotide encoding the chimeric pore monomer can be designed using sequences of at least two different pores. This polynucleotide can be used to express the chimeric pore monomer as a gene fusion. This is discussed in more detail below.

[0047] A region is considered "pore-sourced" or "pore-derived" if it shares significant homology / identity with a region that is sourced from or originates from pores. This region, preferably the cap region (or scaffold) of a constricted region, preferably contains sequences having at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or more preferably at least about 95%, at least about 97%, at least about 98%, or at least about 99% homology with the corresponding sequence of the pore-sourced region, preferably the cap region (or scaffold) or the sequence of the constricted region. This region, preferably a cap region (or scaffold) or constriction region, preferably contains an array having 100% homology to the corresponding region from which the pores originate, preferably the array of the cap region (or scaffold) or constriction region. This region, preferably the cap region (or scaffold) of the constriction region, preferably contains an array having at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, or more preferably at least about 95%, at least about 97%, at least about 98%, or at least about 99% identity to the corresponding region from which these pores originate, preferably the array of the cap region (or scaffold) or constriction region. This region, preferably a capped region (or scaffold) or constricted region, preferably contains an array that has 100% identity with the corresponding region from which the pores originate, preferably the array of the capped region (or scaffold) or constricted region. Homology and / or identity are typically measured along the entire length of the region.

[0048] The capping region, constricting region, or transmembrane region preferably contains sequences having at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or more preferably at least about 95%, at least about 97%, at least about 98%, or at least about 99% homology to the sequence of capping region, constricting region, or transmembrane region from which the pores originate. The capping region, constricting region, or transmembrane region preferably contains sequences having 100% homology to the sequence of capping region, constricting region, or transmembrane region from which the pores originate. The capping, constricting, or transmembrane region preferably contains sequences that have at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, or more preferably at least about 95%, at least about 97%, at least about 98%, or at least about 99% identity with respect to the sequence of capping, constricting, or transmembrane regions from which the pores originate. The capping, constricting, or transmembrane region preferably contains sequences that have 100% identity with respect to the sequence of capping, constricting, or transmembrane regions from which the pores originate. Homology and / or identity are typically measured along the entire length of the region.

[0049] A chimeric pore monomer comprises at least two regions from which at least two different pores are supplied or derived. These at least two different pores are typically at least two different pores that appear in nature. These at least two different pores are typically at least two different wild-type or naturally occurring pores. These at least two different pores are preferably different before any artificial or synthetic modifications such as addition, deletion, and / or substitution are made to them. These at least two different pores are preferably different before any modifications such as addition, deletion, and / or substitution are made to their wild-type or naturally occurring sequences. However, as described below, when constructing a chimeric pore monomer according to the present invention, at least two different pores, or at least two regions, or one or more of the two regions from which two different pores are supplied or derived, may be modified. In particular, at least one of two regions, or one or more of two regions, in the chimeric pore monomer preferably contains one or more modifications that stabilize the chimeric pore and / or improve the ability of the chimeric pore formed from the chimeric pore monomer to characterize the target analyte. Specific modifications are discussed in more detail below.

[0050] At least two distinct pores are preferably homologous, for example, structural homologs. Structural homologs are proteins or molecules that share a similar three-dimensional structure with other proteins or molecules. This can be determined using standard methods in the art (e.g., AlphaFold or PSIPRED). Structural homologs typically have similar sequences. Structural homologs are usually identified in similar species. For example, at least two distinct pores may be at least two PorARc pores selected from Table 2 below. For example, at least two distinct pores may be at least two CsgG pores selected from Table 4 below. These represent structural homologs between different species.

[0051] Each region in a chimeric pore monomer typically shares low homology or identity with a corresponding region in a different pore(s) from which other regions in the chimeric pore monomer are sourced or originated. In the context of the present invention, a region in one pore corresponds to a region in another pore if they share similar structure and / or function. A region in one pore corresponds to a region in another pore if they share similar arrangements. This can be determined as described above. A cap region (or scaffold) in one pore corresponds to a cap region (or scaffold) in another pore. A cap region in one pore corresponds to a cap region in another pore. A constriction region in one pore corresponds to a constriction region in another pore. A transmembrane region in one pore corresponds to a transmembrane region in another pore. A transmembrane beta-barrel region in one pore corresponds to a transmembrane beta-barrel region in another pore.

[0052] Each region in the chimeric pore preferably contains a sequence that is homologous or identical by about 99% or less to the sequence of the corresponding region in the different pore(s) that is the source or origin of the other region(s) in the chimeric pore monomer. Each region in the chimeric pore preferably contains a sequence that is homologous or identical by about 99% or less, about 98% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, or more preferably about 50% or less, about 45% or less, or about 40% or less to the sequence of the corresponding region in the different pore(s) that is the source or origin of the other region(s) in the chimeric pore. Each region preferably contains a cap region, a constriction region, or a transmembrane region. Homology and / or identity are typically measured over the entire length of the region.

[0053] In some embodiments, the chimeric pore monomer comprises a capping region (or scaffold) and a constricting region from two different pores. In some embodiments, the chimeric pore monomer comprises a constricting region formed from two different pores and a capping region (or scaffold) supplied from one of those different pores, i.e., the chimeric pore monomer is formed from / derived from two different pores. The capping region (or scaffold) in the chimeric pore monomer preferably comprises an arrangement that is homologous or identical by about 99% or less to the arrangement of the capping region (or scaffold) of the pore from which the constricting region is supplied or derived. The cap regions (or scaffolds) in the chimeric pore monomer preferably contain sequences that are homologous or identical to the sequences of the cap regions (or scaffolds) in the pore that is the source or origin of the constricted regions by about 98% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, or more preferably about 50% or less, about 45% or less, or about 40% or less. The constricted regions in the chimeric pore monomer preferably contain sequences that are homologous or identical to the sequences of the constricted regions in the pore that is the source or origin of the cap regions (or scaffolds) by about 99% or less. The constricted regions in the chimeric pore monomer more preferably contain sequences that are homologous or identical by about 98% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, or more preferably about 50% or less, about 45% or less, or about 40% or less to the sequence of constricted regions in the pore that is the source or origin of the capped region (or scaffold). Homology and / or identity are typically measured over the entire length of the region.

[0054] In some embodiments, the chimeric pore monomer includes a constricted region formed from one pore and a capped region and a transmembrane region supplied from a different pore; that is, the chimeric pore monomer is formed from / derived from two different pores. In some embodiments, the chimeric pore monomer includes a constricted region formed from two different pores and a capped region and a transmembrane region supplied from one of those different pores; that is, the chimeric pore monomer is formed from / derived from two different pores. The constricted region in the chimeric pore monomer preferably includes an arrangement that is homologous or identical by about 99% or less to the arrangement of the constricted region of the pore that is the source or origin of the capped region and the transmembrane region. The constricted regions in the chimeric pore monomer preferably contain sequences that are homologous or identical to the sequences of the constricted regions in the pores that are the source or origin of the cap regions and transmembrane regions by about 98% or less, about 97% or less, about 95% or less, about 94% or less, about 90% or less, about 85% or less, about 82% or less, about 80% or less, about 75% or less, about 72% or less, about 70% or less, about 69% or less, about 65% or less, about 60% or less, about 55% or less, or more preferably about 50% or less, about 45% or less, or about 40% or less. The cap regions and / or transmembrane regions in the chimeric pore monomer preferably contain sequences (multiple sequences) that are homologous or identical to the sequences of the cap regions and / or transmembrane regions in the pores that are the source or origin of the constricted regions by about 99% or less. Cap regions and / or transmembrane regions in chimeric pore monomers more preferably include sequences(s) that are homologous or identical to about 98% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, or more preferably about 50% or less, about 45% or less, or about 40% or less of sequences(s). Homology and / or identity are typically measured over the entire length of the region.

[0055] In some embodiments, the chimeric pore monomer includes a constricted region, a capped region, and a transmembrane region, each supplied by a different pore; that is, the chimeric pore monomer is formed from / derived from three different pores. In some embodiments, the chimeric pore monomer includes a constricted region formed from two different pores, a transmembrane region supplied by one of those two different pores, and a capped region supplied by a third different pore; that is, the chimeric pore monomer is formed from / derived from three different pores. In some embodiments, the chimeric pore monomer includes a constricted region, a capped region, a landing platform region, a C-terminal region, and a transmembrane region, each supplied by a different pore; that is, the chimeric pore monomer is formed from / derived from five different pores. In some embodiments, the chimeric pore monomer includes a constricted region formed from two different pores, a transmembrane region supplied from one of the two different pores, and a capped region, a landing platform region, and a C-terminal region supplied from three different pores, i.e., the chimeric pore monomer is formed from / derived from five different pores. The constricted region in the chimeric pore monomer preferably includes an arrangement that is homologous or identical by about 99% or less to the arrangement of the constricted region in the pore(s) that are the source or origin of the capped region and / or the transmembrane region. The constricted regions in the chimeric pore monomer preferably contain sequences that are homologous or identical to sequences ofThe cap regions in the chimeric pore monomer preferably contain sequences that are homologous or identical to the sequences of cap regions in the pore(s) that are the source or origin of the constricted regions and / or transmembrane regions by about 98% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, or more preferably about 50% or less, about 45% or less, or about 40% or less. The transmembrane regions in the chimeric pore monomer preferably contain sequences that are homologous or identical to the sequences of transmembrane regions in the pore(s) that are the source or origin of the cap regions and / or constricted regions by about 99% or less. The transmembrane regions in the chimeric pore monomer more preferably contain sequences that are homologous or identical by about 98% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, or more preferably about 50% or less, about 45% or less, or about 40% or less to the sequences of transmembrane regions in the pore(s) that are the source or origin of the capping and / or constricting regions. Homology and / or identity are typically measured along the entire length of the region.

[0056] The constricted regions in the chimeric pore monomer preferably contain at least about one amino acid difference compared to the constricted regions (multiple) in the pores originating from the cap region (or scaffold), or in the pores originating from both the cap region and the transmembrane region, for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 pieces, 47 pieces, 48 ​​pieces, 49 pieces, 50 pieces, 51 pieces, 52 pieces, 53 pieces, 54 pieces, 55 pieces, 56 pieces, 57 pieces, 58 pieces, 59 pieces, 60 pieces, 61 pieces, 62 pieces, 63 pieces, 64 pieces, 65 pieces, 66 pieces, 67 pieces, 68 pieces, 69 pieces, 70 pieces, 71 pieces, 72 pieces, 73 pieces, 74 pieces, 75 pieces, 76 pieces, 77 pieces, 78 pieces, 79 pieces, 80 pieces, 81 pieces, 82 pieces, 83 pieces , including differences of 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 114 amino acids.

[0057] When a constricted region in a chimeric pore monomer is formed from constricted regions of two different pores (i.e., a hybrid constricted region), it preferably contains at least about one amino acid difference compared to the constricted regions in the two different pores, for example, at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 4 7 pieces, 48 ​​pieces, 49 pieces, 50 pieces, 51 pieces, 52 pieces, 53 pieces, 54 pieces, 55 pieces, 56 pieces, 57 pieces, 58 pieces, 59 pieces, 60 pieces, 61 pieces, 62 pieces, 63 pieces, 64 pieces, 65 pieces, 66 pieces, 67 pieces, 68 pieces, 69 pieces, 70 pieces, 71 pieces, 72 pieces, 73 pieces, 74 pieces, 75 pieces, 76 pieces, 77 pieces, 78 pieces, 79 pieces, 80 pieces, 81 pieces, 82 pieces, 83 pieces, Includes differences of 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 114 amino acids.

[0058] The whole or complete chimeric pore monomer preferably contains sequences that are homologous or identical by about 96% or less to the wild-type monomer sequences of at least two different pores. In other words, the chimeric pore monomer preferably contains sequences that are homologous or identical by about 96% or less to the wild-type monomer sequences of the different pores from which the chimeric pore monomer originates. The chimeric pore monomer more preferably contains sequences that are homologous or identical by about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, or more preferably about 50% or less, about 45% or less, or about 40% or less to the wild-type monomer sequences of at least two different pores, or the wild-type monomer sequences of the different pores from which the chimeric pore monomer originates. Homologousity and / or identity are typically measured over the entire length of the chimeric pore monomer.

[0059] The whole or complete chimeric pore monomer preferably contains a sequence that is approximately 99.7% or less homologous or identical to the wild-type monomer sequences of at least two different pores. This is especially true for chimeric pore monomers formed from two different CsgG pores, such as the constricted graft described in Example 2, or from three different CsgG pores. This is also true for chimeric pore monomers formed from five different CsgG pores. In other words, the chimeric pore monomer preferably contains a sequence that is approximately 99.7% or less homologous or identical to the wild-type monomer sequences of the different pores from which the chimeric pore monomer is derived. The chimeric pore monomer more preferably comprises a sequence that is homologous or identical to at least two different wild-type monomer sequences of pores, or to a wild-type monomer sequence of a different pore from which the chimeric pore monomer originates, by about 99.6% or less, about 99.5% or less, about 99.4% or less, about 99.3% or less, about 99.2% or less, about 99.1% or less, about 99.0% or less, about 98.9% or less, about 98.7% or less, about 98.7% or less, about 98.7% or less, about 98.7% or less, about 98.7% or less, about 98.7% or less, about 98.7% or less, about 98.7% or less. Homology and / or identity is typically measured over the entire length of the chimeric pore monomer.

[0060] Standard methods in the art may be used to determine homology or identity. For example, the UWGCG package provides the BESTFIT program, which can be used to calculate homology or identity, for example, by using its default settings (Devereux et al (1984) Nucleic Acids Research 12, pp. 387-395). For example, homology and identity can be calculated or sequences can be aligned (identifying equivalent residues or corresponding sequences, typically in their default settings) using the PILEUP and BLAST algorithms, as described in Altschul SF (1993) J Mol Evol 36:290-300 and Altschul, SF et al (1990) J Mol Biol 215:403-10. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0061] The term “pore” is well known to those skilled in the art and typically refers to a biological pore, i.e., a typical protein structure that defines a channel. In the context of defining the chimeric pore monomer of the present invention, the term “pore” typically refers to a structure that is associated with or transverses a membrane, such as a cell membrane, in its native state, preferably a protein structure. Other ring-shaped or channel-shaped structures are preferably not included. At least two different pores, or two different pores, preferably do not contain a proteasome. At least two different pores, or two different pores, preferably do not contain mouse proteasome activator 28α (also known as REG or 11S activator).

[0062] At least two different pores, or two different pores, are preferably selected from Wza, iota toxin, Bacillus anthrax protective antigen, Vibrio cholerae cytolysin, cytotoxin K (CytK), CELIII, CsgG, aerolidine, alpha-hemolysin, InvG, GspD, MspA, MspB, MspC, PorARr, PorBRr, PorARc, PilQ, necrotizing enterocolitis B-like toxin (NetB), FraC, portal proteins (G20c, P23_45, T4, SPP1, P22, Phi29), gamma-hemolysin, monalisin, lysenin, ClyA, and Clostridium perfringens beta toxin.

[0063] At least two different pores, or two different pores, preferably containing Wza, iota toxin, anthrax protective antigen, cholera cytolysin, cytotoxin K (CytK), CELIII, CsgG, aerolidine, alpha-hemolysin, InvG, GspD, MspA, MspB, MspC, PorARr, PorBRr, PorARc, PilQ, necrotizing enterocolitis B-like toxin (NetB), FraC, portal proteins (G20c, P23_45, T4, SPP1, P22, Phi29), gamma-hemolysin, monalisin, lysenin, ClyA, Clostridium perfringens beta toxin, parasporin-2, epsilon toxin, and the parasitic mushroom Laetiporus The lectins, volvatoxin, Cry toxin, Cyt1Aa, and Cyt2Aa are selected from those derived from *Sulphureus* (LSL).

[0064] At least three different pores are preferably selected from Wza, iota toxin, Bacillus anthrax protective antigen, Vibrio cholerae cytolysin, cytotoxin K (CytK), CELIII, CsgG, aerolidine, alpha-hemolysin, InvG, GspD, MspA, MspB, MspC, PorARr, PorBRr, PorARc, PilQ, necrotizing enterocolitis B-like toxin (NetB), FraC, portal proteins (G20c, P23_45, T4, SPP1, P22, Phi29), gamma-hemolysin, monalisin, lysenin, ClyA, and Clostridium perfringens beta toxin. Five different pores can be selected from any of these pores.

[0065] At least three different pores preferably contain Wza, iota toxin, anthrax protective antigen, cholera cytolysin, cytotoxin K (CytK), CELIII, CsgG, aerolidine, alpha-hemolysin, InvG, GspD, MspA, MspB, MspC, PorARr, PorBRr, PorARc, PilQ, necrotizing enterocolitis B-like toxin (NetB), FraC, portal proteins (G20c, P23_45, T4, SPP1, P22, Phi29), gamma-hemolysin, monalisin, lysenin, ClyA, Clostridium perfringens beta toxin, parasporin-2, epsilon toxin, and the parasitic mushroom Laetiporus Lectins derived from *Sulphureus* (LSL), including Volvatoxin, Cry toxin, Cyt1Aa, and Cyt2Aa, are selected. Five different pores can be selected from any of these pores.

[0066] The pores can be from any species. If a particular pore is found in multiple species, different pores may be selected from any pore of any of the species. For example, two different pores may be two different CsgG pores. For example, three different pores may be three different CsgG pores.

[0067] The at least two different pores may be two different PorARc pores or three different PorARc pores. The at least two different pores may be five different PorARc pores. The PorARc pore or two different PorARc pores preferably include a capping region (or scaffold) (e.g., C in Figure 12) and a constricting region (e.g., D in Figure 12). The PorARc pore, two different PorARc pores, or three different PorARc pores preferably include one or more of (a) a capping region (e.g., A in Figure 12), (b) a constricting region (e.g., D in Figure 12), and (c) a transmembrane beta-barrel region (e.g., B in Figure 12), for example, (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), (a), (b), and (c), etc. The chimeric pore monomer preferably comprises one or more of (a) a capping region, (b) a constricting region, and (c) a transmembrane beta-barrel region, for example, (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), (a), (b), and (c), etc. The chimeric pore monomer preferably comprises (a) to (c). The chimeric pore monomer preferably comprises (a) and (c) supplied from one PorARc pore and (b) supplied from a different PorARc pore. The chimeric pore monomer preferably comprises (a), (b), and (c) each supplied from a different PorARc pore, or (a), (b), and (c) each supplied from three different PorARc pores. The PorARc pores, two different PorARc pores, or three different PorARc pores may have any structure, but preferably have or include the structure of the wild-type PorARc pore (Figure 12). The protein structure of PorARc defines channels or holes that allow for the translocation of molecules and ions from one side of the membrane to the other.

[0068] The PorARc pores, two different PorARc pores, or three different PorARc pores may be of any size, but preferably have the dimensions of wild-type PorARc_Rco (Figure 12). The PorARc pores or at least two different PorARc pores preferably have an outer diameter of about 70 to about 110 Å at their widest point, for example, about 80 to about 100 Å or about 85 to about 95 Å at their widest point. The PoARc pores, two different PoARc pores, or three different PoARc pores preferably have an outer diameter of about 90.7 Å at their widest point. The PorARc pores, two different PorARc pores, or three different PorARc pores preferably have a total length of about 70 to about 110 Å, for example, about 80 to about 100 Å or about 85 to about 95 Å. The PorARc pores, two different PorARc pores, or three different PorARc pores preferably have a total length of about 90.4 Å. "Total length" and "length" refer to the length of the pore or pore region when viewed from the side (see, for example, the side view in Figure 12).

[0069] The capping region (A in Figure 12) preferably has a length of about 25 to about 65 Å, for example, about 35 to about 55 Å, or about 40 to about 50 Å. The capping region preferably has a length of about 44.7 Å. The channel defined by the capping region preferably has an opening with a diameter of about 30 to about 70 Å, for example, about 40 to about 60 Å or about 45 to about 55 Å. The channel defined by the capping region preferably has an opening with a diameter of about 49 Å. The channel defined by the capping region preferably has a diameter of about 20 to about 60 Å at its narrowest point, for example, about 30 to about 50 Å or about 35 to about 45 Å at its narrowest point. The channel defined by the capping region preferably has a diameter of about 41.5 Å at its narrowest point.

[0070] The transmembrane beta-barrel region (B in Figure 12) preferably has a length of about 5 to about 45 Å, for example, about 15 to about 35 Å, or about 20 to about 30 Å. The transmembrane beta-barrel preferably has a length of about 26.2 Å. The channel defined by the transmembrane beta-barrel region preferably has a diameter of about 20 to about 60 Å at its narrowest point, for example, about 30 to about 50 Å or about 35 to about 45 Å at its narrowest point. The channel defined by the transmembrane beta-barrel region preferably has a diameter of about 39.8 Å at its narrowest point.

[0071] The cap region (or scaffold) (C in Figure 12, formed from A and B) preferably has a length of about 55 to about 95 Å, for example, about 65 to about 85 Å or about 70 to about 80 Å. The cap region (or scaffold) preferably has a length of about 73.6 Å. The channel defined by the cap region (or scaffold) (C) preferably has a diameter of about 20 to about 60 Å at its narrowest point, for example, about 30 to about 50 Å or about 35 to about 45 Å at its narrowest point. The channel defined by the cap region (or scaffold) (C) preferably has a diameter of about 39.8 Å at its narrowest point.

[0072] The constricted region (D in Figure 12) is preferably about 5 to about 40 Å, and has a length of, for example, about 10 to about 30 Å, or about 15 to about 25 Å. The constricted region is preferably about 19.7 Å in length. The channel defined by the constricted region is preferably about 10 to about 50 Å in diameter at its narrowest point, for example, about 20 to about 40 Å, about 22 to about 32 Å, or about 25 to about 35 Å in diameter at its narrowest point. The channel defined by the constricted region is preferably about 27.4 Å in diameter at its narrowest point. The channel defined by the constricted region is preferably about 10 to about 50 Å in diameter at its narrowest point, for example, about 15 to about 55 Å, about 25 to about 45 Å, or about 30 to about 40 Å in diameter at the base of the pore structure. The channel defined by the constricted region preferably has a diameter of about 36.1 Å at the base of the pore structure. The constricted region preferably has a diameter of about 20 to about 60 Å, for example, about 30 to about 50 Å or about 35 to about 45 Å. The constricted region preferably has a diameter of about 41.9 Å.

[0073] All of the above measurements are based on backbone-to-backbone measurements of amino acids forming different regions (as shown in Figure 12).

[0074] The capping region (or scaffold) in the PorARc pore, at least two different PorARc pores, or at least three different PorARc pores (e.g., C in Figure 12) is preferably about 67 to about 187 amino acids in length. 7 pieces, 88 pieces, 89 pieces, 90 pieces, 91 pieces, 92 pieces, 93 pieces, 94 pieces, 95 pieces, 96 pieces, 97 pieces, 98 pieces, 99 pieces, 100 pieces, 101 pieces, 102 pieces, 103 pieces, 104 pieces, 105 pieces, 10 6 pieces, 107 pieces, 108 pieces, 109 pieces, 110 pieces, 111 pieces, 112 pieces, 113 pieces, 114 pieces, 115 pieces, 116 pieces, 117 pieces, 118 pieces, 119 pieces, 120 pieces, 121 pieces, 122 pieces, 1 23 pieces, 124 pieces, 125 pieces, 126 pieces, 127 pieces, 128 pieces, 129 pieces, 130 pieces, 131 pieces, 132 pieces, 133 pieces, 134 pieces, 135 pieces, 136 pieces, 137 pieces, 138 pieces, 139 pieces , 140 pieces, 141 pieces, 142 pieces, 143 pieces, 144 pieces, 145 pieces, 146 pieces, 147 pieces, 148 pieces, 149 pieces, 150 pieces, 151 pieces, 152 pieces, 153 pieces, 154 pieces, 155 pieces, 15 The lengths are 6, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, or 187. The cap regions (or scaffolds) of PorARc, at least two different PorARc pores, or at least three different PorARc pores are preferably about 87 to about 167 amino acids in length.The cap regions (or scaffolds) in PorARc, at least two different PorARc pores, or at least three different PorARc pores preferably contain approximately 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, and 120 amino acids. The lengths are 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, and 167. The capping region (or scaffold) in the PorARc pore, at least two different PorARc pores, or at least three different PorARc pores is preferably about 87, 159, 160, 166, or 167 amino acids in length.

[0075] The constricted region in the PorARc pore, at least two different PorARc pores, or at least three different PorARc pores is preferably about 5 to 114 amino acids in length. The constricted region in PorARc, at least two different PorARc pores, or at least three different PorARc pores preferably contains about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, and 54 amino acids. , 55 pieces, 56 pieces, 57 pieces, 58 pieces, 59 pieces, 60 pieces, 61 pieces, 62 pieces, 63 pieces, 64 pieces, 65 pieces, 66 pieces, 67 pieces, 68 pieces, 69 pieces, 70 pieces, 7 1 piece, 72 pieces, 73 pieces, 74 pieces, 75 pieces, 76 pieces, 77 pieces, 78 pieces, 79 pieces, 80 pieces, 81 pieces, 82 pieces, 83 pieces, 84 pieces, 85 pieces, 86 pieces, 87 pieces The lengths are 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, or 114. The constricted region in the PorARc pore, at least two different PorARc pores, or at least three different PorARc pores is preferably about 15 to about 94 amino acids in length.The constricted region in the PorARc pore, at least two different PorARc pores, or at least three different PorARc pores preferably contains about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46 amino acids. The lengths are 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, or 94. The constricted regions in the PorARc pores, at least two different PorARc pores, or at least three different PorARc pores are preferably about 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 39, 50, 52, 86, and 94 amino acids in length.

[0076] PorARc pores are preferably selected from the pores shown in Table 2. Table 2 - Different PorARc pores (aa = amino acid) for use in the present invention [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13] [Table 2-14] [Table 2-15] [Table 2-16]

[0077] PorARc pores are preferably selected from the pores in Table 2. The (fourth) "reference" column contains the GenBank reference information for the wild-type (or naturally occurring) sequence of each pore. PorARc pores may be selected from any of the wild-type pores in Table 2, i.e., from the reference information in column 4. PorARc pores may be selected from any of the wild-type pores in Table 2 from which the signal peptide has been removed and which have methionine (M) at the N-terminus (i.e., position 1). Those skilled in the art can determine these sequences from the reference information in column 4.

[0078] The preferred capping and constricting regions for each pore are shown in columns 5 and 6 of Table 2. The residue numbers in columns 5 and 6 correspond to the wild-type sequence that does not contain a signal peptide and has methionine (M) at the N-terminus (i.e., position 1). The residue numbers of the capping and constricting regions will need to be adjusted to match the wild-type (or naturally occurring) sequence in the reference information in column 4. The capping and constricting regions shown in columns 5 and 6 of Table 2 are preferred capping and constricting regions. The present invention also includes further capping and constricting regions that differ from the regions shown in columns 5 and 6 by only ±10 amino acids. For example, in PorARc_Rco (pore 1), -1 to 86th place includes 1st to 76th place, 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, or 96th place. -107 to 180th place includes 97th, 98th, 99th, 100th, 101st, 102nd, 103rd, 104th, 105th, 106th, 107th, 108th, 109th, 110th, 111th, 112th, 113th, 114th, 115th, 116th, or 117th to 170th place, 171st, 172nd, 173rd, 174th, 175th, 176th, 177th, 178th, 179th, 180th, 181st, 182nd, 183rd, 184th, 185th, 186th, 187th, 188th, 189th, or 190th place. -87 to 106th place implies 77th, 78th, 79th, 80th, 81st, 82nd, 83rd, 84th, 85th, 86th, 87th, 88th, 89th, 90th, 91st, 92nd, 93rd, 94th, 95th, 96th, 97th to 96th, 97th, 98th, 99th, 100th, 101st, 102nd, 103rd, 104th, 105th, 106th, 107th, 108th, 109th, 110th, 111th, 112th, 113th, 114th, 115th, 116th, or 117th place.

[0079] This also applies to pores 2-161 in Table 2. As will be explained in more detail below, at least one of two regions, or one of two regions, in the chimeric pore monomer preferably includes one or more modifications that stabilize the chimeric pore and / or improve the ability of the chimeric pore formed from the chimeric pore monomer to characterize the target analyte. One or more negatively charged amino acids in any of the pores in Table 2 may be removed, for example, by deletion or substitution. One or more negatively charged amino acids in any of the pores in Table 2, such as one or more E and / or D, are preferably deleted or substituted with one or more different amino acids, such as one or more positively charged amino acids and / or one or more uncharged amino acids. This removes the negative charge from the pore arrangement. Any number of negatively charged amino acids, such as one, two, three, four, five, six, seven, eight, nine, ten, or more, may be deleted or substituted. One or more negatively charged amino acids are preferably located in the pore constriction region. The preferred pore constriction region is defined in column 6 of Table 2. This applies to both the wild-type sequence and the sequences in Table 2, including the wild-type sequence that does not contain a signal peptide and has methionine (M) at the N-terminus (i.e., position 1).

[0080] Preferred pore constriction substitutions are shown in column 7. The PorARc pores may be selected from any of the pores in Table 2, which consist of substituting a negatively charged amino acid (such as D or E) with a positively charged amino acid (such as R, H, or K) or an uncharged amino acid (such as S, T, N, or Q) at one or more, preferably all, of the indicated positions. For example, the PorARc pore may be pore 2, in which D at positions 91 and / or 92 is substituted with a positively charged amino acid such as R, H, or K, or an uncharged amino acid such as S, T, N, or Q. The PorARc pores may also be pores 3, pore 8, or pore 19, in which D at positions 91 and / or 92 is substituted with a positively charged amino acid such as R, H, or K, or an uncharged amino acid such as S, T, N, or Q. PorARc pores may also be pores 17 in which D or E at position 91 and / or 101 is substituted with a positively charged amino acid such as R, H, or K, or an uncharged amino acid such as S, T, N, or Q. PorARc pores may also be pores 25 in which D or E at one or more, preferably all, of positions 89, 91, 93, and 100 is substituted with a positively charged amino acid such as R, H, or K, or an uncharged amino acid such as S, T, N, or Q. PorARc pores may also be pores 27 in which D at one or more, preferably all, of positions 90, 95, and 103 is substituted with a positively charged amino acid such as R, H, or K, or an uncharged amino acid such as S, T, N, or Q. This is also true for any of the sequences in Table 2, including the wild-type sequence and the wild-type sequence that does not contain a signal peptide and has methionine (M) at the N-terminus (i.e., position 1). The residue numbers in column 7 correspond to the wild-type sequence, which does not contain a signal peptide and has methionine (M) at the N-terminus (i.e., position 1).

[0081] The PorARc pore may be selected from any of the pores in Table 2 that contain one or more of the substitutions shown in column 7. For example, the PorARc pore may be pore 2 in Table 2 having D91N and / or D92N. The PorARc pore may be pore 3, pore 8, or pore 19 having D91N and / or D92N. The PorARc pore may be pore 17 having D91N and / or E101Q. The PorARc pore may be pore 25 having one or more, preferably all, of E89Q, D91N, D93N, and D100N. The PorARc pore may be pore 27 having one or more, preferably all, of D90N, D95N, and D103N. This is also true for any of the sequences in Table 2, including the wild-type sequence and the wild-type sequence that does not contain a signal peptide and has methionine (M) at the N-terminus (i.e., position 1). The residue numbers in column 7 correspond to wild-type sequences that do not contain a signal peptide and have methionine (M) at the N-terminus (i.e., position 1). PorARc pores preferably contain or consist of (a) sequences shown in SEQ ID NOs. 50, 51, 52, 53, 54, or 55, or (b) sequences having at least about 20% homology or identity with sequences shown in SEQ ID NOs. 50, 51, 52, 53, 54, or 55. Such sequences are discussed in more detail below.

[0082] A PorARc pore may be a pore 1 of Table 2 having one or more cap substitutions. For example, PorARc is a pore 1 of Table 2, preferably having one or more, preferably all, of the 78, 82, 116, 125, and 165 positions replaced with D or E by a positively charged amino acid such as R, H, or K, or an uncharged amino acid such as S, T, N, or Q. A PorARc pore is preferably a pore 1 of Table 2 having one or more, preferably all, of E78R, D82S, E116T, E125A, or D165S. This is also true for any of the pore 1 sequences of Table 2, including the wild-type sequence and the wild-type sequence that does not contain a signal peptide and has methionine (M) at the N-terminus (i.e., position 1). A PorARc pore preferably contains or consists of the sequence shown in Sequence ID No. 1.

[0083] PorARc pores may be pores 1 of Table 2 having one or more stenotic substitutions. For example, PorARc is preferably pores 1 of Table 2 in which D or E at positions 89 and / or 104 is substituted with a positive amino acid such as R, H or K, or an uncharged amino acid such as S, T, N or Q. PorARc pores are preferably pores 1 of Table 2 having E89R, E89Q, E89L or E89A and / or D104S or D104N, for example, pores 1 of Table 2 having E89R / D104S, E89Q / D104S, E89Q / D104N, E89L / D104S, E89L / D104N, E89A / D104S or E89A / D104N. The PorARc pore is preferably pore 1 of Table 2 having E89R and / or D104S, for example, E89R / D104S. This is also true for any of the pore 1 sequences of Table 2, including the wild-type sequence and the wild-type sequence that does not contain a signal peptide and has methionine (M) at the N-terminus (i.e., position 1). These one or more constriction substitutions may be made in addition to the one or more cap substitutions described above.

[0084] At least two different pores preferably include at least two different PorARc pores. These two different pores are preferably two different PorARc pores. At least two different PorARc pores, or two different PorARc pores, may be selected from the pores in Table 2. One of the at least two different pores is preferably PorARc_Rco (pore 1 in Table 2) or PorARc_Mph (pore 2 in Table 2). One of the two different pores is preferably PorARc_Rco (pore 1 in Table 2) or PorARc_Mph (pore 2 in Table 2). These at least two different pores preferably include (a) PorARc_Rco or PorARc_Mph and (b) one of the pores in Table 2, or these two different pores preferably include (a) PorARc_Rco or PorARc_Mph and (b) one of the pores in Table 2. In (b), one of the pores in Table 2 is preferably pore 3, 8, 17, 19, 20, 25, or 27. At least two different pores preferably contain PorArc_Rco and PorARc_Rco_Mph, or these two different pores preferably contain PorArc_Rco and PorARc_Rco_Mph. The reference in this paragraph to a particular pore in Table 2 (e.g., pore 1) encompasses any of the pores described above in relation to that pore in Table 2, which includes any of the wild-type sequences, sequences lacking a signal peptide and containing M at the N-terminus (i.e., position 1), and one or more of the substitutions described above. The PorARc_Rco pore preferably contains or consists of the sequence shown in SEQ ID NO: 1. The PorARc_Mph pore preferably contains or consists of the sequence shown in SEQ ID NO: 2.

[0085] The constriction graft and cap graft (the latter also known as the scaffold graft) of Example 1 are formed from different PorARc pores. In each of these grafts, at least two different pores preferably comprise a combination of two different PorARc pores, or two different pores preferably comprise a combination of two different PorARc pores. At least two different pores preferably comprise any combination shown in the rows of Table 3, or two different pores preferably comprise any combination shown in the rows of Table 3. For any row in Table 3, the cap region (or scaffold) preferably derives from or originates from pores in column A, and the constriction region preferably derives from or originates from pores in column B. For any row in Table 3, the constriction region preferably derives from or originates from pores in column A, and the cap region (or scaffold) preferably derives from or originates from pores in column B. References in Table 3 to a specific pore in Table 2 (e.g., pore 3) encompass any of the pores described above in relation to that pore in Table 2, including any of the wild-type sequences, sequences lacking a signal peptide and containing M at the N-terminus (i.e., position 1), and any of the pores described above containing one or more of the substitutions described above. If preferred pores have sequence identifier numbers, these are also shown in Table 3. Pores preferably contain or consist of sequence identifier numbers. Table 3 - Preferred combinations of PorARc pores used in the present invention [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0086] The chimeric pore monomer preferably contains a sequence having at least about 40% homology or identity with the sequence shown in any one of SEQ ID NOs: 3 to 49. The chimeric pore monomer preferably contains a sequence having at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or more preferably at least about 95%, at least about 97%, at least about 98%, or at least about 99% homology or identity with the sequence shown in any one of SEQ ID NOs: 3 to 49. Any one of sequence numbers 3-49 is, of course, equivalent to sequence numbers 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49. Homology and / or identity are typically measured over the entire length of the chimeric pore monomer.

[0087] The chimeric pore monomer preferably contains a sequence having at least about 20% homology or identity with the sequence shown in any one of SEQ ID NOs: 3 to 49. The chimeric pore monomer preferably contains a sequence having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or more preferably at least about 95%, at least about 97%, at least about 98%, or at least about 99% homology or identity with the sequence shown in any one of SEQ ID NOs: 3 to 49. The chimeric pore monomer preferably contains a sequence having at least 100% identity with the sequence shown in any one of SEQ ID NOs: 3 to 49. Any one of sequence numbers 3-49 is, of course, equivalent to sequence numbers 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49. Homology and / or identity are typically measured over the entire length of the chimeric pore monomer.

[0088] In any of these embodiments, the chimeric pore monomer preferably does not contain the entire sequence of wild-type pores. In any of these embodiments, the chimeric pore monomer preferably does not contain a sequence that is 100% identical to the entire sequence of wild-type pores. In any of these embodiments, the chimeric pore monomer preferably does not contain the entire sequence of any of the different pores used to produce the chimeric pore monomer, or the entire sequence of any of the different pores that are the source or origin of two or more regions, such as two, three, or five regions. In any of these embodiments, the chimeric pore monomer preferably does not contain the entire sequence of any of the different pores used to produce the chimeric pore monomer, or a sequence that is 100% identical to the entire sequence of any of the different pores that are the source or origin of two or more regions, such as two, three, or five regions.

[0089] At least two different pores preferably include (a), (b), (c), or (d) below, or two different pores preferably include (a), (b), (c), or (d) below: (a) PorARc_Rco and MspA, (b) two different CsgG pores or three different CsgG pores, (c) alpha-hemolysin and CytK, or (d) NetB and CytK. At least two different pores preferably include (a), (b), (c), or (d) below, or two different pores preferably include (a), (b), (c), or (d) below: (a) PorARc_Rco and MspA, (b) two different CsgG pores, (c) alpha-hemolysin and CytK, or (d) NetB and CytK. At least two different pores preferably include five different CsgG pores. Two different CsgG pores or three different CsgG pores in (b) are WO2016 / 034591, WO2017 / 149316, WO2017 / 149317, WO2017 / 149318, WO2018 / 211241, WO2019 / 002893, PCT / EP2023 / 059821, PCT / EP2023 / 072113, PCT / EP202 Pores or their variants (all incorporated herein in whole by reference) disclosed in 3 / 072065, PCT / EP2023 / 072106, and PCT / EP2023 / 072068, CN113773373A, CN113896776A, CN113912683A, and CN113754743A may be selected. Five different CsgG pores may be selected from any of the pores disclosed in these documents. Two different CsgG pores may be CsgG from Escherichia coli and CsgG from a different species. Three different CsgG pores may be CsgG from Escherichia coli and CsgG from two different non-E. coli species. Five different CsgG pores may be CsgG from Escherichia coli and CsgG from four different non-E. coli species.

[0090] CsgG pores are well known in the art, particularly from WO2019 / 002893 (which is incorporated herein by reference in its entirety). A CsgG pore, two different CsgG pores, or three different CsgG pores preferably comprises one or more of (a) a capping region, (b) a constricting region, and (c) a transmembrane beta-barrel region, for example, (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), or (a), (b), and (c). This is also true for five different CsgG pores. The capping region (a) in CsgG is also known as a rim. The combination of (a) and (c) is also known as a scaffold. The chimeric pore monomer preferably comprises one or more of (a) a capping region, (b) a constricting region, and (c) a transmembrane beta-barrel region, for example, (a), (b), (c), (a) and (b), (a) and (c), (b) and (c), (a), (b), and (c), etc. The chimeric pore monomer preferably comprises (a) to (c). The chimeric pore monomer preferably comprises (a) and (c) from one CsgG pore and (b) from a different CsgG pore. The chimeric pore monomer preferably comprises (a), (b), and (c) each sourced from a different CsgG pore, or (a), (b), and (c) each sourced from three different CsgG pores. The residues of SEQ ID NOs. 56 to 64 that form these regions are defined below. The CsgG pore, two different CsgG pores, or three different CsgG pores may have any structure, but preferably have or include the structure of the wild-type CsgG pore (Figure 11). This is also true for five different CsgG pores. The protein structure of CsgG defines a channel or hole that allows for the translocation of molecules and ions from one side of the membrane to the other.

[0091] The terms “constriction,” “opening,” “constricted region,” “channel constriction,” or “constricted area,” as used interchangeably herein, refer to an opening defined by the luminal surface of a pore or pore complex, which acts to allow the passage of ions and target molecules (e.g., polynucleotides or not limited to individual nucleotides) but prevents the passage of other non-target molecules through the pore or pore complex channel. A constriction(s) is typically the narrowest opening(s) within a pore or pore complex, or within a channel defined by a pore or pore complex. Constrictions(s) can help limit the passage of molecules through the pore. The size of the constriction is typically a critical factor in determining the suitability of the pore or pore complex for characterizing an analyte. If the constriction is too small, the molecule being characterized will not be able to pass through. However, to achieve the greatest effect on ion flow through the channel, the constriction should not be too large. For example, the constriction should not be wider than the lateral diameter through which the solvent of the target analyte can access. Ideally, any constriction should be as close as possible to the lateral diameter of the analyte passing through it.

[0092] The CsgG pores, two different CsgG pores, or three different CsgG pores may be of any size, but preferably have the dimensions of wild-type CsgG pores (Figure 11). The CsgG pores, two different CsgG pores, or three different CsgG pores preferably have an outer diameter of about 100 to about 150 Å at their widest point, for example, about 110 to about 140 Å or about 115 to about 125 Å at their widest point. The CsgG pores, two different CsgG pores, or three different CsgG pores preferably have an outer diameter of about 120 Å at their widest point. The CsgG pores, two different CsgG pores, or three different CsgG pores preferably have a total length of about 80 to about 120 Å, for example, about 90 to about 110 Å or about 95 to about 105 Å. The CsgG pores, two different CsgG pores, or three different CsgG pores preferably have a total length of approximately 98 Å. "Total length" and "length" refer to the length of the pore or pore region when viewed from the side (see, for example, the side view in Figure 11). These sizes are also the same for five different CsgG pores.

[0093] The capping region preferably has a length of about 20 to about 60 Å, for example, about 30 to about 50 Å or about 35 to about 45 Å. The capping region preferably has a length of about 39 Å. The channel defined by the capping region preferably has an opening with a diameter of about 45 to about 85 Å, for example, about 55 to about 75 Å or about 60 to about 70 Å. The channel defined by the capping region preferably has an opening with a diameter of about 66 Å. The channel defined by the capping region preferably has a diameter of about 30 to about 70 Å at its narrowest point, for example, about 35 to about 60 Å or about 40 to about 50 Å at its narrowest point. The channel defined by the capping region preferably has a diameter of about 43 Å at its narrowest point.

[0094] The constricted region preferably has a length of about 5 to about 40 Å, for example, about 10 to about 30 Å or about 15 to about 25 Å. The constricted region preferably has a length of about 20 Å. The channel defined by the constricted region preferably has a diameter of about 2 to about 40 Å at its narrowest point, for example, about 5 to about 35 Å, about 8 to about 25 Å or about 10 to about 20 Å at its narrowest point. The channel defined by the constricted region preferably has a diameter of about 9 Å or 12 Å. The channel defined by the constricted region preferably has a diameter of about 18.5 Å. The constricted portion preferably has a diameter of about 2 to about 40 Å, for example, about 5 to about 35 Å, about 8 to about 25 Å or about 10 to about 20 Å. The constricted portion preferably has a diameter of about 9 Å or 12 Å. The constricted portion preferably has a diameter of about 12 Å.

[0095] The transmembrane beta-barrel region preferably has a length of about 20 to about 60 Å, for example, about 30 to about 50 Å or about 35 to about 45 Å. The transmembrane beta-barrel preferably has a length of about 39 Å. The channel defined by the transmembrane beta-barrel region preferably has a diameter of about 35 to about 75 Å at its narrowest point, for example, about 45 to about 65 Å or about 50 to about 60 Å at its narrowest point. The channel defined by the transmembrane beta-barrel region preferably has a diameter of about 55 Å at its narrowest point.

[0096] All of the above measurements are based on backbone-to-backbone measurements of amino acids forming different regions (as shown in Figure 11).

[0097] The capping region in the CsgG pore, at least two different CsgG pores, or at least three different CsgG pores is preferably about 164 to about 210 amino acids in length. The capping region (or scaffold) in the CsgG pore, at least two different CsgG pores, or at least three different CsgG pores is preferably about 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 1 The lengths are 80, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, or 210. The capping region (or scaffold) in the CsgG pore, at least two different CsgG pores, or at least three different CsgG pores is preferably about 184 to about 190 amino acids in length. The capping region (or scaffold) in the CsgG pore, at least two different CsgG pores, or at least three different CsgG pores is preferably about 184, 185, 186, 187, 188, 189, or 190 amino acids in length. These lengths are also the same for five different CsgG pores. The capping region preferably includes a landing platform region and a carboxyl-terminal (C-terminal) region.

[0098] The constricted region of a CsgG pore, at least two different CsgG pores, or at least three different CsgG pores is preferably about 6 to about 46 amino acids in length. The constricted region in a CsgG pore, at least two different CsgG pores, or at least three different CsgG pores is preferably about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, or 46 amino acids in length. The constricted region of a CsgG pore, at least two different CsgG pores, or at least three different CsgG pores is preferably about 16 to about 36 amino acids in length. The constricted region of a CsgG pore, at least two different CsgG pores, or at least three different CsgG pores is preferably about 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 amino acids in length. The constricted portion of a CsgG pore, at least two different CsgG pores, or at least three different CsgG pores is preferably about 26 amino acids in length. These lengths are similar for five different CsgG pores.

[0099] The transmembrane beta-barrel regions of the CsgG pore, at least two different CsgG pores, or at least three different CsgG pores are preferably about 28 to 68 amino acids in length. The constricted region within the CsgG pore, at least two different CsgG pores, or at least three different CsgG pores is preferably about 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, or 68 amino acids in length. The transmembrane beta-barrel regions of the CsgG pore, at least two different CsgG pores, or at least three different CsgG pores are preferably about 48 amino acids long. These lengths are similar for five different CsgG pores.

[0100] CsgG pores are highly conserved (as can be easily seen from Figures 45-47 of WO2017 / 149317). A CsgG pore, two different CsgG pores, or three different CsgG pores may have any of the sequences shown in Sequence IDs 68-88 of WO2019 / 002893 (which is incorporated herein by reference in its entirety), and may include any of the modifications or mutations disclosed therein. A CsgG pore, two different CsgG pores, or three different CsgG pores may also have any of the sequences shown in CN113773373A, CN113896776A, CN113912683A, and CN113754743A, or variants thereof. Five different CsgG pores may have any of these sequences. It will be further understood that the present invention also extends to other variant CsgG pores not explicitly specified herein, which represent highly conserved regions.

[0101] CsgG pores, two different CsgG pores, or three different CsgG pores are preferably selected from the pores in Table 4. Five different CsgG pores are preferably selected from the pores in Table 4. Table 4 - Different CsgG pores for use in the present invention (The position numbering in the last three columns excludes the signal peptides shown in SEQ ID NOs. 56-64. That is, positions 1-37 of SEQ ID NO. 56 in column 5 correspond to positions 16-52 of SEQ ID NO. 56 as shown in the sequence listing. TM = transmembrane, aa = amino acid) [Table 4-1] [Table 4-2]

[0102] Column 4 provides sequence numbers for wild-type pores. CsgG pores, two different CsgG pores, or three different CsgG pores may be selected from wild-type pores in Table 4 (i.e., from the sequences in Column 4). CsgG pores, two different CsgG pores, or three different CsgG pores may also be selected from wild-type pores in Table 4 with the signal peptide removed, i.e., from sequence numbers 56-64 with the signal peptide removed, or from sequence numbers 55-64 and 73-75 with the signal peptide removed. Five different CsgG pores may be selected from any of these pores. Those skilled in the art can determine these sequences from the sequences in the sequence listing where the signal peptide is underlined.

[0103] Preferred capping, constricting, and transmembrane beta-barrel regions of each pore are shown in columns 5, 6, and 7 of Table 4. These preferred regions may be used to construct the chimeric pore monomer of the present invention, as described above. The residue numbers in columns 5, 6, and 7 correspond to wild-type sequences that do not contain the signal peptide. Those skilled in the art can determine these sequences from the sequence listings where the signal peptide is underlined. The capping, transmembrane beta-barrel, and constricting regions in columns 5, 6, and 7 of Table 4 are preferred capping and constricting regions. The present invention encompasses capping, transmembrane beta-barrel, and constricting regions that differ from those shown in columns 5, 6, and 7 by approximately ±10 amino acids. For example, in CsgG_Eco_WT: -1 to 37th place means 1st to 27th, 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, or 47th place. -64~134th place includes 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, 73rd, or 74~124th place, 125th, 126th, 127th, 128th, 129th, 130th, 131st, 132nd, 133rd, 134th, 135th, 136th, 137th, 138th, 139th, 140th, 141st, 142nd, 143rd, or 144th place. -155~181 means 145th, 146th, 147th, 148th, 149th, 150th, 151st, 152nd, 153rd, 154th, 155th, 156th, 157th, 158th, 159th, 160th, 161st, 162nd, 163rd, 164th, or 165~171st, 172nd, 173rd, 174th, 175th, 176th, 177th, 178th, 179th, 180th, 181st, 182nd, 183rd, 184th, 185th, 186th, 187th, 188th, 189th, 190th, 191st. -210~262nd place includes 200th, 201st, 202nd, 203rd, 204th, 205th, 206th, 207th, 208th, 209th, 210th, 211th, 212th, 213th, 214th, 215th, 216th, 217th, 218th, 219th, or 220~252nd place, 253rd, 254th, 255th, 256th, 257th, 258th, 259th, 260th, 261st, 262nd, 263rd, 264th, 265th, 266th, 267th, 268th, 269th, 270th, 271st, or 272nd place. -38~63rd place implies 28th, 29th, 30th, 31st, 32nd, 33rd, 34th, 35th, 36th, 37th, 38th, 39th, 40th, 41st, 42nd, 43rd, 44th, 45th, 46th, 47th, or 48th~53rd place, 54th, 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, 65th, 66th, 67th, 68th, 69th, 70th, 71st, 72nd, or 73rd place. -135~154 implies 125th, 126th, 127th, 128th, 129th, 130th, 131st, 132nd, 133rd, 134th, 135th, 136th, 137th, 138th, 139th, 140th, 141st, 142nd, 143rd, 144th, or 145th~144th, 145th, 146th, 147th, 148th, 149th, 150th, 151st, 152nd, 153rd, 154th, 155th, 156th, 157th, 158th, 159th, 160th, 161st, 162nd, 163rd, or 164th, and -182~209 implies 172nd, 173rd, 174th, 175th, 176th, 177th, 178th, 179th, 180th, 181st, 182nd, 183rd, 184th, 185th, 186th, 187th, 188th, 189th, 190th, 191st, or 192nd~199th, 200th, 201st, 202nd, 203rd, 204th, 205th, 206th, 207th, 208th, 209th, 210th, 211th, 212th, 213th, 214th, 215th, 216th, 217th, 218th, or 219th.

[0104] This also applies to the other holes in Table 4.

[0105] The cap region may further contain two sub-regions, namely a landing platform region and a carboxyl-terminal (C-terminal) region. The landing platform region contains helix 2, which forms the Cys-side pore surface, and molecules such as the N22 peptide with a CsgA-like sequence, or enzymes in analyte characterization as discussed below, engage with the channel. This is a distinct structural and functional unit that can be replaced with homologous equivalent sequences. The C-terminal tail can carry further sequences upon fusion with the channel. These may originate from CsgG homologs. These regions correspond to the following residues of the CsgG pore in Table 4 (numbered excluding signal peptides): • Sequence ID 56, CsgG_Eco_WT: Landing platform region = 85~117, C-terminal region = 242~262 • Sequence ID 57, CsgG_Vdi_WT: Landing platform region = 87~119, C-terminal region = 244~263 • Sequence ID 58, CsgG_Vmae_WT: Landing platform region = 87~119, C-terminal region = 244~264 • Sequence ID 59, CsgG_Vsp_WT: Landing platform region = 87~119, C-terminal region = 244~263 • Sequence ID 60, CsgG_Ler_WT: Landing platform region = 85~117, C-terminal region = 242~261 • Sequence ID 61, CsgG_Vcr_WT: Landing platform region = 87~119, C-terminal region = 244~263 • Sequence ID 62, CsgG_Psh_WT: Landing platform region = 86~118, C-terminal region = 243~263 • Sequence ID 63, CsgG_Vhi_WT: Landing platform region = 87~115, C-terminal region = 240~259 • Sequence ID 64, CsgG_Vma_WT: Landing platform region = 87~119, C-terminal region = 244~263 • Sequence ID 73, CsgG_Vfu_WT: Landing platform region = 87~119, C-terminal region = 244~263 • Sequence ID 74, CsgG_Vme_WT: Landing platform region = 87~119, C-terminal region = 244~263

[0106] For Sequence ID No. 75, CsgG_Vge_WT: Landing platform region = 87-119, C-terminal region = 244-263. As is clear from this, the constriction region in Table 4 can be 46 amino acids long (for example, 28-73 in CsgG_Eco_WT). When constructing a chimeric pore monomer from two or three different CsgG pores, the entire constriction region of one CsgG pore may be used, i.e., all 46 amino acids of one CsgG pore may be used. This typically involves the complete replacement (i.e., absence) of the constriction region(s) from another different CsgG pore or two other different CsgG pores. This is also true for chimeric pore monomers constructed from five different CsgG pores. Alternatively, a portion of the constriction region of one CsgG pore may be replaced with all or part of the constriction region of a different CsgG pore. This is an example of the hybrid constriction region described above. At least about six amino acids in the constricted region of one CsgG pore, for example, at least about seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty, twenty-one, twenty-two, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty, thirty-one, thirty-two, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, twenty-eight, twenty-five, thirty-five, thirty-five, thirty-five, thirty-one, thirty-two, thirty-two, thirty-five As described above, the constricted region in the chimeric pore monomer may be longer, shorter, or the same length as the constricted region in the pore(s) from which the cap region and / or transmembrane beta-barrel region in the chimeric pore monomer are supplied or originated.At least about six amino acids in the constricted region of one CsgG pore, for example, at least about seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty, twenty-one, twenty-two, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty, thirty-one, thirty-two, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, twenty-eight, twenty-five, thirty-five, thirty-five, thirty-five, thirty-one, thirty-two, thirty-two, thirty-five At least about six amino acids in the constricted region of one CsgG pore, for example, at least about seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty, twenty-one, twenty-two, twenty-two, twenty-two, twenty-four, twenty-six, twenty-seven, twenty-eight, twenty-nine, thirty, thirty, thirty-one, thirty-two, thirty-two, thirty-three, thirty-four, thirty-five, thirty-six, thirty-seven, thirty-eight, thirty-four, thirty-five At least about six amino acids in the constricted region of the gG pore may be replaced, for example, at least about seven, about eight, about nine, about ten, about eleven, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, or about 46.

[0107] Preferred constriction regions for each pore are shown in column 6 of Table 4. Any of these constriction regions can be used to construct the chimeric pore monomer of the present invention as described above. All preferred constriction regions in Table 4 are 26 amino acids long. When constructing a chimeric pore monomer from two or three different CsgG pores, the entire constriction region of one CsgG pore may be used, i.e., all 26 amino acids of one CsgG pore may be used. This typically involves the complete replacement (i.e., absence) of constriction regions (or more) from other different CsgG pores or two other different CsgG pores. This is also true for chimeric pore monomers constructed from five different CsgG pores. Alternatively, a portion of the constriction region of one CsgG pore may be replaced with all or part of the constriction region of a different CsgG pore. This is an example of the hybrid constriction region described above. At least about six amino acids in the constriction region of one CsgG pore, for example, at least about seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty, twenty-one, twenty-two, twenty-two, twenty-three, twenty-four, or twenty-five, may be replaced with constriction regions from different CsgG pores. As described above, the constriction region in the chimeric pore monomer may be longer, shorter, or the same length as the constriction region in the pore(s) from which the cap region and / or transmembrane beta-barrel region in the chimeric pore monomer are supplied or derived. At least about six amino acids in the constricted region of one CsgG pore, for example, at least about seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty, twenty-one, twenty-two, twenty-three, twenty-four, or twenty-five, may be introduced into the constricted regions of different CsgG pores.At least about six amino acids in the constricted region of one CsgG pore, for example, at least about seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty, twenty-one, twenty-two, twenty-two, twenty-three, twenty-four, or twenty-five, may be replaced with at least about six amino acids in the constricted region of different CsgG pores, for example, at least about seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty-two, twenty-one, twenty-two, twenty-two, twenty-two, twenty-two, twenty-three, twenty-four, or twenty-five. At least about 15 amino acids in the constricted region of one CsgG pore may be replaced with at least about 14 amino acids from the constricted region of a different CsgG pore. At least about 18 amino acids in the constricted region of one CsgG pore may be replaced with at least about 20 amino acids from the constricted region of a different CsgG pore.

[0108] As will be described in more detail below, at least one of two regions, or one of two regions, in the chimeric pore monomer preferably includes one or more modifications that stabilize the chimeric pore and / or improve the ability of the chimeric pore formed from the chimeric pore monomer to characterize the target analyte. One or more negatively charged amino acids in any of the pores in Table 4 may be removed, for example, by deletion or substitution. One or more negatively charged amino acids in any of the pores in Table 4, such as one or more E and / or D, are preferably deleted or substituted with one or more different amino acids, such as one or more positively charged amino acids and / or one or more uncharged amino acids. This removes the negative charge from the pore arrangement. Any number of negatively charged amino acids, such as one, two, three, four, five, six, seven, eight, nine, ten, or more, may be deleted or substituted. These one or more negatively charged amino acids are preferably located in the constricted regions of the pore. The preferred constriction region of each pore is defined in column 6 of Table 4. This applies to all sequences in Table 4, including the wild-type sequence and the wild-type sequence without the signal peptide.

[0109] Sequence ID 56 is a wild-type CsgG pore from the Escherichia coli K-12 strain MC4100 substrain. A CsgG pore, one of two different CsgG pores, or one of three different CsgG pores may contain the sequence of Sequence ID 56, or it may contain Sequence ID 56 having any of the substitutions present in another CsgG homolog. This is also true for five different CsgG pores. Preferred CsgG homologs are shown in WO2019 / 002893 (which is incorporated herein in its entirety by reference) and Sequence IDs 68-88 in Table 4 above. A CsgG pore, one of two different CsgG pores, or one of three different CsgG pores may contain one or more combinations of substitutions present in WO2019 / 002893 (which is incorporated herein by reference in its entirety) or SEQ ID NOs. 68-88, or one or more substitutions, one or more conservative mutations, one or more deletions or one or more insertion mutations, such as deletions or insertions of 1 to 10 amino acids, such as 2 to 8 or 3 to 6 amino acids, in other homologies in Table 4 compared to SEQ ID NO. 56. This also applies to one of five different CsgG pores.

[0110] The chimeric pore monomers of the present invention typically retain the same 3D structure as wild-type CsgG pore monomers, for example, the ability to form the same 3D structure as CsgG pores having the sequence of Sequence ID No. 56. The 3D structure of CsgG is well known in the art and is disclosed, for example, in Goyal et al (2014) Nature 516(7530):250-3. Provided that the chimeric pore monomers retain the improved properties of the present invention, any number of modifications or mutations may be made in the wild-type CsgG sequence in addition to the modifications and mutations described herein.

[0111] Typically, chimeric pore monomers formed from CsgG pores, two different CsgG pores, or three different CsgG pores retain the ability to form structures containing three alpha helices and five beta sheets. This is also true for chimeric pore monomers constructed from five different CsgG pores. One or more modifications may be made without affecting the ability of the chimeric pore monomer to form transmembrane pores capable of dislocating the analyte, at least in the region that is the N-terminus of the first alpha-helix (starting at S63 in SEQ ID NO: 56), in the second alpha-helix (G85-A99 in SEQ ID NO: 56), in the loop between the second alpha-helix and the first beta-sheet (Q100-N120 in SEQ ID NO: 56), in the fourth and fifth beta-sheets (S173-R192 and R198-T107, respectively in SEQ ID NO: 56), and in the loop between the fourth and fifth beta-sheets (F193-Q197 in SEQ ID NO: 56). For chimeric pore monomers formed from CsgG pores, two different CsgG pores, or three different CsgG pores, further modifications may be made to any of these regions without affecting the ability of the chimeric pore monomer to form pores capable of dislocating the analyte. This is also true for chimeric pore monomers constructed from five different CsgG pores.

[0112] Furthermore, it is expected that one or more modifications may be made in other regions, for example, in any of the alpha-helices (S63-R76, G85-A99, or V211-L236 in SEQ ID NO: 56) or in any of the beta-sheets (I121-N133, K135-R142, I146-R162, S173-R192, or R198-T107 in SEQ ID NO: 56), without affecting the ability of the chimeric pore monomer to form pores that can rearrange the analyte. It is also expected that the deletion of one or more amino acids may be made in any of the loop regions connecting the alpha-helices and beta-sheets, and / or in the N-terminal and / or C-terminal regions, without affecting the ability of the chimeric pore monomer to form pores that can rearrange the analyte.

[0113] The chimeric pore monomer may include the region(s) of SEQ ID NO: 56 that cause pore formation. The pore-forming ability of CsgG containing β barrels is provided by the β sheet in each subunit. The chimeric pore monomer may include the region of SEQ ID NO: 56 that forms the β sheet, namely K134-Q154 and S183-S208. One or more modifications can be made to the region of SEQ ID NO: 56 that forms the β sheet, insofar as the resulting variant retains its ability to form pores. Preferably, the chimeric pore monomer includes one or more modifications, such as substitution, addition, or deletion, within the alpha-helix and / or loop region of SEQ ID NO: 56.

[0114] One or more modifications in a CsgG pore, two different CsgG pores, or three different CsgG pores preferably improve the analyte characterization ability of the chimeric pore monomer formed from a CsgG pore, two different CsgG pores, or three different CsgG pores. For example, the modification / mutation / substitution is intended to alter the number, size, shape, arrangement, or orientation of constrictions within the channel of the chimeric pore monomer. CsgG pores, two different CsgG pores, or three different CsgG pores may have any of the specific modifications or substitutions disclosed in WO2016 / 034591, WO2017 / 149316, WO2017 / 149317, WO2017 / 149318, WO2018 / 211241, WO2019 / 002893, PCT / EP2023 / 059821, PCT / EP2023 / 072113, PCT / EP2023 / 072065, PCT / EP2023 / 072106, and PCT / EP2023 / 072068 (all of which are incorporated herein in their entirety by reference). This also applies to chimeric pore monomers constructed from five different CsgG pores.

[0115] Preferred modifications or substitutions in Sequence ID No. 56 include, but are not limited to, one or more of the following: two or more, three or more, four or more, five or more, six or more, seven or more, or all of the following: (a) Replacement at position Y51, e.g., Y51I, Y51L, Y51A, Y51V, Y51T, Y51S, Y51Q or Y51N, (b) Replacement at position N55, e.g., N55I, N55L, N55A, N55V, N55T, N55S or N55Q, (c) Replacement at position F56, for example F56I, F56L, F56A, F56V, F56T, F56S, F56Q or F56N, (d) Replacement at position L90, e.g., L90N, L90D, L90E, L90R or L90K, (e) Replacement at position N91, e.g., N91D, N91E, N91R or N91K, (f) Substitution at position K94, e.g., K94R, K94F, K94Y, K94Q, K94W, K94L, K94S, or K94N, (g) Substitution at position R192, for example R192Q, R192F, R192S, R192D, or R192T, and (i) Replacement at position C215, for example, C215T, C215S, C215I, C215L, C215A, C215V, or C215G.

[0116] Preferred modifications or substitutions in Sequence ID No. 56 include, but are not limited to, one or more of the following: two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, eleven or more, twelve or more, or all of the following: (a) Replacement at position Y51, e.g., Y51I, Y51L, Y51A, Y51V, Y51T, Y51S, Y51Q or Y51N, (b) Replacement at position N55, e.g., N55I, N55L, N55A, N55V, N55T, N55S or N55Q, (c) Replacement at position F56, for example F56I, F56L, F56A, F56V, F56T, F56S, F56Q or F56N, (d) Replacement at position L90, e.g., L90N, L90D, L90E, L90R or L90K, (e) Replacement at position N91, e.g., N91D, N91E, N91R or N91K, (f) Substitution at position K94, e.g., K94R, K94F, K94Y, K94Q, K94W, K94L, K94S, or K94N, (g) Replacement at position R97, for example R97H, R97K, R97A, R97V, R97I, R97L, R97M, R97F, R97W, R97Y, R97S, R97T, R97Q, R97D, R97E, R97N, R97C, R97P, or R97G, (h) Replacement at position Q100, for example, Q100R, Q100H, Q100K, Q100W, Q100A, Q100V, Q100I, Q100L, Q100M, Q100F, Q100Y, Q100T, Q100N, or Q100S, (i) Substitution at position E101, for example E101V, E101I, E101L, E101M, E101A, E101F, E101Y, E101W, E101S, E101T, E101N, E101Q, E101C, E101G, or E101P, (j) Replacement at position N102, for example N102E, N102R, N102H, N102K, N102S, N102T, N102D, N102Q, N102V, N102I, N102L, N102M, N102F, N102Y, N102W, or N102A, (k) Replacement at position T104, for example T104E, T104R, T104H, T104K, T104S, T104T, T104Q, T104V, T104D, T104I, T104L, T104M, T104F, T104Y, T104W, or T104A, (l) Replacement at position R192, for example R192Q, R192F, R192S, R192D, or R192T, (m) Replacement of position C215, for example, C215T, C215S, C215I, C215L, C215A, C215V, or C215G.

[0117] The CsgG pore may contain deletions at one or more positions from sequence number 56, such as deletions at V105-I107, F193-L199, or F195-L199.

[0118] All references to specific positions in sequence number 56 (e.g., V105) above relate to the sequence of sequence number 56 without the signal peptide (underlined in the sequence listing below).

[0119] CsgG pores are highly conserved (as can be easily seen from Figures 45-47 of WO2017 / 149317). Furthermore, knowledge of the modification associated with sequence number 56 makes it possible to determine the equivalent locations of modifications to other CsgG pores, particularly those with the sequences shown in sequence numbers 57-64 in Table 4.

[0120] In addition to those discussed above, amino acid substitutions can be made to any of the amino acid sequences of SEQ ID NOs. 56-64, for example, by 1, 2, 3, 4, 5, 10, 20, or 30 substitutions. Conservative substitutions replace amino acids with other amino acids that have a similar chemical structure, similar chemical properties, or similar side-chain volume. The introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino 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.

[0121] CsgG pores, two different CsgG pores, or three different CsgG pores may be modified to introduce one or more cysteines, one or more hydrophobic amino acids, one or more charged amino acids, one or more unnatural amino acids, one or more polar amino acids, or one or more photoreactive amino acids. Such introductions may be carried out in any number and combination. The introductions are preferably carried out by substitution or addition. This is also true for chimeric pore monomers constructed from five different CsgG pores.

[0122] One or more amino acid residues from any of the amino acid sequences of SEQ ID NOs. 56-64 may be deleted from the polypeptides described above. Up to one, two, three, four, five, ten, twenty, or thirty or more residues may be deleted.

[0123] One or more amino acids may be added to the CsgG polypeptide described above, either substituted or in addition. The elongation portion may be provided at the amino-terminus or carboxy-terminus of any of the amino acid sequences of SEQ ID NOs. 56-64, or at its polypeptide variant or fragment. The elongation portion may be very short, for example, 1 to 10 amino acids in length. Alternatively, the elongation portion may be longer, for example, up to 50 or 100 amino acids.

[0124] The at least two different pores preferably include at least two different CsgG pores or at least three different CsgG pores. The two different pores are preferably two different CsgG pores. The three different pores are preferably three different CsgG pores. The at least two different pores preferably include at least five different CsgG pores or five different CsgG pores.

[0125] At least two different CsgG pores, two different CsgG pores, or three different CsgG pores may be selected from the pores in Table 4. One of the at least two different pores is preferably CsgG_Eco_WT (first row of Table 4). One of the two different pores is preferably CsgG_Eco_WT (first row of Table 4). One of the three different pores is preferably CsgG_Eco_WT (first row of Table 4). One of the five different pores is preferably CsgG_Eco_WT (first row of Table 4).

[0126] At least two different pores preferably include (a) CsgG_Eco_WT, (b) CsgG_Vdi_WT, (c) CsgG_Vmae_WT, (d) CsgG_Vsp_WT, (e) CsgG_Ler_WT, (f) CsgG_Vcr_WT, (g) CsgG_Psh_WT, (h) CsgG_Vhi_WT, or (i) CsgG_Vma_WT. Alternatively, two or three different pores may preferably be selected from (a) to (i) above, which are defined in Table 4. Five different pores may also be selected from (a) to (i). At least two different pores preferably include the following, or two different pores preferably include the following: (a) and (b), (a) and (c), (a) and (d), (a) and (e), (a) and (f), (a) and (g), (a) and (h), (a) and (i), (b) and (c), (b) and (d), (b) and (e), (b) and (f), (b) and (g), (b) and (h), (b) and (i) , (c) and (d), (c) and (e), (c) and (f), (c) and (g), (c) and (h), (c) and (i), (d) and (e), (d) and (f), (d) and (g), (d) and (h), (d) and (i), (e) and (f), (e) and (g), (e) and (h), (e) and (i), (f) and (g), (f) and (h), (f) and (i), (g) and (h), (g) and (i), or (h) and (i).

[0127] At least three different pores preferably include the following, or three different pores preferably include the following: (a) and (b) and (c); (a) and (b) and (d); (a) and (b) and (e); (a) and (b) and (f); (a) and (b) and (g); (a) and (b) and (h); (a) and (b) and (i); (a) and (c) and (d); (a) and (c) and (e); (a) and (c) and (f); (a) and (c) and (g); (a) and (d) (h); (a) and (d) and (i); (a) and (e) and (f); (a) and (e) and (g); (a) and (e) and (h); (a) and (e) and (i); (a) and (f) and (g); (a) and (f) and (h); (a) and (f) and (i); (a) and (g) and (h); (a) and (g) and (i); (a) and (h) and (i); (b) and (c) and (d); (b) and (c) and (e); (b) and (c) and (f); (b) and (c) and (i); (b) and (d) and (e); (b) and (d) and (f); (b) and (d) and (g); (b) and ( d) and (h); (b) and (d) and (i); (b) and (e) and (f); (b) and (e) and (g); (b) and (e) and (h); (b) and (e) and (i); (b) and (f) and (g); (b) and (f) and (h); (b) and (f) and (i); (b) and (g) and (h); (b) and (g) and (i); (b) and (h) and (i); (c) and (d) and (e); (c) and (d) and (f); (c) and (d) and (g); (c) and (d) and (i); (c) and (e) and (f); (c) and (e) and (g); (c) and (e) and (h); (c) and (e) and (i); (c ) and (f) and (g); (c) and (f) and (h); (c) and (f) and (i); (c) and (g) and (h); (c) and (g) and (i); (c) and (h) and (i); (d) and (e) and (f); (d) and (e) and (g); (d) and (e) and (h); (d) and (e) and (i); (d) and (f) and (g); (d) and (f) and (h); (d) and (f) and (i); (d) and (g) and (i); (e) and (f) and (g); (e) and (f) and (h); (e) and (f) and (i); (e) and (g) and (h); (e) and (g) and (i);(e) and (h) and (i); (f) and (g) and (h); (f) and (g) and (i); (f) and (h) and (i); or (g) and (h) and (i).

[0128] At least two different pores preferably include the following, or two or three different pores preferably selected from the following: (a) CsgG_Eco_WT, (b) CsgG_Vdi_WT, (c) CsgG_Vmae_WT, (d) CsgG_Vsp_WT, (e) CsgG_Ler_WT, (f) CsgG_Vcr_WT, (g) CsgG_Psh_WT, (h) CsgG_Vhi_WT, (i) CsgG_Vma_WT, (j) CsgG_Vfu_WT, (k) CsgG_Vme_WT, or (l) CsgG_Vge_WT. (a) to (l) are defined in Table 4. Five different pores may be selected from (a) to (l). At least two different pores preferably include the following, or two different pores preferably include the following: (a) and (b), (a) and (c), (a) and (d), (a) and (e), (a) and (f), (a) and (g), (a) and (h), (a) and (i), (a) and (j), (a) and (k), (a) and (l), (b) and ( c), (b) and (d), (b) and (e), (b) and (f), (b) and (g), (b) and (h), (b) and (i), (b) and (j), (b) and (k), (b) and (l), (c) and (d), (c) and (e), (c) and (f), (c) and (g), (c) and (h), (c) and (i), (c) and (j), (c) and (k), (c) and (l) , (d) and (e), (d) and (f), (d) and (g), (d) and (h), (d) and (i), (d) and (j), (d) and (k), (d) and (l), (e) and (f), (e) and (g), (e) and (h), (e) and (i), (e) and (j), (e) and (k), (e) and (l), (f) and (g), (f) and (h), (f) and (i), ( f) and (j), (f) and (k), (f) and (l), (g) and (h), (g) and (i), (g) and (j), (g) and (k), (g) and (l), (h) and (i), (h) and (j), (h) and (k), (h) and (l), (i) and (j), (i) and (k), (i) and (l), (j) and (k), (j) and (l), or (k) and (l).

[0129] At least three different pores preferably include the following, and the three different pores preferably include the following: (a) and (b) and (c); (a) and (b) and (d); (a) and (b) and (e); (a) and (b) and (f); (a) and (b) and (g); (a) and (b) and (h); (a) and (b) and (i); (a) and (b) and (j); (a) and (b) and (k); (a) and (b) and (l); (a) and (c) and (d); (a) and (c) and (e); (a) and (c) and (f); (a) and (c) and (g); (a) and (c) and (h); (a) and (c) and (i); (a) and (c) and ( j); (a) and (c) and (k); (a) and (c) and (l); (a) and (d) and (e); (a) and (d) and (f); (a) and (d) and (g); (a) and (d) and (h); (a) and (d) and (i); (a) and (d) and (j); (a) and (d) and (k); (a) and (d) and (l); (a) and (e) and (f); (a) and (e) and (g); (a) and (e) and (h); (a) and (e) and (i); (a) and (e) and (j); (a) and (e) and (k); (a) and (e) and (l); (a) and (f) and (g); (a) and (f) and (h); (a) and (f) and (i); (a) and (f ) and (j); (a) and (f) and (k); (a) and (f) and (l); (a) and (g) and (h); (a) and (g) and (i); (a) and (g) and (j); (a) and (g) and (k); (a) and (g) and (l); (a) and (h) and (i); (a) and (h) and (j); (a) and (h) and (k); (a) and (h) and (l); (a) and (i) and (j); (a) and (i) and (k); (a) and (i) and (l); (a) and (j) and (k); (a) and (j) and (l); (a) and (k) and (l); (b) and (c) and (d); (b) and (c) and (e); (b) and (c) and (f); (b) (c) and (g); (b) and (c) and (h); (b) and (c) and (i); (b) and (c) and (j); (b) and (c) and (k); (b) and (c) and (l); (b) and (d) and (e); (b) and (d) and (f); (b) and (d) and (g); (b) and (d) and (h); (b) and (d) and (i); (b) and (d) and (k); (b) and (d) and (l); (b) and (e) and (f); (b) and (e) and (g); (b) and (e) and (h); (b) and (e) and (i); (b) and (e) and (j); (b) and (e) and (k); (b) and (e) and (l);(b) and (f) and (g); (b) and (f) and (h); (b) and (f) and (i); (b) and (f) and (j); (b) and (f) and (k); (b) and (f) and (l); (b) and (g) and (h); (b) and (g) and (i); (b) and (g) and (j); (b) and (g) and (k); (b) and (g) and (l); (b) and (h) and (i); (b) and (h) and (j); (b) and (h) and (k); (b) and (h) and (l); (b) and (i) and (j); (b) and (i) and (k); (b) and (i) and (l); (b) and (j) and (l); (b) and (k) and ( l);(c) and(d) and(e);(c) and(d) and(f);(c) and(d) and(g);(c) and(d) and(h);(c) and(d) and(i);(c) and(d) and(j);(c) and(d) and(k);(c) and(d) and(l);(c) and(e) and(f);(c) and(e) and(g);(c) and(e) and(h);(c) and(e) and(i);(c) and(e) and(j);(c) and(e) and(k);(c) and(e) and(l);(c) and(f) and(g);(c) and(f) and(h);(c) and(f) and(i);(c) and(f) and(j);(c) and(f) and(k);(c) and(f ) and (l); (c) and (g) and (h); (c) and (g) and (i); (c) and (g) and (j); (c) and (g) and (k); (c) and (g) and (l); (c) and (h) and (i); (c) and (h) and (j); (c) and (h) and (k); (c) and (h) and (l); (c) and (i) and (j); (c) and (i) and (k); (c) and (i) and (l); (c) and (j) and (k); (c) and (k) and (l); (d) and (e) and (f); (d) and (e) and (g); (d) and (e) and (h); (d) and (e) and (i); (d) and (e) and (j); (d) (e) and (k); (d) and (e) and (l); (d) and (f) and (g); (d) and (f) and (h); (d) and (f) and (i); (d) and (f) and (j); (d) and (f) and (k); (d) and (f) and (l); (d) and (g) and (h); (d) and (g) and (i); (d) and (g) and (j); (d) and (g) and (k); (d) and (g) and (l); (d) and (h) and (i); (d) and (h) and (j); (d) and (h) and (k); (d) and (h) and (l); (d) and (i) and (j); (d) and (i) and (k); (d) and (i) and (l); (d) and (j) and (k);(d) and (j) and (l); (d) and (k) and (l); (e) and (f) and (g); (e) and (f) and (h); (e) and (f) and (i); (e) and (f) and (j); (e) and (f) and (k); (e) and (f) and (l); (e) and (g) and (h); (e) and (g) and (i); (e) and (g) and (j); (e) and (g) and (k); (e) and (g) and (l); (e) and (h) and (i); (e) and (h ) and (j); (e) and (h) and (k); (e) and (h) and (l); (e) and (i) and (j); (e) and (i) and (k); (e) and (i) and (l); (e) and (j) and (k); (e) and (j) and (l); (e) and (k) and (l); (f) and (g) and (h); (f) and (g) and (i); (f) and (g) and (j); (f) and (g) and (k); (f) and (g) and (l); (f) and (h) and (i); ( f) and (h) and (j); (f) and (h) and (k); (f) and (h) and (l); (f) and (i) and (j); (f) and (i) and (k); (f) and (i) and (l); (f) and (j) and (k); (f) and (j) and (l); (f) and (k) and (l); (g) and (h) and (i); (g) and (h) and (j); (g) and (h) and (k); (g) and (h) and (l); (g) and (i) and (j); (g) and (i) and (k);(g) and (i) and (l);(g) and (j) and (k);(g) and (j) and (l);(g) and (k) and (l);(h) and (i) and (j);(h) and (i) and (k);(h) and (i) and (l);(h) and (j) and (k);(h) and (j) and (l);(h) and (k) and (l);(i) and (j) and (k);(i) and (j) and (l);(i) and (k) and (l);or (j) and (k) and (l).

[0130] The stenosis grafts of Example 2 are formed from two different CsgG pores. In each of these grafts, at least two different pores preferably include a combination of two different CsgG pores, or two different pores preferably are a combination of two different CsgG pores. Using the above definitions of (a) to (i), at least two different pores preferably include (a) and (b), (a) and (c), (a) and (d), (a) and (e), (a) and (f), (a) and (g), (a) and (h), or (a) and (i), or two different pores preferably include (a) and (b), (a) and (c), (a) and (d), (a) and (e), (a) and (f), (a) and (g), (a) and (h), or (a) and (i). In these embodiments, the chimeric pore monomer preferably comprises a capping region and a transmembrane beta-barrel region from (a) and a constricting region from one of (b) to (i).

[0131] Using the above definitions of (a) to (i), at least two different pores preferably include (a) and (c), (a) and (d), (a) and (h), (a) and (i), or (a) and (b), or two different pores preferably include (a) and (c), (a) and (d), (a) and (h), (a) and (i), or (a) and (b). In these embodiments, the chimeric pore monomer preferably includes a capping region and a transmembrane beta-barrel region from (a) and a constricting region from (c), (d), (h), (i), or (d).

[0132] Using the definitions (a) to (i) above, at least two different pores preferably include (a) and (f), or (a) and (g), or two different pores preferably include (a) and (f), or (a) and (g). In these embodiments, the chimeric pore monomer preferably includes a capping region and a transmembrane beta-barrel region from (a), and a constricting region from (f) or (g).

[0133] Using the definitions (a) to (i) above, at least two different pores preferably include (a) and (c), (a) and (h), or (a) and (i), or two different pores preferably include (a) and (c), (a) and (h), or (a) and (i). In these embodiments, the chimeric pore monomer preferably includes a capping region from (a) and a constricting region from (c), (h), or (i).

[0134] The chimeric pore monomer preferably contains a sequence having at least about 20% homology or identity with the sequence shown in any one of SEQ ID NOs. 65-72 or any one of SEQ ID NOs. 76-78 or any one of SEQ ID NOs. 65-72 or any one of SEQ ID NOs. 76-78 The chimeric pore monomer preferably contains a sequence having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or more preferably at least about 95%, at least about 97%, at least about 98%, or at least about 99% homology or identity with respect to any one of the sequences shown in any one of SEQ ID NOs. 65-72, any one of the sequences shown in any one of SEQ ID NOs. 65-72 and 76-78, any one of the sequences shown in any one of SEQ ID NOs. 65-72 and 76-78, any one of the sequences shown in any one of SEQ ID NOs. 75-72 and 76-78, any one of the sequences that does not contain a signal peptide. The chimeric pore monomer preferably includes the sequence shown in any one of SEQ ID NOs. 65 to 72, or the sequence shown in any one of SEQ ID NOs. 65 to 72 that does not include the signal peptide; that is, it includes a sequence that has 100% identity with respect to the sequence shown in any one of SEQ ID NOs. 65 to 72, or with respect to the sequence shown in any one of SEQ ID NOs. 65 to 72 that does not include the signal peptide.The chimeric pore monomer preferably contains the sequence shown in any one of SEQ ID NOs. 65-72 and 76-78, or the sequence shown in any one of SEQ ID NOs. 65-72 and 76-78 without the signal peptide, i.e., it contains a sequence that has 100% identity with respect to any one of SEQ ID NOs. 65-72 and 76-78, or any one of SEQ ID NOs. 65-72 is naturally equivalent to SEQ ID NOs. 65, 66, 67, 68, 69, 70, 71, or 72. 65-72 and 76-78 are naturally equivalent to SEQ ID NOs. 65, 66, 67, 68, 69, 70, 71, 72, 76, 77, or 78. Homology and / or identity are typically measured over the entire length of the chimeric pore monomer.

[0135] The chimeric pore monomer preferably contains a sequence having at least about 20% homology or identity with the sequence shown in any one of SEQ ID NOs. 66, 67, 71, 72, and 65, or with the sequence shown in any one of SEQ ID NOs. 66, 67, 71, 72, and 65 that does not contain the signal peptide. The chimeric pore monomer preferably contains a sequence having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or more preferably at least about 95%, at least about 97%, at least about 98%, or at least about 99% homology or identity with respect to the sequence shown in any one of SEQ ID NOs. 66, 67, 71, 72, and 65, or the sequence shown in any one of SEQ ID NOs. 66, 67, 71, 72, and 65 that does not contain a signal peptide. The chimeric pore monomer preferably contains the sequence shown in any one of SEQ ID NOs: 66, 67, 71, 72, and 65, or the sequence shown in any one of SEQ ID NOs: 66, 67, 71, 72, and 65 without the signal peptide, i.e., it contains a sequence that has 100% identity with respect to the sequence shown in any one of SEQ ID NOs: 66, 67, 71, 72, and 65, or with respect to the sequence shown in any one of SEQ ID NOs: 66, 67, 71, 72, and 65 without the signal peptide. Any one of SEQ ID NOs: 66, 67, 71, 72, and 65 is, of course, equivalent to SEQ ID NOs: 66, 67, 71, 72, or 65. Homology and / or identity are typically measured over the entire length of the chimeric pore monomer.

[0136] The chimeric pore monomer preferably contains a sequence having at least about 20% homology or identity with the sequence shown in any one of SEQ ID NOs. 66, 67, 71, 72, 65, 76, 77, and 78, or with the sequence shown in any one of SEQ ID NOs. 66, 67, 71, 72, 65, 76, 77, and 78 that does not contain the signal peptide. The chimeric pore monomer preferably contains a sequence having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or more preferably at least about 95%, at least about 97%, at least about 98%, or at least about 99% homology or identity with respect to the sequence shown in any one of SEQ ID NOs. 66, 67, 71, 72, 65, 76, 77, and 78, or with respect to the sequence shown in any one of SEQ ID NOs. 66, 67, 71, 72, 65, 76, 77, and 78 that does not contain a signal peptide. The chimeric pore monomer preferably contains the sequence shown in any one of SEQ ID NOs: 66, 67, 71, 72, 65, 76, 77, and 78, or the sequence shown in any one of SEQ ID NOs: 66, 67, 71, 72, 65, 76, 77, and 78 without the signal peptide, i.e., it contains a sequence that has 100% identity with respect to the sequence shown in any one of SEQ ID NOs: 66, 67, 71, 72, 65, 76, 77, and 78, or with respect to the sequence shown in any one of SEQ ID NOs: 66, 67, 71, 72, 65, 76, 77, and 78 without the signal peptide. Any one of SEQ ID NOs: 66, 67, 71, 72, 65, 76, 77, and 78 is, of course, equivalent to SEQ ID NOs: 66, 67, 71, 72, 65, 76, 77, or 78. Homology and / or identity are typically measured over the entire length of the chimeric pore monomer.

[0137] The chimeric pore monomer preferably contains a sequence having at least about 20% homology or identity with the sequence shown in either SEQ ID NO: 69 or 70, or with the sequence shown in either SEQ ID NO: 69 or 70 without the signal peptide. The chimeric pore monomer preferably contains a sequence having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or more preferably at least about 95%, at least about 97%, at least about 98%, or at least about 99% homology or identity with the sequence shown in either SEQ ID NO: 69 or 70, or with the sequence shown in either SEQ ID NO: 69 or 70 without the signal peptide. The chimeric pore monomer preferably contains the sequence shown in either SEQ ID NO: 69 or 70, or the sequence shown in either SEQ ID NO: 69 or 70 without the signal peptide, i.e., it contains a sequence that has 100% identity with respect to either SEQ ID NO: 69 or 70, or with respect to either SEQ ID NO: 69 or 70 without the signal peptide. Either SEQ ID NO: 69 or 70 is, of course, equivalent to SEQ ID NO: 69 or 70. Homology and / or identity are typically measured over the entire length of the chimeric pore monomer.

[0138] The chimeric pore monomer preferably contains a sequence having at least about 20% homology or identity with the sequence shown in any one of SEQ ID NOs. 66, 71, and 72, or with the sequence shown in any one of SEQ ID NOs. 71, 72, or with the sequence shown in any one of SEQ ID NOs. 71, 72, or with the sequence shown in any one of SEQ ID NOs. 71, 72, or with the sequence shown in any one of SEQ ID NOs. 66, 71, and 72, or with the sequence shown in any one of SEQ ID NOs. 66, 71, and 72, or with the sequence shown in any one of SEQ ID NOs. 71, The chimeric pore monomer preferably contains the sequence shown in any one of SEQ ID NOs: 66, 71, and 72, or the sequence shown in any one of SEQ ID NOs: 66, 71, and 72 without the signal peptide, i.e., it contains a sequence that has 100% identity with respect to the sequence shown in any one of SEQ ID NOs: 66, 71, and 72, or the sequence shown in any one of SEQ ID NOs: 66, 71, and 72 without the signal peptide. Any one of SEQ ID NOs: 66, 71, and 72 is, of course, equivalent to SEQ ID NOs: 66, 71, or 72. Homology and / or identity are typically measured over the entire length of the chimeric pore monomer.

[0139] The chimeric pore monomer preferably contains a sequence having at least about 20% homology or identity with the sequence shown in any one of SEQ ID NOs. 66, 71, 72, and 76, or with the sequence shown in any one of SEQ ID NOs. 66, 71, 72, and 76 that does not contain the signal peptide. The chimeric pore monomer preferably contains a sequence having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or more preferably at least about 95%, at least about 97%, at least about 98%, or at least about 99% homology or identity with respect to the sequence shown in any one of SEQ ID NOs. 66, 71, 72, and 76, or the sequence shown in any one of SEQ ID NOs. 66, 71, 72, and 76 that does not contain a signal peptide. The chimeric pore monomer preferably contains the sequence shown in any one of SEQ ID NOs: 66, 71, 72, and 76, or the sequence shown in any one of SEQ ID NOs: 66, 71, 72, and 76 without the signal peptide, i.e., it contains a sequence that has 100% identity with respect to the sequence shown in any one of SEQ ID NOs: 66, 71, 72, and 76, or with respect to the sequence shown in any one of SEQ ID NOs: 66, 71, 72, and 76 without the signal peptide. Any one of SEQ ID NOs: 66, 71, 72, and 76 is, of course, equivalent to SEQ ID NOs: 66, 71, 72, or 76. Homology and / or identity are typically measured over the entire length of the chimeric pore monomer.

[0140] In any of the above embodiments, the chimeric pore monomer comprises a sequence having at least about 20% homology or identity with any one of SEQ ID NOs: 65-72, or any one of SEQ ID NOs: 65-72 and 76-78, or any one of SEQ ID NOs: 65-72 without the signal peptide, or any one of SEQ ID NOs: 65-72 and 76-78, the chimeric pore monomer preferably does not contain the entire sequence of any wild-type pore. In any of these embodiments, the chimeric pore monomer preferably does not contain a sequence having 100% identity with the entire sequence of a wild-type pore. In any of these embodiments, the chimeric pore monomer preferably does not contain the entire sequence of any of the different pores used to produce the chimeric pore monomer, or the entire sequence of any of the different pores that are the source or origin of two or more regions, such as two or three regions. In any of these embodiments, the chimeric pore monomer preferably does not contain any sequence that is 100% identical to the entire sequence of any of the different pores used to produce the chimeric pore monomer, or to the entire sequence of any of the different pores that are the source or origin of two or more regions, such as two-region or three-region regions.

[0141] When defining the chimeric pore monomers of the present invention, at least two different pores do not contain alpha-hemolysin and gamma-hemolysin. Preferably, at least two different pores do not contain two sodium channels.

[0142] The chimeric pore monomer preferably does not contain the sequence shown in SEQ ID NO: 70.

[0143] The present invention also provides a chimeric pore monomer comprising a fusion protein comprising three structural regions, the three structural regions being (1) a cap region, (2) a constriction region, and (5) a transmembrane region, and these three structural regions are derived from at least two different CsgG pores. The present invention also provides a chimeric pore monomer comprising a fusion protein comprising five structural regions, the five structural regions being (1) a cap region, (2) a landing platform region, (3) a C-terminal region, (4) a constriction region, and (5) a transmembrane region, and these five structural regions are derived from at least two different CsgG pores. Any of the embodiments described above are similarly applicable to these chimeric pore monomers of the present invention. In particular, the three or five structural regions may be derived from two, three, or five different CsgG pores.

[0144] Any of the chimeric pore monomers of the present invention may further comprise one or more CsgF peptides. Such peptides and their association with pore monomers are described in WO2019 / 002893, PCT / EP2023 / 059821, PCT / EP2023 / 072113, PCT / EP2023 / 072065, PCT / EP2023 / 072106, and PCT / EP2023 / 072068 (all incorporated herein in their entirety by reference).

[0145] Stabilization and other mutations Preferably, one or more of at least two regions in the chimeric pore monomer, or one or more of the two regions, contains one or more modifications that stabilize the chimeric pore and / or improve the ability of the chimeric pores formed from the chimeric pore monomer to characterize the target analyte. Preferably, all of at least two regions, or both of the two regions, contain one or more modifications that stabilize the chimeric pore and / or improve the ability of the chimeric pores formed from the chimeric pore monomer to characterize the target analyte. Preferably, the cap region (or scaffold) and / or constriction region contain one or more modifications that stabilize the chimeric pore and / or improve the ability of the chimeric pores formed from the chimeric pore monomer to characterize the target analyte. Preferably, one or more of the cap region, constriction region, and transmembrane region, for example, all of them, contain one or more modifications that stabilize the chimeric pore and / or improve the ability of the chimeric pores formed from the chimeric pore monomer to characterize the target analyte. The CsgG pore monomer of the present invention preferably includes one or more modifications that stabilize the CsgG pore and / or improve the ability of the CsgG pore formed from the CsgG pore monomer to characterize a target analyte.

[0146] One or more modifications are preferably (a) one or more deletions, (b) one or more substitutions, (c) one or more additions, or (d) any combination of (a) to (c). (d) Any combination is, for example, (a) and (b), (a) and (c), (b), or (d), or (a), (b) and (c). One or more modifications are preferably one or more substitutions. One or more modifications are preferably one or more of the substitutions for PorARc described above, particularly in relation to Table 2. One or more modifications are preferably one or more of the substitutions for CsgG described above, particularly in relation to Sequence ID No. 56. One or more substitutions described above preferably remove negative charge. One or more substitutions improve the ability of the chimeric pores formed from the chimeric pore monomers to characterize negatively charged target analytes such as polynucleotides. One or more modifications may be any of those described below with reference to Sequence ID Nos. 2, 50, 51, 52, 53, 54, or 55. The one or more modifications are preferably one or more of the modifications to Sequence ID No. 56 described above.

[0147] One or more suitable modifications are well known in the art. For example, modifications to CsgG that improve the ability to characterize target analytes are disclosed in WO2016 / 034591, WO2017 / 149316, WO2017 / 149317, WO2017 / 149318, WO2018 / 211241, WO2019 / 002893, PCT / EP2023 / 059821, PCT / EP2023 / 072113, PCT / EP2023 / 072065, PCT / EP2023 / 072106, and PCT / EP2023 / 072068 (all incorporated herein in their entirety by reference). Preferred modifications are also described in UK Patent Application No. 2118939.4, filed on 23 December 2021 (which is incorporated herein by reference in its entirety).

[0148] PorARc pore monomer The present invention provides various PorARc pore monomers derived from different species, collectively referred to as the PorARc pore monomers of the present invention.

[0149] Sequence ID 2 is a wild-type PorARc pore (PorARc_Mph) derived from Mycolicibacterium phlei, containing the D91N / D92N substitution. This is pore 2 in Table 2 above, with the signal peptide removed and containing methionine (M) and its preferred substitution (column 7) at the N-terminus (i.e., position 1). The present invention provides a PorARc_Mph pore monomer that contains or consists of a sequence having at least about 88% homology or identity with the sequence shown in Sequence ID 2. The PorARc_Mph pore monomer of the present invention preferably contains or consists of a sequence having at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology with the sequence shown in Sequence ID 2. The PorARc_Mph pore monomer of the present invention preferably contains or consists of a sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with respect to the sequence shown in SEQ ID NO: 2. The PorARc_Mph pore monomer of the present invention preferably contains or consists of a sequence having 100% homology or identity with respect to the sequence shown in SEQ ID NO: 2. Homology and / or identity is typically measured over the entire length of the pore monomer. The method for determining homology and / or identity is as described above.

[0150] One or more negative amino acids in SEQ ID NO: 2, such as one or more E and / or D, are preferably deleted or substituted with one or more different amino acids, such as one or more positively charged amino acids, such as R, H, or K, and / or one or more uncharged amino acids, such as S, T, N, or Q. This removes the negative charge from the sequence. Any number of negatively charged amino acids, such as one, two, three, four, five, six, seven, eight, nine, ten, or more, may be deleted or substituted. The one or more negatively charged amino acids are preferably located within the constricted region of SEQ ID NO: 2, as shown in Table 2.

[0151] A sequence homologous or identical to the sequence shown in Sequence ID No. 2 preferably contains a positively charged amino acid such as R, H, or K, or an uncharged amino acid such as S, T, N, or Q, at one or both of the positions corresponding to positions 91 and 92 of Sequence ID No. 2. A sequence homologous or identical to the sequence shown in Sequence ID No. 2 preferably contains N at the positions corresponding to positions 91 and / or 92 of Sequence ID No. 2.

[0152] Sequence ID 50 shows the amino acid sequence of a PorARc pore (PorARc_Msp) from the genus Mycobacterium, containing the D91N / D92N substitution. This is pore 3 in Table 2 above, with the signal peptide removed and containing methionine (M) and its preferred substituent (column 7) at the N-terminus (i.e., position 1). Sequence ID 51 shows the amino acid sequence of a PorARc pore (PorARc_Mrh) from Mycolicibacterium rhodesiae, containing the D91N / D92N substitution. This is pore 8 in Table 2 above, with the signal peptide removed and containing methionine (M) and its preferred substituent (column 7) at the N-terminus (i.e., position 1). Sequence ID 52 is the amino acid sequence of the PorARc pore (PorARc_Mel) from Mycolicibacterium elephantis containing the D91N / E101Q substitution. This is pore 17 in Table 2 above, with the signal peptide removed and methionine (M) and its preferred substitution (column 7) at the N-terminus (i.e., position 1). Sequence ID 53 shows the amino acid sequence of the PorARc pore (PorARc_Mco) from Mycolicibacterium cosmeticum containing the D91N / D92N substitution (column 7). This is pore 19 in Table 2 above, with the signal peptide removed and methionine (M) and its preferred substitution (column 7) at the N-terminus (i.e., position 1). Sequence ID 54 shows the amino acid sequence of a PorARc pore (WP_056447532.1; PorARc_Rsp) from an unclassified Rhodococcus species containing the substitutions E89Q / D91N / D93N / D100N. This is pore 25 in Table 2 above, with the signal peptide removed and containing methionine (M) and its preferred substitutions (column 7) at the N-terminus (i.e., position 1). Sequence ID 55 shows the amino acid sequence of a PorARc pore (WP_206003768.1; PorARc_Rsp) from Rhodococcus sp. PSBB049 containing the substitutions D90N / D95N / D103N.This is pore 27 in Table 2 above, where the signal peptide has been removed and the N-terminus (i.e., position 1) contains methionine (M) and its preferred substitution (column 7).

[0153] The present invention provides PorARc pore monomers comprising or consisting of sequences having at least about 40% homology or identity with the sequences shown in SEQ ID NOs. 50, 51, 52, 53, 54, or 55. A pore monomer based on SEQ ID NOs. 50 may be referred to as PorARc_Msp pore monomer. A pore monomer based on SEQ ID NOs. 51 may be referred to as PorARc_Mrh pore monomer. A pore monomer based on SEQ ID NOs. 52 may be referred to as PorARc_Mel pore monomer. A pore monomer based on SEQ ID NOs. 53 may be referred to as PorARc_Mco pore monomer. A pore monomer based on SEQ ID NOs. 54 may be referred to as PorARc_Rsp pore monomer. A pore monomer based on SEQ ID NOs. 55 may be referred to as PorARc_Rsp pore monomer.

[0154] The PorARc pore monomer of the present invention preferably comprises or consists of a sequence having at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or more preferably at least about 95%, at least about 97%, at least about 98%, or at least about 99% homology to the sequence shown in SEQ ID NOs. 50, 51, 52, 53, 54, or 55. The PorARc pore monomer preferably contains or consists of a sequence having at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, or more preferably at least about 95%, at least about 97%, at least about 98%, or at least about 99% identity with the sequence shown in SEQ ID NOs. The PorARc pore monomer of the present invention preferably contains or consists of a sequence having 100% homology or identity with the sequence shown in SEQ ID NOs.

[0155] The present invention provides PorARc pore monomers that include or consist of sequences having at least about 20% homology or identity with sequences 50, 51, 52, 53, 54, or 55 shown in the sequence numbers. A pore monomer based on sequence number 50 may be called a PorARc_Msp pore monomer. A pore monomer based on sequence number 51 may be called a PorARc_Mrh pore monomer. A pore monomer based on sequence number 52 may be called a PorARc_Mel pore monomer. A pore monomer based on sequence number 53 may be called a PorARc_Mco pore monomer. A pore monomer based on sequence number 54 may be called a PorARc_Rsp pore monomer. A pore monomer based on sequence number 55 may be called a PorARc_Rsp pore monomer.

[0156] The PorARc pore monomer of the present invention preferably contains or comprises a sequence having at least about 25%, at least about 30%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or more preferably at least about 95%, at least about 97%, at least about 98%, or at least about 99% homology to the sequence shown in SEQ ID NOs. 50, 51, 52, 53, 54, or 55. The PorARc pore monomer preferably comprises or consists of a sequence having at least about 25%, at least about 30%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least 75%, at least about 80%, at least about 85%, at least about 90%, or more preferably at least about 95%, at least about 97%, at least about 98%, or at least about 99% identity with the sequence shown in SEQ ID NOs. The PorARc pore monomer of the present invention preferably comprises or consists of a sequence having 100% homology or identity with the sequence shown in SEQ ID NOs.

[0157] One or more negatively charged amino acids in sequence numbers 50, 51, 52, 53, 54, or 55, such as one or more E and / or D, are preferably deleted or substituted with one or more different amino acids, such as one or more positively charged amino acids, such as R, H, or K, and / or one or more uncharged amino acids, such as S, T, N, or Q. This removes the negative charge from the sequence. Any number of negatively charged amino acids, such as one, two, three, four, five, six, seven, eight, nine, ten, or more, may be deleted or substituted. The one or more negatively charged amino acids are preferably located within the constricted region of sequence numbers 50, 51, 52, 53, 54, or 55. These are shown in Table 2.

[0158] Sequences homologous or identical to the sequence shown in SEQ ID NO: 50 preferably contain a positively charged amino acid such as R, H, or K, or an uncharged amino acid such as S, T, N, or Q, at one or both of the positions corresponding to positions 91 and 92 of SEQ ID NO: 50. Sequences homologous or identical to the sequence shown in SEQ ID NO: 50 preferably contain N at the positions corresponding to positions 91 and / or 92 of SEQ ID NO: 50.

[0159] Sequences homologous or identical to the sequence shown in SEQ ID NO: 51 preferably contain a positively charged amino acid such as R, H, or K, or an uncharged amino acid such as S, T, N, or Q, at one or both of the positions corresponding to positions 91 and 92 of SEQ ID NO: 51. Sequences homologous or identical to the sequence shown in SEQ ID NO: 51 preferably contain N at the positions corresponding to positions 91 and / or 92 of SEQ ID NO: 51.

[0160] Sequences homologous or identical to the sequence shown in Sequence ID No. 52 preferably contain a positively charged amino acid such as R, H, or K, or an uncharged amino acid such as S, T, N, or Q, at one or both of the positions corresponding to positions 91 and 101 of Sequence ID No. 52. Sequences homologous or identical to the sequence shown in Sequence ID No. 52 preferably contain Q at the positions corresponding to positions 91 and / or 101 of Sequence ID No. 52.

[0161] Sequences homologous or identical to the sequence shown in Sequence ID No. 53 preferably contain a positively charged amino acid such as R, H, or K, or an uncharged amino acid such as S, T, N, or Q, at one or both of the positions corresponding to positions 91 and 92 of Sequence ID No. 53. Sequences homologous or identical to the sequence shown in Sequence ID No. 53 preferably contain N at the positions corresponding to positions 91 and / or 92 of Sequence ID No. 53.

[0162] Sequences homologous or identical to the sequence shown in SEQ ID NO: 54 preferably contain a positively charged amino acid such as R, H, or K, or an uncharged amino acid such as S, T, N, or Q, at one or more of the positions corresponding to positions 89, 91, 93, and 100 of SEQ ID NO: 54. Sequences homologous or identical to the sequence shown in SEQ ID NO: 54 preferably contain N at the positions corresponding to positions 89, 91, 93, and 100 of SEQ ID NO: 54.

[0163] Sequences homologous or identical to the sequence shown in SEQ ID NO: 55 preferably contain, for example, one or all of, a positively charged amino acid such as R, H, or K, or an uncharged amino acid such as S, T, N, or Q, at one or more of the positions corresponding to positions 90, 95, and 103 of SEQ ID NO: 55. Sequences homologous or identical to the sequence shown in SEQ ID NO: 55 preferably contain N at the positions corresponding to positions 90, 95, and 103 of SEQ ID NO: 55.

[0164] The sequence of the PorARc pore monomer (i.e., a sequence having homology or identity to sequence numbers 2, 50, 51, 52, 53, 54, or 55) may contain any of the substitutions present in other PorARc pores containing any of the pores listed in Table 2.

[0165] PorARc pore monomers typically retain the ability to form the same 3D structure as wild-type PorARc pore monomers, for example, PorARc pore monomers having sequences 2, 50, 51, 52, 53, 54, or 55. PorARc pore monomers can form pores. The measurement method was described above with respect to the chimeric pore monomers of the present invention.

[0166] Amino acid substitutions may be made to the amino acid sequences of SEQ ID NOs. 2, 50, 51, 52, 53, 54, or 55, for example, by 1, 2, 3, 4, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 or more. Conservative substitutions replace amino acids with other amino acids that have a similar chemical structure, similar chemical properties, or similar side-chain volume. The introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino 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.

[0167] The PorARc pore monomer may be modified to introduce one or more cysteines, one or more hydrophobic amino acids, one or more charged amino acids, one or more unnatural amino acids, one or more polar amino acids, or one or more photoreactive amino acids. Such introductions may be carried out in any number and combination. The introductions are preferably carried out by substitution or addition.

[0168] One or more amino acid residues may be additionally deleted from the polypeptide of the amino acid sequence of SEQ ID NOs: 2, 50, 51, 52, 53, 54, or 55. Up to 1, 2, 3, 4, 5, 10, 20, or 30 or more residues may be deleted.

[0169] The PorARc pore monomer may contain fragments of SEQ ID NOs. 2, 50, 51, 52, 53, 54, or 55. Such fragments retain pore-forming activity. The fragments may have lengths of at least about 50, at least about 100, at least about 150, or at least about 200 amino acids. Such fragments can be used to produce the pores of the present invention. The fragment preferably includes the transmembrane beta-barrel region of the relevant sequence, i.e., residues W73-T84 and G113-N122 of SEQ ID NO: 2, residues W73-T84 and G114-N123 of SEQ ID NO: 50, residues W73-T84 and G127-N136 of SEQ ID NO: 51, residues W73-T84 and G111-N120 of SEQ ID NO: 52, residues W73-T84 and G109-N118 of SEQ ID NO: 53, residues A73-S84 and Q104-P113 of SEQ ID NO: 54, residues G74-S85 and Q107-P116 of SEQ ID NO: 55, or variants thereof as described above.

[0170] One or more amino acids may be added to the polypeptide described above, either substituted or in addition. The elongation portion may be provided at the amino-terminus or carboxy-terminus of the amino acid sequence of SEQ ID NOs. 2, 50, 51, 52, 53, 54, or 55, or at its polypeptide variant or fragment. The elongation portion may be very short, for example, 1 to 10 amino acids in length. Alternatively, the elongation portion 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 present invention.

[0171] The sequences of PorARc pore monomers have amino acid sequences that are modified from the sequences of SEQ ID NOs: 2, 50, 51, 52, 53, 54, or 55, and that retain the ability to form pores. These sequences typically contain the regions of SEQ ID NOs: 2, 50, 51, 52, 53, 54, or 55 that are involved in pore formation. The pore-forming ability of PorARc containing β-barrels is provided by β-strands in the transmembrane β-barrel region of each monomer. Variants of SEQ ID NO: 2 typically include the regions of the relevant sequences that form β-strands, namely residues W73-T84 and G113-N122 of SEQ ID NO: 2, residues W73-T84 and G114-N123 of SEQ ID NO: 50, residues W73-T84 and G127-N136 of SEQ ID NO: 51, residues W73-T84 and G111-N120 of SEQ ID NO: 52, residues W73-T84 and G109-N118 of SEQ ID NO: 53, residues A73-S84 and Q104-P113 of SEQ ID NO: 54, residues G74-S85 and Q107-P116 of SEQ ID NO: 55, or any of its variants as described above. One or more modifications can be made to the regions of SEQ ID NO: 2, 50, 51, 52, 53, 54, or 55 that form β-strands, as long as the resulting variant retains its ability to form pores.

[0172] One or more modifications within the PorARc pore monomer preferably improve the ability of the pore containing the pore monomer to characterize the analyte.

[0173] CsgG pore monomer The present invention also provides various CsgG pore monomers, collectively referred to as the CsgG pore monomers of the present invention.

[0174] The present invention also provides a CsgG pore monomer comprising a sequence having at least about 20% homology or identity with the sequence shown in any one of SEQ ID NOs. 65 to 72, or with the sequence shown in any one of SEQ ID NOs. 72 without the signal peptide, the CsgG pore monomer preferably comprises a sequence having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or more preferably at least about 95%, at least about 97%, at least about 98%, or at least about 99% homology or identity with the sequence shown in any one of SEQ ID NOs. 65 to 72, or with the sequence shown in any one of SEQ ID NOs. 65 to 72 without the signal peptide, or at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or more preferably at least about 95%, at least about 97%, at least about 98%, or at least about 99% homology or identity. The CsgG pore monomer preferably contains the sequence shown in any one of SEQ ID NOs. 65-72, or the sequence shown in any one of SEQ ID NOs. 65-72 without the signal peptide, i.e., a sequence having 100% identity with respect to any one of SEQ ID NOs. 65-72 is, of course, equivalent to SEQ ID NOs. 65, 66, 67, 68, 69, 70, 71, or 72. Homology and / or identity are typically measured over the entire length of the chimeric pore monomer.

[0175] The present invention also provides a CsgG pore monomer containing a sequence having at least about 20% homology or identity with the sequence shown in any one of SEQ ID NOs. 65-72 and 76-78, or with the sequence shown in any one of SEQ ID NOs. 65-72 and 76-78 that does not contain a signal peptide. The CsgG pore monomer preferably contains a sequence having at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or more preferably at least about 95%, at least about 97%, at least about 98%, or at least about 99% homology or identity with respect to the sequence shown in any one of SEQ ID NOs. 65-72 and 76-78, or the sequence shown in any one of SEQ ID NOs. 65-72 and 76-78 that does not contain a signal peptide. The CsgG pore monomer preferably contains the sequence shown in any one of SEQ ID NOs. 65-72 and 76-78, or the sequence shown in any one of SEQ ID NOs. 65-72 and 76-78 without the signal peptide, i.e., it contains a sequence that has 100% identity with respect to any one of SEQ ID NOs. 65-72 and 76-78, or any one of SEQ ID NOs. 76-78 is, of course, equivalent to SEQ ID NOs. 65, 66, 67, 68, 69, 70, 71, 72, 76, 77, or 78. Homology and / or identity are typically measured over the entire length of the chimeric pore monomer.

[0176] The CsG pore monomer preferably does not contain the entire sequence of the wild-type pore. The CsgG pore monomer preferably does not contain a sequence that is 100% identical to the entire sequence of the wild-type pore. The CsgG pore monomer preferably does not contain the entire sequence of any of the CsgG pores identified in Table 4, including SEQ ID NO: 58 or 73.

[0177] The present invention also provides a CsgG pore monomer comprising or consisting of a sequence having at least about 68% homology or identity with the sequence shown in SEQ ID NO: 58. The CsgG pore monomer of the present invention preferably comprises or consists of a sequence having at least about 69%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity with the sequence shown in SEQ ID NO: 58. The CsgG pore monomer may comprise or consist of SEQ ID NO: 58. Homology and / or identity is typically measured over the entire length of SEQ ID NO: 58.

[0178] The present invention also provides a CsgG pore monomer comprising or consisting of a sequence having at least about 79% homology or identity with the sequence shown in SEQ ID NO: 73. The CsgG pore monomer of the present invention preferably comprises or consists of a sequence having at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology or identity with the sequence shown in SEQ ID NO: 73. The CsgG pore monomer may comprise or consist of SEQ ID NO: 73. Homology and / or identity is typically measured over the entire length of SEQ ID NO: 73.

[0179] The CsgG pore monomer of the present invention preferably comprises or consists of a sequence having at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% homology to the sequence shown in SEQ ID NO: 58 or 73.

[0180] Each of the CsgG pore monomers of the present invention includes one or more specific modifications or substitutions disclosed in WO2016 / 034591, WO2017 / 149316, WO2017 / 149317, WO2017 / 149318, WO2018 / 211241, WO2019 / 002893, PCT / EP2023 / 059821, PCT / EP2023 / 072113, PCT / EP2023 / 072065, PCT / EP2023 / 072106, and PCT / EP2023 / 072068 (all incorporated herein by reference in their entirety). A CsgG pore monomer may include one or more of the modifications or substitutions described above with respect to Sequence ID No. 56. One or more modifications within the CsgG pore monomer preferably improve the ability of the pore containing the pore monomer to characterize the analyte.

[0181] CsgG pore monomers typically retain the ability to form the same 3D structure as wild-type CsgG pore monomers, for example, CsgG pore monomers having the sequence of Sequence ID No. 58 or 73. CsgG pore monomers can form pores. The measurement method was described above with respect to the chimeric pore monomers of the present invention.

[0182] Amino acid substitutions may involve, for example, one, two, three, four, five, ten, twenty, thirty, forty, fifty, sixty, seventy, eighty, ninety, or even one hundred or more substitutions of the amino acid sequence of SEQ ID NO: 58 or 73. Conservative substitutions replace amino acids with other amino acids that have a similar chemical structure, similar chemical properties, or similar side-chain volume. The introduced amino acids may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality, or charge to the amino 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.

[0183] The CsgG pore monomer may be modified to introduce one or more cysteines, one or more hydrophobic amino acids, one or more charged amino acids, one or more unnatural amino acids, one or more polar amino acids, or one or more photoreactive amino acids. Such introductions may be carried out in any number and combination. The introductions are preferably carried out by substitution or addition.

[0184] One or more amino acid residues may be additionally deleted from the polypeptide of the amino acid sequence of SEQ ID NO: 58 or 73. Up to 1, 2, 3, 4, 5, 10, 20, or 30 or more residues may be deleted.

[0185] The CsgG pore monomer may contain or consist of the fragment of SEQ ID NO: 58 or 73. Such fragments retain pore-forming activity. The fragments may have a length of at least about 50, at least about 100, at least about 150, or at least about 200 amino acids. Pores of the present invention can be produced using such fragments. The fragments preferably contain a transmembrane beta-barrel region of the relevant sequence (shown above).

[0186] One or more amino acids may be added to the polypeptide described above, either substituted or in addition. The elongation portion may be provided at the amino-terminus or carboxy-terminus of the amino acid sequence of SEQ ID NO: 58 or 73, or at a polypeptide variant or fragment thereof. The elongation portion may be very short, for example, 1 to 10 amino acids in length. Alternatively, the elongation portion 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 present invention.

[0187] Structures The present invention also provides constructs comprising two or more covalently attached chimeric pore monomers of the present invention or two or more covalently attached PorARc pore monomers of the present invention. In this and subsequent sections, the chimeric pore monomers of the present invention and the PorARc pore monomers of the present invention are collectively referred to as "pore monomers of the present invention." In this and subsequent sections, the chimeric pore monomers of the present invention, the PorARc pore monomers of the present invention and the CsgG pore monomers of the present invention are collectively referred to as "pore monomers of the present invention." In this and subsequent sections, the chimeric constructs of the present invention and the PorARc constructs of the present invention are collectively referred to as "constructs of the present invention."

[0188] The present invention also provides constructs comprising two or more covalently attached CsgG pore monomers. In this and subsequent sections, the chimeric constructs, PorARc constructs, and CsgG constructs of the present invention are collectively referred to as "constructs of the present invention."

[0189] The constructs of the present invention may contain two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more pore monomers of the present invention. The constructs may contain at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten pore monomers of the present invention. The two or more pore monomers of the present invention may be the same or different. The two or more pore monomers of the present invention may be different based on their arrangement. Preferably, the two or more pore monomers of the present invention are the same (i.e., identical).

[0190] The construct of the present invention preferably comprises two pore monomers of the present invention. The two pore monomers of the present invention may be the same or different. Preferably, the two pore monomers of the present invention are the same (i.e., identical).

[0191] The pore monomers of the present invention may be genetically fused. The pore monomers of the present invention may be attached via a linker, or chemically fused via, for example, a chemical crosslinking agent. Methods for covalently attaching the pore monomers of the present invention are disclosed in WO2017 / 149316, WO2017 / 149317, and WO2017 / 149318 (all of which are incorporated herein by reference). The pore monomers of the present invention may be genetically fused using a linker.

[0192] The linker is preferably an amino acid sequence and / or a chemical crosslinking agent. Suitable amino acid linkers, such as peptide linkers, are well known in the art. The length, flexibility, and hydrophilicity of the amino acid or peptide linker are typically designed to facilitate pore formation from the construct. Preferred flexible peptide linkers are stretches of 2 to 20, for example, 4, 6, 8, 10, or 16 serine and / or glycine amino acids. More preferred flexible linkers are (SG)1, (SG)2, (SG)3, (SG)4, (SG)5, (SG)8, (SG)10 , (SG) 15 , or (SG) 20 It contains, where S is serine and G is glycine. A preferred rigid linker is a stretch of 2 to 30 proline amino acids, for example, 4, 6, 8, 16, or 24. A more preferred rigid linker is where P is proline (P) 12 Includes.

[0193] Suitable chemical crosslinking agents are well known in the art. Suitable chemical crosslinking agents include, but are not limited to, those containing the following functional groups: maleimides, active esters, succinimides, azides, alkynes (such as dibenzocyclooctinol (DIBO or DBCO), difluorocycloalkynes, and linear alkynes), phosphines (such as those used in traceless and non-traceless Staudinger ligation), haloacetyls (such as iodoacetamide), phosgene-type reagents, sulfonyl chloride reagents, isothiocyanates, acyl halides, hydrazines, disulfides, vinyl sulfones, aziridines, and photoreactive reagents (such as aryl azides and diaziridines).

[0194] The reaction between amino acids and functional groups may be spontaneous, such as cysteine / maleimide, or it may require external reagents such as Cu(I) to link azides and linear alkynes.

[0195] The linker may contain any molecule extending over the required distance. Linker lengths can vary from one carbon (phosgene-type linker) to many angstroms. Examples of linker molecules include, but are not limited to, polyethylene glycol (PEG), polypeptides, polysaccharides, deoxyribonucleic acid (DNA), peptide nucleic acid (PNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), saturated and unsaturated hydrocarbons, and polyamides. These linkers may be inert or reactive, and in particular, they may be chemically cleavable at defined positions, or they may be modified with fluorophores or ligands themselves. Preferably, the linker is resistant to reducing agents such as dithiothreitol (DTT) after covalent attachment of the monomer.

[0196] Preferred crosslinking agents include 2,5-dioxopyrrolidine-1-yl3-(pyridine-2-yldisulfanyl)propanoate, 2,5-dioxopyrrolidine-1-yl4-(pyridine-2-yldisulfanyl)butanoate, and 2,5-dioxopyrrolidine-1-yl8-(pyridine-2-yldisulfanyl)octanoate, dimaleimide PEG 1k, dimaleimide PEG 3.4k, dimaleimide PEG 5k, and dimaleimide PEG 10k, bis(maleimide)ethane (BMOE), bis-maleimidehexane (BMH), 1,4-bis-maleimidobutane (BMB), 1,4-bis-maleimidyl-2,3-dihydroxybutane (BMDB), BM[PEO]2 (1,8-bis-maleimidodiethylene glycol), BM[PEO]3 (1,11-bis-maleimidotriethylene glycol), tris[2-maleimidoethyl]amine (TMEA), DTME dithiobismaleimidoethane, bis-maleimide PEG3, bis-maleimide PEG11, DBCO-maleimide, DBCO-PEG4-maleimide, DBCO-PEG4-NH2, DBCO-PEG4-NHS, DBCO-NHS, DBCO-PEG-DBCO 2.8kDa, DBCO-PEG-DBCO The compounds include 4.0 kDa, DBCO-15 atom-DBCO, DBCO-26 atom-DBCO, DBCO-35 atom-DBCO, DBCO-PEG4-SS-PEG3-biotin, DBCO-SS-PEG3-biotin, DBCO-SS-PEG11-biotin, (succinimidyl 3-(2-pyridyldithio)propionic acid (SPDP), and maleimide-PEG(2kDa)-maleimide (alpha, omega-bis-maleimide poly(ethylene glycol)). The most preferred crosslinking agent is maleimide-propyl-SRDFWRS-(1,2-diaminoethane)-propyl-maleimide.

[0197] The linker is preferably resistant to dithiothreitol (DTT). Suitable linkers include, but are not limited to, iodoacetamide and maleimide linkers.

[0198] The pore monomers of the present invention may be linked using two or more linkers, each containing a hybridizable region and a group capable of forming a covalent bond. The hybridizable regions in the linkers hybridize and link the pore monomers of the present invention. The linked pore monomers of the present invention are then bonded via the formation of covalent bonds between the groups. Any of the specific linkers disclosed in WO2010 / 086602 (which is incorporated herein by reference in its entirety) may be used in accordance with the present invention.

[0199] The linker may be labeled. Suitable labels include fluorescent molecules (such as Cy3 or AlexaFluor® 555), for example. 125 I, 35 S, 32 Labels include, but are not limited to, radioactive isotopes such as phosphate (P), enzymes, antibodies, antigens, polynucleotides, and ligands such as biotin. Such labels allow for the quantification of linker amounts. The labels may also be cleavable purified tags such as biotin, or specific sequences that appear in identification methods, such as peptides that are not present in the protein itself but are released by trypsin digestion.

[0200] A preferred method for linking the porous monomers of the present invention is via cysteine ​​bonds. This can be mediated by a bifunctional chemical crosslinking agent or by an amino acid linker having a cysteine ​​residue presented at the terminal.

[0201] Another preferred method of attachment via 4-azidophenylalanine or Faz linkage. This may be mediated by a bifunctional chemical linker or by a polypeptide linker having a 4-azidophenylalanine or Faz residue presented at the terminal. Further preferred linkers are discussed in detail below.

[0202] The pores of the present invention The present invention provides a chimeric pore comprising at least one chimeric pore monomer of the present invention, or at least one construct of the present invention comprising two or more covalently bonded chimeric pore monomers of the present invention. The present invention provides a CsgG pore comprising at least one CsgG pore monomer of the present invention, or at least one construct of the present invention comprising two or more covalently bonded CsgG pore monomers of the present invention. The present invention provides a PorARc pore comprising at least one PorARc pore monomer of the present invention, or at least one construct of the present invention comprising two or more covalently bonded PorARc pore monomers of the present invention. In this section and subsequent sections, the chimeric pores of the present invention and the PorARc pores of the present invention are collectively referred to as "pores of the present invention." In this section and subsequent sections, the chimeric pores of the present invention, the PorARc pores of the present invention, and the CsgG pores of the present invention are collectively referred to as "pores of the present invention."

[0203] The term "pore" refers to an oligomeric pore comprising at least one pore monomer of the present invention (for example, one or more pore monomers of the present invention, such as two or more pore monomers of the present invention, or three or more pore monomers of the present invention). The pores of the present invention have the characteristics of biological pores, i.e., they have a typical protein structure and define channels. When the pores are provided in an environment having membrane components, membranes, cells, or insulating layers, the pores are inserted into the membrane or insulating layer to form "transmembrane pores".

[0204] The chimeric pores of the present invention typically exhibit improved target analyte characterization compared to, or compared to, at least two different pores from which they originate. The PorARc pores of the present invention typically exhibit improved target analyte characterization compared to other pores used in nanopore sensing.

[0205] As described above, the pores of the present invention exhibit one or more of the following: (a) increased signal-to-noise ratio (SNR), (b) increased current range, (c) reduced noise, and (d) increased normalized median absolute deviation (nMAD). The median absolute deviation (MAD) is the median absolute value between the current at a given event and the overall average current of the irregular curve (i.e., the current trace in the analyte dislocation). Normalized MAD (nMAD) is this value normalized over the entire range of waveform currents. The pores of the present invention may be described as (a);(b);(c);(d);(a) and (b);(a) and (c);(a) and (d);(b) and (c);(b) and (d);(c) and (d);(a), (b), and (c);(a), (b), and (d);(a), (c), and (d);(b), (c), and (d); or (a), (b), (c), and (d). For the chimeric pores of the present invention, one or more of (a) to (d) are preferably compared to at least two different pores in which the chimeric pores of the present invention are constructed, or to two different pores in which the chimeric pores of the present invention are constructed. For the PorARc pores of the present invention, one or more of (a) to (d) are preferably compared to other pores used for nanopore sensing, which preferably include any of the pores described herein.

[0206] The pores of the present invention typically include at least one constriction. The constriction and its function are defined above. The pores of the present invention may include two or more constrictions, for example, three or more, four or more, five or more, or six or more constrictions. Further constrictions(s) can increase the contact surface with the analyte passing through, improving the detection and characterization of the analyte. Pores containing the pore monomers of the present invention can improve the characterization of analytes such as polynucleotides, providing a more discriminative direct relationship between the currents observed as the polynucleotide moves through the pore. In particular, by having two stacked constrictions spaced at a defined distance apart, the pore may facilitate the characterization of at least one homopolymer stretch, for example, a polynucleotide containing several consecutive copies of the same nucleotide that would otherwise exceed the interaction length of a single constriction. In addition, by having two stacked constrictions at a defined distance apart, small molecule analytes, including organic or inorganic drugs and contaminants passing through the pore, pass through the two constrictions consecutively. The chemical properties of each constriction can be modified independently, each giving it unique interaction characteristics with the analyte, thus providing further discriminative power during analyte detection.

[0207] The pores of the present invention are preferably homooligomers containing 6 to 20, for example, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 pore monomers of the present invention. The pores of the present invention are preferably homooligomers containing 6 to 10, for example, 6, 7, 8, 9, or 10 pore monomers of the present invention. The pore monomers of the present invention are typically identical. The pores preferably contain 8 or 9 identical pore monomers of the present invention. The pore monomers of the present invention may be any one of those discussed above.

[0208] The present invention provides pores comprising at least one construct of the present invention. A pore typically comprises at least one, two, three, four, or five constructs of the present invention. A pore contains enough monomers to form a pore. For example, an octameric pore may include (a) four constructs each comprising two pore monomers of the present invention, (b) two constructs each comprising four pore monomers of the present invention, (c) one construct comprising two pore monomers of the present invention and six pore monomers of the present invention that do not form part of the construct, (d) three constructs comprising two pore monomers of the present invention and two pore monomers of the present invention that do not form part of the construct, and (e) combinations thereof. For example, a nonameric pore offers similar and further possibilities. Other combinations of constructs and monomers can be envisioned by those skilled in the art. One or more constructs of the present invention can be used to form pores for characterizing polynucleotides, such as for sequencing. A pore preferably comprises two chimeric pore monomers or four constructs of the present invention each comprising pore monomers. Structures are typically the same (i.e., identical).

[0209] The pores of the present invention are preferably homooligomers comprising one to five, for example, one, two, three, four, or five constructs of the present invention. The constructs are typically the same (i.e., identical). The pores preferably comprise four identical constructs of the present invention, each comprising two pore monomers of the present invention. The constructs may be any of those discussed above.

[0210] Preferably, all pore monomers of the present invention within the pores are approximately the same length or the same length. Preferably, the barrels of pore monomers of the present invention within the pores are approximately the same length or the same length. The length can be measured in units of the number and / or length of amino acids.

[0211] Any pore of the present invention may further contain one or more CsgF peptides. Such peptides and their association with pores are described in WO2019 / 002893, PCT / EP2023 / 059821, PCT / EP2023 / 072113, PCT / EP2023 / 072065, PCT / EP2023 / 072106, and PCT / EP2023 / 072068 (all incorporated herein in their entirety by reference).

[0212] The pores of the present invention may be isolated, substantially isolated, purified, or substantially purified. The pores of the present invention are isolated or purified if they do not contain any other components such as lipids or other pores. The pores are substantially isolated if they are mixed with a carrier or diluent that does not interfere with their intended use. For example, the pores are substantially isolated or substantially purified if they exist in a form that contains less than 10%, less than 5%, less than 2%, or less than 1% of other components, such as block copolymers, lipids, or other pores. Alternatively, the pores of the present invention may exist in a membrane. Suitable membranes are considered below.

[0213] The pores of the present invention may exist as individual or single pores. Alternatively, the pores of the present invention may exist as homologous or heterogeneous groups of two or more pores. Other forms in which the pores of the present invention are involved are discussed in detail below.

[0214] Multimer pores The present invention also provides a chimeric pore multimer (multimer, multimer) containing two or more pores, wherein at least one of the pores is the chimeric pore of the present invention. The present invention also provides a PorARc pore multimer containing two or more pores, wherein at least one of the pores is the PorARc pore of the present invention. In this section and subsequent sections, the chimeric pore multimer of the present invention and the PorARc pore multimer of the present invention are collectively referred to as the "pore multimer of the present invention". The present invention also provides a CsgG pore multimer containing two or more pores, wherein at least one of the pores is the CsgG pore of the present invention. In this section and subsequent sections, the chimeric pore multimer of the present invention, the PorARc pore multimer of the present invention, and the CsgG pore multimer of the present invention are collectively referred to as the "pore multimer of the present invention".

[0215] The multimer of the present invention may contain any number of pores, for example, two, three, four, five, six, seven, or eight, or more pores. Any number (including all) of the pores in the multimer may be the pores of the present invention.

[0216] The pore multimers of the present invention may be double pores including the first pore and the second pore of the present invention. The double pores may be in any orientation. The two pores of the double pore may be joined end to end. The double pore may be two pores adjacent (i.e., next to) each other. The second pore may be a pore of the present invention. Both the first pore and the second pore are preferably pores of the present invention. In the double pore, the first pore may be attached to the second pore by hydrophobic interaction and / or one or more disulfide bonds. One or more, for example two, three, four, five, six, eight, nine, for example all, of the monomers in the first pore and / or the second pore (complex) may be modified to enhance such interactions. This may be achieved by any suitable method. A particular method of forming double pores is described in WO2019 / 002893, which is hereby incorporated by reference in its entirety.

[0217] The pore multimers of the present invention may be isolated, substantially isolated, purified, or substantially purified. Such terms are defined above with respect to the pores of the present invention.

[0218] Membrane embodiments The present invention also provides the pores of the present invention or the pore multimers of the present invention contained in a membrane. The present invention also provides a membrane comprising the pores of the present invention or the pore multimers of the present invention. These products are directly applicable for use in molecular sensing such as analyte characterization and polynucleotide sequencing. Suitable membranes are discussed in detail below.

[0219] Methods for producing modified proteins Methods for introducing or substituting naturally occurring amino acids into pore monomers are also well known in the art and are described in WO2019 / 002893 (which is incorporated herein by reference in its entirety). Proteins may be modified to aid in their identification or purification, for example, by the addition of streptavidin tags or by the addition of signal sequences to promote their secretion from cells that do not naturally contain such sequences in monomers. Proteins may also be produced using D-amino acids, or mixtures of L-amino acids and D-amino acids. This is a conventional method in the art for producing such proteins or peptides.

[0220] Chimeric pore monomers, PorARc pore monomers, chimeric constructs, PorARc constructs, chimeric pores, PorARc pores, chimeric pore multimers, or PorARc pore multimers (i.e., any protein of the present invention) may be chemically modified. In this section, these proteins of the present invention are collectively referred to as "proteins." Proteins may be chemically modified in any manner and at any site. Proteins may be chemically modified by attachment of molecules to one or more cysteine ​​molecules (cysteine ​​linkage), attachment of molecules to one or more lysines, attachment of molecules to one or more non-natural amino acids, enzymatic modification of epitopes, or modification of terminals. Suitable methods for carrying out such modifications are well known in the art. Proteins may be chemically modified by attachment of any molecules, such as dyes or fluorophores.

[0221] Proteins may be chemically modified with molecular adapters that facilitate interaction between monomer-containing pores and target nucleotides or target polynucleotide sequences. Suitable adapters, including cyclic molecules, cyclodextrins, hybridizable species, DNA binders or interchelators, peptides or peptide analogs, synthetic polymers, aromatic planar molecules, small positively charged molecules, or small molecules capable of hydrogen bonding, are described in WO2019 / 002893 (which is incorporated herein in its entirety by reference). Molecular adapters may be linked using either the methods described above or linkers.

[0222] The protein may be bound to a polynucleotide-binding protein. This forms a modular sequencing system that can be used in the sequencing method of the present invention. The polynucleotide-binding protein is considered below. The protein may be covalently attached to a monomer using any method known in the art. The monomer and the protein may be chemically fused or genetically fused. Genetic fusion of monomer to polynucleotide-binding protein is considered in WO2010 / 004265 (which is incorporated herein by reference in its entirety). The polynucleotide-binding protein may be bound via cysteine ​​bonds using any of the above methods.

[0223] Polynucleotide-binding proteins may be directly bound to the protein via one or more linkers. The molecule may be bound to the pore monomer using a hybridization linker described as WO2010 / 086602 (which is incorporated herein by reference in its entirety). Alternatively, peptide linkers may be used. Suitable peptide linkers are described above.

[0224] Any protein can be modified to aid in their identification or purification, for example, by adding histidine residues (his tags), aspartic acid residues (asp tags), streptavidin tags, flag tags, SUMO tags, GST tags, or MBP tags, or by adding signal sequences to promote the secretion of polypeptides from cells that do not naturally contain such sequences. An alternative to introducing genetic tags is to chemically react the tags onto the native or engineered sites on the protein. One example of this would be reacting an engineered cysteine ​​outside the protein with a gel shift reagent. This has been shown as a method for separating hemolytic heterooligomers (Chem Biol. 1997 Jul;4(7):497-505).

[0225] Any of the proteins may be labeled with an explicit label. The explicit label can be any suitable label that allows the protein to be detected. Suitable labels include, but are not limited to, fluorescent molecules, radioisotopes such as 125I and 35S, enzymes, antibodies, antigens, polynucleotides, and ligands such as biotin.

[0226] Proteins may also contain other nonspecific modifications, provided they do not interfere with protein function. Many nonspecific side-chain modifications are well known in the art and can be performed on the side chains of proteins. Such modifications include, for example, the reductive alkylation of amino acids by reduction with NaBH4 following a reaction with an aldehyde, amidation with methyl acetimidate, or amidation with acetic anhydride.

[0227] Any of the proteins can be produced using standard methods well known in the art. The polynucleotide sequences encoding the proteins can be obtained and replicated using standard methods in the art. The polynucleotide sequences encoding the proteins can be expressed in bacterial host cells using standard techniques in the art. The proteins can be produced intracellularly by expressing polypeptides in situ from recombinant expression vectors. The expression vectors optionally carry inductive promoters to control polypeptide expression. 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.

[0228] Proteins can be produced on a large scale from protein-producing organisms, either following purification by any protein liquid chromatography system or after recombinant expression. Typical protein liquid chromatography systems include FPLC, AKTA systems, Bio-Cad systems, Bio-Rad BioLogic systems, and Gilson HPLC systems.

[0229] Method for producing chimeric pore monomers The present invention provides a method for producing the chimeric pore monomer of the present invention. This method involves attaching, preferably covalently, at least two regions having at least two different pores as sources or origins. The at least two regions can be attached using one or more linkers as described above, or covalently. In particular, the present invention provides a method for producing the chimeric pore monomer of the present invention, comprising (a) designing a polynucleotide encoding the chimeric pore monomer of the present invention as a gene fusion, and (b) expressing the chimeric pore monomer from the polynucleotide. Methods for designing polynucleotide sequences, constructing polynucleotides, and expressing them are well known in the art.

[0230] With respect to the chimeric pore monomers of the present invention, any of the embodiments described above are similarly applicable to these methods.

[0231] Methods for producing pores The present invention also provides a method for producing the pores of the present invention or the pore multimers of the present invention. The pores of the present invention may be the chimeric pores of the present invention or the PorARc pores of the present invention. The pore multimers of the present invention may be the chimeric pore multimers of the present invention or the PorARc pore multimers of the present invention.

[0232] The above method may involve expressing pores in host cells. In particular, the method may include expressing at least one pore monomer or at least one construct of the present invention and a sufficient amount of the pore monomer or construct to form pores or pore multimers in host cells, and enabling the formation of the pores or pore multimers in the host cells. The sufficient amount of pore monomer or construct is preferably the sufficient amount of the present invention. In this context, the pore monomer(s) of the present invention may be the chimeric pore monomer(s) of the present invention or the PorARc pore monomer(s) of the present invention. The construct(s) of the present invention may be the chimeric construct(s) of the present invention or the PorARc construct(s) of the present invention. The number of pore monomers or constructs required to form the pores or pore multimers of the present invention is considered above. Suitable host cells and expression systems are well known in the art and are considered in the examples.

[0233] The method may involve forming pores in a non-cellular or in vitro environment. In particular, the method may include contacting at least one pore monomer or at least one construct of the present invention with a sufficient number of pore monomers or constructs in vitro, thereby enabling the formation of pores or pore multimers. The pore monomer(s) or construct(s) may be produced individually by in vitro translation and transcription (IVTT) and then incubated with a sufficient number of pore monomers or constructs. The sufficient number of pore monomers or constructs is preferably a sufficient number of pore monomers or constructs of the present invention. In this context, the pore monomer(s) of the present invention may be a chimeric pore monomer(s) or a PorARc pore monomer(s) of the present invention. The construct(s) of the present invention may be a chimeric construct(s) or a PorARc construct(s) of the present invention. The number of pore monomers or constructs required to form pores or pore multimers of the present invention is considered above. The method may be constructed in an "in vitro system," which refers to a system that includes at least the components and environment necessary to carry out the method, utilizing biomolecules, organisms, cells (or parts of cells) outside of their normally naturally occurring environment, enabling more detailed, convenient, or efficient analysis than can be performed on whole organisms. The in vitro system may also include a suitable buffer composition provided in a test tube, to which the protein components for complex formation are added. Those skilled in the art will know the options for providing such a system.

[0234] To facilitate purification, some or all of the components of the pores or pore multimers may be tagged. Purification can also be performed when the components are not tagged. The components of the pores can be purified using methods well known in the art (e.g., ion exchange, gel filtration, hydrophobic interaction column chromatography) alone or in different combinations.

[0235] The pores or pore multimers can be produced before insertion into the membrane or after insertion of the components into the membrane.

[0236] The methods for making the pores and complexes of the present invention, as well as the methods for tagging them, are disclosed in WO2016 / 034591, WO2017 / 149316, WO2017 / 149317, WO2017 / 149318, WO2018 / 211241, WO2019 / 002893, PCT / EP2023 / 059821, PCT / EP2023 / 072113, PCT / EP2023 / 072065, PCT / EP2023 / 072106, and PCT / EP2023 / 072068 (all of which are hereby incorporated by reference in their entirety).

[0237] Method for characterizing an analyte The present invention provides a method for determining the presence, absence, or one or more characteristics of a target analyte. The method involves contacting the target analyte with a pore or pore multimer such that the target analyte moves towards or through the pore or pore multimer, and performing one or more measurements as the target analyte moves relative to the pore or pore multimer, thereby determining the presence, absence, or one or more characteristics of the target analyte. The target analyte may also be referred to as a template analyte or an analyte of interest.

[0238] The pore may be a chimeric pore comprising two or more regions, at least two of which are derived from at least two different pores. All of the above discussions related to chimeric pores, two or more regions, and at least two of said regions being derived from at least two different pores are equally applicable to the methods of the present invention. To avoid doubt, in the context of the methods of the present invention, at least two different pores can include alpha-hemolysin and gamma-hemolysin. The chimeric pore is preferably a chimeric pore of the present invention.

[0239] The pore may be a PorARc pore of the present invention.

[0240] The pores may be the CsgG pores of the present invention.

[0241] The pore multimer may contain two or more pores, at least one of which is a chimeric pore, and the chimeric pore contains two or more regions, of which at least two of the two or more regions are supplied or derived from at least two different pores. All of the above discussion relating to the chimeric pore, two or more regions, and at least two of the regions being supplied or derived from at least two different pores also applies equally to the method of the present invention. The pore multimer is preferably the chimeric pore multimer of the present invention.

[0242] The pore multimer may be the PorARc pore multimer of the present invention.

[0243] The pores may be the CsgG pore multimer of the present invention.

[0244] This method is for determining the presence or absence of a target analyte, or one or more characteristics of it. This method may be for determining the presence or absence of at least one analyte, or one or more characteristics of it. This method may relate to determining the presence or absence of two or more analytes, or one or more characteristics of them. This method may include determining the presence or absence of any number of analytes, for example, two, five, ten, fifteen, twenty, thirty, forty, fifty, one hundred, or more. Any number of characteristics of one or more analytes, for example, one, two, three, four, five, ten, or more characteristics, may be determined.

[0245] The binding of molecules within or near the openings of pores or porous multimers affects the open-channel ion flow through the pores or porous multimers, which is the essence of “molecular sensing.” In a manner similar to nucleic acid sequencing applications, fluctuations in open-channel ion flow can be measured using preferred measurement techniques based on changes in current (e.g., WO2000 / 28312 and D. Stoddart et al., Proc. Natl. Acad. Sci., 2010, 106, 7702-7 or WO2009 / 077734; all of which are incorporated herein by reference in their entirety). The degree of decrease in ion flow, measured by a decrease in current, is related to the size of the obstruction within or near the pore. Thus, the binding of the molecule of interest, also referred to as the “analyte,” within or near the pore provides a detectable and measurable event, thereby forming the basis of a “biological sensor.” Suitable molecules for nanopore sensing include polynucleotides / nucleic acids, proteins, peptides, polynucleotide-polypeptide conjugates, polysaccharides, and small molecules (here referring to low molecular weight organic or inorganic compounds (e.g., <900 Da or <500 Da)), such as pharmaceuticals, toxins, cytokines, and contaminants. By detecting the presence of biomolecules, applications are found in personalized drug development, medicine, diagnostics, life science research, environmental monitoring, and the security and / or defense industries.

[0246] Pores or pore multimers may function as molecular or biological sensors. The target analyte molecule to be detected may bind to either the channel surface or within the lumen of the channel itself. The binding site may be determined by the size of the molecule being sensed.

[0247] The target analytes preferably include or are metal ions, inorganic salts, polymers, amino acids, peptides, polypeptides, proteins, nucleotides, oligonucleotides, polynucleotides, polynucleotide-polypeptide conjugates, monosaccharides, oligosaccharides, polysaccharides, dyes, bleaching agents, pharmaceuticals, diagnostic agents, recreational drugs, explosives, toxic compounds, or environmental pollutants.

[0248] The target analyte may comprise two or more different molecules, such as peptides and polypeptides. The target analyte may also be a polynucleotide-polypeptide conjugate. The method may also relate to determining the presence or absence of two or more analytes of the same type, for example, two or more proteins, two or more nucleotides, or two or more pharmaceuticals, or one or more characteristics. Alternatively, the method may also relate to determining the presence or absence of two or more different types of analytes, for example, one or more proteins, one or more nucleotides, and one or more pharmaceuticals, or one or more characteristics.

[0249] The target analyte may be secreted from cells. Alternatively, the target analyte may be an analyte present within cells, and therefore must be extracted from cells before the method may be performed. The target analyte may be obtained from or extracted from any organism or microorganism. The target analyte may be obtained from humans or animals, for example from urine, lymph, saliva, mucus, semen, or amniotic fluid, or from whole blood, plasma, or serum. The target analyte may be obtained from plants, for example from grains, legumes, fruits, or vegetables.

[0250] Pores or pore multimers may be modified via recombination or chemical methods to increase binding strength, binding location, or binding specificity of the sensed molecule. Typical modifications include the attachment of a specific binding site complementary to the structure of the sensed molecule. If the target analyte molecule contains nucleic acids, this binding site may contain cyclodextrins or oligonucleotides. For small molecules, this may be an antigen-binding site of an antibody or non-antibody molecule containing a known complementary binding site, such as a single-strand variable fragment (scFv) region or antigen-recognition domain from a T cell receptor (TCR); or, for proteins, it may be a known ligand of the target protein. In this way, the pores or pore multimers may be given the ability to act as molecular sensors for detecting the presence of suitable antigens (including epitopes) in a sample, which may include receptors, cell surface antigens including markers of solid tumors or hematological cancer cells (e.g., lymphoma or leukemia), viral antigens, bacterial antigens, protozoan antigens, allergens, allergy-related molecules, albumin (e.g., human, rodent, or bovine), fluorescent molecules (including fluorescein), blood group antigens, small molecules, drugs, enzymes, catalytic sites or substrates of enzymes, and transition state analogs of enzyme substrates. As described above, modifications can be achieved using known genetic engineering and recombinant DNA techniques. The positioning of any fit will depend on the properties of the molecule being sensed, e.g., size, three-dimensional structure, and its biochemical properties. The selection of a fitted structure may utilize computational structural design. The determination and optimization of protein-protein interactions or protein-small molecule interactions can be investigated using technologies such as BIAcore®, which detects molecular interactions using surface plasmon resonance (see also BIAcore, Inc., Piscataway, NJ; www.biacore.com).

[0251] The target analyte preferably comprises, or is, amino acids, peptides, polypeptides, or proteins. The amino acids, peptides, polypeptides, or proteins may or may not be naturally occurring. Polypeptides or proteins may contain synthetic or modified amino acids. Several different types of modifications to amino acids are well known in the art. Preferred amino acids and their modifications are as described above. It should be understood that the target analyte may be modified by any method available in the art.

[0252] The target analyte is preferably a polynucleotide, such as a nucleic acid. A polynucleotide is defined as a macromolecule containing two or more nucleotides. Nucleic acids are particularly suitable for nanopore sequencing. Naturally occurring nucleic acid bases in DNA and RNA may be distinguished by their physical size. As nucleic acid molecules or individual bases pass through nanopore channels, the ion flow through the channels is reduced in a directly correlated manner due to the size differences between the bases. The variation in ion flow can be recorded. Suitable electrical measurement techniques for recording the variation in ion flow are discussed above. With suitable calibration, specific nucleotides and associated bases passing through the channels can be identified in real time using the characteristic reduction in ion flow. In typical nanopore nucleic acid sequencing, the open-channel ion flow is reduced as individual nucleotides of the target nucleotide sequence pass through the nanopore channels in succession due to the partial blockage of channels by nucleotides. It is this reduction in ion flow that is measured using the preferred recording techniques described above. The reduction of ion flow can be calibrated to match the reduction of ion flow measured for known nucleotides passing through the channel, providing a means to determine which nucleotides pass through the channel; thus, if performed sequentially, a method for determining the nucleotide sequence of nucleic acids passing through nanopores is obtained. To accurately determine individual nucleotides, it is typically necessary that the reduction of ion flow through the channel directly correlates with the size of the individual nucleotides passing through the constriction. It will be understood that sequencing may be performed, for example, on intact nucleic acid polymers "inserted" through the pore via the action of the relevant polymerase. Alternatively, sequencing may be determined by the passage of nucleotide triphosphate bases being sequentially removed from the target nucleic acid adjacent to the pore (see, for example, WO2014 / 187924, which is incorporated herein by reference in its entirety).

[0253] 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 cutaneous melanoma. One or more nucleotides in a polynucleotide may be modified, for example, by labels or tags, preferred examples of which are known to those skilled in the art. A polynucleotide may contain one or more spacers. A nucleotide typically contains a nucleic acid base, a sugar, and at least one phosphate group. The nucleic acid base and sugar form a nucleoside. The nucleic acid base is typically heterocyclic. The nucleic acid base includes, but is 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. Polynucleotides preferably contain the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and / or thymidine (dT), deoxyguanosine (dG), and deoxycytidine (dC). Nucleotides are typically ribonucleotides or deoxyribonucleotides. Nucleotides typically contain monophosphate, diphosphate, or triphosphate. Nucleotides may contain more than three phosphate groups, for example, four or five. The phosphate groups may be attached to the 5' or 3' side of the nucleotide. Nucleotides in a polynucleotide may be attached to each other in any manner. Nucleotides are typically attached by their sugar and phosphate groups, similar to nucleic acids. Nucleotides may be linked via nucleic acid bases, similar to pyrimidine dimers. Polynucleotides 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 using a polynucleotide as an alternative 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 or not the polynucleotide is modified.

[0254] A polynucleotide can be of any length (i). For example, a polynucleotide may have a length of 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. A polynucleotide may have a length of 1000 or more nucleotides or nucleotide pairs, 5000 or more nucleotides or nucleotide pairs, or 100,000 or more nucleotides or nucleotide pairs. Any number of polynucleotides can be investigated. For example, this method may relate to the characterization of 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more polynucleotides. If two or more polynucleotides are characterized, they may be different polynucleotides or two examples of the same polynucleotide. Polynucleotides may be naturally occurring or artificial. For example, this method can be used to verify the sequence of a manufactured oligonucleotide. This method is typically performed in vitro.

[0255] Nucleotides may have identity (ii) and may include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), 5-methylcytidine monophosphate, 5-hydroxymethylcytidine monophosphate, cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP), deoxycytidine monophosphate (dCMP), and deoxymethylcytidine monophosphate. Nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP, and dUMP. Nucleotides can be debased (i.e., lacking a nucleic acid base). Nucleotides may also lack both a nucleic acid base and a sugar (i.e., they are C3 spacers). The sequence (iii) of a nucleotide is determined by the continuous identity of the following nucleotides linked to each other in the 5′ to 3′ direction of the chain across the entire polynucleotide strain.

[0256] The movement of polynucleotides to or from pores or pore multimers, such as through pores or pore multimers, is preferably controlled using polynucleotide-binding proteins. Suitable proteins are discussed in detail below. The present invention provides a method for determining the presence, absence, or one or more characteristics of a target polynucleotide, the method is (i)(a) a chimeric pore comprising two or more regions wherein at least two of the two or more regions are supplied or derived from at least two different pores, (b) a chimeric pore multimer comprising two or more pores wherein at least one pore is a chimeric pore as defined in (a), (c) the PorARc pore of the present invention, or (d) the PorARc pore multimer and polynucleotide-binding protein of the present invention, wherein the polynucleotide-binding protein controls the movement of the target analyte as it moves toward the pore or pore multimer, for example, by passing through them. (ii) The step of performing one or more measurements as the polynucleotide moves toward a pore or pore multimer, for example, as it moves through thereto, thereby determining the presence or absence of the polynucleotide or one or more characteristics.

[0257] The chimeric pore in (a) is preferably the chimeric pore of the present invention. The chimeric pore multimer in (b) is preferably the chimeric pore multimer of the present invention.

[0258] The target analyte preferably comprises a polypeptide. Any suitable polypeptide can be characterized. The polypeptide may be an unmodified protein or a part thereof, or a naturally occurring polypeptide or a part thereof. The target polypeptide may be secreted from a cell. Alternatively, since the target polypeptide is produced intracellularly, it needs to be extracted from the cell for characterization. Polypeptides are plasmids, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 4 thThis may include the intracellular expression product of a plasmid used for cloning a protein according to the methods described in ed., Cold Spring Harbor Press, Plainsview, New York (2012), and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016).

[0259] Polypeptides can be provided as impurity mixtures of one or more polypeptides and one or more impurities. The impurities may contain cleavage forms of target polypeptides different from the "target polypeptide" for characterization purposes. For example, the target polypeptide may be a full-length protein, and the impurities may contain fractions of that protein. The impurities may also contain proteins other than the target protein, which may be co-purified from cell cultures or obtained from samples.

[0260] Polypeptides can contain any combination of amino acids, amino acid analogs, and modified amino acids (i.e., amino acid derivatives). Amino acids (and derivatives, analogs, etc.) in a polypeptide can be distinguished by their physical size and charge. Amino acids / derivatives / analogs may be naturally occurring or artificial. Polypeptides may contain any naturally occurring amino acid.

[0261] The polypeptide may be modified. The polypeptide may be modified for detection using the method of the present invention. This method may be for characterizing modifications of a target polypeptide.

[0262] One or more of the amino acids / derivatives / analogs in the polypeptide may be modified. One or more of the amino acids / derivatives / analogs in the polypeptide may be modified after translation. Thus, the method of the present invention can be used to detect the presence, absence, and number of positions of post-translational modifications in a polypeptide. This method can be used to characterize the degree to which a polypeptide is post-translationally modified.

[0263] The polypeptide may have one or more post-translational modifications. Typical post-translational modifications include modification by hydrophobic groups, modification by cofactors, addition of chemical groups, glycosylation (non-enzymatic binding of sugars), biotinylation, pegylation, etc. Post-translational modifications are non-natural and may also be chemical modifications performed in the laboratory for biotechnological and biomedical purposes. This makes it possible to monitor the levels of peptides, polypeptides, or proteins produced in the laboratory in comparison to their natural counterparts.

[0264] Examples of post-translational modifications by hydrophobic groups include 14 myristoylation, which is the attachment of myristic acid, a saturated acid; 16 palmitoylation, which is the attachment of palmitic acid, a saturated acid; isoprenylation or prenylation, which is the attachment of an isoprenoid group; farnesylation, which is the attachment of a farnesol group; geranylgeranylation, which is the attachment of a geranylgeraniol group; and glypiation, which is the formation of a glycosylphosphatidylinositol (GPI) anchor by an amide bond, etc.

[0265] Examples of post-translational modifications by cofactors include lipoylation, which is the attachment of a lipoic acid (C8) functional group; flavinylation, which is the attachment of a flavin moiety (e.g., flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD)); attachment of heme C, for example, via a thioether bond with cysteine; phosphopantetheinylation, which is the attachment of a 4'-phosphopantetheinyl group; and retinylidene Schiff base formation, etc.

[0266] Examples of post-translational modifications by the addition of chemical groups include: acylation, e.g., O-acylation (ester), N-acylation (amide), or S-acylation (thioester); acetylation, e.g., attachment of an acetyl group to the N-terminus or lysine; formylation; alkylation, e.g., addition of alkyl groups such as methyl or ethyl; methylation, e.g., addition of a methyl group to lysine or arginine; amidation; butyrylation; gamma-carboxylation; glycosylation, e.g., enzymatic attachment of a glycosyl group to arginine, asparagine, cysteine, hydroxylysine, serine, threonine, tyrosine, or tryptophan; polysialylation; polysia Attachment of glycerides; malonylation; hydroxylation; iodization; bromination; citrullination; nucleotide addition, attachment of nucleotides as described above, ADP-ribosylation; oxidation; phosphorylation, e.g., attachment of a phosphate group to serine, threonine, or tyrosine (O-bond) or histidine (N-bond); adenylation, e.g., attachment of an adenyl moiety to tyrosine (O-bond) or histidine or lysine (N-bond); propionylation; pyroglutamate formation; S-glutathionylation; smoylation; S-nitrosylation; succinylation, e.g., attachment of a succinyl group to lysine; selenoylation, incorporation of selenium; ubiquitination, addition of a ubiquitin subunit (N-bond).

[0267] Polypeptides may be labeled with molecular labels. The molecular label may be a modification of the polypeptide that facilitates the detection of the polypeptide in the method of the present invention. For example, the label may be a modification of the polypeptide that can alter the signal obtained in the characterization of the conjugate. For example, the label may interfere with the flux of ions through nanopores. In such a way, the label may improve the sensitivity of the method.

[0268] The polypeptide may contain one or more cross-linking sites, such as CC bridges. The polypeptide does not need to be cross-linked before being characterized using this method.

[0269] Polypeptides may contain sulfide-containing amino acids and therefore may form disulfide bonds. Typically, in such embodiments, the polypeptide is reduced using a reagent such as DTT (dithiothreitol) or TCEP (tris(2-carboxyethyl)phosphine) before being characterized using the present method.

[0270] Polypeptides may be full-length proteins or naturally occurring polypeptides. Proteins or naturally occurring polypeptides may be fragmented before being attached to polynucleotides. Proteins or polypeptides may be fragmented chemically or enzymatically. Polypeptides or polypeptide fragments may be linked together to form a longer target polypeptide.

[0271] The length of the polypeptide is any suitable length. Preferably, the polypeptide has a length of about 2 to about 300 peptide units or amino acids. The polypeptide has a length of about 2 to about 100 peptide units, for example, about 2 to about 50 peptide units, for example, about 3 to about 50 peptide units, for example, about 5 to about 25 peptide units, for example, about 7 to about 16 peptide units, for example, about 9 to about 12 peptide units. A "peptide unit" is interchangeable with an "amino acid".

[0272] One or more features of a polypeptide are preferably selected from (i) the length of the polypeptide, (ii) the identity of the polypeptide, (iii) the sequence of the polypeptide, (iv) the secondary structure of the polypeptide, and (v) whether or not the polypeptide is modified. One or more features are the sequence of the polypeptide, or whether or not the polypeptide is modified, for example, whether or not it is modified by one or more post-translational modifications. One or more features are preferably the sequence of the polypeptide.

[0273] Polypeptides may be in a relaxed state. Polypeptides may also be held in a linear state. Holding polypeptides in a linear state prevents "bundling" of polypeptides within nanopores, making it easier to characterize polypeptide residues individually. Polypeptides can be held in a linear state using any suitable means. For example, if the polypeptide is charged, it can be held in a linear state by applying a voltage.

[0274] If the polypeptide is uncharged or only weakly charged, the charge can be altered or controlled by adjusting the pH. For example, by using a high pH to increase the relative negative charge of the polypeptide, the polypeptide can be retained in a linear form. Increasing the negative charge of the polypeptide allows it to be retained in a linear form, for example, under a positive voltage. Alternatively, the polypeptide can also be retained in a linear form by using a low pH to increase the relative positive charge of the polypeptide. Increasing the positive charge of the polypeptide allows it to be retained in a linear form, for example, under a negative voltage. In the disclosed method, a polynucleotide handling protein is used to control the movement of polynucleotides into nanopores. Since polynucleotides are typically negatively charged, it is generally most preferable to promote the linearization of the polypeptide by increasing the pH, thereby making the polypeptide more negatively charged in common with the polynucleotide. In this way, the conjugate retains an overall negative charge and can therefore move easily, for example, under applied voltage.

[0275] Polypeptides can be maintained in a linear form by using preferred modification conditions. Preferred modification conditions include, for example, the presence of a suitable concentration of a denaturing agent such as guanidine HCl and / or urea. The concentration of such a denaturing agent used in the disclosed method depends on the target polypeptide being characterized by the method and can be readily selected by those skilled in the art.

[0276] Polypeptides can be retained in a linear form by using a suitable detergent. Suitable detergents for use in the disclosed method include SDS (sodium dodecyl sulfate). Polypeptides can also be retained in a linear form by carrying out the disclosed method at a high temperature. Increasing the temperature overcomes intrachain bonds, allowing the polypeptide to become linear.

[0277] Polypeptides can be retained in a linear morphology by carrying out this method under strong electroosmotic conditions. Such forces can be provided by using asymmetric salt conditions and / or by providing a suitable charge to the channels of the nanopores. The charge of the pore channels can be altered, for example, by mutagenesis. Altering the charge of the pores is within the capabilities of those skilled in the art. When the charge of the pores is altered, a strong electroosmotic force is generated because cations and anions flow unbalanced through the nanopores when a potential is applied throughout the nanopores.

[0278] Polypeptides can be maintained in a linear morphology by passing through structures such as nanopillar arrays, nanoslits, or nanogaps. These physical constraints of the structure necessitate the polypeptide taking a linear form.

[0279] The target analyte may include polynucleotides and polypeptides. The target analyte may also be a polynucleotide-polypeptide conjugate. The conjugate preferably includes a polypeptide to which a polynucleotide is attached. One or both of the polynucleotide and the polypeptide may be the target and may be characterized according to the present invention.

[0280] Polypeptides can be attached to polynucleotides at any preferred position. For example, a polypeptide can be attached to a polynucleotide at its N-terminus or C-terminus. A polypeptide can be attached to a polynucleotide via side chain groups of residues (e.g., amino acid residues) within the polypeptide. Polypeptides may have naturally occurring reactive functional groups that can be used to facilitate attachment to polynucleotides. For example, cysteine ​​residues can be used to form disulfide bonds to the polynucleotide or a modifying group thereon.

[0281] Polypeptides may be modified to facilitate binding to polynucleotides. For example, a polypeptide may be modified by attaching a moiety containing a reactive functional group for attachment to a polynucleotide. For example, a polypeptide may be extended at the N-terminus or C-terminus by one or more residues (e.g., amino acid residues) containing one or more reactive functional groups for reacting with corresponding reactive functional groups on the polynucleotide. For example, a polypeptide may be extended at the N-terminus and / or C-terminus by one or more cysteine ​​residues. Such residues can be used for binding to the polynucleotide moiety of the conjugate by maleimide chemistry (e.g., by the reaction of cysteine ​​with an azido-maleimide compound such as azido-[Pol]-maleimide, where [Pol] is typically a short-chain polymer such as PEG, e.g., PEG2, PEG3, or PEG4, and then bound to a appropriately functionalized polynucleotide, e.g., a polynucleotide having a BCN group for reacting with the azide). Such chemical reactions are described in Example 2. To avoid any ambiguity, if a polypeptide contains suitable naturally occurring residues at its N-terminus and / or C-terminus (for example, naturally occurring cysteine ​​residues at the N-terminus and / or C-terminus), such residues may be used for binding to the polynucleotide.

[0282] Residues in a polypeptide may be modified to promote adhesion between the polypeptide and the polynucleotide. Residues in a polypeptide (e.g., amino acid residues) may be chemically modified to adhere to the polynucleotide. Residues in a polypeptide (e.g., amino acid residues) may be enzymatically modified to adhere to the polynucleotide.

[0283] The bonding chemistry between the polynucleotide and polypeptide in the conjugate is not particularly limited. Any suitable combination of reactive functional groups can be used. Many suitable reactive groups and their chemical targets are well known in the art. Some exemplary reactive groups and their corresponding targets include aryl azides that can react with amines, carbodiimides that can react with amines and carboxyl groups, hydrazides that can react with carbohydrates, hydroxymethylphosphines that can react with amines, imide esters that can react with amines, isocyanates that can react with hydroxyl groups, carbonyls that can react with hydrazines, maleimides that can react with sulfhydryl groups, NHS esters that can react with amines, PFP esters that can react with amines, psoralens that can react with thymine, pyridyl disulfide that can react with sulfhydryl groups, vinyl sulfones and vinyl sulfonamides that can react with sulfhydrylamines and hydroxyl groups. Another suitable chemical reaction for bonding polypeptides to polynucleotides is click chemistry. Many suitable click chemistry reagents are well known in the art. Suitable examples of click chemistry include, but are not limited to, the following:

[0284] Copper(I) catalyzed azide-alkyne cycloaddition (azide-alkyne hysgene cycloaddition); Strain-accelerated azide-alkyne cycloaddition; [3+2] cycloaddition of alkenes and azides; reverse demand Diels-Alder reaction of alkenes and tetrazines; and photoclic reaction of alkenes and tetrazoles. Copper-free variants of 1,3-dipolar cycloaddition reactions in which azides react with strained alkynes, for example, in a cyclooctane ring, for example, in bicyclic [6.1.0]nonine (BCN); The reaction between an oxygen nucleophile on one linker and an epoxide or aziridine reactive moiety on the other; and A Staudinger linkage can be formed by replacing the alkyne moiety with an arylphosphine, leading to a specific reaction with an azide and conferring an amide bond.

[0285] Any reactive group may be used to form the conjugate. Suitable reactive groups include [1,4-bis[3-(2-pyridyldithio)propionamide]butane]; 1,11-bismaleimidetriethylene glycol; 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide); ethylene glycol-bis(succinate N-hydroxysuccinimide); 4,4'-diisothiocyanatostilbene-2,2'-disodium disodium disulfonate; bis[2-(4-azidosalicylamide)ethyl]disulfide; 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide; 4-maleimoidbutyrate N-hydroxysuccinimide; iodoacetate N-hydroxysuccinimide; S-acetylthioglycolate N-hydroxysuccinimide; azido-PEG-maleimide; and alkyne-PEG-maleimide. The reactive group may be any of those disclosed in WO2010 / 086602, particularly in Table 3 of that application.

[0286] The reactive functional group may be present in the polynucleotide and the target functional group in the polypeptide before the binding step. The reactive functional group may be present in the polypeptide and the target functional group in the polynucleotide before the binding step. The reactive functional group may be directly attached to the polypeptide. The reactive functional group may be attached to the polypeptide via a spacer. Any suitable spacer can be used. Suitable spacers include, for example, alkyldiamines such as ethyldiamine.

[0287] The conjugate may contain multiple polypeptide moieties and / or multiple polynucleotide moieties. For example, the conjugate may contain a structure in the form of ...-PNPNPN..., where P is a polypeptide and N is a polynucleotide. The polynucleotide handling protein may sequentially control the N moiety of the conjugate relative to the pore, thereby sequentially controlling the movement of the P moiety relative to the pore and allowing for sequential characterization of the P moiety. The multiple polynucleotides and polypeptides may be conjugated by the same chemical substance or by different chemical substances.

[0288] The conjugate may include a leader. Any suitable leader may be used. The leader may be a polynucleotide. The leader may be the same type of polynucleotide as the polynucleotide used in the conjugate, or a different type of polynucleotide. For example, the polynucleotide in the conjugate may be DNA and the leader may be RNA, or vice versa.

[0289] The leader may be a charged polymer, such as a negatively charged polymer. The leader may contain polymers such as PEG or polysaccharides. The leader may be 10 to 150 monomer units (e.g., ethylene glycol or sugar units) long, for example, 20 to 120 monomer units (e.g., ethylene glycol or sugar units), for example, 30 to 100 units, for example, 40 to 80 units, for example, 50 to 70 units. A method for characterizing the target polypeptide of the present invention may include conjugating the polypeptide to a polynucleotide.

[0290] In either method, one or more features of the target analyte are preferably measured by electrical and / or optical measurements. Electrical measurements include current measurements, impedance measurements, tunnel effect measurements, or field-effect transistor (FET) measurements. The method preferably includes measuring the current flowing through the pore or pore multimer as the target analyte moves through the pore, for example.

[0291] The general conditions for carrying out the method of the present invention will be discussed in detail below with reference to the kit and system of the present invention.

[0292] The present invention provides polynucleotides The present invention also provides polynucleotides encoding pores or constructs of the present invention, comprising the chimeric pore monomer of the present invention, the PorARc pore monomer of the present invention, the chimeric construct of the present invention, or the PorARc construct of the present invention. The present invention also provides polynucleotides encoding CsgG pores of the present invention. The polynucleotides may be any of those discussed above. The present invention also provides expression vectors comprising the polynucleotides of the present invention. The present invention also provides host cells comprising the polynucleotides of the present invention or host cells of the present invention. Suitable vectors and host cells are well known in the art.

[0293] kit The present invention also provides a kit for characterizing a target analyte. In one embodiment, the kit comprises (a) a pore monomer or construct of the present invention, and (b) a membrane component. The kit preferably comprises (a) a chimeric pore monomer, a PorARc pore monomer, a chimeric construct, or a PorARc construct of the present invention, and (b) a membrane component. The kit preferably comprises (a) a CsgG pore monomer or CsgG construct of the present invention, and (b) a membrane component. Suitable membranes and components are discussed below.

[0294] In another embodiment, the kit is -(a) a chimeric pore comprising two or more regions, wherein at least two of the two or more regions are supplied or derived from at least two different pores, (b) a chimeric pore multimer comprising two or more pores, wherein at least one pore is a chimeric pore as defined in (a), (c) the PorARc pore of the present invention, or (d) the PorARc pore multimer of the present invention, - Contains polynucleotide-binding proteins.

[0295] In another embodiment, the kit is -(a) The CsgG pore of the present invention or the CsgG pore multimer of the present invention, -(b) Polynucleotide-binding proteins, and

[0296] Any of the embodiments described above with respect to pores and pore multimers are similarly applicable to the kit of the present invention.

[0297] The kit preferably further comprises membrane components. The kit may contain components of any type of membrane, such as an amphiphilic layer or a triblock copolymer membrane. Preferred polynucleotide-binding proteins are polymerases, exonucleases, helicases, and topoisomerases, such as gyrases. Preferred enzymes include, but are not limited to, exonuclease I from E. coli, exonuclease III from E. coli, RecJ and bacteriophage lambda exonucleases from Thermus thermophilus, TatD exonuclease, and their variants. Three subunits comprising the RecJ sequence or its variants from Thermus thermophilus interact to form a trimer exonuclease. The polymerase may be PyroPhage® 3173 DNA polymerase (commercially available from Lucigen® Corporation), SD polymerase (commercially available from Bioron®), or a variant thereof. The enzyme may be Phi29 DNA polymerase or a variant thereof. The topoisomerase is preferably a member of either subclassification (EC) group 5.99.1.2 or 5.99.1.3.

[0298] The enzyme is most preferably derived from a helicase such as Hel308 Mbu, Hel308 Csy, Hel308 Tga, Hel308 Mhu, TraI Eco, XPD Mbu, or a variant thereof. Any helicase can be used in the present invention. The helicase may be or derived from Hel308 helicase, RecD helicase, such as TraI helicase or TrwC helicase, XPD helicase, or Dda helicase. The helicase may be any of the helicases, modified helicases, or helicase constructs disclosed in WO2013 / 057495, WO2013 / 098562, WO2013098561, WO2014 / 013260, WO2014 / 013259, WO2014 / 013262, and WO2015 / 055981. All of these are incorporated by reference.

[0299] The kit may further include one or more anchors, such as cholesterol, for coupling a target analyte to a membrane. The kit may further include one or more polynucleotide adapters, which can be attached to a target polynucleotide to facilitate the characterization of the polynucleotide. The anchors, such as cholesterol, are preferably attached to the polynucleotide adapters.

[0300] The kit may further include one or more other reagents or instruments that enable the implementation of any of the embodiments described above. Such reagents or instruments may include one or more of the following: a suitable buffer (aqueous solution), means for obtaining a sample from the subject (such as an instrument including a container or needle), means for amplifying and / or expressing polynucleotides, or one or more of a voltage or patch clamp device. The reagents may be present in the kit in a dry state so that the fluid sample resuspends the reagent. The kit may also optionally include instructions for use to enable the kit to be used in the method of the present invention, or details regarding organisms in which the method may be used. Finally, the kit may also include additional components useful for characterizing the analyte.

[0301] Device The present invention also relates to an apparatus for characterizing a target analyte, such as a target polynucleotide, in a sample. -(a) a plurality of chimeric pores comprising two or more regions, wherein at least two of the two or more regions are supplied or derived from at least two different pores; (b) a plurality of chimeric pore multimers comprising two or more pores, wherein at least one pore is a chimeric pore as defined in (a); (c) a plurality of PorARc pores of the present invention; or (d) a plurality of PorARc pore multimers of the present invention. - Provides a device comprising multiple polynucleotide-binding proteins.

[0302] The present invention also relates to an apparatus for characterizing a target analyte in a sample. -(a) Multiple CsgG pores of the present invention or multiple CsgG pore multimers of the present invention, -(b) Provide an apparatus comprising multiple polynucleotide-binding proteins.

[0303] Multiple pores or multiple pore multimers may be any of those discussed above.

[0304] The present invention also provides an apparatus comprising the pores of the present invention or the pore multimers of the present invention, inserted into an in vitro membrane. The apparatus preferably comprises the chimeric pore monomers of the present invention, the PorARc pore monomers of the present invention, the chimeric constructs of the present invention, or the PorARc constructs of the present invention, inserted into an in vitro membrane. The apparatus preferably comprises the CsgG pores of the present invention or the CsgG pore multimers of the present invention, inserted into an in vitro membrane.

[0305] The present invention also provides an apparatus manufactured by a method comprising (i) obtaining the pores or pore multimers of the present invention, and (ii) contacting the pores or pore multimers with an in vitro membrane. The apparatus is preferably manufactured by a method comprising (i) obtaining the chimeric pore monomers, PorARc pore monomers, chimeric constructs, or PorARc constructs of the present invention, and (ii) contacting the pores or pore multimers with an in vitro membrane so that the pores or pore multimers are inserted into an in vitro membrane. The apparatus is preferably manufactured by a method comprising (i) obtaining the CsgG pores or CsgG pore multimers of the present invention, and (ii) contacting the pores or pore multimers with an in vitro membrane so that the pores or pore multimers are inserted into an in vitro membrane. Any of the specific embodiments discussed above are equally applicable to the apparatus of the present invention.

[0306] array The present invention also provides an array comprising multiple membranes of the present invention. Any of the embodiments discussed above with respect to the membranes of the present invention are similarly applicable to the array of the present invention. The array may be configured to perform any of the methods described below.

[0307] In a preferred embodiment, each membrane in the array contains one pore or pore multimer. Depending on how the array is formed, for example, the array may contain one or more membranes that do not contain a pore or pore multimer, and / or one or more membranes that contain two or more pores or pore multimers. The array may contain about 2 to about 1000 membranes, for example, about 10 to about 800, about 20 to about 600, or about 30 to about 500.

[0308] system The present invention provides a system comprising (a) a film or array of the present invention, (b) means for applying a potential across the film(s), and (c) means for detecting an electrical signal or optical signal across the film(s).

[0309] The pores and membranes may be any of those described above and below.

[0310] In one embodiment, the system further comprises a first chamber and a second chamber, the first and second chambers separated by a membrane(s). When used to characterize a target analyte, the system may further comprise the target analyte, which is transiently located within a continuous channel, with one end of the target analyte located in the first chamber and the other end located in the second chamber. The target analyte is preferably a target polypeptide or a target polynucleotide.

[0311] In one embodiment, the system further includes a conductive solution in contact with the pore(s), electrodes providing a potential across the membrane(s), and a measuring system for measuring the current passing through the pore(s). The voltage applied to the membrane and pores is preferably +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 distinction between different amino acids or nucleotides by the pores. Any suitable conductive solution can be used. For example, the solution may contain charge carriers such as metal salts, e.g., alkali metal salts, halide salts, e.g., chloride salts, e.g., alkali metal chloride salts. Examples of charge carriers include ionic liquids or organic salts, such as tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride, or 1-ethyl-3-methylimidazolium chloride. In exemplary systems, the salt is present in an aqueous solution within the chamber. Potassium chloride (KCl), sodium chloride (NaCl), cesium chloride (CsCl), or mixtures of potassium ferrocyanide and potassium ferricyanide are typically used. KCl, NaCl, and mixtures of potassium ferrocyanide and potassium ferricyanide are preferred. The charge carrier may be asymmetric across the membrane. For example, the type and / or concentration of the charge carrier may differ on each side of the membrane, for example, within each chamber.

[0312] The salt concentration may be saturated. The salt concentration may be 3M or less, and is typically 0.1–2.5M, 0.3–1.9M, 0.5–1.8M, 0.7–1.7M, 0.9–1.6M, or 1M–1.4M. The salt concentration is preferably 150mM–1M. This method is preferably carried out using a salt concentration of at least 0.3M, for example, 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. Higher salt concentrations provide a high signal-to-noise ratio, allowing for the identification of currents indicating the presence of amino acids or nucleotides against a background of normal current fluctuations.

[0313] A buffer may be present in the conductive solution. Typically, the buffer is a phosphate buffer. Other suitable buffers are HEPES and Tris-HCl buffer. 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.

[0314] The system may be included in the apparatus. The apparatus may be any conventional apparatus for analyte analysis, such as an array or a chip. The apparatus is preferably configured to carry out the disclosed method. For example, the apparatus may include a chamber containing an aqueous solution and a barrier separating the chamber into two sections. The barrier typically has openings on which a membrane(s) containing pores(s) is formed. Alternatively, the barrier forms a membrane in which pores are present.

[0315] The apparatus may also include an electrical circuit capable of applying an electric potential and measuring electrical signals across the membrane and pores.

[0316] The apparatus may be any of those described in WO2008 / 102120, WO2009 / 077734, WO2010 / 122293, WO2011 / 067559, or WO00 / 28312 (all of which are incorporated herein by reference in their entirety).

[0317] film Any suitable membrane can be used in the system. The membrane is preferably an amphiphilic layer. The amphiphilic layer is a layer formed from amphiphilic molecules such as phospholipids that have both hydrophilic and lipophilic properties. The amphiphilic molecules may be synthetic or naturally occurring. Amphiphilic substances that do not exist naturally and amphiphilic substances that form monolayers are well known in the art and include, for example, block copolymers (Gonzalez-Perez et al., Langmuir, 2009, 25, 10447-10450). Block copolymers are polymer materials in which two or more monomer subunits are polymerized together to produce a single polymer chain. Block copolymers typically have properties contributed by each monomer subunit. However, block copolymers may have unique properties that polymers formed from individual subunits do not have. Block copolymers can be manipulated in an aqueous medium such that one of the monomer subunits is hydrophobic (i.e., lipophilic) and the other subunit(s) are hydrophilic. In this case, the block copolymer may have amphiphilic properties and can form structures that mimic biological membranes. The block copolymer may be a diblock (consisting of two monomer subunits), but it can also be constructed from more than two monomer subunits to form more complex arrangements that behave as amphiphilic materials. The copolymer may be a triblock, tetrablock, or pentablock copolymer. The membrane is preferably a triblock copolymer membrane.

[0318] The film may include one of the films disclosed in International Application No. 2014 / 064443 or No. 2014 / 064444.

[0319] Amphiphilic molecules can be chemically modified or functionalized to facilitate the coupling of polynucleotides. The amphiphilic layer may be a monolayer or a dilayer. The amphiphilic layer is typically planar. The amphiphilic layer may be curved. The amphiphilic layer may be supported.

[0320] Amphiphilic membranes are typically about 10 -8 cm s -1 It is naturally mobile, essentially acting as a two-dimensional fluid with a lipid diffusion rate. This means that pores and coupled polynucleotides can move typically within amphiphilic membranes.

[0321] The membrane may be a lipid bilayer. Lipid bilayers are a model of cell membranes and serve as an excellent base for a wide range of experimental studies. For example, lipid bilayers can be used for in vitro investigations of membrane proteins by single-channel recording. Alternatively, lipid bilayers can be used as biosensors for detecting the presence of a wide range of substances. The lipid bilayer can be any lipid bilayer. Preferred lipid bilayers include, but are not limited to, planar lipid bilayers, supported bilayers, or liposomes. The lipid bilayer is preferably a planar lipid bilayer. Preferred lipid bilayers are disclosed in WO2008 / 102121, WO2009 / 077734, and WO2006 / 100484 (all of which are incorporated herein by reference in their entirety).

[0322] The film may include a solid state layer. The solid state layer may include, but is 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, and can be formed from both organic and inorganic materials. The solid state layer may also be formed from graphene. A suitable graphene layer is disclosed in WO2009 / 035647 (which is incorporated herein by reference in its entirety). When the film includes a solid state layer, pores are typically present in the amphiphilic film or layer contained within the solid state layer, for example, in holes, wells, gaps, channels, trenches, or slits within the solid state layer. Those skilled in the art can prepare suitable solid-state / amphiphilic hybrid systems. Preferred systems are disclosed in WO2009 / 020682 and WO2012 / 005857 (both of which are incorporated herein by reference in their entirety). Any of the amphiphilic membranes or layers discussed above may be used.

[0323] This method is typically carried out using (i) an artificial amphiphilic layer containing pores, (ii) an isolated, naturally occurring lipid bilayer containing pores, or (iii) cells into which pores have been inserted. This method is typically carried out using an artificial amphiphilic layer such as an artificial diblock copolymer layer or an artificial triblock copolymer layer. In addition to pores, the layer may contain other transmembrane proteins and / or intramembrane proteins, as well as other molecules. Preferred apparatus and conditions are discussed below. The method of the present invention is typically carried out in vitro.

[0324] Sequence List Sequence ID 1 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTRREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPGLELGISSAGAVSGTLSDVLPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSSGSAWSHPQFEK

[0325] Sequence ID 2 MGLDNELSLVDGQGRTLTIQQWDTFLNGVFPLDRNRLTREWFHSGRAKYIVAGEGAEDFEGTLELGYQIGFPWSLGVGINFSYTTPNILLNNVSLFPAFNPLGSVITPNLFPGVSISADLGNGPGIQEVATFSVDVEGPEGGVAVSNAHGTVTGAAGGVLLRPFARLISSAGDSVTTYGEPWNMNGSGGENLYFQGSGSGSAWSHPQFEK

[0326] Sequence ID 3 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPNILLNNVSLFPAFNPLGSV ITPNLFPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0327] Sequence ID 4 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPNILLNNVSIAPGAFNPLGS VITPNLFPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0328] Sequence ID 5 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPNFLFNNAQVYAIPGTPSANNGIG PFNSIITPNILPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0329] Sequence ID 6 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPNVLFNQAPLNPAGYLNPNNG FITTPNFFPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0330] Sequence ID 7 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPNILLNNVSISPTNFNPLAQ VITPNLFPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0331] Sequence ID 8 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPNILLNQAPPNLNPAAGFL TTPNLFPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0332] Sequence ID 9 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTRREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPNLLINNASIAPNLTPGSPFGPTVGTG FAPLGSIITPNLFPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0333] Sequence ID 10 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPNILLNNVNPFPSAWGPLGYQGN GGIITPNLFPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0334] Sequence ID 11 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPNVLVYNASIAPPPLGVGITPL SSVVTPNLFPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0335] Sequence ID 12 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPNVALNAQNVIGTAVGPINFFP PISTPPLLPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0336] Sequence ID 13 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPNILLNNAPLPGFPTAIFGPG FITTPNLFPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSSAWSHPQFEK

[0337] Sequence ID 14 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTTPNILFNQLNPVVPINPNGGLG FITTPNLFPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSSAWSHPQFEK

[0338] Sequence ID 15 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPNVALNAQNVILAPGINLFPP ISTPPLFPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0339] Sequence ID 16 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPNVLLNQAPPPGANFTQFGF LTTPNLFPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0340] Sequence ID 17 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPNVLLNQFSPTAPLGPAFT TPNLFPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0341] Sequence ID 18 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPNILLNSAPFPPVAGQFIT TPNLFPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0342] Sequence ID 19 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTRREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPNILISNAPITNPLSSIITPNLLPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0343] Sequence ID 20 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTRREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPNILLNNATPANPLQVITPNLFPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0344] Sequence ID 21 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPNILINNGNITAPPFGLNSV ITPNLFPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0345] Sequence ID 22 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTRREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPQLQLNAGNIIVTNILPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSSGSAWSHPQFEK

[0346] Sequence ID 23 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTRREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPQLSVNANGGTLSNILPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0347] Sequence ID 24 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTRREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPQLSVSTNNFTVSNILPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0348] Sequence ID 25 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTRREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPGLSISSNNFTLSNVLPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0349] Sequence ID 26 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTRREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPQLQLNFNGGGTVSNILPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0350] Sequence ID 27 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTRREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPQLQIGNGNAFTVSNILPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSSGSAWSHPQFEK

[0351] Sequence ID 28 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTRREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPGLNLSVGNGVAATVTNVLPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0352] Sequence ID 29 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTRREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPGLQVNLPNASATVTNILPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSSGSAWSHPQFEK

[0353] Sequence ID 30 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTRREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPQLQVGNGNAFTVSNILPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0354] Sequence ID 31 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTRREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPGLQINFNGGGTINSLIPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0355] Sequence ID 32 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPGLQVQVGTNTQVNIF NLIPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0356] Sequence ID 33 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTRREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPGINSRPPPGQGIQLKNLIPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0357] Sequence ID 34 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTRREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPGLNVQVFPNVAVGLTNVIPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSSGSAWSHPQFEK

[0358] Sequence ID 35 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPGLQLQLSAPSTLNVF NLIPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGTATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0359] Sequence ID 36 MAVDDSNSVVDGGGNTITVSQSDTFINSVFPLDGSPLTREWFHNGRAIVDVTGPDAEDFSGTVTIGYQVGYPASLGGRLTFSYTTPNILLNNVSLFPAFNPLGSV ITPNLFPQAGVGVTLTPGPGIETVAVASGAASGAHTEIQIANLHGATKIAGNVSVRPYVQVVSSNGDVATTFGQPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0360] Sequence ID 37 MRVEGGDSVVDGKKRKIAALVSDTRVESVVPIDSNPTSRQWRYSGKSTLRITGGDEDEDGAKVEDWGGTVTFGFLIGYPATLTGSIGVSYSTPNILLNNVSLFPAFNP LGSVITPNLFPTVTGNLAVGFGPGVRAVDATTGQAKGAGGVLRVKNFHGAVTGVLGQVTIQPYIRVWSNEGDTIMVYGRKHLLGGSGGENLYFQGSGSGSAWSHPQFEK

[0361] Sequence ID 38 MEVNDQNRIVSNGHEVIVTQEDTFINGVPPIGGSPLSREWFHNGRGIANIVGPEAADFEDSTFQFGYQVAWSAAIDGSVGFNWTSPNILLNNVSLFPAFNPLGS VITPNLFPQLTAGIELTPAPGIEELVVAEGQFDGDYKEVQIANVHGAATGVVGPVSVRPFVRVITENGDNVTTYGQVWTLGSGGENLYFQGSGSGSAWSHPQFEK

[0362] Sequence ID 39 MEVNDQNRIVSNGHEVIVTQEDTFINGVPPIGGSPLSRQWFHNGRGIANVVGPQADEFEDSTFQFGYQVAWPASIDGAIGFSWTSPNILLNNVSLFPAFNPLGGSVITPNLFPQVTASVALTPAPGIAELVVAEGQFDGDYKEVQIANVSGAATGVIGPVSVRPFVRVITHNGDNVTTYGQVWTLGGSGGENLYFQGSGSGSAWSHPQFEK

[0363] Sequence ID 40 MALDDTKQIETGDNLTIEARQSDTDIRFVAPLDGNPLTREWFHDAIAGFHIDGAGADEFLGKITIGYQIGYPATLSGQIKFSYTSPNILLNNVSLFPAFNPLGSVITPNLFPTVGVEISAGFGPGIKSVDIATVAIAGADGWIKIAGVHGTVTGVVGRTTIRPYVTVTSVRGDTVTTYGKDWKAGSGGENLYFQGSGSGSAWSHPQFEK

[0364] Sequence ID 41 MDVDSTDRVIDGKQRTISAIQADTTIRAVPPLDRNPLTRQWFHDVTAKFTVEGDGAEEFAGTIKIGYLVGFPATVDGRIKFGYSPNILLNNVSLFPAFNPLGSVITPNLFPTVTGEIETGFGPGVRQIEAVSGRITGAEGSVRLTNSIGTVTGVIGTATVQPYVTVVSDTGDSVTTLGKPYEVNGSGGENLYFQGSGSGSAWSHPQFEK

[0365] Sequence ID 42 MVVDNADIVVDAQQRTITAIQADTMIRGLAPLDRNPLTRMWEHDGRAEFTVTGDKADEFKGTIKIGYLVGFPATFGGKIRVSYSTPNILLNNVSLFPAFNPLGGSVITPNLFPTVTGEIEVGFGPGVQQVELASGAITGASGHISLVNFVGTVTGVIGPATIQPYVTVIADSGDTVTTLGRPWDIGSGGENLYFQGSGSGSAWSHPQFEK

[0366] Sequence ID 43 MAVDSTNTVVDANGNVITVSLSDTFINSVSPLDGNPLTREWFANGVAGWTVTGPDADDFEGTVAIGYQVGYPMSLGGSITFGYTTPNILLNNVSLFPAFNPLGSVITP NLFPSVGFEAEIIAPGPGIVDATAASGNIQGTEGDAAGPSGTIQIANAHGTATGILGNVRVRPYVSVTSSTGDVAVTYGTPWTFNGSGGENLYFQGSGSGSAWSHPQFEK

[0367] Sequence ID 44 MAVDDSNSVVDGGGNTITVSQADTFINSVFPLDGSPLTRREWFHNGRAIVEVTGPDAADFEGDITLGYQFGYPASLGGELTFSYSTPNILLNNVSLFPAFNPLGGSVITPNLFPQVGTTVTLEPGPGIEDVEVGTGSASGERTEIQISNVHGTATNIAGNVSVRPYVKVVSSNGDTAVTYGKPWRFNGSGGENLYFQGSGSGSAWSHPQFEK

[0368] Sequence ID 45 MRLDDQLSAVDGAGRTLTVQQWDTVVEGVASLDRNPLTREWFYSGKATYAVSGPDAAGFKGKLELGYQVGFPWALGMNVAFTYTTPNILLNNVSLFPAFNPLGSVITPNLFPGVSITSNLSNGPGNQEVSTFSTDVAGADGVVAVSGAHGTVTGVAGGVVLRPFVRLTSNTGATVTTYTHPWNLDGSGGENLYFQGSGSGSAWSHPQFEK

[0369] Sequence ID 46 MELDDEHSLIDAQGRTLKIQQWDTFLNGVAPLDRNRLTREWFYNGRVKYAVEGPGAESFEGSVELGYQIQFPWSMGVGLNFTYTTPNILLNNVSLFPAFNPLGSVITPNLFPGASINVMLENGPGIEDIVVVAIPVSGPSGGTAVSNGHGTVTGAAGGVTLRPFARLVSSTGDTATTYGEIWNMNGSGGENLYFQGSGSGSAWSHPQFEK

[0370] Sequence ID 47 MAVDDQNRIVSTDGYEVVVTQEDTFIQGVPALGGSPFNREFFHNGRGTANLVGADAADAEGTTFQFGYQFAWAGSIDGAIGVTYSTPNILLNNVSLFPAFNPLGS VITPNLFPQAYAELRLTPAPGIEELVVAEGRFDGDFKSVQFSNVHGTASGVLGAVQVRPFVRAITANGDNVTTYGKPWTVGSGGENLYFQGSGSGSAWSHPQFEK

[0371] Sequence ID 48 MGLDNEKSASDRSGHQLTVQQWDTAVHAVPPMDKNRLTREWFYSGKAAYRVTGAGAETFSGTLEFGYQIGIPWTVGVGLNFTYTTPNILLNNVSLFPAFNPLGSVITPNLFPGASISTDIGNSPGVQELVTFAVPVSGHGGAVAVAKAHGTVTGVAGGIQVRPFARLTWPDHASITTYGDLTNVDGGSGGENLYFQGSGSGSAWSHPQFEK

[0372] Sequence ID 49 MGLDDELSLVDGQGRTLTVQQWDIFLDGVSPLDRNRLTREWFHSGQAKYTVSGPGAEDFEGTLILGYEVGFPWSLGVAIGFSYTTPNILLNNVSLFPAFNPLGSVITPNLFPGVNFSADLGNGPGVQEISTFEVDVSGSAGGVAVSKAHGTVTGAAGGVLLRPFARLVASTGDLVTTYGEPWNMNGSGGENLYFQGSGSGSAWSHPQFEK

[0373] Sequence ID 50 MGLDNQQSLVDGKGRTMTIQQWDTFLDGVFPLDRNRLTREWFHSGKAIYAVVGPGASDFAGTLELGYQVGFPWSLGVGINFSYTTPNILLNNVSIAPGAFNPLGSVITPNLFPGVSISSDLGNGPGI

[0374] Sequence ID 51 MGLDNELSLVDGQDRTLTVQQWDTFLNGVFPLDRNRLTREWFHSGRAKYIVAGPGADKFEGTLELGYQIGFPWSLGVGINFSYTTPNLLINNASIAPNLTPGSPFGPTVGTGFAPLGSIITPNLFPGVSISADLGNGPGIQEVATFSVDVAGPQGGVAVSNAHGTVTGAAGGVLLRPFARLISKSGDSVTTYGEPWNMN

[0375] Sequence ID 52 MGLDNELSLIDGRDRTLTIQQWDTFLNGVFPLDRNRLTREWFHSGRAKYIVAGPDAEEFEGTLELGYQIGFPWSLGVGINFSYTTPNILLNSAPFPPVAGQFITTPNLFPGVSISADLGNGPGIQEVATFSVDVAGPNGGVAVSNAHGTVTGAAGGVLLRPFARLIASTGDGLTTYGDPWNMN

[0376] Sequence ID 53 MGLDNELSLVDGKDRTLTIQQWDTFLNGVFPLDRNRLTREWFHSGKAKYIVSGPGADEFEGTLELGYQIGFPWSLGVGINFSYTTPNILLNNATPANPLQVITPNLFPGASISADLGNGPGIQEVATFSTDDVSGANGAVSNAHGTVTGAAGGVLLRPFARLIAKEGDSVTTYGEPWNMN

[0377] Sequence ID 54 MAVDDSNSVVDGGGNTITVSQADTFINSVFPLDGSPLTREWFHNGRAIVEVTGPDAADFEGDITLGYQFGYPASLGGELTFSYSTPQLQLNFNGGGTVSNILPQVGTTVTLEPGPGIEDVEVGTGSASGARTEIQISNVHGTATNIAGNVSVRPYVKVVSSNGDTAVTYGKPWRFN

[0378] Sequence ID 55 MAIDDQNRIVSTDGYEVVVTQENTVIQGVPALGGSPFNREFFHNGRGTTNLIGAGAADAEGTTFQFGYQFAWAGSIDGAIGVTYSTPGLNLSVGNGVAATVTNVLPQAYAELELTPAPGIEELVVAESTFDGDFKSVQLANVHGAASGVLGSVQVRPFVRAVTANGDNVTTYGKAWTI

[0379] Sequence ID 56 MQRLFLVAVMLLSGCLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQFKPYPASNFSTAVPQSATAMLVTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIMV EGSIIGYESNVKSGGVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGEVGYTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPES

[0380] Sequence ID 57 MKKGLCLAVILVLSLTGCSNFMDTPDAEEYPTLAPRGAIYKDLINLPLPKGKIMVSVYDFRDQTGQYKDYPSSTFATAVPQGGTSMLTSSLLDSKWFLPLEREGLQNLLTERKIIRAAQKKDEAPVNIGDDLPALKSANL VIEGGIIGYESDLKSGGHGIGYFGLATYGEYRMDQVTVNLRAVDVRTGQILLSVTTSKTIFSHALSGSVFRYIAYQDLLEMESGYTNNEPVNIAVMSAIDSAVIHMIIDGIQKGLWEPADKKQMESPVMKRYMQESTTIL

[0381] Sequence ID 58 MKAVISLLSVLLISACSTSLSVPDVDEAPQIMQRSSTYTDLLSLPAPKGRILVSVYDFRDQTGQYKSSPASSFSTAVPQGGTALLTTALLESNWFIPLEREGLQNLLTERKIIRAAQGKGETVNNHNNGLPSLNSANIM IEGGVVAYDWNIKTGGAGAKYLGISAAGEYRADQVTVNLRAVDVRSGRILSSVTISKTIYSHQLSMGAFRYIDYQELLEAELGYSNNEPVNIALMSAIDASIIHLIVDGVARGLWQPRDFDDLTKNKIYQKYSSQVREIL

[0382] Sequence ID 59 MKGLILLCASLVLGGCSYSLEIPETSASPKLMQRGSVYTDLTSLPPPVGKIMVSVYDFRDQTGQYKPSPNNSNFSTAVPQGGTSLTTALIDSKWFVPLEREGLQNLLTERKIIRAAQKKDTVVSNHGTDLSSLNSANVV IEGGIVAYDSNLRTGGAGARYLGVGGSGQYRTDQVTVNLRAVDVRTGRVLLSVTTSKTISSHEIGLGAFRFIDYEELLEVELGYSNNEPVNIAVMSAIDAAVIHLIVKGMERGMWSSNDPQAMSHPIIAKYSQATTEIL

[0383] Sequence ID 60 MRHLLIIASLFLLNGCLTAPPKQAAEPTLLPRSQSYQDLVQLPTPKGKIFVSVYNIQDETGQFKPYPASNFSTSVPQSATAMLISALKDSNWFIPLERQGLQNLLNERKIIRAAQENGRVAINNAQPLSSLVAANVLI EGSIIGYESNVKSGGIGARYFGIGGSTQYQLDQIAVNLRVVDVSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGEVGYTSNEPVMMCLMSAIETGVIYLINDGITRNLWQLQNPKDINTPVFERYKNLKVPTA

[0384] Sequence ID 61 MKGLFSIIVILIMTGCSASLDIPDADSEPKLMPRGTTYTDLISLPTPKGKILVSVYDFRDQTGQYKPYPNSTYSTAVPQGGTTLLTNSLLDSQWFIPLEREGLQNLLTERKIIRAAQKKETKISNHGSNLSSLNSANVV IEGGIVAYDSNIKTGGLGAKYLGIGGSGQYRTDQVTVSLRAIDVRTGQVLLSVTTSKTISSHEIGLGAFRFIDYQELLEVELGYSNNEPVNIAVMSAIDAAVIHLIVKGMSLGMWQSNDPNVESNPIIAKYSQATREIL

[0385] Sequence ID 62 MRAMILIIAVLLGGCSITEVPKEAAKPTLMPRASTYKDLVALPKPNGKIIVSVYSVQDETGQFKPLPASNFSTAVPQSGNAMLTSALKDSGWFVPLEREGLQNLLNERKIIRAAQENGTVAANNQQPLPSLLSANVVI EGAIIGYDSDIKTGGAGARYFGIGADGKYRVDQVAVNLRAVDVRTGEVLLSVNTSKTILSSELSAGVFRFIEYQRLLELEAGYTTNEPVMMCMMSALEAGVAHLIVEGIRQNLWSLQNPSDINNPIIQRYMKEDVPLAI

[0386] Sequence ID 63 MKRLFLFIAVIMVAGCSNSLSIPDTDEAPKLMQRGSTYQDLIHLPDPKGKLYVSIYDFRDQTGQYKPQPNSNFSTAVPQGAISLLIMSLIDSKWFVPLEREGLQNLLTERKIIRAAQSSSKGQANIASLRSANVMIE GGIVAYDTNIKTGGAGARYLGVGASTQYRTDQVTVSLRVVDVSSGAILSSVTTSKTIFSQEMQTGAFRFIDYKDLLEVELGYTNNEPVNIALMSAIDAAVIYLVVQGIDQGLWQAGTSDNINNKIYKKYSQNKAEIL

[0387] Sequence ID 64 MKGLISIGLVLLLSGCAYSLDIPDTDASPKLMPRGATYTDLVSLPKPAGKILVSVYDFRDQTGQYKPQPNSNFSTAVPQGGTSLLTTSLLDSQWFVPLEREGLQNLLTERKIIRAAQKKDKVISNHGADLSSLNSANVV IEGGIVAYDSNIRTGGLGAKYLGIGASGQYRTDQVTVNLRAVDVRSGQVLLSITTSKTISSHEMGLGAFRFIDYKELLEVEMGYSNNEPVNIAVMSAIDAAVIHLIVKGMERGMWSASDPQAMSNPIIARYSQAETEIL

[0388] Sequence ID 65 MQRLFLVAVMLLSGCLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQYKDYPSSTFATAVPQSATAMLVTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIMV EGSIIGYESNVKSGGVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGEVGYTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPES

[0389] Sequence ID 66 MQRLFLVAVMLLSGCLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQYKSSPASSFSTAVPQSATAMLVTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIMV EGSIIGYESNVKSGGVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGEVGYTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPES

[0390] Sequence ID 67 MQRLFLVAVMLLSGCLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQYKPSPNSNFSTAVPQSATAMLVTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIMV EGSIIGYESNVKSGGVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGEVGYTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPES

[0391] Sequence ID 68 MQRLFLVAVMLLSGCLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQFKPYPASNFSTSVPQSATAMLVTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIMV EGSIIGYESNVKSGGVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGEVGYTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPES

[0392] Sequence ID 69 MQRLFLVAVMLLSGCLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQYKPYPNSTYSTAVPQSATAMLVTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIMV EGSIIGYESNVKSGGVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGEVGYTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPES

[0393] Sequence ID 70 MQRLFLVAVMLLSGCLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQFKPLPASNFSTAVPQSATAMLVTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIMV EGSIIGYESNVKSGGVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGEVGYTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPES

[0394] Sequence ID 71 MQRLFLVAVMLLSGCLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQYKPQPNSNFSTAVQSATAMLVTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIMV EGSIIGYESNVKSGGVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGEVGYTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPES

[0395] Sequence ID 72 MQRLFLVAVMLLSGCLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDQTGQYKPQPNSNFSTAVPQSATAMLVTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIMVEGSII GYESNVKSGGVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGEVGYTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPESAWSHPQFEK

[0396] Sequence ID 73 MKTGLMLCVALLAGCSNSMSIPDADESPTLTPRGPTYNDLVKLPLPKGKIMVSVYDFRDQTGQYKSPNSFSTAVPQGGTSMLTTALLDSGWFLPLEREGLQNLLTERKIIRAAQKKDQTPANIGDDLPALKSANLV IEGGIIGYESDLKTGGHGAGYLGFAAYGQYRMDQVTVNLRAVDVRTGQIVLSVTTSKTIFSQEVSASVFRYIAYQDLLELESGYTNNEPVNIAIMSAIDSAVIHMVVDGIKRGLWQPADEAQLKNPIIQRYSDETVAIL

[0397] Sequence ID 74 MKLIISCILVLVMTGCSNSMGIPEADSAPTLMPRGATYQDLVRLPEPKGKILVSVYDFRDQTGQYKAQPNSNFSTAVPQGGTALLTTSLLDSRWFIPLEREGLQNLLTERKIIRAAQKKGEGASNHGDDLSSLSSSANVV IEGGIIAYDSNIRTGGLGARYLGVGSSGEYRADQVTVNLRAVNVRTGQILLSITTSKTIFSHQISAGAFRFVDYKDLLEIEMGYSNNEPVNIAVMSAIDAAVIHLVVKGMERGMWQPAATEGEGFDVIERYAAQTQVIL

[0398] Sequence ID 75 MNRLFLLVTLLVLAGCSNSLSVPESDEAPRLMPRGATYSDLIALPKPKGRILVSVYDFRDQTGQYKSSPNSNFSTAVPQGGTALLTTSLLDSNWFTPLEREGLQNLLTERKIIRAAQKKDQSVSNHGADLPSLSSANVV IEGGIVAYDSNVKTGGFGARYLGIGGATEYRSDMVTVNLRVVDVRTGQILLSVTTSKTILSMQVTGDVFRFVDYKDLLEVEAGYTNNEPVNVAVMSAIDASVIHLVIEGIERGMWQPANLEELNSPIIERYAKEKHHIL

[0399] Sequence ID 76 MQRLFLVAVMLLSGCLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQYKSSPNSFSTAVPQSATAMLVTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIMV EGSIIGYESNVKSGGVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGEVGYTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPES

[0400] Sequence ID 77 MQRLFLVAVMLLSGCLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQYKAQPNSNFSTAVQSATAMLVTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIMV EGSIIGYESNVKSGGVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGEVGYTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPES

[0401] Sequence ID 78 MQRLFLVAVMLLSGCLTAPPKEAARPTLMPRAQSYKDLTHLPAPTGKIFVSVYNIQDETGQYKSPNSNFSTAVPQSATAMLVTALKDSRWFIPLERQGLQNLLNERKIIRAAQENGTVAINNRIPLQSLTAANIMV EGSIIGYESNVKSGGVGARYFGIGADTQYQLDQIAVNLRVVNVSTGEILSSVNTSKTILSYEVQAGVFRFIDYQRLLEGEVGYTSNEPVMLCLMSAIETGVIFLINDGIDRGLWDLQNKAERQNDILVKYRHMSVPPES

[0402] The following examples illustrate the present invention. While specific embodiments, specific configurations, and materials and / or molecules have been discussed herein regarding manipulated 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 invention. The following examples are provided to better illustrate specific embodiments and should not be considered as limiting this application. This application is limited solely by the claims. [Examples]

[0403] Detailed methods for preparing and testing pores are described in WO2016 / 034591, WO2017 / 149316, WO2017 / 149317, WO2017 / 149318, WO2018 / 211241, WO2019 / 002893, PCT / EP2023 / 059821, PCT / EP2023 / 072113, PCT / EP2023 / 072065, PCT / EP2023 / 072106, and PCT / EP2023 / 072068 (all incorporated herein in their entirety by reference).

[0404] Example 1 - PORARC Chimera Chimeric pore monomer Two types of chimeras were designed: (1) stenosis graft and (2) cap graft (also called scaffold graft).

[0405] For (1), a different constriction region was transplanted into the cap region (or scaffold) of PorARc derived from Rhodococcus corynebacteroides (PorARc_Rco; SEQ ID NO: 1). The chimeras were named first by the cap region (or scaffold) and then by the constriction region. For example, PorARc_Rco_Mph contained the cap region (or scaffold) derived from PorARc_Rco and the constriction portion derived from the PorARc pore of Mycolicibacterium phlei (PorARc_Mph; SEQ ID NO: 2).

[0406] (2) involves transplanting a different cap region (or scaffold) around the constriction region derived from PorARc_Mph (sequence number 2). The same naming convention was applied. For example, PorARc_Gcr_Mph is a result of transplanting the cap region (or scaffold) of PorARc derived from Gordonia crocea around the constriction region derived from PorARc_Mph (sequence number 2).

[0407] E. coli pore production Chemically competent E. coli cells were transformed with recombinant expression vectors encoding chimeric pore monomers and PorARc pores, each possessing a C-terminal Strep affinity tag and an ampicillin resistance gene. The cells were plated onto LB agar plates containing appropriate antibiotics for selection. Single colonies from the agar plates were inoculated into LB medium containing antibiotics and grown overnight. The cultures were diluted with LB medium and the required antibiotics and incubated at 37°C for 6.5 hours. After incubation at 37°C, glucose was added and the temperature was reduced to 18°C. After incubation at 18°C ​​for 1 hour, lactose was added and incubated for a further 16 hours at 18°C. Cells were harvested by centrifugation before lysis and extracted with 1x Bugbuster extraction reagent (Merck 70921) and 0.1% DDM. Chimeric pore monomers were purified from the supernatant using affinity chromatography and ion-exchange chromatography, and oligomeric nanopores were selected by SDS-PAGE.

[0408] Irregular curves of DNA (i.e., DNA transposition current traces) For comparison, PorARc (PorARc_Rco; SEQ ID NO: 1) derived from Rhodococcus corynebacteroides was tested. PorARc pores (PorARc_Mph; SEQ ID NO: 2) derived from Mycolicibacterium phlei were also tested. The tested chimeric pores are shown in the table below. All tested pores were homooligomeric.

[0409] Electrical measurements were obtained from pores inserted into a MinION flow cell. After inserting a single pore into a block copolymer membrane, any excess nanopores were removed by passing 1 mL of buffer containing 25 mM potassium phosphate, 150 mM potassium(II) ferrocyanide, 150 mM potassium(III) ferricyanide, and pH 8.0 through the system.

[0410] The analyte used to evaluate the irregular curve of DNA was a 3.6 kilobase DNA section from the 3' end of the lambda genome. Analyte preparation, ligation of the analyte to a Y adapter, SPRI-bead cleanup of the ligated analyte, and addition to a minION flow cell were performed using the Oxford Nanopore Technologies Q-SQK-LSK109 protocol.

[0411] Electrical measurements were acquired using the minION Mk1b from Oxford Nanopore Technologies. A standard sequencing script was run at -180mV for 2–6 hours, with static flicking every 5 minutes to remove extended nanopore blocks. Raw data were collected in bulk FAST5 files using MinKNOW software (Oxford Nanopore Technologies). The median number of DNA strands used to calculate the metrics shown in Figures 7 and 8 was 1990.

[0412] Irregular curves of peptide-DNA conjugates (i.e., peptide-DNA transposition current traces) An example of current-versus-time tracing of peptide rearrangement through a nanopore was obtained using a conjugate containing a polypeptide sandwiched between two polynucleotides: a dsDNA Y adapter (DNA1) and a dsDNA tail (DNA2). A polynucleotide handling protein on the cis side of the nanopore controls the movement of the conjugate by first unwinding DNA1, rearranging the 5'-3' on the ssDNA, then sliding across the polypeptide portion, and finally unwinding the DNA2 segment. As this construct moves from the cis side to the trans side of the nanopore, the DNA and polypeptide portions can be visualized in current-versus-time plots.

[0413] The adapter used was from the Oxford Nanopore Technologies Q-SQK-LSK109 kit mentioned above. The DNA tail was created by annealing two DNA oligonucleotides and also includes a side arm for tethering, resulting in two tethering sites per construct, which enhances capture efficiency.

[0414] Polypeptide analytes were obtained using an ethyldiamine spacer along the peptide backbone, resulting in analytes with azide moieties immediately after the N-terminus and C-terminus. Each analyte was then conjugated to a Y-adapter and DNA tail by a copper-free click reaction between the azide and the BCN (bicyclo[6.1.0]nonine) moiety. The samples were purified using Agencourt AMPure XP (Beckman Coulter) beads, washed twice with 28% PEG 8K, 2.5M NaCl, 25mM Tris (pH 8.0) buffer, and eluted in 10mM Tris-Cl, 50mM NaCl (pH 8.0).

[0415] Electroanalysis was performed using Oxford Nanopore Technologies' MinION Mk1b and a custom MinION flow cell with inserted pores. The flow cell was washed with a tether mix containing 50 nM DNA tether and ATP-deficient SQB buffer. An initial 800 μL of the tether mix was added for 5 minutes, followed by an additional 200 μL of the mix flowing through the system with the SpotON port open. DNA-peptide constructs were prepared to a 0.5 nM concentration in a buffer similar to SQB from the Oxford Nanopore Technologies sequencing kit (SQK-LSK109), but with ATP depleted, and prepared in LB from the Oxford Nanopore Technologies sequencing kit (SQK-LSK109) to obtain a "sequencing mix." 75 μL of the sequencing mix was added to the MinION flow cell via the SpotON flow cell port. This mixture was incubated on the flow cell for 5–10 minutes to tether the construct and subsequently capture it by the nanopores. In the absence of ATP, the DNA motor remains stationary in the spacer region of the Y-adapter, and the conjugate is trapped in the nanopore, but no rearrangement occurs. After incubation, 200 μL of SQB from the Oxford Nanopore Technologies sequencing kit (SQK-LSK109) is added, and in the presence of ATP, the trapped DNA-peptide conjugate moves along the nanopore by helicase, yielding a reproducible current footprint.

[0416] Standard sequencing scripts were run at -180mV for 1–6 hours, with static flicking every minute to remove extended nanopore blocks. Raw data were collected in bulk FAST5 files using MinKNOW software (Oxford Nanopore Technologies).

[0417] result The results are shown in Figures 3-6. The results for all the chimeric pores tested, as shown in Figures 7 and 8, are summarized in Tables 5 and 6 below. The last two columns compare the chimeric pores of PorARc_Rco (ONLP21293) and PorARc_Mph (ONLP21323). Table 5 - Stenosis grafts [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4] Table 6 - Cap grafts (also called scaffold grafts) [Table 6-1] [Table 6-2] [Table 6-3]

[0418] Example 2 - CSGG Chimera Various CsgG stenosis transplant chimeras were created and tested in the same manner as in Example 1 above. The chimeras are summarized in Table 7 below. Each chimeric pore contains the cap region and transmembrane beta-barrel region (collectively called the scaffold) of CsgG_Eco_WT, which includes the stenosis of a different CsgG pore from a different species. For example, CsgG-Eco-Vdi contains the cap region and transmembrane beta-barrel region (or scaffold) of CsgG_Eco_WT, which includes the stenosis of CsgG_Vdi_WT. The pores from which the stenosis originates are summarized in Table 4 above. Table 7 - CsgG stenosis, transplanted chimeric pores [Table 7] Table 8-Table 7: Sequence identity of pores (calculated including signal peptides) [Table 8] Sequence identity of the perfectly homologous pores that are the source of the constricted portion of the chimera relative to A=CsgG-Eco-WT Sequence identity of all chimeras relative to B=CsgG-Eco-WT. Sequence identity of the chimeric form relative to C=CsgG-Eco-WT (constriction region only, E44-A59). Sequence identity of the chimera relative to D=CsgG-Eco-WT (constriction region only, F48-A59). E = Sequence identity of the chimera with respect to CsgG-Eco-WT (constriction region only, V38-S63). E indicates the sequence identity of the constriction region in Table 4 with respect to the constriction region of CsgG-Eco-WT in Table 4.

[0419] Representative electrophysiological results for CsgG-Eco-Vmae are shown in Figure 9. Similar traces were obtained for all pores in Table 7 (data not shown). The results for all chimeric pores tested in Figure 10 are summarized in Table 9 below. The last column compares the chimeric pores with CsgG-Eco-WT. Summary of data from Table 9 to Figure 11 [Table 9]

[0420] Example 3 - Other CSGG Chimeras Various CsgG stenosis transplant chimeras were created and tested in the same manner as in Examples 1 and 2 above. The chimeras are summarized in Table 10 below. Each chimeric pore contains the cap region and transmembrane beta-barrel region (collectively called the scaffold) of CsgG_Eco_WT, along with the stenosis of a different CsgG pore from a different species. For example, CsgG-Eco-Vfu contains the cap region and transmembrane beta-barrel region (or scaffold) of CsgG_Eco_WT, along with the stenosis of CsgG_Vfu_WT. The pores from which the stenosis originates are summarized in Table 4 above. Table 10 - CsgG stenosis, transplanted chimeric pores [Table 10] Table 11-Table 10: Sequence identity of pores (calculated including signal peptides) [Table 11] Sequence identity of the perfectly homologous pores that are the source of the constricted portion of the chimera relative to A=CsgG-Eco-WT Sequence identity of all chimeras relative to B=CsgG-Eco-WT. Sequence identity of the chimeric form relative to C=CsgG-Eco-WT (constriction region only, E44-A59). Sequence identity of the chimera relative to D=CsgG-Eco-WT (constriction region only, F48-A59). E = Sequence identity of the chimera relative to CsgG-Eco-WT (constriction region only: V38-S63). E indicates the sequence identity of the constriction region in Table 4 relative to the constriction region of CsgG-Eco-WT in Table 4.

[0421] Representative electrophysiological results for CsgG-Eco-Vfu are shown in Figure 13. Similar traces were obtained for all pores in Table 11 (data not shown). The results for the three tested chimeric pores (the last three from left to right) are summarized in Table 12 below. The last column compares the chimeric pores with CsgG-Eco-WT.

[0422] Summary of data from Table 12 to Figure 14 Table 12

Claims

1. A chimeric pore monomer comprising two or more regions, wherein at least two of the two or more regions are derived from at least two different pores, and the at least two different pores do not contain alpha-hemolysin and gamma-hemolysin.

2. The chimeric pore monomer according to claim 1, wherein the chimeric pore monomer comprises two or three regions derived from two different pores, the two different pores being not alpha-hemolysin and gamma-hemolysin.

3. The chimeric pore monomer according to claim 1 or 2, wherein the chimeric pore monomer comprises a sequence that is identical to at least two different pores or the wild-type monomer sequences of the two different pores by about 99.7% or less, or about 96% or less.

4. The at least two different pores, or the two different pores, contain Wza, iota toxin, anthrax protective antigen, cholera cytolysin, cytotoxin K (CytK), CELIII, CsgG, aerolidine, alpha-hemolysin, InvG, GspD, MspA, MspB, MspC, PorARr, PorBRr, PorARc, PilQ, necrotizing enterocolitis B-like toxin (NetB), FraC, portal proteins (G20c, P23_45, T4, SPP1, P22, Phi29), gamma-hemolysin, monalisin, lysenin, ClyA, Clostridium perfringens beta toxin, parasporin-2, epsilon toxin, and the parasitic mushroom Laetiporus A chimeric pore monomer according to any one of claims 1 to 3, selected from lectins derived from Sulphureus (LSL), Volvatoxin, Cry toxin, Cyt1Aa, and Cyt2Aa.

5. The chimeric pore monomer according to claim 4, wherein the PorARc is selected from the pores in Table 2.

6. The chimeric pore monomer according to claim 4 or 5, wherein the at least two different pores include at least two different PorARc pores, or the two different pores are two different PorARc pores.

7. The chimeric monomer according to any one of claims 4 to 6, wherein one of the at least two different pores is PorARc_Rco or PorARc_Mph, or one of the two different pores is PorARc_Rco or PorARc_Mph.

8. The chimeric pore monomer according to any one of claims 4 to 7, wherein the at least two different pores include (a) PorARc_Rco or PorARc_Mph and (b) one of the pores in Table 2, or the two different pores are (a) PorARc_Rco or PorARc_Mph and (b) one of the pores in Table 2.

9. The chimeric pore monomer according to claim 8, wherein the at least two different pores contain PorArc_Rco and PorArc_Rco_Mph, or the two different pores are PorArc_Rco and PorArc_Rco_Mph.

10. The chimeric pore monomer according to any one of claims 1 to 4, wherein the at least two different pores comprise (a) PorARc_Rco and MspA, (b) two different CsgG pores, three different CsgG pores, or five different CsgG pores, (c) alpha-hemolysin and CytK, or (d) NetB and CytK, or the two different pores comprise (a) PorARc_Rco and MspA, (b) two different CsgG pores, three different CsgG pores, or five different CsgG pores, (c) alpha-hemolysin and CytK, or (d) NetB and CytK.

11. The chimeric pore monomer according to claim 4 or 10, wherein the CsgG pores are selected from the CsgG pores in Table 4, or the two different CsgG pores, the three different CsgG pores, or the five different CsgG pores are selected from the CsgG pores in Table 4.

12. The chimeric pore monomer according to any one of claims 1 to 11, wherein at least two of the regions include a cap region and a constricted region, or the two regions are a cap region and a constricted region, or at least two of the regions include a cap region, a constricted region and a transmembrane region, or the three regions are a cap region, a constricted region and a transmembrane region.

13. The chimeric pore monomer according to claim 12, wherein the chimeric pore monomer comprises a sequence having at least about 20% or at least about 40% identity with the sequence shown in any one of sequence numbers 3 to 49.

14. The chimeric pore monomer according to claim 12, wherein the chimeric pore monomer includes a sequence having at least about 20% identity with the sequence shown in any one of sequence numbers 65-72 and 76-78.

15. A chimeric structure comprising two or more covalently attached chimeric pore monomers according to any one of claims 1 to 14.

16. A chimeric pore comprising at least one chimeric pore monomer according to any one of claims 1 to 14 or at least one construct according to claim 15.

17. A chimeric pore multimer comprising two or more pores, wherein at least one of the pores is the chimeric pore described in claim 16.

18. A PorARc pore monomer comprising a sequence having at least approximately 88% identity with the sequence shown in Sequence ID No. 2, or a sequence having at least approximately 20% or at least approximately 40% identity with the sequence shown in Sequence ID No. 50, 51, 52, 53, 54, or 55.

19. A PorARc construct comprising two or more covalently attached PorARc pore monomers as described in claim 18.

20. A PoRc pore comprising at least one PoRc pore monomer as described in claim 18, or at least one construct as described in claim 19.

21. A PoRc pore multimer comprising two or more pores, wherein at least one of the pores is the PoRc pore described in claim 20.

22. A chimeric pore according to claim 16, a chimeric pore multimer according to claim 17, a PorARc pore according to claim 20, or a PorARc pore multimer according to claim 22, contained in the membrane.

23. A membrane comprising the chimeric pores described in claim 16, the chimeric pore multimer described in claim 17, the PorARc pores described in claim 20, or the PorARc pore multimer described in claim 21.

24. A method for producing a chimeric pore monomer according to any one of claims 1 to 14, comprising attaching at least two regions derived from at least two different pores.

25. A method for determining the presence or absence of a target analyte, or one or more of its characteristics, (i) (a) a chimeric pore comprising two or more regions wherein at least two of the two or more regions are derived from at least two different pores, (b) a chimeric pore multimer comprising two or more pores wherein at least one pore is a chimeric pore as defined in (a), (c) a PorArc pore according to claim 20, or (d) a PorArc pore multimer according to claim 21, a step of contacting a target analyte with the multimer. (ii) A method comprising the step of obtaining one or more measurements as the target analyte moves toward the pore or pore multimer, thereby determining the presence, absence, or one or more features of the target analyte.

26. The method according to claim 25, wherein the target analyte includes metal ions, inorganic salts, polymers, amino acids, peptides, polypeptides, proteins, nucleotides, oligonucleotides, polynucleotides, polynucleotide-polypeptide conjugates, monosaccharides, oligosaccharides, polysaccharides, dyes, bleaching agents, pharmaceuticals, diagnostic agents, recreational drugs, explosives, toxic compounds, environmental pollutants, or metabolites.

27. The method according to claim 26, wherein the target analyte comprises a polynucleotide.

28. The method according to claim 26, wherein the target analyte comprises a peptide.

29. A method for characterizing a target analyte using: (a) a chimeric pore comprising two or more regions wherein at least two of the two or more regions originate from at least two different pores; (b) a chimeric pore multimer comprising two or more pores wherein at least one pore is a chimeric pore as defined in (a); (c) the PorARc pore according to claim 20; or (d) the PorARc pore multimer according to claim 21.

30. Use of (a) a chimeric pore comprising two or more regions wherein at least two of the two or more regions are derived from at least two different pores, (b) a chimeric pore multimer comprising two or more pores wherein at least one pore is a chimeric pore as defined in (a), (c) a PorARc pore according to claim 20, or (d) a PorARc pore multimer according to claim 21, for determining the presence or absence of a target analyte or one or more characteristics.

31. A kit for characterizing target polynucleotides, - (a) a chimeric pore comprising two or more regions wherein at least two of the two or more regions are derived from at least two different pores, (b) a chimeric pore multimer comprising two or more pores wherein at least one pore is a chimeric pore as defined in (a), (c) the PorARc pore according to claim 20, or (d) the PorARc pore multimer according to claim 21, - A kit containing polynucleotide-binding proteins.

32. A device for characterizing target polynucleotides in a sample, - (a) a plurality of chimeric pores comprising two or more regions, wherein at least two of the two or more regions are derived from at least two different pores; (b) a plurality of chimeric pore multimers comprising two or more pores, wherein at least one pore is a chimeric pore as defined in (a); (c) a plurality of PorArc pores according to claim 20; or (d) a plurality of PorArc pore multimers according to claim 21. - A device containing multiple polynucleotide-binding proteins.

33. The method according to any one of claims 25 to 29, the use according to claim 30, the kit according to claim 31, or the apparatus according to claim 32, wherein the at least two different pores are as defined in any one of claims 4 to 12.

34. The method according to any one of claims 25 to 29, the use according to claim 30, the kit according to claim 31, or the apparatus according to claim 32, wherein the chimeric pore is the chimeric pore according to claim 16, the plurality of chimeric pores is the plurality of chimeric pores according to claim 16, the chimeric pore multimer is the chimeric pore multimer according to claim 17, or the plurality of chimeric pore multimers is the plurality of chimeric pore multimers according to claim 17.

35. A polynucleotide encoding a chimeric pore monomer according to any one of claims 1 to 14, a chimeric construct according to claim 15, a PorARc pore monomer according to claim 18, or a PorARc construct according to claim 19.

36. A kit for characterizing a target analyte, comprising (a) the chimeric pore according to claim 16, the chimeric pore multimer according to claim 17, the PorARc pore according to claim 20, or the PorARc pore multimer according to claim 21, and (b) a membrane component.

37. An array comprising multiple membranes according to claim 23.

38. A system comprising: (a) a film according to claim 23 or an array according to claim 37; (b) means for applying a potential across the film(s); and (c) means for detecting an electrical signal or an optical signal across the film(s).

39. An apparatus comprising, inserted into an in vitro membrane, a chimeric pore according to claim 16, a chimeric pore multimer according to claim 17, a PorARc pore according to claim 20, or a PorARc pore multimer according to claim 21.

40. An apparatus manufactured by a method comprising: (i) obtaining a chimeric pore according to claim 16, a chimeric pore multimer according to claim 17, a PorARc pore according to claim 20, or a PorARc pore multimer according to claim 21; and (ii) contacting the chimeric pore or the pore multimer with an in vitro membrane so that the chimeric pore or the pore multimer is inserted into the in vitro membrane.

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