Mutant CSGG pore

Abstract

The present invention relates to a mutated form of lipoprotein CsgG, in particular the modification at one or more positions in Tyr51, Asn55, and Phe56, which is located in an external mode. The invention also relates to analyte detection and characterization using said mutant CsgG.

Description

[0001] This patent application is a divisional application of the patent application with application number 201580059505.8, application date September 1, 2015, and invention name “Mutated CSGG Pore”. Technical Field

[0002] The present invention relates to a novel protein pore and its use, in particular to a biological nanopore in nucleic acid sequencing applications and molecular sensing.

[0003] The present invention relates to mutant forms of CsgG. The present invention also relates to the detection and characterization of analytes using CsgG. Background Art

[0004] Protein pores are transmembrane polypeptides and complexes that form channels in the membrane through which ions and certain molecules can pass. The minimum diameter of the channel is typically in the nanometer range (10 -9 meters), and therefore some of these polypeptides are named "nanopores".

[0005] Nanopores have great potential as biosensors. When a potential is applied to a nanopore bound to a membrane, ions flow through the channel. This ion flow can be measured as an electric current. For example, WO 2000 / 28312 and D. Stoddart et al., Proc. Natl. Acad. Sci., 2010, 106, 7702-7 describe suitable electrical detection techniques using single-channel recording devices. For example, WO 2009 / 077734 describes multi-channel recording techniques.

[0006] Molecules that pass through the pore, or that bind to or are close to the pore, act to block and thereby reduce the flow of ions through the channel. The degree of reduction in ion flow indicates the size of the blockage in or near the pore, and the degree of reduction in ion flow is measured by a drop in current. The measured current can therefore be used to measure the size or degree of channel blockage. Changes in current can be used to identify that a molecule or portion of a molecule has bound in or near the pore (molecular sensing) or in a specific system, which can be used to determine the identity of the molecule located in the pore based on its size (nucleic acid sequencing).

[0007] "Strand sequencing" is a well-known method for sequencing nucleic acids using biological nanopores. Single-stranded polynucleotides are passed through a nanopore. As they transiently pass through the nanopore channel, the bases of each nucleotide are determined by detecting changes in the current. This method significantly reduces the time and cost of traditional nucleic acid sequencing methods.

[0008] Previously reported protein nanopores, such as mutant MspA (Manrao et al., Nature Biotechnology, 2012, 30(4), 349-353) and α-hemolysin nanopores (Nat. Nanotechnol., 2009, 4(4), 265-70), have been used for nucleic acid sequencing using the “strand sequencing” method. Similarly, other pores such as α-hemolysin (J Am Chem Soc, 2012, 134(5), 2781-7) and ClyA (Am. Chem. Soc. Nano. 2014, 8(12), 12826-35) (J. Am. Chem. Soc, 2013, 135(36), 13456-63) have also been used for protein sensing.

[0009] There remains a need for new nanopores that overcome the shortcomings of existing technologies, especially to optimize the dimensions and characteristics of the pores for molecular sensing applications and applications such as nucleic acid sequencing.

[0010] Nanopore sensing is a sensing method that relies on observing the interaction between individual binding or analyte molecules and receptors. Nanopore sensors can be created by placing a single nanometer-sized pore in an insulating membrane and measuring voltage-driven ion transport through the pore in the presence of analyte molecules. Analyte identification is indicated by its unique current signal, the significant duration and range of current blocks, and changes in current levels.

[0011] There is a need for a fast, inexpensive, and widely applicable technology for sequencing nucleic acids (e.g., DNA or RNA). Existing technologies are slow and expensive primarily because they rely on amplification techniques to produce large quantities of nucleic acids and require large quantities of specialized fluorescent chemicals for signal detection. Nanopore sensing has the potential to provide a fast and inexpensive method for nucleic acid sequencing by reducing the amount of nucleotides and reagents required.

[0012] Two essential components of nucleic acid sequencing using nanopore sensing are (1) controlled movement of nucleic acids through the pore; and (2) recognition of nucleotides as nucleic acid polymers move through the pore. Previously, to achieve nucleotide recognition, nucleic acids were passed through mutants of hemolysin. This provided a current signal that was shown to be sequence-dependent. This also suggests that when using hemolysin pores, a large number of nucleotides contribute to the observed current, making the direct relationship between the observed current and the polynucleotide more intriguing.

[0013] While mutations in the hemolysin pore have improved the current range used to identify nucleotides, sequencing systems would benefit from further improvements in the current variability between nucleotides. Furthermore, it has been observed that certain current states exhibit high variability as nucleic acids move through the pore. It has also been shown that certain mutant hemolysin pores exhibit higher variability than others. While these state variations can contain sequence-specific information, it would be desirable to create pores with reduced variability to simplify the system. It would also be desirable to reduce the number of nucleotides that contribute to the observed current. Summary of the Invention

[0014] The inventors have identified the structure of the bacterial amyloid secretion channel, CsgG. The CsgG channel is a transmembrane oligomeric protein that forms a channel with a minimum diameter of approximately 0.9 nm. The structure of the CsgG nanopore makes it suitable for protein sensing applications, particularly nucleic acid sequencing. Modified variants of the CsgG polypeptide can be used to further enhance the channel's suitability for these specific applications.

[0015] The CsgG pore offers advantages over existing protein pores such as ClyA or α-hemolysin because its structure is more suited for DNA sequencing applications. The CsgG pore has a more favorable aspect ratio, including a shorter transmembrane channel than the ClyA pore. The CsgG pore has a wider channel opening than the α-hemolysin pore. For certain applications, this can facilitate enzyme attachment, such as for nucleic acid sequencing applications. In these embodiments, it can also minimize the length of the nucleic acid segment between the enzyme and the read head (defined as the narrowest part of the pore), leading to an improved readout signal. In embodiments of the invention related to nucleic acid sequencing, a narrow internal constriction in the channel of the CsgG pore also facilitates translocation of single-stranded DNA. The constriction is composed of two annular loops formed by the juxtaposition of a tyrosine residue at position 51 (Tyr 51) and a phenylalanine residue and an asparagine residue at positions 56 and 55 (Phe 56 and Asn 55), respectively, in adjacent protein monomers. The dimensions of the constriction can be modified. ClyA has a wider internal constriction that currently prevents double-stranded DNA from passing through for sequencing. The α-hemolysin pore has not only a 1.3 nm wide internal constriction but also a 2 nm wide β-barrel structure that features an additional read head.

[0016] In a first aspect, the present invention relates to a method for molecular sensing comprising:

[0017] a) providing a CsgG biological pore formed by at least one CsgG monomer within an insulating layer;

[0018] b) applying an electric potential across the insulating layer, thereby establishing a current through the biological pore;

[0019] c) contacting the CsgG biological pore with a test substrate; and

[0020] d) measuring the current flowing through the biological pore.

[0021] Typically, the insulating layer is a membrane, such as a phospholipid bilayer. In one embodiment, the current through the pore is carried by a flow of soluble ions from a first side of the insulating layer to a second side of the insulating layer.

[0022] In one embodiment of the invention, the molecular sensing is analyte detection. In a specific embodiment, the method of analyte detection comprises, after step (d), the further step of determining the presence of the test substrate by a decrease in the current through the biological pore compared to the current through the biological pore in the absence of the test substrate.

[0023] In an alternative embodiment of the present invention, the molecular sensing is nucleic acid sequencing. Typically, the nucleic acid sequenced by the method is DNA or RNA. In a specific embodiment of the present invention, the CsgG biological pore is adapted to accommodate additional accessory proteins. Typically, the additional accessory proteins are nucleic acid processing enzymes selected from the group consisting of: DNA or RNA polymerases; isomerases; topoisomerases; gyrases; telomerases; exonucleases; and helicases.

[0024] In embodiments of the present invention, the CsgG biological pore is a modified CsgG pore, wherein the modified CsgG pore comprises at least one modification to a wild-type E. coli CsgG polypeptide sequence in at least one CsgG monomer of the CsgG pore. Typically, the same modification is made to all CsgG monomers forming the CsgG pore. In specific embodiments of the present invention, the modified CsgG monomer comprises a polypeptide sequence from positions 38 to 63 according to SEQ ID NOs: 4 to 388.

[0025] In a second aspect, the present invention relates to a modified CsgG biological pore comprising at least one CsgG monomer, wherein the modified CsgG biological pore has no more than one channel constriction, wherein the constriction has a diameter ranging from 0.5 nm to 1.5 nm. Typically, the modification is between positions 38 and 63 of the CsgG monomer polypeptide sequence. Suitably, the modification is at a position selected from the group consisting of: Tyr51; Asn55; and Phe56. In specific embodiments, the modification is at position Tyr51, or at both Asn55 and Phe56.

[0026] In embodiments of the invention, the modification of the CsgG monomer is selected from the group consisting of: substitution of a naturally occurring amino acid; deletion of a naturally occurring amino acid; and modification of a naturally occurring amino acid side chain. Suitably, the modification reduces or removes steric hindrance of the unmodified amino acid. In specific embodiments, at least one CsgG monomer of the pore has a polypeptide sequence according to positions 38 to 63 of SEQ ID NOs 4 to 388.

[0027] In a third aspect, the present invention relates to an isolated polypeptide encoding at least one CsgG monomer of the modified CsgG biological pore of the second aspect of the invention.

[0028] In a fourth aspect, the present invention relates to an isolated nucleic acid encoding the isolated polypeptide of the third aspect of the invention.

[0029] In a fifth aspect, the present invention relates to a biosensor comprising:

[0030] a) insulation layer;

[0031] b) a CsgG biological pore in the insulating layer; and

[0032] c) A device for measuring the current passing through the pore of said organism.

[0033] In a specific embodiment, the CsgG biological pore in the biosensor is a modified CsgG biological pore according to the second aspect of the present invention.

[0034] In a sixth aspect, the present invention relates to the use of a CsgG biological pore for biosensing applications, wherein the biosensing application is analyte detection or nucleic acid sequencing.

[0035] In one embodiment of the sixth aspect of the present invention, the nucleic acid sequencing is DNA sequencing or RNA sequencing.

[0036] The inventors have unexpectedly demonstrated that CsgG and novel mutants thereof can be used to characterize analytes, such as polynucleotides. The present invention relates to mutant CsgG monomers in which one or more modifications have been made to enhance the monomer's ability to interact with analytes, such as polynucleotides. The inventors have also unexpectedly demonstrated that pores comprising the novel mutant monomers have an enhanced ability to interact with analytes, such as polynucleotides, and thus exhibit improved performance for assessing analyte characteristics (e.g., polynucleotide sequences). The mutant pores unexpectedly exhibit improved nucleotide discrimination. In particular, the mutant pores unexpectedly exhibit an increased current range, making it easier to distinguish between different nucleotides and reducing state changes that would increase the signal-to-noise ratio. In addition, the number of nucleotides contributing to the current as the polynucleotide moves through the pore is reduced. This makes it easier to determine a direct relationship between the current observed as the polynucleotide moves through the pore and the polynucleotide. Furthermore, the mutant pores can exhibit increased flux, e.g., are more likely to interact with analytes, such as polynucleotides. This makes it easier to characterize analytes using the pores. The mutant pores can also be more easily inserted into membranes.

[0037] Accordingly, the present invention provides a mutant CsgG monomer comprising a sequence variant shown in SEQ ID NO: 390, wherein the variant comprises a mutation at one or more of positions Y51, N55 and F56.

[0038] Accordingly, the present invention provides a mutant CsgG monomer comprising a sequence variant of SEQ ID NO: 390, wherein the variant comprises one or more of the following: (i) one or more mutations at the following positions (i.e., mutations at one or more of the following positions) N40, D43, E44, S54, S57, Q62, R97, E101, E124, E131, R142, T150, and R192; (ii) mutations at Y51 / N55, Y51 / F56, N55 / F56, or Y51 / N55 / F56; (iii) Q42R or Q42K; (iv) K49R ; (v) N102R, N102F, N102Y or N102W; (vi) D149N, D149Q or D149R; (vii) E185N, E185Q or E185R; (viii) D195N, D195Q or D195R; (ix) E201N, E201Q or E201R; (x) E203N, E203Q or E203R; and (xi) deletion of one or more of the following positions: F48, K49, P50, Y51, P52, A53, S54, N55, F56 and S57.

[0039] The present invention also provides:

[0040] - a construct comprising two or more covalently linked CsgG monomers, wherein at least one monomer is a mutant monomer described herein;

[0041] - a polynucleotide encoding a mutant monomer of the present invention or a construct of the present invention;

[0042] - a homo-oligomeric pore derived from CsgG comprising the same mutant monomer of the invention or the same construct of the invention;

[0043] - a hetero-oligomeric pore derived from CsgG comprising at least one mutant monomer of the invention or at least one construct of the invention;

[0044] - A method for determining the presence, absence or one or more characteristics of a target analyte, comprising:

[0045] a) contacting a target analyte with a CsgG pore or a mutant thereof such that the target analyte moves relative to the pore; and

[0046] b) obtaining one or more measurements as the analyte moves relative to the pore and thereby determining the presence, absence, or one or more characteristics of the analyte;

[0047] - A method of forming a sensor for characterizing a target polynucleotide, comprising forming a complex between a CsgG pore or a mutant thereof and a polynucleotide binding protein, and thereby forming a sensor for characterizing the target polynucleotide;

[0048] - A sensor for characterizing a target polynucleotide, comprising a complex between a CsgG pore or a mutant thereof and a polynucleotide-binding protein;

[0049] - Use of the CsgG pore or a mutant thereof for determining the presence, absence or one or more characteristics of a target analyte;

[0050] - A kit for characterizing a target analyte, comprising (a) a Csg pore or a mutant thereof, and

[0051] (b) membrane components;

[0052] - A device for characterizing a target analyte in a sample, comprising (a) a plurality of CsgG pores or mutants thereof and (b) a plurality of membranes;

[0053] A method for characterizing a target polynucleotide, comprising:

[0054] a) contacting the polynucleotide with a CsgG pore or a mutant thereof, a polymerase, and labeled nucleosides, such that phosphate-labeled species are sequentially added to the target polynucleotide by the polymerase, wherein the phosphate-labeled specie contains a label specific for each nucleotide; and

[0055] b) detecting the phosphate label using the pore and thereby characterizing the polynucleotide; and

[0056] The method for producing the mutant monomer or the construct of the present invention comprises expressing the polynucleotide of the present invention in a suitable host cell to thereby produce the mutant monomer or construct of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Figure 2 shows a side cross-sectional view of the CsgG nonamer in its channel conformation in ribbon and surface representation.

[0058] Figure 2 A cross-sectional view showing the CsgG channel constriction structure (i.e., the pore read head in the context of nanopore sensing applications) and associated diameter measurements.

[0059] Figure 3 Figure 5 represents the structural motif that forms the pore constriction structure, comprising three stacked concentric side chain layers: Tyr 51, Asn 55, and Phe 56.

[0060] Figure 4 Sequence homology among CsgG homologs is shown, including a multiple sequence alignment of CsgG-like proteins (SEQ ID NO: 442 to SEQ ID NO: 448). Selected sequences were selected from monophyletic clades across a phylogenetic tree of CsgG-like sequences (not shown) to provide a representative view of sequence diversity. Secondary structural elements are shown, represented by arrows or sticks for β-strands and α-helices, respectively, and are based on the E. coli CsgG crystal structure. Importantly, residues corresponding to E. coli Tyr 51, Asn 55, and Phe 56 are highlighted by arrows. These residues form the internal constriction of the pore, i.e., the pore read head, in the aforementioned nanopore sensing applications.

[0061] Figure 5 Representative single-channel current recordings (a) and conductance histograms (b) of CsgG reconstituted in a planar phospholipid bilayer and measured at an electric field of +50 mV (n=33) or −50 mV (n=13).

[0062] Figure 6Figure 3. Single-channel current recordings of PPB-reconstituted CsgG at +50 mV or -50 mV in the presence of increasing concentrations of CsgE. The horizontal scale bar is at 0 pA.

[0063] Figure 7 a. Original negative-stained EM image of CsgG solubilized by C8E4 / LDAO. Arrows indicate the different particle populations marked in the size elution curve shown in g, which are (I) aggregates of CsgG nonamers, (II) CsgG octadecamers, and (III) CsgG nonamers. Scale bar, 20 nm. b, Schematic representation of the class averages of the indicated oligomeric states from top and side views. c, Rotational autocorrelation function of LDAO-solubilized CsgG from the top view, showing nine-fold symmetry. d, CsgG C1S Original negative stain EM images of - arrows indicate hexadecameric (IV) and octameric (V) particles observed by size exclusion chromatography in Figure g. e, CsgG C1S Schematic representation of the class average of the side view of the oligomer. Top view without observation of this construct, f, CsgG observed by size exclusion chromatography shown in g. C1S and the elution volume (EV) of CsgG particles, the calculated molecular mass (MWcalc), the molecular weight corresponding to the oligomeric state of CsgG (CsgG n ) of the expected molecular mass (MW CsgG ) and a table showing the symmetry of the particles as observed by negative stain electron microscopy (EM) and X-ray crystallography. g, CsgG C1S Size exclusion chromatograms of CsgG (black) and C8E4 / LDAO-solubilized CsgG (grey) on a Superdex200 10 / 300GL (GE Healthcare). h, i, Schematic representation of the top and side views of the crystallized oligomers showing the CsgG C1S D8 hexadecamer (h) and D9 octamer (i) in membrane-extracted CsgG. One protomer is colored with rainbow colors from N-terminus (blue) to C-terminus (red). Two C8 octamers (CsgG) trapped in the crystal form a tail-to-tail dimer. C1S ) or C9 nonamer (CsgG) are colored blue and tan. r and θ represent the radius and interprotomer rotation, respectively.

[0064] Figure 8 CsgG C1S exist Electron density map at 1.5 g, calculated using the experimental SAD phase with NCS averaging and density modification, and contoured at 1.5 g. The map shows the region of channel construction (CL; individual protomers are labeled) and is overlaid on the final refined model. CsgG C1S is a mutant CsgG in which the N-terminal Cys of the mature CsgG sequence, i.e., Cys 1, is replaced by Ser, resulting in a lack of lipid modification by the E. coli LOL pathway. This results in a soluble homo-octameric oligomer in a pre-pore conformation (see Figure 42 ), a conformation that is opposite to the membrane-targeting homononameric pore formed by native, ester-modified CsgG ( Figure 43 ).

[0065] Figure 9 Figure 2 shows a top view of the modeled CsgG constriction with a polyalanine chain helically threading through the channel in an extended conformation shown in the C-terminal to N-terminal direction ( Figure 9 a) and side view ( Figure 9 b). Modelled solvation of polypropylamino acid chains, i.e. Figure 9 The position in b, such as Figure 9 c, where the C ring structure is removed for clarity (the solvent molecules are shown as being 1 / 4 of the total polyalanine chain). molecules within).

[0066] Figure 10 : represents CsgG in Escherichia coli.

[0067] Figure 11 : Indicates the size of CsgG.

[0068] Figure 12 :Instructions Translocation of a single G. In E. coli CsgG (CsgG-Eco), guanine entry into the F56 loop is significantly hindered. * = G enters the F56 loop. A = G stops interacting with loop 56. B = G stops interacting with loop 55. C = G stops interacting with loop 51.

[0069] Figure 13 :Instructions Pulling DNA through the constriction of CsgG in E. coli requires a significant force.

[0070] Figure 14 :Instructions Both the CsgG-F56A-N55S and CsG-F56A-N55S-Y51A mutants showed low resistance to ssDNA translocation.

[0071] Figure 15-17 : Mutant pores exhibit increased range compared to wild type (WT).

[0072] Figure 18-19 : Mutant pores exhibit increased flux compared to wild type (WT).

[0073] Figure 20-21 : Mutant pores exhibit enhanced insertion compared to wild type (WT).

[0074] Figure 22 : represents DNA construct X used in Example 18. The region labeled 1 corresponds to 30 SpC3 spacers. The region labeled 2 corresponds to SEQ ID NO: 415. The region labeled 3 corresponds to 4 iSp18 spacers. The region labeled 4 corresponds to SEQ ID NO: 416. The segment labeled 5 corresponds to 4 5-nitroindole. The region labeled 6 corresponds to SEQ ID NO: 417. The region labeled 7 corresponds to SEQ ID NO: 418. The region labeled 8 corresponds to SEQ ID NO: 419, which has 4 iSp18 spacers (region labeled 9) attached to the 3' end of SEQ ID NO: 419. At the other end of the iSp18 spacer is a 3' cholesterol tether (labeled 10). The region labeled 11 corresponds to 4 SpC3 spacers.

[0075] Figure 23 Figure 2 shows an exemplary chromatography trace of Strep trap (GE Healthcare) purification of CsgG protein (x-axis label = elution volume (mL), Y-axis label = absorbance (mAu)). The sample was loaded in 25 mM Tris, 150 mM NaCl, 2 mM EDTA, 0.01% DDM and eluted with 10 mM desthiobiotin. The elution peak of the eluted CsgG protein is labeled E1.

[0076] Figure 24 : An example of a typical SDS-PAGE visualization of CsgG protein after initial strep purification. 4-20% TGX gel (Bio Rad) was run at 300 V for 22 minutes in 1X TGS buffer. The gel was stained with Sypro Ruby stain. Lanes 1-3 represent the major elution peak containing CsgG protein, as indicated by the arrow ( Figure 23 Lanes 4-6 correspond to the main elution peak containing contaminants ( Figure 23 M represents the molecular weight marker used, which is unstained Novex Sharp (unit = kD).

[0077] Figure 25 Figure 2 shows an example of size exclusion chromatography (SEC) of a CsgG protein (120 mL S200GE healthcare, x-axis label = elution volume (mL), y-axis label = absorbance (mAu)). SEC was performed after strep purification and heating of the protein sample. The SEC running buffer was 25 mM Tris (hydroxymethylaminomethane), 150 mM NaCl, 2 mM EDTA, 0.01% DDM, 0.1% SDS, pH 8.0, and the column was run at a rate of 1 mL / min. The trace marked with an X has an absorbance at 220 nm, and the trace marked with a Y has an absorbance at 280 nm. Peaks marked with an asterisk were collected.

[0078] Figure 26 : An example of a typical SDS-PAGE visualization of CsgG protein after SEC. A 4-20% TGX gel (BioRad) was run at 300 V for 22 minutes in 1X TGS buffer and stained with Sypro Ruby stain. Lane 1 represents a CsgG protein sample after strep purification and heating but before SEC. Lanes 2-8 represent Figure 25 The fractions collected from the peak of about 48 mL to 60 mL (middle peak = 55 mL) were run in Figure 25 The molecule is marked with an asterisk in the middle. M indicates the molecular weight marker used, which is unstained Novex Sharp (unit = kD). The band corresponding to the E. coli CsgG pore is indicated by an arrow.

[0079] Figures 27 to 33 : Mutant pores exhibit increased range compared to wild type (WT).

[0080] Figures 34-39 : Mutant pores exhibit increased range compared to wild type (WT).

[0081] Figure 40 Snapshots of the enzyme (T4Dda-(E94C / C109A / C136A / A360C) (SEQ ID NO: 412) with mutations E94C / C109A / C136A / A360C and (ΔM1)G1G2)) located on top of the pore (CsgG-Eco-(Y51T / F56Q)-Strep11(C))9 (SEQ ID NO: 390 with mutations Y51T / F56Q, wherein Step11(C) is SEQ ID NO: 435 and is linked to the pore mutant No. 20 at the C-terminus) taken at 0 and 20 ns in the simulation (run Figures 1 to 3 ).

[0082] Figure 41 Snapshots of the enzyme (T4Dda-(E94C / C109A / C136A / A360C) (SEQ ID NO: 412) with mutations E94C / C109A / C136A / A360C and (ΔM1)G1G2)) located on top of the pore (CsgG-Eco-(Y51T / F56Q)-Strep11(C)9 (SEQ ID NO: 390 with mutations Y51T / F56Q, wherein Step11(C) is SEQ ID NO: 435 and is linked to the pore mutant No. 20 at the C-terminus) taken at 30 and 40 ns in the simulation (run Figures 1 to 3 ).

[0083] Figure 42 Represents CsgG in the prepore conformation C1S X-ray structure of a,CsgG C1S Figure 2. Ribbon diagram of the monomer, colored in rainbow colors from blue to red from the N-terminus to the C-terminus. Secondary structural elements are labeled according to the ABD-like fold, with the additional N-terminal and C-terminal α-helices and the extended loop connecting β1 and α1 labeled αN, αC, and C-loop (CL), respectively. b, CsgG C1S Side view of the C8 octamer with subunits differentiated by color and one subunit oriented and colored as in a.

[0084] Figure 43 The structure of CsgG in its channel conformation is shown. a, CsgG C1S ATR-FTIR spectra of amide I region (1,700–1,600 cm) of CsgG (blue) and membrane-extracted CsgG (red). -1 b, TM1 and TM2 sequences (SEQ ID NO:449 and SEQ ID NO:450) (bilayer-facing residues in blue) and the Congo red binding region of E. coli BW25141ΔcsgG complemented with wild-type csgG (WT), empty vector, or csgG lacking the underlined fragments of TM1 or TM2. Data are presented using three biological replicates. c, Overlays of CsgG monomers in the prepore conformation (light blue; TM1 pink, TM2 purple) and channel conformation (brown; TM1 green; TM2 orange). CL, C-loop. d, e, Side view (d) and cross-section (e) of the CsgG nonamer in faceted representation; helix 2, the core domain, and the TM hairpin are indicated in blue, light blue, and brown, respectively. A single protomer is shown. Figure 42 a Coloring. Magenta spheres indicate the location of Leu 2. OM, outer membrane.

[0085] Figure 44Representation of the CsgG channel constriction. a, Cross-sectional view of the CsgG channel constriction and its solvent-excluded diameter. b, The constriction consists of three stacked concentric side chain layers: Tyr 51, Asn 55, and Phe 56, preceded by Phe 48 from the periplasmic side. c, Topology of the CsgG channel. d, Congo red binding domain of Escherichia coli BW25141ΔcsgG complemented with csgG (WT), empty vector, or csgG harboring a constriction-displaying mutation. Data are presented using six biological replicates. e, f, Representative single-channel current recordings (e) and conductance histograms (f) of CsgG reconstituted in a planar phospholipid bilayer and measured at an electric field of +50 mV (n = 33) or −50 mV (n = 13).

[0086] Figure 45 Model representing the transport mechanism of CsgG. a, Native PAGE of CsgE (E), CsgG (G), and CsgG supplemented with excess CsgG (E+G), showing the formation of the CsgG-CsgE complex (EG*). Data are presented from seven experiments involving four different batches of protein. b, SDS-PAGE of CsgE (E), CsgG (G), and EG* complexes recovered from native PAGE. Data are presented from two replicates. M, molecular weight marker. c, Average of selected species of CsgG-CsgE particles. From left to right: top and side views as seen by cryo-electron microscopy (cryo-EM), and comparison of a negatively stained side view with a CsgG nonamer. d, Average of CsgE particles viewed from the top and oblique side. Rotational autocorrelations indicate nine-fold symmetry. e, 3D reconstruction of CsgG-CsgE (24 Å resolution, 1221 single particles) showing nonamers containing CsgG (blue) and additional density representing CsgE nonamers (orange). f, Single-channel current recordings of PPB-recombined CsgG at +50 mV or −50 mV supplemented with increasing concentrations of CsgE. Horizontal scale bar at 0 pA. g, Tentative model of CsgG-mediated protein secretion. CsgG and CsgE are proposed to form a secretion complex that captures CsgA (in Figure 54 ), generating an entropic potential across the channel. After CsgA is trapped in the channel constriction, DS-rectified Brownian diffusion drives the gradual translocation of the peptide across the outer membrane.

[0087] Figure 46Representation of the Curli biosynthetic pathway in Escherichia coli. The larger curli subunit, CsgA (light green), is secreted from the cell as a soluble monomeric protein. The smaller curli subunit, CsgB (dark green), is associated with the outer membrane (OM) and serves as a nucleator for converting CsgA from a soluble protein to amyloid deposits. CsgG (orange) assembles into oligomeric curli-specific transport channels in the outer membrane. CsgE (purple) and CsgF (light blue) form soluble accessory proteins required for the transport and deposition of prolific CsgA and CsgB. CsgC forms a putative oxidoreductase of unknown function. All curli proteins possess a putative Sec signal sequence for transport across the cytoplasmic (inner) membrane (IM).

[0088] Figure 47 Represents CsgG and CsgG analyzed by size exclusion chromatography and negative stain electron microscopy C1S Oligomeric states in solution. a, Raw negative stain EM image of CsgG solubilized by C8E4 / LDAO. Arrows indicate the different particle populations marked in the size elution curve shown in g, which are (I) aggregates of CsgG nonamers, (II) CsgG octadecamers, and (III) CsgG nonamers. Scale bar, 20 nm. b, Schematic representation of the class averages of the indicated oligomeric states from top and side views. c, Rotational autocorrelation function plot of LDAO-solubilized CsgG from top view, showing nine-fold symmetry. d, CsgG C1S Original negative stain EM image of CsgG. Arrows indicate hexadecanomer (IV) and octamer (V) particles observed by size exclusion chromatography in Figure g. e, CsgG C1S Schematic representation of the class average of the side view of the oligomer. The top view of this construct was not observed. f, CsgG observed by size exclusion chromatography as shown in g. C1S and the elution volume (EV) of CsgG particles, the calculated molecular weight (MWcalc), the corresponding CsgG oligomeric state (CsgG n Table of the expected molecular mass (MWCsgG) of CsgG and the symmetry of the particles observed by negative stain EM and X-ray crystallography. C1S Size exclusion chromatograms of CsgG (black) and C8E4 / LDAO-solubilized CsgG (grey) on a Superdex 200 10 / 300GL (GE Healthcare). h, i, Schematic representation of the ribbons of the crystallized oligomers in top and side views, showing the CsgG C1S(h) D8 hexadecamer and (i) D9 octamer of membrane-extracted CsgG. One protomer is colored in rainbow colors from N-terminus (blue) to C-terminus (red). Two C8 octamers (CsgG) trapped in a crystal forming a tail-to-tail dimer C1S ) or C9 nonamer (CsgG) are stained in blue and tan. r and h represent the radius and interprotomer turning angle, respectively.

[0089] Figure 48 Comparison of CsgG with structural homologs and interprotomer contacts with CsgG. a, b, CsgG C1S Ribbon diagrams of a monomer (e.g., CsgG in the prepore conformation) (a) and the nucleotide-binding domain-like structure of TolB (b) (PDB 2hqs), both colored rainbow from N-terminus (blue) to C-terminus (red). Common secondary structure elements are labeled in the same way. c, CsgG C1S (gray), stacked from left to right with Xanthomonas campestris rare lipoprotein B (PDB 2r76, colored pink), Shewanella oneidensis hypothetical lipoprotein DUF330 (PDB 2iqi, colored pink), and Escherichia coli TolB (PDB 2hqs, with its N-terminal and β-propeller domains colored pink and yellow, respectively). CsgG-specific structural elements are labeled and colored as shown in the upper left panel. d, e, Ribbon diagrams of two adjacent protomers found in the CsgG structure, viewed along the plane of the bilayer from the outside (c) or inside (d) of the oligomer. The first protomer is depicted in rainbow colors (dark blue to red) from N-terminus to C-terminus; the second protomer is depicted in light blue (core domain), blue (helix 2), and brown (TM domain). Four major oligomerization interfaces are evident: interactions of the b6-b39 backbone within the b-barrel structure, the constriction loop (CL), side chain stacking of helix 1 (α1) abutting b1-b3-b4-b5, and helix-helix stacking of helix 2 (α2). An 18-residue N-terminal loop connecting the lipid anchor (magenta spheres indicate the position of Ca in Leu 2) and the N-terminal helix (αN) is also observed to wrap around the two adjacent protomers. The projected position of the lipid anchor is expected to abut the TM1 and TM2 hairpin structures of the +2 protomer (not shown for clarity).

[0090] Figure 49Figure 5. Cys accessibility test of selected surface residues in CsgG oligomers. ac, Schematic representation of the ribbons of a CsgG nonamer shown in periplasmic (a), lateral (b), and extracellular (c) views. One protomer is colored rainbow from the N-terminus (blue) to the C-terminus (red). Cysteine ​​substituents are labeled, and the equivalent positions of the S atoms are shown as spheres, colored according to their accessibility to the MAL-PEG (5,000 Da) label in the E. coli outer membrane. d, Western blot of samples reacted with MAL-PEG analyzed on SDS-PAGE, showing an increase in the binding of the introduced cysteine ​​to MAL-PEG by 5 kDa. Accessible (11 and 111), moderately accessible (1), and inaccessible (2) sites are colored green, orange, and red in ae, respectively. For Arg 97 and Arg 110, a second species at 44 kDa is present, corresponding to a portion of the protein where both the introduced and native cysteines are labeled. Data are presented using four independent biological replicates. e, Side view of the dimerization interface within the D9 octadecamer present in the X-ray structure. Introduced cysteines at the dimerization interface or within the lumen of the D9 particle are labeled. In membrane-bound CsgG, these residues are accessible to MAL-PEG, demonstrating that the D9 particle is a product of concentrated solutions of membrane-extracted CsgG and that C9 complex formation is physiologically relevant. Residues in the C-terminal helix (aC; Lys 242, Asp 248, and His 255) were found to be inaccessible to poorly accessible, suggesting that aC may form additional contacts with the E. coli cell membrane, likely the peptidoglycan layer.

[0091] Figure 50 Figure 2 shows molecular dynamics simulations of the CsgG constriction using a polyalanine chain model. a, b, Top view (a) and side view (b) of the CsgG constriction modeled with a polyalanine chain in an extended conformation helix passing through the channel, shown in the C-terminus to N-terminus orientation. Substrate passage through the CsgG transporter is not sequence-specific. 参考文献16,23. For clarity, a polyalanine chain is used to simulate the hypothetical interactions of a passing polypeptide chain. The simulated region consists of nine concentric CsgG C-loop structures, each containing residues 47-58. The side chains connecting the constriction are represented by stick diagrams, where Phe 51 is colored slate blue, Asn 55 (amide clamp) is colored cyan, and Phe 48 and Phe 56 (Φ clamp) are colored light orange and dark orange, respectively. N, O and H atoms (only hydroxyl or amide H atoms on the side chain are shown) are colored blue, red and white, respectively. The C, N, O and H atoms in the polyalanine chain are colored green, blue, red and white, respectively. The polyalanine residues in the constriction The solvent molecules (water) within the range are shown as red dots. c, Simulated solvation of the polyalanine, in the position shown in b and with the C-ring structure removed for clarity (the solvent molecules shown are within the entire polyalanine At the height of the amide clamp and the Φ clamp, the solvation of the polyalanine chain is reduced to a single water shell bridging the peptide backbone and the amide clamp side chains. Most of the side chains in the Tyre 51 ring are similar to those in CsgG (and CsgG C1S ) X-ray structures, compared to the inward-pointing positions observed in the X-ray structure, are rotated toward the solvent. This model was constructed using GROMACS 参考文献53 Results of 40 ns all-atom explicit solvent molecular dynamics simulations using the AMBER99SB-ILDN54 force field with restrained positions of the Cα atoms of the residues located at the termini of the C-loop (Gin 47 and Thr 58).

[0092] Figure 51Figure 1. Representation of sequence conservation among CsgG homologs. a, Surface schematic of a CsgG nonamer, colored by sequence similarity (colors yellow to blue are scored from low to high conservation) and viewed from the periplasm (far left), side (center left), extracellular environment (center right), or as a cross-sectional view (far right). The figure shows the regions of highest sequence conservation mapping at the entrance of the periplasmic vestibule, the vestibular side of the contractile ring, and the lumenal surface of the TM domain. b, Multiple sequence alignment of CsgG-like lipoproteins. Selected sequences were selected from three monophyletic clades of CsgG-like sequences across phylogeny (not shown) to provide a representative view of sequence diversity. Secondary structural elements, β-strands and α-helices, are indicated by arrows or sticks, respectively, and are based on the E. coli CsgG crystal structure. c, d, Schematic diagram of the secondary structure of the CsgG protomer (c) and a cross-sectional view of a schematic surface diagram of the CsgG nonamer (d), both in gray, with three contiguous regions of high sequence conservation colored red (HCR1), blue (HCR2), and yellow (HCR3). HCR1 and HCR2 form the vestibular side of the constriction; HCR3 corresponds to helix 2 and is located at the entrance to the periplasmic vestibule. Within the constriction, Phe 56 is 100% conserved, while Asn 55 can be conservatively substituted with Ser or Thr, for example, with a small polar side chain that can act as a hydrogen bond donor / acceptor. The concentric side chain ring (Tyr 51) at the exit of the constriction is not conserved. The presence of the Phe ring at the constriction entrance is topologically similar to the Phe 427 ring (termed the Φ clamp) in the anthrax protective antigen PA63, which has been shown to catalyze polypeptide capture and passage. 参考文献20 MST of toxB superfamily proteins reveals a conserved motif D(D / Q)(F)(S / N)S at the height of the Phe ring. This is similar to the motif S(Q / NT)(F)ST seen in curli-like transporters. Although the atomic-resolution structure of PA63 in the pore conformation is not yet available, the available structure suggests that the Phe ring may similarly be followed by conserved hydrogen bond donors / acceptors (Ser / Asn428) as concentric rings in the subsequent translocation channel (note that the orientation of these elements is reversed in both transporters).

[0093] Figure 52Single-channel current analysis of CsgG and CsgG:CsgE pores is shown. a, Under negative field potentials, CsgG pores exhibit two conductance states. The upper left and upper right panels show representative single-channel current traces for the normal (measured at +50, 0, and -50 mV) and low-conductance forms (measured at 0, +50, and -50 mV), respectively. No transitions between the two states were observed during the total observation time (n = 22), indicating that the conductance states have long lifetimes (on the order of seconds to minutes). The lower left panel shows current histograms of the normal and low-conductance forms of the CsgG pore obtained at +50 and -50 mV (n = 33). The IV curves of the CsgG pore at normal and low conductance are shown in the lower right panel. The data represent the mean and standard deviation of at least four independent recordings. The natural or physiological existence of the low-conductance form is unknown. b, Electrophysiological properties of CsgG channels titrated with the cofactor CsgE. The curve shows the proportion of channels in the open, intermediate, and closed states as a function of CsgE concentration. Figure 45 f. Increasing the CsgE concentration to above 10 nM causes CsgG pore closure. This effect occurs at both +50 mV (left panel) and -50 mV (right panel), ruling out pore clogging caused by electrophoresis of CsgE (calculated pI 4.7) into the CsgG pore. A low-probability (5%) intermediate state has approximately half the conductance of the open channel. This may represent incomplete closure of the CsgG channel by CsgE; alternatively, it may represent transient formation of CsgG dimers caused by residual CsgG monomers from the electrolyte solution binding to the membrane-embedded pore. The proportions of these three states can be obtained from full-point histogram analysis of single-channel current traces. The peak areas of up to three states generated by these histograms, and the proportion of a given state can be obtained by dividing the corresponding peak area by the sum of all other states in the recording. At negative field potentials, two open conductance states are identified, similar to those observed for CsgG (see panel a). Because both open channel changes are blocked by higher CsgG concentrations, the "open" trace in (b) combines both conductance forms. Data in the graph represent the mean and standard deviation from three independent recordings. c, Crystal structure, size exclusion chromatography, and EM reveal that detergent-extracted CsgG pores form non-native tail-to-tail stacked dimers (e.g., two nonamers like D9 particles) at higher protein concentrations. Figure 47These dimers can also be observed in single-channel recordings. The top panel shows single-channel current traces of stacked CsgG pores at +50, 0, and -50 mV (from left to right). The bottom left panel shows current histograms of dimeric CsgG pores recorded at +50 and -50 mV. The experimental conductance values ​​of +16.2±1.8 and -16.0±3.0 pA (n=15) at +50 and -50 mV, respectively, are close to the theoretically calculated value of 23 pA. The bottom right panel shows the IV curve of stacked CsgG pores. Data represent the mean and standard deviation of six independent recordings. d, The ability of CsgE to bind to and block stacked CsgG pores was tested by electrophysiology. Shown are single-channel current traces of stacked CsgG pores in the presence of 10 or 100 nM CsgE at +50 mV (top panel) and -50 mV (bottom panel). The current traces indicate that an otherwise saturating concentration of CsgE does not result in pore closure of the stacked CsgG dimer. These observations are consistent with the mapping of the CsgG-CsgE contact region to helix 2 and the entrance to the CsgG periplasmic cavity, as identified by EM and site-directed mutagenesis ( Figure 45 and Figure 52 ).

[0094] Figure 53 Representation of CsgE oligomers and the CsgG-CsgE complex. a, Size exclusion chromatography of CsgE (Superose 6, 16 / 600; running buffer 20 mM Tris-HCl pH 8, 100 mM NaCl, 2.5% glycerol) shows an equilibrium of two oligomeric states, 1 and 2, with an apparent molecular mass ratio of 9.16:1. Negative-stain electron microscopy (EM) of peak 1 reveals discrete CsgE particles (inset: five representative species-averaged images, sorted by increasing tilt angle) comparable in size to nine CsgE replicas. b, Selected species-averaged images of CsgE oligomers observed by cryo-EM from a top view, and their rotational autocorrelograms reveal the presence of C9 symmetry. c, FSC analysis of the CsgG-CsgE cryo-EM model. Using 125 classes corresponding to 1221 particles, the three-dimensional reconstruction was achieved by FSC analysis at a correlation threshold of 0.5. d, Overlay of the CsgG nonamer observed in the CsgG-CsgE cryo-electron microscopy density and X-ray structure. The overlay is shown as a semi-transparent density map (left) or a cross-section from a side view. e, Congo red binding region of E. coli BW25141ΔcsgG complemented with wild-type csgG (WT), empty vector (ΔcsgG), or csgG helix 2 mutants (single amino acid substitutions marked with single-letter codes). Data are presented using four biological replicates. f, Effects of bile salt toxicity on E. coli LSR12 complemented with csgG (WT) or csgG carrying different helix 2 mutations that complement (1) or do not complement (2) csgE. Ten-fold serial dilutions starting from 107 bacteria were spotted on MacConkey agar plates. Expression of CsgG pores in the outer membrane leads to increased bile salt sensitivity, which can be blocked by coexpression of CsgE (n = 6, three biological replicates, two replicates each). g, Cross-sectional view of the X-ray structure of CsgG with molecular surface representation. CsgG mutants with no effect on the Congo red binding region or toxin are shown in blue; mutants that interfere with CsgE-mediated rescue of bile salt sensitivity are shown in red.

[0095] Figure 54 Figure 3 shows the assembly and substrate recruitment of the CsgG secretion complex. The Curli transporter CsgG forms a secretion complex with a 9:9 stoichiometric ratio with the soluble secretion cofactor CsgE, which encloses a 3 compartment, which is thought to capture the CsgA substrate and facilitate its transport across the outer membrane (OM; see above and Figure 45) entropy-driven diffusion. Theoretically, three hypothetical pathways (ac) can be envisioned for substrate recruitment and assembly of the secretory complex. a. The ‘catch-and-cap’ mechanism requires CsgA to bind to the apolipoprotein CsgG translocation channel (1), leading to a conformational change in the latter that exposes a high-affinity binding platform for CsgE binding (2). CsgE binding results in capping of the substrate cage structure. Upon CsgA secretion, CsgG returns to its low-affinity conformation, leading to dissociation of CsgE and release of the secretory channel for a new secretory cycle. b. In the ‘dock-and-trap’ mechanism, CsgE first captures CsgA in the periplasm (1), leading to CsgE adopting a high-affinity complex that docks to the CsgG translocation pore (2), enclosing CsgA in the secretory complex. CsgA can bind directly to CsgE oligomers or CsgE monomers, the latter leading to subsequent oligomerization and CsgG binding. Secretion of CsgA causes CsgE to revert to its low-affinity conformation and dissociate from the secretory pathway. CsgG and CsgE form a constitutive complex, in which CsgE's conformational dynamics cycle between open and closed forms during CsgA recruitment and secretion. Currently available or published data do not allow us to distinguish between these putative recruitment modes or their derivatives, or to propose one of these modes.

[0096] Figure 55 CsgG C1S Data collection statistics and electron density mapping of CsgG. a, CsgG C1S and data collection statistics of CsgG X-ray structures. b, in CsgG C1S Electron density map of the CsgG TM domain (resolutions 3.6, 3.7, and 1.5 Å along the reciprocal vectors a*, b*, and c*, respectively). ), which was calculated from the NCS-averaged and density-modified molecular replacement phase (TM loops were absent in the input model); B-factor sharpening- The contour lines are drawn at 1.0σ. This figure shows the TM1 (Lys 135-Leu 154) and TM2 (Leu 182-Asn 209) regions of a single CsgG protomer, overlaid on the final refined model.

[0097] Figure 56 Single-channel current trace (left) and zoomed-in region (right) of CsgG WT protein interacting with a DNA hairpin carrying a single-stranded DNA overhang. The trace shows the change in current (indicated by arrows) in response to potentials measured at +50 mV or -50 mV intervals. The blockade of the downward current in the final +50 mV segment indicates that both strands of the hairpin enter the pore lumen and spiral the single-stranded hairpin end into the pore's internal structure, resulting in almost complete current blockade. Reversing the electric field to -50 mV results in electrophoretic unblocking of the pore. The new +50 mV phase again results in entry / spiraling of the DNA hairpin and pore blockage. Unwinding of the hairpin over the +50 mV segment can lead to cessation of the current blockade, as indicated by the reversal of the current blockade. The hairpin structure, which has the sequence: 3'GCGGGGA GCGTATT AGAGTTG GATCGGATGCA GCTGGCTACTGACGTCATGACGTCAGTAGCCAGCATGCATCCGATC-5', was added to the cis side of the chamber at a final concentration of 10 nM.

[0098] Figure 57 Shown are the purification and channel properties of CsgG-ΔΡYPA, a mutant CsgG pore in which the PYPA sequence (residues 50-53) is mutated to GG at the constriction residue Y51.

[0099] Description of Sequence Listing

[0100] SEQ ID NO: 1 shows the amino acid sequence of wild-type Escherichia coli CsgG including the signal sequence (Uniprot accession number P0AEA2).

[0101] SEQ ID NO: 2 shows the polynucleotide sequence of wild-type Escherichia coli CsgG including the signal sequence (Gene ID: 12932538).

[0102] SEQ ID NO: 3 represents the amino acid sequence of wild-type E. coli CsgG from positions 53 to 77 of SEQ ID NO: 2. This corresponds to the amino acid sequence from positions 38 to 63 of a mature wild-type E. coli CsgG monomer (i.e., lacking the signal sequence).

[0103] SEQ ID NOs: 4 to 388 represent the amino acid sequences at positions 38 to 63 of modified monomers of CsgG lacking the signal sequence.

[0104] SEQ ID NO: 389 shows a codon-optimized polynucleotide sequence encoding a wild-type CsgG monomer from Escherichia coli Str. K-12 substr. MC4100. This monomer lacks the signal sequence.

[0105] SEQ ID NO: 390 represents the amino acid sequence of a mature, wild-type CsgG monomer from Escherichia coli Str. K-12 substr. MC4100. This monomer lacks the signal sequence. The abbreviation CsgG used herein is CsgG = CsgG-Eco.

[0106] SEQ ID NO: 391 shows the amino acid sequence of YP_001453594.1:1-248 of the hypothetical protein CKO_02032 [Citrobacter koseri ATCC BAA-895], which is 99% identical to the sequence of SEQ ID NO: 390.

[0107] SEQ ID NO: 392 shows the amino acid sequence of WP_001787128.1:16-238 of the curli production assembly / transport component CsgG, part of [Salmonella enterica], which is 98% identical to SEQ ID NO: 390.

[0108] SEQ ID NO: 393 shows the amino acid sequence of KEY44978.1|:16-277 of the curli production assembly / transport protein CsgG [Citrobacter amalonaticus], which is 98% identical to SEQ ID NO: 390.

[0109] SEQ ID NO: 394 shows the amino acid sequence of YP_003364699.1: 16-277 of the curli production assembly / transport component [Citrobacter rodentium ICC168], which is 97% identical to SEQ ID NO: 390.

[0110] SEQ ID NO: 395 shows the amino acid sequence of YP_004828099.1:16-277 of the curli production assembly / transport component CsgG [Enterobacter asburiae LF7a], which is 94% identical to SEQ ID NO: 390.

[0111] SEQ ID NO: 396 represents a polynucleotide sequence encoding Phi29 DNA polymerase.

[0112] SEQ ID NO: 397 shows the amino acid sequence of Phi29 DNA polymerase.

[0113] SEQ ID NO: 398 represents a codon-optimized polynucleotide sequence derived from the sbcB gene of Escherichia coli, encoding the exonuclease I enzyme (EcoExo I) from Escherichia coli.

[0114] SEQ ID NO: 399 shows the amino acid sequence of exonuclease I enzyme (EcoExo I) from Escherichia coli.

[0115] SEQ ID NO: 400 represents the codon-optimized polynucleotide sequence of the xthA gene from Escherichia coli, encoding the exonuclease III enzyme from Escherichia coli.

[0116] SEQ ID NO: 401 represents the amino acid sequence of an exonuclease III enzyme from Escherichia coli. This enzyme digests one strand of double-stranded DNA (dsDNA) in a distributed manner, cleaving 5' nucleoside monophosphates from the 3' to 5' direction. Priming of the enzyme on one strand requires a 5' overhang of approximately 4 nucleotides.

[0117] SEQ ID NO: 402 represents the codon-optimized polynucleotide sequence of the recJ gene from T. thermophilus , encoding the RecJ enzyme (TthRecJ-cd) from T. thermophilus .

[0118] SEQ ID NO: 403 represents the amino acid sequence of the RecJ enzyme from Streptococcus thermophilus (TthRecJ-cd). This enzyme performs processive digestion of 5' monophosphate nucleosides of single-stranded DNA (ssDNA) from the 5' to 3' direction. The enzyme requires at least 4 nucleotides for priming on the strand.

[0119] SEQ ID NO: 404 represents a codon-optimized polynucleotide sequence derived from the bacteriophage lambda exonuclease (redX) gene, encoding the bacteriophage lambda exonuclease.

[0120] SEQ ID NO:405 represents the amino acid sequence of bacteriophage lambda exonuclease. This sequence represents one of three identical subunits that assemble into a trimer. The enzyme is highly processive in cleaving nucleotides from one strand of double-stranded DNA (dsDNA) in the 5' to 3' direction (http: / / www.neb.com / nebecomm / products / productM0262.asp). Priming of the enzyme on a strand preferably requires an overhang of approximately four nucleotides with 5' phosphates.

[0121] SEQ ID NO: 406 shows the amino acid sequence of Hel308Mbu.

[0122] SEQ ID NO:407 shows the amino acid sequence of Hel308Csy.

[0123] SEQ ID NO: 408 shows the amino acid sequence of Hel308Tga.

[0124] SEQ ID NO: 409 shows the amino acid sequence of Hel308Mhu.

[0125] SEQ ID NO: 410 shows the amino acid sequence of Tral Eco.

[0126] SEQ ID NO: 411 shows the amino acid sequence of XPD Mbu.

[0127] SEQ ID NO: 412 shows the amino acid sequence of Dda 1993.

[0128] SEQ ID NO:413 shows the amino acid sequence of Trwc Cba.

[0129] SEQ ID NO: 414 represents the amino acid sequence of WP_006819418.1: 19-280 [Yokenella regensburgei] of the transporter, which is 91% identical to the sequence of SEQ ID NO: 390.

[0130] SEQ ID NO: 415 shows the amino acid sequence of WP_024556654.1:16-277 [Cronobacter pulveris] of the curli production assembly / transport protein CsgG, which is 89% identical to SEQ ID NO: 390.

[0131] SEQ ID NO: 416 shows the amino acid sequence of YP_005400916.1:16-277 [Rahnella aquatilis HX2] of the curli production assembly / transport protein CsgG, which is 84% ​​identical to SEQ ID NO: 390.

[0132] SEQ ID NO: 417 shows the amino acid sequence of KFC99297.1:20-278 of the CsgG family curli production assembly / transport component [Kluyvera ascorbata ATCC 33433], which is 82% identical to SEQ ID NO: 390.

[0133] SEQ ID NO: 418 shows the amino acid sequence of KFC86716.11:16-274 of the CsgG family curli production assembly / transport component [Hafnia alvei ATCC 13337], which is 81% identical to SEQ ID NO: 390.

[0134] SEQ ID NO: 419 shows the amino acid sequence of YP_007340845.1|:16-270 of an uncharacterized protein involved in the formation of curli polymers [Enterobacteriaceae bacterium strain FGI 57], which is 76% identical to SEQ ID NO: 390.

[0135] SEQ ID NO: 420 shows the amino acid sequence of WP_010861740.1: 17-274 of the curli production assembly / transport protein CsgG [Plesiomonas shigelloides], which is 70% identical to SEQ ID NO: 390.

[0136] SEQ ID NO: 421 represents the amino acid sequence of YP_205788.1: 23-270 of the outer membrane lipoprotein component CsgG produced, assembled, and transported by curli [Vibrio fischeri ES1 14], which is 60% identical to SEQ ID NO: 390.

[0137] SEQ ID NO: 422 shows the amino acid sequence of WP_017023479.1:23-270 of the curli production assembly protein CsgG [Aliivibrio logei], which is 59% identical to SEQ ID NO: 390.

[0138] SEQ ID NO: 423 shows the amino acid sequence of WP_007470398.1:22-275 of the Curli production assembly / transport component CsgG [Photobacterium sp. AK15], which is 57% identical to SEQ ID NO: 390.

[0139] SEQ ID NO: 424 shows the amino acid sequence of WP_021231638.1:17-277 of the curli production assembly protein CsgG [Aeromonas veronii], which is 56% identical to SEQ ID NO: 390.

[0140] SEQ ID NO: 425 shows the amino acid sequence of WP_033538267.1:27-265 of the Curli production assembly / transport protein CsgG [Shewanella sp. ECSMB14101], which is 56% identical to SEQ ID NO: 390.

[0141] SEQ ID NO: 426 shows the amino acid sequence of WP_003247972.1:30-262 of the curli production assembly protein CsgG [Pseudomonas putida], which is 54% identical to SEQ ID NO: 390.

[0142] SEQ ID NO: 427 shows the amino acid sequence of YP_003557438.1:1-234 of the curli production assembly / transport component CsgG [Shewanella violacea DSS12], which is 53% identical to SEQ ID NO: 390.

[0143] SEQ ID NO: 428 shows the amino acid sequence of WP_027859066.1:36-280 of the curli production assembly / transport protein CsgG [Marinobacterium jannaschii], which is 53% identical to SEQ ID NO: 390.

[0144] SEQ ID NO: 429 shows the amino acid sequence of CEJ70222.1: 29-262 of the Curli production assembly / transport component CsgG [Chryseobacterium oranimense G311], which is 50% identical to SEQ ID NO: 390.

[0145] SEQ ID NO: 430 represents the polynucleotide sequence used in Example 18.

[0146] SEQ ID NO: 431 represents the polynucleotide sequence used in Example 18.

[0147] SEQ ID NO: 432 represents the polynucleotide sequence used in Example 18.

[0148] SEQ ID NO: 433 represents the polynucleotide sequence used in Example 18.

[0149] SEQ ID NO: 434 represents the polynucleotide sequence used in Example 18. Attached to the 3' end of SEQ ID NO: 434 are six iSp18 spacers, and the other end thereof is attached to two thymines and 3' cholesterol TEG.

[0150] SEQ ID NO: 435 represents the amino acid sequence of Step II (C).

[0151] SEQ ID NO: 436 shows the amino acid sequence of Pro.

[0152] SEQ ID NOs: 437 to 440 represent primers from Example 1.

[0153] SEQ ID NO: 441 represents the hairpin structure from Example 21.

[0154] SEQ ID NOs: 442 to 448 represent Figure 4 sequence.

[0155] SEQ ID NO: 449 to 450 represent Figure 43 sequence. DETAILED DESCRIPTION

[0156] Unless otherwise indicated, the practice of the present invention employs conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA technology and chemical methods, which are within the capabilities of ordinary skill in the art. These techniques are also explained in the literature, for example, in M.R. Green, J. Sambrook, 2012, Molecular Cloning: A Laboratory Manual, 4th ed., vols. 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel, F.M. et al. (1995 and regular supplements; Current Protocols in Molecular Biology, Chapters 9, 13, and 16, John Wiley & Sons, New York, NY); B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Basic Techniques, John Wiley & Sons; J.M. Polk and James O'D. McGee, 1990, In Situ Hybridization: Principles and Practice, Oxford University Press; M.J. Gait (ed.), 1984, Oligonucleotide Synthesis: A Practical Approach, IRL Press; and D.M.J. Lilley and J.E. Dahlberg, 1992, Methods in Enzymology: DNA Structure Part A: Methods of DNA Synthesis and Physical Analysis in Enzymology, Academic Press. The entire contents of these documents are incorporated herein by reference.

[0157] Before describing the present invention, providing a number of definitions will help to understand the present invention. All references cited herein are incorporated herein by reference in their entirety. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the present invention belongs.

[0158] As used herein, the term "comprising" means that any listed elements must be included, and other elements may also be optionally included. "Consisting essentially of" means that any listed elements must be included, elements that have a significant effect on the basic and novel characteristics of the listed elements are excluded, and other elements may be optionally included. "Consisting of" means that all unlisted elements are excluded. Embodiments defined by each of these terms are within the scope of the present invention.

[0159] As used herein, the term "nucleic acid" refers to a single-stranded or double-stranded covalently linked nucleotide sequence in which the 3' and 5' ends of each nucleotide are linked by a phosphodiester bond. The polynucleotide can be composed of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acids can include DNA and RNA and can be synthesized in vitro or isolated from natural resources. Nucleic acids can further include modified DNA or RNA, such as methylated DNA or RNA, or RNA that has been post-translationally modified, such as 5'-capping with 7-methylguanosine, 3'-end processing, such as cleavage and polyadenylation, and splicing. Nucleic acids can also include synthetic nucleic acids (XNA), such as hexitol nucleic acids (HNA), cyclohexene nucleic acids (CeNA), threose nucleic acids (TNA), glycerol nucleic acids (GNA), locked nucleic acids (LNA) and peptide nucleic acids (PNA). The size of nucleic acids (also referred to herein as "polynucleotides") is typically expressed in terms of the number of base pairs (bp) for double-stranded polynucleotides or in terms of the number of nucleotides (nt) for single-stranded polynucleotides. One thousand bp or nt equals one kilobase pair (kb).Polynucleotides less than about 40 nucleotides in length are often referred to as "oligonucleotides," and may comprise primers used in DNA manipulations, such as by the polymerase chain reaction (PCR).

[0160] The term "amino acid" in the present invention is used in its broadest sense and includes naturally occurring Lα-amino acids or residues. As used herein, naturally occurring amino acids are generally abbreviated using single or three letters: A=Ala;C=Cys;D=Asp;E=Glu;F=Phe;G=Gly;H=His;I=Le;K=Lys;L=Leu;M=Met;N=Asn;P=Pro;Q=Gln;R=Arg;S=Ser;T=Thr;V=Val;W=Trp;and Y=Tyr (Lehninger, AL, (1975) Biochemistry, 2nd edition, pp. 71-92, Worth Publishers, New York). The general term "amino acid" also includes D-amino acids, retro-inverso amino acids and chemically modified amino acids, such as amino acid analogs, naturally occurring amino acids that are not normally contained in proteins, such as norleucine, and chemically synthesized compounds having amino acid characteristics known in the art, such as β-amino acids. For example, analogs or mimetics of phenylalanine or proline, which allow the peptide compound to have the same conformational constraints as native Phe or Pro, are included in the definition of an amino acid. Such analogs and mimetics are referred to herein as "functional equivalents" of the corresponding amino acids. Other examples of amino acids are listed in: Roberts and Vellaccio, Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, eds., Vol. 5, p. 341, Academic Press, Inc., NY 1983, which is incorporated herein by reference.

[0161] "Polypeptide" refers to a polymer of amino acid residues linked by peptide bonds, whether produced naturally or in vitro by synthetic methods. Polypeptides of less than about 12 amino acid residues in length are generally referred to as "peptides", and polypeptides between about 12 and about 30 amino acid residues in length may be referred to as "oligopeptides". The term "polypeptide" as used herein refers to a naturally occurring polypeptide, a precursor form, or a product of a preprotein. Polypeptides may also undergo maturation or post-translational modification processes, which may include, but are not limited to, glycosylation, proteolytic cleavage, lipidation, signal peptide cleavage, propeptide cleavage, phosphorylation, etc. The term "protein" as used herein refers to a macromolecule containing one or more polypeptide chains.

[0162] "Biological pore" refers to a transmembrane protein structure that defines a channel or pore that allows molecules and ions to translocate from one side of the membrane to the other. The translocation of ionic species through the pore can be driven by a potential difference applied to either side of the pore. A nanopore is a biological pore in which the minimum diameter of the channel through which a molecule or ion passes is on the order of nanometers (10 -9 rice).

[0163] For all embodiments and aspects of the present invention, the polynucleotide may comprise a polynucleotide having at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% complete sequence identity to wild-type E. coli CsgG, as set forth in SEQ ID NO:2. Similarly, the polypeptide may comprise a polypeptide having at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% complete sequence identity to wild-type E. coli CsgG, as set forth in SEQ ID NO:1. The polypeptide may comprise a polypeptide comprising the PFAM domain PF03783, which is characteristic of CsgG-like proteins. A list of currently known CsgG homologs and CsgG architecture can be found at http: / / pfam.xfam.org / / family / PF03783. Sequence identity may thus be to fragments or portions of the full-length polynucleotide or polypeptide. Thus, a sequence may have only 50% overall sequence identity with a sequence of the present invention, but a specific region, domain or subunit may share 80%, 90% or up to 99% sequence identity with a sequence of the present invention. According to the present invention, homology to the nucleic acid sequence of SEQ ID NO: 2 is not limited to sequence identity. Many nucleic acid sequences can show significant biological homology to each other, despite having significantly low sequence identity. In the present invention, homologous nucleic acid sequences are considered to be sequences that hybridize to each other under low stringency conditions (MR Green, J. Sambrook, 2012, Molecular Cloning: A Laboratory Manual, 4th Edition, Volumes 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0164] The term "vector" is used to refer to a linear or circular DNA molecule into which another nucleic acid (usually DNA) sequence segment of appropriate size can be incorporated. The DNA segment may include additional segments that provide for transcription of the gene encoded by the DNA segment. Such additional segments may include, but are not limited to, promoters, transcription terminators, enhancers, internal ribosome entry sites, untranslated regions, polyadenylation signals, selectable markers, origins of replication, and the like. A variety of promoters suitable for prokaryotic (e.g., β-lactamase and lactose promoter systems, alkaline phosphatase, tryptophan (trp) promoter systems, Escherichia coli lac, tac, T3, T7 promoters) and eukaryotic (e.g., simian virus 40 early or late promoters, Rous sarcoma virus long terminal repeat promoter, cytomegalovirus promoter, adenovirus late promoter, EG-1a promoter) hosts can be used. Expression vectors are typically derived from plasmids, cosmids, viral vectors, and yeast artificial chromosomes; vectors are typically recombinant molecules containing DNA sequences from several sources. Specific embodiments of the present invention provide expression vectors encoding wild-type or modified CsgG polypeptides as described herein. The term "operably linked," when applied to DNA sequences, for example in a nucleic acid vector as described above, means that the sequences are arranged so that they function synergistically to achieve their intended purpose, i.e., a promoter sequence allows initiation of transcription through the associated coding sequence to the termination sequence.

[0165] The transmembrane protein structure of the biological pore can be monomeric or oligomeric in nature. Typically, the pore includes a plurality of polypeptide subunits arranged around a central axis, thereby forming a protein-lined channel, the extension of which is substantially perpendicular to the membrane where the nanopore residue is located. The number of polypeptide subunits is not limited. Typically, the number of subunits is from 5 to a maximum of 30, with a suitable number of subunits being 6 to 10. Alternatively, in the case of perfringolysin or related large membrane pores, the number of subunits is not limited. The protein subunit portion within the nanopore forming the protein-lined channel typically includes a secondary structural motif, which may include one or more transmembrane β-barrel structures, and / or α-helical portions.

[0166] It should be understood that different applications of the disclosed products and methods can be adjusted according to the specific needs of the art. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments of the present invention and are not intended to be limiting.

[0167] Furthermore, as used in this specification and the appended claims, unless the context clearly dictates otherwise, the singular forms "a," "an," and "the" include plural referents. Thus, for example, reference to "a polynucleotide" includes two or more polynucleotides, reference to "a polynucleotide binding protein" includes two or more such proteins, reference to "a helicase" includes two or more helicases, reference to "a monomer" refers to two or more monomers, reference to "a pore" includes two or more pores, etc.

[0168] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0169] The present invention relates in part to bacterial amyloid secretion channel (CsgG), methods for its preparation, and its use in nucleic acid sequencing applications and molecular sensing.

[0170] CsgG is a membrane lipoprotein present in the outer membrane of Escherichia coli (Uniprot accession number P0AEA2; gene ID: 12932538). In this outer lipid membrane, CsgG forms a nanopore comprising an oligomeric complex of nine CsgG monomeric subunits. The CsgG preprotein is translocated through the SEC translocon by a type II (lipoprotein) signal sequence and subsequently triacylated on the N-terminal Cys residue of mature CsgG (i.e., CsgG with a cleaved type II signal sequence). Triacylated or "lipidated" CsgG is transported to the outer membrane of Gram-negative hosts, where it inserts into the bilayer as a nonameric pore. Non-lipidated forms of CsgG, e.g., CsgG C1S Exists in the periplasm as a soluble protein in a forespore conformation ( Figure 42 ).

[0171] The X-ray structure of the wild-type CsgG nanopore (Goyal et al., Nature, 2014, 516(7530), 250-3) shows that it has The width and (Hereinafter, the term "width" of a nanopore refers to its dimension parallel to the membrane surface, while the term "height" of a nanopore refers to its dimension perpendicular to the membrane.) The CsgG pore complex passes through the membrane via a 36-stranded β-barrel structure to provide Inner diameter of the channel ( Figure 1 Each of the assembled monomers of the CsgG channel has a conserved 12-residue ring structure (C-loop, “CL”; Figure 2 ), which work together to form a channel with a diameter of approximately The contraction structure ( Figure 1 and 2The constriction structure of the wild-type CsgG nanopore consists of three stacked concentric rings formed by the side chains of amino acid residues Tyr51, Asn55, and Phe56 of each CsgG monomer present in the CsgG oligomer ( Figure 3 ). This residue numbering is based on the mature protein lacking the native 15 amino acid signal sequence at the N-terminus. Thus, the mature protein corresponds to residues 16 to 277 of SEQ ID NO: 1. Tyre56 is located at position 66 of SEQ ID NO: 1, Asn is located at position 70 of SEQ ID NO: 1, and Phe56 is located at position 71 of SEQ ID NO: 1.

[0172] The role of this constriction is to restrict the path of ions and other molecules through the CsgG channel. Using standard electrolyte conditions and a potential of +50 mV or -50 mV, single-channel current recordings of CsgG reconstituted in a planar phospholipid bilayer resulted in stable currents of 43.1 ± 4.5 pA (n = 33) or -45.1 ± 4.0 pA (n = 13), respectively. Figure 5 ).

[0173] Current flow through the CsgG channel can be effectively blocked by adding a stoichiometric amount of the periplasmic factor CsgE (Uniprot accession number POAE95; Examples 10 to 12). Without wishing to be bound by theory, the evidence for the current points to a mechanism whereby CsgE forms a complex with the CsgG pore, blocking one end of the channel. A significant reduction in ion flux through the CsgG channel can be detected using standard single-channel recording techniques (Examples 12 and 13, Figure 6 The inventors have discovered that, according to one embodiment of the present invention, the measurement parameters of the current (maximum current and the ability to monitor changes in current) make the nanopore suitable for use in nucleic acid sequencing and molecular sensing applications.

[0174] Thus, the present invention relates in part to methods and uses of CsgG nanopore protein complexes for nucleic acid sequencing based on changes in electrical measurements of current flowing through the nanopore.

[0175] Nucleic acids are particularly suitable for nanopore sequencing. The naturally occurring nucleic acid bases in DNA and RNA can be distinguished by their physical size. When a nucleic acid molecule or a single base passes through the channel of a nanopore, the size difference between the bases causes a directly related reduction in the ion flow through the channel. The changes in the ion flow can be recorded. Suitable electrical measurement techniques for recording changes in ion flow are described in, for example, WO 2000 / 28312 and D.Stoddart et al., Proc.Natl.Acad.Sci., 2010, 106, pp 7702-7 (single-channel recording equipment); and, for example, WO 2009 / 077734 (multi-channel recording technology). By appropriate calibration, the reduction in the characteristics of the ion flow can be used to identify specific nucleotides and related bases that pass through the channel in real time.

[0176] The size of the narrowest constriction in a transmembrane channel is often a key factor in determining the suitability of a nanopore for nucleic acid sequencing applications. If the constriction is too small, the molecules to be sequenced will not be able to pass through. However, in order to achieve the maximum impact on the ion flow through the channel, the channel should not be too large at its narrowest point (i.e., at the constriction). Ideally, any constriction should be as close in diameter as possible to the size of the bases passing through. For sequencing of nucleic acids and nucleic acid bases, suitable constriction diameters are in the nanometer range (10 -9 The constriction in wild-type E. coli CsgG has a diameter of approximately The inventors concluded that the size and conformation of the constriction in the CsgG channel are suitable for nucleic acid sequencing.

[0177] For applications related to nucleic acid sequencing, the CsgG nanopore can be used in its wild-type form or can be further modified, for example, by targeted mutagenesis of specific amino acid residues, to further enhance the desired properties of the nanopore in question. For example, in embodiments of the present invention, mutations are contemplated to alter the number, size, shape, location, or orientation of constrictions within the channel. Modified mutant CsgG nanopore complexes can be prepared using known genetic engineering techniques that result in the insertion, substitution, and / or deletion of specifically targeted amino acid residues within the polypeptide sequence. In the case of oligomeric CsgG nanopores, mutations can be made to each monomeric polypeptide subunit, or to any one or all monomers. Suitably, in one embodiment of the present invention, the mutations are made to all monomeric polypeptides within the oligomeric protein structure.

[0178] According to one embodiment of the present invention, a modified mutant CsgG nanopore is provided, wherein the number of channel constrictions in the pore is reduced.

[0179] The wild-type E. coli CsgG pore consists of two channel constrictions (see Figure 1 These constrictions are formed by (i) amino acid residues Phe56 and Asn55, and (ii) amino acid residue Tyr 51, as part of a broader structure that includes additional amino acids from positions 54 to 53 and the C-loop motif ( Figure 2 and Figure 3 ).

[0180] In typical nanopore nucleic acid sequencing, as individual nucleotides of a nucleic acid sequence of interest sequentially pass through the nanopore channel, the ion flux through the open channel is reduced due to the partial blockage of the channel by the nucleotides. This reduction in ion flux is measured using a suitable recording technique as described above. The reduction in ion flux can be calibrated to the reduction in ion flux measured when a known nucleotide passes through the channel, providing a method for determining which nucleotides pass through the channel and, therefore, the nucleotide sequence of a nucleic acid passing through the nanopore as it passes sequentially through the channel. To accurately sequence individual nucleotides, it is generally desirable that the reduction in ion flux through the channel be directly correlated with the size of the individual nucleotides passing through the single constriction (or "reading head"). It will be appreciated that sequencing can be performed on an intact nucleic acid polymer "threaded" through the pore, for example, by the action of a relevant polymerase. Alternatively, sequence can be determined by the passage of nucleotide triphosphates that have been sequentially removed from the target nucleic acid near the pore (see, for example, WO 2014 / 187924).

[0181] When two or more constrictions are present and spaced apart from each other, each constriction can simultaneously read or interact with a separate nucleotide in the nucleic acid chain. In this case, the reduction in ion flow through the channel will be the result of the combined restriction of flow from all nucleotide-containing constrictions. Thus, in some cases, a dual constriction can result in a composite current signal. In some cases, when two such read heads are present, the current reading from one constriction, or "read head," may not be determinable alone.

[0182] The wild-type CsgG pore structure can be redesigned using recombinant genetic techniques to enlarge, alter, or remove one of the two constrictions, leaving a single constriction within the channel, thereby defining a single read head. The constriction motif in the CsgG oligomeric pore is located at amino acid residues 38 to 63 in the wild-type E. coli CsgG polypeptide. The wild-type amino acid sequence of this region is shown in SEQ ID NO:3. When considering this region, mutations at any of the amino acid residues at positions 50 to 53, 54 to 56, and 58 to 59 are considered within the scope of the present invention. Based on sequence similarity to CsgG homologs ( Figure 4 ), amino acid residues at positions 38 to 49, 53, 57, and 61 to 63 are considered highly conserved and therefore may be less amenable to substitution or other modification. Due to the critical positioning of the Tyr51, Asn55, and Phe56 side chains within the channel of the wild-type CsgG structure, mutations at these positions may be advantageous in order to modify or alter the properties of the read head.

[0183] Mutations at designated positions in monomeric CsgG proteins can result in substitution of the wild-type amino acid at that position with any other natural or unnatural amino acid. In one embodiment of the invention, it is desirable to enlarge or remove a constriction; suitably, the amino acid side chains in the modified CsgG protein are selected to provide less steric hindrance than the amino acid side chains in the wild-type structure they replace. The substituted amino acid residue at a designated position can have similar electrostatic properties, or it can have different electrostatic properties. Suitably, the substituted amino acid side chain will have an electrostatic charge similar to that of the wild-type structure it replaces, to minimize disruption of secondary structure or channel properties.

[0184] The selection of the amino acid to be replaced can be based on the BLOSUM62 matrix, which provides a standard method for calculating the probability of an amino acid being replaced by another based on a large number of multiple sequence alignments. Examples of BLOSUM62 matrices are freely available to the skilled person on the Internet; see, for example, the website of the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / blog / blossum62matrices).

[0185] / / www.ncbi.nlm.nih.gov / Class / Structure / aa / aa_explorer.cgi).

[0186] For Tyr51 in the wild-type CsgG structure, which forms the first constriction in the CsgG channel, any amino acid substitution is contemplated. Specifically, in certain embodiments of the invention, Tyr51 can be substituted with alanine, glycine, valine, leucine, isoleucine, asparagine, glutamine, and phenylalanine (SEQ IDs: 39-318). In embodiments of the invention, substitution of Tyr51 with alanine or glycine is particularly suitable (SEQ IDs: 39-108). In embodiments, residues 50 to 53 (PYPA in the wild-type sequence) can be substituted with glycine-glycine (GG) (SEQ IDs: 354-388).

[0187] For Asn55, which contributes to the second constriction in the CsgG channel, any amino acid substitution is provided. In particular, in certain embodiments of the invention, Asn55 can be substituted with alanine, glycine, valine, serine, or threonine (SEQ IDs: 9-33, 44-68, 79-103, 14-14, 138, 149-173, 184-208, 219-243, 254-278, 289-313, and 324-348).

[0188] For Phe56, which forms part of the second constriction in the CsgG channel, any amino acid substitution is provided. In particular, in certain embodiments of the invention, Phe56 can be substituted with alanine, glycine, valine, leucine, isoleucine, asparagine, and glutamine (SEQ ID NO: 1). ID: 5-13,15-18,20-23,25-28,30-33,40-43,45-48,50-53,55-58,60-63,65-68,75-78,80-83,85-88,90-93,95-98,100-103,110- 113,115-118,120-123,125-128,130-133,135-138,145-148,150-153,155-158,160-163,165-168,170-173,180-183,185-188,190 -193,195-198,200-203,205-208,215-218,220-223,225-228,230-233,235-238,240-243,250-253,255-258,260-263,265-268,270-273,275-578,285-288,290-293,295-298,300-303,305-308,310-313,320-323,325-328,330-333,335-338,340-343 and 345-348).In an embodiment of the present invention, substitution of Phe56 with alanine and glycine is particularly suitable (SEQ ID: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 111 15,120,125,130,135,140,145,150,155,160,165,170,175,180,185,190,195,200,205,210,215,220,225,230,235,240,245,250,255,260,265,270,275,280,285,290,295,300,305,310,315,320,325,330,335,340,345,350,355,360,365,370,375,380,385,6,11,16,21,26,31 ,36,41,46,51,56,61,66,71,76,81,86,91,96,101,106,111,116,121,126,131,136,141,146,151,156,161,166,171,176,181,186,191,196,201,206,211,216,221,226,231,236,241,246,251,256,261,266,271,276,281,286,291,296,301,306,311,316,321,326,331,336,341 346, 351, 356, 361, 366, 371, 376, 381 and 386).

[0189] In a given mutant CsgG protein, substitution of Tyr51 can be made simultaneously with any of positions 55 and 56 (SEQ ID NOs: 44, 49, 54, 59, 64, 79, 84, 89, 94, 99, 114, 119, 124, 129, 134, 149, 154, 159, 164, 169, 184, 189, 194, 199, 204, 219, 224, 229, 234, 239, 254, 259, 264, 269, 274, 289, 294, 299, 304, 309, 359, 364, 369, 374, 379, 40, 41, 42, 75, 76 77,110,111,112,145,146,147,180,181,182,215,216,217,250,251,252,285,286 and 287), wherein at least one constriction of appropriate size within the channel is maintained. Alternatively, substitution of Tyr51 is mutually exclusive with substitutions at positions Asn55 and Phe56 (SEQ ID: 39, 74, 109, 144, 179, 214, 249, 284, 354, 10, 11, 12, 15, 16, 17, 20, 21, 22, 25, 26, 27, 30, 31 and 32).

[0190] Alternatively, one or more of Tyr51, Asn55, or Phe56 in the wild-type CsgG protein may be deleted (SEQ ID: 319-353, 34-38, 69-73, 104-108, 139-143, 174-178, 209-213, 244-248, 279-283, 314-318, 384-388, 8, 13, 18, 23, 28, 33, 43, 48, 53, 58, 63, 68, 78, 83, 88, 93, 98, 103, 113, 113 18,123,128,133,138,148,153,158,163,168,173,183,188,193,198,203,208,218,223,228,233,238,243,253,258,263,268,273,278,288,293,298,303,308 and 313). To maintain at least one constriction in the channel, in a given embodiment, deletion of amino acid residue Tyr51 is mutually exclusive with deletion of amino acid residues Asn55 and Phe56 (SEQ ID: 319-322, 324-327, 329-332, 334-337, 339-342, 344-347, 38, 73, 108, 143, 178, 213, 248, 283, and 318). Certain adjacent amino acid residues at positions 53 and 54 and 48 and 49 may also be deleted.

[0191] It should be understood that the present invention provides embodiments in which the above modifications may be made individually or in any combination.

[0192] Removal of either the constriction at Tyr51 or the constriction at Asn55 / Tyr56 results in a single constriction within the CsgG channel. Without wishing to be bound by theory, it is hypothesized that the constriction at Asn55 / Tyr56 has a higher conformational stability than the constriction at Tyr51, which might be desirable. However, the Asn55 / Tyr56 constriction may be too tall (measured along the axis of the central pore) compared to the nucleotide. This may result in poor resolution of individual base pairs during DNA strand displacement.

[0193] The opposite may be true for the Tyr51 constriction. After removal of the Asn55 / Tyr56 constriction, the remaining loop of Tyr51 residues in the oligomer may be conformationally less stable than in the native structure. However, the Tyr51 constriction is shorter (when measured along the axis of the central pore) and may be more capable of providing a constriction within the channel that can discriminate between individual bases.

[0194] In either embodiment, when the pore is used in nucleic acid sequencing applications, the presence of a single narrow constriction (when measured along the central pore axis) may reduce the complexity of current readings. Modulations in the observed current generated as nucleic acid translocates through the pore will therefore only reflect the passage of isolated nucleotides through a single constriction or reading head.

[0195] Effective removal of a constriction can also increase the open-channel current of pore mutants. Increased open-channel current is advantageous because higher background conductance leads to higher resolution of current blockade levels for different nucleic acid base pair signals. In this way, modifications to the read head can improve the suitability of biological pores for nucleic acid sequencing and other molecular sensing applications.

[0196] As an alternative embodiment, or in addition to the above sequence modifications, it is also provided that the Asn55 / Phe56 constriction can be further adapted to adjust its height (measured along the central pore axis). This further adjustment of the Asn55 / Phe56 constriction may be accompanied by or without mutation of Tyr51 or other positions within the CsgG channel. Suitably, further adjustment of the Asn55 / Phe56 constriction is contemplated as part of a mutation that enlarges or removes the constriction formed by the Tyr51 residue.

[0197] In the wild-type form, the Asn55 / Phe56 channel constriction consists of two amino acid rings arranged vertically adjacent to each other. Therefore, the constriction has a length greater than 1 nm. A constriction of 1 nm in length may not allow for the resolution of the electrical signals generated by the ion flow that separates the bases in the nucleic acid chain displacement. Typically, the constriction of known nanopores used for nucleic acid sequencing is generally less than 1 nm in length. For example, the MspA nanopore used for DNA sequencing has a constriction height of 0.6 nm (measured along the central pore axis; Manrao et al., Nature Biotechnology, 2012, 30(4), 349-353).

[0198] To reduce the height of the Asn55 / Phe56 constriction (measured along the central pore axis), either residue can be substituted or deleted, resulting in a widening of the top or bottom of the constriction.

[0199] Any amino acid substitution is contemplated for Asn55, which forms part of the second constriction in the CsgG channel. In particular, substitution with alanine, glycine, valine, serine, or threonine is contemplated (SEQ IDs: 9-33, 44-68, 79-103, 14-14, 138, 149-173, 184-208, 219-243, 254-278, 289-313, and 324-348).

[0200] For Phe56, which forms part of the second constriction in the CsgG channel, substitutions with any amino acid are contemplated. In particular, substitutions with alanine, glycine, valine, leucine, isoleucine, asparagine, and glutamine (SEQ ID: 5-13,15-18,20-23,25-28,30-33,40-43,45-48,50-53,55-58,60-63,65-68,75-78,80-83,85-88,90-93,95-98,100-103,110- 113,115-118,120-123,125-128,130-133,135-138,145-148,150-153,155-158,160-163,165-168,170-173,180-183,185-188,190 -193,195-198,200-203,205-208,215-218,220-223,225-228,230-233,235-238,240-243,250-253,255-258,260-263,265-268,270-273,275-578,285-288,290-293,295-298,300-303,305-308,310-313,320-323,325-328,330-333,335-338,340-343 and 345-348).In an embodiment of the present invention, substitution of Phe56 with alanine and glycine is particularly suitable (SEQ ID: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 111 15,120,125,130,135,140,145,150,155,160,165,170,175,180,185,190,195,200,205,210,215,220,225,230,235,240,245,250,255,260,265,270,275,280,285,290,295,300,305,310,315,320,325,330,335,340,345,350,355,360,365,370,375,380,385,6,11,16,21,26,31 ,36,41,46,51,56,61,66,71,76,81,86,91,96,101,106,111,116,121,126,131,136,141,146,151,156,161,166,171,176,181,186,191,196,201,206,211,216,221,226,231,236,241,246,251,256,261,266,271,276,281,286,291,296,301,306,311,316,321,326,331,336,341 346, 351, 356, 361, 366, 371, 376, 381 and 386).

[0201] Modifications to adjust the minimum diameter of the constriction of the wild-type CsgG pore are also conceivable. The minimum diameter of the two constrictions in the CsgG pore is approximately 0.9 nm. Its diameter is smaller than the 1.2 nm diameter of the constriction in the MspA nanopore known to be effective for DNA sequencing (Manrao et al., Nature Biotechnology, 2012, 30(4), 349-353). Any of the above mutations that can provide a remaining constriction in a modified CsgG pore with a minimum diameter of 0.5 to 1.5 nm is suitable.

[0202] Any of the modifications listed above can advantageously alter the hydrophilicity and charge distribution of amino acids at the constriction to improve passage of and non-covalent interactions with the translocated nucleic acid strand, thereby improving current readout. Any of the mutations listed above can also advantageously alter the hydrophilicity and charge distribution near the channel constriction to optimize the flow of electrolyte ions through the constriction and achieve better discrimination of nucleotides passing through the translocated nucleic acid strand.

[0203] Further modifications of the CsgG protein are thought to alter the surface charge distribution within the channel lumen. In one embodiment of the present invention, these modifications can prevent unwanted electrostatic adsorption of translocated nucleic acids onto the channel walls. Since nucleic acids are negatively charged and the CsgG channel lumen contains some positive charges ( Figure 1 ), hypothesizing that electrostatic interactions may interfere with the helical or translocation of nucleic acids during sequencing. Appropriately positively charged amino acid residues, such as lysine, histidine, and arginine, can be substituted with neutral or negatively charged side chains to further enhance the efficiency of nucleic acid translocation through the pore, thereby improving the clarity of the current readout.

[0204] Embodiments of the present invention also provide for modifications to the lumen of the channel of wild-type E. coli CsgG or mutant CsgG to facilitate translocation or helical entry of nucleic acid chains (or individual nucleotides) into the pore constriction. The transmembrane portion of CsgG with an internal constriction resembles a barrel-like structure with a lid characterized by a central pore. Helical entry can be facilitated by adding an additional loop to the pore lumen closest to the barrel and lid.

[0205] Embodiments of the present invention provide that the CsgG pore may be composed of one or more covalently linked monomers, dimers, or oligomers. As a non-limiting example, the monomers may be genetically fused in any conformation, for example via their terminal amino acids. In this case, the amino terminus of one monomer may be fused to the carboxyl terminus of another monomer.

[0206] According to one embodiment of the present invention, it is also provided that the CsgG pore can be adapted to accommodate additional auxiliary proteins that can have advantageous properties when molecules pass through the pore. Adaptability to the pore may facilitate the anchoring of nucleic acid processing enzymes. Nucleic acid processing enzymes may include DNA or RNA polymerases; isomerases; topoisomerases; gyrases; telomerases; and helicases. Associating one or more of these enzymes with the nanopore can have benefits in terms of enhancing the entry of the nucleic acid helix into the pore and controlling the speed of nucleic acid strand translocation through the pore (Manrao et al., Nature Biotechnology, 2012, 30(4), 349-353). Controlling the speed of nucleic acid strand translocation through the pore has the advantage of providing an improved response in terms of current measurements of ion flow that are more amenable to reading and more consistent.

[0207] In embodiments of the present invention, it is contemplated that modifications to the extracellular region of the nanopore membrane may facilitate the docking of suitable nucleic acid processing enzymes (e.g., DNA polymerases) within or adjacent to the lumen of the channel by providing one or more binding / anchoring sites. Suitable anchoring sites may include electrostatic patches for electrostatic binding of the enzyme; one or more cysteine ​​residues to allow covalent coupling; and / or altered internal width of the transmembrane channel portion to provide a spatial anchor.

[0208] The present invention provides further adaptations of the CsgG wild-type pore for nucleic acid sequencing, as detailed in more detail below.

[0209] In embodiments of the invention, the extracellular region of the CsgG pore (e.g. Figure 1 The bottom portion (shown) can be truncated or removed to facilitate the departure of the nucleic acid chain on the other side of the channel lumen. Truncation or removal of the extramembrane region can also improve the current resolution of the electrical signal from the ion flow in the channel. The latter benefit is brought about by reducing the resistance to ion flow by the extramembrane region. In the CsgG pore of the present invention, the transmembrane channel, the internal constriction, and the cap region represent three continuous resistance regions. Removing or reducing the contribution of one of the above regions will increase the open channel current, thereby improving the current resolution. Such changes can include deletion of α-helix 2 (α2), C-terminal α-helix (αC) ( Figure 1 ), and / or combinations thereof.

[0210] In further embodiments of the present invention, membrane-facing amino acids on the outer surface of the wild-type E. coli CsgG pore can be modified to facilitate insertion of the pore into the membrane. In certain embodiments, single amino acid substitutions can replace wild-type residues with suitable, more hydrophobic analogs. For example, one or more of residues Ser136, Gly138, Gly140, Ala148, Ala188, or Gly202 can be changed to Ala, Val, Leu, or He. Furthermore, aromatic residues such as tyrosine or tryptophan can be substituted for appropriate wild-type amino acids so that they are located at the interface between the hydrophobic membrane and the hydrophilic solvent. For example, one or more of residues Leu154 or Leu182 can be substituted with Tyr, Phe, or Trp.

[0211] In an embodiment of the present invention, the thermal stability of the protein pore can be increased. This leads to a favorable increase in the shelf life of the nanopore in the sequencing device. In an embodiment, the increase in the thermal stability of the protein pore is achieved by modifying the beta-turn sequences or improving the electrostatic interactions on the protein surface. In one embodiment, the β-hairpin structure in the transmembrane region can be stabilized by forming a covalent disulfide across two adjacent β-chains in adjacent β-hairpin structures or between adjacent β-hairpin structures. Examples of such cross-chain cysteine ​​pairs can be Val139-Asp203; Gly139-Gly205; Lys135-Thr207; Glu201-Ala141; Gly147-Gly189; Asp149-Gln187; Gln151-Glu185;

[0212] Thr207-Glu185; Gly205-Gln187; Asp203-Gly189; Ala153-Lys135; Gly137-Gln151;

[0213] Val139-Asp149 or Ala141-Gly147.

[0214] In a further embodiment of the invention, the codons used in the polypeptide sequence can be modified to allow expression of the CsgG protein at high levels and low error rates according to the methods described in Biotechnol. J., 2011, 6(6), 650-659. The modification can also target any secondary structure of the mRNA.

[0215] The present invention also provides changes in the protein sequence to improve its stability to proteases. This can be achieved by removing flexible loop regions, such as deleting α-helix 2 (α2), the C-terminal α-helix (αC) ( Figure 1 ), and / or a combination thereof.

[0216] In embodiments of the invention, the CsgG polypeptide sequence / expression system is altered to avoid potential aggregation of the protein.

[0217] The present invention also provides changes in polypeptide sequences to improve protein folding efficiency. Suitable techniques are provided in Biotechnol. J., 2011, 6(6), 650-659.

[0218] One embodiment of the present invention further provides for the replacement or addition of a bioaffinity tag to facilitate purification of the CsgG protein. The disclosed structure of the CsgG pore includes a StrepII tag (Goyal et al., Nature, 2014, 516(7530), 250-3). Embodiments of the present invention include other bioaffinity tags, such as a histidine tag for facilitating purification by metal chelate affinity chromatography. In alternative embodiments of the present invention, the tag may include a FLAG tag or an epitope tag, such as a Myc tag or an HA tag. In further embodiments of the present invention, the nanopore may be modified by biotinylation with biotin or an analog thereof (e.g., desthiobiotin), thereby facilitating purification via interaction with streptavidin.

[0219] In embodiments of the present invention, negative charges at the protein termini can be added to increase the net charge of the polypeptide and promote protein migration in polyacrylamide gel electrophoresis. This may result in improved separation of CsgG hetero-oligomers in cases where these species are of interest (see Howorka et al. Proc. Nat. Acad. Sci., 2001, 98(23), 12996-13001). Hetero-oligomers can be used to introduce a single cysteine ​​residue per well, which can facilitate the attachment of suitable nucleic acid processing enzymes, such as DNA polymerases as described above.

[0220] The present invention relates in part to the use of wild-type or modified CsgG nanopores in molecular sensing applications based on changes in electrical measurements of current flowing through the nanopore.

[0221] Binding of molecules within the channel of the CsgG pore or near either opening of the channel affects the open-channel ion current through the pore. In a manner similar to the nucleic acid sequencing applications described above, changes in open-channel ion current can be measured by changes in current using suitable measurement techniques (e.g., WO 2000 / 28312 and D. Stoddart et al., Proc. Natl. Acad. Sci., 2010, 106, 7702-7 or WO 2009 / 077734). The extent of the reduction in ion current (measured by a decrease in current) is related to the size of the blockage within or near the pore. Therefore, binding of a molecule of interest, also known as an analyte, within or near the pore provides a detectable and measurable event, thereby forming the basis of a biosensor. Suitable molecules for nanopore sensing include nucleic acids; proteins; peptides; and small molecules such as drugs, toxins, or cytokines.

[0222] Detecting the presence of biomolecules can have applications in personalized drug development, medicine, diagnostics, life science research, environmental monitoring, and the security and / or defense industries.

[0223] In embodiments of the present invention, the wild-type or modified E. coli CsgG nanopores disclosed herein, or homologs thereof, can be used as molecular sensors. The analyte detection process is described in Howorka et al. Nature Biotechnology (2012) Jun 7; 30(6): 506-7. The analyte molecule to be detected can bind to either side of the channel, or to the lumen of the channel itself. The binding location can be determined by the size of the molecule to be sensed. The wild-type CsgG pore can be used as a sensor, or in embodiments of the present invention, the CsgG pore can be modified by recombinant or chemical methods to increase the binding strength, binding location or binding specificity of the molecule to be sensed. Typical modifications include the addition of a specific binding moiety that is structurally complementary to the molecule to be sensed. When the analyte molecule comprises a nucleic acid, the binding moiety can comprise a cyclodextrin or an oligonucleotide; for small molecules, this can be a known complementary binding region, such as the antigen binding portion of an antibody or non-antibody molecule, including a single-chain variable fragment (scFv) region or antigen recognition domain from a T cell receptor (TCR); or for proteins, it can be a known ligand for the target protein. In this way, wild-type or modified E. coli CsgG nanopores or homologs thereof can be shown to be capable of functioning as molecular sensors for detecting the presence of suitable antigens (including epitopes) in a sample, which may include cell surface antigens, including receptors, markers of solid tumors or hematological cancer cells (such as lymphoma or leukemia), viral antigens, bacterial antigens, protozoan antigens, allergens, allergy-related molecules, albumin (such as human, rodent or bovine), fluorescent molecules (including fluorescein), blood group antigens, small molecules, drugs, enzymes, catalytic sites of enzymes or enzyme substrates, and transition state analogs of enzyme substrates.

[0224] Modifications can be achieved using known genetic engineering and recombinant DNA techniques. The location of any adaptation will depend on the properties of the molecule to be sensed, such as size, three-dimensional structure, and its biochemical properties. Selection of the adaptive structure can be achieved using computational structural design. A range of customized CsgG nanopores has been envisioned, each specifically adapted for its intended sensing application. Determination and optimization of protein-protein interactions or protein-small molecule interactions can be achieved using, for example, et al., which use surface plasmon resonance to detect molecular interactions (BIAcore, Inc., Piscataway, NJ; see also www.biacore.com).

[0225] In one embodiment, the CsgG pore can be in the form of a water-soluble octameric form in which the N-terminal Cys residue is replaced with another amino acid to prevent lipidation at the N-terminus of the protein. In an alternative embodiment, the protein can be expressed in the cytoplasm by removing the N-terminal leader sequence to avoid processing by the bacterial lipidation pathway.

[0226] Goyal et al. (Nature, 2014; 516(7530)250-3) describe methods for preparing CsgG monomeric soluble protein, octameric soluble protein, and oligomeric lipidated CsgG pores, which are incorporated herein by reference as well as Examples 1 and 2.

[0227] Mutant CsgG monomer

[0228] The present invention provides mutant CsgG monomers. Mutant CsgG monomers can be used to form pores as described herein. A mutant CsgG monomer is a monomer whose sequence varies from that of a wild-type CsgG monomer and which retains the ability to form a pore. Methods for confirming the pore-forming ability of a mutant monomer are well known in the art and are discussed in more detail below.

[0229] The mutant monomer has the polynucleotide reading property of improvement, i.e., shows improved polynucleotide capture and nucleotide recognition. In particular, the hole constructed by the mutant monomer is easier to capture nucleotides and polynucleotides than the wild type. In addition, the hole constructed by the mutant monomer shows an increased current range (which makes it easier to distinguish different nucleotides) and a reduced state change (which increases the signal-to-noise ratio). In addition, as the polynucleotide moves through the hole constructed by the mutant, the number of nucleotides contributing to the electric current decreases. This makes it easier to identify the direct relationship between the electric current and the polynucleotide sequence observed when the polynucleotide moves through the hole. In addition, the hole constructed by the mutant monomer can show an increased flux, i.e., it is more likely to interact with analytes such as polynucleotides. This makes it easier to characterize analytes using the hole. The hole constructed by the mutant monomer can be more easily inserted into the membrane.

[0230] Mutant monomers described herein include variants of the sequence set forth in SEQ ID NO:390. SEQ ID NO:390 is a wild-type CsgG monomer from Escherichia coli Str. K-12 substrain MC4100. Variants of SEQ ID NO:390 are polypeptides having an amino acid sequence that differs from the sequence of SEQ ID NO:390 and that retain its pore-forming ability. The pore-forming ability of a variant can be determined using any method known in the art. For example, a variant can be inserted into an amphiphilic layer along with other suitable subunits, and the ability of the variant to oligomerize and form a pore can be determined. Methods for inserting subunits into membranes, such as amphiphilic layers, are known in the art. For example, the subunits can be suspended in purified form in a solution containing a triblock copolymer membrane, allowing them to diffuse into the membrane and be inserted by binding to the membrane and assembling into a functional state.

[0231] Throughout this application, standard single-letter codes for amino acids are used. These codes are as follows: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamic acid (E), glutamine (Q), glycine (G), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), and valine (V). Standard substitution symbols are also used, i.e., Q42R indicates that the Q at position 42 is replaced by R.

[0232] In one embodiment, the mutant monomer of the present invention includes a mutant of SEQ ID NO: 390, which comprises one or more of the following: (i) one or more mutations at the following positions (i.e., mutations at one or more of the following positions): N40, D43, E44, S54, S57, Q62, R97, E101, E124, E131, R142, T150 and R192, for example, one or more mutations at the following positions (i.e., mutations at one or more of the following positions): N40, D43, E44, S54, S57, Q62, E101, E131 and T150 or N40, D43, E44, E101 and E131; (ii) one or more mutations at Y51N55, Y51F56, N55F56 or (iii) Q42R or Q42K; (iv) K49R; (v) N102R, N102F, N102Y or N102W; (vi) D149N, D149Q or D149R; (vii) E185N, E185Q or E185R; (viii) D195N, D195Q or D195R; (ix) E201N, E201Q or E201R; (x) E203N, E203Q or E203R; and (xi) deletion of one or more of the following positions: F48, K49, P50, Y51, P52, A53, S54, N55, F56 and S57. The variant may comprise any combination of (i) to (xi). 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ix,x,xi}{iii,iv,v,vi,vii,viii,ix,x,xi}{i,ii,iii,iv,v,vi,vii,viii,ix,x}{i,ii,iii,iv,v,vi,vii,viii,ix,xi}{i,ii,iii,iv,v,vi,vii,viii,x,xi}{i ,ii,iii,iv,v,vi,vii,ix,x,xi}{i,ii,iii,iv,v,vi,viii,ix,x,xi}{i,ii,iii,iv,v,vii,viii,ix,x,xi}{i,ii,iii,iv,vi,vii,viii,ix,x,xi}{i,ii,iii,v,v i,vii,viii,ix,x,xi}{i,ii,iv,v,vi,vii,viii,ix,x,xi}{i,iii,iv,v,vi,vii,viii,ix,x,xi}{ii,iii,iv,v,vi,vii,viii,ix,x,xi} or {i,ii,iii,iv,v,vi,vii viii,ix,x,xi}. ,

[0233] If the variant includes any one of (i) and (iii) to (xi), it may further include a mutation at one or more positions among Y51, N55 and F56, for example, at Y51, N55, F56, Y51N55, Y51 / F56, N55 / F56 or Y51 / N55 / F56.

[0234] In (i), the variant may include any number and combination of mutations in N40, D43, E44, S54, S57, Q62, R97, E101, E124, E131, R142, T150 and R192. In (i), the variant preferably includes one or more mutations located at the following positions (i.e., mutations at one or more of the following positions): N40, D43, E44, S54, S57, Q62, E101, E131 and T150. In (i), the variant preferably includes one or more mutations located at the following positions (i.e., mutations at one or more of the following positions): N40, D43, E44, E101 and E131. In (i), the variant preferably includes a mutation located at S54 and / or S57. In (i), the variant more preferably includes a mutation at (a) S54 and / or S57 and (b) one or more of Y51, N55 and F56, for example at Y51, N55, F56, Y51 / N55, Y51 / F56, N55 / F56 or Y51 / N55 / F56. If S54 and / or S57 are deleted in (xi), they cannot be mutated in (i), and vice versa. In (i), the variant preferably includes a mutation at T150, for example T150I. Alternatively, the variant preferably includes a mutation at (a) T150 and (b) one or more of Y51, N55 and F56, for example at Y51, N55, F56, Y51 / N55, Y51 / F56, N55 / F56 or Y51 / N55 / F56. In (i), the variant preferably comprises a mutation at Q62, such as Q62R or Q62K. Alternatively, the variant preferably comprises a mutation at (a) Q62 and (b) one or more of Y51, N55 and F56, such as Y51, N55, F56, Y51 / N55, Y51 / F56, N55 / F56 or Y51 / N55 / F56. The variant may comprise a mutation at D43, E44, Q62 or any combination thereof, such as D43, E44, Q62, D43 / E44, D43 / Q62, E44 / Q62 or D43 / E44 / Q62. Alternatively, the variant preferably comprises a mutation at one or more of (a) D43, E44, Q62, D43 / E44, D43 / Q62, E44 / Q62 or D43 / E44 / Q62 and (b) Y51, N55 and F56, for example at Y51, N55, F56, Y51 / N55, Y51 / F56, N55 / F56 or Y51 / N55 / F56.

[0235] In (ii) and elsewhere in this application, the symbol / represents "and," e.g., Y51 / N55 refers to Y51 and N55. In (ii), the variant preferably includes a mutation at Y51 / N55. It has been proposed that the constriction in CsgG consists of three stacked concentric rings formed by the side chains of residues Y51, N55, and F56 (Goyal et al, 2014, Nature, 516, 250-253). Mutation of these residues in (ii) can thus reduce the number of nucleotides contributing to the current as the polynucleotide moves through the pore, thereby making it easier to identify a direct relationship between the observed current (as the polynucleotide moves through the pore) and the polynucleotide. Y56 can be mutated in any of the ways discussed below with respect to the variants and pores useful in the methods of the invention.

[0236] In (v), the variant may include N102R, N102F, N102Y or N102W. The variant preferably includes (a) N102R, N102F, N102Y or N102W and (b) a mutation at one or more of Y51, N55 and F56, such as Y51, N55, F56, Y51 / N55, Y51 / F56, N55 / F56 or Y51 / N55 / F56.

[0237] In (xi), any number and combination of the following positions: K49, P50, Y51, P52, A53, S54, N55, F56 and S57 may be deleted. Preferably, one or more of K49, P50, Y51, P52, A53, S54, N55 and S57 may be deleted. If any one of Y51, N55 and F56 is deleted in (xi), it / they cannot be mutated in (ii), and vice versa.

[0238] In (i), the variant preferably comprises one or more of the following substitutions: N40R, N40K, D43N, D43Q, D43R, D43K, E44N, E44Q, E44R, E44K, S54P, S57P, Q62R, Q62K, R97N, R97G, R97L, E101N, E101Q, E101R, E101K, E101F, E101Y, E101W, E124N, E124Q, E124R, E124K, E124F, E124Y, E124W, E131D, R142E, R142N, T150I, R192E and R192N, e.g., N40K. 101N, E101Q, E101R, E101K, E101F, E101Y, E101W, E131D and T150I, or one or more of N40R, N40K, D43N, D43Q, D43R, D43K, E44N, E44Q, E44R, E44K, E101N, E101Q, E101R, E101K, E101F, E101Y, E101W and E131D. The variant may include any number of these substitutions and any combination of these substitutions. In (i), the variant preferably includes S54P and / or S57P. In (i), the mutant preferably includes (a) S54P and / or S57P and (b) a mutation at one or more of Y51, N55, and F56, such as Y51, N55, F56, Y51 / N55, Y51 / F56, N55 / F56, or Y51 / N55 / F56. The mutation at one or more of Y51, N55, and F56 can be any of the mutations described below. In (i), the variant preferably includes F56A / S57P or S54P / F56A. The variant preferably includes T150I. Alternatively, the variant preferably comprises a mutation at one or more of (a) T150I and (b) Y51, N55 and F56, such as a mutation at Y51, N55, F56, Y51 / N55, Y51 / F56, N55 / F56 or Y51 / N55 / F56.

[0239] In (i), the variant preferably comprises Q62R or Q62K. Alternatively, the variant preferably comprises (a) Q62R or Q62K and (b) a mutation at one or more of Y51, N55 and F56, such as Y51, N55, F56, Y51 / N55, Y51 / F56, N55 / F56 or Y51 / N55 / F56. The variant may comprise D43N, E44N, Q62R or Q62K or any combination thereof, such as D43N, E44N, Q62R, Q62K, D43N / E44N, D43N / Q62R, D43N / Q62K, E44N / Q62R, E44N / Q62K, D43N / E44N / Q62R or D43N / E44N / Q62K. Alternatively, the variant preferably includes (a) D43N, E44N, Q62R, Q62K, D43N / E44N, D43N / Q62R, D43N / Q62K, E44N / Q62R, E44N / Q62K, D43N / E44N / Q62R or D43N / E44N / Q62K and (b) a mutation at one or more positions among Y51, N55 and F56, such as at Y51, N55, F56, Y51 / N55, Y51 / F56, N55 / F56 or Y51 / N55 / F56.

[0240] In (i), the variant preferably comprises D43N.

[0241] In (i), the variant preferably comprises E101R, E101S, E101F or E101N.

[0242] In (i), the variant preferably comprises E124N, E124Q, E124R, E124K, E124F, E124Y, E124W or E124D, such as E124N.

[0243] In (i), the variant preferably comprises R142E and R142N.

[0244] In (i), the variant preferably comprises R97N, R97G or R97L.

[0245] In (i), the variant preferably comprises R192E and R192N.

[0246] In (ii), the variants preferably include F56N / N55Q, F56N / N55R, F56N / N55K, F56N / N55S, F56N / N55G, F56N / N55A, F56N / N55T, F56Q / N55Q, F56Q / N55R, F56Q / N55K, F56Q / N55S, F56Q / N55G, F56Q / N55A, F56Q / N55T, F56R / N55Q, F56R / N55R, F56R / N55K, F56R / N55S, F56R / N55G, F56R / N55A, F56R / N55T, F56S / N55Q, F56S / N55R, F56S / N55K, F56S / N55S, F56S / N55G, F56S / N55A, F56S / N55T, F56G / N55Q, F56G / N55R, F56G / N55K, F56G / N55S, F56G / N55G, F56G / N55A, F56G / N55T, F56A / N55Q, F56A / N55R, F56A / N55K, F56A / N55S, F56A / N55G, F56A / N55A, F56A / N55T, F56K / N55Q, F56K / N55R, F56K / N55K, F56K / N55S, F56K / N55G, F56K / N55A, F56K / N55T, F56N / Y51L, F56N / Y51V, F56N / Y51A, F56N / Y51N, F56N / Y51Q, F5…N55Q / Y51V,N55Q / Y51A,N55Q / Y51N,N55Q / Y51Q,N55Q / Y51S / N55Q / Y51G,N55R / Y51L,N55R / Y51V,N55R / Y51A,N55R / Y51N,N55R / Y51Q,N55R / Y51S / N55R / Y51G,N55K / Y51L,N55K / Y51V,N55K / Y51A,N55K / Y51N,N55K / Y51Q,N55K / Y51S / N55K / Y51G,N55S / Y51L,N55S / Y51V,N55S / Y51A,N55S / Y51N,N55S / Y51Q,N55S / Y51S / N55S / Y51G,N55G / Y51L,N55G / Y51V,N55G / Y51A,N55G / Y51N,N55G / Y51Q,N55G / Y51SN55G / Y51G,N55A / Y51L,N55A / Y51V,N55A / T51A,N55A / T51N,N55A / Y51Q,N55AY51S N55A / Y51G,N55T / Y51L,N55T / Y51V,N55T / T51A,N55T / T51N,N55T / Y51Q,N55T / Y51S,N55T / Y51G,F56N / N55Q / Y51L,F56N / N55Q / Y51V,F56N / N55Q / Y51A,F56N / N55Q / Y51N,F56N / N55Q / Y51Q,F56N / N55Q / Y51S,F56N / N55Q / Y51G,F56N / N55R / Y51L,F56N / N55R / Y51V,F56N / N55R / Y51A,F56N / N55R / Y51N,F56N / N55R / Y51Q,F56N / N55R / Y51S,F56N / N55R / Y51G,F56N / N55K / Y51LF56N / N55K / Y51V,F56N / N55K / Y51A,F56N / N55K / Y51N,F56N / N55K / Y51Q,F56N / N55K / Y51S,F56N / N55K / Y51G,F56N / N55S / Y51L,F56N / N55S / Y51V,F56N / N55S / Y51A,F56N / N55S / Y51N,F56N / N55S / Y51Q,F56N / N55S / Y51S,F56N / N55S / Y51G,F56N / N55G / Y51L,F56N / N55G / Y51V,F56N / N55G / Y51A,F56N / N55G / Y51N,F56N / N55G / Y51Q,F56N / N55G / Y51S,F56N / N55G / Y51G,F56N / N55A / Y51L,F56N / N55A / Y51V,F56N / N55A / Y51A,F56N / N55A / Y51N,F56N / N55A / Y51Q,F56N / N55A / Y51S,F56N / N55A / Y51G,F56N / N55T / Y51L,F56N / N55T / Y51V,F56N / N55T / Y51A,F56N / N55T / Y51N,F56N / N55T / Y51Q,F56N / N55T / Y51S,F56N / N55T / Y51G,F56Q / N55Q / Y51L,F56Q / N55Q / Y51V,F56Q / N55Q / Y51A,F56Q / N55Q / Y51N,F56Q / N55Q / Y51Q,F56Q / N55Q / Y51S,F56Q / N55Q / Y51G,F56Q / N55R / Y51L,F56Q / N55R / Y51V,F56Q / N55R / Y51A,F56Q / N55R / Y51N,F56Q / N55R / Y51Q,F56Q / N55R / Y51S,F56Q / N55R / Y51G,F56Q / N55K / Y51L,F56Q / N55K / Y51V,F56Q / N55K / Y51A,F56Q / N55K / T51N,F56Q / N55K / Y51Q,F56Q / N55K / Y51S,F56Q / N55K / Y51G,F56Q / N55S / Y51L,F56Q / N55S / Y51V,F56Q / N55S / Y51A,F56Q / N55S / Y51N,F56Q / N55S / Y51Q,F56Q / N55S / Y51S,F56Q / N55S / Y51G,F56Q / N55G / Y51L,F56Q / N55G / Y51V,F56Q / N55G / Y51A,F56Q / N55G / Y51N,F56Q / N55G / Y51Q,F56Q / N55G / Y51S,F56Q / N55G / Y51G,F56Q / N55A / Y51L,F56Q / N55A / Y51V,F56Q / N55A / Y51A,F56Q / N55A / Y51N,F56Q / N55A / Y51Q,F56Q / N55A / Y51S,F56Q / N55A / Y51G,F56Q / N55T / Y51L,F56Q / N55T / Y51V,F56Q / N55T / Y51A,F56Q / N55T / Y51N,F56Q / N55T / Y51Q,F56Q / N55T / Y51S,F56Q / N55T / Y51G,F56R / N55Q / Y51L,F56R / N55Q / Y51V,F56R / N55Q / Y51A,F56R / N55Q / Y51N,F56R / N55Q / Y51Q,F56R / N55Q / Y51S,F56R / N55Q / Y51G,F56R / N55R / Y51L,F56R / N55R / Y51V,F56R / N55R / Y51A,F56R / N55R / Y51N,F56R / N55R / Y51Q,F56R / N55R / Y51S,F56R / N55R / Y51G,F56R / N55K / Y51L,F56R / N55K / Y51V,F56R / N55K / Y51A,F56R / N55K / Y51N,F56R / N55K / Y51Q,F56R / N55K / Y51S,F56R / N55K / Y51G,F56R / N55S / Y51L,F56R / N55S / Y51V,F56R / N55S / Y51A,F56R / N55S / Y51N,F56R / N55S / Y51Q,F56R / N55S / Y51S,F56R / N55S / Y51G,F56R / N55G / Y51L,F56R / N55G / Y51V,F56R / N55G / Y51A,F56R / N55G / Y51N,F56R / N55G / Y51Q,F56R / N55G / Y51S,F56R / N55G / Y51G;F56R / N55A / Y51L,F56R / N55A / Y51V,F56R / N55A / Y51A,F56R / N55A / Y51N,F56R / N55A / Y51Q,F56R / N55A / Y51S,F56R / N55A / Y51GF56R / N55T / Y51L,F56R / N55T / Y51V,F56R / N55T / Y51A,F56R / N55T / Y51N,F56R / N55T / Y51QF56R / N55T / Y51S,F56R / N55T / Y51G,F56S / N55Q / Y51L,F56S / N55Q / Y51V,F56S / N55Q / Y51AF56S / N55Q / Y51N,F56S / N55Q / Y51Q,F56S / N55Q / Y51S,F56S / N55Q / Y51G,F56S / N55R / Y51LF56S / N55R / Y51V,F56S / N55R / Y51A,F56S / N55R / Y51N,F56S / N55R / Y51Q,F56S / N55R / Y51SF56S / N55R / Y51G,F56S / N55K / Y51L,F56S / N55K / Y51V,F56S / N55K / Y51A,F56S / N55K / Y51NF56S / N55K / Y51Q,F56S / N55K / Y51S,F56S / N55K / Y51G,F56S / N55S / Y51L,F56S / N55S / Y51VF56S / N55S / Y51A,F56S / N55S / Y51N,F56S / N55S / Y51Q,F56S / N55S / Y51S,F56S / N55S / Y51GF56S / N55G / Y51L,F56S / N55G / Y51V,F56S / N55G / Y51A,F56S / N55G / Y51N,F56S / N55G / Y51QF56S / N55G / Y51S,F56S / N55G / Y51G,F56S / N55A / Y51L,F56S / N55A / Y51V,F56S / N55A / Y51AF56S / N55A / Y51N,F56S / N55A / Y51Q,F56S / N55A / Y51S,F56S / N55A / Y51G,F56S / N55T / Y51LF56S / N55T / Y51V,F56S / N55T / Y51A,F56S / N55T / Y51N,F56S / N55T / Y51Q,F56S / N55T / Y51SF56S / N55T / Y51G,F56G / N55Q / Y51L,F56G / N55Q / Y51V,F56G / N55Q / Y51A,F56G / N55Q / Y51NF56G / N55Q / Y51Q,F56G / N55Q / Y51S,F56G / N55Q / Y51G;F56G / N55R / Y51L,F56G / N55R / Y51VF56G / N55R / Y51A,F56G / N55R / Y51N,F56G / N55R / Y51Q,F56G / N55R / Y51S,F56G / N55R / Y51GF56G / N55K / Y51L,F56G / N55K / Y51V,F56G / N55K / Y51A,F56G / N55K / Y51N,F56G / N55K / Y51Q F56G / N55K / Y51S,F56G / N55K / Y51G,F56G / N55S / Y51L,F56G / N55S / Y51V,F56G / N55S / Y51A F56G / N55S / Y51N,F56G / N55S / Y51Q,F56G / N55S / Y51S,F56G / N55S / Y51G,F56G / N55G / Y51L F56G / N55G / Y51V,F56G / N55G / Y51A,F56G / N55G / Y51N,F56G / N55G / Y51Q,F56G / N55G / Y51S F56G / N55G / Y51G,F56G / N55A / Y51L,F56G / N55A / Y51V,F56G / N55A / Y51A,F56G / N55A / Y51N F56G / N55A / Y51Q,F56G / N55A / Y51S,F56G / N55A / Y51G,F56G / N55T / Y51L,F56G / N55T / Y51V,F56G / N55T / Y51A,F56G / N55T / Y51N,F56G / N55T / Y51Q,F56G / N55T / Y51S,F56G / N55T / Y51G,F56A / N55Q / Y51L,F56A / N55Q / Y51V,F56A / N55Q / Y51A,F56A / N55Q / Y51N,F56A / N55Q / Y51Q,F56A / N55Q / Y51S,F56A / N55Q / Y51G,F56A / N55R / Y51L,F56A / N55R / Y51 V,F56A / N55R / Y51A,F56A / N55R / Y51N,F56A / N55R / Y51Q,F56A / N55R / Y51S,F56A / N55R / Y51G,F56A / N55K / Y51L,F56A / N55K / Y51V,F56A / N55K / Y51A,F56A / N55K / Y51N,F56A / N55K / Y51Q,F56A / N55K / Y51S,F56A / N55K / Y51G,F56A / N55S / Y51L,F56A / N55S / Y51V,F56A / N55S / Y51A,F56A / N55S / Y51N,F56A / N55S / Y51Q,F56A / N55S / Y51S,F56A / N55S / Y51G,F56A / N55G / Y51L,F56A / N55G / Y51V,F56A / N55G / Y51A,F56A / N55G / Y51N,F56A / N55G / Y51Q,F56A / N55G / Y51S,F56A / N55G / Y51G,F56A / N55A / Y51L,F56A / N55A / Y51V,F56A / N55A / Y51A,F56A / N55A / Y51N,F56A / N55A / Y51Q,F56A / N55A / Y51S,F56A / N55A / Y51G,F56A / N55T / Y51L,F56A / N55T / Y51V,F56A / N55T / Y51A,F56A / N55T / Y51N,F56A / N55T / Y51Q,F56A / N55T / Y51S,F56A / N55T / Y51G,F56K / N55Q / Y51L,F56K / N55Q / Y51V,F56K / N55Q / Y51A,F56K / N55Q / Y51N,F56K / N55Q / Y51Q,F56K / N55Q / Y51S,F56K / N55Q / Y51G, F56K / N55R / Y51L, F56K / N55R / Y51V, F56K / N55R / Y51A, F56K / N55R / Y51N, F56K / N55R / Y51Q, F56K / N55R / Y51S, F56K / N55R / Y51G, F56K / N55K / Y51L, F56K / N55K / Y51V, F56K / N55K / Y51A, F56K / N55K / Y51N, F56K / N55K / Y51Q, F56K / N55K / Y51S, F56K / N55K / Y51G, F56K / N55S / Y51L, F56K / N55S / Y51V, F56K / N55S / Y51A, F56K / N55S / Y51N, F56K / N55S / Y51Q, F56K / N55S / Y51S, F56K / N55S / Y51G, F56K / N55G / Y51L, F56K / N55G / Y51V, F56K / N55G / Y51A, F56K / N55G / Y51N, F56K / N55G / Y51Q, F56K / N55G / Y51S, F56K / N55G / Y51G, F56K / N55A / Y51L, F56K / N55A / Y51V, F56K / N55A / Y51A, F56K / N55A / Y51N, F56K / N55A / Y51Q, F56K / N55A / Y51S, F56K / N55A / Y51G, F56K / N55T / Y51L, F56K / N55T / Y51V, F56K / N55T / Y51A, F56K / N55T / Y51N, F56K / N55T / Y51Q, F56K / N55T / Y51S, F56K / N55T / Y51G, F56E / N55R, F56E / N55K, F56D / N55R, F56D / N55K, F56R / N55E, F56R / N55D, F56K / N55E or F56K / N55D.,

[0247] In (ii), the variant preferably comprises Y51R / F56Q, Y51N / F56N, Y51M / F56Q, Y51L / F56Q, Y51I / F56Q, Y51V / F56Q, Y51A / F56Q, Y51P / F56Q, Y51G / F56Q, Y51C / F56Q, Y51Q / F56Q, Y51N / F56Q, Y51S / F56Q, Y51E / F56Q, Y51D / F56Q, Y51K / F56Q or Y51H / F56Q.

[0248] In (ii), the variant preferably comprises Y51T / F56Q, Y51Q / F56Q or Y51A / F56Q.

[0249] In (ii), the variant preferably comprises Y51T / F56F, Y51T / F56M, Y51T / F56L, Y51T / F56I, Y51T / F56V, Y51T / F56A, Y51T / F56P, Y51T / F56G, Y51T / F56C, Y51T / F56Q, Y51T / F56N, Y51T / F56T, Y51T / F56S, Y51T / F56E, Y51T / F56D, Y51T / F56K, Y51T / F56H or Y51T / F56R.

[0250] In (ii), the variant preferably comprises Y51T / N55Q, Y51T / N55S or Y51T / N55A.

[0251] In (ii), the variant preferably comprises Y51A / F56F, Y51A / F56L, Y51A / F56I, Y51A / F56V, Y51A / F56A, Y51A / F56P, Y51A / F56G, Y51A / F56C, Y51A / F56Q, Y51A / F56N, Y51A / F56T, Y51A / F56S, Y51A / F56E, Y51A / F56D, Y51A / F56K, Y51A / F56H or Y51A / F56R.

[0252] In (ii), the variant preferably comprises Y51C / F56A, Y51E / F56A, Y51D / F56A, Y51K / F56A, Y51H / F56A, Y51Q / F56A, Y51N / F56A, Y51S / F56A, Y51P / F56A or Y51V / F56A.

[0253] In (xi), the variant preferably comprises deletion of Y51 / P52, Y51 / P52 / A53, P50 to P52, P50 to A53, K49 to Y51, K49 to A53 and substitution with a single proline (P), K49 to S54 and substitution with a single P, Y51 to A53, Y51 to S54, N55 / F56, N55 to S57, N55 / F56 and substitution with a single P, N55 / F56 and substitution with a single glycine (G), N55 / F56 and substitution with a single alanine (A), N55 / F56 and substitution with P and Y Replacement with single P and Y51Q, N55 / F56 and single P and Y51S, N55 / F56 and single glycine (G) and Y51N, N55 / F56 and single G and Y51Q, N55 / F56 and single G and Y51S, N55 / F56 and single alanine (A) and Y51N, N55 / F56 and single alanine (A), Y51Q or N55 / F56 and single alanine (A) and Y51S.

[0254] More preferably, the variant comprises D195N / E203N, D195Q / E203N, D195N / E203Q,

[0255] D195Q / E203Q, E201N / E203N, E201Q / E203N, E201N / E203Q, E201Q / E203Q, E185N / E203Q,

[0256] E185Q / E203Q,E185N / E203N, E185Q / E203N, D195N / E201N / E203N, D195Q / E201N / E203N,

[0257] D195N / E201Q / E203N, D195N / E201N / E203Q, D195Q / E201Q / E203N, D195Q / E201N / E203Q,

[0258] D195N / E201Q / E203Q,D195Q / E201Q / E203Q, D149N / E201N, D149Q / E201N, D149N / E201Q,

[0259] D149Q / E201Q, D149N / E201N / D195N, D149Q / E201N / D195N, D149N / E201Q / D195N,

[0260] D149N / E201N / D195Q,D149Q / E201Q / D195N,D149Q / E201N / D195Q,D149N / E201Q / D195Q,

[0261] D149Q / E201Q / D195Q,D149N / E203N,D149Q / E203N,D149N / E203Q,D149Q / E203Q,

[0262] D149N / E185N / E201N,D149Q / E185N / E201N,D149N / E185Q / E201N,D149N / E185N / E201Q,

[0263] D149Q / E185Q / E201N, D149Q / E185N / E201Q, D149N / E185Q / E201Q, D149Q / E185Q / E201Q,

[0264] D149N / E185N / E203N,D149Q / E185N / E203N,D149N / E185Q / E203N,D149N / E185N / E203Q,

[0265] D149Q / E185Q / E203N, D149Q / E185N / E203Q, D149N / E185Q / E203Q, D149Q / E185Q / E203Q,

[0266] D149N / E185N / E201N / E203N,D149Q / E185N / E201N / E203N,D149N / E185Q / E201N / E203N,

[0267] D149N / E185N / E201Q / E203N,D149N / E185N / E201N / E203Q,D149Q / E185Q / E201N / E203N,

[0268] D149Q / E185N / E201Q / E203N,D149Q / E185N / E201N / E203Q,D149N / E185Q / E201Q / E203N,

[0269] D149N / E185Q / E201N / E203Q, D149N / E185N / E201Q / E203Q, D149Q / E185Q / E201Q / E203Q,

[0270] D149Q / E185Q / E201N / E203Q,D149Q / E185N / E201Q / E203Q,D149N / E185Q / E201Q / E203Q,

[0271] D149Q / E185Q / E201Q / E203N,D149N / E185N / D195N / E201N / E203N,

[0272] D149Q / E185N / D195N / E201N / E203N,D149N / E185Q / D195N / E201N / E203N,

[0273] D149N / E185N / D195Q / E201N / E203N,D149N / E185N / D195N / E201Q / E203N,

[0274] D149N / E185N / D195N / E201N / E203Q,D149Q / E185Q / D195N / E201N / E203N,

[0275] D149Q / E185N / D195Q / E201N / E203N,D149Q / E185N / D195N / E201Q / E203N,

[0276] D149Q / E185N / D195N / E201N / E203Q,D149N / E185Q / D195Q / E201N / E203N,

[0277] D149N / E185Q / D195N / E201Q / E203N,D149N / E185Q / D195N / E201N / E203Q,

[0278] D149N / E185N / D195Q / E201Q / E203N,D149N / E185N / D195Q / E201N / E203Q,

[0279] D149N / E185N / D195N / E201Q / E203Q,D149Q / E185Q / D195Q / E201N / E203N,

[0280] D149Q / E185Q / D195N / E201Q / E203N,D149Q / E185Q / D195N / E201N / E203Q,

[0281] D149Q / E185N / D195Q / E201Q / E203N,D149Q / E185N / D195Q / E201N / E203Q,

[0282] D149Q / E185N / D195N / E201Q / E203Q,D149N / E185Q / D195Q / E201Q / E203N,

[0283] D149N / E185Q / D195Q / E201N / E203Q,D149N / E185Q / D195N / E201Q / E203Q,

[0284] D149N / E185N / D195Q / E201Q / E203Q,D149Q / E185Q / D195Q / E201Q / E203N,

[0285] D149Q / E185Q / D195Q / E201N / E203Q,D149Q / E185Q / D195N / E201Q / E203Q,

[0286] D149Q / E185N / D195Q / E201Q / E203Q,D149N / E185Q / D195Q / E201Q / E203Q,

[0287] D149Q / E185Q / D195Q / E201Q / E203Q,D149N / E185R / E201N / E203N,D149Q / E185R / E201N / E203N,

[0288] D149N / E185R / E201Q / E203N,D149N / E185R / E201N / E203Q,D149Q / E185R / E201Q / E203N,

[0289] D149Q / E185R / E201N / E203Q,D149N / E185R / E201Q / E203Q,D149Q / E185R / E201Q / E203Q,

[0290] D149R / E185N / E201N / E203N,D149R / E185Q / E201N / E203N,D149R / E185N / E201Q / E203N,

[0291] D149R / E185N / E201N / E203Q,D149R / E185Q / E201Q / E203N,D149R / E185Q / E201N / E203Q,

[0292] D149R / E185N / E201Q / E203Q,D149R / E185Q / E201Q / E203Q,D149R / E185N / D195N / E201N / E203N,

[0293] D149R / E185Q / D195N / E201N / E203N,D149R / E185N / D195Q / E201N / E203N,

[0294] D149R / E185N / D195N / E201Q / E203N,D149R / E185Q / D195N / E201N / E203Q,

[0295] D149R / E185Q / D195Q / E201N / E203N,D149R / E185Q / D195N / E201Q / E203N,

[0296] D149R / E185Q / D195N / E201N / E203Q,D149R / E185N / D195Q / E201Q / E203N,

[0297] D149R / E185N / D195Q / E201N / E203Q,D149R / E185N / D195N / E201Q / E203Q,

[0298] D149R / E185Q / D195Q / E201Q / E203N,D149R / E185Q / D195Q / E201N / E203Q,

[0299] D149R / E185Q / D195N / E201Q / E203Q,D149R / E185N / D195Q / E201Q / E203Q,

[0300] D149R / E185Q / D195Q / E201Q / E203Q,D149N / E185R / D195N / E201N / E203N,

[0301] D149Q / E185R / D195N / E201N / E203N,D149N / E185R / D195Q / E201N / E203N,

[0302] D149N / E185R / D195N / E201Q / E203N,D149N / E185R / D195N / E201N / E203Q,

[0303] D149Q / E185R / D195Q / E201N / E203N,D149Q / E185R / D195N / E201Q / E203N,

[0304] D149Q / E185R / D195N / E201N / E203Q,D149N / E185R / D195Q / E201Q / E203N,

[0305] D149N / E185R / D195Q / E201N / E203Q,D149N / E185R / D195N / E201Q / E203Q,

[0306] D149Q / E185R / D195Q / E201Q / E203N,D149Q / E185R / D195Q / E201N / E203Q,

[0307] D149Q / E185R / D195N / E201Q / E203Q,D149N / E185R / D195Q / E201Q / E203Q,

[0308] D149Q / E185R / D195Q / E201Q / E203Q,D149N / E185R / D195N / E201R / E203N,

[0309] D149Q / E185R / D195N / E201R / E203N,D149N / E185R / D195Q / E201R / E203N,

[0310] D149N / E185R / D195N / E201R / E203Q,D149Q / E185R / D195Q / E201R / E203N,

[0311] D149Q / E185R / D195N / E201R / E203Q,D149N / E185R / D195Q / E201R / E203Q,

[0312] D149Q / E185R / D195Q / E201R / E203Q,E131D / K49R,E101N / N102F,E101N / N102Y,E101N / N102W,

[0313] E101F / N102F,E101F / N102Y,E101F / N102W,E101Y / N102F,E101Y / N102Y,E101Y / N102W,

[0314] E101W / N102F, E101W / N102Y, E101W / N102W, E101N / N102R, E101F / N102R, E101Y / N102R or E101W / N102F.

[0315] Preferred variants of the invention that form the pore, in which fewer nucleotides contribute to the current as the polynucleotide moves through the pore, include Y51A / F56A, Y51A / F56N, Y51I / F56A, Y51L / F56A, Y51T / F56A, Y51I / F56N, Y51L / F56N or Y51T / F56N or more preferably Y51I / F56A, Y51L / F56A or Y51T / F56A. As described above, this makes it easier to identify a direct relationship between the current observed (as the polynucleotide moves through the pore) and the polynucleotide.

[0316] Preferred variants that form pores showing increased range include mutations at the following positions:

[0317] Y51, F56, D149, E185, E201, and E203;

[0318] N55 and F56;

[0319] Y51 and F56;

[0320] Y51, N55 and F56; or

[0321] F56 and N102.

[0322] Preferred variations for forming pores that exhibit increased range include:

[0323] Y51N, F56A, D149N, E185R, E201N, and E203N;

[0324] N55S and F56Q;

[0325] Y51A and F56A;

[0326] Y51A and F56N;

[0327] Y51I and F56A;

[0328] Y51L and F56A;

[0329] Y51T and F56A;

[0330] Y51I and F56N;

[0331] Y51L and F56N;

[0332] Y51T and F56N;

[0333] Y51T and F56Q;

[0334] Y51A, N55S, and F56A;

[0335] Y51A, N55S, and F56N;

[0336] Y51T, N55S and F56Q; or

[0337] F56Q and N102R.

[0338] Preferred variants that form the pore, in which fewer nucleotides contribute to the current as the polynucleotide moves through the pore, include mutations at the following positions:

[0339] N55 and F56, e.g., N55X and F56Q, wherein X is any amino acid; or

[0340] Y51 and F56, such as Y51X and F56Q, wherein X is any amino acid.

[0341] Preferred variants that form pores that exhibit increased flux include mutations at the following positions:

[0342] D149, E185 and E203;

[0343] D149, E185, E201, and E203; or

[0344] D149, E185, D195, E201 and E203.

[0345] Preferred variations for forming pores that exhibit increased flux include:

[0346] D149N, E185N and E203N;

[0347] D149N, E185N, E201N and E203N;

[0348] D149N, E185R, D195N, E201N and E203N; or

[0349] D149N, E185R, D195N, E201R and E203N.

[0350] Preferred variants for forming the pore, wherein capture of the polynucleotide is increased, include the following mutations:

[0351] D43N / Y51T / F56Q;

[0352] E44N / Y51T / F56Q;

[0353] D43N / E44N / Y51T / F56Q;

[0354] Y51T / F56Q / Q62R;

[0355] D43N / Y51T / F56Q / Q62R;

[0356] E44N / Y51T / F56Q / Q62R; or

[0357] D43N / E44N / Y51T / F56Q / Q62R.

[0358] Preferred variants include the following mutations:

[0359] D149R / E185R / E201R / E203R or Y51T / F56Q / D149R / E185R / E201R / E203R;

[0360] D149N / E185N / E201N / E203N or Y51T / F56Q / D149N / E185N / E201N / E203N;

[0361] E201R / E203R or Y51T / F56Q / E201R / E203R

[0362] E201N / E203R or Y51T / F56Q / E201N / E203R;

[0363] E203R or Y51T / F56Q / E203R;

[0364] E203N or Y51T / F56Q / E203N;

[0365] E201R or Y51TF56Q / E201R;

[0366] E201N or Y51T / F56Q / E201N;

[0367] E185R or Y51T / F56Q / E185R;

[0368] E185N or Y51T / F56Q / E185N;

[0369] D149R or Y51T / F56Q / D149R;

[0370] D149N or Y51T / F56Q / D149N;

[0371] R142E or Y51T / F56Q / R142E;

[0372] R142N or Y51T / F56Q / R142N;

[0373] R192E or Y51T / F56Q / R192E; or

[0374] R192N or Y51T / F56Q / R192N.

[0375] Preferred variants include the following mutations:

[0376] Y51A / F56Q / E101N / N102R;

[0377] Y51A / F56Q / R97N / N102G;

[0378] Y51A / F56Q / R97N / N102R;

[0379] Y51A / F56Q / R97N;

[0380] Y51A / F56Q / R97G;

[0381] Y51A / F56Q / R97L;

[0382] Y51A / F56Q / N102R;

[0383] Y51A / F56Q / N102F;

[0384] Y51A / F56Q / N102G;

[0385] Y51A / F56Q / E101R;

[0386] Y51A / F56Q / E101F;

[0387] Y51A / F56Q / E101N; or

[0388] Y51A / F56Q / E101G.

[0389] The present invention also provides a mutant CsgG monomer comprising a variant of the sequence shown in SEQ ID NO: 390, wherein the variant comprises a mutation at position T150. Preferred variants that form pores exhibiting increased insertion include T150I. A mutation at position T150, such as T150I, can be combined with any of the above mutations or combinations of mutations.

[0390] The present invention also provides a mutant CsgG monomer comprising a variant of the sequence shown in SEQ ID NO: 390, wherein the variant comprises a combination of mutations present in the variants disclosed in the Examples.

[0391] Methods for introducing or replacing naturally occurring amino acids are well known in the art. For example, methionine (M) can be replaced with arginine (R) by replacing the codon for methionine (ATG) with the codon for arginine (CGT) at the relevant position in a polynucleotide encoding a mutant monomer. The polynucleotide can then be expressed as described below.

[0392] Methods for introducing or substituting non-naturally occurring amino acids are also well known in the art. For example, non-naturally occurring amino acids can be introduced by including synthetic aminoacyl-tRNAs in the IVTT system used to express mutant monomers. Alternatively, they can be introduced by expressing the mutant monomers in E. coli that are auxotrophic for the specific amino acids in the presence of synthetic (i.e., non-naturally occurring) analogs of those specific amino acids. If partial peptide synthesis is used to generate the mutant monomers, they can also be prepared by naked ligation.

[0393] Other monomers of the present invention

[0394] In another embodiment, the present invention provides a mutant CsgG monomer comprising a variant of the sequence set forth in SEQ ID NO:390, wherein the variant comprises a mutation at one or more of positions Y51, N55, and F56. The variant may comprise a mutation at position Y51; N55; F56; Y51 / N55; Y51 / F56; N55 / F56; or Y51 / N55 / F56. The variant preferably comprises a mutation at position Y51, N55, or F56. The variant may comprise any of the specific mutations discussed above at one or more of Y51, N55, and F56, and any combination thereof. One or more of Y51, N55, and F56 may be substituted with any amino acid. Y51 may be substituted with F, M, L, I, V, A, P, G, C, Q, N, T, S, E, D, K, H, or R, for example, A, S, T, N, or Q. N55 may be substituted with F, M, L, I, V, A, P, G, C, Q, T, S, E, D, K, H or R, such as A, S, T, or Q. F56 may be substituted with M, L, I, V, A, P, G, C, Q, N, T, S, E, D, K, H or R, such as A, S, T, N or Q.

[0395] The variant may further include one or more of the following: (i) one or more mutations at the following positions (i.e., mutations at one or more of the following positions): (i) N40, D43, E44, S54, S57, Q62, R97, E101, E124, E131, R142, T150, and R192, (iii) Q42R or Q42K; (iv) K49R; (v) N102R, N102F, N102Y, or N102W; (v)

[0015] The present invention also provides a method for preparing an amino acid sequence of the present invention, wherein the amino acid sequence of the present invention is selected from the group consisting of amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 51, 52, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 51, 52, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 51, 52, 30, 31, 32, 34, 35, 36, 37, 38, 40, 51, 52, 30, 31, 32, 34, 35, 36, 37, 38, 40, 51, 52, 30, 31, 32, 34, 35

[0396] Variants

[0397] In addition to the specific mutations described above, variants may include other mutations. Over the entire length of the amino acid sequence of SEQ ID NO: 390, based on amino acid identity, the variant is preferably at least 50% homologous to the sequence. More preferably, based on amino acid identity, the variant and the amino acid sequence of SEQ ID NO: 390 may be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous over the entire length. Over a continuous stretch of more than 100, for example 125, 150, 175 or more than 200 amino acids, there may be at least 80%, for example at least 85%, 90% or 95% amino acid identity ("strict hard homology").

[0398] Standard methods of the prior art can be used to determine homology. For example, the UWGCG software package provides the BESTFIT program that can be used to calculate homology, for example, using its default settings (Devereux et al. (1984) Nucleic Acids Research 12, p387-395). PILEUP and BLAST algorithms can be used to calculate homology or sequence sorting (for example, identifying equivalent residues or corresponding sequences (usually with their default settings)), for example, as described in Altschul SF (1993) J Mol Evol 36290-300; Altschul, S.F et al (1990) J Mol Biol 215403-10. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov).

[0399] SEQ ID NO: 390 is a wild-type CsgG monomer from Escherichia coli Str. K-12 substr. MC4100. Variants of SEQ ID NO: 390 may include any substitution present in another CsgG homolog. Preferred CsgG homologs are shown in SEQ ID NOs: 391 to 395 and 414 to 429. Variants may include a combination of one or more substitutions present in SEQ ID NOs: 391 to 395 and 414 to 429 compared to SEQ ID NO: 390.

[0400] In addition to the amino acid sequences discussed above, amino acid substitutions may be made to the amino acid sequence of SEQ ID NO: 390, for example, up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions are replacements of amino acids with other amino acids having similar chemical structures, similar chemical properties or similar side chain volumes. The introduced amino acid may have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge compared to the amino acid it replaces. Alternatively, the conservative substitution may introduce another aromatic or aliphatic amino acid to replace the previously existing aromatic or aliphatic amino acid. Conservative amino acid changes are well known in the art and can be selected based on the properties of the 20 main amino acids defined in Table 1 below. When amino acids have similar polarity, this can also be determined by reference to the hydrophilicity levels of the amino acid side chains in Table 2.

[0401] Table 1 - Chemical properties of amino acids

[0402]

[0403] Table 2 - Hydrophilicity Levels

[0404]

[0405]

[0406] One or more amino acid residues of the amino acid sequence of SEQ ID NO: 390 may be deleted from the polypeptide. Up to 1, 2, 3, 4, 5, 10, 20 or more residues may be deleted.

[0407] Variants may comprise fragments of SEQ ID NO: 390. The fragments retain pore-forming activity. The fragments may be at least 50, at least 100, at least 150, at least 200, or at least 250 amino acids in length. The fragments may be used to generate the pore. The fragments preferably include the transmembrane domain of SEQ ID NO: 390, i.e., K135-Q153 and S183-S208.

[0408] One or more amino acids may be added alternatively or additionally to the above polypeptides. Extensions may be provided at the amino-terminus or carboxyl-terminus of the amino acid sequence of SEQ ID NO:390 or a polypeptide variant thereof or a fragment thereof. The extensions may be relatively short, for example, 1 to 10 amino acids in length. Alternatively, the extensions 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. Other fusion proteins are discussed in more detail below.

[0409] As described above, variants are polypeptides having amino acid sequences that vary from the sequence of SEQ ID NO: 390 and retain its pore-forming ability. Variants typically include the regions of SEQ ID NO: 390 responsible for pore formation. The pore-forming ability of CsgG, which comprises a β-barrel structure, is provided by β-sheets in each subunit. Variants of SEQ ID NO: 390 typically include the β-sheet-forming regions of SEQ ID NO: 390, namely K135-Q153 and S183-S208. As long as the resulting variant retains its pore-forming ability, one or more modifications may be made to the β-sheet-forming regions of SEQ ID NO: 390. Variants of SEQ ID NO: 390 preferably include one or more modifications, such as substitutions, additions, or deletions, within its α-helical and / or loop structure regions.

[0410] Monomers derived from CsgG can be modified to facilitate their identification or purification, for example by adding a streptavidin tag or a signal sequence to promote secretion from cells, where the monomer does not naturally contain such a sequence. Other suitable tags are discussed in more detail below. The monomer can be labeled with a display tag. The display tag can be any suitable tag that allows the monomer to be detected. Suitable tags are described below.

[0411] Monomers derived from CsgG can also be prepared using D-amino acids. For example, monomers derived from CsgG can include a mixture of L-amino acids and D-amino acids. This is conventional in the art of preparing such proteins or peptides.

[0412] Monomers derived from CsgG contain one or more specific modifications to facilitate nucleotide discrimination. Monomers derived from CsgG may also contain other nonspecific modifications, provided they do not interfere with pore formation. Numerous nonspecific side chain modifications are known in the art and can be performed on the side chains of monomers derived from CsgG. Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH4, amidination with methylacetimidate, or acylation with acetic anhydride.

[0413] The CsgG-derived monomer can be produced using methods known in the art. The CsgG-derived monomer can be prepared synthetically or by recombinant methods. For example, the monomer can be synthesized by in vitro translation and transcription (IVTT). Suitable methods for producing the pore and monomer are discussed in International Application Nos. PCT / GB09 / 001690 (published as WO 2010 / 004273), PCT / GB09 / 001679 (published as WO 2010 / 004265), or PCT / GB10 / 000133 (published as WO 2010 / 086603). Methods for inserting the pore into the membrane are described below.

[0414] In some embodiments, the mutant monomer is chemically modified. The mutant monomer can be chemically modified in any manner and at any site. The mutant monomer is preferably chemically modified by connecting the molecule to one or more cysteines (cysteine ​​connection), the molecule to one or more lysines, the molecule to one or more non-natural amino acids, enzyme modification of the antigenic epitope or terminal modification. Suitable methods for implementing the above-mentioned modifications are well known in the art. The mutant monomer can be chemically modified by the connection of any molecule. For example, the mutant monomer can be chemically modified by connecting a dye or a fluorophore.

[0415] In some embodiments, the mutant monomer is chemically modified with a molecular adapter that promotes the interaction between the pore comprising the monomer and the target nucleotide or target polynucleotide sequence. The presence of the adapter improves the host-guest chemistry of the pore and the nucleotide or polynucleotide, thereby improving the sequencing capacity of the pore formed by the mutant monomer. The principles of host-guest chemistry are well known in the art. The adapter has an effect on the physical or chemical properties of the pore and can improve its interaction with the nucleotide or polynucleotide sequence. The adapter can change the charge of the barrel structure or channel structure of the pore, or specifically interact or bind to the nucleotide or polynucleotide sequence to promote its interaction with the pore.

[0416] The molecular adaptor is preferably a cyclic molecule, a cyclodextrin, a substance capable of hybridization, a DNA binder or chelator, a peptide or peptide analog, a synthetic polymer, an aromatic planar molecule, a positively charged small molecule or a small molecule capable of hydrogen bonding.

[0417] The adaptor can be circular. A circular adaptor preferably has the same symmetry as the pore. Because CsgG typically has eight or nine subunits around a central axis, the adaptor preferably has eight-fold or nine-fold symmetry. This will be discussed in more detail below.

[0418] The adapter typically interacts with the nucleotide or polynucleotide sequence through host-guest chemistry. The adapter is typically capable of interacting with the nucleotide or polynucleotide sequence. The adapter comprises one or more chemical groups capable of interacting with the nucleotide or polynucleotide sequence. The one or more chemical groups preferably interact with the nucleotide or polynucleotide sequence through non-covalent interactions, such as hydrophobic interactions, hydrogen bonds, van der Waals forces, π-cation interactions, and / or electrostatic forces. The one or more chemical groups capable of interacting with the nucleotide or polynucleotide sequence are preferably positively charged. The one or more chemical groups capable of interacting with the nucleotide or polynucleotide sequence more preferably comprise an amino group. The amino group can be attached to a primary, secondary, or tertiary carbon atom. Even more preferably, the adapter comprises a ring of amino groups, such as a ring of 6, 7, or 8 amino groups. Most preferably, the adapter comprises a ring of 8 amino groups. The protonated ring of amino groups can interact with negatively charged phosphate groups in the nucleotide or polynucleotide sequence.

[0419] Correct positioning of the adapter within the pore can be facilitated by host-guest chemistry between the adapter and the pore comprising the mutant monomer. The adapter preferably comprises one or more chemical groups capable of interacting with one or more amino acids within the pore. More preferably, the adapter comprises one or more chemical groups capable of interacting with one or more amino acids within the pore via non-covalent interactions, such as hydrophobic interactions, hydrogen bonds, van der Waals forces, π-cation interactions, and / or electrostatic forces. The chemical groups capable of interacting with one or more amino acids within the pore are typically hydroxyl groups or amine groups. The hydroxyl groups can be attached to primary, secondary, or tertiary carbon atoms. The hydroxyl groups can form hydrogen bonds with uncharged amino acids within the pore. Any adapter that facilitates the interaction between the pore and the nucleotide or polynucleotide sequence can be used.

[0420] Suitable adapters include, but are not limited to, cyclodextrins, cyclic peptides, and cucurbiturils. The adapter is preferably a cyclodextrin or a derivative thereof. The cyclodextrin or derivative thereof may be any of those disclosed in Eliseev, A.V., and Schneider, H.J. (1994) J. Am. Chem. Soc. 116, 6081-6088. The adapter is more preferably heptakis-6-amino-β-cyclodextrin (am7-βCD), 6-monodeoxy-6-monoamino-β-cyclodextrin (am1-βCD), or heptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu7-βCD). The guanidino group in gu7-βCD has a higher pKa value than the primary amine in am7-βCD, and therefore is more likely to be positively charged. The gu7-βCD adapter can be used to increase the residence time of the nucleotide in the pore, thereby improving the accuracy of the measured residual current and increasing the basal detection rate at high temperatures or low data acquisition rates.

[0421] If the succinimidyl 3-(2-pyridyldithio)propionate (SPDP) cross-linker is used as described in more detail below, the adapter is preferably heptakis(6-deoxy-6-amino)-6-N-mono(2-pyridyl)dithiopropionyl-β-cyclodextrin (am6amPDP1-βCD).

[0422] More suitable adapters include γ-cyclodextrin, which contains nine saccharide units (and thus has nine-fold symmetry). γ-cyclodextrin may contain a linker molecule, or may be modified to contain all or more of the modified saccharide units used in the above-described β-cyclodextrin examples.

[0423] The molecular adapter is preferably covalently attached to the mutant monomer. The adapter can be covalently attached to the pore by any method known in the art. The adapter is typically attached by chemical attachment. If the molecular adapter is attached via a cysteine ​​linker, one or more cysteines are preferably introduced into the mutant by substitution, for example into the barrel structure. The mutant monomer can be chemically modified by attaching a molecular adapter to one or more cysteines in the mutant monomer. The one or more cysteines can be naturally occurring, i.e., located at positions 1 and / or 215 in SEQ ID NO: 390. Alternatively, the mutant monomer can be chemically modified by attaching a molecule to one or more cysteines introduced at other positions. The cysteine ​​at position 215 can be removed, for example, by substitution, to ensure that the molecular adapter does not attach to positions other than the cysteine ​​at position 1 or to a cysteine ​​introduced at another position.

[0424] The reactivity of cysteine ​​residues can be enhanced by modifying their neighboring residues. For example, the addition of a basic group to the side chain of an arginine, histidine, or lysine residue will change the pKa of the cysteine ​​thiol group to the more reactive S - The pKa of the group. The reactivity of the cysteine ​​residue can be protected by a thiol protecting group such as dTNB. These protecting groups can react with one or more cysteine ​​residues of the mutant monomer before attaching the linker.

[0425] The molecule may be directly linked to the mutant monomer. Preferably, the molecule is linked to the mutant monomer via a linker, such as a chemical cross-linker or a peptide linker.

[0426] Suitable chemical cross-linking agents are well known in the art. Preferred cross-linking agents include 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-ylsulfanyl)propionate, ethyl 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-ylsulfanyl)butanoate, and 2,5-dioxopyrrolidin-1-yl 8-(pyridin-2-yldisulfanyl)octanoate. The most preferred cross-linking agent is succinimidyl 3-(2-pyridyldithio)propionate (SPDP). Typically, the molecule is covalently linked to the bifunctional cross-linker before the molecule / cross-linker complex is covalently linked to the mutant monomer, but it is also possible that the bifunctional cross-linker is covalently linked to the monomer before the bifunctional cross-linker / monomer complex is linked to the molecule.

[0427] The linker is preferably resistant to dithiothreitol (DTT). Suitable linkers include, but are not limited to, iodoacetamide-based and maleimide-based linkers.

[0428] In another embodiment, the monomers can be linked to polynucleotide binding proteins. This forms a modular sequencing system that can be used in the sequencing methods of the present invention. The following discusses polynucleotide binding proteins.

[0429] The polynucleotide-binding protein is preferably covalently linked to the mutant monomer. The protein can be covalently linked to the monomer using any method known in the art. The monomer and protein can be chemically fused or genetically fused. If the entire construct is expressed from a single polynucleotide sequence, the monomer and protein can be genetically fused. Genetic fusion of monomers to polynucleotide-binding proteins is discussed in International Application No. PCT / GB09 / 001679 (published as WO 2010 / 004265).

[0430] If the polynucleotide-binding protein is connected through a cysteine ​​linking group, the one or more cysteines are preferably introduced into the mutant by substitution. The one or more cysteines are preferably introduced into a ring region that has low conservation among homologues, which indicates that mutations or insertions can be tolerated. They are therefore suitable for connection to polynucleotide-binding proteins. In this class of embodiments, the naturally occurring cysteine ​​at position 251 can be removed. The reactivity of the cysteine ​​residue can be enhanced by modification as described above.

[0431] The polynucleotide binding protein can be linked to the mutant monomer directly or through one or more linkers. The molecule can be linked to the mutant monomer using a hybrid linker described in International Application No. PCT / GB10 / 000132 (published as WO 2010 / 086602). Alternatively, a peptide linker can be used. The peptide linker is an amino acid sequence. The length, flexibility and hydrophilicity of the peptide linker are generally designed so that it does not disrupt the function of the monomer and molecule. Preferred flexible peptide linkers have a chain length of 2 to 20, such as 4, 6, 8, 10 or 16 serines and / or glycines. More preferred flexible linkers include (SG)1, (SG)2, (SG)3, (SG)4, (SG)5and (SG)8, wherein S is serine and G is glycine. Preferred rigid linkers have a chain length of 2 to 30, such as 4, 6, 8, 16 or 24 prolines. More preferred rigid linkers include (P) 12 , where P is proline.

[0432] The mutant monomers can be chemically modified using molecular adaptors and polynucleotide binding proteins.

[0433] The molecule (with which the monomer is chemically modified) may be linked directly to the monomer or via a linker as disclosed in International Applications PCT / GB09 / 001690 (published as WO 2010 / 004273), PCT / GB09 / 001679 (published as WO 2010 / 004265) or PCT / GB10 / 000133 (published as WO 2010 / 086603).

[0434] Any of the proteins described herein, such as the mutant monomers and pores of the present invention, may be modified to facilitate their identification or purification, for example by the addition of histidine residues (his-tag), aspartic acid residues (asp-tag), streptavidin tags, flag tags, SUMO tags, GST tags or MBP tags, or by the addition of signal sequences that promote their secretion from cells where the polypeptide does not naturally contain such sequences. An alternative to introducing a genetic tag is to chemically react the tag to a native or modified position on the protein. An example of this would be reacting a gel shift reagent with a cysteine ​​engineered on the outside of the protein. This approach has been demonstrated as a method for separating hemolysin hetero-oligomers (Chem Biol. 1997 Jul; 4(7): 497-505).

[0435] Any of the proteins described herein, such as the mutant monomers and pores described herein, can be labeled with a revealing marker. The revealing marker can be any suitable marker that allows the protein to be detected. Suitable markers include, but are not limited to, fluorescent molecules, radioactive isotopes such as 125 I, 35 S, enzymes, antibodies, antigens, polynucleotides and ligands such as biotin.

[0436] Any of the proteins described herein, such as the monomers and pores described herein, can be produced synthetically or by recombinant methods. For example, the proteins can be synthesized by in vitro translation and transcription (IVTT). The amino acid sequence of the protein can be modified to include non-naturally occurring amino acids or to increase the stability of the protein. When the protein is produced synthetically, such amino acids can be introduced during the production process. The protein may also be altered after synthesis or recombinant production.

[0437] Proteins can also be prepared using D-amino acids. For example, the protein can include a mixture of L-amino acids and D-amino acids. This is conventional in the art of preparing such proteins or peptides.

[0438] The protein may also contain other nonspecific modifications, as long as they do not interfere with the function of the protein. Many nonspecific side chain modifications are known in the art and can be performed on the side chains of the protein. Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH4, amidination with methyl iminoacetate, or acylation with acetic anhydride.

[0439] Any of the proteins described herein, including the monomers and pores described herein, can be prepared using standard methods known in the art. Polynucleotide sequences encoding proteins can be derived and replicated using standard methods in the art. Polynucleotide sequences encoding proteins can be expressed in bacterial host cells using standard techniques in the art. The proteins can be prepared from recombinant expression vectors by expressing the polypeptide in situ in cells. The expression vectors optionally contain an inducible promoter to control expression of the polypeptide. These methods are described in Sambrook, J. and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[0440] Proteins can be produced on a large scale after purification from the organism in which they are produced or after recombinant expression using any protein liquid chromatography system. Typical protein liquid chromatography systems include FPLC, AKTA systems, Bio-Cad systems, Bio-Rad BioLogic systems, and Gilson HPLC systems.

[0441] Construct

[0442] The present invention also provides a construct comprising two or more covalently linked CsgG monomers, wherein at least one of the monomers is a mutant monomer as described herein. The construct of the present invention retains its ability to form a pore. This can be determined as described above. One or more constructs of the present invention can be used to form a pore for characterization, such as polynucleotide sequencing. The construct may comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 monomers. The construct preferably comprises two monomers. The two or more monomers may be the same or different.

[0443] At least one monomer in the construct is a mutant monomer as described herein. 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 monomers in the construct may be mutant monomers as described herein. All monomers in the construct are preferably mutant monomers as described herein. The mutant monomers may be the same or different. In a preferred embodiment, the construct comprises two mutant monomers as described herein.

[0444] The mutant monomers of the present invention in the construct preferably have approximately the same length or have the same length. The barrel-shaped structures of the mutant monomers of the present invention in the construct preferably have approximately the same length or have the same length. Length can be measured by number of amino acids and / or unit length.

[0445] The construct may include one or more monomers that are not mutant monomers of the present invention. CsgG mutant monomers that are not mutant monomers of the present invention include those comprising SEQ ID NO:

[0446] 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429 or a comparative variant of SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429, wherein none of the above amino acids / positions are mutated. At least one monomer in the construct may include SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429 or a comparative variant of the sequence shown in SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429. A comparative variant of SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429 is at least 50% homologous to 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429 over its entire sequence based on amino acid identity. More preferably, the control variant is at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous to the amino acid sequence of SEQ ID NO:390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429 based on amino acid identity over its entire length.

[0447] The monomers in the construct are preferably genetically fused. If the entire construct is expressed by a single polynucleotide sequence, the monomers can be genetically fused. The coding sequences of the monomers can be recombined in any manner to form a single polynucleotide sequence encoding the construct.

[0448] The monomers can be genetically fused in any conformation. The monomers can be fused via their terminal amino acids. For example, the amino terminus of one monomer can be fused to the carboxyl terminus of another monomer. The second and subsequent monomers in the construct (from amino to carboxyl) can contain a methionine at their amino termini (each fused to the carboxyl terminus of the previous monomer). For example, if M is a monomer (without an amino-terminal methionine) and mM is a monomer with an amino-terminal methionine, the construct can include the sequence M-mM, M-mM-mM, or M-mM-mM-mM. The presence of these methionines typically results from the expression of a start codon (i.e., ATG) at the 5' end of the polynucleotide encoding the second or subsequent monomer in the polynucleotide encoding the entire construct. The first monomer in the construct (from amino to carboxyl) can also include a methionine (e.g., mM-mM, mM-mM-mM, or mM-mM-mM-mM).

[0449] The two or more monomers can be genetically fused together directly. The monomers are preferably genetically fused using a linker. The linker can be designed to restrict the movement of the monomers. The preferred linker is an amino acid sequence (i.e., a peptide linker). Any of the peptide linkers described above can be used.

[0450] In another preferred embodiment, the monomers are chemically fused. If two monomers are chemically linked, for example, by a chemical cross-linking agent, then the two monomers are chemically fused. Any of the chemical cross-linking agents described below may be used. The linker can be attached to one or more cysteine ​​residues introduced into the mutant monomers of the present invention. Alternatively, the linker can be attached to the end of one of the monomers in the construct.

[0451] If the construct comprises different monomers, cross-linking of the monomers can be prevented by maintaining the concentration of the linker in a large excess of the monomers. Alternatively, a "lock and key" arrangement can be employed when two linkers are used. Only one end of each linker can be reacted together to form a longer linker, and the other end of each linker can be reacted with a different monomer. Such a linker is described in International Application No. PCT / GB10 / 000132 (Publication No. WO 2010 / 086602).

[0452] polynucleotides

[0453] The present invention also provides a polynucleotide sequence encoding a mutant monomer according to the present invention. The mutant monomer can be any one as described above. The polynucleotide sequence preferably includes a sequence based on nucleotide identity and a sequence of SEQ ID NO:389 that is at least 50%, 60%, 70%, 80%, 90% or 95% homologous over the entire sequence. More than 300, for example, 375, 450, 525 or more than 600 continuous nucleotide extension chains can have at least 80%, for example, at least 85%, 90% or 95% nucleotide identity ("strict homology"). Homology can be calculated as described above. The polynucleotide sequence can include a sequence different from SEQ ID NO:389 based on the degeneracy of the genetic code.

[0454] The present invention also provides the polynucleotide sequence encoding any genetic fusion construct of the present invention. The polynucleotide preferably includes two or more variants of the sequence shown in SEQ ID NO:389. The polynucleotide preferably includes two or more sequences with SEQ ID NO:389 sequence based on nucleotide identity having at least 50%, 60%, 70%, 80%, 90% or 95% homology over the entire sequence. More than 600, for example 750, 900, 1050 or 1200 continuous nucleotide extension chains, can have at least 80%, for example at least 85%, 90% or 95% nucleotide identity ("strict homology"). Homology can be calculated as described above.

[0455] Polynucleotide sequences can be derived and replicated using standard methods in the art. Chromosomal DNA encoding wild-type CsgG can be extracted from a pore-producing organism, such as Escherichia coli. The genes encoding the pore subunits can be amplified using PCR using specific primers. The amplified sequences can then be subjected to site-directed mutagenesis. Suitable methods for site-directed mutagenesis are known in the art and include, for example, combinatorial chain reactions. Polynucleotides encoding constructs of the present invention can be prepared using techniques known in the art, such as those described in Sambrook, J. and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[0456] The resulting polynucleotide sequence can then be incorporated into a recombinant replicable vector, such as a cloning vector. The vector can be used to replicate the polynucleotide in a compatible host cell. Therefore, the polynucleotide sequence can be prepared by introducing the polynucleotide into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions that cause the vector to replicate. The vector can be recovered from the host cell. Suitable host cells for polynucleotide cloning are known in the art and are described in more detail below.

[0457] The polynucleotide sequence can be cloned into a suitable expression vector. In the expression vector, the polynucleotide sequence is usually operably linked to a control sequence that can provide a host cell with expression of the coding sequence. The expression vector can be used to express the pore subunit.

[0458] The term "operably linked" refers to a juxtaposition wherein the described components are in a relationship permitting them to function in their intended manner. A control sequence "operably linked" to a coding sequence is linked in such a manner that expression of the coding sequence is achieved under conditions compatible with control sequences. Multiple copies of the same or different polynucleotide sequences may be introduced into the vector.

[0459] The expression vector can then be introduced into a suitable host cell. Therefore, the mutant monomer or construct of the present invention can be prepared by inserting the polynucleotide sequence into an expression vector, introducing the vector into a compatible bacterial host cell, and growing the host cell under conditions that can cause the expression of the polynucleotide sequence. The recombinantly expressed monomer or construct can self-assemble into a hole in the host cell membrane. Alternatively, the recombinant pore prepared in this way can be removed from the host cell and inserted into another membrane. When a hole comprising at least two different monomers or constructs is produced, the different monomers or constructs can be expressed separately in different host cells as described above, removed from the host cell and assembled into the hole in a separate membrane, such as a rabbit cell membrane or a synthetic membrane.

[0460] The vector can be, for example, a plasmid, virus or phage vector provided with an origin of replication, optionally a promoter for expressing the polynucleotide sequence, and optionally a regulator of the promoter. The vector can contain one or more selectable marker genes, for example, a tetracycline resistance gene. The promoter and other expression regulatory signals can be selected to be compatible with the host cell for which the expression vector is designed. T7, trc, lac, ara or lambda are commonly used. L promoter.

[0461] The host cell typically expresses the monomer or construct at high levels. Host cells transformed with the polynucleotide sequence are selected to be compatible with the expression vector used to transform the cell. The host cell is typically a bacterium and preferably Escherichia coli. Any cell with a λDE3 lysogen, such as C41(DE3), BL21(DE3), JM109(DE3), B834(DE3), TUNER, Origami, and Origami B, can express vectors containing a T7 promoter. In addition to the conditions described above, any of the methods cited in Cao et al, 2014, PNAS, Structure of the nonameric bacterial amyloid secretion channel, doi: 1411942111, and Goyal et al, 2014, Nature, 516, 250-253, structural and mechanistic insights into the bacterial amyloid secretion channel CsgG, can also be used to express the CsgG protein.

[0462] The present invention also includes methods for producing mutant monomers of the present invention or constructs of the present invention. The methods include expressing a polynucleotide of the present invention in a suitable host cell. The polynucleotide is preferably part of a vector and is preferably operably linked to a promoter.

[0463] hole

[0464] The present invention also provides various pores. The pores of the present invention are ideal for characterizing, for example, sequencing, polynucleotide sequences because they can distinguish between different nucleotides with high sensitivity. The pores can surprisingly distinguish between the four nucleotides in DNA and RNA. The pores of the present invention can even distinguish between methylated and unmethylated nucleotides. The pores of the present invention have surprisingly high base resolution. The pores exhibit nearly complete separation of all four DNA nucleotides. The pores further distinguish between deoxycytosine monophosphate (dCMP) and methyl-dCMP based on residence time in the pore and current flowing through the pore.

[0465] The pores of the present invention can also discriminate between different nucleotides under a range of conditions. In particular, the pores discriminate between nucleotides under conditions that are conducive to nucleic acid characterization, such as sequencing. The degree to which the pores of the present invention discriminate between different nucleotides can be controlled by varying the applied potential, salt concentration, buffer, temperature, and the presence of additives such as urea, betaine, and DTT. This allows for fine-tuning of pore function, particularly during sequencing. This will be discussed in more detail below. The pores of the present invention can also be used to identify polynucleotide polymers through interactions with one or more monomers, rather than on a nucleotide-by-nucleotide basis.

[0466] The pores of the present invention can be isolated, substantially isolated, purified, or substantially purified. A pore of the present invention is isolated or purified if it is completely free of any other components, such as liposomes or other pores. A pore is substantially isolated if it is mixed with a carrier or diluent that does not interfere with its intended use. For example, a pore is substantially isolated or substantially purified if it is present in a form that contains less than 10%, less than 5%, less than 2%, or less than 1% of other components, such as triblock copolymers, liposomes, or other pores. Alternatively, the pores of the present invention can be present in a membrane. Suitable membranes are described below.

[0467] The pores of the invention may exist as individual or single pores. Alternatively, the pores of the invention may exist in a homogenous or heterogenous population of two or more pores.

[0468] Homo-oligomeric pore

[0469] The present invention also provides a homo-oligomeric pore derived from CsgG comprising the same mutant monomers of the present invention. The homo-oligomeric pore may comprise any of the mutants of the present invention. The homo-oligomeric pore of the present invention is ideal for polynucleotide characterisation, such as sequencing. The homo-oligomeric pore of the present invention may have any of the advantages described above.

[0470] The homo-oligomeric pore may comprise any number of mutant monomers. The pore typically comprises at least 7, at least 8, at least 9 or at least 10 identical mutant monomers, for example, 7, 8, 9 or 10 mutant monomers. The pore preferably comprises 8 or 9 identical mutant monomers. One or more, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the mutant monomers are preferably chemically modified as discussed above.

[0471] The method for making the pores is discussed in more detail below.

[0472] Hetero-oligomeric pore

[0473] The present invention also provides hetero-oligomeric pores derived from CsgG comprising at least one mutant monomer of the present invention. The hetero-oligomeric pores of the present invention are ideal for polynucleotide characterization, such as sequencing. Hetero-oligomeric pores can be prepared using methods known in the art (e.g., Protein Sci. 2002 Jul;11(7):1813-24).

[0474] The hetero-oligomeric pore comprises sufficient monomers to form the pore. The monomers may be of any type. The pore typically comprises at least 7, at least 8, at least 9 or at least 10 monomers, for example 7, 8, 9 or 10 monomers. The pore preferably comprises 8 or 9 monomers.

[0475] In a preferred embodiment, all of the monomers (e.g., 10, 9, 8, or 7 monomers) are mutant monomers of the invention, and at least one of the monomers is different from the others. In a more preferred embodiment, the pore comprises eight or nine mutant monomers of the invention, and at least one of the monomers is different from the others. They may also all be different from each other.

[0476] The mutant monomers of the present invention in the pore preferably have approximately the same length or have the same length. The barrel-shaped structures of the mutant monomers of the present invention in the pore preferably have approximately the same length or have the same length. Length can be measured in terms of number of amino acids and / or length units.

[0477] In another preferred embodiment, at least one of the mutant monomers is not a mutant monomer of the present invention. In this embodiment, the remaining monomers are preferably mutant monomers of the present invention. Thus, the pore may include 9, 8, 7, 6, 5, 4, 3, 2 or 1 mutant monomers of the present invention. Any number of monomers in the pore may not be mutant monomers of the present invention. The pore preferably includes seven or eight mutant monomers of the present invention and one monomer that is not a monomer of the present invention. The mutant monomers of the present invention may be the same or different.

[0478] The mutant monomers of the present invention in the construct preferably have approximately the same length or have the same length. The barrel-shaped structures of the mutant monomers of the present invention in the construct preferably have approximately the same length or have the same length. Length can be measured using amino acid number and / or length units.

[0479] The pore may comprise one or more monomers that are not mutant monomers of the invention. Examples of CsgG monomers that are not mutant monomers of the invention include monomers comprising SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429, or control variants of SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 414, 421, 422, 423, 424, 425, 426, 427, 428 or 429, wherein none of the amino acids / positions described above in relation to the invention have been mutated / substituted. A reference variant of SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429 is typically at least 50% homologous to SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429 over its entire sequence based on amino acid identity. More preferably, the control variant can be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90% and more preferably at least 95%, 97% or 99% homologous to the amino acid sequence of SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429 over the entire sequence based on amino acid identity.

[0480] In all embodiments described above, one or more, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the mutant monomers are preferably chemically modified as described above.

[0481] The method for making the pores is discussed in more detail below.

[0482] Wells containing constructs

[0483] The present invention also provides a pore comprising at least one construct of the present invention. The construct of the present invention comprises two or more covalently linked monomers derived from CsgG, wherein at least one of the monomers is a mutant monomer of the present invention. That is, the construct must comprise more than one monomer. The pore contains sufficient constructs and, if necessary, sufficient monomers to form the pore. For example, an octameric pore may comprise (a) four constructs, each comprising two monomers, (b) two constructs, each comprising four monomers, or (b) one construct comprising two monomers and six monomers that do not form part of the construct. For example, a nonameric pore may comprise (a) four constructs, each comprising two monomers, and one monomer that does not form part of the construct, (b) two constructs, each comprising four monomers, and one monomer that does not form part of the construct, or (b) one construct comprising two monomers and seven monomers that do not form part of the construct. Other combinations of constructs and monomers will be contemplated by those skilled in the art.

[0484] At least two of the monomers in the pore are in the form of a construct of the invention. The construct, and therefore the pore, comprises at least one mutant monomer of the invention. The pore generally comprises at least 7, at least 8, at least 9 or at least 10 monomers in total, for example, 7, 8, 9 or 10 monomers (at least two of which must be in the construct). The pore preferably comprises eight or nine monomers (at least two of which must be in the construct).

[0485] The pore comprising the constructs may be a homo-oligomer (ie comprising identical constructs) or a hetero-oligomer (ie wherein at least one construct is different from the others).

[0486] The pore typically comprises (a) a construct comprising two monomers and (b) 5, 6, 7 or 8 monomers. The construct may be any of those described above. The monomers may be any of those described above, including mutant monomers of the invention, monomers comprising SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429, and mutant monomers comprising a control variant of SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 414, 421, 422, 423, 424, 425, 426, 427, 428 or 429 as described above.

[0487] Another typical pore comprises more than one construct of the invention, for example two, three or four constructs of the invention. If necessary, the pore further comprises sufficient additional monomers or constructs to form the pore. The additional monomer can be any of those described above, including mutant monomers of the present invention, monomers comprising SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429, and mutant monomers comprising a control variant of SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429 as described above. The additional construct may be any as described above or may be a construct comprising two or more covalently linked CsgG monomers, each comprising a monomer comprising SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429 and a monomer comprising a control variant of SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 414, 421, 422, 423, 424, 425, 426, 427, 428 or 429 as described above.

[0488] Further pores of the present invention only comprise constructs comprising two monomers, for example a pore may comprise 4, 5, 6, 7 or 8 constructs comprising two monomers. At least one construct is a construct of the present invention, i.e. at least one monomer in at least one construct, and preferably each monomer in at least one construct, is a mutant monomer of the present invention. All constructs comprising two monomers may be constructs of the present invention.

[0489] A specific pore of the present invention comprises four constructs of the present invention, each construct comprising two monomers, wherein at least one monomer in each construct, and preferably each monomer in each construct, is a mutant monomer of the present invention. The constructs can oligomerize into a pore having a structure such that only one monomer in each construct contributes to the pore channel. Typically, the other monomers in the construct will be outside the channel of the pore. For example, a pore of the present invention can comprise 7, 8, 9, or 10 constructs comprising two monomers, wherein the channel comprises 7, 8, 9, or 10 monomers.

[0490] Mutations can be introduced into constructs as described above. The mutations can be alternating, i.e., the mutations are different for each monomer in a two-monomer construct, and the constructs are assembled into homo-oligomers resulting in alternating modifications. That is, monomers comprising MutA and MutB are fused and assembled to form an AB:AB:AB:AB pore. Alternatively, the mutations can be adjacent, i.e., the same mutation is introduced into two monomers in one construct, which are then oligomerized with different mutant monomers or constructs. That is, a monomer comprising MutA is fused and then oligomerized with a monomer comprising MutB to form AA:B:B:B:B:B:B.

[0491] One or more monomers of the invention within a pore containing a construct may be chemically modified as described above.

[0492] Characterization of analytes

[0493] The present invention provides a method for determining the presence, absence, or one or more properties of a target analyte. The method involves contacting the target analyte with a CsgG pore or a mutant thereof (e.g., a pore of the present invention), causing the target analyte to move relative to, e.g., through, the pore, and obtaining one or more measurements as the analyte moves relative to the pore, thereby determining the presence, absence, or one or more properties of the analyte. The target analyte may also be referred to as a template analyte or an analyte of interest.

[0494] The method comprises contacting the target analyte with a CsgG pore or a mutant thereof (e.g., a pore of the invention), such that the target analyte migrates through the pore. The pore generally comprises at least 7, at least 8, at least 9, or at least 10 monomers, e.g., 7, 8, 9, or 10 monomers. The pore preferably comprises 8 or 9 identical monomers. One or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, of the monomers are preferably chemically modified as discussed above.

[0495] The CsgG pore may be derived from any organism. The CsgG pore may comprise a monomer comprising the sequence shown in SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429. The CsgG pore may comprise at least 7, at least 8, at least 9 or at least 10 monomers, for example 7, 8, 9 or 10 monomers, each comprising the sequence shown in SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429 (i.e. the pore is a homo-oligomer comprising identical monomers from the same organism). The CsgG pore may comprise any combination of monomers, each comprising the sequence shown in SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429. For example, the pore may comprise 7 monomers comprising the sequence shown in SEQ ID NO: 390 and 2 monomers comprising the sequence shown in SEQ ID NO: 391.

[0496] The CsgG mutant may comprise any number of mutant monomers, for example at least 7, at least 8, at least 9 or at least 10 monomers, for example 7, 8, 9 or 10 monomers. The mutant monomers may comprise comparative variants of the sequences shown in SEQ ID NO: 390, 391, 392, 393, 394, 395, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428 or 429. Comparative variants have been discussed above. The comparative variants must be capable of forming a pore and may have any percentage of homology to the pore of the invention as discussed above.

[0497] The CsgG mutant preferably comprises 9 monomers and at least one of the monomers is a variant of the sequence shown in SEQ ID NO: 390, the variant comprising: (a) a mutation at one or more of the following positions: N40, Q42, D43, E44, K49, Y51, S54, N55, F56, S57, Q62, E101, N102, E124, E131, R142, D149, T150, E185, R192, D195, E201 and E203, for example a mutation at one or more of the following positions: N40, Q42, D43, E44, K49, Y51, S54, N55, F56, S57, Q62 , E101, N102, E131, D149, T150, E185, D195, E201 and E203, or one or more mutations at one or more of the following positions: N40, Q42, D43, E44, K49, Y51, N55, F56, E101, N102, E131, D149, T150, E185, D195, E201 and E203; and / or (b) deletions at one or more of the following positions: F48, K49, P50, Y51, P52, A53, S54, N55, F56 and S57. The variant may comprise (a); (b); or (a) and (b). In (a), any number and combination of the following positions: N40, Q42, D43, E44, K49, Y51, S54, N55, F56, S57, Q62, E101, N102, R124, E131, R142, D149, E185, R192, D195, E201 and E203 can be mutated. As discussed above, mutation of one or more of Y51, N55 and F56 can reduce the number of nucleotides that contribute to the current as the polynucleotide moves through the pore, thereby making it easier to identify a direct relationship between the current observed as the polynucleotide moves through the pore and the polynucleotide.

[0498] In (b), any number and combination of the following positions: F48, K49, P50, Y51, P52, A53, S54, N55, F56 and S57 may be deleted. The variant may comprise any of the specific mutations / substitutions discussed above for the mutant monomers of the present invention, or a combination thereof.

[0499] Preferred variants for use in the methods of the invention include one or more of the following substitutions: (a) F56N, F56Q, F56R, F56S, F56G, F56A or F56K or F56A, F56P, F56R, F56H, F56S, F56Q, F56I, F56L, F56T or F56G; (b) N55Q, N55R, N55K, N55S, N55G, N55A or N55T; (c) Y51L, Y51V, Y51A, Y51N, Y51Q, Y51S or Y51G; (d) T150I; (e) S54P; and (f) S57P. The variants may include any number and combination of (a) to (f).

[0500] Preferred variants for use in the methods of the invention comprise Q62R or Q62K.

[0501] Preferred variants for use in the methods of the invention comprise a mutation in D43, E44, Q62 or any combination thereof, such as D43, E44, Q62, D43 / E44, D43 / Q62, E44 / Q62 or D43 / E44 / Q62.

[0502] The variant may comprise a mutation in the following positions:

[0503] Y51, F56, D149, E185, E201, and E203, such as Y51N, F56A, D149N, E185R, E201N, and E203N;

[0504] N55, such as N55A or N55S;

[0505] Y51, such as Y51N or Y51T;

[0506] S54, such as S54P;

[0507] S57, such as S57P;

[0508] F56, such as F56N, F56Q, F56R, F56S, F56G, F56A or F56K or F56A, F56P, F56R, F56H, F56S, F56Q, F56I, F56L, F56T or F56G;

[0509] Y51 and F56, such as Y51A and F56A, Y51A and F56N, Y51I and F56A, Y51L and F56A, Y51T and F56A, Y51T and F56Q, Y51I and F56N, Y51L and F56N or Y51T and F56N, preferably, Y51I and F56A, Y51L and F56A or Y51T and F56A, more preferably Y51T and F56Q or further preferably Y51X and F56Q, wherein X is any amino acid;

[0510] N55 and F56, such as N55X and F56Q, wherein X is any amino acid;

[0511] Y51, N55 and F56, such as Y51A, N55S and F56A, Y51A, N55S and F56N or Y51T, N55S and F56Q;

[0512] S54 and F56, such as S54P and F56A or S54P and F56N;

[0513] F56 and S57, such as F56A and S57P or F56N and S57P;

[0514] D149, E185 and E203, such as D149N, E185N and E203N;

[0515] D149, E185, E201 and E203, such as D149N, E185N, E201N and E203N;

[0516] D149, E185, D195, E201 and E203, for example, D149N, E185R, D195N, E201N and E203N or D149N, E185R, D195N, E201R and E203N;

[0517] F56 and N102, such as F56Q and N102R;

[0518] (a) Q62, such as Q62R or Q62K, and (b) one or more of Y51, N55 and F56, such as Y51, N55, F56, Y51N55, Y51F56, N55F56 or Y51N55F56, such as Y51TF56QQ62R;

[0519] (i) D43, E44, Q62 or any combination thereof, e.g., D43, E44, Q62, D43 / E44, D43 / Q62, E44 / Q62 or D43 / E44 / Q62 and (ii) one or more of Y51, N55 and F56, e.g., Y51, N55, F56, Y51 / N55, Y51 / F56, N55 / F56 or Y51 / N55 / F56, for example, D43N / Y51T / F56Q, E44N / Y51T / F56Q, D43N / E44N / Y51T / F56Q, D43N / Y51T / F56Q / Q62R, E44N / Y51T / F56Q / Q62R or D43N / E44N / Y51T / F56Q / Q62R; or

[0520] T150, for example, T150I.

[0521] Preferred pores for use in the methods of the invention comprise at least 7, at least 8, at least 9 or at least 10 monomers, for example 7, 8, 9 or 10 monomers, each of which comprises a variant of SEQ ID NO: 390 comprising one or more of the following substitutions: (a) F56N, F56Q, F56R, F56S, F56G, F56A or F56K or F56A, F56P, F56R, F56H, F56S, F56Q, F56I, F56L, F56T or F56G; (b) N55Q, N55R, N55K, N55S, N55G, N55A or N55T; (c) Y51L, Y51V, Y51A, Y51N, Y51Q, Y51S or Y51G; (d) T150I; (e) S54P; and (f) S57P. The variant may comprise any number and combination of (a) to (f).The monomers are preferably identical in these preferred pores.

[0522] Preferred pores for use in the methods of the invention comprise at least 7, at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9 or 10 monomers, each of which comprises a variant of SEQ ID NO: 390 comprising:

[0523] Y51N, F56A, D149N, E185, E201N, and E203N;

[0524] N55A;

[0525] N55S;

[0526] Y51N;

[0527] S54P;

[0528] S57P;

[0529] F56N, F56Q, F56R, F56S, F56G, F56A or F56K;

[0530] F56A, F56P, F56R, F56H, F56S, F56Q, F56I, F56L, F56T or F56G;

[0531] Y51A and F56A;

[0532] Y51A and F56N;

[0533] Y51I and F56A;

[0534] Y51L and F56A;

[0535] Y51T and F56A;

[0536] Y51T and F56Q;

[0537] Y51I and F56N;

[0538] Y51L and F56N;

[0539] Y51T and F56N;

[0540] N55S and F56Q;

[0541] Y51A, N55S, and F56A;

[0542] Y51A, N55S, and F56N;

[0543] Y51T, N55S and F56Q;

[0544] S54P and F56A;

[0545] S54P and F56N;

[0546] F56A and S57P;

[0547] F56N and S57P;

[0548] D149N, E185N and E203N;

[0549] D149N, E185N, E201N and E203N;

[0550] D149N, E185R, D195N, E201N and E203N;

[0551] D149N, E185R, D195N, E201R and E203N;

[0552] T150I;

[0553] F56Q and N102R;

[0554] F56 and N102, such as F56Q and N102R;

[0555] Y51T / F56Q / Q62R;

[0556] D43N / Y51T / F56Q;

[0557] E44N / Y51T / F56Q;

[0558] D43N / E44N / Y51T / F56Q;

[0559] D43N / Y51T / F56Q / Q62R;

[0560] E44N / Y51T / F56Q / Q62R; or

[0561] D43N / E44N / Y51T / F56Q / Q62R.

[0562] The CsgG mutant for use in the methods of the invention preferably comprises 9 monomers, at least one of which is a variant of the sequence shown in SEQ ID NO:390, comprising a mutation at one or more of positions Y51, N55, and F56. Preferred pores for use in the methods of the invention comprise at least 7, at least 8, at least 9, or at least 10 monomers, for example 7, 8, 9, or 10 monomers, each comprising a variant of SEQ ID NO:390 comprising a mutation at one or more of positions Y51, N55, and F56. The monomers are preferably identical in these preferred pores. The variant may comprise mutations at positions Y51; N55; F56; Y51 / N55; Y51 / F56; N55 / F56; or Y51 / N55 / F56. The variant may comprise any of the specific mutations at one or more of positions Y51, N55, and F56 discussed above, and any combination thereof. One or more of Y51, N55, and F56 may be substituted with any amino acid. Y51 may be substituted with F, M, L, I, V, A, P, G, C, Q, N, T, S, E, D, K, H or R, for example, A, S, T, N or Q. N55 may be substituted with F, M, L, I, V, A, P, G, C, Q, T, S, E, D, K, H or R, for example, A, S, T, or Q. F56 may be substituted with M, L, I, V, A, P, G, C, Q, N, T, S, E, D, K, H or R, for example, A, S, T, N or Q. The variant may further comprise one or more of the following: (i) one or more mutations at the following positions (i.e., mutations at one or more of the following positions): (i) N40, D43, E44, S54, S57, Q62, R97, E101, E124, E131, R142, T150, and R192; (iii) Q42R or

[0015] The variants may comprise any combination of (i) and (iii) to (xi) discussed above.

[0015] The variants may comprise any of the embodiments discussed above for (i) and (iii) to (xi) described above.

[0563] (1) Preferred variants for use in the methods of the invention or (2) preferred pores for use in the methods of the invention comprise at least 7, at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9 or 10 monomers, each of which comprises a variant of SEQ ID NO:390 comprising: Y51R / F56Q, Y51N / F56N, Y51M / F56Q, Y51L / F56Q, Y51I / F56Q, Y51V / F56Q, Y51A / F56Q, Y51P / F56Q, Y51G / F56Q, Y51C / F56Q, Y51Q / F56Q, Y51N / F56Q, Y51S / F56Q, Y51E / F56Q, Y51D / F56Q, Y51K / F56Q or Y51H / F56Q.

[0564] (1) Preferred mutants for use in the methods of the invention or (2) preferred pores for use in the methods of the invention comprise at least 7, at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9 or 10 monomers, each of which comprises a variant of SEQ ID NO: 390 comprising: Y51TF56Q, Y51QF56Q or Y51A / F56Q.

[0565] (1) Preferred mutants for use in the methods of the invention or (2) Preferred pores for use in the methods of the invention comprising at least 7, at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9 or 10 monomers, each of which comprises SEQ A variant of IDNO: 390, the variant comprising: Y51T / F56F, Y51T / F56M, Y51T / F56L, Y51T / F56I, Y51T / F56V, Y51T / F56A, Y51T / F56P, Y51T / F56G, Y51T / F56C, Y51T / F56Q, Y51T / F56N, Y51T / F56T, Y51T / F56S, Y51T / F56E, Y51T / F56D, Y51T / F56K, Y51T / F56H or Y51T / F56R.

[0566] (1) Preferred variants for use in the methods of the invention or (2) preferred pores for use in the methods of the invention comprise at least 7, at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9 or 10 monomers, each of which comprises a variant of SEQ ID NO: 390 comprising: Y51T / N55Q, Y51T / N55S or Y51T / N55A.

[0567] (1) Preferred variants for use in the methods of the invention comprise or (2) preferred pores for use in the methods of the invention comprise at least 7, at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9 or 10 monomers, each of which comprises a variant of SEQ ID NO:390 comprising: Y51A / F56F, Y51A / F56L, Y51A / F56I, Y51A / F56V, Y51A / F56A, Y51A / F56P, Y51A / F56G, Y51A / F56C, Y51A / F56Q, Y51A / F56N, Y51A / F56T, Y51A / F56S, Y51A / F56E, Y51A / F56D, Y51A / F56K, Y51A / F56H or Y51A / F56R.

[0568] (1) Preferred variants for use in the methods of the invention or (2) preferred pores for use in the methods of the invention comprise at least 7, at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9 or 10 monomers, each of which comprises a variant of SEQ ID NO:390 comprising: Y51C / F56A, Y51E / F56A, Y51D / F56A, Y51K / F56A, Y51H / F56A, Y51Q / F56A, Y51N / F56A, Y51S / F56A, Y51P / F56A or Y51V / F56A.

[0569] (1) Preferred variants for use in the methods of the invention or (2) preferred pores for use in the methods of the invention comprising at least 7, at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9 or 10 monomers, each of which comprises a variant of SEQ ID NO: 390 comprising:

[0570] D149R / E185R / E201R / E203R or Y51T / F56Q / D149R / E185R / E201R / E203R;

[0571] D149N / E185N / E201N / E203N or Y51T / F56Q / D149N / E185N / E201N / E203N;

[0572] E201R / E203R or Y51T / F56Q / E201R / E203R

[0573] E201N / E203R or Y51T / F56Q / E201N / E203R;

[0574] E203R or Y51T / F56Q / E203R;

[0575] E203N or Y51T / F56Q / E203N;

[0576] E201R or Y51T / F56Q / E201R;

[0577] E201N or Y51T / F56Q / E201N;

[0578] E185R or Y51T / F56Q / E185R;

[0579] E185N or Y51T / F56Q / E185N;

[0580] D149R or Y51T / F56Q / D149R;

[0581] D149N or Y51TF56Q / D149N;

[0582] R142E or Y51T / F56Q / R142E;

[0583] R142N or Y51T / F56Q / R142N;

[0584] R192E or Y51T / F56Q / R192E; or

[0585] R192N or Y51T / F56Q / R192N;

[0586] (1) Preferred variants for use in the methods of the invention or (2) preferred pores for use in the methods of the invention comprising at least 7, at least 8, at least 9 or at least 10 monomers, such as 7, 8, 9 or 10 monomers, each of which comprises a variant of SEQ ID NO: 390 comprising:

[0587] Y51A / F56Q / E101N / N102R;

[0588] Y51A / F56Q / R97N / N 102G;

[0589] Y51A / F56Q / R97N / N102R;

[0590] Y51A / F56Q / R97N;

[0591] Y51A / F56Q / R97G;

[0592] Y51A / F56Q / R97L;

[0593] Y51A / F56Q / N102R;

[0594] Y51A / F56Q / N102F;

[0595] Y51A / F56Q / N102G;

[0596] Y51A / F56Q / E101R;

[0597] Y51A / F56Q / E101F;

[0598] Y51A / F56Q / E101N; or

[0599] Y51A / F56Q / E101G.

[0600] The monomers are preferably identical in these preferred pores.

[0601] Preferred pores for use in the methods of the invention comprise at least 7, at least 8, at least 9 or at least 10 monomers, for example 7, 8, 9 or 10 monomers, each of which comprises a variant of SEQ ID NO: 390 comprising F56A, F56P, F56R, F56H, F56S, F56Q, F56I, F56L, F56T or F56G. The monomers are preferably identical in these preferred pores.

[0602] The CsgG mutant may comprise any variant of SEQ ID NO: 390 disclosed in the Examples or may be any pore disclosed in the Examples.

[0603] The CsgG mutant is most preferably a pore of the invention.

[0604] Steps (a) and (b) are preferably performed with an applied potential across the pore. As discussed in more detail below, the applied potential generally results in the formation of a complex between the pore and the polynucleotide binding protein. The applied potential may be a voltage potential. Alternatively, the applied potential may be a chemical potential. An example of this is the use of a salt gradient across the amphiphilic molecule layer. Salt gradients are disclosed in Holden et al., J Am Chem Soc. 2007 Jul 11; 129(27): 8650-5.

[0605] The method is used to determine the presence, absence, or one or more characteristics of a target analyte. The method can be used to determine the presence, absence, or one or more characteristics of at least one target analyte. The method can involve determining the presence, absence, or one or more characteristics of two or more target analytes. The method can include determining the presence, absence, or one or more characteristics of any number of target analytes, such as 2, 5, 10, 15, 20, 30, 40, 50, 100, or more target analytes. Any number of characteristics of one or more target analytes can be determined, such as 1, 2, 3, 4, 5, 10, or more characteristics.

[0606] The target analyte is preferably a metal ion, an inorganic salt, a polymer, an amino acid, a peptide, a polypeptide, a protein, a nucleotide, an oligonucleotide, a polynucleotide, a dye, a bleach, a drug, a diagnostic agent, an explosive, or an environmental pollutant. The method can involve determining the presence, absence, or one or more properties of two or more target analytes of the same class, for example, two or more proteins, two or more nucleotides, or two or more drugs. Alternatively, the method can involve determining the presence, absence, or one or more properties of two or more target analytes of different classes, for example, one or more proteins, one or more nucleotides, and one or more drugs.

[0607] The target analyte may be secreted by the cell. Alternatively, the target analyte may be an analyte present within the cell such that the analyte must be extracted from the cell before performing the present invention.

[0608] The analyte is preferably an amino acid, peptide, polypeptide and / or protein. The amino acid, peptide, polypeptide or protein may be naturally occurring or non-naturally occurring. The polypeptide or protein may contain synthetic or modified amino acids. A large number of different types of amino acid modifications are known in the art. Suitable amino acids and modifications thereof are described above. For the purposes of the present invention, it will be understood that the target analyte may be modified by any method known in the art.

[0609] The protein may be an enzyme, an antibody, a hormone, a growth factor or a growth regulatory protein, such as a cytokine. The cytokine may be selected from interleukins, preferably IFN-1, IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12 and IL-13, interferons, preferably IL-γ, and other cytokines, such as TNF-α. The protein may be a bacterial protein, a fungal protein, a viral protein or a parasitic protein.

[0610] The target analyte is preferably a nucleotide, oligonucleotide or polynucleotide. Nucleotides and polynucleotides are discussed below. Oligonucleotides are short chain nucleotide polymers, generally having 50 or less nucleotides, such as 40 or less, 30 or less, 20 or less, 10 or less or 5 or less nucleotides. The oligonucleotide can include any nucleotide discussed below, including abasic and modified nucleotides.

[0611] The target analyte, eg, target polynucleotide, may be present in any suitable sample as discussed below.

[0612] As described below, the pores are typically present in a membrane. Target analytes can be coupled or delivered to the membrane using methods discussed below.

[0613] Any of the measurement methods discussed below can be used to determine the presence, absence, or one or more characteristics of a target analyte. The method preferably comprises contacting the target analyte with the pore such that the analyte moves relative to (e.g., moves through) the pore, and measuring the current passing through the pore as the analyte moves relative to the pore, thereby determining the presence, absence, or one or more characteristics of the analyte.

[0614] If the current flows through the pore in a manner specific for the analyte (i.e., if a specific current associated with the analyte is detected flowing through the pore), then the target analyte is present. If the current does not flow through the pore in a manner specific for the nucleotide, then the analyte is not present. Control experiments can be performed in the presence of the analyte to determine whether this affects the way the current flows through the pore.

[0615] The present invention can be used to differentiate analytes of similar structure based on their different effects on the current passing through the pore. Individual analytes can be identified at the single molecule level based on their current amplitude when interacting with the pore. The present invention can also be used to determine the presence or absence of a specific analyte in a sample. The present invention can also be used to measure the concentration of a specific analyte in a sample. Analyte characterization using pores other than CsgG is known in the art.

[0616] Characterization of polynucleotides

[0617] The present invention provides a method for characterizing a target polynucleotide, such as for polynucleotide sequencing. There are two main strategies for characterizing or sequencing polynucleotides using nanopores: strand characterization / sequencing and exonuclease characterization / sequencing. The methods of the present invention may involve either approach.

[0618] In strand sequencing, DNA is displaced through a nanopore along or against an applied potential. Exonucleases acting on double-stranded DNA in a progressive or processive manner can be used on the cis side of the pore to force the remaining single strand into and through the pore under an applied potential, or on the trans side of the pore under a reverse potential to force the remaining single strand into and through the pore. Similarly, helicases that unwind double-stranded DNA can be used in a similar manner. Polymerases can also be used. Sequencing applications that require strand displacement against an applied potential are also possible, but the DNA must first be "captured" by the enzyme under a reverse potential or no potential. Then, as the potential switches back after binding, the strand will pass through the pore from cis to trans and be maintained in an extended conformation by the current. Single-stranded DNA exonucleases or single-stranded DNA-dependent polymerases can act as molecular motors, pulling the recently displaced single strand back through the pore from trans to cis in a controlled, stepwise manner against the applied potential.

[0619] In one embodiment, the method for characterizing a target polynucleotide comprises contacting a target sequence with a pore and a helicase. Any helicase can be used in the method. Suitable helicases are discussed below. The helicase can operate in two modes relative to the pore. First, the method is preferably performed using a helicase that controls the movement of the target sequence through the pore by a field generated by an applied voltage. In this mode, the 5' end of the DNA is first captured in the pore, and the enzyme controls the movement of the DNA into the pore, causing the target sequence to pass through the pore under the electric field until it ultimately shifts to the trans side of the bilayer. Alternatively, the method is preferably performed such that the helicase controls the movement of the target sequence through the pore against the field generated by the applied voltage. In this mode, the 3' end of the DNA is first captured in the pore, and the enzyme controls the movement of the DNA through the pore, causing the target sequence to be pulled out of the pore against the applied field until it ultimately rebounds to the cis side of the bilayer.

[0620] In exonuclease sequencing, an exonuclease releases each individual nucleotide from one end of a target polynucleotide and identifies these individual nucleotides as described below. In another embodiment, a method for characterizing a target polynucleotide comprises contacting the target sequence with a hole and an exonuclease. Any exonuclease as described below can be used in the method. The enzyme can be covalently attached to the hole as described below.

[0621] Exonucleases are enzymes that typically latch onto one end of a polynucleotide and digest the sequence one nucleotide at a time from that end. Exonucleases can digest the polynucleotide in either a 5' to 3' direction or a 3' to 5' direction. The end of a polynucleotide to which the exonuclease binds is typically determined by the choice of enzyme used and / or by methods known in the art. Hydroxyl groups or capping structures at either end of a polynucleotide can typically be used to prevent or promote binding of exonucleases to specific ends of the polynucleotide.

[0622] The method comprises contacting the polynucleotide with the exonuclease so that the nucleotides are digested from the ends of the polynucleotide at a rate that allows characterization or identification of a portion of the nucleotides as described above. Methods for performing these steps are well known in the art. For example, Edman degradation is used to sequentially digest individual amino acids from the ends of a polypeptide so that they can be identified using high performance liquid chromatography (HPLC). Methods of homology can be used in the present invention.

[0623] The speed of described exonuclease effect is generally lower than the optimal rate of wild-type exonuclease.The suitable speed of the described exonuclease activity in the method for the invention comprises digestion 0.5 to 1000 Nucleotide per second, digestion 0.6 to 500 Nucleotide per second, digestion 0.7 to 200 Nucleotide per second, digestion 0.8 to 100 Nucleotide per second, digestion 0.9 to 50 Nucleotide per second or digestion 1 to 20 or 10 Nucleotide per second.Described speed is preferably 1,10,100,500 or 1000 Nucleotide per second.The suitable speed of exonuclease activity can be realized in many ways.For example, different exonucleases with the optimal activity rate of reduction can be used according to the present invention.

[0624] In strand characterisation embodiments, the method comprises contacting the polynucleotide with a CsgG pore or a mutant thereof, such as a pore of the invention, such that the polynucleotide moves relative to, e.g., through, the pore, and obtaining one or more measurements as the polynucleotide moves relative to the pore, wherein the measurements are indicative of one or more characteristics of the polynucleotide, thereby characterising the target polynucleotide.

[0625] In embodiments of exonuclease characterisation, the method comprises contacting the polynucleotide with a CsgG pore or a mutant thereof, such as a pore of the invention, and an exonuclease, such that the exonuclease digests individual nucleotides from one end of the target polynucleotide and the individual nucleotides move relative to, such as through, the pore, and obtaining one or more measurements as the individual nucleotides move relative to the pore, wherein the measurements are indicative of one or more characteristics of the individual nucleotides, thereby characterising the target polynucleotide.

[0626] Each nucleotide is a single nucleotide. Each nucleotide is a nucleotide that is not bound to another nucleotide or polynucleotide via a nucleotide bond. A nucleotide bond comprises one of the phosphate groups of a nucleotide bound to the sugar group of another nucleotide. Each nucleotide is typically not bound to another polynucleotide having at least 5, at least 10, at least 20, at least 50, at least 100, at least 200, at least 500, at least 1000, or at least 5000 nucleotides via a nucleotide bond. For example, each nucleotide may have been digested from a target polynucleotide sequence, such as a DNA or RNA strand. The nucleotide may be any of the following.

[0627] The individual nucleotides may interact with the pore in any manner and at any site. The nucleotides are preferably reversibly bound to the pore via an adapter or by attachment to an adapter, as described above. Most preferably, the nucleotides are reversibly bound to the pore via an adapter or by attachment to an adapter as they pass across the membrane through the pore. The nucleotides may also reversibly bind to the barrel or channel of the pore via an adapter or by engagement with the adapter as they pass across the membrane through the pore.

[0628] In the interaction between each nucleotide and the pore, the nucleotide typically affects the current flowing through the pore in a manner specific to the nucleotide. For example, a specific nucleotide will reduce the current flowing through the pore with a specific average time period and a specific degree. In other words, the current flowing through the pore is unique to a specific nucleotide. Control experiments can be performed to determine the effect of a specific nucleotide on the current flowing through the pore. The results obtained by applying the method of the present invention to the test sample are then compared with the results from the above-mentioned control experiments to identify the specific nucleotide in the sample or to determine whether the specific nucleotide is present in the sample. The frequency of the current flowing through the pore in a manner that indicates the presence of a specific nucleotide can be used to determine the concentration of the nucleotide in the sample. The ratio of different nucleotides in the sample can also be calculated. For example, the ratio of dCMP to methyl-dCMP can be calculated.

[0629] The method comprises measuring one or more properties of the target polynucleotide.The target polynucleotide may also be referred to as a template polynucleotide or a polynucleotide of interest.

[0630] This embodiment may also use a CsgG pore or a mutant thereof, such as the pores of the present invention. Any of the pores and embodiments described above for the target analyte may be used.

[0631] polynucleotides

[0632] A polynucleotide, such as a nucleic acid, is a macromolecule comprising two or more nucleotides. The polynucleotide or nucleic acid may comprise any combination of nucleotides. The nucleotides may be naturally occurring or synthetic. One or more nucleotides in the polynucleotide may be oxidized or methylated. One or more nucleotides in the polynucleotide may be damaged. For example, the polynucleotide may comprise a pyrimidine dimer. Such dimers are often associated with damage caused by ultraviolet light and are a major cause of melanoma of the skin. One or more nucleotides in the polynucleotide may be modified, for example with a label or tag. Suitable labels are described below. The polynucleotide may comprise one or more spacers.

[0633] Nucleotides generally contain a nucleobase, a sugar and / or at least one phosphate group. The nucleobase and sugar form a nucleoside.

[0634] The nucleobases are typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines, more specifically adenine (A), guanine (G), thymine (T), uracil (U) and cytosine (C).

[0635] The sugar is typically a pentose. Nucleoside sugars include, but are not limited to, ribose and deoxyribose. The sugar is preferably deoxyribose.

[0636] The polynucleotide preferably comprises the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and / or deoxythymidine (dT), deoxyguanosine (dG) and deoxycytidine (dC).

[0637] The nucleotides are typically ribonucleotides or deoxyribonucleotides. The nucleotides typically contain monophosphate, diphosphate or triphosphate. The nucleotides may contain more than three phosphates, for example 4 or 5 phosphates. The phosphates may be attached to the 3' side or the 5' side of the nucleotide. Nucleotides include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), 5-methylcytidine monophosphate, 5-hydroxymethylcytidine monophosphate, cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP), deoxycytidine monophosphate (dCMP) and deoxymethylcytidine monophosphate. The nucleotide is preferably selected from the group consisting of AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP and dUMP.

[0638] Nucleotides can be abasic (i.e., lack a nucleobase). Nucleotides can also lack a nucleobase and a sugar (i.e., be a C3 spacer).

[0639] The nucleotides in the polynucleotide can be linked to each other in any manner. The nucleotides are typically linked by their sugar and phosphate groups, as in nucleic acids. The nucleotides can be linked by their nucleobases, as in pyrimidine dimers.

[0640] The polynucleotide may be single-stranded or double-stranded. Preferably, at least a portion of the polynucleotide is double-stranded.

[0641] The polynucleotide can be a nucleic acid, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The polynucleotide can comprise an RNA chain hybridized to a DNA chain. The polynucleotide can be any synthetic nucleic acid known in the art, such as peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA), or other synthetic polymers with nucleotide side chains. The PNA backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. The GNA backbone is composed of repeating ethylene glycol units linked by phosphodiester bonds. The TNA backbone is composed of repeating threose groups linked together by phosphodiester bonds. LNA is formed from the above-mentioned ribonucleic acids and has an additional bridge structure connecting the 2' oxygen and 4' carbon of the ribose moiety. Bridged nucleic acids (BNA) are modified RNA nucleotides. They can also be referred to as restricted or inaccessible RNA. BNA monomers can contain 5-, 6-, or even 7-membered bridges with a "fixed" C3'-endo sugar puckering. The bridges are synthetically introduced into the 2',4'-positions of the ribose to produce 2',4'-BNA monomers.

[0642] The polynucleotide is most preferably ribonucleic acid (RNA) or deoxyribonucleic acid (DNA).

[0643] The polynucleotide can be of any length. For example, the polynucleotide can be at least 10, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotides or nucleotide pairs in length. The polynucleotide can be 1,000 or more nucleotides or nucleotide pairs in length, 5,000 or more nucleotides or nucleotide pairs in length, or 100,000 or more nucleotides or nucleotide pairs in length.

[0644] Any number of polynucleotides can be studied. For example, the methods of the present invention can involve characterizing 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more polynucleotides. If two or more polynucleotides are characterized, they can be different polynucleotides or two instances of the same polynucleotide.

[0645] The polynucleotides can be naturally occurring or artificially synthesized. For example, the method can be used to verify the sequence of the prepared oligonucleotides. The method is usually performed in vitro.

[0646] sample

[0647] The polynucleotides are typically present in any suitable sample. The present invention is typically performed on a sample known to contain or suspected of containing the polynucleotides. Alternatively, the present invention can be performed on a sample to confirm the identity of a polynucleotide known to be present or expected to be present in the sample.

[0648] The sample can be a biological sample. The present invention can be implemented in vitro for samples obtained or extracted from any organism or microorganism. The organism or microorganism is typically archaeal, prokaryotic or eukaryotic, and typically belongs to one of the following five kingdoms: plant kingdom, animal kingdom, fungi, prokaryotes and protists. The present invention is implemented in vitro for samples obtained or extracted from any virus. The sample is preferably a fluid sample. The sample typically includes a patient's body fluid. The sample can be urine, lymph, saliva, mucus or amniotic fluid, but is preferably blood, plasma or serum.

[0649] Typically, the sample is derived from a human, but may alternatively be from other mammals, such as commercially raised animals such as horses, cattle, sheep, fish, chickens or pigs, or alternatively may be a pet such as a cat or dog. Alternatively, the sample may be derived from a plant, such as a sample obtained from a commercial crop, such as a cereal, legume, fruit or vegetable, for example wheat, barley, oats, canola, corn, soybean, rice, rhubarb, banana, apple, tomato, potato, grape, tobacco, beans, lentils, sugarcane, cocoa, cotton.

[0650] The sample may be a non-biological sample. The non-biological sample is preferably a fluid sample. Examples of non-biological samples include surgical fluids, water such as drinking water, seawater or river water, and reagents for laboratory tests.

[0651] The sample is typically processed prior to use in the present invention, for example by centrifugation or by filtering through a membrane to remove unwanted molecules or cells, such as red blood cells. The sample can be measured immediately after collection. The sample can also be stored, preferably below -70°C, prior to measurement.

[0652] Characterization

[0653] The method may involve measuring two, three, four or five or more characteristics of a polynucleotide. The one or more characteristics are preferably selected from (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide, and (v) whether the polynucleotide is modified. Any combination of (i) to (v) may be measured according to the present invention, for example {i}, {ii}, {iii}, {iv}, {v}, {i,ii}, {i,iii}, {i,iv}, {i,v}, {ii,iii}, {ii,iv}, {ii,v}, {iii,iv}, {iii,v}, {iv,v}, {i,ii,iii}, {i,ii,iv}, {i,ii,v}, {i,iii,iv}, {i,iii,v}, {i,iv,v}, {ii,iii,iv}, {ii,iii,v}, {iv,v}, {i,ii,iii}, {i,ii,iv}, {i,iii,iv}, {i,iv,v}, {ii,iii,iv}, {ii,iii,v},

[0654] {ii,iv,v},{iii,iv,v},{i,ii,iii,iv},{i,ii,iii,v},{i,ii,iv,v},{i,iii,iv,v},{ii,iii,iv,v}, or {i,ii,iii,iv,v}. Different combinations of (i) to (v) can measure the first polynucleotide compared to the second polynucleotide, including any of the combinations listed above.

[0655] For (i), the length of the polynucleotide may be measured, for example, by determining the number of interactions between the polynucleotide and the pore or the duration of interactions between the polynucleotide and the pore.

[0656] Regarding (ii), the identity of a polynucleotide can be measured in a variety of ways. The identity of a polynucleotide can be measured in conjunction with or without measuring the polynucleotide sequence. The former is relatively simple; the polynucleotide is sequenced and identified. The latter can be accomplished in several different ways. For example, the presence of a specific motif in a polynucleotide can be measured (without measuring the rest of the polynucleotide sequence). Alternatively, the measurement of a specific electrical and / or optical signal in the described methods can identify the polynucleotide as originating from a specific source.

[0657] For (iii), the sequence of the polynucleotide can be determined as previously described. Suitable sequencing methods, in particular sequencing methods using electrical measurement methods, are described in Stoddart D et al., Proc Natl Acad Sci, 12; 106(19): 7702-7, Lieberman KR et al, J Am Chem Soc. 2010; 132(50): 17961-72, and in international application WO 2000 / 28312.

[0658] Regarding (iv), secondary structure can be measured using a variety of methods. For example, if the method involves electrical measurement, changes in residence time or changes in the current flowing through the pore can be used to measure the secondary structure. This allows for differentiation between regions of single-stranded and double-stranded polynucleotides.

[0659] For (v), the presence or absence of any modifications can be measured. The method preferably comprises determining whether the polynucleotide has been modified by methylation, oxidation, damage, modification with one or more proteins, or modification with one or more markers, tags, or spacers. Specific modifications will result in specific interactions with the pore, which can be measured using the methods described below. For example, methylcytosine can be distinguished from cytosine based on the current flowing through the pore during its interaction with each nucleotide.

[0660] The target polynucleotide is contacted with a CsgG pore or a mutant thereof, such as a pore of the present invention. The pore is typically present in a membrane. Suitable membranes are described below. The method can be performed using any device suitable for studying membrane / pore systems in which the pore is present in the membrane. The method can be performed using any device suitable for sensing transmembrane pores. For example, the device comprises a chamber containing an aqueous solution and a barrier dividing the chamber into two parts. The barrier typically has a pore, with a membrane containing the pore formed in the pore. Alternatively, the barrier forms a membrane having a pore present in the membrane.

[0661] The method may be carried out using the apparatus described in International Application No. PCT / GB08 / 000562 (WO 2008 / 102120).

[0662] Various different types of measurements can be performed. This includes, but is not limited to, electrical measurements and optical measurements. Possible electrical measurements include current measurements, impedance measurements, tunneling measurements (Ivanov AP et al., Nano Lett. 2011 Jan 12; 11(1): 279-85) and FET measurements (International Application WO 2005 / 124888). Optical measurements can be combined with electrical measurements (Soni GV et al., Rev Sci Instrum. 2010 Jan; 81(1): 014301). The measurement can be a transmembrane current measurement, such as a measurement of the ionic current flowing through the pore.

[0663] Electrical measurements can be performed using standard single-channel recording equipment as described in Stoddart D et al., Proc Natl Acad Sci, 12; 106(19)7702-7, Lieberman KR et al, J Am Chem Soc. 2010; 132(50)17961-72 and international application WO 2000 / 28312. Alternatively, electrical measurements can be performed using a multi-channel system, such as described in international application WO 2009 / 077734 and international application WO 2011 / 067559.

[0664] The method is preferably performed using an applied potential across the membrane. The applied potential can be a voltage potential. Alternatively, the applied potential can be a chemical potential. One example is the use of a salt gradient across a membrane, such as an amphiphilic layer. Salt gradients are disclosed in Holden et al., J Am Chem Soc. 2007 Jul 11;129(27):8650-5. In some cases, the current flowing through the pore as the polynucleotide moves relative to the pore is used to estimate or determine the sequence of the polynucleotide. This is known as strand sequencing.

[0665] The method may include measuring the current flowing through the pore when the polynucleotide moves relative to the pore. Therefore, the equipment used for the method may also include a circuit capable of applying an electric potential and measuring the electrical signal passing through the membrane and the pore. The method may be performed using a patch clamp or a voltage clamp. The method preferably involves the use of a voltage clamp.

[0666] The method of the present invention may comprise measuring the current flowing through the pore as the polynucleotide moves relative to the pore. Suitable conditions for measuring ion flow through a transmembrane protein pore are known in the art and disclosed in the Examples. The method is typically performed by applying a voltage across the membrane and the pore. The voltage used is typically from +5V to -5V, for example, from +4V to -4V, from +3V to -3V, or from +2V to -2V. The voltage used is typically from -600mV to +600V or -400mV to +400mV. The voltage used is preferably within a range having a lower limit selected from -400mV, -300mV, -200mV, -150mV, -100mV, -50mV, -20mV and 0mV and an upper limit independently selected from +10mV, +20mV, +50mV, +100mV, +150mV, +200mV, +300mV and +400mV. The voltage used is more preferably within a range of 100mV to 240mV and most preferably within a range of 120mV to 220mV. By using an increased applied potential, the recognition of different nucleotides by the pore can be increased.

[0667] The method is generally carried out in the presence of any charge carrier, such as a metal salt such as an alkali metal salt, a halide salt such as a chloride salt, such as an alkali metal chloride salt. The charge carrier can include an ionic liquid or an organic salt, such as tetramethylammonium chloride, trimethylphenylammonium chloride, phenyltrimethylammonium chloride or 1-ethyl-3-methylimidazolium chloride. In the exemplary device described above, the salt is present in an aqueous solution in the chamber. Potassium chloride (KCl), sodium chloride (NaCl), cesium chloride (CsCl) or a mixture of potassium ferrocyanide and potassium ferricyanide are generally used. A mixture of KCl, NaCl and potassium ferrocyanide and potassium ferricyanide is preferred. The charge carrier can be asymmetric on the membrane. For example, the type and / or concentration of the charge carrier can be different on each side of the membrane.

[0668] The concentration of the salt may be saturating. The concentration of the salt may be 3 M or less, and is typically 0.1 to 2.5 M, 0.3 to 1.9 M, 0.5 to 1.8 M, 0.7 to 1.7 M, 0.9 to 1.6 M or 1 M to 1.4 M. The concentration of the salt is preferably 150 mM to 1 M. The method is preferably performed using a salt concentration of at least 0.3 M, such as at least 0.4 M, at least 0.5 M, at least 0.6 M, at least 0.8 M, at least 1.0 M, at least 1.5 M, at least 2.0 M, at least 2.5 M or at least 3.0 M. High salt concentrations provide a high signal-to-noise ratio and allow the presence of the nucleotide to be identified in the context of normal current fluctuations to be indicated by current.

[0669] The method is typically performed in the presence of a buffer. In the exemplary apparatus described above, the buffer is present in an aqueous solution in the chamber. Any buffer can be used in the method of the present invention. Typically, the buffer is a phosphate buffer. Other suitable buffers are HEPES or Tris-HCl buffer. The method is typically performed at a pH of 4.0 to 12.0, 4.5 to 10.0, 5.0 to 9.0, 5.5 to 8.8, 6.0 to 8.7, or 7.0 to 8.8, or 7.5 to 8.5. The pH used is preferably about 7.5.

[0670] The method can be carried out at a temperature of 0°C to 100°C, 15°C to 95°C, 16°C to 90°C, 17°C to 85°C, 18°C ​​to 80°C, 19°C to 70°C or 20°C to 60°C. The method is typically carried out at room temperature. The method is optionally carried out at a temperature that supports enzyme function, such as about 37°C.

[0671] Polynucleotide binding protein

[0672] The strand characterisation method preferably comprises contacting the polynucleotide with a polynucleotide binding protein, such that the protein controls movement of the polynucleotide relative to, eg through, the pore.

[0673] More preferably, the method comprises (a) contacting the polynucleotide with a CsgG pore or mutant thereof (e.g., a pore of the invention) and a polynucleotide binding protein, such that the protein controls movement of the polynucleotide relative to the pore, e.g., through the pore, and (b) obtaining one or more measurements as the polynucleotide moves relative to the pore, wherein the measurements are indicative of one or more characteristics of the polynucleotide, thereby characterizing the polynucleotide.

[0674] More preferably, the method comprises (a) contacting the polynucleotide with a CsgG pore or mutant thereof (e.g., a pore of the invention) and a polynucleotide binding protein, such that the protein controls movement of the polynucleotide relative to the pore, e.g., through the pore, and (b) measuring a current passing through the pore as the polynucleotide moves relative to the pore, wherein the current is indicative of one or more characteristics of the polynucleotide, thereby characterizing the polynucleotide.

[0675] The polynucleotide binding protein can be any protein that can bind to the polynucleotide and control its movement through the hole. It is relatively simple to determine whether a protein is bound to a polynucleotide in this area. Proteins usually interact with the polynucleotide and modify at least one property of the polynucleotide. Proteins can modify the polynucleotides by cracking the polynucleotides to form short chains of individual nucleotides or nucleotides (e.g., dinucleotides or trinucleotides). Proteins can modify the polynucleotides by orienting them or moving them to a specific location, that is, controlling their movement.

[0676] The polynucleotide binding protein is preferably derived from a polynucleotide treatment enzyme. The polynucleotide treatment enzyme is a polypeptide that can interact with a polynucleotide and modify at least one property of the polynucleotide. The enzyme can be modified by cracking the polynucleotide to form a short chain (for example, a dinucleotide or a trinucleotide) of each single nucleotide or nucleotide. The enzyme can be modified by orienting the polynucleotide or moving it to a specific position. The polynucleotide treatment enzyme does not need to show enzymatic activity, as long as it can bind the polynucleotide and control its movement through the hole. For example, the enzyme can be modified to remove its enzymatic activity, or can be used under conditions that prevent it from being used as an enzyme. This condition is discussed in more detail below.

[0677] The polynucleotide handling enzyme is preferably derived from a nucleolytic enzyme. The polynucleotide handling enzyme used in the enzyme construct is more preferably derived from any of the enzyme classification (EC) groups 3.1.11, 3.1.13, 3.1.14, 3.1.15, 3.1.16, 3.1.21, 3.1.22, 3.1.25, 3.1.26, 3.1.27, 3.1.30 and 3.1.31. The enzyme may be any of those disclosed in International Application No. PCT / GB10 / 000133 (published as WO2010 / 086603).

[0678] Preferably, the enzyme is a polymerase, an exonuclease, a helicase and a topoisomerase, for example, a gyrase. Suitable enzymes include, but are not limited to, exonuclease I from Escherichia coli (SEQ ID NO: 399), exonuclease III from Escherichia coli (SEQ ID NO 401), RecJ from Streptococcus thermophilus (SEQ ID NO: 403) and bacteriophage lambda exonuclease (SEQ ID NO: 405), TatD exonuclease and variants thereof. Three subunits comprising the sequence shown in SEQ ID NO: 403 or a variant thereof interact to form a trimer exonuclease. These exonucleases can also be used in the exonuclease method of the present invention. The polymerase can be 3173 DNA polymerase (which can be obtained from Company), SD polymerase (available from The enzyme is preferably Phi29 DNA polymerase (SEQ ID NO: 397) or a variant thereof. The topoisomerase is preferably any one of Enzyme Classification (EC) groups 5.99.1.2 and 5.99.1.3.

[0679] The enzyme is most preferably derived from a helicase, for example, Hel308Mbu (SEQ ID NO: 406), Hel308Csy (SEQ ID NO: 407), Hel308Tga (SEQ ID NO: 408), Hel308Mhu (SEQ ID NO: 409), Tral Eco (SEQ ID NO: 410), XPD Mbu (SEQ ID NO: 411) or a variant thereof. Any helicase can be used in the present invention. The helicase can be or be derived from a Hel308 helicase, a RecD helicase, for example, a Tral helicase or a TrwC helicase, an XPD helicase or a Dda helicase. The helicase may be any one of the helicases, modified helicases or helicase constructs disclosed in the following international applications: PCT / GB2012 / 052579 (published as WO2013 / 057495); PCT / GB2012 / 053274 (published as WO2013 / 098562); PCT / GB2012 / 053273 (published as WO2013098561); PCT / GB2013 / 051925 (published as WO2013 / 057495); PCT / GB2 012 / 053274 (published as WO2013 / 098562); PCT / GB2012 / 053273 (published as WO2013098561); PCT / GB2013 / 051925 (published as WO2014 / 013260); PCT / GB2013 / 051924 (published as WO2014 / 013259); PCT / GB2013 / 051928 (published as WO2014 / 013262) and PCT / GB2014 / 052736.

[0680] The helicase preferably comprises the sequence set forth in SEQ ID NO:413 (Trwc Cba) or a variant thereof, the sequence set forth in SEQ ID NO:406 (Hel308Mbu) or a variant thereof, or the sequence set forth in SEQ ID NO:412 (Dda) or a variant thereof. The variant may differ from the native sequence in any of the ways discussed below for the transmembrane pore. Preferred variants of SEQ ID NO:412 comprise (a) E94C and A360C or (b) E94C, A360C, C109A and C136A, and then, optionally, (ΔM1) G1G2 (i.e., deletion of M1 followed by addition of G1 and G2).

[0681] According to the present invention, any number of helicases can be used. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more helicases can be used. In some embodiments, different numbers of helicases can be used.

[0682] The method of the present invention preferably comprises contacting the polynucleotide with two or more helicases. The two or more helicases are generally the same helicase. The two or more helicases may be different helicases.

[0683] The two or more helicases can be any combination of the above helicases. The two or more helicases can be two or more Dda helicases. The two or more helicases can be one or more Dda helicases and one or more TrwC helicases. The two or more helicases can be different variants of the same helicase.

[0684] The two or more helicases are preferably linked to each other. The two or more helicases are more preferably covalently linked to each other. The helicases can be linked in any order and using any method. Preferred helicase constructs used in the present invention are described in the following international applications: PCT / GB2013 / 051925 (published as WO2014 / 013260); PCT / GB2013 / 051924 (published as WO2014 / 013259); PCT / GB2013 / 051928 (published as WO2014 / 013262) and PCT / GB2014 / 052736.

[0685] Variants of SEQ ID NO: 397, 399, 401, 403, 405, 406, 407, 408, 409, 410, 411, 412, or 413 are enzymes having amino acid sequences that vary from the amino acid sequence of SEQ ID NO: 397, 399, 401, 403, 405, 406, 407, 408, 409, 410, 411, 412, or 413 and that retain polynucleotide binding ability. This can be measured using any method known in the art. For example, the variant can be contacted with a polynucleotide and its ability to bind to and move along the polynucleotide can be measured. The variant can include modifications that enhance polynucleotide binding and / or enhance activity at high salt concentrations and / or room temperature. Variants can be modified such that they bind polynucleotides (i.e., retain polynucleotide binding ability) but do not function as helicases (i.e., do not move along the polynucleotide when provided with all necessary components to facilitate movement, such as ATP and Mg). 2+ ). Such modifications are known in the art. For example, Mg in helicase 2+ Modifications to the binding domain typically result in variants that are unable to function as helicases. These types of variants can act as molecular brakes (see below).

[0686] The variant is preferably at least 50% homologous, based on amino acid identity, to the amino acid sequence of SEQ ID NO: 397, 339, 401, 403, 405, 406, 407, 408, 409, 410, 411, 412 or 413 over the entire length of the sequence. More preferably, the variant polypeptide may be at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, more preferably at least 95%, 97% or 99% homologous, based on amino acid identity, to the amino acid sequence of SEQ ID NO: 397, 399, 401, 403, 405, 406, 407, 408, 409, 410, 411, 412 or 413 over the entire sequence. There may be at least 80%, e.g., at least 85%, 90% or 95% amino acid identity over a stretch of 200 or more, e.g., 230, 250, 270, 280, 300, 400, 500, 600, 700, 800, 900 or 1000 or more consecutive amino acids ("strict homology"). Homology is determined as described above. Variants may differ from the wild-type sequence in any of the ways discussed above for SEQ ID NO: 390. The enzyme may be covalently linked to the pore. Any method may be used to covalently link the enzyme to the pore.

[0687] A preferred molecular brake is TrwC Cba-Q594A (SEQ ID NO: 413 with mutation Q594A). This variant does not function as a helicase (i.e., binds to a polynucleotide but does not move along it when provided with all the necessary components to facilitate movement, e.g., ATP and Mg). 2+ ).

[0688] During chain sequencing, the polynucleotide is displaced through the pore with or against the applied potential. Exonucleases that act gradually or progressively on double-stranded polynucleotides can be used on the cis side of the pore to feed the remaining single strand into and through the pore when a potential is applied, or on the trans side to feed the remaining single strand into and through the pore when a reverse potential is applied. Similarly, helicases that unwind double-stranded DNA can be used in a similar manner. Polymerases can also be used. It is also possible to use sequencing applications that require chain displacement against an applied potential, but the DNA must first be "captured" by the enzyme when a reverse potential or no potential is applied. Then, as the potential switches back after binding, the chain will pass through the pore from cis to trans and be maintained in an extended conformation by the current. Single-stranded DNA exonucleases or single-stranded DNA-dependent polymerases can act as molecular motors, pulling the recently displaced single strand back through the pore in a controlled, step-by-step manner, against the applied potential, from trans to cis.

[0689] Any helicase can be used in the method. The helicase can work in two modes relative to the hole. First, the method is preferably performed using a helicase so that the helicase moves the polynucleotide through the hole by the field generated by the applied voltage. In this mode, the 5' end of the polynucleotide is first captured in the hole, and the helicase moves the polynucleotide into the hole so that it passes through the hole under the electric field until it eventually shifts to the trans side of the membrane. Alternatively, the method is preferably performed like this: the helicase moves the polynucleotide through the hole against the field generated by the applied voltage. In this mode, the 3' end of the polynucleotide is first captured in the hole, and the helicase moves the polynucleotide through the hole so that it is pulled out of the hole against the applied field until it eventually rebounds to the cis side of the membrane.

[0690] The method can also be performed in the reverse direction. The 3' end of the polynucleotide can be captured first in the pore and the helicase can move the polynucleotide into the pore so that it uses the field to pass through the pore until it is eventually translocated to the trans side of the membrane.

[0691] In the case of helicase, the polynucleotide is not provided with the necessary components that promote movement or the helicase is modified to hinder or prevent it from moving. It can be used as a brake to slow down the movement of the polynucleotide in conjunction with the polynucleotide and when it is pulled into the hole by the applied field. In the inactive mode, it is insignificant whether the 3' or 5' end of the polynucleotide is captured, and it is that the applied field is used as the enzyme that pulls the polynucleotide into the hole towards the trans side. When in the inactive mode, the mobile control that the helicase carries out to the polynucleotide can be described in a variety of ways, including ratchet, sliding and braking. The helicase variant lacking helicase activity can also be used in this way.

[0692] The polynucleotide may be contacted with the polynucleotide binding protein and the pore in any order. Preferably, when the polynucleotide is contacted with the polynucleotide binding protein (e.g., a helicase) and the pore, the polynucleotide first forms a complex with the protein. When a voltage is applied across the pore, the polynucleotide / protein complex now forms a complex with the pore and controls the movement of the polynucleotide through the pore.

[0693] Any step in the method employing a polynucleotide binding protein is typically performed in the presence of free nucleotides or free nucleotide analogs and enzyme cofactors that facilitate the action of the polynucleotide binding protein. The free nucleotides may be any one or more of the individual nucleotides discussed above. Free nucleotides include, but are not limited to, adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), guanosine monophosphate (GMP), guanosine diphosphate (GDP), guanosine triphosphate (GTP), thymidine monophosphate (TMP), thymidine diphosphate (TDP), thymidine triphosphate (TTP), uridine monophosphate (UMP), uridine diphosphate (UDP), uridine triphosphate (UTP), cytidine monophosphate (CMP), cytidine diphosphate (CDP), cytidine triphosphate (CTP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (DAMP), deoxyadenosine monophosphate (DAMP), deoxyadenosine monophosphate (DAMP), deoxyadenosine diphosphate (DGP), deoxyadenosine monophosphate (DMP), deoxyadenosine diphosphate (DDP), deoxyadenosine triphosphate (DTP), deoxyadenosine monophosphate (DMP), deoxyadenosine diphosphate (UDP), deoxyadenosine diphosphate (DTP), deoxyadenosine monophosphate (DMP), deoxyadenosine diphosphate (DDP), deoxyadenosine triphosphate (DTP), deoxyadenosine monophosphate (DMP), deoxyadenosine diphosphate (DDP), deoxyadenosine di ... The free nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP or dCMP. The free nucleotides are preferably adenosine triphosphate (ATP). The enzyme cofactor is a factor that enables the construct to function. The enzyme cofactor is preferably a divalent metal cation. The divalent metal cation is preferably Mg. 2+ 、Mn 2+ , Ca 2+ , or Co 2+ The most preferred enzyme cofactor is Mg. 2+ .

[0694] Helicases and molecular brakes

[0695] In a preferred embodiment, the method comprises:

[0696] (a) providing one or more helicases and one or more molecular brakes attached to the polynucleotide to the polynucleotide;

[0697] (b) contacting the polynucleotide with a CsgG pore or a mutant thereof, e.g., a pore of the invention, and applying an electric potential across the pore such that one or more helicases and one or more molecular brakes come together, both of which control movement of the polynucleotide relative to, e.g., through, the pore;

[0698] (c) obtaining one or more measurements as the polynucleotide moves relative to the pore, wherein the measurements are indicative of one or more characteristics of the polynucleotide, thereby characterizing the polynucleotide.

[0699] Such methods are discussed in detail in international application number PCT / GB2014 / 052737.

[0700] The one or more helicases can be any of those discussed above. The one or more molecular brakes can be any compound or molecule that binds to the polynucleotide and slows the movement of the polynucleotide through the pore. The one or more molecular brakes preferably include one or more compounds that bind to the polynucleotide. The one or more compounds are preferably one or more macrocyclic compounds. Suitable macrocyclic compounds include, but are not limited to, cyclodextrins, calixarenes, cyclic peptides, crown ethers, cucurbitacins, pillararenes, derivatives thereof, or combinations thereof. The cyclodextrin or its derivative can be any of those disclosed in Eliseev, A.V. and Schneider, H.J. (1994) J. Am. Chem. Soc 116, 6081-6088. More preferably, the agent is heptakis-6-amino-β-cyclodextrin (am7-βCD), 6-monodeoxy-6-monoamino-β-cyclodextrin (am1-βCD), or heptakis-(6-deoxy-6-guanidino)-cyclodextrin (gu7-βCD).

[0701] The one or more molecular brakes are preferably one or more single-stranded binding proteins (SSBs). The one or more molecular brakes are more preferably single-stranded binding proteins (SSBs) comprising a carboxyl-terminal (C-terminal) region without a net negative charge or (ii) modified SSBs having one or more modifications in their C-terminal region that reduce the net negative charge of the C-terminal region. The one or more molecular brakes are most preferably one of the SSBs disclosed in International Application No. PCT / GB2013 / 051924 (published as WO2014 / 013259).

[0702] The one or more molecular brakes are preferably one or more polynucleotide binding proteins. The polynucleotide binding protein can be any protein that can bind to a polynucleotide and control its movement through the pore. It is easy to determine whether a protein binds to a polynucleotide in the art. Proteins typically interact with polynucleotides and modify at least one property of the polynucleotide. Proteins can modify a polynucleotide by cleaving it to form individual nucleotides or short chains of nucleotides (e.g., dinucleotides or trinucleotides). This moiety can modify a polynucleotide by orienting it or moving it to a specific position, i.e., controlling its movement.

[0703] The polynucleotide binding protein is preferably derived from a polynucleotide handling enzyme. The one or more molecular brakes can be derived from any of the polynucleotide handling enzymes described above. A modified version of Phi29 polymerase (SEQ ID NO: 396) as a molecular brake is disclosed in U.S. Patent No. 5,576,204. The one or more molecular brakes are preferably derived from a helicase.

[0704] Any number of molecular brakes derived from helicases can be used. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more helicases can be used as molecular brakes. If two or more helicases are used as molecular brakes, the two or more helicases are typically the same helicase. The two or more helicases can be different helicases.

[0705] The two or more helicases can be any combination of the above helicases. The two or more helicases can be two or more Dda helicases. The two or more helicases can be one or more Dda helicases and one or more TrwC helicases. The two or more helicases can be different variants of the same helicase.

[0706] The two or more helicases are preferably linked to each other. The two or more helicases are more preferably covalently linked to each other. The helicases can be linked in any order and using any method. Preferably, the one or more molecular brakes derived from the helicase are modified to reduce the size of the opening in the polynucleotide binding domain, wherein the polynucleotide is unwound from the helicase by the polynucleotide binding domain in at least one conformational state. This is disclosed in WO2014 / 013260.

[0707] Preferred helicase constructs for use in the present invention are described in the following International Applications: PCT / GB2013 / 051925 (published as WO2014 / 013260); PCT / GB2013 / 051924 (published as WO2014 / 013259); PCT / GB2013 / 051928 (published as WO2014 / 013262) and PCT / GB2014 / 052736.

[0708] If the one or more helicases are used in active mode (i.e., when the one or more helicases are provided with all necessary components to facilitate movement, e.g., ATP and Mg 2+), the one or more molecular brakes are preferably used (a) in an inactive mode (i.e., in the absence of the necessary components to facilitate movement or incapable of active movement); (b) in an active mode, wherein the one or more molecular brakes move in the opposite direction to the one or more helicases; or (c) in an active mode, wherein the one or more molecular brakes move in the same direction as the one or more helicases and move slower than the one or more helicases.

[0709] If the one or more helicases are in an inactive mode (i.e., when the one or more helicases are not provided with all the necessary components to facilitate movement, e.g., ATP and Mg 2+ The one or more molecular brakes are preferably used (a) in an inactive mode (i.e., in the absence of the necessary components to facilitate movement or incapable of active movement) or (b) in an active mode, wherein the one or more molecular brakes move along the polynucleotide in the same direction as the direction of passage of the polynucleotide through the pore.

[0710] The one or more helicases and one or more molecular brakes can be attached to the polynucleotide at any position such that they are brought together and both control the movement of the polynucleotide through the pore. The one or more helicases and one or more molecular brakes are at least one separate nucleotide, for example, at least 5, at least 10, at least 50, at least 100, at least 500, at least 1000, at least 5000, at least 10,000, at least 50,000 or more separate nucleotides. If the method involves characterizing a double-stranded polynucleotide having a Y adapter at one end and a hairpin adapter at the other end, the one or more helicases are preferably attached to the Y adapter, and the one or more molecular brakes are preferably attached to the hairpin adapter. In this embodiment, the one or more molecular brakes are preferably one or more helicases that have been modified so that they bind to the polynucleotide but do not function as a helicase. The one or more helicases attached to the Y adapter are preferably arrested at a spacer, which is discussed in more detail below. The one or more molecular brakes attached to the hairpin adapter are preferably not arrested at the spacer. When the one or more helicases reach the hairpin loop, the one or more helicases and the one or more molecular brakes are preferably brought together. The one or more helicases can be connected to the Y adapter before or after the Y adapter is connected to the polynucleotide. The one or more molecular brakes can be connected to the hairpin loop adapter before or after the hairpin loop adapter is connected to the polynucleotide.

[0711] The one or more helicases and the one or more molecular brakes are preferably not linked to each other. The one or more helicases and the one or more molecular brakes are more preferably not covalently linked to each other. As described in International Applications PCT / GB2013 / 051925 (published as WO2014 / 013260); PCT / GB2013 / 051924 (published as WO2014 / 013259); PCT / GB2013 / 051928 (published as WO2014 / 013262) and PCT / GB2014 / 052736, the one or more helicases and the one or more molecular brakes are preferably not linked to each other.

[0712] spacer

[0713] The one or more helicases may be stalled at one or more spacers as described in International Application PCT / GB2014 / 050175. Any conformation of the one or more helicases and one or more spacers disclosed in that International Application may be used in the present invention.

[0714] When a portion of the polynucleotide enters the pore and moves through the pore along the field generated by the applied potential, the one or more helicases move through the pore through the spacer as the polynucleotide moves through the pore. This is because the polynucleotide (including the one or more spacers) moves through the pore while the one or more helicases remain at the top of the pore.

[0715] The one or more spacers are preferably part of the polynucleotide, eg they interrupt the polynucleotide sequence.The one or more spacers are preferably not part of one or more blocking molecules, eg speed bumps, that hybridize to the polynucleotide.

[0716] Any number of spacers may be present in a polynucleotide, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more spacers. Preferably, two, four, or six spacers are present in a polynucleotide. One or more spacers may be present in different regions of a polynucleotide, for example, one or more spacers in a Y adaptor and / or a hairpin loop adaptor.

[0717] Each of the one or more spacers provides an energy barrier that the one or more helicases cannot overcome even in active mode. The one or more spacers can retard the one or more helicases by reducing the pulling force of the helicase (e.g., by removing bases of nucleotides in the polynucleotide) or physically blocking the movement of the one or more helicases (e.g., using bulky chemical groups).

[0718] The one or more spacers can comprise any molecule or combination of molecules that make the one or more helicases stagnate. The one or more spacers can comprise any molecule or combination of molecules that prevent the one or more helicases from moving along the polynucleotide. In the absence of a transmembrane pore and applied potential, directly determine whether the one or more helicases stagnate at one or more spacers. For example, the ability of the helicase to move through the spacer and substitute the DNA complementary chain can be measured by PAGE.

[0719] The one or more spacers generally include linear molecules, for example, polymers. The one or more spacers generally have a structure different from that of the polynucleotide. For example, if the polynucleotide is DNA, the one or more spacers are not DNA generally. In particular, if the polynucleotide is deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), the one or more spacers preferably include peptide nucleic acid (PNA), glycerol nucleic acid (GNA), threose nucleic acid (TNA), locked nucleic acid (LNA) or a synthetic polymer with nucleotide side chains. The one or more spacers may include one or more nucleotides in the opposite direction to the polynucleotide. For example, when the polynucleotide is in a 5' to 3' direction, the one or more spacers may include one or more nucleotides in a 3' to 5' direction. Nucleotide may be any of those discussed above.

[0720] The one or more spacers preferably include one or more nitroindoles, for example, one or more 5-nitroindoles, one or more inosines, one or more acridines, one or more 2-aminopurines, one or more 2-6-diaminopurines, one or more 5-bromo-deoxyuridines, one or more trans-thymidines (trans-dT), one or more trans-dideoxythymidines (ddT), one or more dideoxycytidines (ddC), one or more 5-methylcytidines, one or more 5-hydroxymethylcytidines, one or more 2'-O-methyl RNA bases, one or more isodeoxycytidines (Iso-dC), one or more isodeoxyguanosines (Iso-dG), one or more iSpC3 groups (i.e., nucleotides lacking a sugar and a base), one or more photocleavable (PC) groups, one or more hexanediol groups, one or more spacer 9 (iSp9) groups, one or more spacer 18 (iSp18) groups, a polymer, or one or more thiol linkages. The one or more spacers may include any combination of these groups. Many of these groups may be selected from (Integrated DNA ) purchased.

[0721] The one or more spacers can include any number of these groups. For example, for 2-aminopurine, 2-6-diaminopurine, 5-bromo-deoxyuridine, trans-dT, ddT, ddC, 5-methylcytidine, 5-hydroxymethylcytidine, 2'-O-methyl RNA bases, Iso-dC, Iso-dG, iSpC3 groups, PC groups, hexanediol groups and thiol connections, the one or more spacers preferably include 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more. The one or more spacers preferably include 2, 3, 4, 5, 6, 7, 8 or more iSp9 groups. The one or more spacers preferably include 2, 3, 4, 5 or 6 or more iSp18 groups. The most preferred spacer is four iSp18 groups.

[0722] The polymer is preferably a polypeptide or polyethylene glycol (PEG). The polypeptide preferably comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more amino acids. The PEG preferably comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more monomer units.

[0723] The one or more spacers preferably include one or more abasic nucleotides (i.e., nucleotides lacking a core base), for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more abasic nucleotides. The core base can be replaced by -H (idSp) or -OH in the abasic nucleotide. By removing the core base from one or more adjacent nucleotides, the abasic spacer can be inserted into the polynucleotide. For example, the polynucleotide can be modified to include 3-methyladenine, 7-methylguanine, 1, N6-ethenoadenine inosine or hypoxanthine, and human alkyladenine DNA glycosylase (hAAG) can be used to remove the core base from these nucleotides. Alternatively, the polynucleotide can be modified to include uracil and the core base removed with uracil-DNA glycosylase (UDG). In one embodiment, the one or more spacers do not include any abasic nucleotides.

[0724] The one or more helicases can be blocked by each linear molecule spacer (i.e., before the spacer) or on each linear molecule spacer. If a linear molecule spacer is used, the polynucleotide is preferably provided with a polynucleotide double-stranded region adjacent to each spacer end, and the one or more helicases move through each spacer end. The double-stranded region generally helps to block the one or more helicases on the adjacent spacer. If the method is performed at a salt concentration of about 100mM or lower, it is particularly preferred that a double-stranded region be present. Each double-stranded region is generally at least 10, for example, at least 12, nucleotides in length. If the polynucleotide used in the present invention is single-stranded, a double-stranded region can be formed by hybridizing a shorter polynucleotide with a region adjacent to the spacer. The shorter polynucleotide is generally formed by the same nucleotides as the polynucleotide, but can also be formed by different nucleotides. For example, the shorter polynucleotide can be formed by LNA.

[0725] If use linear molecule spacer, then polynucleotide is preferably provided with blocking molecule at the end of each spacer, and described end is relative with the end that described one or more helicases move through.This can help to guarantee that described one or more helicases keep stagnation on each spacer.Can also help when described one or more helicases diffuse in solution, make it / they remain on the polynucleotide.Described blocking molecule can be arbitrary chemical group discussed below, and these chemical groups physically cause described one or more helicases to stagnate.Described blocking molecule can be the double-stranded region of polynucleotide.

[0726] The one or more spacers preferably include one or more chemical groups that physically cause the one or more helicases to stagnate. The one or more chemical groups are preferably one or more side chain chemical groups. The one or more chemical groups can be connected to one or more core bases in a polynucleotide. The one or more chemical groups can be connected to a polynucleotide backbone. Any number of these chemical groups can be present, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more. Suitable groups include but are not limited to fluorophores, streptavidin and / or biotin, cholesterol, methylene blue, dinitrophenol (DNP), digoxin and / or anti-digoxin and dibenzylcyclooctyne groups.

[0727] Different spacers in the polynucleotide can in...

Claims

1. A method for determining the presence, absence or one or more characteristics of a target analyte, wherein the target analyte is an oligonucleotide or a polynucleotide, the method comprising: a. a step of contacting the target analyte with a CsgG pore or a mutant thereof in an insulating membrane, so that the target analyte binds within or near the pore, or moves through the pore, wherein the mutant has an amino acid sequence having at least 90% identity with the amino acid sequence of SEQ ID NO: 390, and maintains the ability to form a pore; as well as b. the step of performing one or more electrical measurements while the analyte is bound within or near the pore, or moves through the pore, thereby determining the presence, absence, or one or more characteristics of the analyte, The characteristic determined is at least one of (i) the length of the analyte, (ii) the identity of the analyte, and (iii) the sequence of the analyte.

2. The method according to claim 1, wherein: The target analyte is a target polynucleotide, step a) further comprises contacting the polynucleotide with a polynucleotide binding protein such that the protein controls movement of the polynucleotide through the pore, optionally the polynucleotide binding protein is a helicase or is derived from a helicase.

3. The method according to claim 1 or 2, wherein: The CsgG mutants include: (i) a mutant CsgG monomer comprising a sequence variant as shown in SEQ ID NO: 390, wherein the variant comprises a mutation at one or more of positions Y51, N55 and F56; or (ii) nine monomers, and at least one of the monomers is a variant of SEQ ID NO 390, comprising: (a) a mutation at one or more of the following positions: N40, Q42, D43, E44, K49, Y51, S54, N55, F56, S57, Q62, E101, N102, E124, E131, R142, D149, T150, E185, R192, D195, E201 and E203; and / or (b) a deletion at one or more of the following positions: F48, K49, P50, Y51, P52, A53, S54, N55, F56 and S57.

4. The method according to claim 3(ii), wherein: The variants include one or more of the following: (i) one or more mutations at the following positions: N40, D43, E44, S54, S57, Q62, R97, E101, E124, E131, R142, T150 and R192; (ii) mutations at Y51 / N55, Y51 / F56, N55 / F56 or Y51 / N55 / F56; (iii) Q42R or Q42K; (iv) K49R; (v) N102R, N102F, N102Y or N102W; (vi) D149N, D149Q or D149R; (vii) E185N, E185Q or E185R; (viii) D195N, D195Q or D195R; (ix) E201N, E201Q or E201R; (x) E203N, E203Q or E203R; and (xi) deletion of one or more of the following positions: F48, K49, P50, Y51, P52, A53, S54, N55, F56 and S57, optionally, (a) the variant comprises in (i) one or more of the following substitutions: N40R, N40K, D43N, D43Q, D43R, D43K, E44N, E44Q, E44R, E44K, S54P, S57P, Q62R, Q62K, R97N, R97G, R97L, E101 N, E101Q, E101R, E101K, E101F, E101Y, E101W, E124N, E124Q, E124R, E124K, E124F, E124Y, E124W, E131D, R142E, R142N, T150I, R192E, and R192N; (b) The variants are included in the following (ii): (ii) F56N / N55Q, F56N / N55R, F56N / N55K, F56N / N55S, F56N / N55G, F56N / N55A, F56N / N55T, F56Q / N55Q, F56Q / N55R, F56Q / N55K, F56Q / N55S, F56Q / N55G,F56Q / N55A,F56Q / N55T,F56R / N55Q,F56R / N55R,F56R / N55K,F56R / N55S, F56R / N55G,F56R / N55A,F56R / N55T,F56S / N55Q,F56S / N55R,F56S / N55K,F56S / N55S, F56S / N55G,F56S / N55A,F56S / N55T,F56G / N55Q,F56G / N55R,F56G / N55K,F56G / N55S, F56G / N55G,F56G / N55A,F56G / N55T,F56A / N55Q,F56A / N55R,F56A / N55K,F56A / N55S,F56A / N55G,F56A / N55A,F56A / N55T,F56K / N55Q,F56K / N55R,F56K / N55K,F56K / N55S, F56K / N55G,F56K / N55A,F56K / N55T,F56N / Y51L,F56N / Y51V,F56N / Y51A,F56N / Y51N, F56N / Y51Q,F56N / Y51S,F56N / Y51G,F56Q / Y51L,F56Q / Y51V,F56Q / Y51A,F56Q / Y51N, F56Q / Y51Q,F56Q / Y51S,F56Q / Y51G,F56R / Y51L,F56R / Y51V,F56R / Y51A,F56R / Y51N, F56R / Y51Q,F56R / Y51S,F56R / Y51G,F56S / Y51L,F56S / Y51V,F56S / Y51A,F56S / Y51N, F56S / Y51Q,F56S / Y51S,F56S / Y51G,F56G / Y51L,F56G / Y51V,F56G / Y51A,F56G / Y51N, F56G / Y51Q,F56G / Y51S,F56G / Y51G,F56A / Y51L,F56A / Y51V,F56A / Y51A,F56A / Y51N, F56A / Y51Q,F56A / Y51S,F56A / Y51G,F56K / Y51L,F56K / Y51V,F56K / Y51A,F56K / Y51N, F56K / Y51Q,F56K / Y51S,F56K / Y51G,N55Q / Y51L,N55Q / Y51V,N55Q / Y51A,N55Q / Y51N, N55Q / Y51Q,N55Q / Y51S,N55Q / Y51G,N55R / Y51L,N55R / Y51V,N55R / Y51A,N55R / Y51N,N55R / Y51Q,N55R / Y51S,N55R / Y51G,N55K / Y51L,N55K / Y51V,N55K / Y51A,N55K / Y51N,N55K / Y51Q,N55K / Y51S,N55K / Y51G,N55S / Y51L,N55S / Y51V,N55S / Y51A,N55S / Y51N, N55S / Y51Q,N55S / Y51S,N55S / Y51G,N55G / Y51L,N55G / Y51V,N55G / Y51A,N55G / Y51N, N55G / Y51Q,N55G / Y51S,N55G / Y51G,N55A / Y51L,N55A / Y51V,Ν55Α / Υ51Α,Ν55Α / Υ51Ν,N55A / Y51Q,N55A / Y51S,N55A / Y51G,N55T / Y51L,N55T / Y51V,Ν55Τ / Υ51Α,Ν55Τ / Υ51Ν, N55T / Y51Q,N55T / Y51S,N55T / Y51G,F56N / N55Q / Y51L,F56N / N55Q / Y51V, F56N / N55Q / Y51A,F56N / N55Q / Y51Ν,F56N / N55Q / Y51Q,F56N / N55Q / Y51S, F56N / N55Q / Y51G,F56N / N55R / Y51L,F56N / N55R / Y51V,F56N / N55R / Y51A,F56N / N55R / Y51N,F56N / N55R / Y51Q,F56N / N55R / Y51S,F56N / N55R / Y51G,F56N / N55K / Y51L,F56N / N55K / Y51V,F56N / N55K / Y51A,F56N / N55K / Y51N,F56N / N55K / Y51Q,F56N / N55K / Y51S, F56N / N55K / Y51G,F56N / N55S / Y51L,F56N / N55S / Y51V,F56N / N55S / Y51A,F56N / N55S / Y51N,F56N / N55S / Y51Q,F56N / N55S / Y51S,F56N / N55S / Y51G,F56N / N55G / Y51L,F56N / N55G / Y51V,F56N / N55G / Y51A,F56N / N55G / Y51N,F56N / N55G / Y51Q,F56N / N55G / Y51S, F56N / N55G / Y51G,F56N / N55A / Y51L,F56N / N55A / Y51V,F56N / N55A / Y51A, F56N / N55A / Y51N,F56N / N55A / Y51Q,F56N / N55A / Y51S,F56N / N55A / Y51G,F56N / N55T / Y51L,F56N / N55T / Y51V,F56N / N55T / Y51A,F56N / N55T / Y51N,F56N / N55T / Y51Q,F56N / N55T / Y51S,F56N / N55T / Y51G,F56Q / N55Q / Y51L,F56Q / N55Q / Y51V,F56Q / N55Q / Y51A,F56Q / N55Q / Y51N,F56Q / N55Q / Y51Q,F56Q / N55Q / Y51S,F56Q / N55Q / Y51G,F56Q / N55R / Y51L,F56Q / N55R / Y51V,F56Q / N55R / Y51A,F56Q / N55R / Y51N,F56Q / N55R / Y51Q,F56Q / N55R / Y51S,F56Q / N55R / Y51G,F56Q / N55K / Y51L,F56Q / N55K / Y51V,F56Q / N55K / Y51A,F56Q / Ν55K / Υ51Ν, F56Q / N55K / Y51Q,F56Q / N55K / Y51S,F56Q / N55K / Y51G,F56Q / N55S / Y51L,F56Q / N55S / Y51V,F56Q / N55S / Y51Α,F56Q / N55S / Y51N,F56Q / N55S / Y51Q,F56Q / N55S / Y51S,F56Q / N55S / Y51G,F56Q / N55G / Y51L,F56Q / N55G / Y51V,F56Q / N55G / Y51A,F56Q / N55G / Y51N, F56Q / N55G / Y51Q,F56Q / N55G / Y51S,F56Q / N55G / Y51G,F56Q / N55A / Y51L,F56Q / N55A / Y51V,F56Q / N55A / Y51A,F56Q / N55A / Y51N,F56Q / N55A / Y51Q,F56Q / N55A / Y51S,F56Q / N55A / Y51G,F56Q / N55T / Y51L,F56Q / N55T / Y51V,F56Q / N55T / Y51A,F56Q / N55T / Y51N,F56Q / N55T / Y51Q,F56Q / N55T / Y51S,F56Q / N55T / Y51G,F56R / N55Q / Y51L,F56R / N55Q / Y51V,F56R / N55Q / Y51A,F56R / N55Q / Y51N,F56R / N55Q / Y51Q,F56R / N55Q / Y51S,F56R / N55Q / Y51G,F56R / N55R / Y51L,F56R / N55R / Y51V,F56R / N55R / Y51A,F56R / N55R / Y51N,F56R / N55R / Y51Q,F56R / N55R / Y51S,F56R / N55R / Y51G,F56R / N55K / Y51L,F56R / N55K / Y51V,F56R / N55K / Y51A,F56R / N55K / Y51N,F56R / N55K / Y51Q,F56R / N55K / Y51S,F56R / N55K / Y51G,F56R / N55S / Y51L,F56R / N55S / Y51V,F56R / N55S / Y51A,F56R / N55S / Y51N,F56R / N55S / Y51Q,F56R / N55S / Y51S,F56R / N55S / Y51G,F56R / N55G / Y51L,F56R / N55G / Y51V,F56R / N55G / Y51A,F56R / N55G / Y51N,F56R / N55G / Y51Q,F56R / N55G / Y51S,F56R / N55G / Y51G,F56R / N55A / Y51L,F56R / N55A / Y51V,F56R / N55A / Y51A,F56R / N55A / Y51N,F56R / N55A / Y51Q,F56R / N55A / Y51S,F56R / N55A / Y51G,F56R / N55T / Y51L,F56R / N55T / Y51V,F56R / N55T / Y51A,F56R / N55T / Y51N,F56R / N55T / Y51Q,F56R / N55T / Y51S,F56R / N55T / Y51G,F56S / N55Q / Y51L,F56S / N55Q / Y51V,F56S / N55Q / Y51A,F56S / N55Q / Y51N,F56S / N55Q / Y51Q,F56S / N55Q / Y51S,F56S / N55Q / Y51G,F56S / N55R / Y51L,F56S / N55R / Y51V,F56S / N55R / Y51A,F56S / N55R / Y51N,F56S / N55R / Y51Q,F56S / N55R / Y51S,F56S / N55R / Y51G,F56S / N55K / Y51L,F56S / N55K / Y51V,F56S / N55K / Y51A,F56S / N55K / Y51N,F56S / N55K / Y51Q,F56S / N55K / Y51S,F56S / N55K / Y51G,F56S / N55S / Y51L,F56S / N55S / Y51V,F56S / N55S / Y51A,F56S / N55S / Y51N,F56S / N55S / Y51Q,F56S / N55S / Y51S,F56S / N55S / Y51G,F56S / N55G / Y51L,F56S / N55G / Y51V,F56S / N55G / Y51A,F56S / N55G / Y51N,F56S / N55G / Y51Q,F56S / N55G / Y51S,F56S / N55G / Y51G,F56S / N55A / Y51L,F56S / N55A / Y51V,F56S / N55A / Y51A,F56S / N55A / Y51N,F56S / N55A / Y51Q,F56S / N55A / Y51S,F56S / N55A / Y51G,F56S / N55T / Y51L,F56S / N55T / Y51V,F56S / N55T / Y51A,F56S / N55T / Y51N,F56S / N55T / Y51Q,F56S / N55T / Y51S,F56S / N55TY51G,F56G / N55QY51L,F56G / N55QY51V,F56G / N55Q / Y51A,F56G / N55Q / Y51N,F56G / N55Q / Y51Q,F56G / N55Q / Y51S,F56G / N55Q / Y51G,F56G / N55R / Y51L,F56G / N55R / Y51V,F56G / N55R / Y51A,F56G / N55R / Y51N,F56G / N55R / Y51Q,F56G / N55R / Y51S,F56G / N55R / Y51G,F56G / N55K / Y51L,F56G / N55K / Y51V,F56G / N55K / Y51A,F56G / N55K / Y51N,F56G / N55K / Y51Q, F56G / N55K / Y51S, F56G / N55K / Y51G, F56G / N55S / Y51L, F56G / N55S / Y51V, F56G / N55S / Y51A, F56G / N55S / Y51N, F56G / N55S / Y51Q, F56G / N55S / Y51S, F56G / N55S / Y51G, F56G / N55G / Y51L, F56G / N55G / Y51V, F56G / N55G / Y51A, F56G / N55G / Y51N, F56G / N55G / Y51Q, F56G / N55G / Y51S, F56G / N55G / Y51G, F56G / N55A / Y51L, F56G / N55A / Y51V, F56G / N55A / Y51A, F56G / N55A / Y51N, F56G / N55A / Y51Q, F56G / N55A / Y51S, F56G / N55A / Y51G, F56G / N55T / Y51L, F56G / N55T / Y51V, F56G / N55T / Y51A, F56G / N55T / Y51N, F56G / N55T / Y51Q, F56G / N55T / Y51S, F56G / N55T / Y51G, F56A / N55Q / Y51L, F56A / N55Q / Y51V, F56A / N55Q / Y51A, F56A / N55Q / Y51N, F56A / N55Q / Y51Q, F56A / N55Q / Y51S, F56A / N55Q / Y51G, F56A / N55R / Y51L, F56A / N55R / Y51V, F56A / N55R / Y51A, F56A / N55R / Y51N, F56A / N55R / Y51Q, F56A / N55R / Y51S, F56A / N55R / Y51G, F56A / N55K / Y51L, F56A / N55K / Y51V, F56A / N55K / Y51A, F56A / N55K / Y51N, F56A / N55K / Y51Q,F56A / N55K / Y51S,F56A / N55K / Y51G,F56A / N55S / Y51L,F56A / N55S / Y51V,F56A / N55S / Y51A,F56A / N55S / Y51N,F56A / N55S / Y51Q,F56A / N55S / Y51S,F56A / N55S / Y51G,F56A / N55G / Y51L F56A / N55G / Y51V,F56A / N55G / Y51A,F56A / N55G / Y51N,F56A / N55G / Y51Q,F56A / N55G / Y51S,F56A / N55G / Y51G,F56A / N55A / Y51L,F56A / N55A / Y51V,F56A / N55A / Y51A,F56A / N55A / Y51N,F56A / N55A / Y51Q,F56A / N55A / Y51S,F56A / N55A / Y51G,F56A / N55T / Y51L,F56A / N55T / Y51V,F56A / N55T / Y51A,F56A / N55T / Y51N,F56A / N55T / Y51Q,F56A / N55T / Y51S,F56A / N55T / Y51G,F56K / N55Q / Y51L,F56K / N55Q / Y51V,F56K / N55Q / Y51A,F56K / N55Q / Y51N,F56K / N55Q / Y51Q,F56K / N55Q / Y51S,F56K / N55Q / Y51G,F56K / N55R / Y51L,F56K / N55R / Y51V,F56K / N55R / Y51A,F56K / N55R / Y51N,F56K / N55R / Y51Q,F56K / N55R / Y51S,F56K / N55R / Y51G,F56K / N55K / Y51L F56K / N55K / Y51V,F56K / N55K / Y51A,F56K / N55K / Y51N,F56K / N55K / Y51Q,F56K / N55K / Y51S F56K / N55K / Y51G,F56K / N55S / Y51L,F56K / N55S / Y51V,F56K / N55S / Y51A,F56K / N55S / Y51N F56K / N55S / Y51Q,F56K / N55S / Y51S,F56K / N55S / Y51G,F56K / N55G / Y51L,F56K / N55G / Y51V F56K / N55G / Y51A,F56K / N55G / Y51N,F56K / N55G / Y51Q,F56K / N55G / Y51S,F56K / N55G / Y51G F56K / N55A / Y51L,F56K / N55A / Y51V,F56K / N55A / Y51A,F56K / N55A / Y51N,F56K / N55A / Y51Q,F56K / N55A / Y51S,F56K / N55A / Y51G,F56K / N55T / Y51L, F56K / N55T / Y51V, F56K / N55T / Y51A, F56K / N55T / Y51N, F56K / N55T / Y51Q, F56K / N55T / Y51S, F56K / N55T / Y51G, F56E / N55R, F56E / N55K, F56D / N55R, F56D / N55K, F56R / N55E, F56R / N55D, F56K / N55E or F56K / N55D; and / or (c) The variants include those in (xi): (xi) deletion of Y51 / P52, deletion of Y51 / P52 / A53, deletion of P50 to P52, deletion of P50 to A53, deletion of K49 to Y51, deletion of K49 to A53 and substitution with a single proline (P), deletion of K49 to S54 and substitution with a single P, deletion of Y51 to A53, deletion of Y51 to S54, deletion of N55 / F56, deletion of N55 to S57, deletion of N55 / F56 and substitution with a single P, deletion of N55 / F56 and substitution with a single glycine (G), deletion of N55 / F56 and substitution with a single 10 A and 10 A, deletion of N55 / F56 and replacement with a single alanine (A), deletion of N55 / F56 and replacement with a single P and Y51N, deletion of N55 / F56 and replacement with a single P and Y51Q, deletion of N55 / F56 and replacement with a single P and Y51S, deletion of N55 / F56 and replacement with a single G and Y51N, deletion of N55 / F56 and replacement with a single G and Y51Q, deletion of N55 / F56 and replacement with a single G and Y51S, deletion of N55 / F56 and replacement with a single A and Y51N, deletion of N55 / F56 and replacement with a single A / Y51Q, or deletion of N55 / F56 and replacement with a single A and Y51 S.

5. The method according to claim 4, wherein: The variants include: D195N / E203N, D195Q / E203N, D195N / E203Q, D195Q / E203Q, E201N / E203N, E201Q / E203N, E201N / E203Q, E201Q / E203Q, E185N / E203Q, E185Q / E203Q, E185N / E203N, E185Q / E203N, D195N / E201 N / E203N,D195Q / E201N / E203N,D195N / E201Q / E203N,D195N / E201N / E203Q, D195Q / E201Q / E203N,D195Q / E201N / E203Q,D195N / E201Q / E203Q,D195Q / E201Q / E203Q, D149N / E201N,D149Q / E201N,D149N / E201Q,D149Q / E201Q,D149N / E201N / D195N, D149Q / E201N / D195N,D149N / E201Q / D195N,D149N / E201N / D195Q,D149Q / E201Q / D19 5N,D149Q / E201N / D195Q,D149N / E201Q / D195Q,D149Q / E201Q / D195Q,D149N / E203N, D149Q / E203N,D149N / E203Q,D149Q / E203Q,D149N / E185N / E201N,D149Q / E185N / E201N, D149N / E185Q / E201N,D149N / E185N / E201Q,D149Q / E185Q / E201N,D149Q / E185N / E201Q, D149N / E185Q / E201Q,D149Q / E185Q / E201Q,D149N / E185N / E203N,D149Q / E185N / E203N, D149N / E185Q / E203N,D149N / E185N / E203Q,D149Q / E185Q / E203N,D149Q / E185N / E203Q, D149N / E185Q / E203Q,D149Q / E185Q / E203Q,D149N / E185N / E201N / E203N, D149Q / E185N / E201N / E203N,D149N / E185Q / E201N / E203N,D149N / E185N / E201Q / E203N, D149N / E185N / E201N / E203Q,D149Q / E185Q / E201N / E203N,D149Q / E185N / E201Q / E203N, D149Q / E185N / E201N / E203Q,D149N / E185Q / E201Q / E203N,D149N / E185Q / E201N / E203Q, D149N / E185N / E201Q / E203Q,D149Q / E185Q / E201Q / E203Q,D149Q / E185Q / E201N / E203Q, D149Q / E185N / E201Q / E203Q,D149N / E185Q / E201Q / E203Q,D149Q / E185Q / E201Q / E203N, D149N / E185N / D195N / E201N / E203N,D149Q / E185N / D195N / E201N / E203N, D149N / E185Q / D195N / E201N / E203N,D149N / E185N / D195Q / E201N / E203N,D149N / E185N / D195N / E201Q / E203N,D149N / E185N / D195N / E201N / E203Q, D149Q / E185Q / D195N / E201N / E203N,D149Q / E185N / D195Q / E201N / E203N, D149Q / E185N / D195N / E201Q / E203N,D149Q / E185N / D195N / E201N / E203Q, D149N / E185Q / D195Q / E201N / E203N,D149N / E185Q / D195N / E201Q / E203N, D149N / E185Q / D195N / E201N / E203Q,D149N / E185N / D195Q / E201Q / E203N, D149N / E185N / D195Q / E201N / E203Q,D149N / E185N / D195N / E201Q / E203Q, D149Q / E185Q / D195Q / E201N / E203N,D149Q / E185Q / D195N / E201Q / E203N, D149Q / E185Q / D195N / E201N / E203Q,D149Q / E185N / D195Q / E201Q / E203N, D149Q / E185N / D195Q / E201N / E203Q,D149Q / E185N / D195N / E201Q / E203Q, D149N / E185Q / D195Q / E201Q / E203N,D149N / E185Q / D195Q / E201N / E203Q, D149N / E185Q / D195N / E201Q / E203Q,D149N / E185N / D195Q / E201Q / E203Q, D149Q / E185Q / D195Q / E201Q / E203N,D149Q / E185Q / D195Q / E201N / E203Q, D149Q / E185Q / D195N / E201Q / E203Q,D149Q / E185N / D195Q / E201Q / E203Q, D149N / E185Q / D195Q / E201Q / E203Q,D149Q / E185Q / D195Q / E201Q / E203Q, D149N / E185R / E201N / E203N,D149Q / E185R / E201N / E203N,D149N / E185R / E201Q / E203N,D149N / E185R / E201N / E203Q,D149Q / E185R / E201Q / E203N,D149Q / E185R / E201N / E203Q,D149N / E185R / E201Q / E203Q,D149Q / E185R / E201Q / E203Q,D149R / E185N / E201N / E203N,D149R / E185Q / E201N / E203N,D149R / E185N / E201Q / E203N,D149R / E185N / E201N / E203Q,D149R / E185Q / E201Q / E203N,D149R / E185Q / E201N / E203Q,D149R / E185N / E201Q / E203Q,D149R / E185Q / E201Q / E203Q, D149R / E185N / D195N / E201N / E203N,D149R / E185Q / D195N / E201N / E203N, D149R / E185N / D195Q / E201N / E203N,D149R / E185N / D195N / E201Q / E203N, D149R / E185Q / D195N / E201N / E203Q,D149R / E185Q / D195Q / E201N / E203N, D149R / E185Q / D195N / E201Q / E203N,D149R / E185Q / D195N / E201N / E203Q, D149R / E185N / D195Q / E201Q / E203N,D149R / E185N / D195Q / E201N / E203Q, D149R / E185N / D195N / E201Q / E203Q,D149R / E185Q / D195Q / E201Q / E203N, D149R / E185Q / D195Q / E201N / E203Q,D149R / E185Q / D195N / E201Q / E203Q, D149R / E185N / D195Q / E201Q / E203Q,D149R / E185Q / D195Q / E201Q / E203Q, D149N / E185R / D195N / E201N / E203N,D149Q / E185R / D195N / E201N / E203N, D149N / E185R / D195Q / E201N / E203N,D149N / E185R / D195N / E201Q / E203N, D149N / E185R / D195N / E201N / E203Q,D149Q / E185R / D195Q / E201N / E203N, D149Q / E185R / D195N / E201Q / E203N,D149Q / E185R / D195N / E201N / E203Q, D149N / E185R / D195Q / E201Q / E203N,D149N / E185R / D195Q / E201N / E203Q, D149N / E185R / D195N / E201Q / E203Q,D149Q / E185R / D195Q / E201Q / E203N, D149Q / E185R / D195Q / E201N / E203Q,D149Q / E185R / D195N / E201Q / E203Q, D149N / E185R / D195Q / E201Q / E203Q,D149Q / E185R / D195Q / E201Q / E203Q, D149N / E185R / D195N / E201R / E203N,D149Q / E185R / D195N / E201R / E203N, D149N / E185R / D195Q / E201R / E203N,D149N / E185R / D195N / E201R / E203Q, D149Q / E185R / D195Q / E201R / E203N,D149Q / E185R / D195N / E201R / E203Q,D149N / E185R / D195Q / E201R / E203Q,D149Q / E185R / D195Q / E201R / E203Q,E131D / K49R, E101N / N102F,E101N / N102Y,E101N / N102W,E101F / N102F,E101F / N102Y,E101F / N102W, E101Y / N102F, E101Y / N102Y, E101Y / N102W, E101W / N102F, E101W / N102Y, E101W / N102W, E101N / N102R, E101F / N102R, E101Y / N102R or E101W / N102F.

6. The method according to claim 4, wherein: The variant comprises a mutation at T150.

7. The method according to claim 3(ii), wherein: The variant comprises one or more of the following substitutions: (i) F56N, F56Q, F56R, F56S, F56G, F56A or F56K or F56A, F56P, F56R, F56H, F56S, F56Q, F56I, F56L, F56T or F56G; (ii) N55Q, N55R, N55K, N55S, N55G, N55A or N55T; (iii) Y51L, Y51V, Y51A, Y51N, Y51Q, Y51S or Y51G; (iv) T150I; (v) S54P; and (vi) S57P.

8. A sensor for use in the method of any one of claims 1 to 7, for characterizing a target polynucleotide, the sensor comprising a complex between a CsgG pore or a mutant thereof and a polynucleotide binding protein.

9. The sensor according to claim 8, wherein: The CsgG mutant is as defined in any one of claims 3 to 7.

10. A kit for use in the method of any one of claims 1 to 7 for characterizing a target analyte, the kit comprising: (a) CsgG pore or its mutants, and (b) components of the membrane.

11. The kit according to claim 10, wherein The CsgG mutant is as defined in any one of claims 3 to 7.

12. A composition for use in the method of any one of claims 1 to 7, comprising a mutant CsgG monomer comprising a variant of the sequence shown in SEQ ID NO 390, (a) the variant comprises a mutation in: (i) Y51, where the mutation is replaced by F, M, L, I, V, A, P, G, C, N, T, S, E, D, K, H or R, (ii) N55, where the mutation is replaced by F, M, L, I, V, P, G, C, Q, T, E, D, K, H or R, (iii) F56, (iv) Y51 and N55, the mutations here are not Y51Q and N55S, (v) Y51 and F56, (vi) N55 and F56, or (vii) Y51, N55 and F56, or (b) The variant is a variant as defined in claim 4 or 5 other than Y51Q and N55S.

13. A composition for use in the method of any one of claims 1 to 7, comprising a construct comprising two or more covalently linked CsgG monomers, wherein: At least one of the monomers is a mutant monomer as defined in claim 12.

14. A composition for use in the method of any one of claims 1 to 7, comprising a modified CsgG pore comprising at least one CsgG monomer, wherein: The monomer comprises a modification between positions 38 to 63 of the sequence shown in SEQ ID NO 390.

15. The modified CsgG composition according to claim 14, wherein (a) the pore comprises a monomer as defined in claim 12(a), or (b) The monomer has a polypeptide sequence from position 38 to position 63 of SEQ ID NO: 4 to 388.

16. A composition for use in the method of any one of claims 1 to 7, comprising the same mutant monomer as defined in claim 12 or the same construct as defined in claim 13.

17. A composition for use in the method of any one of claims 1 to 7, comprising a polynucleotide encoding the mutant CsgG monomer defined in claim 12.

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