Novel modified protein pores and enzymes

Abstract

The present invention relates to modified Dda helicases that can be used to control the movement of analytes, such as polynucleotides. The modified Dda helicases are useful for analyte detection and characterization. The invention also relates to novel protein pores and their use in analyte detection and characterization. In particular, the present invention relates to isolated pore complexes formed from CsgG-like pores and a modified CsgF peptide or a homolog or mutant thereof, thereby incorporating an additional channel shrinkage structure or read head into the nanopore.

Description

Technical Field

[0001] The present invention relates to modified Dda helicases that can be used to control the movement of analytes such as polynucleotides. The modified Dda helicases are useful for analyte detection and characterization. The present invention also relates to novel protein pores or pore complexes and their use in analyte detection and characterization. Background Art

[0002] Two of the essential components of characterizing analytes, particularly polymers, using nanopore sensing are (1) controlling the movement of the polymer through the pore, and (2) discriminating between the constituent building blocks as the polymer moves through the pore. During nanopore sensing, the narrowest portion of the pore generally corresponds to the most discriminating portion of the nanopore, relative to the change in the measured signal as a function of analyte movement relative to the nanopore. CsgG was identified as a non-gated, non-selective protein secretion channel from Escherichia coli (Goyal et al., 2014) and has been used as a nanopore for detecting and characterizing analytes. Mutations of the wild-type CsgG pore that improve the properties of the pore in this context have also been disclosed (WO2016 / 034591, WO2017 / 149316, WO2017 / 149317, WO2017 / 149318, WO2018 / 211241, and WO2019 / 002893, which are incorporated herein by reference in their entireties). WO2015 / 055981, WO2015 / 166276 and WO2016 / 055777, which are also incorporated herein by reference in their entirety, describe polynucleotide binding proteins, in particular Dda helicases, which can be used to control the movement of analytes relative to transmembrane protein pores, such as the CsgG pore described herein. Summary of the Invention

[0003] The inventors have surprisingly identified specific Dda mutants, which have the ability of improved control analyte movement through holes. When using holes to sequence polynucleotides, the system estimates the quantity and identity of the bases / nucleotides passing through holes together. Better controlling the variability of movement speed can reduce one of the sources of statistical noise and simplify the estimation task. The operation of polynucleotides that stay for a short time in holes may cause the failure of calling bottom nucleotides / bases, thereby causing deletion errors. Abnormal long-term stays may cause insertion errors. Ensuring that each nucleotide / base spends enough time intervals in the hole helps to solve the statistical uncertainty of the nucleotide / base identity from the noise signal level. Further information can be extracted from the dependence of the dwell time on the nucleotide / base identity, for example, via the interaction with the motility enzyme. Reducing the overall variability of the dwell time helps to extract more accurate information by this channel. In the region (for example, long homopolymer region) that provides limited information about movement at the signal level, the polynucleotide / base dwell time can be used to infer the quantity of the bases passing through holes. Reducing the variability of the dwell time can make these inferences more accurate.

[0004] In some embodiments, when used to control the method for the movement of analytes through transmembrane pores and for characterizing analytes using transmembrane pores, the mutants of the present invention show improved accuracy. In the case of analyte characterization (particularly polynucleotides), accuracy is interpreted as meaning the original reading simplex accuracy; i.e., the single passage of a single molecule through a transmembrane pore. Accuracy is a useful measurement for tracking platform improvements of sequencing devices. Accuracy can also refer to consensus accuracy or the accuracy of detecting a specific thing (e.g., such as a mutation in a polynucleotide analyte). Additionally or alternatively, accuracy is interpreted as meaning the percentage of bases above a specific confidence level, wherein the confidence level has been pre-calibrated. In some embodiments, the mutants of the present invention show improved accuracy, while the speed change is minimal or even unchanged. In some embodiments, accuracy is improved to give an error of less than 10%, an error of less than 5%, an error of less than 4%, an error of less than 3%, an error of less than 2%, an error of less than 1%, an error of less than 0.1%. The mutants identified by the present inventors generally include a combination of mutations, i.e., one or more modifications in the part that interacts with the transmembrane pore in the mutant. Accuracy can also be affected by the speed at which the polymer translocates through the pore under enzyme control, and this speed can be altered by varying the concentration of ATP supplied to the enzyme. The inventors surprisingly recognized that during successive polymer translocations in the same sequencing run under identical conditions, the enzyme can exhibit variations in speed, which can lead to reduced accuracy.

[0005] Accuracy can be affected by many factors, such as the shape and composition of the nanopore, the enzyme, and the interaction between the enzyme and the nanopore. It is also affected by the speed at which the polymer translocates through the pore under the control of the enzyme, and the translocation speed can be increased or decreased by varying the concentration of ATP provided to the enzyme. The inventors surprisingly recognized that speed variations occur during the translocation of successive polymers in the same sequencing run under the same sequencing conditions, which can lead to a reduction in sequencing accuracy. The variation in sequencing speed of multiple polymers can be measured to obtain a normalized speed distribution, and the inventors surprisingly recognized that some modified enzymes can produce a lower normalized speed distribution and thus improve sequencing accuracy.

[0006] The present invention provides:

[0007] - a modified DNA-dependent ATPase (Dda) helicase, wherein the helicase comprises a modification or substitution at one or more positions corresponding to amino acid positions 55, 114, 156, 177, 210, 221, 350 and 358 in Dda 1993;

[0008] - a construct comprising a helicase of the invention and an additional polynucleotide binding moiety, wherein the helicase is attached to the polynucleotide binding moiety and the construct has the ability to control the movement of an analyte;

[0009] A polynucleotide comprising a sequence encoding a helicase according to the invention or a construct according to the invention;

[0010] - A vector comprising a polynucleotide of the present invention operably linked to a promoter;

[0011] - A host cell comprising the vector of the present invention;

[0012] - A method for preparing the helicase of the present invention or the construct of the present invention, the method comprising: expressing the polynucleotide of the present invention, transfecting a cell with the vector of the present invention, or culturing the host cell of the present invention;

[0013] - a method of controlling the movement of an analyte, the method comprising contacting the analyte with a helicase of the invention or a construct of the invention, and thereby controlling the movement of the analyte;

[0014] A method for characterizing a target analyte, the method comprising:

[0015] (a) contacting a target analyte with a transmembrane pore and a helicase of the invention or a construct of the invention such that the helicase or construct controls movement of the target analyte through the pore; and

[0016] (b) taking one or more measurements as the polynucleotide moves relative to the pore, wherein the measurements are indicative of one or more characteristics of the target analyte and thereby characterize the target analyte;

[0017] - a method of forming a sensor for characterising a target analyte, the method comprising forming a complex between (a) a pore and (b) a helicase of the invention or a construct of the invention, and thereby forming a sensor for characterising a target analyte;

[0018] - A sensor for characterising an analyte of interest, the sensor comprising a complex between (a) a pore and (b) a helicase of the invention or a construct of the invention;

[0019] - Use of a helicase of the invention or a construct of the invention for controlling the movement of an analyte of interest through a pore;

[0020] - A kit for characterizing a target analyte, the kit comprising

[0021] (a) a pore and a helicase of the invention or a construct of the invention; or

[0022] (b) a helicase of the invention or a construct of the invention and one or more cargo moieties;

[0023] - A device for characterizing a target analyte in a sample, comprising (a) a plurality of pores and (b) a plurality of helicases of the invention or a plurality of constructs of the invention;

[0024] A method for producing the helicase of the present invention, the method comprising:

[0025] (a) providing a helicase; and

[0026] (b) modifying a helicase to produce a helicase of the invention;

[0027] - a method of producing a construct of the invention, the method comprising attaching a helicase of the invention to a further polynucleotide binding moiety, and thereby producing the construct;

[0028] - a series of two or more helicases attached to a polynucleotide, wherein at least one of the two or more helicases is a helicase of the invention; and

[0029] - A method for improving the movement of a target analyte relative to a transmembrane pore when the movement of the target analyte relative to the transmembrane pore is controlled by a DNA-dependent ATPase (Dda) helicase, wherein the DNA-dependent ATPase (Dda) helicase is modified to contain a substitution at one or more positions corresponding to amino acid positions 55, 114, 156, 177, 210, 221, 350 and 358 in Dda 1993 and / or at amino acid position 40 in Dda 1993 that improves the movement of the target analyte relative to the transmembrane pore.

[0030] The inventors have also surprisingly identified new transmembrane pore mutations that improve or alter the speed at which an analyte passes through / relative to it, preferably wherein the movement of the analyte is under the control of a polynucleotide binding protein. In one embodiment of the invention, the transmembrane pore mutation increases the speed at which an analyte passes through / relative to it. In another embodiment, the transmembrane pore mutation decreases the speed at which an analyte passes through / relative to it. The speed at which an analyte passes through / relative to the pore may be increased by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 100%, 150%, 200% or 300% or more relative to the speed at which the analyte moves relative to a pore that does not contain the mutations of the invention. The speed at which the analyte moves through / relative to the pore can be reduced by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% relative to the speed at which the analyte moves relative to the pore that does not contain the mutations of the present invention. The inventors have surprisingly found that these changes in speed caused by modifications to the pore, such as an increase or decrease in speed, have minimal or no effect on the accuracy reading. This is particularly advantageous in methods of characterizing analytes, wherein the analyte is contacted with a pore and a polynucleotide binding protein (such as a helicase of the present invention) such that the polynucleotide binding protein controls the movement of the target analyte through / relative to the pore. In one embodiment, the mutant pore interacts with the polynucleotide binding protein in a different manner than other transmembrane pores that do not contain the mutations. The pore mutant can alter the velocity profile of DNA translocation through the pore so that the velocity profile is tighter than other transmembrane pores that do not contain the mutations, resulting in reduced sequencing errors. In a preferred embodiment of the invention, the modified DNA-dependent ATPase (Dda) helicase of the invention is used to control the movement of an analyte (such as a polynucleotide) through the transmembrane pore of the invention.

[0031] The present invention provides an isolated CsgG pore, or a homologue or mutant thereof, or an isolated pore complex comprising a CsgG pore, or a homologue or mutant thereof, and a modified CsgF peptide, or a homologue or mutant thereof, wherein the CsgG pore comprises at least one monomer comprising a modification at one or more of positions W97, Q100, E101, N102 and T104 of SEQ ID NO: 117;

[0032] In one aspect, the CsgF peptide comprises a CsgG binding region and a region that forms a constriction in the pore. In one aspect, the CsgF peptide is a truncated CsgF peptide lacking the C-terminal head domain of CsgF. In another aspect, the CsgF peptide is a truncated CsgF peptide lacking a portion of the C-terminal head and neck domains of CsgF. In another aspect, the CsgF peptide is a truncated CsgF peptide lacking the C-terminal head and neck domains of CsgF. The CsgG / CsgF pore is also referred to herein as a pore complex and an isolated pore complex. The isolated pore complex comprises the CsgG pore, or a homolog or mutant thereof, and a modified CsgF peptide, or a homolog or mutant thereof, particularly a truncated CsgF fragment, or a homolog or mutant thereof. In one embodiment, the modified CsgF peptide, or a homolog or mutant thereof, is located within the lumen of the CsgG pore, or a homolog or mutant thereof. In another embodiment, the isolated pore complex has two or more channel constrictions, one positioned or provided by the CsgG pore formed by its constriction loop, and another additional channel constriction or readerhead introduced by a modified CsgF peptide, or a homolog or mutant thereof. In one embodiment, the CsgG pore or CsgG-like pore is not a wild-type pore, but rather a mutant CsgG pore. In specific embodiments, the mutation is present, for example, in the channel constriction loop. In other embodiments, the mutation is alternatively or additionally present at the top of the pore, at the region where the pore interacts with the polynucleotide-binding protein. The mutation can affect how the polynucleotide-binding protein interacts with the pore and / or how the pore interacts with the polynucleotide-binding protein. In another embodiment, the isolated pore complex comprising the modified CsgF peptide, or a homolog or mutant thereof, has a CsgF channel constriction with a diameter ranging from 0.5 nm to 2.0 nm. In one embodiment, the pore complex comprises: (i) a CsgG pore comprising a first opening, a middle section comprising a β-barrel, a second opening, and a lumen extending from the first opening through the middle section to the second opening, wherein the lumenal surface of the middle section defines a CsgG constriction; and (ii) a plurality of modified CsgF peptides, each having a CsgF constriction region and a CsgF binding region (also referred to herein as a CsgG binding domain or a region of CsgF), wherein the modified CsgF peptides form a CsgF constriction within the β-barrel of the CsgG pore, and wherein the CsgG constriction and the CsgF constriction are coaxially spaced within the β-barrel of the CsgG pore. The lumenal surface of the CsgG pore may comprise one or more loop regions of a CsgG monomer that define the CsgG constriction. The CsgF constriction region and the CsgF binding region generally correspond to the N-terminal portion of the mature CsgF peptide. In one embodiment, the pore complex does not include CsgA, CsgB, and CsgE.

[0033] One embodiment relates to a pore comprising a CsgG pore and a modified CsgF peptide, wherein the modified CsgF peptide binds to the CsgG and forms a constriction in the pore, and wherein the pore is mutated to alter its interaction with a polynucleotide-binding enzyme and / or the pore is mutated to increase the rate at which an analyte passes through the pore. In one embodiment of the invention, the rate at which the analyte passes through the pore is increased. In another embodiment of the invention, the rate at which the analyte passes through the pore is decreased.

[0034] Another embodiment relates to an isolated pore complex wherein a modified CsgF peptide is covalently coupled to a CsgG pore or a monomer of said pore, or a homologue or mutant thereof, and even more particularly, the coupling is via a cysteine ​​residue or via a non-naturally reactive or photoreactive amino acid or a homologue thereof in the CsgG monomer at a position corresponding to 132, 133, 136, 138, 140, 142, 144, 145, 147, 149, 151, 153, 155, 183, 185, 187, 189, 191, 201, 203, 205, 207 or 209 of SEQ ID NO: 117 or SEQ ID NO: 3.

[0035] The present invention also provides an isolated transmembrane pore or pore complex, or a membrane composition comprising the isolated pore or pore complex of the present invention and a component of a membrane. In particular, the transmembrane pore or pore complex or membrane composition comprises the isolated pore or pore complex of the present invention and a component of a membrane or an insulating layer.

[0036] The present invention also provides:

[0037] - a membrane comprising a pore or pore complex according to the invention;

[0038] - an array comprising a plurality of membranes according to the invention;

[0039] - a system comprising (a) a membrane according to the invention or an array according to the invention, (b) means for applying an electric potential across the membrane,

[0040] and (c) a device for detecting electrical or optical signals across the membrane.

[0041] The present invention also provides a method for producing a transmembrane pore complex of the present invention, the method comprising co-expressing a CsgG pore or a homologue or mutant thereof, and a modified CsgF peptide or a homologue or mutant thereof in a suitable host cell, thereby allowing the formation of a transmembrane pore complex in vivo.

[0042] The present invention also provides a method for producing an isolated pore complex of the present invention, the method comprising contacting a CsgG monomer, or a homologue or mutant thereof, with a modified CsgF peptide, or a homologue or mutant thereof, thereby allowing for in vitro reconstitution of the isolated pore complex. The modified CsgF peptide may be a peptide comprising an enzymatic cleavage site at a suitable position in the amino acid sequence, which is cleaved before or after pore formation.

[0043] In specific embodiments, the modified CsgF peptide or a homologue or mutant thereof comprises SEQ ID NO: 12 or SEQ ID NO: 14, or a homologue or mutant thereof. In particular embodiments, the modified CsgF peptide of the method comprises SEQ ID NO: 15 or SEQ ID NO: 16, or a homologue or mutant thereof.

[0044] The present invention also provides a method for determining the presence, absence, or one or more characteristics of a target analyte, the method comprising the steps of:

[0045] (i) contacting a target analyte with an isolated pore or an isolated pore complex of the invention or a transmembrane pore complex of the invention such that the target analyte moves into the pore channel; and

[0046] (ii) one or more measurements are performed as the analyte moves through the pore channel, and the presence, absence or one or more characteristics of the analyte are determined thereby. In one embodiment, the analyte is a polynucleotide. In particular, the method using polynucleotides as analytes alternatively includes determining one or more characteristics selected from the following: (i) the length of the analyte or polynucleotide, (ii) the identity of the analyte or polynucleotide, (iii) the sequence of the analyte or polynucleotide, (iv) the secondary structure of the analyte or polynucleotide and (v) whether the analyte or polynucleotide is modified.

[0047] In another embodiment, the analyte is a protein, (poly)peptide or peptide. In a further embodiment, the analyte is a polymer, oligosaccharide, polysaccharide or small organic or inorganic compound, such as, for example but not limited to, pharmacologically active compounds, toxic compounds and pollutants.

[0048] The present invention also provides a method for characterizing a polynucleotide or (poly)peptide using the isolated pore or isolated pore complex of the present invention or the transmembrane pore complex of the present invention. In particular, the CsgG pore or homologue or mutant thereof comprises six to ten CsgG monomers forming the CsgG pore channel.

[0049] The present invention also provides the use of an isolated pore or isolated pore complex of the present invention or a transmembrane pore complex of the present invention for determining the presence, absence or one or more characteristics of a target analyte. Furthermore, the present invention relates to a kit for characterizing a target analyte, comprising (a) the isolated pore or pore complex and (b) a component of a membrane.

[0050] The present invention also provides:

[0051] - a method of altering the rate at which a target analyte passes through a pore, the method comprising contacting the target analyte with an isolated pore or an isolated pore complex of the invention or with a transmembrane pore complex of the invention, such that the target analyte moves relative to or into the pore complex;

[0052] - a kit for characterising a target analyte comprising (a) an isolated pore or isolated pore complex of the invention, and one or both of (b) a component of a membrane and (c) a polynucleotide binding protein;

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

[0054] (a) contacting a target analyte with an isolated pore or isolated pore complex of the invention and a DNA-dependent ATPase (Dda) helicase of the invention or a helicase construct of the invention, such that the helicase or construct controls movement of the target analyte through the pore or pore complex; and

[0055] (b) taking one or more measurements as the polynucleotide moves relative to the pore or pore complex, wherein the measurements are indicative of one or more characteristics of the target analyte and thereby characterize the target analyte;

[0056] - A kit for characterising a target analyte comprising (a) a DNA-dependent ATPase (Dda) helicase of the invention or a helicase construct of the invention (b) an isolated CsgG pore or an isolated pore complex of the invention;

[0057] - a device comprising a pore or pore complex according to the invention inserted into an extracorporeal membrane;

[0058] - A device produced by a method comprising: (i) obtaining an isolated pore or an isolated pore complex of the invention, and (ii) contacting the isolated pore or isolated pore complex with an extracorporeal membrane such that the pore is inserted into the extracorporeal membrane. DETAILED DESCRIPTION

[0059] All publications, patents, and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety. All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent that an incorporated by reference publication, patent, or patent application contradicts the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.

[0060] The present invention will be described with respect to specific embodiments and with reference to certain figures, but the invention is not limited thereto but only by the claims. Any reference signs in the claims should not be construed as limiting the scope. Of course, it should be understood that not all aspects or advantages need be achieved according to any particular embodiment of the present invention. Thus, for example, those skilled in the art will recognize that the present invention can be implemented or carried out in a manner that achieves or optimizes one advantage or group of advantages taught herein without necessarily achieving other aspects or advantages taught or suggested herein.

[0061] The organization and method of operation of the present invention, as well as its features and advantages, can be best understood with reference to the following detailed description when read in conjunction with the accompanying drawings. Aspects and advantages of the present invention will be apparent from and elucidated with reference to the embodiments described hereinafter. References throughout the specification to "one embodiment" or "an embodiment" mean that the particular features, structures, or characteristics described in conjunction with that embodiment are included in at least one embodiment of the present invention. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing throughout this specification do not necessarily refer to the same embodiment, but may refer to the same embodiment. Similarly, it should be understood that in the description of exemplary embodiments of the present invention, various features of the present invention are sometimes combined in a single embodiment or its description for the purpose of simplifying the disclosure and aiding understanding of one or more various inventive aspects. However, this method of disclosure should not be interpreted as reflecting an intention that the claimed invention requires more features than the features explicitly recited in each claim. On the contrary, as reflected in the appended claims, inventive aspects lie in less than all the features of a single aforementioned disclosed embodiment.

[0062] In addition, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to a "polynucleotide" includes two or more polynucleotides, reference to a "polynucleotide binding protein" includes two or more such proteins, reference to a "helicase" includes two or more helicases, reference to a "monomer" refers to two or more monomers, reference to a "pore" includes two or more pores, etc.

[0063] In all discussions herein, the standard single-letter codes for amino acids are used. They are as follows: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V). Standard substitution symbols are also used, i.e., Q42R means that the Q at position 42 is replaced by R.

[0064] In the paragraphs herein where different amino acids at a particular position are separated by a / symbol, the / symbol represents "or". For example, Q87R / K represents Q87R or Q87K. In the paragraphs herein where different positions are separated by a / symbol, the / symbol represents "and", such that Y51 / N55 is Y51 and N55.

[0065] definition

[0066] The following terms or definitions are provided only to assist in understanding the present invention. Unless specifically defined herein, all terms used herein have the same meaning as understood by those skilled in the art. Practitioners are particularly interested in Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th edition, Cold Spring Harbor Press, Plainsview, New York (2012); and Ausubel et al., Current Protocols in Molecular Biology (Supplement 114), John Wiley & Sons, New York (2016). The definitions provided herein should not be construed as having a scope less than that understood by those of ordinary skill in the art.

[0067] As used herein, "about" when referring to a measurable value such as an amount, duration, etc., is meant to encompass deviations of ±20% or ±10%, more preferably ±5%, even more preferably ±1%, and still more preferably ±0.1% from the specified value, as such deviations are suitable for performing the disclosed methods.

[0068] As used herein, "nucleotide sequence", "DNA sequence" or "nucleic acid molecule" refers to a polymeric form of nucleotides of any length, whether ribonucleotides or deoxyribonucleotides. The term refers only to the primary structure of the molecule. Therefore, the term includes double-stranded and single-stranded DNA and RNA. As used herein, the term "nucleic acid" is a single-stranded or double-stranded covalently linked sequence of nucleotides, wherein the 3' and 5' ends of each nucleotide are linked by a phosphodiester bond. A polynucleotide can be composed of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acids can be synthesized in vitro or isolated from natural sources. Nucleic acids can further include modified DNA or RNA, such as methylated DNA or RNA, or RNA that has been post-translationally modified, such as 5'-capping with 7-methylguanosine, 3'-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 a nucleic acid (also referred to herein as a "polynucleotide") is typically expressed as the number of base pairs (bp) for double-stranded polynucleotides or as the number of nucleotides (nt) in the case of single-stranded polynucleotides. One thousand bp or nt equals a kilobase (kb). Polynucleotides less than about 40 nucleotides in length are often referred to as "oligonucleotides" and may include primers for use in manipulating DNA, such as via the polymerase chain reaction (PCR).

[0069] As used herein, "gene" includes the promoter region and coding sequence of a gene. It refers to both the genomic sequence (including possible introns) and the cDNA derived from the spliced ​​message that is operably linked to the promoter sequence.

[0070] A "coding sequence" is a nucleotide sequence that is transcribed into mRNA and / or translated into a polypeptide when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a translation start codon at the 5' terminus and a translation stop codon at the 3' terminus. A coding sequence may include, but is not limited to, mRNA, cDNA, a recombinant nucleotide sequence, or genomic DNA, and in some cases may also contain introns.

[0071] In the context of the present disclosure, the term "amino acid" is used in its broadest sense and is intended to include organic compounds containing amine (NH2) and carboxyl (COOH) functional groups and side chains (e.g., R groups) specific to each amino acid. In some embodiments, amino acids refer to naturally occurring Lα-amino acids or residues. Common one-letter and three-letter abbreviations for naturally occurring amino acids are used herein: A=Ala;C=Cys;D=Asp;E=Glu;F=Phe;G=Gly;H=His;I=Ile;K=Lys;L=Leu;M=Met;N=Asn;P=Pro;Q=Gln;R=Arg;S=Ser;T=Thr;V=Val;W=Trp;And Y=Tyr (Lehninger, AL, (1975) Biochemistry, 2nd edition, pp. 71-92, Worth Publishers, New York). The general term "amino acid" further includes D-amino acids, retro-inverso amino acids, and chemically modified amino acids (such as amino acid analogs), naturally occurring amino acids that are not normally incorporated into proteins (such as norleucine), and chemically synthesized compounds with properties unique to amino acids known in the art (such as β-amino acids). For example, analogs or mimetics of phenylalanine or proline that allow conformational restriction of peptide compounds identical to natural Phe or Pro are included within 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, The Peptides: Analysis, Synthesis, Biology, Gross and Meiehofer, eds., Vol. 5, p. 341, Academic Press, Inc., NY 1983, which is incorporated herein by reference.

[0072] The terms "protein," "polypeptide," and "peptide" are further used interchangeably herein to refer to polymers of amino acid residues, as well as variants and synthetic analogs thereof. Thus, these terms apply to amino acid polymers in which one or more amino acid residues is a synthetic non-naturally occurring amino acid (such as a chemical analog of a corresponding naturally occurring amino acid), as well as naturally occurring amino acid polymers. 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, and the like. A "recombinant polypeptide" refers to a polypeptide prepared using recombinant techniques, such as by expression of a recombinant or synthetic polynucleotide. When a chimeric polypeptide or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, e.g., culture medium comprises less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. "Isolated" means material that is substantially or essentially free from components that normally accompany it in its native state. For example, as used herein, an "isolated polypeptide" refers to a polypeptide purified from molecules that flank it in its naturally occurring state, e.g., a protein complex or CsgF peptide that has been removed from molecules adjacent to the polypeptide in the production host. The isolated CsgF peptide (optionally a truncated CsgF peptide) can be produced by amino acid chemical synthesis or by recombinant production. The isolated complex can be produced by in vitro reconstitution after purification of the components of the complex (e.g., the CsgG pore and the CsgF peptide), or can be produced by recombinant co-expression.

[0073] The terms "orthologs" and "paralogs" encompass evolutionary concepts used to describe the ancestral relationships of genes. Paralogs are genes within the same species that originated through duplication of an ancestral gene; orthologs are genes from different organisms that originated through speciation and also derive from a common ancestral gene.

[0074] " Homologues " of a protein include peptides, oligopeptides, polypeptides, proteins and enzymes, which have amino acid substitutions, deletions and / or insertions relative to the unmodified or wild-type protein in question, and have biological and functional activities similar to the unmodified protein from which they are derived. As used herein, the term "amino acid identity" refers to the degree to which a sequence is identical on an amino acid-by-amino acid basis within a comparison window. Therefore, "percentage of sequence identity" is calculated by comparing two optimally aligned sequences within a comparison window, determining the number of positions at which identical amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) appear in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size), and multiplying the result by 100 to produce percentage of sequence identity.

[0075] The term "CsgG pore" is defined as a pore comprising a plurality of CsgG monomers. Each CsgG monomer can be a wild-type monomer from Escherichia coli (SEQ ID NO: 3), a wild-type homolog of E. coli CsgG, such as, for example, a monomer having any of the amino acid sequences shown in SEQ ID NOs: 68 to 88, or any variant thereof (e.g., a variant of any of SEQ ID NOs: 3, 117, and 68 to 88). Variant CsgG monomers may also be referred to as modified CsgG monomers or mutated CsgG monomers. Modifications or mutations in the variants include, but are not limited to, any one or more modifications disclosed herein, or combinations of such modifications.

[0076] For all aspects and embodiments of the present invention, a CsgG homolog refers to a polypeptide having at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, or 99% complete sequence identity to wild-type E. coli CsgG as shown in SEQ ID NO: 117 or SEQ ID NO: 3. CsgG homologs are also referred to as polypeptides containing the PFAM domain PF03783, which is characteristic of CsgG-like proteins. A list of currently known CsgG homologs and CsgG structures can be found at http: / / pfam.xfam.org / / family / PF0378 3. Similarly, a CsgG homologous polynucleotide may comprise a polynucleotide having at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% complete sequence identity to wild-type E. coli CsgG as shown in SEQ ID NO: 1. Examples of CsgG homologs shown in SEQ ID NO: 3 have the sequences shown in SEQ ID NOs: 68 to 88.

[0077] The term "modified CsgF peptide" or "CsgF peptide" defines a CsgF peptide that has been truncated from its C-terminus (e.g., is an N-terminal fragment) and / or modified to include a cleavage site. The CsgF peptide may be a fragment of wild-type E. coli CsgF (SEQ ID NO: 5 or SEQ ID NO: 6), or a fragment of a wild-type homolog of E. coli CsgF, such as, for example, a peptide comprising any one of the amino acid sequences shown in SEQ ID NOs: 17 to 36, or any variant thereof (e.g., a variant modified to include a cleavage site).

[0078] For all aspects and embodiments of the present invention, a CsgF homolog refers to a polypeptide having at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% complete sequence identity to wild-type E. coli CsgF as shown in SEQ ID NO:6. In some embodiments, a CsgF homolog is also referred to as a polypeptide containing the PFAM domain PF10614, which is characteristic of CsgF-like proteins. A list of currently known CsgF homologs and CsgF structures can be found at http: / / pfam.xfam.org / / family / PF10614 Similarly, a CsgF homologous polynucleotide may comprise a polynucleotide having at least 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% complete sequence identity to wild-type E. coli CsgF as shown in SEQ ID NO: 4. Examples of truncated regions of the CsgF homolog shown in SEQ ID NO: 6 have the sequences shown in SEQ ID NOs: 17 to 36.

[0079] The term "N-terminal portion of the mature CsgF peptide" refers to a peptide having an amino acid sequence corresponding to the first 60, 50, or 40 amino acid residues from the N-terminus of the mature CsgF peptide (without the signal sequence). The mature CsgF peptide can be wild-type or mutant (e.g., having one or more mutations).

[0080] Sequence identity can also be with respect to fragments or portions of a full-length polynucleotide or polypeptide. Thus, a sequence may have only 50% overall sequence identity with a full-length reference sequence, but the sequence of a specific region, domain, or subunit may share 80%, 90%, or up to 99% sequence identity with the reference sequence. Homology to the nucleic acid sequence of SEQ ID NO: 1 for a CsgG homolog or SEQ ID NO: 4 for a CsgF homolog, respectively, is not limited to sequence identity. Many nucleic acid sequences can exhibit biologically significant homology to one another despite having significantly lower sequence identity. Homologous nucleic acid sequences are considered to hybridize to one another under low stringency conditions (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).

[0081] The term "wild type" refers to a gene or gene product isolated from a naturally occurring source. A wild-type gene is the gene most commonly observed in a population and is therefore arbitrarily designed to be the "normal" or "wild-type" form of the gene. In contrast, the terms "modified," "mutant," or "variant" refer to a gene or gene product that exhibits sequence modifications (e.g., substitutions, truncations, or insertions), post-translational modifications, and / or functional properties (e.g., altered characteristics) when compared to a wild-type gene or gene product. It is noteworthy that naturally occurring mutants can be isolated; these mutants are identified by the fact that they have altered characteristics when compared to a wild-type gene or gene product. Methods for introducing or replacing naturally occurring amino acids are well known in the art. For example, methionine (M) can be replaced with an arginine codon (CGT) by replacing a methionine (ATG) codon at the relevant position in the polynucleotide encoding the mutant monomer. Methods for introducing or replacing non-naturally occurring amino acids are also well known in the art. For example, non-naturally occurring amino acids can be introduced by including a synthetic aminoacyl-tRNA in the IVTT system for expressing the mutant monomer. Alternatively, they can be introduced by expressing mutant monomers in E. coli, which are auxotrophic to specific amino acids in the presence of those specific amino acid synthesis (i.e., non-naturally occurring) analogs. If mutant monomers are produced using partial peptide synthesis, they can also be produced by naked connection. Conservative substitutions substitute amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side chain volumes. The amino acid introduced can have similar polarity, hydrophilicity, hydrophobicity, alkalinity, acidity, neutrality or electric charge with the amino acid they replace. Alternatively, conservative substitutions can introduce another aromatic or aliphatic amino acid to replace pre-existing aromatic or aliphatic amino acids. Conservative amino acid changes are well known in the art and can be selected according to the properties of 20 kinds of main amino acids defined in the following table 1. When amino acid has similar polarity, this can also be determined with reference to the hydrophilicity grade of the amino acid side chain in Table 2.

[0082] Table 1 – Chemical properties of amino acids

[0083]

[0084]

[0085] Table 2 - Hydrophilicity Grade

[0086]

[0087] It is also possible to chemically modify the mutant or modified protein, monomer or peptide in any way and at any site. Preferably, the mutant or modified monomer or peptide is chemically modified by attaching the molecule to one or more cysteines (cysteine ​​bond), attaching the molecule to one or more lysines, attaching the molecule to one or more non-natural amino acids, enzymatic modification of the epitope or modification of the end. Suitable methods for performing such modifications are well known in the art. The mutant of the modified protein, monomer or peptide can be chemically modified by attaching any molecule. For example, the mutant of the modified protein, monomer or peptide can be chemically modified by attaching a dye or a fluorophore. In certain embodiments, the mutant or modified monomer or peptide is chemically modified with a molecular adapter that promotes the interaction between the hole comprising the monomer or peptide and the target nucleotide or target polynucleotide sequence. The molecular adapter is preferably a cyclic molecule, a cyclodextrin, a substance capable of hybridization, a DNA binder or a mutual chelating agent, a peptide or peptide analog, a synthetic polymer, an aromatic planar molecule, a positively charged small molecule or a molecule capable of forming a hydrogen bond.

[0088] The presence of the adaptor improves the host-guest chemistry between the pore and the nucleotide or polynucleotide sequence, thereby improving the sequencing capability of the pore formed from the mutant monomer. The principles of host-guest chemistry are well known in the art. The adaptor has an effect on the physical or chemical properties of the pore, thereby improving its interaction with the nucleotide or polynucleotide sequence. The adaptor can alter the charge of the barrel or channel of the pore, or specifically interact or bind to the nucleotide or polynucleotide sequence, thereby promoting its interaction with the pore. Thus, the modified CsgF peptides provided in the present disclosure can be coupled to enzymes or proteins, thereby providing better accessibility of the protein or enzyme to the pore, which can facilitate certain applications of pore complexes comprising the modified CsgF peptide.

[0089] In this context, a protein may also be a fusion protein, in particular referring to a gene fusion produced, for example, by recombinant DNA technology. A protein may also be conjugated, or "conjugated to", as used herein, which in particular refers to chemical and / or enzymatic conjugation resulting in a stable covalent linkage.

[0090] When several polypeptides or protein monomers bind to or interact with each other, proteins can form a protein complex. "Binding" means any interaction, whether direct or indirect. Direct interaction means contact between binding partners, for example, through covalent bonding or coupling. Indirect interaction means any interaction in which interaction partners interact in a complex of more than two compounds. This interaction can be completely indirect (with the help of one or more bridging molecules) or partially indirect, where there is still direct contact between the partners, which is stabilized by the additional interaction of one or more compounds. The "complex" mentioned in this disclosure is defined as a group of two or more related proteins, which may have different functions. The association between different polypeptides in a protein complex can be through non-covalent interactions, such as hydrophobic forces or ionic forces, or it can be covalent binding or coupling, such as disulfide bridges or peptide binding. Covalent "binding" or "coupling" are used interchangeably herein and can also refer to "cysteine ​​coupling" or "reactive or photoreactive amino acid coupling", which refers to biological conjugation between cysteines or (photo) reactive amino acids, respectively, which is a chemical covalent connection that forms a stable complex. Examples of photoreactive amino acids include azidohomoalanine, homopropargylglycine, homoisoglycine, p-acetyl-Phe, p-azido-Phe, p-propargyloxy-Phe, and p-benzoyl-Phe (Wang et al. 2012, in Protein Engineering, DOI: 10.5772 / 28719; Chin et al. 2002, Proc. Nat. Acad. Sci. USA 99(17); 11020-24).

[0091] A "biological pore" is a transmembrane protein structure that defines a channel or hole 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 molecules or ions pass is on the order of nanometers (10-9 nanometers). In some embodiments, the biological pore can be a transmembrane protein pore. The transmembrane protein structure of the biological pore can be monomeric or oligomeric in nature. Typically, the pore contains a plurality of polypeptide subunits arranged around a central axis, thereby forming a protein-lined channel extending substantially perpendicular to the membrane in which the nanopore is located. The number of polypeptide subunits is not limited. Typically, the number of subunits is 5 to a maximum of 30, and suitably, the number of subunits is 6 to 10. Alternatively, the number of subunits is not defined as in the case of perfringens or related large membrane pores. The portion of the protein subunit that forms the protein-lined channel within the nanopore typically contains a secondary structure motif, which may include one or more transmembrane β-barrels and / or α-helical segments.

[0092] The terms "pore," "pore complex," or "composite pore," used interchangeably herein, refer to an oligomeric pore in which, for example, at least one CsgG monomer (including, for example, one or more CsgG monomers, such as two or more CsgG monomers, three or more CsgG monomers) or a CsgG pore (composed of CsgG monomers) and a CsgF peptide (e.g., a modified or truncated CsgF peptide) associate in a complex and together form a pore or nanopore. The pore complex of the present invention has the characteristics of a biological pore, i.e., it has a typical transmembrane protein structure. When the pore complex is provided in an environment having a membrane component, a membrane, a cell, or an insulating layer, the pore complex will insert into the membrane or insulating layer and form a "transmembrane pore complex."

[0093] The pores, pore complexes, transmembrane pores, or transmembrane pore complexes of the present disclosure are suitable for analyte characterization. In some embodiments, the pores, pore complexes, transmembrane pores, or transmembrane pore complexes described herein can be used to sequence polynucleotide sequences, for example, because they can distinguish different nucleotides with high sensitivity. A pore or pore complex can be isolated, substantially isolated, purified, or substantially purified. A pore or pore complex is "isolated" or purified if it is completely free of any other components, such as lipids or other pores, or other proteins with which it is normally associated in its native state, such as CsgE, CsgA, or CsgB, or if it is substantially enriched from a membrane compartment. A pore or pore complex is substantially isolated if it is mixed with a carrier or diluent that does not interfere with its intended use. For example, a pore or pore complex 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 any other components, such as triblock copolymers, lipids, or other pores. Alternatively, when present in a membrane, the pore complex of the present disclosure may be a transmembrane pore or transmembrane pore complex. The present disclosure provides isolated pores and isolated pore complexes comprising a homo-oligomeric pore derived from CsgG comprising identical mutant monomers, which may also contain mutant forms of CsgG monomers as homologs thereof. Alternatively, isolated pores or isolated pore complexes comprising a hetero-oligomeric CsgG pore are provided, which may be a CsgG pore composed of mutant and wild-type CsgG monomers, or a CsgG pore composed of different forms of CsgG variants, mutants, or homologs. The isolated pore complex typically comprises at least 7, at least 8, at least 9, or at least 10 CsgG monomers, and one or more (modified) CsgF peptides, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10 CsgF peptides. The pore complex may comprise any ratio of CsG monomers to CsgF peptides. In one embodiment, the ratio of CsG monomer:CsgF peptide is 1:1.

[0094] As used interchangeably herein, "constriction," "pore opening," "constriction region," "channel constriction," or "constriction site" refers to an aperture defined by the lumen surface of a pore or pore complex that functions to allow ions and target molecules (such as, but not limited to, polynucleotides or single nucleotides) but not other non-target molecules to pass through the pore or pore complex channel. In some embodiments, the constriction is the narrowest aperture within the pore or pore complex. In this embodiment, the constriction can be used to restrict the passage of molecules through the pore. The size of the constriction 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 effect on the flow of ions through the channel, the constriction should not be too large. For example, the constriction should not be wider than the solvent-accessible lateral diameter of the target analyte. Ideally, the diameter of any constriction should be as close as possible to the lateral diameter of the analyte being passed. For sequencing of nucleic acids and nucleic acid bases, suitable constriction diameters are in the nanometer range (10-9 meter range). Suitably, the diameter should be in the range of 0.5 to 2.0 nm, and typically, the diameter is in the range of 0.7 to 1.2 nm. The constricted structure of wild-type E. coli CsgG has a size of approximately The CsgF constriction formed in the pore complex comprising the CsgG-like pore and the modified CsgF peptide or its homologue or mutant has a diameter in the range of 0.5 to 2 nm or in the range of 0.7 to 1.2 nm and is therefore suitable for nucleic acid sequencing.

[0095] When two or more constrictions are present and spaced apart, each can simultaneously interact with or "read" individual nucleotides within a nucleic acid chain. In this case, a reduction in ion flux through the channel would be the result of a combined restriction of flux from all nucleotide-containing constrictions. Thus, in some cases, a dual constriction may 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" alone may not be determinable. The constriction of wild-type Escherichia coli CsgG (SEQ ID NO: 3) consists of two cyclic loops formed by the juxtaposition of a tyrosine residue at position 51 (Tyr 51) and phenylalanine and asparagine residues at positions 56 and 55, respectively (Phe 56 and Asn 55), in adjacent protein monomers. In most cases, the wild-type pore structure of CsgG is reengineered via recombinant genetic techniques to widen, alter, or remove one of the two cyclic loops that comprise the CsgG constriction (referred to herein as the "CsgG channel constriction"), leaving a single, well-defined read head. The constriction motif in the CsgG oligomeric pore is located at amino acid residues 38 to 63 in the wild-type monomeric E. coli CsgG polypeptide, as shown in SEQ ID NO: 3. In considering this region, mutations at any of amino acid residue positions 50 to 53, 54 to 56, and 58 to 59, as well as the key to the side chain positioning of Tyr51, Asn55, and Phe56 within the channel of the wild-type CsgG structure, have been shown to be advantageous for modifying or altering the properties of the readhead. The present disclosure relates to a pore complex comprising a CsgG pore and a modified CsgF peptide, or a homolog or mutant thereof, that surprisingly adds another constriction (referred to herein as a "CsgF channel constriction") to the CsgG-containing pore complex, forming a suitable additional second readhead in the pore via complex formation with the modified CsgF peptide. The additional CsgF channel constriction or readhead is positioned adjacent to the constriction loop of the CsgG pore or mutant CsgG pore. The additional CsgF channel constriction or readhead is positioned about 10 nm or less, such as 5 nm or less, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 nm, from the constriction ring of the CsgG pore or mutant GcsG pore. The pore complex or transmembrane pore complex of the present disclosure includes a pore complex with two readheads, which means that the channel constriction is positioned in a manner that provides a suitable individual readhead without interfering with the accuracy of the other constriction channel readhead. The pore complex may thus include CsgG mutant pores WO2016 / 034591, WO2017 / 149316, WO2017 / 149317, WO2019 / 002893, WO2017 / 149318, WO 2018 / 211241, WO2019 / 002893(all of which are incorporated herein by reference in their entirety), each of which lists mutations of the wild-type CsgG pore that improve the properties of the pore) as well as the wild-type CsgG pore, or a homologue thereof, and a modified CsgF peptide, or a homologue or mutant thereof, wherein the CsgF peptide has another constriction channel that forms the readhead.

[0096] Pores and pore complexes

[0097] The present invention provides an isolated CsgG pore, or a homologue or mutant thereof, or an isolated pore complex comprising a CsgG pore, or a homologue or mutant thereof, and a modified CsgF peptide, or a homologue or mutant thereof, wherein the CsgG pore comprises at least one mutant CsgG monomer comprising a modification at one or more of positions W97, Q100, E101, N102 and T104 of SEQ ID NO: 117. The CsgG pore may be the pore of SEQ ID NO: 3 or 117, or a homologue or mutant thereof. The at least one mutant monomer preferably comprises a variant of SEQ ID NO: 117 comprising a modification at one or more of positions W97, Q100, E101, N102 and T104.

[0098] The present invention provides an isolated CsgG pore, or a homologue or mutant thereof, wherein the CsgG pore comprises at least one mutant CsgG monomer comprising a modification at one or more of positions W97, Q100, E101, N102 and T104 of SEQ ID NO: 117. The CsgG pore may be the pore of SEQ ID NO: 3 or 117, or a homologue or mutant thereof. The at least one mutant monomer preferably comprises a variant of SEQ ID NO: 117 comprising a modification at one or more of positions W97, Q100, E101, N102 and T104.

[0099] The present invention provides an isolated pore complex comprising a CsgG pore, or a homologue or mutant thereof, and a modified CsgF peptide, or a homologue or mutant thereof, wherein the CsgG pore comprises at least one mutant CsgG monomer comprising a modification at one or more of positions W97, Q100, E101, N102 and T104 of SEQ ID NO: 117. The CsgG pore may be the pore of SEQ ID NO: 3 or 117, or a homologue or mutant thereof. The at least one mutant monomer preferably comprises a variant of SEQ ID NO: 117 comprising a modification at one or more of positions W97, Q100, E101, N102 and T104.

[0100] The at least one mutant monomer or variant may comprise any number and combination of modifications at one or more of positions (a) W97, (b) Q100, (c) E101, (d) N102, and (e) T104 in SEQ ID NO: 117. For example, the at least one mutant monomer may comprise (a); (b); (c); (d); (e); (a) and (b); (a) and (c); (a) and (d); (a) and (e); (b) and (c); (b) and (d); (b) and (e); (c) and (d); (c) and (e); (d) and (e); (a), (b) and (c); (a), (b) and (d); (a), (b) and (e); (a), (c) and (d); (a), (c) and (d); and (e). At least one mutant monomer or variant preferably comprises modifications at one or more positions (a) W97, (b) Q100, (c) E101, and (d) N102 of SEQ ID NO: 117, including all combinations of (a) to (d) listed above.

[0101] The modification at one or more of positions W97, Q100, E101, N102, and T104 in SEQ ID NO: 117 can be any modification discussed in more detail below. The modification can be a deletion, such as a deletion of E101. The deletion of E101 increases the speed of movement through the pore (Example 1). The modification is preferably a substitution.

[0102] The W at position 97 is preferably replaced by R, H, K, A, V, I, L, M, F, Y, S, T, Q, D, E, N, C, P or G. The W at position 97 is more preferably replaced by D, G, N, R or S. These replacements increase the speed of the analyte through / relative to the hole (Example 1). The W at position 97 is more preferably replaced by D or R. These replacements increase the speed of the analyte through / relative to the hole, and increase the normalized velocity distribution (Example 1). The W at position 97 is more preferably replaced by G, N or S. These replacements increase the speed of the analyte through / relative to the hole, and reduce the normalized velocity distribution (Example 1).

[0103] The Q at position 100 is preferably replaced by R, H, K, W, A, V, I, L, M, F, Y, T, N or S. The Q at position 100 is more preferably replaced by A, K or S. These replacements increase the speed of the analyte through / relative to the hole (Example 1). The Q at position 100 is more preferably replaced by K (Q100K). This replacement increases the speed of the analyte through / relative to the hole, and increases the normalized velocity distribution (Example 1). The Q at position 100 is more preferably replaced by A or S (Q100A or Q100S). These replacements increase the speed of the analyte through / relative to the hole, and reduce the normalized velocity distribution (Example 1). The Q at position 100 is most preferably replaced by A (Q100A). When used together with the modified Dda helicase of the present invention, this replacement also has the effect shown in Example 4.

[0104] The E at position 101 is preferably substituted with A, V, I, L, M, F, Y, or W. More preferably, the E at position 101 is substituted with A (E101A). This substitution reduces the velocity of the analyte through / relative to the pore and reduces the normalized velocity distribution (Example 1).

[0105] The E at position 101 is preferably substituted with S, T, N, Q, C, G or P. The E at position 101 is more preferably substituted with G or S. These substitutions increase the speed of the analyte through / relative to the hole (Example 1). The E at position 101 is more preferably substituted with G (E101G). This substitution increases the speed of the analyte through / relative to the hole and reduces the normalized velocity distribution (Example 1). The E at position 101 is more preferably substituted with S (E101S). This substitution increases the speed of the analyte through / relative to the hole and increases the normalized velocity distribution (Example 1).

[0106] The N at position 102 is preferably replaced by D, E, R, H, K, S, T, Q, V, I, L, M, F, Y, W or A. The N at position 102 is more preferably replaced by A, D, R, S or W. These replacements increase the speed of the analyte through / relative to the hole (Example 1). The N at position 102 is preferably replaced by A, R or S. These replacements increase the speed of the analyte through / relative to the hole, and reduce the normalized velocity distribution (Example 1). The N at position 102 is preferably replaced by D or W (N102D or N102W). These replacements increase the speed of the analyte through / relative to the hole, and increase the normalized velocity distribution (Example 1). The N at position 102 is most preferably replaced by A or S (N102A or N102S). When used together with the modified Dda helicase of the present invention, these replacements also have the effect shown in Example 4.

[0107] T at position 104 is preferably substituted with R, H or K.

[0108] As explained in more detail below, the pore preferably comprises six to ten monomers. Any number of these, such as 6, 7, 8, 9 or 10, may be mutant monomers comprising modifications at one or more of positions W97, Q100, E101, N102 and T104 of SEQ ID NO: 117, or may comprise a variant of SEQ ID NO: 117 comprising modifications at one or more of positions W97, Q100, E101, N102 and T104. All six to ten monomers may comprise modifications at one or more of positions W97, Q100, E101, N102 and T104 of SEQ ID NO: 117, or may comprise a variant of SEQ ID NO: 117 comprising modifications at one or more of positions W97, Q100, E101, N102 and T104.

[0109] A mutant CsgG monomer is a monomer whose sequence differs from that of a wild-type CsgG monomer and that retains the ability to form a pore. Mutant monomers may also be referred to herein as variants. Methods for confirming the ability of mutant monomers to form a pore are well known in the art and are discussed in more detail below. The at least one mutant monomer or variant may have homology / sequence identity to any of SEQ ID NO: 117 or SEQ ID NO: 3 listed below. The at least one mutant monomer may contain any of the additional modifications, mutations, or substitutions described below, including the types of modifications and substitutions described with reference to the Dda helicase of the present invention. The at least one mutant monomer may contain any of the additional modifications, mutations, or substitutions described in WO2016 / 034591, WO2017 / 149316, WO2017 / 149317, WO2017 / 149318, WO2018 / 211241, and WO2019 / 002893 (all of which are incorporated herein by reference in their entireties).

[0110] The present invention relates to a CsgG pore, optionally complexed with an extracellularly located CsgF peptide, which surprisingly introduces an additional channel constriction or readhead into the pore complex. Furthermore, the present disclosure provides information on the position of the constriction within the pore complex generated by the CsgF peptide, which is inserted into the lumen of the CsgG pore, and the constriction site is located at the N-terminal portion of the CsgF protein. Furthermore, the modified or truncated CsgF peptides of the present disclosure have been shown to be sufficient to form a pore complex and provide means and methods for biosensing applications. The present disclosure encompasses wild-type and mutant CsgG pores (as disclosed, for example, in WO2016 / 034591, WO2017 / 149316, WO2017 / 149317, WO2017 / 149318, and International Patent Application No. PCT / GB2018 / 051191), or homologs or mutants thereof, optionally in combination with modified or truncated CsgF peptides and mutants or homologs thereof, all of which together enhance the ability of the CsgG pore or CsgG-like pore complex to interact with analytes (such as polynucleotides). By forming a complex with the (modified or truncated) CsgF peptide, an additional constriction is introduced into the CsgG-like nanopore channel, expanding the contact surface with the passing analyte and serving as a second readhead for analyte detection and characterization. Pores containing mutant CsgG monomers in combination with novel mutant or modified forms of CsgF can improve the characterization of analytes, such as polynucleotides, providing a more discriminating direct relationship between the current observed as the polynucleotide moves through the pore. In particular, by spacing the two stacked read heads at a defined distance, the CsgG:CsgF pore complex can facilitate the characterization of polynucleotides containing at least one homopolymeric stretch, e.g., several consecutive copies of the same nucleotide, which would otherwise exceed the interaction length of a single CsgG read head. Additionally, by positioning the two stacked constrictions at a defined distance, small molecule analytes (including organic or inorganic drugs and pollutants) passing through the CsgG:CsgF composite pore will pass sequentially through two separate read heads. The chemistry of either read head can be independently modified, each imparting unique interaction properties with the analyte, thereby providing additional discriminatory power during analyte detection.

[0111] The present invention relates to an isolated pore complex comprising a CsgG pore, or a homologue or mutant thereof, or a CsgG-like pore, and a modified CsgF peptide, or a homologue or mutant thereof. Indeed, the present disclosure relates to a modified CsgG biological pore comprising a modified CsgF peptide, which may be a truncated, mutant, and / or variant thereof. In one embodiment, the interaction region between the modified CsgF peptide, or a homologue or mutant thereof, is located within the lumen of the CsgG pore, or a homologue or mutant thereof. In another embodiment, the pore complex has two or more constriction sites or read heads, provided by at least one constriction site of the CsgG pore and by at least one introduced CsgF peptide, thereby forming a complex with the CsgG pore. N-terminal CsgF positions encompassing amino acid residues 39-64 of SEQ ID NO:5, or more specifically, positions within the range of amino acid residues 49-64 of SEQ ID NO:5, have been shown to allow for detectable amounts of stable CsgG:CsgF complexes. In one embodiment, the CsgF constriction generated by a modified CsgF peptide (e.g., those described herein) is adjacent to or head-to-head with the first constriction in the CsgG pore of the pore complex. For CsgG or CsgG-like protein pores, it has been determined that the constriction site is formed by a loop region of the β strand.

[0112] In one embodiment, a modified CsgF peptide is a peptide, wherein the modification specifically refers to a truncated CsgF protein or fragment comprising an N-terminal CsgF peptide fragment defined by a restriction to contain a constriction region and bind to a CsgG monomer or a homolog or mutant thereof. The modified CsgF peptide may further comprise a mutation or homologous sequence that may promote certain properties of the pore complex. In a specific embodiment, the modified CsgF peptide comprises a truncation of the CsgF protein compared to the wild-type proprotein (SEQ ID NO: 5) or mature protein (SEQ ID NO: 6) sequence or a homolog thereof. These modified peptides are intended to function as pore complex components, introducing an additional constriction site or read head within the CsgG-like pore formed by CsgG and the modified or truncated CsgF peptide. Examples of truncated modified peptides are described below.

[0113] Examples of modified homologs of CsgF peptides are disclosed in WO2019 / 002893 (incorporated herein by reference in its entirety) and disclose CsgF-like proteins or CsgF peptides containing homologous or similar constriction domains in different bacterial strains that can be used to construct similar pore complexes. Structural features and CsgG binding elements are conserved in CsgF peptides derived from various CsgF homologs, allowing the CsgF peptides to be used in combination with different wild-type or mutant CsgG pores. This includes complexes of the CsgG pore with non-homologous CsgFs, meaning that the parent CsgF homolog from which the CsgG pore and CsgF are derived need not originate from the same operon, bacterial species, or strain.

[0114] In alternative embodiments, the CsgG pore within the pore complex is not a wild-type pore, but also contains mutations or modifications to enhance pore properties. The isolated pore complexes of the present disclosure formed from a CsgG pore or homolog thereof and a modified CsgF peptide or homolog thereof can be formed from a wild-type form of the CsgG pore or can be further modified within the CsgG pore, such as by targeted mutagenesis of specific amino acid residues, to further enhance the desired properties of the CsgG pore within the pore complex. For example, in embodiments of the present invention, mutations are contemplated to alter the number, size, shape, placement, or orientation of constrictions within the channel. Pore complexes comprising modified mutant CsgG pores can be prepared by known genetic engineering techniques that result in the insertion, substitution, and / or deletion of specific target amino acid residues within the polypeptide sequence. In the case of an oligomeric CsgG pore, mutations can be made in each monomeric polypeptide subunit, in any one monomer, or in all monomers. Suitably, in one embodiment of the present invention, the described mutations are made to all monomeric polypeptides within the oligomeric protein structure. A mutant CsgG monomer is a monomer whose sequence differs from that of a wild-type CsgG monomer and which retains the ability to form a pore. Methods for confirming the ability of mutant monomers to form pores are well known in the art. The present disclosure encompasses wild-type and mutant CsgG pores (e.g., as disclosed in WO2016 / 034591, WO2017 / 149316, WO2017 / 149317, WO2017 / 149318, and International Patent Application No. PCT / GB2018 / 051191), or homologs thereof, in combination with modified or truncated CsgF peptides and mutants or homologs thereof, all of which together enhance the ability of the CsgG-like pore complex to interact with an analyte (such as a polynucleotide). A mutant CsgG pore may comprise one or more mutant monomers. A CsgG pore may be a homopolymer comprising the same monomer, or a heteropolymer comprising two or more different monomers. A monomer may have one or more of the following mutations, in any combination.

[0115] Nanopore complexes comprising modified CsgF peptides differ from the wild-type CsgF protein depicted in SEQ ID NO:6 in that, in certain embodiments, the modified CsgF peptide comprises only an N-terminal fragment or truncation of the wild-type CsgF protein. However, the modified CsgF peptide may additionally or alternatively be a mutant CsgF peptide, in which case the mutation, as an amino acid substitution, is performed to allow for a better second constriction site in the pore formed by the complex comprising the CsgF pore and the modified CsgF peptide. When the complex is used for nucleotide sequencing, the mutant peptide may therefore have improved polynucleotide readout properties, i.e., exhibit improved polynucleotide capture and nucleotide discrimination, in addition to the improved characteristics of the complex comprising two read heads. In particular, pores constructed with the mutant peptides may more readily capture nucleotides and polynucleotides than the wild-type peptide. Furthermore, pores constructed with the mutant peptides may exhibit an increased current range, which makes it easier to distinguish different nucleotides, and reduced state variance, which increases the signal-to-noise ratio. In addition, the number of nucleotides that contribute to the current when the polynucleotide moves through the pore constructed by the mutant may be reduced. This makes it easier to identify a direct relationship between the current observed when the polynucleotide moves through the pore and the polynucleotide sequence. In addition, the pore constructed by the mutant peptide can show increased flux, for example, it is more likely to interact with the analyte (such as a polynucleotide). This makes it easier to use the pore to characterize the analyte. The pore constructed by the mutant peptide can be more easily inserted into the membrane, or can provide a simpler method to retain additional proteins near the pore complex.

[0116] In alternative embodiments, the CsgF constriction site provided in the pore complex of the invention has a diameter in the range of 0.5 nm to 2.0 nm, thereby providing a pore complex suitable for nucleic acid sequencing, as described above.

[0117] The CsgG pore can be stabilized by covalently attaching a CsgF peptide to the pore. The covalent linkage can be, for example, a disulfide bond or click chemistry. The CsgF peptide and the CsgG pore can be covalently linked, for example, via residues at one or more of the following pairs of positions corresponding to SEQ ID NO: 6 and SEQ ID NO: 3 or SEQ ID NO: 117, respectively: 1 and 153, 4 and 133, 5 and 136, 8 and 187, 8 and 203, 9 and 203, 11 and 142, 11 and 201, 12 and 149, 12 and 203, 26 and 191, and 29 and 144.

[0118] In the pore, the interaction between the CsgF peptide and the CsgG pore can be stabilized, for example, by hydrophobic interactions or electrostatic interactions at one or more of the following pairs of positions corresponding to SEQ ID NO: 6 and SEQ ID NO: 3 or SEQ ID NO: 117, respectively: 1 and 153, 4 and 133, 5 and 136, 8 and 187, 8 and 203, 9 and 203, 11 and 142, 11 and 201, 12 and 149, 12 and 203, 26 and 191, and 29 and 144.

[0119] Residues in CsgF and / or CsgG at one or more of the positions listed above may be modified to enhance the interaction between CsgG and CsgF in the pore.

[0120] In one embodiment, the pores of the present invention can be isolated, substantially isolated, purified or substantially purified. If the pores of the present invention are completely free of any other components, such as lipids or other pores, then they are isolated or purified. If the pores are mixed with a carrier or diluent that does not interfere with their intended use, then they are substantially isolated. For example, if the pores are present in a form comprising less than 10%, less than 5%, less than 2% or less than 1% of other components (such as triblock copolymers, lipids or other pores), then they are substantially isolated or substantially purified. Alternatively, the pores of the present invention can be present in a membrane. Suitable membranes are discussed below.

[0121] 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.

[0122] CsgF peptide

[0123] The isolated pore complex of the present invention comprises a modified CsgF monomer (peptide), or a truncated CsgF protein, or a modified or truncated peptide of a CsgF homologue or mutant. These novel modified CsgF peptides can be used in the pore complex to integrate a second or additional read head. The modification or truncation preferably produces a fragment, more preferably an N-terminal fragment, of a wild-type CsgF or mutant or homologue CsgF protein. The modified CsgF peptide of SEQ ID NO 5 or a homologue or mutant thereof may be any of those disclosed in WO2016 / 034591, WO2017 / 149316, WO2017 / 149317, WO2017 / 149318, WO2018 / 211241 and WO2019 / 002893 (all of which are incorporated herein by reference in their entirety).

[0124] The CsgF peptides forming part of the present invention are truncated CsgF peptides that lack the C-terminal head; lack the C-terminal head and a portion of the neck domain of CsgF (e.g., the truncated CsgF peptide may comprise only a portion of the neck domain of CsgF); or lack the C-terminal head and neck domain of CsgF. The CsgF peptide may lack a portion of the neck domain of CsgF, for example, the CsgF peptide may comprise a portion of the neck domain, such as, for example, amino acid residue 36 (see SEQ ID NO: 6) from the N-terminus of the neck domain (e.g., residues 36-40, 36-41, 36-42, 36-43, 36-45, 36-46 up to residues 36-50 or 36-60 of SEQ ID NO: 6). The CsgF peptide preferably comprises a CsgG binding region and a region that forms a constriction in the pore. The CsgG binding region typically comprises residues 1 to 8 and / or 29 to 32 of the CsgF protein (SEQ ID NO: 6 or a homolog from another species) and may include one or more modifications. The region that forms the constriction in the pore typically comprises residues 9 to 28 of the CsgF protein (SEQ ID NO: 6 or a homolog from another species) and may include one or more modifications. Residues 9 to 17 contain the conserved motif N9PXFGGXXX17 and form a turn region. Residues 9 to 28 form an α-helix. X17 (N17 in SEQ ID NO: 6) forms the apex of the constriction region, corresponding to the narrowest part of the CsgF constriction in the pore. The CsgF constriction region also forms stabilizing contacts with the CsgG β-barrel primarily at residues 9, 11, 12, 18, 21, and 22 of SEQ ID NO: 6.

[0125] The CsgF peptide typically has a length of 28 to 50 amino acids, such as 29 to 49, 30 to 45, or 32 to 40 amino acids. Preferably, the CsgF peptide comprises 29 to 35 amino acids, or 29 to 45 amino acids. The CsgF peptide comprises all or part of the FCP, which corresponds to residues 1 to 35 of SEQ ID NO: 6. In cases where the CsgF peptide is shorter than the FCP, it is preferably truncated at the C-terminus.

[0126] The CsgF fragment of SEQ ID NO: 6 or a homologue or mutant thereof may have a length of 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54 or 55 amino acids.

[0127] The CsgF peptide may comprise the amino acid sequence of SEQ ID NO: 6, from residue 1 up to any one of residues 25 to 60, such as 27 to 50, for example 28 to 45, of SEQ ID NO: 6, or the corresponding residues from a homologue of SEQ ID NO: 6 or any variant thereof. More specifically, the CsgF peptide may comprise SEQ ID NO: 39 (residues 1 to 29 of SEQ ID NO: 6) or a homologue or variant thereof.

[0128] Examples of such CsgF peptides include, consist essentially of, or consist of SEQ ID NO: 15 (residues 1 to 34 of SEQ ID NO: 6), SEQ ID NO: 54 (residues 1 to 30 of SEQ ID NO: 6), SEQ ID NO: 40 (residues 1 to 45 of SEQ ID NO: 6), or SEQ ID NO: 55 (residues 1 to 35 of SEQ ID NO: 6), and any homologs or variants thereof. Other examples of CsgF peptides include, consist essentially of, or consist of SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 16.

[0129] In the CsgF peptide, for example, one or more residues in SEQ ID NO: 15, SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 54, or SEQ ID NO: 55 may be modified.

[0130] For example, the CsgF peptide may comprise modifications at positions corresponding to one or more of the following positions in SEQ ID NO: 6: G1, T4, F5, R8, N9, N11, F12, A26, and Q29.

[0131] The CsgF peptide can be modified to introduce cysteine, a hydrophobic amino acid, a charged amino acid, a non-natural reactive amino acid, or a photoreactive amino acid, for example, at positions corresponding to one or more of the following positions in SEQ ID NO: 6: G1, T4, F5, R8, N9, N11, F12, A26, and Q29.

[0132] For example, the CsgF peptide may comprise modifications at positions corresponding to one or more of the following positions in SEQ ID NO: 6: N15, N17, A20, N24, and A28. The CsgF peptide may comprise a modification at a position corresponding to D34 to stabilize the CsgG-CsgF complex. In certain embodiments, the CsgF peptide comprises one or more of the following substitutions: N15S / A / T / Q / G / L / V / I / F / Y / W / R / K / D / C, N17S / A / T / Q / G / L / V / I / F / Y / W / R / K / D / C, A20S / T / Q / N / G / L / V / I / F / Y / W / R / K / D / C, N24S / T / Q / A / G / L / V / I / F / Y / W / R / K / D / C, A28S / T / Q / N / G / L / V / I / F / Y / W / R / K / D / C, and D34F / Y / W / R / K / N / Q / C. The CsgF peptide may, for example, comprise one or more of the following substitutions: G1C, T4C, N17S, and D34Y or D34N.

[0133] CsgF peptides can be produced by enzymatically cleaving longer proteins (such as full-length CsgF). Cleavage at specific sites can be performed by modifying longer proteins (such as full-length CsgF) to include an enzymatic cleavage site at an appropriate position. Examples of CsgF amino acid sequences that have been modified to include such enzymatic cleavage sites are shown in SEQ ID NOs: 56 to 67. After cleavage, all or part of the added enzymatic cleavage site may be present in the CsgF peptide that associates with CsgG to form a pore. Therefore, the CsgF peptide may further include all or part of the enzymatic cleavage site at its C-terminus.

[0134] Some examples of suitable CsgF peptides are shown in Table 3 below:

[0135] Table 3: CsgF peptides

[0136]

[0137]

[0138]

[0139] In certain embodiments, the CsgF fragment comprises the amino acid sequence of SEQ ID NO:39 or a mutant or homolog thereof. In particular, SEQ ID NO:39 comprises the first 29 amino acids of the mature CsgF peptide (SEQ ID NO:6). In another embodiment, the modified CsgF peptide of the present invention is a truncated peptide comprising SEQ ID NO:40. In particular, SEQ ID NO:40 comprises the first 45 amino acids of the mature CsgF peptide (SEQ ID NO:6). In particular, the CsgF constriction site and the binding site for CsgG are located within the N-terminal CsgF peptide region, further characterized by amino acids 39 to 64 of SEQ ID NO:5 (present in both SEQ ID NO:39 and SEQ ID NO:40), or in particular, amino acids 49 to 64 of SEQ ID NO:5 (present in SEQ ID NO:40 but not in SEQ ID NO:39; the latter fragment encoded by SEQ ID NO:39 exhibits a weaker interaction with CsgG (see Examples)), conferring greater stability to the complex. Thus, the present disclosure provides for modification of the CsgF protein by truncation of the protein to the described peptides, or peptides comprising the described N-terminal fragment or constriction site region, to allow for in vivo complex formation with the CsgG pore, or homologs or mutants thereof. Further limitations are provided in one embodiment involving a modified CsgF peptide comprising SEQ ID NO: 37 or SEQ ID NO: 38. Finally, the identification of CsgF homologous peptides, particularly peptides aligned within the constriction region (FCP peptides), also provides for modified CsgF peptide homologs that can form part of the isolated complex.

[0140] Further embodiments relate to modified or truncated CsgF peptides comprising SEQ ID NO: 15, wherein SEQ ID NO: 15 contains a region of the CsgF protein, including several residues from the CsgG binding and / or constriction site region, sufficient to reconstitute a composite pore comprising CsgG or a homolog thereof and the modified CsgF peptide in vitro, to produce an isolated pore complex comprising the CsgF channel constriction. Another embodiment describes the modified CsgF peptide comprising SEQ ID NO: 16, which contains an N-terminal fragment of the CsgF protein, as well as two additional amino acids (KD), which also increase the solubility and stability of the (synthetic) peptide and allow for in vitro reconstitution of the composite pore. Further embodiments are provided, wherein the modified CsgF peptide comprises SEQ ID NO: 15, SEQ ID NO: 16, or a homologue or mutant thereof, wherein the modified CsgF peptide is further mutated but still retains a minimum of 35% amino acid identity, e.g., 40%, 50%, 60%, 70%, 80%, 85%, 90% amino acid identity, to SEQ ID NO: 15 or SEQ ID NO: 16, respectively, within the region of the modified CsgF peptide corresponding to SEQ ID NO: 15 or 16. Further embodiments are provided wherein the modified CsgF peptide comprises SEQ ID NO: 15, SEQ ID NO: 16, or a homologue or mutant thereof, wherein the modified CsgF peptide is further mutated but retains a minimum of 40%, 45%, 50%, 60%, 70%, 80%, 85%, or 90% amino acid identity to SEQ ID NO: 15 or SEQ ID NO: 16, respectively, within the region of the modified CsgF peptide corresponding to SEQ ID NO: 15 or 16. These mutated regions are intended to alter and / or improve the characteristics of the CsgF constriction site, as described above, thereby, for example, enabling more accurate target analysis. Another embodiment discloses a modified CsgF peptide, wherein one or more positions in the region comprising SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 54, or SEQ ID NO: 55 are modified, and wherein in the peptide fragment corresponding to the region comprising SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 54, or SEQ ID NO: 55, the mutations retain a minimum of 35% amino acid identity, or 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% amino acid identity with SEQ ID NO: 39, SEQ ID NO: 40, SEQ ID NO: 54, or SEQ ID NO: 55.

[0141] Another embodiment relates to an isolated pore complex in which the CsgG pore is coupled to a modified CsgF peptide via covalent bonding, at least via one monomer. In one instance, the covalent bond or bonding can be via a cysteine ​​linkage, wherein the sulfhydryl side group of the cysteine ​​is covalently bonded to another amino acid residue or moiety. In a second possibility, the covalent bond is achieved via interactions between non-natural (photo)reactive amino acids. (Photo)reactive amino acids are artificial analogs of natural amino acids that can be used to cross-link protein complexes and can be incorporated into proteins and peptides in vivo or in vitro. Commonly used photoreactive amino acid analogs include photoreactive diazirine analogs of leucine and methionine and p-benzoyl-phenyl-alanine, as well as azidohomoalanine, homopropargylglycine, homoisoglycine, p-acetyl-Phe, p-azido-Phe, p-propargyloxy-Phe, and p-benzoyl-Phe (Wang et al. 2012; Chin et al. 2002). When exposed to ultraviolet light, they become activated and covalently bind to the interacting protein within a few angstroms of the photoreactive amino acid analog. However, the position within the CsgG monomer where this covalent attachment occurs depends on the exposure to the modified CsgF peptide. Several amino acids are located in positions that provide for covalent attachment, namely positions 132, 133, 136, 138, 140, 142, 144, 145, 147, 149, 151, 153, 155, 183, 185, 187, 189, 191, 201, 203, 205, 207, or 209 of SEQ ID NO: 3 or SEQ ID NO: 117, or a homologue thereof.

[0142] Another aspect of the invention relates to constructs comprising the modified CsgF peptide, wherein the peptide is covalently attached. A "construct" comprises two or more covalently attached monomers derived from modified CsgF and / or CsgG or homologs thereof. In other words, a construct may contain more than one monomer. In another aspect, the invention also provides a pore complex comprising at least one construct of the invention. The pore complex contains sufficient constructs and, if necessary, 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, (c) one construct comprising two monomers and six monomers that do not form part of the construct, or (d) one construct comprising one or two CsgF monomers and one construct comprising six to seven CsgG monomers, or even (e) a construct comprising CsgF and CsgG monomers in addition to another construct comprising only CsgG monomers. For example, the same and additional possibilities are provided for non-polymeric pores. Other combinations of constructs and monomers can be envisioned by the skilled artisan. One or more constructs of the invention can be used to form a pore complex for characterizing (such as sequencing) a polynucleotide. The construct can 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 can be the same or different and can be CsgF, CsgG, a CsgG / CsgF fusion monomer, or homologs thereof, or any combination thereof.

[0143] Another embodiment relates to a polynucleotide or nucleic acid molecule encoding the pore or pore complex of the invention, or a homologue or mutant thereof, or a polynucleotide encoding a construct as described above.

[0144] Certain embodiments relate to an isolated transmembrane pore complex comprising an isolated pore complex or an isolated pore complex of the present invention, and a membrane component. The isolated transmembrane pore complex can be directly applied to molecular sensing, such as nucleic acid sequencing. Alternatively, a membrane composition is provided comprising a modified CsgG / CsgF biological pore as described herein of an isolated pore complex of the present invention, and a membrane, a membrane component, or an insulating layer. One embodiment relates to an isolated transmembrane pore complex comprising an isolated pore complex of the present invention and a membrane component.

[0145] Although the CsgG:CsgF complex is very stable, when CsgF is truncated, the stability of the CsgG:CsgF complex is reduced compared to a complex containing full-length CsgF. Therefore, disulfide bonds can be formed between CsgG and CsgF to make the complex more stable, for example, after introducing cysteine ​​residues at the positions identified herein. The pore complex can be prepared using any of the aforementioned methods, and disulfide bond formation can be induced by using an oxidizing agent (e.g., copper-o-phenanthroline). Other interactions (e.g., hydrophobic interactions, charge-charge interactions / electrostatic interactions) can also be used at these positions instead of cysteine ​​interactions.

[0146] In another embodiment, non-natural amino acids can also be incorporated into those positions. In this embodiment, covalent bonds are formed by click chemistry. For example, non-natural amino acids with an azide or alkyne or with a dibenzocyclooctyne (DBCO) group and / or a bicyclo [6.1.0] nonyne (BCN) group can be introduced into one or more of these positions.

[0147] Such stabilizing mutations can be combined with any other modifications to CsgG and / or CsgF, such as those disclosed herein.

[0148] The CsgG pore may comprise at least one, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10, CsgG monomers modified to facilitate attachment to a CsgF peptide. For example, cysteine ​​residues may be introduced at one or more of the positions corresponding to positions 132, 133, 136, 138, 140, 142, 144, 145, 147, 149, 151, 153, 155, 183, 185, 187, 189, 191, 201, 203, 205, 207, and 209 of SEQ ID NO: 3 or SEQ ID NO: 117 to facilitate covalent attachment to CsgG. As an alternative to or in addition to covalent attachment via cysteine ​​residues, the pore may be stabilized by hydrophobic interactions or electrostatic interactions. To facilitate such interactions, a non-naturally reactive or photoreactive amino acid is present at positions corresponding to one or more of positions 132, 133, 136, 138, 140, 142, 144, 145, 147, 149, 151, 153, 155, 183, 185, 187, 189, 191, 201, 203, 205, 207, and 209 of SEQ ID NO: 3 or SEQ ID NO: 117.

[0149] The CsgF peptide can be modified to facilitate attachment to the CsgG pore. For example, a cysteine ​​residue can be introduced at one or more of the positions corresponding to positions 1, 4, 5, 8, 9, 11, 12, 26, or 29 of SEQ ID NO: 6 to facilitate covalent attachment to CsgG. As an alternative to or in addition to covalent attachment via cysteine ​​residues, the pore can be stabilized by hydrophobic or electrostatic interactions. To facilitate such interactions, a non-naturally reactive or photoreactive amino acid is present at one or more of the positions corresponding to positions 1, 4, 5, 8, 9, 11, 12, 26, or 29 of SEQ ID NO: 6.

[0150] Preferred exemplary CsgF peptides include those corresponding to SEQ ID NO:6 comprises the following mutations: N15X1 / N17X2 / A20X3 / N24X4 / A28X5 / D34X6, wherein X1 is N / S / A / T / Q / G / L / V / I / F / Y / W / R / K / D / C, X2 is N / S / A / T / Q / G / L / V / I / F / Y / W / R / K / D / C, X3 is A / S / T / Q / N / G / L / V / I / F / Y / W / R / K / D / C, X4 is N / S / T / Q / A / G / L / V / I / F / Y / W / R / K / D / C, X5 is A / S / T / Q / N / G / L / V / I / F / Y / W / R / K / D / C and X6 is D / F / Y / W / R / K / N / Q / C. Mutations at positions N15, N17, A20, N24, and A28 are constriction mutations, and the mutation at position 34 affects the interaction of CsgF with the bottom of the CsgG pore to stabilize the interaction.

[0151] CsgG pore

[0152] The CsgG pore may be a homo-oligomeric pore comprising identical mutant monomers of the invention. The CsgG pore may be a hetero-oligomeric pore derived from CsgG, for example comprising at least one mutant monomer as disclosed herein.

[0153] The CsgG pore can contain 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, such as 7, 8, 9, or 10 mutant monomers. The CsgG pore preferably comprises eight or nine identical mutant monomers.

[0154] In preferred embodiments, all monomers (such as 10, 9, 8 or 7 monomers) in the hetero-oligomeric CsgG pore are mutant monomers as disclosed herein, wherein at least one of them differs from the others. They may differ from each other.

[0155] The mutant monomers in the CsgG pore preferably all have approximately the same length or the same length. The barrels of the mutant monomers of the invention in the pore preferably have approximately the same length or the same length. Length can be measured in terms of number of amino acids and / or length units.

[0156] The mutant monomer may be a variant of SEQ ID NO: 3 or SEQ ID NO: 117 comprising a modification at one or more of positions W97, Q100, E101, N102, and T104. Over the entire length of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 117, the variant is preferably at least 40% homologous to the sequence based on amino acid identity. More preferably, the variant may be at least 45%, at least 50%, 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: 3 or SEQ ID NO: 117 based on amino acid identity over the entire sequence. Over the entire length of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 117, the variant is preferably at least 40% identical to the sequence. More preferably, the variant may be at least 45%, at least 50%, 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% identical across the entire sequence to SEQ ID NO: 3 or SEQ ID NO: 117. At least 80%, such as at least 85%, 90% or 95% amino acid identity may exist over a stretch of 100 or more, such as 125, 150, 175 or 200 or more contiguous amino acids ("hard homology").

[0157] The CsgG monomer is highly conserved (as can be readily understood from Figures 45 to 47 of WO2017 / 149317). Furthermore, based on knowledge of mutations related to SEQ ID NO: 3 or SEQ ID NO: 117, equivalent positions for mutations in CsgG monomers other than SEQ ID NO: 3 or SEQ ID NO: 117 can be determined.

[0158] Thus, references to mutant CsgG monomers comprising variants of the sequences shown in SEQ ID NO:3 or SEQ ID NO:117, and the specific amino acid mutations thereof as listed in the claims and elsewhere in the specification, also encompass mutant CsgG monomers comprising variants of the sequences shown in SEQ ID NOs:68 to 88, and the corresponding amino acid mutations thereof. Similarly, references to constructs, pores, or methods involving the use of pores associated with mutant CsgG monomers comprising variants of the sequences shown in SEQ ID NO:3 or SEQ ID NO:117, and the specific amino acid mutations thereof as listed in the claims and elsewhere in the specification, also encompass constructs, pores, or methods associated with mutant CsgG monomers comprising variants of the sequences according to the SEQ ID NOs disclosed above, and the corresponding amino acid mutations thereof. It will further be understood that the present invention extends to other variant CsgG monomers that exhibit highly conserved regions not explicitly identified in this specification.

[0159] Homology can be determined using the standard methods in the art. For example, the UWGCG package provides the BESTFIT program that can be used to calculate homology, for example, using (Devereux et al. (1984) Nucleic Acids Research 12, pp. 387-395) on its default settings. PILEUP and BLAST algorithms can be used to calculate homology or arrange sequences (such as identifying equivalent residues or corresponding sequences (usually under its default settings), for example, as Altschul SF (1993) J Mol Evol 36: 290-300; Altschul, SF et al. (1990) J Mol Biol 215: described in 403-10. Software for performing BLAST analysis can be publicly available through the National Center for Biotechnology Information (http: / / www.ncbi.nlm.nih.gov / ).

[0160] SEQ ID NO: 3 is a wild-type CsgG monomer from Escherichia coli Str. K-12 substr. MC4100. Variants of SEQ ID NO: 3 or SEQ ID NO: 117 may comprise any substitution found in another CsgG homolog. Preferred CsgG homologs are shown in SEQ ID NOs: 68 to 88. Variants may comprise a combination of one or more substitutions found in SEQ ID NOs: 68 to 88 compared to SEQ ID NO: 3 or SEQ ID NO: 117. For example, a mutation may be made at any one or more positions in SEQ ID NO: 3 or SEQ ID NO: 117 that differ from SEQ ID NO: 3 or SEQ ID NO: 117 and any of SEQ ID NOs: 68 to 88. Such a mutation may be a substitution of an amino acid in SEQ ID NO: 3 or SEQ ID NO: 117 with an amino acid at the corresponding position in any of SEQ ID NOs: 68 to 88. Alternatively, the mutation at any of these positions may be a substitution of any amino acid, or may be a deletion or insertion mutation, such as a deletion or insertion of 1 to 10 amino acids, such as a deletion or insertion of 2 to 8 or 3 to 6 amino acids. In addition to the mutations disclosed herein, amino acids conserved between SEQ ID NO: 3 or SEQ ID NO: 117 and all SEQ ID NOs: 66 to 88 are preferably present in the variants of the present invention. However, conservative mutations may be made at any one or more of these positions conserved between SEQ ID NO: 3 or SEQ ID NO: 117 and all SEQ ID NOs: 66 to 88.

[0161] The present invention provides a pore-forming CsgG mutant monomer comprising any one or more amino acids described herein, wherein these amino acids are substituted into specific positions in SEQ ID NO: 3 or SEQ ID NO: 117 at positions corresponding to specific positions in the CsgG monomer structure. The corresponding positions can be determined using standard techniques in the art. For example, the PILEUP and BLAST algorithms described above can be used to align the sequence of the CsgG monomer with SEQ ID NO: 3 or SEQ ID NO: 117 to identify corresponding residues.

[0162] The pore-forming mutant monomer generally retains the ability to form the same 3D structure as a wild-type CsgG monomer, such as the same 3D structure as a CsgG monomer having the sequence of SEQ ID NO: 3 or SEQ ID NO: 117. The 3D structure of CsgG is known in the art and is disclosed, for example, in Goyal et al. (2014) Nature 516(7530):250-3. In addition to the mutations described herein, any number of mutations can be made in the wild-type CsgG sequence, provided that the CsgG mutant monomer retains the improved properties conferred upon it by the mutations of the present invention.

[0163] Typically, a CsgG monomer will retain the ability to form a structure comprising three α-helices and five β-sheets. Mutations can be made in at least the N-terminus of the first α-helix (which begins at S63 in SEQ ID NO: 3), in the second α-helix (from G85 to A99 of SEQ ID NO: 3 or SEQ ID NO: 117), in the loop between the second α-helix and the first β-sheet (from Q100 to N120 of SEQ ID NO: 3 or SEQ ID NO: 117), in the fourth and fifth β-sheets (S173 to R192 and R198 to T107 of SEQ ID NO: 3 or SEQ ID NO: 117, respectively), and in the loop between the fourth and fifth β-sheets (F193 to Q197 of SEQ ID NO: 3 or SEQ ID NO: 117) without affecting the ability of the CsgG monomer to form a transmembrane pore capable of polypeptide translocation. Therefore, it is envisioned that further mutations can be made in any of these regions in any CsgG monomer without affecting the monomer's ability to form a pore that can translocate a polynucleotide. It is also contemplated that mutations can be made in other regions, such as in any α-helix (S63 to R76, G85 to A99, or V211 to L236 of SEQ ID NO: 3 or SEQ ID NO: 117) or in any β-sheet (I121 to N133, K135 to R142, I146 to R162, S173 to R192, or R198 to T107 of SEQ ID NO: 3 or SEQ ID NO: 117) without affecting the monomer's ability to form a pore that can translocate a polynucleotide. It is also contemplated that deletions of one or more amino acids can be made in any loop region connecting the α-helix and β-sheet and / or in the N-terminal and / or C-terminal regions of the CsgG monomer without affecting the monomer's ability to form a pore that can translocate a polynucleotide.

[0164] In addition to those discussed above, amino acid substitutions can also be made to the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:117, for example, up to 1, 2, 3, 4, 5, 10, 20 or 30 substitutions. Conservative substitutions replace amino acids with other amino acids of similar chemical structure, similar chemical properties or similar side chain volume. The introduced amino acids can have similar polarity, hydrophilicity, hydrophobicity, basicity, acidity, neutrality or charge as the amino acids they replace. Alternatively, conservative substitutions can introduce another aromatic or aliphatic amino acid to replace a pre-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 major amino acids as defined in Table 1 above. In the case where amino acids have similar polarity, this can also be determined by reference to the hydrophilicity rating of the amino acid side chains in Table 2.

[0165] One or more amino acid residues of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 117 may additionally be deleted from the above polypeptides. Up to 1, 2, 3, 4, 5, 10, 20 or 30 or more residues may be deleted.

[0166] Variants may include fragments of SEQ ID NO: 3 or SEQ ID NO: 117. Such fragments retain pore-forming activity. Fragments may be at least 50, at least 100, at least 150, at least 200, or at least 250 amino acids in length. Such fragments may be useful for generating pores. Fragments preferably comprise the transmembrane domain of SEQ ID NO: 3 or SEQ ID NO: 117, i.e., K135-Q153 and S183-S208.

[0167] Alternatively or additionally, one or more amino acids may be added to the above polypeptides. Extensions may be provided at the amino terminus or carboxyl terminus of the amino acid sequence of SEQ ID NO:3 or SEQ ID NO:117, or a polypeptide variant or fragment thereof. The extensions may be very 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 will be discussed in more detail below.

[0168] The CsgG pore described herein includes the wild-type CsgG pore or a homologue or mutant / variant thereof. A variant is a polypeptide having an amino acid sequence that differs from that of SEQ ID NO: 3 or SEQ ID NO: 117 and that retains its pore-forming ability. Variants typically contain the regions of SEQ ID NO: 3 or SEQ ID NO: 117 responsible for pore formation. CsgG contains a β-barrel, whose pore-forming ability is provided by a β-sheet in each subunit. Variants of SEQ ID NO: 3 or SEQ ID NO: 117 typically contain the β-sheet-forming regions of SEQ ID NO: 3 or SEQ ID NO: 117, namely K134-Q154 and S183-S208. One or more modifications may be made to the β-sheet-forming regions of SEQ ID NO: 3 or SEQ ID NO: 117, as long as the resulting variant retains its pore-forming ability. Variants of SEQ ID NO: 3 or SEQ ID NO: 117 preferably contain one or more modifications, such as substitutions, additions or deletions, within their α-helical and / or loop regions.

[0169] The mutant CsgG monomer may be a mutant CsgG monomer, which is a monomer that differs in sequence from a wild-type CsgG monomer and retains the ability to form a pore. Mutant monomers may also be referred to herein as variants. Methods for confirming the ability of mutant monomers to form a pore are well known in the art and are discussed in more detail below.

[0170] At least one monomer, or any or all of the six to ten monomers in the CsgG pore or pore complex of the invention may have any of the specific modifications or substitutions disclosed in WO2016 / 034591, WO2017 / 149316, WO2017 / 149317, WO2017 / 149318, WO2018 / 211241, and WO2019 / 002893 (all of which are herein incorporated by reference in their entirety).

[0171] Preferred additional modifications of at least one monomer / variant of SEQ ID NO: 117 in the pore or pore complex of the invention include, but are not limited to, one or more, such as 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, or all of the following:

[0172] (a) a substitution at position Y51, such as Y51A, Y51I, Y51L or Y51T;

[0173] (b) a substitution at position N55, such as N55V, N55Q, N55S or N55A;

[0174] (c) a substitution at position F56, such as F56Q, F56A or F56N;

[0175] (d) a substitution at position L90, such as L90N, L90R or L90K;

[0176] (e) a substitution at position N91, such as N91N, N91R or N91K;

[0177] (f) a substitution at position K94, such as K94Q, K94R, K94F, K94Y, K94W, K94L, K94S or K94N;

[0178] (g) substitution at position R192, such as R192D, R192Q, R192F, R192S or R192T;

[0179] (h) a substitution at position Q153, such as Q153C; and

[0180] (i) Substitution at position C215, such as C215T.

[0181] At least one monomer / variant of SEQ ID NO: 117 may further comprise a deletion of one or more positions, such as a deletion of V105-I107, a deletion of F193-L199, or a deletion of F195-L199.

[0182] Any number of monomers in the pore or pore complex, such as 6, 7, 8, 9 or 10, may be mutant monomers / variants of SEQ ID NO: 117, further comprising one or more of these additional substitutions, in addition to modifications at one or more of positions W97, Q100, E101, N102 and T104 in SEQ ID NO: 117. All six to ten monomers in the pore or pore complex may be mutant monomers / variants of SEQ ID NO: 117, further comprising one or more of these additional substitutions, in addition to modifications at one or more of positions W97, Q100, E101, N102 and T104 in SEQ ID NO: 117.

[0183] Double pore complex

[0184] The pore or pore complex of the present invention may be a dual pore complex comprising a first pore or complex and a second pore or complex. The dual pore complex may comprise a pore-pore, a pore-pore complex, or a pore complex-pore complex. Either of the pores or complexes may be a pore or complex of the present invention. In one embodiment, both the first pore complex and the second pore complex are CsgG / CsgF pore complexes of the present invention. In another embodiment, both the first pore and the second pore are CsgG pores of the present invention. The first pore and the second pore or pore complex may be the same or different. In addition to any mutations disclosed herein, at least one CsgG monomer in the dual pore may comprise one or more of the additional mutations described in WO2016 / 034591, WO2017 / 149316, WO2017 / 149317, WO2017 / 149318, WO2018 / 211241, and WO2019 / 002893 (all of which are incorporated herein by reference in their entireties). At least one CsgG monomer preferably comprises any of the additional substitutions disclosed above.

[0185] Methods for preparing modified proteins

[0186] 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 mutant monomers in E. coli that are auxotrophic for specific amino acids in the presence of synthetic (i.e., non-naturally occurring) analogs of those specific amino acids. If the mutant monomers are produced using partial peptide synthesis, they can also be produced by naked ligation.

[0187] Monomers derived from CsgG can be modified to aid their identification or purification, for example by adding a streptavidin tag or by adding a signal sequence to facilitate their secretion from cells, where the monomers do not naturally contain such a sequence. Other suitable tags are discussed in more detail below. The monomers can be labeled with a revealing marker. The revealing marker can be any suitable marker that allows for detection of the monomer. Suitable markers are described below.

[0188] Monomers derived from CsgG can also be produced using D-amino acids. For example, monomers derived from CsgG can comprise a mixture of L-amino acids and D-amino acids. This is conventional in the art of producing such proteins or peptides.

[0189] Monomers derived from CsgG contain one or more specific modifications to facilitate nucleotide discrimination. Monomers derived from CsgG may also contain other nonspecific modifications, as long as they do not interfere with pore formation. Many 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 methyl acetimidate, or acylation with acetic anhydride.

[0190] Monomers derived from CsgG can be produced using standard methods known in the art. Monomers derived from CsgG can be synthesized or recombinantly prepared. For example, monomers can be synthesized by in vitro translation and transcription (IVTT). Suitable methods for producing pores and monomers are discussed in International Applications WO 2010 / 004273, WO 2010 / 004265, or WO 2010 / 086603 (incorporated herein by reference in their entireties). Methods for inserting pores into membranes are known.

[0191] Two or more CsgG monomers in a pore may be covalently attached to each other. For example, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 monomers may be covalently attached. The covalently attached monomers may be the same or different.

[0192] Monomers can optionally be fused genetically via a linker, or chemically fused, for example, via a chemical cross-linker. Methods for covalently attaching monomers are disclosed in WO2017 / 149316, WO2017 / 149317, and WO2017 / 149318 (incorporated herein by reference in their entirety).

[0193] In certain embodiments, the mutant monomer is chemically modified. The mutant monomer can be chemically modified in any way and at any site. The mutant monomer is preferably chemically modified by attaching the molecule to one or more cysteines (cysteine ​​connection), attaching the molecule to one or more lysines, attaching the molecule to one or more non-natural amino acids, enzyme modifications of epi-positions or terminal modifications. Suitable methods for carrying out such modifications are well known in the art. Mutant monomers can be chemically modified by attaching any molecule. For example, mutant monomers can be chemically modified by attaching dyes or fluorophores.

[0194] 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 polynucleotide sequence. The presence of the adapter improves the host-guest chemistry of the pore and the nucleotide or polynucleotide sequence, 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, thereby improving its interaction with the nucleotide or polynucleotide sequence. The adapter can change the charge of the barrel or channel of the pore, or specifically interact or bind to the nucleotide or polynucleotide sequence, thereby promoting its interaction with the pore.

[0195] The molecular adaptor is preferably a cyclic molecule, a cyclodextrin, a substance capable of hybridization, a DNA binder or mutual chelator, a peptide or peptide analog, a synthetic polymer, an aromatic planar molecule, a positively charged small molecule or a molecule capable of forming hydrogen bonds.

[0196] The adaptor can be circular. Circular adaptors preferably have the same symmetry as the pore. The adaptor preferably has eight-fold or nine-fold symmetry, as CsgG typically has eight or nine subunits arranged around a central axis. This will be discussed in more detail below.

[0197] Adaptors typically interact with nucleotide or polynucleotide sequences through host-guest chemistry. Adaptors are typically capable of interacting with nucleotide or polynucleotide sequences. Adaptors comprise one or more chemical groups capable of interacting with nucleotide or polynucleotide sequences. One or more chemical groups preferably interact with nucleotide or polynucleotide sequences through non-covalent interactions, such as hydrophobic interactions, hydrogen bonds, van der Waals forces, π-cation interactions, and / or electrostatic forces. One or more chemical groups capable of interacting with nucleotide or polynucleotide sequences are preferably positively charged. One or more chemical groups capable of interacting with nucleotide or polynucleotide sequences more preferably comprise amino groups. Amino groups can be attached to primary, secondary, or tertiary carbon atoms. Adaptors even more preferably comprise an amino ring, such as a ring of 6, 7, or 8 amino groups. Adaptors most preferably comprise an eight amino ring. The protonated amino ring can interact with negatively charged phosphate groups in the nucleotide or polynucleotide sequence.

[0198] The correct positioning of the adapter in the hole can be promoted by host-guest chemistry between the adapter and the hole containing the mutant monomer. The adapter preferably comprises one or more chemical groups that can interact with one or more amino acids in the hole. The adapter more preferably comprises one or more chemical groups that can interact with one or more amino acids in the hole through non-covalent interactions, such as hydrophobic interactions, hydrogen bonds, van der Waals forces, π-cation interactions and / or electrostatic forces. The chemical groups that can interact with one or more amino acids in the hole are typically hydroxyls or amines. The hydroxyl group can be attached to a primary, secondary or tertiary carbon atom. The hydroxyl group can form hydrogen bonds with uncharged amino acids in the hole. Any adapter that promotes interaction between the hole and the nucleotide or polynucleotide sequence can be used.

[0199] Suitable adapters include, but are not limited to, cyclodextrins, cyclic peptides, and cucurbiturils. The adapter is preferably cyclodextrin or a derivative thereof. Cyclodextrin or a derivative thereof may be any of those disclosed in Eliseev, AV and Schneider, HJ. (1994) J. Am. Chem. Soc. 116, 6081-6088. The adapter is more preferably seven-6-amino-β-cyclodextrin (am7-βCD), 6-monodeoxy-6-monoamino-β-cyclodextrin (am1-βCD), or seven-(6-deoxy-6-guanidino)-cyclodextrin (gu7-βCD). The guanidino group in gu7-βCD has a much higher pKa than the primary amine in am7-βCD, and therefore it carries more positive charges. The gu7-βCD adapter can be used to increase the residence time of the nucleotide in the pore to increase the accuracy of the measured residual current, as well as to increase the base detection rate at high temperatures or low data acquisition rates.

[0200] If the succinimidyl 3-(2-pyridyldithio) propionate (SPDP) cross-linker is used as discussed in more detail below, the linker is preferably heptakis(6-deoxy-6-amino)-6-N-mono(2-pyridyl)dithiopropionyl-β-cyclodextrin (am6amPDP1-βCD).

[0201] More suitable adaptors include γ-cyclodextrin, which comprises nine saccharide units (thus having nine-fold symmetry). γ-cyclodextrin may contain a linker molecule or may be modified to include all or more of the modified saccharide units used in the β-cyclodextrin example above.

[0202] The molecular adapter can be covalently attached to the mutant monomer. The adapter can be covalently attached to the hole using any method known in the art. The adapter is usually attached by chemical connection. If the molecular adapter is attached via a cysteine ​​connection, one or more cysteines are preferably introduced into the mutant by substitution, for example in a barrel. The mutant monomer can be chemically modified by attaching the molecular adapter to one or more cysteines in the mutant monomer. One or more cysteines can be naturally occurring, i.e., at positions 1 and / or 215 in SEQ ID NO:3 or SEQ ID NO:117. Alternatively, the mutant monomer can be chemically modified by attaching the 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 is not attached to this position, rather than the cysteine ​​at position 1 or the cysteine ​​introduced at another position.

[0203] The reactivity of cysteine ​​residues can be enhanced by modification of adjacent residues. For example, the basic groups of flanking arginine, histidine or lysine residues will change the pKa of the cysteine ​​thiol group to that of a more reactive S-group. The reactivity of cysteine ​​residues can be protected by thiol protecting groups such as dTNB. Before the linker is attached, they can react with one or more cysteine ​​residues of the mutant monomer.

[0204] The molecule may be attached directly to the mutant monomer.The molecule is preferably attached to the mutant monomer using a linker such as a chemical cross-linker or a peptide linker.

[0205] Suitable chemical cross-linking agents are well known in the art. Preferred cross-linking agents include 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldithio) propionate, 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldithio) butyrate, and 2,5-dioxopyrrolidin-1-yl 8-(pyridin-2-yldithio) octanoate. The most preferred cross-linking agent is succinimidyl 3-(2-pyridyldithio) propionate (SPDP). Typically, the molecule is covalently attached to the bifunctional cross-linker before the molecule / cross-linker complex is covalently attached to the mutant monomer, but the bifunctional cross-linker can also be covalently attached to the monomer before the bifunctional cross-linker / monomer complex is attached to the molecule.

[0206] The linker is preferably resistant to dithiothreitol (DTT). Suitable linkers include, but are not limited to, iodoacetamide-based and maleimide-based linkers.

[0207] In other embodiments, the monomers can be attached to polynucleotide binding proteins. This forms a modular sequencing system that can be used in the sequencing methods of the present invention. Polynucleotide binding proteins are discussed below.

[0208] The polynucleotide binding protein is preferably covalently attached to the mutant monomer. Protein can be covalently attached to the monomer using any method known in the art. Monomer and protein can be chemically fused or genetically fused. If the entire construct is expressed by a single polynucleotide sequence, the monomer and protein are genetically fused. Genetic fusion of monomers and polynucleotide binding proteins is discussed in WO 2010 / 004265 (incorporated herein by reference in its entirety).

[0209] If the polynucleotide binding protein is attached via a cysteine ​​connection, one or more cysteines are preferably introduced into a mutant by substitution. Preferably, one or more cysteines are introduced into a loop region with low conservation in a homologue, which indicates that mutation or insertion can be tolerated. Therefore, they are suitable for attaching a polynucleotide binding protein. In such 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.

[0210] The polynucleotide binding protein can be directly attached to the mutant monomer or connected via one or more joints. The molecule can be attached to the mutant monomer using a hybridization joint as described in WO 2010 / 086602 (incorporated herein by reference in its entirety). Alternatively, a peptide joint can be used. The peptide joint is an amino acid sequence. The length, flexibility and hydrophilicity of the peptide joint are generally designed so that it does not interfere with the function of the monomer and the molecule. Preferred flexible peptide joints are 2 to 20, such as 4, 6, 8, 10 or 16 serine and / or glycine amino acid extensions. Preferred flexible joints include (SG) 1, (SG) 2, (SG) 3, (SG) 4, (SG) 5 and (SG) 8, wherein S is serine and G is glycine. Preferred rigid joints are 2 to 30, such as 4, 6, 8, 16 or 24 proline amino acid extensions. Preferred rigid joints include (P) 12, wherein P is proline.

[0211] Chemical modification

[0212] Mutant CsgG monomers or CsgF peptides can be chemically modified using molecular adaptors and polynucleotide binding proteins.

[0213] The molecule with which the monomer or peptide is chemically modified may be attached directly to the monomer or peptide or via a linker as disclosed in WO 2010 / 004273, WO 2010 / 004265 or WO 2010 / 086603 (incorporated herein by reference in their entirety).

[0214] Any of the proteins described herein, such as the CsgG monomer and / or the CsgF peptide, can be modified to aid in their identification or purification, for example by the addition of a histidine residue (his tag), an aspartic acid residue (asp tag), a streptavidin tag, a flag tag, a SUMO tag, a GST tag, or an MBP tag, or by the addition of a signal sequence to promote their secretion from cells in which 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 engineered position on the protein. An example of this is reacting a gel shift reagent with a cysteine ​​engineered on the outside of the protein. This has been demonstrated as a method for separating hemolysin hetero-oligomers (ChemBiol. 1997 Jul;4(7):497-505).

[0215] Any of the proteins described herein, such as the CsgG monomer and / or the CsgF peptide, can be labeled with a revealing marker. The revealing marker can be any suitable marker that allows for detection of the protein. Suitable markers include, but are not limited to, fluorescent molecules, radioisotopes (e.g., 125I, 35S), enzymes, antibodies, antigens, polynucleotides, and ligands (such as biotin).

[0216] Any of the proteins described herein, such as the CsgG monomer and / or CsgF peptide, can be synthesized or recombinantly prepared. For example, the protein 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 production. The protein can also be altered after synthesis or recombinant production.

[0217] Proteins can also be produced using D-amino acids. For example, a protein can contain a mixture of L-amino acids and D-amino acids. This is conventional in the art of producing such proteins or peptides.

[0218] Proteins 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 proteins. Such modifications include, for example, reductive alkylation of amino acids by reaction with an aldehyde followed by reduction with NaBH4, amidination with methyl acetimidate, or acylation with acetic anhydride.

[0219] Any of the proteins described herein, such as the CsgG monomer and / or the CsgF peptide, can be produced using standard methods known in the art. Polynucleotide sequences encoding the proteins can be derived and replicated using standard methods in the art. Polynucleotide sequences encoding the proteins can be expressed in bacterial host cells using standard techniques in the art. Proteins can be produced in cells by in situ expression of the polypeptides from recombinant expression vectors. The expression vectors optionally carry an inducible promoter to control expression of the polypeptides. These methods are described in Sambrook, J. and Russell, D. (2001). Molecular Cloning: A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

[0220] Proteins can be produced on a large scale after purification from the organism that produces the protein by any protein liquid chromatography system or after recombinant expression. Typical protein liquid chromatography systems include FPLC, AKTA system, Bio-Cad system, Bio-Rad BioLogic system and Gilson HPLC system.

[0221] Method for generating holes

[0222] The present invention provides methods for producing CsgG:modified CsgF pore complexes containing two or more constriction sites in vivo and in vitro. One embodiment provides a method for co-expressing a transmembrane pore complex comprising a CsgG pore, or a homolog or mutant thereof, and a modified CsgF peptide, or a homolog or mutant thereof. The method comprises the steps of expressing a CsgG monomer (expressed as a proprotein provided in SEQ ID NO: 2, or a homolog or mutant thereof) and a modified or truncated CsgF monomer in a suitable host cell, thereby allowing for in vivo composite pore formation. The complex comprises a modified CsgF peptide complexed with the CsgG pore to provide an additional read head for the pore. The resulting pore complex produced by the method using the modified CsgF peptide provides a structure sufficient for characterization of target analytes, such as nucleic acid sequencing, as it allows passage of the analyte (particularly a polynucleotide chain) and comprises two or more read heads for improved reading of the polynucleotide sequence when used in an appropriate setting for the application. Methods for preparing the pores and complexes of the present invention and ways of labeling them are disclosed in WO2016 / 034591, WO2017 / 149316, WO2017 / 149317 and WO2017 / 149318, WO2018 / 211241 and WO2019 / 002893 (all of which are incorporated herein by reference in their entirety).

[0223] Methods for characterizing analytes

[0224] The present invention provides a method for determining the presence, absence, or one or more characteristics of a target analyte. The method comprises contacting the target analyte with an isolated pore complex or transmembrane pore (such as a pore of the present invention), causing the target analyte to move relative to the pore channel, such as into or through the pore channel, and performing one or more measurements as the analyte moves relative to the pore, thereby determining the presence, absence, or one or more characteristics of the analyte. The target analyte may also be referred to as a template analyte or an analyte of interest. The isolated pore complex typically comprises at least 7, at least 8, at least 9, or at least 10 monomers, such as 7, 8, 9, or 10 CsgG monomers. The isolated pore complex preferably comprises eight or nine identical CsgG monomers. One or more, such as 2, 3, 4, 5, 6, 7, 8, 9, or 10, CsgG monomers are preferably chemically modified, or the CsgF peptide is chemically modified. Isolated pore complex monomers, such as CsgG monomers or homologs or mutants thereof, and modified CsgF monomers or homologs or mutants thereof, can be derived from any organism. The analyte can pass through the CsgG constriction and then through the CsgF constriction. In alternative embodiments, the analyte can pass through the CsgF constriction and then through the CsgG constriction, depending on the orientation of the CsgG / CsgF complex in the membrane.

[0225] 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 analyte. The method can relate to determining the presence, absence or one or more characteristics of two or more analytes. The method can include determining the presence, absence or one or more characteristics of any number of analytes (such as 2, 5, 10, 15, 20, 30, 40, 50, 100 or more analytes). Any number of characteristics of one or more analytes can be determined, such as 1, 2, 3, 4, 5, 10 or more characteristics.

[0226] The binding of molecules in the channel of the pore complex or near any opening of the channel will affect the open channel ion flow through the pore, which is the essence of "molecular sensing" of the pore channel. In a manner similar to nucleic acid sequencing applications, changes in open channel ion flow 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; all of which are incorporated herein by reference in their entirety). The degree of reduction in ion flow, measured by the reduction in current, is related to the size of the obstruction in or near the pore. Therefore, the binding of a molecule of interest (also referred to as an "analyte") in 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, polysaccharides, and small molecules (herein, low molecular weight (e.g., <900 Da or <500 Da) organic or inorganic compounds) such as drugs, toxins, cytokines, and pollutants. Detecting the presence of biomolecules has applications in personalized drug development, medicine, diagnostics, life science research, environmental monitoring, and the security and / or defense industries.

[0227] In another aspect, isolated pore complexes or transmembrane pore complexes containing a wild-type or modified E. coli CsgG nanopore, or a homolog or mutant thereof, and a modified CsgF peptide that provides a channel constriction to the pore within the complex can be used as molecular or biosensors. In some embodiments, the CsgG nanopore can be derived from or isolated from a bacterial protein (e.g., E. coli, Salmonella typhi). In some embodiments, the CsgG nanopore can be recombinantly produced. The procedure for analyte detection is described in Howorka et al., Nature Biotechnology (2012) Jun 7;30(6):506-7. The analyte molecule to be detected can be bound to either side of the channel or within the lumen of the channel itself. The location of binding can be determined by the size of the molecule to be sensed.

[0228] 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 polysaccharide, a dye, a bleach, a drug, a diagnostic agent, a recreational drug, an explosive, a toxic compound, or an environmental pollutant. The method can involve determining the presence, absence, or one or more characteristics of two or more analytes of the same type (such as two or more proteins, two or more nucleotides, or two or more drugs). Alternatively, the method can involve determining the presence, absence, or one or more characteristics of two or more different types of analytes (such as one or more proteins, one or more nucleotides, and one or more drugs).

[0229] The target analyte may be secreted from the cell. Alternatively, the target analyte may be an analyte present inside the cell, such that the analyte must be extracted from the cell before the method can be performed.

[0230] Wild-type pores can act as sensors, but are typically 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, this binding moiety may comprise a cyclodextrin or oligonucleotide; for small molecules, this may 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 an antigen recognition domain from a T-cell receptor (TCR); or for proteins, it may be a known ligand for the protein of interest. In this way, wild-type or modified E. coli CsgG nanopores or homologs thereof can be made to function as molecular sensors for detecting the presence of suitable antigens (including epitopes) in a sample. These antigens can include cell surface antigens, including receptors, markers of solid tumors or hematological cancer cells (e.g., lymphomas or leukemias), viral antigens, bacterial antigens, protozoal antigens, allergens, allergy-related molecules, albumin (e.g., human, rodent, or bovine), fluorescent molecules (including fluorescein), blood group antigens, small molecules, drugs, enzymes, catalytic sites of enzymes or enzyme substrates, and transition state analogs of enzyme substrates. As described above, modifications can be achieved using known genetic engineering and recombinant DNA techniques. The positioning of any adaptation will depend on the properties of the molecule to be sensed, such as size, three-dimensional structure, and its biochemical properties. The selection of suitable structures can utilize computational structure design. The determination and optimization of protein-protein interactions or protein-small molecule interactions can be performed using methods such as The studies were conducted using a technique such as BIAcore, which uses surface plasmon resonance to detect molecular interactions (BIAcore, Inc., Piscataway, NJ; see also www.biacore.com).

[0231] In one embodiment, the analyte is an amino acid, peptide, polypeptide, or protein. The amino acid, peptide, polypeptide, or protein may be naturally occurring or non-naturally occurring. The polypeptide or protein may include synthetic or modified amino acids. Several different types of amino acid modifications are known in the art. Suitable amino acids and modifications thereof are described above. It should be understood that the target analyte can be modified by any method available in the art.

[0232] In another embodiment, the analyte is a polynucleotide, such as a nucleic acid, which is defined as a macromolecule comprising two or more nucleotides. Nucleic acids are particularly well-suited 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 results in a directly correlated reduction in the ion flow through the channel. This change in ion flow can be recorded. Suitable electrical measurement techniques for recording changes in ion flow are discussed above. With appropriate calibration, the characteristic reduction in ion flow can be used to identify specific nucleotides and related bases passing through the channel in real time. In typical nanopore nucleic acid sequencing, due to partial blockage of the channel by nucleotides, the open-channel ion flow decreases as each nucleotide of the nucleic acid sequence of interest sequentially passes through the nanopore channel. This reduction in ion flow is measured using the appropriate recording techniques described above. The reduction in ion flow can be calibrated to the reduction in measured ion flow of known nucleotides passing through the channel, thereby providing a means for determining which nucleotide is passing through the channel and, therefore, providing a way to determine the nucleotide sequence of the nucleic acid passing through the nanopore as the sequence proceeds. In order to accurately determine a single nucleotide, it is generally necessary that the reduction in ion flow through the channel is directly related to the size of the single nucleotide passing through the constriction (or "reading head"). It will be understood that the complete nucleic acid polymer that "passes through the hole" via the action of, for example, an associated polymerase can be sequenced. Alternatively, the sequence can be determined by the passage of triphosphate nucleotide bases that have been sequentially removed from the target nucleic acid near the hole (see, for example, WO 2014 / 187924, which is incorporated herein by reference in its entirety).

[0233] A polynucleotide or nucleic acid may comprise any combination of nucleotides. Nucleotides may be naturally occurring or artificial. One or more nucleotides in a polynucleotide may be oxidized or methylated. One or more nucleotides in a polynucleotide may be damaged. For example, a polynucleotide may comprise a pyrimidine dimer. Such dimers are often associated with UV damage and are a major cause of cutaneous melanoma. One or more nucleotides in a polynucleotide may be modified, for example, with a marker or tag, suitable examples of which are known to those skilled in the art. A polynucleotide may comprise one or more spacers. Nucleotides typically contain a nucleobase, a sugar, and at least one phosphate group. The nucleobase and sugar form a nucleoside. Nucleobases are typically heterocyclic. Nucleobases include, but are not limited to, purines and pyrimidines, and more specifically, adenine (A), guanine (G), thymine (T), uracil (U), and cytosine (C). The sugar is typically a pentose. Nucleotide sugars include, but are not limited to, ribose and deoxyribose. The sugar is preferably deoxyribose. Polynucleotides preferably comprise the following nucleosides: deoxyadenosine (dA), deoxyuridine (dU) and / or thymidine (dT), deoxyguanosine (dG) and deoxycytidine (dC). Nucleotides are typically ribonucleotides or deoxyribonucleotides. Nucleotides typically contain monophosphates, diphosphates or triphosphates. Nucleotides can contain more than three phosphates, such as 4 or 5 phosphates. Phosphates can be attached to the 5' side or 3' side of the nucleotide. The nucleotides in a polynucleotide can be attached to each other in any way. Nucleotides are typically attached through their sugar and phosphate groups, as in nucleic acids. Nucleotides can be connected via their core bases, as in pyrimidine dimers. Polynucleotides can be single-stranded or double-stranded. At least a portion of a polynucleotide is preferably double-stranded. The polynucleotide is most preferably ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). In particular, the method of using a polynucleotide as an analyte alternatively comprises determining one or more characteristics selected from the group consisting of: (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.

[0234] Polynucleotide can have any length (i).For example, the length of 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.The length of polynucleotide can be 1000 or more nucleotides or nucleotide pairs, 5000 or more nucleotides or nucleotide pairs or 100000 or more nucleotides or nucleotide pairs.The polynucleotide of any number can be studied.For example, the method can relate to characterize 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 50, 100 or more polynucleotides.If characterize two or more polynucleotides, then they can be two examples of different polynucleotides or identical polynucleotides.Polynucleotide can be naturally occurring or artificial.For example, the method can be used for verifying the sequence of the oligonucleotide manufactured.The method is usually carried out in vitro.

[0235] The nucleotides may have any identity (ii) and include, but are not limited to, adenosine monophosphate (AMP), guanosine monophosphate (GMP), thymidine monophosphate (TMP), uridine monophosphate (UMP), 5-methylcytidine monophosphate, 5-hydroxymethylcytidine monophosphate, cytidine monophosphate (CMP), cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), deoxyadenosine monophosphate (dAMP), deoxyguanosine monophosphate (dGMP), deoxythymidine monophosphate (dTMP), deoxyuridine monophosphate (dUMP), deoxycytidine monophosphate (dCMP), and deoxymethylcytidine monophosphate. The nucleotides are preferably selected from AMP, TMP, GMP, CMP, UMP, dAMP, dTMP, dGMP, dCMP, and dUMP. The nucleotides may be abasic (i.e., lack a nucleobase). The nucleotides may also lack a nucleobase and a sugar (i.e., be a C3 spacer). The sequence of nucleotides (iii) is determined by the consecutive identity of subsequent nucleotides attached to each other in the 5' to 3' direction of the chain throughout the polynucleotide strain.

[0236] Wells comprising a CsgG pore and a CsgF peptide are particularly useful for analyzing homopolymers. For example, the well can be used to determine the sequence of a polynucleotide comprising two or more, such as at least 3, 4, 5, 6, 7, 8, 9, or 10, identical contiguous nucleotides. For example, the well can be used to sequence a polynucleotide comprising polyA, polyT, polyG, and / or polyC regions.

[0237] The CsgG pore constriction consists of residues at positions 51, 55, and 56 of SEQ ID NO:3 or SEQ ID NO:117. Readheads for CsgG and its constriction mutants are typically sharp. As DNA passes through the constriction, approximately five bases of DNA interacting with the pore's readhead at any given time dominate the current signal. While these sharper readheads are very good at reading mixed sequence regions of DNA (when A, T, G, and C are mixed), the signal becomes flat and lacks information when homopolymeric regions (e.g., polyT, polyG, polyA, polyC) are present within the DNA. Because these five bases dominate the signal for CsgG and its constriction mutants, photopolymers longer than five bases are difficult to distinguish without additional residence time information. However, if the DNA is passing through a second readhead, more DNA bases will interact with the combined readheads, increasing the length of homopolymers that can be distinguished.

[0238] Modified Dda helicase

[0239] The present invention provides a modified Dda helicase. One or more specific modifications are discussed in more detail below. Modifications according to the present invention include one or more substitutions as described below.

[0240] One or more of positions 55, 114, 156, 177, 210, 221, 350, and 358 of DDA 1993

[0241] The present invention provides a modified DNA-dependent ATPase (Dda) helicase in which one or more positions corresponding to amino acid positions 55, 114, 156, 177, 210, 221, 350, and 358 in Dda 1993 are modified or substituted. The Dda helicase and the positions corresponding to positions 55, 114, 156, 177, 210, 221, 350, and 358 in Dda 1993 are discussed in more detail below. Positions 55, 114, 156, and 177 are located in the 1A domain of Dda 1993. Positions 210 and 221 are located in the 2A domain of Dda 1993. Positions 350 and 358 are located in the tower domain of Dda 1993. The modified Dda helicase of the present invention may comprise any number and combination of modifications or substitutions at positions corresponding to amino acid positions (a) 55, (b) 114, (c) 156, (d) 177, (e) 210, (f) 221, (g) 350, and (h) 358, including those in (a); (b); (c); (d); (e); (f); (g); (h); (a) and (b); (a) and (c); (a) and (d); (a) and (e); (a) and (f); (a) and (g); (a) and (h); (h); (b) and (c); (b) and (d); (b) and (e); (b) and (f); (b) and (g); (b) and (h); (c) and (d); (c) and (e); (c) and (f); (c) and (g); (c) and (h); (d) and (e); (d) and (f); (d) and (g); (d) and (h); (e) and (f); (e) and (g); (e) and (h); (f) and (g); (f) and (h); (g) and (h); (a), (b) and (c); ( a), (b) and (d); (a), (b) and (e); (a), (b) and (f); (a), (b) and (g); (a), (b) and (h); (a), (c) and (d); (a), (c) and (e); (a), (c) and (f); (a), (c) and (g); (a), (c) and (h); (a), (d) and (e); (a), (d) and (f); (a), (d) and (g); (a), (d) and (h); (a), (e) and (f); (a), (e ) and (g); (a), (e) and (h); (a), (f) and (g); (a), (f) and (h); (a), (g) and (h); (b), (c) and (d); (b), (c) and (e); (b), (c) and (f); (b), (c) and (g); (b), (c) and (h); (b), (d) and (e); (b), (d) and (f); (b), (d) and (g); (b), (d) and (h); (b), (e) and (f); (b), (e) and (g);(b), (e), and (h); (b), (f), and (g); (b), (f), and (h); (b), (g), and (h); (c), (d), and (e); (c), (d), and (f); (c), (d), and (g); (c), (d), and (h); (c), (e), and (f); (c), (e), and (g); (c), (f), and (g); (c), (f), and (h); (c), (g), and (h); (d), (e), and (f); (d), (e), and (g); (d), (e), and (h); (d), (f), and (g); (d), (f), and (h); (d), (g), and (h); (e), (f) and (g); (e), (f) and (h); (e), (g) and (h); (f), (g) and (h); (a), (b), (c) and (d); (a), (b), (c) and (e); (a), (b), (c) and (f); (a), (b), (c) and (g); (a), (b), (c) and (h); (a), (b), (d) and (e); (a), (b), (d) and (f); (a), (b), (d) and (g); (a), (b), (d) and (h); (a), (b), (e) and (f); (a), (b), (e) and (g); (a), (b), (e) and (h); (a), (b), (f) and (g); (a), (b), (f), and (h); (a), (b), (g), and (h); (a), (c), (d), and (e); (a), (c), (d), and (f); (a), (c), (d), and (g); (a), (c), (d), and (h); (a), (c), (e), and (f); (a), (c), (e), and (g); (a), (c), (e), and (h); (a), (c), (f), and (g); (a), (c), (f), and (h); (a), (c), (e), and (f); (a), (d), (e), and (g); (a), (d), (e), and (h); (a), (d), (f) and (g); (a), (d), (f) and (h); (a), (d), (g) and (h); (a), (e), (f) and (g); (a), (e), (f) and (h); (a), (e), (g) and (h); (a), (f), (g) and (h); (b), (c), (d) and (e); (b), (c), (d) and (f); (b), (c), (d) and (g); (b), (c), (d) and (h); (b), (c), (e) and (f); (b), (c), (e) and (g); (b), (c), (e) and (h); (b), (c), (f) and (g); (b), (c), (f) and (h);(b), (c), (g), and (h); (b), (d), (e), and (f); (b), (d), (e), and (g); (b), (d), (e), and (h); (b), (d), (f), and (g); (b), (d), (g), and (h); (b), (d), (g), and (h); (b), (e), (f), and (g); (b), (e), (f), and (h); (b), (e), (g), and (h); (b), (f), (g), and (h); (c), (d), (e), and (f); (c), (d), (e), and (g); (c), (d), (e), and (h); (c), (d), (f), and (g); (c), (d), ( f) and (h); (c), (d), (g) and (h); (c), (e), (f) and (g); (c), (e), (f) and (h); (c), (e), (g) and (h); (c), (f), (g) and (h); (d), (e), (f) and (g); (d), (e), (f) and (h); (d), (e), (g) and (h); (d), (f), (g) and (h); (e), (f), (g) and (h); (a), (b), (c), (d) and (e); (a), (b), (c), (d) and (f); (a), (b), (c), (d) and (g); (a), (b), (c), (d) and (h); (a ), (b), (c), (e) and (f); (a), (b), (c), (e) and (g); (a), (b), (c), (e) and (h); (a), (b), (c), (f) and (g); (a), (b), (c), (f) and (h); (a), (b), (c), (g) and (h); (a), (b), (d), (e) and (f); (a), (b), (d), (e) and (g); (a), (b), (d), (f) and (g); (a), (b), (d), (f) and (h); (a), (b), (d), (g) and (h); (a), (b), (e) , (f) and (g); (a), (b), (e), (f) and (h); (a), (b), (e), (g) and (h); (a), (b), (f), (g) and (h); (a), (c), (d), (e) and (f); (a), (c), (d), (e) and (g); (a), (c), (d), (e) and (h); (a), (c), (d), (f) and (g); (a), (c), (d), (g) and (h); (a), (c), (e), (f) and (g); (a), (c), (e), (f) and (h); (a), (c), (e), (g) and (h);(a), (c), (f), (g), and (h); (a), (d), (e), (f), and (g); (a), (d), (e), (f), and (h); (a), (d), (e), (g), and (h); (a), (d), (f), (g), and (h); (a), (e), (f), (g), and (h); (b), (c), (d), (e), and (f); (b), (c), (d), (e), and (g); (b), (c), (d), (e), and (h); (b), (c), (d), (f), and (g); (b), (c), (d), (f), and (g); (b), (c), (d), (f), and (h); (b), (c), ( e), (f) and (g); (b), (c), (e), (f) and (h); (b), (c), (e), (g) and (h); (b), (c), (f), (g) and (h); (b), (d), (e), (f) and (g); (b), (d), (e), (f) and (h); (b), (d), (e), (g) and (h); (b), (d), (e), (f), (g) and (h); (b), (e), (f), (g) and (h); (c), (d), (e), (f) and (g); (c), (d), (e), (f) and (h); (c), (d), (e), (g) and (h); (c), (d), (f), (g) and (h ); (c), (e), (f), (g) and (h); (d), (e), (f), (g) and (h); (a), (b), (c), (d), (e) and (f); (a), (b), (c), (d), (e) and (g); (a), (b), (c), (d), (e) and (h); (a), (b), (c), (d), (f) and (g); (a), (b), (c), (d), (e) and (h); (a), (b), (c), (d), (f) and (g); (a), (b), (c), (e), (f) and (g); (a), (b), (c), (e), (f) and (h); (a), (b), (c), (e) , (g) and (h); (a), (b), (c), (f), (g) and (h); (a), (b), (d), (e), (f) and (g); (a), (b), (d), (e), (f) and (h); (a), (b), (d), (e), (g) and (h); (a), (b), (d), (e), (g) and (h); (a), (b), (d), (f), (g) and (h); (a), (c), (d), (e), (f) and (g); (a), (c), (d), (e), (f) and (h); (a), (c), (d), (e), (g) and (h); (a), (c), (d), (f), (g) and (h);(a), (c), (e), (f), (g), and (h); (a), (d), (e), (f), (g), and (h); (b), (c), (d), (e), (f), and (g); (b), (c), (d), (e), (f), and (h); (b), (c), (d), (e), (g), and (h); (b), (c), (d), (e), (f), (g), and (h); (b), (c), (d), (e), (f), (g), and (h); (b), (c), (d), (e), (f), (g), and (h); (c), (d), (e), (f), (g), and (h); f) and (g); (a), (b), (c), (d), (e), (f), and (h); (a), (b), (c), (d), (e), (g), and (h); (a), (b), (c), (d), (f), (g), and (h); (a), (b), (c), (d), (f), (g), and (h); (a), (b), (c), (e), (f), (g), and (h); (a), (c), (d), (e), (f), (g), and (h); (b), (c), (d), (e), (f), (g), and (h); or (a), (b), (c), (d), (e), (f), (g), and (h). ;

[0242] The present invention provides a modified DNA-dependent ATPase (Dda) helicase in which one or more positions corresponding to amino acid positions 114, 177, 350, and 358 in Dda 1993 are modified or substituted. The Dda helicase and the positions corresponding to positions 114, 177, 350, and 358 in Dda 1993 are discussed in more detail below. Positions 114 and 177 are located in the 1A domain of Dda 1993. Positions Y350 and K358 are located in the tower domain of Dda 1993. The modified Dda helicases of the invention may comprise any number and combination of modifications or substitutions at positions corresponding to amino acid positions (a) 114, (b) 177, (c) 350, and (d) 358 in Dda 1993, including at (a); (b); (c); (d); (a) and (b); (a) and (c); (a) and (d); (b) and (c); (b) and (d); (c) and (d); (a), (b) and (c); (a), (b) and (d); (a), (c) and (d); (b), (c) and (d); or (a), (b), (c), and (d).

[0243] The position corresponding to amino acid position 55 in Dda 1993 is preferably substituted with D, E, K, N, or S. The position corresponding to amino acid position 114 in Dda 1993 is preferably substituted with K (T55K). When used to characterize polynucleotide analytes, these substitutions increased speed and improved accuracy (Example 5). When used to characterize polynucleotide analytes, these substitutions also decreased the normalized velocity distribution (Example 5).

[0244] The position corresponding to amino acid position 114 in Dda 1993 is preferably substituted with A, V, I, L, M, F, Y, W, G, P, S, T, N, or Q. The position corresponding to amino acid position 114 in Dda 1993 is preferably substituted with A, G, I, L, M, P, S, T, or V. The position corresponding to amino acid position 114 in Dda 1993 is preferably substituted with G, L, S, or T. When used to characterize polynucleotide analytes, these substitutions reduce speed (Example 2). The position corresponding to amino acid position 114 in Dda 1993 is preferably substituted with A, I, M, P, or V. When used to characterize polynucleotide analytes, these substitutions increase speed (Example 2). The position corresponding to amino acid position 114 in Dda 1993 is preferably substituted with G (C11G). When used to characterize polynucleotide analytes, this substitution reduces speed and increases accuracy (Example 2). The position corresponding to amino acid position 114 in Dda 1993 is preferably substituted with I or P. When used to characterize polynucleotide analytes, these substitutions increased speed and decreased accuracy (Example 2). The position corresponding to amino acid position 114 in Dda 1993 is preferably substituted with G, I, or P. When used to characterize polynucleotide analytes, these substitutions decreased the normalized speed distribution (Example 2). The position corresponding to amino acid position 114 in Dda 1993 is most preferably substituted with I (C114I).

[0245] The position corresponding to amino acid position 156 in Dda 1993 is preferably substituted with A, E, F, G, I, L, M, P, S, V, Y, D, K, or N. The position corresponding to amino acid position 156 in Dda 1993 is preferably substituted with F (T156F). When used to characterize polynucleotide analytes, this substitution increases speed and improves accuracy (Example 5). When used to characterize polynucleotide analytes, this substitution also decreases the normalized velocity distribution (Example 5).

[0246] The position corresponding to amino acid position 177 in Dda 1993 is preferably substituted with D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, or Y. The position corresponding to amino acid position 177 in Dda 1993 is preferably substituted with F, G, S, V, W, or Y. When used to characterize polynucleotide analytes, these substitutions reduce velocity (Example 2). The position corresponding to amino acid position 177 in Dda 1993 is preferably substituted with D, E, G, H, I, L, M, N, Q, R, or T. When used to characterize polynucleotide analytes, these substitutions increase velocity (Example 2). The position corresponding to amino acid position 177 in Dda 1993 is preferably substituted with F, H, I, L, M, N, or W. When used to characterize polynucleotide analytes, these substitutions reduce accuracy and normalized velocity distribution (Example 2). They have different effects on velocity (Example 2). The position corresponding to amino acid position 177 in Dda 1993 is preferably substituted with N (K177N). This substitution reduces accuracy and increases normalized velocity distribution when used to characterize polynucleotide analytes (Example 2). The position corresponding to amino acid position 177 in Dda 1993 is most preferably substituted with M (K177M).

[0247] The position corresponding to amino acid position 210 in Dda 1993 is preferably substituted with D, E, K, S, N, R, H, or Y. The position corresponding to amino acid position 210 in Dda 1993 is preferably substituted with R (T210R), H (T210H), or K (T210K). The position corresponding to amino acid position 210 in Dda 1993 is preferably substituted with K (T210K). When used to characterize polynucleotide analytes, this substitution increases speed and improves accuracy (Example 5). When used to characterize polynucleotide analytes, this substitution also decreases the normalized velocity distribution (Example 5).

[0248] The position corresponding to amino acid position 221 in Dda 1993 is preferably substituted with D, K, E, Q, R, A, H, L, T, or Y. The position corresponding to amino acid position 221 in Dda 1993 is preferably substituted with D (N221D) or E (N221E). The position corresponding to amino acid position 221 in Dda 1993 is preferably substituted with E (N221E). When used to characterize polynucleotide analytes, this substitution increases speed and improves accuracy (Example 5). When used to characterize polynucleotide analytes, this substitution also decreases the normalized velocity distribution (Example 5).

[0249] The position corresponding to amino acid position 350 in Dda 1993 is preferably substituted with D, E, A, V, I, L, M, F, W, R, H, K, L, S, T, N, or Q. The position corresponding to amino acid position 350 in Dda 1993 is preferably substituted with I, F, W, or S. The position corresponding to amino acid position 350 in Dda 1993 is preferably substituted with I or S (Y350I or Y350S). The position corresponding to amino acid position 350 in Dda 1993 is preferably substituted with I (Y350I). When used to characterize polynucleotide analytes, this substitution increases speed and decreases accuracy and normalized speed distribution (Example 3). The position corresponding to amino acid position 350 in Dda 1993 is preferably substituted with I or S (Y350I or Y350S). These substitutions have the effects shown in Example 4 when used with the pore complexes of the present invention.

[0250] The position corresponding to amino acid position 350 in Dda 1993 is preferably substituted with A, D, E, G, K, L, N, Q, R, T, V, H, or M. The position corresponding to amino acid position 350 in Dda 1993 is preferably substituted with D (Y350D) or E (Y350E). The position corresponding to amino acid position 350 in Dda 1993 is preferably substituted with E (Y350E). When used to characterize polynucleotide analytes, this substitution increases speed and improves accuracy (Example 5). When used to characterize polynucleotide analytes, this substitution also decreases the normalized velocity distribution (Example 5).

[0251] The position corresponding to amino acid position 358 in Dda 1993 is preferably substituted with D, E, A, V, I, L, M, F, Y, W, R, H, L, S, T, N or Q.

[0252] The position corresponding to amino acid position 358 in Dda 1993 is preferably substituted with E, I, L, or M. When used to characterize polynucleotide analytes, these substitutions reduce velocity (Example 2). The position corresponding to amino acid position 358 in Dda 1993 is preferably substituted with I or M. When used to characterize polynucleotide analytes, these substitutions reduce velocity and improve accuracy (Example 2). The position corresponding to amino acid position 358 in Dda 1993 is preferably substituted with M (K358M). When used to characterize polynucleotide analytes, this substitution reduces velocity and improves accuracy and normalized velocity distribution (Example 2). The position corresponding to amino acid position 358 in Dda 1993 is preferably substituted with I (K358I). When used to characterize polynucleotide analytes, this substitution reduces velocity and normalized velocity distribution and improves accuracy (Example 2). Example 2 uses a CsgG pore without a CsgF peptide.

[0253] The position corresponding to amino acid position 358 in Dda 1993 is preferably substituted with A, E, F, I, M, or S. When used to characterize polynucleotide analytes, these substitutions improve accuracy (Example 3). The position corresponding to amino acid position 358 in Dda 1993 is preferably substituted with A, E, F, I, or M. When used to characterize polynucleotide analytes, these substitutions reduce velocity (Example 3). The position corresponding to amino acid position 358 in Dda 1993 is preferably substituted with S (K358S). When used to characterize polynucleotide analytes, this substitution increases velocity (Example 3). The position corresponding to amino acid position 358 in Dda 1993 is preferably substituted with A, E, I, M, or S. When used to characterize polynucleotide analytes, these substitutions reduce the normalized velocity distribution (Example 3). The position corresponding to amino acid position 358 in Dda 1993 is preferably substituted with (K358F). When used to characterize polynucleotide analytes, these substitutions improve the normalized velocity distribution (Example 3). Example 3 used a CsgG pore complex containing a CsgF peptide.

[0254] The position corresponding to amino acid position 358 in Dda 1993 is preferably substituted with I, L or Q. These substitutions reduce velocity and increase accuracy and normalized velocity distribution when used to characterize polynucleotide analytes (Example 4). Example 4 uses a pore complex of the invention.

[0255] The position corresponding to amino acid position 358 in Dda 1993 is most preferably substituted with I (K358I).

[0256] The modified helicases of the present invention may further comprise any of the modifications, mutations or substitutions discussed below.

[0257] The modified Dda helicase according to the present invention may be any one of SEQ ID NOs: 118 to 133. SEQ ID NO: 118 is Dda 1993. The modified helicase preferably comprises a variant of any one of SEQ ID NOs: 118 to 133. The variant may have any % sequence homology / identity with any one of SEQ ID NOs: 118 to 113 listed below.

[0258] Table 4 below summarizes preferred Dda helicases that can be modified according to the present invention.

[0259]

[0260]

[0261]

[0262] Table 5 shows the amino acids in SEQ ID NOs: 119 to 133 corresponding to positions 40, 55, 114, 156, 177, 210, 221, 350 and 358 in SEQ ID NO: 118.

[0263]

[0264]

[0265] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118, which comprises one or more of the following (a)-(h):

[0266] (a)

[0267] T55D, T55E, T55K, T55N or T55S, or T55K.

[0268] (b)

[0269] C114A, C114V, C114I, C114L, C114M, C114F, C114Y, C114W, C114G, C114P, C114S, C114T, C114N, or C114Q,

[0270] C114A, C114G, C114I, C114L, C114M, C114P, C114S, C114T, or C114V,

[0271] C114G, C114L, C114S, or C114T,

[0272] C114A, C114I, C114M, C114P, or C114V

[0273] C11G,

[0274] C114I or C114P

[0275] C114G, C114I, or C114P, or

[0276] C114I;

[0277] (c)

[0278] T156A, T156E, T156F, T156G, T156I, T156L, T156M, T156P, T156S, T156V, T156Y, T156D, T156K, or T156N, or

[0279] T156F;

[0280] (d)

[0281] K177D, K177E, K177F, K177G, K177H, K177I, K177L, K177M, K177N, K177Q, K177R, K177S, K177T, K177V, K177W, or K177Y,

[0282] K177F, K177G, K177S, K177V, K177W or K177Y,

[0283] K177D, K177E, K177G, K177H, K177I, K177L, K177M, K177N, K177Q, K177R, or K177T,

[0284] K177F, K177H, K177I, K177L, K177M, K177N, or K177W,

[0285] K177N, or

[0286] K177M;

[0287] (e)

[0288] T210D, T210E, T210K, T210S, T210N, T210R, T210H, or T210Y,

[0289] T210R, T210H, or T210Y, or

[0290] T210K;

[0291] (f)

[0292] N221D, N221K, N221E, N221Q, N221R, N221A, N221H, N221L, N221T, or N221Y,

[0293] N221D or N221E, or

[0294] N221E;

[0295] (g)

[0296] Y350D, Y350E, Y350A, Y350V, Y350I, Y350L, Y350M, Y350F, Y350W, Y350R, Y350H, Y350K, Y350L, Y350S, Y350T, Y350N or Y350Q,

[0297] Y350i or Y350S,

[0298] Y350i,

[0299] Y350S,

[0300] Y350I, Y350F, Y350W or Y350S,

[0301] Y350A, Y350D, Y350E, Y350G, Y350K, Y350L, Y350N, Y350Q, Y350R, Y350T, Y350V, Y350H or Y350M,

[0302] Y350D or Y350E, or

[0303] Y350E; and

[0304] (h)

[0305] K358D, K358E, K358A, K358V, K358I, K358L, K358M, K358F, K358Y, K358W, K358R, K358H, K358L, K358S, K358T, K358N or K358Q,

[0306] K358E, K358I, K358L or K358M,

[0307] K358I or K358M,

[0308] K358M,

[0309] K358I,

[0310] K358A, K358E, K358F, K358I, K358M or K358S,

[0311] K358A, K358E, K358F, K358I or K358M,

[0312] K358S,

[0313] K358A, K358E, K358I, K358M or K358S,

[0314] K358F,

[0315] K358I, K358L, or K358Q, or

[0316] K358I.

[0317] The variation may include any combination and permutation of (a)-(h) as described above.

[0318] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118, which comprises one or more of the following (a), (b), (c) and (d):

[0319] (a)

[0320] C114A, C114V, C114I, C114L, C114M, C114F, C114Y, C114W, C114G, C114P, C114S, C114T, C114N, or C114Q,

[0321] C114A, C114G, C114I, C114L, C114M, C114P, C114S, C114T, or C114V,

[0322] C114G, C114L, C114S, or C114T,

[0323] C114A, C114I, C114M, C114P, or C114V

[0324] C11G,

[0325] C114I or C114P

[0326] C114G, C114I, or C114P, or

[0327] C114I;

[0328] (b)

[0329] K177D, K177E, K177F, K177G, K177H, K177I, K177L, K177M, K177N, K177Q, K177R, K177S, K177T, K177V, K177W, or K177Y,

[0330] K177F, K177G, K177S, K177V, K177W or K177Y,

[0331] K177D, K177E, K177G, K177H, K177I, K177L, K177M, K177N, K177Q, K177R, or K177T,

[0332] K177F, K177H, K177I, K177L, K177M, K177N, or K177W,

[0333] K177N, or

[0334] K177M;

[0335] (c)

[0336] Y350D, Y350E, Y350A, Y350V, Y350I, Y350L, Y350M, Y350F, Y350W, Y350R, Y350H, Y350K, Y350L, Y350S, Y350T, Y350N or Y350Q,

[0337] Y350I or Y350S,

[0338] Y350I, or

[0339] Y350S;

[0340] (d)

[0341] K358D, K358E, K358A, K358V, K358I, K358L, K358M, K358F, K358Y, K358W, K358R, K358H, K358L, K358S, K358T, K358N or K358Q,

[0342] K358E, K358I, K358L or K358M,

[0343] K358I or K358M,

[0344] K358M,

[0345] K358I,

[0346] K358A, K358E, K358F, K358I, K358M or K358S,

[0347] K358A, K358E, K358F, K358I or K358M,

[0348] K358S,

[0349] K358A, K358E, K358I, K358M or K358S,

[0350] K358F,

[0351] K358I, K358L or K358Q, or

[0352] K358I.

[0353] The variant may include (a); (b); (c); (d); (a) and (b); (a) and (c); (a) and (d); (b) and (c); (b) and (d); (c) and (d); (a), (b) and (c); (a), (b) and (d); (a), (c) and (d); (b), (c) and (d); or (a), (b), (c) and (d).

[0354] Preferred variants of SEQ ID NO: 118 comprise: C114I; K177M; Y350I; K358I; C114I and K177M; C114I and Y350I; C114I and K358I; K177M and Y350I; K177M and K358I; Y350I and K358I; C114I, K177M and Y350I; C114I, K177M and K358I; C114I, Y350I and K358I; K177M, Y350I and K358I; or C114I, K177M, Y350I and K358I.

[0355] The helicase preferably comprises a variant of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133, wherein one or more positions corresponding to amino acid positions 55, 114, 156, 177, 210, 221, 350 and 358 in Dda 1993 are modified or substituted (including specific substitutions) as defined above. Various combinations and permutations of one or more of positions 55, 114, 156, 177, 210, 221, 350 and 358 in Dda 1993 are defined above with reference to (a) to (h).

[0356] The helicase preferably comprises a variant of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133, wherein one or more positions corresponding to amino acid positions 114, 177, 350 and 358 in Dda 1993 are modified or substituted (including specific substitutions) as defined above. Various combinations and permutations of one or more of positions 114, 177, 350 and 358 in Dda 1993 are defined above with reference to (a) to (d).

[0357] The helicase preferably comprises a variant of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133, wherein one or more positions corresponding to amino acid positions 114, 177 and 358 in Dda 1993 are modified or substituted (including specific substitutions) as defined above.

[0358] Position 40 in Dda 1993

[0359] Any modified helicase of the present invention may further comprise a modification or substitution at a position corresponding to amino acid position 40 in Dda 1993. Position 40 or a corresponding position may be substituted with A, V, I, L, M, F, Y, or W. The position corresponding to position T40 in Dda 1993 is shown in Table 5 above. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118, which, in addition to the above modifications / substitutions, further comprises a substitution at T40, such as T40A, T40V, T40I, T40L, T40M, T40F, T40Y, or T40W. The substitution is preferably T40Y.

[0360] The present invention provides a modified DNA-dependent ATPase (Dda) helicase in which the position corresponding to amino acid position 40 in Dda 1993 is modified or substituted. Position T40 is located in the tower domain of Dda 1993. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118 comprising a substitution at T40, such as T40A, T40V, T40I, T40L, T40M, T40F, T40Y or T40W. The substitution is preferably T40Y. The modified Dda helicase of the present invention may further comprise modifications or substitutions at one or more positions corresponding to amino acid positions (a) 55, (b) 114, (c) 156, (d) 177, (e) 210, (f) 221, (g) 350 and (h) 358, including any combination and permutation of (a) to (h). The modified Dda helicase of the invention may further comprise modifications or substitutions at one or more positions corresponding to amino acid positions (a) 114, (b) 177, (c) 350, and (d) 358 in Dda 1993, including at (a); (b); (c); (d); (a) and (b); (a) and (c); (a) and (d); (b) and (c); (b) and (d); (c) and (d); (a), (b) and (c); (a), (b) and (d); (a), (c) and (d); (b), (c) and (d); or (a), (b), (c), and (d). The helicase preferably comprises a variant of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133, wherein the position corresponding to amino acid position 40 in Dda 1993 is modified or substituted (including specific substitutions) as defined above.

[0361] The modified helicases of the invention may further comprise any modification, substitution, combination of modifications, or combination of substitutions discussed below.

[0362] Other helicases of the present invention

[0363] The present invention also provides a modified DNA-dependent ATPase (Dda) helicase having any modification, substitution, combination of modifications, or combination of substitutions discussed individually below. In other words, these helicases of the present invention do not necessarily have a substitution at a position corresponding to any of amino acid positions 40, 55, 114, 156, 177, 210, 221, 350, and 358 in Dda 1993 or any of amino acid positions 40, 114, 350, 177, and K358 in Dda 1993. Such modified helicases of the present invention are preferably variants of any of SEQ ID NOs: 118 to 133. The variant may have any % sequence homology / identity with any of SEQ ID NOs: 118 to 133 listed below.

[0364] The use of Dda helicases in analyte characterization is described in WO 2015 / 055981, WO 2015 / 166276 and WO 2016 / 055777 (all incorporated by reference).

[0365] The modified helicase of the present invention provides the more consistent movement of target analyte relative to transmembrane pore (such as by transmembrane pore), thereby causing the accuracy of raising.When target analyte (such as polynucleotide) moves (such as by hole) relative to hole, helicase preferably provides the more consistent movement from one k aggregate to another k aggregate or from k aggregate to k aggregate. Helicase allows target analyte (such as target polynucleotide) to move more smoothly relative to transmembrane pore (such as by transmembrane pore). Helicase preferably provides target analyte (such as target polynucleotide) relative to transmembrane pore (such as by transmembrane pore) more regular or more irregular movement.

[0366] Modifications typically improve accuracy by at least 0.1%, at least 0.5%, at least 1%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the unmodified helicase.

[0367] The ability of a helicase to control the movement of a polynucleotide can be determined as described in the Examples.

[0368] The ability of the helicase to control the movement of the polynucleotide can be measured using any method known in the art. For example, the helicase can be contacted with the polynucleotide, and the position of the polynucleotide can be determined using standard methods. The ability of the helicase to control the movement of the polynucleotide can be measured typically in a nanopore system, such as a system described below, particularly a system as described in the examples.

[0369] The modified helicases of the present invention can be isolated, substantially isolated, purified, or substantially purified. If the helicase is completely free of any other components, such as lipids, polynucleotides, pore monomers, or other proteins, it is isolated or purified. If the helicase is mixed with a carrier or diluent that does not interfere with its intended use, it is substantially isolated. For example, if the helicase 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 lipids, polynucleotides, pore monomers, or other proteins), it is substantially isolated or substantially purified.

[0370] Dda helicase

[0371] Any Dda helicase can be modified according to the present invention. Preferred Dda helicases are discussed below and described in WO2015 / 055981, WO2015 / 166276 and WO2016 / 055777 (all incorporated by reference).

[0372] Dda helicases typically contain the following five domains: 1A (RecA-like motor) domain, 2A (RecA-like motor) domain, tower domain, pin domain, and hook domain (Xiaoping He et al., 2012, Structure; 20: 1189-1200). Protein modeling, X-ray diffraction measurements of proteins in a crystalline state (Rupp B (2009). Biomolecular Crystallography: Principles, Practice and Application to Structural Biology. New York: Garland Science.), nuclear magnetic resonance (NMR) spectroscopy of proteins in solution (Mark Rance; Cavanagh, John; Wayne J. Fairbrother; Arthur W. Hunt III; Skelton, N. Nicholas J. (2007). Protein NMR spectroscopy: principles and practice (2nd ed.). Boston: Academic Press.) or cryo-electron microscopy of proteins in a frozen hydrated state (van Heel M, Gowen B, Matadeen R, Orlova EV, Finn R, Pape T, Cohen D, Stark H, Schmidt R, Schatz M, Patwardhan A (2000). "Single-particle electron cryo-microscopy: towards atomic resolution.". Q Rev. 2006). Biophys. 33: 307–69) to identify these domains. The structural information of proteins determined by the above methods is publicly available from the Protein Database (PDB).

[0373] In addition to modifications or substitutions at one or more positions corresponding to amino acid positions 55, 114, 156, 177, 210, 221, 350 and 358 in Dda 1993, modifications or substitutions at one or more positions corresponding to amino acid positions 114, 177, 350 and 358 in Dda 1993, modifications or substitutions at one or more positions corresponding to amino acid positions 114, 177 and 350 in Dda 1993 and / or a modification or substitution at a position corresponding to position 40 in Dda 1993, the modified helicase of the present invention preferably comprises any of the following additional modifications, substitutions, combinations of modifications or combinations of substitutions. As described above, the present invention also provides a modified helicase having any modification, substitution, combination of modifications, or combination of substitutions listed individually below (i.e., not necessarily having a substitution at any of positions 40, 55, 114, 156, 177, 210, 221, 350, and 358 of Dda 1993 or any of positions 40, 114, 177, 350, and 358 of Dda 1993).

[0374] Modifications of the Invention

[0375] The helicase of the present invention can be a helicase in which at least one amino acid that interacts with the transmembrane pore is substituted. Any number of amino acids can be substituted, such as 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more amino acids. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids can be substituted. Amino acids that interact with the transmembrane pore can be identified using protein modeling as described above.

[0376] Base and / or sugar interactions

[0377] The helicase of the present invention is preferably a helicase in which at least one amino acid that interacts with the sugar and / or base of one or more nucleotides in single-stranded DNA (ssDNA) is replaced by an amino acid that contains a larger side chain (R group). Any number of amino acids can be substituted, such as 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more amino acids. Each amino acid can interact with a base, a sugar, or a base and a sugar. Amino acids that interact with the sugar and / or base of one or more nucleotides in single-stranded DNA can be identified using protein modeling as described above.

[0378] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118, wherein at least one amino acid that interacts with the sugar and / or base of one or more nucleotides in ssDNA is at least one of H82, N88, P89, F98, D121, V150, P152, F240, F276, S287, H396, and Y415. These numbers correspond to the relevant positions in SEQ ID NO: 118 and may need to be changed in the case of variants in which one or more amino acids have been inserted or deleted compared to SEQ ID NO: 118. A skilled person can determine the corresponding positions in the variants as described above. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118, wherein at least one amino acid that interacts with the sugar and / or base of one or more nucleotides in ssDNA is F98, and one or more of H82, N88, P89, D121, V150, P152, F240, F276, S287, H396 and Y415, such as F98 / H82, F98 / N88, F98 / P89, F98 / D121, F98 / V150, F98 / P152, F98 / F240, F98 / F276, F98 / S287 or F98 / H396.

[0379] The helicase of the present invention is preferably a variant of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133, wherein at least one amino acid that interacts with the sugar and / or base of one or more nucleotides in ssDNA is at least one of the amino acids corresponding to H82, N88, P89, F98, D121, V150, P152, F240, F276, S287, H396 and Y415 in SEQ ID NO: 118. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133, wherein at least one amino acid that interacts with the sugar and / or base of one or more nucleotides in ssDNA is the amino acid corresponding to F98 in SEQ ID NO: 118 and the amino acid corresponding to F99 in SEQ ID NO: 129. One or more of the amino acids H82, N88, P89, D121, V150, P152, F240, F276, S287, H396, and Y415 of NO: 118, such as amino acids corresponding to F98 / H82, F98 / N88, F98 / P89, F98 / D121, F98 / V150, F98 / P152, F98 / F240, F98 / F276, F98 / S287, or F98 / H396.

[0380] Table 6 shows the amino acids in SEQ ID NOs: 119 to 133 that correspond to H82, N88, P89, F98, D121, V150, P152, F240, F276, S287, H396, and Y415 in SEQ ID NO: 118.

[0381]

[0382]

[0383] The at least one amino acid that interacts with the sugar and / or base of one or more nucleotides in the ssDNA is preferably at least one amino acid inserted between nucleotides in the ssDNA. The amino acid inserted between nucleotides in the ssDNA can be modeled as described above. The at least one amino acid inserted between nucleotides in the ssDNA is preferably at least one of P89, F98, and V150 in SEQ ID NO: 118, such as P89, F98, V150, P89 / F98, P89 / V150, F98 / V150, or P89 / F98 / V150.

[0384] At least one amino acid inserted between the nucleotides in the ssDNA in SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, or 133 is preferably at least one of the amino acids corresponding to P89, F98, and V150 in SEQ ID NO: 118, such as P89, F98, V150, P89 / F98, P89 / V150, F98 / V150, or P89 / F98 / V150. The corresponding amino acids are shown in Table 6 above.

[0385] Larger R groups

[0386] Larger side chains (R groups) preferably (a) contain an increased number of carbon atoms, (b) have an increased length, (c) have an increased molecular volume and / or (d) have an increased van der Waals volume. Larger side chains (R groups) preferably are (a); (b); (c); (d); (a) and (b); (a) and (c); (a) and (d); (b) and (c); (b) and (d); (c) and (d); (a), (b) and (c); (a), (b) and (d); (a), (c) and (d); (b), (c) and (d); or (a), (b), (c) and (d). Each of (a) to (d) can be measured using standard methods in the art.

[0387] The larger side chain (R group) preferably increases (i) electrostatic interactions, (ii) hydrogen bonding, and / or (iii) cation-π (cation-π) interactions between at least one amino acid and one or more nucleotides in the ssDNA, such as increasing (i); (ii); (iii); (i) and (ii); (i) and (iii); (ii) and (iii); and (i), (ii), and (iii). A skilled artisan can determine whether an R group increases any of these interactions. For example, in (i), positively charged amino acids such as arginine (R), histidine (H), and lysine (K) have R groups that increase electrostatic interactions. For example, in (ii), amino acids such as asparagine (N), serine (S), glutamine (Q), threonine (T), and histidine (H) have R groups that increase hydrogen bonding. For example, in (iii), aromatic amino acids such as phenylalanine (F), tryptophan (W), tyrosine (Y), or histidine (H) have R groups that increase cation-π (cation-π) interactions. The specific substitutions below are labeled (i) to (iii) to reflect these changes. Other possible substitutions are labeled (iv). These (iv) substitutions typically increase the length of the side chain (R group).

[0388] The amino acid comprising a larger side chain (R) may be an unnatural amino acid. The unnatural amino acid may be any of those discussed below.

[0389] The amino acid comprising a larger side chain (R group) is preferably not alanine (A), cysteine ​​(C), glycine (G), selenocysteine ​​(U), methionine (M), aspartic acid (D) or glutamic acid (E).

[0390] Histidine (H) is preferably substituted with (i) arginine (R) or lysine (K), (ii) glutamine (Q) or asparagine (N), or (iii) phenylalanine (F), tyrosine (Y) or tryptophan (W). Histidine (H) is more preferably substituted with (a) N, Q or W, or (b) Y, F, Q or K.

[0391] Asparagine (N) is preferably substituted by (i) arginine (R) or lysine (K), (ii) glutamine (Q) or histidine (H), or (iii) phenylalanine (F), tyrosine (Y) or tryptophan (W). Asparagine (N) is more preferably substituted by R, H, W or Y.

[0392] Proline (P) is preferably substituted by (i) arginine (R) or lysine (K), (ii) glutamine (Q), asparagine (N), threonine (T) or histidine (H), (iii) tyrosine (Y), phenylalanine (F) or tryptophan (W), or (iv) leucine (L), valine (V) or isoleucine (I). Proline (P) is more preferably substituted by (i) arginine (R) or lysine (K), (ii) glutamine (Q), asparagine (N), threonine (T) or histidine (H), (iii) phenylalanine (F) or tryptophan (W), or (iv) leucine (L), valine (V) or isoleucine (I). Proline (P) is more preferably substituted by (a) F, (b) L, V, I, T or F, or (c) W, F, Y, H, I, L or V.

[0393] Valine (V) is preferably substituted by (i) arginine (R) or lysine (K), (ii) glutamine (Q), asparagine (N) or histidine (H), (iii) phenylalanine (F), tyrosine (Y) or tryptophan (W), or (iv) isoleucine (I) or leucine (L). Valine (V) is more preferably substituted by (i) arginine (R) or lysine (K), (ii) glutamine (Q), asparagine (N) or histidine (H), (iii) tyrosine (Y) or tryptophan (W), or (iv) isoleucine (I) or leucine (L). Valine (V) is more preferably substituted by I or H or I, L, N, W or H.

[0394] Phenylalanine (F) is preferably substituted by (i) arginine (R) or lysine (K), (ii) histidine (H) or (iii) tyrosine (Y) or tryptophan (W). Phenylalanine (F) is more preferably substituted by (a) W, (b) W, Y or H, (c) W, R or K or (d) K, H, W or R.

[0395] Glutamine (Q) is preferably substituted by (i) arginine (R) or lysine (K) or (iii) phenylalanine (F), tyrosine (Y) or tryptophan (W).

[0396] Alanine (A) is preferably substituted by (i) arginine (R) or lysine (K), (ii) glutamine (Q), asparagine (N) or histidine (H), (iii) phenylalanine (F), tyrosine (Y) or tryptophan (W), or (iv) isoleucine (I) or leucine (L).

[0397] Serine (S) is preferably substituted by (i) arginine (R) or lysine (K), (ii) glutamine (Q), asparagine (N) or histidine (H), (iii) phenylalanine (F), tyrosine (Y) or tryptophan (W), or (iv) isoleucine (I) or leucine (L). Serine (S) is preferably substituted by K, R, W or F

[0398] Lysine (K) is preferably substituted by (i) arginine (R) or (iii) tyrosine (Y) or tryptophan (W).

[0399] Arginine (R) is preferably substituted with (iii) tyrosine (Y) or tryptophan (W).

[0400] Methionine (M) is preferably substituted by (i) arginine (R) or lysine (K), (ii) glutamine (Q), asparagine (N) or histidine (H), or (iii) phenylalanine (F), tyrosine (Y) or tryptophan (W).

[0401] Leucine (L) is preferably substituted by (i) arginine (R) or lysine (K), (ii) glutamine (Q) or asparagine (N), or (iii) phenylalanine (F), tyrosine (Y) or tryptophan (W).

[0402] Aspartic acid (D) is preferably substituted by (i) arginine (R) or lysine (K), (ii) glutamine (Q), asparagine (N) or histidine (H), or (iii) phenylalanine (F), tyrosine (Y) or tryptophan (W). Aspartic acid (D) is more preferably substituted by H, Y or K.

[0403] Glutamic acid (E) is preferably substituted by (i) arginine (R) or lysine (K), (ii) glutamine (Q), asparagine (N) or histidine (H), or (iii) phenylalanine (F), tyrosine (Y) or tryptophan (W).

[0404] Isoleucine (I) is preferably substituted by (i) arginine (R) or lysine (K), (ii) glutamine (Q), asparagine (N) or histidine (H), (iii) phenylalanine (F), tyrosine (Y) or tryptophan (W), or (iv) leucine (L).

[0405] Tyrosine (Y) is preferably substituted with (i) arginine (R) or lysine (K) or (iii) tryptophan (W). Tyrosine (Y) is more preferably substituted with W or R.

[0406] The helicase more preferably comprises a variant of SEQ ID NO: 118 and comprises (a) P89F, (b) F98W, (c) V150I, (d) V150H, (e) P89F and F98W, (f) P89F and V150I, (g) P89F and V150H, (h) F98W and V150I, (i) F98W and V150H, (j) P89F, F98W and V150I or (k) P89F, F98W and V150H.

[0407] More preferably, the helicase comprises a variant of SEQ ID NO: 118 comprising: H82N; H82Q; H82W; N88R; N88H; N88W; N88Y; P89L; P89V; P89I; P89E; P89T; P89F; D121H; D121Y; D121K; V150I; V150L; V150N; V150W; V150H; P152W; P152F; P152Y; P152H; P152I; P152L; P152V; F240W; F240Y; F240Y; 40H; F276W; F276R; F276K; F276H; S287K; S287R; S287W; S287F; H396Y; H396F; H396Q; H396K; Y415W; Y415R; F98 W / H82N; F98W / H82Q; F98W / H82W; F98W / N88R; F98W / N88H; F98W / N88W; F98W / N88Y; F98W / P89L; F98W / P89V; F98W / P89I; F98W / P89T; F98W / P89F; F98W / D121H; F98W / D121Y; F98W / D121K; F98W / V150I; F98W / V150L; F98W / V150N; F98W / V150W; F98W / V150H; F98W / P152W; F98W / P152F; F98W / P152Y; F98W / P152H; F98W / P152I; F98W / P152L; F98W / P152V;F98W / F240W;F98W / F240Y;F98W / F240H;F98W / F276W;F98W / F276R;F98W / F276K;F98W / F276H;F98W / S287K;F98W / S287R;F98W / S287W;F98W / S287F;F98W / H396Y;F98W / H396F;F98W / H396Q;F98W / Y415W;or F98W / Y415R.

[0408] Phosphate interactions

[0409] The helicase of the present invention is preferably a helicase in which at least one amino acid that interacts with one or more phosphate groups in one or more nucleotides in the ssDNA is substituted. Any number of amino acids can be substituted, such as 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more amino acids. Each nucleotide in the ssDNA contains three phosphate groups. Each substituted amino group can interact with any number of phosphate groups at a time, such as one, two, or three phosphate groups at a time. Amino acids that interact with one or more phosphate groups can be identified using protein modeling as described above.

[0410] The substitution preferably increases (i) electrostatic interactions, (ii) hydrogen bonding, and / or (iii) cation-π (cation-π) interactions between at least one amino acid and one or more phosphate groups in the ssDNA. Preferred substitutions that increase (i), (ii), and (iii) are discussed below using the labels (i), (ii), and (iii).

[0411] The substitution preferably increases the net positive charge at that position. The net charge at any position can be measured using methods known in the art. For example, the isoelectric point can be used to define the net charge of an amino acid. The measured net charge is typically about 7.5. The substitution preferably replaces a negatively charged amino acid with a positively charged, uncharged, non-polar, or aromatic amino acid. A negatively charged amino acid is an amino acid with a net negative charge. Negatively charged amino acids include, but are not limited to, aspartic acid (D) and glutamic acid (E). A positively charged amino acid is an amino acid with a net positive charge. Positively charged amino acids can be naturally occurring or non-naturally occurring. Positively charged amino acids can be synthetic or modified. For example, modified amino acids with a net positive charge can be specifically designed for use in the present invention. Many different types of amino acid modifications are well known in the art. Preferred naturally occurring positively charged amino acids include, but are not limited to, histidine (H), lysine (K), and arginine (R).

[0412] Uncharged amino acids, non-polar amino acids, or aromatic amino acids can be naturally occurring or non-naturally occurring. They can be synthetic or modified. Uncharged amino acids have no net charge. Suitable uncharged amino acids include, but are not limited to, cysteine ​​(C), serine (S), threonine (T), methionine (M), asparagine (N), and glutamine (Q). Non-polar amino acids have non-polar side chains. Suitable non-polar amino acids include, but are not limited to, glycine (G), alanine (A), proline (P), isoleucine (I), leucine (L), and valine (V). Aromatic amino acids have aromatic side chains. Suitable aromatic amino acids include, but are not limited to, histidine (H), phenylalanine (F), tryptophan (W), and tyrosine (Y).

[0413] The helicase preferably comprises a variant of SEQ ID NO: 118, wherein the at least one amino acid that interacts with one or more phosphate groups in one or more nucleotides in the ssDNA is at least one of H64, T80, S83, N242, K243, N293, T394, and K397. These numbers correspond to the relevant positions in SEQ ID NO: 89 and may need to be changed in the case of a variant in which one or more amino acids have been inserted or deleted compared to SEQ ID NO: 118. A skilled person can determine the corresponding positions in the variant as described above.

[0414] The helicase preferably comprises a variant of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133, and wherein the at least one amino acid that interacts with one or more phosphate groups in one or more nucleotides in the ssDNA is at least one of the amino acids corresponding to H64, T80, S83, N242, K243, N293, T394 and K397 in SEQ ID NO: 118.

[0415] Table 7 shows the amino acids in SEQ ID NOs: 119 to 133, which correspond to H64, T80, S83, N242, K243, N293, T 394, and K397 in SEQ ID NO: 118.

[0416]

[0417]

[0418] Histidine (H) is preferably substituted by (i) arginine (R) or lysine (K), (ii) asparagine (N), serine (S), glutamine (Q) or threonine (T), (iii) phenylalanine (F), tryptophan (W) or tyrosine (Y). Histidine (H) is preferably substituted by (a) N, Q, K or F or (b) N, Q or W.

[0419] Threonine (T) is preferably substituted by (i) arginine (R), histidine (H) or lysine (K), (ii) asparagine (N), serine (S), glutamine (Q) or histidine (H), or (iii) phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H). Threonine (T) is more preferably substituted by (a) K, Q or N, or (b) K, H or N.

[0420] Serine (S) is preferably substituted by (i) arginine (R), histidine (H) or lysine (K), (ii) asparagine (N), glutamine (Q), threonine (T) or histidine (H) or (iii) phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H). Serine (S) is more preferably substituted by H, N, K, T, R or Q.

[0421] Asparagine (N) is preferably substituted with (i) arginine (R), histidine (H) or lysine (K), (ii) serine (S), glutamine (Q), threonine (T) or histidine (H), or (iii) phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H). Asparagine (N) is more preferably substituted with (a) H or Q or (b) Q, K or H.

[0422] Lysine (K) is preferably substituted with (i) arginine (R) or histidine (H), (ii) asparagine (N), serine (S), glutamine (Q), threonine (T) or histidine (H), or (iii) phenylalanine (F), tryptophan (W), tyrosine (Y) or histidine (H). Lysine (K) is more preferably substituted with (a) Q or H or (b) R, H or Y.

[0423] The helicase more preferably comprises a variant of SEQ ID NO: 118 and comprises one or more, such as all, of the following: (a) H64N, H64Q, H64K or H64F, (b) T80K, T80Q or T80N, (c) S83H, S83N, S83K, S83T, S83R or S83Q, (d) N242H or N242Q, (e) K243Q or K243H, (f) N293Q, N293K or N293H, (g) T394K, T394H or T394N or (h) K397R, K397H or K397Y.

[0424] combination

[0425] The helicase is preferably a variant of SEQ ID NO: 118 comprising a substitution at:

[0426] -F98 / H64, such as F98W / H64N, F98W / H64Q, F98W / H64K or F98W / H64F;

[0427] -F98 / T80, such as F98W / T80K, F98W / T80Q, F98W / T80N;

[0428] -F98 / H82, such as F98W / H82N, F98W / H82Q or F98W / H82W;

[0429] -F98 / S83, such as F98W / S83H, F98W / S83N, F98W / S83K, F98W / S83T,

[0430] F98W / S83R or F98W / S83Q;

[0431] F98 / N242, such as F98W / N242H, F98W / N242Q, F98W / K243Q or F98W / K243H;

[0432] -F98 / N293, such as F98W / N293Q, F98W / N293K, F98W / N293H, F98W / T394K,

[0433] F98W / T394H, F98W / T394N, F98W / H396Y, F98W / H396F, F98W / H396Q, or F98W / H396K; or

[0434] -F98 / K397, such as F98W / K397R, F98W / K397H or F98W / K397Y.

[0435] Preferred combinations of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133 include amino acid combinations corresponding to the combinations in SEQ ID NO: 118 listed above.

[0436] Pore ​​interactions

[0437] The helicase of the present invention is another helicase in which the helicase portion interacting with the transmembrane pore comprises one or more modifications, preferably one or more substitutions. The portion of the helicase interacting with the transmembrane pore is typically a portion of the helicase that interacts with the transmembrane pore when the helicase is used to control the movement of the polynucleotide by the hole, such as as discussed in more detail below. The portion typically comprises an amino acid that interacts with the hole or contacts the hole when the helicase is used to control the movement of the polynucleotide by the hole, such as as discussed in more detail below. The portion typically comprises an amino acid that interacts with the hole or contacts the hole when the helicase is combined with or attached to an analyte (such as a polynucleotide) that moves through the hole under an applied potential.

[0438] In SEQ ID NO: 118, the portion that interacts with the transmembrane pore typically comprises positions 1, 2, 3, 4, 5, 6, 51, 176, 177, 178, 179, 180, 181, 185, 189, 191, 193, 194, 195, 197, 198, 199, 200, 201, 202, 203, 204, 207, 208, 209, 210, 211, 212, 213, 118. The amino acids at positions 216, 219, 220, 221, 223, 224, 226, 227, 228, 229, 247, 254, 255, 256, 257, 258, 259, 260, 261, 298, 300, 304, 308, 318, 319, 321, 337, 347, 350, 351, 405, 415, 422, 434, 437, 438 are preferably present in the sequence of SEQ ID NO: 118. These numbers correspond to the relevant positions in SEQ ID NO: 118 and may need to be changed in the case of variants in which one or more amino acids have been inserted or deleted compared to SEQ ID NO: 118. The skilled person can determine the corresponding positions in the variants as described above. The portion that interacts with the transmembrane pore preferably comprises amino acids at the following positions:

[0439] (a) positions 1, 2, 4, 51, 177, 178, 179, 180, 185, 193, 195, 197, 198, 199, 200, 202, 203, 204, 207, 208, 209, 210, 211, 212, 216, 221, 223, 224, 226, 227, 228, 229, 254, 255, 256, 257, 258, 260, 304, 318, 321, 347, 350, 351, 405, 415, 422, 434, 437, and 438 of SEQ ID NO: 118; or

[0440] (b) Positions 1, 2, 178, 179, 180, 185, 195, 197, 198, 199, 200, 202, 203, 207, 209, 210, 212, 216, 221, 223, 226, 227, 255, 258, 260, 304, 350 and 438 of SEQ ID NO: 118.

[0441] The portion that interacts with the transmembrane pore preferably comprises one or more, such as 2, 3, 4 or 5, of the amino acids at positions K194, W195, K198, K199 and E258 in SEQ ID NO: 118. Variants of SEQ ID NO: 118 preferably comprise modifications at one or more of (a) K194, (b) W195, (c) D198, (d) K199 and (d) E258. Variants of SEQ ID NO: 118 preferably comprise substitutions at one or more of: (a) K194, such as K194L, (b) W195, such as W195A, (c) D198, such as D198V, (d) K199, such as K199L and (e) E258, such as E258L. The variant can contain {a}; {b}; {c}; {d}; {e}; {a, b}; {a, c}; {a, d}; {a, e}; {b, c}; {b, d}; {b, e}; , d}; {a, c, e}; {a, d, e}; {b, c, d}; {b, c, e}; {b, d, e}; {c, d, e}; {a, b, c, d}; {a, b, c, e}; {a, b, d, e}; The modifications or substitutions listed in this paragraph are preferred when the modified polynucleotide binding protein interacts with a pore derived from MspA, particularly any of the modified pores discussed below.

[0442] The portion of the polynucleotide binding protein that interacts with the transmembrane pore preferably comprises the amino acid at position 194 or 199 of SEQ ID NO: 118. The variant preferably comprises K194A, K194V, K194F, K194D, K194S, K194W or K194L and / or K199A, K199V, K199F, K199D, K199S, K199W or K199L. In SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133, the portion that interacts with the transmembrane pore typically comprises amino acids at positions corresponding to those in SEQ ID NO: 118 above. The alignment in Table 8 below can be used to identify amino acids in SEQ ID NOs: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, and 133 that correspond to these positions in SEQ ID NO: 118.

[0443]

[0444]

[0445] Preferred combination

[0446] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118 comprising a substitution at F98, such as F98R, F98K, F98Q, F98N, F98H, F98Y, F98F or F98W, and a substitution at K194, such as K194A, K194V, K194F, K194D, K194S, K194W or K194L and / or K199, such as K199A, K199V, K199F, K199D, K199S, K199W or K199L. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133 comprising a substitution at a position corresponding to F98 in SEQ ID NO: 118 and a substitution at a position corresponding to K194 and / or K199 in SEQ ID NO: 118. These corresponding positions can be substituted with any of the amino acids listed above for F98, K194 and K119 in SEQ ID NO: 118.

[0447] The helicase is preferably a variant of SEQ ID NO: 118 comprising a substitution at:

[0448] F98 / K194 / H64, such as F98W / K194L / H64N, F98W / K194L / H64Q, F98W / K194L / H64K or F98W / K194L / H64F;

[0449] -F98 / K194 / T80, such as F98W / K194L / T80K, F98W / K194L / T80Q or F98W / K194L / T80N;

[0450] -F98 / K194 / H82, such as F98W / K194L / H82N, F98W / K194L / H82Q, or F98W / K194L / H82W

[0451] F98 / S83 / K194, such as F98W / S83H / K194L, F98W / S83T / K194L, F98W / S83R / K194L, F98W / S83Q / K194L, F98W / S83N / K194L, F98W / S83K / K194L, F98W / N88R / K194L, F98W / N88H / K194L, F98W / N88W / K194L or F98W / N88Y / K194L;

[0452] -F98 / S83 / K194 / F276, such as F98W / S83H / K194L / F276K;

[0453] F98 / P89 / K194, such as F98W / P89L / K194L, F98W / P89V / K194L, F98W / P89I / K194L or F98W / P89T / K194L;

[0454] F98 / D121 / K194, such as F98W / D121H / K194L, F98W / D121Y / K194L or F98W / D121K / K194L;

[0455] F98 / V150 / K194, such as F98W / V150I / K194L, F98W / V150L / K194L, F98W / V150N / K194L, F98W / V150W / K194L or F98W / V150H / K194L;

[0456] F98 / P152 / K194, such as F98W / P152W / K194L, F98W / P152F / K194L, F98W / P152Y / K194L, F98W / P152H / K194L, F98W / P152I / K194L, F98W / P152L / K194L or F98W / P152V / K194L;

[0457] F98 / F240 / K194, such as F98W / F240W / K194L, F98W / F240Y / K194L or F98W / F240H / K194L;

[0458] F98 / N242 / K194, such as F98W / N242H / K194L or F98W / N242Q / K194L;

[0459] F98 / K194 / F276, such as F98W / K194L / F276K, F98W / K194L / F276H, F98W / K194L / F276W or F98W / K194L / F276R;

[0460] F98 / K194 / S287, such as F98W / K194L / S287K, F98W / K194L / S287R, F98W / K194L / S287W or F98W / K194L / S287F;

[0461] F98 / N293 / K194, such as F98W / N293Q / K194L, F98W / N293K / K194L or F98W / N293H / K194L;

[0462] F98 / T394 / K194, such as F98W / T394K / K194L, F98W / T394H / K194L or F98W / T394N / K194L;

[0463] F98 / H396 / K194, such as F98W / H396Y / K194L, F98W / H396F / K194L, F98W / H396Q / K194L or F98W / H396K / K194L;

[0464] F98 / K397 / K194, such as F98W / K397R / K194L, F98W / K397H / K194L or F98W / K397Y / K194L; or

[0465] -F98 / Y415 / K194, such as F98W / Y415W / K194L or F98W / Y415R / K194L.

[0466] In any of the above combinations, K194 may be replaced with any one of W195, D198, K199 and E258.

[0467] The modified helicase preferably comprises a modification or substitution at positions corresponding to amino acid positions 98 and / or 194 in Dda 1993. Preferably, in addition to modifications or substitutions at one or more positions corresponding to amino acid positions 55, 114, 156, 177, 210, 221, 350, and 358 in Dda 1993, modifications or substitutions at one or more positions corresponding to amino acid positions 114, 177, 350, and 358 in Dda 1993, and / or modifications or substitutions at a position corresponding to position 40 in Dda 1993. Position 98 or a corresponding position may be substituted with R, H, K, S, T, N, Q, A, V, I, L, M, Y, or W. Position 98 or a corresponding position is preferably substituted with R, K, Q, N, H, Y, or W. Position 194 or a corresponding position may be substituted with A, V, I, L, M, F, Y, W, D, E, S, T, N, or Q. Position 194 or the corresponding position is preferably substituted with A, V, F, D, S, W or L. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118 comprising a substitution at F98, such as F98R, F98K, F98Q, F98N, F98H, F98Y or F98W, and / or a substitution at K194, such as K194A, K194V, K194F, K194D, K194S, K194W or K194L. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118 comprising F98W and K194L. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133, which comprises a substitution at a position corresponding to F98 in SEQ ID NO: 118 and / or a substitution at a position corresponding to K194 in SEQ ID NO: 118.

[0468] In any of the above combinations, K194 may be replaced with any one of W195, D198, K199 and E258.

[0469] Modifications in the tower domain and / or pin domain and / or 1A domain

[0470] The modified helicase preferably comprises a modification or substitution at the position corresponding to amino acid position 360 in Dda 1993. This can include modifications or substitutions at one or more positions corresponding to amino acid positions 55, 114, 156, 177, 210, 221, 350, and 358 in Dda 1993, as well as modifications or substitutions at one or more positions corresponding to amino acid positions 114, 177, 350, and 358 in Dda 1993 and / or at a position corresponding to position 40 in Dda 1993. A360, like Y350 and K358, is located in the tower domain of Dda 1993. Position 360, or a corresponding position, can be substituted with C, G, P, A, V, I, L, M, F, Y, or W. Position 360, or a corresponding position, is preferably substituted with C or Y. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118 comprising a substitution at A360, such as A360C or A360Y. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118 comprising K358I and A360C. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133 comprising a substitution at a position corresponding to A360 in SEQ ID NO: 118.

[0471] The modified helicase preferably comprises a modification or substitution at one or more positions corresponding to amino acid positions 94, 98, and 109 in Dda 1993 (such as positions 94, 98, 109, 94 and 98, 94 and 109, 98 and 109, and 94, 98, and 109). This can be in addition to modifications or substitutions at one or more positions corresponding to amino acid positions 55, 114, 156, 177, 210, 221, 350, and 358 in Dda 1993, as well as modifications or substitutions at one or more positions corresponding to amino acid positions 114, 177, 350, and 358 in Dda 1993 and / or at a position corresponding to position 40 in Dda 1993. These positions are all within the pin domain. Position 94 or a corresponding position can be substituted with C, G, P, A, V, I, L, M, F, Y, or W. Position 94 or a corresponding position is preferably substituted with C or Y. Position 98 or the corresponding position can be substituted with R, H, K, S, T, N, Q, A, V, I, L, MY or W. Position 98 or the corresponding position is preferably substituted with R, K, Q, N, H, Y or W. Position 109 or the corresponding position can be substituted with A, V, I, L, M, F, Y or W. Position 109 or the corresponding position is preferably substituted with A or V. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118 comprising a substitution at one or more of E94, F98 and C109 (including all combinations listed above). Preferred variants comprise substitutions at the following positions:

[0472] -E94 and F98, such as E94C or E94Y and F98R, F98K, F98Q, F98N, F98H, F98Y,

[0473] F98F or F98W;

[0474] - E94 and C109, such as E94C or E94Y and C109A or C109V;

[0475] - F98 and C109, such as F98R, F98K, F98Q, F98N, F98H, F98Y, F98F or F98W and C109A or C109V; or

[0476] -E94, F98 and C109, such as E94C or E94Y and F98R, F98K, F98Q, F98N, F98H,

[0477] F98Y, F98F, or F98W and C109A or C109V.

[0478] More preferred variants include: E94C and F98W; E94C and C109A; F98W and C109A; or E94C, F98W and C109A. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133, which comprises a substitution at one or more positions corresponding to E94, F98 and C109 in SEQ ID NO: 118.

[0479] Table 9 includes information for E94, C109, C136, and A360 (reference is made to the modified helicases disclosed above and below).

[0480]

[0481]

[0482] The helicase of the present invention is preferably wherein at least one cysteine ​​residue (i.e. one or more cysteine ​​residues) and / or at least one alpha-non-natural amino acid (i.e. one or more alpha-non-natural amino acids) has been introduced into the helicase in (i) tower domain and / or (ii) pin domain and / or (iii) 1A (RecA sample motor) domain, wherein the helicase has the ability to control the movement of polynucleotides. These types of modifications are disclosed in WO 2015 / 055981 (incorporated herein by reference in its entirety). At least one cysteine ​​residue and / or at least one alpha-non-natural amino acid can be introduced into tower domain, pin domain, 1A domain, tower domain and pin domain, tower domain and 1A domain or tower domain, pin domain and 1A domain.

[0483] The helicase of the present invention is preferably a helicase in which at least one cysteine ​​residue and / or at least one unnatural amino acid has been introduced into each of (i) the tower domain and (ii) the pin domain and / or the 1A (RecA-like motor) domain (i.e., into the tower domain and the pin domain, the tower domain and the 1A domain, or the tower domain, the pin domain and the 1A domain).

[0484] Any number of cysteine ​​residues and / or non-natural amino acids can be introduced into each domain. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cysteine ​​residues and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-natural amino acids can be introduced. Only one or more cysteine ​​residues can be introduced. Only one or more non-natural amino acids can be introduced. A combination of one or more cysteine ​​residues and one or more non-natural amino acids can be introduced.

[0485] Preferably at least one cysteine ​​residue and / or at least one unnatural amino acid is introduced by substitution. Methods for doing so are known in the art.

[0486] These modifications do not prevent the helicase from binding to the polynucleotide. These modifications reduce the ability of the polynucleotide to debind or dissociate from the helicase. In other words, one or more modifications increase the processivity of the helicase by preventing it from dissociating from the polynucleotide chain. The thermostability of the enzyme is also generally increased by one or more modifications, thereby giving it improved structural stability, which is beneficial in chain sequencing.

[0487] Non-natural amino acids are amino groups that are not naturally present in helicases. Non-natural amino acids are preferably not histidine, alanine, isoleucine, arginine, leucine, asparagine, lysine, aspartic acid, methionine, cysteine, phenylalanine, glutamic acid, threonine, glutamine, tryptophan, glycine, valine, proline, serine, or tyrosine. Non-natural amino acids are more preferably not any of the twenty amino acids in the preceding sentence or selenocysteine.

[0488] Preferred unnatural amino acids for use in the present invention include, but are not limited to, 4-azido-L-phenylalanine (Faz), 4-acetyl-L-phenylalanine, 3-acetyl-L-phenylalanine, 4-acetoacetyl-L-phenylalanine, O-allyl-L-tyrosine, 3-(phenylseleno)-L-alanine, O-2-propyn-1-yl-L-tyrosine, 4-(dihydroxyboryl)-L-phenylalanine, 4-[(ethylthio)carbonyl]-L-phenylalanine, (2S)-2-amino-3-4-[(prop-2-ylthio)carbonyl]phenyl; propionic acid, (2S)-2-amino-3-4-[(2-amino-3-thiopropionyl)amino]phenyl; propionic acid, O-methyl-L-tyrosine, 4-amino 4-Chloro-L-phenylalanine, 4-cyano-L-phenylalanine, 3-cyano-L-phenylalanine, 4-fluoro-L-phenylalanine, 4-iodo-L-phenylalanine, 4-bromo-L-phenylalanine, O-(trifluoromethyl)tyrosine, 4-nitro-L-phenylalanine, 3-hydroxy-L-tyrosine, 3-amino-L-tyrosine, 3-iodo-L-tyrosine, 4-isopropyl-L-phenylalanine, 3-(2-naphthyl)-L-alanine, 4-phenyl-L-phenylalanine, (2S)-2-amino-3-(naphthyl-2-ylamino)propionic acid, 6-(methylthio)norleucine, 6-oxo-L-lysine, D-tyrosine, (2R)-2-hydroxy-3-(4-hydroxyphenyl)propionic acid, (2R)-2- ammonium octanoate, 3-(2,2'-bipyridin-5-yl)-D-alanine, 2-amino-3-(8-hydroxy-3-quinolinyl)propionic acid, 4-benzoyl-L-phenylalanine, S-(2-nitrobenzyl)cysteine, (2R)-2-amino-3-[(2-nitrobenzyl)thio]propionic acid, (2S)-2-amino-3-[(2-nitrobenzyl)oxy]propionic acid, O-(4,5-dimethoxy-2-nitrobenzyl)-L-serine, (2S)-2-amino-6-([(2-nitrobenzyl)oxy]carbonyl; amino)hexanoic acid, O-(2-nitrobenzyl)-L-tyrosine, 2-nitrophenylalanine, 4-[(E)-phenyldiazenyl]-L-phenylalanine, 4-[3- (Trifluoromethyl)-3H-diazinyl-3-yl]-D-phenylalanine, 2-amino-3-[[5-(dimethylamino)-1-naphthyl]sulfonylamino]propionic acid, (2S)-2-amino-4-(7-hydroxy-2-oxo-2H-benzo-4-yl)butanoic acid, (2S)-3-[(6-acetylnaphth-2-yl)amino]-2-aminopropionic acid, 4-(carboxymethyl)phenylalanine, 3-nitro-L-tyrosine, O-sulfo-L-tyrosine, (2R)-6-acetylamino-2-aminohexanoic acid, 1-methylhistidine, 2-aminononanoic acid, 2-aminodecanoic acid, L-homocysteine, 5-thionorvaline, 6-thio-L-norleucine, 5-(methylthio)-L-norvaline, N 6-[(2R,3R)-3-methyl-3,4-dihydro-2H-pyrrol-2-yl]carbonyl; -L-lysine, N 6 -[(Benzyloxy)carbonyl]lysine, (2S)-2-amino-6-[(cyclopentylcarbonyl)amino]hexanoic acid, N 6 -[(cyclopentyloxy)carbonyl]-L-lysine, (2S)-2-amino-6-[(2R)-tetrahydrofuran-2-ylcarbonyl]amino; hexanoic acid, (2S)-2-amino-8-[(2R,3S)-3-ethynyltetrahydrofuran-2-yl]-8-oxooctanoic acid, N 6 -(tert-Butoxycarbonyl)-L-lysine, (2S)-2-hydroxy-6-([(2-methyl-2-propyl)oxy]carbonyl; amino)hexanoic acid, N 6 -[(allyloxy)carbonyl]lysine, (2S)-2-amino-6-([(2-azidobenzyl)oxy]carbonyl; amino)hexanoic acid, N 6 -L-prolyl-L-lysine, (2S)-2-amino-6-[(prop-2-yn-1-yloxy)carbonyl]amino; hexanoic acid and N 6 -[(2-azidoethoxy)carbonyl]-L-lysine. The most preferred unnatural amino acid is 4-azido-L-phenylalanine (Faz).

[0489] Table 10 below (divided into two parts) identifies the residues that make up each domain in each of the Dda homologs (SEQ ID NOs: 118 to 133).

[0490] Homologs SEQ ID NO 1A 2A Dda-Rma-DSM 119 M1-I84+R113-Y211 FR212-E294+G422-S678 Dda-Csp 120 M1-L147+S166-V240 R241-N327+A449-G496 Dda-Sru 121 M1-L90+E108-H173 R174-D260+A371-V421 Dda-Sgo 122 M1-L115+N136-V205 R206-K293+I408-L500 Dda-Vph12B8 123 M1-L96+F114-V194 R195-D287+V394-Q450 Dda-Vph 124 M1-L77+V96-V166 R167-T249+L372-N421 Dda-Aph65 125 M1-M81+L99-M171 R172-T254+L381-K434 Dda-AphCC2 126 M1-M68+M86-M158 R159-T241+L367-K420 Dda-Cph 127 M1-L87+A108-M181 R182262+L393-V443 Dda-Kph 128 M1-L87+A108-M181 R182262+L392-V442 Dda-SphIME13 129 M1-L85+T103-K176 R177-N257+L387-V438 Dda-AphAc42 130 M1-L91+V109-M183 R184-T265+L393-I442 Dda-SphSP18 131 M1-L87+M105-M179 R180-T261+L393-V442 Dda-Yph 132 M1-L86+V104-K178 R179-T260+L390-I439 Dda-SphS16 133 M1-L86+V104-M178 R179-T260+L391-V441 Dda-1993 118 M1-L85+V103-K177 R178-T259+L390-V439

[0491] Homologs SEQ ID NO: tower pin hook Dda-Rma-DSM 119 G295-N309+F316-Y421 Y85-L112 A310-L315 Dda-Csp 120 V328-P342+N360-Y448 K148-N165 V343-L359 Dda-Sru 121 A261-T275+T285-Y370 G91-E107 W276-L284 Dda-Sgo 122 G294-I307+T314-Y407 G116-T135 R308-Y313 Dda-Vph12B8 123 V288-E301+N307-N393 G97-P113 M302-W306 Dda-Vph 124 S250-P264+E278-S371 K78-E95 V265-I277 Dda-Aph65 125 K255-P269+T284-S380 K82-K98 V270-F283 Dda-AphCC2 126 D242-P256+T271-S366 K69-K85 V257-F270 Dda-Cph 127 T263-P277+N295-P392 K88-K107 L278-Y294 Dda-Kph 128 D263-P277+N295-A391 K88-K107 L278-Y294 Dda-SphIME13 129 A258-P272+N290-P386 K86-G102 L273-F289 Dda-AphAc42 130 L266-P280+N298-A392 K92-D108 L281-F297 Dda-SphSP18 131 D262-P276+N294-A392 K88-E104 H277-F293 Dda-Yph 132 D261-P275+N293-A389 K87-E103 L276-F292 Dda-SphS16 133 E261-P275+T293-A390 K87-E103 L276-F292 Dda-1993 118 D260-P274+N292-A389 K86-E102 L275-F291

[0492] The helicases of the present invention preferably comprise variants of SEQ ID NO: 118, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been introduced into (i) the tower domain (residues D260-P274 and N292-A389) and / or (ii) the pin domain (residues K86-E102) and / or (iii) the 1A domain (residues M1-L85 and V103-K177). Preferably, the at least one cysteine ​​residue and / or at least one unnatural amino acid is introduced into residues N292-A389 of the tower domain.

[0493] The helicases of the present invention preferably comprise variants of SEQ ID NO: 119, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been introduced into (i) the tower domain (residues G295-N309 and F316-Y421) and / or (ii) the pin domain (residues Y85-L112) and / or (iii) the 1A domain (residues M1-I84 and R113-Y211). Preferably, at least one cysteine ​​residue and / or at least one unnatural amino acid is introduced into residues F316-Y421 of the tower domain.

[0494] The helicases of the present invention preferably comprise variants of SEQ ID NO: 120, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been introduced into (i) the tower domain (residues V328-P342 and N360-Y448) and / or (ii) the pin domain (residues K148-N165) and / or (iii) the 1A domain (residues M1-L147 and S166-V240). Preferably, at least one cysteine ​​residue and / or at least one unnatural amino acid is introduced into residues N360-Y448 of the tower domain.

[0495] The helicases of the present invention preferably comprise variants of SEQ ID NO: 121, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been introduced into (i) the tower domain (residues A261-T275 and T285-Y370) and / or (ii) the pin domain (residues G91-E107) and / or (iii) the 1A domain (residues M1-L90 and E108-H173). Preferably, the at least one cysteine ​​residue and / or at least one unnatural amino acid is introduced into residues T285-Y370 of the tower domain.

[0496] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 122, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been introduced into (i) the tower domain (residues G294-I307 and T314-Y407) and / or (ii) the pin domain (residues G116-T135) and / or (iii) the 1A domain (residues M1-L115 and N136-V205). Preferably, at least one cysteine ​​residue and / or at least one unnatural amino acid is introduced into residues T314-Y407 of the tower domain.

[0497] The helicases of the present invention preferably comprise variants of SEQ ID NO: 123, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been introduced into (i) the tower domain (residues V288-E301 and N307-N393) and / or (ii) the pin domain (residues G97-P113) and / or (iii) the 1A domain (residues M1-L96 and F114-V194). Preferably, at least one cysteine ​​residue and / or at least one unnatural amino acid is introduced into residues N307-N393 of the tower domain.

[0498] The helicases of the present invention preferably comprise variants of SEQ ID NO: 124, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been introduced into (i) the tower domain (residues S250-P264 and E278-S371) and / or (ii) the pin domain (residues K78-E95) and / or (iii) the 1A domain (residues M1-L77 and V96-V166). Preferably, at least one cysteine ​​residue and / or at least one unnatural amino acid is introduced into residues E278-S371 of the tower domain.

[0499] The helicases of the present invention preferably comprise variants of SEQ ID NO: 125, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been introduced into (i) the tower domain (residues K255-P269 and T284-S380) and / or (ii) the pin domain (residues K82-K98) and / or (iii) the 1A domain (residues M1-M81 and L99-M171). Preferably, at least one cysteine ​​residue and / or at least one unnatural amino acid is introduced into residues T284-S380 of the tower domain.

[0500] The helicases of the present invention preferably comprise variants of SEQ ID NO: 126, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been introduced into (i) the tower domain (residues D242-P256 and T271-S366) and / or (ii) the pin domain (residues K69-K85) and / or (iii) the 1A domain (residues M1-M68 and M86-M158). Preferably, at least one cysteine ​​residue and / or at least one unnatural amino acid is introduced into residues T271-S366 of the tower domain.

[0501] The helicases of the present invention preferably comprise variants of SEQ ID NO: 127, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been introduced into (i) the tower domain (residues T263-P277 and N295-P392) and / or (ii) the pin domain (residues K88-K107) and / or (iii) the 1A domain (residues M1-L87 and A108-M181). Preferably, the at least one cysteine ​​residue and / or at least one unnatural amino acid is introduced into residues N295-P392 of the tower domain.

[0502] The helicases of the present invention preferably comprise variants of SEQ ID NO: 128, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been introduced into (i) the tower domain (residues D263-P277 and N295-A391) and / or (ii) the pin domain (residues K88-K107) and / or (iii) the 1A domain (residues M1-L87 and A108-M181). Preferably, the at least one cysteine ​​residue and / or at least one unnatural amino acid is introduced into residues N295-A391 of the tower domain.

[0503] The helicases of the present invention preferably comprise variants of SEQ ID NO: 129, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been introduced into (i) the tower domain (residues A258-P272 and N290-P386) and / or (ii) the pin domain (residues K86-G102) and / or (iii) the 1A domain (residues M1-L85 and T103-K176). Preferably, at least one cysteine ​​residue and / or at least one unnatural amino acid is introduced into residues N290-P386 of the tower domain.

[0504] The helicases of the present invention preferably comprise variants of SEQ ID NO: 130, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been introduced into (i) the tower domain (residues L266-P280 and N298-A392) and / or (ii) the pin domain (residues K92-D108) and / or (iii) the 1A domain (residues M1-L91 and V109-M183). Preferably, the at least one cysteine ​​residue and / or at least one unnatural amino acid is introduced into residues N298-A392 of the tower domain.

[0505] The helicases of the present invention preferably comprise variants of SEQ ID NO: 131, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been introduced into (i) the tower domain (residues D262-P276 and N294-A392) and / or (ii) the pin domain (residues K88-E104) and / or (iii) the 1A domain (residues M1-L87 and M105-M179). Preferably, the at least one cysteine ​​residue and / or at least one unnatural amino acid is introduced into residues N294-A392 of the tower domain.

[0506] The helicases of the present invention preferably comprise variants of SEQ ID NO: 132, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been introduced into (i) the tower domain (residues D261-P275 and N293-A389) and / or (ii) the pin domain (residues K87-E103) and / or (iii) the 1A domain (residues M1-L86 and V104-K178). Preferably, the at least one cysteine ​​residue and / or at least one unnatural amino acid is introduced into residues N293-A389 of the tower domain.

[0507] The helicases of the present invention preferably comprise variants of SEQ ID NO: 133, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been introduced into (i) the tower domain (residues E261-P275 and T293-A390) and / or (ii) the pin domain (residues K87-E103) and / or (iii) the 1A domain (residues M1-L86 and V104-M178). Preferably, at least one cysteine ​​residue and / or at least one unnatural amino acid is introduced into residues T293-A390 of the tower domain.

[0508] The helicase of the present invention preferably comprises a variant of any one of SEQ ID NOs: 118 to 133, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been introduced into each of (i) the tower domain and (ii) the pin domain and / or the 1A domain. The helicase of the present invention more preferably comprises a variant of any one of SEQ ID NOs: 118 to 133, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been introduced into each of (i) the tower domain, (ii) the pin domain and (iii) the 1A domain. As described above, any number and combination of cysteine ​​residues and unnatural amino acids can be introduced.

[0509] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118 comprising (i) E94C and / or A360C; (ii) E93C and / or K358C; (iii) E93C and / or A360C; (iv) E93C and / or E361C; (v) E93C and / or K364C; (vi) E94C and / or L354C; (vii) E94C and / or K358C; (viii) E93C and / or L354C; (ix) E94C and / or E361C; (x) E94C and / or K364C; (xi) L97C and / or L354C; (xii) L97C and / or K358C; (xiii) L97C and / or A360C; (xiv) L97C and / or E361C; (xv) L97C and / or K364C; (xvi) K123C and / or L354C; (xvii) K123C and / or K358C; (xviii) K123C and / or A360C; (xix) K123C and / or E361C; (xx) K123C and / or K364C; (xxi) N155C and / or L354C; (xxii) N155C and / or K358C; (xxiii) N155C and / or A360C; (xxiv) N155C and / or E361C; (xxv) N155C and / or K364C; (xxvi) (i) to (xxv) any one and G357C; any one of (xxvii)(i) to (xxv) and Q100C; any one of (xxviii)(i) to (xxv) and I127C; any one of (xxix)(i) to (xxv) and Q100C and I127C; (xxx) E94C and / or F377C; (xxxi) N95C; (xxxii) T91C; (xxxiii) Y92L, E94Y, Y350N, A360C and Y363N; (xxxiv) E94Y and A360C; (xxxv) A360C; (xxxvi) Y92L, E94C, Y350N, A360Y and Y363N; (xxxvii) Y92L, E94C, and A360Y; (xxxviii) E94C and / or A360C and F276A; (xxxix) E94C and / or L356C; (xl) E93C and / or E356C; (xli) E93C and / or G357C; (xlii) E93C and / or A360C; (xliii) N95C and / or W378C; (xliv) T91C and / or S382C; (xlv) T91C and / or W378C; (xlvi) E93C and / or N353C; (xlvii) E93C and / or S382C; (xlviii) E93C and / or K381C;(xlix) E93C and / or D379C; (l) E93C and / or S375C; (li) E93C and / or W378C; (lii) E93C and / or W374C; (liii) E94C and / or N353C; (liv) E94C and / or S382C; (lv) E94C and / or K381C; (lvi) E94C and / or D379C; (lvii) E94C and / or S375C; (lviii) E94C and / or W378C; (lix) E94C and / or W374C; (lx) E94C and A360Y; (lxi) E94C, G357C and A360C or (lxii) T2C, E94C and A360C. In any one of (i) to (lxii), and / or is preferably and. ;

[0510] The helicase of the present invention preferably comprises a variant of any one of SEQ ID NO: 119 to 133 comprising a cysteine ​​residue at a position corresponding to those positions in SEQ ID NO: 118 as defined in any one of (i) to (lxii). The positions of any one of SEQ ID NO: 119 to 133 corresponding to those positions in SEQ ID NO: 118 can be identified using the comparison of SEQ ID NO: 118 to 133 below. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 92 comprising (a) D99C and / or L341C, (b) Q98C and / or L341C or (d) Q98C and / or A340C. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 96 comprising D90C and / or A349C. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 102 comprising D96C and / or A362C.

[0511] The helicase of the present invention preferably comprises a variant of any one of SEQ ID NOs: 118 to 133 as defined in any one of (i) to (lxii), wherein Faz is introduced instead of cysteine ​​at one or more specific positions. Faz can be introduced instead of cysteine ​​at each specific position. The helicase of the present invention preferably comprises a variant of SEQ ID NO:118, which comprises (i) E94Faz and / or A360C; (ii) E94C and / or A360Faz; (iii) E94Faz and / or A360Faz; (iv) Y92L, E94Y, Y350N, A360Faz and Y363N; (v) A360Faz; (vi) E94Y and A360Faz; (vii) Y92L, E94Faz, Y350N, A360Y and Y363N; (viii) Y92L, E94Faz and A360Y; (ix) E94Faz and A360Y; and (x) E94C, G357Faz and A360C.

[0512] The helicase of the present invention preferably further comprises one or more single amino acid deletions from the pin domain. Any number of single amino acid deletions can be performed, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. The helicase more preferably comprises a variant of SEQ ID NO: 118 comprising a deletion of E93, a deletion of E95, or a deletion of E93 and E95. The helicase more preferably comprises a variant of SEQ ID NO: 118 comprising (a) a deletion of E94C, N95, and A360C; (b) a deletion of E93, a deletion of E94, a deletion of N95, and A360C; (c) a deletion of E93, E94C, a deletion of N95, and A360C, or (d) E93C, a deletion of N95, and A360C. The helicase of the present invention preferably comprises a variant of any one of SEQ ID NOs: 119 to 133, comprising a deletion of the position corresponding to E93 in SEQ ID NO: 118, a deletion of the position corresponding to E95 in SEQ ID NO: 118, or a deletion of the positions corresponding to E93 and E95 in SEQ ID NO: 118.

[0513] The helicase of the present invention preferably further comprises one or more single amino acid deletions from the hook domain. Any number of single amino acid deletions can be made, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more. The helicase more preferably comprises a variant of SEQ ID NO: 118 comprising any number of deletions from positions T278 to S287. The helicase more preferably comprises a variant of SEQ ID NO: 118 comprising (a) E94C, a deletion of Y279 to K284 and A360C, (b) E94C, a deletion of T278, Y279, V286 and S287 and A360C, (c) E94C, a deletion of I281 and K284 and replacement with a single G and A360C, (d) E94C, a deletion of K280 and P2845 and replacement with a single G and A360C, or (e) a deletion of Y279 to K284, E94C, F276A and A230C. The helicase of the present invention preferably comprises a variant of any one of SEQ ID NOs: 119 to 133 comprising a deletion of any number of positions corresponding to 278 to 287 in SEQ ID NO: 118.

[0514] The helicase of the present invention preferably further comprises one or more single amino acid deletions from the pin domain and one or more single amino acid deletions from the hook domain.

[0515] The helicase of the present invention is preferably one in which at least one cysteine ​​residue and / or at least one unnatural amino acid has been further introduced into the hook domain and / or the 2A (RecA-like) domain. As discussed above for the tower, pin, and 1A domains, any number and combination of cysteine ​​residues and unnatural amino acids may be introduced.

[0516] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been further introduced into the hook domain (residues L275-F291) and / or the 2A (RecA-like) domain (residues R178-T259 and L390-V439).

[0517] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 119, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been further introduced into the hook domain (residues A310-L315) and / or the 2A (RecA-like) domain (residues R212-E294 and G422-S678).

[0518] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 120, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been further introduced into the hook domain (residues V343-L359) and / or the 2A (RecA-like) domain (residues R241-N327 and A449-G496).

[0519] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 121, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been further introduced into the hook domain (residues W276-L284) and / or the 2A (RecA-like) domain (residues R174-D260 and A371-V421).

[0520] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 122, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been further introduced into the hook domain (residues R308-Y313) and / or the 2A (RecA-like) domain (residues R206-K293 and I408-L500).

[0521] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 123, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been further introduced into the hook domain (residues M302-W306) and / or the 2A (RecA-like) domain (residues R195-D287 and V394-Q450).

[0522] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 124, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been further introduced into the hook domain (residues V265-I277) and / or the 2A (RecA-like) domain (residues R167-T249 and L372-N421).

[0523] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 125, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been further introduced into the hook domain (residues V270-F283) and / or the 2A (RecA-like) domain (residues R172-T254 and L381-K434).

[0524] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 126, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been further introduced into the hook domain (residues V257-F270) and / or the 2A (RecA-like) domain (residues R159-T241 and L367-K420).

[0525] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 127, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been further introduced into the hook domain (residues L278-Y294) and / or the 2A (RecA-like) domain (residues R182-T262 and L393-V443).

[0526] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 128, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been further introduced into the hook domain (residues L278-Y294) and / or the 2A (RecA-like) domain (residues R182-T262 and L392-V442).

[0527] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 129, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been further introduced into the hook domain (residues L273-F289) and / or the 2A (RecA-like) domain (residues R177-N257 and L387-V438).

[0528] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 130, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been further introduced into the hook domain (residues L281-F297) and / or the 2A (RecA-like) domain (residues R184-T265 and L393-I442).

[0529] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 131, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been further introduced into the hook domain (residues H277-F293) and / or the 2A (RecA-like) domain (residues R180-T261 and L393-V442).

[0530] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 132, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been further introduced into the hook domain (residues L276-F292) and / or the 2A (RecA-like) domain (residues R179-T260 and L390-I439).

[0531] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 133, wherein at least one cysteine ​​residue and / or at least one unnatural amino acid has been further introduced into the hook domain (residues L276-F292) and / or the 2A (RecA-like) domain (residues R179-T260 and L391-V441).

[0532] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118 comprising one or more of the following: (i) I181C; (ii) Y279C; (iii) I281C; and (iv) E288C. The helicase may comprise any combination of (i) to (iv), such as (i); (ii); (iii); (iv); (i) and (ii); (i) and (iii); (i) and (iv); (ii) and (iii); (ii) and (iv); (iii) and (iv); or (i), (ii), (iii) and (iv). The helicase more preferably comprises a variant of SEQ ID NO: 118 comprising (a) E94C, I281C and A360C or (b) E94C, I281C, G357C and A360C. The helicase of the present invention preferably comprises a variant of any one of SEQ ID NOs: 119 to 133 comprising a cysteine ​​residue at one or more positions corresponding to those positions in SEQ ID NO: 118 as defined in (i) to (iv), (a) and (b). The helicase may comprise any of these variants wherein Faz is introduced instead of cysteine ​​at one or more specific positions (or each specific position).

[0533] The helicase of the present invention is further modified to reduce its surface negative charge. Surface residues can be identified in the same manner as for the Dda domain disclosed above. Surface negative charge is typically a surface negatively charged amino acid, such as aspartic acid (D) and glutamic acid (E).

[0534] Preferably, the helicase is modified to replace one or more negatively charged amino acids with one or more positively charged amino acids, uncharged amino acids, non-polar amino acids and / or aromatic amino acids, or by introducing one or more positively charged amino acids, preferably adjacent to one or more negatively charged amino acids to neutralize one or more surface negative charges. Suitable positively charged amino acids include but are not limited to histidine (H), lysine (K) and arginine (R). Uncharged amino acids have no net charge. Suitable uncharged amino acids include but are not limited to cysteine ​​(C), serine (S), threonine (T), methionine (M), asparagine (N) and glutamine (Q). Non-polar amino acids have non-polar side chains. Suitable non-polar amino acids include but are not limited to glycine (G), alanine (A), proline (P), isoleucine (I), leucine (L) and valine (V). Aromatic amino acids have aromatic side chains. Suitable aromatic amino acids include but are not limited to histidine (H), phenylalanine (F), tryptophan (W) and tyrosine (Y).

[0535] Preferred substitutions include, but are not limited to, E substituted by R, E substituted by K, E substituted by N, D substituted by K, and D substituted by R.

[0536] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118, and the one or more negatively charged amino acids are one or more of D5, E8, E23, E47, D167, E172, D202, D212 and E273. Any number of these amino acids can be neutralized, such as 1, 2, 3, 4, 5, 6, 7 or 8 of them. Any combination can be neutralized. The helicase of the present invention preferably comprises a variant of any one of SEQ ID NO: 119 to 133, and the one or more negatively charged amino acids correspond to one or more of D5, E8, E23, E47, D167, E172, D202, D212 and E273 in SEQ ID NO: 118. The amino acids in SEQ ID NOs: 119 to 133 corresponding to D5, E8, E23, E47, D167, E172, D202, D212 and E273 in SEQ ID NO: 118 can be determined using the alignment in WO 2015 / 055981. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118 comprising (a) E94C, E273G and A360C or (b) E94C, E273G, N292G and A360C.

[0537] The helicase of the present invention is preferably further modified by removing one or more native cysteine ​​residues. Any number of native cysteine ​​residues can be removed. One or more cysteine ​​residues are preferably removed by substitution. One or more cysteine ​​residues are preferably replaced by alanine (A), serine (S) or valine (V). The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118, and the one or more native cysteine ​​residues are one or more of C109, C114, C136, C171 and C412. Any number and combination of these cysteine ​​residues can be removed. For example, SEQ ID Variants of NO:118 may include C109; C114; C136; C171; C412; C109 and C114; C109 and C136; C109 and C171; C109 and C412; C114 and C136; C114 and C171; C114 and C412; C136 and C171; C136 and C412; C171 and C412; C109, C114 and C136; C109, C114 and C171; C109, C136 and C171; and C412; C109, C171, and C412; C114, C136, and C171; C114, C136, and C412; C114, C171, and C412; C136, C171, and C412; C109, C114, C136, and C171; C109, C114, C136, and C412; C109, C114, C171, and C412; C109, C136, C171, and C412; C114, C136, C171, and C412; or C109, C114, C136, C171, and C412.

[0538] The modified helicase preferably comprises a modification or substitution at positions corresponding to amino acid positions 109 and / or 136 in Dda 1993. This removes one or two cysteine ​​residues. This can be in addition to modifications or substitutions at one or more positions corresponding to amino acid positions 55, 114, 156, 177, 210, 221, 350, and 358 in Dda 1993, as well as modifications or substitutions at one or more positions corresponding to amino acid positions 114, 177, 350, and 358 in Dda 1993 and / or at a position corresponding to position 40 in Dda 1993. Position 109 or a corresponding position may be substituted with A, V, I, L, M, F, Y, or W. Position 109 or a corresponding position is preferably substituted with A or V. Position 136 or a corresponding position may be substituted with A, V, I, L, M, F, Y, or W. Position 136 or a corresponding position is preferably substituted with A or V. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118 comprising a substitution at C109, such as C109A, C109V, C109I, C109L, C109M, C109F, C109Y or C109W, and / or a substitution at C136, such as C136A, C136V, C136I, C136L, C136M, C136F, C136Y or C136W. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118 comprising C109A and / or C136A. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132 or 133 comprising a substitution at a position corresponding to C109 and / or C136 in SEQ ID NO: 118. The helicase of the present invention is preferably one in which at least one cysteine ​​residue (i.e., one or more cysteine ​​residues) and / or at least one unnatural amino acid (i.e., one or more unnatural amino acids) is introduced only into the tower domain. Suitable modifications are discussed above.

[0539] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118 comprising the following mutations: E93C and K364C; E94C and K364C; E94C and A360C; L97C and E361C; L97C and E361C and C412A; K123C and E361C; K123C, E361C and C412A; N155C and K358C; N155C, K358C and C412A; N155C and L354C; N155C, L354C and C412A; deltaE93, E94C, deltaN95 and A360C; E94C, deltaN95 and A360C; E94C, Q100C, I127C and A360C ; L354C; G357C; E94C, G357C and A360C; E94C, Y279C and A360C; E94C, I281C and A360C; E94C, Y279Faz and A360C; Y279C and G357C; I281C and G357C; E94C, Y279C, G357C and A360C; E94C, I281C, G357C and A360C; E8R, E47K, E94C, D202K and A360C; D5K, E23N, E94C, D167K, E172R, D212R and A360C; D5K, E8R, E23N, E47K, E94C, D167K, E172R, D202K, D212R, and A360C; E94C, C114A, C171A, A360C, and C412D; E94C, C114A, C171A, A360C, and C412S; E94C, C109A, C136A, and A360C; E94C, C109A, C114A, C136A, C171A, A360C, and C412S; E94C, C109V, C114V, C171A, A360C, and C412S; C109A, C114A, C136A, G153C, C171A, E361C, and C412A; C109A, C114A , C136A, G153C, C171A, E361C, and C412D; C109A, C114A, C136A, G153C, C171A, E361C, and C412S; C109A, C114A, C136A, G153C, C171A, K358C, and C412A; C109A, C114A, C136A, G153C, C171A, K358C, and C412D; C109A, C114A, C136A, G153C, C171A, K358C, and C412S; C109A, C114A, C136A, N155C, C171A, K358C, and C412A;C109A, C114A, C136A, N155C, C171A, K358C, and C412D; C109A, C114A, C136A, N155C, C171A, K358C, and C412S; C109A, C114A, C136A, N155C, C171A, L354C, and C412A; C109A, C114A, C136A, N155C, C171A, L354C, and C412D; C109A, C114A, C136A, N 155C, C171A, L354C, and C412S; C109A, C114A, K123C, C136A, C171A, E361C, and C412A; C109A, C114A, K123C, C136A, C171A, E361C, and C412D; C109A, C114A, K123C, C136A, C171A, E361C, and C412S; C109A, C114A, K123C, C136A, C171A, K358C, and C41 2A; C109A, C114A, K123C, C136A, C171A, K358C, and C412D; C109A, C114A, K123C, C136A, C171A, K358C, and C412S; C109A, C114A, C136A, G153C, C171A, E361C, and C412A; E94C, C109A, C114A, C136A, C171A, A360C, and C412D; E94C, C109A, C114V, C 136A, C171A, A360C, and C412D; E94C, C109V, C114A, C136A, C171A, A360C, and C412D; L97C, C109A, C114A, C136A, C171A, E361C, and C412A; L97C, C109A, C114A, C136A, C171A, E361C, and C412D; or L97C, C109A, C114A, C136A, C171A, E361C, and C412S. ;

[0540] Modifications in the hook domain and / or 2A domain

[0541] In one embodiment, the helicase of the present invention is a helicase wherein at least one cysteine ​​residue and / or at least one non-natural amino acid has been introduced into the hook domain and / or the 2A (RecA-like motor) domain, wherein the helicase has the ability to control the movement of a polynucleotide. Preferably, at least one cysteine ​​residue and / or at least one non-natural amino acid is introduced into the hook domain and the 2A (RecA-like motor) domain.

[0542] Any number of cysteine ​​residues and / or non-natural amino acids can be introduced into each domain. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more cysteine ​​residues and / or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-natural amino acids can be introduced. Only one or more cysteine ​​residues can be introduced. Only one or more non-natural amino acids can be introduced. A combination of one or more cysteine ​​residues and one or more non-natural amino acids can be introduced.

[0543] Preferably, at least one cysteine ​​residue and / or at least one unnatural amino acid is introduced by substitution. Methods for doing so are known in the art. Suitable modifications of the hook domain and / or the 2A (RecA-like motor) domain are discussed above.

[0544] The helicase of the present invention is preferably a variant of SEQ ID NO: 118 comprising (a) Y279C, I181C, E288C, Y279C and I181C, (b) Y279C and E288C, (c) I181C and E288C or (d) Y279C, I181C and E288C. The helicase of the present invention preferably comprises a variant of any one of SEQ ID NOs: 199 to 133 comprising a mutation at one or more positions corresponding to those positions in SEQ ID NO: 118 as defined in (a) to (d).

[0545] Surface modification

[0546] In one embodiment, the helicase is modified to reduce its surface negative charge, wherein the helicase has the ability to control the movement of the polynucleotide. Suitable modifications are discussed above. Any amount of surface negative charge can be neutralized.

[0547] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118 comprising the following mutations: E273G; E8R, E47K and D202K; D5K, E23N, D167K, E172R and D212R; or D5K, E8R, E23N, E47K, D167K, E172R, D202K and D212R.

[0548] Other modified helicases

[0549] In one embodiment, the helicase of the invention comprises a variant of SEQ ID NO: 118 comprising: A360K; Y92L and / or A360Y; Y92L, Y350N, and Y363N; Y92L and / or Y363N; or Y92L.

[0550] Other modifications

[0551] In addition to the specific mutations disclosed above, variants of SEQ ID NO: 118 may comprise one or more of the following mutations: K38A; T91F; T91N; T91Q; T91W; V96E; V96F; V96L; V96Q; V96R; V96W; V96Y; P274G; V286F; V286W; V286Y; F291G; N292F; N292G; N292P; N292Y; G294Y; G294F; K364A; and W378A.

[0552] In addition to the specific mutations disclosed above, SEQ ID Variants of NO:118 may include: K38A, E94C and A360C; H64K; E94C and A360C; H64N; E94C and A360C; H64Q; E94C and A360C; H64S; E94C and A360C; H64W, E94C and A360C; T80K, E94C and A360C; T80K, S83K, E94C, N242K, N293K and A360C; T80K, S83K, E94C, N242K, N293K and A360C; C and T394K; T80K, S83K, E94C, A360C and T394N; T80K, E94C, N242K and A360C; T80K, E94C, N242K, N293K and A360C; T80K, E94C, N293K and A360C; T80N, E94C and A360C; H82A, E94C and A360C; H82A, P89A, E94C, F98A and A360C; H82F, E94C and A360C; H82Q, E94C, A360C; H82R, E94C and A360C; H82W, E94C and A360C; H82W, P89W, E94C, F 98W and A360C; H82Y, E94C and A360C; S83K, E94C and A360C; S83K, T80K, E94C, A360C and T394K; S83N, E94C and A360C; S83T, E94C and A360C; N88H, E94C and A360C; N88Q, E94C and A360C; P89A, E94C and A360C; P89A, F98W, E94C and A360C; P89A, E94C, F98Y and A360C; P89A, E94C, F98A and A360C; P89F, E94C and A360C; P89S, E94C and A360C; P89T, E94C and A360C; P89W, E94C, F98W and A360C; P89Y, E94C and A360C; T91F, E94C and A360C; T91N, E94C and A360C; T91Q, E94C and A360C; T91W, E94C and A360C; E94C, V96E and A360C; E94C, V96F and A360C; E94C, V96L and A360C; E94C, V96Q and A360C; E94C, V96R and A360C; E94C, V96W and A360C; E94C, V96Y and A360C; E94C, F98A and A360C;E94C, F98L and A360C; E94C, F98V and A360C; E94C, F98Y and A360C; E94C; F98W and A360C; E94C, V150A and A360C; E94C, V150F and A360C; E94C, V150I and A360C; E94C, V150K and A360C; E94C, V150L and A360C; E94C, V150S and A360C; E94C, V150T and A360C; E94C, V150W and A360C; E94C, V150Y and A360C; E94C , F240Y and A360C; E94C, F240W and A360C; E94C, N242K and A360C; E94C, N242K, N293K and A360C; E94C, P274G and A360C; E94C, L275G and A360C; E94C, F276A and A360C; E94C, F276I and A360C; E94C, F276M and A360C; E94C, F276V and A360C; E94C, F276W and A360C; E94C, F276Y and A360C; E94C, V286F and A360C E94C, V286W and A360C; E94C, V286Y and A360C; E94C, S287F and A360C; E94C, S287W and A360C; E94C, S287Y and A360C; E94C, F291G and A360C; E94C, N292F and A360C; E94C, N292G and A360C; E94C, N292P and A360C; E94C, N292Y and A360C; E94C, N293F and A360C; E94C, N293K and A360C; E94C, N293Q and A360C ; E94C, N293Y, and A360C; E94C, G294F, and A360C; E94C, G294Y, and A360C; E94C, A36C, and K364A; E94C, A360C, W378A; E94C, A360C, and T394K; E94C, A360C, and H396Q; E94C, A360C, and H396S; E94C, A360C, and H396W; E94C, A360C, and Y415F; E94C, A360C, and Y415K; E94C, A360C, and Y415M; or E94C, A360C, and Y415W. ;

[0553] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118, which comprises (a) E94C / A360C / W378A or (b) E94C / A360C / C109A / C136A / W378A or (d) E94C / A360C / C109A / C136A / W378A, and then (ΔM1)G1G2 (i.e., deletion of M1, followed by addition of G1 and G2).

[0554] Preferred variants of any of SEQ ID NOs: 118 to 133 have (in addition to the modifications of the present invention) an N-terminal methionine (M) substituted with a glycine residue (G). In the examples, this is shown as (ΔM1)G1. It may also be referred to as M1G. Any of the variants discussed above may further comprise M1G.

[0555] The most preferred helicases of the present invention comprise variants of SEQ ID NO: 118 comprising (a) E94C / F98W / A360C / C109A / C136A / K194L, (b) M1G / E94C / F98W / A360C / C109A / C136A / K194L; (c) E94C / F98W / A360C / C109A / C136A / K199L; or (d) M1G / E94C / F98W / A360C / C109A / C136A / K199L.

[0556] Other preferred helicases

[0557] Other preferred helicases of the present invention include variants of SEQ ID NO: 118 comprising a substitution at:

[0558] T40 / E94 / F98, such as T40Y / E94C / F98W;

[0559] T40 / E94 / F98 / C114, such as T40Y / E94C / F98W / C114I;

[0560] T40 / E94 / F98 / K177, such as T40Y / E94C / F98W / K177M;

[0561] T40 / E94 / F98 / Y350, such as T40Y / E94C / F98W / Y350, T40Y / E94C / F98W / Y350I or T40Y / E94C / F98W / Y350E;

[0562] T40 / E94 / F98 / K358, such as T40Y / E94C / F98W / K358I;

[0563] E94 / F98 / C114, such as E94C / F98W / C114I;

[0564] E94 / F98 / K177, such as E94C / F98W / K177M;

[0565] E94 / F98 / Y350, such as E94C / F98W / Y350I or E94C / F98W / Y350E;

[0566] E94 / F98 / K358, such as E94C / F98W / K358I;

[0567] T40 / E94 / F98 / C114 / K177, such as T40Y / E94C / F98W / C114I / K177M;

[0568] T40 / E94 / F98 / C114 / Y350, such as T40Y / E94C / F98W / C114I / Y350I or T40Y / E94C / F98W / C114I / Y350E;

[0569] T40 / E94 / F98 / C114 / K358, such as T40Y / E94C / F98W / C114I / K358I;

[0570] T40 / E94 / F98 / K177 / Y350, such as T40Y / E94C / F98W / K177M / Y350I or T40Y / E94C / F98W / K177M / Y350E;

[0571] T40 / E94 / F98 / K177 / K358, such as T40Y / E94C / F98W / K177M / K358I;

[0572] T40 / E94 / F98 / Y350 / K358, such as T40Y / E94C / F98W / Y350I / K358I or T40Y / E94C / F98W / Y350E / K358I;

[0573] E94 / F98 / C114 / K177, such as E94C / F98W / C114I / K177M;

[0574] E94 / F98 / C114 / Y350, such as E94C / F98W / C114I / Y350I or E94C / F98W / C114I / Y350E;

[0575] E94 / F98 / C114 / K358, such as E94C / F98W / C114I / K358I;

[0576] E94 / F98 / K177 / Y350, such as E94C / F98W / K177M / Y350I or E94C / F98W / K177M / Y350E;

[0577] E94 / F98 / K177 / K358, such as E94C / F98W / K177M / K358I;

[0578] E94 / F98 / Y350 / K358, such as E94C / F98W / Y350I / K358I or E94C / F98W / Y350E / K358I;

[0579] T40 / E94 / F98 / C114 / K177 / Y350, such as T40Y / E94C / F98W / C114I / K177M / Y350I or T40Y / E94C / F98W / C114I / K177M / Y350E;

[0580] T40 / E94 / F98 / C114 / K177 / K358, such as T40Y / E94C / F98W / C114I / K177M / K358I;

[0581] T40 / E94 / F98 / C114 / Y350 / K358, such as T40Y / E94C / F98W / C114I / Y350I / K358I or T40Y / E94C / F98W / C114I / Y350E / K358I;

[0582] T40 / E94 / F98 / K177 / Y350 / K358, such as T40Y / E94C / F98W / K177M / Y350I / K358I or T40Y / E94C / F98W / K177M / Y350E / K358I;

[0583] E94 / F98 / C114 / K177 / Y350, such as E94C / F98W / C114I / K177M / Y350I or E94C / F98W / C114I / K177M / Y350E;

[0584] E94 / F98 / C114 / K177 / K358, such as E94C / F98W / C114I / K177M / K358I;

[0585] E94 / F98 / C114 / Y350 / K358, such as E94C / F98W / C114I / Y350I / K358I or E94C / F98W / C114I / Y350E / K358I;

[0586] E94 / F98 / K177 / Y350 / K358, such as E94C / F98W / K177M / Y350I / K358I or E94C / F98W / K177M / Y350E / K358I;

[0587] T40 / E94 / F98 / C114 / K177 / Y350 / K358, such as T40Y / E94C / F98W / C114I / K177M / Y350I / K358I or T40Y / E94C / F98W / C114I / K177M / Y350E / K358I;

[0588] E94 / F98 / C114 / K177 / Y350 / K358, such as E94C / F98W / C114I / K177M / Y350I / K358I or E94C / F98W / C114I / K177M / Y350E / K358I;

[0589] T40 / F98 / K194, such as T40Y / E94C / F98W / K194L;

[0590] T40 / F98 / C114 / K194, such as T40Y / E94C / F98W / C114I K194L;

[0591] T40 / F98 / K177 / K194, such as T40Y / E94C / F98W / K177M K194L;

[0592] T40 / F98 / K194 / Y350, such as T40Y / E94C / F98W / K194L / Y350I or T40Y / E94C / F98W / K194L / Y350E;

[0593] T40 / F98 / K194 / K358, such as T40Y / E94C / F98W / K194L / K358I;

[0594] F98 / C114 / K194, such as E94C / F98W / C114I / K194L;

[0595] F98 / K177 / K194, such as E94C / F98W / K177M / K194L;

[0596] F98 / K194 / Y350, such as E94C / F98W / K194L / Y350I or E94C / F98W / K194L / Y350E;

[0597] F98 / K194 / K358, such as E94C / F98W / K194L / K358I;

[0598] T40 / F98 / C114 / K177 / K194, such as T40Y / E94C / F98W / C114I / K177M / K194L;

[0599] T40 / F98 / C114 / K194 / Y350, such as T40Y / E94C / F98W / C114I / K194L / Y350I or T40Y / E94C / F98W / C114I / K194L / Y350E;

[0600] T40 / F98 / C114 / K194 / K358, such as T40Y / E94C / F98W / C114I / K194L / K358I;

[0601] T40 / F98 / K177 / K194 / Y350, such as T40Y / E94C / F98W / K177M / K194L / Y350I or T40Y / E94C / F98W / K177M / K194L / Y350E;

[0602] T40 / F98 / K177 / K194 / K358, such as T40Y / E94C / F98W / K177M / K194L / K358I;

[0603] T40 / F98 / K194 / Y350 / K358, such as T40Y / E94C / F98W / K194L / Y350I / K358I or T40Y / E94C / F98W / K194L / Y350E / K358I;

[0604] F98 / C114 / K177 / K194, such as E94C / F98W / C114I / K177M / K194L;

[0605] F98 / C114 / K194 / Y350, such as E94C / F98W / C114I / K194L / Y350I or E94C / F98W / C114I / K194L / Y350E;

[0606] F98 / C114 / K194 / K358, such as E94C / F98W / C114I / K194L / K358I;

[0607] F98 / K177 / K194 / Y350, such as E94C / F98W / K177M / K194L / Y350I or E94C / F98W / K177M / K194L / Y350E;

[0608] F98 / K177 / K194 / K358, such as E94C / F98W / K177M / K194L / K358I;

[0609] F98 / K194 / Y350 / K358, such as E94C / F98W / K194L / Y350I / K358I or E94C / F98W / K194L / Y350E / K358I;

[0610] T40 / F98 / C114 / K177 / K194 / Y350, such as T40Y / E94C / F98W / C114I / K177M / K194L / Y350I or T40Y / E94C / F98W / C114I / K177M / K194L / Y350E;

[0611] T40 / F98 / C114 / K177 / K194 / K358, such as T40Y / E94C / F98W / C114I / K177M / K194L / K358I;

[0612] T40 / F98 / C114 / K194 / Y350 / K358, such as T40Y / E94C / F98W / C114I / K194L / Y350I / K358I or T40Y / E94C / F98W / C114I / K194L / Y350E / K358I;

[0613] T40 / F98 / K177 / K194 / Y350 / K358, such as T40Y / E94C / F98W / K177M / K194L / Y350I / K358I or T40Y / E94C / F98W / K177M / K194L / Y350E / K358I;

[0614] F98 / C114 / K177 / K194 / Y350, such as E94C / F98W / C114I / K177M / K194L / Y350I or E94C / F98W / C114I / K177M / K194L / Y350E;

[0615] F98 / C114 / K177 / K194 / K358, such as E94C / F98W / C114I / K177M / K194L / K358I;

[0616] F98 / C114 / K194 / Y350 / K358, such as E94C / F98W / C114I / K194L / Y350I / K358I or E94C / F98W / C114I / K194L / Y350E / K358I;

[0617] F98 / K177 / K194 / Y350 / K358, such as E94C / F98W / K177M / K194L / Y350I / K358I or E94C / F98W / K177M / K194L / Y350E / K358I;

[0618] T40 / F98 / C114 / K177 / K194 / Y350 / K358, such as T40Y / E94C / F98W / C114I / K177M / K194L / Y350I / K358I or T40Y / E94C / F98W / C114I / K177M / K194L / Y350E / K358I;

[0619] F98 / C114 / K177 / K194 / Y350 / K358, such as E94C / F98W / C114I / K177M / K194L / Y350I / K358I or E94C / F98W / C114I / K177M / K194L / Y350E / K358I;

[0620] T40 / A360, such as T40Y / A360C;

[0621] T40 / C114 / A360, such as T40Y / C114I / A360C;

[0622] T40 / K177 / A360, such as T40Y / K177M / A360C;

[0623] T40 / Y350 / A360, such as T40Y / Y350I / A360C or T40Y / Y350E / A360C;

[0624] T40 / K358 / A360, such as T40Y / K358I / A360C;

[0625] C114 / A360, such as C114I / A360C;

[0626] K177 / A360, such as K177M / A360C;

[0627] Y350 / A360, such as Y350I / A360C or Y350E / A360C;

[0628] K358 / A360, such as K358I / A360C;

[0629] T40 / C114 / K177 / A360, such as T40Y / C114I / K177M / A360C;

[0630] T40 / C114 / Y350 / A360, such as T40Y / C114I / Y350I / A360C or T40Y / C114I / Y350E / A360C;

[0631] T40 / C114 / K358 / A360, such as T40Y / C114I / K358I / A360C;

[0632] T40 / K177 / Y350 / A360, such as T40Y / K177M / Y350I / A360C or T40Y / K177M / Y350E / A360C;

[0633] T40 / K177 / K358 / A360, such as T40Y / K177M / K358I / A360C;

[0634] T40 / Y350 / K358 / A360, such as T40Y / Y350I / K358I / A360C or T40Y / Y350E / K358I / A360C;

[0635] C114 / K177 / A360, such as C114I / K177M / A360C;

[0636] C114 / Y350 / A360, such as C114I / Y350I / A360C or C114I / Y350E / A360C;

[0637] C114 / K358 / A360, such as C114I / K358I / A360C;

[0638] K177 / Y350 / A360, such as K177M / Y350I / A360C or K177M / Y350E / A360C;

[0639] K177 / K358 / A360, such as K177M / K358I / A360C;

[0640] Y350 / K358 / A360, such as Y350I / K358I / A360C or Y350E / K358I / A360C;

[0641] T40 / C114 / K177 / Y350 / A360, such as T40Y / C114I / K177M / Y350I / A360C or T40Y / C114I / K177M / Y350E / A360C;

[0642] T40 / C114 / K177 / K358 / A360, such as T40Y / C114I / K177M / K358I / A360C;

[0643] T40 / C114 / Y350 / K358 / A360, such as T40Y / C114I / Y350I / K358I / A360C or T40Y / C114I / Y350E / K358I / A360C;

[0644] T40 / K177 / Y350 / K358 / A360, such as T40Y / K177M / Y350I / K358I / A360C or T40Y / K177M / Y350E / K358I / A360C;

[0645] C114 / K177 / Y350 / A360, such as C114I / K177M / Y350I / A360C or C114I / K177M / Y350E / A360C;

[0646] C114 / K177 / K358 / A360, such as C114I / K177M / K358I / A360C;

[0647] C114 / Y350 / K358 / A360, such as C114I / Y350I / K358I / A360C or C114I / Y350E / K358I / A360C;

[0648] K177 / Y350 / K358 / A360, such as K177M / Y350I / K358I / A360C or K177M / Y350E / K358I / A360C;

[0649] T40 / C114 / K177 / Y350 / K358 / A360, such as T40Y / C114I / K177M / Y350I / K358I / A360C or K177M / Y350E / K358I / A360C;

[0650] C114 / K177 / Y350 / K358 / A360, such as C114I / K177M / Y350I / K358I / A360C or C114I / K177M / Y350E / K358I / A360C;

[0651] T40 / E94 / F98 / C109, such as T40Y / E94C / F98W / C109A;

[0652] T40 / E94 / F98 / C109 / C114, such as T40Y / E94C / F98W / C109A / C114I;

[0653] T40 / E94 / F98 / C109 / K177, such as T40Y / E94C / F98W / C109A / K177M;

[0654] T40 / E94 / F98 / C109 / Y350, such as T40Y / E94C / F98W / C109A / Y350I or T40Y / E94C / F98W / C109A / Y350E;

[0655] T40 / E94 / F98 / C109 / K358, such as T40Y / E94C / F98W / C109A / K358I;

[0656] E94 / F98 / C109 / C114, such as E94C / F98W / C109A / C114I;

[0657] E94 / F98 / C109 / K177, such as E94C / F98W / C109A / K177M;

[0658] E94 / F98 / C109 / Y350, such as E94C / F98W / C109A / Y350I or E94C / F98W / C109A / Y350E;

[0659] E94 / F98 / C109 / K358, such as E94C / F98W / C109A / K358I;

[0660] T40 / E94 / F98 / C109 / C114 / K177, such as T40Y / E94C / F98W / C109A / C114I / K177M;

[0661] T40 / E94 / F98 / C109 / C114 / Y350, such as T40Y / E94C / F98W / C109A / C114I / Y350I or T40Y / E94C / F98W / C109A / C114I / Y350E;

[0662] T40 / E94 / F98 / C109 / C114 / K358, such as T40Y / E94C / F98W / C109A / C114I / K358I;

[0663] T40 / E94 / F98 / C109 / K177 / Y350, such as T40Y / E94C / F98W / C109A / K177M / Y350I or T40Y / E94C / F98W / C109A / K177M / Y350E;

[0664] T40 / E94 / F98 / C109 / K177 / K358, such as T40Y / E94C / F98W / C109A / K177M / K358I;

[0665] T40 / E94 / F98 / C109 / Y350 / K358, such as T40Y / E94C / F98W / C109A / Y350I / K358I or T40Y / E94C / F98W / C109A / Y350E / K358I;

[0666] E94 / F98 / C109 / C114 / K177, such as E94C / F98W / C109A / C114I / K177M;

[0667] E94 / F98 / C109 / C114 / Y350, such as E94C / F98W / C109A / C114I / Y350I or E94C / F98W / C109A / C114I / Y350E;

[0668] E94 / F98 / C109 / C114 / K358, such as E94C / F98W / C109A / C114I / K358I;

[0669] E94 / F98 / C109 / K177 / Y350, such as E94C / F98W / C109A / K177M / Y350I or E94C / F98W / C109A / K177M / Y350E;

[0670] E94 / F98 / C109 / K177 / K358, such as E94C / F98W / C109A / K177M / K358I;

[0671] E94 / F98 / C109 / Y350 / K358, such as E94C / F98W / C109A / Y350I / K358I or E94C / F98W / C109A / Y350E / K358I;

[0672] T40 / E94 / F98 / C109 / C114 / K177 / Y350, such as T40Y / E94C / F98W / C109A / C114I / K177M / Y350I or T40Y / E94C / F98W / C109A / C114I / K177M / Y350E;

[0673] T40 / E94 / F98 / C109 / C114 / K177 / K358, such as T40Y / E94C / F98W / C109A / C114I / K177M / K358I;

[0674] T40 / E94 / F98 / C109 / C114 / Y350 / K358, such as T40Y / E94C / F98W / C109A / C114I / Y350I / K358I or T40Y / E94C / F98W / C109A / C114I / Y350E / K358I;

[0675] T40 / E94 / F98 / C109 / K177 / Y350 / K358, such as T40Y / E94C / F98W / C109A / K177M / Y350I / K358I or T40Y / E94C / F98W / C109A / K177M / Y350E / K358I;

[0676] E94 / F98 / C109 / C114 / K177 / Y350, such as E94C / F98W / C109A / C114I / K177M / Y350I or E94C / F98W / C109A / C114I / K177M / Y350E;

[0677] E94 / F98 / C109 / C114 / K177 / K358, such as E94C / F98W / C109A / C114I / K177M / K358I;

[0678] E94 / F98 / C109 / C114 / Y350 / K358, such as E94C / F98W / C109A / C114I / Y350I / K358I or E94C / F98W / C109A / C114I / Y350E / K358I;

[0679] E94 / F98 / C109 / K177 / Y350 / K358, such as E94C / F98W / C109A / K177M / Y350I / K358I or E94C / F98W / C109A / K177M / Y350E / K358I;

[0680] T40 / E94 / F98 / C109 / C114 / K177 / Y350 / K358, such as T40Y / E94C / F98W / C109A / C114I / K177M / Y350I / K358I or T40Y / E94C / F98W / C109A / C114I / K177M / Y350E / K358I;

[0681] E94 / F98 / C109 / C114 / K177 / Y350 / K358, such as E94C / F98W / C109A / C114I / K177M / Y350I / K358I or E94C / F98W / C109A / C114I / K177M / Y350E / K358I;

[0682] T40 / C109 / C136, such as T40Y / C109A / C136A;

[0683] T40 / C109 / C114 / C136, such as T40Y / C109A / C114I / C136A;

[0684] T40 / C109 / C136 / K177, such as T40Y / C109A / C136A / K177M;

[0685] T40 / C109 / C136 / Y350, such as T40Y / C109A / C136A / Y350I or T40Y / C109A / C136A / Y350E;

[0686] T40 / C109 / C136 / K358, such as T40Y / C109A / C136A / K358I;

[0687] C109 / C114 / C136, such as C109A / C136A / C114I;

[0688] C109 / C136 / K177, such as C109A / C136A / K177M;

[0689] C109 / C136 / Y350, such as C109A / C136A / Y350I or C109A / C136A / Y350E;

[0690] C109 / C136 / K358, such as C109A / C136A / K358I;

[0691] T40 / C109 / C114 / C136 / K177, such as T40Y / C109A / C114I / C136A / K177M;

[0692] T40 / C109 / C114 / C136 / Y350, such as T40Y / C109A / C114I / C136A / Y350 or T40Y / C109A / C114I / C136A / Y350I I;

[0693] T40 / C109 / C114 / C136 / K358, such as T40Y / C109A / C114I / C136A / K358I;

[0694] T40 / C109 / C136 / K177 / Y350, such as T40Y / C109A / C136A / K177M / Y350E or T40Y / C109A / C136A / K177M / Y350I;

[0695] T40 / C109 / C136 / K177 / K358, such as T40Y / C109A / C136A / K177M / K358I;

[0696] T40 / C109 / C136 / Y350 / K358, such as T40Y / C109A / C136A / Y350I / K358I or T40Y / C109A / C136A / Y350E / K358I;

[0697] C109 / C114 / C136 / K177, such as C109A / C114I / C136A / K177M;

[0698] C109 / C114 / C136 / Y350, such as C109A / C114I / C136A / Y350I or C109A / C114I / C136A / Y350E;

[0699] C109 / C114 / C136 / K358, such as C109A / C114I / C136A / K358I;

[0700] C109 / C136 / K177 / Y350, such as C109A / C136A / K177M / Y350I or C109A / C136A / K177M / Y350E;

[0701] C109 / C136 / K177 / K358, such as C109A / C136A / K177M / K358I;

[0702] C109 / C136 / Y350 / K358, such as C109A / C136A / Y350I / K358I or C109A / C136A / Y350E / K358I;

[0703] T40 / C109 / C114 / C136 / K177 / Y350, such as T40Y / C109A / C114I / C136A / K177M / Y350I or T40Y / C109A / C114I / C136A / K177M / Y350E;

[0704] T40 / C109 / C114 / C136 / K177 / K358, such as T40Y / C109A / C114I / C136A / K177M / K358I;

[0705] T40 / C109 / C114 / C136 / Y350 / K358, such as T40Y / C109A / C114I / C136AY350I / K358I or T40Y / C109A / C114I / C136AY350E / K358I;

[0706] T40 / C109 / C136 / K177 / Y350 / K358, such as T40Y / C109A / C136A / K177M / Y350I / K358I or T40Y / C109A / C136A / K177M / Y350E / K358I;

[0707] C109 / C114 / C136 / K177 / Y350, such as C109A / C114I / C136A / K177M / Y350I or C109A / C114I / C136A / K177M / Y350E;

[0708] C109 / C114 / C136 / K177 / K358, such as C109A / C114I / C136A / K177M / K358I;

[0709] C109 / C114 / C136 / Y350 / K358, such as C109A / C114I / C136AY350I / K358I or C109A / C114I / C136AY350E / K358I;

[0710] C109 / C136 / K177 / Y350 / K358, such as C109A / C136A / K177M / Y350I / K358I or C109A / C136A / K177M / Y350E / K358I;

[0711] T40 / C109 / C114 / C136 / K177 / Y350 / K358, such as T40Y / C109A / C114I / C136A / K177M / Y350I / K358I or T40Y / C109A / C114I / C136A / K177M / Y350E / K358I;

[0712] C109 / C114 / C136 / K177 / Y350 / K358, such as C109A / C114I / C136A / K177M / Y350I / K358I / C109A / C114I / C136A / K177M / Y350E / K358I;

[0713] T40 / E94 / F98 / C109 / K194, such as T40Y / E94C / F98W / C109A / K194L;

[0714] T40 / E94 / F98 / C109 / C114 / K194, such as T40Y / E94C / F98W / C109A / C114I / K194L;

[0715] T40 / E94 / F98 / C109 / K177 / K194, such as T40Y / E94C / F98W / C109A / K177M / K194L;

[0716] T40 / E94 / F98 / C109 / K194 / Y350, such as T40Y / E94C / F98W / C109A / K194L / Y350I or T40Y / E94C / F98W / C109A / K194L / Y350E;

[0717] T40 / E94 / F98 / C109 / K194 / K358, such as T40Y / E94C / F98W / C109A / K194L / K358I;

[0718] E94 / F98 / C109 / C114 / K194, such as E94C / F98W / C109A / C114I / K194L;

[0719] E94 / F98 / C109 / K177 / K194, such as E94C / F98W / C109A / K177M / K194L;

[0720] E94 / F98 / C109 / K194 / Y350, such as E94C / F98W / C109A / K194L / Y350I or E94C / F98W / C109A / K194L / Y350E;

[0721] E94 / F98 / C109 / K194 / K358, such as E94C / F98W / C109A / K194L / K358I;

[0722] T40 / E94 / F98 / C109 / C114 / K177 / K194, such as T40Y / E94C / F98W / C109A / C114I / K194L / K177M;

[0723] T40 / E94 / F98 / C109 / C114 / K194 / Y350, such as T40Y / E94C / F98W / C109A / C114I / K194L / Y350I or T40Y / E94C / F98W / C109A / C114I / K194L / Y350E;

[0724] T40 / E94 / F98 / C109 / C114 / K194 / K358, such as T40Y / E94C / F98W / C109A / C114I / K194L / K358I;

[0725] T40 / E94 / F98 / C109 / K177 / K194 / Y350, such as T40Y / E94C / F98W / C109A / K177M / K194L / Y350I or T40Y / E94C / F98W / C109A / K177M / K194L / Y350E;

[0726] T40 / E94 / F98 / C109 / K177 / K194 / K358, such as T40Y / E94C / F98W / C109A / K177M / K194L / K358I;

[0727] T40 / E94 / F98 / C109 / K194 / Y350 / K358, such as T40Y / E94C / F98W / C109A / K194L / Y350I / K358I or T40Y / E94C / F98W / C109A / K194L / Y350E / K358I;

[0728] E94 / F98 / C109 / C114 / K177 / K194, such as E94C / F98W / C109A / C114I / K194L / K177M;

[0729] E94 / F98 / C109 / C114 / K194 / Y350, such as E94C / F98W / C109A / C114I / K194L / Y350I or E94C / F98W / C109A / C114I / K194L / Y350E;

[0730] E94 / F98 / C109 / C114 / K194 / K358, such as E94C / F98W / C109A / C114I / K194L / K358I;

[0731] E94 / F98 / C109 / K177 / K194 / Y350, such as E94C / F98W / C109A / K177M / K194L / Y350I or E94C / F98W / C109A / K177M / K194L / Y350E;

[0732] E94 / F98 / C109 / K177 / K194 / K358, such as E94C / F98W / C109A / K177M / K194L / K358I;

[0733] E94 / F98 / C109 / K194 / Y350 / K358, such as E94C / F98W / C109A / K194L / Y350I / K358I or E94C / F98W / C109A / K194L / Y350E / K358I;

[0734] T40 / E94 / F98 / C109 / C114 / K177 / K194 / Y350, such as T40Y / E94C / F98W / C109A / C114I / K177M / K194L / Y350I or T40Y / E94C / F98W / C109A / C114I / K177M / K194L / Y350E;

[0735] T40 / E94 / F98 / C109 / C114 / K177 / K194 / K358, such as T40Y / E94C / F98W / C109A / C114I / K177M / K194L / K358I;

[0736] T40 / E94 / F98 / C109 / C114 / K194 / Y350 / K358, such as T40Y / E94C / F98W / C109A / C114I / K194L / Y350I / K358I or T40Y / E94C / F98W / C109A / C114I / K194L / Y350E / K358I;

[0737] T40 / E94 / F98 / C109 / K177 / K194 / Y350 / K358, such as T40Y / E94C / F98W / C109A / K177M / K194L / Y350I / K358I or T40Y / E94C / F98W / C109A / K177M / K194L / Y350E / K358I;

[0738] E94 / F98 / C109 / C114 / K177 / K194 / Y350, such as E94C / F98W / C109A / C114I / K177M / K194L / Y350I or E94C / F98W / C109A / C114I / K177M / K194L / Y350E;

[0739] E94 / F98 / C109 / C114 / K177 / K194 / K358, such as E94C / F98W / C109A / C114I / K177M / K194L / K358I;

[0740] E94 / F98 / C109 / C114 / K194 / Y350 / K358, such as E94C / F98W / C109A / C114I / K194L / Y350I / K358I or E94C / F98W / C109A / C114I / K194L / Y350E / K358I;

[0741] E94 / F98 / C109 / K177 / K194 / Y350 / K358, such as E94C / F98W / C109A / K177M / K194L / Y350I / K358I or E94C / F98W / C109A / K177M / K194L / Y350E / K358I;

[0742] T40 / E94 / F98 / C109 / C114 / K177 / K194 / Y350 / K358, such as T40Y / E94C / F98W / C109A / C114I / K177M / K194L / Y350I / K358I or T40Y / E94C / F98W / C109A / C114I / K177M / K194L / Y350E / K358I;

[0743] E94 / F98 / C109 / C114 / K177 / K194 / Y350 / K358, such as E94C / F98W / C109A / C114I / K177M / K194L / Y350I / K358I or E94C / F98W / C109A / C114I / K177M / K194L / Y350E / K358I;

[0744] T40 / E94 / F98 / C109 / C136, such as T40Y / E94C / F98W / C109A / C136A;

[0745] T40 / E94 / F98 / C109 / C114 / C136, such as T40Y / E94C / F98W / C109A / C114I / C136A;

[0746] T40 / E94 / F98 / C109 / C136 / K177, such as T40Y / E94C / F98W / C109A / C136A / K177M;

[0747] T40 / E94 / F98 / C109 / C136 / Y350, such as T40Y / E94C / F98W / C109A / C136A / Y350I or T40Y / E94C / F98W / C109A / C136A / Y350E;

[0748] T40 / E94 / F98 / C109 / C136 / K358, such as T40Y / E94C / F98W / C109A / C136A / K358I;

[0749] E94 / F98 / C109 / C114 / C136, such as E94C / F98W / C109A / C114I / C136A;

[0750] E94 / F98 / C109 / C136 / K177, such as E94C / F98W / C109A / C136A / K177M;

[0751] E94 / F98 / C109 / C136 / Y350, such as E94C / F98W / C109A / C136A / Y350I or T40Y / E94C / F98W / C109A / C136A / Y350E;

[0752] E94 / F98 / C109 / C136 / K358, such as E94C / F98W / C109A / C136A / K358I;

[0753] T40 / E94 / F98 / C109 / C114 / C136 / K177, such as T40Y / E94C / F98W / C109A / C114I / C136A / K177M;

[0754] T40 / E94 / F98 / C109 / C114 / C136 / Y350, such as T40Y / E94C / F98W / C109A / C114I / C136A / Y350I or T40Y / E94C / F98W / C109A / C114I / C136A / Y350E;

[0755] T40 / E94 / F98 / C109 / C114 / C136 / K358, such as T40Y / E94C / F98W / C109A / C114I / C136A / K358I;

[0756] T40 / E94 / F98 / C109 / C136 / K177 / Y350, such as T40Y / E94C / F98W / C109A / C136AK177M / Y350I or T40Y / E94C / F98W / C109A / C136AK177M / Y350E;

[0757] T40 / E94 / F98 / C109 / C136 / K177 / K358, such as T40Y / E94C / F98W / C109A / C136AK177M / K358I;

[0758] T40 / E94 / F98 / C109 / C136 / Y350 / K358, such as T40Y / E94C / F98W / C109A / C136AY350I / K358I or T40Y / E94C / F98W / C109A / C136AY350E / K358I;

[0759] E94 / F98 / C109 / C114 / C136 / K177, such as E94C / F98W / C109A / C114I / C136A / K177M;

[0760] E94 / F98 / C109 / C114 / C136 / Y350, such as E94C / F98W / C109A / C114I / C136A / Y350I or E94C / F98W / C109A / C114I / C136A / Y350E;

[0761] E94 / F98 / C109 / C114 / C136 / K358, such as E94C / F98W / C109A / C114I / C136A / K358I;

[0762] E94 / F98 / C109 / C136 / K177 / Y350, such as E94C / F98W / C109A / C136AK177M / Y350I or E94C / F98W / C109A / C136AK177M / Y350E;

[0763] E94 / F98 / C109 / C136 / K177 / K358, such as E94C / F98W / C109A / C136AK177M / K358I;

[0764] E94 / F98 / C109 / C136 / Y350 / K358, such as E94C / F98W / C109A / C136AY350I / K358I or E94C / F98W / C109A / C136AY350E / K358I;

[0765] T40 / E94 / F98 / C109 / C114 / C136 / K177 / Y350, such as T40Y / E94C / F98W / C109A / C114I / C136A / K177M / Y350I or T40Y / E94C / F98W / C109A / C114I / C136A / K177M / Y350E;

[0766] T40 / E94 / F98 / C109 / C114 / C136 / K177 / K358, such as T40Y / E94C / F98W / C109A / C114I / C136A / K177M / K358I;

[0767] T40 / E94 / F98 / C109 / C114 / C136 / Y350 / K358, such as T40Y / E94C / F98W / C109A / C114I / C136A / Y350I / K358I or T40Y / E94C / F98W / C109A / C114I / C136A / Y350E / K358I;

[0768] T40 / E94 / F98 / C109 / C136 / K177 / Y350 / K358, such as T40Y / E94C / F98W / C109A / C136A / K177M / Y350I / K358I or T40Y / E94C / F98W / C109A / C136A / K177M / Y350E / K358I;

[0769] E94 / F98 / C109 / C114 / C136 / K177 / Y350, such as E94C / F98W / C109A / C114I / C136A / K177M / Y350I or E94C / F98W / C109A / C114I / C136A / K177M / Y350E;

[0770] E94 / F98 / C109 / C114 / C136 / K177 / K358, such as E94C / F98W / C109A / C114I / C136A / K177M / K358I;

[0771] E94 / F98 / C109 / C114 / C136 / Y350 / K358, such as E94C / F98W / C109A / C114I / C136A / Y350I / K358I or E94C / F98W / C109A / C114I / C136A / Y350E / K358I;

[0772] E94 / F98 / C109 / C136 / K177 / Y350 / K358, such as E94C / F98W / C109A / C136A / K177M / Y350I / K358I or E94C / F98W / C109A / C136A / K177M / Y350E / K358I;

[0773] T40 / E94 / F98 / C109 / C114 / C136 / K177 / Y350 / K358, such as T40Y / E94C / F98W / C109A / C114I / C136A / K177M / Y350I / K358I or T40Y / E94C / F98W / C109A / C114I / C136A / K177M / Y350E / K358I;

[0774] E94 / F98 / C109 / C114 / C136 / K177 / Y350 / K358, such as E94C / F98W / C109A / C114I / C136A / K177M / Y350I / K358I or E94C / F98W / C109A / C114I / C136A / K177M / Y350E / K358I;

[0775] T40 / E94 / F98 / C109 / A360, such as T40Y / E94C / F98W / C109A / A360C;

[0776] T40 / E94 / F98 / C109 / C114 / A360, such as T40Y / E94C / F98W / C109A / C114I / A360C;

[0777] T40 / E94 / F98 / C109 / K177 / A360, such as T40Y / E94C / F98W / C109A / K177M / A360C;

[0778] T40 / E94 / F98 / C109 / Y350 / A360, such as T40Y / E94C / F98W / C109A / Y350I / A360C or T40Y / E94C / F98W / C109A / Y350E / A360C;

[0779] T40 / E94 / F98 / C109 / K358 / A360, such as T40Y / E94C / F98W / C109A / K358I / A360C;

[0780] E94 / F98 / C109 / C114 / A360, such as E94C / F98W / C109A / C114I / A360C;

[0781] E94 / F98 / C109 / K177 / A360, such as E94C / F98W / C109A / K177M / A360C;

[0782] E94 / F98 / C109 / Y350 / A360, such as E94C / F98W / C109A / Y350I / A360C or E94C / F98W / C109A / Y350E / A360C;

[0783] E94 / F98 / C109 / K358 / A360, such as E94C / F98W / C109A / K358I / A360C;

[0784] T40 / E94 / F98 / C109 / C114 / K177 / A360, such as T40Y / E94C / F98W / C109A / C114I / K177M / A360C;

[0785] T40 / E94 / F98 / C109 / C114 / Y350 / A360, such as T40Y / E94C / F98W / C109A / C114I / Y350I / A360C or T40Y / E94C / F98W / C109A / C114I / Y350E / A360C;

[0786] T40 / E94 / F98 / C109 / C114 / K358 / A360, such as T40Y / E94C / F98W / C109A / C114I / K358I / A360C;

[0787] T40 / E94 / F98 / C109 / K177 / Y350 / A360, such as T40Y / E94C / F98W / C109A / K177M / Y350I / A360C or T40Y / E94C / F98W / C109A / K177M / Y350E / A360C;

[0788] / A360C

[0789] T40 / E94 / F98 / C109 / Y350 / K358 / A360, such as T40Y / E94C / F98W / C109A / Y350I / K358I / A360C or T40Y / E94C / F98W / C109A / Y350E / K358I / A360C;

[0790] E94 / F98 / C109 / C114 / K177 / A360, such as E94C / F98W / C109A / C114I / K177M / A360C;

[0791] E94 / F98 / C109 / C114 / Y350 / A360, such as E94C / F98W / C109A / C114I / Y350I / A360C or E94C / F98W / C109A / C114I / Y350E / A360C;

[0792] E94 / F98 / C109 / C114 / K358 / A360, such as E94C / F98W / C109A / C114I / K358I / A360C;

[0793] E94 / F98 / C109 / K177 / Y350 / A360, such as E94C / F98W / C109A / K177M / Y350I / A360C or E94C / F98W / C109A / K177M / Y350E / A360C;

[0794] E94 / F98 / C109 / Y350 / K358 / A360, such as E94C / F98W / C109A / Y350I / K358I / A360C or E94C / F98W / C109A / Y350E / K358I / A360C;

[0795] T40 / E94 / F98 / C109 / C114 / K177 / Y350 / A360, such as T40Y / E94C / F98W / C109A / C114I / K177M / Y350I / A360C or T40Y / E94C / F98W / C109A / C114I / K177M / Y350E / A360C;

[0796] T40 / E94 / F98 / C109 / C114 / K177 / K358 / A360, such as T40Y / E94C / F98W / C109A / C114I / K177M / K358I / A360C;

[0797] T40 / E94 / F98 / C109 / C114 / Y350 / K358 / A360, such as T40Y / E94C / F98W / C109A / C114I / Y350I / K358I / A360C or T40Y / E94C / F98W / C109A / C114I / Y350E / K358I / A360C;

[0798] T40 / E94 / F98 / C109 / K177 / Y350 / K358 / A360, such as T40Y / E94C / F98W / C109A / K177M / Y350I / K358I / A360C or T40Y / E94C / F98W / C109A / K177M / Y350E / K358I / A360C;

[0799] E94 / F98 / C109 / C114 / K177 / Y350 / A360, such as E94C / F98W / C109A / C114I / K177M / Y350I / A360C or E94C / F98W / C109A / C114I / K177M / Y350E / A360C;

[0800] E94 / F98 / C109 / C114 / K177 / K358 / A360, such as E94C / F98W / C109A / C114I / K177M / K358I / A360C;

[0801] E94 / F98 / C109 / C114 / Y350 / K358 / A360, such as E94C / F98W / C109A / C114I / Y350I / K358I / A360C or E94C / F98W / C109A / C114I / Y350E / K358I / A360C;

[0802] E94 / F98 / C109 / K177 / Y350 / K358 / A360, such as E94C / F98W / C109A / K177M / Y350I / K358I / A360C or E94C / F98W / C109A / K177M / Y350E / K358I / A360C;

[0803] T40 / E94 / F98 / C109 / C114 / K177 / Y350 / K358 / A360, such as T40Y / E94C / F98W / C109A / C114I / K177M / Y350I / K358I / A360C or T40Y / E94C / F98W / C109A / C114I / K177M / Y350E / K358I / A360C; or

[0804] E94 / F98 / C109 / C114 / K177 / Y350 / K358 / A360, such as E94C / F98W / C109A / C114I / K177M / Y350I / K358I / A360C or E94C / F98W / C109A / C114I / K177M / Y350E / K358I / A360C.

[0805] Any of these variants of SEQ ID NO: 118 may further comprise modifications or substitutions at any number and combination of positions (a) 55, (b) 156, (c) 210, and (d) 221, including at (a); (b); (c); (d); (a) and (b); (a) and (c); (a) and (d); (b) and (c); (b) and (d); (c) and (d); (a), (b) and (c); (a), (b) and (d); (a), (c) and (d); (b), (c) and (d); or (a), (b), (c), and (d). Any of these variants of SEQ ID NO: 118 may further comprise any number and combination of (a) T55K, (b) T156F, (c) T210K, and (d) N221E, including (a); (b); (c); (d); (a) and (b); (a) and (c); (a) and (d); (b) and (c); (b) and (d); (c) and (d); (a), (b) and (c); (a), (b) and (d); (a), (c) and (d); (b), (c) and (d); or (a), (b), (c), and (d).

[0806] Additional modifications / substitutions

[0807] The present invention also provides a modified DNA-dependent ATPase (Dda) helicase, wherein one or more positions corresponding to the following amino acid positions in Dda1993 are modified or substituted: 86, 90, 92, 97, 101, 102, 273, 293, 300, 301, 303, 305, 308, 310, 312, 317, 323, 328, 332, 334, 335, 336, 337, 339, 351, 354, 359, 361, 364, 366, 368, 371, 374, 376, 377, 379 and 388. The present invention also provides a modified DNA-dependent ATPase (Dda) helicase, wherein one or more positions corresponding to the following amino acid positions in Dda1993 are modified or substituted: 351, 354 and 361. Any number and combination of these modifications / substitutions can be made, including 351, 354, 361, 351 and 354, 351 and 361, 354 and 361, or 351, 354 and 361. These positions can be modified or substituted individually or in combination with any modification or substitution of the invention described above.

[0808] The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118 comprising one or more of the following substitutions: K86A, V90T, Y92A, Y92D, Y92F, Y92G, Y92H, Y92N, Y92Q, Y92S, Y92T, Y92V, Y92W, L97H, K101A, E102G, E273A, N293H, I200A, I300F, E301I, E303I, Y305L, F308I, F308L, K310A, K310I, K310L, R312I, R312L, R312M, E317I, E317Y, W323H, E328I, D332A, D333 2L, E334A, E334I, E334Y, Y335A, Y335I, Y335L, Y336L, R337I, R337L, R337M, K339A, K339I, K339L, K351I, K351Q, L354Q, L354A, T359L, E361 T, E361I, E361Q, K364A, K364R, W366L, K368A, K368I, K368L, K371I, K371L, K371M, W374A, W374L, W374L, D376S, F377A, D379A, D379L or K388R. The helicase of the present invention preferably comprises a variant of SEQ ID NO: 118 comprising one or more of the following substitutions: (a) K351I or K351Q, (b) L354A or L354Q and (c) E361I or E361Q. The variant may comprise (a), (b), (c), (a) and (b), (a) and (c), (b) and (c) or (a), (b) and (c). These substitutions may be made alone or in combination with any of the modifications or substitutions of the present invention described above.

[0809] Variants

[0810] The variant of helicase is an enzyme having an amino acid sequence different from the wild-type helicase and having polynucleotide binding activity. In particular, the variant of any one of SEQ ID NO:118 to 133 is an enzyme having an amino acid sequence different from any one of SEQ ID NO:118 to 133 and having polynucleotide binding activity. The polynucleotide binding activity can be determined using methods known in the art. Suitable methods include but are not limited to fluorescence anisotropy, tryptophan fluorescence and electrophoretic mobility shift assay (EMSA). For example, the ability of the variant to bind to single-stranded polynucleotides can be determined as described in the examples.

[0811] The variant has helicase activity. This can be measured in a variety of ways. For example, the ability of the variant to translocate along a polynucleotide can be measured using electrophysiology, fluorescence assays, or ATP hydrolysis.

[0812] The variant may include modifications that facilitate handling of the polynucleotide encoding the helicase and / or facilitate its activity at high salt concentrations and / or at room temperature.

[0813] The variant is preferably at least 20% homologous to the amino acid sequence of any one of SEQ ID NOs: 118 to 133 based on amino acid similarity or identity over the entire length of the sequence. More preferably, the variant polypeptide may be at least 30%, at least 40%, at least 45%, at least 50%, 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 based on amino acid identity to the amino acid sequence of any one of SEQ ID NOs: 118 to 133 over the entire sequence. More preferably, the variant polypeptide may be at least 30%, at least 40%, at least 45%, at least 50%, 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% identical to the amino acid sequence of any one of SEQ ID NOs: 118 to 133 over the entire sequence. At least 70%, such as at least 80%, at least 85%, at least 90% or at least 95% amino acid identity may exist over a stretch of 100 or more, such as 150, 200, 300, 400 or 500 or more consecutive amino acids ("hard homology"). Homology is determined as described below. In particular, in addition to the specific modifications discussed above, variants of any one of SEQ ID NOs: 118 to 133 may comprise one or more substitutions, one or more deletions and / or one or more additions, as discussed below.

[0814] Preferred variants of any one of SEQ ID NOs: 118 to 133 have an unnatural amino acid at the amino-(N-) terminus and / or the carboxyl (C-) terminus, such as Faz. Preferred variants of any one of SEQ ID NOs: 118 to 133 have a cysteine ​​residue at the amino-(N-) terminus and / or the carboxyl (C-) terminus. Preferred variants of any one of SEQ ID NOs: 118 to 133 have a cysteine ​​residue at the amino-(N-) terminus and an unnatural amino acid at the carboxyl (C-) terminus, such as Faz, or vice versa.

[0815] Preferred variants of SEQ ID NO: 118 contain one or more, such as all, of the following modifications E54G, D151E, I196N and G357A.

[0816] No connection

[0817] In a preferred embodiment, none of the introduced cysteines and / or unnatural amino acids in the modified helicase of the invention are linked to each other.

[0818] Linking two or more of the introduced cysteine ​​and / or unnatural amino acids

[0819] In another preferred embodiment, two or more of the introduced cysteine ​​and / or unnatural amino acids in the modified helicase of the invention are linked to each other. This generally reduces the ability of the helicase of the invention to debind from a polynucleotide.

[0820] Any number and combination of cysteine ​​and / or non-natural amino acids can be connected to one another. For example, 3, 4, 5, 6, 7, 8 or more cysteines and / or non-natural amino acids can be connected to one another. One or more cysteines can be connected to one or more cysteines. One or more cysteines can be connected to one or more non-natural amino acids, such as Faz. One or more non-natural amino acids, such as Faz, can be connected to one or more non-natural amino acids, such as Faz.

[0821] The two or more cysteines and / or unnatural amino acids can be linked in any manner. The linking can be transient, such as non-covalent. Even transient linking can reduce the unbinding of the polynucleotide to the helicase.

[0822] The two or more cysteines and / or unnatural amino acids are preferably linked via an affinity molecule. Suitable affinity molecules are known in the art. The affinity molecule is preferably (a) a complementary polynucleotide (WO 2010 / 086602, which is incorporated herein by reference in its entirety), (b) an antibody or fragment thereof and a complementary epitope (Biochemistry 6th edition, WH Freeman and co (2007) pp953-954), (c) a peptide zipper (O'Shea et al., Science 254(5031):539-544), (d) capable of enhancing the interaction through β-sheets (Remaut and Waksman Trends Biochem. Sci. (2006) 31 436-444), (e) capable of hydrogen bonding, π stacking or salt bridge formation, (f) a rotaxane (Xiang Ma and He Tian Chem. Soc. Rev., 2010, 39, 70-80), (g) an aptamer and a complementary protein (James, W. in Encyclopedia of Analytical Chemistry, R. A. Meyers (ed.) pp. 4848–4871 John Wiley & Sons Ltd, Chichester, 2000) or (h) hemichelates (Hammerstein et al. J Biol Chem. 2011 Apr 22;286(16):14324–14334). For (e), hydrogen bonding occurs between a proton bound to an electronegative atom and another electronegative atom. Pi stacking requires two aromatic rings that can be stacked together with the planes of the rings parallel. Salt bridges occur between groups that can delocalize their electrons over several atoms, such as between aspartic acid and arginine.

[0823] Two or more parts can be transiently linked via hexa-his tags or Ni-NTA.

[0824] The two or more cysteines and / or unnatural amino acids are preferably permanently linked. In the context of the present invention, a link is permanent if it is not broken when a helicase is used, or cannot be broken without intervention on the part of the user (such as using reduction to open the -SS- bond).

[0825] The two or more cysteines and / or unnatural amino acids are preferably covalently attached.The two or more cysteines and / or unnatural amino acids can be covalently attached using any method known in the art.

[0826] Two or more cysteines and / or non-natural amino acids can be covalently attached via their naturally occurring amino acids (such as cysteine, threonine, serine, aspartate, asparagine, glutamate and glutamine). Naturally occurring amino acids can be modified to promote attachment. For example, naturally occurring amino acids can be modified by acylation, phosphorylation, glycosylation or farnesylation. Other suitable modifications are known in the art. Modifications to naturally occurring amino acids can be post-translational modifications. Two or more cysteines and / or non-natural amino acids can be attached via amino acids introduced into their sequence. Such amino acids are preferably introduced by substitution. The amino acids introduced can be cysteine ​​or non-natural amino acids that promote attachment. Suitable non-natural amino acids include, but are not limited to, 4-azido-L-phenylalanine (Faz), any of the amino acids numbered 1-71 included in Figure 1 of Liu C.C. and Schultz PG, Annu. Rev. Biochem., 2010, 79, 413-444, or any of the amino acids listed below. The introduced amino acids can be modified as described above.

[0827] In a preferred embodiment, two or more cysteines and / or non-natural amino acids are connected using a joint. The joint molecule will be discussed in more detail below. A suitable method of connection is cysteine ​​connection. This will be discussed in more detail below. Two or more cysteines and / or non-natural amino acids are preferably connected using one or more (such as two or three) joints. One or more joints can be designed to reduce the size of the opening as described above or to close the opening. If one or more joints are used to close the opening as described above, at least a portion of the one or more joints is preferably oriented so that it is not parallel to the polynucleotide when bound by the helicase. More preferably, all joints are oriented in this way. If one or more joints are used to close the opening as described above, when the polynucleotide is bound by the helicase, at least a portion of the one or more joints is preferably passed through the opening in an orientation that is not parallel to the polynucleotide. More preferably, all joints are passed through the opening in this way. In these embodiments, at least a portion of the one or more joints can be perpendicular to the polynucleotide. This type of orientation effectively closes the opening so that the polynucleotide cannot be debonded by the helicase through the opening.

[0828] Each connector may have two or more functional ends, such as two, three or four functional ends. Suitable configurations of ends in connectors are well known in the art.

[0829] One or more ends of the one or more linkers are preferably covalently attached to the helicase. If one end is covalently attached, the one or more linkers can transiently connect the two or more cysteines and / or unnatural amino acids, as discussed above. If two or all ends are covalently attached, the one or more linkers permanently connect the two or more cysteines and / or unnatural amino acids.

[0830] The one or more linkers are preferably amino acid sequences and / or chemical cross-linking agents.

[0831] Suitable amino acid linkers, such as peptide linkers, are known in the art. The length, flexibility and hydrophilicity of the amino acid or peptide linker are generally designed so that it reduces the size of the opening but does not interfere with the function of the helicase. Preferred flexible peptide linkers are stretches of 2 to 20, such as 4, 6, 8, 10 or 16 serine and / or glycine amino acids. More preferred flexible linkers include (SG)1, (SG)2, (SG)3, (SG)4, (SG)5, (SG)8, (SG) 10 、(SG) 15 or (SG) 20 , wherein S is serine and G is glycine. Preferred rigid linkers are stretches of 2 to 30, such as 4, 6, 8, 16 or 24 proline amino acids. More preferred rigid linkers include (P) 12 , wherein P is proline. The amino acid sequence of the linker preferably comprises a polynucleotide binding portion. Such portions and the advantages associated with their use are discussed below.

[0832] Suitable chemical cross-linking agents are well known in the art. Suitable chemical cross-linking agents include, but are not limited to, those comprising the following functional groups: maleimide, active ester, succinimide, azide, alkyne (such as dibenzocyclooctynol (DIBO or DBCO), difluorocycloalkyne and linear alkyne), phosphine (such as those used for traceless and non-traceless Staudinger linkage), haloacetyl (such as iodoacetamide), phosgene-type reagents, sulfonyl chloride reagents, isothiocyanate, acyl halide, hydrazine, disulfide, vinyl sulfone, aziridine and photoreactive reagents (such as aryl azide, diaziridine).

[0833] The reaction between amino acids and functional groups may be spontaneous, such as cysteine / maleimide, or may require external reagents, such as Cu(I) for attachment of azides and linear alkynes.

[0834] The joint can comprise any molecule extending across the desired distance. The length of the joint can vary from one carbon (phosgene type joint) to many angstroms. The example of linear molecule includes but is not limited to polyethylene glycol (PEG), polypeptide, polysaccharide, deoxyribonucleic acid (DNA), peptide nucleic acid (PNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), saturated nucleic acid and unsaturated hydrocarbon, polyamide. These joints can be inert or reactive, particularly they can be chemically cleaved at the position of limitation, or they themselves can be modified with fluorophore or ligand. The joint is preferably resistant to dithiothreitol (DTT).

[0835] Preferred crosslinking agents include 2,5-dioxopyrrolidin-1-yl 3-(pyridin-2-yldithio) propionate, 2,5-dioxopyrrolidin-1-yl 4-(pyridin-2-yldithio) butyrate and 2,5-dioxopyrrolidin-1-yl 8-(pyridin-2-yldithio) octanoate, bismaleimide PEG 1k, bismaleimide PEG 3.4k, bismaleimide PEG 5k, bismaleimide PEG 10k, bis(maleimido)ethane (BMOE), bismaleimidohexane (BMH), 1,4-bismaleimidobutane (BMB), 1,4-bismaleimido-2,3-dihydroxybutane (BMDB), BM[PEO]2(1,8-bismaleimidodiethylene glycol), BM[PEO]3(1,11-bismaleimidotriethylene glycol), tris[2-maleimidoethyl]amine (TMEA), DTME dithiobismaleimidoethane, bismaleimide PEG3, bismaleimide PEG11, DBCO-maleimide, DBCO-PEG4-maleimide, DBCO-PEG4-NH2, DBCO-PEG4-NHS, DBCO-NHS, DBCO-PEG-DBCO 2.8kDa, DBCO-PEG-DBCO 4.0 kDa, DBCO-15 atoms-DBCO, DBCO-26 atoms-DBCO, DBCO-35 atoms-DBCO, DBCO-PEG4-SS-PEG3-biotin, DBCO-SS-PEG3-biotin, DBCO-SS-PEG11-biotin, (succinimidyl 3-(2-pyridyldithio) propionate (SPDP), and maleimide-PEG (2 kDa)-maleimide (ALPHA, OMEGA-bismaleimide poly (ethylene glycol)). The most preferred crosslinker is maleimido-propyl-SRDFWRS-(1,2-diaminoethane)-propyl-maleimide.

[0836] One or more linkers may be cleavable. This will be discussed in more detail below.

[0837] Two or more cysteines and / or non-natural amino acids can be connected using two different joints that are specific to each other. One in the joint is attached to a part, and the other is attached to another part. The joint should react to form a modified helicase of the present invention. Two or more cysteines and / or non-natural amino acids can be connected using the hybridization joint described in WO 2010 / 086602 (incorporated herein by reference in its entirety). In particular, two or more cysteines and / or non-natural amino acids can be connected using two or more joints, each joint comprising a hybridizable region and a group that can form a covalent bond. The hybridizable region in the joint hybridizes and connects two or more cysteines and / or non-natural amino acids. Then, the cysteines and / or non-natural amino acids connected are coupled via forming a covalent bond between the groups. Any specific joint disclosed in WO 2010 / 086602 (incorporated herein by reference in its entirety) can be used according to the present invention.

[0838] Two or more cysteines and / or unnatural amino acids can be modified and then attached using a chemical cross-linker that is specific for both modifications.Any of the cross-linkers discussed above can be used.

[0839] The linker may be labeled. Suitable labels include, but are not limited to, fluorescent molecules (such as Cy3 or 555), radioisotopes (e.g. 125 I. 35 The present invention relates to a method for the identification of a protein, wherein the protein is expressed as a target site, such as a target protein, ...

[0840] A preferred method of linking two or more cysteines is via cysteine ​​ligation. This can be mediated by bifunctional chemical cross-linkers or by amino acid linkers with terminal cysteine ​​residues.

[0841] The length, reactivity, specificity, rigidity and solubility of any bifunctional linker can be designed to fully reduce the size of the opening and retain the function of the helicase. Suitable linkers include bismaleimide cross-linkers, such as 1,4-bis (maleimido) butane (BMB) or bis (maleimido) hexane. A shortcoming of bifunctional linkers is that if attachment at a specific site is preferred, the helicase is required to be free of other surface accessible cysteine ​​residues, because the combination of bifunctional linkers and surface accessible cysteine ​​residues may be difficult to control and may affect substrate binding or activity. If the helicase does contain several accessible cysteine ​​residues, it may be necessary to modify the helicase to remove them while ensuring that modification does not affect the folding or activity of the helicase. This is discussed in WO 2010 / 086603 (incorporated herein by reference in its entirety). The reactivity of cysteine ​​residues can be enhanced by the modification of adjacent residues (such as residues on a peptide linker). For example, basic groups flanking arginine, histidine, or lysine residues will alter the pKa of the cysteine ​​thiol group to the more reactive S - The pKa of the group. The reactivity of cysteine ​​residues can be protected by thiol protecting groups such as 5,5'-dithiobis-(2-nitrobenzoic acid) (dTNB). Before the linker is attached, they can react with one or more cysteine ​​residues of the helicase. Surface accessible cysteines can be selectively deprotected using a reducing agent immobilized on the beads (e.g., immobilized tris(2-carboxyethyl)phosphine, TCEP). Cysteine ​​ligation is discussed in more detail below.

[0842] Another preferred method of attachment is via Faz linkage. This can be mediated by a bifunctional chemical linker or by a polypeptide linker with a terminally present Faz residue.

[0843] Other modifications

[0844] The helicase of the present invention can also be modified to increase the attraction between (i) the tower domain and (ii) the pin domain and / or the 1A domain. Any known chemical modification can be performed according to the present invention. These types of modifications are disclosed in WO2015 / 055981 (incorporated herein by reference in its entirety).

[0845] In particular, the invention provides helicases of the invention wherein at least one charged amino acid has been introduced into (i) the tower domain and / or (ii) the pin domain and / or (iii) the 1A (RecA-like motor) domain, wherein the helicase has the ability to control the movement of a polynucleotide. The ability of the helicase to control the movement of a polynucleotide can be measured as discussed above. The invention preferably provides helicases of the invention wherein at least one charged amino acid has been introduced into (i) the tower domain and (ii) the pin domain and / or the 1A domain.

[0846] At least one charged amino acid can be negatively charged or positively charged.At least one charged amino acid is preferably oppositely charged to any amino acid that interacts with it in the helicase.For example, at least one positively charged amino acid can be introduced into the tower domain at a position that interacts with the negatively charged amino acid in the pin domain.Usually, at least one charged amino acid is introduced into a non-charged position in a wild-type (i.e., unmodified) helicase.At least one charged amino acid can be used to replace at least one oppositely charged amino acid in the helicase.For example, a positively charged amino acid can be used to replace a negatively charged amino acid.

[0847] Suitable charged amino acids are discussed above. At least one charged amino acid can be natural, such as arginine (R), histidine (H), lysine (K), aspartic acid (D) or glutamic acid (D). Alternatively, at least one charged amino acid can be artificial or non-natural. Any number of charged amino acids can be introduced into each domain. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more charged amino acids can be introduced into each domain.

[0848] The helicase preferably comprises a variant of SEQ ID NO: 118 comprising a positively charged amino acid at one or more of the following positions: (i) 93; (ii) 354; (iii) 360; (iv) 361; (v) 94; (vi) 97; (vii) 155; (viii) 357; (ix) 100; and (x) 127. The helicase preferably comprises a variant of SEQ ID NO: 118 comprising a negatively charged amino acid at one or more of the following positions: (i) 354; (ii) 358; (iii) 360; (iv) 364; (v) 97; (vi) 123; (vii) 155; (viii); 357; (ix) 100; and (x) 127. The helicase preferably comprises a variant of any one of SEQ ID NOs: 119 to 133 comprising a positively charged amino acid or a negatively charged amino acid at a position corresponding to those positions in SEQ ID NO: 118 as defined in any one of (i) to (x). Positions in any one of SEQ ID NOs: 119 to 133 corresponding to those positions in SEQ ID NO: 118 can be identified using the following alignment of SEQ ID NOs: 118 to 133.

[0849] The helicase preferably comprises a variant of SEQ ID NO: 118, which has been modified by introducing at least one charged amino acid such that it comprises oppositely charged amino acids at the following positions: (i) 93 and 354; (ii) 93 and 358; (iii) 93 and 360; (iv) 93 and 361; (v) 93 and 364; (vi) 94 and 354; (vii) 94 and 358; (viii) 94 and 360; (ix) 94 and 361; (x) 94 and 364; (xi) 97 and 354; (xii) 361; (xxv) 155 and 364. The helicase of the present invention preferably comprises a variant of any one of SEQ ID NOs: 119 to 133, which comprises oppositely charged amino acids at positions corresponding to those in SEQ ID NO: 118 as defined in any one of (i) to (xxv).

[0850] The present invention also provides a helicase, wherein (i) at least one charged amino acid has been introduced into the tower domain, and (ii) at least one oppositely charged amino acid has been introduced into the pin domain and / or the 1A (RecA-like motor) domain, wherein the helicase has the ability to control the movement of polynucleotides. At least one charged amino acid can be ...

Claims

1. A modified DNA-dependent ATPase (Dda) helicase, wherein the helicase comprises a modification or substitution at one or more of the positions corresponding to amino acid positions 55, 114, 156, 177, 210, 221, 350 and 358 in Dda1993.

2. The modified DNA-dependent ATPase (Dda) helicase of claim 1, wherein (a) the amino acid corresponding to position 55 is substituted by D, E, K, N or S, (b) the amino acid corresponding to position 114 is substituted by A, V, I, L, M, F, Y, W, G, P, S, T, N or Q, (c) the amino acid corresponding to position 156 is substituted by A, E, F, G, I, L, M, P, S, V, Y, D, K or N, (d) the amino acid corresponding to position 177 is substituted by D, E, F, G, H, I, L, M, N, Q, R, S, T, V, W or Y, or (e) the amino acid corresponding to position 210 is substituted by D, E, K, S, N, R, H or Y, (f) the amino acid at position 221 is substituted by D, K, E, Q, R, A, H, L, T or Y, (g) the amino acid corresponding to position 350 is substituted by A, D, E, G, K, L, N, Q, R, T, V, H or M, or by D, E, A, V, I, L, M, F, W, R, H, K, L, S, T, N or Q, and / or (h) the amino acid corresponding to position 358 is substituted by D, E, A, V, I, L, M, F, Y, W, R, H, L, S, T, N or Q.

3. The modified Dda helicase according to claim 1 or 2, wherein the helicase further comprises a modification or substitution at the position corresponding to amino acid position 40 in Dda1993.

4. The modified DNA-dependent ATPase (Dda) helicase according to claim 3, wherein the amino acid corresponding to position 40 is substituted by A, V, I, L, M, F, Y or W.

5. A construct comprising a helicase according to any one of claims 1 to 4 and an additional polynucleotide binding portion, wherein the helicase is attached to the polynucleotide binding portion and the construct has the ability to control the movement of an analyte.

6. The construct according to claim 5, wherein the construct comprises two or more helicases according to any one of claims 1 to 4.

7. A polynucleotide comprising a sequence encoding the helicase according to any one of claims 1 to 4 or the construct according to claim 5 or 6.

8. A vector comprising the polynucleotide according to claim 7, which is operably linked to a promoter.

9. A host cell comprising the vector according to claim 8.

10. A method for preparing a helicase according to any one of claims 1 to 4 or a construct according to claim 5 or 6, the method comprising: Expressing the polynucleotide according to claim 7, transfecting cells with the vector according to claim 8, or culturing the host cell according to claim 9.

11. A method of controlling the movement of an analyte, the method comprising contacting the analyte with a helicase according to any one of claims 1 to 4 or a construct according to claim 5 or 6, and thereby controlling the movement of the analyte.

12. The method of claim 11, wherein the method is used to control the movement of an analyte through a transmembrane pore.

13. A method for characterizing a target analyte, the method comprising: (a) contacting the target analyte with a transmembrane pore and a helicase according to any one of claims 1 to 4 or a construct according to claim 5 or 6, such that the helicase or construct controls the movement of the target analyte through the pore; and (b) taking one or more measurements as the polynucleotide moves relative to the pore, wherein the measurements are indicative of one or more characteristics of the target analyte and thereby characterise the target analyte.

14. The method of claim 13, wherein the one or more features are selected from (i) the length of the target analyte, (ii) the identity of the target analyte, (iii) the sequence of the target analyte, (iv) the secondary structure of the target analyte, and (v) whether the target analyte is modified.

15. The method of claim 14, wherein the target analyte is modified by methylation, oxidation, damage, with one or more proteins, or with one or more labels, tags or spacers.

16. The method according to any one of claims 13 to 15, wherein the one or more characteristics of the target analyte are measured by electrical measurement and / or optical measurement.

17. The method of claim 16, wherein the electrical measurement is a current measurement, an impedance measurement, a tunneling measurement, or a field effect transistor (FET) measurement.

18. The method according to claim 17, wherein the method comprises: (a) contacting the target analyte with a transmembrane pore and a helicase according to any one of claims 1 to 4 or a construct according to claim 5 or 6, such that the helicase or the construct controls the movement of the target analyte through the pore; and (b) measuring an electric current passing through the pore as the polynucleotide moves relative to the pore, wherein the electric current is indicative of one or more characteristics of the target analyte and thereby characterizes the target analyte.

19. A method according to any one of claims 13 to 18, wherein the method further comprises the step of applying a voltage across the pore to form a complex between the pore and the helicase or construct.

20. The method of any one of claims 13 to 20, wherein at least a portion of the polynucleotide is double-stranded.

21. A method according to any one of claims 13 to 20, wherein the pore is a transmembrane protein pore or a solid state pore.

22. The method of claim 21, wherein the transmembrane protein pore is derived from hemolysin, leukocidin, Mycobacterium smegmatis porin A (MspA), MspB, MspC, MspD, lysin, outer membrane porin F (OmpF), outer membrane porin G (OmpG), outer membrane phospholipase A, Neisseria autotransporter lipoprotein (NalP) and WZA, CsgG, CsgG / CsgF, lysin, ClyA, Sp1 or hemolysin fragaceatoxin C (FraC).

23. The method according to claim 22, wherein the transmembrane protein is formed by six to ten identical subunits as shown in SEQ ID NO: 3, or (b) a variant thereof, wherein one or more of the subunits have at least 40% identity with SEQ ID NO: 3 over the entire sequence, and has pore activity.

24. A method of forming a sensor for characterizing a target analyte, the method comprising forming a complex between (a) a pore and (b) a helicase according to any one of claims 1 to 4 or a construct according to claim 5 or 6, and thereby forming a sensor for characterizing the target analyte.

25. The method of claim 24, wherein the complex is formed by: (a) contacting the pore with the helicase or construct in the presence of the target analyte, and (a) applying an electric potential across the pore.

26. The method of claim 25, wherein the potential is a voltage potential or a chemical potential.

27. The method of claim 26, wherein the complex is formed by covalently attaching the pore to the helicase or construct.

28. A sensor for characterising a target analyte, the sensor comprising a complex between (a) a pore and (b) a helicase according to any one of claims 1 to 4 or a construct according to claim 5 or 6.

29. Use of a helicase according to any one of claims 1 to 4 or a construct according to claim 5 or 6 to control the movement of an analyte of interest through a pore.

30. A kit for characterizing a target analyte, the kit comprising (a) a pore and a helicase according to any one of claims 1 to 4 or a construct according to claim 5 or 6; or (b) A helicase according to any one of claims 1 to 4 or a construct according to claim 5 or 6 and one or more cargo moieties.

31. A device for characterizing a target analyte in a sample, the device comprising (a) a plurality of pores and (b) a plurality of helicases according to any one of claims 1 to 4 or a plurality of constructs according to claim 5 or 6.

32. A method for producing a helicase according to any one of claims 1 to 4, the method comprising: (a) providing a helicase; as well as (b) modifying the helicase to produce the helicase according to any one of claims 1 to 4.

33. The method of claim 32, wherein the method further comprises (c) determining whether the resulting helicase is capable of controlling the movement of a polynucleotide.

34. A method of producing a construct according to claim 5 or 6, the method comprising attaching a helicase according to any one of claims 1 to 4 to a further polynucleotide binding moiety, and thereby producing the construct.

35. The method of claim 34, wherein the method further comprises determining whether the resulting construct is capable of controlling the movement of a polynucleotide.

36. A series of two or more helicases attached to a polynucleotide, wherein at least one of the two or more helicases is a helicase according to any one of claims 1 to 4.

37. A method for improving the movement of a target analyte relative to a transmembrane pore when the movement of the target analyte relative to the transmembrane pore is controlled by a DNA-dependent ATPase (Dda) helicase, wherein the DNA-dependent ATPase (Dda) helicase is modified to contain a substitution at one or more of the positions corresponding to amino acid positions 55, 114, 156, 177, 210, 221, 350 and 358 in Dda 1993 and / or at a position corresponding to amino acid position 40 in Dda 1993, which improves the movement of the target analyte relative to the transmembrane pore.

38. An isolated CsgG pore, or a homologue or mutant thereof, or an isolated pore complex comprising a CsgG pore, or a homologue or mutant thereof, and a modified CsgF peptide, or a homologue or mutant thereof, wherein the CsgG pore comprises at least one monomer comprising a modification at one or more of positions W97, Q100, E101, N102 and T104 in SEQ ID NO:

117.

39. The isolated pore complex of claim 38, wherein the modified CsgF peptide, or a homologue or mutant thereof, is inserted into the lumen of the CsgG pore, or a homologue or mutant thereof.

40. The isolated pore complex of claim 38 or 39, wherein the pore complex has two or more channel constrictions, including a CsgG channel constriction and a CsgF channel constriction.

41. The isolated pore complex of claim 40, wherein the CsgF channel constriction has a diameter in the range of 0.5 nm to 2.0 nm.

42. An isolated transmembrane pore complex comprising a pore or pore complex according to any one of claims 38 to 41 and components of a membrane.

43. A membrane comprising a pore or pore complex according to any one of claims 38 to 41.

44. An array comprising a plurality of the membranes of claim 43.

45. A system comprising (a) a membrane according to claim 43 or an array according to claim 44, (b) a device for applying an electric potential across the membrane, and (c) a device for detecting an electrical signal or an optical signal across the membrane.

46. ​​A method for producing a transmembrane pore complex according to claim 42, the method comprising co-expressing the CsgG pore or a homologue or mutant thereof, and the modified CsgF peptide or a homologue or mutant thereof in a suitable host cell, thereby allowing transmembrane pore complex formation in vivo.

47. A method for producing an isolated pore complex according to any one of claims 38 to 41, the method comprising contacting a CsgG monomer, or a homologue or mutant thereof, with the modified CsgF peptide, or a homologue or mutant thereof, thereby allowing reconstitution of the isolated pore complex in vitro.

48. A method for determining the presence, absence, or one or more characteristics of a target analyte, the method comprising the steps of: (a) contacting the target analyte with an isolated pore or an isolated pore complex according to any one of claims 38 to 41 or with a transmembrane pore complex according to claim 42, such that the target analyte moves relative to or into the pore complex; as well as (b) taking one or more measurements as the analyte moves through the pore complex and thereby determining the presence, absence or one or more characteristics of the analyte.

49. The method of claim 48, wherein the movement of the target analyte relative to the pore complex is controlled by a polynucleotide binding protein, a Dda helicase according to any one of claims 1 to 4, or a construct according to claim 5 or 6.

50. The method of claim 48 or 49, wherein the analyte is a polynucleotide.

51. The method according to claim 48 or 49, wherein the analyte is a (poly)peptide.

52. The method of claim 48 or 49, wherein the analyte is a polysaccharide.

53. The method of claim 50, comprising determining one or more characteristics selected from: (i) the length of the polynucleotide, (ii) the identity of the polynucleotide, (iii) the sequence of the polynucleotide, (iv) the secondary structure of the polynucleotide, and (v) whether the polynucleotide is modified.

54. A method of characterising a polynucleotide or (poly)peptide using the isolated pore or isolated pore complex according to any one of claims 38 to 41 or the transmembrane pore complex according to claim 42.

55. The method of any one of claims 46 to 54, wherein the CsgG pore or homologue or mutant thereof comprises six to ten monomers.

56. Use of an isolated pore or an isolated pore complex according to any one of claims 38 to 41 or a transmembrane pore complex according to claim 42 for determining the presence, absence or one or more characteristics of a target analyte.

57. A method for changing the speed at which a target analyte passes through a pore, the method comprising contacting the target analyte with a separated pore or separated pore complex according to any one of claims 38 to 41 or with a transmembrane pore complex according to claim 42, so that the target analyte moves relative to the pore complex or moves into the pore complex.

58. The method of claim 57, wherein the movement of the target analyte relative to the pore complex is controlled by the Dda helicase according to any one of claims 1 to 4 or the construct according to claim 5 or 6.

59. A kit for characterizing a target analyte, the kit comprising (a) an isolated pore or an isolated pore complex according to any one of claims 38 to 41, and one or both of (b) components of a membrane and (c) a polynucleotide binding protein.

60. A method of characterizing a target analyte, the method comprising: (a) contacting the target analyte with an isolated pore or an isolated pore complex according to any one of claims 38 to 41 and a DNA-dependent ATPase (Dda) helicase according to any one of claims 1 to 4 or a helicase construct according to claim 5 or 6, such that the helicase or construct controls movement of the target analyte through the pore or pore complex; and (b) taking one or more measurements as the polynucleotide moves relative to the pore or pore complex, wherein the measurements are indicative of one or more characteristics of the target analyte and thereby characterize the target analyte.

61. A kit for characterising a target analyte, the kit comprising (a) a DNA-dependent ATPase (Dda) helicase according to any one of claims 1 to 4 or a helicase construct according to claim 5 or 6, and (b) an isolated CsgG pore or an isolated pore complex according to any one of claims 38 to 41.

62. A device comprising a transmembrane protein pore or pore complex according to any one of claims 38 to 41 inserted into an extracorporeal membrane.

63. A device produced by a method comprising: (i) obtaining an isolated pore or an isolated pore complex according to any one of claims 38 to 41, and (ii) contacting the isolated pore or isolated pore complex with an extracorporeal membrane such that the pore is inserted into the extracorporeal membrane.

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