Thermostable mismatch endonuclease mutants

Abstract

The present invention provides a GG-specific mismatch endonuclease variant, a TT-specific mismatch endonuclease variant, and a GT / TG-specific mismatch endonuclease variant. The present invention also provides a mismatch specific cleaving reaction using said variant, a method for removing errors in a nucleic acid amplification reaction using a mismatch nuclease, a method for suppressing amplification of a nucleic acid having a specific base sequence during a nucleic acid amplification reaction, and a method for detecting a nucleic acid having a single base polymorphic mutation using said suppression method.

Description

[Technical Field]

[0001] The present invention relates to a heat-stable mismatch endonuclease mutant that recognizes and cleaves mismatched base pairs (hereinafter sometimes simply referred to as mismatches) in double-stranded nucleic acids, a composition containing the mismatch endonuclease mutant, and a method using the mismatch endonuclease mutant. [Background technology]

[0002] Recent advances in biotechnology have been remarkable, particularly in large-scale genome analysis, which has led to the accumulation of vast amounts of genome sequence information. Furthermore, by combining this information with various physiological function analyses, numerous functional gene mutations have been identified. These mutation analyses are being used not only for the improvement of agricultural crops and the isolation and creation of useful microorganisms, but also for human genetic diagnosis, bringing great benefits to everyday life.

[0003] In addition to direct analysis of genome sequences, methods for analyzing these mutations have included the use of enzymes that recognize mismatches. One method involves pairing mutant and wild-type DNA to detect mismatches by binding a factor with specific binding ability. A typical example is the search for mutation sites using the MutS, MutT, and MutL complex of Escherichia coli (Patent Document 1).

[0004] Another method is to use a mismatch endonuclease that specifically cleaves mismatch sites. In this case, the presence or absence of a mutation and its location are detected by analyzing DNA fragments cleaved near the mismatch using the mismatch endonuclease. A typical example is a method that uses the CelI gene product derived from celery (Patent Document 2), which has actually been used to analyze mutant bases. However, this enzyme is not heat-resistant and cannot be directly used in methods that involve high-temperature reaction processes, such as PCR. Therefore, detecting mutant bases requires four steps: amplification, mismatch formation, mismatch cleavage, and detection.

[0005] Other known mismatch endonucleases are derived from Pyrococcus furiosus, Methanocaldococcus jannaschii, Thermococcus barophilus, and Thermococcus kodakarensis (Patent Documents 3 and 4). However, these mismatch endonucleases recognize and cleave multiple mismatches in mismatch recognition (substrate specificity), and their substrate specificity poses a problem.

[0006] In addition, there are cases where the genetic mutation to be detected does not match the substrate specificity of the mismatch endonuclease and cannot be analyzed as is. In such cases, the only way to specifically detect the genetic mutation is to add an appropriately designed nucleic acid called an inhibitory oligonucleotide to the reaction system (Patent Document 5).

[0007] Another biotechnology technology that has had a major impact beyond mutation analysis is nucleic acid amplification technology. Polymerase chain reaction (PCR), a representative example of this nucleic acid amplification technology, allows for the simple amplification of desired nucleic acid fragments in a test tube. It has become an essential experimental method not only in genetic research but also in a wide range of fields, including biology, medicine, and agriculture. PCR is also used to detect mutant genes and analyze DNA methylation. Isothermal nucleic acid amplification methods such as LAMP and ICAN do not require special equipment and are therefore used as less expensive nucleic acid detection methods. Furthermore, in the recent trend toward whole-genome structural analysis, whole genome amplification is an important technique, especially when analyzing rare samples.

[0008] A problem with these nucleic acid amplification methods is the probability of incorrect base incorporation. Although this probability has been reduced through improvements in polymerases, it still poses a hurdle to precise analysis.

[0009] Nucleic acid amplification techniques are used not only to amplify DNA with a specific base sequence, but also to amplify a mixture of DNA that shares a common base sequence at both ends. Specific examples include the construction of genomic and cDNA libraries, but in this process, DNA molecules with a high content are preferentially amplified, which can hinder the analysis and screening of diverse types of DNA.

[0010] To solve this problem, normalization using self-hybridization has been used to reduce the proportion of DNA with a high content (Non-Patent Document 1). SSH-PCR, which combines PCR and self-hybridization, has also been used (Non-Patent Document 2), but this carries the risk of removing DNA that is homologous to the DNA with a high content.

[0011] Furthermore, in DNA detection using nucleic acid amplification methods, the amplification of the target DNA may compete with the amplification of non-target DNA. In other words, non-target DNA may also be amplified simultaneously, making it difficult to detect the target DNA. Although real-time PCR using probes such as cycling probes or TaqMan probes can sometimes solve the above problem by detecting only the amplification of the target DNA, if the non-target DNA is present in large excess relative to the target DNA, it becomes difficult to detect the target DNA due to false positive reactions caused by the non-target DNA.

[0012] Such problems can arise, for example, in the detection of a small number of mutant alleles in the presence of normal alleles (e.g., detection of circulating tumor DNA in the blood), the detection of a small number of methylated or unmethylated alleles in epigenetic assays, and the detection of small amounts of fetal DNA sequences circulating in maternal blood.

[0013] To solve the above problems, a method called enrichment PCR or restriction endonuclease-mediated selective polymerase chain reaction (REMS PCR) has been developed (Non-Patent Document 3). This method utilizes a heat-stable restriction enzyme and, for example, primers designed to cleave the template with the normal nucleotide sequence only, to selectively detect DNA with a mutant nucleotide sequence. However, depending on the DNA to be detected, there may be no heat-stable restriction enzyme with a recognition sequence suitable for selective detection by REMS PCR. [Prior art documents] [Patent documents]

[0014] [Patent Document 1] U.S. Patent No. 5,922,539 [Patent Document 2] International Publication No. 01 / 062974 Brochure [Patent Document 3] International Publication No. 2014 / 142261 Brochure [Patent Document 4] International Publication No. 2016 / 039377 Brochure [Patent Document 5] International Publication No. 2016 / 152812 Brochure [Non-patent literature]

[0015] [Non-Patent Document 1] “Nucleic Acids Research”, February 2004, Volume 32, Issue 3, e37 [Non-patent document 2] "Methods in Molecular Biology", 2009, Vol. 496, No. 2, pp. 223-243 [Non-patent document 3] “American Journal of Pathology”, August 1998, Volume 153, Issue 2, p.373-379 Summary of the Invention [Problem to be solved by the invention]

[0016] An object of the present invention is to provide a heat-resistant mismatch endonuclease mutant that has improved specificity for mismatches compared to conventional mismatch endonucleases, a composition containing the mismatch endonuclease mutant, and a method for using the mismatch endonuclease mutant. [Means for solving the problem]

[0017] In light of the above circumstances, the present inventors have made extensive efforts and have succeeded in creating a mismatch endonuclease mutant capable of specifically cleaving a GG mismatch and a mismatch endonuclease mutant capable of specifically cleaving a TT mismatch. Furthermore, although mismatch endonucleases are originally homodimers, by connecting the monomers of these two mutants with a linker, they have succeeded in creating a mismatch endonuclease mutant capable of specifically cleaving a GT / TG mismatch. Thus, the present invention has been completed.

[0018] That is, the present invention is [1] A polypeptide represented by the following (A) or (B): (A) A polypeptide selected from the group consisting of the following (1) to (4), which has an activity of cleaving a nucleic acid strand having a guanine (G) that forms a mismatched base pair in a double-stranded nucleic acid: (1) In the amino acid sequence set forth in SEQ ID NO: 1, Serine at position 47 becomes a basic amino acid residue, and Asparagine at position 76 becomes an acidic amino acid residue a polypeptide comprising each substituted amino acid sequence; (2) A polypeptide comprising the substituted amino acid sequence according to (1) above, in which 1 to 10 amino acid residues are further mutated, provided that the mutations are substitutions, deletions, insertions, and / or additions, and the amino acid substitutions in the substituted amino acid sequence according to (1) above are retained; (3) A polypeptide comprising an amino acid sequence having 50% or more homology to the substituted or mutated amino acid sequence described in (1) or (2) above, provided that the amino acid substitutions or mutations in the substituted or mutated amino acid sequence described in (1) or (2) above are retained; (4) A polypeptide having an amino acid sequence that is 50% or more identical to the substituted or mutated amino acid sequence described in (1) or (2) above, provided that the amino acid substitutions or mutations in the substituted or mutated amino acid sequence described in (1) or (2) above are retained; or (B) A polypeptide selected from the group consisting of the following (5) to (8), which has the activity of cleaving a nucleic acid strand having a thymine (T) that forms a mismatched base pair in a double-stranded nucleic acid: (5) In the amino acid sequence set forth in SEQ ID NO: 1, a substitution of glutamine at position 78 with a basic amino acid residue; and a polypeptide comprising an amino acid sequence in which leucine at position 123 is substituted with a neutral (polar, uncharged) amino acid residue or a sulfur-containing amino acid residue, or leucine at position 125 is substituted with a hydrophobic amino acid residue or a sulfur-containing amino acid residue; (6) A polypeptide comprising the substituted amino acid sequence according to (5) above, in which 1 to 10 amino acid residues are further mutated, provided that the mutations are substitutions, deletions, insertions, and / or additions, and the amino acid substitutions in the substituted amino acid sequence according to (5) above are retained; (7) A polypeptide comprising an amino acid sequence having 50% or more homology to the substituted or mutated amino acid sequence described in (5) or (6) above, provided that the amino acid substitutions or mutations in the substituted or mutated amino acid sequence described in (5) or (6) above are retained; (8) A polypeptide comprising an amino acid sequence having 50% or more identity to the substituted or mutated amino acid sequence described in (5) or (6) above, provided that the amino acid substitution or mutation in the substituted or mutated amino acid sequence described in (5) or (6) above is maintained. [2] The polypeptide according to [1], wherein the basic amino acid residue is lysine, arginine, or histidine, the acidic amino acid residue is aspartic acid or glutamic acid, the neutral (polar, uncharged) amino acid is threonine, serine, asparagine, glutamine, tyrosine, or cysteine, the sulfur-containing amino acid is cysteine ​​or methionine, and the hydrophobic (non-polar) amino acid is alanine, glycine, valine, leucine, isoleucine, proline, tryptophan, phenylalanine, or methionine. [3] The polypeptide according to [1] or [2], which comprises an amino acid sequence selected from SEQ ID NOs: 6 to 15 in the sequence listing. [4] A nucleic acid encoding the polypeptide according to any one of [1] to [3]. [5] The nucleic acid according to [4], which contains a base sequence selected from SEQ ID NOs: 21 to 30 in the sequence listing. [6] An expression vector comprising the nucleic acid according to [4] or [5] and an expression control sequence. [7] A cell that expresses a polypeptide having mismatch endonuclease activity, which is transformed with the expression vector according to [6]. [8] A method for cleaving a double-stranded nucleic acid having a mismatch, comprising a step of allowing the polypeptide according to any one of [1] to [3] to act on the double-stranded nucleic acid. [9] A composition comprising the following (a) to (d): (a) the polypeptide according to any one of [1] to [3]; (b) an oligonucleotide that contains at least one mismatch when hybridized with a non-target nucleic acid to produce a double-stranded nucleic acid that is cleaved by the polypeptide of (a), and that, when hybridized with a target nucleic acid, produces a double-stranded nucleic acid that is not cleaved by the polypeptide of (a); (c) at least one pair of oligonucleotide primers; and (d) a polypeptide having DNA polymerase activity;

[10] A method for amplifying nucleic acid, comprising the following steps (1) and (2): (1) preparing a composition comprising a template nucleic acid molecule and the following (a) to (d): (a) the polypeptide according to any one of [1] to [3]; (b) an oligonucleotide that contains at least one mismatch when hybridized with a non-target nucleic acid to produce a double-stranded nucleic acid that is cleaved by the polypeptide of (a), and that, when hybridized with a target nucleic acid, produces a double-stranded nucleic acid that is not cleaved by the polypeptide of (a); (c) at least one pair of oligonucleotide primers; and (d) a polypeptide having DNA polymerase activity; and (2) reacting the composition obtained in step (1) under appropriate conditions to amplify nucleic acids;

[11] The method according to

[10] , wherein the nucleic acid amplification is carried out by polymerase chain reaction (PCR), isothermal nucleic acid amplification, or multiple displacement amplification (MDA).

[12] A method for inhibiting amplification of a nucleic acid containing a specific base sequence, comprising the step of carrying out a nucleic acid amplification reaction in the presence of the following (a) to (d): (a) an oligodeoxynucleotide designed to generate one or several mismatches when hybridized with a nucleic acid containing the specific base sequence or a complementary strand thereof; (b) DNA polymerase; (c) at least one pair of oligonucleotide primers; and (d) the polypeptide according to any one of [1] to [3];

[13] The method according to

[12] , wherein the nucleic acid amplification reaction is a polymerase chain reaction (PCR) method or an isothermal nucleic acid amplification method.

[14] A method for preferentially amplifying a target DNA, characterized in that the amplification of DNA having a base sequence that differs from the target DNA by one or several bases is suppressed by the method according to

[12] or

[13] .

[15] The method according to

[14] , which is used for the purpose of distinguishing and amplifying DNA of a wild-type base sequence from DNA in which a single nucleotide polymorphism has occurred in the DNA;

[16] The method according to

[15] , wherein the single nucleotide polymorphism is a single nucleotide polymorphism that correlates with canceration or the therapeutic effect of a cancer therapeutic drug; and

[17] A dimeric polypeptide selected from the group consisting of: (1) A homodimer having the polypeptide according to [1] (A) as a subunit. (2) A homodimer having the polypeptide according to [1] (B) as a subunit, and (3) A heterodimer having the polypeptides according to [1] (A) and [1] (B) as subunits. Regarding. [Effects of the Invention]

[0019] INDUSTRIAL APPLICABILITY The present invention provides thermostable mismatch endonuclease mutants that are highly useful in biotechnology, compositions containing the mismatch endonuclease mutants, and methods using the mismatch endonuclease mutants. DETAILED DESCRIPTION OF THE INVENTION

[0020] In the present invention, "mismatch" refers to a base pairing other than the Watson-Crick base pairs present in a double-stranded nucleic acid, i.e., a base pairing other than a combination of G (guanine base)-C (cytosine base), A (adenine base)-T (thymine base), or U (uracil base) base pairing.

[0021] In the present invention, "GG-specific" means specificity for a GG mismatch, "TT-specific" means specificity for a TT mismatch, and "GT / TG-specific" means specificity for a GT or TG mismatch. In other words, it means that it does not substantially recognize mismatches other than the mismatch.

[0022] As used herein, a "polypeptide having mismatch endonuclease activity" (sometimes simply referred to as a mismatch endonuclease) refers to a nuclease that has the activity of cleaving a mismatch site in a double-stranded nucleic acid. The mismatch endonuclease activity includes the activity of cleaving a phosphodiester bond adjacent to a nucleotide that forms a mismatch, as well as the activity of cleaving a phosphodiester bond adjacent to a nucleotide that is 1 to 5 base pairs, preferably 1 to 3 base pairs, away from the mismatch. In the present invention, a mismatch endonuclease that specifically recognizes a specific mismatch and cleaves a double-stranded nucleic acid is preferred. Furthermore, as used herein, a thermostable mismatch endonuclease refers to a nuclease that exhibits the activity of cleaving a mismatch site in a double-stranded nucleic acid at a temperature of 50°C or higher, and the use of a thermostable mismatch endonuclease is preferred in the present invention.

[0023] The mismatch endonuclease of the present invention is an enzyme that exhibits activity by forming a dimer. It is known that when monomers of this protein (monomers; also called "subunits," which indicate the components of a dimer) are expressed in a host such as Escherichia coli, they spontaneously associate to form a dimer (homodimer). Furthermore, in this specification, "a polypeptide having the activity of cleaving a nucleic acid strand containing a guanine (G) that forms a mismatched base pair in a double-stranded nucleic acid" and "a polypeptide having the activity of cleaving a nucleic acid strand containing a thymine (T) that forms a mismatched base pair in a double-stranded nucleic acid" each refer to the monomer.

[0024] Amino acids can be classified based on their chemical properties as hydrophilic (polar) and hydrophobic (non-polar). Hydrophilic (polar) amino acids include acidic amino acids, basic amino acids, neutral (polar, uncharged) amino acids, tyrosine among amino acids having an aromatic ring, and cysteine ​​among sulfur-containing amino acids. Hydrophobic (non-polar) amino acids include amino acids having an aliphatic side chain, tryptophan and phenylalanine among the amino acids having an aromatic ring, and methionine among the sulfur-containing amino acids.

[0025] Acidic amino acids include aspartic acid (Asp, D) and glutamic acid (Glu, E). Basic amino acids include lysine (Lys, K), arginine (Arg, R), and histidine (His, H). Neutral (polar, uncharged) amino acids include threonine (Thr, T), serine (Ser, S), asparagine (Asn, N), glutamine (Gln, Q), tyrosine (Tyr, Y), and cysteine ​​(Cys, C). Amino acids with aromatic rings include tyrosine (Tyr, Y), tryptophan (Trp, W), and phenylalanine (Phe, F). Sulfur-containing amino acids include cysteine ​​(Cys, C) and methionine (Met, M). Amino acids with aliphatic side chains include alanine (Ala, A), glycine (Gly, G), valine (Val, V), leucine (Leu, L), isoleucine (Ile, I), and proline (Pro, P), which is an imino acid with a cyclized side chain.

[0026] In the present invention, "similar amino acids" refer to amino acids that fall into the same classification based on the chemical properties of the amino acids. For example, lysine, arginine, and histidine are all classified as basic amino acids, and therefore are similar to each other.

[0027] In the present invention, "amino acid sequence homology" refers to the similarity of polypeptide sequences, taking into account the above-mentioned similar amino acids. Therefore, in the classification based on the chemical properties of amino acids, amino acids belonging to the same classification are considered to be "homologous."

[0028] In the present invention, "amino acid sequence identity" refers to the identity of polypeptide sequences without taking into account the similar amino acids. Also, in the present invention, "nucleotide sequence identity" refers to the identity of polynucleotide sequences.

[0029] To calculate the scores of the amino acid sequence homology, amino acid sequence identity, and nucleotide sequence identity, known programs can be used, including, but not limited to, BLAST, BLAT, FASTA, SSEARCH, and MPsrch.

[0030] The amino acid numbers (also referred to as amino acid positions) used in the present invention are based on the amino acid sequence of SEQ ID NO: 1. Therefore, the amino acid positions identified by the amino acid numbers herein may differ from the positions indicated by these numbers when counted from the N-terminus of homologous proteins derived from other organisms or proteins in which mutations have been introduced into the polypeptide of SEQ ID NO: 1. In other words, in the present invention, "a position equivalent to (or corresponding to) position 47 in the amino acid sequence of a wild-type mismatch endonuclease" refers to the amino acid residue that is deemed to be at the same position as the amino acid residue at position 47 in the wild-type amino acid sequence, as determined by comparing and aligning the wild-type amino acid sequence (SEQ ID NO: 1) with a mutant amino acid sequence and the amino acid sequence of a mismatch endonuclease derived from another organism using a known algorithm (BLASTp, ClustalOmega, etc.).

[0031] In the present invention, a "target nucleic acid" or "target DNA" refers to a nucleic acid that is not cleaved by the mismatch endonuclease mutant of the present invention. In other words, it is a nucleic acid that serves as a target (template) for amplification in the amplification method of the present invention. On the other hand, a "non-target nucleic acid" or "non-target DNA" refers to a nucleic acid that is cleaved by the mismatch endonuclease mutant of the present invention. In other words, it is a nucleic acid that is cleaved in the cleavage method of the present invention and therefore is not a target for amplification in the amplification method of the present invention. The "target nucleic acid" or "target DNA" and the "non-target nucleic acid" or "non-target DNA" are not particularly limited and may be any nucleic acids that are desired to be distinguished from each other.

[0032] This will be explained in detail below.

[0033] 1. Mismatch endonuclease mutant of the present invention The first aspect of the present invention relates to a mismatch endonuclease mutant, i.e., a mismatch endonuclease mutant with altered substrate specificity. The mismatch endonuclease mutant is characterized by an alteration in the amino acid sequence of a site contributing to substrate recognition and cleavage in a wild-type mismatch endonuclease derived from Pyrococcus furiosus (sometimes referred to as PfuNucS) or its homologues, or a mutant having the mismatch endonuclease activity of PfuNucS.

[0034] Although the present invention is not particularly limited thereto, the mismatch endonuclease mutant of the present invention can be prepared by introducing into the polypeptide PF_RS00065 (RefSeq ID: WP_011011124.1, SEQ ID NO: 1, former name: PF0012, former RefSeq ID: NP_577741) derived from Pyrococcus furiosus (i) an amino acid substitution of serine at position 47 with a basic amino acid and asparagine at position 76 with an acidic amino acid, or (ii) an amino acid substitution of glutamine at position 78 with a basic amino acid and leucine at position 123 with a neutral (polar, uncharged) amino acid or a sulfur-containing amino acid, or leucine at position 125 with a hydrophobic amino acid or a sulfur-containing amino acid.

[0035] Furthermore, mismatch endonuclease mutants of the present invention can be prepared by similarly introducing a mutation corresponding to the above amino acid substitution (i) or (ii) into polypeptide W77F (SEQ ID NO: 2), in which the tryptophan residue at position 77 of PF_RS00065 is replaced with phenylalanine, polypeptide MJ_RS01180 (RefSeq ID: NP_247194, SEQ ID NO: 3, former name: MJ_0225) derived from Methanocaldococcus jannaschii, which is a homolog of PF_RS00065, polypeptide TERMP_01877 (RefSeq ID: YP_004072075, SEQ ID NO: 4) derived from Thermococcus barophilus, and polypeptide (SEQ ID NO: 5) derived from Thermococcus kodakarensis KOD1 strain (JCM 12380T).

[0036] The mismatch endonuclease mutant of the present invention is preferably a thermostable enzyme. While not particularly limiting the present invention, a polypeptide that stably retains its activity even during thermal cycling such as PCR is preferred, and a polypeptide that is not inactivated even at temperatures above 50°C, preferably above 60°C, further preferably above 70°C, and even more preferably above 90°C can be used in the method of the present invention.

[0037] Although the present invention is not particularly limited thereto, for example, the mismatch endonuclease mutant of the present invention comprises the polypeptide shown in (A) and / or (B) below. A preferred example is a mismatch endonuclease mutant consisting of the polypeptide shown in (A) and / or (B) below.

[0038] (A) A polypeptide selected from the group consisting of the following (1) to (4), which has the activity of specifically cleaving a nucleic acid strand having a guanine (G) that forms a mismatched base pair in a double-stranded nucleic acid: (1) In the amino acid sequence set forth in SEQ ID NO: 1, Serine at position 47 becomes a basic amino acid residue, and Asparagine at position 76 becomes an acidic amino acid residue a polypeptide comprising each substituted amino acid sequence; (2) A polypeptide comprising the substituted amino acid sequence according to (1) above, in which 1 to 10 amino acid residues are further mutated, provided that the mutations are substitutions, deletions, insertions, and / or additions, and the amino acid substitutions in the substituted amino acid sequence according to (1) above are retained; (3) A polypeptide comprising an amino acid sequence having 50% or more homology to the substituted or mutated amino acid sequence described in (1) or (2) above, provided that the amino acid substitutions or mutations in the substituted or mutated amino acid sequence described in (1) or (2) above are retained; (4) A polypeptide comprising an amino acid sequence having 50% or more identity to the substituted or mutated amino acid sequence described in (1) or (2) above, provided that the amino acid substitutions or mutations in the substituted or mutated amino acid sequence described in (1) or (2) above are retained.

[0039] (B) A polypeptide selected from the group consisting of the following (5) to (8), which has the activity of specifically cleaving a nucleic acid strand having a thymine (T) forming a mismatched base pair in a double-stranded nucleic acid: (5) In the amino acid sequence set forth in SEQ ID NO: 1, a substitution of glutamine at position 78 with a basic amino acid residue; and a polypeptide comprising an amino acid sequence in which leucine at position 123 is substituted with a neutral (polar, uncharged) amino acid residue or a sulfur-containing amino acid residue, or leucine at position 125 is substituted with a hydrophobic amino acid residue or a sulfur-containing amino acid residue; (6) A polypeptide comprising the substituted amino acid sequence according to (5) above, in which 1 to 10 amino acid residues are further mutated, provided that the mutations are substitutions, deletions, insertions, and / or additions, and the amino acid substitutions in the substituted amino acid sequence according to (5) above are retained; (7) A polypeptide comprising an amino acid sequence having 50% or more homology to the substituted or mutated amino acid sequence described in (5) or (6) above, provided that the amino acid substitutions or mutations in the substituted or mutated amino acid sequence described in (5) or (6) above are retained; (8) A polypeptide comprising an amino acid sequence having 50% or more identity to the substituted or mutated amino acid sequence described in (5) or (6) above, provided that the amino acid substitutions or mutations in the substituted or mutated amino acid sequence described in (5) or (6) above are retained.

[0040] The present invention provides polypeptides having GG-specific mismatch endonuclease activity. Examples of such polypeptides include the polypeptides described in (A) above. While not particularly limited, preferred examples include polypeptides comprising the amino acid sequence of SEQ ID NO: 1, in which the serine at position 47 is substituted with a basic amino acid residue and the asparagine at position 76 is substituted with an acidic amino acid residue, as described in (A)(1) above. Furthermore, examples of polypeptides having GG-specific mismatch endonuclease activity include polypeptides comprising an amino acid sequence in which, in addition to the amino acid substitutions at positions 47 and 76 of SEQ ID NO: 1, the leucine at position 123 is substituted with an amino acid residue having an aliphatic side chain. Even more preferred examples include polypeptides in which the serine at position 47 of SEQ ID NO: 1 is substituted with lysine, arginine, or histidine, and the asparagine at position 76 is substituted with aspartic acid or glutamic acid. More preferred examples include polypeptides in which, in addition to the amino acid substitutions at positions 47 and 76, leucine at position 123 is substituted with alanine, glycine, valine, leucine, isoleucine, or proline.

[0041] Furthermore, the present invention provides a polypeptide having TT-specific mismatch endonuclease activity. Examples of the polypeptide having TT-specific mismatch endonuclease activity include the polypeptides described in (B) above. While not particularly limited, preferred examples include polypeptides comprising the amino acid sequence described in SEQ ID NO: 1, in which glutamine at position 78 is substituted with a basic amino acid residue and leucine at position 123 is substituted with a neutral (polar, uncharged) amino acid residue or a sulfur-containing amino acid residue, or leucine at position 125 is substituted with a hydrophobic amino acid residue or a sulfur-containing amino acid residue, as described in (B)(5) above. More preferred examples include polypeptides comprising an amino acid sequence in which, in the amino acid sequence of SEQ ID NO: 1, glutamine at position 78 is substituted with lysine, arginine, or histidine, and leucine at position 123 is substituted with threonine, serine, asparagine, glutamine, tyrosine, cysteine, or methionine, or leucine at position 125 is substituted with alanine, glycine, valine, isoleucine, proline, tryptophan, phenylalanine, cysteine, or methionine. Even more preferably, leucine at position 125 may be substituted with an amino acid having an aliphatic side chain other than leucine.

[0042] The polypeptide having GG-specific mismatch endonuclease activity and the polypeptide having TT-specific mismatch endonuclease activity may be in the form of a homodimer. When a monomer of the polypeptide having GG-specific mismatch endonuclease activity is expressed in a host such as E. coli, the polypeptide spontaneously associates to form a dimer (homodimer) and exhibits the activity. The same applies to the polypeptide having TT-specific mismatch endonuclease activity.

[0043] Furthermore, the present invention also provides polypeptides having GT / TG-specific mismatch endonuclease activity. Examples of such polypeptides having GT / TG-specific mismatch endonuclease activity include heterodimeric proteins comprising the polypeptides shown in (A) and (B) above. A preferred example is a heterodimeric protein comprising the polypeptides having GG-specific mismatch endonuclease activity and TT-specific mismatch endonuclease activity, although this is not particularly limited. Such heterodimeric proteins can be prepared, for example, by expressing the polypeptides having GG-specific mismatch endonuclease activity and the polypeptides having TT-specific mismatch endonuclease activity in a host such as Escherichia coli, so that they form a dimer (heterodimer). However, there are no limitations on the method for preparing such heterodimeric proteins. The polypeptides having GG-specific mismatch endonuclease activity and the polypeptides having TT-specific mismatch endonuclease activity may be expressed separately, dissociated into monomers by any method, and then mixed to reassociate. In a preferred method for producing the heterodimeric protein, a polypeptide having GG-specific mismatch endonuclease activity and a polypeptide having TT-specific mismatch endonuclease activity are expressed as a single polypeptide via a linker. There is no limitation on the arrangement within the single polypeptide. It may be [polypeptide having GG-specific mismatch endonuclease activity]-[linker]-[polypeptide having TT-specific mismatch endonuclease activity], or it may be [polypeptide having TT-specific mismatch endonuclease activity]-[linker]-[polypeptide having GG-specific mismatch endonuclease activity]. The linker is preferably a peptide linker. There are no limitations on the amino acid sequence or the number of residues (length) of the peptide linker. Any sequence and number of residues that do not inhibit heterodimerization and cleavage of double-stranded nucleic acids can be suitably used in the present invention.Furthermore, a recognition sequence for a protease that cleaves the fusion polypeptide, such as Factor Xa, PreScission Protease, thrombin, enterokinase, or TEV protease (Tobacco Etch Virus Protease), may be present between the polypeptide having mismatch endonuclease activity and the linker.

[0044] Furthermore, in addition to the amino acid substitutions described in (A)(1) and (B)(5) above, the mismatch endonuclease mutants of the present invention may contain mutations at other amino acid positions, as long as the substrate specificity of the mutants (GG, TT, or GT / TG mismatch specificity) is not lost. Examples of mutations at other amino acid positions include, but are not limited to, mutations that improve thermostability, mutations that improve resistance to inhibitors, and amino acid mutations that improve the properties of the mismatch endonuclease, as long as the substrate specificity of the mismatch endonuclease mutants of the present invention is maintained. Here, the "mutation" may be any of an amino acid substitution mutation, an amino acid insertion mutation, an amino acid deletion mutation, or an amino acid addition mutation.

[0045] For example, without particularly limiting the present invention, the mismatch endonuclease mutant of the present invention containing mutations at other amino acid positions may contain an amino acid sequence in which 1 to 10 amino acid residues have been mutated in addition to the amino acid substitutions described above in (A)(1) and (B)(5).

[0046] Furthermore, for example, mismatch endonuclease mutants of the present invention containing mutations at other amino acid positions may contain an amino acid sequence that is 50% or more homologous to the amino acid sequence of SEQ ID NO: 1 containing the amino acid substitutions described above in (A)(1) and (B)(5), or to an amino acid sequence in which 1 to 10 amino acid residues have been further mutated in the amino acid sequence of SEQ ID NO: 1 containing the amino acid substitutions. The above-mentioned 50% or more homology includes, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, or 75% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, and even more preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence homology.

[0047] Furthermore, for example, mismatch endonuclease mutants of the present invention containing mutations at other amino acid positions may contain an amino acid sequence that is 50% or more identical to the amino acid sequence of SEQ ID NO: 1 containing the amino acid substitutions described above in (A)(1) and (B)(5), or to the amino acid sequence of SEQ ID NO: 1 containing the amino acid substitutions with an additional 1 to 10 amino acid residues mutated. The above-mentioned 50% or more identity includes, for example, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, or 75% or more, preferably 80% or more, more preferably 85% or more, even more preferably 90% or more, and even more preferably 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more amino acid sequence identity.

[0048] Furthermore, the present invention also encompasses mismatch endonuclease mutants with affinity tags added to the N- or C-terminus for the purpose of facilitating purification. Examples of affinity tags include well-known histidine (His) tags (containing 4 to 8 consecutive His residues), Flag tags, HA tags, c-myc tags, glutathione S-transferase (GST) tags, maltose binding protein (MBP) tags, and Strep (II) tags consisting of 8 amino acid residues (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys). If desired, these tags can be linked to the mutants of the present invention via linkers containing 1 to 15 amino acids. Furthermore, a recognition sequence for a fusion polypeptide-cleaving protease, such as Factor Xa, PreScission Protease, thrombin, enterokinase, or Tobacco Etch Virus Protease (TEV) may be present between the polypeptide having mismatch endonuclease activity and the linker. Therefore, polypeptides containing such affinity tags, linkers, and / or recognition sequences for fusion polypeptide-cleaving proteases are also exemplified as mismatch endonuclease mutants of the present invention that contain mutations at other amino acid positions. For example, mismatch endonuclease mutants of the present invention that contain mutations at other amino acid positions may contain an affinity tag, linker, and / or recognition sequence for a fusion polypeptide-cleaving protease within the above-mentioned range of 1 to 10 mutated amino acid residues, amino acid homology, or amino acid identity.

[0049] Furthermore, the mismatch endonuclease mutant of the present invention may contain an artificial amino acid (also referred to as a non-natural amino acid). Examples of artificial amino acids include halogenated tyrosine (chloro, bromo, iodo), tyrosine sulfate, azidotyrosine, acetyllysine, azidophenylalanine, and fluorophenylalanine. There are no particular limitations on the method for substituting a natural amino acid with an artificial amino acid, and known methods can be used.

[0050] The mismatch endonuclease mutant of the present invention is not particularly limited, but includes mutants consisting of any of the amino acid sequences set forth in SEQ ID NOs: 6 to 15.

[0051] Such mismatch endonuclease mutants of the present invention are suitable for use in various applications described below, for example, in methods for eliminating DNA containing a specific DNA sequence and amplifying and detecting other DNA.

[0052] Here, the activity of a mismatch endonuclease can be measured using a double-stranded nucleic acid containing a mismatch as a substrate. Specifically, activity is measured by reacting an excess amount of double-stranded nucleic acid containing a mismatch with the mismatch endonuclease and measuring the amount of cleaved nucleic acid per unit time. The cleaved double-stranded nucleic acid can be separated from uncleaved nucleic acid by electrophoresis or other methods and quantified. If a double-stranded nucleic acid doubly labeled with a fluorescent substance and a quencher is used so that an increase in fluorescence intensity can be detected only upon cleavage, activity can be easily measured by measuring the fluorescence intensity in the reaction solution at appropriate time intervals. It is also possible to examine the cleavage activity for a specific mismatch by changing the mismatched base in the double-stranded nucleic acid substrate.

[0053] 2. Nucleic acids encoding the mismatch endonuclease mutants of the present invention The present invention can provide a nucleic acid encoding a mismatch endonuclease mutant. Specifically, the present invention provides a nucleic acid encoding the above-described mismatch endonuclease mutant of the present invention.

[0054] Nucleic acids encoding the mismatch endonuclease mutants of the present invention are not particularly limited, but include nucleic acids comprising a nucleotide sequence encoding the amino acid sequence set forth in any of SEQ ID NOs: 6 to 15. More preferably, nucleic acids encoding the mismatch endonuclease mutants of the present invention include nucleic acids comprising a nucleotide sequence set forth in any of SEQ ID NOs: 21 to 30.

[0055] Although not intended to limit the present invention in any way, the amino acid sequences of the polypeptides prepared in the Examples of the present invention as examples of the mismatch endonuclease mutants of the present invention and the nucleic acid sequences encoding them are shown in Table 1.

[0056] [Table 1]

[0057] The nucleic acid encoding the mismatch endonuclease mutant of the present invention is not particularly limited as long as it is expressible in the host used and is composed of codons that encode a protein having reverse transcriptase activity, and codons may be optimized to enable expression in the host used or to increase the expression level. The codon optimization is preferably performed by a method commonly used in this field.

[0058] 3. Expression vector containing a nucleic acid encoding the mismatch endonuclease mutant of the present invention The expression vector of the present invention comprises a nucleic acid encoding the mismatch endonuclease mutant of the present invention and an expression control sequence operably linked to the nucleic acid.

[0059] The expression vector used in the present invention is not particularly limited as long as it is an expression vector commonly used in this field. Vectors that can autonomously replicate in host cells or vectors that can be integrated into host chromosomes can be used. Any vector compatible with the host may be used.

[0060] Examples of expression vectors that can be used for inserting nucleic acids encoding the mismatch endonuclease mutants of the present invention include plasmid vectors, phage vectors, and viral vectors. Plasmid vectors suitable for the host used, such as Escherichia coli-derived plasmids, Bacillus bacteria-derived plasmids, and yeast-derived plasmids, are well known to those skilled in the art and many of these are commercially available. These known plasmids and their modifications can be used in the present invention. Phage vectors that can be used include, for example, λ phage, and viral vectors that can be used include, for example, retroviruses, animal viruses such as vaccinia virus, and insect viruses such as baculovirus. Furthermore, many heterologous protein expression systems using yeast, insect cells, or mammalian cells as hosts have been constructed and are already commercially available. These expression systems can be used to produce the mismatch endonuclease mutants of the present invention.

[0061] The promoter to be incorporated into the expression vector of the present invention can be selected depending on the host, and for example, in E. coli, promoters derived from E. coli or phages, such as the trp promoter, lac promoter, PL promoter, or PR promoter, or modified versions thereof, can be used, but are not limited to the above. Furthermore, an expression system combining a phage-derived promoter with an RNA polymerase gene (e.g., the pET expression system) can also be used.

[0062] To facilitate purification of the expressed polypeptide, the expression vector of the present invention may further contain a nucleic acid encoding an affinity tag. The nucleic acid encoding the affinity tag is inserted into the vector so that a fusion protein of the mismatch endonuclease mutant of the present invention and the affinity tag is expressed. Although not intended to limit the present invention, examples of affinity tags include nucleic acids encoding a histidine (His) tag, a glutathione S-transferase (GST) tag, a maltose binding protein (MBP) tag, and a Strep (II) tag consisting of eight amino acid residues (Trp-Ser-His-Pro-Gln-Phe-Glu-Lys). The tag may be added to either the 5'-end or / and the 3'-end of the nucleic acid encoding the mismatch endonuclease mutant of the present invention, as long as it is added appropriately to a position that does not interfere with expression or tag function. Preferably, the tag can be cleaved during or after the purification of the expressed polypeptide. Although not intended to limit the present invention in any way, examples of such cleavable tags include tags comprising a nucleic acid encoding a recognition sequence for a protease for cleaving the fusion polypeptide, such as Factor Xa, PreScission Protease, thrombin, enterokinase, or TEV protease (Tobacco Etch Virus Protease).

[0063] The expression vector of the present invention further comprises one or more expression regulatory sequences. Examples of such expression regulatory sequences include, but are not limited to, promoters and genes involved in promoter control, ribosome binding sequences, polyadenylation signals, transcription termination sequences (transcription terminators), enhancers, etc. Furthermore, the vector may also comprise genes encoding replication origins and markers (drug resistance genes, fluorescent markers, luminescent markers) used to select transformants, and nucleotide sequences for increasing translation efficiency.

[0064] 4. Cells transformed with the expression vector of the present invention The cells (hosts) to be transformed with the vectors expressing the mismatch endonuclease mutants of the present invention are not particularly limited as long as they are hosts commonly used in this field, such as bacteria (Escherichia coli, Bacillus subtilis, etc.), yeast, filamentous fungi, insect cells, eukaryotic cells, and animal cells (such as mammalian cells including human cells).

[0065] When a prokaryotic host cell is used, bacteria belonging to the genus Escherichia such as Escherichia coli, the genus Bacillus such as Bacillus subtilis, the genus Pseudomonas such as Pseudomonas putida, or the genus Rhizobium such as Rhizobium meliloti can be used as the host cell. E. coli strains that can be used to produce heterologous proteins are well known to those skilled in the art, and many are commercially available (e.g., Escherichia coli BL21T1R, Escherichia coli BL21, E. coli XL1-Blue, E. coli XL2-Blue, E. coli DH1, E. coli JM109, E. coli HB101, etc.). In addition, Bacillus bacteria such as Bacillus subtilis MI114 and B. subtilis 207-21, and Brevibacillus bacteria such as Brevibacillus choshinensis, are known as hosts for producing heterologous proteins. These host cells can be combined with an appropriate expression vector and used to produce the mismatch endonuclease mutant of the present invention. While not particularly limited, E. coli strains of the BL21 lineage, such as E. coli BL21T1R and BL21DE3, are suitable.

[0066] The method for introducing an expression vector into a host is not particularly limited as long as it is a method that can introduce nucleic acid into the host, and for example, a method using calcium ions, electroporation, the spheroplast method, the lithium acetate method, etc. can be used. The method for introducing an expression vector into insect cells is not particularly limited as long as it can introduce DNA into insect cells, and for example, the calcium phosphate method, lipofection, electroporation, etc. can be used. Phage vectors and viral vectors can be used to infect host cells using a method appropriate for the vector, and a transformant that expresses the mismatch endonuclease mutant of the present invention can be obtained.

[0067] The transformant is cultured, and the mismatch endonuclease mutant of the present invention can be isolated from the culture. Culture conditions are not particularly limited as long as they are appropriate for the expression vector, host, and other factors used. For example, when E. coli BL21DE3 is transformed with a pET vector, the transformant is inoculated into LB medium and cultured with shaking at 37°C. When the OD of the culture reaches 0.2 to 0.8, IPTG is added, and the culture is cultured with shaking for 2 to 5 hours, for example, at 15 to 30°C, and preferably at 25°C for 4 to 5 hours, to induce expression of the target protein. The culture is then centrifuged, and the resulting cells are washed and then disrupted by sonication or lysozyme lysis to obtain a disrupted product containing the mutant of the present invention. Because the disrupted product contains a large amount of impurities, it is desirable to purify the mutant of the present invention by an appropriate combination of purification methods commonly used in the art, such as ammonium sulfate precipitation, anion exchange columns, cation exchange columns, gel filtration columns, affinity chromatography columns, and dialysis. Affinity-tagged mutants can be easily purified using an affinity carrier that corresponds to the properties of the affinity tag. Depending on the type of host or expression vector used, other necessary inducers, such as L-arabinose, may be added in addition to IPTG at an appropriate time.

[0068] 5. Method for producing nucleic acids encoding mismatch endonuclease mutants of the present invention The method for producing a nucleic acid of the present invention includes substituting, for example, a codon encoding serine at position 47 in the amino acid sequence of SEQ ID NO: 1 in a nucleic acid encoding polypeptide PF_RS00065 or its mutant polypeptide W77F, or a mismatch endonuclease that is a homolog of RS_00065, with a codon encoding a basic amino acid. Although not particularly limited, the substitution of the codon corresponding to serine is preferably a codon corresponding to arginine or lysine. Furthermore, substitution with a codon corresponding to arginine is particularly preferred. Similar codon substitutions are also possible for amino acid residues at other positions.

[0069] As explained in 1 above, the mismatch endonuclease mutant of the present invention may be combined with mutations at other amino acid positions. In this case, codon substitutions can be similarly performed.

[0070] For example, nucleic acids encoding the mismatch endonuclease mutants into which the above-mentioned codon substitutions have been introduced are preferably the nucleic acids of SEQ ID NOS: 16 to 30 in the sequence listing.

[0071] The codon conversion may be performed by a known method, and is not particularly limited. For example, it can be performed by mutagenesis using a known method such as site-directed mutagenesis using a mutagenesis primer, or by artificial synthesis of a nucleic acid having the mutated sequence (or a part of the sequence). Furthermore, codon optimization may be performed to enable expression in the host used or to increase the expression level. The codon optimization can be performed by a method commonly used in this field.

[0072] Furthermore, in the method for producing a nucleic acid of the present invention, a nucleic acid encoding a mismatch endonuclease mutant may be combined with codon substitution to stabilize and improve protein production in a host.

[0073] The nucleic acid described in 2 above can be applied to the method for producing a nucleic acid of the present invention.

[0074] 6. Method for cleaving double-stranded nucleic acid of the present invention The method of the present invention for cleaving double-stranded nucleic acids involves allowing the mismatch endonuclease mutant of the present invention described in 1 above to act on double-stranded nucleic acids containing mismatches in an appropriate buffer containing divalent metal ions (e.g., magnesium ions). The method of the present invention can cleave double-stranded nucleic acids containing GG mismatches, TT mismatches, GT mismatches, or TG mismatches.

[0075] The double-stranded nucleic acid having mismatches in the method of cleaving double-stranded nucleic acid of the present invention is not limited to a single mismatch in the double-stranded nucleic acid, as long as a mismatch exists within the double-stranded nucleic acid (between two base pairs that undergo normal base pairing); it may have multiple mismatches spaced apart, or two or more consecutive mismatches. Examples of mismatches in the method of cleaving double-stranded nucleic acid of the present invention include preferably 1 to 8 consecutive mismatches present within the double-stranded nucleic acid, more preferably 1 to 4 consecutive mismatches, and even more preferably two consecutive mismatches or one mismatch. When multiple mismatches exist in the double-stranded nucleic acid in the method of cleaving double-stranded nucleic acid of the present invention, the multiple mismatches may be the same type or different types of mismatches.

[0076] Furthermore, oligodeoxynucleotides disclosed in WO 2014 / 142261 and the like may be combined. Here, the oligodeoxynucleotide is an oligodeoxynucleotide designed to generate one or several mismatches when hybridized with a nucleic acid having a specific base sequence (e.g., a target gene mutation). By labeling both ends of the oligodeoxynucleotide with a fluorescent substance and a quencher, it can be used as a mutation detection probe. The chain length of the oligodeoxynucleotide can be appropriately determined so that the oligodeoxynucleotide can hybridize with the nucleic acid having the specific base sequence under the reaction conditions to be performed. Furthermore, it is desirable that the positions at which mismatches occur when hybridized with a nucleic acid having the specific base sequence be at least three nucleotides away from both the 5' and 3' ends of the oligodeoxynucleotide.

[0077] Furthermore, if the genetic mutation to be detected does not match the substrate specificity of the mismatch endonuclease and cannot be analyzed as is, a suppressor oligonucleotide, as disclosed in International Publication No. WO 2016 / 152812, may be combined. Here, a suppressor oligonucleotide is an oligonucleotide that generates at least one mismatch when hybridized with a non-target nucleic acid and generates more mismatches when hybridized with a target nucleic acid than when hybridized with the non-target nucleic acid. The number of mismatches generated when a suppressor oligonucleotide hybridizes with a non-target nucleic acid is not particularly limited as long as it results in selective cleavage of the non-target nucleic acid. Depending on the length of the suppressor oligonucleotide, the number of mismatches may be, for example, 1 to 7, 1 to 5, or 1 to 3. Furthermore, the number of mismatches, expressed as a percentage of the length of the suppressor oligonucleotide, is, for example, in the range of 1 to 20%, 3 to 15%, or 4 to 8%. This embodiment will be described using, as an example, a combination of a target nucleic acid and a non-target nucleic acid that differ in only one base in their base sequences. When the suppressor oligonucleotide is hybridized with the target nucleic acid, a mismatch (hereinafter sometimes referred to as the first mismatch) occurs between the different bases in the target nucleic acid and the non-target nucleic acid, and another mismatch (hereinafter sometimes referred to as the second mismatch) also occurs.

[0078] The first mismatched base relates to a base that differs between the target nucleic acid and the non-target nucleic acid, regardless of whether it is recognized and cleaved by a coexisting polypeptide having mismatch endonuclease activity.

[0079] On the other hand, the second mismatch is a mismatch base that is recognized and cleaved by a coexisting polypeptide having mismatch endonuclease activity. For example, without limitation, in the case of a polypeptide having mismatch endonuclease activity that recognizes and cleaves guanine base-guanine base, guanine base-thymine base, or thymine base-thymine base, the suppressor oligonucleotide is designed to form a mismatch selected from these.

[0080] Furthermore, when the suppressor oligonucleotide hybridizes with a non-target nucleic acid, the second mismatch occurs, but the first mismatch does not occur.

[0081] By designing and using an inhibitory oligonucleotide with the above properties, selective cleavage of non-target nucleic acids by a polypeptide with mismatch endonuclease activity becomes possible. The present invention is not limited to the use of inhibitory oligonucleotides capable of forming one or two mismatches as described above; inhibitory oligonucleotides that form three or more mismatches may also be designed and used, as long as selective cleavage of the desired nucleic acid occurs. In this case, it is sufficient that at least one mismatch formed between the target nucleic acid and the non-target nucleic acid is the second mismatch; it is not important whether other mismatches can be recognized and cleaved by a polypeptide with mismatch endonuclease activity. Preferably, the three or more mismatches are recognized and cleaved by a polypeptide with mismatch endonuclease activity.

[0082] The oligodeoxynucleotides disclosed in WO 2014 / 142261 and the suppressor oligonucleotides disclosed in WO 2016 / 152812 that can be used in the methods of the present invention are composed of DNA, but may contain a nucleotide analog or RNA as a portion thereof. That is, they are not particularly limited as long as they have a structure that forms a double-stranded nucleic acid having at least one mismatch when hybridized with a non-target nucleic acid, and further, they are recognized and cleaved by a polypeptide having mismatch endonuclease activity in which the mismatch coexists.

[0083] Examples of double-stranded nucleic acids having mismatches that can be cleaved by the method of the present invention include PCR products, nucleic acids derived from biological samples such as genomic DNA and fragments thereof, and synthetic nucleic acids. Furthermore, double-stranded nucleic acids having mismatches may be nucleic acid mixtures produced by melting and reannealing a mixture derived from a biological sample or a mixture of nucleic acids derived from a biological sample and synthetic nucleic acids. For example, when a nucleic acid having a mutation and a wild-type nucleic acid are mixed and melted and reannealed, mismatches are formed, and the mismatched nucleic acids are cleaved by the mismatch endonuclease at these sites. The presence and location of mutations can be verified by observing the size of the resulting nucleic acid fragments cleaved by the mismatch endonuclease. By utilizing the mismatch endonuclease of the present invention, mutation analysis can be performed simply by adding this mismatch endonuclease to the reaction solution of a nucleic acid amplification method such as PCR. It is known that in PCR, even if the number of cycles is increased beyond a certain number, the amplification effect is no longer obtained. The main causes of this are depletion of added primers or substrate dNTPs, and competition between primers and reaction products. However, annealing between reaction products also occurs at this time. If a template containing a mutation and a wild-type template are present, annealing between the reaction products amplified from these templates will result in mismatches at the mutation site. Therefore, mutation analysis is possible simply by performing PCR in the presence of the mismatch endonuclease of the present invention at a higher cycle number than usual. Specifically, the present invention provides a mutation analysis method comprising reacting the mismatch endonuclease described in 1. above with double-stranded nucleic acid.

[0084] The method for cleaving double-stranded nucleic acids of the present invention can be carried out in the presence of an acidic polymer. It has been confirmed that the coexistence of such an acidic polymer suppresses the mismatch recognition and cleavage activity of the polypeptide. This effect is more pronounced when the amount of nucleic acid in a sample is low. The acidic polymer is preferably a polyanion. Acidic polysaccharides having a sugar backbone or linear carbon chains are also preferred. The acidic polymer can be one or more selected from the group consisting of fucose sulfate-containing polysaccharides, dextran sulfate, carrageenan, heparin, rhamnan sulfate, dermatan sulfate (chondroitin sulfate B), heparan sulfate, hyaluronic acid, alginic acid, pectin, polyglutamic acid, polyacrylic acid, polyvinyl sulfate, polystyrene sulfate, and salts thereof, or nucleic acids different from the target nucleic acid and non-target nucleic acid.

[0085] The method of cleaving double-stranded nucleic acid of the present invention may be carried out in combination with proliferating cell nuclear antigen (PCNA) or a mutant thereof.

[0086] 7. Method for amplifying double-stranded nucleic acid of the present invention The method for cleaving double-stranded nucleic acids of the present invention can also be carried out during a nucleic acid amplification reaction. By adding a mismatch endonuclease to the nucleic acid amplification reaction solution, double-stranded nucleic acids having mismatches resulting from the incorporation of incorrect nucleotides during the amplification process are cleaved. As a result, amplification of nucleic acids with sequences different from those of the template nucleic acid before the start of the reaction is suppressed. This enables nucleic acid amplification with a reduced error rate.

[0087] That is, the present invention provides a nucleic acid amplification method comprising the step of cleaving a double-stranded nucleic acid having a mismatch using the mismatch endonuclease mutant of the present invention described in 1. The step of cleaving the double-stranded nucleic acid having a mismatch may be carried out simultaneously with the nucleic acid amplification step. Furthermore, one aspect of the present invention is a composition comprising (a) a DNA polymerase, (b) at least one pair of oligonucleotide primers, and (c) the mismatch endonuclease described in 1.

[0088] Similarly, another aspect of the present invention is a method for amplifying nucleic acids, further comprising the steps of preparing a composition containing the above-mentioned composition for nucleic acid amplification reaction and a template nucleic acid molecule, and reacting the obtained composition under appropriate conditions to amplify the nucleic acid.

[0089] The composition may further contain at least one selected from a reaction buffer, a divalent metal ion, deoxyribonucleotides, an oligonucleotide probe, and an intercalating dye. When the composition is used in a nucleic acid amplification reaction, it may further contain a nucleic acid serving as a template for the nucleic acid amplification reaction. In the composition, the deoxyribonucleotide may contain a nucleotide analog. The reaction buffer refers to a compound or mixture that acts to moderate fluctuations in the hydrogen ion concentration (pH) of the reaction solution. Generally, mixed solutions of weak acids and their salts, or weak bases and their salts, have strong buffering properties and are widely used as reaction buffers for pH control. Examples of reaction buffers that can be used in the present invention include Good's buffers such as Tris-HCl and HEPES-KOH, and phosphate buffers such as sodium phosphate buffer. Examples of the divalent metal ions include magnesium ions, manganese ions, zinc ions, and cobalt ions. Divalent metal ions may be supplied in the form of salts such as chlorides, sulfates, or acetates.

[0090] Although the present invention is not particularly limited by the method for amplifying double-stranded nucleic acids, examples of the method for amplifying DNA include polymerase chain reaction (PCR), multiple displacement amplification (MDA), and isothermal nucleic acid amplification methods such as ICAN and LAMP.

[0091] The concentration of the polypeptide having mismatch endonuclease activity in the composition for the nucleic acid amplification reaction may be determined by testing a concentration that does not inhibit the DNA amplification reaction in each reaction system and a concentration that is effective in cleaving double-stranded nucleic acids containing mismatches.

[0092] As the at least one pair of primers used in the composition for nucleic acid amplification reaction, two or more primers suitable for various nucleic acid amplification methods are selected. These may be DNA, RNA, or so-called chimeric primers in which part of DNA is replaced with RNA, as long as they result in the desired amplification. Furthermore, primers containing known nucleic acid analogs or primers labeled with fluorescent dyes for detection purposes may also be used.

[0093] In another embodiment, the composition of the present invention may further contain components for preventing carryover, such as dUTP and uracil N-glycosidase.

[0094] The method for amplifying double-stranded nucleic acid of the present invention can be carried out in the presence of the aforementioned acidic polymeric substance. The effect of the acidic polymeric substance is more pronounced when the amount of nucleic acid in the sample is small. Suitable examples of the acidic polymeric substance include those exemplified in the description of the method for selectively cleaving non-target nucleic acids of the present invention. The method of the present invention may also be carried out in combination with the aforementioned proliferating cell nuclear antigen (PCNA) or a mutant thereof.

[0095] 8. Method of inhibiting nucleic acid amplification of the present invention The method for inhibiting nucleic acid amplification of the present invention is carried out by using the mismatch endonuclease mutant of the present invention described in 1. above and an appropriately designed oligodeoxynucleotide or inhibitory oligonucleotide to inhibit the amplification of nucleic acid having a specific base sequence in a nucleic acid amplification reaction.

[0096] Therefore, one aspect of the present invention is a method for suppressing the amplification of a nucleic acid having a specific base sequence in a nucleic acid amplification reaction, comprising the steps of: (a) an oligodeoxynucleotide or suppressor oligonucleotide designed to generate one or more mismatches when hybridized with a nucleic acid having the specific base sequence; (b) a DNA polymerase; (c) at least one pair of primers; and (d) a polypeptide having mismatch endonuclease activity. Another aspect of the present invention is a method for preferentially amplifying a target nucleic acid by using this method to suppress the amplification of a nucleic acid having a specific base sequence that differs from the base sequence of the target nucleic acid by one or more bases.

[0097] The oligodeoxynucleotide or suppressor oligonucleotide (a) above is not particularly limited as long as it is a single-stranded DNA having a base sequence designed to generate one or more mismatches when hybridized with a nucleic acid having a specific base sequence. It may also be a so-called chimeric oligodeoxynucleotide in which a portion of the DNA is replaced with RNA. Furthermore, although this does not limit the present invention, the 3' end of the oligodeoxynucleotide or suppressor oligonucleotide may be modified to inhibit the extension reaction of the single-stranded DNA by DNA polymerase. Examples of such modifications include amination. Furthermore, the oligodeoxynucleotide or suppressor oligonucleotide may be protected from deoxyribonuclease cleavage by phosphorothioation or other modifications, as long as the nucleic acid to which the oligodeoxynucleotide or suppressor oligonucleotide binds is susceptible to cleavage by a polypeptide having mismatch endonuclease activity. Furthermore, the oligodeoxynucleotide or suppressor oligonucleotide may be labeled with a fluorescent dye or quencher for detection purposes.

[0098] The chain length of the oligodeoxynucleotide or suppressor oligonucleotide can be determined appropriately so that the oligodeoxynucleotide or suppressor oligonucleotide can hybridize with a nucleic acid having the specific base sequence under the conditions of the reaction to be carried out. Furthermore, it is desirable that the position at which a mismatch occurs when hybridized with a nucleic acid having the specific base sequence be at least three nucleotides away from both the 5'-end and the 3'-end of the oligodeoxynucleotide or suppressor oligonucleotide.

[0099] For example, when the method of the present invention is used to suppress the amplification of a nucleic acid having a specific base sequence in a method such as PCR, which involves a reaction at high temperatures, it is preferable to use a mismatch endonuclease that is also heat-resistant. The mismatch endonuclease described in 1 above includes heat-resistant enzymes that are not inactivated even in PCR, and is suitable for such applications.

[0100] Therefore, the present invention further provides a composition for a nucleic acid amplification reaction, comprising the following (a) to (d): (a) an oligodeoxynucleotide or suppressor oligonucleotide designed to generate one or several mismatches when hybridized with a nucleic acid having a specific base sequence or its complementary strand; (b) DNA polymerase; (c) at least one pair of oligonucleotide primers; and (d) The mismatch endonuclease mutant of the present invention described in 1 above.

[0101] The composition may further include at least one selected from a reaction buffer, a divalent metal ion, a deoxyribonucleotide, an oligonucleotide probe, and an intercalating dye. The composition may also include a nucleic acid serving as a template for a nucleic acid amplification reaction. The composition may also include dUTP and uracil N-glycosidase.

[0102] The method of the present invention for suppressing amplification of a nucleic acid having a specific base sequence in a nucleic acid amplification reaction can be carried out in any nucleic acid amplification method. While the present invention is not particularly limited, it is particularly suitable for methods of amplifying DNA. For example, the present invention can be carried out in isothermal nucleic acid amplification methods such as PCR, MDA, and ICAN and LAMP.

[0103] The method of the present invention for suppressing the amplification of nucleic acids having specific base sequences in nucleic acid amplification reactions can be performed on any nucleic acid. Examples of DNA to be amplified include artificially produced DNA mixtures, environmental samples, biological samples, and DNA present in DNA mixtures prepared from such samples. While not limiting the present invention, examples of biological samples include samples from mammals such as humans. Furthermore, while not limiting the present invention, examples of DNA mixtures include mixtures of fragmented genomic DNA, cDNA mixtures generated by reverse transcription of mRNA, and mixtures of multiple PCR products. Examples of DNA having specific base sequences whose amplification is suppressed include reverse transcription products derived from rRNA that remain unseparated and low-molecular-weight DNA generated by primer pairing. When amplifying a gene library for subsequent functional screening, suppressing the amplification of DNA containing the sequences of known positive genes enables the creation of a library that can more efficiently search for unknown genes.

[0104] The concentration of the polypeptide having mismatch endonuclease activity in the method of the present invention may be determined by testing a concentration that does not inhibit the DNA amplification reaction in each reaction system or a concentration that is effective for cleaving mismatched double-stranded nucleic acids. The concentration of the oligonucleotide or inhibitory oligodeoxynucleotide may be determined by optimizing the concentration used, taking into account the amount of template and the amplification efficiency of the target DNA. For example, the method can be carried out at a concentration 0.1 to 10 times the concentration of the primer used in the amplification reaction.

[0105] As described above, the present invention provides a method for preferentially amplifying a target nucleic acid. The method may further include a step of detecting the amplified target DNA. In this specification, this aspect of the present invention may be referred to as the "nucleic acid detection method of the present invention." For example, according to the detection method of the present invention in which DNA is the detection target, even when non-target DNA (DNA having a specific base sequence) is present in large excess relative to the target DNA (target DNA), amplification of DNA using the non-target DNA as a template is suppressed by the oligonucleotide or inhibitory oligodeoxynucleotide and the polypeptide having mismatch endonuclease activity in the method of suppressing amplification of a nucleic acid having a specific base sequence in a nucleic acid amplification reaction of the present invention, thereby enabling detection of the target DNA.

[0106] The method of the present invention for inhibiting nucleic acid amplification can be carried out in the presence of the aforementioned acidic polymeric substance. The effect of the acidic polymeric substance is more pronounced when the amount of nucleic acid in the sample is small. Suitable examples of the acidic polymeric substance include those exemplified in the description of the aforementioned method for selectively cleaving non-target nucleic acids of the present invention. The method of the present invention may also be carried out in combination with the aforementioned proliferating cell nuclear antigen (PCNA) or a mutant thereof.

[0107] 9. Method for detecting nucleic acids of the present invention The nucleic acid detection method of the present invention includes the steps of suppressing the amplification of non-target nucleic acids using the method for suppressing nucleic acid amplification of the present invention described in 8 above, thereby preferentially amplifying the target nucleic acid, and detecting the amplified target nucleic acid. The nucleic acid detection method of the present invention enables, for example, the detection of wild-type and mutant forms of nucleic acids corresponding to genes known to have mutations. By performing the detection method of the present invention using DNA containing a wild-type nucleotide sequence as a nucleic acid having a specific nucleotide sequence (non-target nucleic acid), it becomes possible to detect a small number of mutant alleles in the presence of a large excess of normal alleles (i.e., DNA containing a wild-type nucleotide sequence). For example, the method of the present invention is useful for detecting circulating tumor DNA in the blood or detecting small amounts of fetal DNA sequences contained in maternal blood. Examples of such mutations include microdeletions and point mutations. Polymorphisms resulting from point mutations are called single nucleotide polymorphisms (SNPs). Herein, DNA containing a mutant nucleotide sequence among SNPs may be referred to as DNA having a single nucleotide polymorphism mutation.

[0108] Although the present invention is not particularly limited, the small number of mutant alleles is preferably a nucleic acid containing at least one single nucleotide polymorphism selected from the group consisting of single nucleotide polymorphisms used as tumor markers, single nucleotide polymorphisms correlated with the therapeutic effect of cancer therapeutic drugs, and single nucleotide polymorphisms known to correlate with cellular carcinogenesis. SNPs include those frequently found in tumor cells and those known to correlate with the therapeutic effect of cancer therapeutic drugs or carcinogenesis. Examples of such SNPs include SNPs in the K-ras gene, B-raf gene, and epidermal growth factor receptor (EGFR) gene. Somatic mutations in the K-ras gene are frequently found in colorectal cancer, lung adenocarcinoma, thyroid cancer, etc. Somatic mutations in the B-raf gene are frequently found in colorectal cancer, malignant melanoma, papillary thyroid carcinoma, non-small cell lung cancer, lung adenocarcinoma, etc. Furthermore, somatic mutations in the EGFR gene are frequently found in various solid tumors. It is known that cancer treatment with EGFR inhibitors such as gefitinib and erlotinib is likely to be effective when the EGFR gene in cancer tissues has a specific single nucleotide polymorphism mutation, whereas cancer tissues with a single nucleotide polymorphism mutation in the K-ras gene are likely to exhibit resistance to EGFR inhibitors.

[0109] The detection method of the present invention may be carried out using DNA obtained by treating a composition containing methylated DNA extracted from a biological sample with bisulfite. The detection method of the present invention allows for the detection of a minority of methylated alleles in the presence of a large excess of unmethylated alleles, or a minority of unmethylated alleles in the presence of a large excess of methylated alleles.

[0110] The bisulfite treatment can be performed using the well-known bisulfite method (bisulfite method) used to detect methylated DNA. This treatment converts unmethylated cytosine to uracil, while leaving methylated cytosine unchanged. Furthermore, when the bisulfite-treated reaction mixture is amplified by PCR, uracil is converted to thymine, and methylated cytosine becomes cytosine. Thus, detecting a minority of methylated alleles in the presence of a large excess of unmethylated alleles at a specific site, or detecting a minority of unmethylated alleles in the presence of a large excess of methylated alleles, is essentially verifying the presence of cytosine in the presence of a large excess of thymine, or thymine in the presence of a large excess of cytosine, respectively. If amplification from DNA containing thymine or cytosine present in a large excess can be suppressed, the presence of a minority of methylated or unmethylated alleles can be easily verified.

[0111] The step of detecting the target nucleic acid in the detection method of the present invention can utilize electrophoresis, base sequence analysis, or real-time PCR using a probe such as a cycling probe or TaqMan probe. These assay methods can be performed using common techniques. In particular, when HRM (High Resolution Melting) analysis is used, amplification and detection of the target DNA can be performed in a single step, enabling rapid and simple verification of the target DNA.

[0112] The nucleic acid detection method of the present invention can be carried out in the presence of the aforementioned acidic polymeric substance. The effect of the acidic polymeric substance is more pronounced when the amount of nucleic acid in the sample is small. Suitable examples of the acidic polymeric substance include those exemplified in the description of the selective cleavage method of non-target nucleic acids of the present invention. The method of the present invention may also be carried out in combination with the aforementioned proliferating cell nuclear antigen (PCNA) or a mutant thereof. [Example]

[0113] The present invention will be described in more detail below with reference to examples, but the scope of the present invention is not limited to these examples.

[0114] Experimental Method 1 (1-1) Construction of mismatch endonuclease mutants The nucleotide sequence of the gene encoding the Pyrococcus furiosus-derived polypeptide PF_R00065 (RefSeq ID: WP_011011124.1, SEQ ID NO: 1) is shown in SEQ ID NO: 16. Herein, an artificial gene was constructed by standard methods using this nucleotide sequence to introduce a mutation into a specific site. The resulting artificial gene was introduced into the plasmid pET6xHN-C (Takara Bio USA) using the In-Fusion (registered trademark) HD ​​Cloning Kit (Takara Bio USA). The resulting plasmid contains a nucleotide sequence encoding a mismatch endonuclease mutant with a histidine tag added to the C-terminus.

[0115] Next, Escherichia coli BL21 DE3 strain (Takara Bio) was transformed with the plasmid and cultured overnight at 37°C on a 1.5% agarose LB plate containing 100 μg / ml ampicillin. Three single colonies were selected from this plate and inoculated into LB medium containing 100 μg / ml ampicillin (hereinafter referred to as LB-AP medium) and cultured overnight with shaking at 37°C. 300 μl of the culture was then inoculated into 6 ml of LB-AP medium and cultured overnight with shaking at 37°C. When the OD600 value reached 0.6, IPTG was added to the culture to a final concentration of 1 mM, and induction culture was continued for another 4 hours at 25°C. The cells were then harvested when the OD600 value reached 4.

[0116] The cells obtained above were suspended in 400 μl of a solution containing 50 mM Tris·HCl (pH 7.5), 300 mM NaCl, 5% glycerol, and 0.15% Triton X-100 (hereafter referred to as Buffer S). The suspension was sonicated three times for 30 seconds at 4°C using an ultrasonicator (Sonic & Materials). This procedure resulted in a clear suspension. The sonicated suspension was centrifuged at 11,000 × g for 10 minutes at 4°C, and the supernatant was collected. The crude extract thus obtained was subjected to Ni resin purification.

[0117] Ni resin purification was performed as follows: 50 μl of Ni-NTA agarose (Qiagen) was placed in a 1.5 ml tube, washed twice with 250 μl of sterile distilled water, and then equilibrated twice with 250 μl of Buffer A (50 mM Tris·HCl pH 7.5, 300 mM NaCl, 5% glycerol, and 5 mM imidazole). The equilibrated Ni-NTA agarose was suspended in 400 μl of crude extract and allowed to stand for 30 minutes. The suspension was then centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was removed, and the Ni-NTA agarose was washed three times with 100 μl of Buffer A. The adsorbed material was then eluted from the Ni-NTA agarose with 100 μl of Buffer B (50 mM Tris·HCl pH 7.5, 300 mM NaCl, 5% glycerol, and 300 mM imidazole). The resulting eluate was used as a mismatch endonuclease mutant solution in the tests described below.

[0118] (1-2) Construction of heterodimeric mismatch endonuclease mutants Based on the genes encoding the mutants prepared in 1-1 above, genes encoding heterodimeric mismatch endonuclease mutants were prepared. An artificial gene was prepared in the same manner as in 1-1, in which the genes encoding the GG-specific mismatch endonuclease mutant and the TT-specific mismatch endonuclease mutant were connected in series via a linker (SEQ ID NO: 35).

[0119] (2) Substrate specificity evaluation test of mismatch endonuclease mutants (2-1) Nucleic acid As nucleic acids forming double-stranded DNA substrates, NucS-Template-T (SEQ ID NO: 31) and NucS-Template-G (SEQ ID NO: 32), as well as NucS-Probe-T (SEQ ID NO: 33) and NucS-Probe-G (SEQ ID NO: 34) were chemically synthesized by known methods. Double-stranded DNAs combining these templates and probes were used as substrates. FAM was attached to the 5' end of each of the two probes, and Eclipse® Dark Quencher was attached to the 3' end.

[0120] (2-2) Cleavage specificity 2.5 μl of 10x buffer, 1 μl of 25 μM template, 1.5 μl of 5 μM probe, 5 μl of mismatch endonuclease mutant solution, and 15 μl of sterile water were mixed to a total volume of 25 μl. Sixty cycles of 60 seconds at 55°C were performed in a thermal cycler. Alternatively, sixty cycles of 15 seconds at 55°C were performed in a thermal cycler. The composition of the 10x buffer was 500 mM Tris-HCl (pH 9.2), 140 mM (NH4)2SO4, 100 mM KCl, 25 mM MgCl2, and 0.1% BSA. The enzyme dilution buffer consisted of 25 mM Tris-HCl (pH 8.0), 50 mM KCl, 0.1 mM DTT, 0.01% gelatin, 0.5% Nonidet P-40, 0.5% Tween-20, and 0.09% BSA.

[0121] Example 1: Construction of mismatch endonuclease mutants (1) Mismatch endonuclease mutant S47K+N76D According to Experimental Method 1 (1-1), an artificial gene encoding a mutant protein in which serine at position 47 in the Pyrococcus furiosus-derived polypeptide PF_RS00065 was replaced with lysine and asparagine at position 76 was replaced with aspartic acid was constructed, and a mismatch endonuclease mutant solution was prepared. The amino acid sequence of the mutant protein is shown in SEQ ID NO:6, and the nucleic acid sequence is shown in SEQ ID NO:21.

[0122] (2) Mismatch endonuclease mutant S47R+N76D+L123A According to Experimental Method 1 (1-1), an artificial gene encoding a mutant protein was created in which the serine at position 47 in the Pyrococcus furiosus-derived polypeptide PF_RS00065 was replaced with arginine, the asparagine at position 76 with aspartic acid, and the leucine at position 123 with alanine, and a mismatch endonuclease mutant solution was prepared. The amino acid sequence of the mutant protein is shown in SEQ ID NO:7, and the nucleic acid sequence is shown in SEQ ID NO:22.

[0123] (3) Mismatch endonuclease mutant S47R+N76D+L123G According to Experimental Method 1 (1-1), an artificial gene encoding a mutant protein in which serine at position 47 in the Pyrococcus furiosus-derived polypeptide PF_RS00065 was replaced with arginine, asparagine at position 76 with aspartic acid, and leucine at position 123 with glycine was constructed, and a mismatch endonuclease mutant solution was prepared. The amino acid sequence of the mutant protein is shown in SEQ ID NO:8, and the nucleic acid sequence is shown in SEQ ID NO:23.

[0124] (4) Mismatch endonuclease mutant Q78R+L123S According to Experimental Method 1 (1-1), an artificial gene encoding a mutant protein in which glutamine at position 78 in the Pyrococcus furiosus-derived polypeptide PF_RS00065 was replaced with arginine and leucine at position 123 was replaced with serine was constructed, and a mismatch endonuclease mutant solution was prepared. The amino acid sequence of the mutant protein is shown in SEQ ID NO:9, and the nucleic acid sequence is shown in SEQ ID NO:24.

[0125] (5) Mismatch endonuclease mutant Q78R+L123T According to Experimental Method 1 (1-1), an artificial gene encoding a mutant protein in which glutamine at position 78 in the Pyrococcus furiosus-derived polypeptide PF_RS00065 was replaced with arginine and leucine at position 123 was replaced with threonine was constructed, and a mismatch endonuclease mutant solution was prepared. The amino acid sequence of the mutant protein is shown in SEQ ID NO: 10, and the nucleic acid sequence is shown in SEQ ID NO: 25.

[0126] (6) Mismatch endonuclease mutant Q78R+L123M According to Experimental Method 1 (1-1), an artificial gene encoding a mutant protein in which glutamine at position 78 in the Pyrococcus furiosus-derived polypeptide PF_RS00065 was replaced with arginine and leucine at position 123 was replaced with methionine was constructed, and a mismatch endonuclease mutant solution was prepared. The amino acid sequence of the mutant protein is shown in SEQ ID NO: 11, and the nucleic acid sequence is shown in SEQ ID NO: 26.

[0127] (7) Mismatch endonuclease mutant Q78R+L123C According to Experimental Method 1(1), an artificial gene encoding a mutant protein was constructed in which glutamine at position 78 in the Pyrococcus furiosus-derived polypeptide PF_RS00065 was replaced with arginine and leucine at position 123 was replaced with cysteine, and a mismatch endonuclease mutant solution was prepared. The amino acid sequence of the mutant protein is shown in SEQ ID NO: 12, and the nucleic acid sequence is shown in SEQ ID NO: 27.

[0128] (8) Mismatch endonuclease mutant Q78R+L125V According to Experimental Method 1 (1-1), an artificial gene encoding a mutant protein in which glutamine at position 78 in the Pyrococcus furiosus-derived polypeptide PF_RS00065 was replaced with arginine and leucine at position 125 was replaced with valine was constructed, and a mismatch endonuclease mutant solution was prepared. The amino acid sequence of the mutant protein is shown in SEQ ID NO: 13, and the nucleic acid sequence is shown in SEQ ID NO: 28.

[0129] (9) Mismatch endonuclease mutant Q78R+L125A According to Experimental Method 1 (1-1), an artificial gene encoding a mutant protein in which glutamine at position 78 in the Pyrococcus furiosus-derived polypeptide PF_RS00065 was substituted with arginine and leucine at position 125 with alanine was constructed, and a mismatch endonuclease mutant solution was prepared. The amino acid sequence of the mutant protein is shown in SEQ ID NO: 14, and the nucleic acid sequence is shown in SEQ ID NO: 29.

[0130] (10) Heterodimeric mismatch endonuclease mutants According to Experimental Method 1 (1-2), an artificial gene was constructed to express Mutant 1 (S47R+N76D+L123A; SEQ ID NO: 7) and Mutant 2 (Q78R+L123M; SEQ ID NO: 11) as a single polypeptide via a linker (SEQ ID NO: 35), and a mismatch endonuclease mutant solution was prepared. The amino acid sequence of the mutant protein is shown in SEQ ID NO: 15, and the nucleic acid sequence is shown in SEQ ID NO: 30.

[0131] Example 2: Substrate specificity evaluation test of mismatch endonuclease mutants The mismatch endonuclease mutants prepared in Example 1 (1) to (9), as well as the wild-type mismatch endonuclease (PfuNucS) and its W77F mutant, were subjected to a substrate specificity evaluation test according to Experimental Method 1 (2). The results are shown in Table 2.

[0132] [Table 2]

[0133] As shown in Table 2, GG-specific mismatch endonuclease mutants and TT-specific mismatch endonuclease mutants were obtained, which have altered substrate specificities from the wild-type mismatch endonuclease.

[0134] Example 3: Substrate specificity evaluation test of heterodimeric mismatch endonuclease mutants The heterodimeric mismatch endonuclease mutants prepared in Example 1(10) were subjected to a substrate specificity evaluation test according to Experimental Method 1(2).

[0135] As a result, the heterodimeric mismatch endonuclease mutant exhibited substrate specificity specific to GT / TG mismatches, which was different from that of the wild-type mismatch endonuclease and was also different from the mismatch endonuclease mutants prepared in Example 1(1) to (9). [Industrial Applicability]

[0136] The present invention is useful in a wide range of fields, including genetic engineering, biology, medicine, and agriculture. [Sequence List Free Text]

[0137] SEQ ID NO: 1: Amino acid sequence of mismatch endonuclease PF0012 from Pyrococcus furiosus SEQ ID NO: 2; Amino acid sequence of mismatch endonuclease PF0012 W77F variant SEQ ID NO: 3: Amino acid sequence of mismatch endonuclease TERMP_01877 from Thermococcus barophilus SEQ ID NO: 4: Amino acid sequence of mismatch endonuclease MJ_0225 from Methanocaldococcus jannaschii SEQ ID NO: 5: Amino acid sequence of mismatch endonuclease TKO NucS from Thermococcus kodakarensis SEQ ID NO: 6: Amino acid sequence of mismatch endonuclease variant S47K+N76D from PF0012 SEQ ID NO: 7: Amino acid sequence of mismatch endonuclease variant S47R+N76D+L123A from PF0012 SEQ ID NO: 8: Amino acid sequence of mismatch endonuclease variant S47R+N76D+L123G from PF0012 SEQ ID NO: 9: Amino acid sequence of mismatch endonuclease variant Q78R+L123S from PF0012 SEQ ID NO: 10: Amino acid sequence of mismatch endonuclease variant Q78R+L123T from PF0012 SEQ ID NO: 11: Amino acid sequence of mismatch endonuclease variant Q78R+L123M from PF0012 SEQ ID NO: 12: Amino acid sequence of mismatch endonuclease variant Q78R+L123C from PF0012 SEQ ID NO: 13: Amino acid sequence of mismatch endonuclease variant Q78R+L125V from PF0012 SEQ ID NO: 14: Amino acid sequence of mismatch endonuclease variant Q78R+L125A from PF0012 SEQ ID NO: 15: Amino acid sequence of mismatch endonuclease variant hetero dimer from variant S47R+N76D+L123A and variant Q78R+L123M SEQ ID NO: 16: Nucleic acid sequence encoding gene of mismatch endonuclease PF0012 from Pyrococcus furiosus SEQ ID NO: 17: Nucleic acid sequence encoding gene of mismatch endonuclease PF0012 W77F variant SEQ ID NO: 18: Nucleic acid sequence encoding gene of mismatch endonuclease TERMP_01877 from Thermococcus barophilus SEQ ID NO: 19: Nucleic acid sequence encoding gene of mismatch endonuclease MJ_0225 from Methanocaldococcus jannaschii SEQ ID NO: 20: Nucleic acid sequence encoding gene of mismatch endonuclease TKO NucS from Thermococcus kodakarensis SEQ ID NO: 21: Nucleic acid sequence encoding gene of mismatch endonuclease variant S47K+N76D from PF0012 SEQ ID NO: 22: Nucleic acid sequence encoding gene of mismatch endonuclease variant S47R+N76D+L123A from PF0012 SEQ ID NO: 23: Nucleic acid sequence encoding gene of mismatch endonuclease variant S47R+N76D+L123G from PF0012 SEQ ID NO: 24: Nucleic acid sequence encoding gene of mismatch endonuclease variant Q78R+L123S from PF0012 SEQ ID NO: 25: Nucleic acid sequence encoding gene of mismatch endonuclease variant Q78R+L123T from PF0012 SEQ ID NO: 26: Nucleic acid sequence encoding gene of mismatch endonuclease variant Q78R+L123M from PF0012 SEQ ID NO: 27: Nucleic acid sequence encoding gene of mismatch endonuclease variant Q78R+L123C from PF0012 SEQ ID NO: 28: Nucleic acid sequence encoding gene of mismatch endonuclease variant Q78R+L125V from PF0012 SEQ ID NO: 29: Nucleic acid sequence encoding gene of mismatch endonuclease variant Q78R+L125A from PF0012 SEQ ID NO: 30: Nucleic acid sequence encoding gene of mismatch endonuclease variant hetero dimer from variant S47R+N76D+L123A and variant Q78R+L123M SEQ ID NO: 31: Synthetic oligo nucleotide "NucS-Template-T" SEQ ID NO: 32: Synthetic oligo nucleotide "NucS-Template-G" SEQ ID NO: 33: Synthetic oligo nucleotide "NucS-Probe-T". 5'-end is labeled with FAM and 3'-end is labeled with Eclipse. SEQ ID NO: 34: Synthetic oligo nucleotide "NucS-Probe-G". 5'-end is labeled with FAM and 3'-end is labeled with Eclipse. SEQ ID NO: 35: Amino acid sequence of Linker SEQ ID NO: 36: Nucleic acid sequence encoding Linker

Claims

1. A polypeptide represented by the following (A) or (B): (A) A polypeptide selected from the group consisting of the following (1) to (3), which has an activity of cleaving a nucleic acid strand having a guanine (G) forming a G-G or G-T / T-G mismatch base pair in a double-stranded nucleic acid: (1) In the amino acid sequence set forth in SEQ ID NO: 1, Serine at position 47 to lysine or arginine, and Asparagine at position 76 becomes aspartic acid a polypeptide comprising each substituted amino acid sequence; (2) A polypeptide comprising an amino acid sequence in which 1 to 10 amino acid residues are further mutated in the substituted amino acid sequence according to (1) above, wherein the mutations are substitutions, deletions, insertions, and / or additions, and the amino acid substitutions in the substituted amino acid sequence according to (1) above are retained; (3) A polypeptide comprising an amino acid sequence having 90% or more identity to the substituted or mutated amino acid sequence described in (1) or (2) above, provided that the amino acid substitutions or mutations in the substituted or mutated amino acid sequence described in (1) or (2) above are retained; or (B) A polypeptide selected from the group consisting of the following (4) to (6), which has an activity of cleaving a nucleic acid strand having thymine (T) forming a T-T or GT / T-G mismatch base pair in a double-stranded nucleic acid: (4) In the amino acid sequence set forth in SEQ ID NO: 1, glutamine at position 78 is substituted with arginine, and a polypeptide comprising an amino acid sequence in which leucine at position 123 is substituted with serine, threonine, methionine, or cysteine, or leucine at position 125 is substituted with valine or alanine; (5) A polypeptide comprising an amino acid sequence in which 1 to 10 amino acid residues are further mutated in the substituted amino acid sequence according to (4) above, wherein the mutations are substitutions, deletions, insertions, and / or additions, and the amino acid substitutions in the substituted amino acid sequence according to (4) above are retained; (6) A polypeptide having an amino acid sequence that is 90% or more identical to the substituted or mutated amino acid sequence described in (4) or (5) above, provided that the amino acid substitutions or mutations in the substituted or mutated amino acid sequence described in (4) or (5) above are retained.

2. The polypeptide of claim 1, comprising an amino acid sequence selected from SEQ ID NOs: 6 to 15.

3. A nucleic acid encoding the polypeptide of claim 1 or 2.

4. The nucleic acid according to claim 3, comprising a base sequence selected from SEQ ID NOs: 21 to 30.

5. An expression vector comprising the nucleic acid of claim 3 or 4 and an expression control sequence.

6. A cell transformed with the expression vector according to claim 5, which expresses a polypeptide having mismatch endonuclease activity.

7. A method for cleaving a double-stranded nucleic acid having a mismatch, comprising the step of allowing the polypeptide according to claim 1 or 2 to act on the double-stranded nucleic acid.

8. A composition for cleaving double-stranded nucleic acid, amplifying double-stranded nucleic acid, controlling nucleic acid amplification, or detecting nucleic acid, comprising the following (a) to (d): (a) a polypeptide according to claim 1 or 2; (b) an oligonucleotide that contains at least one mismatch when hybridized with a non-target nucleic acid to produce a double-stranded nucleic acid that is cleaved by the polypeptide of (a), and that, when hybridized with a target nucleic acid, produces a double-stranded nucleic acid that is not cleaved by the polypeptide of (a); (c) at least one pair of oligonucleotide primers; and (d) a polypeptide having DNA polymerase activity.

9. A method for amplifying nucleic acid, comprising the following steps (1) and (2): (1) preparing a composition comprising a template nucleic acid molecule and the following (a) to (d): (a) a polypeptide according to claim 1 or 2; (b) an oligonucleotide that contains at least one mismatch when hybridized with a non-target nucleic acid to produce a double-stranded nucleic acid that is cleaved by the polypeptide of (a), and that, when hybridized with a target nucleic acid, produces a double-stranded nucleic acid that is not cleaved by the polypeptide of (a); (c) at least one pair of oligonucleotide primers; and (d) a polypeptide having DNA polymerase activity; and (2) A step of reacting the composition obtained in step (1) under appropriate conditions to amplify nucleic acids.

10. 10. The method of claim 9, wherein the nucleic acid amplification is performed by polymerase chain reaction (PCR), isothermal nucleic acid amplification, or multiple displacement amplification (MDA).

11. A method for inhibiting amplification of a nucleic acid containing a specific base sequence, comprising the step of carrying out a nucleic acid amplification reaction in the presence of the following (a) to (d): (a) an oligodeoxynucleotide designed to generate one or several mismatches when hybridized with a nucleic acid containing the specific base sequence or a complementary strand thereof; (b) a DNA polymerase; (c) at least one pair of oligonucleotide primers; and (d) A polypeptide according to claim 1 or 2.

12. The method according to claim 11, wherein the nucleic acid amplification reaction is a polymerase chain reaction (PCR) method or an isothermal nucleic acid amplification method.

13. A method for preferentially amplifying a target DNA, characterized in that the amplification of DNA having a base sequence that differs from the target DNA by one or several bases is suppressed by the method described in claim 11 or 12.

14. The method according to claim 13, which is used for the purpose of distinguishing and amplifying DNA having a wild-type base sequence from DNA in which a single nucleotide polymorphism has occurred in said DNA.

15. The method according to claim 14 , wherein the single nucleotide polymorphism is a single nucleotide polymorphism that correlates with canceration or the therapeutic effect of a cancer therapeutic drug.

16. A dimeric form of a polypeptide selected from the group consisting of: （１） A homodimer having the polypeptide according to claim 1 (A) as a subunit. （２） A homodimer having the polypeptide of claim 1(B) as a subunit, and (3) A heterodimer having the polypeptides according to claim 1(A) and claim 1(B) as subunits.

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