Ligase mutants

Abstract

To provide ligase variants having excellent properties.SOLUTION: Disclosed is a ligase variant being one of the following (1), (2) and (3): (1) a ligase variant comprising an amino acid sequence having 95% or more identity to a specific amino acid sequence and having nucleic acid-ligating activity; (2) a ligase variant comprising an amino acid sequence having 90% or more identity to another specific amino acid sequence and having nucleic acid-ligating activity; and (3) a ligase variant comprising an amino acid sequence having 97% or more identity to a still another specific amino acid sequence and having nucleic acid-ligating activity.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to ligase mutants and the like. [Background technology]

[0002] T4 RNA ligase 2 is a type of RNA ligase (EC6.5.1.3) capable of linking ribonucleotides in the presence of ATP, and is an enzyme derived from the T4 bacteriophage capable of infecting Escherichia (NP_049790). T4 RNA ligase 2 has the ability to link a phosphate group (donor) at the 5' end of a nucleic acid to a hydroxyl group (acceptor) at the 3' end by forming a phosphodiester bond. T4 RNA ligase 2 is used in reactions such as linking double-stranded RNA with protruding ends and linking nicks in double-stranded RNA. It is also known that T4 RNA ligase 2 can use not only RNA, but also DNA and modified nucleic acids other than DNA and RNA as substrates. In addition, several mutants of T4 RNA ligase 2 are known. The prior art of T4 RNA ligase 2 can be found below. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2008 / 094599 [Non-patent literature]

[0004] [Non-Patent Document 1] [online],INTERNET,NCBI Protein Database,August 13, 2018,NP_049790,Retrieved date:February 27, 2020,URL,https: / / www.ncbi.nlm.nih.gov / protein / NP_049790 [Non-Patent Document 2] Chauleau , M. , & Shuman , S. (2013). Kinetic mechanism of nick sealing by T4 RNA ligase 2 and effects of 3′-OH base mispairs and damaged base lesions. RNA, 19(12): 1840-1847.

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[0005] An object of the present invention is to provide a ligase mutant having excellent properties. [Means for solving the problem]

[0006] As a result of intensive research, the present inventors have succeeded in developing three ligase mutants Mut1 to 3 (SEQ ID NOs: 1 to 3) that show 93%, 87% and 95% amino acid sequence identity to T4 RNA ligase 2 (NP_049790), respectively (see Table 1 below) and have superior properties compared to T4 RNA ligase 2, thereby completing the present invention. The above prior art does not teach or suggest such three ligase mutants.

[0007] That is, the present invention is as follows. [1] A ligase mutant of the following (1), (2), or (3): (1) a ligase mutant comprising an amino acid sequence showing 95% or more identity to the amino acid sequence of SEQ ID NO:1 and having nucleic acid ligation activity; (2) a ligase mutant having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO:2 and having nucleic acid ligation activity; or (3) A ligase mutant comprising an amino acid sequence that is 97% or more identical to the amino acid sequence of SEQ ID NO:3 and having nucleic acid ligation activity. [2] The ligase mutant of [1], wherein the nucleic acid is single-stranded RNA or double-stranded RNA which may contain DNA and / or modified nucleic acid. [3] ligating nucleic acid material in the presence of the ligase mutant of [1] or [2] to produce a product nucleic acid; A method for producing a nucleic acid product, wherein the nucleic acid material is selected from the group consisting of single-stranded nucleic acid material, double-stranded nucleic acid material, and mixtures thereof. [4] The method of [3], wherein the nucleic acid material is RNA. [5] The method according to [3] or [4], wherein the nucleic acid material is four or more single-stranded RNAs. [6] Any of the methods according to [3] to [5], wherein the nucleic acid product comprises a complementary portion having a length of 12 to 27 bases. [7] Any of the methods according to [3] to [6], wherein the nucleic acid material comprises DNA and / or modified nucleic acid. [8] Any of the methods according to [3] to [7], wherein the concentration of the nucleic acid material is 1 μM or more. [9] Any of the methods according to [3] to [8], wherein the nucleic acid product is siRNA.

[10] A polynucleotide encoding the ligase mutant of [1] or [2].

[11] An expression vector comprising the polynucleotide of

[10] .

[12] A transformed microorganism comprising an expression unit comprising a polynucleotide encoding the ligase mutant of [1] or [2] and a promoter operably linked thereto.

[13] A method for producing a ligase mutant, comprising producing the ligase mutant of [1] or [2] by using the transformed microorganism of

[12] . Effect of the Invention

[0008] According to the present invention, a nucleic acid product (eg, a modified nucleic acid such as siRNA or a heteroduplex nucleic acid) can be efficiently produced from a nucleic acid material. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows the amino acid sequences (SEQ ID NOs: 1 to 3) of the ligase mutants of the present invention (Mut1 to 3). [Diagram 2] 2 shows the double-stranded oligonucleotide produced by ligation of the four fragment single-stranded oligonucleotides. The designations of modified nucleotide residues are the same as those in Table 2. [Diagram 3] 3 shows (A) a double-stranded oligonucleotide produced by ligation of four single-stranded oligonucleotide fragments, and (B) a time course of the production of the double-stranded oligonucleotide. The designations of modified nucleotide residues are the same as those in Table 4. [Figure 4] 4 shows (A) the double-stranded oligonucleotide produced by the ligation reaction of three single-stranded oligonucleotide fragments, and (B) the amount of the double-stranded oligonucleotide (reaction time 15 min). Oligonucleotides in which the 2'-positions of the nucleotide residues at positions -2, -1, +1, and +2 from the ligation point were modified with a fluorine atom (F), O-methyl (Ome), or O-methoxyethyl (MOE), or substituted with a hydrogen atom (DNA), were used as substrates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] 1. Ligase Mutants The present invention provides a ligase mutant of the following (1), (2) or (3): (1) a ligase mutant comprising an amino acid sequence showing 95% or more identity to the amino acid sequence of SEQ ID NO:1 and having nucleic acid ligation activity; (2) a ligase mutant having an amino acid sequence that is 90% or more identical to the amino acid sequence of SEQ ID NO:2 and having nucleic acid ligation activity; or (3) A ligase mutant comprising an amino acid sequence that is 97% or more identical to the amino acid sequence of SEQ ID NO:3 and having nucleic acid ligation activity.

[0011] The amino acid sequence of SEQ ID NO: 1 shows 93% identity to the amino acid sequence of the known T4 RNA ligase 2 (NP_049790). The ligase mutant of (1) is specified by an amino acid sequence showing 95% or more identity to the amino acid sequence of SEQ ID NO: 1, and is therefore sufficiently differentiated from the known T4 RNA ligase 2. The degree of identity % to the amino acid sequence of SEQ ID NO: 1 in the ligase mutant of (1) may be preferably 96% or more, more preferably 97% or more, even more preferably 98% or more, and most preferably 99% or more.

[0012] The amino acid sequence of SEQ ID NO: 2 shows 87% identity to the amino acid sequence of the known T4 RNA ligase 2 (NP_049790). The ligase mutant (2) is specified by an amino acid sequence showing 90% or more identity to the amino acid sequence of SEQ ID NO: 2, and is therefore sufficiently differentiated from the known T4 RNA ligase 2. The degree of identity % to the amino acid sequence of SEQ ID NO: 2 in the ligase mutant (1) may be preferably 91% or more, more preferably 92% or more, even more preferably 93% or more, and most preferably 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more.

[0013] The amino acid sequence of SEQ ID NO: 3 shows 95% identity to the amino acid sequence of the known T4 RNA ligase 2 (NP_049790). The ligase mutant of (3) is specified by an amino acid sequence showing 97% or more identity to the amino acid sequence of SEQ ID NO: 3, and is therefore sufficiently differentiated from the known T4 RNA ligase 2. The degree of identity % to the amino acid sequence of SEQ ID NO: 3 in the ligase mutant of (3) may be preferably 97.5% or more, more preferably 98% or more, even more preferably 98.5% or more, and most preferably 99% or more or 99.5% or more.

[0014] The percent identity of amino acid sequences can be calculated using Genetyx Corporation's software GENETYX Ver13.1.1, using the full length of the polypeptide portion encoded by the ORF, and then performing Muscle alignment, ClustalW alignment, or Multiple sequence alignment, followed by calculation with the setting "Gaps are taken into account." The number of modifications (e.g., substitutions, deletions, additions, or insertions, or a combination thereof) of amino acid residues that can achieve the above percent identity in SEQ ID NO:1 (total length 332 amino acid residues), SEQ ID NO:2 (total length 330 amino acid residues), and (total length 332 amino acid residues) may be 1 to 33 for 90% or more, 1 to 29 for 91% or more, 1 to 26 for 92% or more, 1 to 23 for 93% or more, 1 to 19 for 94% or more, 1 to 16 for 95% or more, 1 to 13 for 96% or more, 1 to 9 for 97% or more, 1 to 6 for 98% or more, and 1 to 3 for 99% or more.

[0015] The ligase mutant of the present invention has a nucleic acid ligation activity. Examples of nucleic acids include single-stranded nucleic acids and double-stranded nucleic acids. Examples of nucleic acids include RNA, DNA, modified nucleic acids other than RNA and DNA, and mixed nucleic acids thereof. Preferably, the nucleic acid may be single-stranded RNA or double-stranded RNA that may contain DNA and / or modified nucleic acids. Details of the nucleic acid are the same as those of the nucleic acid material in the method for producing a nucleic acid product described below.

[0016] The nucleic acid ligation activity of the ligase mutant of the present invention is not particularly limited as long as it is superior to that of T4 RNA ligase 2 (NP_049790), but may be preferably 1.2 times or more, more preferably 1.5 times or more, even more preferably 1.8 times or more, and particularly preferably 2.0 times or more, of T4 RNA ligase 2. Such nucleic acid ligation activity can be measured by a predetermined reaction as described in the Examples. For example, such a reaction may be carried out by the following steps a) to c) (see, for example, Example 2): a) Prepare 20 μL of reaction solution containing 10 μM nucleic acid material (e.g., single-stranded RNA), 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM dithiothreitol, and 0.4 mM ATP at final concentrations; b) Add 50 μL of the purified mutant ligase solution to the reaction mixture to a final concentration of 0.36 μg / mL to initiate the reaction; c) React at 25°C for 1 hour.

[0017] The ligase mutant of the present invention may also have excellent temperature stability. For example, when the ligase mutant containing the amino acid sequence of the present invention is subjected to heat treatment and the activity is subsequently measured, it is preferable that the residual activity is 30% or more, 40% or more, 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or 95% or more of the activity measured under the condition without heat treatment. Such conditions may be 23 hours of incubation at 25°C or 4 hours of incubation at 37°C (e.g., incubation in a solution containing 54 mM Tris-HCl (pH 7.5), 2.2 mM MgCl2, 1.1 mM dithiothreitol, 0.43 mM ATP, and 0.78 μg / mL of enzyme), followed by activity measurement. Activity measurement may be performed under the conditions of a) to c) in the above paragraph. Alternatively, activity measurement may be performed under conditions in which activity is measured in a 15-minute reaction at 25°C or 37°C. The reaction solution composition used under such conditions may be, for example, 10 μM oligonucleotide, 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM dithiothreitol, 0.4 mM ATP, and 0.72 μg / mL enzyme. Since the temperature stability of an enzyme generally correlates with its liquid stability and / or long-term storage stability, it can be said that the ligase mutant of the present invention having excellent temperature stability has excellent liquid stability and / or long-term storage stability. Therefore, the ligase mutant of the present invention is useful as a reagent.

[0018] The ligase mutant of the present invention may have one or more amino acid residue mutations within a range in which the desired percent identity and ligation activity are maintained. The position of the amino acid residue into which the mutation may be introduced is clear to those skilled in the art. For example, those skilled in the art can 1) compare the amino acid sequences of multiple proteins having similar properties (e.g., SEQ ID NOs: 1 to 3), 2) clarify the relatively conserved regions and the relatively non-conserved regions, and then 3) predict the regions that may play an important role in function and the regions that may not play an important role in function from the relatively conserved regions and the relatively non-conserved regions, respectively, and thus recognize the correlation between structure and function. Thus, those skilled in the art can identify the positions of amino acid residues into which the mutation may be introduced in the amino acid sequence of the ligase mutant of the present invention. The amino acid residue after mutation at such a position is a desired natural α-amino acid residue different from the amino acid residue before mutation. Such desired natural α-amino acid residues are residues of L-alanine (A), L-asparagine (N), L-cysteine ​​(C), L-glutamine (Q), L-isoleucine (I), L-leucine (L), L-methionine (M), L-phenylalanine (F), L-proline (P), L-serine (S), L-threonine (T), L-tryptophan (W), L-tyrosine (Y), L-valine (V), L-aspartic acid (D), L-glutamic acid (E), L-arginine (R), L-histidine (H), L-lysine (K), or glycine (G). For example, in an amino acid sequence showing 95% or more identity to the amino acid sequence of SEQ ID NO: 1, 1 to 16 amino acid residues may be mutated into the amino acid sequence of SEQ ID NO: 1. In an amino acid sequence that shows 90% or more identity to the amino acid sequence of SEQ ID NO: 2, mutations of 1 to 33 amino acid residues may be introduced into the amino acid sequence of SEQ ID NO: 2. In an amino acid sequence that shows 97% or more identity to the amino acid sequence of SEQ ID NO: 3, mutations of 1 to 9 amino acid residues may be introduced into the amino acid sequence of SEQ ID NO: 3. The mutations of amino acid residues are selected from the group consisting of substitution, deletion, addition, and insertion of amino acid residues.

[0019] When an amino acid residue is mutated by substitution, the substitution of the amino acid residue may be a conservative substitution. As used herein, the term "conservative substitution" refers to replacing a given amino acid residue with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains are well known in the art. For example, such families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with beta-branched side chains (e.g., threonine, valine, isoleucine), amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), amino acids with hydroxyl (e.g., alcoholic, phenolic)-containing side chains (e.g., serine, threonine, tyrosine), and amino acids with sulfur-containing side chains (e.g., cysteine, methionine). Preferably, conservative amino acid substitutions may be between aspartic acid and glutamic acid, between arginine, lysine and histidine, between tryptophan and phenylalanine, between phenylalanine and valine, between leucine, isoleucine and alanine, and between glycine and alanine.

[0020] The ligase mutant of the present invention may also contain other peptide components (e.g., tag moieties) at the C-terminus or N-terminus. Examples of other peptide components that may be contained in the ligase mutant of the present invention include peptide components that facilitate purification of a target protein (e.g., tag moieties such as histidine tag and Strep-tag II; proteins commonly used in the purification of target proteins such as glutathione-S-transferase and maltose-binding protein), peptide components that improve the solubility of a target protein (e.g., Nus-tag), peptide components that act as chaperones (e.g., trigger factor), proteins or protein domains with other functions, or peptide components that serve as linkers connecting them to the ligase mutant.

[0021] 2. Inventions that may be related to the production of the ligase mutant of the present invention The ligase mutant of the present invention can be prepared using a transformed microorganism containing an expression unit comprising a polynucleotide encoding the ligase mutant of the present invention and a promoter operably linked thereto, or using a cell-free system, etc. The present invention also provides such polynucleotides and transformed microorganisms, as well as expression vectors that can be used to prepare transformants.

[0022] The polynucleotide of the present invention is a polynucleotide that encodes the ligase mutant of the present invention. The polynucleotide of the present invention may be DNA or RNA, but is preferably DNA.

[0023] The transformed microorganism of the present invention can be prepared, for example, by a method using an expression vector containing the polynucleotide of the present invention (e.g., competent cell method, electroporation method), or by genome modification technology. When the expression vector is an integrative vector that undergoes homologous recombination with the genomic DNA of the host cell, the expression unit can be integrated into the genomic DNA of the host cell by transformation. On the other hand, when the expression vector is a non-integrative vector that does not undergo homologous recombination with the genomic DNA of the host cell, the expression unit is not integrated into the genomic DNA of the host cell by transformation, and can exist in the host cell as an expression vector, independent of the genomic DNA. Alternatively, by genome editing technology (e.g., CRISPR / Cas system, Transcription Activator-Like Effector Nucleases (TALEN)), it is possible to integrate the expression unit into the genomic DNA of the host cell and to modify the expression unit inherent to the host cell.

[0024] The present invention also provides an expression vector comprising the polynucleotide of the present invention. The expression vector of the present invention may further comprise elements such as a terminator, a ribosome binding site, and a drug resistance gene that function in a host cell. Examples of the drug resistance gene include resistance genes to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin.

[0025] The expression vector may further include a region that allows homologous recombination with the genome of the host cell for homologous recombination with the genomic DNA of the host cell. For example, the expression vector may be designed so that the expression unit contained therein is located between a pair of homologous regions (e.g., homology arms that are homologous to a specific sequence in the genome of the host cell, loxP, FRT). The genomic region of the host cell into which the expression unit is to be introduced (target of the homologous region) is not particularly limited, and may be the locus of a gene that is highly expressed in the host cell.

[0026] The expression vector may be a plasmid, a virus vector, a phage, or an artificial chromosome. The expression vector may also be an integrative vector or a non-integrative vector. The integrative vector may be a type of vector that is integrated into the genome of a host cell in its entirety. Alternatively, the integrative vector may be a type of vector that is integrated into the genome of a host cell only in part (e.g., an expression unit). The expression vector may further be a DNA vector or an RNA vector (e.g., a retrovirus). The expression vector may also be a commonly used expression vector. Examples of such expression vectors include pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC (e.g., pACYC177, pACYC184), pMW (e.g., pMW119, pMW118, pMW219, pMW218), pQE (e.g., pQE30), and derivatives thereof.

[0027] As a host for expressing the ligase mutant of the present invention, various prokaryotic cells including bacteria of the genus Escherichia such as Escherichia coli, bacteria of the genus Corynebacterium (e.g., Corynebacterium glutamicum), and bacteria of the genus Bacillus (e.g., Bacillus subtilis), and various eukaryotic cells including bacteria of the genus Saccharomyces (e.g., Saccharomyces cerevisiae), bacteria of the genus Pichia (e.g., Pichia stipitis), and bacteria of the genus Aspergillus (e.g., Aspergillus oryzae) can be used. As a host, a strain lacking a specific gene may be used. Examples of transformed microorganisms include transformed microorganisms carrying an expression vector in the cytoplasm and transformed microorganisms into which a target gene has been introduced into the genome.

[0028] The transformed microorganism of the present invention can be cultured in a medium having the composition described below, for example, using a predetermined culture device (e.g., test tube, flask, jar fermenter). Culture conditions can be appropriately set. Specifically, the culture temperature may be 10°C to 37°C, the pH may be 6.5 to 7.5, and the culture time may be 1h to 100h. The culture may be performed while controlling the dissolved oxygen concentration. In this case, the dissolved oxygen concentration (DO value) in the culture solution may be used as an index of control. Aeration and agitation conditions can be controlled so that the relative dissolved oxygen concentration (DO value) when the oxygen concentration in the atmosphere is 21% does not fall below, for example, 1 to 10%, preferably 3% to 8%. The culture may be batch culture or fed-batch culture. In the case of fed-batch culture, the culture can be continued by successively adding a solution serving as a sugar source or a solution containing phosphoric acid to the culture solution continuously or discontinuously.

[0029] The host to be transformed is as described above, but in particular, E. coli can be selected from Escherichia coli K12 subspecies JM109 strain, DH5α strain, HB101 strain, BL21(DE3) strain, etc. Methods for transformation and selection of transformed microorganisms are also described in Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press (2001 / 01 / 15), etc. Below, a more specific example will be given of a method for producing a transformed E. coli and using it to produce a specific enzyme.

[0030] The promoter for expressing the polynucleotide of the present invention may be a promoter generally used for heterologous protein production in E. coli, and examples of such strong promoters include PhoA, PhoC, T7 promoter, lac promoter, trp promoter, trc promoter, tac promoter, lambda phage PR promoter, PL promoter, and T5 promoter, with PhoA, PhoC, and lac being preferred. Also, examples of vectors that may be used include pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSF1010), pACYC (e.g., pACYC177, pACYC184), pMW (e.g., pMW119, pMW118, pMW219, pMW218), pQE (e.g., pQE30), and derivatives thereof. As other vectors, phage DNA vectors may be used.Furthermore, expression vectors that contain a promoter and can express the inserted DNA sequence may be used.Preferably, the vector may be pUC, pSTV, or pMW.

[0031] Furthermore, a terminator, which is a transcription termination sequence, may be linked downstream of the polynucleotide of the present invention. Examples of such terminators include the T7 terminator, the fd phage terminator, the T4 terminator, the tetracycline resistance gene terminator, and the E. coli trpA gene terminator.

[0032] The vector for introducing the polynucleotide of the present invention into E. coli is preferably a so-called multicopy type, and examples thereof include a plasmid having a replication origin derived from ColE1, such as a pUC-series plasmid or a pBR322-series plasmid, or a derivative thereof, where "derivative" refers to a plasmid modified by base substitution, deletion, insertion and / or addition.

[0033] In order to select transformed microorganisms, it is preferable that the vector has a marker such as an ampicillin resistance gene. As such a plasmid, expression vectors having strong promoters are commercially available (e.g., pUC series (manufactured by Takara Bio), pPROK series (manufactured by Clontech), and pKK233-2 (manufactured by Clontech)).

[0034] The resulting expression vector of the present invention can be used to transform E. coli, and the E. coli can be cultured to obtain the ligase mutant of the present invention.

[0035] The medium may be a medium that is usually used for culturing E. coli, such as M9-casamino acid medium or LB medium. The medium may contain a predetermined carbon source, nitrogen source, and coenzyme (e.g., pyridoxine hydrochloride). Specifically, peptone, yeast extract, NaCl, glucose, MgSO4, ammonium sulfate, potassium dihydrogen phosphate, ferric sulfate, manganese sulfate, and the like may be used. The culture conditions and production induction conditions are appropriately selected depending on the type of marker, promoter, host bacteria, and the like of the vector used.

[0036] The ligase mutant of the present invention can be recovered by the following methods. The ligase mutant of the present invention can be obtained as a disruptant or lysate by recovering the transformed microorganism of the present invention and then disrupting (e.g., sonication or homogenization) or lysing (e.g., lysozyme treatment) the cells. The ligase mutant of the present invention can be obtained by subjecting such disruptant or lysate to a technique such as extraction, precipitation, filtration, column chromatography, etc.

[0037] 3. Method for producing nucleic acid product The present invention also provides a method for producing a product nucleic acid, comprising ligating nucleic acid material in the presence of a mutant ligase of the present invention to produce a product nucleic acid, the nucleic acid material being selected from the group consisting of single-stranded nucleic acid material, double-stranded nucleic acid material, and mixtures thereof.

[0038] (nucleic acid) Nucleic acids in nucleic acid materials and nucleic acid products can be classified into natural nucleic acids and modified nucleic acids. Natural nucleic acids are nucleic acids (RNA and DNA) composed of nucleotide residues (adenosine (A), guanosine (G), cytidine (C), uridine (U), deoxyadenosine (dA), deoxyguanosine (dG), deoxycytidine (dC), and thymidine (dT) (hereinafter referred to as "natural nucleotide residues") that constitute polynucleotides contained in cells. Modified nucleic acids are nucleic acids other than natural nucleic acids, and contain nucleotide residues other than natural nucleotide residues (hereinafter referred to as "modified residues"). Modified residues include, for example, modified nucleotide residues, amino acid residues, and linkers. Modified nucleotide residues include, for example, nucleotide residues containing the modifications described below. Amino acids include amino acid derivatives. Examples of amino acids include glycine, alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan, serine, threonine, asparagine, glutamine, tyrosine, cysteine, aspartic acid, glutamic acid, histidine, lysine, arginine, and derivatives thereof. An amino acid derivative refers to an amino acid in which any atom or group in the amino acid is replaced with another atom or group, such as an amino acid in which a hydrogen atom in an amino group, a hydrogen atom in a carboxyl group, an oxygen atom, a hydroxyl group, any atom or group in a side chain, or a hydrogen atom bonded to a backbone carbon atom (e.g., α-, β-, γ-, δ-carbon atom) is replaced with another atom (e.g., a halogen atom such as a fluorine atom, a chlorine atom, a bromine atom, or an iodine atom) or group (e.g., a substituent after substitution in a chemical modification described below).

[0039] Modifications in a modified nucleotide residue include substitutions of atoms or groups in the sugar moiety (ribose or deoxyribose) of the nucleotide residue, substitutions in the sugar moiety itself (sugar backbone) of the nucleotide residue, and modifications of the nucleobase portion of the nucleotide residue (e.g., substitution of a substituent group in the nucleobase moiety).

[0040] Substitutions of atoms or groups in the sugar moiety of a nucleotide residue include, for example, substitutions of 1'-H, 2'-OH (only ribose), 2'-H, 3'-OH, 3'-NH2, 3'-H, 3'-phosphate group, 4'-H, 5'-phosphate group, or combinations thereof. Here, the phosphate group includes not only -OP(O)(OH)2, but also groups in which an oxygen atom is replaced with a sulfur atom or ΝH (e.g., -O-Ρ(S)(OH)2, -NH-Ρ(O)(OH)2, -NH-Ρ(S)(OH)2). In addition, the hydroxyl group (-OH) in the phosphate group may be replaced with OR * (In the formula, R * represents an organic group such as a protecting group for the phosphate group) (e.g., a protected phosphate group). Such substitutions include, for example, 1', 2', 3', or 4'-chemical modifications (substitution of the 1', 2', 3', or 4' position with another substituent), 5'- or 3'-phosphate group modifications (substitution of the 5'- or 3'-phosphate group with another substituent), bridge modifications (substitution that bridges two of the 1', 2', 3', or 4' positions), and carrier addition modifications (substitution of the 1', 2', 3', 4', or 5' position with a carrier).

[0041] The chemical modification may be introduced, for example, to improve the degradation resistance of the oligonucleotide. Substituents after substitution in the chemical modification include, for example, C 1~6 Alkyloxy C 1~6 Alkylene (e.g., methoxyethyl: MOE), -OC 1~6 Alkyl (e.g., -O-Me), -OC 6~14 Aryl (e.g., -O-phenyl), -C-aryl (e.g., -C-phenyl), halogen atom (e.g., fluorine atom), -OC 1~6 Alkyl N-amide C 1~6 Alkylene (e.g., -ON-methylacetamide, -O-NMA), -OC 1~6 Alkyl-(C 1~6 Alkyl-)amino-C 1~6 Alkylene (e.g., -O-dimethylaminoethoxyethyl, -O-DMAEOE), and -O-aminoC 1~6Examples of the chemical modification include alkyl (e.g., -O-aminopropyl, -O-AP). The chemical modification is preferably a 2'-chemical modification (substitution at the 2' site) or a 3'-chemical modification (substitution at the 3' site), and among these, the 2'-chemical modification (substitution at the 2' site) is more preferred. Examples of the substituent after the substitution in the 2'-chemical modification include 2'-C 1~6 Alkyloxy C 1~6 Alkylene (e.g., 2'-methoxyethyl), 2'-OC 1~6 Alkyl (e.g., 2'-O-Me), 2'-OC 6~14 Aryl (e.g., 2'-O-phenyl), 2'-C-aryl (e.g., 2'-C-phenyl), 2'-halogen atom (e.g., 2'-F), 2'-OC 1~6 Alkyl N-amide C 1~6 Alkylene (e.g., 2'-ON-methylacetamide, 2'-O-NMA), 2'-OC 1~6 Alkyl-(C 1~6 Alkyl-)amino-C 1~6 Alkylene (e.g., 2'-O-dimethylaminoethoxyethyl, 2'-O-DMAEOE), and 2'-O-amino C 1~6 Alkyl (e.g., 2'-O-aminopropyl, 2'-O-AP) is included. Substituents after substitution in 3'-chemical modification include, for example, 3'-OP(O)(OH). 2、 3'-O-Ρ(S)(OH)2, 3'-NH-Ρ(O)(OH)2, 3'-NH-Ρ(S)(OH)2), and the hydroxyl group (-OH) in the phosphate group are OR * (In the formula, R * represents an organic group such as a protecting group for a phosphate group, as described below.

[0042] The 5'- or 3'-phosphate group modification may be introduced, for example, to improve the resistance of the oligonucleotide to degradation. Examples of the 5'- or 3'-phosphate group modification include the substitution of a phosphate group (-OP(O)(OH)2) with a group in which the oxygen atom in the phosphate group is replaced with a sulfur atom or ΝH. Examples of such groups include -O-Ρ(S)(OH)2 (thiophosphate group: phosphorothioate modification), -NH-Ρ(O)(OH)2, and -NH-Ρ(S)(OH)2. In addition, the 5'- or 3'-phosphate group modification includes the substitution of a hydroxyl group (-OH) in the phosphate group with an OR * (In the formula, R * represents an organic group such as a protecting group for a phosphate group) (e.g., a protected phosphate group). Examples of the protecting group for a phosphate group include a trityl (Tr) group, a p-methoxyphenyldiphenylmethyl (MMTr) group, a di(p-methoxyphenyl)phenylmethyl (DMTr) group, and a cyanoethyl group (CN-C2H4-).

[0043] Bridging modifications may be introduced, for example, to improve the conformational stability of nucleotide residues. Examples of bridging modifications include 2'4'-bridging modifications (substitutions that bridge 2'-OH and 4'-H), 3'5'-bridging modifications (substitutions that bridge 3'-H and 5'-H), etc. Examples of 2'4'-bridging modifications include 2'-OC of 2'-OH and 4'-H. 1~6 Substitution with alkylene-4' (e.g., 2'-O-methylene-4' (locked nucleic acid: LNA), 2'-O-ethylene-4' (ethylene-bridged nucleic acid: ENA), substitution with 2'-O-methyl-substituted methylene-4' (constrained ethyl-bridged nucleic acid: a type of BNA (cEt-BNA)), 2'-OH and 4'-H in 2'-OC 1~6 Alkylene-OC 1~6 Substitution with alkylene-4' (e.g., 2'-O-methylene-O-methylene-4' (Bridged nucleic acid: a type of BNA (BNA COC )), 2'-OH and 4'-H 2'-ON(R)-C 1~6 Substitution with alkylene-4' (e.g., 2'-ON(R)-methylene-4' (Bridged nucleic acid: a type of BNA (BNA NC), where R represents methyl, hydrogen atom or benzyl), replacement of 2'-NH2 and 4'-H with 2'-N(R)-C(O)-4' (e.g., 2'-N(methyl)-C(O)-4' (amide bridged nucleic acid: AmNA), replacement of 2'-NH2 and 4'-H with 2'-NH-C 1~6 Substitution to alkylene-4' (e.g., 2'-NH-methylene-4'), 2'-H and 4'-H of 2'-C 1~6 Examples of 3'-5'-bridge modifications include 3'-C of 3'-H and 5'-H. 1~6 Substitution with alkylene-5' (eg, 3'-ethylene-5' (bicyclonucleic acid: Bc nucleic acid), a type of Bc nucleic acid: tc nucleic acid, etc.)) can be mentioned.

[0044] The carrier in the carrier addition modification may be a carrier for improving or imparting performance such as stability, targeting, or efficacy to the desired modified oligonucleotide. Such a carrier can be appropriately selected from known carriers depending on the intended use. Examples of the carrier include N-acetylgalactosamine (GalNAc), peptide, phosphate, cholesterol, tocopherol, aliphatic chain, and folic acid. The addition site in the carrier addition modification is preferably the 3' or 5' site corresponding to the end of the desired modified oligonucleotide.

[0045] Examples of nucleotide residues containing a substitution in the sugar moiety of the nucleotide residue itself include modified nucleotide residues containing a substitution of a 5-membered sugar with a 6-membered pseudosugar. Examples of such modified nucleotide residues include hexitol nucleic acid (HNA) and cyclohexenyl nucleic acid (CeNA). In addition, examples of modified nucleotide residues containing a substitution in the sugar moiety of the nucleotide residue itself also include morpholino nucleic acid (PMO) residues, which are nucleotide-like artificial compounds having a morpholino ring structure that are not decomposed by enzymes in vivo (e.g., nucleases such as RNase) and do not induce immune responses.

[0046] Modifications of the nucleobase moiety of the nucleotide residue include, for example, alkyl substitution of the nucleobase moiety of the nucleotide residue (eg, substitution of a methyl group at the 5-position of a cytosyl group).

[0047] (Nucleic acid material) The nucleic acid material may be single or multiple. For example, when a double-stranded nucleic acid having a protruding end is used as a single nucleic acid material, the method of the present invention can be used for cyclization of the double-stranded nucleic acid. When multiple nucleic acid materials are used, the method of the present invention can be used for linking multiple double-stranded nucleic acids having protruding ends, linking a single or multiple double-stranded nucleic acid having a protruding end with a single or multiple single-stranded nucleic acid, linking multiple single-stranded nucleic acids, etc. When multiple nucleic acid materials are used, the number of nucleic acid materials is not particularly limited as long as it is 2 or more, and may be a relatively small number such as 2 to 10, 2 to 8, 3 to 7, or 4 to 6, but may also be more than 10.

[0048] The length of the nucleic acid material is not particularly limited. For example, a long nucleic acid material having a length of more than 1000 bases can be used. Alternatively, when production of a short nucleic acid product is desired, a short nucleic acid material can be used. The short nucleic acid material may be, for example, 5 bases or more, preferably 6 bases or more, more preferably 7 bases or more, even more preferably 8 bases or more, and particularly preferably 9 bases or more. The short nucleic acid material may also be 19 bases or less, preferably 18 bases or less, more preferably 17 bases or less, even more preferably 16 bases or less, and particularly preferably 15 bases or less.

[0049] A protruding end may be used in the linkage. Examples of the protruding end used in the linkage include a protruding end in a double-stranded nucleic acid (nucleic acid material) having a protruding end, and a protruding end formed by annealing between nucleic acid materials (e.g., between a double-stranded nucleic acid and a single-stranded nucleic acid, or between single-stranded nucleic acids). The length of the protruding end is not particularly limited, but when production of a short nucleic acid product is desired, the protruding end may be, for example, 1 to 10 bases long, preferably 1 to 8 bases long, more preferably 1 to 6 bases long, and even more preferably 2 to 6 bases long, 3 to 6 bases long, or 4 to 6 bases long. Therefore, as the nucleic acid material, a double-stranded nucleic acid having such a protruding end, or a combination of multiple nucleic acid materials (e.g., a combination of a double-stranded nucleic acid and a single-stranded nucleic acid, or a combination of multiple single-stranded nucleic acids) may be selected.

[0050] The nucleic acid material may be in free form, fixed to a solid phase, or complexed to a functional moiety at its corresponding site, where complexation of the product nucleic acid with the functional moiety is desired.

[0051] The nucleic acid material can be produced by chemical synthesis (e.g., solid-phase synthesis, liquid-phase synthesis) or enzymatic synthesis, such as those described in WO 2012 / 157723 and WO 2005 / 070859.

[0052] Preferably, the nucleic acid material may be single-stranded or double-stranded RNA, which may contain DNA and / or modified nucleic acid. More preferably, such nucleic acid material may be double-stranded RNA having a protruding end as described above, or a combination of multiple RNA materials that form a protruding end by annealing as described above (e.g., a combination of double-stranded RNA and single-stranded RNA, a combination of multiple single-stranded RNAs).

[0053] (nucleic acid product) The nucleic acid product includes a complementary portion in which bases are paired. Examples of such nucleic acid products include double-stranded nucleic acids and single-stranded nucleic acids including a double-stranded structure portion (e.g., loop-type nucleic acids such as hairpin-type nucleic acids and dumbbell-type nucleic acids). The double-stranded nucleic acid may be a double-stranded nucleic acid in which each strand is the above-mentioned nucleic acid, and examples thereof include double-stranded RNA, double-stranded DNA, heteroduplex nucleic acid consisting of RNA and DNA, double-stranded nucleic acid consisting of RNA and RNA-DNA hybrid nucleic acid, double-stranded nucleic acid consisting of DNA and RNA-DNA hybrid nucleic acid, and double-stranded nucleic acid consisting of RNA-DNA hybrid nucleic acid. Examples of double-stranded nucleic acids include siRNA and heteroduplex nucleic acids.

[0054] In certain embodiments, the nucleic acid product may contain the above-mentioned modified residue in the complementary portion. Examples of such nucleic acid products include double-stranded or loop-type nucleic acids containing modified nucleotide residues (e.g., double-stranded or loop-type nucleic acids containing modified nucleotide residues in the complementary portion), and loop-type nucleic acids containing modified nucleotide residues or residues other than nucleotide residues (e.g., amino acid residues, linkers, etc.) in the loop portion (e.g., International Publication No. 2012 / 005368). In such nucleic acid products, some nucleotide residues may be modified nucleotide residues, or all nucleotide residues may be modified nucleotide residues, but when the modified nucleotide residues are morpholino nucleic acid (PMO) residues, it is preferable that some nucleotide residues are morpholino nucleic acid (PMO) residues in the modified nucleic acid of interest. Such nucleic acid products also include gapmers, which are nucleic acids having modified nucleotide residues at both ends of their sequence and a gap region in the center of their sequence that is recognized by RNase, as well as nucleic acids that do not induce RNase activity, such as mixmers, which are nucleic acids whose sequence contains modified nucleotide residues, and fully modified nucleic acids, which are nucleic acids whose sequence contains all modified nucleotide residues.

[0055] The nucleic acid product may be a nucleic acid consisting of only a complementary portion where bases are paired, or may be a nucleic acid containing a non-complementary portion where bases are not paired in addition to the complementary portion. The length of the complementary portion and / or the non-complementary portion is not particularly limited, but the complementary portion and / or the non-complementary portion may be short. For example, the short complementary portion may be 11 to 27 bases long, 12 to 27 bases long, 15 to 27 bases long, or 18 to 27 bases long. For example, the short non-complementary portion may be 1 to 16 bases long, 1 to 10 bases long, 1 to 5 bases long, or 1, 2, or 3 bases long. When the nucleic acid product has a non-complementary portion in addition to the complementary portion, the complementary portion may be in a continuous form, or may be in a non-continuous form interrupted by the non-complementary portion. The length of the nucleic acid product is not particularly limited, but the nucleic acid product may be short. The short nucleic acid product may be, for example, 20 to 80 bases long, or 24 to 74 bases long.

[0056] Preferably, the nucleic acid product may be DNA and / or single-stranded or double-stranded RNA, which may contain modified nucleic acids. More preferably, such nucleic acid product may contain modified residues in the complementary portion, as described above.

[0057] (Reaction conditions for linking) An aqueous solution can be used as the reaction system. A buffer solution is preferable as the aqueous solution. Examples of the buffer solution include phosphate buffer, Tris buffer, carbonate buffer, acetate buffer, and citrate buffer. The pH may be, for example, about 5 to 9. For example, when efficient mass production of the target nucleic acid product is particularly desired, the pH may be 7.5 to 9.0 (e.g., 8.0 to 8.5).

[0058] The concentration of each nucleic acid material in the ligation reaction may be sufficient so long as the nucleic acid material dissolves and produces the desired nucleic acid product. The concentration of each nucleic acid material may be, for example, 1 μM or more, 10 μM or more, 50 μM or more, 100 μM or more, 300 μM or more, 500 μM or more, or 1000 μM or more. The concentration of each nucleic acid material may also be, for example, 1 M, 100 mM, or 10 mM or less. When efficient mass production of the desired nucleic acid product is particularly desired, it is preferable to use each nucleic acid material at a concentration of 100 μM or more among the above concentrations.

[0059] When multiple nucleic acid materials are used in a ligation reaction, the molar amount of all nucleic acid materials is preferably approximately equal from the viewpoint of improving production efficiency by reducing the amount of unreacted nucleic acid materials. In order for the molar amount of all nucleic acid materials to be approximately equal, the total molar ratio of any two nucleic acid materials selected from the multiple nucleic acid materials may be within the range of, for example, 0.5 to 2, preferably 1 / 1.8 to 1.8, more preferably 1 / 1.5 to 1.5, even more preferably 1 / 1.2 to 1.2, and particularly preferably 1 / 1.1 to 1.1.

[0060] The concentration of the ligase mutant of the present invention in the ligation reaction may be sufficient to produce the desired nucleic acid product. The concentration of the ligase mutant may be, for example, 0.01 U / μL or more, preferably 0.02 U / μL or more, more preferably 0.03 U / μL or more, and even more preferably 0.04 U / μL or more. The concentration of the ligase mutant may also be, for example, 1 U / μL or less, preferably 0.5 U / μL or less, more preferably 0.2 U / μL or less, and even more preferably 0.1 U / μL or less. Here, 1 unit (U) is defined as the amount of enzyme required to produce 1 μmol of nucleic acid product in 1 hour in the reaction described in Example 2.

[0061] The reaction system may contain a cofactor. Examples of the cofactor include ATP and divalent metal salts (e.g., magnesium salts such as magnesium chloride). The reaction system may contain a stabilizer for ligase. Examples of the stabilizer for ligase include antioxidants (e.g., reducing agents such as dithiothreitol and mercaptoethanol). The reaction system may contain a surfactant for the purpose of stably maintaining the enzyme and improving the reaction rate. Examples of the surfactant include nonionic surfactants (e.g., Triton series surfactants such as Triton X-100) and ionic surfactants. Examples of the ionic surfactant include cationic surfactants, anionic surfactants, and amphoteric surfactants. The reaction system may also contain polyethylene glycol for the purpose of improving the reaction rate.

[0062] The reaction system may contain a low concentration of monovalent cationic salt or may be substantially free of monovalent cationic salt. When the reaction system contains a monovalent cationic salt, the concentration of the monovalent cationic salt in the reaction system may be, for example, 10 mM or less, preferably 1 mM or less, more preferably 0.1 mM or less, and even more preferably 0.01 mM or less. Particularly preferably, the reaction system may be substantially free of monovalent cationic salt. Examples of the monovalent cationic salt include salts of monovalent cations such as lithium ion, sodium ion, potassium ion, rubidium ion, cesium ion, and ammonium ion and anions such as fluoride ion, chloride ion, bromide ion, and iodide ion.

[0063] The reaction temperature may be any temperature sufficient for activating the ligase mutant of the present invention, for example, 2 to 50°C, preferably 16 to 50°C, and more preferably 25 to 50°C.

[0064] The reaction time may be any time sufficient to produce the desired nucleic acid product, and may be, for example, 1 to 72 hours. EXAMPLES

[0065] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0066] Example 1: Design and preparation of RNA ligase 1) Design of RNA ligase Using the known function of T4 RNA ligase 2 (NP_049790.1) as a template, 425 similar sequences were obtained from Blastp. The analysis conditions for Blastp were Max target sequences of 10000 and Expected threshold of 1.0E -6 The obtained similar sequences were analyzed, and duplicated sequences and sequences whose sequence length was clearly different from the template were removed, and 172 sequences were selected. This was used as the sequence library. Then, as described in Reference 1 (Nakano, S., Motoyama, T., Miyashita, Y., Ishizuka, Y., Matsuo, N., Tokiwa, H., Shinoda, S., Asano, Y., and Ito, S. (2018) Benchmark Analysis of Native and Artificial NAD +By applying the method used in (Nakano, S., Niwa, M., Asano, Y., and Ito, S. (2019) Following the Evolutionary Track of a Highly Specific l-Arginine Oxidase by Reconstruction and Biochemical Analysis of Ancestral and Native Enzymes, Appl Environ Microbiol 85, e00459-00419.), we identified motif-like sequences of T4 RNA ligase 2 (Ala at position 32, Phe at position 116, Ser at position 170, and Ile at position 274). Only sequences with these motif-like sequences were selected from the sequence library, and data on 21 sequences was finally obtained. Artificial design was performed using 21 sequences, and artificial RNA ligase sequences designated Mut1, Mut2, and Mut3, which are represented by the amino acid sequences of SEQ ID NOs: 1 to 3, were designed (FIG. 1).

[0067] [Table 1]

[0068] 2) Construction of an expression strain for artificial RNA ligase The designed RNA ligase was optimized for Escherichia coli codons, and expression plasmids were synthesized by linking it to the Nde I and Bam HI sites of pET-16b (Merck Millipore) at Eurofins Genomics. Each artificially designed RNA ligase expression plasmid having the base sequences of SEQ ID NOs: 4 to 6 as ORFs corresponding to Mut1, Mut2, and Mut3 was transformed into Escherichia coli BL21 (DE3) strain, spread on an LB agar plate containing 100 mg / L ampicillin, and cultured overnight at 37 ° C. The resulting colonies were isolated to obtain expression strains of each RNA ligase. In this expression strain, each RNA ligase with a His-tag attached to the N-terminus is expressed.

[0069] 3) Expression of artificial RNA ligase Each RNA ligase expression strain was spread on an LB agar plate containing 100 mg / L ampicillin and cultured overnight at 37°C. The grown cells were scraped off with an agar plate and inoculated into a 500-mL Sakaguchi flask containing 150 mL of LB medium containing 100 mg / L ampicillin. The OD 600 After shaking culture at 120 rpm for 3 hours until the β-amino acid ratio reached 0.5, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM, and the culture was continued at 37°C and 120 rpm for another 3 hours. After the culture was completed, the cells were collected from 150 ml of the resulting culture medium by centrifugation at 8000 rpm for 30 minutes.

[0070] 4) Purification of RNA ligase The collected cells were suspended in 15 mL of 50 mM tris(hydroxymethyl)aminomethane-HCl buffer (pH 7.5) containing 250 mM NaCl, 10% sucrose, and 15 mM imidazole. Lysozyme (Sigma-Aldrich) was added to a final concentration of 50 μg / mL, and 10% Triton-X100 was added to a final concentration of 0.1%, and the mixture was left to stand on ice for 30 minutes. After 30 minutes, the cells were disrupted using an ultrasonic homogenizer (Insonator 201M) (Kubota), and the cell debris was removed by centrifugation at 10,000 × g for 10 minutes, and the supernatant was used as the soluble fraction.

[0071] The resulting soluble fraction was loaded onto a HisTALON Superflow Cartridge (5 mL) (Takara Bio) equilibrated with the above buffer using AKTA Pure (GE Healthcare Life Sciences). Non-adsorbed proteins were washed with 30 mL of Tris-HCl buffer (pH 7.5) containing 250 mM NaCl, 10% sucrose, and 15 mM imidazole, and then eluted with 50 mM Tris-HCl (pH 8.0) buffer containing 250 mM NaCl, 10% glycerol, and 200 mM imidazole.

[0072] Elution of the protein was detected by absorbance at 280 nm, and fractions containing RNA ligase expressed as a His-tag fusion protein were collected. 4 ml of the eluted fraction was concentrated using an Amicon Ultra-15 10 kDa (Merck Millipore), and the buffer was replaced with 10 mM Tris-HCl (pH 7.5) buffer containing 50 mM KCl, 35 mM (NH4)2SO4, 0.1 mM EDTA, 0.1 mM DTT, and 50% glycerol to make a 50 μL solution. The purified enzyme was stored in this buffer solution at -20°C. The artificial RNA ligases consisting of the amino acid sequences shown in sequences 1 to 3 were named Mut1, Mut2, and Mut3, respectively. 4.2 mg, 1.1 mg, and 0.65 mg of each artificial RNA ligase Mut1, Mut2, and Mut3 were obtained from 150 ml of culture solution, respectively.

[0073] [Example 2] Ligation reaction of four fragments using each artificial RNA ligase 1) RNA ligation reaction conditions The ligase activity of the three artificial RNA ligases prepared was measured under the following conditions. The four oligonucleotide fragments in Table 2 were used as substrates, and the four oligonucleotide fragments were ligated as shown in Figure 2 to generate two complementary nucleotide fragments. As a control, commercially available T4 RNA ligase 2 (New England Biolabs) was used. For convenience, the oligonucleotides generated by the ligation reaction of MOD1-S-12U and MOD1-S-12D were designated as the sense strand, and the oligonucleotides generated by MOD1-A-13U and MOD1-A-13D were designated as the antisense strand.

[0074] A reaction solution (20 μL) consisting of 10 μM of each oligonucleotide, 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM dithiothreitol, and 0.4 mM ATP was prepared on ice and placed in a 200 μL microtube. 50 μL of each purified enzyme solution was added to a final concentration of 0.36 μg / mL to start the reaction. The reaction was kept at 25°C in a thermal cycler, and 1 hour after the start of the reaction, the temperature was raised to 80°C and heated for 5 minutes to stop the reaction. The concentration of the ligation product was quantified by HPLC under the following conditions.

[0075] [Table 2]

[0076] 2) Analysis by HPLC The oligonucleotides of the ligation products were quantified by HPLC using an ACQUITY UPLC® Oligonucleotide BEH C18 Column (Waters, 1.7 μm 2.1×50 mm). HPLC was performed under the following conditions: column temperature 60° C., detection wavelength 260 nm, injection volume 10 μL, flow rate 0.4 mL / min, and the mobile phase was eluent A containing 100 mM hexafluoroisopropanol, 8 mM triethylamine, and 0.004% phosphoric acid, and eluent B containing 10% methanol, with a linear gradient as shown in Table 3. Oligonucleotides of the same sequence as the products synthesized by each ligation were separately synthesized, and the concentrations of the ligation products were quantified using these as standards.

[0077] [Table 3]

[0078] 3) Results The results are shown in Table 4. For convenience, the oligonucleotides produced in the ligation reaction of MOD1-S-12U and MOD1-S-12D are referred to as the sense strand. In the reaction using the control T4 RNA ligase 2, 0.47 μM of the sense strand ligation product was produced after 1 hour of reaction. The reaction rate increased with all artificial RNA ligases, and in the reaction using Mut1, 1.98 μM of ligation product was produced compared to the reaction using T4 RNA ligase 2, a significant improvement of about 4.2 times. In addition, improved activity was observed with Mut2 and Mut3, and the amount of ligation product after 1 hour of reaction was about 2.2 times higher than that of T4 RNA ligase 2. If the amount of enzyme required to produce 1 μmol of ligation product in 1 hour in this reaction is defined as 1 unit, the most active Mut1 was calculated to be 5.50 units / mg.

[0079] [Table 4]

[0080] [Example 3] Ligation reaction of four fragments using artificial RNA ligase under high substrate concentration conditions Ligation reactions were performed using Mut1 RNA ligase, which had the highest activity, by increasing the substrate concentration. The four oligonucleotide fragments in Table 5 were used as substrates. As a control, reactions were performed using T4 RNA ligase 2 (New England Biolabs). As shown in Figure 3(A), the oligonucleotides generated by the ligation reaction of MOD5-S-11U and MOD5-S-11D were designated as the sense strand, and the oligonucleotides generated by MOD5-A-12U and MOD5-A-12D were designated as the antisense strand. 10 μL of reaction solution containing 500 μM of each oligonucleotide, 50 mM Tris-HCl (pH 8.0), 2 mM MgCl2, 1 mM dithiothreitol, 1.4 mM ATP, and 7.2 μg / mL of each enzyme was added to a 200 μL microtube and incubated at 25° C. in a thermal cycler. After the start of the reaction, 1 μl of the reaction solution was sampled at 0.5, 1, 2, 4, 6, and 24 hours, and 49 μL of 10 mM EDTA solution was added to stop the reaction. The concentration of the ligation product was analyzed by HPLC under the conditions described in Example 2. The time course of the formation of the ligation product is shown in Figure 3(B). With the control T4 RNA ligase 2, the formation rate of the ligation product on the antisense side was slow, and the amounts of the ligation products of the antisense and sense strands after 24 hours of reaction were 320 μM and 120 μM, respectively, and some of the four oligonucleotide fragments added as substrates remained unreacted. On the other hand, Mut1, which has improved ligation activity, significantly improved the ligation rates of both sense and antisense oligonucleotides. After 24 hours of reaction, the amounts of ligated products of the antisense and sense strands were 470 μM and 450 μM, respectively, and the four oligonucleotide fragments added as substrates were almost completely consumed.

[0081] [Table 5]

[0082] [Example 4] Substrate specificity of artificial RNA ligase As shown in Figure 4(A), the change in substrate specificity of artificial RNA ligase was examined by a reaction in which three-fragment oligonucleotides were used as substrates and two-fragment oligonucleotides were ligated. Oligonucleotides in which the vicinity of the ligation point of the oligonucleotide to be ligated was replaced with modified RNA, specifically, oligonucleotides in which the 2'-positions of the -2, -1, +1, and +2 positions from the ligation point were modified with fluorine atoms (F), O-methyl, or O-methoxyethyl, or replaced with hydrogen atoms (DNA), as shown in Table 6, were used as substrates. Mut1 RNA ligase, which had the highest activity, and T4 RNA ligase 2 (New England Biolabs) were used as a control. 20 μL of reaction solution containing 10 μM oligonucleotide at final concentration, 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM dithiothreitol, 0.4 mM ATP, and 1.78 μg / mL of each enzyme were added to a 200 μL microtube and kept at 25°C. After the start of the reaction, 3 μl of the reaction solution was sampled 15 minutes later, and 27 μL of 10 mM EDTA solution was added to stop the reaction. The concentration of the ligation product contained in the reaction solution was analyzed by HPLC under the conditions described in Example 2, and the product was quantified using a ligation product not containing modified nucleic acid as a standard.

[0083] The amount of ligation product generated in a 15-minute reaction is shown in Figure 4(B). In the case of the control T4 RNA ligase 2, the amount of ligation product was significantly reduced by 5-50% when the substrate was an oligonucleotide with 2'-F, 2'-MOE, or DNA substituted at the -2 position, or 2'-O-Me, 2'-MOE, or DNA substituted at the -1 position, compared to when the substrate was a fragment consisting entirely of natural RNA. On the other hand, in the case of Mut1, which has improved ligation activity, the amount of ligation product was comparable to that when the substrate was an oligonucleotide with 2'-F and DNA substituted at the -2 position. Moreover, Mut1 showed more than twice the amount of ligation product as T4 RNA ligase 2, regardless of the oligonucleotide substrate.

[0084] [Table 6]

[0085] [Example 5] Temperature stability of artificial RNA ligase The temperature stability of the artificially designed RNA ligase was evaluated. Mut1 RNA ligase, which had the highest activity, and T4 RNA ligase 2 (New England Biolabs) were used as a control. The four oligonucleotide fragments in Table 7 were used as substrates. As shown in Table 7, the oligonucleotides generated from RNA1-A-13U and RNA1-A-13D were designated as antisense strands. 9.2 μL of a solution containing 54 mM Tris-HCl (pH 7.5), 2.2 mM MgCl2, 1.1 mM dithiothreitol, 0.43 mM ATP, and 0.78 μg / mL of each enzyme were kept at 25°C in a 200 μL microtube. 23 hours after the start of the incubation, 0.2 μL of each 500 μM substrate was added to start the ligation reaction. The reaction solution consisted of 10 μM oligonucleotide, 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM dithiothreitol, 0.4 mM ATP, and 0.72 μg / mL of each enzyme. After the start of the reaction, 3 μL was sampled at 15 minutes, and the reaction was stopped by adding 27 μL of 10 mM EDTA. The amount of the antisense strand product was quantified by HPLC under the conditions described in Example 2.

[0086] The amount of ligation product and residual activity produced in a 15-minute reaction are shown in Table 8. When the control T4 RNA ligase 2 was used, the amount of ligation product was significantly reduced to 19% compared to the untreated case when incubated at 25°C for 23 hours. On the other hand, when Mut1, which has improved ligation activity, was used, the amount of ligation product was 89% of the untreated case even when incubated at 25°C for 23 hours, demonstrating improved stability.

[0087] Similarly, activity after incubation at 37°C for 4 hours was also measured. As shown in Table 9, when the control T4 RNA ligase 2 was incubated at 37°C for 4 hours, the amount of ligation product was not detected and activity was significantly reduced. On the other hand, when Mut1, which has improved ligation activity, was incubated at 37°C for 4 hours, the amount of ligation product was 83% of that of the untreated sample, indicating improved stability.

[0088] [Table 7]

[0089]

Table 8

[0090]

Table 9

Claims

1. A ligase mutant of the following (1), (2) or (3): (1) A ligase mutant comprising an amino acid sequence showing 95% or more identity to the amino acid sequence of SEQ ID NO:1 and having a nucleic acid ligation activity that is 1.5-fold or more higher than that of T4 RNA ligase 2 (NP_049790); (2) a ligase mutant comprising an amino acid sequence showing 90% or more identity to the amino acid sequence of SEQ ID NO: 2 and having a nucleic acid ligation activity that is 1.5 times or more higher than that of T4 RNA ligase 2 (NP_049790); or (3) A ligase mutant comprising an amino acid sequence showing 97% or more identity to the amino acid sequence of SEQ ID NO: 3 and having nucleic acid ligation activity that is 1.5-fold or more higher than that of T4 RNA ligase 2 (NP_049790).

2. The ligase mutant of claim 1, wherein the ligase mutant is: (1) A ligase mutant comprising an amino acid sequence showing 97% or more identity to the amino acid sequence of SEQ ID NO:1 and having a nucleic acid ligation activity that is 1.5-fold or more higher than that of T4 RNA ligase 2 (NP_049790); (2) a ligase mutant comprising an amino acid sequence showing 97% or more identity to the amino acid sequence of SEQ ID NO: 2 and having a nucleic acid ligation activity that is 1.5 times or more higher than that of T4 RNA ligase 2 (NP_049790); or (3) A ligase mutant comprising an amino acid sequence showing 97% or more identity to the amino acid sequence of SEQ ID NO: 3 and having nucleic acid ligation activity that is 1.5-fold or more higher than that of T4 RNA ligase 2 (NP_049790).

3. The ligase mutant according to claim 1 or 2, wherein the nucleic acid is single-stranded or double-stranded RNA which may contain DNA and / or modified nucleic acid.

4. ligating nucleic acid material in the presence of a ligase mutant according to any one of claims 1 to 3 to produce a product nucleic acid, A method for producing a nucleic acid product, wherein the nucleic acid material is selected from the group consisting of single-stranded nucleic acid material, double-stranded nucleic acid material, and mixtures thereof.

5. The method of claim 4, wherein the nucleic acid material is RNA.

6. 6. The method of claim 4 or 5, wherein the nucleic acid material is four or more single-stranded RNA strands.

7. The method of any one of claims 4 to 6, wherein the product nucleic acid comprises a complementary portion 12 to 27 bases in length.

8. The method of any one of claims 4 to 7, wherein the nucleic acid material comprises DNA and / or modified nucleic acids.

9. The method of any one of claims 4 to 8, wherein the concentration of the nucleic acid material is 1 μM or greater.

10. The method of any one of claims 4 to 9, wherein the nucleic acid product is an siRNA.

11. A polynucleotide encoding the ligase mutant according to any one of claims 1 to 3.

12. An expression vector comprising the polynucleotide of claim 11.

13. A transformed microorganism comprising an expression unit comprising a polynucleotide encoding the ligase mutant according to any one of claims 1 to 3 and a promoter operably linked thereto.

14. A method for producing a ligase mutant, comprising producing the ligase mutant according to any one of claims 1 to 3 using the transformed microorganism according to claim 13.

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