Ligase mutants

Abstract

To provide a ligase mutant having excellent properties.SOLUTION: 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 a nucleic acid-linking activity; (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-linking activity; 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 a nucleic acid-linking 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 (EC 6.5.1.3) capable of ligating ribonucleotides in the presence of ATP and is derived from the T4 bacteriophage, which infects Escherichia (NP_049790). T4 RNA ligase 2 is capable of linking the phosphate group at the 5' end of a nucleic acid (donor) to the hydroxyl group at the 3' end (acceptor) by forming a phosphodiester bond. T4 RNA ligase 2 has been used in reactions such as ligating double-stranded RNA with overhanging ends and ligating nicks in double-stranded RNA. T4 RNA ligase 2 is also known to be able to use not only RNA but also DNA and modified nucleic acids other than DNA and RNA as substrates. Several variants of T4 RNA ligase 2 are also known. For prior art on T4 RNA ligase 2, see 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.

Outdoor Tools3

Outdoor Tools 4

Direct Environment 5

Outdoor Configuration6

Direct Environment 7

[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 extensive research, the present inventors have succeeded in developing three ligase mutants, Mut1 to Mut3 (SEQ ID NOs: 1 to 3), which show 93%, 87%, and 95% amino acid sequence identity to T4 RNA ligase 2 (NP_049790), respectively (see Table 1 below), and which have superior properties compared to T4 RNA ligase 2. The above-mentioned prior art neither teaches nor suggests these three ligase mutants, thereby completing the present invention.

[0007] That is, the present invention is as follows. [1] A ligase mutant of the following (1), (2), or (3): (1) A ligase mutant having an amino acid sequence that is 95% or more identical 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 that contains an amino acid sequence that is 97% or more identical to the amino acid sequence of SEQ ID NO: 3 and has 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 nucleic acid product; 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 of [3] or [4], wherein the nucleic acid material is four or more single-stranded RNAs. [6] Any of the methods [3] to [5], wherein the nucleic acid product contains a complementary portion having a length of 12 to 27 bases. [7] Any of the methods [3] to [6], wherein the nucleic acid material comprises DNA and / or modified nucleic acids. [8] Any of the methods [3] to [7], wherein the concentration of the nucleic acid material is 1 μM or more. [9] Any of the methods [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 containing 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] using the transformed microorganism of

[12] . [Effects of the Invention]

[0008] According to the present invention, nucleic acid products (eg, modified nucleic acids such as siRNA and heteroduplex nucleic acids) can be efficiently produced from nucleic acid materials. [Brief explanation 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). [Figure 2] 2 shows the double-stranded oligonucleotides produced by ligation of four single-stranded oligonucleotide fragments. The designations of modified nucleotide residues are the same as those in Table 2. [Figure 3] 3 shows (A) the double-stranded oligonucleotide produced by the ligation reaction of four single-stranded oligonucleotide fragments, and (B) the 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] Figure 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 minutes). The oligonucleotides used as substrates were those 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), O-methoxyethyl (MOE), or substituted with a hydrogen atom (DNA). DETAILED DESCRIPTION OF THE INVENTION

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

[0011] The amino acid sequence of SEQ ID NO: 1 shows 93% identity to the amino acid sequence of known T4 RNA ligase 2 (NP_049790). The ligase mutant (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 known T4 RNA ligase 2. The percent identity of the ligase mutant (1) to the amino acid sequence of SEQ ID NO: 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 known T4 RNA ligase 2 (NP_049790). The ligase variant (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 known T4 RNA ligase 2. The percent identity of the ligase variant (1) to the amino acid sequence of SEQ ID NO: 2 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 known T4 RNA ligase 2 (NP_049790). The ligase variant (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 known T4 RNA ligase 2. The percent identity of the ligase variant (3) to the amino acid sequence of SEQ ID NO: 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 Ver. 13.1.1, using the full length of the polypeptide portion encoded by the ORF, and then performing Muscle alignment, ClustalW alignment, or Multiple sequence alignment with the setting "Gaps are taken into account." The number of amino acid residue modifications (e.g., substitutions, deletions, additions, or insertions, or a combination thereof) 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 nucleic acid ligation activity. Examples of nucleic acids include single-stranded nucleic acids and double-stranded nucleic acids. Examples of nucleic acids also include RNA, DNA, modified nucleic acids other than RNA and DNA, and mixtures thereof. Preferably, the nucleic acid may be single-stranded RNA or double-stranded RNA, which 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, which will be described later.

[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 it 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, e.g., 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) 50 μL of a solution containing the purified mutant ligase is added 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 mutants of the present invention may also have excellent temperature stability. For example, when a ligase mutant comprising an amino acid sequence of the present invention is subjected to heat treatment and subsequent activity measurement, it preferably retains 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 residual activity measured without heat treatment. Such conditions may include incubation at 25°C for 23 hours or at 37°C for 4 hours (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 described in a) to c) in the above paragraphs. Alternatively, activity measurement may be performed under conditions in which activity is measured after a 15-minute reaction at 25°C or 37°C. The reaction solution composition used under these conditions may be, for example, 10 μM oligonucleotide, 50 mM Tris-HCl (pH 7.5), 2 mM MgCl, 1 mM dithiothreitol, 0.4 mM ATP, and 0.72 μg / mL enzyme. Because the temperature stability of an enzyme generally correlates with its liquid stability and / or long-term storage stability, a ligase mutant of the present invention with excellent temperature stability can be said to have 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 that maintains the desired percent identity and ligation activity. The positions of amino acid residues that may be mutated are readily apparent to those skilled in the art. For example, a skilled artisan can 1) compare the amino acid sequences of multiple proteins with similar properties (e.g., SEQ ID NOS: 1-3), 2) identify relatively conserved and relatively non-conserved regions, and then 3) predict regions that may play important roles in function and regions that may not, respectively, from the relatively conserved and non-conserved regions, thereby recognizing the correlation between structure and function. Thus, a skilled artisan can identify the positions of amino acid residues that may be mutated in the amino acid sequence of the ligase mutant of the present invention. The mutated amino acid residue at such a position is a desired natural α-amino acid residue that is different from the unmutated amino acid residue. Such desired natural α-amino acid residues are 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 residue mutations may be introduced 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 β-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 can be contained in the ligase mutant of the present invention include peptide components that facilitate the purification of the 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 the 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 produced, for example, by a method using an expression vector containing a polynucleotide of the present invention (e.g., a competent cell method, an electroporation method), or by genome modification techniques. When the expression vector is an integrative vector that undergoes homologous recombination with the genomic DNA of a 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 a 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, genome editing techniques (e.g., CRISPR / Cas system, Transcription Activator-Like Effector Nucleases (TALEN)) can be used to integrate an expression unit into the genomic DNA of a host cell and modify an 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 that function in host cells, such as a terminator, a ribosome binding site, and a drug resistance gene. Examples of drug resistance genes include genes that are resistant to drugs such as tetracycline, ampicillin, kanamycin, hygromycin, and phosphinothricin.

[0025] The expression vector may further comprise a region that enables 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 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 viral vector, a phage, or an artificial chromosome. The expression vector may also be an integrative vector or a non-integrative vector. An integrative vector may be a vector that is integrated in its entirety into the genome of a host cell. Alternatively, an integrative vector may be a vector that is integrated only in part (e.g., an expression unit) into the genome of a host cell. 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] Hosts for expressing the ligase mutants of the present invention include various prokaryotic cells, such as Escherichia bacteria (e.g., Escherichia coli), Corynebacterium bacteria (e.g., Corynebacterium glutamicum), and Bacillus bacteria (e.g., Bacillus subtilis), as well as various eukaryotic cells, such as Saccharomyces bacteria (e.g., Saccharomyces cerevisiae), Pichia bacteria (e.g., Pichia stipitis), and Aspergillus bacteria (e.g., Aspergillus oryzae). Host strains lacking a specific gene may also be used. Transformed microorganisms include, for example, transformed microorganisms harboring an expression vector in their cytoplasm and transformed microorganisms into which a gene of interest has been introduced into their genome.

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

[0029] The host to be transformed is as described above, but in particular, Escherichia coli can be selected from Escherichia coli K12 subspecies strains such as JM109, DH5α, HB101, and BL21(DE3). Methods for transformation and screening for 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 preparing transformed Escherichia coli and using it to produce a predetermined enzyme.

[0030] Promoters used to express the polynucleotides of the present invention include those typically used in heterologous protein production in E. coli, such as 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. 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. Other vectors may include phage DNA vectors. Furthermore, expression vectors containing a promoter and capable of expressing the inserted DNA sequence may also be used. Preferably, the vector may be pUC, pSTV, or pMW.

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

[0032] Vectors for introducing the polynucleotides of the present invention into E. coli are preferably multicopy vectors, including plasmids having a replication origin derived from ColE1, such as pUC-series plasmids and pBR322-series plasmids, or their derivatives. Here, "derivatives" refers to plasmids that have been modified by base substitution, deletion, insertion, and / or addition.

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

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

[0035] The medium may be a medium typically used for culturing E. coli, such as M9-casamino acids 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, etc. may be used. The culture conditions and production induction conditions are appropriately selected depending on the type of marker, promoter, host bacterium, etc. of the vector used.

[0036] The ligase mutant of the present invention can be recovered by the following methods. After recovering the transformed microorganism of the present invention, the ligase mutant of the present invention can be obtained as a disruptant or lysate by 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 techniques such as extraction, precipitation, filtration, column chromatography, etc.

[0037] 3. Method for producing nucleic acid products The present invention also provides a method for producing a nucleic acid product, comprising ligating nucleic acid materials in the presence of a ligase mutant of the present invention to produce a nucleic acid product, The nucleic acid material can be selected from the group consisting of single-stranded nucleic acid materials, double-stranded nucleic acid materials, 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 refer to 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 refer to nucleic acids other than natural nucleic acids, and are nucleic acids that 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 has been replaced with another atom or group, such as a hydrogen atom in the amino group, a hydrogen atom in the carboxyl group, an oxygen atom, a hydroxyl group, any atom or group in the side chain, or a hydrogen atom bonded to a backbone carbon atom (e.g., an α-, β-, γ-, or δ-carbon atom) replaced with another atom (e.g., a halogen atom such as a fluorine atom, chlorine atom, bromine atom, or iodine atom) or group (e.g., a substituent group after substitution in a chemical modification, as described below).

[0039] Modifications in modified nucleotide residues include substitutions of atoms or groups in the sugar moiety (ribose or deoxyribose) of the nucleotide residue, substitutions of 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 portion).

[0040] Substitutions of atoms or groups in the sugar moiety of a nucleotide residue include, for example, 1'-H, 2'-OH (ribose only), 2'-H, 3'-OH, 3'-NH, 3'-H, 3'-phosphate group, 4'-H, 5'-phosphate group, or combinations thereof. Here, the phosphate group includes not only -OP(O)(OH) but also groups in which an oxygen atom is replaced with a sulfur atom or NH (e.g., -O-P(S)(OH)2, -NH-P(O)(OH)2, -NH-P(S)(OH)2). In addition, the hydroxyl group (-OH) in a 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' position) or a 3'-chemical modification (substitution at the 3' position), and among these, the 2'-chemical modification (substitution at the 2' position) is more preferred. Examples of the substituent after substitution in the 2'-chemical modification include, for example, 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 Examples of the substituent after substitution in the 3'-chemical modification include 3'-OP(O)(OH). 2、 3'-O-P(S)(OH)2, 3'-NH-P(O)(OH)2, 3'-NH-P(S)(OH)2), and the hydroxyl group (-OH) in the phosphate group is OR * (In the formula, R * represents an organic group such as a protecting group for a phosphate group, as described below).

[0042] 5'- or 3'-phosphate group modifications may be introduced, for example, to improve the degradation resistance of oligonucleotides. Examples of 5'- or 3'-phosphate group modifications include 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 NH. Examples of such groups include -O-P(S)(OH)2 (thiophosphate group: phosphorothioate modification), -NH-P(O)(OH)2, and -NH-P(S)(OH)2. Furthermore, 5'- or 3'-phosphate group modifications include 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). Protecting groups for a phosphate group include, for example, 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 modifications of 2'-OH and 4'-H. 1~6 Substitution of alkylene-4' (e.g., 2'-O-methylene-4' (locked nucleic acid: LNA), 2'-O-ethylene-4' (ethylene-bridged nucleic acid: ENA), substitution of 2'-O-methyl-substituted methylene-4' (constrained ethyl-bridged nucleic acid: a type of BNA (cEt-BNA)), 2'-OH and 4'-H to 2'-OC 1~6 Alkylene-OC 1~6 Substitution to 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), substitution 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)), substitution 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 2'-C 1~6 Examples of 3'-5'-bridge modifications include 3'-C of 3'-H and 5'-H. 1~6 Examples include substitution with alkylene-5' (for example, 3'-ethylene-5' (bicyclonucleic acid: Bc nucleic acid), a type of Bc nucleic acid: tc nucleic acid, etc.).

[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 carriers include N-acetylgalactosamine (GalNAc), peptides, phosphate, cholesterol, tocopherol, fatty chains, and folic acid. The addition site in the carrier-addition modification is preferably the 3' or 5' site corresponding to the terminus of the desired modified oligonucleotide.

[0045] Examples of nucleotide residues containing a substitution in the sugar moiety of the nucleotide residue include modified nucleotide residues containing a substitution of a five-membered ring sugar with a six-membered ring pseudosugar. Examples of such modified nucleotide residues include hexitol nucleic acid (HNA) and cyclohexenyl nucleic acid (CeNA). Furthermore, modified nucleotide residues containing a substitution in the sugar moiety of the nucleotide residue also include morpholino nucleic acid (PMO) residues, which are nucleotide-like artificial compounds with a morpholino ring structure that are not degraded by in vivo enzymes (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 the cytosyl group).

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

[0048] The length of the nucleic acid material is not particularly limited. For example, a long nucleic acid material exceeding 1,000 bases in length can be used. Alternatively, if 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 in length, preferably 6 bases or more in length, more preferably 7 bases or more in length, even more preferably 8 bases or more in length, and particularly preferably 9 bases or more in length. The short nucleic acid material may also be 19 bases or less in length, preferably 18 bases or less in length, more preferably 17 bases or less in length, even more preferably 16 bases or less in length, and particularly preferably 15 bases or less in length.

[0049] A protruding end may be used in the ligation. Examples of protruding ends used in the ligation include protruding ends in double-stranded nucleic acids (nucleic acid materials) having protruding ends, and protruding ends 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. However, 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, 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 as the nucleic acid material.

[0050] The nucleic acid material may be in free form, immobilized on a solid phase, or complexed to a functional moiety at its corresponding site, if complexation of the nucleic acid product 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, for example, as described in International Publication Nos. 2012 / 157723 and 2005 / 070859.

[0052] Preferably, the nucleic acid material may be single-stranded RNA or double-stranded RNA, which may contain DNA and / or modified nucleic acids. 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 the protruding ends by annealing as described above (e.g., a combination of double-stranded RNA and single-stranded RNA, or a combination of multiple single-stranded RNAs).

[0053] (nucleic acid product) The nucleic acid product contains a complementary portion where the bases are paired. Examples of such nucleic acid products include double-stranded nucleic acids and single-stranded nucleic acids containing 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 acids consisting of RNA and DNA, double-stranded nucleic acids consisting of RNA and RNA-DNA hybrid nucleic acids, double-stranded nucleic acids consisting of DNA and RNA-DNA hybrid nucleic acids, and double-stranded nucleic acids consisting of RNA-DNA hybrid nucleic acids. Examples of double-stranded nucleic acids include siRNA and heteroduplex nucleic acids.

[0054] In certain embodiments, the nucleic acid product may contain the above-described modified residues 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., WO 2012 / 005368). In such nucleic acid products, some or all of the nucleotide residues may be modified nucleotide residues. However, when the modified nucleotide residues are morpholino nucleic acid (PMO) residues, it is preferable that some of the nucleotide residues in the modified nucleic acid of interest are morpholino nucleic acid (PMO) residues. 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 in which modified nucleotide residues are mixed into the sequence, and fully modified nucleic acids, which are nucleic acids in which all nucleotide residues in the sequence are modified nucleotide residues.

[0055] The nucleic acid product may be a nucleic acid consisting only of a complementary portion where bases are paired, or may contain 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 a complementary portion, the complementary portion may be continuous or may be discontinuous, interrupted by a 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 as described above in the complementary portion.

[0057] (Reaction conditions for ligation) An aqueous solution can be used as the reaction system. A buffer solution is preferable as the aqueous solution. Examples of buffer solutions 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 to dissolve the nucleic acid material and produce 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 amounts of all nucleic acid materials are preferably approximately equal, from the viewpoint of improving production efficiency by reducing the amount of unreacted nucleic acid materials. To achieve approximately equal molar amounts of all nucleic acid materials, the total molar ratio of any two nucleic acid materials selected from the multiple nucleic acid materials may be, for example, within the range of 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 per hour in the reaction described in Example 2.

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

[0062] The reaction system may contain a low concentration of monovalent cation salt or may be substantially free of monovalent cation salt. When the reaction system contains a monovalent cation salt, the concentration of the monovalent cation 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 cation salt. Examples of monovalent cation salts include salts of monovalent cations such as lithium ions, sodium ions, potassium ions, rubidium ions, cesium ions, and ammonium ions with anions such as fluoride ions, chloride ions, bromide ions, and iodide ions.

[0063] The reaction temperature may be any temperature sufficient for activating the ligase mutant of the present invention, and may be, 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. [Example]

[0065] The present invention will be described in more 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 functionally known 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 = 10000, Expected threshold = 1.0E -6 The obtained similar sequences were analyzed, and duplicated sequences and sequences whose sequence lengths were clearly different from the template were removed, resulting in a selection of 172 sequences. This was used as a sequence library. 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 same method used in [1] and [2] (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 in T4 RNA ligase 2 (Ala at position 32, Phe at position 116, Ser at position 170, and Ile at position 274). From the sequence library, we selected only sequences with these motif-like sequences, ultimately obtaining data on 21 sequences. Artificial design was carried out 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 codon-optimized for Escherichia coli and ligated into the NdeI and BamHI sites of pET-16b (Merck Millipore) to synthesize expression plasmids at Eurofins Genomics. Each artificially designed RNA ligase expression plasmid, which contains the ORFs corresponding to Mut1, Mut2, and Mut3 (SEQ ID NOs: 4-6), was transformed into Escherichia coli BL21(DE3), plated onto LB agar plates containing 100 mg / L ampicillin, and grown overnight at 37°C. The resulting colonies were then isolated to obtain expression strains for each RNA ligase. These expression strains expressed each RNA ligase with a His-tag at the N-terminus.

[0069] 3) Expression of artificial RNA ligase Each RNA ligase-expressing 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 and inoculated into a 500 mL Sakaguchi flask containing 150 mL of LB medium containing 100 mg / L ampicillin. The OD 600 After culturing for 3 hours with shaking at 120 rpm until the β-glucan content reached 0.5, isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 0.1 mM, and culturing was continued for another 3 hours at 37°C and 120 rpm. After culturing, the resulting 150 ml of culture medium was centrifuged at 8000 rpm for 30 minutes to recover the bacterial cells.

[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 on ice for 30 minutes. After 30 minutes, the cells were disrupted using an ultrasonic homogenizer (Insonator 201M) (Kubota). 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 5 mL HisTALON Superflow Cartridge (Takara Bio) equilibrated with the buffer solution using an AKTA Pure (GE Healthcare Life Sciences). Unadsorbed 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) containing 250 mM NaCl, 10% glycerol, and 200 mM imidazole.

[0072] Eluted protein was detected by absorbance at 280 nm, and fractions containing RNA ligase expressed as a His-tagged fusion protein were collected. The 4 ml eluted fraction was concentrated using an Amicon Ultra-15 10 kDa (Merck Millipore) and buffer exchanged 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 designated Mut1, Mut2, and Mut3, respectively. From 150 ml of culture, 4.2 mg, 1.1 mg, and 0.65 mg of each artificial RNA ligase, Mut1, Mut2, and Mut3, were obtained, 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 listed 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. Commercially available T4 RNA ligase 2 (New England Biolabs) was used as a control. For convenience, the oligonucleotides generated by the ligation reaction of MOD1-S-12U and MOD1-S-12D are referred to as the sense strand, and the oligonucleotides generated by MOD1-A-13U and MOD1-A-13D are referred to as the antisense strand.

[0074] A 20 μL reaction mixture 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 initiate the reaction. The mixture was kept at 25°C in a thermal cycler, and after 1 hour, the temperature was raised to 80°C and the reaction was stopped by heating for 5 minutes. The concentration of the ligation product was quantified by HPLC under the following conditions.

[0075] [Table 2]

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

[0077] [Table 3]

[0078] 3) Results The results are shown in Table 4. For convenience, the oligonucleotides produced in the ligation reactions of MOD1-S-12U and MOD1-S-12D are referred to as the sense strand. In the control reaction using T4 RNA ligase 2, 0.47 μM of sense strand ligation product was produced after 1 hour of reaction. The reaction rate increased with all artificial RNA ligases. Mut1 produced 1.98 μM of ligation product, a significant improvement of approximately 4.2-fold compared to T4 RNA ligase 2. Mut2 and Mut3 also showed improved activity, producing approximately 2.2-fold more ligation product after 1 hour of reaction than T4 RNA ligase 2. If the amount of enzyme required to produce 1 μmol of ligation product in 1 hour of reaction is defined as 1 unit, the most active reaction, Mut1, was calculated to produce 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 showed the highest activity, by increasing the substrate concentration. The four oligonucleotide fragments listed 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 the sense strand, and the oligonucleotides generated by the ligation reaction of MOD5-A-12U and MOD5-A-12D were designated the antisense strand. Ten microliters of a reaction mixture containing 500 μM of each oligonucleotide, 50 mM Tris-HCl (pH 8.0), 2 mM MgCl, 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 initiation of the reaction, 1 μL of the reaction mixture was sampled at 0.5, 1, 2, 4, 6, and 24 hours, and the reaction was stopped by adding 49 μL of 10 mM EDTA solution. The concentration of the ligation product was analyzed by HPLC under the conditions described in Example 2. The time course of ligation product formation is shown in Figure 3(B). With the control T4 RNA ligase 2, the rate of antisense ligation product formation was slow; after 24 hours of reaction, the amounts of antisense and sense ligation products were 320 μM and 120 μM, respectively. 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 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), changes in the substrate specificity of artificial RNA ligase were investigated by ligating two oligonucleotide fragments using three oligonucleotide fragments as substrates. Oligonucleotides in which the ligation point was modified with modified RNA were used near the ligation point. Specifically, oligonucleotides in which the 2'-positions at positions -2, -1, +1, and +2 from the ligation point were modified with fluorine (F), O-methyl, or O-methoxyethyl, or substituted with hydrogen (DNA), as shown in Table 6, were used as substrates. Mut1 RNA ligase, which showed the highest activity, and T4 RNA ligase 2 (New England Biolabs) were used as a control. A 20 μL reaction mixture containing 10 μM oligonucleotides (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) was added to a 200 μL microtube and incubated at 25°C. 15 minutes after the start of the reaction, 3 μL of the reaction solution was sampled 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 containing no modified nucleic acid as a standard.

[0083] Figure 4(B) shows the amount of ligation product generated after 15 minutes of reaction. With the control T4 RNA ligase 2, the amount of ligation product significantly decreased by 5–50% compared to when an all-natural RNA fragment was used as a substrate. When oligonucleotides with 2'-F, 2'-MOE, or DNA at the -2 position or 2'-O-Me, 2'-MOE, or DNA at the -1 position were used as substrates, the amount of ligation product was significantly reduced by 5–50% compared to when an all-natural RNA fragment was used as a substrate. On the other hand, with Mut1, which has improved ligation activity, the amount of ligation product was comparable to that when an all-natural RNA fragment was used as a substrate, even when an oligonucleotide with 2'-F and DNA at the -2 position was used as substrate. Furthermore, Mut1 produced more than twice the amount of ligation product compared to T4 RNA ligase 2, regardless of the oligonucleotide used as substrate.

[0084] [Table 6]

[0085] [Example 5] Temperature stability of artificial RNA ligase The thermal stability of engineered RNA ligases was evaluated. Mut1 RNA ligase, which showed the highest activity, and T4 RNA ligase 2 (New England Biolabs) were used as a control. The four oligonucleotide fragments listed 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 was incubated at 25°C in a 200 μL microtube. After 23 hours of incubation, 0.2 μL of each 500 μM substrate was added to initiate the ligation reaction. The reaction mixture consisted of 10 μM oligonucleotide, 50 mM Tris-HCl (pH 7.5), 2 mM MgCl, 1 mM dithiothreitol, 0.4 mM ATP, and 0.72 μg / mL of each enzyme. After 15 minutes, a 3 μL aliquot was taken and the reaction was stopped by adding 27 μL of 10 mM EDTA. The amount of antisense strand product was quantified by HPLC under the conditions described in Example 2.

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

[0087] Similarly, activity was measured after incubation at 37°C for 4 hours. As shown in Table 9, with the control T4 RNA ligase 2, no detectable ligation product was produced after incubation at 37°C for 4 hours, indicating a significant decrease in activity. In contrast, with Mut1, which has improved ligation activity, the amount of ligation product was 83% of that of the untreated sample, even after incubation at 37°C for 4 hours, demonstrating 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, maintaining an alanine residue at position 32, a phenylalanine residue at position 116, and a serine residue at position 170, and having a nucleic acid ligation activity that is 1.2 times 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, maintaining an alanine residue at position 32, a phenylalanine residue at position 116, and a serine residue at position 170, and having a nucleic acid ligation activity that is 1.2 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, maintaining an alanine residue at position 32, a phenylalanine residue at position 116, and a serine residue at position 170, and having nucleic acid ligation activity that is 1.2 times 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, maintaining an alanine residue at position 32, a phenylalanine residue at position 116, and a serine residue at position 170, and having a nucleic acid ligation activity that is 1.2 times 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, maintaining an alanine residue at position 32, a phenylalanine residue at position 116, and a serine residue at position 170, and having a nucleic acid ligation activity that is 1.2 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, maintaining an alanine residue at position 32, a phenylalanine residue at position 116, and a serine residue at position 170, and having nucleic acid ligation activity that is 1.2 times 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. 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 RNAs.

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

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