Mutant ligase

Abstract

The present application provides a ligase mutant having excellent properties. The present application provides a ligase mutant of (1), (2), or (3) below: (1) a ligase mutant comprising an amino acid sequence showing 95% or greater identity with the amino acid sequence of SEQ ID NO: 1 and having a ligation activity of a nucleic acid; (2) a ligase mutant comprising an amino acid sequence showing 90% or greater identity with the amino acid sequence of SEQ ID NO: 2 and having a ligation activity of a nucleic acid; or (3) a ligase mutant comprising an amino acid sequence showing 97% or greater identity with the amino acid sequence of SEQ ID NO: 3 and having a ligation activity of a nucleic acid.

Description

TECHNICAL FIELD

[0001] The present application relates to a ligase mutant. BACKGROUND

[0002] T4 RNA ligase 2 is an RNA ligase (EC 6.5.1.3) having a ligation ability of ribonucleotides in the presence of ATP, and is an enzyme derived from a T4 bacteriophage having an infection ability to Escherichia (NP_049790). T4 RNA ligase 2 has an ability to ligate a phosphate group at the 5' end of a nucleic acid (donor) to a hydroxyl group at the 3' end (acceptor) by forming a phosphodiester bond. T4 RNA ligase 2 is used for reactions such as ligation of double-stranded RNA having protruding ends, and ligation of a nick in double-stranded RNA. It is known that not only RNA but also DNA and modified nucleic acids other than DNA and RNA can be used as a substrate for T4 RNA ligase 2. In addition, it is also known that there are several mutants of T4 RNA ligase 2. For the prior art of T4 RNA ligase 2, the following documents can be referred to.

[0003] Prior Art Documents

[0004] Patent Documents

[0005] Patent Document 1: International Publication No. 2008 / 094599

[0006] Non-Patent Documents

[0007] Non-Patent Document 1: 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.

[0008] NPL 3: Nandakumar, J., Shuman, S., Lima, CD. (2006). RNA ligase structures reveal the basis for RNA specificity and conformational changes that drive ligation forward. Cell, 6; 127(1): 71-84.

[0009] NPL 4: Ho, CK., & Shuman, S. (2002). Bacteriophage T4 RNA ligase 2 (gp24.1) exemplifies a family of RNA ligases found in all phylogenetic domains. PNAS, 1; 99(20): 12709-14.

[0010] NPL 5: Nandakumar, J., Ho, CK., Lima, CD., Shuman, S. (2004). RNA substrate specificity and structure-guided mutational analysis of bacteriophage T4 RNA ligase 2. J. Biol. Chem., 23; 279(30): 31337-47.

[0011] NPL 6: Nandakumar, J. & Shuman, S. (2004). How an RNA ligase discriminates RNA versus DNA damage. Mol. Cell., 22; 16(2): 211-21.

[0012] NPL 7: Nandakumar, J. & Shuman, S. (2005). Dual mechanisms whereby a broken RNA end assists the catalysis of its repair by T4 RNA ligase 2. J. Biol. Chem., 24; 280(25): 23484-9.

[0013] Non-patent literature 8: Yin, S., Ho, C. K., Shuman, S. (2003). Structure-function analysis of T4 RNA ligase 2. J. Biol. Chem., 16; 278(20): 17601-8.

[0014] Non-patent literature 9: Yin, S., Kiong Ho, C., Miller, E. S., Shuman, S. (2004). Characterization of bacteriophage KVP40 and T4 RNA ligase 2. Virology, 5; 319(1): 141-51. SUMMARY

[0015] PROBLEMS TO BE SOLVED BY THE INVENTION

[0016] An object of the present application is to provide a ligase mutant having excellent properties.

[0017] MEANS OF SOLVING THE PROBLEMS

[0018] The present inventors etc. have made earnest studies, and as a result, have successfully developed three kinds of ligase mutants Mutl to 3 (SEQ ID NO: 1 to 3) which show 93%, 87% and 95% of amino acid sequence identity, respectively, to T4 RNA ligase 2 (NP_049790) (see Table 1 below) and have more excellent properties than the T4 RNA ligase 2, thereby completing the present application. There is no suggestion or teaching about such three kinds of ligase mutants in the above-mentioned prior art.

[0019] That is, the present application is as follows;

[0020] [1] A ligase mutant of the following (1), (2) or (3):

[0021] (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 ligation activity of nucleic acid;

[0022] (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 ligation activity of nucleic acid; or

[0023] (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 ligation activity of nucleic acid;

[0024] [2] The ligase mutant according to [1], wherein the nucleic acid is single-stranded RNA or double-stranded RNA which can contain DNA and / or modified nucleic acid;

[0025] [3] A method for producing a nucleic acid product, comprising ligating a nucleic acid material in the presence of the ligase mutant according to [1] or [2] to produce the nucleic acid product,

[0026] the nucleic acid material is selected from the group consisting of single-stranded nucleic acid material, double-stranded nucleic acid material, and a mixture thereof;

[0027] [4] The method according to [3], wherein the nucleic acid material is RNA;

[0028] [5] The method according to [3] or [4], wherein the nucleic acid material is four or more single-stranded RNAs;

[0029] [6] The method according to any one of [3] to [5], wherein the nucleic acid product comprises a complementary portion having a length of 12 to 27 bases;

[0030] [7] The method according to any one of [3] to [6], wherein the nucleic acid material contains DNA and / or modified nucleic acid;

[0031] [8] The method according to any one of [3] to [7], wherein the concentration of the nucleic acid material is 1 μM or more;

[0032] [9] The method according to any one of [3] to [8], wherein the nucleic acid product is siRNA;

[0033]

[10] A polynucleotide encoding the ligase mutant according to [1] or [2];

[0034]

[11] An expression vector comprising the polynucleotide according to

[10] ;

[0035]

[12] A transformed microorganism comprising an expression unit,

[0036] the expression unit comprises a polynucleotide encoding the ligase mutant according to [1] or [2], and a promoter operably linked thereto;

[0037]

[13] A method for producing a ligase mutant, comprising using the transformed microorganism according to

[12] to produce the ligase mutant according to [1] or [2].

[0038] Effects of the Invention

[0039] According to the present invention, a nucleic acid product (e.g., siRNA or modified nucleic acid such as heteroduplex nucleic acid) can be efficiently produced from a nucleic acid material.

[0040] Brief description of drawings

[0041] Figure 1 is a graph showing the amino acid sequences (SEQ ID NO: 1 to 3) of the ligase mutants (Mut 1 to 3) of the present application;

[0042] Figure 2 is a graph showing a double-stranded oligonucleotide generated by a ligation reaction of single-stranded oligonucleotides of 4 fragments. The notation of modified nucleotide residues is the same as in Table 2;

[0043] Figure 3 is a graph showing (A) a double-stranded oligonucleotide generated by a ligation reaction of single-stranded oligonucleotides of 4 fragments, and (B) a time course of the generation of the double-stranded oligonucleotide. The notation of modified nucleotide residues is the same as in Table 4;

[0044] Figure 4 is a graph showing (A) a double-stranded oligonucleotide generated by a ligation reaction of single-stranded oligonucleotides of 3 fragments, and (B) the amount of the double-stranded oligonucleotide (reaction time 15 minutes). Oligonucleotides in which the 2' position of the nucleotide residues at -2 position, -1 position, +1 position, +2 position from the ligation point are modified with a fluorine atom (F), O-methyl (Ome), O-methoxyethyl (MOE), or substituted with a hydrogen atom (DNA) were used as substrates. DETAILED DESCRIPTION

[0045] 1. Ligase mutant

[0046] The present application provides a ligase mutant of (1), (2), or (3) described below:

[0047] (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 ligation activity of nucleic acids;

[0048] (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 ligation activity of nucleic acids; or

[0049] (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 ligation activity of nucleic acids.

[0050] 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 identified as an amino acid sequence showing 95% or more identity to the amino acid sequence of SEQ ID NO: 1, and thus is sufficiently different from the known T4 RNA ligase 2. The degree of the % identity to the amino acid sequence of SEQ ID NO: 1 in the ligase mutant of (1) can be preferably 96% or more, more preferably 97% or more, further more preferably 98% or more, and most preferably 99% or more.

[0051] 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 of (2) is identified as an amino acid sequence showing 90% or more identity to the amino acid sequence of SEQ ID NO: 2, and thus is sufficiently different from the known T4 RNA ligase 2. The degree of the % identity to the amino acid sequence of SEQ ID NO: 2 in the ligase mutant of (2) can be preferably 91% or more, more preferably 92% or more, further 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.

[0052] 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 identified as an amino acid sequence showing 97% or more identity to the amino acid sequence of SEQ ID NO: 3, and thus is sufficiently different from the known T4 RNA ligase 2. The degree of the % identity to the amino acid sequence of SEQ ID NO: 3 in the ligase mutant of (3) can be preferably 97.5% or more, more preferably 98% or more, further more preferably 98.5% or more, and most preferably 99% or more or 99.5% or more.

[0053] The calculation of the % identity of the amino acid sequence can be performed using the following values: using the software GENETYX Ver 13.1.1 of GENETYX Corporation, using the entire length of the polypeptide encoded by the ORF, after the Muscle alignment or the ClustalW alignment or the multiple sequence alignment, and the values obtained when the calculation is performed with the gaps taken into account. As for the number of modifications (for example, substitutions, deletions, additions, or insertions, or a combination thereof) of the amino acid residues in SEQ ID NO: 1 (full length of 332 amino acid residues), SEQ ID NO: 2 (full length of 330 amino acid residues), and SEQ ID NO: 3 (full length of 332 amino acid residues) that can achieve the % identity described above, it can 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.

[0054] The ligase mutant of the present application has a nucleic acid ligation activity. As the nucleic acid, for example, single-stranded nucleic acid and double-stranded nucleic acid can be mentioned. As the nucleic acid, for example, RNA, DNA, modified nucleic acid other than RNA and DNA, and mixed-type nucleic acid thereof can also be mentioned. Preferably, the nucleic acid can be single-stranded RNA or double-stranded RNA that can contain DNA and / or modified nucleic acid. The 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 later.

[0055] The nucleic acid ligation activity possessed by the ligase mutant of the present application is not particularly limited as long as it is more superior than that of T4 RNA ligase 2 (NP_049790), and can have a nucleic acid ligation activity that is 1.2 times or more, more preferably 1.5 times or more, further more preferably 1.8 times or more, particularly preferably 2.0 times or more, as compared to T4 RNA ligase 2. Such a nucleic acid ligation activity can be determined by a prescribed reaction as described in the examples. For example, such a reaction can be performed by the steps of a) to c) described below (see, for example, Example 2):

[0056] a) preparing a 20 μL reaction solution containing a nucleic acid material (for example, single-stranded RNA) at a final concentration of 10 μM, 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM dithiothreitol, 0.4 mM ATP;

[0057] b) To the reaction solution, 50 μL of a solution containing the purified ligase mutant was added to start the reaction, so that the final concentration became 0.36 μg / mL;

[0058] c) The reaction was performed at 25°C for 1 hour.

[0059] The ligase mutant of the present application can also have excellent temperature stability. For example, the ligase mutant comprising the amino acid sequence of the present application, in the case where heat treatment is performed and then activity is measured, preferably has a residual activity of 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, based on the activity measured under the condition without heat treatment. Such a condition can be a condition where incubation at 25°C for 23 hours, or incubation at 37°C for 4 hours (for example, 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 enzyme) is performed, and then activity is measured. The activity measurement can be performed under the conditions of a) to c) in the above paragraph. Alternatively, the activity measurement can be a condition where activity is measured in a reaction at 25°C or 37°C for 15 minutes. The reaction solution composition used under such a condition can 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. The temperature stability of the enzyme can generally have a correlation with the liquid stability and / or long-term storage stability, so the ligase mutant of the present application having excellent temperature stability can be considered to have excellent liquid stability and / or long-term storage stability. Therefore, the ligase mutant of the present application can be used as a reagent.

[0060] The ligase mutant of the present application can have a mutation of one or more amino acid residues within a range of maintaining the desired % identity and ligation activity. The positions of the amino acid residues into which mutations can be introduced are obvious to one skilled in the art. For example, one skilled in the art can recognize the correlation of structure and function by 1) comparing the amino acid sequences of various proteins having the same property (e.g., SEQ ID NOs: 1 to 3), 2) specifying the regions that are relatively conserved and the regions that are relatively not conserved, and then 3) predicting from the regions that are relatively conserved and the regions that are relatively not conserved, respectively, the regions that can play an important role for the function and the regions that can not play an important role for the function. Thus, one skilled in the art can identify the positions of the amino acid residues in the amino acid sequence of the ligase mutant of the present application into which mutations can be introduced. The amino acid residue after mutation in such a position is a desired natural alpha-amino acid residue different from the amino acid residue before mutation. As such a desired natural alpha-amino acid residue is a residue 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 of the amino acid sequence of SEQ ID NO: 1 can be introduced into mutations. In an amino acid sequence showing 90% or more identity to the amino acid sequence of SEQ ID NO: 2, 1 to 33 amino acid residues of the amino acid sequence of SEQ ID NO: 2 can be introduced into mutations. In an amino acid sequence showing 97% or more identity to the amino acid sequence of SEQ ID NO: 3, 1 to 9 amino acid residues of the amino acid sequence of SEQ ID NO: 3 can be introduced into mutations. The mutation of the amino acid residue is selected from substitution, deletion, addition, and insertion of the amino acid residue.

[0061] In the case of substitution mutation of an amino acid residue, the substitution of the amino acid residue can be a conservative substitution. The term "conservative substitution" as used in the present specification means substitution of a prescribed 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 field. For example, as such families, there are families of amino acid having basic side chains (e.g., lysine, arginine, histidine), families of amino acid having acidic side chains (e.g., aspartic acid, glutamic acid), families of amino acid having non-charged polar side chains (e.g., asparagine, glutamine, serine, threonine, tyrosine, cysteine), families of amino acid having non-polar side chains (e.g., glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), families of amino acid having β-position branched side chains (e.g., threonine, valine, isoleucine), families of amino acid having aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine), families of amino acid having hydroxyl-containing (e.g., alcoholic, phenolic) side chains (e.g., serine, threonine, tyrosine), and families of amino acid having sulfur-containing side chains (e.g., cysteine, methionine). Preferably, the conservative substitution of the amino acid can be substitution between aspartic acid and glutamic acid; substitution between arginine, lysine and histidine; substitution between tryptophan and phenylalanine; substitution between phenylalanine and valine; substitution between leucine, isoleucine and alanine; and substitution between glycine and alanine.

[0062] The ligase mutant of the present application can further comprise other peptide components (e.g., tag moieties) at the C-terminal or N-terminal. As the other peptide components that can be comprised in the ligase mutant of the present application, there are, for example, peptide components that make purification of a target protein easy (e.g., histidine tag, Strep-tag II and the like tag moieties; glutathione S-transferase, maltose binding protein and the like proteins that are widely used for purification of target proteins), peptide components that improve the solubility of a target protein (e.g., Nus-tag), peptide components that function as chaperones (e.g., trigger factor), proteins or domains of proteins having other functions, or peptide components that function as linkers for linking them to the ligase mutant.

[0063] 2. Inventions that can be related to production of the ligase mutant of the present application

[0064] The ligase mutant of the present application can be produced using a transformed microorganism comprising an expression unit comprising a polynucleotide encoding the ligase mutant of the present application and a promoter operably linked thereto, or using a cell-free system, and the like. The present application also provides such polynucleotides and transformed microorganisms, and expression vectors that can be used for production of the transformants.

[0065] The polynucleotide of the present application is a polynucleotide encoding the ligase mutant of the present application. The polynucleotide of the present application can be DNA or RNA, preferably DNA.

[0066] The transformed microorganism of the present application can be produced by, for example, a method using an expression vector containing the polynucleotide of the present application (e.g., competent cell method, electroporation method), or a genome editing technique. 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 state of the expression vector in the host cell, independently of the genomic DNA. Alternatively, according to the genome editing technique (e.g., CRISPR / Cas system, Transcription Activator-Like Effector Nucleases (TALEN)), the expression unit can be integrated into the genomic DNA of the host cell, and can edit the expression unit inherently possessed by the host cell.

[0067] The present application also provides an expression vector containing the polynucleotide of the present application. The expression vector of the present application can further contain, for example, a terminator that functions in the host cell, a ribosome binding site, and a drug resistance gene, and the like. As the drug resistance gene, for example, a gene having resistance to tetracycline, ampicillin, kanamycin, hygromycin, glufosinate, and the like can be mentioned.

[0068] With respect to the expression vector, a region capable of achieving homologous recombination with the genome of the host cell can be further contained for homologous recombination with the genomic DNA of the host cell. For example, the expression vector can be designed such 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). As the region of the genome of the host cell to which the expression unit is to be introduced (target of the homologous region), there is no particular limitation, and can be the locus of a gene that is expressed in large amounts in the host cell.

[0069] The expression vector can be a plasmid, a viral vector, a phage, or an artificial chromosome. The expression vector can also be an integrative vector or a non-integrative vector. The integrative vector can be a vector of the type that is integrated as a whole into the genome of the host cell. Alternatively, the integrative vector can also be a vector of the type that is integrated only a part thereof (e.g., an expression unit) into the genome of the host cell. The expression vector can also be a DNA vector or an RNA vector (e.g., a retrovirus). The expression vector can also be a widely used expression vector. As such expression vectors, there can be mentioned, for example: pUC (e.g., pUC19, pUC18), pSTV, pBR (e.g., pBR322), pHSG (e.g., pHSG299, pHSG298, pHSG399, pHSG398), RSF (e.g., RSFlOlO), pACYC (e.g., pACYCl77, pACYCl 84), pMW (e.g., pMWl 19, pMWl 18, pMW2l9, pMW2l8), pQE (e.g., pQE30), and derivatives thereof.

[0070] As the host for expressing the ligase mutant of the present application, there can be used, for example: various prokaryotic cells typified by Escherichia coli and the like, Corynebacterium bacteria [e.g., Corynebacterium glutamicum], and Bacillus bacteria [e.g., Bacillus subtilis]; and various eukaryotic cells typified by Saccharomyces bacteria [e.g., Saccharomyces cerevisiae], Pichia bacteria [e.g., Pichia stipitis], and Aspergillus bacteria [e.g., Aspergillus oryzae]. As the host, there can be used a strain in which a prescribed gene is deleted. As the transformed microorganism, there can be mentioned, for example, a transformed microorganism that retains an expression vector in the cytoplasm, and a transformed microorganism in which a target gene is introduced into the genome.

[0071] The transformed microorganism of the present application can be cultured in a medium having, for example, the composition described later, using a prescribed culture device (e.g., test tube, flask, fermenter). The culture conditions can be appropriately set. Specifically, the culture temperature can be from 10°C to 37°C, the pH can be from 6.5 to 7.5, and the culture time can be from 1 h to 100 h. Furthermore, the culture can be performed while managing the dissolved oxygen concentration. In this case, the dissolved oxygen concentration (DO value) in the culture solution is sometimes used as an index for control. The aeration and agitation conditions can be controlled so that the relative dissolved oxygen concentration DO value when the oxygen concentration in the atmosphere is set to 21% is, for example, not less than 1 to 10%, and more preferably 3 to 8%. Furthermore, the culture can be batch culture, or fed-batch culture. In the case of fed-batch culture, a solution to be a sugar source or a solution containing phosphoric acid can also be added to the culture solution continuously or discontinuously in stages, and the culture can be continued.

[0072] The host to be transformed can be selected from, for example, Escherichia coli JM109 strain, DH5α strain, HB101 strain, BL21(DE3) strain, and the like, of the Escherichia coli K12 strain subspecies, if the Escherichia coli is specifically described. The method of transformation and the method of selecting the transformed microorganism are also described in Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor press (2001 / 01 / 15), and the like. Hereinafter, the method of producing the transformed Escherichia coli and using it to produce a prescribed enzyme will be described more specifically as an example.

[0073] As the promoter for expressing the polynucleotide of the present application, a promoter generally used for the production of a heterologous protein in E. coli can be used, and strong promoters such as PhoA, PhoC, T7 promoter, lac promoter, trp promoter, trc promoter, tac promoter, PR promoter of lambda phage, PL promoter, T5 promoter, and the like can be exemplified, and preferably PhoA, PhoC, and lac. In addition, as the vector, 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, and the like can be used. As other vectors, vectors of phage DNA can be used. In addition, an expression vector including a promoter and capable of expressing an inserted DNA sequence can also be used. Preferably, the vector can be pUC, pSTV, or pMW.

[0074] In addition, a terminator as a transcription termination sequence can be ligated downstream of the polynucleotide of the present application. As such a terminator, T7 terminator, fd phage terminator, T4 terminator, terminator of the tetracycline resistance gene, terminator of the E. coli trpA gene, and the like can be exemplified.

[0075] As the vector for introducing the polynucleotide of the present application into E. coli, a so-called multicopy type vector can be preferably used, and plasmids having a replication origin derived from ColE1, such as plasmids of the pUC series or plasmids of the pBR322 series or derivatives thereof can be exemplified. Here, the "derivative" means a derivative obtained by modifying the plasmid by substitution, deletion, insertion, and / or addition of bases, and the like.

[0076] In addition, in order to screen the transformed microorganism, the vector preferably has a marker such as an ampicillin resistance gene. As such a plasmid, expression vectors having a strong promoter are commercially available [e.g., pUC series (manufactured by Takara Bio Inc.), pPROK series (manufactured by Clontech), pKK233-2 (manufactured by Clontech)].

[0077] Using the obtained expression vector of the present application, E. coli is transformed, and the E. coli is cultured, whereby the ligase mutant of the present application can be obtained.

[0078] As the medium, M9-casein amino acid medium, LB medium, and the like, which are generally used for culturing E. coli, can be used. The medium can contain a prescribed carbon source, nitrogen source, coenzyme (e.g., pyridoxine hydrochloride). Specifically, proteose peptone, yeast extract, NaCl, glucose, MgSO4, ammonium sulfate, potassium dihydrogen phosphate, ferric sulfate, manganese sulfate, and the like can be used. Furthermore, the culture conditions, production induction conditions are appropriately selected depending on the kind of the marker, promoter, host bacterium, and the like of the vector used.

[0079] The following methods and the like can be used to recover the ligase mutant of the present application. With regard to the ligase mutant of the present application, after recovering the transformed microorganism of the present application, the bacterial cells can be disrupted (e.g., sonicated, homogenized) or lysed (e.g., lysozyme treatment), thereby obtaining as the disrupted matter and lysate. Such disrupted matter and lysate can be subjected to extraction, precipitation, filtration, column chromatography, and the like, thereby obtaining the ligase mutant of the present application.

[0080] 3. Method for producing nucleic acid product

[0081] The present application also provides a method for producing a nucleic acid product, which includes ligating a nucleic acid material in the presence of the ligase mutant of the present application to produce a nucleic acid product. As the nucleic acid material, a material selected from the group consisting of single-stranded nucleic acid material, double-stranded nucleic acid material, and mixtures thereof can be used.

[0082] (Nucleic acid)

[0083] Nucleic acids in nucleic acid materials and nucleic acid products can be classified into natural nucleic acids and modified nucleic acids. The 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), thymidine (dT)) constituting polynucleotides contained in cells. Hereinafter, these are referred to as "natural nucleotide residues". The modified nucleic acids refer to nucleic acids other than the natural nucleic acids, and are nucleic acids containing nucleotide residues other than the natural nucleotide residues (hereinafter, referred to as "modified residues"). As the modified residues, for example, modified nucleotide residues, amino acid residues, linkers can be given. As the modified nucleotide residues, for example, nucleotide residues containing modifications described later can be given. The amino acids include derivatives of amino acids. As the amino acids, for example, 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 can be given. The derivatives of amino acids refer to amino acids in which any atom or group in the amino acid is replaced with other atom or group, and for example, amino acids in which hydrogen atom in the amino acid, hydrogen atom in the carboxyl group, oxygen atom, hydroxyl group, any atom or group in the side chain, or hydrogen atom bonded to the skeleton carbon atom (for example, a-, β-, γ-, δ-carbon atom) is replaced with other atom (for example, halogen atom such as fluorine atom, chlorine atom, bromine atom, iodine atom) or group (for example, substituted substituent after substitution in the chemical modification described later) can be given.

[0084] The modification in the modified nucleotide residue includes substitution of atom or group of the sugar moiety (ribose or deoxyribose) of the nucleotide residue, substitution of the sugar moiety itself (sugar backbone) of the nucleotide residue, and modification of the nucleic acid base moiety of the nucleotide residue (for example, substitution of substituent of the nucleic acid base moiety).

[0085] As the substitution of atom or group of the sugar moiety of the nucleotide residue, for example, substitution of 1'-H, 2'-OH (only for ribose), 2'-H, 3'-OH, 3'-NH2, 3'-H, 3'-phosphate group, 4'-H, 5'-phosphate group, or a combination thereof can be given. Here, the phosphate group includes not only -O-P(O)(OH)2, but also a group in which the oxygen atom is replaced with sulfur atom or NH, for example, -O-P(S)(OH)2, -NH-P(O)(OH)2, -NH-P(S)(OH)2. Further, a group in which the hydroxyl group (-OH) in the phosphate group is substituted with OR * (for example, a protected phosphate group) is also included in the phosphate group, in the formula, R *A protecting group for a phosphate group or the like organic group. As such substitution, there can be mentioned, for example: 1', 2', 3' or 4'-chemical modification (substitution for other substituent at 1', 2', 3' or 4' position), 5'- or 3'-phosphate group modification (substitution for other substituent for 5'- or 3'-phosphate group), bridging modification (bridging of two substituents at 1', 2', 3' or 4' position with each other), and carrier addition modification (substitution for carrier at 1', 2', 3', 4' or 5' position).

[0086] For example, a chemical modification can be introduced to improve the resistance to decomposition of the oligonucleotide. As the substituent after substitution in the chemical modification, there can be mentioned, for example: C 1～6 alkyl (e.g., -O-Me), -O-C 1～6 alkylene (e.g., methoxyethyl: MOE), -O-C 1～6 alkyl (e.g., -O-Me), -O-C 6～14 aryl (e.g., -O-phenyl), -C-aryl (e.g., -C-phenyl), halogen atom (e.g., fluorine atom), -O-C 1～6 alkyl N-amide C 1～6 alkylene (e.g., -O-N-methylacetamide, -O-NMA), -O-C 1～6 alkyl- (C 1～6 alkyl-)amino-C 1～6 alkylene (e.g., -O-dimethylaminoethyloxyethyl, -O-DMAEOE), and -O-amino C 1～6 alkyl (e.g., -O-amino propyl, -O-AP). The chemical modification is preferably 2'-chemical modification (substitution at 2' position), 3'-chemical modification (substitution at 3' position), of which, more preferably, 2'-chemical modification (substitution at 2' position). As the substituent after substitution in the 2'-chemical modification, there can be mentioned, for example: 2'-C 1～6 alkyl (e.g., -O-Me), -O-C 1～6 alkylene (e.g., 2'-methoxyethyl), 2'-O-C 1～6 alkyl (e.g., 2'-O-Me), 2'-O-C 6～14 aryl (e.g., 2'-O-phenyl), 2'-C-aryl (e.g., 2'-C-phenyl), 2'-halogen atom (e.g., 2'-F), 2'-O-C 1～6 alkyl N-amide C 1～6 alkylene (e.g., 2'-O-N-methylacetamide, 2'-O-NMA), 2'-O-C 1～6 alkyl- (C 1～6 alkyl-)amino-C 1～6 alkylene (e.g., 2'-O-dimethylaminoethyloxyethyl, 2'-O-DMAEOE), and 2'-O-amino C1～6 alkyl group (e.g., 2'-0-aminopropyl, 2'-0-AP). As a substituent after substitution in 3'-chemical modification, there can be mentioned, for example, 3'-0-P(0)(OH)2, 3'-0-P(S)(OH)2, 3'-NH-P(0)(OH)2, 3'-NH-P(S)(OH)2, and a group in which the hydroxyl group (-OH) in the phosphate group is substituted with OR * , wherein R * represents an organic group such as a protecting group of a phosphate group described later.

[0087] For example, a 5'- or 3'-phosphate group modification can be introduced to improve the decomposition resistance of the oligonucleotide. As the 5'- or 3'-phosphate group modification, there can be mentioned, for example, substitution of the phosphate group (-0-P(0)(OH)2) with a group in which the oxygen atom in the phosphate group is replaced with a sulfur atom or NH. As such a group, there can be mentioned, for example: -0-P(S)(OH)2(thiophosphate group: phosphorothioate modification), -NH-P(0)(OH)2, -NH-P(S)(OH)2. Further, the 5'- or 3'-phosphate group modification also includes a group in which the hydroxyl group (-OH) in the phosphate group is substituted with OR * , for example, a protected phosphate group, wherein R * represents an organic group such as a protecting group of a phosphate group. As the protecting group of the phosphate group, there can be mentioned, for example, trityl (Tr), p-methoxyphenyldiphenylmethyl (MMTr), di(p-methoxyphenyl)phenylmethyl (DMTr), cyanoethyl (CN-C2H4-).

[0088] For example, a bridging modification can be introduced to improve the stereoscopic structure stability of the nucleotide residue. As the bridging modification, there can be mentioned, for example: 2'4'-bridging modification (substitution of 2'-OH and 4'-H), 3'5'-bridging modification (substitution of 3'-H and 5'-H), and the like. As the 2'4'-bridging modification, there can be mentioned, for example: 2'-OH and 4'-H are substituted with 2'-0-C 1～6 alkylene-4' (e.g., 2'-0-methylene-4' (locked nucleic acid: LNA), 2'-0-ethylene-4' (ethylene-bridged nucleic acid: ENA), 2'-0-methyl-substituted methylene-4' (constrained ethyl bridged nucleic acid: BNA), 2'-OH and 4'-H are substituted with 2'-0-C 1～6 alkylene-0-C 1～6alkylene-4' (e.g., 2'-0-methylene-0-methylene-4' (one of bridged nucleic acids: BNA COC )) and 4'-H to 2'-0-N(R)-C 1～6 alkylene-4' (e.g., 2'-0-N(R)-methylene-4' (one of bridged nucleic acids: BNA NC ), where R represents a methyl group, a hydrogen atom, or a benzyl group), 2'-NH2and 4'-H to 2'-N(R)-C(0)-4' (e.g., 2'-N(methyl)-C(0)-4' (amide bridged nucleic acid: AmNA)), 2'-NH2and 4'-H to 2'-NH-C 1～6 alkylene-4' (e.g., 2'-NH-methylene-4'), 2'-H and 4'-H to 2'-C 1～6 alkylene-4' (e.g., 2'-methyl-substituted ethylene-4'). In addition, as the 3'5'-bridging modification, for example, 3'-H and 5'-H to 3'-C 1～6 alkylene-5' (e.g., 3'-ethylene-5' (one of bicyclic nucleic acids: Bc nucleic acid), Bc nucleic acid: tc nucleic acid, etc.).

[0089] The carrier in the carrier addition modification can be a carrier for improving or imparting stability, targeting, drug efficacy, and the like to the target modification oligonucleotide. Such a carrier can be appropriately selected from known carriers according to the purpose of use. As the carrier, for example, N-acetylgalactosamine (GalNAc), a peptide, a phosphate, cholesterol, tocopherol, a fatty chain, and folic acid can be mentioned. The addition site in the carrier addition modification is preferably 3' or 5' position (site) corresponding to the terminal of the target modification oligonucleotide.

[0090] As the nucleotide residue including substitution of the sugar moiety itself, for example, a modified nucleotide residue including substitution of a pseudo-sugar having a six-membered ring for a sugar having a five-membered ring can be mentioned. As such a modified nucleotide residue, for example, a hexitol nucleic acid (HNA), a cyclohexenyl nucleic acid (CeNA) can be mentioned. In addition, as the nucleotide residue including substitution of the sugar moiety itself, a morpholino nucleic acid (PMO) residue as a nucleotide analog artificial compound having a morpholine ring structure which is not decomposed by enzymes (e.g., RNase and the like) in vivo and does not induce an immune response can be mentioned.

[0091] As the modification of the nucleic acid base moiety of the nucleotide residue, for example, alkyl substitution (e.g., substitution of a methyl group at the 5-position of a cytosine base) of the nucleic acid base moiety of the nucleotide residue can be mentioned.

[0092] (Nucleic acid materials)

[0093] The nucleic acid material can be single or multiple. For example, when using a double-stranded nucleic acid with protruding ends as the single nucleic acid material, the method of the present invention can be used for the circularization of this double-stranded nucleic acid. When using multiple nucleic acid materials, the method of the present invention can be used for the following ligation: ligation of multiple double-stranded nucleic acids with protruding ends, ligation of a single or multiple double-stranded nucleic acid with protruding ends to a single or multiple single-stranded nucleic acid, ligation of multiple single-stranded nucleic acids, etc. The number of nucleic acid materials used when using multiple nucleic acid materials is not particularly limited as long as it is two or more; it can be, for example, a smaller number such as 2 to 10, 2 to 8, 3 to 7, 4 to 6, etc., but it can also be more than 10.

[0094] There is no particular limitation on the length of the nucleic acid material. For example, longer nucleic acid materials exceeding 1000 bases in length can be used. Alternatively, shorter nucleic acid materials can be used when it is desired to produce shorter nucleic acid products. For shorter nucleic acid materials, for example, the length can be 5 bases or more, preferably 6 bases or more, more preferably 7 bases or more, further preferably 8 bases or more, and particularly preferably 9 bases or more. In addition, for shorter nucleic acid materials, the length can also be 19 bases or less, preferably 18 bases or less, more preferably 17 bases or less, further preferably 16 bases or less, and particularly preferably 15 bases or less.

[0095] Protruding ends can be used in the ligation. Examples of protruding ends used in the ligation include: protruding ends in double-stranded nucleic acids (nucleic acid materials) with protruding ends, and protruding ends formed by annealing (renaturation) between nucleic acid materials (e.g., between double-stranded and single-stranded nucleic acids, or between single-stranded nucleic acids). The length of the protruding ends is not particularly limited. When it is desired to produce a shorter nucleic acid product, the length of the protruding ends can be, for example, 1 to 10 bases, preferably 1 to 8 bases, more preferably 1 to 6 bases, and even more preferably 2 to 6 bases, 3 to 6 bases, or 4 to 6 bases. Therefore, double-stranded nucleic acids with protruding ends or combinations of multiple nucleic acid materials (e.g., combinations of double-stranded and single-stranded nucleic acids, or combinations of multiple single-stranded nucleic acids) can be selected as nucleic acid materials.

[0096] Nucleic acid materials can be in a free form or immobilized in a solid phase. Furthermore, when it is desirable to form a complex between the nucleic acid product and the functional portion, the nucleic acid material can form a complex with the functional portion at its corresponding part.

[0097] The nucleic acid material can be manufactured by a chemical synthesis method (e.g., solid phase synthesis method, liquid phase synthesis method) or an enzymatic synthesis method. As such a synthesis method, for example, the methods described in International Publication No. 2012 / 157723, International Publication No. 2005 / 070859 can be mentioned.

[0098] Preferably, the nucleic acid material can be single-stranded RNA or double-stranded RNA which can contain DNA and / or modified nucleic acids. More preferably, such a nucleic acid material can be double-stranded RNA having overhanging ends as described above, or a combination of various RNA materials forming overhanging ends by annealing as described above (e.g., a combination of double-stranded RNA and single-stranded RNA, a combination of various single-stranded RNAs).

[0099] (Nucleic acid product)

[0100] The nucleic acid product contains a complementary portion of base pairing. As such a nucleic acid product, for example, a double-stranded nucleic acid, a single-stranded nucleic acid containing a double-stranded-like structure portion (e.g., a hairpin type nucleic acid, a dumbbell type nucleic acid, and the like, a circular nucleic acid) can be mentioned. The double-stranded nucleic acid can be a double-stranded nucleic acid in which each strand is the above-described nucleic acid, and for example, a double-stranded RNA, a double-stranded DNA, a hetero double-stranded nucleic acid formed of RNA and DNA, a double-stranded nucleic acid formed of RNA and RNA-DNA hybrid nucleic acid, a double-stranded nucleic acid formed of DNA and RNA-DNA hybrid nucleic acid, and a double-stranded nucleic acid formed of RNA-DNA hybrid nucleic acid each other can be mentioned. As the double-stranded nucleic acid, for example, an siRNA and a hetero double-stranded nucleic acid can be mentioned.

[0101] In a particular embodiment, the nucleic acid product can include the modified residue described above in the complementary portion. As such a nucleic acid product, there can be mentioned, for example, a double-stranded nucleic acid or a circular nucleic acid including a modified nucleotide residue (for example, a double-stranded nucleic acid or a circular nucleic acid including a modified nucleotide residue in the complementary portion), a circular nucleic acid including a modified nucleotide residue or a residue other than a nucleotide residue (for example, an amino acid residue, a linker, etc.) in the circular portion (for example, International Publication No. 2012 / 005368). In such a nucleic acid product, a part of the nucleotide residues can be modified nucleotide residues, or all of the nucleotide residues can be modified nucleotide residues, and in the case where the modified nucleotide residue is a morpholino nucleic acid (PMO) residue, it is preferable that a part of the nucleotide residues be morpholino nucleic acid (PMO) residues in the target modified nucleic acid. Further, such a nucleic acid product includes a gapmer which is a nucleic acid having a modified nucleotide residue at both ends of the sequence and having a gap region which accepts recognition by RNase in the central portion of the sequence, and further includes a mixmer which is a nucleic acid having a modified nucleotide residue mixed in the sequence, a fully modified nucleic acid which is a nucleic acid in which all of the nucleotide residues in the sequence are modified nucleotide residues, and the like, which do not induce RNase activity.

[0102] The nucleic acid product can be a nucleic acid formed only by the complementary portion of base pairing, or can be a nucleic acid including a non-complementary portion of base non-pairing in addition to the complementary portion. The length of the complementary portion and / or the non-complementary portion is not particularly limited, and the complementary portion and / or the non-complementary portion can be short. For example, the shorter complementary portion can be 11 to 27 bases in length, 12 to 27 bases in length, 15 to 27 bases in length, or 18 to 27 bases in length. For example, the shorter non-complementary portion can be 1 to 16 bases in length, 1 to 10 bases in length, 1 to 5 bases in length, or 1, 2, or 3 bases in length. In the case where the nucleic acid product has a non-complementary portion in addition to the complementary portion, the complementary portion can be in a continuous form, or can be in a non-continuous form interrupted by the non-complementary portion. The length of the nucleic acid product is not particularly limited, and the nucleic acid product can be short. The shorter nucleic acid product can be, for example, 20 to 80 bases in length, or 24 to 74 bases in length.

[0103] Preferably, the nucleic acid product can be a single-stranded RNA or a double-stranded RNA which can include DNA and / or a modified nucleic acid. More preferably, such a nucleic acid product can include a modified residue as described above in the complementary portion.

[0104] (Reaction conditions for ligation)

[0105] As the reaction system, an aqueous solution can be used. As the aqueous solution, a buffer solution is preferable. As the buffer solution, for example, a phosphate buffer solution, a Tris buffer solution, a carbonate buffer solution, an acetate buffer solution, a citrate buffer solution can be mentioned. The pH can be, for example, about 5 to 9. For example, in a case where it is particularly desired to efficiently mass-produce the target nucleic acid product, the pH can be 7.5 to 9.0 (for example, 8.0 to 8.5).

[0106] The concentration of each nucleic acid material in the ligation reaction can be a concentration at which the nucleic acid material is dissolved and is sufficient to produce the target nucleic acid product. The concentration of each nucleic acid material can 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. In addition, the concentration of each nucleic acid material can be, for example, 1 M, 100 mM, or 10 mM or less. In a case where it is particularly desired to efficiently mass-produce the target nucleic acid product, among the above concentrations, it is preferable to use each nucleic acid material at a concentration of 100 μM or more.

[0107] In a case where a plurality of nucleic acid materials is used in the ligation reaction, from the viewpoint of improving the manufacturing efficiency by reducing the amount of unreacted nucleic acid material, the number of moles of all the nucleic acid materials is preferably approximately equal. In order to make the number of moles of all the nucleic acid materials approximately equal, the total molar ratio of any two nucleic acid materials selected from the plurality of nucleic acid materials can be, for example, in the range of 0.5 to 2, preferably 1 / 1.8 to 1.8, more preferably 1 / 1.5 to 1.5, further more preferably 1 / 1.2 to 1.2, particularly preferably 1 / 1.1 to 1.1.

[0108] The concentration of the ligation enzyme mutant of the present application in the ligation reaction can be a concentration sufficient to produce the target nucleic acid product. The concentration of the ligation enzyme mutant can be, for example, 0.01 U / μL or more, preferably 0.02 U / μL or more, more preferably 0.03 U / μL or more, further more preferably 0.04 U / μL or more. In addition, the concentration of the ligation enzyme mutant can be, for example, 1 U / μL or less, preferably 0.5 U / μL or less, more preferably 0.2 U / μL or less, further 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 a nucleic acid product in 1 hour in the reaction described in Example 2.

[0109] The reaction system can contain a co-factor. As the co-factor, for example, ATP, a divalent metal salt (e.g., magnesium salt such as magnesium chloride) can be given. The reaction system can contain a stabilizer for the ligase. As the stabilizer for the ligase, for example, an antioxidant (e.g., a reducing agent such as dithiothreitol, mercaptoethanol) can be given. The reaction system can contain a surfactant for the purpose of maintaining stability of the enzyme and increasing the reaction speed, and the like. As the surfactant, for example, a nonionic surfactant (e.g., a surfactant of Triton series such as Triton X-100), and an ionic surfactant can be given. As the ionic surfactant, for example, a cationic surfactant, an anionic surfactant, an amphoteric surfactant (zwitterionic surfactant) can be given. In addition, the reaction system can contain polyethylene glycol for the purpose of increasing the reaction speed, and the like.

[0110] The reaction system can contain a low concentration of a monovalent cation salt, or substantially contain no monovalent cation salt. In the case where the reaction system contains a monovalent cation salt, the concentration of the monovalent cation salt in the reaction system can be, for example, 10 mM or less, preferably 1 mM or less, more preferably 0.1 mM or less, and further more preferably 0.01 mM or less. Particularly preferably, the reaction system can substantially contain no monovalent cation salt. As the monovalent cation salt, for example, a salt of a monovalent cation such as lithium ion, sodium ion, potassium ion, rubidium ion, cesium ion, ammonium ion, and a halide ion such as fluoride ion, chloride ion, bromide ion, and iodide ion can be given.

[0111] The reaction temperature can be a temperature sufficient to activate the ligase mutant of the present application. Such a temperature can be, for example, 2 to 50°C, preferably 16 to 50°C, and more preferably 25 to 50°C.

[0112] The reaction time can be a time sufficient to generate the target nucleic acid product. Such a time can be, for example, 1 to 72 hours.

[0113] Examples

[0114] Hereinafter, the present application will be described in detail by way of examples, but the present application is not limited to these examples.

[0115] [Example 1] Design and preparation of RNA ligase

[0116] 1) Design of RNA ligase

[0117] As a template, T4 RNA ligase 2 (NP_049790.1) of which the function is known was used, and 425 similar sequences were obtained by Blastp. It should be noted that, as the analysis conditions of Blastp, Max target sequences was set to 10000, and the value of Expected threshold was set to 1.0E-6 The obtained similar sequences were analyzed, and sequences that were duplicated and sequences that were significantly different in length from the template were removed, thereby screening 172 sequences. These were used as a sequence library. Then, by applying the method employed in Literature 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+-Dependent Enzymes Generated by a Sequence-Based Design Method with or without Phylogenetic Data, Biochemistry 57, 3722-3732.) and Literature 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.), 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) were identified. From the sequence library, only sequences having these motif-like sequences were selected, and finally, data of 21 sequences were obtained. Using the 21 sequences, artificial design was performed, and artificial RNA ligase sequences represented by the amino acid sequences of SEQ ID NOs: 1 to 3, which were named Mutl, Mut2, and Mut3, were designed. Figure 1

[0118] [Table 1]

[0119] Table 1. Amino acid sequence identity (%) with T4 RNA ligase 2 (NP_049790)

[0120] Identity (%) Mut 1 (SEQ ID NO: 1) 93 Mut 2 (SEQ ID NO: 2) 87 Mut 3 (SEQ ID NO: 3) 95 .

[0121] 2) Construction of artificial RNA ligase expression strains​

[0122] The designed RNA ligases were codon-optimized for Escherichia coli, and expression plasmids ligated to Nde I and Bam HI sites of pET-16b (Merck Millipore) were synthesized by Eurofins Genomics. Each of the artificial designed RNA ligase expression plasmids having a base sequence of SEQ ID NO: 4 to 6 as the ORF corresponding to Mutl, Mut2, and Mut3 was transformed into an Escherichia coli BL21(DE3) strain, spread on an LB agar plate containing 100 mg / L ampicillin, and the colonies obtained by culturing at 37°C overnight were isolated to obtain an expression strain of each RNA ligase. In the expression strain, each RNA ligase having a His-tag at the N terminus was expressed.

[0123] 3) Expression of artificial RNA ligases

[0124] Each of the RNA ligase expression strains was spread on an LB agar plate containing 100 mg / L ampicillin and cultured at 37°C overnight. The grown bacterial cells were scraped with a inoculating loop and inoculated into a 500-ml Sakaguchi flask containing 150 mL of LB medium containing 100 mg / L ampicillin. The culture was performed at 37°C with shaking at 120 rpm for 3 hours until the OD 600 became 0.5, and then isopropyl-β-D-thiogalactopyranoside (IPTG) was added at a final concentration of 0.1 mM, and the culture was further continued at 37°C with shaking at 120 rpm for 3 hours. After the culture, the bacterial cells were recovered from 150 mL of the obtained culture solution by centrifugal separation at 8000 rpm for 30 minutes.

[0125] 4) Purification of RNA ligases

[0126] The recovered bacterial cells were suspended in 15 mL of 50 mM Tris-HCl buffer (pH 7.5) containing 250 mM NaCl, 10% sucrose, and 15 mM imidazole. Then, lysozyme (Sigma-Aldrich) was added at a final concentration of 50 μg / mL, and 10% Triton-X100 was added at a final concentration of 0.1%, and the mixture was left on ice for 30 minutes. After 30 minutes, the bacterial cells were disrupted by an ultrasonic disrupter (Insonator 201M) (KUBOTA Corporation), and the bacterial cell debris was removed by centrifugal separation at 10,000 x g for 10 minutes, and the supernatant was used as a soluble fraction.

[0127] The obtained soluble fraction was added to a HisTALON Superflow Cartridge (5 mL) (Takara Bio Inc.) equilibrated with the above-described buffer and subjected to adsorption. The unadsorbed proteins were washed with 30 mL of a 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.

[0128] The elution of the proteins was detected at 280 nm, and the fraction containing the RNA ligase expressed as a His-tag fusion protein was collected. For 4 mL of the eluted fraction, concentration was performed using Amicon Ultra-15 10 kDa (Merck Millipore), the buffer was replaced with 10 mM Tris-HCl (pH 7.5) containing 50 mM KCl, 35 mM (NH4)2SO4, 0.1 mM EDTA, 0.1 mM DTT, and 50% glycerol, and a 50-μL solution was prepared. The purified enzyme was stored at -20°C in the form of this buffer solution. Each of the artificial RNA ligases formed of the amino acid sequences represented by SEQ ID NOs: 1 to 3 was designated Mutl, Mut2, and Mut3, respectively. From 150 mL of the culture solution, 4.2 mg of the artificial RNA ligase Mutl, 1.1 mg of the artificial RNA ligase Mut2, and 0.65 mg of the artificial RNA ligase Mut3 were obtained, respectively.

[0129] [Example 2] Ligation Reaction Using Four Fragments of Each Artificial RNA Ligase

[0130] 1) RNA Ligation Reaction Conditions

[0131] The ligase activities of the three artificial RNA ligases prepared were measured under the following conditions. As the substrates, the four-fragment oligonucleotides of Table 2 were used, and the four-fragment oligonucleotides were ligated to perform a reaction to generate complementary two-fragment nucleotides, as shown in Table 3. Figure 2 As a control, commercially available T4 RNA ligase 2 (New England Biolabs) was used. For convenience, the oligonucleotide generated by the ligation reaction of MOD1-S-12U and MOD1-S-12D was designated as the sense strand, and the oligonucleotide generated by MOD1-A-13U and MOD1-A-13D was designated as the antisense strand.

[0132] A reaction solution 20 μL containing each oligonucleotide at a final concentration of 10 μM, 50 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 1 mM dithiothreitol, 0.4 mM ATP was prepared on ice, and 50 μL of each purified enzyme solution was added to a micro test tube of 200 μL volume, and the reaction was started (initiated) by adding 50 μL of each purified enzyme solution under conditions to become 0.36 μg / mL in final concentration. The reaction was incubated at 25°C by a thermal cycler, and after 1 hour from the start of the reaction, 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.

[0133] [Table 2]

[0134] Table 2. Oligonucleotides used in the ligation reaction of 4 fragments

[0135]

[0136] Pho: indicates a modification using a 5' terminal phosphate group,

[0137] (F): indicates a modification using a 2'-fluoro group,

[0138] (Me): indicates a modification using a 2'-O-methyl group,

[0139] ^: indicates substitution of a phosphate group with a phosphorothioate group.

[0140] 2) Analysis by HPLC

[0141] The oligonucleotide of the ligation product was quantified by HPLC using an ACQUITY UPLC (registered trademark) oligonucleotide BEH C18 column (Waters, 1.7 μm 2.1 x 50 mm). With respect to HPLC, analysis was performed using a column temperature of 60°C, a detection wavelength of 260 nm, an injection amount of 10 μL, a flow rate of 0.4 mL / minute, using eluent A containing 100 mM hexafluoroisopropanol, 8 mM triethylamine, 0.004% phosphoric acid and eluent B containing 10% methanol as the mobile phase, and a linear gradient shown in Table 3. In addition, an oligonucleotide having the same sequence as the product synthesized in each ligation was synthesized, and this was used as a standard to quantify the concentration of the ligation product.

[0142] [Table 3]

[0143] Table 3. Gradient conditions

[0144] Time (min) A％ B％ Curve 0.00 95 5 - 8.00 75 25 6 8.10 10 90 6 10.50 10 90 6 10.60 95 5 6 14.00 95 5 6 .

[0145] 3) Results

[0146] The results are shown in Table 4. For convenience, the oligonucleotide produced by the ligation reaction of MOD1-S-12U and MOD1-S-12D is referred to as the sense strand. In the reaction using the control T4 RNA ligase 2, 0.47 μM of the ligation product of the sense strand was produced by a 1 hour reaction. As for the artificial RNA ligases, the reaction rate increased, and in the case of Mutl, 1.98 μM of the ligation product was produced, which was about 4.2 times higher than in the case of T4 RNA ligase 2. Furthermore, an increase in activity was also observed in the cases of Mut2 and Mut3, and the amount of the ligation product produced by a 1 hour reaction was about 2.2 times higher than in the case of T4 RNA ligase 2. If the amount of the enzyme required to produce 1 μmol of the ligation product by a 1 hour reaction in this reaction is defined as 1 unit (U), then the case of Mutl, which had the highest activity, was calculated to be 5.50 units / mg.

[0147] [Table 4]

[0148] Table 4. Amount of the ligation product and specific activity using the artificial RNA ligases

[0149]

[0150] [Example 3] Ligation reaction of four fragments using the artificial RNA ligase under a high concentration condition of the substrate The ligation reaction using Mutl RNA ligase, which had the highest increase in activity, was performed with an increase in the concentration of the substrate. As the substrates, the four oligonucleotides of Table 5 were used. As a control, a reaction was performed using T4 RNA ligase 2 (New England Biolabs). The reaction was performed as described in Example 2, and the concentration of the ligation product was analyzed by HPLC. Figure 3 (A), the oligonucleotide produced by the ligation reaction of MOD5-S-11U and MOD5-S-11D is referred to as the sense strand, and the oligonucleotide produced by MOD5-A-12U and MOD5-A-12D is referred to as the antisense strand;

[0151] A 10 μL reaction solution containing each oligonucleotide at a final concentration of 500 μM, 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 micro test tube with a volume of 200 μL, and incubated at 25°C by a thermal cycler. One μL of the reaction solution was sampled at 0.5, 1, 2, 4, 6, and 24 hours after the start of the reaction, and the reaction was stopped by adding 49 μL of a 10 mM EDTA solution. The concentration of the ligation product was analyzed by HPLC under the conditions described in Example 2;

[0152] The time course of the production of the ligation product is shown in Figure 3(B). For the control T4 RNA ligase 2, the rate of production of the ligated product on the antisense strand side was slow, and the amounts of the ligated products of the antisense strand and the sense strand after 24 hours of the reaction were 320 μM and 120 μM, respectively, as a part of the oligonucleotide of the four fragments added as a substrate remained in an unreacted state;

[0153] On the other hand, for Mutl having improved ligation activity, the rate of ligation of the oligonucleotides on the sense strand side and the antisense strand side was greatly improved. The amounts of the ligated products of the antisense strand and the sense strand after 24 hours of the reaction were 470 μM and 450 μM, respectively, and the oligonucleotide of the four fragments added as a substrate was substantially completely consumed.

[0154] [Table 5]

[0155] Table 5. Oligonucleotides used in the ligation reaction of the four fragments at a high concentration

[0156] Fragment Name Sequence SEQ ID NO 5' side of sense strand MOD5-S-11U GU(Me)AAC(Me)C(Me)AAGAG 12 3' side of sense strand MOD5-S-11D Pho_U(Me)AU(Me)U(Me)C(Me)C(Me)AU(Me)tt 13 5' side of antisense strand MOD5-A-12U AUGGAAU(Me)ACUCU 14 3' side of antisense strand MOD5-A-12D Pho_UGGUU(Me)ACtt -

[0157] Pho: indicates modification using a 5' end of a phosphate group,

[0158] (Me): indicates modification using a 2'-0-methyl group,

[0159] English lowercase letter: indicates DNA.

[0160] [Example 4] Substrate specificity of artificial RNA ligase

[0161] As Figure 4(A) The change in substrate specificity of artificial RNA ligase was examined in a reaction for ligating two kinds of oligonucleotides, using three kinds of oligonucleotides as substrates, as shown in Fig. 1. As substrates, oligonucleotides in which the vicinity of the ligation site of the ligated oligonucleotide was substituted with modified RNA, specifically, oligonucleotides in which the 2'-position at -2, -1, +1, +2 from the ligation site were modified with a fluorine atom (F) or O-methyl, O-methoxyethyl, or substituted with a hydrogen atom (DNA) as shown in Table 6 were used. Mutl RNA ligase, which showed the most increased activity, and T4 RNA ligase 2 (New England Biolabs) as a control were used. A 20 μL reaction solution containing oligonucleotides at a final concentration of 10 μM, 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 micro test tube with a volume of 200 μL, and incubated at 25°C. Three μL of the reaction solution was sampled 15 minutes after the start of the reaction, and the reaction was stopped by adding 27 μL of a 10 mM EDTA solution. The concentration of the ligated product contained in the reaction solution was analyzed by HPLC under the conditions described in Example 2, and the product was quantified using a ligated product containing no modified nucleic acid as a standard.

[0162] The amount of the ligated product produced by a 15 minute reaction is shown in Figure 4 (B). For the control T4 RNA ligase 2, the amount of the ligated product was greatly reduced to 5 to 50% when oligonucleotides in which the -2 position was substituted with 2'-F, 2'-MOE, DNA, or the -1 position was substituted with 2'-O-Me, 2'-MOE, DNA were used as substrates, compared to when all of the fragments containing natural RNA were used as substrates. On the other hand, for Mutl, in which the ligation activity was increased, the amount of the ligated product was equivalent even when oligonucleotides in which the -2 position was substituted with 2'-F and DNA were used as substrates. Furthermore, for Mutl, the amount of the ligated product was more than twice that of T4 RNA ligase 2 regardless of the oligonucleotides used as substrates.

[0163] [Table 6]

[0164] Table 6. Oligonucleotides used in the ligation reaction of three fragments

[0165]

[0166] Pho: indicates modification using a 5' terminal phosphate group,

[0167] (F): indicates modification using a 2'-fluoro group,

[0168] (Me): Indicates modification using a 2'-O-methyl group.

[0169] (m): indicates modification with 2'-O-methoxyethyl,

[0170] Lowercase letters in English: indicate the use of DNA modification.

[0171] [Example 5] Temperature stability of artificial RNA ligase

[0172] The temperature stability of the artificially designed RNA ligases was evaluated. Mut1 RNA ligase, which showed the greatest enhanced activity, and T4 RNA ligase 2 (New England Biolabs) were used as controls. 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 are designated as antisense strands. 9.2 μL solutions 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 incubated in 200 μL microtubes at 25 °C. After 23 hours of incubation, 0.2 μL of 500 μM substrate was added to each microtube to initiate the ligation reaction. The reaction solution consisted of 10 μM oligonucleotides, 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. A 3 μL sample was taken 15 minutes after the reaction started, and the reaction was stopped by adding 27 μL of 10 mM EDTA. The amount of antisense product was quantified by HPLC under the conditions described in Example 2.

[0173] The amount of ligation product and residual activity generated from the 15-minute reaction are shown in Table 8. For the control T4 RNA ligase 2, the amount of ligation product was significantly reduced to 19% compared to the untreated sample after incubation at 25°C for 23 hours. On the other hand, for Mut1, which showed improved ligation activity, the amount of ligation product was 89% of the untreated sample even after incubation at 25°C for 23 hours, indicating improved stability.

[0174] The activity was also measured after incubation at 37°C for 4 hours. As shown in Table 9, for the control T4 RNA ligase 2, the amount of ligation product could not be determined after incubation at 37°C for 4 hours, and the activity decreased significantly. On the other hand, for Mut1, which showed improved ligation activity, even after incubation at 37°C for 4 hours, the amount of ligation product was 83% of that of the untreated product, indicating improved stability.

[0175] [Table 7]

[0176] Table 7. Sequences used in ligation reactions for temperature stability of 4 fragments

[0177] Use Name Sequence SEQ ID NO 5' side fragment of sense strand RNA1-S-12U AACAGUGUUCUU 17 3' side fragment of sense strand RNA1-S-12D Pho-GCUCUAUAA - 5' side fragment of antisense strand RNA1-A-13U UUAUAGAGCAAGA 28 3' side fragment of antisense strand RNA1-A-13D Pho-ACACUGUUUU 29

[0178] Pho: indicates modification of 5' end with a phosphate group,

[0179] English capital letter: indicates RNA.

[0180] [Table 8]

[0181] Table 8. Product amount and residual activity based on ligation reactions for temperature stability (25°C · 23h)

[0182]

[0183] [Table 9]

[0184] Table 9. Product amount and residual activity based on ligation reactions for temperature stability (37°C · 4h)

[0185] SEQUENCE LISTING <110> Ajinomoto Co., Inc. <120> Ligase mutant <130> PAMA-20897 <150> JP2020-055033 <151> 2020-03-25 <150> JP2021-010565 <151> 2021-01-26 <160> 29 <170> PatentIn version 3.5 <210> 1 <211> 332 <212> PRT <213> Artificial sequence <220> <223> Ligase mutant <400> 1 Met Phe Lys Lys Tyr Ser Ser Leu Glu Asn His Tyr Asn Ser Lys Phe 1 5 10 15 Ile Glu Lys Leu Tyr Ser Leu Gly Leu Thr Gly Gly Glu Trp Val Ala 20 25 30 Arg Glu Lys Ile His Gly Thr Asn Phe Ser Leu Ile Ile Glu Arg Asp 35 40 45 Lys Val Thr Cys Ala Lys Arg Thr Gly Pro Ile Leu Pro Ala Glu Asp 50 55 60 Phe Tyr Gly Tyr Glu Ile Val Leu Lys Lys Tyr Asp Asp Ser Ile Lys 65 70 75 80 Ala Val Gln Asp Ile Met Glu Thr Ser Ala Ala Val Ser Tyr Gln Val 85 90 95 Phe Gly Glu Phe Ala Gly Gly Gly Ile Gln Lys Gly Val Asp Tyr Gly 100 105 110 Glu Lys Asp Phe Tyr Val Phe Asp Ile Ile Val Asn Thr Glu Ser Gly 115 120 125 Asp Val Thr Tyr Val Asp Asp Tyr Met Met Glu Ser Phe Cys Asn Thr 130 135 140 Phe Gly Phe Lys Met Ala Pro Leu Leu Gly Arg Gly Thr Phe Glu Glu 145 150 155 160 Leu Ile Lys Leu Pro Asn Asp Leu Asp Ser Val Val Gln Asp Tyr Asn 165 170 175 Val Thr Val Asp Ala Asp Leu Val Glu Ala Asn Lys Cys Val Phe Asp 180 185 190 Ala Glu Ala Lys Gly Glu Asn Thr Ala Glu Gly Tyr Val Leu Lys Pro 195 200 205 Cys Tyr Pro Lys Trp Leu Pro Asn Gly Asn Arg Val Ala Ile Lys Cys 210 215 220 Lys Asn Ser Lys Phe Ser Glu Lys Lys Lys Ser Asp Lys Pro Ile Lys 225 230 235 240 Ala Lys Val Glu Leu Ser Glu Ala Asp Asn Lys Leu Val Gly Ile Leu 245 250 255 Ala Cys Tyr Val Thr Leu Asn Arg Val Asn Asn Val Ile Ser Lys Ile 260 265 270 Gly Glu Ile Gly Pro Lys Asp Phe Gly Lys Val Met Gly Leu Thr Val 275 280 285 Gln Asp Ile Leu Glu Glu Thr Ser Arg Glu Gly Ile Thr Leu Thr Gln 290 295 300 Ala Asp Asn Pro Ser Leu Ile Lys Lys Glu Leu Val Lys Met Val Gln 305 310 315 320 Asp Val Leu Arg Pro Ala Trp Ile Glu Leu Val Ser 325 330 <210> 2 <211> 330 <212> PRT <213> Artificial Sequence <220> <223> Ligase Mutant <400> 2 Met Phe Lys Lys Tyr Ser Ser Leu Glu Asn His Tyr Asn Ser Lys Phe 1 5 10 15 Ile Glu Lys Leu Tyr Ser Leu Gly Leu Thr Ser Gly Glu Trp Val Ala 20 25 30 Arg Glu Lys Ile His Gly Thr Asn Phe Ser Leu Ile Ile Glu Arg Asp 35 40 45 Lys Val Thr Cys Ala Lys Arg Thr Gly Pro Ile Leu Pro Ala Glu Asp 50 55 60 Phe Tyr Gly Tyr Glu Ile Ile Met Lys Lys Tyr Asp Asp Ala Ile Lys 65 70 75 80 Ala Val Gln Asp Ile Met Glu Thr Ser Ala Ala Val Ser Tyr Gln Val 85 90 95 Phe Gly Glu Phe Ala Gly Gly Gly Ile Gln Lys Gly Val Asp Tyr Gly 100 105 110 Asp Lys Asp Phe Tyr Val Phe Asp Ile Ile Val Thr Thr Glu Asp Gly 115 120 125 Glu Val Ser Tyr Met Asp Asp Tyr Glu Met Glu Ser Phe Cys Asn Thr 130 135 140 Phe Gly Phe Lys Met Ala Pro Leu Leu Gly Arg Gly Ser Phe Glu Asp 145 150 155 160 Leu Ile Lys Leu Pro Asn Asp Leu Asp Ser Val Val Asn Asp Tyr Asn 165 170 175 Val Thr Val Asp Ala Asp Leu Val Glu Ala Asn Lys Cys Val Phe Asp 180 185 190 Ala Glu Ala Lys Gly Glu Asn Thr Ala Glu Gly Tyr Val Leu Lys Pro 195 200 205 Cys Tyr Pro Lys Trp Leu Pro Asn Gly Asn Arg Val Ala Ile Lys Cys 210 215 220 Lys Asn Ser Lys Phe Ser Glu Lys Lys Lys Ser Asp Lys Pro Ile Lys 225 230 235 240 Ala Lys Val Glu Leu Ser Glu Ala Asp Asn Asp Leu Val Gly Ile Leu 245 250 255 Ala Glu Tyr Val Thr Trp Asn Arg Val Ser Asn Val Ile Ser Lys Ile 260 265 270 Gly Glu Val Gly Pro Lys Asp Phe Gly Lys Val Met Gly Leu Thr Val 275 280 285 Gln Asp Ile Leu Glu Glu Ala Ser Arg Glu Gly Ile Thr Leu Thr Gln 290 295 300 Ala Glu Asn Pro Ser Leu Val Lys Lys Glu Leu Val Lys Met Val Met 305 310 315 320 Asp Thr Leu Arg Glu Ala Trp Ile Glu Leu 325 330 <210> 3 <211> 332 <212> PRT <213> Artificial Sequence <220> <223> Ligase Mutant <400> 3 Met Phe Lys Lys Tyr Ser Ser Leu Glu Asn His Tyr Asn Ser Lys Phe 1 5 10 15 Ile Glu Lys Leu Tyr Ser Leu Gly Leu Thr Gly Gly Glu Trp Val Ala 20 25 30 Arg Glu Lys Ile His Gly Thr Asn Phe Ser Leu Ile Ile Ser Asp Asp 35 40 45 Lys Val Thr Cys Ala Lys Arg Ser Gly Pro Ile Leu Pro Ala Glu Asp 50 55 60 Phe Phe Gly Tyr Glu Ile Ile Val Lys Asn Tyr Ala Asp Ala Ile Arg 65 70 75 80 Ala Val Gln Asp Ile Met Glu Thr Ser Ala Val Val Ser Tyr Gln Val 85 90 95 Phe Gly Glu Phe Ala Gly Pro Gly lie Gin Lys Asn Val Asp Tyr Gly 100 105 110 Asp Lys Asp Phe Tyr Val Phe Asp lie lie Val Thr Thr Glu Ser Gly 115 120 125 Asp Val Thr Tyr Val Asp Asp Tyr Met Met Glu Ser Phe Cys Asn Thr 130 135 140 Phe Lys Phe Lys Met Ala Pro Leu Leu Gly Arg Gly Lys Phe Glu Glu 145 150 155 160 Leu lie Lys Leu Pro Asn Asp Leu Asp Ser Val Val Asn Asp Tyr Asn 165 170 175 Phe Thr Val Asp His Ala Gly Leu Val Asp Ala Asn Lys Cys Val Phe 180 185 190 Asn Ala Glu Ala Lys Gly Glu Val Phe Thr Ala Glu Gly Tyr Val Leu 195 200 205 Lys Pro Cys Tyr Pro Ser Trp Leu Arg Asn Gly Asn Arg Val Ala lie 210 215 220 Lys Cys Lys Asn Ser Lys Phe Ser Glu Lys Lys Lys Ser Asp Lys Arg 225 230 235 240 Ile Lys Ala Lys Val Glu Leu Ser Glu Ala Asp Asn Glu Leu Val Gly 245 250 255 Ile Leu Ala Cys Tyr Val Thr Leu Asn Arg Val Asn Asn Val Ile Ser 260 265 270 Lys Ile Gly Glu Val Gly Pro Lys Asp Phe Gly Lys Val Met Gly Leu 275 280 285 Thr Val Gln Asp Ile Leu Glu Glu Ala Ser Arg Glu Gly Ile Thr Leu 290 295 300 Thr Gln Ala Asp Asn Trp Ser Leu Ile Lys Lys Glu Leu Val Lys Met 305 310 315 320 Val Gln Asp Val Val Arg Glu Ala Trp Ile Glu Leu 325 330 <210> 4 <211> 996 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence encoding ligase mutant <400> 4 atgttcaaga aatacagtag ccttgagaat cactacaaca gcaaattcat cgaaaaactc 60 tactccttag gcctcactgg tggtgaatgg gttgcgcgtg agaaaatcca tggtacgaac 120 ttctctctta tcattgaacg cgataaagtg acctgtgcga aacgcacggg acctattctg 180 ccggctgaag acttttatgg gtatgagatt gtgctgaaga aatacgacga ctcgataaaa 240 gccgtacagg atatcatgga aacctctgca gcagtgagct atcaggtctt tggcgaattt 300 gcagggggtg gtattcagaa aggcgtggat tacggagaga aagacttcta cgtgttcgat 360 attatcgtga acacagaatc aggggatgtg acctatgtcg atgactatat gatggagtcg 420 ttttgcaaca cctttggctt caaaatggct ccgctgttag gtcgtggcac gtttgaagag 480 ctgatcaaac tgccgaatga tctggactct gttgtacagg attataacgt caccgttgat 540 gcggacttgg tagaagcgaa taaatgtgtg tttgatgccg aagcgaaagg tgagaatacc 600 gctgaagggt atgttctgaa accgtgctat ccgaaatggc tgccaaatgg caatcgcgtt 660 gccatcaaat gcaagaactc caagtttagc gaaaagaaaa aatcggacaa accgattaaa 720 gcgaaagttg aactgagtga agcagacaat aaactggtag gaatcttggc ctgttatgtt 780 accctcaatc gggtaaacaa cgtgatttcc aagattggcg aaattggtcc caaggatttt 840 ggcaaagtga tgggccttac agttcaggat atactggaag aaacgtcacg tgaaggcatt 900 actctgactc aagccgataa ccctagcctg atcaaaaagg aattagtcaa aatggtgcaa 960 gatgtcttgc gaccagcgtg gattgagcta gtcagt 996 <210> 5 <211> 990 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence encoding a ligase mutant <400> 5 atgtttaaaa agtacagttc cctggaaaac cactacaact ctaagttcat cgagaaactg 60 tatagccttg gactgacttc tggtgaatgg gttgctcggg agaaaattca tggcaccaac 120 ttctccctga ttattgagcg cgataaagtc acgtgtgcaa aacgcacagg tccgatactt 并确保翻译后的内容符合专利文本的专业性和准确性要求。180 cctgccgaag atttctatgg ctacgagatc atcatgaaaa agtacgacga tgcgatcaaa 240 gccgtgcagg atattatgga gacaagcgca gcagtgagtt atcaggtctt tggcgaattt 300 gcgggtggag gaatccagaa aggggtagat tatggcgaca aagacttcta tgtctttgac 360 atcatcgtta ctacggaaga tggcgaagtt tcgtacatgg atgattacga gatggaatcc 420 ttctgtaaca cctttgggtt caaaatggct ccgttattag gtcgtggtag ctttgaggat 480 ctgattaaac tcccaaacga cttggatagc gtagtgaatg actataacgt gaccgtggat 540 gcggatctag tggaagccaa caaatgcgtg tttgatgcgg aagccaaagg cgaaaacacg 600 gctgaaggct atgtcctcaa gccatgctat ccgaaatggt tgcccaatgg caatcgagtg 660 gcaattaagt gcaaaaattc gaaatttagc gaaaagaaaa aaagcgataa gcctatcaaa 720 gcgaaagtag agctgtcaga agccgataat gacttggttg gtattctggc cgagtatgtt 780 acctggaatc gcgtgtctaa cgttatctcg aaaattgggg aagtaggtcc gaaagacttt 840 ggcaaagtta tgggcttaac cgtccaagac atactggaag aagcgtcacg tgaaggtatt 900 accctgaccc aagcggaaaa tccgagtctg gtgaagaaag aactcgtcaa aatggtgatg 960 gatacgctgc gtgaagcgtg gattgaactg 990 <210> 6 <211> 996 <212> DNA <213> Artificial Sequence <220> <223> Nucleotide sequence encoding ligase mutant <400> 6 atgttcaaga aatatagcag cctcgagaat cactacaact ctaagttcat agagaaactg 60 tattctctgg gactcacagg tggtgaatgg gtagctcggg agaaaatcca tggcacgaat 120 ttttcgctga tcatttccga tgataaagtc acctgtgcaa aacgcagtgg tccaatactt 180 ccagcagagg acttttttgg gtacgaaatc atcgtcaaaa attacgccga tgcgattcga 240 gcagtccaag acattatgga aactagtgcg gttgtttcgt atcaggtgtt cggtgagttt 300 gccggacctg gcattcagaa aaacgtcgac tatggcgata aagacttcta cgtgtttgat 360 atcattgtta ccacggaatc cggagatgtc acctatgtag atgactacat gatggagagc 420 ttctgcaata cgttcaaatt caaaatggct ccgttgttag gtcgtgggaa atttgaagaa 480 ctgatcaaac tgccgaatga cctggatagt gtagtgaacg actacaactt taccgtggat 540 catgcgggct tagttgatgc caacaaatgc gtgtttaatg ccgaagcgaa aggcgaagtg 600 tttaccgctg aaggctatgt actgaaaccg tgttatccgt catggttgcg taacggtaat 660 cgtgtggcca ttaaatgcaa gaacagcaaa ttcagcgaga aaaagaaatc cgataagcgc 720 atcaaagcga aagtggaact gtctgaagcg gataacgagc ttgtaggcat tttagcgtgt 780 tatgtgactc taaatcgcgt gaacaacgtg atctcgaaaa ttggcgaagt tggccccaaa 840 gactttggga aagttatggg tctgacggtt caggacattc tggaagaagc ctcacgcgaa 900 ggtattacct tgacacaggc ggataattgg agcctgatta agaaggaact cgtcaaaatg 960 gtgcaagatg ttgtccgtga agcatggatc gaactg 996 <210> 7 <211> 12 <212> RNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 7 aacaguguuc uu 12 <210> 8 <211> 13 <212> RNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 8 uuauagagca aga 13 <210> 9 <211> 10 <212> RNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 9 acacuguuuu 10 <210> 10 <211> twenty one <212> RNA <213> Artificial sequence <220> <223> Oligonucleotides <400> 10 aacaguguuc uugcucuaua a 21 <210> 11 <211> twenty three <212> RNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 11 uuauagagca agaacacugu uuu 23 <210> 12 <211> 11 <212> RNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 12 guaaccaaga g 11 <210> 13 <211> 10 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 13 uauuccautt 10 <210> 14 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 14 auggaauacu cu 12 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 15 guaaccaaga guauuccaut t 21 <210> 16 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 16 auggaauacu cuugguuact t 21 <210> 17 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 17 aacaguguuc uu 12 <210> 18 <211> 23 <212> RNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 18 uuauagagca agaacacugu uuu 23 <210> 19 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 19 aacaguguuc uu 12 <210> 20 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 20 aacaguguuc uu 12 <210> 21 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 21 aacaguguuc uu 12 <210> 22 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 22 aacaguguuc tu 12 <210> 23 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 23 aacaguguuc uu 12 <210> 24 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 24 aacaguguuc uu 12 <210> 25 <211> 12 <212> RNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 25 aacaguguuc uu 12 <210> 26 <211> 12 <212> DNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 26 aacaguguuc ut 12 <210> 27 <211> 21 <212> RNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 27 aacaguguuc uugcucuaua 21 <210> 28 <211> 13 <212> RNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 28 uuauagagca aga 13 <210> 29 <211> 10 <212> RNA <213> Artificial Sequence <220> <223> Oligonucleotide <400> 29 acacuguuuu 10

Claims

1. The ligase mutants of (1), (2), or (3) below: (1) A ligase mutant consisting of an amino acid sequence of SEQ ID NO:1 that may have a histidine tag at the N-terminus and has nucleic acid ligation activity; (2) A ligase mutant consisting of an amino acid sequence of SEQ ID NO:2 that may have a histidine tag at the N-terminus and possessing nucleic acid ligation activity; or (3) A ligase mutant consisting of an amino acid sequence of SEQ ID NO:3 that may have a histidine tag at the N-terminus and has nucleic acid ligation activity.

2. The ligase mutant according to claim 1, wherein, Nucleic acids are single-stranded or double-stranded RNA. The single-stranded or double-stranded RNA may contain DNA and / or modified nucleic acids.

3. A method for manufacturing a nucleic acid product, wherein, The method includes: ligating nucleic acid materials in the presence of the ligase mutant as described in claim 1 or 2 to generate a nucleic acid product. Nucleic acid materials are selected from single-stranded RNA, double-stranded RNA, and mixtures thereof.

4. The method according to claim 3, wherein, The nucleic acid material consists of four or more single-stranded RNA molecules.

5. The method according to claim 3, wherein, Nucleic acid products contain complementary portions of 12 to 27 bases in length.

6. The method according to claim 3, wherein, The single-stranded RNA, double-stranded RNA, and mixtures thereof contain DNA and / or modified nucleic acids.

7. The method according to claim 3, wherein, The concentration of the nucleic acid material is above 1 μM.

8. The method according to claim 3, wherein, The nucleic acid product is siRNA.

9. A polynucleotide encoding the ligase mutant of claim 1 or 2.

10. An expression vector comprising the polynucleotide of claim 9.

11. A transforming microorganism comprising an expression unit, The expression unit comprises: a polynucleotide encoding the ligase mutant of claim 1 or 2, and a promoter operatively linked thereto.

12. A method for manufacturing a ligase mutant, the method comprising: The ligase mutant is generated using a transforming microorganism containing an expression unit comprising a polynucleotide encoding a ligase mutant of claim 1 or 2 and a promoter operatively linked thereto.

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