Ligase variant

Abstract

The present invention provides a ligase variant having excellent properties. More specifically, the present invention provides: a ligase variant (i) which comprises a mutated amino acid sequence having a structure such that a mutation of an amino acid residue selected from the group consisting of amino acid residues located at Positions 3, 18, 19, 25, 36, 39, 54, 56, 64, 72, 76, 79, 82, 92, 103, 104, 105, 114, 116, 125, 129, 157, 158, 196, 207, 219, 226, 231, 235, 239, 241, 279, 305, 311, 313, and 318, and combinations thereof is included in the amino acid sequence encoding wild-type T4 RNA ligase 2 and (ii) which has an improved nucleic acid linking activity compared with an enzyme comprising wild-type T4 RNA ligase 2; and others.

Description

Ligase mutants

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

[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 capable of infecting 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 capable of using 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.

[0003] International Publication No. WO 2008 / 094599 JP 2021-153573 A International Publication No. WO 2024 / 134502 International Publication No. WO 2024 / 138200

[0004] [[online]], INTERNET, NCBI Protein Database, August 13, 2018, NP_049790, Search date: February 27, 2020, URL, https: / / www.ncbi.nlm.nih.gov / protein / NP_049790Chauleau, 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.Nandakumar, J., Shuman, S., Lima, C. D. (2006). RNA ligase structures reveal the basis for RNA specificity and conformational changes that drive ligation forward. Cell, 6;127(1):71 - 84.Ho, C. K., & 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.Nandakumar, J., Ho, C. K., Lima, C. D., 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.Nandakumar, J. & Shuman, S. (2004). How an RNA ligase discriminates RNA versus DNA damage. Mol. Cell., 22;16(2):211-21. 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. Yin, S. , Ho, C. K. , Shuman, S. (2003). Structure-function analysis of T4 RNA ligase 2. J. Biol. Chem. , 16;278(20):17601-8. 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. ;

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

[0006] As a result of extensive research, the present inventors have succeeded in developing a ligase mutant with excellent properties, thereby completing the present invention.

[0007] That is, the present invention is as follows. [1] A ligase mutant comprising a mutant amino acid sequence containing a mutation in an amino acid residue selected from the group consisting of positions 3, 18, 19, 25, 36, 39, 54, 56, 64, 72, 76, 79, 82, 92, 103, 104, 105, 114, 116, 125, 129, 157, 158, 196, 207, 219, 226, 231, 235, 239, 241, 279, 305, 311, 313, and 318, and combinations thereof, based on the amino acid sequence encoding wild-type T4 RNA ligase 2, and the ligase mutant has improved nucleic acid ligation activity compared to an enzyme consisting of wild-type T4 RNA ligase 2. [2] The ligase mutant of [1], wherein the ligase mutant comprises the mutation in the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 1. [3] The ligase mutant of [1] or [2], which is any of the following: (A1) a mutant amino acid sequence comprising mutations of amino acid residues selected from the group consisting of K3, E18, K19, L25, I36, T39, K54, T56, D64, L72, A76, I79, V82, V92, P103, G104, I105, K114, F116, T125, D129, K157, F158, A196, V207, G219, C226, F231, K235, K239, I241, P279, T305, S311, I313, and V318 in the amino acid sequence of SEQ ID NO: 1, and combinations thereof; (B1) an amino acid sequence in which the mutant amino acid sequence contains additional mutations of 1 to 30 amino acid residues selected from the group consisting of substitution, deletion, insertion, and addition of amino acid residues (wherein the mutation (A1) in the modified amino acid sequence is maintained); or (C1) an amino acid sequence having 90% or more identity to the mutant amino acid sequence (wherein the mutation (A1) in the modified amino acid sequence is maintained); and a ligase mutant having improved nucleic acid ligation activity compared to an enzyme consisting of the amino acid sequence of SEQ ID NO: 1.[4] The ligase mutant of [3], wherein the mutation (A1) in the mutant amino acid sequence is selected from the group consisting of K3, E18, K19, K54, L72, P103, and F158, and combinations thereof. [5] The ligase mutant of [3] or [4], wherein the mutation (A1) in the mutant amino acid sequence is one of the following: (1) a mutation at K3: K3Q; (2) a mutation at E18: E18K; (3) a mutation at K19: K19R; (4) a mutation at L25: L25H; (5) a mutation at I36: I36V; (6) a mutation at T39: T39G or T39M; (7) a mutation at K54: K54R; (8) a mutation at T56: T56S; (9) a mutation at D64: D64K; (10) a mutation at L72: L72P; (11) a mutation at A76: A76V; (12) a mutation at I79: I79V; (13) a mutation at V82: V82I; (14) a mutation at V92. (15) P103 mutation P103C or P103D; (16) G104 mutation G104N or G104Q; (17) I105 mutation I105L; (18) K114 mutation K114R; (19) F116 mutation F116L; (20) T125 mutation T125A; (21) D129 mutation D129V; (22) T157 mutation T157S; (23) F158 mutation F158Y; (24) A196 mutation A196T; (25) V207 mutation V207I; (26) G219 mutation G219K; (27) C226 mutation C226S; (28) F231 mutation F231L; (29) K235 mutation K235I; (30) K239 mutation K239E; (31) I241 mutation I241L; (32) P279 mutation P279R; (33) T305 mutation T305S; (34) S311 mutation S311R; (35) I313 mutation I313V; and (36) V318 mutation V318L.[6] The ligase mutant of [1] or [2], which is the following: (A2) a mutant amino acid sequence comprising mutations of amino acid residues selected from the group consisting of K3, E18, K19, L25, I36, T39, K54, T56, D64, L72, D76, I79, V82, V92, G103, G104, I105, K114, F116, T125, D129, T157, F158, A195, V205, G217, C224, F229, K233, K237, I239, P277, T303, S309, I311, and V316 in the amino acid sequence of SEQ ID NO: 2, and combinations thereof; (B2) an amino acid sequence in which the mutant amino acid sequence contains additional mutations of 1 to 30 amino acid residues selected from the group consisting of substitution, deletion, insertion, and addition of amino acid residues (wherein the mutation (A2) in the modified amino acid sequence is maintained); or (C2) an amino acid sequence having 90% or more identity to the mutant amino acid sequence (wherein the mutation (A2) in the modified amino acid sequence is maintained); and a ligase mutant having improved nucleic acid ligation activity compared to an enzyme consisting of the amino acid sequence of SEQ ID NO: 2.[7] The ligase mutant of [6], wherein the mutation (A2) in the mutant amino acid sequence is one of the following: (1) a mutation at K3: K3Q; (2) a mutation at E18: E18K; (3) a mutation at K19: K19R; (4) a mutation at L25: L25H; (5) a mutation at I36: I36V; (6) a mutation at T39: T39G or T39M; (7) a mutation at K54: K54R; (8) a mutation at T56: T56S; (9) a mutation at D64: D64K; (10) a mutation at L72: L72P; (11) a mutation at D76: D76V; (12) a mutation at I79: I79V; (13) a mutation at V82: V82I; (14) a mutation at V92. (15) G103 mutation G103C or G103D; (16) G104 mutation G104N or G104Q; (17) I105 mutation I105L; (18) K114 mutation K114R; (19) F116 mutation F116L; (20) T125 mutation T125A; (21) D129 mutation D129V; (22) T157 mutation T157S; (23) F158 mutation F158Y; (24) A195 mutation A195T; (25) V205 mutation V205I; (26) G217 mutation G217K; (27) a mutation at C224: C224S; (28) a mutation at F229: F229L; (29) a mutation at K233: K233I; (30) a mutation at K237: K237E; (31) a mutation at I239: I239L; (32) a mutation at P277: P277R; (33) a mutation at T303: T303S; (34) a mutation at S309: S309R; (35) a mutation at I311: I311V; and (36) a mutation at V316: V316L. [8] The ligase mutant of any of [1] to [7], wherein the nucleic acid is a single-stranded or double-stranded nucleic acid that may contain a modified nucleic acid. [9] The ligase mutant of [8], wherein the modified nucleic acid is a 2'-modified nucleic acid.

[10] The 2'-modified nucleic acid is 2'-O-C. １～６ Alkyloxy-C １～６ Alkyl, or 2'-O-C １～６

[11] The ligase mutant of any one of [1] to

[10] , wherein the nucleic acid ligation activity is the activity of ligating a nucleic acid comprising the following first nucleic acid and second nucleic acid: (A) the first nucleic acid; (A1) a single-stranded nucleic acid comprising a 2'-OH or 2'-H moiety as the 5'-terminal nucleotide residue; (A2) a single-stranded nucleic acid comprising a 2'-O-C moiety as the 5'-terminal nucleotide residue; １～６ Alkyloxy-C １～６ Alkyl, or 2'-O-C １～６ (A3) a double-stranded nucleic acid comprising the single-stranded nucleic acid of (A1) or (A2) above and a complementary strand comprising a portion complementary thereto; and (B) a second nucleic acid: (B1) a single-stranded nucleic acid comprising a 2'-OH or 2'-H moiety as the 3'-terminal nucleotide residue; (B2) a 2'-O-C moiety as the 3'-terminal nucleotide residue; １～６ Alkyloxy-C １～６ Alkyl, or 2'-O-C １～６ (B3) a double-stranded nucleic acid comprising the single-stranded nucleic acid of (B1) or (B2) above and a complementary strand comprising a portion complementary thereto.

[12] A method for producing a nucleic acid product, comprising contacting two or more nucleic acid materials with the ligase mutant of any of [1] to

[11] to produce a nucleic acid product in which the two or more nucleic acid materials are ligated, wherein the nucleic acid materials are selected from the group consisting of single-stranded nucleic acid materials, double-stranded nucleic acid materials, and mixtures thereof.

[13] The method of

[12] , wherein the concentration of the nucleic acid materials is 1 μM or more.

[14] The method of

[13] , wherein the nucleic acid product is a single-stranded or double-stranded nucleic acid which may contain a modified nucleic acid.

[15] A polynucleotide encoding the ligase mutant of any of [1] to

[11] .

[16] An expression vector comprising the polynucleotide of

[15] .

[17] A transformed microorganism containing an expression unit comprising a polynucleotide encoding the ligase mutant of any one of [1] to

[11] and a promoter operably linked thereto.

[0008] According to the present invention, a nucleic acid product can be efficiently produced from a nucleic acid material.

[0009] FIG. 1 shows the amino acid sequences of SEQ ID NO: 1 and SEQ ID NO: 2. The amino acid sequence of SEQ ID NO: 1 is the amino acid sequence of T4 RNA ligase 2 (NP_049790) (total length 334 aa). The amino acid sequence of SEQ ID NO: 2 is the same as SEQ ID NO: 1 described in JP 2021-153573 A (T4 RNA ligase 2-derived sequence / total length 332 aa). FIG. 2 shows an alignment of the amino acid sequences of SEQ ID NO: 1 (T4Rnl2) and SEQ ID NO: 2 (Anc2) (sequence identity 93.4%).

[0010] The present invention provides a ligase mutant comprising a mutant amino acid sequence containing mutations in amino acid residues selected from the group consisting of positions 3, 18, 19, 25, 36, 39, 54, 56, 64, 72, 76, 79, 82, 92, 103, 104, 105, 114, 116, 125, 129, 157, 158, 196, 207, 219, 226, 231, 235, 239, 241, 279, 305, 311, 313, and 318, and combinations thereof, relative to the amino acid sequence encoding wild-type T4 RNA ligase 2, and the ligase mutant has improved nucleic acid ligation activity compared to an enzyme consisting of wild-type T4 RNA ligase 2.

[0011] Wild-type T4 RNA ligase 2 is the RNA ligase 2 of the T4 bacteriophage capable of infecting Escherichia (NP_049790 / SEQ ID NO: 1). Whether a ligase mutant contains a mutant amino acid sequence containing the above-mentioned amino acid residue mutations can be determined by whether the amino acid sequence encoding the ligase mutant shows a significant percent identity to the amino acid sequence encoding wild-type T4 RNA ligase 2. Such a significant percent identity is 50% or greater. A significant percent identity may also be 55% or greater, 60% or greater, 65% or greater, 70% or greater, 75% or greater, 80% or greater, 85% or greater, 90% or greater, 91% or greater, 92% or greater, 93% or greater, 94% or greater, 95% or greater, 96% or greater, 97% or greater, 98% or greater, or 99% or greater. Examples of ligases derived from wild-type T4 RNA ligase 2 that show such a significant percent identity include mutant ligases containing the amino acid sequences of SEQ ID NOs: 2 and 3 described in JP 2021-153573 A, mutant ligases containing the amino acid sequence of SEQ ID NO: 2 described in WO 2024 / 134502, and mutant ligases containing the amino acid sequence of SEQ ID NO: 14 described in WO 2024 / 138200.

[0012] The percent identity of the amino acid sequence can be calculated using the software GENETYX Ver. 13.1.1 from Genetics Corporation, using the full length of the polypeptide portion encoded by the ORF, by performing Muscle alignment, ClustalW alignment, or Multiple sequence alignment, and then using the numerical value calculated with the Gaps are take into account setting.

[0013] The ligase mutants of the present invention include mutant amino acid sequences containing mutations at amino acid residues selected from the group consisting of positions 3, 18, 19, 25, 36, 39, 54, 56, 64, 72, 76, 79, 82, 92, 103, 104, 105, 114, 116, 125, 129, 157, 158, 196, 207, 219, 226, 231, 235, 239, 241, 279, 305, 311, 313, and 318, and combinations thereof, relative to the amino acid sequence encoding wild-type T4 RNA ligase 2. The positions of these amino acid residues relative to the amino acid sequence encoding wild-type T4 RNA ligase 2 will be apparent to those skilled in the art. Those skilled in the art can identify a ligase of interest by comparing the amino acid sequence alignment with wild-type T4 RNA ligase 2 (NP_049790 / SEQ ID NO: 1). The amino acid sequence alignment can be performed simultaneously with the calculation of percent identity described above.

[0014] In the present invention, the mutated amino acid residue is a desired natural α-amino acid residue different from the original amino acid residue, such as 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).

[0015] In certain embodiments, the mutations may be at positions selected from the group consisting of positions 3, 18, 19, 54, 72, 103, and 158, and combinations thereof.

[0016] In another specific embodiment, the mutations may be: (1) a mutation at position 3, 3Q; (2) a mutation at position 18, 18K; (3) a mutation at position 19, 19R; (4) a mutation at position 25, 25H; (5) a mutation at position 36, I36V; (6) a mutation at position 39, 39G or 39M; (7) a mutation at position 54, 54R; (8) a mutation at position 56, 56S; (9) a mutation at position 64, 64K; (10) a mutation at position 72, 72P; (11) a mutation at position 76, 76V; (12) a mutation at position 79, 79V; (13) a mutation at position 82, 82I; (14) a mutation at position 92, 92A; (15) a mutation at position 103, 103C or 103D; (16) Mutation at position 104: G104N or G104Q; (17) Mutation at position 105: 105L; (18) Mutation at position 114: 114R; (19) Mutation at position 116: 116L; (20) Mutation at position 125: 125A; (21) Mutation at position 129: 129V; (22) Mutation at position 157: 157S; (23) Mutation at position 158: 158Y; (24) Mutation at position 196: 196T; (25) Mutation at position 207: 207I; (26) Mutation at position 219: 219K; (27) Mutation at position 226: 226S; (28) Mutation at position 231: 231L; (29) Mutation at position 235: 235I; (30) mutation at position 239, 239E; (31) mutation at position 241, 241L; (32) mutation at position 279, 279R; (33) mutation at position 305, 305S; (34) mutation at position 311, 311R; (35) mutation at position 313, 313V; and (36) mutation at position 318.

[0017] In certain embodiments, the ligase mutant may be a ligase mutant comprising the above-described mutation in the following amino acid sequence: (a) the amino acid sequence encoding wild-type T4 RNA ligase 2 (NP_049790 / SEQ ID NO: 1); or (b) a mutant amino acid sequence derived from the amino acid sequence encoding wild-type T4 RNA ligase 2. Such mutant amino acid sequences include, for example, amino acid sequences encoding mutant ligases that exhibit a significant percent identity as described above.

[0018] In one embodiment, the ligase mutant may be: (A) a mutant amino acid sequence comprising a mutation in an amino acid residue selected from the group consisting of positions 3, 18, 19, 25, 36, 39, 54, 56, 64, 72, 76, 79, 82, 92, 103, 104, 105, 114, 116, 125, 129, 157, 158, 196, 207, 219, 226, 231, 235, 239, 241, 279, 305, 311, 313, and 318, and combinations thereof, in the amino acid sequence of wild-type T4 RNA ligase 2 or a mutant T4 RNA ligase 2 derived therefrom, based on the amino acid sequence encoding the wild-type T4 RNA ligase 2; (B) an amino acid sequence comprising, in the mutant amino acid sequence, additional mutations of 1 to 30 amino acid residues selected from the group consisting of amino acid substitution, deletion, insertion, and addition (wherein the mutation (A) in the modified amino acid sequence is maintained); or (C) an amino acid sequence having 90% or more identity to the mutant amino acid sequence (wherein the mutation (A) in the modified amino acid sequence is maintained); and a ligase mutant having improved nucleic acid ligation activity relative to wild-type T4 RNA ligase 2 or a mutant T4 RNA ligase 2 derived therefrom. Examples of mutant T4 RNA ligase 2 derived from wild-type T4 RNA ligase 2 include mutant ligases exhibiting significant % identity as described above. The amino acid residue at the above position may be an amino acid residue selected as described above. Furthermore, the amino acid residue after mutation at the above position may be one described above.

[0019] In one embodiment, the ligase mutant may be: (A1) a mutant amino acid sequence comprising a mutation of an amino acid residue selected from the group consisting of K3, E18, K19, L25, I36, T39, K54, T56, D64, L72, A76, I79, V82, V92, P103, G104, I105, K114, F116, T125, D129, K157, F158, A196, V207, G219, C226, F231, K235, K239, I241, P279, T305, S311, I313, and V318 in the amino acid sequence of SEQ ID NO: 1, and a combination thereof; (B1) an amino acid sequence in which the mutant amino acid sequence contains additional mutations of 1 to 30 amino acid residues selected from the group consisting of substitution, deletion, insertion, and addition of amino acid residues (wherein the mutation (A1) in the modified amino acid sequence is maintained); or (C1) an amino acid sequence having 90% or more identity to the mutant amino acid sequence (wherein the mutation (A1) in the modified amino acid sequence is maintained); and a ligase mutant having improved nucleic acid ligation activity compared to an enzyme consisting of the amino acid sequence of SEQ ID NO: 1.

[0020] In certain embodiments, the mutation (A1) in the mutant amino acid sequence may be selected from the group consisting of K3, E18, K19, K54, L72, P103, and F158, and combinations thereof.

[0021] In another specific embodiment, the mutation (A1) in the mutant amino acid sequence may be: (1) a mutation at K3, K3Q; (2) a mutation at E18, E18K; (3) a mutation at K19, K19R; (4) a mutation at L25, L25H; (5) a mutation at I36, I36V; (6) a mutation at T39, T39G or T39M; (7) a mutation at K54, K54R; (8) a mutation at T56, T56S; (9) a mutation at D64, D64K; (10) a mutation at L72, L72P; (11) a mutation at A76, A76V; (12) a mutation at I79, I79V; (13) a mutation at V82, V82I; (14) a mutation at V92, V92A; (15) P103 mutation P103C or P103D; (16) G104 mutation G104N or G104Q; (17) I105 mutation I105L; (18) K114 mutation K114R; (19) F116 mutation F116L; (20) T125 mutation T125A; (21) D129 mutation D129V; (22) T157 mutation T157S; (23) F158 mutation F158Y; (24) A196 mutation A196T; (25) V207 mutation V207I; (26) G219 mutation G219K; (27) C226 mutation (28) F231 mutation F231L; (29) K235 mutation K235I; (30) K239 mutation K239E; (31) I241 mutation I241L; (32) P279 mutation P279R; (33) T305 mutation T305S; (34) S311 mutation S311R; (35) I313 mutation I313V; and (36) V318 mutation V318L.

[0022] In another embodiment, the ligase mutant may be: (A2) a mutant amino acid sequence comprising a mutation of an amino acid residue selected from the group consisting of K3, E18, K19, L25, I36, T39, K54, T56, D64, L72, D76, I79, V82, V92, G103, G104, I105, K114, F116, T125, D129, T157, F158, A195, V205, G217, C224, F229, K233, K237, I239, P277, T303, S309, I311, and V316 in the amino acid sequence of SEQ ID NO: 2, and combinations thereof; (B2) an amino acid sequence in which the mutant amino acid sequence contains additional mutations of 1 to 30 amino acid residues selected from the group consisting of substitution, deletion, insertion, and addition of amino acid residues (wherein the mutation (A2) in the modified amino acid sequence is maintained); or (C2) an amino acid sequence having 90% or more identity to the mutant amino acid sequence (wherein the mutation (A2) in the modified amino acid sequence is maintained); and a ligase mutant having improved nucleic acid ligation activity compared to an enzyme consisting of the amino acid sequence of SEQ ID NO: 2.

[0023] The amino acid sequence of SEQ ID NO: 2 is the same as the amino acid sequence of SEQ ID NO: 2 described in JP 2021-153573 A and shows 93% identity to the amino acid sequence of the known T4 RNA ligase 2 (NP_049790). The amino acid sequence of SEQ ID NO: 2 consists of a total of 332 amino acid residues.

[0024] In certain embodiments, the mutation (A2) in the mutant amino acid sequence may be selected from the group consisting of K3, E18, K19, L25, I36, T39, K54, T56, D64, D76, I79, V82, V92, G103, G104, I105, K114, F116, T125, D129, T157, F158, A195, V205, G217, C224, F229, K233, K237, I239, P277, T303, S309, I311, and V316, and combinations thereof.

[0025] In another specific embodiment, the mutation (A2) in the mutant amino acid sequence may be: (1) a mutation at K3, K3Q; (2) a mutation at E18, E18K; (3) a mutation at K19, K19R; (4) a mutation at L25, L25H; (5) a mutation at I36, I36V; (6) a mutation at T39, T39G or T39M; (7) a mutation at K54, K54R; (8) a mutation at T56, T56S; (9) a mutation at D64, D64K; (10) a mutation at L72, L72P; (11) a mutation at D76, D76V; (12) a mutation at I79, I79V; (13) a mutation at V82, V82I; (14) a mutation at V92, V92A; (15) G103 mutation G103C or G103D; (16) G104 mutation G104N or G104Q; (17) I105 mutation I105L; (18) K114 mutation K114R; (19) F116 mutation F116L; (20) T125 mutation T125A; (21) D129 mutation D129V; (22) T157 mutation T157S; (23) F158 mutation F158Y; (24) A195 mutation A195T; (25) V205 mutation V205I; (26) G217 mutation G217K; (27) C224 mutation (28) F229 mutation F229L; (29) K233 mutation K233I; (30) K237 mutation K237E; (31) I239 mutation I239L; (32) P277 mutation P277R; (33) T303 mutation T303S; (34) S309 mutation S309R; (35) I311 mutation I311V; and (36) V316 mutation V316L.

[0026] The relationship between SEQ ID NO: 1 and SEQ ID NO: 2 will be explained. SEQ ID NO: 1 (also referred to as "T4Rnl2" in the Examples) is an amino acid sequence encoding wild-type T4 RNA ligase 2. SEQ ID NO: 2 (also referred to as "Anc2" in the Examples) is a mutant ligase derived from wild-type T4 RNA ligase 2 (approximately 93% identical to wild-type T4 RNA ligase 2). The amino acid sequence of SEQ ID NO: 1 (T4Rnl2) corresponds to the amino acid sequence of SEQ ID NO: 2 (Anc2) in which a histidine residue (H) is inserted between the amino acid residues at positions 180 and 181, and a phenylalanine residue (F) is inserted between the amino acid residues at positions 199 and 200. Thus, the amino acid residues at positions 181 to 199 in the amino acid sequence of SEQ ID NO: 2 (Anc2) correspond to the amino acid residues at positions 182 to 200 in the amino acid sequence of SEQ ID NO: 1 (T4Rnl2), and the amino acid residues at positions 200 to 332 in the amino acid sequence of SEQ ID NO: 2 (Anc2) correspond to the amino acid residues at positions 202 to 334 in the amino acid sequence of SEQ ID NO: 1 (T4Rnl2) (Table A).

[0027]

[0028] The combination of amino acid residues in (A), (A1), and (A2) may be 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, or 28. Such combinations may also be 28 or less, 27 or less, 26 or less, 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, 13 or less, 12 or less, 11 or less, 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. Such combinations may further include 2 to 28, 3 to 28, 4 to 28, 5 to 28, 6 to 28, 7 to 28, or 8 to 28, and the upper and lower limits can be appropriately modified or set to the above-mentioned numbers.

[0029] Examples of combinations of amino acid residues in (A) include combinations of amino acid residues at positions described below. Examples of combinations of amino acid residues in (A1) and (A2) can also be set so as to follow the combinations of amino acid residues corresponding to the positions described below.

[0030] Combinations of amino acid residues at position 3 and other positions include, for example, combinations of amino acid residues at positions 3 and 54; combinations of amino acid residues at positions 3 and 158; combinations of amino acid residues at positions 3 and 233; combinations of amino acid residues at positions 3 and 235; combinations of amino acid residues at positions 3, 54 and 158; combinations of amino acid residues at positions 3, 54 and 233; combinations of amino acid residues at positions 3, 54 and 235; combinations of amino acid residues at positions 3, 158 and 233; combinations of amino acid residues at positions 3, 158 and 235; combinations of amino acid residues at positions 3, 54, 158 and 233; combinations of amino acid residues at positions 3, 54, 158 and 235; combinations of amino acid residues at positions 3, 54, 158 and 233; and combinations of amino acid residues at positions 3, 54, 158 and 235.

[0031] Combinations of amino acid residues at position 18 and other positions include, for example, the combination of amino acid residues at positions 18 and 54; the combination of amino acid residues at positions 18 and 158; the combination of amino acid residues at positions 18 and 233; the combination of amino acid residues at positions 18, 54 and 158; the combination of amino acid residues at positions 18, 54 and 233; the combination of amino acid residues at positions 18, 158 and 233; and the combination of amino acid residues at positions 18, 54, 158 and 233.

[0032] Combinations of amino acid residues at position 19 and other positions include, for example, the combination of amino acid residues at positions 19 and 54; the combination of amino acid residues at positions 19 and 158; the combination of amino acid residues at positions 19 and 233; the combination of amino acid residues at positions 19 and 235; the combination of amino acid residues at positions 19, 54 and 158; the combination of amino acid residues at positions 19, 54 and 233; the combination of amino acid residues at positions 19, 158 and 233; the combination of amino acid residues at positions 19, 54 and 235; the combination of amino acid residues at positions 19, 54, 158 and 233; the combination of amino acid residues at positions 19, 54, 158 and 235.

[0033] Combinations of amino acid residues at position 36 and other positions include, for example, the combination of amino acid residues at positions 3 and 36; the combination of amino acid residues at positions 18 and 36; the combination of amino acid residues at positions 19 and 36; the combination of amino acid residues at positions 36 and 39; the combination of amino acid residues at positions 36 and 54; the combination of amino acid residues at positions 36 and 72; the combination of amino acid residues at positions 36 and 103; the combination of amino acid residues at positions 36 and 105; the combination of amino acid residues at positions 36 and 158; the combination of amino acid residues at positions 36 and 233; the combination of amino acid residues at positions 36 and 235; the combination of amino acid residues at positions 36 and 277; and the combination of amino acid residues at positions 36 and 279.

[0034] Combinations of amino acid residues at position 39 and other positions include, for example, the combination of amino acid residues at positions 39 and 54; the combination of amino acid residues at positions 39 and 82; the combination of amino acid residues at positions 39 and 116; the combination of amino acid residues at positions 39 and 125; the combination of amino acid residues at positions 39, 54 and 82; the combination of amino acid residues at positions 39, 54 and 116; the combination of amino acid residues at positions 39, 54 and 125; and the combination of amino acid residues at positions 39, 82 and 116. combinations of amino acid residues at positions 39, 82, and 125; combinations of amino acid residues at positions 39, 116, and 125; combinations of amino acid residues at positions 39, 54, 82, and 116; combinations of amino acid residues at positions 39, 54, 82, and 125; combinations of amino acid residues at positions 39, 82, 116, and 125; combinations of amino acid residues at positions 39, 54, 116, and 125; combinations of amino acid residues at positions 39, 54, 82, 116, and 125.

[0035] Combinations of amino acid residues at position 54 and other positions include, for example, the combination of amino acid residues at positions 3 and 54; the combination of amino acid residues at positions 18 and 54; the combination of amino acid residues at positions 19 and 54; the combination of amino acid residues at positions 39 and 54; the combination of amino acid residues at positions 54 and 72; the combination of amino acid residues at positions 54 and 103; the combination of amino acid residues at positions 54 and 105; the combination of amino acid residues at positions 54 and 158; the combination of amino acid residues at positions 54 and 233; the combination of amino acid residues at positions 54 and 240; a combination of amino acid residues at positions 35; a combination of amino acid residues at positions 54 and 277; a combination of amino acid residues at positions 54 and 279; a combination of amino acid residues at positions 3, 54 and 158; a combination of amino acid residues at positions 3, 54 and 233; a combination of amino acid residues at positions 3, 54 and 235; a combination of amino acid residues at positions 18, 54 and 158; a combination of amino acid residues at positions 18, 54 and 233; a combination of amino acid residues at positions 19, 54 and 158; a combination of amino acid residues at positions 19, 54 and 233 combination of amino acid residues at positions 19, 54, and 235; combination of amino acid residues at positions 39, 54, and 82; combination of amino acid residues at positions 39, 54, and 116; combination of amino acid residues at positions 39, 54, and 125; combination of amino acid residues at positions 54, 72, and 158; combination of amino acid residues at positions 54, 72, and 233; combination of amino acid residues at positions 54, 72, and 235; combination of amino acid residues at positions 54, 103, and 158; combination of amino acid residues at positions 54, 103, and 23 a combination of amino acid residues at positions 3; a combination of amino acid residues at positions 54, 105, and 158; a combination of amino acid residues at positions 54, 105, and 233; a combination of amino acid residues at positions 54, 158, and 233; a combination of amino acid residues at positions 54, 158, and 235; a combination of amino acid residues at positions 54, 158, and 277; a combination of amino acid residues at positions 54, 158, and 279; a combination of amino acid residues at positions 54, 233, and 277; a combination of amino acid residues at positions 54, 235, and 279;The combination of amino acid residues at positions 3, 54, 158 and 233; the combination of amino acid residues at positions 3, 54, 158 and 235; the combination of amino acid residues at positions 18, 54, 158 and 233; the combination of amino acid residues at positions 19, 54, 158 and 233; the combination of amino acid residues at positions 19, 54, 158 and 235; the combination of amino acid residues at positions 39, 54, 82 and 116; the combination of amino acid residues at positions 39, 54, 82 and 125; the combination of amino acid residues at positions 39, 54, 116 and 125; the combination of amino acid residues at positions 39, 54, 82, 116 and 125.

[0036] Combinations of amino acid residues at position 72 and other positions include, for example, the above-mentioned combinations that include combinations of amino acid residues at position 72 and other positions, and therefore, such combinations can be identified by extracting from the above-mentioned combinations of amino acid residues at position 72 and other positions.

[0037] Examples of combinations of amino acid residues at position 103 and other positions include those combinations of amino acid residues at position 103 and other positions among the above combinations, and therefore, such combinations can be identified by extracting from the above combinations of amino acid residues at position 103 and other positions.

[0038] Examples of combinations of amino acid residues at position 105 and other positions include those combinations of amino acid residues at position 105 and other positions among the above combinations. Therefore, such combinations can be identified by extracting from the above combinations of amino acid residues at position 105 and other positions.

[0039] Examples of combinations of amino acid residues at position 116 and other positions include those combinations described above that include combinations of amino acid residues at position 116 and other positions. Therefore, such combinations can be identified by extracting from the above combinations of amino acid residues at position 116 and other positions.

[0040] Examples of combinations of amino acid residues at position 125 and other positions include those combinations of amino acid residues at position 125 and other positions among the above combinations. Therefore, such combinations can be identified by extracting from the above combinations of amino acid residues at position 125 and other positions.

[0041] Examples of combinations of amino acid residues at position 157 and other positions include those combinations of amino acid residues at position 157 and other positions among the above combinations, and therefore, such combinations can be identified by extracting from the above combinations of amino acid residues at position 157 and other positions.

[0042] Combinations of amino acid residues at position 158 and other positions include, for example, the above-mentioned combinations that include combinations of amino acid residues at position 158 and other positions. Therefore, such combinations can be identified by extracting from the above-mentioned combinations of amino acid residues at position 158 and other positions.

[0043] Combinations of amino acid residues at position 196 and other positions include, for example, the above-mentioned combinations that include combinations of amino acid residues at position 196 and other positions. Therefore, such combinations can be identified by extracting from the above-mentioned combinations of amino acid residues at position 196 and other positions.

[0044] Examples of combinations of amino acid residues at position 219 and other positions include those combinations of amino acid residues at position 219 and other positions among the above combinations. Therefore, such combinations can be identified by extracting from the above combinations of amino acid residues at position 219 and other positions.

[0045] Examples of combinations of amino acid residues at position 226 and other positions include those combinations of amino acid residues at position 226 and other positions among the above combinations, and therefore, such combinations can be identified by extracting from the above combinations of amino acid residues at position 226 and other positions.

[0046] Examples of combinations of amino acid residues at position 231 and other positions include those combinations described above that include combinations of amino acid residues at position 231 and other positions. Therefore, such combinations can be identified by extracting from the above combinations of amino acid residues at position 231 and other positions.

[0047] Examples of combinations of amino acid residues at position 235 and other positions include those combinations described above that include combinations of amino acid residues at position 235 and other positions. Therefore, such combinations can be identified by extracting from the above combinations of amino acid residues at position 235 and other positions.

[0048] Examples of combinations of amino acid residues at position 239 and other positions include those combinations described above that include combinations of amino acid residues at position 239 and other positions. Therefore, such combinations can be identified by extracting from the above combinations of amino acid residues at position 239 and other positions.

[0049] Examples of combinations of amino acid residues at position 279 and other positions include those combinations of amino acid residues at position 279 and other positions among the above combinations, and therefore, such combinations can be identified by extracting from the above combinations of amino acid residues at position 279 and other positions.

[0050] Examples of combinations of amino acid residues at position 305 and other positions include those combinations described above that include combinations of amino acid residues at position 305 and other positions. Therefore, such combinations can be identified by extracting from the above combinations of amino acid residues at position 305 and other positions.

[0051] Examples of combinations of amino acid residues at position 318 and other positions include those combinations of amino acid residues at position 318 and other positions among the above combinations. Therefore, such combinations can be identified by extracting from the above combinations of amino acid residues at position 318 and other positions.

[0052] In certain embodiments, the ligase mutant may not contain any combination of amino acid residues at the above positions (i.e., single mutations).

[0053] In a preferred embodiment, the mutation of an amino acid residue selected from the group consisting of positions 3, 18, 19, 25, 54, 56, 64, 72, 76, 79, 82, 92, 103, 105, 116, 125, 157, 158, 196, 226, 231, 235, 279, 305, and 318, and combinations thereof, may be a mutation selected from the group consisting of positions 3, 25, 39, 54, 56, 64, 72, 76, 79, 82, 92, 103, 116, 125, 157, 196, 226, 231, 235, 305, and 318, and combinations thereof.

[0054] In the amino acid sequences of (B), (B1), and (B2), the number of additionally mutated amino acid residues is 1 to 30. The number of additionally mutated amino acid residues may be preferably 1 to 25, more preferably 1 to 20, even more preferably 1 to 15, and most preferably 1 to 12, 1 to 10, 1 to 8, 1 to 6, 1 to 5, 1 to 3, or 1 or 2.

[0055] In the amino acid sequences of (C), (C1) and (C2), the percent identity of the amino acid sequences of (C), (C1) and (C2) to the variant amino acid sequences of (A), (A1) and (A2) 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.

[0056] The positions of amino acid residues that may be mutated relative to the above amino acid sequences will be clear to those skilled in the art. For example, those skilled in the art can 1) compare the amino acid sequences of multiple proteins with similar properties (e.g., the amino acid sequence of SEQ ID NO: 1 (T4 RNA ligase 2 / NP_049790) and the amino acid sequence of SEQ ID NO: 2), 2) identify relatively conserved and relatively non-conserved regions, and then 3) predict regions that may play an important role in function and regions that may not play an important role in function from the relatively conserved and relatively non-conserved regions, respectively, thereby recognizing the correlation between structure and function. Thus, those skilled in the art 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 amino acid residue before the mutation. Such a desired natural α-amino acid residue is the same as the amino acid residue described above.

[0057] 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.

[0058] 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.

[0059] The ligase mutant of the present invention has the ligation activity of nucleic acid.Nucleic acid includes, for example, single-stranded nucleic acid and double-stranded nucleic acid, and double-stranded nucleic acid is preferred.Nucleic acid also includes, for example, RNA, DNA, modified nucleic acid other than RNA and DNA, and the mixed nucleic acid thereof.Preferably, nucleic acid can be siRNA.

[0060] Nucleic acids for which ligation activity is evaluated can also 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"). Modified nucleic acids refer to nucleic acids other than natural nucleic acids, and are nucleic acids containing 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 substituted 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) substituted 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).

[0061] 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).

[0062] Substitution of an atom or group in the sugar moiety of a nucleotide residue includes, for example, 1'-H, 2'-OH (ribose only), 2'-H, 3'-OH, 3'-NH２ , 3'-H, 3'-phosphate group, 4'-H, 5'-phosphate group, or a combination thereof, where the phosphate group is -O-P(O)(OH) ２ as well as groups in which an oxygen atom is replaced by a sulfur atom or NH (e.g., —O—P(S)(OH) ２ , -NH-Ρ(O)(OH) ２ , -NH-Ρ(S)(OH) ２ ) is also included. ＊ (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).

[0063] 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 alkyl, such as alkyl, C １～６ Alkyloxy such as alkyloxy, C ６～１４ aryl such as aryl, amino C １～６ alkyl, aminoalkyl, etc., and combinations thereof.

[0064] C １～６ Alkyl is alkyl having 1 to 6 carbon atoms. １～６ The alkyl is preferably an alkyl having 1 to 4 carbon atoms, more preferably an alkyl having 1 to 3 carbon atoms, and particularly preferably an alkyl having 1 or 2 carbon atoms. １～６ Examples of alkyl include methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, isobutyl, t-butyl, pentyl, and hexyl.

[0065] C ６～１４ The aryl is an aryl having 6 to 14 carbon atoms. ６～１４ Aryl includes, for example, phenyl and naphthyl.

[0066] More specifically, examples of the substituent after substitution in chemical modification include C １～６ Alkyloxy C １～６ Alkyl (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 Alkyl (e.g., —O—N-methylacetamide, —O—NMA), —O—C １～６ Alkyl-(C １～６ Alkyl-)amino-C １～６ Alkyl (e.g., —O-dimethylaminoethoxyethyl, —O-DMAEOE), and —O-aminoC １～６ Alkyl (eg, —O-aminopropyl, —O-AP) is exemplified.

[0067] 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, 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 Alkyl (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 Alkyl (e.g., 2'-O-N-methylacetamide, 2'-O-NMA), 2'-O-C １～６ Alkyl-(C １～６ Alkyl-)amino-C １～６Alkyl (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'-O-P(O)(OH). ２、 3'-O-Ρ(S)(OH) ２ ,3'-NH-Ρ(O)(OH) ２ ,3'-NH-Ρ(S)(OH) ２ ), and the hydroxyl group (—OH) in the phosphate group is OR ＊ (In the formula, R ＊ represents an organic group such as a protecting group for a phosphate group, which will be described later).

[0068] The 5'- or 3'-phosphate group modification may be introduced, for example, to improve the degradation resistance of the oligonucleotide. Examples of the 5'- or 3'-phosphate group modification include a phosphate group (-O-P(O)(OH) ２ ) to a group in which the oxygen atom in the phosphate group is replaced with a sulfur atom or NH. Such a group includes, for example, —O—P(S)(OH) ２ (thiophosphate group: phosphorothioate modification), —NH—P(O)(OH) ２ , -NH-Ρ(S)(OH) ２ In addition, the 5'- or 3'-phosphate group modification may be carried out by replacing the hydroxyl group (-OH) in the phosphate group with OR. ＊ (In the formula, R ＊ represents an organic group such as a protecting group for a phosphate group) (for example, a protected phosphate group). Examples of the protecting group for a phosphate group include a trityl (Tr) group, a p-methoxyphenyldiphenylmethyl (MMTr) group, a di(p-methoxyphenyl)phenylmethyl (DMTr) group, a cyanoethyl group (CN-C ２ H ４ -) are listed.

[0069] 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'-O-C substitutions between 2'-OH and 4'-H. １～６ Substitution with alkylene-4' (e.g., 2'-O-methylene-4' (locked nucleic acid: LNA), 2'-O-ethylene-4' (ethylene-bridged nucleic acid: ENA), substitution with 2'-O-methyl-substituted methylene-4' (constrained ethyl-bridged nucleic acid: a type of BNA (cEt-BNA)), 2'-OH and 4'-H 2'-O-C １～６ Alkylene -O-C １～６ Substitution with alkylene-4' (e.g., 2'-O-methylene-O-methylene-4' (a type of bridged nucleic acid: BNA (BNA ＣＯＣ )), 2'-OH and 4'-H 2'-O-N(R)-C １～６ Substitution with alkylene-4' (e.g., 2'-O-N(R)-methylene-4' (bridged nucleic acid: a type of BNA (BNA ＮＣ ), where R represents methyl, hydrogen atom or benzyl), 2'-NH ２ and substitution of 4'-H with 2'-N(R)-C(O)-4' (e.g., 2'-N(methyl)-C(O)-4' (amide-bridged nucleic acid: AmNA)), 2'-NH ２ and 4'-H 2'-NH-C １～６ Substitution to alkylene-4' (e.g., 2'-NH-methylene-4'), 2'-C of 2'-H and 4'-H １～６ Examples of the 3'-5'-bridge modification include a 3'-C bond between 3'-H and 5'-H. １～６ Examples include substitution with alkylene-5' (eg, 3'-ethylene-5' (bicyclonucleic acid: Bc nucleic acid), a type of Bc nucleic acid: tc nucleic acid, etc.).

[0070] 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.

[0071] Examples of nucleotide residues containing a substitution in the sugar moiety of the nucleotide residue itself include modified nucleotide residues containing a substitution of a 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, examples of modified nucleotide residues containing a substitution in the sugar moiety of the nucleotide residue itself also include morpholino nucleic acid (PMO) residues, which are nucleotide-like artificial compounds having a morpholino ring structure that are not degraded by in vivo enzymes (e.g., nucleases such as RNase) and do not induce an immune response.

[0072] The modification of the nucleic acid base portion of the nucleotide residue may be, for example, a modification of the nucleic acid base portion of the nucleotide residue to C １～６ Examples include those substituted with alkyl such as alkyl (for example, those substituted with a methyl group at the 5-position of a cytosyl group).

[0073] In certain embodiments, the nucleic acid for which ligation activity is assessed may be a single-stranded or double-stranded nucleic acid, which may contain modified nucleic acids, as described above.

[0074] In certain embodiments, the modified nucleic acid may be a 2'-modified nucleic acid. The 2'-modified nucleic acid is preferably a 2'-chemically modified nucleic acid as described above, and may be a 2'-O-C １～６ Alkyloxy-C １～６ Alkyl, or 2'-O-C １～６ Nucleic acids containing alkyl modifications are more preferred. １～６ Alkyloxy-C １～６A preferred example of alkyl is 2'-O-methoxyethyl. １～６ A preferred example of alkyl is 2'-O-methyl.

[0075] In certain embodiments, the nucleic acid ligation activity may be a ligation activity of a nucleic acid comprising the following first and second nucleic acids: (A) a first nucleic acid; (A1) a single-stranded nucleic acid comprising a 2'-OH or 2'-H moiety as the 5'-terminal nucleotide residue; (A2) a single-stranded nucleic acid comprising a 2'-O-C moiety as the 5'-terminal nucleotide residue; １～６ Alkyloxy-C １～６ Alkyl, or 2'-O-C １～６ (A3) a double-stranded nucleic acid comprising the single-stranded nucleic acid of (A1) or (A2) above and a complementary strand comprising a portion complementary thereto; and (B) a second nucleic acid: (B1) a single-stranded nucleic acid comprising a 2'-OH or 2'-H moiety as the 3'-terminal nucleotide residue; (B2) a 2'-O-C moiety as the 3'-terminal nucleotide residue; １～６ Alkyloxy-C １～６ Alkyl, or 2'-O-C １～６ (B3) a double-stranded nucleic acid comprising the single-stranded nucleic acid of (B1) or (B2) above and a complementary strand comprising a portion complementary thereto. When single-stranded nucleic acids are used as the first nucleic acid and the second nucleic acid, a complementary strand of the single-stranded nucleic acid corresponding to the concatenation of the first nucleic acid and the second nucleic acid may be present in the reaction system.

[0076] The nucleic acid ligation activity of the ligase mutant of the present invention is not particularly limited as long as it exhibits improved nucleic acid ligation activity relative to the enzyme consisting of the amino acid sequence of SEQ ID NO: 2. However, the nucleic acid ligation activity may be preferably at least 1.1 times, more preferably at least 1.2 times, even more preferably at least 1.5 times, and particularly preferably at least 1.6 times, at least 1.8 times, or at least 2.0 times that of the enzyme consisting of the amino acid sequence of SEQ ID NO: 2. The above-mentioned mutations corresponding to the generic concepts of the specific mutations in the Examples that exhibit such nucleic acid ligation activity can be appropriately selected as mutations that exhibit such nucleic acid ligation activity. 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 procedure (see, e.g., the Examples): a) A final concentration of 10 μM nucleic acid material (oligonucleotide), 50 mM Tris-HCl (pH 7.5), 2 mM MgCl ２ A reaction solution containing 1 mM dithiothreitol (DTT), 0.4 mM ATP, and 0.01 mg / ml enzyme is prepared and reacted at 25° C. for 30 minutes; b) The reaction is stopped by adding 10 mM EDTA.

[0077] 2. Inventions 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 produce transformants.

[0078] 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.

[0079] 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 technology. If 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, if the expression vector is a non-integrative vector that does not undergo homologous recombination with the genomic DNA of the host cell, the expression unit is not integrated into the genomic DNA of the host cell by transformation, and can exist in the host cell as an expression vector, independent of the genomic DNA. Alternatively, genome editing technology (e.g., CRISPR / Cas system, Transcription Activator-Like Effector Nucleases (TALEN)) can be used to integrate the expression unit into the genomic DNA of the host cell and modify the expression unit inherently possessed by the host cell.

[0080] 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.

[0081] 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, loxP, FRT, which are homologous to a specific sequence in the genome of the host cell). The genomic region of the host cell into which the expression unit is to be introduced (the 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.

[0082] 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.

[0083] Hosts for expressing the ligase mutant of the present invention include various prokaryotic cells such as Escherichia bacteria such as Escherichia coli, Corynebacterium bacteria (e.g., Corynebacterium glutamicum), and Bacillus bacteria (e.g., Bacillus subtilis), as well as Saccharomyces bacteria (e.g., Saccharomyces cerevisiae), Pichia bacteria (e.g., Pichia stipitis), and Aspergillus bacteria (e.g., Aspergillus oryzae). Various eukaryotic cells, including E. oryzae, can be used. A strain lacking a specific gene may also be used as the host. Examples of transformed microorganisms include transformed microorganisms that carry an expression vector in their cytoplasm and transformed microorganisms that have a target gene introduced into their genome.

[0084] The transformed microorganism of the present invention can be cultured in a medium having the composition described below, for example, using a predetermined culture device (e.g., test tube, flask, or jar fermenter). Culture conditions can be set appropriately. Specifically, the culture temperature may be 10°C to 37°C, the pH may be 6.5 to 7.5, and the culture time may be 1 hour to 100 hours. Culture may also be performed while controlling the dissolved oxygen concentration. In this case, the dissolved oxygen concentration (DO value) in the culture solution may be used as a control index. Aeration and agitation conditions can be controlled so that the relative dissolved oxygen concentration (DO value) when the atmospheric oxygen concentration is 21% does not fall below, for example, 1% to 10%, preferably 3% to 8%. Culture may be performed by batch or fed-batch culture. In fed-batch culture, the culture can be continued by sequentially adding a solution serving as a sugar source or a solution containing phosphate to the culture solution, either continuously or discontinuously.

[0085] 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 selection of transformed microorganisms are also described in "Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press (2001 / 01 / 15)" and the like. Below, a method for preparing transformed Escherichia coli and using it to produce a predetermined enzyme will be described in more detail as an example.

[0086] As a promoter for expressing the polynucleotide of the present invention, a promoter typically used for heterologous protein production in E. coli can be used, and examples thereof include strong promoters 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. Also, examples of vectors that can 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 that can be used include phage DNA vectors. Furthermore, expression vectors that contain a promoter and can express the inserted DNA sequence can also be used. Preferably, the vectors can be pUC, pSTV, or pMW.

[0087] 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.

[0088] The vector for introducing the polynucleotide of the present invention into E. coli is preferably a so-called multicopy type, and examples thereof include plasmids having a replication origin derived from ColE1, such as pUC-based plasmids and pBR322-based plasmids, or derivatives thereof. Here, "derivatives" refer to plasmids that have been modified by base substitution, deletion, insertion, and / or addition.

[0089] Furthermore, in order to select transformed microorganisms, it is preferable that the vector has a marker such as an ampicillin resistance gene. As such a plasmid, expression vectors having strong promoters are commercially available (e.g., pUC series (manufactured by Takara Bio Inc.), pPROK series (manufactured by Clontech), and pKK233-2 (manufactured by Clontech)).

[0090] 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.

[0091] The medium may be a medium commonly used for culturing Escherichia coli, such as M9-casamino acid medium or LB medium. The medium may contain a predetermined carbon source, nitrogen source, and coenzyme (e.g., pyridoxine hydrochloride). Specifically, the medium may contain peptone, yeast extract, NaCl, glucose, MgSO4, etc. ４ ammonium sulfate, potassium dihydrogen phosphate, ferric sulfate, manganese sulfate, etc. may also 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.

[0092] 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 disrupted or lysed product 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 disrupted or lysed products to techniques such as extraction, precipitation, filtration, column chromatography, etc.

[0093] 3. Method for Producing a Nucleic Acid Product The present invention also provides a method for producing a nucleic acid product, comprising contacting two or more nucleic acid materials with a mutant ligase of the present invention to produce a nucleic acid product in which two or more nucleic acid materials are ligated. The nucleic acid material can be selected from the group consisting of single-stranded nucleic acid material, double-stranded nucleic acid material, and a mixture thereof.

[0094] (Nucleic Acid Material) The nucleic acid material may be a single or multiple. 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. For example, it may be a relatively small number, such as 2 to 10, 2 to 8, 2 to 6, 2 to 4, 3 to 7, or 4 to 6, or it may be more than 10. When multiple single-stranded nucleic acids are ligated, complementary strands of the single-stranded nucleic acids corresponding to the ligated products may be present in the reaction system.

[0095] In certain embodiments, the nucleic acid material may be a plurality of single-stranded or double-stranded nucleic acids, which may include modified nucleic acids, as described above.

[0096] In certain embodiments, the modified nucleic acid may be a 2'-modified nucleic acid. The 2'-modified nucleic acid is preferably a 2'-chemically modified nucleic acid as described above, and may be a 2'-O-C １～６ Alkyloxy-C １～６ Alkyl, or 2'-O-C １～６ Nucleic acids containing alkyl modifications are more preferred. １～６ Alkyloxy-C １～６ Alkyl and 2'-O-C １～６ Preferred examples of alkyl are as described above.

[0097] The length of the nucleic acid material is not particularly limited. For example, a long nucleic acid material exceeding 1,000 bases 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 or more bases long, 6 or more bases long, 7 or more bases long, 8 or more bases long, or 9 or more bases long. The short nucleic acid material may also be 25 or less bases long, 24 or less bases long, 23 or less bases long, 22 or less bases long, 21 or less bases long, 20 or less bases long, 19 or less bases long, 18 or less bases long, 17 or less bases long, 16 or less bases long, or 15 or less bases long. More specifically, the short nucleic acid material may have a base length of 5 to 25 bases, 6 to 25 bases, 7 to 25 bases, 8 to 25 bases, 9 to 25 bases, 5 to 24 bases, 5 to 23 bases, 5 to 22 bases, 5 to 21 bases, 5 to 20 bases, 5 to 19 bases, 5 to 18 bases, 5 to 17 bases, 5 to 16 bases, or 5 to 15 bases.

[0098] When at least one double-stranded nucleic acid is used as the nucleic acid material, a protruding end may be used in the ligation. Examples of protruding ends used in the ligation include protruding ends in a double-stranded nucleic acid (nucleic acid material) having a protruding end, 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.

[0099] When the desired nucleic acid product is a single-stranded nucleic acid (e.g., an antisense oligonucleotide), it is desirable to have, in addition to two or more nucleic acid materials, complementary strands (single strands) of the single-stranded nucleic acid corresponding to the ligation product of these nucleic acid materials coexist in the reaction system. Therefore, the method of the present invention may include contacting two or more single-stranded nucleic acid materials with a mutant ligase of the present invention to produce a single-stranded nucleic acid product (e.g., an antisense oligonucleotide) in which two or more single-stranded nucleic acid materials are ligated, and the contacting may be performed in the presence of a complementary strand. The complementary strand may be perfectly complementary to each of the two or more single-stranded nucleic acid materials (i.e., the material of the single-stranded nucleic acid product) (i.e., a perfectly complementary strand). Alternatively, the complementary strand may contain a non-complementary portion (e.g., an incompletely complementary strand) as long as it can form a duplex with each of the two or more single-stranded nucleic acid materials and promote the ligation reaction. The nucleic acid constituting the complementary strand is the same as the nucleic acid described above. The concentration of the complementary strand is not particularly limited as long as the desired single-stranded nucleic acid product is produced. For example, the concentration of the complementary strand can be set in the same manner as the concentration of the nucleic acid material described below.

[0100] The nucleic acid material may be in free form, immobilized on a solid phase, or complexed to a functional moiety if complexation of the nucleic acid product with the functional moiety is desired.

[0101] 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.

[0102] (Nucleic Acid Product) The nucleic acid product includes, for example, double-stranded nucleic acid and single-stranded nucleic acid.

[0103] The double-stranded nucleic acid as the nucleic acid product may be a double-stranded nucleic acid in which each strand is composed of the above-mentioned nucleic acid, and examples thereof include double-stranded RNA, double-stranded DNA, a heteroduplex nucleic acid composed of RNA and DNA, a double-stranded nucleic acid composed of RNA and an RNA-DNA hybrid nucleic acid, a double-stranded nucleic acid composed of DNA and an RNA-DNA hybrid nucleic acid, and a double-stranded nucleic acid composed of two RNA-DNA hybrid nucleic acids. Examples of double-stranded nucleic acids include siRNA and heteroduplex nucleic acids.

[0104] The single-stranded nucleic acid as the nucleic acid product may be a single-stranded nucleic acid composed of the above-mentioned nucleic acid, for example, single-stranded RNA, single-stranded DNA, and hetero-single-stranded nucleic acid composed of RNA and DNA. Such single-stranded nucleic acid may or may not contain a double-stranded structural portion (e.g., two or more complementary portions capable of pairing). Examples of single-stranded nucleic acids containing a double-stranded structural portion include loop-type nucleic acids such as hairpin-type nucleic acids and dumbbell-type nucleic acids. Examples of single-stranded nucleic acids not containing a double-stranded structural portion include linear nucleic acids (e.g., antisense oligonucleic acids).

[0105] 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.

[0106] The nucleic acid product may be a nucleic acid consisting only of complementary portions where bases are paired, or may contain, in addition to complementary portions, non-complementary portions where bases are not paired. The length of the complementary portions and / or non-complementary portions is not particularly limited, but the complementary portions and / or non-complementary portions may be short. For example, the short complementary portions 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 portions 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 non-complementary portions in addition to complementary portions, the complementary portions may be contiguous or may be discontinuous, separated by non-complementary portions. 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.

[0107] Preferably, the product nucleic acid may comprise a modified nucleic acid, which may be a single-stranded or double-stranded nucleic acid. Modified nucleic acids are as described above. More preferably, such product nucleic acids may comprise modified residues, as described above, in the complementary portion.

[0108] (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 desired nucleic acid product is particularly desired, the pH may be 7.5 to 9.0 (e.g., 8.0 to 8.5).

[0109] 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, or 500 μM or more. The concentration of each nucleic acid material may also be, for example, 1 M or less, 100 mM or less, or 50 mM or less. More specifically, the concentration of each nucleic acid material may be, for example, 1 μM to 1 M, 10 μM to 1 M, 50 μM to 1 M, 100 μM to 1 M, 300 μM to 1 M, 500 μM to 1 M, 1 μM to 100 mM, or 1 μM to 50 mM.

[0110] 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 molar ratio of any two (and preferably all) 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.

[0111] 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 minute.

[0112] The reaction system may contain a cofactor. Examples of the cofactor include ATP and divalent metal salts (e.g., magnesium salts such as magnesium chloride). The reaction system may contain a stabilizer for the ligase. Examples of the stabilizer for the ligase include antioxidants (e.g., reducing agents such as dithiothreitol and mercaptoethanol). The reaction system may contain a surfactant for purposes such as maintaining the stability of the enzyme and improving the reaction rate. Examples of the surfactant include nonionic surfactants (e.g., Triton series surfactants such as Triton X-100) and ionic surfactants. Examples of the ionic surfactant include cationic surfactants, anionic surfactants, and zwitterionic surfactants. The reaction system may also contain polyethylene glycol for purposes such as improving the reaction rate.

[0113] 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.

[0114] The reaction temperature may be any temperature sufficient for activating the ligase mutant of the present invention, such as 2 to 50°C, preferably 16 to 45°C, and more preferably 20 to 40°C.

[0115] The reaction time may be any time sufficient to produce the desired nucleic acid product. Such a time may be, for example, from 10 minutes to 72 hours. The reaction time may also be from 15 minutes to 60 hours, or from 30 minutes to 48 hours.

[0116] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0117] Example 1: Construction of a ligase mutant expression plasmid (1-1) Construction of a ligase mutant expression plasmid for an enzyme consisting of the amino acids of SEQ ID NO: 2 A plasmid was prepared by inserting a gene encoding an enzyme consisting of the amino acids of SEQ ID NO: 2 (also called Anc2) into the NdeI and BamHI sites in the multicloning site of pET-16b (Novagen) (purchased from GenScript Japan Co., Ltd.). The amino acid sequence of the enzyme consisting of the amino acids of SEQ ID NO: 2 was determined by reference to SEQ ID NO: 1 published in JP 2021-153573 A.

[0118] Using the prepared plasmid as a template plasmid, a gene sequence was designed to introduce site-specific amino acid mutations into the amino acid sequence of SEQ ID NO: 2. The mutant numbers and the introduced mutation sites are as follows:

[0119]

[0120]

[0121] (1-2) Construction of a Plasmid Expressing a Ligase Mutant of the Enzyme Composed of the Amino Acids of SEQ ID NO: 1 Next, a gene encoding the enzyme composed of the amino acids of SEQ ID NO: 1 (also referred to as T4Rnl2) was inserted into the NdeI and BamHI sites in the multicloning site of pET-16b (Novagen) to prepare a plasmid (purchased from GenScript Japan Co., Ltd.). The amino acid sequence of the enzyme composed of the amino acids of SEQ ID NO: 1 was determined by reference to the amino acid sequence of T4 RNA ligase 2 (NP_049790).

[0122] Using the prepared plasmid as a template plasmid, a gene sequence was designed to introduce site-specific amino acid mutations into the amino acid sequence of SEQ ID NO: 1. The mutant numbers and the introduced mutation sites are as follows:

[0123]

[0124] Example 2: Purification of the enzyme

[0125] (1) Preparation of enzyme-producing strain E. coli BL21(DE3) was transformed with the plasmid prepared in Example 1, and transformants having the plasmid were obtained from ampicillin-resistant strains and identified by the strain name with an ID number.

[0126] (2) Preparation of cell-free extract The prepared strain was inoculated into 3 mL of LB medium containing 100 mg / L ampicillin and cultured in a test tube with shaking at 37°C for 16 hours to prepare a seed culture. 0.03 mL of the seed culture was inoculated into 3 mL of Overnight Express™ Instant TB Medium (Novagen) containing 100 mg / L ampicillin placed in a test tube, and cultured with shaking at 25°C for 24 hours to prepare a culture solution. Starting with 1 mL of culture medium, cells were harvested by centrifugation (12,000 × g, 10 minutes, 4°C) and resuspended in 0.3 mL of 50 mmol / L Tris-HCl (pH 7.5), 0.25 mmol / L NaCl, 10% (wt / vol) sucrose, and 0.1% Triton X-100. A disrupted cell solution was obtained by ultrasonic disruption (BioRaptor (registered trademark), manufactured by BM Kiki Co., Ltd., 4°C, treatment time: 30 minutes). The resulting disrupted cell solution was centrifuged (15,000 × g, 20 minutes, 4°C) to separate the insoluble fraction. The resulting supernatant was used as a cell-free extract.

[0127] (3) Affinity chromatography: Starting from the cell-free extract, affinity chromatography was performed using a TALON® Spin Column (Takara Bio Inc.) according to the manufacturer's protocol. The equilibration buffer was 50 mM Tris HCl (pH 7.5), 0.25 mM NaCl, 10% (wt / vol) sucrose, and 0.1% Triton X-100. The elution buffer was 50 mmol / L Tris HCl (pH 7.5), 0.25 mmol / L NaCl, 10% (wt / vol) sucrose, and 0.1% Triton X-100, and 200 mmol / L imidazole. The resulting eluate was collected and purified using an Amicon Ultra 0.5 10 kda (Merck) in a solution of 10 mmol / L Tris-HCl (pH 7.5), 50 mmol / L KCl, 35 mmol / L (NH ４ ) ２ SO ４ The buffer was exchanged with 0.1 mmol / L DTT, 0.1 mmol / L EDTA, and 50% (vol / vol) glycerol to obtain a purified enzyme solution.

[0128] (4) Quantification of Enzyme Concentration The protein concentration of the enzyme solution was quantified according to the method described in the instruction manual for Protein Assay CBB Solution (5x concentrated) (Nacalai Tesque, Inc.). A calibration curve was prepared using Quick Start Bovine Serum Albumin Standard (Bio-Rad) as the standard protein, and the protein concentration in the sample was calculated.

[0129] Example 3: Measurement of condensation activity of ligase mutants in the ligation reaction of 2'-O-methoxyethyl modified nucleic acid and 2'-O-methoxyethyl modified nucleic acid

[0130] The enzyme activity was measured using oligonucleotide as a substrate under the following conditions: 50 mmol / L Tris HCl (pH 7.5), 2 mmol / L MgCl ２ The reaction mixture was incubated at 25°C for 30 minutes, and the reaction was stopped with 10 mmol / L EDTA (pH 8.0). The reaction solution was subjected to HPLC analysis to quantify the amount of oligonucleotides produced.

[0131] The HPLC analysis conditions are as follows: (Analysis Condition 1) Column: Waters ACQUITY PRMST BEH130 (1.7μ, 2.1×100 mm) (Japan Waters) Mobile phase A: 8 mM TEA, 100 mM HFIP Mobile phase B: methanol:mobile phase A=80:20 Flow rate: 0.4 mL / min Column temperature: 80° C. Detection: UV 210 nm

[0132]

[0133]

[0134]

[0135]

[0136] The results confirmed that the ligase mutants exhibited superior activity compared to the control (enzyme before mutation) (Table 3). Many ligase mutants with mutations at other amino acid residue positions either exhibited activity equivalent to that of the control or tended to exhibit reduced activity compared to the control. The activities of the ligase mutants were further examined below.

[0137] Example 4: Measurement of condensation activity of ligase mutants in ligation reaction of 2'-O-methoxyethyl-modified nucleic acid with natural DNA Enzyme activity was measured using oligonucleotide as a substrate under the following conditions: 50 mmol / L Tris HCl (pH 7.5), 2 mmol / L MgCl ２ The reaction mixture was incubated at 25°C for 30 minutes, and the reaction was stopped with 10 mmol / L EDTA (pH 8.0). The reaction solution was subjected to HPLC analysis to quantify the amount of oligonucleotides produced.

[0138]

[0139]

[0140]

[0141]

[0142] Example 5: Measurement of condensation activity of ligase mutants in the reaction for producing compound 2 Enzyme activity was measured using oligonucleotide as a substrate under the following conditions: 50 mmol / L Tris HCl (pH 8.0), 2 mmol / L MgCl ２ The reaction mixture was incubated at 25°C for 2 hours, and the resulting solution was stopped with 10 mmol / L EDTA (pH 8.0). The resulting solution was analyzed by HPLC to quantify the amount of oligonucleotides produced.

[0143]

[0144]

[0145]

[0146]

[0147] Example 6: Measurement of condensation activity of ligase mutants in ligation reaction of 2'-O-methyl modified nucleic acids Enzyme activity was measured using oligonucleotides as substrates under the following conditions: 50 mmol / L Tris HCl (pH 7.5), 2 mmol / L MgCl ２ The reaction mixture was incubated at 25°C for 30 minutes, and the resulting solution was stopped with 10 mmol / L EDTA (pH 8.0). The resulting solution was analyzed by HPLC to quantify the amount of oligonucleotides produced.

[0148]

[0149]

[0150]

[0151]

[0152] Example 7: Measurement of condensation activity of ligase mutants in siRNA production reaction. The target compound was 21-residue siRNA consisting of a sense strand and an antisense strand. Oligonucleotides were synthesized by splitting the sense strand and antisense strand into two. The enzyme activity was measured using these oligonucleotides as substrates under the following conditions: 50 mmol / L Tris HCl (pH 7.5), 2 mmol / L MgCl ２ The reaction mixture was incubated at 25°C for 30 minutes, and the resulting solution was stopped with 10 mmol / L EDTA (pH 8.0). The resulting solution was analyzed by HPLC to quantify the amount of oligonucleotides produced.

[0153]

[0154]

[0155]

[0156]

Claims

1. A ligase mutant comprising a mutant amino acid sequence containing mutations in amino acid residues selected from the group consisting of positions 3, 18, 19, 25, 36, 39, 54, 56, 64, 72, 76, 79, 82, 92, 103, 104, 105, 114, 116, 125, 129, 157, 158, 196, 207, 219, 226, 231, 235, 239, 241, 279, 305, 311, 313, and 318, and combinations thereof, based on the amino acid sequence encoding wild-type T4 RNA ligase 2, and having improved nucleic acid ligation activity compared to an enzyme consisting of wild-type T4 RNA ligase 2.

2. The ligase mutant of claim 1, wherein the ligase mutant comprises the mutation in the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:

1.

3. The ligase mutant of claim 1, wherein the ligase mutant is: (A1) a mutant amino acid sequence comprising mutations of amino acid residues selected from the group consisting of K3, E18, K19, L25, I36, T39, K54, T56, D64, L72, A76, I79, V82, V92, P103, G104, I105, K114, F116, T125, D129, K157, F158, A196, V207, G219, C226, F231, K235, K239, I241, P279, T305, S311, I313, and V318 in the amino acid sequence of SEQ ID NO: 1, and combinations thereof; (B1) an amino acid sequence in which the mutant amino acid sequence contains additional mutations of 1 to 30 amino acid residues selected from the group consisting of substitution, deletion, insertion, and addition of amino acid residues (wherein the mutation (A1) in the modified amino acid sequence is maintained); or (C1) an amino acid sequence having 90% or more identity to the mutant amino acid sequence (wherein the mutation (A1) in the modified amino acid sequence is maintained); and a ligase mutant having improved nucleic acid ligation activity compared to an enzyme consisting of the amino acid sequence of SEQ ID NO:

1.

4. The ligase mutant according to claim 3, wherein the mutation (A1) in the mutant amino acid sequence is selected from the group consisting of K3, E18, K19, K54, L72, P103, and F158, and combinations thereof.

5. The ligase mutant according to claim 3, wherein the mutation (A1) in the mutant amino acid sequence is one of the following: (1) a mutation at K3: K3Q; (2) a mutation at E18: E18K; (3) a mutation at K19: K19R; (4) a mutation at L25: L25H; (5) a mutation at I36: I36V; (6) a mutation at T39: T39G or T39M; (7) a mutation at K54: K54R; (8) a mutation at T56: T56S; (9) a mutation at D64: D64K; (10) a mutation at L72: L72P; (11) a mutation at A76: A76V; (12) a mutation at I79: I79V; (13) a mutation at V82: V82I; (14) a mutation at V92: V92A; (15) P103 mutation P103C or P103D; (16) G104 mutation G104N or G104Q; (17) I105 mutation I105L; (18) K114 mutation K114R; (19) F116 mutation F116L; (20) T125 mutation T125A; (21) D129 mutation D129V; (22) T157 mutation T157S; (23) F158 mutation F158Y; (24) A196 mutation A196T; (25) V207 mutation V207I; (26) G219 mutation G219K; (27) C226 mutation (28) F231 mutation F231L; (29) K235 mutation K235I; (30) K239 mutation K239E; (31) I241 mutation I241L; (32) P279 mutation P279R; (33) T305 mutation T305S; (34) S311 mutation S311R; (35) I313 mutation I313V; and (36) V318 mutation V318L.

6. The ligase mutant of claim 1, wherein the ligase mutant is: (A2) a mutant amino acid sequence comprising mutations of amino acid residues selected from the group consisting of K3, E18, K19, L25, I36, T39, K54, T56, D64, L72, D76, I79, V82, V92, G103, G104, I105, K114, F116, T125, D129, T157, F158, A195, V205, G217, C224, F229, K233, K237, I239, P277, T303, S309, I311, and V316 in the amino acid sequence of SEQ ID NO: 2, and combinations thereof; (B2) an amino acid sequence in which the mutant amino acid sequence contains additional mutations of 1 to 30 amino acid residues selected from the group consisting of substitution, deletion, insertion, and addition of amino acid residues (wherein the mutation (A2) in the modified amino acid sequence is maintained); or (C2) an amino acid sequence having 90% or more identity to the mutant amino acid sequence (wherein the mutation (A2) in the modified amino acid sequence is maintained); and a ligase mutant having improved nucleic acid ligation activity compared to an enzyme consisting of the amino acid sequence of SEQ ID NO:

2.

7. The ligase mutant according to claim 6, wherein the mutation (A2) in the mutant amino acid sequence is one of the following: (1) a mutation at K3: K3Q; (2) a mutation at E18: E18K; (3) a mutation at K19: K19R; (4) a mutation at L25: L25H; (5) a mutation at I36: I36V; (6) a mutation at T39: T39G or T39M; (7) a mutation at K54: K54R; (8) a mutation at T56: T56S; (9) a mutation at D64: D64K; (10) a mutation at L72: L72P; (11) a mutation at D76: D76V; (12) a mutation at I79: I79V; (13) a mutation at V82: V82I; (14) a mutation at V92: V92A; (15) G103 mutation G103C or G103D; (16) G104 mutation G104N or G104Q; (17) I105 mutation I105L; (18) K114 mutation K114R; (19) F116 mutation F116L; (20) T125 mutation T125A; (21) D129 mutation D129V; (22) T157 mutation T157S; (23) F158 mutation F158Y; (24) A195 mutation A195T; (25) V205 mutation V205I; (26) G217 mutation G217K; (27) C224 mutation (28) F229 mutation F229L; (29) K233 mutation K233I; (30) K237 mutation K237E; (31) I239 mutation I239L; (32) P277 mutation P277R; (33) T303 mutation T303S; (34) S309 mutation S309R; (35) I311 mutation I311V; and (36) V316 mutation V316L.

8. The ligase mutant of claim 1, wherein the nucleic acid is a single-stranded or double-stranded nucleic acid that may contain a modified nucleic acid.

9. The ligase mutant according to claim 8, wherein the modified nucleic acid is a 2'-modified nucleic acid.

10. The 2'-modified nucleic acid is 2'-O-C １～６ Alkyloxy-C １～６ Alkyl, or 2'-O-C １～６ The ligase mutant of claim 9, which is a nucleic acid containing an alkyl modification.

11. The ligase mutant according to claim 1, wherein the nucleic acid ligation activity is an activity to ligate nucleic acids comprising the following first and second nucleic acids: (A) the first nucleic acid; (A1) a single-stranded nucleic acid comprising a 2'-OH or 2'-H moiety as the nucleotide residue at the 5' end; (A2) a single-stranded nucleic acid comprising a 2'-O-C moiety as the nucleotide residue at the 5' end; １～６ Alkyloxy-C １～６ Alkyl, or 2'-O-C １～６ (A3) a double-stranded nucleic acid comprising the single-stranded nucleic acid of (A1) or (A2) above and a complementary strand comprising a portion complementary thereto; and (B) a second nucleic acid: (B1) a single-stranded nucleic acid comprising a 2'-OH or 2'-H moiety as the 3'-terminal nucleotide residue; (B2) a 2'-O-C moiety as the 3'-terminal nucleotide residue; １～６ Alkyloxy-C １～６ Alkyl, or 2'-O-C １～６ (B3) a double-stranded nucleic acid comprising the single-stranded nucleic acid of (B1) or (B2) above and a complementary strand comprising a portion complementary thereto; or (B4) a single-stranded nucleic acid comprising a nucleotide residue containing an alkyl-modified moiety; or (B5) a double-stranded nucleic acid comprising the single-stranded nucleic acid of (B1) or (B2) above and a complementary strand comprising a portion complementary thereto.

12. A method for producing a nucleic acid product, comprising contacting two or more nucleic acid materials with the ligase mutant of any one of claims 1 to 11 to produce a nucleic acid product in which the two or more nucleic acid materials are ligated, wherein the nucleic acid materials are selected from the group consisting of single-stranded nucleic acid materials, double-stranded nucleic acid materials, and mixtures thereof.

13. The method of claim 12, wherein the concentration of nucleic acid material is 1 μM or greater.

14. The method of claim 13, wherein the product nucleic acid is a single-stranded or double-stranded nucleic acid, which may contain modified nucleic acids.

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

16. An expression vector comprising the polynucleotide of claim 15.

17. A transformed microorganism comprising an expression unit comprising a polynucleotide encoding the ligase mutant of any one of claims 1 to 11 and a promoter operably linked thereto.

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