Modified double-stranded ligases and uses thereof

Abstract

The present disclosure relates to the field of biotechnology, and in particular to modified double-stranded RNA (dsRNA) ligases and their applications in industrial biocatalysis. The present disclosure also relates to a process for producing the modified dsRNA ligases and a method for producing oligonucleotides by contacting oligonucleotide fragments with the modified dsRNA ligases.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of European Patent Application No. 22215201.9, filed December 20, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] Sequence Listing This application contains a Sequence Listing that has been submitted electronically in .XML format and is incorporated herein by reference in its entirety. The .XML copy, created on December 2, 2023, has the file name PAT059445-WO-PCT_SL.xml and is 1.24 MB in size.

[0003] The present disclosure relates to the field of biotechnology, and in particular to modified double-stranded RNA (dsRNA) ligases and their applications in industrial biocatalysis. The present invention also relates to a process for producing the modified dsRNA ligases and a method for producing oligonucleotides by contacting oligonucleotide fragments with the modified dsRNA ligases. [Background technology]

[0004] Therapeutic oligonucleotides, including small interfering RNA (siRNA) and inhibitory antisense oligonucleotides (ASO), have the potential to treat a variety of life-threatening diseases. In recent years, the number of approved oligonucleotide-based drugs has increased significantly, as has the number of therapeutic oligonucleotides in clinical investigation (Roberts, T.C., Langer, R., & Wood, M.J. Nature Reviews Drug Discovery 2020 19:10 19, 673-694 (2020)).

[0005] In support of green synthesis initiatives across the pharmaceutical industry, there is a great need for next-generation oligonucleotide synthesis methods that are sustainable and economical at the scale required to reach broader patient populations (Mishra, M. et al. Current Research in Green and Sustainable Chemistry 4, (2021)).

[0006] To this end, biocatalysis is being applied more frequently in the production of active pharmaceutical ingredients (APIs) because enzymes are capable of highly selective transformations under mild reaction conditions and in aqueous media (Mann, G. & Stanger, FV Chimia (Aarau) 74, 407-417 (2020)). Biocatalysis of short oligonucleotide fragments offers a sustainable and economical alternative to the currently used solid-phase chemical synthesis of full-length therapeutic oligonucleotides.

[0007] Shorter oligonucleotides can be synthesized more easily and with higher purity than longer oligonucleotides, simplifying downstream processing and reducing solvent waste. These short oligonucleotide fragments can then be combined using nucleic acid ligase to generate the oligonucleotide product. Nucleic acid ligases have shown remarkable tolerance to non-natural DNA / RNA containing pharmaceutically relevant chemical modifications (Kestemont, D., Herdewijn, P. & Renders, M. Curr Protoc Chem Biol 11, e62 (2019); Kestemont, D. et al. Chemical Communications 54, 6408-6411 (2018); and Nandakumar, J. & Shuman, S. Molecular Cell 16, 211-221 (2004)). The use of dsRNA ligases to synthesize siRNA products starting from short fragments (≤9 nt) containing a wide range of chemical modifications, including 2'-OMe, 2'-F modified nucleotides, phosphorothioate backbone modified nucleotides, and terminal fragments functionalized with bulky N-acetylgalactosamine (GalNAc) moieties, has already been described (Mann, G. et al. Tetrahedron Letters 93,153696(2022)).

[0008] To achieve cost-effective and sustainable industrial-scale biocatalysis of oligonucleotides, enzymes that exhibit high ligase activity are required. There is an urgent unmet need for modified ligase enzymes that exhibit improved ligase activity compared to wild-type enzymes. There is also an unmet need for biocatalytic methods for producing oligonucleotides from oligonucleotide fragments. [Brief explanation of the drawings]

[0009] [Figure 1]dsRNA ligase catalyzed the following ligations: (A) oligonucleotide fragments 6:9, 7:10, and 11:12 were used to generate oligonucleotide 2:3 (= siRNA1); and (B) oligonucleotide fragments 6:9, 7:10, and 8:11 were used to generate oligonucleotide 5:3 (= siRNA4). The sequences of oligonucleotides 2, 3, and 5-12 are provided in Table 1. [Figure 2-1] Comparative data showing the relative % peak area of ​​siRNA (1) present in reaction samples containing different concentrations of wild-type enzyme (SEQ ID NO: 2) and modified enzymes (SEQ ID NOs: 288, 290, and 292) assayed under the following conditions: (A) Condition 1; and (B) Condition 2 (described in Example 13). Enzyme concentrations are provided as g / L of shake flask powder (SFP) produced by lyophilization of frozen clarified lysate, as described in the Examples. [Figure 2-2] (As mentioned above.) [Figure 3-1] (A) Comparative data showing the relative % peak area of ​​siRNA (1) present in reaction samples containing different concentrations of wild-type enzyme (SEQ ID NO: 2) and modified enzyme (SEQ ID NOs: 288 and 632) after 4 hours of enzyme preincubation at 4°C or 37°C. (B) Comparative data showing the residual enzyme activity after 4 hours of SFP preincubation at 37°C. Expressed relative to the ligation activity of SFP preincubated for 4 hours at 4°C. Enzyme concentrations are provided as g / L of shake flask powder (SFP) produced by lyophilization of frozen clarified lysate, as described in the Examples. [Figure 3-2] (As mentioned above.) Summary of the Invention

[0010] The present disclosure provides modified double-stranded RNA (dsRNA) ligase polypeptides. The disclosure also provides gene sequences of the modified polypeptides, recombinant expression vectors containing the genes, modified host strains, and efficient methods for their production, as well as biocatalytic reaction processes for oligonucleotides using the modified polypeptides.

[0011] The modified double-stranded RNA (dsRNA) ligase polypeptides described herein have improved catalytic activity compared to the wild-type dsRNA ligase from which they are derived. The modified polypeptides provided herein were derived from the wild-type dsRNA ligase from bacteriophage RB69 by substitution and / or deletion of amino acid residues in a directed evolution process. The wild-type dsRNA ligase consists of 332 amino acids and has the amino acid sequence set forth in SEQ ID NO: 302 (also accessible at UniProt under accession number Q7Y4V8).

[0012] The present disclosure provides SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 442, 444, 446, 448, 350, 352, 354, 2, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 540, 541, 54 6, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 63 Provided is a modified double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of 80, 582, 584, 586, 588, 590, 592, 594, 596, 598, and 600; wherein the modified dsRNA ligase polypeptide (a) has dsRNA ligase activity; and (b) does not comprise the amino acid sequence of SEQ ID NO: 302.

[0013] The present disclosure provides a modified double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668, wherein the modified dsRNA ligase polypeptide (a) has dsRNA ligase activity; and (b) does not comprise the amino acid sequence of SEQ ID NO: 302.

[0014] The present disclosure provides SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400 , 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 636, 638, Provided is a modified double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668, wherein the modified dsRNA ligase polypeptide (a) has dsRNA ligase activity; and (b) does not comprise the amino acid sequence of SEQ ID NO: 302.

[0015] The present disclosure provides SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400 , 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 636, 638, Provided is a modified double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 85% sequence identity to an amino acid sequence selected from the group consisting of 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668, wherein the modified dsRNA ligase polypeptide (a) has dsRNA ligase activity; and (b) does not comprise the amino acid sequence of SEQ ID NO: 302.

[0016] The present disclosure provides modified double-stranded RNA (dsRNA) ligase polypeptides, which are the following polypeptides: (a) SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382 , 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490 , 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598 or (b) a polypeptide having dsRNA ligase activity, comprising an amino acid sequence selected from the group consisting of (i)(a), (ii)(a), (iii)(a), (iv), (v), (vi), (vii), (viii), (viiii), (viiii), (v ...

[0017] The present disclosure provides modified double-stranded RNA (dsRNA) ligase polypeptides, which are: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668; or (b) a polypeptide having dsRNA ligase activity, comprising an amino acid sequence having at least 80% sequence identity to one of the polypeptides set forth in (i)(a) and (ii) having one or more amino acid residue substitutions, deletions, additions, or insertions relative to the one amino acid sequence set forth in (a), wherein the modified dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302.

[0018] The present disclosure provides modified double-stranded RNA (dsRNA) ligase polypeptides, which are the following polypeptides: (a) SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 5 08, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 65 or (b) a polypeptide having dsRNA ligase activity, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, and 368; or (b) a polypeptide having dsRNA ligase activity, comprising an amino acid sequence having at least 80% sequence identity to one of the polypeptides set forth in (i)(a) and having one or more amino acid substitutions, deletions, additions, or insertions relative to said one amino acid sequence set forth in (ii)(a), wherein the modified dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302.

[0019] The present disclosure provides SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 4 24, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 48 6, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668. provided, wherein (a) the modified dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X6 is G or E; X7 is Q; X15 is R, D, or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X44 is V; X45 is V; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; and X57 is S;X60 is T, G, or P; X63 is S, Q, or G; X64 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; X87 is T, P, K, or absent; X88 is C; X89 is T; X91 is S; X92 is D; X93 is G, C, or A; X103 is V, C, Y, or T; X105 is V; X107 is R or T; X114 is N; X122 is W; X126 is G; X129 is N; X130 is R, S or Y; X131 is R; X137 is V or C; X144 is N; X146 is R; X158 is W; X163 is G; X173 is L; X178 is R; X185 is K; X190 is Q; X196 is S or C; X216 is L or R; X221 is I; X228 is R; X230 is T; X232 is R; X235 is A, T, or G; X236 is S, L, or F; X237 is S, Q, R, L, or G; X238 is F; X239 is G or R; X242 is R or M; X243 is N, S, G, or M; X244 is G or K; X251 is D or L; X252 is V; and X254 is K. X255 is C; X258 is V; X269 is L; X280 is W; X284 is A; X285 is A; X293 is R; X296 is R; X301 is G, L, E, or F; X303 is Q; X305 is G; X313 is A; X314 is A or V; X325 is R; and X328 is R (numbering refers to SEQ ID NO: 302).

[0020] In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590, and 592. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 666. In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590, 592, and 666.

[0021] The present disclosure provides a modified double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590, 592, and 666; wherein (a) the modified dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.

[0022] The present disclosure provides a modified double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 666; wherein (a) the modified dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all ten) of the following amino acid residues: X15 is D; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.

[0023] The present disclosure provides a modified double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 370; wherein (a) the modified dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more (e.g., two or more, three or more, or all four) of the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I.

[0024] The present disclosure provides a modified double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 488; wherein (a) the modified dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more (e.g., two or more, or all three) of the following amino acid residues: X39 is A; X218 is N; and X221 is I.

[0025] The present disclosure provides a modified double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 526; wherein (a) the modified dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more (e.g., two or more, three or more, or all four) of the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C.

[0026] The present disclosure provides a modified double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 578; wherein (a) the modified dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, or all eight) of the following amino acid residues: X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.

[0027] The present disclosure provides a modified double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 588 or 590; wherein (a) the modified dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all nine) of the following amino acid residues: X15 is D or E; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.

[0028] The present disclosure provides a modified double-stranded RNA (dsRNA) ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 592; wherein (a) the modified dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all nine) of the following amino acid residues: X19 is D; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.

[0029] In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668. In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 642, 646, 664, and 666.

[0030] The present disclosure also provides a modified dsRNA ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 302, wherein the modified dsRNA ligase polypeptide produces at least 5% more oligonucleotide products under the same ligation reaction conditions than a dsRNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO: 302, wherein the modified dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302.

[0031] In some embodiments, ligation reaction conditions include about 1 μM to about 10 mM of oligonucleotide fragment, an ATP source, about 5 mM to about 100 mM divalent cations, and about 0.5 g / L to about 10 g / L of modified dsRNA ligase polypeptide, a pH of about 4.0 to about 8.0, and a temperature of about 10° C. to about 50° C. In some embodiments, the ATP source includes ATP, optionally including a stoichiometric excess of ATP. In some embodiments, the ATP source includes: (a) polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and / or ATP.

[0032] In some embodiments, the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from the following: X6, X7, X15, X19, X29, X36, X39, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X91, X93, X103, X105, X107, X114, X122, X126, X129, X130, X131 , X137, X144, X146, X158, X163, X173, X178, X190, X196, X216, X218, X221, X228, X230, X232, X235, X236, X237, X238, X239, X242, X243, X244, X251, X252, X254, X255, X258, X269, X280, X284, X285, X293, X296, X301, X303, X305, X314, X325, and X328 (numbering refers to SEQ ID NO: 302).

[0033] In some embodiments, the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from the following: X6, X7, X15, X19, X29, X36, X39, X44, X45, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X89, X91, X92, X93, X103, X105, X107, X114, X122, X126, X129, X130, X131, X132, X133, X134, X135, X136, X137, X138, X139, X140, X141, X142, X143, X144, X145, X146, X147, X148, X149, X150, X151, X152, X153, X154, X155, X156, X157, X158, X159, X160, X161, X162, X163, X164, X165, X166, X167, X168, X169, X170, X171, X172, X173, X174, X175, X176, X177, X178, X179, X180, X181, X182, X183, X184, X185, X186, X187, X188, X189, X191, X192, X193, X1 131, X137, X144, X146, X158, X163, X173, X178, X185, X190, X196, X216, X218, X221, X228, X230, X232, X235, X236, X237, X238, X239, X242, X243, X244, X251, X252, X254, X255, X258, X269, X280, X284, X285, X293, X296, X301, X303, X305, X313, X314, X325, and X328 (numbering refers to SEQ ID NO: 302).

[0034] In some embodiments, the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X6 is G; X7 is Q; X15 is R, D, or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; X57 is S; X60 is T, G, or P; X63 is S, Q, or G; X6 X4 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; X87 is T, P, K, or absent; X88 is C; X91 is S; X93 is G, C, or A; X103 is V, C, Y, or T; X105 is V; X107 is R or T; X114 is N; X122 is W; X126 is G; X129 is N; X130 is R, S, or Y; X131 is R; X137 is V or C; X144 is N; X146 is X158 is W; X163 is G; X173 is L; X178 is R; X190 is Q; X196 is S or C; X216 is L or R; X218 is N; X221 is I; X228 is R; X230 is T; X232 is R; X235 is A, T, or G; X236 is S, L, or F; X237 is S, Q, or R; X238 is F; X239 is G or R; X242 is R or M; X243 is N, S, G or M; X244 is G or K; X251 is D or L; X252 is V; X254 is K; X255 is C; X258 is V; X269 is L; X280 is W; X284 is A; X285 is A; X293 is R; X296 is R; X301 is G, L, E, or F; X303 is Q; X305 is G; X314 is A or V; X325 is R; X328 is R; (numbering refers to SEQ ID NO: 302).

[0035] In some embodiments, the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X6 is G or E; X7 is Q; X15 is R, D, or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X44 is V; X45 is V; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; X57 is S; X60 is T, G, or P; and X63 is S, Q, or G. X64 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; X87 is T, P, K or absent; X88 is C; X89 is T; X91 is S; X92 is D; X93 is G, C, or A; X103 is V, C, Y, or T; X105 is V; X107 is R or T; X114 is N; X122 is W; X126 is G; X129 is N; X130 is R, S, or Y; X131 is R; X137 is V or C; and X144 is N. Yes; X146 is R; X158 is W; X163 is G; X173 is L; X178 is R; X185 is K; X190 is Q; X196 is S or C; X216 is L or R; X218 is N; X221 is I; X228 is R; X230 is T; X232 is R; X235 is A, T, or G; X236 is S, L, or F; X237 is S, Q, R, L, or G; X238 is F; X239 is G or R; X242 is R or M; X243 is N, S, G, or M; X244 is G or K; X251 is D or L; X252 is V; X254 is K; X255 is C; X258 is V; X269 is L; X280 is W; X284 is A; X285 is A; X293 is R; X296 is R; X301 is G, L, E, or F; X303 is Q; X305 is G; X313 is A; X314 is A or V; X325 is R; and X328 is R (numbering refers to SEQ ID NO: 302).

[0036] In some embodiments, the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from the following: X15, X19, X36, X39, X53, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302); wherein the modified dsRNA ligase polypeptide has dsRNA ligase activity.

[0037] In some embodiments, the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A (numbering refers to SEQ ID NO: 302).

[0038] In some embodiments, the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from the following: X15, X19, X36, X39, X53, X185, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302); wherein the modified dsRNA ligase polypeptide has dsRNA ligase activity.

[0039] In some embodiments, the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A (numbering refers to SEQ ID NO: 302).

[0040] In some embodiments, the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more (e.g., two or more, three or more, or all four) amino acid residues selected from the following: X36, X39, X218, and X221 (numbering refers to SEQ ID NO: 302); wherein the modified dsRNA ligase polypeptide has dsRNA ligase activity, and optionally the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more (e.g., two or more, three or more, or all four) of the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I.

[0041] In some embodiments, the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more (e.g., two or more, or all three) amino acid residues selected from the following: X39, X218, and X221 (numbering refers to SEQ ID NO: 302); wherein the modified dsRNA ligase polypeptide has dsRNA ligase activity, and optionally the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more (e.g., two or more, or all three) of the following amino acid residues: X39 is A; X218 is N; and X221 is I.

[0042] In some embodiments, the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more (e.g., two or more, three or more, or all four) amino acid residues selected from the following: X39, X218, X221, and X255 (numbering refers to SEQ ID NO: 302); wherein the modified dsRNA ligase polypeptide has dsRNA ligase activity, and optionally the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more (e.g., two or more, three or more, or all four) of the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C.

[0043] In some embodiments, the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, or all eight) amino acid residues selected from the following: X39, X53, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302); The gauze polypeptide has dsRNA ligase activity, and optionally, the amino acid sequence of the modified dsRNA ligase polypeptide includes one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, or all eight) of the following amino acid residues: X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.

[0044] In some embodiments, the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all nine) amino acid residues selected from the following: X15, X39, X53, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302); The modified dsRNA ligase polypeptide has dsRNA ligase activity, and optionally, the amino acid sequence of the modified dsRNA ligase polypeptide includes one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all nine) of the following amino acid residues: X15 is E; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.

[0045] In some embodiments, the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all nine) amino acid residues selected from the following: X19, X39, X53, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302); The modified dsRNA ligase polypeptide has dsRNA ligase activity, and optionally, the amino acid sequence of the modified dsRNA ligase polypeptide includes one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, or all nine) of the following amino acid residues: X19 is D; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.

[0046] In some embodiments, the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all ten) amino acid residues selected from the following: X15, X39, X53, X185, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302); The peptide has dsRNA ligase activity, and optionally, the amino acid sequence of the modified dsRNA ligase polypeptide includes one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or all ten) of the following amino acid residues: X15 is D; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.

[0047] In some embodiments, the modified dsRNA ligase polypeptide comprises a purification tag. In some embodiments, the modified dsRNA ligase polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 138, 140, 142, 144, 146, 148, 149, 150, 151, 152, 153 2, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 62, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236 , 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, and 300. In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, and 634.In some embodiments, the modified dsRNA ligase polypeptide is selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178 , 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262 , 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, and 634.

[0048] The present disclosure also provides a polypeptide immobilized on a solid material by chemical bonding or physical adsorption methods, wherein the polypeptide comprises a modified dsRNA ligase polypeptide described herein.

[0049] The present disclosure also provides polynucleotides that encode the modified dsRNA ligase polypeptides described herein.

[0050] In some embodiments, the polynucleotide is selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219 41, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 20 3, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265 , 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 392, 393, 394, 395, 396, 397, 398, 399, 399, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 93, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 45 5, 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517,519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, and 599.

[0051] In some embodiments, the polynucleotide comprises a nucleic acid sequence selected from SEQ ID NOs: 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, and 667.

[0052] In some embodiments, the polynucleotide comprises a nucleic acid sequence selected from the following: (a) SEQ ID NOs: 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367; 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 45 7, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, and 667;and / or (b) SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 139, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 1 01, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 1 79, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 2 57, 259, 261, 263, 265, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, and 633. ;

[0053] The present disclosure also provides expression vectors comprising the polynucleotides described herein. In some embodiments, the vector comprises a plasmid, cosmid, bacteriophage, or viral vector.

[0054] The present disclosure also provides host cells comprising the polynucleotides described herein or the expression vectors described herein. In some embodiments, the host cell is E. coli.

[0055] The present disclosure also provides a method for preparing a modified dsRNA ligase polypeptide, comprising culturing a host cell described herein and obtaining the modified dsRNA ligase polypeptide from the culture.

[0056] The present disclosure also provides modified dsRNA ligase catalysts obtained by culturing the host cells described herein or according to the methods described herein, including cells or culture media containing the modified dsRNA ligase polypeptide, or articles treated therewith, wherein the article refers to an extract obtained from the culture of the host cells, an isolated product obtained by isolating or purifying the modified dsRNA ligase from the extract, or an immobilized product obtained by immobilizing the host cells, the extract, or an isolated product of the extract.

[0057] The present disclosure further provides a method for producing an oligonucleotide from two or more oligonucleotide fragments, the method comprising contacting (i) the two or more oligonucleotide fragments; (ii) a modified dsRNA ligase polypeptide described herein; (iii) a source of ATP; and (iv) a divalent cation to obtain an oligonucleotide.

[0058] In some embodiments, the ATP source comprises ATP.

[0059] In some embodiments, the ATP source comprises: (a) polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and / or ATP. In some embodiments, the PPK is selected from PPK12 or ajPAP.

[0060] In some embodiments, the method is performed using sub-stoichiometric concentrations of AMP and / or ATP.

[0061] In some embodiments, the polyphosphate is a polyphosphate salt. In some embodiments, the polyphosphate salt is sodium polyphosphate (Madrell's salt) or sodium hexametaphosphate (Graham's salt).

[0062] In some embodiments, the divalent cation cofactor is Mg 2+ or Mn 2+ is.

[0063] In some embodiments, the method is carried out at a divalent cation concentration of 5 to 100 mM, optionally 30 to 50 mM.

[0064] In some embodiments, the method further comprises purifying the oligonucleotide.

[0065] The present disclosure also provides the use of a modified dsRNA ligase polypeptide described herein in generating an oligonucleotide from two or more oligonucleotide fragments.

[0066] In some embodiments, the oligonucleotide is up to 60 nucleotides in length.

[0067] In some embodiments, the oligonucleotide fragments are each 4-16 nucleotides in length, optionally 6-9 nucleotides in length.

[0068] In some embodiments, one or more of the oligonucleotide fragments comprises one or two overhangs.

[0069] In some embodiments, one or more of the oligonucleotide fragments comprises a chemical modification selected from: (a) a modified backbone, optionally selected from phosphorothioate (e.g., chiral phosphorothioate) or methylphosphonate internucleotide linkages; (b) optionally 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON modified nucleotides selected from 2'-O-methylacetamide (2'-O-NMA), locked nucleic acid (LNA), glycol nucleic acid (GNA), phosphoramidate (e.g., mesyl phosphoramidate), 2',3'-seconucleotide mimics, 2'-F-arabinonucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, vinyl phosphonates (e.g., 5' vinyl phosphonate), and cyclopropyl phosphonate deoxyribonucleotides; and / or (c) conjugation to a ligand (optionally, the ligand comprises one or more N-acetylgalactosamine (GalNAc) derivatives).

[0070] The present disclosure also provides a composition comprising: i. a modified dsRNA ligase polypeptide described herein; ii. a source of ATP; and iii. a divalent cation.

[0071] In some embodiments, the composition further comprises two or more oligonucleotide fragments.

[0072] The present disclosure also provides a kit comprising: i. a modified dsRNA ligase polypeptide described herein; ii. a source of ATP; iii. a divalent cation; and iv. instructions for use in a method of generating an oligonucleotide from two or more oligonucleotide fragments.

[0073] In some embodiments, the ATP source comprises ATP.

[0074] In some embodiments, the ATP source comprises: (a) polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and / or ATP.

[0075] In some embodiments, the PPK is selected from PPK12 or ajPAP.

[0076] In some embodiments, the polyphosphate is a polyphosphate salt.

[0077] In some embodiments, the polyphosphate is sodium polyphosphate (Madrell's salt) or sodium hexametaphosphate (Graham's salt).

[0078] In some embodiments, the divalent cation cofactor is Mg 2+ or Mn 2+ is.

[0079] definition Unless otherwise clearly defined, the technical and scientific terms used in this disclosure have the meanings commonly understood by those skilled in the art to which this invention belongs. The following references provide those skilled in the art with general definitions of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The HarperCollins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless otherwise specified.

[0080] As used throughout this disclosure, the articles "a" and "an" refer to one or to more than one (at least one) of the grammatical object of the article.

[0081] The term "and / or" means "and" or "or" unless otherwise indicated.

[0082] As used herein, the term "about" typically refers to the value that immediately follows the term "about." For example, "about 15 or more nucleotides" typically refers to 15 or more nucleotides. In some embodiments, the term "about" encompasses values ​​that are + / - 1, 2, or 3 of the stated value. For example, "about 15 or more nucleotides" can refer to 15 + / - 3 nucleotides, e.g., 12, 13, 14, 15, 16, 17, or 18 nucleotides.

[0083] The terms "double-stranded RNA ligase" and "dsRNA ligase" are used interchangeably herein to refer to an enzyme having dsRNA ligase activity. A dsRNA ligase polypeptide may also be referred to herein as a "dsRNA ligase catalyst."

[0084] The dsRNA ligase of the present invention is an ATP-dependent nucleic acid ligase. As used herein, dsRNA ligase activity typically involves the ATP-dependent formation of a covalent bond between the 3'-OH of a ribonucleotide and the 5'-PO4 of a ribonucleotide or deoxyribonucleotide through the following steps: (1) the dsRNA ligase reacts with ATP to form a covalent dsRNA ligase-AMP intermediate and release pyrophosphate; (2) AMP is transferred from the dsRNA ligase-AMP intermediate to the 5'-phosphate of the 3' oligonucleotide fragment to form an adenylated oligonucleotide intermediate; and (3) the 3'-OH of the 5' oligonucleotide fragment attacks the 5' phosphate of the adenylated intermediate, thereby forming a phosphodiester bond and releasing AMP.

[0085] The stoichiometric concentration of a cofactor is the theoretical concentration required to achieve complete ligation in a given ligation reaction. Those skilled in the art can easily derive the stoichiometric concentration of ATP required to achieve complete ligation based on the concentration of the oligonucleotide fragments and the number of ligation reactions required to produce the oligonucleotide product. For example, a ligation reaction using 1 mM substrate that requires four ligation reactions has a stoichiometric ATP concentration of 4 mM. A stoichiometric excess of ATP can help ensure complete ligation. In some embodiments, the stoichiometric excess includes at least 105% of the theoretical stoichiometric concentration of ATP required to achieve complete ligation, such as at least 110%, at least 115%, at least 120%, at least 125%, at least 130%, at least 135%, at least 140%, at least 145%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, or at least 200%.

[0086] The terms "modified dsRNA ligase," "modified dsRNA ligase polypeptide," "improved dsRNA ligase polypeptide," and "modified polypeptide" are used interchangeably herein.

[0087] As used herein, the term "oligonucleotide" refers to a nucleic acid typically containing up to 100 nucleotides. As used herein, the term "oligonucleotide product" refers to an oligonucleotide formed by ligation of two or more oligonucleotide fragments with a dsDNA ligase described herein. An oligonucleotide product is also referred to herein simply as an oligonucleotide. It is understood that the oligonucleotide products described herein include RNA. It is also understood that the oligonucleotide products described herein include a double-stranded region. In some embodiments, the oligonucleotide products described herein include RNA and DNA. For example, a portion of the oligonucleotide product can be double-stranded DNA, and another portion is double-stranded RNA forming a DNA-RNA chimera.

[0088] The term "therapeutic oligonucleotide" refers to an oligonucleotide that can provide a therapeutic effect, for example, by interacting with a biomolecule and / or regulating gene expression. Therapeutic oligonucleotides include, but are not limited to, RNA interference (RNAi) agents and antisense oligonucleotides (ASOs). RNAi is a post-transcriptional targeted gene silencing technique that uses an RNAi agent to degrade messenger RNA (mRNA) containing the same sequence as the RNAi agent. ASOs are single-stranded nucleic acids that can be used to target mRNA derived from a gene of interest. ASOs can alter gene expression through many mechanisms, including direct steric blocking of mRNA and RNase H-mediated degradation of mRNA.

[0089] Non-limiting examples of RNAi agents include siRNA (small interfering RNA), dsRNA (double-stranded RNA), shRNA (short hairpin RNA), and miRNA (microRNA). RNAi agents also include, by way of further non-limiting example, locked nucleic acids (LNA), morpholinos, UNAs, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), and fluoro-arabino nucleic acids (FANAs). RNAi agents also include molecules in which one or more strands are a mixture of RNA, DNA, LNAs, morpholinos, UNAs (unlocked nucleic acids), TNAs, GNAs, and / or FANAs. As a non-limiting example, one or both strands of an RNAi agent can be RNA, except that, for example, one or more RNA nucleotides are replaced with DNA, LNAs, morpholinos, UNAs, TNAs, GNAs, and / or FANAs. In some embodiments, one or both strands of the RNAi agent can be nicked, and both strands can be the same length, or one strand can be shorter than the other. The oligonucleotides of the invention can be any of the RNAi agents described herein.

[0090] As used herein, the term "oligonucleotide fragment" refers to a nucleic acid that can be ligated to one or more additional oligonucleotide fragments to provide an oligonucleotide (or oligonucleotide product). Each oligonucleotide fragment corresponds to a portion of an oligonucleotide product. An oligonucleotide fragment may be referred to herein as a "substrate" for a ligation reaction.

[0091] As described above, dsRNA ligase activity involves ligating a 5' oligonucleotide fragment with a 3' oligonucleotide fragment. In the context of oligonucleotide fragments, the prefixes 5' and 3' refer to the relative positions of each oligonucleotide fragment in the oligonucleotide product after ligation, with the 5' oligonucleotide fragment being located upstream of the 3' oligonucleotide fragment (when the oligonucleotide product is presented in the 5' to 3' direction). As used herein, a "5' oligonucleotide fragment" typically comprises a 3'-terminal ribonucleotide having a 3'-hydroxyl group. As used herein, a "3' oligonucleotide fragment" comprises a 5'-phosphate, where the 5'-terminal nucleotide is a deoxyribonucleotide or a ribonucleotide.

[0092] It will be appreciated that in some embodiments, the oligonucleotide fragments can be a 3' oligonucleotide fragment and a 5' oligonucleotide fragment (e.g., ligation reactions occur at the 5' and 3' ends of the oligonucleotide fragment). For example, the oligonucleotide fragments can provide: (i) a 3' oligonucleotide fragment in a ligation reaction with a 5' oligonucleotide fragment; and (ii) a 5' oligonucleotide fragment in a ligation reaction with a 3' oligonucleotide fragment. For example, oligonucleotide fragment 7 in Figure 1A can provide (i) a 3' oligonucleotide fragment in a ligation reaction with 5' oligonucleotide fragment 6, and (ii) a 5' oligonucleotide fragment in a ligation reaction with 3' oligonucleotide fragment 12 to provide oligonucleotide product 2.

[0093] As used herein, a "terminal oligonucleotide fragment" refers to a nucleic acid corresponding to a terminal (e.g., 5'- or 3'-terminal) portion of an oligonucleotide product. The 5'-terminal oligonucleotide fragment typically provides a 3'-oligonucleotide fragment for ligation to a 5'-oligonucleotide fragment. The 3'-terminal oligonucleotide fragment typically provides a 5'-oligonucleotide fragment for ligation to a 3'-oligonucleotide fragment. In some embodiments, the 5'-terminal oligonucleotide is ligated directly to the 3'-terminal oligonucleotide. In some embodiments, the 5'-terminal oligonucleotide and the 3'-terminal oligonucleotide are separated by one or more oligonucleotide fragments.

[0094] In some embodiments, the oligonucleotide fragments described herein comprise RNA and DNA, for example, one portion of the oligonucleotide fragment can be double-stranded DNA and another portion is double-stranded RNA forming a DNA-RNA chimera.

[0095] The term "overhang" or "nucleotide overhang" as used herein refers to at least one unpaired nucleotide that protrudes from the end of at least one of the two strands of a double-stranded oligonucleotide. In some embodiments, when the 3'-end of one strand extends beyond the 5'-end of the other strand, or vice versa, a nucleotide overhang is formed, for example, an unpaired nucleotide forms an overhang. An overhang that is complementary to the overhang of a second oligonucleotide fragment may be referred to as a "sticky end." The oligonucleotide fragments described herein may have one or two sticky ends.

[0096] "Blunt" or "blunt-ended" refers to the absence of unpaired nucleotides at the ends of a double-stranded oligonucleotide, i.e., the absence of nucleotide overhangs. A "blunt-ended" oligonucleotide or oligonucleotide fragment is an oligonucleotide that is double-stranded throughout its entire length, i.e., has no nucleotide overhangs at either end of the molecule.

[0097] A double-stranded nucleic acid comprises two antiparallel and substantially complementary nucleic acid strands, referred to as "sense" and "antisense" strands. In the context of double-stranded RNAi agents, "antisense strand" refers to the strand of RNAi that comprises a region that is substantially complementary to a target sequence, such as an mRNA sequence. "Sense strand" refers to the strand of RNAi that comprises a region that is substantially complementary to a region of the antisense strand. The sense strand and antisense strand of an RNAi agent can be referred to as passenger strand and guide strand, respectively.

[0098] "Substantially complementary" sequences may be perfectly complementary or may contain one or more mismatches upon hybridization, while retaining the ability to hybridize under conditions most relevant to their end use.

[0099] "Conversion" refers to the enzymatic conversion of a substrate to a corresponding product. "Percent conversion" or "conversion" refers to the proportion of oligonucleotide fragments that are converted to oligonucleotide products under specific conditions within a given time period. Thus, the "enzymatic activity" or "activity" of a ligase can be expressed as the "conversion rate" of oligonucleotide fragments to oligonucleotide products.

[0100] Ideally, to compare activity between ligation reactions and account for natural variations in peak intensity between injections, the percent conversion to product can be calculated for each sample analyzed using the following formula:

number

number

[0101] "Improved enzyme properties" refer to enzyme properties that are better or more desirable for a particular purpose compared to a reference dsRNA ligase, such as a wild-type dsRNA ligase, or another modified dsRNA ligase under the same reaction conditions. Improved enzyme properties are exhibited by the modified dsRNA ligase polypeptides of the present disclosure. The modified dsRNA ligase polypeptides described herein exhibit increased enzyme activity (which can be expressed as substrate conversion rate). Additional enzyme properties that may be improved include, but are not limited to, thermal stability, pH activity characteristics, cofactor requirements, and tolerance to inhibitors (e.g., inhibition of reaction components, substrates, or products).

[0102] An "isolated polypeptide" refers to a polypeptide that has been substantially separated from other substances with which it is naturally associated, such as proteins, lipids, and polynucleotides. This term includes polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., a host cell or in vitro synthesis). Modified dsRNA ligase polypeptides may be present intracellularly, in cell culture medium, or prepared in various forms, such as a lysate or isolated preparation. Thus, in some embodiments, a modified dsRNA ligase polypeptide may be an isolated polypeptide.

[0103] "Wild type" refers to the form found in nature.For example, wild type polypeptide or polynucleotide sequence can be isolated from natural source, and is the sequence present in organism, not intentionally modified by manual procedure.The polypeptide sequence of wild type dsRNA ligase described herein is provided in SEQ ID NO: 302.As used herein, wild type sequence can also include purification tag, and can be provided by SEQ ID NO: 2.

[0104] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0105] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently joined by amide bonds, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.).

[0106] "Recombinant" or "modified," for example, when used with respect to a cell, nucleic acid, or polypeptide, refers to material that does not otherwise occur in nature or that corresponds to a native or naturally occurring form of the material that is identical to but has been modified in a way that is produced or derived from synthetic material and / or by manipulation using recombinant techniques.

[0107] The abbreviations used for the genetically encoded amino acids are conventional and are as follows:

[0108] [Table 1]

[0109] When three-letter abbreviations are used, the amino acid may be in either the L- or D-configuration about the α-carbon (Cα) unless specifically preceded by "L" or "D" or unless otherwise clear from the context in which the abbreviation is used. For example, "Ala" refers to alanine without specifying configuration about the α-carbon, while "D-Ala" and "L-Ala" refer to D-alanine and L-alanine, respectively.

[0110] When single-letter abbreviations are used, an uppercase letter indicates an amino acid in the L-configuration about the α-carbon, and a lowercase letter indicates an amino acid in the D-configuration about the α-carbon. For example, "A" indicates L-alanine and "a" indicates D-alanine. When a polypeptide sequence is presented as a string of one-letter or three-letter abbreviations (or mixtures thereof), the sequence is presented in amino (N) to carboxy (C) orientation, according to common convention.

[0111] The abbreviations used for genetically encoded nucleotides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically indicated, abbreviated nucleotides may be ribonucleotides or 2'-deoxyribonucleotides. Nucleotides may be identified as ribonucleotides or 2'-deoxyribonucleotides, individually or collectively. When nucleic acid sequences are presented as strings of single-letter abbreviations, the sequences are presented in the 5' to 3' direction, according to common convention, and phosphodiester bonds are not shown.

[0112] Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be substituted with other moieties without substantially changing the base pairing properties of the oligonucleotide that comprises the nucleotide with such substituted moieties.For example, but not limited to, the nucleotide that comprises inosine as its base can base pair with the nucleotide that contains adenine, cytosine or uracil.Therefore, the nucleotide that contains uracil, guanine or adenine can be replaced by the nucleotide that contains inosine, for example, in the nucleotide sequence of the oligonucleotide that is characterized in the present disclosure.In another example, adenine and cytosine can be replaced by guanine and uracil, respectively, at any position in oligonucleotide, to form wobble base pairs with target mRNA.

[0113] An "amino acid difference" or "residue difference" refers to an amino acid residue difference at a position in a polypeptide sequence relative to the amino acid residue at the corresponding position in a reference sequence. The position of an amino acid difference is generally referred to herein as "Xn," where n refers to the corresponding position in the reference sequence on which the residue difference is based. For example, a "residue difference at position X6 compared to SEQ ID NO:302" refers to an amino acid residue difference at a polypeptide position corresponding to position 6 of SEQ ID NO:302. Thus, if a reference polypeptide of SEQ ID NO:302 has a serine at position 6, then a "residue difference at position X2 compared to SEQ ID NO:302" refers to an amino acid substitution to a residue other than serine at a polypeptide position corresponding to position 6 of SEQ ID NO:302.

[0114] A specific amino acid residue difference at that position may be indicated as "XnY" or "Xn is Y," where "Xn" designates the corresponding position in the reference sequence above and "Y" is the one-letter identifier of the residue present at that position in the modified polypeptide. A specific amino acid difference may be denoted by the conventional notation "AnY," where A is the one-letter identifier of the residue in the reference sequence, "n" is the number of the residue position in the reference sequence, and "Y" is the one-letter identifier of the residue present at that position in the modified polypeptide.

[0115] In some examples, a modified polypeptide of the disclosure may contain one or more amino acid residue differences relative to a reference sequence, as indicated in the list of particular positions where the residue difference relative to the reference sequence occurs. In some embodiments, more than one amino acid residue may be used at a particular residue position in a modified polypeptide, and various amino acid residues may be listed as alternatives (e.g., "X19 is Q or D").

[0116] An amino acid deletion may be represented by "-", for example, an "amino acid sequence comprising Xn-" indicates that the amino acid sequence contains a deletion at the position corresponding to "Xn" in the reference sequence. "Deletion" refers to modification of a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions can include removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty or more amino acids, up to 10% of the total number of amino acids in the enzyme, or up to 20% of the total number of amino acids comprising the reference enzyme, while retaining the enzymatic activity of the modified dsRNA ligase and / or retaining improved properties of the modified dsRNA ligase. Deletions can include internal and / or terminal portions of the polypeptide. In various embodiments, deletions can include continuous segments or can be discontinuous.

[0117] In the context of the numbering of a given amino acid or polynucleotide sequence, "corresponding to," "reference to," or "compared to" refers to the numbering of residues in a particular reference when comparing a given amino acid or polynucleotide sequence to a reference sequence. In other words, residue numbers or residue positions in a given sequence are specified relative to the reference sequence, not by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as a modified dsRNA ligase, can be aligned with a reference sequence by introducing gaps to optimize residue matching between the two sequences. In such cases, despite the gaps, the numbering of residues in a given amino acid or polynucleotide sequence is done relative to the aligned reference sequence.

[0118] A "reference sequence" refers to a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset of a larger sequence, such as a full-length gene or a fragment of a polypeptide sequence. In some embodiments, a "reference sequence" is a wild-type sequence. In some embodiments, a "reference sequence" is an altered or modified sequence.

[0119] Methods for determining the percentage of sequence identity are known in the art. As an example, when assessing sequence identity, a sequence having a specified number of consecutive nucleotides or amino acids can be aligned with a nucleic acid or peptide sequence (having the same number of consecutive nucleotides or amino acids) from a corresponding portion of the nucleic acid or peptide sequence disclosed herein. The percentage of sequence identity can be calculated by determining the number of positions in both sequences where the same nucleic acid base or amino acid residue exists or where the nucleic acid base or amino acid residue aligns with a gap to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the sequence, and multiplying the result by 100 to obtain the percentage of sequence identity. Those skilled in the art will understand that there are many established algorithms available for aligning two sequences. Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Package), or by visual inspection (see generally Current Protocols in Molecular Biology, F.M. Ausubel et al. eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1995 Supplement) (Ausubel)).Examples of suitable algorithms for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms described in Altschul et al., 1990, J. Mol. Biol. 215:403-410 and Altschul et al., 1977, Nucleic Acids Res. 3389-3402, respectively. Software for performing BLAST analyses is publicly available from the website of the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that, when aligned with words of the same length in a database sequence, match or satisfy a certain positive threshold score T. T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. Word hits are extended in either direction along each sequence for as far as the cumulative alignment score can be increased. For nucleotide sequences, cumulative scores are calculated using the parameters M (reward score for a pair of matching residues; always > 0) and N (penalty score for mismatching residues; always < 0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is halted when the cumulative alignment score falls by an amount X from its achieved maximum value; when the cumulative score falls to 0 or below due to the accumulation of alignment of one or more negative-scoring residues; or when either end of the sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses defaults of a word length (W) of 11, an expectation (E) of 10, M=5, and N=-4, and performs a comparison of both strands.The BLASTP program for amino acid sequences uses as defaults a word length of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:10915). Exemplary sequence alignments and determination of percent sequence identity can use the BESTFIT or GAP programs using the default parameters provided in the GCG Wisconsin software package (Accelrys, Madison WI).

[0120] It will be understood that regardless of the percent sequence identity to the reference sequence, the modified dsRNA ligase has dsRNA ligase activity.

[0121] "Suitable reaction conditions" refer to conditions in a reaction system (e.g., enzyme load, substrate load, temperature, pH, etc.) under which a substrate is converted into a desired product. Suitable reaction conditions can be readily identified by one skilled in the art. Examples of "suitable reaction conditions" are provided in the present disclosure and illustrated by the Examples.

[0122] Modified dsRNA ligase polypeptides The present disclosure provides SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 442, 80, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542 , 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, and 600.

[0123] The present disclosure provides a modified dsRNA ligase polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668.

[0124] The present disclosure provides SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 442, 444, 446, 448, 350, 352, 8, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 477 8, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 567 8, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668.

[0125] The present invention also relates to oligonucleotides having dsRNA ligase activity and selected from the group consisting of SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 428, 429, 430, 431, 432, 433, 434, 435, 436, 438, 440, 2, 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474 , 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566 , 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, and 600, wherein the modified dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302.

[0126] The present invention also provides a modified dsRNA ligase polypeptide having dsRNA ligase activity and comprising an amino acid sequence having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668, wherein the modified dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302.

[0127] The present invention also relates to a method for the preparation of oligonucleotides having dsRNA ligase activity and comprising administering to the adult human anthraquinone-containing polypeptides SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 428, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352 , 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 540, 541, 542 92, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 59 2, 594, 596, 598, 600, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668, wherein the modified dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302.

[0128] The present disclosure provides modified double-stranded RNA (dsRNA) ligase polypeptides, which are the following polypeptides: (a) SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382 , 384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490 , 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598 or (b) a polypeptide having dsRNA ligase activity, comprising an amino acid sequence selected from the group consisting of (i)(a), (ii)(a), (iii)(a), (iv), (v), (vi), (vii), (viii), (viiii), (viiii), (v ...

[0129] The present disclosure provides modified double-stranded RNA (dsRNA) ligase polypeptides, which are: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668; or (b) a polypeptide having dsRNA ligase activity, comprising an amino acid sequence having at least 80% sequence identity to one of the polypeptides set forth in (i)(a) and (ii) having one or more amino acid residue substitutions, deletions, additions, or insertions relative to the one amino acid sequence set forth in (a), wherein the modified dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302.

[0130] The present disclosure provides modified double-stranded RNA (dsRNA) ligase polypeptides, which are the following polypeptides: (a) SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 5 08, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 65 or (b) a polypeptide having dsRNA ligase activity, comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, and 368; or (b) a polypeptide having dsRNA ligase activity, comprising an amino acid sequence having at least 80% sequence identity to one of the polypeptides set forth in (i)(a) and having one or more amino acid substitutions, deletions, additions, or insertions relative to said one amino acid sequence set forth in (ii)(a), wherein the modified dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302.

[0131] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence having at least 85% sequence identity to the even-numbered sequence identifiers of SEQ ID NOs:304-600, and optionally at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the even-numbered sequence identifiers of SEQ ID NOs:304-600. In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence having at least 80% sequence identity to the even-numbered sequence identifiers of SEQ ID NOs:304-600, and optionally at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the even-numbered sequence identifiers of SEQ ID NOs:304-600.

[0132] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence having at least 80% sequence identity to the even-numbered sequence identifiers of SEQ ID NOs:636-668, and optionally at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the even-numbered sequence identifiers of SEQ ID NOs:636-668.

[0133] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence having at least 80% sequence identity to the even-numbered sequence identifiers of SEQ ID NOs: 304-600 or 636-668, and optionally at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to the even-numbered sequence identifiers of SEQ ID NOs: 304-600 or 636-668.

[0134] As shown in the Examples, modified dsRNA ligase polypeptides represented by the even-numbered sequence identifiers of SEQ ID NOS: 304-600 and 636-668 exhibit higher activity than the polypeptide of SEQ ID NOS: 302. The dsRNA ligase polypeptides used in the Examples (represented by the even-numbered sequence identifiers of SEQ ID NOS: 4-300 and 602-634, respectively) contain the even-numbered sequence identifiers of SEQ ID NOS: 304-600 and 636-668 and an N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NOS: 669)). For example, SEQ ID NOS: 4 contains: (i) the N-terminal purification tag MHHHHHENLYFQS (SEQ ID NOS: 669); and (ii) SEQ ID NOS: 304. The dsRNA ligase polypeptides represented by the even-numbered sequence identifiers of SEQ ID NOS: 304-600 and 636-668 do not contain the N-terminal purification tag represented by SEQ ID NOS: 669.

[0135] The wild-type dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 302 (also accessible under UniProt accession number Q7Y4V8). SEQ ID NO: 2 comprises: (i) the N-terminal purification tag MHHHHHENLYFQS (SEQ ID NO: 669); and (ii) SEQ ID NO: 302. It is readily understood that both SEQ ID NOs: 2 and 302 comprise the wild-type dsRNA ligase polypeptide sequence, and both sequences may be referred to herein as the wild-type sequence.

[0136] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590, and 592. In some embodiments, the modified dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 666. In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590, 592, and 666. In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 188, 226, 278, 288, 290, and 292. In some embodiments, the modified dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 632. In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 70, 188, 226, 278, 288, 290, 292, and 632. SEQ ID NOs: 70, 188, 226, 278, 288, 290, 292, and 632 comprise: (i) an N-terminal purification tag MHHHHHHENLYFQS (SEQ ID NO: 669); and (ii) the amino acid sequences provided in SEQ ID NOs: 370, 488, 526, 578, 588, 590, 592, and 666, respectively.

[0137] In some embodiments, the modified dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 370. In some embodiments, the modified dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 488. In some embodiments, the modified dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 526. In some embodiments, the modified dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 578. In some embodiments, the modified dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 588. In some embodiments, the modified dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 590. In some embodiments, the modified dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 592. In some embodiments, the modified dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 666.

[0138] The present disclosure also provides a modified dsRNA ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 302, wherein the modified dsRNA ligase polypeptide produces at least 5% more oligonucleotide products under the same ligation reaction conditions than a dsRNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO: 302, wherein the modified dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302. In some embodiments, the modified dsRNA ligase polypeptide produces at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% more oligonucleotide products under the same ligation reaction conditions than a dsRNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO: 302. In some embodiments, the ligation reaction conditions are as described herein.

[0139] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO:302, and optionally at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO:302.

[0140] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO:302, and optionally at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% sequence identity to SEQ ID NO:302.

[0141] In some embodiments, ligation reaction conditions include about 1 μM to about 10 mM of oligonucleotide fragment, an ATP source, about 5 mM to about 100 mM divalent cations, and about 0.5 g / L to about 10 g / L of modified dsRNA ligase polypeptide, a pH of about 4.0 to about 8.0, and a temperature of about 10° C. to about 50° C. In some embodiments, the ATP source is a stoichiometric concentration of ATP or a stoichiometric excess of ATP. In some embodiments, the ATP source includes: (a) polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and / or ATP.

[0142] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 by one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) amino acid residues selected from the following: X6, X7, X15, X19, X29, X36, X39, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X91, X93, X103, X105, X107, X114, X1 22, X126, X129, X130, X131, X137, X144, X146, X158, X163, X173, X178, X190, X196, X216, X218, X221, X228, X230, X232, X235, X236, X237, X238, X239, X242, X243, X244, X251, X252, X254, X255, X258, X269, X280, X284, X285, X293, X296, X301, X303, X305, X314, X325, and X328 (numbering refers to SEQ ID NO: 302). In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 by one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) amino acid residues selected from the following: X6, X7, X15, X19, X29, X36, X39, X44, X45, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X89, X91, X92, X93, X103, X105, X107, X114 ,X122,X126,X129,X130,X131,X137,X144,X146,X158,X163,X173,X178,X18 5, X190, X196, X216, X218, X221, X228, X230, X232, X235, X236, X237, X238, 39, X242, X243, X244, X251, X252, X254, X255, X258, X269, X280, X284, X285, X293, X296, X301, X303, X305, X313, X314, X325, and X328 (numbering refers to SEQ ID NO: 302).

[0143] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that includes one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more) of the following amino acid residues: X6 is G; X7 is Q; X15 is R, D, or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; X57 is S; X60 is T, G, or P; X63 is S, Q, or G; X64 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; X87 is T, P, K, or absent; X88 is C; X91 is S; X93 is G, C, or A; X103 is V, C, Y, or T; X105 is V; X107 is R or T; X114 is N; X122 is W; X126 is G; X129 is N; X130 is R, S, or Y and X131 is R; X137 is V or C; X144 is N; X146 is R; X158 is W; X163 is G; X173 is L; X178 is R; X190 is Q; X196 is S or C; X216 is L or R; X218 is N; X221 is I; X228 is R; X230 is T; X232 is R; X235 is A, T, or G; X236 is S, L, or F; X237 is S, Q, or R; X238 is F; X239 is G or R; X242 is R or M; X243 is N, S, G, or M; X244 is G or K; X251 is D or L; X252 is V; X254 is K; X255 is C; X258 is V; X269 is L; X280 is W; X284 is A; X285 is A; X293 is R; X296 is R; X301 is G, L, E, or F; X303 is Q; X305 is G; X314 is A or V; X325 is R;X328 is R (numbering refers to SEQ ID NO: 302). In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that includes one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more) of the following amino acid residues: X6 is G or E; X7 is Q; X15 is R, D or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X44 is V; X45 is V; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; X57 is S; X60 is T, G, or P; X63 is S, Q, or G; X64 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; X87 is T, P, K, or absent; X88 is C; X89 is T; X91 is S; X92 is D; X93 is G, C, or A; X103 is V, C, Y, or T; X105 is V; X107 is R or T; X114 is N; X122 is W; X126 is G; X 129 is N; X130 is R, S or Y; X131 is R; X137 is V or C; X144 is N; X146 is R; X158 is W; X163 is G; X173 is L; X178 is R; X185 is K; X190 is Q; X196 is S or C; X216 is L or R; X218 is N; X221 is I; X228 is R; X230 is T; X232 is R; X235 is A, T, or G; X236 is S, L, or F; X237 is S , Q, R, L or G; X238 is F; X239 is G or R; X242 is R or M; X243 is N, S, G, or M; X244 is G or K; X251 is D or L; X252 is V; X254 is K; X255 is C; X258 is V; X269 is L; X280 is W; X284 is A; X285 is A; X293 is R; X296 is R; X301 is G, L, E, or F; X303 is Q; X305 is G; X313 is A;X314 is A or V; X325 is R; and X328 is R (numbering refers to SEQ ID NO: 302);

[0144] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 by one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, or eleven) amino acid residues selected from the following: X15, X19, X36, X39, X53, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302). In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that includes one or more (e.g., 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, or 11) of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A (numbering refers to SEQ ID NO: 302).

[0145] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 by one or more (e.g., 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, or 12) amino acid residues selected from the following: X15, X19, X36, X39, X53, X185, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302). In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that includes one or more (e.g., 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, or 12) of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A (numbering refers to SEQ ID NO: 302).

[0146] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 by one or more (e.g., two or more, or more) amino acid residues selected from the following: X36, X39, X218, and X221 (numbering refers to SEQ ID NO: 302). In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that includes one or more (e.g., two or more, or more) of the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I (numbering refers to SEQ ID NO: 302).

[0147] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at the following amino acid residues: X36, X39, X218, and X221 (numbering refers to SEQ ID NO: 302). In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that includes the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I (numbering refers to SEQ ID NO: 302).

[0148] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 by one or more (e.g., two or more) amino acid residues selected from the following: X39, X218, and X221 (numbering refers to SEQ ID NO: 302). In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that includes one or more (e.g., two or more) of the following amino acid residues: X39 is A; X218 is N; and X221 is I (numbering refers to SEQ ID NO: 302).

[0149] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at the following amino acid residues: X39, X218, and X221 (numbering refers to SEQ ID NO: 302). In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that includes the following amino acid residues: X39 is A; X218 is N; and X221 is I (numbering refers to SEQ ID NO: 302).

[0150] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 by one or more (e.g., two or more, or more) amino acid residues selected from the following: X39, X218, X221, and X255 (numbering refers to SEQ ID NO: 302). In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that includes one or more (e.g., two or more, or more) of the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C (numbering refers to SEQ ID NO: 302).

[0151] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at the following amino acid residues: X39, X218, X221, and X255 (numbering refers to SEQ ID NO: 302). In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that includes the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C (numbering refers to SEQ ID NO: 302).

[0152] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 by one or more (e.g., two or more, three or more, four or more, five or more, six or more, or seven or more) amino acid residues selected from the following: X39, X53, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302). In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that includes one or more (e.g., two or more, three or more, four or more, five or more, six or more, or seven or more) of the following amino acid residues: X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A (numbering refers to SEQ ID NO: 302).

[0153] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at the following amino acid residues: X39, X53, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302). In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that includes the following amino acid residues: X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A (numbering refers to SEQ ID NO: 302).

[0154] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 by one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more) amino acid residues selected from the following: X15, X39, X53, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302). In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that includes one or more (e.g., two or more, three or more, four or more, five or more, six or more, seven or more, or eight or more) of the following amino acid residues: X15 is D or E; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A (numbering refers to SEQ ID NO: 302).

[0155] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at the following amino acid residues: X15, X39, X53, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302). In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence that includes the following amino acid residues: X15 is D; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A (numbering refers to SEQ ID NO: 302). In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence comprising the following amino acid residues: X15 is E; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A (numbering refers to SEQ ID NO: 302).

[0156] In some embodiments, the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from the following: X15, X39, X53, X185, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302); wherein the modified dsRNA ligase polypeptide has dsRNA ligase activity, and optionally the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.

[0157] In some embodiments, the modified dsRNA ligase polypeptide comprises a purification tag. Purification tags are typically added to polypeptides to enable their purification from crude biological sources using affinity techniques. In some embodiments, the purification tag comprises a polyhistidine tag. The polyhistidine tag can be bound to a matrix with immobilized metal ions and used to purify the polypeptide by affinity chromatography. In some embodiments, the purification tag further comprises a protease recognition site for removing the purification tag. In some embodiments, the protease recognition site comprises a Tobacco Etch Virus (TEV) protease recognition sequence. In some embodiments, the purification tag comprises the amino acid sequence MHHHHHHENLYFQS (SEQ ID NO: 669).

[0158] In some embodiments, the modified dsRNA ligase polypeptide is selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 2, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 62, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236 , 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298 and 300.

[0159] In some embodiments, the modified dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, and 634.

[0160] In some embodiments, the modified dsRNA ligase polypeptide is selected from the group consisting of SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178 , 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262 , 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, and 634.

[0161] immobilization The present disclosure also provides a polypeptide immobilized on a solid support material by chemical bonding or physical adsorption methods, wherein the polypeptide comprises a modified dsRNA ligase polypeptide disclosed herein.

[0162] Immobilization of a polypeptide by physical absorption typically involves physically adsorbing or attaching the polypeptide to a solid support material. Adsorption can occur through weak, non-specific forces such as van der Waals forces, hydrophobic interactions, and hydrogen bonds. Physical adsorption can be achieved by immersing the support material in a solution of the polypeptide and incubating until physical adsorption occurs. Immobilization of a polypeptide by chemical bonding typically involves attaching the polypeptide to the support material by a covalent bond.

[0163] In some embodiments, the dsRNA ligase polypeptide is immobilized via a spacer located between the dsRNA ligase polypeptide and the solid material. In some embodiments, the spacer is a peptide (e.g., a peptide containing 2 or more, 3 or more, 4 or more, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 75 or more, or 100 or more amino acids).

[0164] In some embodiments, the modified dsRNA ligase polypeptide is immobilized using affinity immobilization. In some embodiments, the modified dsRNA ligase polypeptide is immobilized using metal affinity immobilization, for example, by contacting the His-tagged modified dsRNA ligase polypeptide with an immobilized metal such as nickel, zinc, cobalt, or copper.

[0165] In some embodiments, the solid support material comprises a membrane, resin, solid carrier, or other solid phase material. The solid support material can be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, polymethacrylate, and polyacrylamide, as well as copolymers and grafts thereof. The solid support material can also be inorganic, such as glass, silica, controlled pore glass (CPG), reverse-phase silica, or metal, such as gold or platinum. The solid support material can be in the form of beads, spheres, particles, granules, gels, membranes, or surfaces. Surfaces can be planar, substantially planar, or non-planar. The solid support material can be porous or non-porous and can have swelling or non-swelling properties. The solid support material can be configured in the form of wells, depressions, or other containers, vessels, features, or locations. Solid support materials useful for immobilizing dsRNA ligase polypeptides to perform the ligase reaction include, but are not limited to, beads or resins such as polymethacrylates, e.g., epoxy-functionalized polymethacrylates, aminoepoxy-functionalized polymethacrylates, polymethacrylates, styrene / DVB copolymers, or octadecyl-functionalized polymethacrylates.

[0166] Exemplary solid supports include chitosan beads, Eupergit C, IB-150, IB-350, IB-C435, IB-A369, IB-A161, IB-A171, IBS500, IB-S861, SEPABEADS (Mitsubishi), such as Sepabeads EC-EP, Sepabeads EC-HFA, Sepabeads EC-HG, Sepabeads EC-BU, Sepabeads EC-OD, Sepabeads EC-CM, Sepabeads EC-IDA, Sepabeads EC-EA, Sepabeads EC-HA, Sepabeads EC-QA, Sepabeads EXE, Sepabeads EXA, Dilbeads-TA, Amberzyme Oxirane, Amberlite XAD-7HP, Amberlite FPA98Cl, Amberlite IRA958Cl, Amberlite IRA67, Amberlite FPA90Cl, Amberlite FPA40Cl, Amberlite XAD18, Accurel EP100, ECR8206F / 5730, ECR8206 / 5803, ECR8206M / 5749, ReliZyme EP403, ReliZyme EP113, Lewatit VP OC 1600, Diaion WA20, Diaion WA21J, Diaion WA30, Dowex 66, Diaion HPA-25L, Lewatit VP OC 1064 MD PH, Lewatit VP OC 1163, Lifetech ECR8304F, Lifetech ECR8309F, Lifetech ECR8315F, Lifetech ECR8204F, Lifetech Chromalite MIDA / M, Chromalite MIDA / M / Fe, Chromalite MIDA / M / Co, Chromalite MIDA / M / Ni, Chromalite MIDA / M / Cu and Chromalite MIDA / M / Zn.

[0167] Polynucleotides, control sequences, expression vectors and host cells that can be used to produce modified dsRNA ligase polypeptides In another aspect, the present disclosure provides a polynucleotide that encodes the modified polypeptide that has dsRNA ligase activity described herein.Polynucleotide can be linked to one or more heterologous regulatory sequences that control gene expression to produce recombinant polynucleotides that can express modified polypeptides.The expression construct that comprises the heterologous polynucleotide that encodes modified dsRNA ligase can be introduced into suitable host cells to express the corresponding modified dsRNA ligase polypeptide.

[0168] As will be apparent to those skilled in the art, the availability of protein sequences and knowledge of the codons corresponding to various amino acids provides an example of all possible polynucleotides encoding a protein sequence of interest. The degeneracy of the genetic code, in which the same amino acid is coded for by alternative or synonymous codons, allows for the production of an extremely large number of polynucleotides, all of which encode the modified dsRNA ligase polypeptides disclosed herein. Thus, once a particular amino acid sequence is determined, one skilled in the art can generate any number of different polynucleotides by modifying one or more codons in a manner that does not alter the amino acid sequence of the protein. In this regard, the present disclosure specifically contemplates all possible modifications of polynucleotides that can be made by selecting combinations based on possible codon choices for any of the polypeptides disclosed herein, including the amino acid sequences of the exemplary modified polypeptides listed in Examples 7-12, any of the polypeptides disclosed as even-numbered sequence identifiers SEQ ID NOS: 304-600 and 636-668, and any of the polypeptides disclosed as even-numbered sequence identifiers SEQ ID NOS: 4-300 and 602-634.

[0169] In various embodiments, codons are preferably selected to be compatible with the host cell in which the recombinant protein will be produced, for example, bacterially preferred codons are used to express genes in bacteria, yeast preferred codons are used to express genes in yeast, and mammalian preferred codons are used to express genes in mammalian cells.

[0170] In some embodiments, the present disclosure provides polynucleotides encoding the above-described modified dsRNA ligase polypeptides.

[0171] In some embodiments, the polynucleotide encodes a polypeptide comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a reference sequence that is an even-numbered sequence identifier of SEQ ID NOs: 304-600 or 636-668, wherein the polypeptide has dsRNA ligase activity and exhibits greater enzymatic activity than a polypeptide comprising the amino acids of SEQ ID NOs: 2 and / or 302.

[0172] In some embodiments, the polynucleotide encodes a modified dsRNA ligase polypeptide described herein and comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a reference polynucleotide selected from sequences having odd-numbered sequence identifiers SEQ ID NOs: 303-599 or 635-667, where (i) the polynucleotide does not comprise SEQ ID NO: 301; (ii) the polynucleotide does not encode a dsRNA ligase polypeptide having the amino acid sequence of SEQ ID NO: 302.

[0173] In some embodiments, the polynucleotide encodes a modified dsRNA ligase polypeptide described herein and comprises a nucleic acid sequence having at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a reference polynucleotide selected from sequences having odd-numbered sequence identifiers SEQ ID NOs: 3-299 or 601-633, where (i) the polynucleotide does not comprise SEQ ID NO: 1; (ii) the polynucleotide does not encode a dsRNA ligase polypeptide having the amino acid sequence of SEQ ID NO: 2. It will be readily understood that polynucleotides having odd-numbered sequence identifiers of SEQ ID NOs: 3-299 or 601-633 encode modified dsRNA ligase polypeptides that include an N-terminal purification tag (SEQ ID NO: 669).

[0174] An isolated polynucleotide encoding a modified dsRNA ligase polypeptide can be manipulated in a variety of ways to express the modified polypeptide, which may include further modification of the sequence by codon optimization to improve expression, insertion into a suitable expression element with or without additional regulatory sequences, and transformation into a host cell suitable for expression and production of the modified polypeptide.

[0175] Depending on the expression vector, it may be desirable or necessary to manipulate the isolated polynucleotide before inserting it into the vector. The techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. Guidance is provided in: Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Ausubel, F. Eds., Greene Pub. Associates, 1998, 2010 Year update.

[0176] The present disclosure also provides an expression vector comprising the polynucleotide described herein. In some embodiments, the vector is selected from a plasmid, cosmid, bacteriophage, or viral vector. Recombinant expression vectors typically include one or more expression control regions, such as a promoter and a terminator, a replication origin, and the like.

[0177] A polynucleotide encoding a modified dsRNA ligase polypeptide described herein can be expressed by inserting the polynucleotide or a nucleic acid construct containing the polynucleotide sequence into an appropriate expression vector. To create an expression vector, a coding sequence is placed in a vector so that it is linked to appropriate control sequences for expression. Recombinant expression vectors can be any vector (e.g., a plasmid or virus) that can be conveniently used in recombinant DNA procedures and can result in the expression of a polynucleotide sequence. The vector is generally selected based on its compatibility with the host cell into which it will be introduced. The vector can be a linear or circular plasmid. The expression vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, such as a plasmid, extrachromosomal element, minichromosome, or artificial chromosome. The vector can contain any tool that ensures self-replication. Alternatively, the vector can be a vector that, upon introduction into a host cell, integrates into the genome and replicates along with the chromosome into which it is integrated. Furthermore, a single vector or plasmid, or two or more vectors or plasmids, can be used that together contain the total DNA to be introduced into the genome of the host cell.

[0178] Many expression vectors useful for embodiments of the present disclosure are commercially available. Exemplary expression vectors can be prepared by inserting a polynucleotide encoding a modified dsRNA ligase polypeptide into the plasmid pACYC-Duet-1 (Novagen), pBR322 vector (New England Biolabs), pUC19 vector (New England Biolabs), or pET T7 expression vector (Novagen).

[0179] The present disclosure also provides host cells capable of expressing the modified dsRNA ligase polypeptides described herein. In some embodiments, the host cells comprise the nucleic acid molecules described herein or the vectors described herein. In some embodiments, the host cells are Escherichia coli.

[0180] In some embodiments, the polynucleotide encoding the polypeptide is linked to one or more control sequences for expression of the polypeptide in a host cell. Host cells for expressing the polypeptide encoded by the expression vectors of the present disclosure are well known in the art and include, but are not limited to, bacterial cells such as E. coli, Streptomyces, and Salmonella typhimurium; fungal cells (e.g., Saccharomyces cerevisiae or Pichia pastoris); insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. An exemplary host cell is E. coli BL21(DE3). Host cells may be wild-type or modified by genome editing. Appropriate culture media and growth conditions for the above host cells are well known in the art.

[0181] The polynucleotides or vectors used to express the polypeptides can be introduced into cells by a variety of methods known in the art. Techniques include, among others, electroporation, bioparticle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion. Various methods for introducing polynucleotides into cells are known to those skilled in the art.

[0182] Host cells can be used to express and isolate the polypeptides described herein.

[0183] Methods for producing modified dsRNA ligase polypeptides The modified dsRNA ligase can be obtained by subjecting a polynucleotide encoding the dsRNA ligase to mutagenesis and / or directed evolution. An exemplary directed evolution technique is described in "Biocatalysis for the Pharmaceutical Industry: Discovery, Development, and Manufacturing" (2009 John Wiley & Sons Asia (Pte) Ltd. ISBN: 978-0-470-82314-9).

[0184] If the sequence of a modified polypeptide is known, encoding polynucleotides can be prepared by standard solid-phase methods according to known synthesis methods. In some embodiments, fragments of up to about 100 bases can be synthesized separately and then ligated (e.g., by enzymatic or chemical ligation or polymerase-mediated methods) to form any desired contiguous sequence. For example, polynucleotides and oligonucleotides of the present disclosure can be prepared by chemical synthesis using the classical phosphoramidite method, as described, for example, in Beaucage et al., 1981, Tet Lett 22:1859-69, or Matthes et al., People, 1984, EMBO J. 3:801-05, commonly practiced in automated synthesis methods. Oligonucleotides are synthesized according to the phosphoramidite method, purified, annealed, ligated, and cloned into a suitable vector, for example, in an automated DNA synthesizer. Furthermore, essentially any nucleic acid is available from any of a variety of commercial sources.

[0185] The present disclosure provides methods for preparing a modified dsRNA ligase polypeptide, comprising culturing a host cell described herein and obtaining the modified dsRNA ligase polypeptide from the culture. In some embodiments, the process for preparing the polypeptide further comprises isolating the polypeptide. The modified polypeptide may be expressed in a suitable cell and isolated (or recovered) from the host cell and / or culture medium using any one or more well-known techniques for purifying proteins, including, among others, lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography.

[0186] The present invention also provides modified dsRNA ligase catalysts obtained by culturing the host cells described herein or from the methods for preparing the modified dsRNA ligase polypeptides described herein, wherein the modified dsRNA ligase catalysts include cells or culture media containing the modified dsRNA ligase polypeptides, or articles treated therewith, and the articles refer to extracts obtained from cultures of the host cells, isolated products obtained by isolating or purifying the modified dsRNA ligase from the extracts, or immobilized products obtained by immobilizing the host cells, extracts thereof, or isolated products of the extracts.

[0187] Ligation Reaction The present disclosure provides a method for generating an oligonucleotide from two or more oligonucleotide fragments, the method comprising contacting (i) the two or more oligonucleotide fragments; (ii) a modified dsRNA ligase polypeptide disclosed herein; (iii) a source of ATP; and (iv) a divalent cation to obtain an oligonucleotide.

[0188] Oligonucleotide Products and Fragments The method of the present invention produces an oligonucleotide by ligating two or more oligonucleotide fragments. The produced oligonucleotide (also referred to herein as "oligonucleotide product") is typically a nucleic acid containing up to 100 nucleotides. It is understood that the oligonucleotide described herein includes RNA. It is also understood that the oligonucleotide described herein includes a double-stranded region.

[0189] As used herein, an "oligonucleotide fragment" refers to a nucleic acid that can be ligated to one or more additional oligonucleotide fragments to provide an oligonucleotide product, where each oligonucleotide fragment corresponds to a portion of the oligonucleotide product.

[0190] In some embodiments, the oligonucleotide is a therapeutic oligonucleotide. In some embodiments, the therapeutic oligonucleotide is a small interfering RNA (siRNA) or an antisense oligonucleotide (ASO). In some embodiments, the oligonucleotide is an aptamer.

[0191] In some embodiments, the oligonucleotide comprises an overhang. In some embodiments, the oligonucleotide comprises a 3' overhang. In some embodiments, the oligonucleotide comprises a 5' overhang. In some embodiments, the overhang comprises 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides. In some embodiments, the oligonucleotide comprises a blunt end. In some embodiments, the oligonucleotide comprises two blunt ends.

[0192] In some embodiments, the oligonucleotide is up to 20 nucleotides in length. In some embodiments, the oligonucleotide is up to 25, up to 30, up to 35, up to 40, up to 45, up to 50, up to 55, up to 60, up to 65, up to 70, up to 75, up to 80, up to 85, up to 90, up to 95, or up to 100 nucleotides in length. In some embodiments, the oligonucleotide is up to 60 nucleotides in length.

[0193] In some embodiments, the oligonucleotide is at least 20 nucleotides in length, hi some embodiments, the oligonucleotide is at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 55, at least 60, at least 65, at least 70, at least 75, at least 80, at least 85, at least 90, at least 95, or 100 nucleotides in length.

[0194] In some embodiments, the oligonucleotide is 10 to 100 nucleotides in length. In some embodiments, the oligonucleotide is 10 to 80, 10 to 70, 10 to 60, 10 to 50, 10 to 40, 10 to 30, 10 to 25, 15 to 80, 15 to 70, 15 to 60, 15 to 50, 15 to 40, 15 to 30, or 15 to 25 nucleotides in length. In some embodiments, the oligonucleotide is 15 to 25 nucleotides in length.

[0195] As used herein, two or more oligonucleotide fragments include one or more 3' oligonucleotide fragments and one or more 5' oligonucleotide fragments, wherein the one or more 3' oligonucleotide fragments each include a 5'-phosphate group and the one or more 5' oligonucleotide fragments each typically include a 3'-terminal ribonucleotide having a 3'-hydroxyl group.

[0196] In some embodiments, one or more of the oligonucleotide fragments comprise one or more mismatches. In some embodiments, one or more of the oligonucleotide fragments comprise an overhang. In some embodiments, one or more of the oligonucleotide fragments comprise a 3' overhang. In some embodiments, one or more of the oligonucleotide fragments comprise a 5' overhang. In some embodiments, one or more of the oligonucleotide fragments comprise a 3' overhang and a 5' overhang. In some embodiments, the overhang comprises 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides.

[0197] In some embodiments, the two or more oligonucleotide fragments comprise a first oligonucleotide fragment having an overhang complementary to an overhang of a second oligonucleotide fragment, hi some embodiments, the two or more oligonucleotide fragments comprise a first oligonucleotide fragment having a 3' overhang and a 5' overhang, where the 3' overhang is complementary to the 5' overhang of the second oligonucleotide fragment and the 5' overhang is complementary to the 3' overhang of a third oligonucleotide.

[0198] In some embodiments, one or more of the oligonucleotide fragments comprise a blunt end. In some embodiments, one or more of the oligonucleotide fragments comprise a 3' overhang and a 5' blunt end. In some embodiments, one or more of the oligonucleotide fragments comprise a 5' overhang and a 3' blunt end. In some embodiments, the 5'-terminal oligonucleotide fragment comprises a 3' overhang and a 5' blunt end. In some embodiments, the 3'-terminal oligonucleotide fragment comprises a 5' overhang and a 3' blunt end.

[0199] In some embodiments, the two or more oligonucleotide fragments comprise two or more RNA oligonucleotide fragments. In some embodiments, the two or more RNA oligonucleotide fragments comprise double-stranded RNA (dsRNA) oligonucleotide fragments.

[0200] In some embodiments, one or more of the oligonucleotide fragments comprises DNA and RNA, for example, one portion of the oligonucleotide fragment can be double-stranded DNA and another portion is double-stranded RNA forming a DNA-RNA chimera.

[0201] In some embodiments, one or more of the oligonucleotide fragments comprise one or two strands that are RNA or a mixture of RNA, DNA, LNA, morpholino, UNA (unlocked nucleic acid), TNA (threose nucleic acid), GNA (glycol nucleic acid) and / or FANA (fluoro-arabino nucleic acid), modified RNA, etc. As a non-limiting example, one or both strands can be RNA in which, for example, one or more nucleotides have been replaced with DNA, LNA, morpholino, UNA, TNA, GNA, and / or FANA, and / or modified RNA (e.g., any modified RNA disclosed herein or known in the art, such as 2'-modified RNA, including but not limited to 2'-F, 2'-OMe, 2'-O-MOE RNA, etc.).

[0202] In some embodiments, two or more oligonucleotide fragments are the same length. In some embodiments, two or more oligonucleotide fragments are different lengths. In some embodiments, two or more oligonucleotide fragments are each 3 to 20 nucleotides in length. In some embodiments, two or more oligonucleotide fragments are each 4 to 16 nucleotides in length. In some embodiments, two or more oligonucleotide fragments are each 4 to 16, 4 to 15, 5 to 15, 6 to 15, 4 to 14, 4 to 13, 4 to 12, 4 to 11, 4 to 10, 4 to 9, 5 to 9, or 6 to 9 nucleotides in length.

[0203] In some embodiments, each of the two or more oligonucleotide fragments is at least 3 nucleotides in length, hi some embodiments, each of the two or more oligonucleotide fragments is at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, or at least 15 nucleotides in length.

[0204] In some embodiments, the two or more oligonucleotide fragments comprise three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more oligonucleotide fragments.

[0205] In some embodiments, one or more ligation reactions are required to produce the oligonucleotide product, hi some embodiments, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more ligation reactions are required to produce the oligonucleotide product.

[0206] In some embodiments, one or more of the oligonucleotide fragments and / or oligonucleotides comprise a chemical modification. In some embodiments, one or more of the oligonucleotide fragments and / or oligonucleotides comprise at least one modified backbone modification. In some embodiments, one or more of the oligonucleotide fragments and / or oligonucleotides comprise at least one modified nucleotide modification. In some embodiments, one or more of the oligonucleotide fragments and / or oligonucleotides comprises at least one sugar modification (e.g., at the 2' or 4' position). In some embodiments, one or more of the oligonucleotide fragments and / or oligonucleotides comprises: (i) at least one modified backbone modification; (ii) and at least one modified nucleotide modification; and / or (iii) at least one sugar modification.

[0207] In some embodiments, one or more of the oligonucleotide fragments and / or oligonucleotides comprise a modification selected from the group consisting of 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxy (2'-O-DMAOE), 2'-O-dimethylaminopropyloxy (2'-O-DMAP), 2'-O-dimethylaminoethyloxy (2'-O-DMAOE), 2'-O-dimethylaminopropyloxy (2'-O-DMAP), 2'-O-dimethylaminoethyloxy (2'-O-DMAOE), 2'-O-dimethylaminoethyl ...ethyloxy (2'-O-DMAP), 2'-O-dimethylaminoethyloxy (2'-O-DMAOE), 2'-O-dimethylaminoethyloxy (2'-O-DMAP), 2'-O-dimethylaminoethyloxy (2'-O-DMAO dimethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acids (LNA), glycol nucleic acids (GNAs), phosphoramidates (e.g., mesyl phosphoramidate), 2',3'-seconucleotide mimics, 2'-F-arabinonucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, vinyl phosphonates (e.g., 5' vinyl phosphonate), and cyclopropyl phosphonate deoxyribonucleotides. In some embodiments, one or more of the oligonucleotide fragments and / or oligonucleotides comprises a 2'-modification selected from the group consisting of 2'-OMe, 2'-F, and 2'-deoxy.

[0208] In some embodiments, one or more of the oligonucleotide fragments and / or oligonucleotides comprises at least one phosphorothioate or methylphosphonate internucleotide linkage, hi some embodiments, the oligonucleotide comprises at least one chiral phosphorothioate linkage.

[0209] In some embodiments, one or more of the oligonucleotide fragments and / or oligonucleotides are conjugated to at least one ligand, which may be conjugated to the sense strand, the antisense strand, or both strands in any configuration, e.g., at the 3' end, the 5' end, non-end, or a combination.

[0210] In some embodiments, the ligand comprises one or more N-acetylgalactosamine (GalNAc) derivatives. GalNAc is an amino sugar derivative of galactose and can be used as a targeting ligand in oligonucleotides intended to target the liver, binding to the asialoglycoprotein receptor on hepatocytes. In some embodiments, the ligand comprises one or more GalNAc derivatives conjugated via a bivalent or trivalent branched carrier. In some embodiments, the ligand is a peptide or peptidomimetic.

[0211] In some embodiments, the ligand is conjugated to the sense strand. In some embodiments, the ligand is conjugated to the 3' end of the sense strand. In some embodiments, the ligand is conjugated to the 5' end of the sense strand. In some embodiments, the ligand is conjugated to a non-terminal end of the sense strand.

[0212] In some embodiments, the ligand is conjugated to the antisense strand. In some embodiments, the ligand is conjugated to the 3' end of the antisense strand. In some embodiments, the ligand is conjugated to a non-terminal end of the antisense strand.

[0213] In some embodiments, the oligonucleotide is an RNAi agent comprising at least one 2'-modified nucleotide selected from the group consisting of 2'-OMe, 2'-F, 2'-deoxy, 2'-deoxy-2'-fluoro, and 2'-O-MOE. In some embodiments, the oligonucleotide is an RNAi agent in which the sense strand is conjugated to one or more GalNAc ligands. In some embodiments, one or more of the oligonucleotide fragments comprises at least one 2'-modified nucleotide selected from the group consisting of 2'-OMe, 2'-F, 2'-deoxy, 2'-deoxy-2'-fluoro, and 2'-O-MOE. In some embodiments, one or more of the oligonucleotide fragments is a dsRNA in which the sense strand is conjugated to one or more GalNAc ligands.

[0214] In some embodiments, the method is performed at an oligonucleotide fragment concentration of at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, or at least 10 mM. In some embodiments, the method is performed with at least 1 mM, at least 2 mM, at least 3 mM, at least 4 mM, at least 5 mM, at least 6 mM, at least 7 mM, at least 8 mM, at least 9 mM, or at least 10 mM of each oligonucleotide fragment. In some embodiments, the method is performed using equimolar amounts of each of two or more oligonucleotide fragments.

[0215] In some embodiments, the method produces at least 15 g of oligonucleotide product per liter of reaction mixture, hi some embodiments, the method produces at least 16 g, at least 17 g, at least 18 g, at least 19 g, at least 20 g, at least 30 g, at least 40 g, at least 50 g, at least 60 g, at least 70 g, at least 80 g, at least 90, or at least 100 g of oligonucleotide product per liter of reaction mixture.

[0216] Modified dsRNA ligase polypeptides The method is carried out using the modified dsRNA ligase described herein.

[0217] In some embodiments, the method comprises administering about 1 g / L of modified dsRNA ligase, optionally 1.1 g / L, 1.15 g / L, 1.2 g / L, 1.25 g / L, 1.3 g / L, 1.35 g / L, 1.4 g / L, 1.45 g / L, 1.5 g / L, 1.55 g / L, 1.6 g / L, 1.65 g / L, 1.7 g / L, 1.75 g / L, 1.8 g / L, 1. The reaction is carried out using 85 g / L, 1.9 g / L, 1.95 g / L, 2 g / L, 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L, 2.5 g / L, 2.6 g / L, 2.7 g / L, 2.8 g / L, 2.9 g / L, 3 g / L, 3.25 g / L, 3.5 g / L, 3.75 g / L, 4 g / L, 4.5 g / L or 5 g / L of modified dsRNA ligase.

[0218] ATP source The enzymatic activity of dsRNA ligase requires ATP as a cofactor. One molecule of ATP is converted to AMP per ligation reaction. The catalytic mechanism of dsRNA ligase and the role of ATP in nucleic acid ligation reactions are described above.

[0219] In some embodiments, the ATP source is ATP. In some embodiments, the method is performed using a stoichiometric concentration of ATP. In some embodiments, the method is performed using a stoichiometric excess of ATP. One skilled in the art can easily determine the stoichiometric concentration of ATP required for a given ligation based on the concentration of the oligonucleotide fragments and the number of ligation reactions required to produce the oligonucleotide product.

[0220] In some embodiments, the method is performed using an ATP and / or AMP concentration of about 0.5 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, or about 30 mM.

[0221] In some embodiments, the ATP source is an ATP regeneration system. In some embodiments, the ATP regeneration system comprises: (a) polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and / or ATP. Advantageously, the use of an ATP regeneration system overcomes the need for high concentrations of ATP to achieve complete ligation. The ATP regeneration system described herein comprises PPK and polyphosphate. PPK generates ATP from AMP using polyphosphate as a phosphate donor. ATP converted to AMP during the ligation reaction can be regenerated by PPK to ATP, which can be used as a cofactor in subsequent ligation reactions. This recycling of ATP eliminates the need for high ATP concentrations in the starting reaction. Instead, the reaction can be performed using substoichiometric concentrations of ATP and / or using the cheaper alternative, AMP.

[0222] "Polyphosphate kinases" or "PPKs" are a family of enzymes that catalyze the formation of ATP from AMP and polyphosphate.

[0223] In some embodiments, the PPK is PPK12. In some embodiments, the PPK comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 670, the amino acid sequence of PPK12. In some embodiments, the PPK comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 670.

[0224] In some embodiments, the PPK comprises an amino acid sequence having at least 70% sequence identity to the amino acid sequence of optimized PPK12, SEQ ID NO: 671. In some embodiments, the PPK comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 671.

[0225] In some embodiments, the PPK is Acinetobacter johnsonii polyphosphate:AMP phosphotransferase (AjPAP) (UniProt ID: Q83XD3). In some embodiments, the PPK comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 672, the amino acid sequence of AjPAP. In some embodiments, the PPK comprises an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 672.

[0226] In some embodiments, the PPK is used in the form of a whole cell, a crude extract (e.g., a cell-free lyophilized extract or a cell lysate), an isolated polypeptide, or a purified polypeptide. In some embodiments, the PPK polypeptide is used in an immobilized form, e.g., immobilized on a resin, as described herein.

[0227] In some embodiments, the method comprises administering a PPK concentration of about 1 g / L, optionally 1.1 g / L, 1.15 g / L, 1.2 g / L, 1.25 g / L, 1.3 g / L, 1.35 g / L, 1.4 g / L, 1.45 g / L, 1.5 g / L, 1.55 g / L, 1.6 g / L, 1.65 g / L, 1.7 g / L, 1.75 g / L, 1.8 g / L, 1. This is carried out using PPKs of 85g / L, 1.9g / L, 1.95g / L, 2g / L, 2.1g / L, 2.2g / L, 2.3g / L, 2.4g / L, 2.5g / L, 2.6g / L, 2.7g / L, 2.8g / L, 2.9g / L, 3g / L, 3.25g / L, 3.5g / L, 3.75g / L, 4g / L, 4.5g / L or 5g / L.

[0228] In some embodiments, the polyphosphate is a polyphosphate salt. In some embodiments, the polyphosphate salt is sodium polyphosphate (Madrell's salt) or sodium hexametaphosphate (Graham's salt).

[0229] In some embodiments, the method is carried out using a stoichiometric excess of polyphosphate, hi some embodiments, the method is carried out using a polyphosphate concentration of at least 5 mM, at least 10 mM, at least 15 mM, at least 20 mM, at least 25 mM, at least 30 mM, at least 35 mM, at least 40 mM, at least 45 mM, at least 50 mM, 55 mM, at least 60 mM, at least 65 mM, at least 70 mM, at least 75 mM, at least 80 mM, at least 85 mM, at least 90 mM, at least 95 mM, or at least 100 mM.

[0230] In some embodiments, the method is performed in the presence of PPK and polyphosphate, and the method is performed in the presence of AMP. In some embodiments, the method is performed in the presence of PPK and polyphosphate, and the method is performed using sub-stoichiometric concentrations of ATP and / or AMP.

[0231] divalent cations The enzymatic activity of dsRNA ligase requires the presence of divalent cations. The enzymatic activity of PPK requires the presence of divalent cations. In some embodiments, the divalent cation is Mg 2+ and / or Mn 2+ Includes.

[0232] In some embodiments, the method is carried out at a divalent cation concentration of 5-100 mM, 10-100 mM, 15-100 mM, 20-100 mM, 30-100 mM, 5-90 mM, 5-80 mM, 5-70 mM, 5-60 mM, 5-50 mM, or 30-50 mM. In some embodiments, the method is carried out at a divalent cation concentration of at least 5 mM, at least 10 mM, at least 15 mM, at least 20 mM, at least 25 mM, at least 30 mM, at least 35 mM, at least 40 mM, at least 45 mM, at least 50 mM, 55 mM, at least 60 mM, at least 65 mM, at least 70 mM, at least 75 mM, at least 80 mM, at least 85 mM, at least 90 mM, at least 95 mM, or at least 100 mM.

[0233] In some embodiments, the method further comprises purifying the oligonucleotide product from the reaction mixture. In some embodiments, the oligonucleotide product is at least 80% pure, optionally at least 85% pure, at least 90% pure, at least 95% pure, optionally at least 98% pure, optionally at least 99% pure, optionally at least 99.5% pure, or optionally at least 99.9% pure. A pure oligonucleotide product does not contain oligonucleotide fragments, intermediate ligation products, or by-products resulting from non-specific ligation. The oligonucleotide product can be purified or isolated using any method known in the art, for example, using gel extraction or using a cellulose-based matrix.

[0234] The present disclosure also provides oligonucleotides produced by the methods described herein. The oligonucleotides can be present in any suitable buffer solution. In some embodiments, the buffer solution is selected from Tris buffer (e.g., Tris-HCl), phosphate buffer, HEPES, MOPS (3(N-morpholino)propanesulfonic acid), and triethanolamine (TEOA) buffer. In some embodiments, the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In some embodiments, the buffer solution further comprises an agent for controlling the osmolality of the solution so that the osmolality is maintained at a desired value, e.g., the physiological value of human plasma. Solutes that can be added to the buffer solution to control the osmolality include, but are not limited to, proteins, peptides, amino acids, non-metabolized polymers, vitamins, ions, sugars, metabolites, organic acids, lipids, or salts. In some embodiments, the agent for controlling the osmolality of the solution is a salt. In some embodiments, the agent for controlling the osmolality of the solution is sodium chloride or potassium chloride.

[0235] Reaction conditions As disclosed herein and illustrated in the Examples, the present disclosure contemplates a range of suitable reaction conditions that may be used in the methods described herein, including, but not limited to, pH, temperature, buffer, substrate load, enzyme load, cofactor load, pressure, and reaction time. Additional suitable reaction conditions for the ligation reactions described herein can be readily optimized by routine experimentation, e.g., by performing the methods described herein under experimental reaction conditions of varying reagent concentrations, pH, and temperature, and detecting the rate of formation of oligonucleotide products.

[0236] In any of the process embodiments disclosed herein, the reaction conditions can include a suitable pH. As noted above, the desired pH or desired pH range can be maintained using an acid or base, a suitable buffer, or a combination of a buffer and added acid or base. The pH of the reaction mixture can be controlled before and / or during the reaction. In some embodiments, suitable reaction conditions include a solution pH of about 4 to about 8, a pH of about 5 to about 8, a pH of about 6 to about 8, or a pH of about 7 to about 8. In some embodiments, the reaction conditions include a solution pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8.

[0237] In any of the embodiments of the methods disclosed herein, a temperature appropriate for the reaction conditions can be used, taking into account, for example, increased reaction rate at higher temperatures and enzyme activity over a sufficient reaction time. Thus, in some embodiments, suitable reaction conditions include temperatures of about 10°C to about 60°C, about 10°C to about 50°C, about 25°C to about 50°C, about 25°C to about 40°C, about 25°C to about 30°C, or about 10°C to about 30°C. In some embodiments, suitable reaction temperatures include about 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, or 60°C. In some embodiments, the temperature during the enzymatic reaction can be maintained at a specific temperature throughout the reaction. In some embodiments, the temperature during the enzymatic reaction can be adjusted along a temperature profile during the course of the reaction.

[0238] The reaction can be carried out in any suitable buffer solution. In some embodiments, the buffer solution is selected from Tris buffer (e.g., Tris-HCl), phosphate buffer, HEPES, MOPS (3(N-morpholino)propanesulfonic acid), and triethanolamine (TEOA) buffer. In some embodiments, the buffer solution comprises acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In some embodiments, the buffer solution is phosphate-buffered saline (PBS).

[0239] In some embodiments, the reaction mixture further comprises a reducing agent, optionally DTT (dithiothreitol).

[0240] In carrying out the ligation reactions described herein, the modified dsRNA ligase polypeptide biocatalyst can be added to the reaction mixture in different formulations: as frozen or lyophilized whole cells (FWC or LWC) transformed with a gene encoding the modified dsRNA ligase polypeptide, and / or as a cell lysate or lyophilized cell lysate of such cells, so-called shake flask powder (SFP) (in which cellular debris has been removed and / or further purified as fermentation powder (FP)). Whole cells or cell extracts transformed with a gene encoding the modified dsRNA ligase polypeptide, their lysates, and isolated enzymes can be used in a variety of forms, including solids (e.g., lyophilized, spray-dried, etc.) or semi-solids (e.g., crude pastes). Cell extracts or cell lysates can be partially purified by precipitation (e.g., ammonium sulfate, polyethyleneimine, heat treatment, etc.) followed by desalting procedures (e.g., ultrafiltration, dialysis, etc.) prior to lyophilization. Any enzyme preparation can be immobilized on a solid-phase material (e.g., resin).

[0241] In any of the embodiments of the processes disclosed herein in which the modified polypeptide is expressed in the form of a secreted polypeptide, the culture medium containing the secreted polypeptide can be used in the processes herein.

[0242] In any of the process embodiments disclosed herein, solid reactants (e.g., enzymes, salts, etc.) can be provided to the reactants in a variety of forms, including powders (e.g., lyophilized, spray-dried, etc.), solutions, emulsions, suspensions, etc. The reactants can be readily lyophilized or spray-dried using methods and equipment known to those of skill in the art. For example, a protein solution can be frozen in aliquots at −80° C. and then placed in a pre-cooled lyophilization chamber, after which a vacuum can be applied.

[0243] In any of the process embodiments disclosed herein, the order of addition of the reactants is not important: the reactants may be added together to the solvent simultaneously, or some reactants may be added separately, and some may be added together at different times.

[0244] The method for performing a ligation reaction may further include a step of isolating the oligonucleotide product of the enzymatic reaction. In particular, this step is typically performed after the enzymatic reaction is completed. The oligonucleotide is typically separated from one or more components of the reaction mixture, particularly from substantially all other components. For example, the oligonucleotide is typically separated from remaining substrates, by-products, and / or enzymes. Isolation of the oligonucleotide can be achieved by means and techniques known in the art, such as gel electrophoresis and gel extraction, or by separating the oligonucleotide based on its size using a cellulose-based matrix. In some embodiments, the method further includes purifying the oligonucleotide by ultrafiltration and chromatography.

[0245] qualification In some embodiments, the oligonucleotide fragments and / or oligonucleotides contain modifications, such as chemical modifications. As used herein, the term "oligonucleotide fragment" refers to one or more oligonucleotide fragments. It will be understood that modifications present in an oligonucleotide fragment are typically present in an oligonucleotide produced from said oligonucleotide fragment. In some embodiments, modifications are introduced into and / or removed from the oligonucleotide product.

[0246] In some embodiments, the oligonucleotide fragments and / or oligonucleotides comprise chemical modifications. In some embodiments, the oligonucleotide fragments and / or oligonucleotides comprise at least one backbone modification. In some embodiments, the oligonucleotide fragments and / or oligonucleotides comprise at least one nucleotide modification. In some embodiments, the oligonucleotide fragments and / or oligonucleotides comprise at least one sugar modification (e.g., at the 2' or 4' position). In some embodiments, the oligonucleotide fragments and / or oligonucleotides comprise: (i) at least one backbone modification; (ii) at least one nucleotide modification; and / or (iii) at least one sugar modification.

[0247] Modifications include, but are not limited to, terminal modifications of terminal oligonucleotide fragments, such as 5'-end modifications (phosphorylation, conjugation, reverse linkage) or 3'-end modifications (conjugation, reverse linkage, etc.); base modifications, such as substitution with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; or backbone modifications, including modification or substitution of phosphodiester linkages.

[0248] In some embodiments, the terminal oligonucleotide fragment and / or oligonucleotide comprises a cap. Terms such as "cap" are used herein to include chemical moieties attached to the terminus of a double-stranded nucleotide duplex, but to exclude chemical moieties that are nucleotides or nucleosides. A "3' cap" is attached to the 3' end of a nucleotide or oligonucleotide to protect the molecule from degradation, e.g., from nucleases such as those found in serum or intestinal fluids. A non-nucleotide 3' cap can replace the TT or UU dinucleotide at the end of a blunt-ended oligonucleotide, rather than a nucleotide. In some embodiments, non-nucleotide 3' terminal caps are as disclosed, for example, in WO 2005 / 021749 and WO 2007 / 128477; and U.S. Pat. Nos. 8,097,716, 8,084,600, and 8,344,128. A "5' cap" is attached to the 5' end of a nucleotide or oligonucleotide. The cap should not interfere (or unduly interfere) with oligonucleotide activity.

[0249] In some embodiments, the oligonucleotide fragment and / or oligonucleotide contains one or more mismatches. A mismatch is defined herein as a difference in base sequence or length when two sequences are maximally aligned and compared. In the context of a double-stranded oligonucleotide (two sequences aligned antiparallel to each other), a mismatch is defined as a position where a base in one sequence is not complementary to a base in the other sequence. Thus, for example, when a first and a second sequence are aligned antiparallel to each other, a mismatch is counted if a position in the first sequence has a specific base (e.g., A) and the corresponding position in the second sequence has a base (e.g., G) that is not complementary to the base in the first sequence. Note, however, that on a given RNA strand, U can be replaced by T (either as RNA or preferably DNA, e.g., 2'-deoxy-thymidine); since either U or T can pair with A on the opposite strand, the substitution of U for T is not a mismatch as used herein. Thus, an RNA oligonucleotide can contain one or more DNA bases, such as T. If base pairing occurs (e.g., between A, G, C, or T of the DNA portion and the corresponding U, C, G, or A, respectively, in the mRNA), the mismatch is not counted between the DNA portion of the RNAi agent and the corresponding target mRNA.

[0250] A mismatch is also counted, for example, when a position in one sequence has a base (e.g., A) and the corresponding position on the other sequence does not have a base (e.g., the position is an abasic nucleotide, which contains a phosphate-sugar backbone but no base). A single-stranded nick in either sequence (or in the sense or antisense strand) is not counted as a mismatch. Thus, as a non-limiting example, if one sequence (5'→3' direction) contains the sequence AG and the complementary sequence (3'→5' direction) contains the sequence TC with a single-stranded nick between the T and C, no mismatch is counted. Nucleotide modifications in the sugar or phosphate are also not considered mismatches. Thus, if one sequence contains G and the complementary sequence contains a modified C (e.g., a 2'-modification) at the same position, no mismatch is counted.

[0251] Therefore, if the sugar, phosphate, or backbone of an oligonucleotide is modified without modifying the base, the mismatch is not counted. Therefore, in the context of double-stranded RNAi, a strand having a given sequence as RNA does not have mismatches with its complementary sequence, such as PNA; or morpholino; or LNA; or TNA; or GNA; or FANA; or a mixture or chimera of RNA and DNA, TNA, GNA, FANA, morpholino, UNA, LNA, and / or PNA. A mismatch does not occur between a T nucleotide and an A nucleotide with a 5'-modification and / or 2'-modification. The key feature of a mismatch (base substitution) is that it cannot base-pair with the corresponding base on the opposite strand. Additionally, when counting the number of mismatches, terminal overhangs such as "UU" or "dTdT" are not counted. In such cases, a mismatch is defined as a position where a base in one sequence does not match a base in the other sequence.

[0252] Note that dTdT (2'-deoxy-thymidine-5'-phosphate and 2'-deoxy-thymidine-5'-phosphate), or possibly TT or UU, can be added to one or both 3' ends of an oligonucleotide as a terminal dinucleotide cap or extension, but this cap or extension is not included in the calculation of the total number of mismatches and is not considered part of the target sequence. This is because the terminal dinucleotide protects the end from nuclease degradation but does not contribute to target specificity (Elbashir et al. 2001 Nature 411:494-498; Elbashir et al. 2001 EMBO J. 20:6877-6888; and Kraynack et al. 2006 RNA 12:163-176).

[0253] There are several examples in the art describing sugar, base, phosphate, and backbone modifications that can be introduced into nucleic acid molecules to significantly improve nuclease stability and effectiveness. For example, oligonucleotides are modified to increase stability and / or biological activity by modification with nuclease-resistant groups, such as 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-O-allyl, 2'-H, and nucleotide base modifications. Sugar modifications of nucleic acid molecules have been extensively described in the art.

[0254] Additional modifications and conjugation of oligonucleotides have also been described. Soutschek et al. 2004 Nature 432:173-178 presents a method of conjugating cholesterol to the 3' end of the sense strand of siRNA molecules using a pyrrolidine linker to produce a covalently linked irreversible conjugate. Chemical modifications of oligonucleotides (including conjugation with other molecules) can also be carried out to improve the retention time and efficiency of in vivo pharmacokinetics.

[0255] In some embodiments, the oligonucleotide fragments and / or oligonucleotides comprise modified bases. The present disclosure encompasses oligonucleotides and oligonucleotide fragments in which a single nucleotide at a given position is replaced with a modified version of the same nucleotide.Thus, the nucleotides (A, G, C, or U) include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, and the like. , 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylqueosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosin, pseudouracil, queosin, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil uracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl)uracil, 2,6-diaminopurine, 5-hydroxymethylcytosine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiothymine, 5-propynyl (-C=C-CH3) uracil and cytosine and pyrimidine Modified bases may be substituted by other alkynyl derivatives of thymine bases, 6-azo uracil, cytosine and thymine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methyladenine, 2-F-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.

[0256] Additional modified variants include the addition of any other moiety (e.g., a radioactive label or other tag or conjugate) to the oligonucleotide or oligonucleotide fragment; providing the base sequence is identical, the addition of the other moiety will produce a "modified variant" (with no mismatches).

[0257] In addition to these modifications and patterns of modifications (e.g., formats), general knowledge of nucleic acid modifications can be used to generate other modifications or sets of modifications of the provided sequences. These various embodiments and embodiments of the oligonucleotides of the present disclosure can be used for RNA interference.

[0258] In some embodiments, the oligonucleotides and / or oligonucleotide fragments comprise modifications that increase the stability of the oligonucleotides in a biological sample or environment (e.g., cytoplasm, interstitial fluid, serum, lung or intestinal lavage fluid).

[0259] In some embodiments, the oligonucleotide and / or oligonucleotide fragment comprises a modification that facilitates cleavage by the RNA-induced silencing complex (i.e., a "RISC cleavage site"). The RISC cleavage site is the site on the target where cleavage occurs. In some embodiments, the antisense strand comprises a RISC cleavage site. For RNAi agents having a duplex region 17-23 nucleotides in length, the cleavage site on the antisense strand is typically approximately positions 10, 11, and 12 from the 5' end. As used herein, the term "cleavage region" refers to a region located immediately adjacent to the cleavage site. In some embodiments, the cleavage region is located on either end of the cleavage site and includes three bases immediately adjacent to the cleavage site. In some embodiments, the cleavage region is located on either end of the cleavage site and includes two bases immediately adjacent to the cleavage site. In some embodiments, the cleavage site specifically occurs at the site bounded by nucleotides 10 and 11 of the antisense strand, and the cleavage region includes nucleotides 11, 12, and 13 of the antisense strand.

[0260] In some embodiments, the oligonucleotide fragments and / or oligonucleotides contain modified backbones. As used herein, an unmodified backbone is composed of 3' to 5' phosphodiester linkages. Modified backbones may contain non-natural internucleoside linkages. Oligonucleotides with modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone.

[0261] Oligonucleotide fragments containing modified backbones include, but are not limited to, those that do not have a phosphorus atom in the backbone. Modified backbones include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates (e.g., 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (e.g., mesylphosphoramidate, 3'-aminophosphoramidate, and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, their 2'-5' linked analogs, and those with reverse polarity where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'.

[0262] Oligonucleotide fragments containing modified backbones that do not contain a phosphorus atom therein can have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include those with morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide, and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH2 moieties.

[0263] In some embodiments, the oligonucleotide and / or oligonucleotide fragment comprises at least one phosphonate linkage, wherein the phosphonate is a modified phosphonate selected from the group consisting of: phosphorothioate (which may be the Rp or Sp isomer): [ka] Phosphorodithioates: [ka] Methylphosphonates: [ka] Methoxypropylphosphonate: [ka] 5'-(E)-vinylphosphonate: [ka] 5'-Methylphosphonate: [ka] (S)-5'-C-methyl bearing phosphonate: [ka] 5'-phosphorothioate: [ka] and peptide nucleic acids: [ka]

[0264] In some embodiments, the oligonucleotide and / or oligonucleotide fragment comprises at least one 5'-uridine-adenine-3' (5'-ua-3') dinucleotide (wherein the uridine is a 2'-modified nucleotide); at least one 5'-uridine-guanine-3' (5'-ug-3') dinucleotide (wherein the 5'-uridine is a 2'-modified nucleotide); at least one 5'-cytidine-adenine-3' (5'-ca-3') dinucleotide (wherein the 5'-cytidine is a 2'-modified nucleotide); or at least one 5'-uridine-uridine-3' (5'-uu-3') dinucleotide (wherein the 5'-uridine is a 2'-modified nucleotide). These dinucleotide motifs are particularly susceptible to serum nuclease degradation (e.g., RNase A). Chemical modification at the 2' position of the first pyrimidine nucleotide in the motif prevents or slows such cleavage. This modified recipe is also known by the term "endolite."

[0265] In some embodiments, the oligonucleotide and / or oligonucleotide fragment comprises a modified nucleobase, wherein the modified nucleobase is difluorotolyl, nitroindolyl, nitropyrrolyl, or nitroimidazolyl. In certain embodiments, the modified nucleobase is difluorotolyl. In some embodiments, the oligonucleotide and / or oligonucleotide fragment is double-stranded, and only one of the two strands contains a modified nucleobase. In some embodiments, the oligonucleotide and / or oligonucleotide fragment is double-stranded, and both strands contain a modified nucleobase.

[0266] In some embodiments, the oligonucleotide fragments and / or oligonucleotides comprise modified sugars. Sugar modifications typically involve chemical modifications of the sugar moiety of RNA or DNA. Sugar modifications include, but are not limited to, one of the following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S-, or N-alkynyl; or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl and alkynyl. Exemplary modifications include O[(CH) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)]2, where n and m are from 1 to about 10. Oligonucleotide fragments for use in the methods described herein can include one of the following at the 2' position: C1 to C 10lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH, OCN, Cl, Br, CN, CF, OCF, SOCH, SOCH, ONO, NO, N, NH, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group for improving the pharmacokinetic properties of a therapeutic RNA, or group for improving the pharmacodynamic properties of a therapeutic RNA. In some embodiments, modifications include 2'-methoxyethoxy (also known as 2'-O-(2-methoxyethyl) or 2'-O-MOE), 2'-dimethylaminooxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE). Further exemplary modifications include: 5'-Me-2'-F nucleotides, 5'-Me-2'-Ome nucleotides, 5'-Me-2'-deoxynucleotides, 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0267] Other modifications include 2'-methoxy (2'-OCH), 2'-aminopropoxy (2'-OCHCHCHNH), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the RNA, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNA and the 5' position of the 5' terminal nucleotide.

[0268] In some embodiments, the oligonucleotide fragment and / or oligonucleotide comprises at least one modified nucleotide, in some embodiments, the modification is selected from the group consisting of 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE). , 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acid (LNA), glycol nucleic acid (GNA), phosphoramidate (e.g., mesyl phosphoramidate), 2',3'-seconucleotide mimics, 2'-F-arabinonucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, vinyl phosphonates (e.g., 5' vinyl phosphonate), and cyclopropyl phosphonate deoxyribonucleotides. In some embodiments, one or more of the oligonucleotide fragments comprises a 2'-modification selected from the group consisting of: 2'-Ome, 2'-F, and 2'-deoxy. In some embodiments, the oligonucleotide and / or oligonucleotide fragment comprises one or more 3'-O-methyl nucleotides.

[0269] In some embodiments, the oligonucleotides and / or oligonucleotide fragments comprise a 2'-modification selected from the group consisting of 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylamino Ethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acids (LNA), phosphoramidates (e.g., mesyl phosphoramidate), 2',3'-seconucleotide mimics, 2'-F-arabinonucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, vinyl phosphonates (e.g., 5' vinyl phosphonate), deoxyribonucleotides, and cyclopropyl phosphonates. In some embodiments, the oligonucleotides and / or oligonucleotide fragments comprise one or more 3'-O-methyl nucleotides.

[0270] In some embodiments, the oligonucleotides and / or oligonucleotide fragments comprise a bridged nucleic acid. In some embodiments, the bridged nucleic acid is a locked nucleic acid. In some embodiments, the bridged nucleic acid is a constrained ethyl bridged nucleic acid: [ka] is.

[0271] In some embodiments, all pyrimidines (uridine and cytidine) are 2'O-methyl modified nucleosides.

[0272] In some embodiments, the sense and / or antisense strands are conjugated to one or more diagnostic compounds, reporter groups, crosslinkers, moieties that confer nuclease resistance, modified or unmodified nucleobases, lipophilic molecules, cholesterol, lipids, lectins, steroids, uvaol, hecigenin, diosgenin, terpenes, triterpenes, sarsasapogenin, friedelin, epifriedelol-derivatized lithocholic acid, vitamins, carbohydrates, dextran, pullulan, chitin, chitosan, synthetic carbohydrates, oligolactate 15-mers, natural polymers, low or medium molecular weight polymers, inulin, cyclodextrins, hyaluronic acid, proteins, protein-binding agents, integrin-targeting molecules, polycations, peptides, polyamines, peptidomimetics, and / or transferrin.

[0273] In some embodiments, the antisense strand comprises at least one 2'-OMe modified nucleotide. In some embodiments, the antisense strand comprises at least one 2'-F modified nucleotide. In some embodiments, the antisense strand comprises at least one 2'-deoxy modified nucleotide. In some embodiments, the antisense strand comprises at least one 2'-OMe modified nucleotide, at least one 2'-F modified nucleotide, or at least one 2'-deoxy modified nucleotide, or any combination thereof. In some embodiments, the antisense strand comprises alternating 2'-OMe and 2'-F modified nucleotides. In some embodiments, the antisense strand comprises at least one 5' vinyl phosphonate. In some embodiments, the antisense strand comprises at least one chiral phosphorothioate linkage. In some embodiments, the antisense strand comprises at least one GNA. In some embodiments, the sense strand comprises at least one 2'-OMe modified nucleotide. In some embodiments, the sense strand comprises at least one 2'-F modified nucleotide. In some embodiments, the sense strand comprises at least one 2'-deoxy modified nucleotide. In some embodiments, the sense strand comprises at least one 2'-OMe modified nucleotide, at least one 2'-F modified nucleotide, or at least one 2'-deoxy modified nucleotide, or any combination thereof. In some embodiments, the sense strand comprises alternating 2'-OMe and 2'-F modified nucleotides. In some embodiments, the antisense strand and the sense strand each comprise at least one 2'-OMe modified nucleotide. In some embodiments, the antisense strand and the sense strand each comprise at least one 2'-F modified nucleotide. In some embodiments, the antisense strand and the sense strand each comprise alternating 2'-OMe and 2'-F modified nucleotides. In some embodiments, the sense strand comprises at least one 5' vinyl phosphonate. In some embodiments, the sense strand comprises at least one chiral phosphorothioate linkage. In some embodiments, the sense strand comprises at least one GNA.

[0274] In some embodiments, the sense strand comprises alternating 2'-OMe and 2'-F modified nucleotides along the entire length of the sense strand, hi some embodiments, the sense strand comprises alternating 2'-OMe and 2'-F modified nucleotides along a portion of the length of the sense strand, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides of the sense strand.

[0275] In some embodiments, the antisense strand comprises alternating 2'-OMe and 2'-F modified nucleotides along the entire length of the antisense strand, hi some embodiments, the antisense strand comprises alternating 2'-OMe and 2'-F modified nucleotides along a portion of the length of the antisense strand, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides of the antisense strand.

[0276] In some embodiments, the sense and antisense strands comprise alternating 2'-OMe and 2'-F modified nucleotides along the entire length of the sense and antisense strands, respectively. In some embodiments, the sense and antisense strands comprise alternating 2'-OMe and 2'-F modified nucleotides along a portion of the length of the sense and antisense strands, for example, at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides of the sense and antisense strands.

[0277] Ligand In some embodiments, one or more of the oligonucleotide fragments are conjugated to at least one ligand. In some embodiments, the oligonucleotide product is conjugated to at least one ligand. The ligand can be conjugated to the sense strand, the antisense strand, or both strands in any configuration, such as the 3' end, the 5' end, a non-end, or a combination.

[0278] In some embodiments, the ligand comprises one or more N-acetylgalactosamine (GalNAc) derivatives, hi some embodiments, the ligand comprises one or more GalNAc derivatives conjugated via a bivalent or trivalent branched carrier.

[0279] In some embodiments, the ligand is [ka] is.

[0280] In some embodiments, the ligand is [ka] is.

[0281] In some embodiments, the ligand is [ka] is.

[0282] In some embodiments, the ligand is [ka] is.

[0283] In some embodiments, the ligand is [ka] is.

[0284] In some embodiments, the ligand is [ka] is.

[0285] In some embodiments, a ligand alters the distribution, targeting, or lifetime of a molecule into which it is incorporated. In some embodiments, a ligand provides enhanced affinity for a selected target, e.g., a molecule, a cell or cell type, a compartment, a receptor, e.g., a cell or organ compartment, a tissue, an organ, or a region of the body, compared to a species in which such ligand is not present. Ligands that provide enhanced affinity for a selected target are also referred to as targeting ligands.

[0286] Some ligands may have endosomal properties. An endosomal ligand promotes endosomal lysis and / or transport of an oligonucleotide or a composition comprising the oligonucleotide from an endosome into the cytoplasm of a cell. An endosomal ligand may be a polyanionic peptide or peptidomimetic that exhibits pH-dependent membrane activity and fusogenicity. In some embodiments, an endosomal ligand adopts its active conformation at endosomal pH. An "active" conformation is one in which the endosomal ligand promotes endosomal lysis and / or transport of an oligonucleotide or a composition comprising the oligonucleotide from an endosome into the cytoplasm of a cell. Exemplary endosomolytic ligands include GALA peptides (Subbarao et al., Biochemistry, 1987, 26:2964-2972), EALA peptides (Vogel et al., J. Am. Chem. Soc., 1996, 118:1581-1586), and their derivatives (Turk et al., Biochem. Biophys. Acta, 2002, 1559:56-68). The endosomolytic component may contain a chemical group (e.g., an amino acid) that undergoes a change in charge or protonation in response to a change in pH. The endosomolytic component may be linear or branched.

[0287] Ligands can improve the transport, hybridization and specificity properties, as well as the nuclease resistance of the resulting natural or modified oligonucleotides.

[0288] Ligands may generally include therapeutic modifiers, such as those for promoting uptake; diagnostic compounds or reporter groups, such as those for monitoring distribution; cross-linking agents; and nuclease resistance-conferring moieties. Common examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptidomimetics.

[0289] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers (e.g., synthetic polyamino acids), oligonucleotides (e.g., aptamers), etc. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-glycolic acid copolymer), divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacrylic acid), N-isopropylacrylamide polymer, or polyphosphazine. Examples of polyamines include: polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0290] The ligand can also include a targeting group, such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid, or protein, such as an antibody, which binds to a specific cell type. The targeting group can be thyroid-stimulating hormone, melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetylgalactosamine, N-acetylglucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acid, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, biotin, RGD peptide, RGD peptidomimetic, or aptamer.

[0291] Other examples of ligands include dyes, intercalating agents (e.g., acridine), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or chelating agents (EDTA, etc.), lipophilic molecules (e.g., cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic ... 3-(oleoyl)cholenoic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0292] Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules with specific affinity for a co-ligand, or antibodies, e.g., antibodies that bind to specific cell types, such as cancer cells, endothelial cells, or bone cells. Ligands can also include hormones and hormone receptors. They can also include non-peptide species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetylgalactosamine, N-acetylglucosamine, multivalent mannose, multivalent fucose, or aptamers. Ligands can be, for example, lipopolysaccharides, activators of p38 MAP kinase, or activators of NF-κB.

[0293] In some embodiments, the ligand is a lipid or lipid-based molecule. Such lipid or lipid-based molecule preferably binds to a serum protein, such as human serum albumin (HSA). The HSA-binding ligand allows the conjugate to be distributed to a target tissue. The lipid or lipid-based ligand can (a) increase the resistance of the conjugate to degradation, (b) increase targeting or transport to a target cell or cell membrane, and / or (c) be used to adjust binding to a serum protein, such as HSA. The lipid-based ligand can be used to regulate, for example, control, the binding of the conjugate to a target tissue.

[0294] In some embodiments, the ligand is a peptide or peptidomimetic. A peptidomimetic is a molecule that can fold into a defined three-dimensional structure similar to a natural peptide. The peptide or peptidomimetic moiety can be about 5 to 50 amino acids in length, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length. The peptide or peptidomimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). The peptide moiety can be a "delivery" peptide, which can transport large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. Peptides or peptidomimetics can be encoded by random sequences of DNA, such as peptides identified from phage display libraries or one-bead-one-compound (OBOC) combinatorial libraries (Lam et al., Nature, 354:82-84, 1991).

[0295] As used herein, a "peptide moiety" can range in length from about 5 amino acids to about 50 amino acids. The peptide moiety can have structural modifications, such as to enhance stability or direct structural properties. Any of the structural modifications described below can be utilized. Arginine-glycine-aspartic acid (RGD) peptide moieties can be used to target tumor cells, such as endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res., 62:5139-43, 2002). RGD peptides can facilitate targeting of oligonucleotides to tumors in various other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). RGD peptides can be linear or cyclic and can be modified, e.g., glycosylated or methylated, to facilitate targeting to specific tissues. Peptides that target markers enriched in proliferating cells can be used. For example, RGD-containing peptides and peptidomimetics can target cancer cells, particularly those that express integrins. Thus, the ligand may comprise an RGD peptide, a cyclic peptide containing RGD, an RGD peptide containing D-amino acids, or a synthetic RGD mimetic.

[0296] Peptide and peptidomimetic ligands include natural or modified peptides, e.g., D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides in which one or more amide (i.e., peptide) bonds are replaced with one or more urea, thiourea, carbamate, or sulfonylurea bonds; or cyclic peptides.

[0297] Ligands can be coupled to oligonucleotide fragments and / or oligonucleotides at various locations, e.g., the 3'-terminus, 5'-terminus, and / or internal ("non-terminal") positions. In some embodiments, the ligand is attached via an intervening tether, e.g., a carrier described herein. The ligand or tethered ligand can be present on the monomer when the monomer is incorporated into the oligonucleotide fragment and / or oligonucleotide. In some embodiments, the ligand can be incorporated via coupling to a "precursor" monomer after the "precursor" monomer is incorporated into the oligonucleotide fragment and / or oligonucleotide. For example, a monomer bearing an amino-terminated tether (i.e., no associated ligand), e.g., Tap-(CH2)nNH2, can be incorporated into a growing oligonucleotide fragment. In subsequent manipulations, i.e., after incorporation of the precursor monomer into the oligonucleotide fragment, a ligand bearing an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can be attached to the precursor monomer by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor monomer's tether.

[0298] In another example, monomers bearing chemical groups suitable for participating in click chemistry reactions, such as azide or alkyne terminal tethers / linkers, may be incorporated. Subsequent manipulation, i.e., after incorporation of the precursor monomer into an oligonucleotide fragment and / or oligonucleotide, a ligand bearing a complementary chemical group, e.g., an alkyne or azide, can be attached to the precursor monomer by coupling the alkyne and azide together.

[0299] In some embodiments, the ligand is conjugated to the nucleobase, sugar moiety, or internucleoside linkage of the oligonucleotide fragment and / or oligonucleotide. Conjugation to a purine nucleobase or a derivative thereof can occur at any position, including endocyclic and exocyclic atoms. In some embodiments, the 2-, 6-, 7-, or 8-position of a purine nucleobase is bound to a conjugate moiety. Conjugation to a pyrimidine nucleobase or a derivative thereof can also occur at any position. In some embodiments, the 2-, 5-, and 6-positions of a pyrimidine nucleobase can be substituted with a conjugate moiety. Conjugation to the sugar moiety of a nucleoside can occur at any carbon atom. Exemplary carbon atoms of the sugar moiety that can be bound to a conjugate moiety include the 2', 3', and 5' carbon atoms. In addition, the 1' position can be bound to a conjugate moiety, such as in an abasic residue. The internucleoside linkage can also have a conjugate moiety. In the case of phosphorus-containing linkages (e.g., phosphodiesters, phosphorothioates (e.g., chiral phosphorothioates), phosphorodithioates, phosphoramidates, etc.), the conjugate moiety can be attached directly to the phosphorus atom or to an O, N, or S atom attached to the phosphorus atom. In the case of amine- or amide-containing internucleoside linkages (e.g., PNA), the conjugate moiety can be attached to the nitrogen atom or adjacent carbon atom of the amine or amide.

[0300] In some embodiments, the ligand is conjugated to the sense strand. In some embodiments, the ligand is conjugated to the 3' end of the sense strand. In some embodiments, the ligand is conjugated to the 5' end of the sense strand. In some embodiments, the ligand is conjugated to a non-terminal end of the sense strand.

[0301] In some embodiments, the ligand is conjugated to the antisense strand. In some embodiments, the ligand is conjugated to the 3' end of the antisense strand. In some embodiments, the ligand is conjugated to a non-terminal end of the antisense strand.

[0302] The ligand can be attached via the carrier. The carrier comprises (i) at least one "backbone attachment point," preferably two "backbone attachment points," and (ii) at least one "tether attachment point." As used herein, "backbone attachment point" refers to a functional group, such as a hydroxyl group, or generally a bond suitable for incorporation of the carrier into the backbone of a nucleic acid, such as a phosphate, or a modified phosphate, such as a sulfur-containing backbone. In some embodiments, a "tether attachment point" (TAP) refers to a ring atom, such as a carbon atom or heteroatom (different from the atom providing the backbone attachment point), of the cyclic carrier to which the selected moiety is attached. This moiety can be, for example, a carbohydrate, such as a monosaccharide, disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide. Optionally, the selected moiety is linked to the cyclic carrier by an intervening tether. Thus, the cyclic carrier often comprises a functional group, such as an amino group, or generally provides a bond suitable for incorporation or tethering of another chemical entity, such as a ligand, to the ring.

[0303] When the oligonucleotide fragment is a dsRNA, the sense strand and / or the antisense strand may be conjugated to the ligand via a carrier, which may be a cyclic group or an acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol skeleton or a diethanolamine skeleton.

[0304] In some embodiments, one or more of the oligonucleotide fragments contain the sequence "TT," "dTdT," "dTsdT," or "UU" as a 3'-terminal single-stranded overhang, also referred to herein as a terminal dinucleotide or 3'-terminal dinucleotide. dT is 2'-deoxy-thymidine-5'-phosphate, and sdT is 2'-deoxy-thymidine-5'-phosphorothioate. The terminal dinucleotide "UU" is UU or 2'-OMe-U 2'-OMe-U, and the terminal TT and terminal UU can be in an inverted / reverse orientation. Terminal dinucleotides (e.g., UU) are modified variants of dithymidine dinucleotides that are commonly placed as overhangs to protect the ends of siRNAs from nucleases (see, for example, Elbashir et al. 2001 Nature 411:494-498; Elbashir et al. 2001 EMBO J. 20:6877-6888; and Kraynack et al. 2006 RNA 12:163-176). From these references, it is known that terminal dinucleotides enhance nuclease resistance but do not contribute to target recognition.

[0305] In some embodiments, one or both of the terminal oligonucleotide fragments contain a 3-terminal cap instead of or in addition to a terminal dinucleotide to stabilize the termini from nuclease degradation, provided that the 3-terminal cap is capable of both stabilizing the oligonucleotide (e.g., against nucleases) and not unduly interfering with its desired activity.

[0306] When the oligonucleotide fragment is a dsRNA, the sense strand and / or the antisense strand may be conjugated to the ligand via a carrier, which may be a cyclic group or an acyclic group; preferably, the cyclic group is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl, and decalin; preferably, the acyclic group is selected from a serinol skeleton or a diethanolamine skeleton.

[0307] Additional Embodiments Embodiment 1. SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 440, 442, 444, 446, 84, 386, 388, 390, 392, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 5 70, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 555 56, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, and 600; the modified dsRNA ligase polypeptide (a) has dsRNA ligase activity; and (b) does not contain the amino acid sequence of SEQ ID NO: 302; Modified dsRNA polypeptides. Embodiment 2. SEQ ID NOs: 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 446, 2, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486 , 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578, 580, 582, 584, 586, 588, 590, 592, 594, 596, 598, 600, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668; the modified dsRNA ligase polypeptide is (a) has dsRNA ligase activity; and (b) does not contain the amino acid sequence of SEQ ID NO: 302; Modified dsRNA polypeptides. Embodiment 3. The modified dsRNA ligase polypeptide of embodiment 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590, and 592. Embodiment 4. The modified dsRNA ligase polypeptide of embodiment 2, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 370, 488, 526, 578, 588, 590, 592, and 666. Embodiment 5. A modified dsRNA ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 302, wherein the modified dsRNA ligase polypeptide produces at least 5% more oligonucleotide products under the same ligation reaction conditions than a dsRNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO: 302, wherein the modified dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302. Embodiment 6. The modified dsRNA ligase polypeptide of embodiment 5, wherein the ligation reaction conditions comprise about 1 μM to about 10 mM of the oligonucleotide fragment, an ATP source, about 5 mM to about 100 mM of divalent cations, and about 0.5 g / L to about 10 g / L of the modified dsRNA ligase polypeptide, a pH of about 4.0 to about 8.0, and a temperature of about 10°C to about 50°C. Embodiment 7. The amino acid sequence of the modified dsRNA ligase polypeptide is selected from the group consisting of X6, X7, X15, X19, X29, X36, X39, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X91, X93, X103, X105, X107, X114, X122, X126, X129, X130, X131, X137, X144, X146, X158, X163, X173, X178, X190, X196, X216, X218, X221, X228, 7. The modified dsRNA ligase polypeptide of embodiment 5 or 6, comprising an amino acid sequence which differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from: X230, X232, X235, X236, X237, X238, X239, X242, X243, X244, X251, X252, X254, X255, X258, X269, X280, X284, X285, X293, X296, X301, X303, X305, X314, X325, and X328 (numbering refers to SEQ ID NO: 302). Embodiment 8. The amino acid sequence of the modified dsRNA ligase polypeptide comprises the following amino acid residues: X6 is G; X7 is Q; X15 is R, D or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; X57 is S; X60 is T, G or P; X63 is S, Q or G; X64 is R, T, Q, F, G, or M; X 66 is F or W; X67 is N; X87 is T, P, K or absent; X88 is C; X91 is S; X93 is G, C, or A; X103 is V, C, Y, or T; X105 is V; X107 is R or T; X114 is N; X122 is W; X126 is G; X129 is N; X130 is R, S, or Y; X131 is R; X137 is V or C; X144 is N; X146 is R; X158 is W; X163 is G; X173 is L; X178 is R; X190 is Q; X196 is S or C; X216 is L or R; X218 is N; X221 is I; X228 is R; X230 is T; X232 is R; X235 is A, T, or G; X236 is S, L, or F; X237 is S, Q, or R; X238 is F; X239 is G or R; X242 is R or M; X243 is N, S, G, or M; X244 is G or K; and X251 is D or L. X252 is V; X254 is K; X255 is C; X258 is V; X269 is L; X280 is W; X284 is A; X285 is A; X293 is R; X296 is R; X301 is G, L, E, or F; X303 is Q; X305 is G; X314 is A or V; X325 is R; X328 is R; (numbering refers to SEQ ID NO: 302). Embodiment 9. The amino acid sequence of the modified dsRNA ligase polypeptide is selected from the group consisting of X6, X7, X15, X19, X29, X36, X39, X44, X45, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X89, X91, X92, X93, X103, X105, X107, X114, X122, X126, X129, X130, X131, X137, X144, X146, X158, X163, X173, X178, X185, X190, X196, X216, X218, X221, 9. The modified dsRNA ligase polypeptide of any one of embodiments 5 to 8, comprising an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from X228, X230, X232, X235, X236, X237, X238, X239, X242, X243, X244, X251, X252, X254, X255, X258, X269, X280, X284, X285, X293, X296, X301, X303, X305, X313, X314, X325, and X328 (numbering refers to SEQ ID NO: 302). Embodiment 10. The amino acid sequence of the modified dsRNA ligase polypeptide comprises the following amino acid residues: X6 is G or E; X7 is Q; X15 is R, D or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X44 is V; X45 is V; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; X57 is S; X60 is T, G or P; X63 is S, Q or G; X64 is R, T, Q, F, G, or is M; X66 is F or W; X67 is N; X87 is T, P, K or absent; X88 is C; X89 is T; X91 is S; X92 is D; X93 is G, C, or A; X103 is V, C, Y, or T; X105 is V; X107 is R or T; X114 is N; X122 is W; X126 is G; X129 is N; X130 is R, S, or Y; X131 is R; X137 is V or C; X144 is N; X146 is R; X158 is W; X 163 is G; X173 is L; X178 is R; X185 is K; X190 is Q; X196 is S or C; X216 is L or R; X218 is N; X221 is I; X228 is R; X230 is T; X232 is R; X235 is A, T, or G; X236 is S, L, or F; X237 is S, Q, R, L, or G; X238 is F; X239 is G or R; X242 is R or M; X243 is N, S, G, or M; X244 is G or K; X25 10. The modified dsRNA ligase polypeptide of embodiment 9, comprising one or more of: X1 is D or L; X252 is V; X254 is K; X255 is C; X258 is V; X269 is L; X280 is W; X284 is A; X285 is A; X293 is R; X296 is R; X301 is G, L, E, or F; X303 is Q; X305 is G; X313 is A; X314 is A or V; X325 is R; and X328 is R; (numbering refers to SEQ ID NO: 302). Embodiment 11. The modified dsRNA ligase polypeptide of any one of embodiments 5 to 10, wherein the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from X15, X19, X36, X39, X53, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302), and wherein the modified dsRNA ligase polypeptide has dsRNA ligase activity. Embodiment 12. The modified dsRNA ligase polypeptide of embodiment 11, wherein the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; X285 is A; (numbering refers to SEQ ID NO: 302). Embodiment 13. The modified dsRNA ligase polypeptide of any one of embodiments 5 to 12, wherein the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from X15, X19, X36, X39, X53, X185, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302), and wherein the modified dsRNA ligase polypeptide has dsRNA ligase activity. Embodiment 14. The modified dsRNA ligase polypeptide of embodiment 13, wherein the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; X285 is A; (numbering refers to SEQ ID NO: 302). Embodiment 15. The modified dsRNA ligase polypeptide of any one of embodiments 5 to 14, wherein the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence which differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from X36, X39, X218 and X221 (numbering refers to SEQ ID NO: 302), and wherein the modified dsRNA ligase polypeptide has dsRNA ligase activity, and optionally the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I. Embodiment 16. The modified dsRNA ligase polypeptide of any one of embodiments 5 to 15, wherein the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from X39, X218 and X221 (numbering refers to SEQ ID NO: 302), and wherein the modified dsRNA ligase polypeptide has dsRNA ligase activity, and optionally the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X39 is A; X218 is N; and X221 is I. Embodiment 17. The modified dsRNA ligase polypeptide of any one of embodiments 5 to 16, wherein the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence which differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from X39, X218, X221 and X255 (numbering refers to SEQ ID NO: 302), and wherein the modified dsRNA ligase polypeptide has dsRNA ligase activity, and optionally the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C. Embodiment 18. The modified dsRNA ligase polypeptide of any one of embodiments 5 to 17, wherein the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence which differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from X39, X53, X218, X221, X237, X251, X255 and X285 (numbering refers to SEQ ID NO: 302), and wherein the modified dsRNA ligase polypeptide has dsRNA ligase activity, and optionally the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A. Embodiment 19. The modified dsRNA ligase polypeptide of any one of embodiments 5 to 18, wherein the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence which differs from that of SEQ ID NO: 302 at one or more amino acid residues selected from X15, X39, X53, X218, X221, X237, X251, X255 and X285 (numbering refers to SEQ ID NO: 302), and wherein the modified dsRNA ligase polypeptide has dsRNA ligase activity, and optionally the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is E; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A. Embodiment 20. The modified dsRNA ligase polypeptide of any one of embodiments 5 to 19, wherein the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence which differs from that of SEQ ID NO: 302 at one or more amino acid residues selected from X19, X39, X53, X218, X221, X237, X251, X255 and X285 (numbering refers to SEQ ID NO: 302), wherein the modified dsRNA ligase polypeptide has dsRNA ligase activity, and optionally the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X19 is D; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A. Embodiment 21. The modified dsRNA ligase polypeptide of any one of embodiments 5 to 20, wherein the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence which differs from that of SEQ ID NO: 302 at one or more amino acid residues selected from X15, X39, X53, X185, X218, X221, X237, X251, X255 and X285 (numbering refers to SEQ ID NO: 302), and wherein the modified dsRNA ligase polypeptide has dsRNA ligase activity, and optionally the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A. Embodiment 22 The modified dsRNA ligase polypeptide of any one of embodiments 1 to 21, wherein the modified dsRNA ligase polypeptide comprises a purification tag. Embodiment 23. SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98 , 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174 , 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 25 23. The modified dsRNA ligase polypeptide of embodiment 22, comprising an amino acid sequence selected from the group consisting of: 0, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298 and 300. Embodiment 24. SEQ ID NOs: 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 246, 248, 250, 251, 252, 253, 92, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 270, 272, 274, 276 23. The modified dsRNA ligase polypeptide of embodiment 22, comprising an amino acid sequence selected from the group consisting of: 6, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, and 634. Embodiment 25. A polypeptide immobilized on a solid material by chemical bonding or physical adsorption, wherein the polypeptide comprises a modified dsRNA ligase polypeptide according to any one of embodiments 1 to 24. Embodiment 26. A polynucleotide encoding the modified dsRNA ligase polypeptide of any one of embodiments 1 to 24. Embodiment 27. The polynucleotide sequence is selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141 , 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 2 67, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 3 31, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 39 3, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455 , 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517,27. The polynucleotide of embodiment 26, which is 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, or 599. Embodiment 28. The polynucleotide sequence is selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141 , 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 265, 2 67, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 3 31, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 381, 383, 385, 387, 389, 391, 39 3, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455 , 457, 459, 461, 463, 465, 467, 469, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517,519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 557, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 59 9, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, 633, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, and 667. Embodiment 29: An expression vector comprising the polynucleotide according to any one of embodiments 26 to 28. Embodiment 30. The expression vector of embodiment 29, comprising a plasmid, cosmid, bacteriophage or viral vector. Embodiment 31. A host cell comprising a polynucleotide according to any one of embodiments 26 to 28, or an expression vector according to embodiment 29 or 30, optionally wherein the host cell is Escherichia coli (E. coli). Embodiment 32 A method for preparing a modified dsRNA ligase polypeptide, comprising culturing the host cell of embodiment 31 and obtaining the modified dsRNA ligase polypeptide from the culture. Embodiment 33. A modified dsRNA ligase catalyst obtained by culturing the host cells of embodiment 31 or according to the method of embodiment 32, including cells or culture media containing the modified dsRNA ligase polypeptide, or articles treated therewith, wherein the article refers to an extract obtained from the culture of the host cells, an isolated product obtained by isolating or purifying the modified dsRNA ligase from the extract, or an immobilized product obtained by immobilizing the host cells, the extract, or an isolated product of the extract. Embodiment 34. A method for generating an oligonucleotide from two or more oligonucleotide fragments, comprising: (i) two or more oligonucleotide fragments; (ii) a modified dsRNA ligase polypeptide according to any one of embodiments 1 to 24; (iii) a source of ATP; and (iv) divalent cations to obtain an oligonucleotide. Embodiment 35. The method of embodiment 34, wherein the ATP source comprises ATP. Embodiment 36. The ATP source comprises: (a) Polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and / or ATP 36. The method of embodiment 34 or 35, comprising: Embodiment 37. The method of embodiment 36, wherein the PPK is selected from PPK12 or ajPAP. Embodiment 38. The method of any one of embodiments 36 or 37, carried out using sub-stoichiometric concentrations of AMP and / or ATP. Embodiment 39. The method of any one of embodiments 36 to 38, wherein the polyphosphoric acid is a polyphosphate salt. Embodiment 40. The method of embodiment 39, wherein the polyphosphate salt is sodium polyphosphate (Madrell's salt) or sodium hexametaphosphate (Graham's salt). Embodiment 41. The divalent cation cofactor is Mg 2+ or Mn 2+ 41. The method of any one of embodiments 34 to 40, wherein Embodiment 42. The method of any one of embodiments 34 to 41, carried out at a divalent cation concentration of 5 to 100 mM, optionally 30 to 50 mM. Embodiment 43. The method of any one of embodiments 34 to 42, further comprising purifying the oligonucleotide. Embodiment 44. Use of a modified dsRNA ligase polypeptide according to any one of embodiments 1 to 24 in generating an oligonucleotide from two or more oligonucleotide fragments. Embodiment 45. The method according to any one of embodiments 34 to 43 or the use according to embodiment 44, wherein the oligonucleotide is at most 60 nucleotides in length. Embodiment 46. The method according to any one of embodiments 34 to 43 or 45, or the use according to embodiment 44 or 45, wherein the oligonucleotide fragments are each 4 to 16 nucleotides in length, optionally 6 to 9 nucleotides in length. Embodiment 47. The method according to any one of embodiments 34 to 43, 45 or 46, or the use according to any one of embodiments 44 to 46, wherein one or more of the oligonucleotide fragments comprises one or two overhangs. Embodiment 48. The method according to any one of embodiments 34 to 43 or 45 to 47, or the use according to any one of embodiments 44 to 47, wherein one or more of the oligonucleotide fragments comprises a chemical modification. Embodiment 49. The chemical modification is (a) optionally a modified backbone selected from phosphorothioate (e.g., chiral phosphorothioate) or methylphosphonate internucleotide linkages; (b) optionally, 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'- modified nucleotides selected from O-NMA, locked nucleic acids (LNA), glycol nucleic acids (GNAs), phosphoramidates (e.g., mesyl phosphoramidate), 2',3'-seconucleotide mimics, 2'-F-arabinonucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, vinyl phosphonates (e.g., 5' vinyl phosphonate), and cyclopropyl phosphonate deoxyribonucleotides; and / or (c) conjugation to a ligand, optionally wherein the ligand comprises one or more N-acetylgalactosamine (GalNAc) derivatives; 49. The method or use according to embodiment 48, selected from: Embodiment 50. i. a modified dsRNA ligase polypeptide according to any one of embodiments 1 to 24; ii. a source of ATP; and iii. Divalent cations A composition comprising: Embodiment 51. The composition of embodiment 50, further comprising two or more oligonucleotide fragments. Embodiment 52. i. a modified dsRNA ligase polypeptide according to any one of embodiments 1 to 24; ii.ATP source; iii. divalent cations; and iv. Instructions for use in a method for generating an oligonucleotide from two or more oligonucleotide fragments. Includes a kit. Embodiment 53. The composition of embodiment 50 or 51, or the kit of embodiment 52, wherein the ATP source comprises ATP. Embodiment 54. The ATP source comprises: (a) Polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and / or ATP 54. The composition of any one of embodiments 50, 51 or 53, or the kit of embodiment 52 or 53, comprising: Embodiment 55. A composition or kit according to embodiment 54, wherein the PPK is selected from PPK12 or ajPAP. Embodiment 56. The composition of any one of embodiments 50, 51, or 53 to 55, or the kit of any one of embodiments 52 to 55, wherein the polyphosphoric acid is a polyphosphate salt. Embodiment 57. The composition or method of embodiment 56, wherein the polyphosphate salt is sodium polyphosphate (Madrell's salt) or sodium hexametaphosphate (Graham's salt). Embodiment 58. The divalent cation cofactor is Mg 2+ or Mn 2+ The composition of any one of embodiments 50, 51, or 53 to 57, or the kit of any one of embodiments 52 to 57, wherein

[0308] Various features and embodiments of the present disclosure are illustrated in the following representative examples, which are intended to be illustrative and not limiting. [Example]

[0309] The following examples (including experiments and results achieved) are provided for illustrative purposes only and are not to be construed as limiting the invention.

[0310] In the examples below, the following abbreviations apply: ppm (parts per million); M (molar); mM (millimolar); uM and μM (micromolar); nM (nanomolar); mol (mole); gm and g (grams); mg (milligrams); ug and μg (micrograms); L and l (liters); ml and mL (milliliters); cm (centimeters); mm (millimeters); um and μm (micrometers); sec. (seconds); min (minutes); h and hr (hours); U (units); M W (molecular weight); rpm (revolutions per minute); psi and PSI (pounds per square inch); °C (degrees Celsius); RT and rt (room temperature); OD600 (optical density at 600 nm); CAM and cam (chloramphenicol); DMSO (dimethyl sulfoxide); FP (fermentation powder); FWC (frozen whole cells); LWC (lyophilized whole cells); PMBS (polymyxin B sulfate); IPTG (isopropyl β-D-1-thiogalactopyranoside); LB (lysogeny broth); TB (Terrific Broth; 12 g / L Bactotryptone, 24 g / L yeast extract, 4 mL / L glycerol, 65 mM potassium phosphate, pH 7.0, 1 mM MgSO4), TEoA (triethanolamine buffer), HEPES (HEPES zwitterionic buffer; 4-(2-hydroxyethyl)-piperazineethanesulfonic acid); SFP (shake flask powder); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); Escherichia coli (E. coli) W3110 (a commonly used laboratory strain of E. coli, available from the Coli Genetic Stock Center [CGSC], New Haven, CT); HTP (high throughput), HPLC (high pressure liquid chromatography); FIOP (fold improvement over positive control); Microfluidics (Microfluidics, Corp., Westwood, MA); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Difco (Difco Laboratories, BD Diagnostics) Systems,Detroit,MI);Agilent(Agilent Technologies,Inc.Corning (Corning, Inc., Palo Alto, CA); Dow Corning (Dow Corning, Corp., Midland, MI); and Gene Oracle (Gene Oracle, Inc., Mountain View, CA). .

[0311] The sequences of the oligonucleotides referred to in parentheses throughout the examples (eg, "siRNA(1)" and "oligonucleotide(2)") are provided in Table 1.

[0312] Example 1 Preparation of isolated enzyme A polynucleotide encoding a polypeptide having ligase activity was cloned into the pCK110900 vector system (see, e.g., Figure 3 of U.S. Patent Application Publication No. 2006 / 0195947A1, which is incorporated herein by reference in its entirety) and then expressed in E. coli W3110fhuA under the control of the lac promoter. The expression vector also contained a P15a origin of replication and a chloramphenicol (CAM) resistance gene.

[0313] E. coli W3110fhuA cells were transformed with the pCK110900 plasmid, which contains the gene encoding the ligase. Transformed cells were plated on lysogeny broth (LB) agar plates containing 1% glucose and 30 μg / mL CAM and grown overnight at 37°C. A single colony was then inoculated into 25 mL of LB supplemented with 30 μg / mL CAM and 1% glucose in a 250 mL baffled shake flask. The culture was grown overnight (16-20 h) and at an optical density (OD ) of 100 kJ / mL in a 37°C incubator with shaking at 250 rpm. 600 )>3.8). 5 mL of the overnight culture was inoculated into a 1 L shake flask containing 250 mL of Terrific Broth (TB) medium containing 30 μg / mL of CAM. The 250 mL culture was incubated at 30°C, 250 rpm, and OD 600The culture was incubated for 3–3.5 hours until the pH reached 0.6–0.8. Expression of the ligase gene was induced by adding isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 1 mM, and growth was continued for an additional 18–20 hours. Cells were harvested by transferring the culture to a centrifuge bottle, which was then centrifuged at 7,000 rpm for 5 minutes at 4°C. The supernatant was discarded, and the remaining cell pellet was lysed. For lysis, the cell pellet was resuspended in 30 mL of 50 mM Tris buffer, pH 7.5, and lysed using an LM20 MICROFLUIDIZER® Processor System (Microfluidics). Cell debris was removed by centrifugation at 14,000 rpm for 30 minutes at 4°C. The ligase enzyme was then isolated from the clarified lysate using standard techniques known in the art, including immobilized metal affinity chromatography.

[0314] Example 2 Identification of dsRNA ligase activity for siRNA(1) generation To identify an enzyme with dsRNA ligase activity for the production of siRNA (1), which contains oligonucleotides (2) and (3), a collection of ligases was first screened for the production of siRNA (4), a surrogate product containing oligonucleotides (3) and (5). Oligonucleotide (5) has the same sequence as oligonucleotide (2), but does not contain the 3'-GalNAc moiety. siRNAs (1) and (4) and oligonucleotides (2), (3), and (5) are shown in Figure 1; the sequences of oligonucleotides (2), (3), and (5) are provided in Table 1.

[0315] Screening of isolated ligases was performed in 20 μL reaction volumes in PCR tubes. Each tube contained 10 μM (each) of substrate oligonucleotides (6–11) with 50 mM Tris buffer pH 7.5, 1 mM ATP or 1 mM NAD, 10 mM MgCl2, 5 mM DTT, and 50% (v / v) isolated ligase enzyme. Reactions were incubated at 16°C for 2 hours in a thermocycler and analyzed using standard techniques known in the art, including electrophoresis. The ligase with SEQ ID NO:2 exhibited the highest dsRNA ligase activity for the formation of siRNA (4). The activity of SEQ ID NO:2 for the production of siRNA (1) was subsequently confirmed using multiple enzyme preparations, including isolated enzyme (Example 1), clarified lysate (Example 4), and shake-flask powder (SFP; Example 5).

[0316] [Table 2]

[0317] Example 3 Preparation of cell pellets for high-throughput (HTP) screening Single colonies were picked in a 96-well format and grown in 190 μL LB medium containing 1% glucose and 30 μg / mL CAM at 30°C, 200 rpm, and 85% humidity. After overnight growth, 20 μL of the grown culture was transferred to a deep-well plate containing 380 μL TB medium with 30 μg / mL CAM. The culture was grown at 30°C, 250 rpm, and 85% humidity for approximately 2.5 hours. The OD of the culture was 600 When the RI reached 0.4–0.8, expression of the ligase gene was induced by adding IPTG to a final concentration of 1 mM. After induction, growth continued for 18–20 h at 30°C, 250 rpm, and 85% humidity. Cells were harvested by centrifugation at 4,000 rpm and 4°C for 10 min, and the supernatant was then discarded. The cell pellet was stored at -80°C until ready for use.

[0318] Example 4 Lysis and clarification lysate preparation Prior to performing the assay, the cell pellets were thawed and resuspended in 300 μL of lysis buffer (1 g / L lysozyme, 0.5 g / L PMBS, and 0.1 μL / mL or 0.2 U / mL commercial DNAse (New England BioLabs, M0303L) in 50 mM Tris buffer, pH 7.5). The plates were shaken and agitated at medium speed on a microtiter plate shaker for 2.5 hours at room temperature. The plates were then centrifuged at 4,000 rpm for 10 minutes at 4°C, and the clarified supernatant was used in the HTP assay reaction for activity determination, as described in the Examples below.

[0319] Example 5 Preparation of shake flask powder (SFP) and fermentation powder (FP) The shake flask procedure can be used to generate a modified dsRNA ligase polypeptide shake flask powder (SFP), which is useful for use in secondary screening assays and / or the biocatalytic processes described herein. The shake flask powder preparation of the enzyme provides a more concentrated preparation of the modified enzyme compared to the cell lysate used in the HTP assay.

[0320] The clarified lysate produced according to Example 1 was collected, frozen at -80°C, and then lyophilized using standard methods known in the art. Lyophilization of the frozen clarified lysate provides a dried SFP containing the crude wild-type or modified dsRNA ligase polypeptide.

[0321] Example 6 Analytical methods for activity and selectivity evaluation The improved activity of the engineered dsRNA ligase was analyzed by high-pressure liquid chromatography (HPLC) using the method described in Tables 6-1 and 6-2. An HPLC method with UV detection was developed to analyze the formation of product oligonucleotides (2) and (3). The analytical method aimed for the shortest run time possible to allow good resolution of product oligonucleotides (2) and (3). Consequently, it was not possible to successfully resolve all six substrate oligonucleotides (6-7, 9-12) and four intermediate oligonucleotides (13-16) from each other. However, it was possible to resolve well-defined GalNAc-containing oligonucleotides, including substrate oligonucleotide (12), reaction intermediate oligonucleotide (14), and product oligonucleotide (2). Therefore, the pseudo% conversion, expressed in arbitrary units (AU), can be calculated by considering only these well-resolved species according to the following formula:

number

[0322] [Table 3]

[0323] To improve the separation of the product oligonucleotide (3) and the substrate oligonucleotide (12), HPLC method 2 (Table 6-2) was developed from HPLC method 1 (Table 6-1).

[0324] [Table 4]

[0325] The improved activity of the modified dsRNA ligase of Example 12 was also analyzed by RapidFire® mass spectrometry (RF-MS) using the method described in Table 6-3. RF-MS aims to shorten analysis time compared to HPLC analysis. Selective detection of product oligonucleotides (2) and (3) is achieved by the specific mass of each product oligonucleotide analyzed in multiple single ion monitoring (SIM) mode. Relative dsRNA ligase activity is determined by comparing the sum of the MS signals of the five specific masses (shown in Table 6-3) corresponding to each product oligonucleotide (2) and (3).

[0326] [Table 5]

[0327] The methods provided herein are used to analyze variants generated using the present invention, however, it is not intended that the present invention be limited to the methods described herein, as there are other suitable methods known in the art that are applicable to the analysis of variants provided herein and / or generated using the methods provided herein.

[0328] Example 7 Round 1 evolution and screening of modified polypeptides derived from SEQ ID NO:2 to improve production of siRNA products (1) A modified polynucleotide (SEQ ID NO: 1) encoding a polypeptide having the dsRNA ligase activity of SEQ ID NO: 2 was used to generate the modified polypeptides in Table 7-1. These polypeptides exhibited improved dsRNA ligase activity under desired conditions, e.g., improved formation of either oligonucleotide product (2) or (3), or preferably both oligonucleotide products (2) and (3), generated in situ from substrate oligonucleotides (6-7, 9-12), compared to the starting polypeptide. As shown in Table 7-1, several polypeptides exhibited improved product formation of either oligonucleotide product (2) or (3), or both oligonucleotide products (2) and (3), compared to the starting polypeptide. The sequences of oligonucleotides (2), (3), (6), (7), and (9-12) are provided in Table 1.

[0329] Modified polypeptides having amino acid sequences of even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO:2 as described below, along with the analytical methods set forth in Table 6-1. Directed evolution began with the polynucleotide set forth in SEQ ID NO:1. Libraries of modified polypeptides were generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using the HTP assay and analytical methods described below, which measure the ability of the polypeptides to generate oligonucleotide products (2) and (3).

[0330] Enzyme assays were performed in a 96-well PCR plate with a total reaction volume of 50 μL per well. Reactions contained 2.5% (v / v) undiluted dsRNA ligase lysate prepared as described in Example 4, 100 μM (each) of substrate oligonucleotides (6-7, 9-12), 50 mM Tris buffer (pH 7.5), 1 mM ATP, 10 mM MgCl2, and 5 mM DTT. The reaction plate was heat-sealed and incubated at 30°C for 2 hours in a thermocycler.

[0331] After incubation, the plate was subjected to a heat inactivation step (95°C, 20 min) to quench the reaction and precipitate the proteinaceous contents of the added lysate. The plate was then centrifuged at 4,000 rpm for 5 min. Subsequently, a 2 μL aliquot of the supernatant was removed from each well and added to a shallow-well 96-well plate containing 98 μL of 5 mM EDTA solution (pH 7.0). These samples were analyzed by HPLC to determine the activity of the enzyme variants using the analytical method described in Table 6-1. Selected ligase variants that showed increased product formation of oligonucleotides (2) and (3) compared to SEQ ID NO: 2 are shown in Table 7-1.

[0332] [Table 6]

[0333] [Table 7]

[0334] The modified dsRNA ligase polypeptides represented by the even-numbered sequence identifiers of SEQ ID NOs:4-106 each contain an even-numbered sequence identifier of SEQ ID NOs:304-406, respectively, and a 14 amino acid N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO:669)). For example, SEQ ID NO:4 contains: (i) the 14 amino acid N-terminal purification tag of SEQ ID NO:669; and (ii) the dsRNA ligase polypeptide of SEQ ID NO:304.

[0335] Throughout the examples, the location of a given mutation is provided relative to SEQ ID NO: 2, which includes (i) a 14-amino acid N-terminal purification tag of SEQ ID NO: 669, and (ii) a wild-type dsRNA ligase polypeptide of SEQ ID NO: 302. The location of a given mutation relative to SEQ ID NO: 302 (i.e., a wild-type dsRNA ligase polypeptide without a purification tag) can be obtained by subtracting the 14-amino acid N-terminal purification tag from the SEQ ID NO set forth in the examples. For example, position X251 in SEQ ID NO: 2 corresponds to position X237 in SEQ ID NO: 302.

[0336] Example 8 Round 2 evolution and screening of modified polypeptides derived from SEQ ID NO: 70 to improve production of siRNA product (1) The polynucleotide from SEQ ID NO:69 in Example 7, encoding the most active polypeptide with dsRNA ligase activity of SEQ ID NO:70, was used to generate the modified polypeptides in Table 8-1. These polypeptides exhibited improved dsRNA ligase activity under desired conditions, e.g., improved formation of either oligonucleotide product (2) or (3), or preferably both oligonucleotide products (2) and (3), generated in situ from substrate oligonucleotides (6-7, 9-12), compared to the starting polypeptide. Several polypeptides that exhibited improved product formation of both oligonucleotide products (2) and (3), compared to the starting polypeptide, are listed in Table 8-1. Modified polypeptides having amino acid sequences with even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO:70, as described below, along with the analytical methods described in Table 6-1.

[0337] Directed evolution began with the polynucleotide set forth in SEQ ID NO: 69. Libraries of modified polypeptides were generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using the HTP assay and analytical methods described below to measure the ability of the polypeptides to generate oligonucleotides (2) and (3).

[0338] Enzyme assays were performed in a 96-well PCR plate with a total reaction volume of 50 μL per well. Reactions contained 1.25 or 2.5% (v / v) undiluted dsRNA ligase lysate prepared as described in Example 4, 100 μM (each) of substrate oligonucleotides (6-7, 9-12), 50 mM Tris buffer (pH 7.5), 1 mM ATP, 10 mM MgCl2, and 5 mM DTT. The reaction plate was heat-sealed and incubated at 30°C for 2 hours in a thermocycler.

[0339] After incubation, the plate was subjected to a heat inactivation step (95°C, 20 minutes) to quench the reaction and precipitate the proteinaceous contents of the added lysate. The plate was then centrifuged at 4,000 rpm for 5 minutes. Subsequently, a 2 μL aliquot of the supernatant was removed from each well and added to a shallow-well 96-well plate containing 98 μL of 5 mM EDTA solution (pH 7.0). These samples were analyzed by HPLC to determine the activity of the enzyme variants using the analytical method described in Table 6-1. Selected ligase variants that exhibited faster product formation of oligonucleotides (2) and (3) compared to SEQ ID NO: 70 are listed in Table 8-1.

[0340] [Table 8]

[0341] [Table 9]

[0342] The modified dsRNA ligase polypeptides represented by the even-numbered sequence identifiers of SEQ ID NOs: 108-216 each comprise an even-numbered sequence identifier of SEQ ID NOs: 408-516, respectively, and a 14 amino acid N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO: 669)). For example, SEQ ID NO: 108 comprises: (i) the 14 amino acid N-terminal purification tag of SEQ ID NO: 669; and (ii) the dsRNA ligase polypeptide of SEQ ID NO: 408.

[0343] Example 9 Round 3 evolution and screening of modified polypeptides derived from SEQ ID NO: 188 to improve production of siRNA product (1) The polynucleotide from SEQ ID NO:187 in Example 8, encoding the most active polypeptide having dsRNA ligase activity of SEQ ID NO:188, was used to generate the modified polypeptides in Table 9-1. These polypeptides exhibited improved dsRNA ligase activity under desired conditions, e.g., improved formation of either oligonucleotide product (2) or (3), or preferably both oligonucleotide products (2) and (3), generated in situ from substrate oligonucleotides (6-7, 9-12), compared to the starting polypeptide. Several polypeptides that exhibited improved product formation of both oligonucleotide products (2) and (3), compared to the starting polypeptide, are listed in Table 9-1. Modified polypeptides having amino acid sequences identified by even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO:188 as described below, along with the analytical methods described in Table 6-2.

[0344] Directed evolution began with the polynucleotide set forth in SEQ ID NO: 187. Libraries of modified polypeptides were generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using the HTP assay and analytical methods described below to measure the ability of the polypeptides to generate oligonucleotides (2) and (3).

[0345] Enzyme assays were performed in a 96-well PCR plate with a total reaction volume of 100 μL per well. Reactions contained 20% (v / v) undiluted dsRNA ligase lysate prepared as described in Example 4, 1 mM (each) of substrate oligonucleotides (6-7, 9-12), 50 mM Tris buffer (pH 7.0), 10 mM ATP, 20 mM MgCl2, 5 mM DTT, and 10% (v / v) DMSO. The reaction plate was heat-sealed and incubated at 30°C in a thermocycler for 2 hours.

[0346] After incubation, the plate was subjected to a heat inactivation step (95°C, 20 minutes) to quench the reaction and precipitate the proteinaceous contents of the added lysate. The plate was then centrifuged at 4,000 rpm for 5 minutes. Subsequently, a 50 μL aliquot of the supernatant was removed from each well and added to a 96-well deep-well plate containing 450 μL of 5 mM EDTA solution (pH 7.0). The samples were further diluted by transferring 50 μL of the diluted sample to a 96-well deep-well plate containing 950 μL of 5 mM EDTA solution (pH 7.0). These samples were analyzed by HPLC to determine the activity of the enzyme variants using the analytical method described in Table 6-2. Selected ligase variants that exhibited faster product formation of oligonucleotides (2) and (3) compared to SEQ ID NO: 188 are listed in Table 9-1.

[0347] [Table 10]

[0348] The modified dsRNA ligase polypeptides represented by the even-numbered sequence identifiers of SEQ ID NOs:218-246 each comprise an even-numbered sequence identifier of SEQ ID NOs:518-546, respectively, and a 14 amino acid N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO:669)). For example, SEQ ID NO:218 comprises: (i) the 14 amino acid N-terminal purification tag of SEQ ID NO:669; and (ii) the dsRNA ligase polypeptide of SEQ ID NO:518.

[0349] Example 10 Round 4 evolution and screening of modified polypeptides derived from SEQ ID NO: 226 to improve production of siRNA product (1) The polynucleotide from SEQ ID NO:225 in Example 9, encoding the most active polypeptide having the dsRNA ligase activity of SEQ ID NO:226, was used to generate the modified polypeptides in Table 10-1. These polypeptides exhibited improved dsRNA ligase activity under desired conditions, e.g., improved formation of either oligonucleotide product (2) or (3), or preferably both oligonucleotide products (2) and (3), generated in situ from substrate oligonucleotides (6-7, 9-12), compared to the starting polypeptide. Several polypeptides that exhibited improved product formation of both oligonucleotide products (2) and (3), compared to the starting polypeptide, are listed in Table 10-1. Modified polypeptides having amino acid sequences identified by even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO:226 as described below, along with the analytical methods described in Table 6-2.

[0350] Directed evolution began with the polynucleotide set forth in SEQ ID NO: 225. Libraries of modified polypeptides were generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using the HTP assay and analytical methods described below to measure the ability of the polypeptides to generate oligonucleotides (2) and (3).

[0351] Enzyme assays were performed in a 96-well PCR plate with a total reaction volume of 100 μL per well. Reactions contained 2.5% (v / v) undiluted dsRNA ligase lysate prepared as described in Example 4, 1 mM (each) of substrate oligonucleotides (6-7, 9-12), 50 mM Tris buffer (pH 7.0), 10 mM ATP, 20 mM MgCl2, 5 mM DTT, and 10% (v / v) DMSO. The reaction plate was heat-sealed and incubated at 30°C in a thermocycler for 24 hours.

[0352] After incubation, the plate was subjected to a heat inactivation step (95°C, 20 minutes) to quench the reaction and precipitate the proteinaceous contents of the added lysate. The plate was then centrifuged at 4,000 rpm for 5 minutes. Subsequently, a 50 μL aliquot of the supernatant was removed from each well and added to a 96-well deep-well plate containing 450 μL of 5 mM EDTA solution (pH 7.0). The samples were further diluted by transferring 50 μL of the diluted sample to a 96-well deep-well plate containing 950 μL of 5 mM EDTA solution (pH 7.0). These samples were analyzed by HPLC to determine the activity of the enzyme variants using the analytical method described in Table 6-2. Selected ligase variants that exhibited faster product formation of oligonucleotides (2) and (3) compared to SEQ ID NO: 226 are listed in Table 10-1.

[0353] [Table 11]

[0354] The modified dsRNA ligase polypeptides represented by the even-numbered sequence identifiers of SEQ ID NOs:248-282 comprise the even-numbered sequence identifiers of SEQ ID NOs:548-582, respectively, and a 14 amino acid N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO:669)). For example, SEQ ID NO:248 comprises: (i) the 14 amino acid N-terminal purification tag of SEQ ID NO:669; and (ii) the dsRNA ligase polypeptide of SEQ ID NO:548.

[0355] Example 11 Round 5 evolution and screening of modified polypeptides derived from SEQ ID NO: 278 to improve production of siRNA product (1) The polynucleotide from SEQ ID NO:277 in Example 10, encoding the most active polypeptide having dsRNA ligase activity of SEQ ID NO:278, was used to generate the modified polypeptides in Table 11-1. These polypeptides exhibited improved dsRNA ligase activity under desired conditions, e.g., improved formation of either oligonucleotide product (2) or (3), or preferably both oligonucleotide products (2) and (3), generated in situ from substrate oligonucleotides (6-7, 9-12), compared to the starting polypeptide. Several polypeptides that exhibited improved product formation of both oligonucleotide products (2) and (3), compared to the starting polypeptide, are listed in Table 11-1. Modified polypeptides having amino acid sequences with even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO:278 as described below, along with the analytical methods described in Table 6-2.

[0356] Directed evolution began with the polynucleotide set forth in SEQ ID NO: 277. Libraries of modified polypeptides were generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using the HTP assay and analytical methods described below to measure the ability of the polypeptides to generate oligonucleotides (2) and (3).

[0357] Enzyme assays were performed in a 96-well PCR plate with a total reaction volume of 100 μL per well. Reactions contained 10% (v / v) undiluted dsRNA ligase lysate prepared as described in Example 4, 5 mM (each) of substrate oligonucleotides (6-7, 9-12), 50 mM Tris buffer (pH 7.0), 30 mM ATP, 60 mM MgCl2, and 10% (v / v) DMSO. The reaction plate was heat-sealed and incubated at 30°C in a thermocycler for 24 hours.

[0358] After incubation, the plate was subjected to a heat inactivation step (95°C, 20 min) to quench the reaction and precipitate the proteinaceous contents of the added lysate. The plate was then centrifuged at 4,000 rpm for 5 min. Subsequently, a 50 μL aliquot of the supernatant was removed from each well and added to a 96-deep-well plate containing 950 μL of 5 mM EDTA solution (pH 7.0). The samples were further diluted by transferring 50 μL of the diluted sample to a 96-deep-well plate containing 450 μL of 5 mM EDTA solution (pH 7.0). A third dilution of the sample was performed by transferring 160 μL of the diluted sample to a 96-deep-well plate containing 640 μL of 5 mM EDTA solution (pH 7.0). Final dilutions of samples were made by transferring 75 μL of diluted sample to a shallow-well 96-well plate containing 75 μL of 5 mM EDTA solution (pH 7.0). These samples were analyzed by HPLC to determine the activity of the enzyme variants using the analytical method described in Table 6-2. Selected ligase variants that exhibit faster product formation for oligonucleotides (2) and (3) compared to SEQ ID NO: 278 are listed in Table 11-1.

[0359] [Table 12]

[0360] The modified dsRNA ligase polypeptides represented by the even-numbered sequence identifiers of SEQ ID NOs:284-300 each comprise an even-numbered sequence identifier of SEQ ID NOs:584-600, respectively, and a 14 amino acid N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO:669)). For example, SEQ ID NO:284 comprises: (i) the 14 amino acid N-terminal purification tag of SEQ ID NO:669; and (ii) the dsRNA ligase polypeptide of SEQ ID NO:584.

[0361] Example 12 Round 6 evolution and screening of modified polypeptides derived from SEQ ID NO: 288 to improve production of siRNA product (1) and improve thermal stability The polynucleotide from SEQ ID NO:287 in Example 11, encoding the most active polypeptide with dsRNA ligase activity of SEQ ID NO:288, was used to generate the modified polypeptides in Table 12-1. These polypeptides exhibited improved dsRNA ligase activity under desired conditions, e.g., improved formation of either oligonucleotide product (2) or (3), or preferably both oligonucleotide products (2) and (3), generated in situ from substrate oligonucleotides (6-7, 9-12), compared to the starting polypeptide. As shown in Table 12-1, some polypeptides exhibited improved product formation of both oligonucleotide products (2) and (3), compared to the starting polypeptide. Furthermore, some polypeptides exhibited improved thermostability, resulting in higher residual activity after incubating the dsRNA ligase solution at 30°C for 1 hour prior to reaction setup (Table 12-2). Modified polypeptides having the amino acid sequences of the even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO:288 as described below, along with the analytical methods described in Table 6-3.

[0362] Directed evolution began with the polynucleotide set forth in SEQ ID NO: 287. Libraries of modified polypeptides were generated using a variety of well-known techniques (e.g., saturation mutagenesis, recombination of previously identified beneficial amino acid differences) and screened using the HTP assay and analytical methods described below to measure the ability of the polypeptides to generate oligonucleotides (2) and (3).

[0363] Enzyme assays were performed in a 96-well PCR plate with a total reaction volume of 100 μL per well. Cells were lysed as described in Example 4, except that 100 mM MOPS buffer, pH 7.2, was used instead of 50 mM Tris, pH 7.5. To heat-challenge and identify more thermostable hits, cell lysates were either undiluted or diluted 1:1 in 100 mM MOPS buffer (pH 7.2) and incubated for 1 h at 30°C and 4°C, respectively. Reactions contained a final dsRNA ligase concentration of 40% (v / v) for lysates incubated at 30°C and 20% (v / v) for lysates incubated at 4°C. Additionally, ligation reactions contained 3 mM (each) of substrate oligonucleotides (6–7, 9–12), 100 mM MOPS buffer, pH 7.2, 30 mM ATP, 60 mM MgCl2, and 10% (v / v) DMSO. The reaction plate was heat sealed and incubated in a thermocycler at 30°C for 24 hours.

[0364] After incubation, the plate was subjected to a heat inactivation step (95°C, 20 min) to quench the reaction and precipitate the proteinaceous contents of the added lysate. The plate was then centrifuged at 4,000 rpm for 5 min. Subsequently, a 50 μL aliquot of the supernatant was removed from each well and added to a 96-well deep-well plate containing 950 μL of 5 mM EDTA solution (pH 7.0). The samples were further diluted by transferring 20 μL of the diluted sample to a 96-well deep-well plate containing 180 μL of 5 mM EDTA solution (pH 7.0). A third dilution of the sample was performed by transferring 30 μL of the diluted sample to a 96-well deep-well plate containing 150 μL of 5 mM EDTA solution (pH 7.0). These samples were analyzed by RF-MS to determine the activity of the enzyme variants using the analytical method described in Table 6-3. Selected ligase variants that exhibit faster product formation for oligonucleotides (2) and (3) compared to SEQ ID NO: 288 after pre-incubation at 4° C. are shown in Table 12-1. Selected ligase variants that exhibit faster product formation for oligonucleotides (2) and (3) compared to SEQ ID NO: 288 after pre-incubation at 30° C. are shown in Table 12-2.

[0365] [Table 13]

[0366] [Table 14]

[0367] The modified dsRNA ligase polypeptides represented by the even-numbered sequence identifiers of SEQ ID NOs:602-634 each comprise an even-numbered sequence identifier of SEQ ID NOs:636-668, respectively, and a 14 amino acid N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO:669)). For example, SEQ ID NO:602 comprises: (i) the 14 amino acid N-terminal purification tag of SEQ ID NO:669; and (ii) the dsRNA ligase polypeptide of SEQ ID NO:636.

[0368] Example 13 Comparison of catalytic activity of wild-type polypeptide SEQ ID NO:2 and modified polypeptides SEQ ID NO:288, SEQ ID NO:290 and SEQ ID NO:292 The polynucleotide of SEQ ID NO: 1 encoding the wild-type dsRNA ligase from bacteriophage RB69, Uniprot ID: Q7Y4V8 having SEQ ID NO: 2, and the modified polynucleotides of SEQ ID NO: 287, SEQ ID NO: 289, and SEQ ID NO: 291 encoding the most improved variants from Example 11 having polypeptide sequences of SEQ ID NO: 288, SEQ ID NO: 290, and SEQ ID NO: 292 were used for SFP generation as described in Example 5.

[0369] The catalytic activity of substrate oligonucleotides (6-7, 9-12) to convert them to the desired siRNA product (1) was evaluated under two reaction conditions: condition 1 (50 mM Tris buffer at pH 7.5, 1 mM ATP, 5 mM MgCl, and 5 mM DTT, containing either 0 g / L, 0.0020 g / L, 0.0039 g / L, 0.0078 g / L, 0.0156 g / L, 0.0313 g / L, 0.0625 g / L, 0.125 g / L, 0.25 g / L, 0.5 g / L, 1 g / L, or 2 g / L of SFP), and condition 2 (50 mM Tris buffer at pH 7.0, 30 mM ATP, 60 mM MgCl2, and 10% (v / v) DMSO, containing either 0 g / L, 0.0049 g / L, 0.0098 g / L, 0.0195 g / L, 0.0391 g / L, 0.0781 g / L, 0.1563 g / L, 0.3125 g / L, 0.625 g / L, 1.25 g / L, 2.5 g / L, or 5 g / L of SFP. Enzyme assays were performed in a 96-well PCR plate with a total reaction volume of 100 μL per well; condition 1 for reaction plate 1 and condition 2 for reaction plate 2. Both reaction plates were heat-sealed and incubated at 30°C in a thermocycler. Reaction plate 1 was incubated for 2 hours, and reaction plate 2 was incubated for 24 hours.

[0370] After incubation, the plates were subjected to a heat inactivation step (95°C, 20 min) to quench the reaction and precipitate the proteinaceous contents of the added SFP. The plates were then centrifuged at 4,000 rpm for 5 min. A 50 μL aliquot of the supernatant was removed from each well of each plate and subsequently diluted with 50 mM EDTA solution (pH 7.0). Reaction Plate 1 was diluted 40-fold, and Reaction Plate 2 was diluted 400-fold. These samples were analyzed by HPLC to determine the activity of the enzyme variants using the analytical method described in Table 6-2.

[0371] The comparative data in Figures 2A and 2B show the relative peak area % of siRNA (1) present in reaction samples assayed under conditions 1 and 2, respectively. Under both conditions 1 and 2, polypeptides SEQ ID NO:288, SEQ ID NO:290, and SEQ ID NO:292 exhibit improved dsRNA ligase activity compared to the wild-type polypeptide SEQ ID NO:2.

[0372] Example 14 Comparison of catalytic activity of wild-type polypeptide SEQ ID NO: 2 and modified polypeptides SEQ ID NOs: 288 and 632 The polynucleotide of SEQ ID NO: 1 encoding the wild-type dsRNA ligase from bacteriophage RB69, Uniprot ID: Q7Y4V8 having SEQ ID NO: 2, and the modified polynucleotides SEQ ID NO: 287 and SEQ ID NO: 631 encoding the most improved variants from Example 11 and Example 12 having polypeptide sequences SEQ ID NO: 288 and SEQ ID NO: 632, respectively, were used for SFP generation as described in Example 5.

[0373] The catalytic activity and thermostability of the two enzymes for converting substrate oligonucleotides (6-7, 9-12) to the desired siRNA product (1) were assessed by incubating stock solutions of SFP at 4 or 37 °C for 4 h before setting up the following ligation reactions: 6 mM (each) of substrate oligonucleotides (6-7, 9-12), 100 mM MOPS buffer at pH 7.2, 30 mM ATP, 60 mM MgCl2, and 10% (v / v) DMSO. Additionally, the ligation reactions contained 0 g / L, 0.156 g / L, 0.313 g / L, 0.625 g / L, 1.25 g / L, 2.5 g / L, 5 g / L, or 10 g / L of SFP. Enzyme assays were performed in a 96-well PCR plate with a total reaction volume of 100 μL per well. The reaction plate was heat-sealed and incubated at 30 °C in a thermocycler for 24 h.

[0374] After incubation, the plates were subjected to a heat inactivation step (95°C, 20 min) to quench the reaction and precipitate the proteinaceous contents of the added SFP. The plates were then centrifuged at 4,000 rpm for 5 min. A 50 μL aliquot of the supernatant was removed from each well of each plate and subsequently diluted 400-fold with 50 mM EDTA solution (pH 7.0). These samples were analyzed by HPLC to determine the activity of the enzyme variants using the analytical method described in Table 6-2.

[0375] The comparative data in Figure 3A shows the relative peak area % of siRNA (1) present in the reaction samples. Figure 3B shows the residual enzyme activity after preincubation of SFPs at 37°C for 4 hours, expressed relative to the ligation activity of SFPs preincubated at 4°C for 4 hours. Under all conditions, polypeptide SEQ ID NO:632 exhibits improved dsRNA ligase activity and thermostability over wild-type polypeptide SEQ ID NO:2 and modified polypeptide SEQ ID NO:288.

[0376] Overview of engineered dsRNA ligase polypeptide sequences Table 13 provides a summary of the nucleic acid and amino acid sequences of the wild-type and modified dsRNA ligase sequences described herein. The purification tag used in the Examples and references in Table 13 is the N-terminal purification tag MHHHHHENLYFQS (SEQ ID NO: 669).

[0377] [Table 15]

[0378] [Table 16]

[0379] [Table 17]

[0380] [Table 18]

[0381] [Table 19]

[0382] [Table 20]

Claims

1. SEQ ID NOs: 666, 304, 306, 308, 310, 312, 314, 316, 318, 320, 322, 324, 326, 328, 330, 332, 334, 336, 338, 340, 342, 344, 346, 348, 350, 352, 354, 356, 358, 360, 362, 364, 366, 368, 370, 372, 374, 376, 378, 380, 382, ​​384, 386, 388, 390, 391 2, 394, 396, 398, 400, 402, 404, 406, 408, 410, 412, 414, 416, 418, 420, 422, 424, 426, 428, 430, 432, 434, 436, 438, 440, 442, 444, 446, 448, 450, 452, 454, 456, 458, 460, 462, 464, 466, 468, 470, 472, 474, 476, 478, 480, 482, 484, 486, 488, 490, 500, 502, 504, 506, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 540, 542, 544, 546, 548, 549, 550, 551, 552, 553, 554, 556, 558, 559, 560, 561, 562, 56 86, 488, 490, 492, 494, 496, 498, 500, 502, 504, 506, 508, 510, 512, 514, 516, 518, 520, 522, 524, 526, 528, 530, 532, 534, 536, 538, 540, 542, 544, 546, 548, 550, 552, 554, 556, 558, 560, 562, 564, 566, 568, 570, 572, 574, 576, 578 600, 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, and 668, the modified dsRNA ligase polypeptide (a) has dsRNA ligase activity; and (b) does not contain the amino acid sequence of SEQ ID NO: 302; The modified dsRNA polypeptide.

2. 2. The modified dsRNA ligase polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 666, 370, 488, 526, 578, 588, 590 and 592.

3. 1. A modified dsRNA ligase polypeptide comprising an amino acid sequence having at least 85% sequence identity to SEQ ID NO:302, wherein the modified dsRNA ligase polypeptide produces at least 5% more oligonucleotide products under the same ligation reaction conditions than a dsRNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO:302, wherein the modified dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO:

302.

4. 4. The modified dsRNA ligase polypeptide of claim 3, wherein the ligation reaction conditions comprise about 1 μM to about 10 mM of oligonucleotide fragments, an ATP source, about 5 mM to about 100 mM divalent cations, and about 0.5 g / L to about 10 g / L of the modified dsRNA ligase polypeptide, a pH of about 4.0 to about 8.0, and a temperature of about 10°C to about 50°C.

5. (a) the amino acid sequence of the modified dsRNA ligase polypeptide is selected from the group consisting of X6, X7, X15, X19, X29, X36, X39, X44, X45, X46, X47, X49, X51, X53, X56, X57, X60, X63, X64, X66, X67, X87, X88, X89, X91, X92, X93, X103, X105, X107, X114, X122, X126, X129, X130, X131, X137, X144, X146, X158, X163, X173, X178, X185, X190, X196, X216, X218, X221, X222, X223, X224, X225, X226, X227, X228, X229, X230, X231, X232, X233, X234, X235, X236, X237, X238, X239, X240, X241, X242, X243, X244, X245, X246, X247, X248, X249, X251, X252, X253, X254, X255, X256, X257, X258, X259, X260, X261, X262, X263, X264, X265, X266, X267, X268, X269, X270, X271, X272, X273, X274, X275, X276, and X328, X228, X230, X232, X235, X236, X237, X238, X239, X242, X243, X244, X251, X252, X254, X255, X258, X269, X280, X284, X285, X293, X296, X301, X303, X305, X313, X314, X325, and X328 (numbering refers to SEQ ID NO: 302); optionally, the amino acid sequence of the modified dsRNA ligase polypeptide comprises: The following amino acid residues are present: X6 is G or E; X7 is Q; X15 is R, D or E; X19 is Q or D; X29 is N or L; X36 is V; X39 is A; X44 is V; X45 is V; X46 is Y; X47 is E; X49 is G; X51 is L; X53 is Y; X56 is R or A; X57 is S; X60 is T, G or P; X63 is S, Q or G; X64 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; and X87 is T, P, K or absent; X88 is C; X89 is T; X91 is S; X92 is D; X93 is G, C, or A; X103 is V, C, Y, or T; X105 is V; X107 is R or T; X114 is N; X122 is W; X126 is G; X129 is N; X130 is R, S, or Y; X131 is R; X137 is V or C; X144 is N; X146 is R; X158 is W; X163 is G; X173 is L; X178 is R; X185 is K;X190 is Q; X196 is S or C; X216 is L or R; X218 is N; X221 is I; X228 is R; X230 is T; X232 is R; X235 is A, T, or G; X236 is S, L, or F; X237 is S, Q, R, L, or G; X238 is F; X239 is G or R; X242 is R or M; X243 is N, S, G, or M; X244 is G or K; and X251 is D or L. and / or; X252 is V; X254 is K; X255 is C; X258 is V; X269 is L; X280 is W; X284 is A; X285 is A; X293 is R; X296 is R; X301 is G, L, E, or F; X303 is Q; X305 is G; X313 is A; X314 is A or V; X325 is R; X328 is R; (numbering refers to SEQ ID NO: 302); and / or; (b) the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from X15, X19, X36, X39, X53, X185, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302), and the modified dsRNA ligase polypeptide has dsRNA ligase activity; optionally wherein the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D or E; X19 is D; X36 is V; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; X285 is A; (numbering refers to SEQ ID NO: 302); and / or (c) the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from X36, X39, X218, and X221 (numbering refers to SEQ ID NO: 302), wherein the modified dsRNA ligase polypeptide has dsRNA ligase activity; optionally, the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I; and / or (d) the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from X39, X218, and X221 (numbering refers to SEQ ID NO: 302), wherein the modified dsRNA ligase polypeptide has dsRNA ligase activity; optionally, the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X39 is A; X218 is N; and X221 is I; and / or (e) the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from X39, X218, X221, and X255 (numbering refers to SEQ ID NO: 302), wherein the modified dsRNA ligase polypeptide has dsRNA ligase activity; optionally, the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C; and / or (f) the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from X39, X53, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302), wherein the modified dsRNA ligase polypeptide has dsRNA ligase activity; optionally, the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; and / or (g) the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from X15, X39, X53, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302), and the modified dsRNA ligase polypeptide has dsRNA ligase activity; optionally, the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is E; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; and / or (h) the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from X19, X39, X53, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302), and the modified dsRNA ligase polypeptide has dsRNA ligase activity; optionally, the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X19 is D; X39 is A; X53 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A; and / or (i) the amino acid sequence of the modified dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 at one or more amino acid residues selected from X15, X39, X53, X185, X218, X221, X237, X251, X255, and X285 (numbering refers to SEQ ID NO: 302), and the modified dsRNA ligase polypeptide has dsRNA ligase activity; optionally, the amino acid sequence of the modified dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X15 is D; X39 is A; X53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A. The modified dsRNA ligase according to any one of claims 1 to 4.

6. the modified dsRNA ligase polypeptide comprises a purification tag; and optionally, the modified dsRNA ligase polypeptide is selected from the group consisting of SEQ ID NOs: 632, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178 , 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, 204, 206, 208, 210, 212, 214, 216, 218, 220, 222, 224, 226, 228, 230, 232, 234, 236, 238, 240, 242, 244, 246, 248, 250, 252, 254, 256, 258, 260, 262, 264, 266, 268, 27 6. The modified dsRNA ligase polypeptide of any one of claims 1-5, comprising an amino acid sequence selected from the group consisting of: 0, 272, 274, 276, 278, 280, 282, 284, 286, 288, 290, 292, 294, 296, 298, 300, 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, and 634.

7. 10. A polypeptide immobilized on a solid material by chemical bonding or physical adsorption, said polypeptide comprising the modified dsRNA ligase polypeptide of any one of claims 1 to 6.

8. A polynucleotide encoding the modified dsRNA ligase polypeptide of any one of claims 1 to 6, optionally comprising: (a) the polynucleotide is selected from the group consisting of SEQ ID NOs: 303, 305, 307, 309, 311, 313, 315, 317, 319, 321, 323, 325, 327, 329, 331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357, 359, 361, 363, 365, 367, 369, 371, 373, 375, 377, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433, 434, 435, 436, 437, 438, 4 81, 383, 385, 387, 389, 391, 393, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, 435, 437, 439, 441, 443, 445, 447, 449, 451, 453, 455, 457, 459, 461, 463, 465, 467, 4 69, 471, 473, 475, 477, 479, 481, 483, 485, 487, 489, 491, 493, 495, 497, 499, 501, 503, 505, 507, 509, 511, 513, 515, 517, 519, 521, 523, 525, 527, 529, 531, 533, 535, 537, 539, 541, 543, 545, 547, 549, 551, 553, 555, 5 and / or comprising a nucleic acid sequence selected from: 57, 559, 561, 563, 565, 567, 569, 571, 573, 575, 577, 579, 581, 583, 585, 587, 589, 591, 593, 595, 597, 599, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, and 667; and / or (b) the polynucleotide is selected from the group consisting of SEQ ID NOs: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 10 1, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 182 3, 185, 187, 189, 191, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233, 235, 237, 239, 241, 243, 245, 247, 249, 251, 253, 255, 257, 259, 261, 263, 26 5, 267, 269, 271, 273, 275, 277, 279, 281, 283, 285, 287, 289, 291, 293, 295, 297, 299, 601, 603, 605, 607, 609, 611, 613, 615, 617, 619, 621, 623, 625, 627, 629, 631, and 633.

9. 9. An expression vector comprising the polynucleotide of claim 8, optionally wherein the expression vector comprises a plasmid, cosmid, bacteriophage or viral vector.

10. 10. A host cell comprising the polynucleotide of claim 8 or the expression vector of claim 9, optionally wherein the host cell is E. coli.

11. 11. A method for preparing a modified dsRNA ligase polypeptide, comprising culturing the host cell of claim 10 and obtaining the modified dsRNA ligase polypeptide from the culture.

12. 12. A modified dsRNA ligase catalyst obtained by culturing the host cell of claim 10 or according to the method of claim 11, comprising cells or culture media containing the modified dsRNA ligase polypeptide, or an article treated therewith, wherein the article refers to an extract obtained from a culture of the host cells, an isolated product obtained by isolating or purifying the modified dsRNA ligase from the extract, or an immobilized product obtained by immobilizing the host cells, the extract, or an isolated product of the extract.

13. 1. A method for generating an oligonucleotide from two or more oligonucleotide fragments, comprising: (i) two or more oligonucleotide fragments; (ii) a modified dsRNA ligase polypeptide according to any one of claims 1 to 6; (iii) an ATP source; and (iv) divalent cations to obtain an oligonucleotide; Optionally, (a) the method further comprises purifying the oligonucleotide; and / or (b) the method is performed using substoichiometric concentrations of AMP and / or ATP; and / or (c) the method is carried out at a divalent cation concentration of 5 to 100 mM, optionally 30 to 50 mM; The method.

14. 10. Use of the modified dsRNA ligase polypeptide of any one of claims 1 to 6 in generating an oligonucleotide from two or more oligonucleotide fragments.

15. (a) the oligonucleotide is at most 60 nucleotides in length; and / or (b) each of said oligonucleotide fragments is 4 to 16 nucleotides in length, optionally 6 to 9 nucleotides in length; and / or (c) one or more of the oligonucleotide fragments comprises one or two overhangs; and / or (d) one or more of said oligonucleotide fragments comprises a chemical modification; optionally, said chemical modification comprises: (i) a modified backbone, optionally selected from phosphorothioate (e.g., chiral phosphorothioate) or methylphosphonate internucleotide linkages; (ii) optionally, 2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-O-N-methylacetamide (2' -O-NMA), locked nucleic acids (LNA), glycol nucleic acids (GNA), phosphoramidates (e.g., mesyl phosphoramidate), 2',3'-seconucleotide mimics, 2'-F-arabinonucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, vinyl phosphonates (e.g., 5' vinyl phosphonate), and cyclopropyl phosphonate deoxyribonucleotides; and / or (iii) Conjugation to a ligand, optionally wherein the ligand comprises one or more N-acetylgalactosamine (GalNAc) derivatives.

15. The method according to claim 13 or the use according to claim 14, wherein the

16. i. a modified dsRNA ligase polypeptide according to any one of claims 1 to 6; ii. an ATP source; and iii. Divalent cations wherein optionally, said composition further comprises two or more oligonucleotide fragments.

17. i. a modified dsRNA ligase polypeptide according to any one of claims 1 to 6; ii. ATP source; iii. a divalent cation; and iv. Instructions for use in a method for generating an oligonucleotide from two or more oligonucleotide fragments. Includes a kit.

18. (A) the ATP source comprises ATP; and / or (B) An ATP source is (a) polyphosphate kinase (PPK); (b) polyphosphoric acid; and (c) AMP and / or ATP and optionally (i) the PPK is selected from PPK12 or ajPAP; and / or (ii) the polyphosphoric acid salt is a polyphosphate, optionally the polyphosphate is sodium polyphosphate (Madrell's salt) or sodium hexametaphosphate (Graham's salt); and / or (C) the divalent cation cofactor is Mg ２＋ or Mn ２＋ That is, 18. The method of claim 13 or 15, the composition of claim 16, or the kit of claim 17.

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