Engineered double-stranded RNA ligases and uses thereof

Abstract

The present disclosure relates to the field of biotechnology, in particular to engineered double-stranded RNA (dsRNA) ligases and their use in industrial biocatalysis. The disclosure also relates to processes of producing the engineered dsRNA ligases, and methods of producing oligonucleotides by contacting oligonucleotide fragments with the engineered dsRNA ligases.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of European application No. 22215201.9, filed on December 20, 2022, the contents of which are incorporated herein by reference in their entirety.

[0003] Sequence Listing

[0004] 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, is named PAT059445-WO-PCT_SL.xml and is 1.24 MB in size. Technical Field

[0005] The present disclosure relates to the field of biotechnology, in particular to engineered double-stranded RNA (dsRNA) ligases and their applications in industrial biocatalysis. The present disclosure also relates to processes for producing engineered dsRNA ligases, and methods for producing oligonucleotides by contacting oligonucleotide fragments with engineered dsRNA ligases. Background Art

[0006] Therapeutic oligonucleotides, including small interfering RNA (siRNA) and inhibitory antisense oligonucleotides (ASOs), have the potential to treat a variety of life-threatening diseases. In recent years, the number of approved oligonucleotide-based drugs has increased significantly, and the number of therapeutic oligonucleotides in clinical research has also increased significantly (Roberts, TC, Langer, R. and Wood, MJA Nature Reviews Drug Discovery 2020 19:10 19, 673–694 (2020)).

[0007] To support green synthesis initiatives across the pharmaceutical industry, sustainable and economical next-generation oligonucleotide synthesis methods are urgently needed at the scale required to reach a wider patient population (Mishra, M. et al. Current Research in Green and Sustainable Chemistry 4, (2021)).

[0008] For this reason, biocatalysis is increasingly being used in the production of active pharmaceutical ingredients (APIs) because enzymes are able to carry out highly selective transformations under mild reaction conditions and in aqueous media (Mann, G. and Stanger, FV Chimia (Aarau) [Chemistry] 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.

[0009] Shorter oligonucleotides are easier to synthesize and have higher purity than longer oligonucleotides, simplifying downstream processing and reducing solvent waste. These short oligonucleotide fragments can then be combined using nucleic acid ligases to produce oligonucleotide products. Nucleic acid ligases show remarkable tolerance to non-natural DNA / RNA containing pharmaceutically relevant chemical modifications (Kestemont, D., Herdewijn, P. and Renders, M. Curr Protoc Chem Biol 11, e62 (2019); Kestemont, D. et al. Chemical Communications 54, 6408–6411 (2018); and Nandakumar, J. and Shuman, S. Molecular Cell 16, 211–221 (2004)), and the use of dsRNA ligases to synthesize siRNA products starting from short fragments (≤9 nt) has been previously described, containing a wide range of chemical modifications, including 2′-OMe, 2′-F modified nucleotides, phosphorothioate backbone modified nucleotides, and terminal fragments functionalized with a large number of N-acetylgalactosamine (GalNAc) moieties (Mann, G. et al. Tetrahedron Tetrahedron Letters 93, 153696 (2022).

[0010] In order to achieve cost-effective and sustainable industrial-scale oligonucleotide biocatalysis, enzymes that exhibit high ligase activity are needed. There is an urgent and unmet need for engineered ligases that exhibit improved ligase activity relative to the wild-type enzyme. There is also an unmet need for biocatalytic methods for producing oligonucleotides from oligonucleotide fragments. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1. dsRNA ligase-catalyzed ligation of: (A) oligonucleotide fragments 6:9, 7:10, and 11:12 to generate oligonucleotide 2:3 (=siRNA 1); and (B) oligonucleotide fragments 6:9, 7:10, and 8:11 to generate oligonucleotide 5:3 (=siRNA 4). The sequences of oligonucleotides 2, 3, and 5-12 are provided in Table 1.

[0012] Figure 2. Comparative data showing the relative peak area % of siRNA (1) present in reaction samples containing varying concentrations of wild-type enzyme (SEQ ID NO: 2) and engineered enzymes (SEQ ID NOs: 288, 290, and 292) assayed under the following conditions: (A) Condition 1; and (B) Condition 2, as described in Example 13. Enzyme concentrations are provided as g / L shake flask powder (SFP), which was produced by lyophilization of frozen clarified lysate, as described in the Examples.

[0013] Figure 3. (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 engineered enzymes (SEQ ID NO: 288 and 632) after enzyme preincubation for 4 h at 4°C or 37°C. (B) Comparative data showing the residual enzyme activity after SFP preincubation for 4 h at 37°C, expressed as ligation activity relative to SFP preincubated for 4 h at 4°C. Enzyme concentrations are provided as g / L shake flask powder (SFP), which was produced by lyophilization of frozen clarified lysate, as described in the Examples. Summary of the Invention

[0014] The present disclosure provides engineered double-stranded RNA (dsRNA) ligase polypeptides. The present disclosure also provides gene sequences of the engineered polypeptides, recombinant expression vectors comprising the genes, engineered host strains, and effective methods for their production, as well as reaction processes using the engineered polypeptides to biocatalyze oligonucleotides.

[0015] The engineered double-stranded RNA (dsRNA) ligase polypeptides described herein have improved catalytic activity compared to the wild-type dsRNA ligase from which they are derived. By substitution and / or deletion of amino acid residues in the directed evolution process, the engineered polypeptides provided herein are derived from the wild-type dsRNA ligase of bacteriophage RB69. The wild-type dsRNA ligase consists of 332 amino acids and has the amino acid sequence shown in SEQ ID NO:302 (also available according to the accession number Q7Y4V8 in UniProt).

[0016] The present disclosure provides an engineered 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 NO: 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 82, 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, 461 2, 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, 54 2, 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 engineered dsRNA ligase polypeptide: (a) has dsRNA ligase activity; and (b) does not comprise the amino acid sequence of SEQ ID NO: 302.

[0017] The present disclosure provides an engineered 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 NO: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668; wherein the engineered dsRNA ligase polypeptide: (a) has dsRNA ligase activity; and (b) does not comprise the amino acid sequence of SEQ ID NO: 302.

[0018] The present disclosure provides an engineered 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 NO: 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 0, 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, 5 wherein the engineered dsRNA ligase polypeptide: (a) has dsRNA ligase activity; and (b) does not comprise the amino acid sequence of SEQ ID NO: 302.

[0019] The present disclosure provides an engineered 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: SEQ ID NO: 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 0, 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, 5 wherein the engineered dsRNA ligase polypeptide: (a) has dsRNA ligase activity; and (b) does not comprise the amino acid sequence of SEQ ID NO: 302.

[0020] The present disclosure provides engineered double-stranded RNA (dsRNA) ligase polypeptides, which are: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 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, 48 4, 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, 5 or (b) a polypeptide having dsRNA ligase activity, the polypeptide comprising an amino acid sequence having (i) at least 80% sequence identity to one of the polypeptides recited in (a), and (ii) one or more amino acid residues substituted, deleted, added or inserted relative to the one amino acid sequence recited in (a); wherein the engineered dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302.

[0021] The present disclosure provides engineered double-stranded RNA (dsRNA) ligase polypeptides, which are: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 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, which polypeptide comprises an amino acid sequence: having (i) at least 80% sequence identity to one of the polypeptides listed in (a), and (ii) one or more amino acid residues substituted, deleted, added or inserted relative to the one amino acid sequence listed in (a); wherein the engineered dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302.

[0022] The present disclosure provides engineered double-stranded RNA (dsRNA) ligase polypeptides, which are: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 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, or (b) a polypeptide having dsRNA ligase activity, the polypeptide comprising an amino acid sequence having (i) at least 80% sequence identity to one of the polypeptides recited in (a), and (ii) one or more amino acid residues substituted, deleted, added or inserted relative to the one amino acid sequence recited in (a); wherein the engineered dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302.

[0023] The present disclosure provides an engineered 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 NO: NO: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 ,418,420,422,424,426,428,430,432,434,436,438,440,442,444,446,448,450,452,454,456,458,460,462,464,466,468,470,472,474,476,478,480,482,484,486,488,490,492,494,496,498,500,502,504,506,508,510,512,514,516,518,520,522,524,526,528,530,53 2, 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; wherein: (a) the engineered dsRNA ligase polypeptide has a dsR dsRNA ligase activity; and (b) the amino acid sequence of the engineered 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; 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; 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; wherein numbering refers to SEQ ID NO: 302.

[0024] 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.

[0025] The present disclosure provides engineered double-stranded RNA (dsRNA) ligase polypeptides 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 engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered 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.

[0026] The present disclosure provides engineered double-stranded RNA (dsRNA) ligase polypeptides comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 666; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises 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 all 10) 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.

[0027] The present disclosure provides engineered double-stranded RNA (dsRNA) ligase polypeptides comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 370; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g., 2 or more, 3 or more, or all 4) of the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I.

[0028] The present disclosure provides an engineered 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 engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g., 2 or more, or all 3) of the following amino acid residues: X39 is A; X218 is N; and X221 is I.

[0029] The present disclosure provides engineered double-stranded RNA (dsRNA) ligase polypeptides comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 526; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g., 2 or more, 3 or more, or all 4) of the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C.

[0030] The present disclosure provides engineered double-stranded RNA (dsRNA) ligase polypeptides comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 578; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all 8) 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.

[0031] The present disclosure provides engineered double-stranded RNA (dsRNA) ligase polypeptides 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 engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises 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, or all 9) 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.

[0032] The present disclosure provides engineered double-stranded RNA (dsRNA) ligase polypeptides comprising an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 592; wherein: (a) the engineered dsRNA ligase polypeptide has dsRNA ligase activity; and (b) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises 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, or all 9) 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.

[0033] In some embodiments, the polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 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 NO: 636, 638, 642, 646, 664, and 666.

[0034] The present disclosure also provides an engineered dsRNA ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 302, which produces at least 5% more oligonucleotide product under the same ligation reaction conditions than a dsRNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO: 302, wherein the engineered dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302.

[0035] In some embodiments, the ligation reaction conditions include about 1 μM to about 10 mM 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 engineered 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 comprises ATP, optionally in a stoichiometric excess of ATP. In some embodiments, the ATP source comprises: (a) polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and / or ATP.

[0036] In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues 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, 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, wherein the numbering refers to SEQ ID NO: 302.

[0037] In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues 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, X164, X165, X166, X170, X171, X172, X173, X174, X175, X176, X177, X178, X179, X180, X181, X182, X183, X184, X185, X186, X187, X188, X189, X190, X191, X192, X193 X305, X313, X314, X325, and X328, wherein the numbering refers to SEQ ID NO: 302.

[0038] In some embodiments, the amino acid sequence of the engineered 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; X 64 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; X87 is T, P, K, or not present; 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; X 146 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; and X328 is R; wherein numbering refers to SEQ ID NO: 302.

[0039] In some embodiments, the amino acid sequence of the engineered 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; 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 not present; 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; X wherein 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; wherein numbering refers to SEQ ID NO: 302.

[0040] In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of X15, X19, X36, X39, X53, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity.

[0041] In some embodiments, the amino acid sequence of the engineered 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; wherein numbering refers to SEQ ID NO: 302.

[0042] In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of X15, X19, X36, X39, X53, X185, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity.

[0043] In some embodiments, the amino acid sequence of the engineered 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; wherein numbering refers to SEQ ID NO: 302.

[0044] In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g., 2 or more, 3 or more, or all 4) amino acid residues selected from: X36, X39, X218, and X221, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g., 2 or more, 3 or more, or all 4) of the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I.

[0045] In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g., 2 or more, or all 3) amino acid residues selected from: X39, X218, and X221, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g., 2 or more, or all 3) of the following amino acid residues: X39 is A; X218 is N; and X221 is I.

[0046] In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g., 2 or more, 3 or more, or all 4) amino acid residues selected from: X39, X218, X221, and X255, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g., 2 or more, 3 or more, or all 4) of the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C.

[0047] In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all 8) amino acid residues selected from the group consisting of: X39, X53, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more (e.g., 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, or all 8) of the following amino acid residues: X39 is A; X53 is A; X218 is A; X221 is A; X237 is A; X251 is A. S3 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.

[0048] In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in 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, or all 9) amino acid residues selected from the group consisting of: X15, X39, X53, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises 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, or all 9) of the following amino acid residues: X15 is E; X39 is A; X251 is A; S3 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.

[0049] In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in 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, or all 9) amino acid residues selected from the group consisting of: X19, X39, X53, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises 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, or all 9) of the following amino acid residues: X19 is D; X39 is A; X251 is D; X255 is A; S3 is Y; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.

[0050] In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in 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 all 10) amino acid residues selected from the group consisting of: X15, X39, X53, X185, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises 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 all 10) of the following amino acid residues: X15 is D; X39 is A; X251 is A. 53 is Y; X185 is K; X218 is N; X221 is I; X237 is R; X251 is L; X255 is C; and X285 is A.

[0051] In some embodiments, the engineered dsRNA ligase polypeptide comprises a purification tag. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 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 ,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,16 4, 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 engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, and 634.In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 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, 103, 104, 105, 106, 107, 108, 109, 110 4, 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, 257 8, 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.

[0052] The present disclosure also provides a polypeptide immobilized on a solid material by chemical bonding or physical adsorption methods, wherein the polypeptide comprises the engineered dsRNA ligase polypeptide described herein.

[0053] The present disclosure also provides polynucleotides encoding the engineered dsRNA ligase polypeptides described herein.

[0054] In some embodiments, the polynucleotide comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO:3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 4 3, 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, 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, 3 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, 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 and 599.

[0055] In some embodiments, the polynucleotide comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 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.

[0056] In some embodiments, the polynucleotide comprises a nucleic acid sequence selected from the group consisting of: (a) SEQ ID NO: 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, 457, 459, 461, 463, 465, 467 7, 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, 5 51, 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 NO: 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、1 81, 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, 256 7, 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. ;

[0057] The present disclosure also provides expression vectors comprising the polynucleotides described herein. In some embodiments, the vector comprises a plasmid, a cosmid, a phage, or a viral vector.

[0058] The present disclosure also provides a host cell comprising a polynucleotide described herein or an expression vector described herein. In some embodiments, the host cell is Escherichia coli (E. coli).

[0059] The present disclosure also provides a method for preparing an engineered dsRNA ligase polypeptide, the method comprising the steps of culturing the host cell described herein and obtaining the engineered dsRNA ligase polypeptide from the culture.

[0060] The present disclosure also provides an engineered dsRNA ligase catalyst obtainable by culturing a host cell described herein or according to a method described herein, wherein the engineered dsRNA ligase catalyst comprises cells or culture fluid containing an engineered dsRNA ligase polypeptide, or an article processed therewith, wherein the article refers to an extract obtained from a culture of the host cell, an isolated product obtained by isolating or purifying the engineered dsRNA ligase from the extract, or an immobilized product obtained by immobilizing the host cell, an extract thereof, or an isolated product of the extract.

[0061] The present disclosure further provides a method of producing an oligonucleotide from two or more oligonucleotide fragments, wherein the method comprises contacting: (i) two or more oligonucleotide fragments; (ii) an engineered dsRNA ligase polypeptide described herein; (iii) a source of ATP; and (iv) a divalent cation; to obtain the oligonucleotide.

[0062] In some embodiments, the source of ATP comprises ATP.

[0063] 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.

[0064] In some embodiments, the methods are performed using substoichiometric concentrations of AMP and / or ATP.

[0065] In some embodiments, the polyphosphoric acid is a polyphosphate salt. In some embodiments, the polyphosphate salt is sodium polyphosphate (Madrell's salt) or sodium hexametaphosphate (Graham's salt).

[0066] In some embodiments, the divalent cation cofactor is Mg 2+ or Mn 2+ .

[0067] In some embodiments, the method is performed at a divalent cation concentration of 5-100 mM, optionally 30-50 mM.

[0068] In some embodiments, the method further comprises the step of purifying the oligonucleotide.

[0069] The present disclosure also provides for the use of the engineered dsRNA ligase polypeptides described herein in generating an oligonucleotide from two or more oligonucleotide fragments.

[0070] In some embodiments, the oligonucleotide is up to 60 nucleotides in length.

[0071] In some embodiments, each oligonucleotide fragment is 4-16 nucleotides in length, optionally 6-9 nucleotides in length.

[0072] In some embodiments, one or more oligonucleotide fragments comprise one or two overhangs.

[0073] In some embodiments, one or more oligonucleotide fragments comprise a chemical modification. In some embodiments, the chemical modification is selected from: (a) a modified backbone optionally selected from phosphorothioate (e.g., chiral phosphorothioate) or methylphosphonate internucleotide linkages; (b) a modified nucleotide optionally selected from 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 (c) conjugation to a ligand, optionally wherein the ligand comprises one or more N-acetylgalactosamine (GalNAc) derivatives.

[0074] The present disclosure also provides compositions comprising: i. an engineered dsRNA ligase polypeptide described herein; ii. a source of ATP; and iii. a divalent cation.

[0075] In some embodiments, the composition further comprises two or more oligonucleotide fragments.

[0076] The present disclosure also provides kits comprising: i. an engineered dsRNA ligase polypeptide described herein; ii. a source of ATP; iii. divalent cations; and iv. instructions for use in a method of generating an oligonucleotide from two or more oligonucleotide fragments.

[0077] In some embodiments, the source of ATP comprises ATP.

[0078] In some embodiments, the source of ATP comprises: (a) polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and / or ATP.

[0079] In some embodiments, the PPK is selected from PPK12 or ajPAP.

[0080] In some embodiments, the polyphosphoric acid is a polyphosphate salt.

[0081] In some embodiments, the polyphosphate is sodium polyphosphate (Madrell's salt) or sodium hexametaphosphate (Graham's salt).

[0082] In some embodiments, the divalent cation cofactor is Mg 2+ or Mn 2+ .

[0083] definition

[0084] Unless otherwise clearly defined, the technical and scientific terms used in this disclosure have the meanings generally understood by those skilled in the art. The following references provide general definitions of many terms used in the present invention for those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology [microbiology and molecular biology dictionary] (2nd edition 1994); The Cambridge Dictionary of Science and Technology [Cambridge science and technology dictionary] (Walker edited, 1988); The Glossary of Genetics [genetics vocabulary], 5th edition, R. Rieger et al. (ed.), Springer Verlag [Springer Publishing Company] (1991); and Hale & Marham, The Harper Collins Dictionary of Biology [Harper Collins biological dictionary] (1991). As used herein, unless otherwise indicated, the following terms have the following meanings given.

[0085] As used throughout this disclosure, articles such as "a" and "an" refer to one or more (eg, to at least one) of the grammatical objects of the article.

[0086] Unless stated otherwise, the term "and / or" means "and" or "or".

[0087] As used herein, the term "about" typically refers to the value immediately following the term "about." For example, "about 15 or more nucleotides" typically refers to 15 or more nucleotides. In some embodiments, the term "about" includes + / - 1, 2, or 3 values ​​of the stated value. For example, "about 15 or more nucleotides" can refer to 15 + / - 3 nucleotides, such as 12, 13, 14, 15, 16, 17, or 18 nucleotides.

[0088] 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."

[0089] The dsRNA ligases of the present invention are ATP-dependent nucleic acid ligases. 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 by the following steps: (1) the dsRNA ligase reacts with ATP to form a covalent dsRNA ligase-AMP intermediate and releases 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, resulting in the formation of a phosphodiester bond and the release of AMP.

[0090] The stoichiometric concentration of the cofactor is the theoretical concentration required to achieve complete ligation in a given ligation reaction. A skilled person 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 requires four ligation reactions, and its stoichiometric ATP concentration is 4 mM. A stoichiometric excess of ATP helps ensure complete ligation. In some embodiments, the stoichiometric excess is at least 105%, e.g., 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% of the theoretical stoichiometric concentration of ATP required to achieve complete ligation.

[0091] The terms "engineered dsRNA ligase," "engineered dsRNA ligase polypeptide," "improved dsRNA ligase polypeptide," and "engineered polypeptide" are used interchangeably herein.

[0092] As used herein, the term "oligonucleotide" refers to a nucleic acid, typically comprising up to 100 nucleotides. As used herein, the term "oligonucleotide product" refers to an oligonucleotide formed by connecting two or more oligonucleotide fragments by a dsDNA ligase as described herein. Oligonucleotide products are also referred to herein as oligonucleotides. It should be understood that the oligonucleotide products described herein comprise RNA. It should also be understood that the oligonucleotide products described herein comprise double-stranded regions. In certain embodiments, the oligonucleotide products described herein comprise RNA and DNA. For example, a portion of the oligonucleotide product can be double-stranded DNA, while another portion is double-stranded RNA, forming a DNA-RNA chimera.

[0093] The term "therapeutic oligonucleotide" refers to an oligonucleotide that can provide a therapeutic effect, for example, by interacting with a biomolecule and / or by 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 technology that uses RNAi agents 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 change gene expression through many mechanisms, including direct spatial blocking of mRNA and ribonuclease H (RNase H)-mediated mRNA degradation.

[0094] As non-limiting examples, RNAi agents include siRNA (small interfering RNA), dsRNA (double-stranded RNA), shRNA (short hairpin RNA) and miRNA (microRNA). As other non-limiting examples, RNAi agents also include locked nucleic acid (LNA), morpholino, UNA, threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA) and fluoroarabinoic acid (FANA). RNAi agents also include molecules in which one or more chains are mixtures of RNA, DNA, LNA, morpholino, UNA (unlocking nucleic acid), TNA, GNA and / or FANA. As non-limiting examples, one or two chains of the RNAi agent can be, for example, RNA, except that one or more RNA nucleotides are replaced by DNA, LNA, morpholino, UNA, TNA, GNA and / or FANA. In certain embodiments, one or two chains of the RNAi agent can be nicked, and the two chains can have the same length, or one chain can be shorter than the other chain. The oligonucleotides of the present invention can be any RNAi agent as described herein.

[0095] The term "oligonucleotide fragment" as used herein refers to a nucleic acid that can be connected to one or more other oligonucleotide fragments to provide an oligonucleotide (or oligonucleotide product). Each oligonucleotide fragment corresponds to a portion of an oligonucleotide product. Oligonucleotide fragments may be referred to as "substrate" for ligation in this article.

[0096] As mentioned above, dsRNA ligase activity is related to connecting 5' oligonucleotide fragments with 3' oligonucleotide fragments.In the context of oligonucleotide fragments, prefixes 5' and 3' refer to the relative positions of each oligonucleotide fragment in the oligonucleotide product after connection, wherein the 5' oligonucleotide fragment is located at the upstream of the 3' oligonucleotide fragment (when the oligonucleotide product is presented in 5' to 3' direction). As used herein, "5' oligonucleotide fragment" typically includes 3' terminal ribonucleotides with 3'-hydroxyl groups. As used herein, "3' oligonucleotide fragment" includes 5'-phosphate, wherein the 5' terminal nucleotides are deoxyribonucleotides or ribonucleotides.

[0097] It should be understood that in some embodiments, the oligonucleotide fragments can be 3' oligonucleotide fragments and 5' oligonucleotide fragments (e.g., wherein the ligation reaction occurs at the 5' and 3' ends of the oligonucleotide fragments). For example, the oligonucleotide fragments can provide: (i) ligation reaction of the 3' oligonucleotide fragment with the 5' oligonucleotide fragment; and (ii) ligation reaction of the 5' oligonucleotide fragment with the 3' oligonucleotide fragment. For example, Figure 1A The oligonucleotide fragment 7 in provides: (i) a ligation reaction between the 3' oligonucleotide fragment and the 5' oligonucleotide fragment 6, and (ii) a ligation reaction between the 5' oligonucleotide fragment and the 3' oligonucleotide fragment 12 to provide an oligonucleotide product 2.

[0098] " terminal oligonucleotide fragment " herein refers to the nucleic acid corresponding to the end (such as 5 ' or 3 ' end) part of the oligonucleotide product.5 ' terminal oligonucleotide fragment is typically provided for being connected to the 5 ' oligonucleotide fragment of 3 ' oligonucleotide fragment.3 ' terminal oligonucleotide fragment is typically provided for being connected to the 3 ' oligonucleotide fragment of 5 ' oligonucleotide fragment.In certain embodiments, 5 ' terminal oligonucleotide is directly connected to 3 ' terminal oligonucleotide.In certain embodiments, 5 ' terminal oligonucleotide and 3 ' terminal oligonucleotide are separated by one or more oligonucleotide fragments.

[0099] In some embodiments, the oligonucleotide fragments described herein comprise RNA and DNA. For example, a portion of the oligonucleotide fragment can be double-stranded DNA, while another portion is double-stranded RNA, forming a DNA-RNA chimera.

[0100] 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, a nucleotide overhang is formed when the 3'-end of one strand extends beyond the 5'-end of the other strand, or vice versa. For example, an unpaired nucleotide overhang forms an overhang. An overhang that is complementary to an overhang of a second oligonucleotide fragment can be referred to as a "sticky end." The oligonucleotide fragments described herein can have one or two sticky ends.

[0101] "Blunt" or "blunt end" means that there are no unpaired nucleotides at that end of a double-stranded oligonucleotide, i.e., there are no nucleotide overhangs. A "blunt-end" oligonucleotide or oligonucleotide fragment is an oligonucleotide that is double-stranded throughout its entire length, i.e., there are no nucleotide overhangs at either end of the molecule.

[0102] Double-stranded nucleic acids comprise two antiparallel and substantially complementary nucleic acid chains, which are referred to as "sense" and "antisense" strands. In the context of double-stranded RNAi agents, "antisense strand" refers to a strand of RNAi that includes a region that is substantially complementary to a target sequence (e.g., an mRNA sequence). "Sense strand" refers to a strand of an RNAi agent that includes a region that is substantially complementary to a region of the antisense strand. The sense and antisense strands of an RNAi agent can be referred to as a passenger strand and a guide strand, respectively.

[0103] Sequences that are "substantially complementary" may be fully complementary or may contain one or more mismatches upon hybridization while retaining the ability to hybridize under conditions most relevant to their ultimate application.

[0104] "Conversion" refers to the enzymatic conversion of a substrate into the corresponding product. "Percent conversion" or "conversion" refers to the percentage of oligonucleotide fragments that are converted to oligonucleotide products within a defined time period under specified conditions. Thus, the "enzyme activity" or "activity" of a ligase can be expressed as the "percent conversion" of oligonucleotide fragments to oligonucleotide products.

[0105] Ideally, to compare activity between ligation reactions and account for natural variations in peak intensity between injections, the % product conversion for each analyzed sample would be calculated using the following formula:

[0106]

[0107] where ε p , ε s and ε i= the extinction coefficient of product, substrate and intermediate oligonucleotide respectively. In some cases, for example, using analytical method as described herein, it is impossible to separate all substrates, reaction intermediates and products. Therefore, it is impossible to determine the conversion % according to the above formula. However, in some cases, for example, using analytical method as described herein, it is possible to separate at least one substrate, reaction intermediate and product, for example, the oligonucleotide containing GalNAc clearly defined, including the substrate fragment (for example oligonucleotide (12) containing GalNAc, as used in the example as described herein), reaction intermediate (for example, oligonucleotide (14) as demonstrated herein) and product chain (for example, product oligonucleotide (2) as demonstrated herein). Therefore, pseudo conversion % can be calculated, represented by arbitrary units (AU), which only considers these substances that are well separated according to the following formula:

[0108]

[0109] where ε (2) , ε (12) and ε (14) are the extinction coefficients for oligonucleotides (2), (12), and (14), respectively. Using this calculation, an AU = 1.0 would mean that there are no longer any GalNAc-containing substrate or intermediate oligonucleotides in the reaction, and that they have all been converted to the GalNAc-containing product (2). In fact, for the sample with AU = 1.0, the only other peak present in the chromatogram corresponds to the product oligonucleotide (2), and no other intermediates or starting materials can be identified. In addition, the ratio of product oligonucleotides (2) and (3) is consistent with that of an authentic standard of siRNA product (1). In summary, it can be concluded that AU = 1.0 is an approximate value that is essentially equivalent to 100% conversion.

[0110] "Improved enzyme properties" refers to enzyme properties that are better or more ideal for a particular purpose compared to a reference dsRNA ligase (e.g., a wild-type dsRNA ligase or another engineered dsRNA ligase) under identical reaction conditions. The engineered dsRNA ligase polypeptides disclosed herein exhibit improved enzyme properties. The engineered dsRNA ligase polypeptides described herein exhibit increased enzymatic activity (which can be expressed as a percentage of substrate conversion). Additional enzyme properties that may be improved include, but are not limited to, thermal stability, pH activity profile, cofactor requirements, and tolerance to inhibitors (e.g., inhibition by reaction components, substrates, or products).

[0111] "Isolated polypeptide" refers to a polypeptide that is substantially separated from other substances with which it is naturally associated (e.g., proteins, lipids, and polynucleotides). The term includes polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., in a host cell or synthesized in vitro). The engineered dsRNA ligase polypeptide can be present in cells, in cell culture medium, or prepared in various forms, such as lysates or isolated preparations. Therefore, in some embodiments, the engineered dsRNA ligase polypeptide can be an isolated polypeptide.

[0112] "Wild-type" refers to the form found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a natural source and has not been intentionally modified by manual procedures. The polypeptide sequence of the wild-type dsRNA ligase described herein is provided by SEQ ID NO: 302. As used herein, a wild-type sequence may also include a purification tag and may be provided by SEQ ID NO: 2.

[0113] The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0114] The terms "protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by amide bonds, regardless of its length or post-translational modifications (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, etc.).

[0115] "Recombinant" or "engineered" when used with reference to, for example, a cell, nucleic acid, or polypeptide, refers to a material or material corresponding to a native or native form of that material that has been modified in a manner not found in nature, or is identical thereto, but is produced or derived from synthetic materials and / or manipulation through the use of recombinant techniques.

[0116] The abbreviations used for the genetically encoded amino acids are conventional and are as follows:

[0117]

[0118]

[0119] When three-letter abbreviations are used, unless specifically preceded by "L" or "D" or clear from the context in which the abbreviation is used, the amino acid can be in the L- or D-configuration about the α-carbon (Cα). For example, where "Ala" represents alanine, but the configuration about the α-carbon is not specified, "D-Ala" and "L-Ala" represent D-alanine and L-alanine, respectively.

[0120] When single-letter abbreviations are used, capital letters represent amino acids in the L-configuration about the α-carbon, and lowercase letters represent amino acids in the D-configuration about the α-carbon. For example, "A" represents L-alanine and "a" represents D-alanine. When a polypeptide sequence is presented as a string of single-letter or three-letter abbreviations (or a mixture thereof), the sequence is presented in the amino (N) to carboxyl (C) direction according to common convention.

[0121] Abbreviations for genetically encoded nucleotides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless otherwise specified, abbreviated nucleotides may be ribonucleotides or 2'-deoxyribonucleotides. Nucleotides may be designated as ribonucleotides or 2'-deoxyribonucleotides on an individual basis or on a collective basis. When a nucleic acid sequence is presented as a string of single-letter abbreviations, the sequence is presented in a 5' to 3' direction according to common convention, and the phosphodiester bond is not indicated.

[0122] Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be replaced by other parts without substantially changing the base pairing properties of the oligonucleotide comprising the nucleotide with such a replacement part. For example, but not limited to, a nucleotide comprising inosine as its base can be base paired with a nucleotide comprising adenine, cytosine or uracil. Therefore, in the nucleotide sequence of the oligonucleotide appearing in this disclosure, a nucleotide containing uracil, guanine or adenine can be replaced by a nucleotide containing, for example, inosine. In another example, an adenine and a cytosine at any position in the oligonucleotide can be replaced by guanine and uracil, respectively, to form a wobble base pairing with the target mRNA.

[0123] "Amino acid difference" or "residue difference" refers to the difference in the amino acid residue at a position in a polypeptide sequence relative to the amino acid residue at the corresponding position in a reference sequence. The position of the 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, "the residue difference at position X6 compared to SEQ ID NO: 302" refers to the difference in the amino acid residue at the polypeptide position corresponding to position 6 of SEQ ID NO: 302. Thus, if the reference polypeptide of SEQ ID NO: 302 has serine at position 6, then "the residue difference at position X2 compared to SEQ ID NO: 302" refers to an amino acid substitution of any residue other than serine at the polypeptide position corresponding to position 6 of SEQ ID NO: 302.

[0124] The specific amino acid residue difference at that position can be represented as "XnY" or "Xn is Y," where "Xn" designates the corresponding position in the reference sequence described above, and "Y" is the one-letter identifier for the residue present at that position in the engineered polypeptide. The specific amino acid difference can also be represented using the conventional notation "AnY," where A is the one-letter identifier for 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 for the residue present at that position in the engineered polypeptide.

[0125] In some examples, the engineered polypeptides disclosed herein may comprise one or more amino acid residue differences relative to a reference sequence, as indicated by a listing of specific positions at which residue differences exist relative to the reference sequence. In some embodiments, more than one amino acid residue may be used at a specific residue position in the engineered polypeptide, and multiple amino acid residues may be listed as alternatives, e.g., "X19 is Q or D."

[0126] The deletion of an amino acid can 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 the modification of a polypeptide by removing one or more amino acids from a reference polypeptide. The deletion can include removing 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 constituting the reference enzyme, while retaining the enzymatic activity of the engineered dsRNA ligase and / or retaining the improved properties of the engineered dsRNA ligase. The deletion can involve an internal portion and / or a terminal portion of the polypeptide. In various embodiments, the deletion can include a continuous segment or can be discontinuous.

[0127] In the context of a given amino acid or polynucleotide sequence numbering, "corresponding to," "with reference to," or "relative to" refers to the numbering of the residues of a given amino acid or polynucleotide sequence when the given amino acid or polynucleotide sequence is compared to a reference sequence. In other words, the residue numbers or residue positions of a given sequence are specified relative to a reference sequence, rather than being specified by the actual numerical positions of the residues within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence (e.g., an engineered dsRNA ligase) can be aligned with a reference sequence by introducing gaps to optimize the matching of residues between the two sequences. In these cases, despite the presence of gaps, the numbering of residues in a given amino acid or polynucleotide sequence is relative to the reference sequence to which it is aligned.

[0128] "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 fragment of a full-length gene or polypeptide sequence. In some embodiments, a "reference sequence" is a wild-type sequence. In some embodiments, a "reference sequence" is an engineered or altered sequence.

[0129] Methods for determining sequence identity percentages are known in the art. For example, when assessing sequence identity, a sequence with a determined number of continuous nucleotides or amino acids can be compared with a nucleic acid or peptide sequence (having the same number of continuous nucleotides or amino acids) from the corresponding portion of a nucleic acid or peptide sequence disclosed herein. Percentage sequence identity can be calculated by the following method: determine the number of positions where the same nucleic acid base or amino acid residue occurs in the two sequences, or compare the nucleic acid base or amino acid residue with a gap to derive the number of matching positions, divide the number of matching positions by the total number of positions in the sequence, and multiply the result by 100, thereby deriving the percentage of sequence identity. It will be appreciated by those skilled in the art that there are many established algorithms that can be used to compare two sequences. Optimal alignment of sequences for comparison can be achieved, for example, by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by searching by the similarity method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85:2444, by computerized 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, FM Ausubel et al. (eds.), Current Protocols, a Joint Venture between Greene Publishing Associates, Inc. and John The method of determining percent sequence identity and percent sequence similarity is performed by Wiley & Sons, Inc. (a joint venture between Green Publishing Associates, Inc. and John Wiley & Sons, Inc.), (1995 supplement) (Ausubel). Examples of algorithms suitable for determining percent sequence identity and percent sequence similarity are the BLAST and BLAST 2.0 algorithms, which are 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 through the National Center for Biotechnology Information website.The algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is called 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. The word hits are then extended in both directions along each sequence as far as the cumulative alignment score can be increased. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a matching residue pair; 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 terminated when: the cumulative alignment score falls by the amount X from its maximum achieved value; the cumulative score goes to zero or lower due to the accumulation of one or more negative-scoring residue alignments; or the end of either 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 by default a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both chains as defaults. For amino acid sequences, the BLASTP program uses by default a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, Proc Natl Acad Sci USA 89:109-15). Sequence alignments and exemplary determinations of percent sequence identity can be performed using the BESTFIT or GAP programs in the GCG Wisconsin software package (Accelrys, Madison, Wisconsin) using the default parameters provided.

[0130] It is understood that the engineered dsRNA ligase has dsRNA ligase activity regardless of the percent sequence identity to the reference sequence.

[0131] "Suitable reaction conditions" refer to those conditions in the reaction system under which the substrate is converted to the desired product (e.g., enzyme loading, substrate loading, temperature, pH, etc.). Suitable reaction conditions can be readily determined by one skilled in the art. Exemplary "suitable reaction conditions" are provided in this disclosure and illustrated by the examples.

[0132] Engineered dsRNA ligase polypeptide

[0133] The present disclosure provides engineered dsRNA ligase polypeptides comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 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, 377, 378, 379, 380, 381, 382, ​​384, 386, 387, 388, 389, 390, 401, 402, 403, 404, 405 6, 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 6, 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.

[0134] The present disclosure provides engineered dsRNA ligase polypeptides comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668.

[0135] The present disclosure provides engineered dsRNA ligase polypeptides comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 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, 467 8, 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, 551 52, 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.

[0136] The present disclosure also provides an engineered 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 NO: 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、53 4, 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 engineered dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302.

[0137] The present disclosure also provides an engineered 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 NO: 636, 638, 640, 642, 644, 646, 648, 650, 652, 654, 656, 658, 660, 662, 664, 666, and 668, wherein the engineered dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302.

[0138] The present disclosure also provides an engineered 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 NO: 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, 387 88, 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, 473 74, 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 60, 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, wherein the engineered dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302.

[0139] The present disclosure provides engineered double-stranded RNA (dsRNA) ligase polypeptides, which are: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 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, 48 4, 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, 5 or (b) a polypeptide having dsRNA ligase activity, the polypeptide comprising an amino acid sequence having (i) at least 80% sequence identity to one of the polypeptides recited in (a), and (ii) one or more amino acid residues substituted, deleted, added or inserted relative to the one amino acid sequence recited in (a); wherein the engineered dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302.

[0140] The present disclosure provides engineered double-stranded RNA (dsRNA) ligase polypeptides, which are: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 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, which polypeptide comprises an amino acid sequence: having (i) at least 80% sequence identity to one of the polypeptides listed in (a), and (ii) one or more amino acid residues substituted, deleted, added or inserted relative to the one amino acid sequence listed in (a); wherein the engineered dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302.

[0141] The present disclosure provides engineered double-stranded RNA (dsRNA) ligase polypeptides, which are: (a) a polypeptide comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 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, or (b) a polypeptide having dsRNA ligase activity, the polypeptide comprising an amino acid sequence having (i) at least 80% sequence identity to one of the polypeptides recited in (a), and (ii) one or more amino acid residues substituted, deleted, added or inserted relative to the one amino acid sequence recited in (a); wherein the engineered dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302.

[0142] In some embodiments, the engineered 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, 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 engineered 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, 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.

[0143] In some embodiments, the engineered 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, 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.

[0144] In some embodiments, the engineered 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, 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.

[0145] The engineered dsRNA ligase polypeptides represented by the even-numbered sequence identifiers of SEQ ID NOs: 304 to 600 and 636 to 668 exhibited greater activity than SEQ ID NO: 302, as shown in the Examples. The dsRNA ligase polypeptides used in the Examples (represented by the even-numbered sequence identifiers of SEQ ID NOs: 4 to 300 and 602 to 634, respectively) comprise the even-numbered sequence identifiers of SEQ ID NOs: 304 to 600 and 636 to 668 and an N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO: 669)). For example, SEQ ID NO: 4 comprises: (i) an N-terminal purification tag MHHHHHHENLYFQS (SEQ ID NO: 669); and (ii) SEQ ID NO: 304. The dsRNA ligase polypeptides represented by the even-numbered sequence identifiers of SEQ ID NOs: 304 to 600 and 636 to 668 do not comprise the N-terminal purification tag represented by SEQ ID NO: 669.

[0146] The wild-type dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 302 (also available under UniProt accession number Q7Y4V8). SEQ ID NO: 2 comprises: (i) an N-terminal purification tag MHHHHHHENLYFQS (SEQ ID NO: 669); and (ii) SEQ ID NO: 302. It will be readily understood that both SEQ ID NOs: 2 and 302 comprise wild-type dsRNA ligase polypeptide sequences, and therefore, these two sequences may be referred to herein as wild-type sequences.

[0147] In some embodiments, the engineered 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 engineered dsRNA ligase polypeptide comprises an amino acid sequence of SEQ ID NO: 666. In some embodiments, the engineered 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 engineered 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 engineered dsRNA ligase polypeptide comprises an amino acid sequence of SEQ ID NO: 632. In some embodiments, the engineered 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 by SEQ ID NOs: 370, 488, 526, 578, 588, 590, 592, and 666, respectively.

[0148] In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 370. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 488. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 526. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 578. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 588. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 590. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 592. In some embodiments, the engineered dsRNA ligase polypeptide comprises the amino acid sequence of SEQ ID NO: 666.

[0149] The present disclosure also provides an engineered dsRNA ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 302 that produces at least 5% more oligonucleotide product under the same ligation reaction conditions than a dsRNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO: 302, wherein the engineered dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302. In some embodiments, the engineered 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 product 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.

[0150] In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 302, 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.

[0151] In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 302, 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.

[0152] In some embodiments, the ligation reaction conditions include about 1 μM to about 10 mM 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 engineered 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 comprises: (a) polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and / or ATP.

[0153] In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in 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 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, 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, wherein the numbering refers to SEQ ID NO: 302.In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid residue that differs from SEQ ID NO: 1 in 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) selected from the group consisting of: The amino acid sequence of NO:302 is 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, X X305, X313, X314, X325, and X328, wherein the numbering refers to SEQ ID NO: 302.

[0154] In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising 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; X 49 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 not present; 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; 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; and X328 is R; wherein numbering refers to SEQ ID NO: 302.In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising 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 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 not present; 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; X12 9 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; X wherein 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; wherein numbering refers to SEQ ID NO: 302.

[0155] In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in 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) amino acid residues selected from X15, X19, X36, X39, X53, X218, X221, X237, X251, X255, and X285; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising 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; wherein numbering refers to SEQ ID NO: 302.

[0156] In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in 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 X15, X19, X36, X39, X53, X185, X218, X221, X237, X251, X255, and X285; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising 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; wherein numbering refers to SEQ ID NO: 302.

[0157] In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g., two or more or three or more) amino acid residues selected from the group consisting of: X36, X39, X218, and X221; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g., two or more or three or more) of the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I; wherein the numbering refers to SEQ ID NO: 302.

[0158] In some embodiments, the engineered 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; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising the following amino acid residues: X36 is V; X39 is A; X218 is N; and X221 is I; wherein the numbering refers to SEQ ID NO: 302.

[0159] In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g., two or more) amino acid residues selected from the group consisting of: X39, X218, and X221; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g., two or more) of the following amino acid residues: X39 is A; X218 is N; and X221 is I; wherein the numbering refers to SEQ ID NO: 302.

[0160] In some embodiments, the engineered 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; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising the following amino acid residues: X39 is A; X218 is N; and X221 is I; wherein the numbering refers to SEQ ID NO: 302.

[0161] In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more (e.g., two or more or three or more) amino acid residues selected from the group consisting of: X39, X218, X221, and X255; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising one or more (e.g., two or more or three or more) of the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C; wherein the numbering refers to SEQ ID NO: 302.

[0162] In some embodiments, the engineered 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; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising the following amino acid residues: X39 is A; X218 is N; X221 is I; and X255 is C; wherein the numbering refers to SEQ ID NO: 302.

[0163] In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in 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: X39, X53, X218, X221, X237, X251, X255, and X285; wherein the numbering is to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising 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; wherein the numbering is to SEQ ID NO: 302.

[0164] In some embodiments, the engineered 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; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising 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; wherein the numbering refers to SEQ ID NO: 302.

[0165] In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in 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: X15, X39, X53, X218, X221, X237, X251, X255, and X285; wherein the numbering is to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising 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; wherein the numbering is to SEQ ID NO: 302.

[0166] In some embodiments, the engineered 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; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence comprising 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; wherein the numbering refers to SEQ ID NO: 302. In some embodiments, the engineered 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; wherein numbering refers to SEQ ID NO: 302.

[0167] In some embodiments, the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of X15, X39, X53, X185, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the amino acid sequence of the engineered 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.

[0168] In some embodiments, the engineered dsRNA ligase polypeptide comprises a purification tag. Purification tags are typically attached to polypeptides so that they can be purified from their crude biological sources using affinity techniques. In some embodiments, the purification tag comprises a polyhistidine tag. The polyhistidine tag is bound to a matrix containing fixed metal ions and can be 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).

[0169] In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 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 ,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,16 4, 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.

[0170] In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 602, 604, 606, 608, 610, 612, 614, 616, 618, 620, 622, 624, 626, 628, 630, 632, and 634.

[0171] In some embodiments, the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 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, 103, 104, 105, 106, 107, 108, 109, 110 4, 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, 257 8, 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.

[0172] fixed

[0173] The present disclosure also provides a polypeptide immobilized on a solid support material by chemical bonding or physical adsorption, wherein the polypeptide comprises the engineered dsRNA ligase polypeptide disclosed herein.

[0174] Immobilization of polypeptides by physical adsorption typically involves physical adsorption or attachment of the polypeptide to a solid support material. Adsorption can occur through weak non-specific forces (e.g., van der Waals, hydrophobic interactions, and hydrogen bonds). Physical adsorption can be achieved by immersing the support material in a polypeptide solution and incubating the support material for a period of time to allow physical adsorption to occur. Immobilization of polypeptides by chemical bonds typically involves attachment of the polypeptide to a support material by a covalent bond.

[0175] In some embodiments, the dsRNA ligase polypeptide is immobilized by a spacer positioned between the dsRNA ligase polypeptide and the solid material. In some embodiments, the spacer is a peptide (e.g., a peptide comprising 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).

[0176] In some embodiments, the engineered dsRNA ligase polypeptide is immobilized using affinity immobilization. In some embodiments, the engineered dsRNA ligase polypeptide is immobilized using metal affinity immobilization, for example, by contacting a His-tagged engineered dsRNA ligase polypeptide with an immobilized metal (e.g., nickel, zinc, cobalt, or copper).

[0177] In certain embodiments, solid support material comprises film, resin, solid carrier or other solid phase material.Solid support material can be made up of organic polymer, for example polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethylene oxide, polymethacrylate and polyacrylamide and copolymer and graft thereof.Solid support material can also be inorganic, for example glass, silicon dioxide, controlled porous glass (CPG), reversed phase silica or metal, as gold or platinum.The configuration of solid support material can be the form of bead, ball, microparticle, particle, gel, film or surface.Surface can be planar, substantially planar or non-planar.Solid support material can be porous or non-porous, and can have swelling or non-swelling characteristics.Solid support material can be configured as the form of hole, depression or other container, vessel, feature or position. Solid support materials that can be used to immobilize the dsRNA ligase polypeptide for ligase reactions include, but are not limited to, microbeads or resins, for example, polymethacrylates, such as polymethacrylates with epoxy functional groups, polymethacrylates with aminoepoxy functional groups, polymethacrylates, styrene / DVB copolymers, or polymethacrylates with octadecyl functional groups.

[0178] Exemplary solid supports include, but are not limited to, chitosan microbeads, Eupergit C, IB-150, IB-350, IB-C435, IB-A369, IB-A161, IB-A171, IBS500, IB-S861, SEPABEADS (Mitsubishi) (e.g., 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, AmberliteIRA67, Amberlite FPA90Cl, Amberlite FPA40Cl, Amberlite XAD18, Accurel EP100, ECR8206F / 5730, ECR8206 / 5803, ECR8206M / 5749, ReliZyme EP403, ReliZymeEP113, LewatitVP OC 1600, Diaion WA20, Diaion WA21J, Diaion WA30, Dowex66, Diaion HPA-25L, Lewatit VP OC 1064MD PH, Lewatit VP OC 1163, Lifetech ECR8304F, LifetechECR8309F, Lifetech ECR8315F, Lifetech ECR8204F, Lifetech ECR8285, LifetechECR1090M, Lifetech ECR1030M, Lifetech ECR8806M, Chromalite(MAM2 / F)D6591, Chromalite MIDA / M, Chromalite MIDA / M / Fe, Chromalite MIDA / M / Co, Chromalite MIDA / M / Ni, Chromalite MIDA / M / Cu and Chromalite MIDA / M / Zn.

[0179] Polynucleotides, control sequences, expression vectors, and host cells useful for producing engineered dsRNA ligase polypeptides

[0180] On the other hand, the present disclosure provides polynucleotides encoding engineered polypeptides having dsRNA ligase activity as described herein. The polynucleotides can be linked to one or more heterologous regulatory sequences that control gene expression to produce recombinant polynucleotides capable of expressing the engineered polypeptide. An expression construct comprising a heterologous polynucleotide encoding an engineered dsRNA ligase can be introduced into a suitable host cell to express the corresponding engineered dsRNA ligase polypeptide.

[0181] As will be apparent to those skilled in the art, the availability of protein sequences and the knowledge corresponding to the codons of various amino acids provide the description of all possible polynucleotides encoding the protein sequence of interest. The degeneracy of the genetic code (wherein identical amino acids are encoded by selectable or synonymous codons) allows the generation of a large number of polynucleotides, all of which encode the engineered dsRNA ligase polypeptides disclosed herein. Therefore, after determining the specific amino acid sequence, those skilled in the art can generate any number of different polynucleotides by modifying one or more codons in a manner that does not change the amino acid sequence of the protein. In this regard, the present disclosure particularly contemplates that for any polypeptide disclosed herein, each possible change of polynucleotides is carried out by selecting a combination based on possible codon selections, including those amino acid sequences of the exemplary engineered polypeptides listed in Examples 7 to 12, any polypeptide disclosed as the even-numbered sequence identifiers of SEQ ID NOs: 304 to 600 and 636 to 668, and any polypeptide disclosed as the even-numbered sequence identifiers of SEQ ID NOs: 4 to 300 and 602 to 634.

[0182] In various embodiments, codons are preferably selected to suit the host cell in which the recombinant protein is produced. For example, codons preferred for bacteria are used to express genes in bacteria; codons preferred for yeast are used to express genes in yeast; and codons preferred for mammals are used to express genes in mammalian cells.

[0183] In some embodiments, the present disclosure provides polynucleotides encoding the above-described engineered dsRNA ligase polypeptides.

[0184] 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 of 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.

[0185] In some embodiments, the polynucleotide encodes an engineered 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 a sequence having an odd-numbered sequence identifier of SEQ ID NOs: 303-599 or 635-667, wherein: (i) the polynucleotide does not comprise SEQ ID NO: 301; and (ii) the polynucleotide does not encode a dsRNA ligase polypeptide having the amino acid sequence of SEQ ID NO: 302.

[0186] In some embodiments, the polynucleotide encodes an engineered 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 a sequence having an odd-numbered sequence identifier of SEQ ID NOs: 3-299 or 601-633, wherein: (i) the polynucleotide does not comprise SEQ ID NO: 1; and (ii) the polynucleotide does not encode a dsRNA ligase polypeptide having the amino acid sequence of SEQ ID NO: 2. It will be readily appreciated that the polynucleotides having odd-numbered sequence identifiers of SEQ ID NOs: 3 to 299 or 601 to 633 encode engineered dsRNA ligase polypeptides comprising an N-terminal purification tag (SEQ ID NO: 669).

[0187] An isolated polynucleotide encoding an engineered dsRNA ligase polypeptide can be manipulated in a variety of ways to achieve expression of the engineered polypeptide, including further modification of the sequence by codon optimization to improve expression, insertion into appropriate expression elements with or without additional control sequences, and transformation into a host cell suitable for the expression and production of the engineered polypeptide.

[0188] Depending on the expression vector, it may be desirable or necessary to manipulate the isolated polynucleotide prior to insertion into the vector. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. Guidelines are provided by Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press; and Current Protocols in Molecular Biology, Ausubel. F., ed., Greene Pub. Associates, 1998, updated 2010.

[0189] The present disclosure also provides expression vectors comprising the polynucleotides described herein. In some embodiments, the vector is selected from a plasmid, a cosmid, a phage, or a viral vector. The recombinant expression vector typically comprises one or more expression regulatory regions, such as a promoter and terminator, an origin of replication, and the like.

[0190] The polynucleotides encoding the engineered dsRNA ligase polypeptides described herein can be expressed by inserting the polynucleotides or nucleic acid constructs comprising the polynucleotide sequences into appropriate expression vectors. When generating an expression vector, the coding sequence is located in the vector so that the coding sequence is connected to a suitable expression control sequence. The recombinant expression vector can be any vector (e.g., plasmid or virus) that can be easily used for recombinant DNA procedures and can result in the expression of the polynucleotide sequences. The selection of the vector will generally depend on the compatibility of the vector with the host cell to be introduced into the vector. The vector can be a linear or closed circular plasmid. The expression vector can be an autonomously replicating vector, i.e., a vector existing as an extrachromosomal entity, which replicates independently of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome or an artificial chromosome. The vector can include any tool for ensuring self-replication. Alternatively, the vector can be a vector that can be integrated into the genome when introduced into the host cell and replicated together with the chromosome to which it is integrated. In addition, a single vector or plasmid or two or more vectors or plasmids (these vectors or plasmids together comprise the total DNA to be introduced into the genome of the host cell) can be used.

[0191] Many expression vectors that can be used in embodiments of the present disclosure are commercially available. Exemplary expression vectors can be prepared by inserting a polynucleotide encoding an engineered dsRNA ligase polypeptide into a plasmid pACYC-Duet-1 (Novagen), a pBR322 vector (New England Biolabs), a pUC19 vector (New England Biolabs), or a pET T7 expression vector (Novagen).

[0192] The present disclosure also provides host cells capable of expressing the engineered dsRNA ligase polypeptides described herein. In some embodiments, the host cell comprises a nucleic acid molecule as described herein or a vector as described herein. In some embodiments, the host cell is Escherichia coli.

[0193] In some embodiments, the polynucleotide encoding the polypeptide is connected to one or more control sequences for expressing the polypeptide in a host cell. Host cells for expressing the polypeptide encoded by the expression vector disclosed herein are well known in the art, including but not limited to bacterial cells, such as Escherichia coli, Streptomyces and Salmonella typhimurium; fungal cells (such as Saccharomyces cerevisiae or Pichia pastoris); insect cells, such as Drosophila S2 and Spodoptera litura Sf9; animal cells, such as CHO, COS, BHK, 293 and Bowes melanoma cells; and plant cells. An exemplary host cell is Escherichia coli BL21 (DE3). The host cell can be wild type or can be engineered by genome editing. Culture media and growth conditions suitable for the above-mentioned host cells are well known in the art.

[0194] Polynucleotides or vectors for expressing polypeptides can be introduced into cells by various methods known in the art. Techniques include 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.

[0195] Host cells can be used to express and isolate the polypeptides described herein.

[0196] Process for producing engineered dsRNA ligase polypeptides

[0197] Engineered dsRNA ligases can be obtained by mutagenesis and / or directed evolution of polynucleotides encoding dsRNA ligases. Exemplary directed evolution techniques can be found in "Biocatalysis for the Pharmaceutical Industry: Discovery, Development, and Manufacturing" (2009 John Wiley & Sons Asia (Pte) Ltd. ISBN: 978-0-470-82314-9).

[0198] When the sequence of engineered polypeptide is known, encoding polynucleotide can be prepared by standard solid phase method according to known synthetic method.In certain embodiments, the fragment of about 100 bases at most can be synthesized separately, then connected (for example, by enzyme or chemical connection method or polymerase-mediated method) to form any desired continuous sequence.For example, the polynucleotide of present disclosure and oligonucleotide can use such as Beaucage et al., 1981, Tet Lett [tetrahedron express] 22:1859-69 or Matthes et al., 1984, EMBO J. [European Molecular Biology Association magazine] 3:801-05 description classical phosphoramidite method is prepared by chemical synthesis, as typically practiced in automatic synthesis method.According to phosphoramidite method, oligonucleotide is synthesized, purified, annealed, connected and cloned into suitable vector in such as DNA automatic synthesizer.In addition, substantially any nucleic acid can be obtained from any one of multiple commercial sources.

[0199] The present disclosure provides a method for preparing an engineered dsRNA ligase polypeptide, the method comprising culturing a host cell as described herein and obtaining an engineered dsRNA ligase polypeptide from the culture. In some embodiments, the method for preparing the polypeptide further comprises isolating the polypeptide. The engineered polypeptide can 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 protein purification techniques, including lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography.

[0200] The present invention also provides an engineered dsRNA ligase catalyst obtainable by culturing the host cells described herein or according to the method for preparing the engineered dsRNA ligase polypeptide described herein, wherein the engineered dsRNA ligase catalyst comprises cells or culture fluid containing the engineered dsRNA ligase polypeptide, or an article processed 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 engineered dsRNA ligase from the extract, or an immobilized product obtained by immobilizing the host cells, an extract thereof, or an isolated product of the extract.

[0201] Ligation reaction

[0202] The present disclosure provides a method of producing an oligonucleotide from two or more oligonucleotide fragments, wherein the method comprises contacting: (i) two or more oligonucleotide fragments; (ii) an engineered dsRNA ligase polypeptide disclosed herein; (iii) a source of ATP; and (iv) a divalent cation; to obtain the oligonucleotide.

[0203] Oligonucleotide products and fragments

[0204] The method of the present invention produces oligonucleotide by connecting two or more oligonucleotide fragments.The oligonucleotide produced (also referred to herein as " oligonucleotide product ") is a nucleic acid typically comprising up to 100 nucleotides.It should be understood that oligonucleotide as herein described comprises RNA.It should also be understood that oligonucleotide as herein described comprises double-stranded region.

[0205] As used herein, "oligonucleotide fragment" refers to a nucleic acid that can be ligated to one or more additional oligonucleotide fragments to provide an oligonucleotide product. Each oligonucleotide fragment corresponds to a portion of the oligonucleotide product.

[0206] 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.

[0207] 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.

[0208] In some embodiments, the oligonucleotide is at most 20 nucleotides in length. In some embodiments, the oligonucleotide is at most 25, at most 30, at most 35, at most 40, at most 45, at most 50, at most 55, at most 60, at most 65, at most 70, at most 75, at most 80, at most 85, at most 90, at most 95, or at most 100 nucleotides in length. In some embodiments, the oligonucleotide is at most 60 nucleotides in length.

[0209] In some embodiments, the oligonucleotide is at least 20 nucleotides in length. In 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.

[0210] In certain embodiments, the oligonucleotide is 10-100 nucleotides in length. In certain embodiments, the oligonucleotide is 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-25, 15-80, 15-70, 15-60, 15-50, 15-40, 15-30 or 15-25 nucleotides in length. In certain embodiments, the oligonucleotide is 15-25 nucleotides in length.

[0211] As used herein, the two or more oligonucleotide fragments comprise one or more 3' oligonucleotide fragments and one or more 5' oligonucleotide fragments, wherein each of the one or more 3' oligonucleotide fragments comprises a 5'-phosphate group and each of the one or more 5'-phosphate groups typically comprises a 3'-terminal ribonucleotide having a 3'-hydroxyl group.

[0212] In some embodiments, one or more oligonucleotide fragments comprise one or more mismatches. In some embodiments, one or more oligonucleotide fragments comprise overhangs. In some embodiments, one or more oligonucleotide fragments comprise 3' overhangs. In some embodiments, one or more oligonucleotide fragments comprise 5' overhangs. In some embodiments, one or more oligonucleotide fragments comprise 3' overhangs and 5' overhangs. In some embodiments, the overhangs comprise 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides.

[0213] In some embodiments, the two or more oligonucleotide fragments comprise a first oligonucleotide fragment having an overhang that is complementary to an overhang of a second oligonucleotide fragment. In some embodiments, the two or more oligonucleotide fragments comprise a first oligonucleotide fragment having a 3' overhang and a 5' overhang, wherein 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.

[0214] In some embodiments, one or more oligonucleotide fragments comprise blunt ends. In some embodiments, one or more oligonucleotide fragments comprise a 3' overhang and a 5' blunt end. In some embodiments, one or more oligonucleotide fragments comprise a 5' overhang and a 3' blunt end. In some embodiments, the 5' end of the oligonucleotide fragment comprises a 3' overhang and a 5' blunt end. In some embodiments, the 3' end of the oligonucleotide fragment comprises a 5' overhang and a 3' blunt end.

[0215] 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.

[0216] In some embodiments, one or more oligonucleotide fragments comprise DNA and RNA. For example, a portion of the oligonucleotide fragment can be double-stranded DNA, while another portion can be double-stranded RNA, forming a DNA-RNA chimera.

[0217] In some embodiments, one or more 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 non-limiting examples, one or both strands can be, for example, RNA (except that one or more nucleotides are 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.).

[0218] In certain embodiments, the length of two or more oligonucleotide fragments is the same.In certain embodiments, the length of two or more oligonucleotide fragments is different.In certain embodiments, the length of each in two or more oligonucleotide fragments is 3-20 nucleotides.In certain embodiments, the length of each in two or more oligonucleotide fragments is 4-16 nucleotides.In certain embodiments, the length of each in two or more oligonucleotide fragments is 4-16,4-15,5-15,6-15,4-14,4-13,4-12,4-11,4-10,4-9,5-9 or 6-9 nucleotides.

[0219] In some embodiments, each of the two or more oligonucleotide fragments is at least 3 nucleotides in length. In 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.

[0220] In some embodiments, the two or more oligonucleotide fragments comprise 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 oligonucleotide fragments.

[0221] In some embodiments, one or more ligations are required to generate oligonucleotide products. In 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 ligations are required to generate oligonucleotide products.

[0222] In some embodiments, one or more oligonucleotide fragments and / or oligonucleotides include chemical modifications. In some embodiments, one or more oligonucleotide fragments and / or oligonucleotides include at least one modified backbone modification. In some embodiments, one or more oligonucleotide fragments and / or oligonucleotides include at least one modified nucleotide modification. In some embodiments, one or more oligonucleotide fragments and / or oligonucleotides include at least one sugar modification (e.g., at the 2' position or the 4' position). In some embodiments, one or more oligonucleotide fragments and / or oligonucleotides include: (i) at least one modified backbone modification; (ii) and at least one modified nucleotide modification; and / or (iii) at least one sugar modification.

[0223] In some embodiments, one or more 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 ... In some embodiments, the oligonucleotide fragments and / or oligonucleotides comprise a 2'-modification selected from the group consisting of 2'-OMe, 2'-F, and 2'-deoxy.

[0224] In some embodiments, one or more oligonucleotide fragments and / or oligonucleotides comprise at least one phosphorothioate or methylphosphonate internucleotide linkage. In some embodiments, the oligonucleotide comprises at least one chiral phosphorothioate linkage.

[0225] In some embodiments, one or more oligonucleotide fragments and / or oligonucleotides are conjugated to at least one ligand. The ligand can be conjugated to the sense strand, the antisense strand, or both strands in any configuration (e.g., 3'-end, 5'-end, non-terminal, or a combination).

[0226] 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 for targeting the liver, where it binds to asialoglycoprotein receptors on hepatocytes. In some embodiments, the ligand comprises one or more GalNAc derivatives conjugated to a bivalent or trivalent branched carrier. In some embodiments, the ligand is a peptide or peptidomimetic.

[0227] 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.

[0228] 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.

[0229] In some embodiments, the oligonucleotide is a 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 a RNAi agent wherein the sense strand is conjugated to one or more GalNAc ligands. In some embodiments, the one or more oligonucleotide fragments comprise 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 one or more oligonucleotide fragments are dsRNA wherein the sense strand is conjugated to one or more GalNAc ligands.

[0230] In some embodiments, the methods are performed using 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 methods are performed using 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 methods are performed using equimolar amounts of each of two or more oligonucleotide fragments.

[0231] In some embodiments, the method produces at least 15 g of oligonucleotide product per liter of reaction mixture. In 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.

[0232] Engineered dsRNA ligase polypeptide

[0233] The methods are performed using the engineered dsRNA ligases described herein.

[0234] In some embodiments, the methods are performed using about 1 g / L of the engineered dsRNA ligase polypeptide, 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, 1.85 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 the engineered dsRNA ligase polypeptide.

[0235] ATP sources

[0236] The enzymatic activity of dsRNA ligase requires ATP as a cofactor. Each ligation reaction converts one ATP molecule into AMP. The catalytic mechanism of dsRNA ligase and the role of ATP in nucleic acid ligation reactions are described above.

[0237] In certain embodiments, the source of ATP is ATP. In certain embodiments, the method is carried out using a stoichiometric concentration of ATP. In certain embodiments, the method is carried out using a stoichiometric excess of ATP. Technicians can easily determine the stoichiometric concentration of ATP required for a given connection based on the concentration of the oligonucleotide fragments and the quantity of the ligation reaction required for the production of the oligonucleotide product.

[0238] In some embodiments, the methods are performed using 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 ATP and / or AMP concentration.

[0239] In some embodiments, the ATP source is an ATP regeneration system. In some embodiments, the ATP regeneration system includes: (a) polyphosphate kinase (PPK); (b) polyphosphate; and (c) AMP and / or ATP. Advantageously, the use of the ATP regeneration system overcomes the requirement to use high concentrations of ATP to achieve complete ligation. The ATP regeneration system described herein includes PPK and polyphosphate. PPK uses polyphosphate as a phosphate donor to generate ATP from AMP. The ATP converted to AMP during the ligation reaction can be regenerated into ATP by PPK and used as a cofactor in subsequent ligation reactions. This ATP cycle avoids the need to use high ATP concentrations in the initial reaction. Instead, substoichiometric concentrations of ATP and / or the use of a cheaper alternative, AMP, can be used for the reaction.

[0240] "Polyphosphate kinases" or "PPKs" are a family of enzymes that catalyze the formation of ATP from AMP and polyphosphate.

[0241] In some embodiments, the PPK is PPK 12. In some embodiments, the PPK comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 670 (amino acid sequence of PPK 12). 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.

[0242] In some embodiments, the PPK comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO: 671 (the amino acid sequence of optimized 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: 671.

[0243] 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 (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.

[0244] In some embodiments, PPK is used in the form of whole cells, crude extracts (e.g., cell-free lyophilized extracts or cell lysates), isolated polypeptides, or purified polypeptides. In some embodiments, the PPK polypeptide is used in an immobilized form as described herein, e.g., immobilized on a resin.

[0245] In some embodiments, the methods are performed using about 1 g / L PPK, 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, 1.85 g / 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 / LPPK.

[0246] In some embodiments, the polyphosphoric acid is a polyphosphate salt. In some embodiments, the polyphosphate salt is sodium polyphosphate (Madrell's salt) or sodium hexametaphosphate (Graham's salt).

[0247] In some embodiments, the methods are performed using a stoichiometric excess of polyphosphoric acid. In some embodiments, the methods are performed using a polyphosphoric acid 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.

[0248] In some embodiments, wherein the method is performed in the presence of PPK and polyphosphate, the method is performed in the presence of AMP. In some embodiments, wherein the method is performed in the presence of PPK and polyphosphate, the method is performed using substoichiometric concentrations of ATP and / or AMP.

[0249] Divalent cations

[0250] The enzymatic activity of the dsRNA ligase requires the presence of divalent cations. The enzymatic activity of the PPK requires the presence of divalent cations. In some embodiments, the divalent cations comprise Mg 2+ and / or Mn 2+ .

[0251] In some embodiments, the methods are performed using 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 methods are performed using 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.

[0252] In certain embodiments, method further comprises purification of oligonucleotide product from reaction mixture.In certain embodiments, oligonucleotide product is at least 80% pure, optionally wherein oligonucleotide product is at least 85%, at least 90%, at least 95% pure, optionally wherein oligonucleotide product is at least 98% pure, optionally wherein oligonucleotide product is at least 99% pure, optionally wherein oligonucleotide product is at least 99.5% pure, optionally wherein oligonucleotide product is at least 99.9% pure.Pure oligonucleotide product does not comprise the by product that oligonucleotide fragment, intermediate connect product or non-specific connection produce.Oligonucleotide product can use any method known in the art to purify or separate, for example, use gel extraction or use cellulose-based matrix.

[0253] The present disclosure also provides the oligonucleotide produced by the method described herein. The oligonucleotide can be in any suitable buffer. In certain embodiments, the buffer is selected from Tris buffer (such as Tris-HCl), phosphate buffer, HEPES, MOPS (3-(N-morpholino) propanesulfonic acid) and triethanolamine (TEOA) buffer. In certain embodiments, the buffer comprises acetate, citrate, prolamin, carbonate or phosphate or any combination thereof. In certain embodiments, the buffer further comprises a reagent for controlling the osmotic pressure of the solution so that the osmotic pressure remains at a desired value, such as the physiological value of human plasma. The solute that can be added to the buffer to control osmotic pressure includes but is not limited to protein, peptide, amino acid, non-metabolizable polymer, vitamin, ion, sugar, metabolite, organic acid, lipid or salt. In certain embodiments, the reagent for controlling the osmotic pressure of the solution is salt. In certain embodiments, the reagent for controlling the osmotic pressure of the solution is sodium chloride or potassium chloride.

[0254] Reaction conditions

[0255] As disclosed herein and illustrated in the Examples, the present disclosure contemplates a range of suitable reaction conditions that can be used in the methods described herein, including but not limited to pH, temperature, buffer, substrate loading, enzyme loading, cofactor loading, pressure, and reaction time. Additional suitable reaction conditions for the ligation reactions described herein can be readily optimized by routine experimentation, e.g., performing the methods described herein under experimental reaction conditions of varying reagent concentrations, pH, and temperature, and detecting the rate of oligonucleotide product formation.

[0256] In any embodiment of the process disclosed herein, reaction conditions may include suitable pH. As described above, desired pH or desired pH range can be maintained by using an acid or base, a suitable buffer or a combination of a buffer and an added acid or base. The pH of the reaction mixture can be controlled before and / or during the reaction. In certain 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 certain embodiments, reaction conditions include a solution pH of about 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8.

[0257] In any embodiment of the method disclosed herein, suitable temperature can be used for reaction conditions, considering, for example, the increase of reaction rate at higher temperatures, the activity of enzyme in sufficient reaction duration.Therefore, in some embodiments, suitable reaction conditions include a temperature of about 10 ℃ to about 60 ℃, about 10 ℃ to about 50 ℃, about 25 ℃ to about 50 ℃, about 25 ℃ to about 40 ℃, about 25 ℃ to about 30 ℃ or about 10 ℃ to about 30 ℃.In certain embodiments, suitable reaction temperature includes a temperature of about 10 ℃, 15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ or 60 ℃.In certain embodiments, the temperature during the enzymatic reaction can be maintained at a certain temperature during the entire reaction process.In certain embodiments, the temperature during the enzymatic reaction can be regulated with a certain temperature spectrum during the reaction process.

[0258] The reaction can be carried out in any suitable buffer solution. In some embodiments, the buffer 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 comprises acetate, citrate, prolamin, carbonate or phosphate, or any combination thereof. In some embodiments, the buffer is phosphate buffered saline (PBS).

[0259] In some embodiments, the reaction mixture further comprises a reducing agent, optionally DTT (dithiothreitol).

[0260] When performing the ligation reaction described herein, the engineered dsRNA ligase polypeptide can be added to the reaction mixture in various formulations, such as frozen or lyophilized whole cells (FWC or LWC) transformed with a gene encoding the engineered dsRNA ligase polypeptide and / or cell lysates or lysed cell lysates of such cells, i.e., so-called shake flask powders (SFP), wherein cell debris is removed and / or further purified to form a fermentation powder (FP). Whole cells transformed with a gene encoding the engineered dsRNA ligase polypeptide, or cell extracts, lysates, and isolated enzymes thereof can be used in a variety of different forms, including solids (e.g., freeze-dried, 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.) prior to lyophilization, followed by desalting procedures (e.g., ultrafiltration, dialysis, etc.). Any enzyme preparation can be immobilized on a solid phase material (e.g., a resin).

[0261] In any of the embodiments of the processes disclosed herein, wherein the engineered polypeptide is expressed as a secreted polypeptide, culture medium containing the secreted polypeptide can be used in the processes herein.

[0262] In any embodiment of the process disclosed herein, solid reactants (e.g., enzymes, salts, etc.) can be provided to the reaction in a variety of different forms, including powders (e.g., freeze-dried, spray-dried, etc.), solutions, emulsions, suspensions, etc. The reactants can be readily freeze-dried or spray-dried using methods and instruments known to those skilled in the art. For example, the protein solution can be frozen in small aliquots at -80°C and then added to a pre-freeze-drying chamber, and then vacuum is applied.

[0263] In any embodiment of the process disclosed herein, the order of addition of the reactants is not critical. The reactants can be added to the solvent together at the same time, or alternatively, some reactants can be added separately and some reactants can be added together at different time points.

[0264] The method for carrying out ligation reaction can comprise the further step of separating the oligonucleotide product of enzymatic reaction.Especially, this step is typically carried out after enzymatic reaction is completed.Especially, oligonucleotide is typically separated with one or more (particularly substantially all) other components of reaction mixture.For example, oligonucleotide is typically separated with remaining substrate, by product and / or enzyme.The separation of oligonucleotide can be realized by means known in the art and technology, for example, by separating based on the size of oligonucleotide, as by gel electrophoresis and gel extraction or using cellulose-based matrix.In certain embodiments, method further comprises by ultrafiltration and chromatography purification oligonucleotide.

[0265] Modification

[0266] In certain embodiments, oligonucleotide fragments and / or oligonucleotide comprise modification, for example chemical modification.As used herein, term " oligonucleotide fragment " means one or more oligonucleotide fragments.Should be understood that the modification existing in the oligonucleotide fragment is typically present in the oligonucleotide produced by the oligonucleotide fragment.In certain embodiments, in the oligonucleotide product, introduce modification and / or remove modification from the oligonucleotide product.

[0267] In certain embodiments, oligonucleotide fragments and / or oligonucleotides include chemical modifications. In certain embodiments, oligonucleotide fragments and / or oligonucleotides include at least one backbone modification. In certain embodiments, oligonucleotide fragments and / or oligonucleotides include at least one nucleotide modification. In certain embodiments, oligonucleotide fragments and / or oligonucleotides include at least one sugar modification (e.g., at the 2'-position or the 4'-position). In certain embodiments, oligonucleotide fragments and / or oligonucleotides include: (i) at least one backbone modification; (ii) at least one nucleotide modification; and / or (iii) at least one sugar modification.

[0268] Modifications include, but are not limited to, terminal modifications of the terminal oligonucleotide fragments, for example, 5'-terminal modifications (phosphorylation, conjugation, inverted linkages) or 3'-terminal modifications (conjugation, inverted linkages, etc.); base modifications, such as replacement with stable bases, unstable bases, or bases that base pair with an extended partner library, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2'-position or the 4'-position) or sugar replacements; or backbone modifications, including modification or replacement of phosphodiester bonds.

[0269] In some embodiments, the terminal oligonucleotide fragment and / or oligonucleotide comprises a cap. The term "cap" etc. includes a chemical moiety attached to the end of a double-stranded nucleotide duplex, but is used herein to exclude a chemical moiety that is a nucleotide or nucleoside. A "3' cap" is attached to the 3' end of a nucleotide or oligonucleotide and protects the molecule from being degraded by, for example, nucleases (such as those in serum or intestinal fluid). A non-nucleotide 3' cap is not a nucleotide and can replace the TT or UU dinucleotide at the end of a blunt-ended oligonucleotide. In some embodiments, a non-nucleotide 3' end cap is as disclosed, for example, in WO 2005 / 021749 and WO 2007 / 128477; and U.S. Patent No. 8,097,716; U.S. Patent No. 8,084,600; and U.S. Patent No. 8,344,128. A "5' cap" is attached to the 5' end of a nucleotide or oligonucleotide. The cap should not interfere with (or excessively interfere with) oligonucleotide activity.

[0270] In certain embodiments, oligonucleotide fragments and / or oligonucleotides include one or more mispairings. Mispairing is defined herein as the difference between base sequence or length when two sequences are compared and compared to the greatest extent. In the context of double-stranded oligonucleotides (wherein two sequences are antiparallel to each other), mispairing is defined as the position where the base of a sequence is not complementary to the base of another sequence. Therefore, for example, when the first and second sequences are antiparallel to each other, if a certain position in the first sequence has a specific base (for example, A), and the base at the corresponding position in the second sequence is not complementary to the base in the first sequence (for example, G), it is counted as mispairing. However, it should be noted that on a given RNA chain, U can be replaced by T (as RNA or preferably DNA, such as 2'-deoxythymidine); Replacing U with T is not a mispairing as used herein, because U or T can be paired with the A on the opposite chain. Therefore, RNA oligonucleotides can include one or more DNA bases, such as T. If base pairing occurs, it is not counted as a mismatch between the DNA portion of the RNAi agent and the corresponding target mRNA (e.g., between an A, G, C, or T in the DNA portion and the corresponding U, C, G, or A in the mRNA).

[0271] For example, if a position in one sequence has a base (e.g., A) and the corresponding position in the other sequence does not have a base (e.g., that position is an abasic nucleotide, which contains a phosphate-sugar backbone but no base), it is also counted as a mismatch. Single-stranded nicks in either sequence (or sense or antisense strand) are not counted as mismatches. Thus, as a non-limiting example, if a sequence (in the 5'→3' direction) contains the sequence AG, but the complementary sequence (in the 3'→5' direction) contains the sequence TC with a single-stranded nick between T and C, it is not counted as a mismatch. Nucleotide modifications in sugars or phosphates are also not considered mismatches. Thus, if a sequence contains G and the complementary sequence contains a modified C (e.g., 2'-modification) at the same position, it is not counted as a mismatch.

[0272] Therefore, if the sugar, phosphate or backbone of the oligonucleotide is modified without modifying the base, it is not counted as a mismatch. Thus, in the context of double-stranded RNAi, there are zero mismatches between a strand of a given sequence that is RNA and its complementary sequence that is a PNA; or a morpholino; or an LNA; or a TNA; or a GNA; or a FANA; or a mixture or chimera of RNA and DNA, TNA, GNA, FANA, morpholino, UNA, LNA and / or PNA, etc. Mismatches do not occur between T nucleotides and A nucleotides that have 5' modifications and / or 2' modifications. The main characteristic of a mismatch (base substitution) is that it cannot base pair with the corresponding base on the opposite strand. In addition, terminal overhangs such as "UU" or "dTdT" are not counted when calculating the number of mismatches. In this case, a mismatch is defined as a position where a base of one sequence does not match a base of the other sequence.

[0273] It should be noted that dTdT (2'-deoxy-thymidine-5'-phosphate and 2'-deoxy-thymidine-5'-phosphate), or in some cases, TT or UU, can be added to one or both 3'-ends of the oligonucleotide as a terminal dinucleotide cap or extension, but such caps or extensions are not included in the calculation of the total number of mismatches and are not considered part of the target sequence. This is because the terminal dinucleotide can protect the termini 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).

[0274] There are multiple examples in the art describing sugars, bases, phosphates and backbone modifications that can be introduced into nucleic acid molecules and significantly enhance their nuclease stability and effectiveness. For example, oligonucleotides are modified with nuclease-resistant groups (e.g., 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-O-allyl, 2'-H, nucleotide base modifications) to enhance stability and / or enhance biological activity. Sugar modifications of nucleic acid molecules are widely described in the art.

[0275] Additional modifications and conjugations of oligonucleotides have also been described. Soutschek et al. (2004 Nature 432:173-178) proposed conjugating cholesterol to the 3'-end of the sense strand of an siRNA molecule via a pyrrolidine linker, thereby generating a covalent and irreversible conjugate. Oligonucleotides can also be chemically modified (including conjugation to other molecules) to improve their pharmacokinetic retention and efficiency in vivo.

[0276] In some embodiments, the oligonucleotide fragments and / or oligonucleotides comprise modified bases. The present disclosure encompasses an oligonucleotide and oligonucleotide fragments in which a single nucleotide at a given position is replaced by a modified version of the same nucleotide. Thus, a nucleotide (A, G, C, or U) can be replaced by a modified base selected from the group consisting of 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylquinoline, inosine, N6-isopentenyl adenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyl Adenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-mannosylquinoside, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-thioguanosine, 5-thioguanosine, 2-thioguanos ... -methyl-2-thiouracil, 2-thiouracil, 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—C H3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azouracil, cytosine and thymine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines, 5-halo, especially 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.

[0277] Additional modified variants include the addition of any other moiety (eg, a radiolabel or other tag or conjugate) to the oligonucleotide or oligonucleotide fragment; if the base sequence is identical, the addition of the other moiety will result in a "modified variant" (no mismatches).

[0278] In addition to these modifications and patterns (e.g., forms) for modification, other modifications or sets of modifications of the provided sequences can also be generated using general knowledge of nucleic acid modification. These various embodiments and embodiments of the oligonucleotides disclosed herein can be used for RNA interference.

[0279] In some embodiments, an oligonucleotide and / or oligonucleotide fragment comprises a modification that confers increased stability to the oligonucleotide in a biological sample or environment (eg, cytoplasm, interstitial fluid, serum, lung or intestinal lavage fluid).

[0280] In certain embodiments, oligonucleotides and / or oligonucleotide fragments include modifications (i.e., "RISC cleavage sites") that promote cutting by RNA-induced silencing complexes. RISC cleavage sites are sites where cutting occurs on a target. In certain embodiments, the antisense strand includes a RISC cleavage site. For RNAi agents with a duplex region of 17-23 nucleotides in length, the cleavage site of the antisense strand is typically located near positions 10, 11, and 12 from the 5'-end. As used herein, the term "cleavage region" refers to the region adjacent to the cleavage site. In certain embodiments, the cleavage region includes three bases located at either end of the cleavage site and adjacent to the cleavage site. In certain embodiments, the cleavage region includes two bases located at either end of the cleavage site and adjacent to the cleavage site. In certain embodiments, the cleavage site particularly occurs at the site where nucleotides 10 and 11 of the antisense strand are combined, and the cleavage region includes nucleotides 11, 12, and 13 of the antisense strand.

[0281] In certain embodiments, oligonucleotide fragments and / or oligonucleotides include modified backbones. As used herein, unmodified backbones are composed of 3' to 5' phosphodiester bonds. Modified backbones may include non-natural internucleoside bonds. Oligonucleotides with modified backbones include those that retain phosphorus atoms in the backbone and those that do not have phosphorus atoms in the backbone.

[0282] The oligonucleotide fragment comprising the modified main chain includes but is not limited to those not having phosphorus atoms in the main chain.The modified main chain includes but is not limited to phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates (for example, 3'-alkylene phosphonates and chiral phosphonates), phosphinate, amido phosphate (for example, amido phosphate methanesulfonyl ester, 3'-aminoamido phosphate and aminoalkylamido phosphate), thioamido phosphate, thioalkylphosphonates, thioalkylphosphotriester and the borane phosphate with normal 3'-5' key, these 2'-5' connection analogs and those with inverted polarity, wherein adjacent nucleoside unit pairs are 3'-5' to 5'-3' or 2'-5' to 5'-2' connection.

[0283] Oligonucleotide fragments comprising modified backbones in which no phosphorus atoms are present may have backbones formed from 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 backbones having morpholino linkages (formed in part by the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methyleneformacetyl and thioformacetyl backbones; backbones containing olefins; sulfamate backbones; methyleneimino and methylenehydrazine backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S, and CH2 components.

[0284] In some embodiments, the oligonucleotide and / or oligonucleotide fragment comprises at least one phosphonate bond, wherein the phosphonate is a modified phosphonate selected from the group consisting of: phosphorothioate (which can be either the Rp isomer or the Sp isomer):

[0285]

[0286] Phosphorodithioates:

[0287]

[0288] Methylphosphonate:

[0289]

[0290] Methoxypropylphosphonate:

[0291]

[0292] 5'-(E)-vinylphosphonate:

[0293]

[0294] 5'-Methylphosphonate:

[0295]

[0296] (S)-5'-C-Methyl with phosphonate:

[0297]

[0298] 5'-phosphorothioate;

[0299]

[0300] and peptide nucleic acids:

[0301]

[0302] In some embodiments, the oligonucleotides and / or oligonucleotide fragments comprise: 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 degradation by serum nucleases (e.g., RNase A). Chemical modification at the 2'-position of the first pyrimidine nucleotide in the motif can prevent or slow such cleavage. This modification scheme is also known by the term "endo light."

[0303] In some embodiments, the oligonucleotide and / or oligonucleotide fragment comprises a modified nucleobase, wherein the modified nucleobase is difluorotolyl, nitroindolyl, nitropyrrolyl, or nitroimidazolyl. In specific embodiments, the modified nucleobase is difluorotolyl. In some embodiments, wherein the oligonucleotide and / or oligonucleotide fragment is double-stranded, only one of the two strands contains a modified nucleobase. In some embodiments, wherein the oligonucleotide and / or oligonucleotide fragment is double-stranded, both strands contain a modified nucleobase.

[0304] 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, wherein the alkyl, alkenyl, and alkynyl groups can be substituted or unsubstituted C1 to C 10 Alkyl or C2 to C 10 Alkenyl and alkynyl. Exemplary modifications include O[(CH2) 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, wherein n and m are 1 to about 10. The oligonucleotide fragment used in the methods described herein may comprise one of the following at the 2' position: C1 to C 10Lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of therapeutic RNA, or group for improving the pharmacodynamic properties of therapeutic RNA. In some embodiments, the modification comprises 2'-methoxyethoxy (also known as 2'-O-(2-methoxyethyl) or 2'-O-MOE), 2'-dimethylaminooxyethoxy (also known as 2'-DMAOE) 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).

[0305] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropyloxy (2'-OCH2CH2CH2NH2), 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 2'-5' linked dsRNA and the 5' position of the 5' terminal nucleotide.

[0306] In some embodiments, the oligonucleotide fragments and / or oligonucleotides comprise 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-MP ... In some embodiments, the oligonucleotides and / or oligonucleotide fragments comprise 2'-O-DMAEOE), 2'-ON-methylacetamido (2'-O-NMA), locked nucleic acids (LNA), glycol nucleic acids (GNA), amidophosphates (e.g., amidophosphomethanesulfonyl esters), 2',3'-split nucleotide mimetics, 2'-F-arabinonucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, vinylphosphonates (e.g., 5' vinylphosphonate), and cyclopropylphosphonate deoxyribonucleotides. In some embodiments, the one or more oligonucleotide fragments comprise a 2'-modification selected from the group consisting of 2'-Ome, 2'-F, and 2'-deoxy. In some embodiments, the oligonucleotides and / or oligonucleotide fragments comprise one or more 3'-O-methyl nucleotides.

[0307] In some embodiments, the oligonucleotide and / or oligonucleotide fragment comprises 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- Dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamido (2'-O-NMA), locked nucleic acid (LNA), amidophosphate (e.g., amidophosphate methylsulfonyl ester), 2',3'-split nucleotide mimics, 2'-F-arabinonucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, vinylphosphonates (e.g., 5' vinylphosphonate), deoxyribonucleotides, and cyclopropylphosphonates. In some embodiments, the oligonucleotides and / or oligonucleotide fragments comprise one or more 3'-O-methyl nucleotides.

[0308] In some embodiments, the oligonucleotide and / or oligonucleotide fragment comprises 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:

[0309]

[0310] In some embodiments, all pyrimidines (uridine and cytidine) are 2'O-methyl-modified nucleosides.

[0311] In some embodiments, the sense and / or antisense strand is conjugated to one or more diagnostic compounds, reporter groups, cross-linkers, nuclease resistance-conferring moieties, modified or unmodified nucleobases, lipophilic molecules, cholesterol, lipids, lectins, steroids, uvaol, agave sapogenin, diosgenin, terpenes, triterpenes, sarsasapogenin, aconitol, epiconitol-derived lithocholic acid, vitamins, carbohydrates, dextran, pullulan, chitin, chitosan, synthetic carbohydrates, oligolactic acid 15-mers, natural polymers, low or medium molecular weight polymers, inulin, cyclodextrin, hyaluronic acid, proteins, protein-binding agents, integrin targeting molecules, polycations, peptides, polyamines, peptidomimetics, and / or transferrin.

[0312] 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. 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' vinylphosphonate. In some embodiments, the sense strand comprises at least one chiral phosphorothioate bond. In some embodiments, the sense strand comprises at least one GNA.

[0313] In some embodiments, the sense strand comprises alternating 2'-OMe and 2'-F modified nucleotides over the entire length of the sense strand. In some embodiments, the sense strand comprises alternating 2'-OMe and 2'-F modified nucleotides over a portion of the length of the sense strand (e.g., 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).

[0314] In some embodiments, the antisense strand comprises alternating 2'-OMe and 2'-F modified nucleotides over the entire length of the antisense strand. In some embodiments, the antisense strand comprises alternating 2'-OMe and 2'-F modified nucleotides over a portion of the length of the antisense strand (e.g., 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).

[0315] In some embodiments, the sense strand and the antisense strand each comprise alternating 2'-OMe and 2'-F modified nucleotides over the full length of the sense strand and the antisense strand. In some embodiments, the sense strand and the antisense strand each comprise alternating 2'-OMe and 2'-F modified nucleotides over a portion of the length of the sense strand and the antisense strand (e.g., 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 and the antisense strand).

[0316] ligand

[0317] In some embodiments, one or more 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, antisense strand, or two strands in any configuration (e.g., 3'-end, 5'-end, non-end, or combination).

[0318] In some embodiments, the ligand comprises one or more N-acetylgalactosamine (GalNAc) derivatives. In some embodiments, the ligand comprises one or more GalNAc derivatives conjugated to a divalent or trivalent branched carrier.

[0319] In some embodiments, the ligand is:

[0320]

[0321] In some embodiments, the ligand is:

[0322]

[0323] In some embodiments, the ligand is:

[0324]

[0325] In some embodiments, the ligand is:

[0326]

[0327] In some embodiments, the ligand is:

[0328]

[0329] In some embodiments, the ligand is:

[0330]

[0331] 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, such as a cell or organ compartment, a tissue, an organ, or a body region, e.g., compared to a substance in the absence of such a ligand. Ligands that provide enhanced affinity for a selected target are also referred to as targeting ligands.

[0332] Some ligands may have endosomal lysis (endosomolytic) properties. Endosomal lysis ligands promote the dissolution of the endosomal and / or the transport of the oligonucleotide or the composition comprising the oligonucleotide from the endosome to the cytoplasm of the cell. Endosomal lysis ligands can be polyanionic peptides or peptidomimetics that exhibit pH-dependent membrane activity and fusogenicity. In certain embodiments, the endosomal lysis ligand exhibits its active conformation at endosomal pH. An "active" conformation is one in which the endosomal lysis ligand promotes the dissolution of the endosomal and / or the transport of the oligonucleotide or the composition comprising the oligonucleotide from the endosome to the cytoplasm of the cell. Exemplary endosomolytic ligands include GALA peptide (Subbarao et al., Biochemistry, 1987, 26: 2964-2972), EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586), and derivatives thereof (Turk et al., Biochem. Biophys. Acta, 2002, 1559: 56-68). Endosomolytic components may contain chemical groups (e.g., amino acids) that undergo charge changes or protonation in response to changes in pH. Endosomolytic components may be linear or branched.

[0333] Ligands can improve transport, hybridization, and specificity properties, and can also improve the nuclease resistance of the resulting native or modified oligonucleotide.

[0334] Ligands can typically include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups, e.g., for monitoring distribution; cross-linking agents; and nuclease resistance-conferring moieties. Common examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptidomimetics.

[0335] The ligand 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. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer (e.g., a synthetic polyamino acid), an oligonucleotide (e.g., an aptamer). Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly-(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly-(2-ethyl acrylic acid), N-isopropylacrylamide polymer or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha helical peptides.

[0336] The ligand can also include a targeting group that binds to a specified cell type, such as a cell or tissue targeting agent, such as a lectin, a glycoprotein, a lipid, or a protein (e.g., an antibody). The targeting group can be thyrotropin, melanocyte stimulating hormone, a lectin, a glycoprotein, surfactant protein A, a mucin carbohydrate, a multivalent lactose, a multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, a multivalent fucose, a glycosylated polyamino acid, a multivalent galactose, transferrin, a bisphosphonate, a polyglutamate, a polyaspartate, a lipid, cholesterol, a steroid, a bile acid, folic acid, vitamin B12, biotin, an RGD peptide, an RGD peptidomimetic, or an aptamer.

[0337] Other examples of ligands include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases or chelators (e.g., EDTA), lipophilic molecules (e.g., cholesterol, bile acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propylene glycol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl or phenoxazine) and peptide conjugates (e.g., Drosophila melanogaster antennapedia peptide, Tat peptide), alkylating agents, phosphates, amino groups, sulfhydryl groups, 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, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP.

[0338] The ligand can be a protein, such as a glycoprotein or peptide, such as a molecule with a specific affinity for the coligand, or an antibody, such as an antibody that binds to a specified cell type (such as a cancer cell, an endothelial cell, or a bone cell). The ligand can also include hormones and hormone receptors. They can also include non-peptide substances, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, or aptamers. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

[0339] In some embodiments, the ligand is a lipid or lipid-based molecule. Such lipid or lipid-based molecules preferably bind serum proteins, such as human serum albumin (HSA). HSA binding ligands allow the conjugate to be distributed to the target tissue. The lipid or lipid-based ligand can (a) increase resistance to conjugate degradation, (b) increase targeting or transport to target cells or cell membranes, and / or (c) can be used to regulate binding to serum proteins (such as HSA). Lipid-based ligands can be used to regulate, for example, control the binding of the conjugate to the target tissue.

[0340] In certain embodiments, the part is a peptide or peptidomimetic. Peptidomimetic is a molecule that can be folded into a definite three-dimensional structure similar to a natural peptide. Peptide or peptidomimetic portion can be about 5-50 amino acid long, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acid long. Peptide or peptidomimetic can be, for example, cell permeability peptides, cationic peptides, amphipathic peptides or hydrophobic peptides (for example, mainly composed of Tyr, Trp or Phe). Peptide portion can be a dendritic peptide, constrained peptide or cross-linked peptide. In another alternative, peptide portion can include a hydrophobic membrane translocation sequence (MTS). Peptide portion can be a "delivery" peptide, which can carry large polar molecules, including peptides, oligonucleotides and proteins, through the cell membrane. The peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage display library or a one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991).

[0341] As used herein, a "peptide portion" may range in length from about 5 amino acids to about 50 amino acids. The peptide portion may have structural modifications, for example to increase stability or direct conformational properties. Any structural modification as described below may be utilized. Arginine-glycine-aspartic acid (RGD)-peptide portions may be used to target tumor cells, such as endothelial tumor cells or breast cancer tumor cells (Zitzmann et al., Cancer Res. [Cancer Research], 62:5139-43, 2002). RGD peptides can facilitate oligonucleotide targeting of tumors in a variety of other tissues, including lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy [Cancer Gene Therapy] 8:783-787, 2001). RGD peptides may be linear or cyclic and may be modified, for example, by glycosylation or methylation, to facilitate targeting specific tissues. Peptides targeting markers enriched in proliferating cells may be used. For example, peptides and peptidomimetics containing RGD may target cancer cells, particularly cells that exhibit integrins. Thus, the ligand may comprise an RGD peptide, a cyclic peptide containing RGD, an RGD peptide containing a D-amino acid, or a synthetic RGD mimetic.

[0342] Peptide and peptidomimetic ligands include those having naturally occurring or modified peptides, such as D or L peptides; α, β or γ peptides; N-methyl peptides; azapeptides; peptides having one or more amide (i.e., peptide) bonds replaced with one or more urea, thiourea, carbamate or sulfonylurea bonds; or cyclic peptides.

[0343] Part can be at multiple positions (for example, 3 '-end, 5 '-end and / or in interior (" non-terminal ") position) place and oligonucleotide fragment and / or oligonucleotide coupling.In certain embodiments, part is connected by intervening tether (for example carrier as described herein).When monomer is incorporated in oligonucleotide fragment and / or oligonucleotide, part or the part of tethering can be present on monomer.In certain embodiments, part can be incorporated by being coupled with " precursor " monomer after " precursor " monomer has been incorporated in oligonucleotide fragment and / or oligonucleotide.For example, there is the monomer (for example TAP-(CH2)nNH2) of for example amino-terminated tether (that is, without relevant part) can be incorporated in the oligonucleotide fragment of growth.In subsequent operation, namely after precursor monomer is incorporated in oligonucleotide fragment, by coupling the electrophilic group of part and the terminal nucleophilic group of precursor monomer chain, the part with electrophilic group (for example pentafluorophenyl ester or aldehyde group) can be attached to precursor monomer subsequently.

[0344] In another example, monomers with chemical groups suitable for participating in click chemistry reactions (e.g., azide- or alkyne-terminated tethers / linkers) can be incorporated. In a subsequent operation, i.e., after the precursor monomers are incorporated into oligonucleotide fragments and / or oligonucleotides, ligands with complementary chemical groups (e.g., alkyne or azide) can be attached to the precursor monomers by coupling alkynes and azides together.

[0345] In certain embodiments, the ligand is conjugated to a core base, sugar moiety, or internucleoside bond of an oligonucleotide fragment and / or oligonucleotide. Conjugation to a purine core base or its derivatives can occur at any position, including endo- and exo-ring atoms. In certain embodiments, the 2-, 6-, 7-, or 8-position of a purine core base is attached to the conjugated moiety. Conjugation to a pyrimidine core base or its derivatives can also occur at any position. In certain embodiments, the 2-, 5-, and 6-positions of a pyrimidine core base can be replaced by the conjugated moiety. Conjugation to the sugar moiety of a nucleoside can occur at any carbon atom. Example carbon atoms of the sugar moiety that can be attached to the conjugated moiety include 2', 3', and 5' carbon atoms. The 1' position can also be attached to the conjugated moiety, for example, in abasic residues. The internucleoside bond can also carry the conjugated moiety. For phosphorus-containing linkages (e.g., phosphodiester, phosphorothioate (e.g., chiral phosphorothioate), phosphorodithioate, amidophosphoroate, etc.), the conjugated moiety can be attached directly to the phosphorus atom or to an O, N, or S atom bound to the phosphorus atom. For amine- or amide-containing internucleoside linkages (e.g., PNA), the conjugated moiety can be attached to the nitrogen atom of the amine or amide or to an adjacent carbon atom.

[0346] 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.

[0347] 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.

[0348] The ligand can be attached by a carrier. The carrier includes (i) at least one "main chain attachment point", preferably two "main chain attachment points" and (ii) at least one "tethered attachment point". As used herein, a "main chain attachment point" refers to a functional group, such as a hydroxyl group, or a key that is generally useful for and suitable for incorporating the carrier into a nucleic acid backbone (e.g., a phosphate or modified phosphate (e.g., sulfur-containing) backbone). In some embodiments, a "tethered attachment point" (TAP) refers to a constituent ring atom of the cyclic carrier connecting the selected portion, such as a carbon atom or a heteroatom (different from the atom providing the main chain attachment point). A portion can be, for example, a carbohydrate, such as a monosaccharide, a disaccharide, a trisaccharide, a tetrasaccharide, an oligosaccharide, and a polysaccharide. Optionally, the selected portion is connected to the cyclic carrier by an intervening tether. Therefore, the cyclic carrier generally includes a functional group, such as an amino group, or generally provides a key that is suitable for incorporating or tethering another chemical entity (e.g., a ligand) into the constituent ring.

[0349] Wherein, the oligonucleotide fragment is dsRNA, and the sense strand and / or antisense strand can be conjugated to the ligand through a carrier, wherein the carrier can 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, pyridazinyl, tetrahydrofuranyl and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.

[0350] In some embodiments, one or more oligonucleotide segments comprise the sequence "TT," "dTdT," "dTsdT," or "UU" as a single-stranded overhang at the 3' terminus, also referred to herein as a terminal dinucleotide or 3' terminal dinucleotide. dT is 2'-deoxythymidine-5'-phosphate, and sdT is 2'-deoxythymidine 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 and are often placed as overhangs to protect the ends of siRNAs from nucleases (see, e.g., 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.

[0351] In some embodiments, one or both terminal oligonucleotide segments comprise a 3' terminal cap in place of or in addition to the terminal dinucleotide to stabilize the termini against nuclease degradation, provided that the 3' terminal cap stabilizes the oligonucleotide (e.g., against nucleases) and does not unduly interfere with its desired activity.

[0352] Wherein, the oligonucleotide fragment is dsRNA, and the sense strand and / or antisense strand can be conjugated to the ligand through a carrier, wherein the carrier can 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, pyridazinyl, tetrahydrofuranyl and decalin; preferably, the acyclic group is selected from serinol backbone or diethanolamine backbone.

[0353] Other embodiments

[0354] Example 1. An engineered 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 NO: 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, 6, 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 6, 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;

[0355] The engineered dsRNA ligase polypeptide:

[0356] (a) having dsRNA ligase activity; and

[0357] (b) does not comprise the amino acid sequence of SEQ ID NO: 302.

[0358] Example 2. An engineered 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 NO: 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, 467 8, 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, 551 52, 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;

[0359] The engineered dsRNA ligase polypeptide:

[0360] (a) having dsRNA ligase activity; and

[0361] (b) does not comprise the amino acid sequence of SEQ ID NO: 302.

[0362] Embodiment 3. The engineered dsRNA ligase polypeptide of embodiment 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 370, 488, 526, 578, 588, 590 and 592.

[0363] Embodiment 4. The engineered dsRNA ligase polypeptide of embodiment 2, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 370, 488, 526, 578, 588, 590, 592, and 666.

[0364] Example 5. An engineered dsRNA ligase polypeptide comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 302, which produces at least 5% more oligonucleotide product under the same ligation reaction conditions than a dsRNA ligase polypeptide comprising the amino acid sequence of SEQ ID NO: 302, wherein the engineered dsRNA ligase polypeptide does not comprise the amino acid sequence of SEQ ID NO: 302.

[0365] Example 6. The engineered dsRNA ligase polypeptide of Example 5, wherein the ligation reaction conditions comprise about 1 μM to about 10 mM 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 engineered dsRNA ligase polypeptide, a pH of about 4.0 to about 8.0, and a temperature of about 10° C. to about 50° C.

[0366] Embodiment 7. The engineered dsRNA ligase polypeptide of embodiment 5 or 6, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues 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, 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, wherein the numbering refers to SEQ ID NO: 302.

[0367] Example 8. The engineered dsRNA ligase polypeptide of Example 7, wherein the amino acid sequence of the engineered 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; X64 is R, T, Q, F, G, or M; X66 is F or W; X67 is N; X87 is T, P, K, or not present; 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; 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; and X328 is R; wherein numbering refers to SEQ ID NO: 302.

[0368] 7, X87, X88, X89, X91, X92, X93, X103, X105, X107, X114, X122, X126, X129, X130, X131, X137, X144, X146, X158, X163, X164, X165, X166, X170, X171, X172, X173, X174, X175, X176, X177, X178, X179, X180, X181, X182, X183, X184, X185, X186, X187, X188, X189, X190, X191, X192, X193, X201, X202, X203, X204, X205, X206, X207, X208, X209, X210, X211, X212, X213, X214, X215, X216, X217 X305, X313, X314, X325, and X328, wherein the numbering refers to SEQ ID NO: 302.

[0369] Embodiment 10. The engineered dsRNA ligase polypeptide of embodiment 9, wherein the amino acid sequence of the engineered 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; 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 not present; 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; X 137 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; wherein numbering refers to SEQ ID NO: 302.

[0370] Embodiment 11. The engineered dsRNA ligase polypeptide of any one of embodiments 5-10, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of X15, X19, X36, X39, X53, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity.

[0371] Embodiment 12. The engineered dsRNA ligase polypeptide of embodiment 11, wherein the amino acid sequence of the engineered 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; wherein numbering refers to SEQ ID NO: 302.

[0372] Embodiment 13. The engineered dsRNA ligase polypeptide of any one of embodiments 5-12, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of X15, X19, X36, X39, X53, X185, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity.

[0373] Embodiment 14. The engineered dsRNA ligase polypeptide of embodiment 13, wherein the amino acid sequence of the engineered 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; wherein numbering refers to SEQ ID NO: 302.

[0374] Embodiment 15. The engineered dsRNA ligase polypeptide of any one of embodiments 5-14, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of: X36, X39, X218, and X221, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the amino acid sequence of the engineered 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.

[0375] Embodiment 16. The engineered dsRNA ligase polypeptide of any one of embodiments 5-15, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of: X39, X218, and X221, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises one or more of the following amino acid residues: X39 is A; X218 is N; and X221 is I.

[0376] Embodiment 17. The engineered dsRNA ligase polypeptide of any one of embodiments 5-16, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of: X39, X218, X221, and X255, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the amino acid sequence of the engineered 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.

[0377] Embodiment 18. The engineered dsRNA ligase polypeptide of any one of embodiments 5-17, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of: X39, X53, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the amino acid sequence of the engineered 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.

[0378] Embodiment 19. The engineered dsRNA ligase polypeptide of any one of embodiments 5-18, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of: X15, X39, X53, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the amino acid sequence of the engineered 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.

[0379] Embodiment 20. The engineered dsRNA ligase polypeptide of any one of embodiments 5-19, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of: X19, X39, X53, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the amino acid sequence of the engineered 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.

[0380] Embodiment 21. The engineered dsRNA ligase polypeptide of any one of embodiments 5-20, wherein the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of: X15, X39, X53, X185, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the amino acid sequence of the engineered 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.

[0381] Embodiment 22. The engineered dsRNA ligase polypeptide of any one of embodiments 1-21, wherein the engineered dsRNA ligase polypeptide comprises a purification tag.

[0382] Example 23. The engineered dsRNA ligase polypeptide of Example 22, comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 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 ,96,98,100,102,104,106,108,110,112,114,116,118,120,122,124,126,128,130,132,134,136,138,140,142,144,146,148,150,152,154,156,158,160,162,16 4, 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.

[0383] Embodiment 24. The engineered dsRNA ligase polypeptide of embodiment 22, comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 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, 103, 104, 105, 106, 107, 108, 109, 110 4, 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, 257 8, 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.

[0384] Embodiment 25. A polypeptide immobilized on a solid material by chemical bonding or physical adsorption, wherein the polypeptide comprises the engineered dsRNA ligase polypeptide according to any one of embodiments 1-24.

[0385] Embodiment 26. A polynucleotide encoding the engineered dsRNA ligase polypeptide of any one of embodiments 1-24.

[0386] Embodiment 27. The polynucleotide of embodiment 26, wherein the polynucleotide sequence is SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 4 3, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 8 3, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 11 7, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 14 7, 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, 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, 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, 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, or 599.

[0387] Embodiment 28. The polynucleotide of embodiment 26, wherein the polynucleotide sequence is SEQ ID NO: 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 4 3, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 8 3, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 11 7, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 14 7, 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, 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, 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, 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, 588 9, 591, 593, 595, 597, 599, 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.

[0388] Embodiment 29. An expression vector comprising the polynucleotide of any one of embodiments 26-28.

[0389] Embodiment 30. The expression vector of embodiment 29, comprising a plasmid, a cosmid, a phage or a viral vector.

[0390] Embodiment 31. A host cell comprising the polynucleotide of any one of embodiments 26-28 or the expression vector of embodiment 29 or 30, optionally wherein the host cell is Escherichia coli.

[0391] Example 32. A method for preparing an engineered dsRNA ligase polypeptide, comprising the steps of culturing the host cell according to Example 31 and obtaining the engineered dsRNA ligase polypeptide from the culture.

[0392] Example 33. An engineered dsRNA ligase catalyst obtainable by culturing the host cell of Example 31 or the method of Example 32, wherein the engineered dsRNA ligase catalyst comprises cells or culture fluid containing the engineered dsRNA ligase polypeptide, or an article processed therewith, wherein the article is an extract obtained from the culture of the host cell, an isolated product obtained by isolating or purifying the engineered dsRNA ligase from the extract, or an immobilized product obtained by immobilizing the host cell, an extract thereof, or an isolated product of the extract.

[0393] Example 34. A method of producing an oligonucleotide from two or more oligonucleotide fragments, wherein the method comprises contacting:

[0394] (i) two or more oligonucleotide fragments;

[0395] (ii) the engineered dsRNA ligase polypeptide according to any one of embodiments 1-24;

[0396] (iii) ATP source; and

[0397] (iv) divalent cations;

[0398] to obtain oligonucleotides.

[0399] Embodiment 35. The method of embodiment 34, wherein the ATP source comprises ATP.

[0400] Embodiment 36. The method of embodiment 34 or 35, wherein the ATP source comprises:

[0401] (a) Polyphosphate kinase (PPK);

[0402] (b) polyphosphoric acid; and

[0403] (c) AMP and / or ATP.

[0404] Embodiment 37. The method of embodiment 36, wherein the PPK is selected from PPK12 or ajPAP.

[0405] Embodiment 38. The method of any one of embodiments 36 or 37, wherein the method is performed using substoichiometric concentrations of AMP and / or ATP.

[0406] Embodiment 39. The method of any one of embodiments 36-38, wherein the polyphosphoric acid is a polyphosphate salt.

[0407] Embodiment 40. The method of embodiment 39, wherein the polyphosphate is sodium polyphosphate (Madrell salt) or sodium hexametaphosphate (Graham salt).

[0408] Embodiment 41. The method of any one of embodiments 34-40, wherein the divalent cation cofactor is Mg 2+ or Mn 2+ .

[0409] Embodiment 42. The method of any one of embodiments 34-41, wherein the method is performed at a divalent cation concentration of 5-100 mM, optionally 30-50 mM.

[0410] Example 43. The method of any one of Examples 34-42, further comprising the step of purifying the oligonucleotide.

[0411] Example 44. Use of the engineered dsRNA ligase polypeptide of any one of Examples 1-24 in generating an oligonucleotide from two or more oligonucleotide fragments.

[0412] Embodiment 45. The method of any one of embodiments 34-43 or the use of embodiment 44, wherein the oligonucleotide is at most 60 nucleotides in length.

[0413] Embodiment 46. The method of any one of embodiments 34-43 or 45, or the use of embodiment 44 or 45, wherein each of the oligonucleotide fragments is 4-16 nucleotides in length, optionally 6-9 nucleotides in length.

[0414] Embodiment 47. The method of embodiment 34-43, 45 or 46, or the use of any one of embodiments 44-46, wherein one or more of the oligonucleotide fragments comprises one or two overhangs.

[0415] Embodiment 48. The method of any one of embodiments 34-43 or 45-47, or the use of any one of embodiments 44-47, wherein one or more of the oligonucleotide fragments comprises a chemical modification.

[0416] Embodiment 49. The method or use of embodiment 48, wherein the chemical modification is selected from:

[0417] (a) a modified backbone, optionally selected from phosphorothioate (e.g., chiral phosphorothioate) or methylphosphonate internucleotide linkages;

[0418] (b) modified nucleotides, optionally selected from 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-methylacetamido (2'-O-NMA), locked nucleic acid (LNA), glycol nucleic acid (GNA), phosphamidonoesters (e.g., phosphamidonomethylsulfonate), 2',3'-split nucleotide mimetics, 2'-F-arabinonucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, vinylphosphonate (e.g., 5' vinylphosphonate), and cyclopropylphosphonate deoxyribonucleotides; and / or

[0419] (c) conjugation to a ligand, optionally wherein the ligand comprises one or more N-acetylgalactosamine (GalNAc) derivatives.

[0420] Embodiment 50. A composition comprising:

[0421] i. An engineered dsRNA ligase polypeptide according to any one of embodiments 1-24;

[0422] ii. Source of ATP; and

[0423] iii. Divalent cations.

[0424] Embodiment 51. The composition of embodiment 50, further comprising two or more oligonucleotide fragments.

[0425] Example 52. A kit comprising:

[0426] i. An engineered dsRNA ligase polypeptide as described in any one of embodiments 1-24;

[0427] ii. Source of ATP;

[0428] iii. divalent cations; and

[0429] iv. Instructions for use of a method for generating an oligonucleotide from two or more oligonucleotide fragments.

[0430] Embodiment 53. The composition of embodiment 50 or 51 or the kit of embodiment 52, wherein the ATP source comprises ATP.

[0431] Embodiment 54. The composition of any one of embodiments 50, 51, or 53, or the kit of embodiments 52 or 53, wherein the ATP source comprises:

[0432] (a) Polyphosphate kinase (PPK);

[0433] (b) polyphosphoric acid; and

[0434] (c) AMP and / or ATP.

[0435] Embodiment 55. The composition or kit of embodiment 54, wherein the PPK is selected from PPK12 or ajPAP.

[0436] Embodiment 56. The composition of any one of embodiments 50, 51, or 53-55, or the kit of any one of embodiments 52-55, wherein the polyphosphoric acid is a polyphosphate salt.

[0437] Embodiment 57. The composition or kit of embodiment 56, wherein the polyphosphate is sodium polyphosphate (Madrell's salt) or sodium hexametaphosphate (Graham's salt).

[0438] Embodiment 58. The composition of any one of embodiments 50, 51, or 53-57, or the kit of any one of embodiments 52-57, wherein the divalent cation cofactor is Mg 2+ or Mn 2+ .

[0439] Various features and embodiments of the present disclosure are exemplified in the following representative examples, which are intended to be illustrative rather than limiting.

[0440] Examples

[0441] The following examples, including the experiments and the results achieved, are provided for illustrative purposes only and should not be construed as limiting the present invention.

[0442] In the examples that follow, the following abbreviations apply: ppm (parts per million); M (mole); mM (millimole), uM, and μM (micromole); nM (nanomole); mol (mole); gm and g (gram); mg (milligram); ug and μg (microgram); L and l (liter); ml and mL (milliliter); cm (centimeter); mm (millimeter); um and μm (micrometer); sec. (second); min (minute); h and hr (hour); U (unit); MW (molecular weight); rpm (revolutions per minute); psi and PSI (pounds per hour). 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 (lysing broth); TB (super broth; 12 g / L tryptone, 24 g / L yeast extract, 4 mL / L glycerol, 65 mM potassium phosphate (pH 7.0)). 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 W3110 (a commonly used laboratory strain of E. coli, available from the Center for Escherichia coli Genetic Collection [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, Inc. Laboratories, BD Diagnostic Systems, Detroit, MI); Agilent Technologies, Inc., Santa Clara, CA; Corning, Inc., Palo Alto, CA; Dow Corning, Corp.), Midland, MI; and Gene Oracle, Inc., Mountain View, CA.

[0443] The sequences of oligonucleotides referenced within parentheses throughout the Examples are provided in Table 1 (eg, "siRNA (1)" and "Oligonucleotide (2)").

[0444] Example 1

[0445] Preparation of isolated enzyme

[0446] The polynucleotide encoding the polypeptide having ligase activity is cloned into the pCK110900 vector system (see, e.g., FIG. 3 of U.S. Patent Application No. 2006 / 0195947A1, which is incorporated herein by reference in its entirety) and subsequently expressed in E. coli W3110fhuA under the control of the lac promoter. The expression vector also contains a P15a origin of replication and a chloramphenicol (CAM) resistance gene.

[0447] E. coli W3110fhuA cells were transformed with the pCK110900 plasmid containing the ligase encoding gene. The transformed cells were plated on lysing broth (LB) agar plates containing 1% glucose and 30 μg / mL CAM and grown overnight at 37°C. Subsequently, a single colony was inoculated into 25 mL LB supplemented with 30 μg / mL CAM and 1% glucose in a 250 ml baffled shake flask. The culture was grown overnight (16-20 hours at optical density (OD)) in an incubator shaken at 37°C, 250 rpm. 600 )>3.8). 5 mL of overnight culture was inoculated into a 1 L shake flask containing 250 mL of super broth (TB) medium (containing 30 μg / mL CAM). The 250 mL culture was incubated at 30°C, 250 rpm for 3-3.5 hours until the OD 600 The expression of the ligase gene was induced by adding isopropyl-β-D-thiogalactopyranoside (IPTG) at a final concentration of 1 mM and the cells were grown for another 18-20 hours. The culture was transferred to a centrifuge bottle and then harvested by centrifugation 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 LM20 Lysis was performed using a FLASH® processor system (Microfluidics, Inc.). Cell debris was removed by centrifugation at 14,000 rpm for 30 minutes at 4°C. Ligase was then isolated from the clarified lysate using standard techniques known in the art, including immobilized metal affinity chromatography.

[0448] Example 2

[0449] Characterization of dsRNA ligase activity for the production of siRNA(1)

[0450] To identify an enzyme with dsRNA ligase activity for producing siRNA (1) comprising oligonucleotides (2) and (3), a collection of ligases was first screened to produce an alternative product, siRNA (4), comprising oligonucleotides (3) and (5). The sequence of oligonucleotide (5) is identical to that of oligonucleotide (2), but it does not contain a 3'-GalNAc moiety. siRNAs (1) and (4) and oligonucleotides (2), (3), and (5) are depicted in FIG1 ; the sequences of oligonucleotides (2), (3), and (5) are provided in Table 1 .

[0451] The isolated ligases were screened in 20 μL reaction volumes in PCR tubes containing 50 mM Tris-buffer (pH 7.5), 1 mM ATP or 1 mM NAD+, 10 mM MgCl2, 5 mM DTT and 10 μM (each) substrate oligonucleotide (6-11) and 50% (v / v) isolated ligase. The reactions were incubated in a thermal cycler at 16°C for 2 h and analyzed using standard techniques known in the art (including electrophoresis). The ligase with SEQ ID NO: 2 showed 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 a variety of enzyme preparations, including isolated enzyme (Example 1), clarified lysate (Example 4) and shake flask powder (SFP; Example 5).

[0452] Table 1 Oligonucleotide sequences

[0453]

[0454] Example 3

[0455] Preparation of cell pellets for high-throughput (HTP) screening

[0456] 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 LB medium (containing 30 μg / mL CAM). The culture was grown at 30°C, 250 rpm, and 85% humidity for approximately 2.5 hours. When the OD of the culture reached 0.05, the culture was plated. 600When the p-value reaches 0.4-0.8, induce expression of the ligase gene by adding IPTG to a final concentration of 1 mM. Following induction, continue growth at 30°C, 250 rpm, and 85% humidity for 18-20 hours. Harvest the cells by centrifugation at 4,000 rpm for 10 minutes at 4°C; discard the supernatant. Store the cell pellet at -80°C until ready for use.

[0457] Example 4

[0458] Preparation of lysis and clarified lysate

[0459] Prior to the assay, the cell pellet was thawed and resuspended in 300 μL of lysis buffer (containing 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 plate was agitated by shaking at medium speed on a microtiter plate shaker for 2.5 hours at room temperature. The plate was then centrifuged at 4,000 rpm for 10 minutes at 4°C, and the clear supernatant was used in the HTP assay reaction for activity determination, as described in the following examples.

[0460] Example 5

[0461] Preparation of shake flask powder (SFP) and fermentation powder (FP)

[0462] The shake flask procedure can be used to generate engineered dsRNA ligase polypeptide shake flask powder (SFP), which can be used in secondary screening assays and / or in the biocatalytic processes described herein. Compared to cell lysates used in HTP assays, shake flask powder preparations of the enzyme can provide a more concentrated engineered enzyme preparation.

[0463] The clarified lysate produced according to Example 1 was collected using standard methods known in the art, frozen at -80°C, and then lyophilized. Lyophilization of the frozen clarified lysate yielded dried SFP containing crude wild-type or engineered dsRNA ligase polypeptide.

[0464] Example 6

[0465] Analytical methods for activity and selectivity evaluation

[0466] The improved activity of the engineered dsRNA ligase was analyzed by high pressure liquid chromatography (HPLC) using the methods 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 goal of the analytical method was to achieve good separation of product oligonucleotides (2) and (3) in the shortest possible run time. As a result, the six substrate (6-7, 9-12) and four intermediate (13-16) oligonucleotides could not be well separated from each other. However, well-defined GalNAc-containing oligonucleotides could be separated, including substrate oligonucleotide (12), reaction intermediate oligonucleotide (14) and product oligonucleotide (2). Therefore, the pseudo-conversion % can be calculated, expressed in arbitrary units (AU), which only takes into account these well-separated substances according to the following formula:

[0467]

[0468] where ε (2) , ε (12) and ε (14) are the extinction coefficients for oligonucleotides (2), (12), and (14), respectively. Using this calculation, an AU = 1.0 would mean that there are no longer any GalNAc-containing substrate or intermediate oligonucleotides in the reaction, and that they have all been converted to the GalNAc-containing product (2). In fact, for the sample with AU = 1.0, the only other peak present in the chromatogram corresponds to the product oligonucleotide (2), and no other intermediates or starting materials can be identified. In addition, the ratio of product oligonucleotides (2) and (3) is consistent with that of an authentic standard of siRNA product (1). In summary, it can be concluded that AU = 1.0 is an approximate value that is essentially equivalent to 100% conversion.

[0469] Table 6-1: HPLC method used for activity determination 1.

[0470]

[0471]

[0472] HPLC Method 2 (Table 6-2) was developed based on HPLC Method 1 (Table 6-1) to improve the separation between the product oligonucleotide (3) and the substrate oligonucleotide (12).

[0473] Table 6-2: HPLC method used for activity determination 1.

[0474]

[0475]

[0476] Also use the method described in Table 6-3 by The improved activity of the engineered dsRNA ligase of Example 12 was analyzed by mass spectrometry (RF-MS). RF-MS is intended to shorten analysis time compared to HPLC analysis. Selective detection of product oligonucleotides (2) and (3) was obtained by analyzing the specific mass of each product oligonucleotide in multiple single ion monitoring (SIM) mode. Relative dsRNA ligase activity was determined by comparing the sum of the MS signals of the five specific masses (given in Table 6-3) corresponding to each product oligonucleotide (2) and (3).

[0477] Table 6-3: RF-MS methods used for activity determination.

[0478]

[0479]

[0480] The methods provided herein can be used to analyze variants produced using the present invention. However, the present invention is not intended to be limited to the methods described herein, as other suitable methods known in the art are also applicable to analyzing variants provided herein and / or variants produced using the methods provided herein.

[0481] Example 7

[0482] First round of evolution and screening of engineered polypeptides derived from SEQ ID NO: 2 for improved production of siRNA products (1)

[0483] The engineered polypeptides of Table 7-1 were generated using engineered polynucleotides (SEQ ID NO: 1) encoding polypeptides having the dsRNA ligase activity of SEQ ID NO: 2. These polypeptides exhibit improved dsRNA ligase activity under desired conditions compared to the starting polypeptides, for example, improved formation of oligonucleotide products (2) or (3) or preferably oligonucleotide products (2) and (3) generated in situ from substrate oligonucleotides (6-7, 9-12). Some polypeptides exhibit improved product formation of oligonucleotide products (2) or (3) or oligonucleotide products (2) and (3) compared to the starting polypeptides, as shown in Table 7-1. The sequences of oligonucleotides (2), (3), (6), (7) and (9-12) are provided in Table 1.

[0484] Engineered polypeptides having amino acid sequences with even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 2, as described below and using the analytical methods described in Table 6-1. Directed evolution was initiated from the polynucleotide set forth in SEQ ID NO: 1. Libraries of engineered 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 HTP assays and analytical methods described below that measure the ability of the polypeptides to produce oligonucleotide products (2) and (3).

[0485] The enzyme assay was performed in a 96-well PCR plate with a total reaction volume of 50 μL per well. The reaction contained 2.5% (v / v) undiluted dsRNA ligase lysate (prepared as described in Example 4), 100 μM (each) substrate oligonucleotide (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 in a thermal cycler at 30°C for 2 h.

[0486] After incubation, the plate was subjected to a heat inactivation step (95°C, 20min) to quench the reaction and precipitate the protein content of the added lysate. The plate was then centrifuged at 4,000rpm for 5min. 2 μL supernatant aliquots were subsequently taken out from each well and added to a shallow 96-well plate containing 98 μL 5mM EDTA solution (pH 7.0). The sample was analyzed by HPLC using the analytical method described in Table 6-1 to determine the activity of the enzyme variant. The selected ligase variants that show more oligonucleotide products (2) and (3) relative to SEQ ID NO:2 are shown in Table 7-1.

[0487]

[0488]

[0489]

[0490] The engineered dsRNA ligase polypeptides represented by the even-numbered sequence identifiers of SEQ ID NOs: 4 to 106 comprise the even-numbered sequence identifiers of SEQ ID NOs: 304 to 406, respectively, and a 14-amino acid N-terminal purification tag (MHHHHHHENLYFQS (SEQ ID NO: 669)). For example, SEQ ID NO: 4 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: 304.

[0491] Throughout the Examples, the position of a given mutation relative to SEQ ID NO: 2 is provided, which includes (i) the 14 amino acid N-terminal purification tag of SEQ ID NO: 669 and (ii) the wild-type dsRNA ligase polypeptide of SEQ ID NO: 302. The position of a given mutation relative to SEQ ID NO: 302 (i.e., the wild-type dsRNA ligase polypeptide without the purification tag) can be obtained by subtracting the 14 amino acid N-terminal purification tag from the SEQ ID NO described in the Examples. For example, position X251 of SEQ ID NO: 2 corresponds to position X237 of SEQ ID NO: 302.

[0492] Example 8

[0493] Second round of evolution and screening of engineered polypeptides derived from SEQ ID NO: 70 for improved production of siRNA products (1)

[0494] The engineered polypeptides of Table 8-1 were generated using polynucleotides from Example 7, SEQ ID NO: 69, which encodes the most active polypeptide having the dsRNA ligase activity of SEQ ID NO: 70. These polypeptides exhibit improved dsRNA ligase activity under desired conditions compared to the starting polypeptide, for example, improved formation of oligonucleotide products (2) or (3) or preferably oligonucleotide products (2) and (3) generated in situ from substrate oligonucleotides (6-7, 9-12). Some polypeptides exhibit improved product formation of oligonucleotide products (2) and (3) compared to the starting polypeptide, as shown in Table 8-1. Engineered 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 and using the analytical methods described in Table 6-1.

[0495] Directed evolution was initiated from the polynucleotide set forth in SEQ ID NO: 69. Libraries of engineered 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 assays that measure the ability of the polypeptides to generate oligonucleotides (2) and (3) as described below.

[0496] The enzyme assay was performed in a 96-well PCR plate with a total reaction volume of 50 μL per well. The reaction contained 1.25 or 2.5% (v / v) undiluted dsRNA ligase lysate (prepared as described in Example 4), 100 μM (each) substrate oligonucleotide (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 in a thermal cycler at 30°C for 2 h.

[0497] After incubation, the plate was subjected to a heat inactivation step (95°C, 20min) to quench the reaction and precipitate the protein content of the added lysate. The plate was then centrifuged at 4,000rpm for 5min. 2 μL supernatant aliquots were subsequently taken out from each well and added to a shallow 96-well plate containing 98 μL 5mM EDTA solution (pH 7.0). Using the analytical method described in Table 6-1, the samples were analyzed by HPLC to determine the activity of the enzyme variants. The selected ligase variants that show faster formation of oligonucleotide products (2) and (3) relative to SEQ ID NO: 70 are shown in Table 8-1.

[0498]

[0499]

[0500]

[0501] The engineered dsRNA ligase polypeptides represented by the even-numbered sequence identifiers of SEQ ID NOs: 108 to 216 comprise the even-numbered sequence identifiers of SEQ ID NOs: 408 to 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.

[0502] Example 9

[0503] 3rd round of evolution and screening of engineered polypeptides derived from SEQ ID NO: 188 for improved production of siRNA products (1)

[0504] The engineered polypeptides of Table 9-1 were generated using polynucleotides from Example 8, SEQ ID NO: 187, which encodes the most active polypeptide having the dsRNA ligase activity of SEQ ID NO: 188. These polypeptides exhibit improved dsRNA ligase activity under desired conditions compared to the starting polypeptide, for example, improved formation of oligonucleotide products (2) or (3) or preferably oligonucleotide products (2) and (3) generated in situ from substrate oligonucleotides (6-7, 9-12). Some polypeptides exhibit improved product formation of oligonucleotide products (2) and (3) compared to the starting polypeptide, as shown in Table 9-1. Engineered polypeptides having amino acid sequences with even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 188 as described below and using the analytical methods described in Table 6-2.

[0505] Directed evolution was initiated from the polynucleotide set forth in SEQ ID NO: 187. Libraries of engineered 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 assays that measure the ability of the polypeptides to generate oligonucleotides (2) and (3) as described below.

[0506] The enzyme assay was performed in a 96-well PCR plate with a total reaction volume of 100 μL per well. The reaction contained 20% (v / v) undiluted dsRNA ligase lysate (prepared as described in Example 4), 1 mM (each) substrate oligonucleotide (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 in a thermal cycler at 30°C for 2 h.

[0507] After incubation, the plate was subjected to a heat inactivation step (95°C, 20min) to quench the reaction and precipitate the protein content of the added lysate. The plate was then centrifuged at 4,000rpm for 5min. 50 μL supernatant aliquots were subsequently taken out from each well and added to a deep-well 96-well plate containing 450 μL 5mM EDTA solution (pH 7.0). The sample was further diluted by transferring 50 μL diluted samples to a deep-well 96-well plate containing 950 μL 5mM EDTA solution (pH 7.0). The sample was analyzed by HPLC using the analytical method described in Table 6-2 to determine the activity of the enzyme variant. The selected ligase variants that show faster formation of oligonucleotide products (2) and (3) relative to SEQ ID NO: 188 are shown in Table 9-1.

[0508]

[0509]

[0510] The engineered dsRNA ligase polypeptides represented by the even-numbered sequence identifiers of SEQ ID NOs: 218 to 246 comprise the even-numbered sequence identifiers of SEQ ID NOs: 518 to 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.

[0511] Example 10

[0512] 4th round of evolution and screening of engineered polypeptides derived from SEQ ID NO: 226 for improved production of siRNA products (1)

[0513] The engineered polypeptides of Table 10-1 were generated using polynucleotides from Example 9, SEQ ID NO: 225, which encodes the most active polypeptide having the dsRNA ligase activity of SEQ ID NO: 226. These polypeptides exhibit improved dsRNA ligase activity under desired conditions compared to the starting polypeptide, for example, improved formation of oligonucleotide products (2) or (3) or preferably oligonucleotide products (2) and (3) generated in situ from substrate oligonucleotides (6-7, 9-12). Some polypeptides exhibit improved product formation of oligonucleotide products (2) and (3) compared to the starting polypeptide, as shown in Table 10-1. Engineered polypeptides having amino acid sequences with even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 226 as described below and using the analytical methods described in Table 6-2.

[0514] Directed evolution was initiated from the polynucleotide set forth in SEQ ID NO: 225. Libraries of engineered 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 assays that measure the ability of the polypeptides to generate oligonucleotides (2) and (3) as described below.

[0515] The enzyme assay was performed in a 96-well PCR plate with a total reaction volume of 100 μL per well. The reaction contained 2.5% (v / v) undiluted dsRNA ligase lysate (prepared as described in Example 4), 1 mM (each) substrate oligonucleotide (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 in a thermal cycler at 30°C for 24 h.

[0516] After incubation, the plate was subjected to a heat inactivation step (95°C, 20min) to quench the reaction and precipitate the protein content of the added lysate. The plate was then centrifuged at 4,000rpm for 5min. 50 μL supernatant aliquots were subsequently taken out from each well and added to a deep-well 96-well plate containing 450 μL 5mM EDTA solution (pH 7.0). The sample was further diluted by transferring 50 μL diluted samples to a deep-well 96-well plate containing 950 μL 5mM EDTA solution (pH 7.0). The sample was analyzed by HPLC using the analytical method described in Table 6-2 to determine the activity of the enzyme variant. The selected ligase variants that show faster formation of oligonucleotide products (2) and (3) relative to SEQ ID NO: 226 are shown in Table 10-1.

[0517]

[0518]

[0519] The engineered dsRNA ligase polypeptides represented by the even-numbered sequence identifiers of SEQ ID NOs: 248 to 282 comprise the even-numbered sequence identifiers of SEQ ID NOs: 548 to 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.

[0520] Example 11

[0521] Fifth round of evolution and screening of engineered polypeptides derived from SEQ ID NO: 278 for improved production of siRNA products (1)

[0522] The engineered polypeptides of Table 11-1 were generated using polynucleotides from Example 10 SEQ ID NO: 277, which encodes the most active polypeptide having the dsRNA ligase activity of SEQ ID NO: 278. These polypeptides exhibit improved dsRNA ligase activity under desired conditions compared to the starting polypeptide, for example, improved formation of oligonucleotide products (2) or (3) or preferably oligonucleotide products (2) and (3) generated in situ from substrate oligonucleotides (6-7, 9-12). Some polypeptides exhibit improved product formation of oligonucleotide products (2) and (3) compared to the starting polypeptide, as shown in Table 11-1. Engineered 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 and using the analytical methods described in Table 6-2.

[0523] Directed evolution was initiated from the polynucleotide set forth in SEQ ID NO: 277. Libraries of engineered 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 assays that measure the ability of the polypeptides to generate oligonucleotides (2) and (3) as described below.

[0524] The enzyme assay was performed in a 96-well PCR plate with a total reaction volume of 100 μL per well. The reaction contained 10% (v / v) undiluted dsRNA ligase lysate (prepared as described in Example 4), 5 mM (each) substrate oligonucleotide (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 in a thermal cycler at 30°C for 24 h.

[0525] After incubation, the plate is subjected to a heat inactivation step (95°C, 20min) to quench the reaction and precipitate the protein content of the added lysate. The plate is then centrifuged at 4,000rpm for 5min. 50 μL supernatant aliquots are subsequently taken out from each well and added to a deep-well 96-well plate containing 950 μL 5mM EDTA solution (pH 7.0). The sample is further diluted by transferring 50 μL of diluted samples to a deep-well 96-well plate containing 450 μL 5mM EDTA solution (pH 7.0). The sample is diluted for the third time by transferring 160 μL of diluted samples to a deep-well 96-well plate containing 640 μL 5mM EDTA solution (pH 7.0). The sample is diluted for the last time by transferring 75 μL of diluted samples to a deep-well 96-well plate containing 75 μL 5mM EDTA solution (pH 7.0). The samples were analyzed by HPLC using the analytical method described in Table 6-2 to determine the activity of the enzyme variants. Selected ligase variants that showed faster formation of oligonucleotide products (2) and (3) relative to SEQ ID NO: 278 are shown in Table 11-1.

[0526]

[0527] The engineered dsRNA ligase polypeptides represented by the even-numbered sequence identifiers of SEQ ID NOs: 284 to 300 comprise the even-numbered sequence identifiers of SEQ ID NOs: 584 to 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.

[0528] Example 12

[0529] Sixth round of evolution and screening of engineered polypeptides derived from SEQ ID NO: 288 for improved siRNA product (1) production and improved thermal stability

[0530] The engineered polypeptides of Table 12-1 were generated using polynucleotides from Example 11, SEQ ID NO: 287, which encode the most active polypeptide having the dsRNA ligase activity of SEQ ID NO: 288. These polypeptides exhibit improved dsRNA ligase activity under desired conditions compared to the starting polypeptide, for example, improved formation of oligonucleotide products (2) or (3) or preferably oligonucleotide products (2) and (3) generated in situ from substrate oligonucleotides (6-7, 9-12). Some polypeptides exhibit improved product formation of oligonucleotide products (2) and (3) compared to the starting polypeptide, as shown in Table 12-1. In addition, some polypeptides exhibit improved thermal stability, with higher residual activity obtained after incubating the dsRNA ligase solution at 30° C. for 1 hour before setting up the reaction (Table 12-2). Engineered polypeptides with amino acid sequences of even-numbered sequence identifiers were generated from the "backbone" amino acid sequence of SEQ ID NO: 288 as described below and using the analytical methods described in Table 6-3.

[0531] Directed evolution was initiated from the polynucleotide set forth in SEQ ID NO: 287. Libraries of engineered 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 assays that measure the ability of the polypeptides to generate oligonucleotides (2) and (3) as described below.

[0532] Enzyme assay is carried out in 96-well PCR plates, and the total reaction volume of each well is 100 μ L.According to Example 4 lysis cells, but 50 mM Tris (pH 7.5) is replaced with 100 mM MOPS-buffer (pH 7.2).In order to provide thermal challenge and identify more heat stable hit point, cell lysate is undiluted or is diluted by 1: 1 in 100 mM MOPS buffer (pH 7.2), and is hatched 1h at 30 DEG C and 4 DEG C respectively.For the lysate hatched at 30 DEG C, the final dsRNA ligase concentration in the reactant is 40% (v / v), and for the lysate hatched at 4 DEG C, the final dsRNA ligase concentration in the reactant is 20% (v / v). In addition, the ligation reaction also contained 3 mM (each) substrate oligonucleotide (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 thermal cycler at 30°C for 24 h.

[0533] After incubation, the plate was subjected to a heat inactivation step (95°C, 20min) to quench the reaction and precipitate the protein content of the added lysate. The plate was then centrifuged at 4,000rpm for 5min. 50 μL supernatant aliquots were subsequently taken out from each well and added to a deep-well 96-well plate containing 950 μL 5mM EDTA solution (pH 7.0). The sample was further diluted by transferring 20 μL of diluted samples to a deep-well 96-well plate containing 180 μL 5mM EDTA solution (pH 7.0). The sample was diluted for the third time by transferring 30 μL of diluted samples to a deep-well 96-well plate containing 150 μL 5mM EDTA solution (pH 7.0). The analytical method described in Table 6-3 was used to analyze the sample to determine the activity of the enzyme variant. Selected ligase variants that showed faster formation of oligonucleotide products (2) and (3) relative to SEQ ID NO: 288 after preincubation at 4°C are shown in Table 12-1. Selected ligase variants that showed faster formation of oligonucleotide products (2) and (3) relative to SEQ ID NO: 288 after preincubation at 30°C are shown in Table 12-2.

[0534]

[0535]

[0536]

[0537]

[0538] The engineered dsRNA ligase polypeptides represented by the even-numbered sequence identifiers of SEQ ID NOs: 602 to 634 comprise the even-numbered sequence identifiers of SEQ ID NOs: 636 to 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.

[0539] Example 13

[0540] Comparison of catalytic activity of wild-type polypeptide SEQ ID NO: 2 and engineered polypeptides SEQ ID NO: 288, SEQ ID NO: 290 and SEQ ID NO: 292

[0541] The polynucleotides SEQ ID NO: 1 together with SEQ ID NO: 2 encoding the wild-type dsRNA ligase from bacteriophage RB69 (Uniprot ID: Q7Y4V8) and the engineered polynucleotides SEQ ID NO: 287, SEQ ID NO: 289 and SEQ ID NO: 291 together with the polypeptide sequences SEQ ID NO: 288, SEQ ID NO: 290 and SEQ ID NO: 292 encoding the most improved variant from Example 11 were used for SFP production as described in Example 5.

[0542] The catalytic activity of converting substrate oligonucleotides (6-7, 9-12) into the desired siRNA product (1) was evaluated under the following two reaction conditions: condition 1 (50 mM Tris-buffer (pH 7.5), 1 mM ATP, 5 mM MgCl2, and 5 mM DTT, containing 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 SFP) and condition 2 (50 mM Tris-buffer (pH 7.0), 30 mM ATP, 60 mM MgCl2, and 10% (v / v) DMSO, containing 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 SFP. The enzyme assay was performed in a 96-well PCR plate with a total reaction volume of 100 μL per well; condition 1 in reaction plate 1 and condition 2 in reaction plate 2. Both reaction plates were heat-sealed and incubated at 30°C in a thermal cycler. Reaction plate 1 was incubated for 2 h, and reaction plate 2 was incubated for 24 h.

[0543] After incubation, the plates were subjected to a heat inactivation step (95°C, 20 min) to quench the reaction and precipitate the protein content of the added SFP. The plates were then centrifuged at 4,000 rpm for 5 min. A 50 μL aliquot of the supernatant was taken from each well of each plate and diluted in a 50 mM EDTA solution (pH 7.0). Reaction plate 1 was diluted 40-fold and reaction plate 2 was diluted 2400-fold. The samples were analyzed by HPLC using the analytical method described in Table 6-2 to determine the activity of the enzyme variants.

[0544] Figure 2A and Figure 2BThe comparative data in show the relative peak area % of siRNA (1) present in the reaction samples measured under conditions 1 and 2, respectively. Under conditions 1 and 2, the polypeptides SEQ ID NO: 288, SEQ ID NO: 290 and SEQ ID NO: 292 exhibited improved dsRNA ligase activity over the wild-type polypeptide SEQ ID NO: 2.

[0545] Example 14

[0546] Comparison of catalytic activity of wild-type polypeptide SEQ ID NO: 2 and engineered polypeptides SEQ ID NO: 288 and 632

[0547] The polynucleotides SEQ ID NO: 1 together with SEQ ID NO: 2 encoding the wild-type dsRNA ligase from bacteriophage RB69 (Uniprot ID: Q7Y4V8) and the engineered polynucleotides SEQ ID NO: 287 and SEQ ID NO: 631 encoding the most improved variants from Examples 11 and 12 together with the polypeptide sequences SEQ ID NO: 288 and SEQ ID NO: 632 were used for SFP production as described in Example 5.

[0548] The catalytic activity of the substrate oligonucleotides (6-7, 9-12) in converting them to the desired siRNA products (1) and the thermal stability of the two enzymes were evaluated by incubating the stock solutions of SFP at 4°C or 37°C for 4 h. The following ligation reactions were then set up: 6 mM (each) substrate oligonucleotide (6-7, 9-12), 100 mM MOPS-buffer (pH 7.2), 30 mM ATP, 60 mM MgCl2, and 10% (v / v) DMSO. In addition, the ligation reactions also 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. The enzyme assays were performed in 96-well PCR plates with a total reaction volume of 100 μL per well. The reaction plates were heat-sealed and incubated in a thermal cycler at 30°C for 24 h.

[0549] After incubation, the plates were subjected to a heat inactivation step (95°C, 20 min) to quench the reaction and precipitate the protein content of the added SFP. The plates were then centrifuged at 4,000 rpm for 5 min. A 50 μL aliquot of the supernatant was taken from each well of each plate and then diluted 400-fold in a 50 mM EDTA solution (pH 7.0). The samples were analyzed by HPLC using the analytical method described in Table 6-2 to determine the activity of the enzyme variants.

[0550] Figure 3AThe comparative data in show the relative peak area % of siRNA (1) present in the reaction samples. Figure 3B The residual enzyme activity of SFP after preincubation for 4 hours at 37° C. is shown, expressed as the ligation activity relative to SFP preincubated for 4 hours at 4° C. Under all conditions, polypeptide SEQ ID NO: 632 exhibited improved dsRNA ligase activity and thermostability compared to wild-type polypeptide SEQ ID NO: 2 and engineered polypeptide SEQ ID NO: 288.

[0551] Summary of engineered dsRNA ligase polypeptide sequences

[0552] A summary of the nucleic acid and amino acid sequences of the wild-type and engineered dsRNA ligase sequences described herein is provided in Table 13. The purification tag used in the examples and references in Table 13 is the N-terminal purification tag MHHHHHHENLYFQS (SEQ ID NO: 669).

[0553] Table 13.

[0554]

[0555]

[0556]

[0557]

[0558]

[0559]

[0560]

[0561]

Claims

1. An engineered 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: SEQ ID NO: 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, 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, 465 6, 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, 5 50, 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 and 668; wherein the engineered dsRNA ligase polypeptide: (a) having dsRNA ligase activity; and (b) does not comprise the amino acid sequence of SEQ ID NO:

302.

2. The engineered dsRNA ligase polypeptide of claim 1, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 666, 370, 488, 526, 578, 588, 590, and 592.

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

302.

4. The engineered dsRNA ligase polypeptide of claim 3, wherein the ligation reaction conditions comprise about 1 μM to about 10 mM 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 engineered 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. The engineered dsRNA ligase of any one of the preceding claims, wherein: (a) the amino acid sequence of the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues 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, X164, X165, X166, X170, X171, X172, X173, X174, X175, X176, X177, X178, X179, X180, X181, X182, X183, X184, X185 73, 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, wherein the numbering refers to SEQ ID NO: NO: 302; Optionally, the amino acid sequence of the engineered 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; X 53 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 not present; 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; X1 07 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; X21 8 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; wherein said numbering refers to SEQ ID NO: 302; and / or; (b) the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of: X15, X19, X36, X39, X53, X185, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of: X15, X19, X36, X39, X53, X185, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the amino acid sequence of the engineered 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; wherein the numbering refers to SEQ ID NO: 302; and / or (c) the engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of: X36, X39, X218, and X221, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the engineered 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 engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of: X39, X218, and X221, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the engineered 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 engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of: X39, X218, X221, and X255, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the engineered 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 engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of: X39, X53, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the engineered 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 engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of: X15, X39, X53, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the engineered 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 engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of: X19, X39, X53, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the engineered 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 engineered dsRNA ligase polypeptide comprises an amino acid sequence that differs from the sequence of SEQ ID NO: 302 in one or more amino acid residues selected from the group consisting of: X15, X39, X53, X185, X218, X221, X237, X251, X255, and X285, wherein the numbering refers to SEQ ID NO: 302, and wherein the engineered dsRNA ligase polypeptide has dsRNA ligase activity; optionally, wherein the engineered 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.

6. The engineered dsRNA ligase polypeptide of any one of claims 1-5, wherein the engineered dsRNA ligase polypeptide comprises a purification tag; optionally, wherein the engineered dsRNA ligase polypeptide comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 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, 10 2, 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 6, 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 and 634.

7. A polypeptide immobilized on a solid material by chemical bonding or physical adsorption, wherein the polypeptide comprises the engineered dsRNA ligase polypeptide according to any one of claims 1 to 6.

8. A polynucleotide encoding the engineered dsRNA ligase polypeptide of any one of claims 1 to 6; optionally wherein: (a) the polynucleotide comprises a nucleic acid sequence selected from the group consisting of SEQ ID NO: 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, 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, 599, 635, 637, 639, 641, 643, 645, 647, 649, 651, 653, 655, 657, 659, 661, 663, 665, and 667; and / or (b) the polynucleotide comprises a nucleic acid sequence selected from the group consisting of: SEQ ID NO: 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、1 81, 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, 256 7, 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.

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

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

11. A method for preparing an engineered dsRNA ligase polypeptide, the method comprising the steps of culturing the host cell according to claim 10 and obtaining the engineered dsRNA ligase polypeptide from the culture.

12. An engineered dsRNA ligase catalyst, which can be obtained by culturing the host cell according to claim 10 or by the method according to claim 11, wherein the engineered dsRNA ligase catalyst comprises cells or culture fluid containing the engineered dsRNA ligase polypeptide, or an article processed therewith, wherein the article is an extract obtained from the culture of the host cell, an isolated product obtained by isolating or purifying the engineered dsRNA ligase from the extract, or an immobilized product obtained by immobilizing the host cell, its extract, or an isolated product of the extract.

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

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

15. The method of claim 13 or the use of claim 14, wherein: (a) the oligonucleotide is at most 60 nucleotides in length; and / or (b) each of the oligonucleotide fragments is 4-16 nucleotides in length, optionally 6-9 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 the oligonucleotide fragments comprises a chemical modification; optionally, wherein the chemical modification is selected from: (i) a modified backbone, optionally selected from phosphorothioate (e.g., chiral phosphorothioate) or methylphosphonate internucleotide linkages; (ii) modified nucleotides, which are optionally selected from 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-D MAEOE), 2'-ON-methylacetamido (2'-O-NMA), locked nucleic acid (LNA), glycol nucleic acid (GNA), phosphamidonate (e.g., phosphamidonyl methyl ester), 2',3'-split nucleotide mimetics, 2'-F-arabinonucleotides, abasic nucleotides, 2'-amino-modified nucleotides, 2'-alkyl-modified nucleotides, morpholino nucleotides, vinylphosphonate (e.g., 5' vinylphosphonate), and cyclopropylphosphonate deoxyribonucleotides; and / or (iii) conjugation to a ligand, optionally wherein the ligand comprises one or more N-acetylgalactosamine (GalNAc) derivatives.

16. A composition comprising: i. The engineered dsRNA ligase polypeptide according to any one of claims 1 to 6; ii. Source of ATP; and iii. divalent cations; Optionally, the composition further comprises two or more oligonucleotide fragments.

17. A kit comprising: i. The engineered dsRNA ligase polypeptide according to any one of claims 1 to 6; ii. Source of ATP; iii. divalent cations; and iv. Instructions for use of a method for generating an oligonucleotide from two or more oligonucleotide fragments.

18. The method of claim 13 or claim 15, the composition of claim 16, or the kit of claim 17, wherein: (A) the ATP source comprises ATP; and / or (B) The ATP source comprises: (a) Polyphosphate kinase (PPK); (b) polyphosphoric acid; and (c) AMP and / or ATP; Optionally wherein: (i) the PPK is selected from PPK12 or ajPAP; and / or (ii) the polyphosphoric acid is a polyphosphate salt, optionally wherein the polyphosphate salt 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+ .

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