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US 12,703,860 B2Granted
Terminal deoxynucleotidyl transferase variants and uses thereof
AssigneeDNA Script
InventorsMikhael Soskine, Elise Champion
Life of this patent
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Abstract
The present invention is directed to terminal deoxynucleotidyl transferase (TdT) variants from a variety of species which display enhanced efficiency in incorporating reversibly blocked nucleoside triphosphates into a polynucleotide, and to the use of such TdTs in synthesizing polynucleotides of any predetermined sequence.
Claims9
A terminal deoxynucleotidyl transferase (TdT) variant comprising an amino acid sequence at least ninety percent identical to an amino acid sequence selected from SEQ ID NO:
- 2, 5, 8, 11, 14, 17, 20, 23 26, 29, 32, 35, 38, 41, 44 or 47, wherein:
- with respect to SEQ ID NO:
- 2 leucine at position 61 is substituted, cysteine at position 170 is substituted, arginine at position 204 is substituted, arginine at position 326 is substituted and glycine at position 329 is substituted;
- with respect to SEQ ID NO:
- 5 leucine at position 48 is substituted, cysteine at position 158 is substituted, arginine at position 192 is substituted, arginine at position 314 is substituted and glycine at position 317 is substituted;
- with respect to SEQ ID NO:
- 8 leucine at position 61 is substituted, tyrosine at position 171 is substituted, arginine at position 205 is substituted, arginine at position 327 is substituted and glutamic acid at position 329 is substituted;
- with respect to SEQ ID NO:
- 11 leucine at position 61 is substituted, tyrosine at position 171 is substituted, arginine at position 205 is substituted, arginine at position 324 is substituted and glutamic acid at position 327 is substituted;
- with respect to SEQ ID NO:
- 14 glycine at position 61 is substituted, arginine at position 205 is substituted, arginine at position 327 is substituted and glutamic acid at position 330 is substituted;
- with respect to SEQ ID NO:
- 17 arginine at position 61 may or may not be substituted, alanine at position 158 is substituted, arginine at position 192 is substituted, arginine at position 311 is substituted and glutamic acid at position 314 is substituted;
- with respect to SEQ ID NO:
- 20 arginine at position 61 may or may not be substituted, proline at position 171 may or may not be substituted, arginine at position 205 is substituted, threonine at position 321 is substituted and glutamic acid at position 324 is substituted;
- with respect to SEQ ID NO:
- 23 arginine at position 61 may or may not be substituted, alanine at position 171 may or may not be substituted, arginine at position 205 is substituted, alanine at position 322 may or may not be substituted and glutamic acid at position 325 is substituted;
- with respect to SEQ ID NO:
- 26 arginine at position 61 may or may not be substituted, alanine at position 171 may or may not be substituted, arginine at position 205 is substituted, arginine at position 322 is substituted and glutamic acid at position 325 is substituted;
- with respect to SEQ ID NO:
- 29 methionine at position 48 is substituted, cysteine at position 158 is substituted, arginine at position 192 is substituted, arginine at position 310 is substituted and glutamic acid at position 313 is substituted;
- with respect to SEQ ID NO:
- 32 methionine at position 61 is substituted, cysteine at position 171 is substituted, arginine at position 205 is substituted, arginine at position 323 is substituted and glutamic acid at position 326 is substituted;
- with respect to SEQ ID NO:
- 35 methionine at position 61 is substituted, cysteine at position 171 is substituted, arginine at position 205 is substituted, arginine at position 323 is substituted and glutamic acid at position 326 is substituted;
- with respect to SEQ ID NO:
- 38 methionine at position 61 is substituted, cysteine at position 171 is substituted, arginine at position 205 is substituted, arginine at position 323 is substituted and glutamic acid at position 326 is substituted;
- with respect to SEQ ID NO:
- 41 methionine at position 61 is substituted, cysteine at position 171 is substituted, arginine at position 205 is substituted, arginine at position 323 is substituted and glutamic acid at position 326 is substituted;
- with respect to SEQ ID NO:
- 44 methionine at position 48 is substituted, cysteine at position 158 is substituted, arginine at position 192 is substituted, arginine at position 309 is substituted and glutamic acid at position 312 is substituted;
- or
- with respect to SEQ ID NO:
- 47 methionine at position 61 is substituted, cysteine at position 171 is substituted, arginine at position 205 is substituted, arginine at position 323 is substituted and glutamic acid at position 326 is substituted;
- and
- wherein the TdT variant
- (i) is capable of synthesizing a nucleic acid fragment without a template and
- (ii) (ii) is capable of incorporating a 3′-O-modified nucleotide onto a free 3′-hydroxyl of a nucleic acid fragment.
35 more elements
A terminal deoxynucleotidyl transferase (TdT) variant comprising an amino acid sequence at least ninety percent identical to an amino acid sequence selected from SEQ ID NO:
- 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45 or 48, wherein:
- with respect to SEQ ID NO:
- 3 amino acid position 61 is R, amino acid position 171 is R, amino acid position 205 is L, amino acid position 327 is A, and amino acid position 330 is N;
- with respect to SEQ ID NO:
- 6 amino acid position 48 is R, amino acid position 158 is R, amino acid position 192 is L, amino acid position 314 is P, and amino acid position 317 is N;
- with respect to SEQ ID NO:
- 9 amino acid position 61 is R, amino acid position 171 is R, amino acid position 205 is L, amino acid position 327 is A, and amino acid position 330 is N;
- with respect to SEQ ID NO:
- 12 amino acid position 61 is R, amino acid position 171 is A, amino acid position 205 is L, amino acid position 324 is P, and amino acid position 327 is N;
- with respect to SEQ ID NO:
- 15 amino acid position 61 is R, amino acid position 171 is R, amino acid position 205 is L, amino acid position 328 is P, and amino acid position 331 is N;
- with respect to SEQ ID NO:
- 18 amino acid position 48 is R, amino acid position 158 is R, amino acid position 192 is L, amino acid position 311 is P, and amino acid position 314 is N;
- with respect to SEQ ID NO:
- 21 amino acid position 61 is R, amino acid position 171 is P, amino acid position 205 is L, amino acid position 321 is A, and amino acid position 324 is N;
- with respect to SEQ ID NO:
- 24 amino acid position 61 is R, amino acid position 171 is A, amino acid position 205 is L, amino acid position 322 is A, and amino acid position 325 is N;
- with respect to SEQ ID NO:
- 27 amino acid position 61 is R, amino acid position 171 is R, amino acid position 205 is L, amino acid position 327 is A, and amino acid position 330 is N;
- with respect to SEQ ID NO:
- 30 amino acid position 48 is R, amino acid position 158 is R, amino acid position 192 is L, amino acid position 310 is P, and amino acid position 313 is N;
- with respect to SEQ ID NO:
- 33 amino acid position 61 is R, amino acid position 171 is R, amino acid position 205 is L, amino acid position 323 is P, and amino acid position 326 is N;
- with respect to SEQ ID NO:
- 36 amino acid position 61 is R, amino acid position 171 is R, amino acid position 205 is L, amino acid position 323 is A, and amino acid position 326 is N;
- with respect to SEQ ID NO:
- 39 amino acid position 61 is R, amino acid position 171 is R, amino acid position 205 is L, amino acid position 323 is P, and amino acid position 326 is N;
- with respect to SEQ ID NO:
- 42 amino acid position 61 is R, amino acid position 171 is R, amino acid position 205 is L, amino acid position 323 is A, and amino acid position 326 is N;
- with respect to SEQ ID NO:
- 45 amino acid position 48 is R, amino acid position 158 is R, amino acid position 192 is L, amino acid position 310 is A, and amino acid position 313 is N;
- or
- with respect to SEQ ID NO:
- 48 amino acid position 61 is R, amino acid position 171 is R, amino acid position 205 is L, amino acid position 323 is P, and amino acid position 326 is N;
- and
- wherein the TdT variant
- (i) is capable of synthesizing a nucleic acid fragment without a template and
- (ii) (ii) is capable of incorporating a 3′-O-modified nucleotide onto a free 3′-hydroxyl of a nucleic acid fragment.
35 more elements
A terminal deoxynucleotidyl transferase (TdT) variant comprising an amino acid sequence at least ninety percent identical to an amino acid sequence selected from SEQ ID NO:
- 3, 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36, 39, 42, 45 or 48, and wherein the TdT variant
- (i) is capable of synthesizing a nucleic acid fragment without a template and
- (ii) is capable of incorporating a 3′-O-modified nucleotide onto a free 3′-hydroxyl of a polynucleotide.
Dependencies are inferred from the claim text.