Terminal deoxynucleotidyl transferase variants and uses thereof
The present invention is directed to terminal deoxynucleotidyltransferase (TdT) variants that (i) comprise an amino acid sequence that is at least a specified percent identical to an indicated SEQ ID NOs and have at least one substitution at Q455 or at least Q455 plus at least one further substitution at G186, S248, T331, Q390, K394 or H466 (where positions are with respect to SEQ ID NO 1 and functionally equivalent positions in indicated SEQ ID NOs), (ii) are capable of template-free extension of a polynucleotide, and (iii) exhibit enhanced stability or enhanced efficiency in incorporating 3′-0-blocked nucleoside triphosphates into a polynucleotide. The invention is also directed to the use of these TdT variants for synthesizing polynucleotides of any predetermined sequence.
1. A terminal deoxynucleotidyl transferase (TdT) variant comprising an amino acid sequence at least ninety percent identical to the amino acid sequence of SEQ ID NO: 3 with a substitution at positions Q326 and K265, 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 nucleic acid fragment.
2. The TdT variant of claim 1 further comprising one or more of:
(i) a substitution at position H337 with respect to SEQ ID NO 3;
(ii) a substitution at position W377 with respect to SEQ ID NO 3;
(iii) a substitution at position A17 with respect to SEQ ID NO 3;
(iv) a substitution at position G57 with respect to SEQ ID NO 3; or
(v) a substitution at position Q261 with respect to SEQ ID NO: 3.
3. The TdT variant of claim 1 wherein:
(i) said substitution at position Q326 with respect to SEQ ID NO 3 is selected from the group consisting of T, F, L, M, I, V and Y;
(ii) said substitution at position K265 with respect to SEQ ID NO 3 is selected from the group consisting of E, T, A and R.
4. The TdT variant of claim 1 further comprising one or more of:
(i) a substitution at position L52 with respect to SEQ ID NO: 3;
(ii) a substitution at position M63 with respect to SEQ ID NO: 3;
(iii) a substitution at position A108 with respect to SEQ ID NO: 3;
(iv) a substitution at position L131 with respect to SEQ ID NO: 3;
(v) a substitution at position C173 with respect to SEQ ID NO: 3;
(vi) a substitution at position R207 with respect to SEQ ID NO: 3;
(vii) a substitution at position G284 with respect to SEQ ID NO: 3;
(viii) a substitution at position E289 with respect to SEQ ID NO: 3;
(ix) a substitution at position R325 with respect to SEQ ID NO: 3;
(x) a substitution at position E328 with respect to SEQ ID NO: 3; or
(xi) a substitution at position R351 with respect to SEQ ID NO: 3.
5. The TdT variant of claim 1 further comprising one or more of:
(i) a substitution Q37E with respect to SEQ ID NO: 3;
(ii) a substitution D41R with respect to SEQ ID NO: 3;
(iii) a substitution C59R with respect to SEQ ID NO: 3;
(iv) a substitution L60D with respect to SEQ ID NO: 3;
(v) a substitution S94R with respect to SEQ ID NO: 3;
(vi) a substitution G98E with respect to SEQ ID NO: 3;
(vii) a substitution S119A with respect to SEQ ID NO: 3;
(viii) a substitution S146E with respect to SEQ ID NO: 3;
(ix) a substitution Q149R with respect to SEQ ID NO: 3;
(x) a substitution F193Y with respect to SEQ ID NO: 3;
(xi) a substitution V199M with respect to SEQ ID NO: 3; or
(xii) a substitution M201V with respect to SEQ ID NO: 3.
6. The TdT variant of claim 1 comprising the following combination of substitutions with respect to SEQ ID NO: 3:
(i) A17V+Q37E+D41R+L52F+G57E+M63R+S94R+G98E+A108V+S119A+L131R+S 146E+Q149R+C173G+R207L+K265T+G284P+E289V+R325P+Q326F+E328N+H337D+R351K+W377R;
(ii) A17V+Q37E+D41R+L52F+G57E+M63R+S94R+G98E+A108V+S146E+Q149R+C173G+F193Y+V199M+M201V+R207L+K265T+G284P+E289V+R325A+Q326F+E328N+R351K;
(iii) L52F+A108V+R351K+A17V+Q37E+D41R+G57E+C59R+L60D+M63R+S94R+G98E+S119A+L131R+5146E+Q149R+C173G+R207L+K265T+G284P+E289V+R325A+Q326F+E328N;
(iv) L52F+A108V+R351K+A17V+Q37E+D41R+G57E+C59R+L60D+M63R+S94R+G98E+K118Q+S119A+L131R+S146E+Q149R+C173G+R207L+K265T+G284P+E289V+R325A+Q326F+E328N+W377R;
(v) A17V+Q37E+D41R+L52F+G57E+C59R+L60D+M63R+S94R+G98E+A108V+S119A+L131R+5146E+Q149R+C173G+R207L+F259S+Q261L, G284P+E289V+R325A+Q326F+E328N+R351K+W377R;
(vi) A17V+Q37E+D41R+L52F+G57E+C59R+L60D+M63R+S94R+G98E+A108V+S119A+L131R+S146E+Q149R+C173G+R207L+E257D+F259S+K260R+Q261L+G284P+E289V+R325A+Q326F+E328N+R351K+W377R;
(vii) A17V+D41R+L53F+G57E+C59R+L60D+M63R+S94R+G98E+K118Q+S119A+L131R+S146E+Q149R+C173G+R207L+K265T+G284P+E289V+R325A+Q326F+R351K+W377R;
(viii) A17V+D41R+L52F+G57E+C59R+L60D+M63R+S94R+G98E+A108V+S119A+L131R+S146E+Q149R+R207L+K265T+G284P+E289V+R325A+Q326F+R351K;
(ix) A17V+L52F+M63R+A108V+C173G+R207L+K265T+G284P+E289V+R325P+E328N+R351K;
(x) A17V+D41R+L52F+G57E+M63R+S94R+G98E+A108V+S146E+Q149R+C173G+M184T+R207L+K209H+G284L+E289A+R325V+E328K+R351K;
(xi) A17V+L52F+G57E+M63R+A108V+C173G+R207L+K265T+G284P+E289V+R325P+E328N+R351K; or
(xii) A17V+L32T+Q37R+D41R+L52F+G57E+C59R+L60D+M63R+S67A+S94R+G98E+A108V+S119A+L131R+5146E+Q149R+V171A+5172E+C173R+V182I+5183E+R207L+K209H+M210K+T211I+E223G+A224P+E228D+Q261L+G284P+E289V+R325A+Q326F+E328N+R351K+D372E.
7. A method of synthesizing a polynucleotide having a predetermined sequence, the method comprising the steps of:
a) providing an initiator having a 3′-terminal nucleotide having a free 3′-hydroxyl;
b) repeating cycles of (i) contacting under elongation conditions the initiator or elongated fragments having free 3′-O-hydroxyls with a 3′-O-blocked nucleoside triphosphate and a TdT variant according to claim 1 , so that the initiator or elongated fragments are elongated by incorporation of a 3′-O-blocked nucleoside triphosphate to form 3′-O-blocked elongated fragments, and (ii) deblocking the elongated fragments to form elongated fragments having free 3′-hydroxyls, until the polynucleotide is formed.
8. The method of claim 7 , wherein said 3′-O-blocked nucleoside triphosphate is a 3′-O-NH2-nucleoside triphosphate, a 3′-O-azidomethyl-nucleoside triphosphate, a 3′-O-allyl-nucleoside triphosphate, or a 3′-O-(2-nitrobenzyl)-nucleoside triphosphate.
9. A kit for performing a nucleotide incorporation reaction comprising:
a) a variant of TdT according to claim 1 ,
b) one or more nucleotides, preferably one or more 3′O-modified nucleotides, and
c) optionally at least one nucleic acid primer.
10. The TdT variant of claim 2 , wherein:
(i) said substitution of at position H337 with respect to SEQ ID NO: 3 is selected from the group consisting of Y, F, N and D;
(ii) said substitution at position W377 with respect to SEQ ID NO: 3 is R;
(iii) said substitution at position A17 with respect to SEQ ID NO: 3 is V, I or L;
(iv) said substitution at position G57 with respect to SEQ ID NO: 3 is E; and
(v) said substitution at position Q261 with respect to SEQ ID NO: 3 is R.