Variants of terminal deoxynucleotidyl transferase and uses thereof

• US 10,435,676 B2
• Filed: 01/08/2019
• Issued: 10/08/2019
• Est. Priority Date: 01/08/2018
• Status: Active Grant

• Current Assignee: DNA Script SAS, Institut Pasteur, National Centre For Scientific Research
• Original Assignee: DNA Script
• Inventors: Champion, Elise, Soskine, Mikhael, Ybert, Thomas, Delarue, Marc
• Primary Examiner(s): Saidha, Tekchand
• Application Number:
  US16/242,904
• Publication Number:
  US 20190211315A1
• Time in Patent Office: 273 Days
• Field of Search: 435174, 435193
• CPC Class Codes: C12N 9/1264 DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal nucleotidyl transferase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12P 21/02 having a known sequence of two or more amino acids, e.g. glutathione
• CPC Class Codes: C12Y 207/07031 DNA nucleotidylexotransferase (2.7.7.31), i.e. terminal deoxynucleotidyl transferase

Abstract

The present invention relates to a variant of Terminal deoxynucleotidyl Transferase (TdT) which (i) comprises the amino acid sequence as set forth in SEQ ID Nº 2 or a functionally equivalent sequence, with at least an amino acid substitution at position corresponding to residue C302 or functionally equivalent residue, wherein the position is numbered by reference to the amino acid sequence set forth in SEQ ID Nº 1, (ii) is able to synthesize a nucleic acid fragment without template and (iii) is able to incorporate a modified nucleotide into the nucleic fragment.

Claims

1. A variant of Terminal deoxynucleotidyl Transferase (TdT) which (i) comprises amino acids 130 to 510 of the amino acid sequence as set forth in SEQ ID NO:

1, with an amino acid substitution selected from C302G/R/P/AN/S/N/O/D, wherein the position is numbered by reference to the amino acid sequence set forth in SEQ ID NO;

1, (ii) is capable of synthesizing a nucleic acid fragment without template and (iii) is capable of incorporating a modified nucleotide into the nucleic acid fragment.

2. The variant of TdT according to claim 1, wherein the variant further comprises at least two amino acid substitutions corresponding to residues selected from M192, L260, R336, D379, R454 and E457.

3. The variant of TdT according to claim 2, wherein the substitutions are selected from M192R/Q/G/A/V/D/N/H/E, L260P/M/E/N/F/K/D/A/G, R336N/L/K/H/G/D/A/P, D379V/A/G/N/E/R/H/K/T, R454P/N/A/L/K/H/G/D, and E457N/T/S/L/V/K/H/G/D.

4. The variant of TdT according to claim 2, wherein the substitution is selected from M192R/Q, L260P, R336L/N, D379V, R454P/N and E457N/L/T/S.

5. The variant of TdT according to claim 1, wherein the variant further comprises at least one substitution at position corresponding to residues selected from T340, G413, H416, E418, W450, and A510.

6. The variant of TdT according to claim 5, wherein the substitution is selected from T340S/N/Q/C/G/M/K/D, G413L/S/P/R, H416D, E418A/V, W450Y/F/P/L/I/V/A/G/E, and A510V/T/G.

7. The variant of TdT according to claim 1, wherein the variant further comprises at least one substitution at position corresponding to residues selected from L181, A237, L260, T340, G413, H416, E418, W450, R480 and A510.

8. The variant of TdT according to claim 7, wherein the substitution is selected from L181F+A237V+R480K and G413L/S+H416D+E418A.

9. The variant of TdT according to claim 1, wherein the variant further comprises at the N-terminal end or C-terminal end of SEQ ID NO:

1 a tag-sequence.

10. The variant of TdT according to claim 1, wherein the tag-sequence is a His-tag sequence.

11. The variant of TdT according to claim 1, wherein the variant comprises at least a combination of substitution selected from the combinations of substitutions disclosed in table 1.