Terminal deoxynucleotidyl transferase variants and uses thereof
Inventors
Champion, Elise • Loc'h, Jéröme • SOSKINE, Mikhael • Sune, Elodie
Assignees
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Publication Number
US-11859217-B2
Publication Date
2024-01-02
Expiration Date
Abstract
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.
Core Innovation
The disclosed invention relates to terminal deoxynucleotidyl transferase (TdT) variants engineered for template-free polynucleotide synthesis. The variants are defined by amino-acid sequences that are at least 90% identical to SEQ ID NO: 3, with substitutions at positions including Q326 and K265, and they synthesize a nucleic acid fragment without a template while incorporating a 3′-O-modified nucleotide onto a nucleic acid fragment.
A central feature is improved incorporation efficiency of reversibly 3′-O-blocked nucleoside triphosphates and increased enzyme stability. The disclosed variant definitions are based on amino-acid sequence identity to specified SEQ ID NOs, together with substitutions at key residues such as Q455, K394, H466, W506, A146, G186, and Q390 relative to SEQ ID NO: 1, and corresponding substitutions at defined positions relative to other SEQ IDs.
The disclosure also describes predetermined-sequence synthesis by cyclic addition of 3′-O-blocked nucleotides followed by deblocking to regenerate free 3′-hydroxyls. Embodiments include isolated TdT variants, engineered nucleic acid or expression constructs, host cells, and kits comprising TdT variants together with 3′-O-blocked dNTP/rNTP and optional deprotection, solid support, and cleavage reagents.
Claims Coverage
The independent claims cover TdT variants defined by sequence identity to SEQ ID NO: 3 plus specific substitutions, where the variants enable template-free nucleic acid synthesis with incorporation of 3′-O-modified nucleotides. The claims also cover a predetermined-sequence method using iterative cyclic addition of 3′-O-blocked nucleoside triphosphates followed by deblocking.
Template-free TdT variant with Q326 and K265 substitutions
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 is capable of synthesizing a nucleic acid fragment without a template and is capable of incorporating a 3′-O-modified nucleotide onto a nucleic acid fragment.
Predetermined-sequence cyclic synthesis using 3′-O-blocked additions and deblocking
A method synthesizes a predetermined-sequence polynucleotide by using an initiator with a 3′-hydroxyl and repeatedly adding 3′-O-blocked nucleoside triphosphates with a TdT variant under elongation conditions, followed by deblocking to regenerate free 3′-hydroxyls until the polynucleotide is formed.
Selection of 3′-O-blocked nucleoside triphosphate protecting groups
The method uses a 3′-O-blocked nucleoside triphosphate selected from 3′-O-NH2-, 3′-O-azidomethyl-, 3′-O-allyl-, or 3′-O-(2-nitrobenzyl)-nucleoside triphosphates.
Overall, the claims focus on TdT variants defined by sequence identity and substitutions at Q326 and K265 enabling template-free synthesis with incorporation of 3′-O-modified nucleotides, and on predetermined-sequence synthesis by cyclic 3′-O-blocked nucleotide addition followed by deblocking.
Stated Advantages
Enhanced incorporation efficiency of reversibly 3′-O-blocked nucleoside triphosphates.
Increased enzyme stability.
Template-free extension at elevated temperatures.
Documented Applications
Template-free polynucleotide synthesis for predetermined-sequence polynucleotide formation using iterative cyclic addition of 3′-O-blocked nucleosides followed by deblocking.
Use of TdT variants in a kit format comprising the TdT variant together with 3′-O-blocked dNTP/rNTP and optional deprotection, solid support, and cleavage reagents.
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