Dna Script

Institut Pasteur

Centre National De La Recherche Scientifique

Dna Script Sas

Other

National Center for Scientific Research (cnrs)

Katholieke Universiteit Leuven

Rijksuniversiteit Groningen

Mikhael Soskine

Elise Champion

Thomas Ybert

Marc Delarue

Annemie Biesemans

Veerle Van Meervelt

Jéröme Loc'h

Elodie Sune

Weidong Wu

Ilya Aparin

Adeline Veillet

Jérôme Loc'h

Giovanni Maglia

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.

Terminal deoxynucleotidyl transferase variants and uses thereof

The present invention relates to variants of Terminal deoxynucleotidyl Transferase (TdT), each of which (i) has an amino acid sequence similarity to SEQ ID NO: 2, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 with corresponding amino acid substitutions, (ii) is capable of synthesizing a nucleic acid fragment without a template and (iii) is capable of incorporating a modified nucleotide into the nucleic acid fragment.

Variants of terminal deoxynucleotidyl transferase and uses thereof

This disclosure relates to an enzymatic method of synthesizing a polynucleotide, comprising a deprotecting step which uses as a deprotecting agent a specific phosphonate compound. It also pertains to a method for deprotecting 3'-O-amino elongated fragments of a polynucleotide in an enzymatic method of synthesizing a polynucleotide, comprising contacting the elongated fragments with this phosphonate compound. This disclosure further relates to a kit for synthesizing a polynucleotide comprising one or more vials of synthesis reagents, at least one of which contains an effective amount of a phosphonate compound and to a specific method for preparing said phosphonate compound.

Reagents and methods used in deprotection of 3'-O-amino polynucleotides

The present invention relates to variants of Terminal deoxynucleotidyl Transferase (TdT), each of which (i) has an amino acid sequence similarity to SEQ ID NO: 2. 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33 or 35 with corresponding amino acid substitutions, (ii) is capable of synthesizing a nucleic acid fragment without a template and (iii) is capable of incorporating a modified nucleotide into the nucleic acid fragment.

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.

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 N&#xba; 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 N&#xba; 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.

Growing community of inventors

Franconville, France

Mikhael Soskine