Modified template-independent enzymes for polydeoxynucleotide synthesis
AssigneeMolecular Assemblies, Inc.
InventorsJ. William Efcavitch, Juliesta E. Sylvester
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Abstract
The invention includes methods for identifying polymerases, such as modified terminal nucleotidyl transferases (TdT), that are capable of binding nucleotides comprising removable 3′-O-blocking moieties to a nucleic acid initiator, without the use of a template. The invention further includes the identified polymerases, and methods of using the polymerases for de novo synthesis of predetermined oligonucleotide sequences.
Claims32
A modified terminal deoxynucleotidyl transferase (TdT) comprising a mutation in the GGFRR or TGSR motifs and capable of adding a nucleotide analog comprising a removable 3′-O-blocking moiety to the 3′-OH of a nucleic acid initiator in the absence of a nucleic acid template.
A composition comprising:
- (a) modified terminal deoxynucleotidyl transferase (TdT) comprising a mutation in the GGFRR or TGSR motifs and capable of adding a nucleotide analog comprising a removable 3′-O-blocking moiety to the 3′-OH of a nucleic acid initiator in the absence of a nucleic acid template;
- and
- (b) a nucleoside triphosphate having a removable 3′-O-blocking moiety.
A method for synthesizing a polynucleotide with a predetermined sequence, comprising:
- (a) providing a nucleic acid linked to a solid support, said nucleic acid comprising a terminal nucleotide having an unprotected 3′-hydroxyl group;
- and
- (b) reacting said nucleic acid linked to a solid support with a nucleoside 5′ triphosphate comprising a removable 3′-O-blocking moiety in the presence of a modified terminal deoxynucleotidyl transferase (TdT) comprising a mutation in the GGFRR or TGSR motifs, thereby adding said nucleotide 5′ triphosphate comprising a removable 3′-O-blocking moiety to said nucleic acid linked to a solid support.
Dependencies are inferred from the claim text.