Template-free enzymatic synthesis of polynucleotides using poly(A) and poly(U) polymerases
AssigneeDNA Script
InventorsTillmann Heinisch, Elise Champion, Elodie Sune, Mikhael Soskine
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Abstract
The present invention is directed to methods and compositions for template-free enzymatic synthesis of a polyribonucleotide of a predetermined sequence from 3′-O-reversibly blocked nucleoside triphosphates using poly(A) and poly(U) polymerases.
Claims7
A kit for performing template-free synthesis of a polyribonucleotide having a predetermined sequence, the kit comprising a poly (A) polymerase (PAP), an initiator attached to a solid support, and 3′-O-blocked ribonucleoside triphosphate monomers,
- wherein the PAP comprises a PAP variant having an amino acid sequence that is at least 90 percent identical to the amino acid sequence of SEQ ID NO:
- 3, wherein the PAP variant comprises a substitution of the methionine at position 318 and a substitution of the valine at position 240 with respect to SEQ ID NO:
- 3, and
- wherein the PAP variant is capable of (a) synthesizing a ribonucleic acid fragment without a template and (b) incorporating a 3′-O-azidomethyl-ribonucleoside triphosphate into a ribonucleic acid fragment or a 3′-O-azidomethyl-2′-deoxyribonucleoside triphosphate into a deoxyribonucleic acid fragment.
A poly (A) polymerase (PAP) variant comprising an amino acid sequence that is at least 90 percent identical to SEQ ID NO:
- 3 wherein the PAP variant comprises a substitution of the methionine
- at position 318 and a substitution of the valine at position 240 with respect to SEQ ID NO:
- 3,
- wherein the PAP variant is capable of (a) synthesizing a ribonucleic acid fragment without a template and (b) incorporating a 3′-O-azidomethyl-ribonucleoside triphosphate into a ribonucleic acid fragment or a 3′-O-azidomethyl-2′-deoxyribonucleoside triphosphate into a deoxyribonucleic acid fragment.
Dependencies are inferred from the claim text.