IP Library › Granted Patent US 12,584,154
Granted Patent B2
US 12,584,154 · App. 17/629,316 · Granted Mar 24, 2026

Template-free enzymatic synthesis of polynucleotides using poly(A) and poly(U) polymerases

Inventors: Tillmann Heinisch (Le Kremlin-Bicêtre, FR); Elise Champion (Paris, FR); Elodie Sune (Le Kremlin-Bicêtre, FR); Mikhael Soskine (Franconville, FR)
Assignee: DNA Script
C12P19/34C12N9/1264C12Y207/07031
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Quick Facts
Patent No.
US 12,584,154
App. No.
17/629,316
Granted
Mar 24, 2026
Kind
B2
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.

Claims (11)

1 . 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.

2 . The kit of claim 1 , wherein said 3′-O-protected ribonucleoside triphosphate monomers comprise one or more of 3′-O-azidomethyl-riboadenosine triphosphate, 3′-O-azidomethyl-riboguanosine triphosphate, 3′-O-azidomethyl-ribocytidine triphosphate, 3′-O-azidomethyl-ribothymidine triphosphate and 3′-O-amino-ribouridine triphosphate.

3 . 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.

4 . The PAP variant of claim 3 , wherein said substitution of the methionine at position 318 is selected from F, Y, V, E or T.

5 . The PAP variant of claim 3 wherein said substitution of the valine at position 240 is alanine or glycine.

6 . The PAP variant of claim 3 , wherein said substitution of the methionine at position 318 is T and said substitution of the valine at position 240 is A.

7 . The PAP variant of claim 6 , further comprising a substitution of the alanine at position 410 relative to SEQ ID NO: 3.

Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Jun 7, 2022
From: HEINISCH, TILLMANN; CHAMPION, ELISE; SUNE, ELODIE; SOSKINE, MIKHAEL
To: DNA SCRIPT SAS
Reel/Frame 060125/0323 →
Priority Claims (2)
EP 19189192 · Jul 30, 2019 · regional
EP 20170323 · Apr 20, 2020 · regional
Continuity (1)
Related Publication 20220403434A1 · Dec 22, 2022
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