IP Library › Granted Patent US 12,398,173
Granted Patent B2
US 12,398,173 · App. 17/001,236 · Granted Aug 26, 2025

Enzymatic RNA capping method

Inventors: G. Brett Robb (Somerville, MA); Siu-Hong Chan (Georgetown, MA); Bijoyita Roy (Medford, MA)
Assignee: New England Biolabs, Inc.
C07H19/20C07H21/04C12P19/34C12Q1/6816
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Quick Facts
Patent No.
US 12,398,173
App. No.
17/001,236
Granted
Aug 26, 2025
Kind
B2
Abstract

Provided herein is a method for efficiently capping RNA in vitro. In some embodiments the capping reaction may be done at high temperature using Vaccinia capping enzyme or a variant thereof. In other embodiments, the capping reactions may comprise a capping enzyme from a large virus of amoeba, e.g., Faustovirus, mimivirus or moumouvirus, or a variant thereof. Compositions and kits for practicing the method are also provided.

Claims (31)

1. A composition comprising:

(i) a polynucleotide;

a single-chain RNA capping enzyme that has RNA triphosphatase (TPase), guanylyltransferase (GTase) and guanine-N7 methyltransferase (N7 MTase) activities and comprises an amino acid sequence at least 90% identical to (a) SEQ ID NO:7, (b) SEQ ID NO:8, (c) SEQ ID NO:9, and/or (d) SEQ ID NO:10;

(iii) guanosine triphosphate (GTP);

(iv) a buffering agent; and

(v) a methyl group donor.

2. The composition of claim 1 , wherein the composition has a temperature in the range of 23° C.-60° C.

3. The composition of claim 1 , wherein the composition is RNase-free and optionally comprises (vi) one or more RNase inhibitors.

4. The method of claim 1 , wherein the single-chain RNA capping enzyme comprises an amino acid sequence at least 90% identical to (a) SEQ ID NO:2, (b) SEQ ID NO:3, and/or (c) SEQ ID NO:4.

5. The composition of claim 1 , wherein the polynucleotide comprises a DNA template and the composition further comprises a bacteriophage polymerase and ribonucleotide triphosphates for transcribing the DNA template to form an uncapped target RNA.

6. The composition of claim 1 , wherein the composition optionally comprises (vii) S-adenosyl methionine (SAM) and (viii) a cap 2′O methyltransferase enzyme.

7. The composition of claim 1 , wherein the polynucleotide comprises an uncapped target, the uncapped target RNA comprising one or more pseudouridines and/or one or more m1pseudouridines.

8. The composition of claim 1 further comprising one or more detergents, dyes, solvents and/or preservatives.

9. The composition of claim 1 , wherein the single-chain RNA capping enzyme comprises an amino acid sequence at least 95% identical to (a) SEQ ID NO:7, (b) SEQ ID NO:8, (c) SEQ ID NO:9, and/or (d) SEQ ID NO: 10.

10. A kit comprising:

a single-chain RNA capping enzyme that has RNA triphosphatase (TPase), guanylyltransferase (GTase) and guanine-N7 methyltransferase (N7 MTase) activities and comprises an amino acid sequence at least 90% identical to (a) SEQ ID NO:7, (b) SEQ ID NO:8, (c) SEQ ID NO:9, and/or (d) SEQ ID NO:10, wherein the enzyme is in a storage buffering agent; and a reaction buffering agent.

11. The kit of claim 10 , wherein the kit further comprises a bacteriophage polymerase and ribonucleotides, for transcribing a template polynucleotide encoding a target RNA.

12. The kit of claim 10 , wherein the kit further comprises S-adenosyl methionine (SAM), cap 2′O methyltransferase enzyme (2′OMTase), or both SAM and 2′OMTase.

13. The kit of claim 10 , wherein the single-chain RNA capping enzyme comprises an amino acid sequence at least 90% identical to (a) SEQ ID NO:2, (b) SEQ ID NO:3, and/or (c) SEQ ID NO:4.

14. The kit of claim 10 , wherein the kit further comprises one or more detergents, dyes, solvents and/or preservatives.

15. The kit of claim 10 , wherein the single-chain RNA capping enzyme comprises an amino acid sequence that is at least 95% identical to (a) SEQ ID NO:7, (b) SEQ ID NO:8, (c) SEQ ID NO:9, and/or (d) SEQ ID NO:10.

16. A method for efficiently capping RNA in vitro, comprising:

contacting:

(i) an RNA sample comprising an uncapped target RNA;

a single-chain RNA capping enzyme that has RNA triphosphatase (TPase), guanylyltransferase (GTase) and guanine-N7 methyltransferase (N7 MTase) activities and comprises an amino acid sequence at least 90% identical to (a) SEQ ID NO:7, (b) SEQ ID NO:8, (c) SEQ ID NO:9, and/or (d) SEQ ID NO: 10;

(iii) guanosine triphosphate (GTP) or modified GTP

(iv) a buffering agent; and

(v) a methyl group donor,

at a temperature of 23° C.-60° C., to form a capped target RNA.

17. The method of claim 16 , wherein the single-chain RNA capping enzyme comprises an amino acid sequence at least 90% identical to (a) SEQ ID NO:2, (b) SEQ ID NO:3, and/or (c) SEQ ID NO:4.

18. The method according to claim 16 , further comprising detecting capped target RNA.

Assignments (2)
NOTICE OF GRANT OF SECURITY INTEREST IN PATENTS Recorded Sep 27, 2023
From: NEW ENGLAND BIOLABS, INC.
To: BANK OF AMERICA, N.A., AS ADMINISTRATIVE AGENT
Reel/Frame 065044/0729 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Sep 10, 2020
From: ROBB, G. B.; CHAN, SIU-HONG; ROY, BIJOYITA
To: NEW ENGLAND BIOLABS, INC.
Reel/Frame 053731/0230 →
Continuity (4)
Continuation PCTUS2020047521 · Aug 21, 2020
Continuation PCTUS2020047533 · Aug 21, 2020
Provisional Application 62890821 · Aug 23, 2019
Related Publication 20210054016A1 · Feb 25, 2021
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Cited By (1)
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