Splint-assisted enzymatic synthesis of polyribounucleotides

• US 20050130201A1
• Filed: 10/13/2004
• Published: 06/16/2005
• Est. Priority Date: 10/14/2003
• Status: Abandoned Application

• Current Assignee: Dharmacon Incorporated (Revvity, Inc.)
• Original Assignee: Dharmacon Incorporated (Revvity, Inc.)
• Inventors: Deras, Michael, Pleiss, Jeffrey A., Scaringe, Stephen
• Application Number:
  US10/965,547
• Publication Number:
  US 20050130201A1
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C12Q 1/6816 characterised by the detection means C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 2521/501 Ligase
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone

Abstract

The present invention comprises methods and compositions for splint-assisted enzymatic synthesis of polyribonucleotides using an RNA polymerizing enzyme. The invention provides ligating ribonucleotides comprising ligating a donor RNA molecule to an acceptor RNA molecule in the presence of RNA ligase and a splint, wherein the donor RNA molecule is comprised of at least one nucleotide and a ligation linker moiety, the acceptor RNA molecule is comprised of at least one nucleotide and a ligation linker moiety and the splint is comprised of a polyribonucleotide. The invention also provides splints for use in splint-assisted enzymatic synthesis using an RNA polymerizing enzyme.

Claims

1. A method of ligating ribonucleotides comprising:

ligating a donor RNA molecule to an acceptor RNA molecule in the presence of RNA ligase and a splint, wherein the donor RNA molecule is comprised of at least one nucleotide and a ligation linker moiety, the acceptor RNA molecule is comprised of at least one nucleotide and a ligation linker moiety and the splint is comprised of a polyribonucleotide.

2. The method of claim 1, wherein the donor RNA molecule further comprises at least one modified nucleotide.

3. The method of claim 2, wherein the at least one modified nucleotide is an orthoester modified nucleotide.

4. The method of claim 3, wherein the orthoester modified nucleotide comprises an orthoester moiety bonded to a 2′

carbon of said at least one nucleotide of the donor RNA.

5. The method of claim 4, wherein the orthoester modified nucleotide comprises an orthoester moiety bonded to the 2′

carbon of every nucleotide the donor RNA.

6. The method of claim 1, wherein the splint further comprises at least one modified nucleotide.

7. The method of claim 6, wherein the at least one modified nucleotide is an orthoester modified nucleotide.

8. The method of claim 7, wherein the orthoester modified nucleotide comprises an orthoester moiety bonded to a 2′

carbon of said at least one nucleotide of the splint.

9. The method according to claim 1, wherein the splint comprises an orthoester moiety bonded to the 2′

carbon of each nucleotide of the splint.

10. The method of claim 1, wherein said ligating proceeds to at least 80% completion.

11. The method of claim 10, wherein said ligating proceeds to at least 85% completion.

12. The method according to claim 1, wherein the splint further comprises a spacer.

13. The method according to claim 16, wherein the spacer is comprised of purines.

14. The method of claim 1, wherein the acceptor RNA molecule further comprises at least one modified nucleotide.

15. The method according to claim 18, wherein the at least one modified nucleotide is an orthoester modified nucleotide.

16. The method according to claim 1, wherein the acceptor RNA molecule has an orthoester moiety bonded to the 2′

carbon of each ribosyl moiety.

17. The method according to claim 1, wherein the acceptor RNA molecule further comprises a 3′

terminal —

OH.

18. The method according to claim 1, wherein the donor RNA molecule further comprises a 5′

terminal phosphate moiety.

19. The method according to claim 1, wherein the length of the ligation linker of the acceptor RNA is greater than or equal to four nucleotide bases.

20. The method according to claim 1, wherein the length of the ligation linker of the donor RNA molecule is greater than or equal to two nucleotide bases.

21. The method according to claim 25, wherein the length of the ligation linker of the donor RNA molecule is eleven to twelve nucleotide bases.

22. The method according to claim 1, wherein the donor RNA molecule and the acceptor RNA molecule each have a Tm with respect to the splint of at least 45 degrees Centigrade.

23. The method according to claim 1, wherein said ligating is carried out in the presence of a pyrophosphatase.

24. The method according to claim 1, wherein the acceptor RNA molecule further comprises a first modified nucleotide, and the donor RNA molecule comprises a second modified nucleotide that is different from the first modified nucleotide.

25. The method according to claim 1, wherein the splint comprises a polyribonucleotide having a blocked 3′

terminus.

26. A splint for use in ligating RNA molecules, comprising:

a polyribonucleotide, wherein said polyribonucleotide has at least one orthoester modified nucleotide base.

27. The splint according to claim 31, further comprising a spacer.

28. The splint according to claim 31, wherein the at least one orthoester modified nucleotide base is a 2′

orthoester modified nucleotide base.