2'-nitrobenzyl-modified ribonucleotides

• US 20110053154A1
• Filed: 06/21/2010
• Published: 03/03/2011
• Est. Priority Date: 06/02/2004
• Status: Active Grant

• Current Assignee: Abbott Diagnostics Scarborough, Inc. (Abbott Laboratories Incorporated)
• Original Assignee: TwistDx, Inc.
• Inventors: Piepenburg, Olaf, Armes, Niall A., Stemple, Derek L.
• Granted Patent: US 8,022,194 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids

Abstract

This disclosure provides novel reversibly terminated ribonucleotides which can be used as a reagent for DNA sequencing reactions. Methods of sequencing nucleic acids using the disclosed nucleotides are also provided.

Claims

1. A ribonucleoside having a formula SM-BASE wherein SM is a ribose, BASE is a pyrimidine or purine, and wherein said ribose comprises a reversible chain terminating moiety at a 2′

position in said ribose.

2. The method of claim 1 wherein said base is selected from the group consisting of adenine, guanine, cytosine and uracil.

3. The ribonucleoside of claim 1 which is a ribonucleotide selected from the group consisting of ribonucleoside 5′

-phosphate, a ribonucleoside 5′

-diphosphate or a ribonucleoside 5′

-triphosphate.

5. The ribonucleoside of claim 1 wherein said reversible chain terminating moiety is connected to said ribose by a reversible linkage.

6. The ribonucleoside of claim 5 wherein said reversible linkage is a bond that is cleavable by electromagnetic radiation, chemical treatment, or a combination thereof.

7. The ribonucleoside of claim 6 wherein said electromagnetic radiation is light.

8. The ribonucleoside of claim 1 wherein the reversible chain terminating moiety is a 2-Nitrobenzyl group.

9. The ribonucleoside of claim 1 wherein reversible chain terminating moiety is a Desyl group.

10. The ribonucleoside of claim 1 wherein reversible chain terminating moiety is a p-hydroxyphenacyl caging group.

11. The ribonucleoside of claim 1 wherein said ribonucleotide is labeled with a detectable label.

12. The ribonucleoside of claim 11 wherein said detectable label is a moiety is selected from the group consisting of green fluorescent protein, blue fluorescent protein, red fluorescent protein, beta-galactosidase, chloramphenicol acetyltransferase, beta-glucoronidase, luciferases, b-lactamase, digoxygenin, fluorescent dye molecule, fluorescein, cy3, cy5, alkaline phosphatase and horse radish peroxidase.

13. The ribonucleoside of claim 11 wherein said detectable label may be removed by photobleaching.

14. The ribonucleoside of claim 11 wherein said detectable label is connected to the ribonucleotide by a reversible linkage.

4. (canceled)

15. (canceled)

16. A method of producing a reversibly terminating ribonucleoside comprising the steps of:

(a) providing a ribonucleoside having a formula SM-BASE wherein SM is a ribose, BASE is a pyrimidine or purine;

(b) attaching a reversible chain terminating moiety at a 2′

position of said ribose.

17. The method of claim 16 further comprising the step of attaching a detectable moiety to said ribonucleoside before or after step (b).

18. The method of claim 16 wherein said reversible chain terminating moiety is selected from the group consisting of a 2-Nitrobenzyl group, a Desyl group and a p-hydroxyphenacyl caging group.

19. A method of sequencing a target nucleic acid comprising the steps of

(a) elongating a primer which is complexed with a target nucleic acid to form an incorporated ribonucleotide with a RNA polymerase and at least one first species of ribonucleotide having the formula PM-SM-BASE wherein PM is a phosphate moiety, SM is a ribose, BASE is a pyrimidine or purine, wherein said ribose comprises a chain terminating moiety connected by a reversible linkage at a 2″

position of said ribose;

(b) detecting said incorporated ribonucleotide to determine a sequence of said target nucleic acid.

20. The method of claim 19 further comprising the step of:

(c) removing said chain terminating moiety of said incorporated ribonucleotide by breaking said reversible linkage;

(d) repeating steps (a), (b) and (c) with at least one second species of ribonucleotide having the formula PM-SM-BASE wherein PM is a phosphate moiety, SM is a ribose, BASE is a pyrimidine or purine, wherein said ribose comprises a chain terminating moiety connected by a reversible linkage at a 2′

position of said ribose.

21. The method of claim 19 wherein said complex between said primer and said target nucleic acid is formed by hybridization or synthesis by RNA polymerase.

22. The method of claim 19 wherein said RNA polymerase is a phage-encoded RNA polymerase selected from the group consisting of T3 RNA polymerase, T7 RNA polymerase and SP6 RNA polymerase.

23-28. -28. (canceled)