3′-OH unblocked, fast photocleavable terminating nucleotides and methods for nucleic acid sequencing

• US 8,889,860 B2
• Filed: 09/13/2012
• Issued: 11/18/2014
• Est. Priority Date: 09/13/2011
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: LaserGen, Inc. (Agilent Technologies, Inc.)
• Inventors: Li, Hong, Wu, Weidong, Hersh, Megan N., Hertzog, David, Morris, Sidney E., Metzker, Michael L., Stupi, Brian
• Primary Examiner(s): Murray, Jeffrey H
• Application Number:
  US13/615,284
• Publication Number:
  US 20130122489A1
• Time in Patent Office: 796 Days
• Field of Search: None
• US Class Current: 544/262
• CPC Class Codes: B01L 2300/0654 Lenses; Optical fibres
• CPC Class Codes: B01L 2300/168 Specific optical properties, e.g. reflective coatings
• CPC Class Codes: B01L 3/5027 by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip B01L3/5023 takes precedence; micromixers B01F33/30; microreactors for synthesis B01J19/0093; microcapillary devices in general B81B1/00
• CPC Class Codes: C07H 19/073 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 19/173 with 2-deoxyribosyl as the saccharide radical
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6874 involving nucleic acid arrays, e.g. sequencing by hybridisation
• CPC Class Codes: C12Q 1/6883 for diseases caused by alterations of genetic material
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2600/156 Polymorphic or mutational markers
• CPC Class Codes: C12Q 2600/16 Primer sets for multiplex assays

Abstract

The present invention relates generally to 3′-OH unblocked, labeled and unlabeled nucleotides and nucleosides with 5-methoxy-substituted nitrobenzyl-based photocleavable terminating groups for use in methods and systems related to DNA and RNA sequencing and analysis. These compounds, which may be represented by the following formulas:

wherein the variables are defined herein, may be used as reversible terminators as they exhibit fast nucleotide incorporation kinetics, single-base termination, high nucleotide selectivity, and rapid terminating group cleavage that results in a naturally occurring nucleotide.

Claims

1. A compound of the formula:

2. The compound of claim 1, further defined as a compound of formula I.

3. The compound of claim 1, further defined as a compound of formula II.

4. The compound of claim 1, further defined as a compound of formula III.

5. The compound of claim 1, further defined as a compound of formula IV.

6. The compound of claim 1, further defined as a compound of formula V.

7. The compound of claim 1, further defined as a compound of formula VI.

8. The compound of claim 1, further defined as a compound of formula VII.

9. The compound of claim 1, wherein R₁ is hydroxy.

10. The compound of claim 1, wherein R₁ is a triphosphate.

11. The compound of claim 1, wherein R₁ is an α

-thiotriphosphate.

12. The compound of claim 1, wherein R₂ is hydrogen.

13. The compound of claim 1, wherein R₂ is hydroxy.

14. The compound of claim 1, wherein R₃ is alkyl_(C≦

8).

15. The compound of claim 1, wherein R₃ is isopropyl.

16. The compound of claim 1, wherein R₃ is tert-butyl.

17. The compound of claim 1, wherein R₄ is hydrogen.

18. The compound of claim 1, wherein R₄ is nitro.

19. The compound of claim 1, wherein R₅ is hydrogen.

20. The compound of claim 1, wherein R₅ is iodo.

21. The compound of claim 1, wherein R₅ is alkoxy_(C≦

6).

22. The compound of claim 21, wherein R₅ is methoxy.

23. The compound of claim 1, wherein R₅ is a -linker-reporter.

24. The compound of claim 1, wherein R₅ is a -linker-reporter, wherein the linker is:

25. The compound of claim 24, wherein X is alkynediyl_(C2-8).

26. The compound of claim 25, wherein X is —

C≡

C—

.

27. The compound of claim 24, wherein n is zero.

28. The compound of claim 24, wherein the reporter is:

29. The compound of claim 1, wherein R₆ is hydrogen.

30. The compound of claim 1, wherein the starred carbon atom is in the S configuration.

31. The compound of claim 1, wherein the starred carbon atom is in the R configuration.

32. The compound of claim 1, further defined as:

33. The compound of claim 1, further defined as:

34. The compound of claim 1, further defined as:

35. The compound of claim 1, further defined as: