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

• US 10,041,115 B2
• Filed: 05/23/2017
• Issued: 08/07/2018
• Est. Priority Date: 09/13/2011
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: LaserGen, Inc. (Agilent Technologies, Inc.)
• Inventors: Stupi, Brian P., Li, Hong, Wu, Weidong, Hersh, Megan N., Hertzog, David, Morris, Sidney E., Metzker, Michael L.
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US15/602,400
• Publication Number:
  US 20170327886A1
• Time in Patent Office: 441 Days
• Field of Search: 435 61, 536 243
• 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 nucleotides and nucleosides labeled and unlabeled 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 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 method comprising:

exposing a compound, a biological component, or a non-biological component to a light source to remove a photocleavable moiety of the formula;

2. The method of claim 1, wherein about 85% to about 100% of the photocleavable moieties are removed by exposure to the light source.

3. The method of claim 1, wherein the light source emits UV light.

4. The method of claim 3, wherein the UV light comprises 365 nm wavelength light.

5. The method of claim 1, wherein the compound, the biological component, or the non-biological component is a nucleobase, a nucleoside, a nucleotide, or a nucleic acid molecule, each of which modified with the photocleavable moiety.

6. The method of claim 1, wherein a product of the photocleavage step comprises a hydroxymethyl nucleoside, hydroxymethyl nucleotide or a hydroxymethyl nucleotide incorporated into a nucleic acid molecule.

7. The method of claim 1, wherein the compound, the biological component, or the non-biological component is a protein that has been modified with the photocleavable moiety.

8. The method of claim 1, wherein R₃ is iso-propyl.

9. The method of claim 1, wherein R₃ is tert-butyl.

10. The method of claim 1, wherein R₄ is hydrogen.

11. The method of claim 1, wherein R₅ is hydrogen, iodo, or methoxy.

12. The method of claim 1, wherein R₅ is a group of formula:

13. The method of claim 12, wherein X is —

C≡

C— and

n is zero.

14. The method of claim 1, wherein R₅ is a -linker-reporter.

15. The method of claim 14, wherein the linker is:

16. The method of claim 15, wherein X is —

C≡

C— and

n is zero.

17. The method of claim 14, wherein the reporter is based on a dye, wherein the dye is zanthene, fluorescein, rhodamine, BODIPY, cyanine, coumarin, pyrene, phthalocyanine, phycobiliprotein, or a squaraine dye.

18. The method of claim 14, wherein the reporter is:

19. The method of claim 1, wherein R₆ is hydrogen.

20. A product prepared by a process comprising the method of claim 1, wherein the product is derived from the photocleavable moiety and comprises a nitroso group and a ketone group.

21. A compound of the formula:

22. The compound of claim 21, wherein R₃ is alkyl_(C3-4).

23. The compound of claim 22, wherein R₃ is iso-propyl.

24. The compound of claim 22, wherein R₃ is tert-butyl.

25. The compound of claim 22, wherein R₄ is hydrogen.

26. The compound of claim 22, wherein R₅ is hydrogen, iodo, or methoxy.