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

• US 9,689,035 B2
• Filed: 06/21/2016
• Issued: 06/27/2017
• Est. Priority Date: 09/13/2011
• Status: Active Grant

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: LaserGen, Inc. (Agilent Technologies, Inc.)
• Inventors: Morris, Sidney E., Metzker, Michael L., Stupi, Brian P., Li, Hong, Wu, Weidong, Hersh, Megan N., Hertzog, David
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US15/188,283
• Publication Number:
  US 20160369336A1
• Time in Patent Office: 371 Days
• Field of Search: 435 612
• 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 system comprising:

a flowcell comprising a plurality of beads, wherein;

each bead attached to a DNA or RNA molecule, wherein a compound according to any one of formulas I-VII has been incorporated into the DNA or RNA molecule using a polymerase, wherein formulas I-VII are defined as follows;

2. The system of claim 1, wherein the flowcell is a microfluidic flowcell.

3. The system of claim 1, further comprising an objective lens between the filter wheel and the flowcell.

4. The system of claim 1, further comprising a mirror configured to direct the light path to the imaging device.

5. The system of claim 1, wherein the compound is further defined as a compound of formula (I).

6. The system of claim 1, wherein the compound is further defined as a compound of formula (II).

7. The system of claim 1, wherein the compound is further defined as a compound of formula (III).

8. The system of claim 1, wherein the compound is further defined as a compound of formula (IV).

9. The system of claim 1, wherein the compound is further defined as a compound of formula (V).

10. The system of claim 1, wherein the compound is further defined as a compound of formula (VI).

11. The system of claim 1, wherein the compound is further defined as a compound of formula (VII).

12. The system of claim 1, wherein R₁ is a triphosphate.

13. The system of claim 1, wherein R₁ is an α

-thiotriphosphate.

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

8).

15. The system of claim 14, wherein R₃ is alkyl_(C3-4).

16. The system of claim 15, wherein R₃ is tert-butyl.

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

18. The system of claim 1, wherein the linker of the -linker-reporter is:

19. The system of claim 18, wherein X is —

C≡

C—

.

20. The system of claim 18, wherein n is zero.

21. The system of claim 1, wherein the dye is a zanthene, fluorescein, rhodamine, BODIPY, cyanine, coumarin, pyrene, phthalocyanine, phycobiliprotein, or a squaraine dye.

22. The system of claim 1, wherein the reporter is:

23. The system of claim 1, wherein R₆ is hydrogen.

24. The system of claim 1, wherein the starred carbon atom is in the S configuration.