Nucleotide analogs

• US 9,163,053 B2
• Filed: 12/04/2009
• Issued: 10/20/2015
• Est. Priority Date: 05/18/2007
• Status: Active Grant

• Current Assignee: Standard Biotools Incorporated
• Original Assignee: Fluidigm Corporation
• Inventors: Siddiqi, Suhaib, Efcavitch, J. William, Mitchell, Judith, Marappan, Subramanian, Bowers, Jayson, Jaroza, Mirna
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US12/631,074
• Publication Number:
  US 20110081647A1
• Time in Patent Office: 2,146 Days
• Field of Search: 536/4.1, 536/23.1, 536/26.6, 435/6.1, 435/91.1
• US Class Current: 1/1
• CPC Class Codes: A61K 49/0041 Xanthene dyes, used in vivo, e.g. administered to a mice, e.g. rhodamines, rose Bengal in vivo G01N
• CPC Class Codes: A61K 49/0052 Small organic molecules oligomers, polymers, dendrimers A61K49/0054
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 1/6876 Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
• CPC Class Codes: C12Q 2563/107 fluorescence

Abstract

The invention generally relates to nucleotide analogs and methods of their use in sequencing-by-synthesis reactions. In certain embodiments, the invention provides a nucleotide analog including a detectable label attached to a nitrogenous base portion of a nucleotide analog by a cleavable linker, in which contact of the analog with at least one activating agent results in cleavage of the label and elimination of the linker, thereby producing a natural nucleotide, a 9-deaza-G, 9-deaza-A, or ψ-uridine.

Claims

1. A nucleotide analog comprising a detectable label attached to the nitrogenous base portion of the nucleotide by a cleavable linker,

wherein contact of the analog with a reducing agent results in release of the label and elimination of the linker, thereby producing a natural nucleotide, 9-deaza-G, 9-deaza-A, or ψ

-uridine, characterized in that no portion of the label or the linker remains attached to the nitrogenous base after cleavage.

2. The analog according to claim 1, wherein the

reducing agent is selected from dithiothreitol (DTT), β

-mercaptoethanol, dithioerythretol (DTE), GSH, cysteine, cysteamine, tricarboxyethyl phosphine (TCEP), tris(3-hydroxypropyl)phosphine (THPP), glutathione, and salts of sulfurous acid.

3. The analog according to

claim 1, wherein the reducing agent is dithiothreitol (DTT).

4. The analog according to

claim 1, wherein the reducing agent is tricarboxyethyl phosphine (TCEP).

5. The analog according to claim 1, wherein the linker is a Staudinger linker.

6. The analog according to claim 1, wherein the linker comprises a disulfide bond.

7. The analog according to claim 1, wherein after cleavage of the linker by contact with the reducing agent, a portion of the linker temporarily remains attached to the analog and is thereafter spontaneously eliminated by a cyclization reaction, thereby producing the natural nucleotide, the 9-deaza-G, the 9-deaza-A, or the ψ

-uridine.

8. The analog according to claim 1, wherein the linker is attached to the nitrogenous base portion of the analog as follows:

to N3 of the base when the base is thymine or uracil;

to N4 of the base when the base is cytosine or adenine;

to N2 of the base when the base is guanine;

to N7 of the base when the base is 9-deaza-G or 9-deaza-A;

or

to the N5 when the base is ψ

-uridine.

9. The analog according to claim 1, wherein the linker is attached to the nitrogenous base portion of the analog as follows:

to C5 of the base when the base is thymine, cytosine, or uracil;

to C7 when the base is adenine or guanine;

to N7 when the base is 9-deaza-guanine or 9-deaza-adenine;

or

to N5 when the base is ψ

-uridine.

10. The analog according to claim 1, wherein the detectable label is a fluorescent label.

11. The analog according to claim 10, wherein the fluorescent label is selected from cyanine, rhodamine, fluorescein, coumarin, BODIPY, alexa, Atto and conjugated multi-dyes.

12. The analog according to claim 1, wherein the detectable label is a non-optically detectable label.

14. A method of sequencing a nucleic acid, the method comprising:

directly or indirectly anchoring a nucleic acid duplex to a surface, the duplex comprising a template portion and a primer portion hybridized thereto;

exposing the duplex to a nucleotide analog according to claim 1 in the presence of a polymerase capable of catalyzing the addition of the nucleotide analog to the primer portion in a template-dependent manner;

detecting incorporation of the nucleotide analog into the primer portion;

contacting the nucleic acid duplex with at least one activating agent, thereby cleaving the detectable label and eliminating the linker to produce a natural nucleotide or a 9-deaza-G; and

repeating the exposing, detecting, and contacting steps at least once.

15. The method according to claim 14, further comprising:

removing unincorporated nucleotide analog and polymerase in all or some repetitions of the exposing, detecting, and contacting steps.

16. The analog according to claim 1, wherein the nitrogenous base is a purine, the linker comprises a disulfide bond (—

S—

S—

), and the linker is attached to the purine through a nitrogen atom in a ring of the purine.

17. The analog according to claim 1, comprising a structure selected from:

18. The analog according to claim 1, wherein the nitrogenous base is a purine, the linker comprises a disulfide bond, and the linker is attached to the purine through a carbon atom in a ring of the purine.

19. The analog according to claim 1, comprising a structure selected from:

20. The analog according to claim 1, wherein the nitrogenous base is a pyrimidine, the linker comprises a disulfide bond, and the linker is attached to the pyrimidine through a nitrogen atom in the ring of the pyrimidine.

21. The analog according to claim 1, comprising a structure selected from:

22. The analog according to claim 1, wherein the nitrogenous base is a pyrimidine, the linker comprises a disulfide bond, and the linker is attached to the pyrimidine through a carbon atom in the ring of the pyrimidine.

23. The analog according to claim 1, comprising a structure selected from:

24. The analog according to claim 1, wherein the linker comprises an azide group (—

N₃).

25. The analog according to claim 1, comprising a structure selected from:

26. The analog according to claim 1, wherein the linker comprises a sulfenamide bond (—

S—

N—

).

27. The analog according to claim 1, comprising a structure selected from:

28. The analog according to claim 1, comprising an inhibitor that is configured so that the analog may be added to a nucleic acid primer by a polymerase, whereupon it inhibits the polymerase from adding a further nucleotide to the primer.

29. The analog according to claim 28, configured so that the inhibitor is released along with the detectable label upon cleavage of the cleavable linker.

30. The analog according to claim 1, wherein the natural nucleotide is adenine, guanine, thymine, cytosine, or uracil.

31. The analog according to claim 1, incorporated into an oligonucleotide.

32. A method of releasing a label from an analog according to claim 1, comprising contacting the analog with a reducing agent, thereby producing a natural nucleotide, 9-deaza-G, 9-deaza-A, or ψ

-uridine with no portion of the label or the linker remaining after cleavage.

33. A method of releasing a label from an analog according to claim 31, comprising contacting the oligonucleotide with a reducing agent, thereby producing a natural nucleotide, 9-deaza-G, 9-deaza-A, or ψ

-uridine from the analog with no portion of the label or the linker remaining after cleavage.

34. A method of sequencing a nucleic acid, comprising iteratively carrying out the steps of adding a nucleotide analog according to claim 1 in a template-dependent manner to a primer that is duplexed with the nucleic acid, detecting incorporation of the nucleotide analog into the duplex, and then contacting the duplex with a reducing agent, thereby producing a natural nucleotide, 9-deaza-G, 9-deaza-A, ψ

-uridine from the added nucleotide analog with no portion of the label or the linker remaining after cleavage.

35. The method of claim 34, wherein the nucleic acid primer duplex is attached to a surface.

36. The method of claim 34, wherein no more than one nucleotide analog is added to the duplex in any single iteration.

37. The analog according to any one of claims 17, 19, 21, 23, 25, and 27, comprising an inhibitor that is configured so that the analog may be added to a nucleic acid primer by a polymerase, whereupon it inhibits the polymerase from adding a further nucleotide to the primer.

38. The analog according to claim 37, configured so that the inhibitor is released along with the detectable label upon cleavage of the cleavable linker.

13. A nucleotide analog comprising a structure selected from: