NUCLEOTIDE ANALOGS

• US 20110081647A1
• Filed: 12/04/2009
• Published: 04/07/2011
• Est. Priority Date: 05/18/2007
• Status: Active Grant

• Current Assignee: Standard Biotools Incorporated
• Original Assignee: Helicos Biosciences Corporation
• Inventors: Marappan, Subramanian, Jaroza, Mirna, Mitchell, Judith, Bowers, Jayson, Efcavitch, J. William, Siddiqi, Suhaib
• Granted Patent: US 9,163,053 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 a nitrogenous base portion by a cleavable linker, wherein 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.

2. The analog according to claim 1, wherein the activating agent is light.

3. The analog according to claim 1, wherein the activating agent is a chemical agent.

4. The analog according to claim 3, wherein the chemical is an alkylating agent.

5. The analog according to claim 4, wherein the alkylating agent is selected from the group consisting of iodoacetamide, haloalkane, haloalkene, haloalkyne, α

-haloketones, α

-haloesters, halopolyetheneglycols, and a combination thereof.

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

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

8. The analog according to claim 1, wherein after cleavage of the linker, a portion of the linker that remains attached to the analog is eliminated by a cyclization reaction, thereby producing the natural nucleotide, the 9-deaza-G, 9-deaza-A, or 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 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; and

to the N5 when the base is ψ

-uridine.

10. 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; and

to C7 when the base is adenine or guanine; and

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

to N5 when the base is ψ

-uridine.

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

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

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

14. The analog according to claim 1, wherein the analog is selected from the group consisting of:

21. 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.

22. The method according to claim 21, further comprising:

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

15. A nucleotide analog comprising a detectable label attached to a nitrogenous base portion of a nucleotide analog by a cleavable linker, wherein contact of the analog with at least one activating agent results in cleavage of the label and a first portion of the linker, wherein a second portion of the linker that remains attached to the analog is neutral in charge.

16. The analog according to claim 15, wherein the second portion of the linker does not contain either an oxygen atom or a nitrogen atom.

17. The analog according to claim 15, wherein the linker comprises a carbamate group.

18. The analog according to claim 15, wherein the detectable label is a fluorescent label.

19. The analog according to claim 18, wherein the fluorescent label is selected from the group consisting of cyanine, rhodamine, fluorescien, coumarin, BODIPY, alexa, Atto and conjugated multi-dyes.

20. The analog according to claim 15, wherein the analog is selected from the group consisting of:

23. 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 15 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 a first portion of the linker, and producing a nucleotide analog wherein a second portion of the linker that remains attached to the analog is neutral in charge; and

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

24. The method according to claim 23, further comprising:

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