Propargyl substituted nucleoside compounds and methods

• US 7,998,706 B2
• Filed: 12/17/2004
• Issued: 08/16/2011
• Est. Priority Date: 12/23/2003
• Status: Active Grant

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Inventors: Jeon, Geun-sook, Khan, Shaheer H., Rosenblum, Barnett B.
• Primary Examiner(s): Crane; Lawrence E
• Application Number:
  US11/015,319
• Publication Number:
  US 20050170388A1
• Time in Patent Office: 2,433 Days
• Field of Search: 536/25.32, 536/26.23, 536/26.26, 536/26.7, 536/26.8, 536/28.5, 536/28.53, 435/6
• US Class Current: 435/91.1
• CPC Class Codes: C07H 19/00 Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/02 with ribosyl as saccharide radical
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

Disclosed, among other things, are compounds having the structure

wherein X comprises a bond or a linker, LABEL comprises at least one detectable label, W₁ taken alone is —H or —OH, W₂ is —OH or a non-extendable moiety, W₃ when taken alone is —H or when taken together with W₁ is —CH₂—O—, and W₄ is OH, monophosphate, diphosphate, or triphosphate. Also disclosed are labeled polynucleotide compounds and methods of use thereof.

Claims

1. A method of extending a polynucleotide comprising

forming a hybridization complex between a 3′

-extendable polynucleotide and a complementary sequence in a template polynucleotide, and

extending the 3′

-extendable polynucleotide in the presence of at least one labeled nucleotide 5′

-triphosphate and a template-dependent nucleic acid polymerase under conditions effective to append at least one labeled nucleotide to the 3′

-end of the extendable polynucleotide,

wherein said at least one labeled nucleotide 5′

-triphosphate comprises a labeled nucleotide 5′

-triphosphate having the structure

2. The method of claim 1, wherein the 3′

-extendable polynucleotide formed is at least 10-500 nucleotides in length.

3. The method of claim 1, wherein W₂ is non-extendable.

4. The method of claim 1, wherein the polymerase comprises a DNA polymerase.

5. The method of claim 1, wherein W₁ is H.

6. The method of claim 1, wherein W₂ is H.

7. The method of claim 1, wherein W₂ is azido.

8. The method of claim 1, wherein W₂ is amino.

9. The method of claim 1, wherein W₂ is fluoro.

10. The method of claim 1, wherein W₂ is chloro.

11. The method of claim 1, wherein W₂ is methoxy.

12. The method of claim 1, wherein W₁ is —

OH.

13. The method of claim 1, wherein W₂ is —

OH.

14. The method of claim 1, wherein LABEL is the detectable label selected from the group consisting of a fluorescent dye, an energy transfer dye, a quencher, and biotin.

15. The method of claim 14, wherein the fluorescent dye is a fluorescein dye, a rhodamine dye or an energy transfer dye.

16. The method of claim 1, wherein LABEL is a fluorescent dye selected from the group consisting of a rhodamine dye, a fluorescein dye, a rhodol dye, an energy transfer dye, a cyanine dye, a phthalocyanine or a squaraine.

17. The method of claim 1, wherein LABEL is an energy transfer dye, said energy transfer dye is a donor dye covalently attached to an acceptor dye, wherein either the donor dye or the acceptor dye is covalently attached to the linker, and the acceptor dye is capable of absorbing light emitted by the donor dye.

18. The method of claim 17, wherein the donor dye is selected from a fluorescein dye and a rhodamine dye.

19. The method of claim 18, wherein the acceptor dye is selected from the group consisting of a rhodamine dye, a fluorescein dye, a cyanine dye and a non-fluorescent quencher.