Nucleotide compounds including a rigid linker

• US 6,653,462 B2
• Filed: 02/26/2002
• Issued: 11/25/2003
• Est. Priority Date: 05/29/1998
• Status: Expired due to Term

• Current Assignee: Applied Biosystems LLC (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applera Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Menchen, Steven M., Khan, Shaheer H., Rosenblum, Barnett B., Zhen, Weiguo
• Primary Examiner(s): Riley, Jezia
• Application Number:
  US10/085,561
• Publication Number:
  US 20020151711A1
• Time in Patent Office: 637 Days
• Field of Search: 435/6, 536/23.1, 536/22.1, 536/25.3, 536/25.32, 536/26.6
• US Class Current: 536/23.1
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/14 Pyrrolo-pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12Q 1/6869 Methods for sequencing
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety

Abstract

A nucleoside/tide compound having the structure:

NUC-L-S-LB/LG

is described wherein NUC is a nucleoside/tide having a nucleobase portion B, L is a rigid linkage, S is a spacer; and LB/LG is a member of a linkage pair or a label. NUC is attached to L through B such that when B is a purine, L is attached to the 8-position of the purine, when B is 7-deazapurine, L is attached to the 7-position of the 7-deazapurine, and when B is pyrimidine, L is attached to the 5-position of the pyrimidine. In an important aspect of the invention, L has the structure:

wherein each of n, o and p are integers ranging from 0 to 3, and the sum of n, o and p is at least 2, and each of W, X, Y and Z is selected from the group consisting of carbon and nitrogen. The invention further includes polynucleotide compounds comprising the nucleoside/tide, and primer extension methods utilizing the nucleoside/tide, particularly when used in combination with certain mutant polymerase enzymes.

Claims

1. A nucleoside/tide compound comprising the structure:

wherein;

NUC is a nucleoside/tide having a nucleobase B;

L is a rigid linkage having the structure;

wherein

each of n, o and p are integers ranging from 0 to 3, and the sum of n, o and p is at least 2; and

each of W, X, Y and Z is independently selected from CH and N, including substituted forms thereof;

S is a spacer selected from C₁-C₈ lower alkyl, lower alkylene oxide, amide, carbamate, and sulfonamide;

wherein lower alkylene oxide is selected from the structures;

2. The compound of claim 1 wherein NUC is selected from 2′

-deoxyribonucleotide, 3′

-deoxyribonucleotide, 2′

,3′

-dideoxyribonucleotide, 2′

,3′

-dideoxy-3′

-fluoro-ribonucleotide, 2′

,3′

-dideoxy-2′

-fluoro-ribonucleotide, 2′

,3′

-dideoxy-3′

-azido-ribonucleotide, 2′

,3′

-dideoxy-2′

-azido-ribonucleotide, 2′

,3′

-dideoxy-2′

-amino-ribonucleotide, 2′

,3′

-dideoxy-3′

-amino-ribonucleotide, 2′

,3′

-dehydroribonucleotide and ribonucleotide.

3. The compound of claim 1 wherein NUC is 2′

-deoxyribonucleotide.

4. The compound of claim 1 wherein B is selected from adenine, guanine, cytosine, uracil, thymine, deazaadenine, and deazaguanosine.

5. The compound of claim 1 wherein B is selected from 2,6-diaminopurine, hypoxanthine, pseudouridine, C-5-propyne, isocytosine, isoguanine, and 2-thiopyrimidine.

6. The compound of claim 1 wherein NUC is selected from 2′

,3′

-dideoxy-3′

-fluoro-ribonucleotide.

7. The compound of claim 1 wherein NUC is a 5′

hydroxyl nucleoside.

8. The compound of claim 1 wherein NUC is a 5′

triphosphate nucleotide.

9. The compound of claim 1 wherein n is 1 or 2.

10. The compound of claim 1 wherein o is 1 or 2.

11. The compound of claim 1 wherein p is 0 or 1.

12. The compound of claim 1 wherein n is 1 or 2, o is 1 or 2, and p is 0 or 1.

13. The compound of claim 1 wherein S has the structure:

14. The compound of claim 13 wherein n is 1 and LB/LG is NH₂.

15. The compound of claim 13 wherein n is 1 and LB/LG is NHC(O)CF₃.

16. The compound of claim 13 wherein n is 1 and LB/LG is a fluorescein-type dye.

17. The compound of claim 13 wherein n is 1 and LB/LG is a rhodamine-type dye.

18. The compound of claim 1 wherein S has the structure:

19. The compound of claim 18 wherein n is 2 and LB/LG is NH₂.

20. The compound of claim 18 wherein n is 2 and LB/LG is NHC(O)CF₃.

21. The compound of claim 18 wherein n is 2 and LB/LG is a fluorescein-type dye.

22. The compound of claim 18 wherein n is 2 and LB/LG is a rhodamine-type dye.

23. The compound of claim 18 wherein n is 3 and LB/LG is NH₂.

24. The compound of claim 18 wherein n is 3 and LB/LG is NHC(O)CF₃.

25. The compound of claim 18 wherein n is 3 and LB/LG is a fluorescein-type dye.

26. The compound of claim 18 wherein n is 3 and LB/LG is a rhodamine-type dye.

27. The compound of claim 1 wherein S has the structure:

28. The compound of claim 27 wherein n is 2 and LB/LG is NH_2.

29. The compound of claim 27 wherein n is 2 and LB/LG is NHC(O)CF₃.

30. The compound of claim 27 wherein n is 2 and LB/LG is a fluorescein-type dye.

31. The compound of claim 27 wherein n is 2 and LB/LG is a rhodamine-type dye.

32. The compound of claim 1 wherein L has the structure:

33. The compound of claim 1 wherein L has the structure:

34. The compound of claim 1 wherein L has the structure:

35. The compound of claim 1 wherein L has the structure:

36. The compound of claim 1 wherein L has the structure:

37. The compound of claim 1 wherein L has the structure: