Tm leveling methods

• US 6,683,173 B2
• Filed: 12/21/2001
• Issued: 01/27/2004
• Est. Priority Date: 04/03/1998
• Status: Expired due to Term

• Current Assignee: ELITechGroup B.V. (Bruker Corporation)
• Original Assignee: Epoch Biosciences Inc. (Ngen, Inc.)
• Inventors: Gall, Alexander A., Dempcy, Robert O., Vermeulen, Nicolaas M. J., Lokhov, Sergey G., Kutyavin, Igor V., Singer, Michael J., Afonina, Irina A.
• Primary Examiner(s): Horlick, Kenneth R.
• Application Number:
  US10/032,307
• Publication Number:
  US 20030224359A1
• Time in Patent Office: 767 Days
• Field of Search: 536/25.3, 435/6, 436/94
• US Class Current: 536/25.3
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• 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 19/16 Purine radicals
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/23 Heterocyclic radicals containing two or more heterocyclic rings condensed among themselves or condensed with a common carbocyclic ring system, not provided for in groups C07H19/14 - C07H19/22
• 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 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 2525/117 incorporating modified base
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures
• CPC Class Codes: G16B 30/00 ICT specially adapted for sequence analysis involving nucleotides or amino acids

Abstract

Modified oligonucleotides are provided containing bases selected from unsubstituted and 3-substituted pyrazolo[3,4-d]pyrimidines and 5-substituted pyrimidines, and optionally have attached minor groove binders and reporter groups.

Claims

1. A method for designing an oligonucleotide-MGB conjugate having a leveled T_m, said method comprising:

a) providing an oligonucleotide with a known sequence having n bases and a known T_m;

b) determining

i.) the sequence dependent thermodynamic contribution of a minor groove binder (MGB) attached to said oligonucleotide, and

ii.) the sequence dependent thermodynamic contribution of said oligonucleotide; and

c) designing an oligonucleotide-MGB conjugate using said thermodynamic contributions, wherein said oligonucleotide-MGB conjugate has fewer than n bases and a T_m that is level with said known T_m.

2. The method of claim 1, wherein the thermodynamic contribution of said MGB attached to said oligonucleotide is determined by comparing the duplex stability of a first duplex of said oligonucleotide to the stability a second duplex of said oligonucleotide with an attached MGB.

3. The method of claim 1, wherein said known T_m of said oligonucleotide is empirically determined.

4. The method of claim 1, wherein said known T_m of said oligonucleotide is determined using nearest neighbor parameters.

5. The method of claim 1, wherein said known sequence having n bases contributes to said known T_m.

6. The method of claim 1, wherein said oligonucleotide is derived from Genbank.

7. The method of claim 1, wherein said MGB has a formula selected from the group consisting of:

wherein

the subscript m is an integer of from 2 to 5;

the subscript r is an integer of from 2 to 10; and

each R^a and R^b is independently a linking group to said modified oligonucleotide, H, —

OR^c, —

NR^cR^d, —

COOR^c and —

CONR^cR^d wherein each R^c and R^d is selected from the group consisting of H, (C₁-C₁₂)heteroalkyl, (C₂-C₁₂)heteroalkenyl, (C₂-C₁₂)heteroalkynyl, (C₁-C₁₂)alkyl, (C₂-C₁₂)alkenyl, (C₂-C₁₂)alkynyl, aryl(C₁-C₁₂)alkyl and aryl.

8. The method of claim 1, wherein said MGB is attached to the 3′

-end of said oligonucleotide-MGB conjugate.

9. The method of claim 1, wherein said MGB is attached to the 5′

-end of said oligonucleotide-MGB conjugate.

10. The method of claim 1, wherein said MGB is attached to other than the 3′

-end or 5′

-end of said oligonucleotide-MGB conjugate.

11. The method of claim 1, wherein said T_m of said oligonucleotide-MGB conjugate is within about ±

5°

C. of said known T_m.

12. The method of claim 1, wherein said oligonucleotide-MGB conjugate has at least one modified base.

13. The method of claim 12, wherein said oligonucleotide-MGB conjugate has at least one modified base wherein said base is selected from the group consisting of a base attached to an amino acid, a locked-nucleic acid base and a universal base.

14. The method of claim 12, wherein said at least one modified base is a member selected from the group consisting of PPA, PPG, PPPA, PPPG, PU, PC, HOPU, HOBuU, HOBuC, (NH₂)₂PPPA, (NH₂)₂PPPAOH, (NH₂)₂BuPPAOH, (NH₂)₂PPAI, and HOBuPPG.

15. The method of claim 1, further comprising repeating step (c) to generate a plurality of different oligonucleotide-MGB conjugates each having a T_m that is level with said known T_m.

16. The method of claim 15, wherein each of said plurality of different oligonucleotide-MGB conjugates are within about 1-2 bases from each other.

17. The method of claim 15, wherein said plurality of different oligonucleotide-MGB conjugates are immobilized on a substrate.

18. The method of claim 15, wherein said plurality of different oligonucleotide-MGB conjugates are synthesized on a substrate.

19. The method of claim 17, wherein said substrate is a member selected from the group consisting of glass, polystyrene, nylon, nitrocellulose, glass, silicon wafers, optical fibers and plastic.

20. A method for designing a modified oligonucleotide having a leveled T_m, said method comprising:

a) providing an oligonucleotide with a known sequence having n bases and a known T_m; and

b) designing a modified oligonucleotide having at least one modified base using the sequence dependent thermodynamic contribution of said at least one modified base determined using nearest neighbor parameters, wherein said modified oligonucleotide has fewer than n bases and has a T_m that is level with said known T_m.

21. The method of claim 20, wherein said oligonucleotide is derived from Genbank.

22. The method of claim 20, wherein said at least one modified base is a member selected from the group consisting of a base attached to an amino acid, a locked-nucleic acid base and a universal base.

23. The method of claim 20, wherein said at least one modified base is a member selected from the group consisting of PPA, PPG, PPPA, PPPG, PU, PC, HOPU, HOBuU, HOBuC, (NH₂)₂PPPA, (NH₂)₂PPPAOH, (NH₂)₂BuPPAOH, (NH₂)₂PPAI, and HOBuPPG.

24. The method of claim 20, wherein said modified oligonucleotide has a MGB attached thereto.

25. The method of claim 24, wherein said MGB has a formula selected from the group consisting of:

wherein

the subscript m is an integer of from 2 to 5;

the subscript r is an integer of from 2 to 10; and

each R^a and R^b is independently a linking group to said modified oligonucleotide, H, —

OR^c, —

NR^cR^d, —

COOR^c and —

CONR^cR^d wherein each R^c and R^d is selected from the group consisting of H, (C₁-C₁₂)heteroalkyl, (C₂-C₁₂)heteroalkenyl, (C₂-C₁₂)heteroalkynyl, (C₁-C₁₂)alkyl, (C₂-C₁₂)alkenyl, (C₂-C₁₂)alkynyl, aryl(C₁-C₁₂)alkyl and aryl.

26. The method of claim 24, wherein said MGB is attached to the 3′

-end of said modified oligonucleotide.

27. The method of claim 24, wherein said MGB is attached to the 5′

-end of said modified oligonucleotide.

28. The method of claim 24, wherein said MGB is attached to other than the 3′

-end or 5′

-end of said oligonucleotide-MGB conjugate.

29. The method of claim 20, wherein said at least one modified base decreases stability of said modified oligonucleotide.

30. The method of claim 20, wherein said at least one modified base increases stability of said modified oligonucleotide.

31. The method of claim 20, further comprising repeating step (b) to generate a plurality of different modified oligonucleotides each having a T_m that is level with said known T_m.

32. The method of claim 31, wherein said plurality of different modified oligonucleotides each having a T_m that is level with said known T_m has at least one oligonucleotide without a modified base.

33. The method of claim 31, wherein said plurality of different oligonucleotides are immobilized on a substrate.

34. The method of claim 20, wherein designing said modified oligonucleotide further comprises using the sequence dependent thermodynamic contribution of the natural bases in said modified oligonucleotide.

35. The method of claim 20, wherein said oligonucleotide and said modified oligonucleotide have similar hybridization properties.

36. The method of claim 31, wherein said plurality of different modified oligonucleotides have MGB conjugates attached thereto.

37. The method of claim 36, wherein said plurality of different modified oligonucleotide-MGB conjugates are synthesized on a substrate.