Inhibition of molecular and biological processes using modified oligonucleotides

• US 6,548,251 B1
• Filed: 09/05/2000
• Issued: 04/15/2003
• Est. Priority Date: 09/05/2000
• Status: Expired due to Fees

• Current Assignee: United States Department of Energy (Government of the United States of America)
• Original Assignee: Fidelity Systems, Inc.
• Inventors: Kozyavkin, Sergei A., Slesarev, Alexei I., Malykh, Andrei G., Polouchine, Nikolai N.
• Primary Examiner(s): Myers, Carla J.
• Application Number:
  US09/655,804
• Time in Patent Office: 952 Days
• Field of Search: 536/23.1, 536/25.3, 536/25.31, 536/23.5, 536/24.3, 536/24.33, 536/24.2, 536/24.5, 435/6, 435/91.1, 435/91.2, 514/44
• US Class Current: 435/6.13
• CPC Class Codes: C12Q 1/6832 Enhancement of hybridisation reaction
• CPC Class Codes: C12Q 1/6853 using modified primers or templates
• CPC Class Codes: C12Q 2525/113 incorporating modified backbone
• CPC Class Codes: C12Q 2527/107 Temperature of melting, i.e. Tm

Abstract

A method of inhibiting at least one molecular process in a sample, comprising administering to the sample an oligonucleotide or polynucleotide containing at least one monomeric unit having formula (I):

A—X_n  (I)

wherein A is an organic moiety, n is at least 1, and each X is independently selected from the group consisting of —NRCOCONu, —NHCOCR₂CR₂CONu, —NHCOCR═CRCONu, and —NHCOSSCONu, wherein each R independently represents H or a substituted or unsubstituted alkyl group, and Nu represents a nucleophile, or a salt of the compound.

Claims

1. A method of inhibiting at least one molecular process involving the interaction between nucleic acids in a sample capable of undergoing said at least one molecular process, comprising:

administering to said sample an oligonucleotide or polynucleotide containing at least one monomeric unit having formula (I);

2. The method of claim 1, wherein said at least one molecular process is selected from the group consisting of nucleic acid hybridization and nucleic acid extension.

3. The method of claim 2, wherein said method inhibits hydridization.

4. The method of claim 3, wherein said monomeric unit decreases the melting temperature of a duplex formed between the oligonucleotide or polynucleotide and a nucleotide sequence in said sample.

5. The method of claim 3, wherein the monomeric unit prevents intramolecular hybridization within the oligonucleotide or polynucleotide.

6. The method of claim 5, wherein the oligonucleotide or polynucleotide contains at least one base complementary to the 3′

terminal base and at least one said monomeric unit within 4 monomers of the complementary base.

7. The method of claim 3, wherein the monomeric unit prevents the oligonucleotide or polynucleotide from hybridizing to its duplicate to form a dimer.

8. The method of claim 7, wherein the oligonucleotide or polynucleotide contains at least one base complementary to the 3′

terminal base and at least one said monomeric unit within 4 monomers of the complementary base.

9. The method of claim 2, wherein said method inhibits nucleic acid extension.

10. The method of claim 9, comprising contacting the oligonucleotide or polynucleotide with a nucleic acid strand in the sample, wherein said monomeric unit prevents a polymerase from extending the 3′

end of the oligonucleotide or polynucleotide.

11. The method of claim 10, wherein the oligonucleotide or polynucleotide contains at least one said monomeric unit at any site within 4 monomers of the 3′

end.

12. The method of claim 9, wherein said oligonucleotide or polynucleotide is a nucleic acid template and wherein said monomeric unit prevents extension beyond said monomeric unit.

13. The method of claim 9, wherein said oligonucleotide or polynucleotide is a template strand, further comprising:

contacting said oligonucleotide or polynucleotide with a primer; and

extending said primer not beyond said monomeric unit.

14. The method of claim 9, further comprising:

contacting said oligonucleotide or polynucleotide with a template nucleic acid strand in said sample;

extending said oligonucleotide or polynucleotide to form an extended oligonucleotide or polynucleotide;

contacting said extended oligonucleotide or polynucleotide with a second primer; and

extending said second primer not beyond said monomeric unit.

15. The method of claim 14, wherein at least one said monomeric unit is located between 4 and 9 monomers from the 3′

end of said oligonucleotide or polynucleotide.

16. The method of claim 1, wherein A is not a nucleotide or nucleoside.

17. The method of claim 16, wherein A is a substituted or unsubstituted alkane, a substituted or unsubstituted cycloalkane, or a substituted or unsubstituted heterocyclic compound.

18. The method of claim 16, wherein A is a substituted or unsubstituted alkane having 3 to 100 carbon atoms, a substituted or unsubstituted cycloalkane having 3 to 12 carbon atoms in a cycle, or a substituted or unsubstituted heterocyclic compound having 3 to 20 carbon atoms in a cycle.

19. The method of claim 18, wherein A is a substituted or unsubstituted cycloalkane or heterocyclic compound having 4 to 8 carbon atoms in a cycle.

20. The method of claim 1, wherein A is a nucleotide or nucleoside having a ribose or deoxyribose sugar, wherein each X is attached to a carbon atom of the ribose or deoxyribose sugar.

21. The method of claim 20, wherein said nucleotide or nucleoside is substituted by said X at the 2′

carbon of said ribose or deoxyribose sugar.

22. The method of claim 1, wherein said oligonucleotide or polynucleotide contains at least one monomeric unit having the formula (II):

wherein B is purine or pyrimidine moiety, and each Y independently represents H, a group that protects a hydroxy group, a (PO₃)_m<sup>−

2</sup> group wherein m is an integer of 1-3, a group reactive to link hydroxy groups, or a phosphodiester linkage to another monomer of said oligonucleotide or polynucleotide, and X is selected from the group consisting of —

NRCOCONu, —

NHCOCR₂CR₂CONu, —

NHCOCR═

CRCONu, and —

NHCOSSCONu, wherein each R independently represents H or a substituted or unsubstituted alkyl group, and Nu represents a nucleophile.