Substituted purines and oligonucleotide cross-linking

• US 6,232,463 B1
• Filed: 08/04/1998
• Issued: 05/15/2001
• Est. Priority Date: 10/09/1997
• Status: Expired due to Fees

• Current Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Original Assignee: ISIS Pharmaceuticals Incorporated (Ionis Pharmaceuticals, Inc.)
• Inventors: Ramasamy, Kanda S., Manoharan, Muthiah, Cook, Phillip Dan
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US09/128,508
• Time in Patent Office: 1,015 Days
• Field of Search: 536/26.26, 536/26.7, 536/25.3, 536/25.31, 536/25.33, 536/25.34, 536/25.4, 544/264
• US Class Current: 536/25.3
• 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

Abstract

This invention is directed to novel purine-based compounds for inclusion into oligonucleotides. The compounds of the invention, when incorporated into oligonucleotides are especially useful as “antisense” agents—agents that are capable of specific hybridization with a nucleotide sequence of an RNA. The compounds of the invention may also be used for cross-linking oligonucleotides. Oligonucleotides are used for a variety of therapeutic and diagnostic purposes, such as treating diseases, regulating gene expression in experimental systems, assaying for RNA and for RNA products through the employment of antisense interactions with such RNA, diagnosing diseases, modulating the production of proteins, and cleaving RNA in site specific fashions. The compounds of the invention include novel heterocyclic bases, nucleosides, and nucleotides. when incorporated into oligonucleotides, the compounds of the invention can be useful for modulating the activity of RNA.

Claims

1. A method of forming a crosslinked oligonucleotide comprising the steps of:

providing an oligonucleotide having a first crosslinking site and a second crosslinking site, wherein said first crosslinking site is located on a nucleobase of said oligonucleotide, and said second crosslinking site is located on a sugar moiety of said nucleobase; and

reacting said first crosslinking site with said second crosslinking site to form said crosslinked nucleotide;

wherein said first and second crosslinking sites each consist of either

(a) a space spanning arm having a reactive functionality thereon;

or

(b) a reactive functionality.

2. The method of claim 1 wherein said crosslinking of said oligonucleotide forms a hairpin loop, a stem loop, an interior loop, or a bulge structure.

3. The method of claim 1 wherein said reactive functionalities are selected from the group consisting of a thiol, an aldehyde, a protected aldehyde, an aldehyde precursor, an amine, hydrazine, hydroxylamine, semicarbazide, thiosemicarbazide, hydrazide, and an alcohol.

4. The method of claim 1 wherein each of said first and second crosslinking sites consist of a reactive functionality.

5. The method of claim 1 wherein each of said first and second crosslinking sites consist of a space spanning arm having a reactive functionality thereon.

6. The method of claim 1 wherein one of said first or said second crosslinking sites consists of a space spanning arm having a reactive functionality thereon, and the other of said first or said second crosslinking sites consists of a reactive functionality.

7. The method of claim 3 wherein one of said reactive functionalities is an amino group, and the other of said reactive functionalities is an aldehyde group or an alcohol group.

8. The method of claim 1 wherein said oligonucleotide contains a plurality of crosslinking sites located on nucleobases of said oligonucleotide, and a plurality of crosslinking sites located on sugar moieties of said nucleobase;

and

each of said plurality of crosslinking sites located on said nucleobases of said oligonucleotide are reacted with each of said a plurality of crosslinking sites located on said sugar moieties of said nucleobase to form said crosslinked nucleotide.

9. A method of fixing the secondary structure of an oligonucleotide comprising the steps of:

providing an oligonucleotide having a first crosslinking site and a second crosslinking site, wherein said first crosslinking site is located on a nucleobase of said oligonucleotide, and said second crosslinking site is located on a sugar moiety of said nucleobase; and

reacting said first crosslinking site with said second crosslinking site to form said crosslinked nucleotide;

wherein said first and second crosslinking sites each consist of either

(a) a space spanning arm having a reactive functionality thereon;

or

(b) a reactive functionality.

10. The method of claim 9 wherein said crosslinking of said oligonucleotide forms a hairpin loop, a stem loop, an interior loop, or a bulge structure.

11. The method of claim 9 wherein said reactive functionalities are selected from the group consisting of a thiol, an aldehyde, a protected aldehyde, an aldehyde precursor, an amine, hydrazine, hydroxylamine, semicarbazide, thiosemicarbazide, hydrazide, and an alcohol.

12. The method of claim 9 wherein each of said first and second crosslinking sites consist of a reactive functionality.

13. The method of claim 9 wherein each of said first and second crosslinking sites consist of a space spanning arm having a reactive functionality thereon.

14. The method of claim 9 wherein one of said first or said second crosslinking sites consists of a space spanning arm having a reactive functionality thereon, and the other of said first or said second crosslinking sites consists of a reactive functionality.

15. The method of claim 11 wherein one of said reactive functionalities is an amino group, and the other of said reactive functionalities is an aldehyde group or an alcohol group.

16. The method of claim 9 wherein said oligonucleotide contains a plurality of crosslinking sites located on nucleobases of said oligonucleotide, and a plurality of crosslinking sites located on sugar moieties of said nucleobase;

and

each of said plurality of crosslinking sites located on said nucleobases of said oligonucleotide are reacted with each of said a plurality of crosslinking sites located on said sugar moieties of said nucleobase to form said crosslinked nucleotide.

17. A method of fixing an oligonucleotide in a stem loop structure comprising the steps of:

providing an oligonucleotide having a first crosslinking site and a second crosslinking site, wherein said first crosslinking site is located on a nucleobase of said oligonucleotide, and said second crosslinking site is located on a sugar moiety of said nucleobase; and

reacting said first crosslinking site with said second crosslinking site to form said stem loop structure;

wherein said first and second crosslinking sites each consist of either

(a) a space spanning arm having a reactive functionality thereon;

or

(b) a reactive functionality.

18. The method of claim 17 wherein said reactive functionalities are selected from the group consisting of a thiol, an aldehyde, a protected aldehyde, an aldehyde precursor, an amine, hydrazine, hydroxylamine, semicarbazide, thiosemicarbazide, hydrazide, and an alcohol.

19. The method of claim 17 wherein each of said first and second crosslinking sites consist of a reactive functionality.

20. The method of claim 17 wherein each of said first and second crosslinking sites consist of a space spanning arm having a reactive functionality thereon.

21. The method of claim 17 wherein one of said first or said second crosslinking sites consists of a space spanning arm having a reactive functionality thereon, and the other of said first or said second crosslinking sites consists of a reactive functionality.

22. The method of claim 19 wherein one of said reactive functionalities is an amino group, and the other of said reactive functionalities is an aldehyde group or an alcohol group.

23. The method of claim 17 wherein said oligonucleotide contains a plurality of crosslinking sites located on nucleobases of said oligonucleotide, and a plurality of crosslinking sites located on sugar moieties of said nucleobase;

and

each of said plurality of crosslinking sites located on said nucleobases of said oligonucleotide are reacted with each of said a plurality of crosslinking sites located on said sugar moieties of said nucleobase to form said crosslinked nucleotide.