Oligonucleotide N3'-P5' phosphoramidates: hybridization and nuclease resistance properties

• US 5,599,922 A
• Filed: 03/18/1994
• Issued: 02/04/1997
• Est. Priority Date: 03/18/1994
• Status: Expired due to Term

• Current Assignee: Geron Corporation
• Original Assignee: Lynx Therapeutics, Inc. (Illumina Incorporated)
• Inventors: Schultz, Ronald G., Gryaznov, Sergei M., Chen, Jer-kang
• Primary Examiner(s): Elliott, George C.
• Assistant Examiner(s): PRIEBE, SCOTT DAVID
• Application Number:
  US08/214,599
• Time in Patent Office: 1,054 Days
• Field of Search: 536/23.1, 536/25.3, 435/6
• US Class Current: 536/25.3
• CPC Class Codes: C07H 19/06 Pyrimidine radicals
• CPC Class Codes: C07H 19/16 Purine radicals
• 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

Modified oligonucleotide 3'"'"'-NHP(O)(O^-)O-5'"'"' phosphoramidates were synthesized on a solid phase support. The phosphoramidate analogs were found to have significantly increased resistance toward phosphodiesterase digestion. Thermal dissociation experiments demonstrated that these compounds form more stable duplexes than phosphodiesters with complementary DNA and particularly RNA strands. Further, the phosphoramidate analogs can also form stable triplexes with double-stranded DNA target, where under similar conditions parent phosphodiester compounds failed to do so.

Claims

1. A method of enhancing resistance of an oligodeoxyribonucleotide to snake venom phosphodiesterase, comprising:

forming an oligodeoxyribonucleotide having at least 9 inter-subunit linkages, wherein at least 50% of said inter-subunit linkages are N3'"'"'→

P5'"'"' phosphoramidate inter-subunit linkages of the formula;

space="preserve" listing-type="equation">'"'"'-- NH--P(═

O)(--X)--O--5'"'"'

where X is O^- or --OR, and R is a 1 to 5 carbon alkyl,

wherein said oligodeoxyribonucleotide is more resistant to snake venom phosphodiestrase digestion than a corresponding oligodeoxyribonucleotide having only phosphodiester inter-subunit linkages.

2. The method of claim 1, wherein all of said inter-subunit linkages of said oligodeoxyfibonucleotide are N3'"'"'→

N5'"'"' phosphorsinitiate linkages.

3. The method of claim 2, wherein said oligodeoxyribonuclcotide has 10 to 50 nucleosides.

4. The method of claim 1, wherein said inter-subunit linkages alternate between N3'"'"'→

P5'"'"' phosphommidate linkages and phosphodiester linkages.

5. The method of claim 4, wherein said oligodeoxyribonucleotide has 10 to 50 nucleosides.

6. The method of claim 1, wherein said oligodeoxyribonucleotide has 10 to 24 nucleosides.

7. The method of claim 1, wherein said N3'"'"'→

P5'"'"' phosphoramidatc inter-subunit linkages are of the formula;

space="preserve" listing-type="equation">3'"'"'--NH--P(═

O)(--X)--O--5'"'"'

where X is O^-, methoxy or ethoxy and said oligodeoxyribonucleotide has 10 to 50 nucleosides.

8. The method of claim 7 wherein all of said inter-subunit linkages of said oligodeoxyribonucleotide are N3'"'"'→

N5'"'"' phosphorsinitiate linkages.

9. The method of claim 7, wherein said inter-subunit linkages alternate between N3'"'"'→

P5'"'"' phosphoramidate linkages and phosphodiester linkages.

10. The method of claim 7, wherein X is O^-.

11. The method of claim 9, wherein said oligodeoxyribonucleotide has 10 to 24 nucleosides.

12. The method of claim 7, wherein said oligodeoxyribonucleotide hybridizes to mRNA from a proto-oncogene.

13. The method of claim 12, wherein said mRNA is from c-myc.

14. The method of claim 12, wherein said mRNA is from c-fos.

15. The method of claim 7, wherein said oligodeoxyribonucleotide hybridizes to RNA or a polynucleotide that is complementary to said RNA, wherein said RNA is derived from an organism selected from the group consisting of a virus, a bacteria, and a fungus.

16. The method of claim 15, wherein said virus is HIV, papillomavirus, or herpes virus.

17. The method of claim 7, wherein said oligodeoxyribonucleotide hydridizes to a single-stranded DNA target.

18. A method of enhancing resistance of an oligodeoxyribonucleotide to snake whom phosphodiesterase, comprising:

forming an oligodeoxyribonucleotide having inter-subunit linkages and 11 to 50 nucleosides, wherein at least 50% of said inter-subunit linkages are N3'"'"'→

P5'"'"' phosphoramidate inter-subunit linkages of the formula;

space="preserve" listing-type="equation">3'"'"'--NH--P(═

O)(--X)--O--5'"'"'

where X is O^- or --OR, and R is a 1 to 5 carbon all, wherein said oligodeoxyfibonucleotide is more resistant to snake venom phosphodiesterase digestion than a corresponding oligodeoxyfibonucleotide having only phosphodiester inter-subunit linkages.

19. The method of claim 18, wherein X is O^- and said oligodeoxyribonucleotide has 11 to 24 nucleosides.

20. The method of claim 18, wherein said snake venom phosphodiesterase is from Crotalus durissus.

21. The method of claim 18, wherein said inter-subunit linkages alternate between N3'"'"'→

P5'"'"' phosphoramidate linkages and phosphodiester linkages.

22. The method of claim 21, wherein said oligodeoxyribonucleotide has 11 to 24 nucleotides.

23. The method of claim 18, wherein said said oligodeoxyribonucleotide has 11 to 24 nucleosides.

24. The method of claim 18, wherein said oligodeoxyribonucleotide carries a reporter moiety.

25. The method of claim 24, wherein said reporter moiety is selected from the group consisting of radioactive labels, biotin labels, and fluorescent labels.

26. A method of enhancing resistance of an oligodeoxyfibonucleotide to snake venom phosphodiesterase, comprising:

forming an oligodeoxyribonucleotide comprising a defined sequence of nucleoside subunits joined by inter-subunit linkages and defined by the formula;

##STR1## wherein B is a purine or pyrimidine or an analog thereof and n is between 4 and 50,

wherein said oligodeoxyribonucleotide is more resistant to snake venom phosphodiesterase digestion than a corresponding oligodeoxyribonucleotide having only phosphodiester inter-subunit linkages.

27. The method of claim 26, wherein B of said oligonucleotide is selected from the group consisting of uracil, thymine, adenine, guanine, cytosine, 5-methylcytosine, 5-bromouracil, arid inosine.

28. The method of claim 27, wherein B of said oligonucleotide is selected from the group consisting of uracil, thymine, adenine, guanine, and cytosine.

29. The method of claim 28, wherein said oligodeoxyribonucleotide hybridizes to a nuleic acid target sequence.

30. The method of claim 29, wherein said oligodeoxyfibonucleotide hybridizes to an RNA target.

31. The method of claim 30, wherein said oligodeoxyribonucleotide carries a reporter moiety.

32. The method of claim 31, wherein said reporter moiety is selected from the group consisting of radioactive labels, biotin labels, and fluorescent labels.