Oligonucleotide N3'.fwdarw.P5' phosphoramidates: hybridization and nuclease resistance properties

• US 5,631,135 A
• Filed: 06/06/1995
• Issued: 05/20/1997
• 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., Chen, Jer-Kang, Gryaznov, Sergei M.
• Primary Examiner(s): Chambers, Jasemine C.
• Assistant Examiner(s): PRIEBE, SCOTT DAVID
• Application Number:
  US08/473,015
• Time in Patent Office: 714 Days
• Field of Search: 435/6, 435/91.1, 536/23.1, 536/24.5, 536/24.3, 536/25.4
• US Class Current: 435/6.14
• 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 oligonucleotides 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 hybridizing an oligodeoxyribonucleotide to an RNA target, comprising:

(a) forming an oligodeoxyribonucleotide comprising a sequence of nucleoside subunits joined by inter-subunit linkages, wherein;

(i) at least 50% of the inter-subunit linkages of said oligodeoxyribonucleotide are N3'"'"'→

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

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

O)(-O.sup.-)-O-5'"'"'; and

(ii) said oligodeoxyribonucleotide has at least 11 nucleotides; and

(b) contacting the oligodeoxyribonucleotide with said RNA target to allow formation of a hybridization complex between said oligodeoxyribonucleotide and said RNA target.

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

N5'"'"' phosphoramidate linkages.

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

P5'"'"' phosphoramidate linkage and a phosphodiester inter-subunit linkage.

4. The method of claim 1, wherein said oligodeoxyribonucleotide has 11 to 24 nucleosides.

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

P5'"'"' phosphoramidate linkages.

6. The method of claim 5, wherein said oligodeoxyribonucleotide has 11 to 50 nucleosides.

7. A kit for the isolation of a target RNA from a sample, the kit comprising:

an oligodeoxyribonucleotide comprising a sequence of nucleoside subunits joined by inter-subunit linkages wherein at least 50% of the inter-subunit linkages of said oligodeoxyribonueleotide are N3'"'"'→

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

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

O)(-O.sup.-)-O-5'"'"'

wherein

said oligodeoxyribonucleotide hybridizes to said target RNA, and

said oligodeoxyribonucleotide has at least 11 nucleosides.

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

P5'"'"' phosphoramidate inter-subunit linkages.

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

P5'"'"' phosphoramidate inter-subunit linkages and a phosphodiester inter-subunit linkage.

10. The kit of claim 7, wherein said oligodeoxyribonucleotide has 11 to 24 nucleosides.

11. The kit of claim 7 further including a solid support attached to said oligodeoxyribonucleotide.

12. A diagnostic method to detect in a sample the presence of an RNA having a selected target sequence comprising:

(a) contacting an oligodeoxyribonucleotide with said RNA to allow formation of a hybridization complex between said oligodeoxyribonucleotide and said selected target sequence, the oligodeoxyribonucleotide comprising a defined sequence of nucleoside subunits and inter-subunit linkages wherein;

(i) at least 50% of the inter-subunit linkages or said oligodeoxyribonucleotide are N3'"'"'→

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

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

O)(-O.sup.-)-O-5'"'"',

and

(ii) said oligodeoxyribonucleotide has at least 11 nucleosides; and

(b) detecting the presence of said hybridization complex, thereby detecting the presence of said RNA.

13. The method of claim 12, wherein said oligodeoxyribonucleotide carries a reporter moiety and said detecting includes detection of said reporter moiety.

14. The method of claim 13, wherein said reporter moiety is selected from the group consisting of radioactive labels, biotin labels, and fluorescent labels.

15. The method of claim 12, wherein all of said inter-subunit linkages of said oligodeoxyribonucleotide are N3'"'"'→

P5'"'"' phosphoramidate linkages.

16. The method of claim 12 wherein said inter-subunit linkages alternate N3'"'"'→

P5'"'"' and a phosphodiester inter-subunit linkage.

17. The method of claim 12, wherein said oligodeoxyribonucleotide has 11 to 24 nucleotides.

18. A method of hybridizing an oligodeoxyribonucleotide to an DNA target, comprising:

(a) forming an oligodeoxyribonucleotide comprising a sequence of nucleoside subunits joined by inter-subunit linkages, wherein;

(i) 100% of the inter-subunit linkages of said oligodeoxyribonucleotide are N3'"'"'→

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

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

O)(-O.sup.-)-O-5'"'"'; and

(ii) said oligodeoxyribonucleotide has at least 11 nucleotides; and

(b) contacting the oligodeoxyribonucleotide with said DNA target to allow formation of a hybridization complex between said oligodeoxyribonucleotide and said DNA target.

19. The method of claim 18, wherein said oligodeoxyribonucleotide carries a reporter moiety and said detecting includes detection of said reporter moiety.

20. The method of claim 19, wherein said reporter moiety is selected from the group consisting of radioactive labels, biotin labels, and fluorescent labels.

21. The method of claim 18, wherein said oligodeoxyribonucleotide has 11 to 24 nucleotides.

22. The method of claim 18, wherein said oligodeoxyribonucleotide has 11 to 50 nucleosides.

23. The method of claim 18, wherein said DNA target is single-stranded DNA.

24. A diagnostic method to detect the presence of a single-stranded DNA having a selected target sequence in a sample, comprising:

(a) contacting an oligodeoxyribonucleotide with said single-stranded DNA to allow formation of a hybridization complex between said oligodeoxyribonucleotide and said selected target sequence, the oligodeoxyribonucleotide comprising a defined sequence of nucleoside subunits and inter-subunit linkages wherein;

(i) 100% of the inter-subunit linkages of said oligodeoxyribonucleotide are N3'"'"'→

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

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

O)(-O.sup.-)-O-5'"'"',

and

(ii) said oligodeoxyribonucleotide has at least 11 nucleosides; and

(b) detecting the presence of said hybridization complex, thereby detecting the presence of said DNA.

25. The method of claim 24, wherein said oligodeoxyribonucleotide carries a reporter moiety and said detecting includes detection of said reporter moiety.

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

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

28. The method of claim 24, wherein said oligodeoxyribonucleotide has 11 to 50 nucleosides.

29. A method of hybridizing an oligodeoxyribonucleotide to a polynucleotide target sequence, the method comprising:

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

##STR1## wherein B is a purine or pyrimidine or an analog thereof and n is between 2 and 48; and

(b) contacting the oligodeoxyribonucleotide with said polynucleotide target sequence to allow formation of a hybridization complex between said oligodeoxyribonucleotide and said polynucleotide target sequence.

30. The method of claim 29, wherein B of said oligodeoxyribonucleotide is selected from the group consisting of uracil, thymine, adenine, guanine, cytosine, 5-methylcytosine, 5-bromouracil, and inosine.

31. The method of claim 30, wherein B of said oligodeoxyribonucleotide is selected from the group consisting of uracil, thymine, adenine, guanine, and cytosine.

32. A kit for the isolation of a target polynucleotide from a sample, comprising:

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

##STR2## wherein B is a purine or pyrimidine or an analog thereof and n is between 2 and 48 and wherein said oligodeoxyribonucleotide hybridizes to said target polynucleotide.

33. The kit of claim 32, wherein B of said oligodeoxyribonucleotide is selected from the group consisting of uracil, thymine, adenine, guanine, cytosine, 5-methylcytosine, 5-bromouracil, and inosine.

34. The kit of claim 32, wherein B of said oligodeoxyribonucleotide is selected from the group consisting of uracil, thymine, adenine, guanine, and cytosine.

35. The kit of claim 32 further including a solid support attached to said oligodeoxyribonucleotide.

36. A diagnostic method to detect the presence of a polynucleotide having a selected target sequence in a sample comprising:

(a) contacting an oligodeoxyribonucleotide with said polynucleotide to allow formation of a hybridization complex between said oligodeoxyribonucleotide and said target sequence, wherein said oligodeoxyribonucleotide comprises a defined sequence of nucleoside subunits joined by inter-subunit linkages defined by the formula;

##STR3## wherein B is a purine or pyrimidine or an analog thereof and n is between 2 and 48; and

(b) detecting the presence of said hybridization complex, thereby detecting said polynucleotide.

37. The method of claim 36, wherein B of said oligodeoxyribonucleotide is selected from the group consisting of uracil, thymine, adenine, guanine, cytosine, 5-methylcytosine, 5-bromouracil, and inosine.

38. The method of claim 37, wherein B of said oligodeoxyribonucleotide is selected from the group consisting of uracil, thymine, adenine, guanine, and cytosine.

39. The method of claim 36, wherein said oligodeoxyribonucleotide carries a reporter moiety and said detecting includes detection of said reporter moiety.

40. The method of claim 39, wherein said reporter moiety is selected from the group consisting of radioactive labels, biotin labels, and fluorescent labels.