Oligonucleotide N3′→N5′Phosphoramidate Duplexes

• US 6,169,170 B1
• Filed: 09/03/1997
• Issued: 01/02/2001
• 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): PRIEBE, SCOTT DAVID
• Application Number:
  US08/923,386
• Time in Patent Office: 1,217 Days
• Field of Search: 435/6, 536/23.1
• US Class Current: 536/23.1
• 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 duplex oligodeoxyribonucleotide comprising two complementary strands, each complementary strand containing nucleoside subunits joined by intersubunit linkages, wherein at least one of the complementary strands comprises a non-homopolymeric defined sequence of nucleoside subunits in which (i) at least fifty percent of the intersubunit linkages are N3′

→

P5′

phosphoramidate linkages and at least two of the N3′

→

N5′

phosphoramidate linkages are consecutive,

wherein said N3′

→

P5′

phosphoramidate linkages are defined by the formula;

3′

-[—

NP—

P(═

O)(X)—

O—

]-5═

, wherein X is OH or OR, and R is C₁ to C₅ alkyl.

2. The duplex oligodeoxyribonucleotide of claim 1 wherein said N3′

→

P5 ′

phosphoramidate linkages are defined by the formula;

3. The duplex oligodeoxyribonucleotide of claim 1 wherein at least fifty percent of the intersubunit linkages in each complementary strand are N3′

→

P5′

phosphoramidate linkages, and at least two of the N3′

→

P5′

phosphoramidate linkages in each complementary strand are consecutive.

4. The duplex oligodeoxyribonucleotide of claim 3 wherein all of said intersubunit linkages in each complementary strand are N3′

→

N5′

phosphoramidate linkage.

5. The duplex oligodeoxyribonucleotide of claim 4 wherein said complementary strands are at least ten nucleotide subunits in length.

6. The duplex oligodeoxyribonucleotide of claim 4 wherein said complementary strands are from 4 to 50 nucleotide subunits in length.

7. An oligodeoxyribonucleotide comprising a defined sequence of nucleoside subunits joined by intersubunit linkages wherein the oligodeoxyribonucleotide is capable of forming a hairpin oligomer consisting of a duplex region and a loop region, wherein at least fifty percent of the intersubunit linkages in the duplex region of the hairpin oligomer are N3′

→

P5′

phosphoramidate linkages defined by the formula;

8. The oligodeoxyribonucleotide of claim 7 wherein said N3′

→

P5′

phosphoramidate linkages are defined by the formula;

9. The oligodeoxyribonucleotide of claim 8 wherein all of said intersubunit linkages are N3′

→

P5′

phosphoramidate linkages.

10. The oligodeoxyribonucleotide of claim 9 wherein said defined sequence of nucleotide subunits has a length of from 4 to 50 nucleotide subunits.

11. The oligodeoxyribonucleotide of claim 8 wherein said intersubunit linkages include a second linkage selected from the group consisting of phosphodiester, phosphotriester, methylphosphonate, P3′

→

N5′

phosphoramidate, and phosphorothioate.

12. The oligodeoxyribonucleotide of claim 11 wherein said second linkage is a phosphodiester linkage.

13. The oligodeoxyribonucleotide of claim 12, wherein said defined sequence of nucleotide subunits has a length of from 4 to 50 nucleotide subunits.