Capped synthetic RNA, analogs, and aptamers

• US 6,369,208 B1
• Filed: 03/30/1998
• Issued: 04/09/2002
• Est. Priority Date: 06/07/1995
• Status: Expired due to Term

• Current Assignee: Merck Sharp & Dohme Corporation (Merck & Co., Inc.)
• Original Assignee: Merck & Co., Inc.
• Inventors: Olsen, David B., Benseler, Fritz, Kuo, Lawrence C., Cole, James L.
• Primary Examiner(s): ZITOMER, STEPHANIE W
• Application Number:
  US08/973,137
• Time in Patent Office: 1,471 Days
• Field of Search: 536/23.1, 536/25.3, 536/25.33, 536/25.34, 536/24.32, 435/196, 435/235.1
• US Class Current: 536/23.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61P 31/12 Antivirals
• CPC Class Codes: A61P 31/16 for influenza or rhinoviruses
• 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
• CPC Class Codes: C12N 15/1131 against viruses
• CPC Class Codes: C12N 15/115 Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2310/317 with an inverted bond, e.g. a cap structure
• CPC Class Codes: C12N 2310/321 2'-O-R Modification
• CPC Class Codes: C12N 2310/322 2'-R Modification
• CPC Class Codes: C12N 2310/3513 Protein; Peptide
• CPC Class Codes: C12N 2310/3521 Methyl

Abstract

A method is provided for making synthetic capped RNAs. These compounds serve as substrates for the virally encoded endonuclease associated with influenza virus. We are able to assay for this unique and specific viral activity of cleavage of a capped RNA in vitro. Therefore, screening of inhibitors of this activity is possible. In addition, short non-extendible (due to their length or because of the modification of the 3′-end of the oligo, i.e. 3′-dA) RNAs are potent inhibitors of the cleavage of capped RNAs by influenza endonuclease. Finally, these compounds may be used to investigate viral and cellular mechanisms of transcription/translation or mRNA maturation.

Claims

1. An influenza endonuclease aptamer having the structure of a capped RNA analog, wherein said aptamer inhibits influenza endonuclease activity and comprises a modified endonuclease cleavage site that is resistant to cleavage by influenza endonuclease activity.

2. The aptamer of claim 1, wherein said aptamer comprises a phosphorothioate at said modified endonuclease cleavage site.

3. The aptamer of claim 1, wherein the 5′

penultimate base of said capped RNA analog has the structure m⁷G(5′

)ppp(5′

)Pu(2′

-OMe).

4. The aptamer of claim 3, wherein said capped RNA analog consists of said 5′

penultimate base and RNA containing one or more modifications, wherein each of said modifications is independently selected from group consisting of;

2′

deoxy, 2′

halo, 2′

amino, 2′

mono, di or tri-halomethyl, 2′

-O-alkyl, 2′

-O-halo-substituted alkyl, 2′

-alkyl, azido, sulfhydryl, non-extendable analogs at the 3′

-end, phosphorothioate, methylphosphonate, 2.6-diamino purine, 2-hydroxy-6-mercaptopurine, pyrimidine bases substituted at the 6-position with sulfur or 5 position with halo or C_1-5, alkyl groups, and abasic linkers.

5. The aptamer of claim 3, wherein said capped RNA analog consists of said 5′

penultimate base and RNA containing one or more modifications, wherein each of said modifications is independently selected from group consisting of;

2′

-deoxy, 2′

-fluoro, 2′

-OMe, phosphorothioate, and 3′

deoxy-adenosine.

6. The aptamer of claim 3, wherein said aptamer is up to approximately 50 bases in length.

7. The aptamer of claim 3, wherein said aptamer is up to 50 bases in length.

8. The aptamer of claim 7, wherein said aptamer is up to 20 bases in length.

9. The aptamer of claim 2, wherein said aptamer is up to approximately 50 bases in length.

10. The aptamer of claim 3, wherein said aptamer is up to 50 bases in length.

11. The a aptamer of claim 10, wherein said aptamer is up to 20 bases in length.

12. The aptamer of claim 5, wherein said aptamer is up to approximately 50 bases in length.

13. The aptamer of claim 5, wherein said aptamer is up to 50 bases in length.

14. The aptamer of claim 13, wherein said aptamer is up to 20 bases in length.

15. The aptamer of claim 7, wherein said aptamer is at least 19 bases in length.

16. The aptamer of claim 8, wherein said aptamer is at least 19 bases in length.

17. The aptamer of claim 10, wherein said aptamer is at least 19 bases in length.

18. The aptamer of claim 11, wherein said aptamer is at least 19 bases in length.

19. The aptamer of claim 13, wherein said aptamer is at least 19 bases in length.

20. The aptamer of claim 14, wherein said aptamer is at least 19 bases in length.