Thio-modified aptamer synthetic methods and compositions

• US 6,867,289 B1
• Filed: 10/25/1999
• Issued: 03/15/2005
• Est. Priority Date: 10/26/1998
• Status: Expired due to Term

• Current Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Original Assignee: Board of Regents of the University of Texas System (University of Texas System)
• Inventors: Aronson, Judy, Luxon, Bruce A., Gorenstein, David G., Herzog, Norbert
• Primary Examiner(s): Zitomer, Stephanie
• Application Number:
  US09/425,804
• Time in Patent Office: 1,968 Days
• Field of Search: 536/23.1, 536/25.4, 435/6, 435/91.2, 530/350, 436/501, 514/44
• 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: 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 15/1048 SELEX
• 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 2310/313 Phosphorodithioates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C40B 40/00 Libraries per se, e.g. arrays, mixtures

Abstract

This invention generally relates the generation of aptamers and to the use of aptamers as diagnostic and therapeutic agents. More particularly, the present invention relates to methods using combinatorial chemistry to prepare aptamers having controlled thiophosphate replacement in the phosphate backbone for improved binding efficiencies to a target and to RNA and/or DNA products having novel nucleotide sequences and enhanced target binding efficacies.

Claims

1. An aptamer that binds to one or more NF-κ

B/Rel proteins selected from the group consisting of;

a) single stranded oligonucleotides identified by SEQ ID NO.;

39, wherein between one and six of the nucleotides are dithiophosphates;

b) double stranded oligonucleotides in accordance with the sequence identified SEQ ID NO.;

39, wherein between one and ten of the nucleotides are dithiophosphates;

c) single stranded oligonucleotides identified by SEQ ID NO.;

39, wherein between one and six of the nucleotides are dithiophosphates; and

d) double stranded oligonucleotides in accordance with the sequence identified by SEQ ID NO.;

39, wherein between one and ten of the nucleotides are dithiophosphates.

2. The aptamer of claim 1, having the thioate and sequence substitutions of the oligonucleotides identified by SEQ ID NO.:

30-39.

3. The aptamer of claim 1, having the sequence of the formula:

5′

-CCAGGAGAT_S2T_S2CCAC-3′

3′

-GG_S2TCC_S2TC_S2TAAGG_S2TG-5′

 

 

(SEQ ID NO.;

     39).

4. The aptamer of claim 1, further comprising one or more pharmaceutically acceptable salts.

5. The aptamer of claim 1, further comprising a diluent.

6. The aptamer of claim 1, wherein the aptamer is achiral.

7. The aptamer of claim 1, wherein the one or more NF-κ

B/Rel proteins comprises and NF-κ

B homodimer.

8. The aptamer of claim 1, wherein the one or more NF-κ

B/Rel proteins comprises an NF-κ

B heterodimer.

9. The aptamer of claim 1, further comprising an NF-κ

B/Rel protein bound to the aptamer.

10. The aptamer of claim 1, wherein the aptamer comprises one or more achiral thiomonophosphates.

11. The aptamer of claim 1, wherein the aptamer comprises one or more dithiophosphates.

12. A partially thio-modified aptamer that binds to an NF-κ

B/Rel protein.

13. A aptamer of claim 12, wherein the NF-κ

B/Rel protein comprises a human NF-κ

B.

14. The aptamer of claim 12, wherein the NF-κ

B/Rel protein comprises an NF-κ

B homodimer.

15. The aptamer of claim 12, wherein the NF-κ

B/Rel protein comprises and NF-κ

B heterodimer.

16. The aptamer of claim 12, wherein the aptamer comprises one or more thio-modifications as set forth in SEQ ID NOS.:

32, 33 and 38.

17. The aptamer of claim 12, wherein the aptamer further comprises a fluorescent label.

18. The aptamer of claim 12, further comprising one or more pharmaceutically acceptable salts.

19. The aptamer of claim 12, further comprising a diluent.

20. The aptamer of claim 12, wherein the aptamer is achiral.

21. A partially thio-modified aptamer the binds to human NF-κ

B protein complex.