Combinatorial selection of phosphorothioate aptamers for RNases

• US 20060281702A1
• Filed: 05/17/2006
• Published: 12/14/2006
• Est. Priority Date: 05/18/2005
• Status: Abandoned Application

• 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: Gorenstein, David G.
• Application Number:
  US11/435,907
• Publication Number:
  US 20060281702A1
• Time in Patent Office: Days
• US Class Current: 514/44.A
• 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: C12N 15/111 General methods applicable to biologically active non-coding nucleic acids
• 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 2310/16 Aptamers
• CPC Class Codes: C12N 2310/313 Phosphorodithioates
• CPC Class Codes: C12N 2310/315 Phosphorothioates
• CPC Class Codes: C12N 2320/11 for the determination of target sites, i.e. of active nucleic acids
• CPC Class Codes: C12N 2320/13 in a process of directed evolution, e.g. SELEX, acquiring a new function
• CPC Class Codes: C12N 2330/31 Libraries, arrays
• CPC Class Codes: C12N 2740/16211 concerning HIV gagpol

Abstract

The present invention includes the selection and isolation of thioaptamers that target the ribonuclease domains of enzymes, e.g., HIV reverse transcriptase.

Claims

1. A partially thio-modified aptamer that binds to a protein comprising RNase H activity.

2. The aptamer of claim 1, having the thioate and sequence substitutions of the oligonucleotides identified by SEQ ID NOS.:

1, 2 and the combination thereof.

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

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 protein comprises a Reverse Transcriptase.

8. The aptamer of claim 1, wherein the protein comprises an HIV Reverse Transcriptase.

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

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

11. A partially thio-modified aptamer that inhibits a Reverse Transcriptase.

12. The aptamer of claim 11, wherein Reverse Transcriptase comprises an HIV Reverse Transcriptase.

13. The aptamer of claim 11, wherein the aptamer comprises one or more thio-modifications as set forth in SEQ ID NOS.:

1-32.

14. The aptamer of claim 12, wherein the aptamer comprises a short interfering RNA (siRNA);

a micro, interfering RNA (miRNA);

a small, temporal RNA (stRNA);

or a short, hairpin RNA (shRNA).

15. A method of identifying an RNase H specific-thioaptamer comprising the steps of:

synthesizing a random phosphodiester oligonucleotide combinatorial library;

contacting the partially thiophosphate-modified oligonucleotide combinatorial library with a protein having RNaseH activity; and

isolating a subset of oligonucleotides binding to the target molecule.

16. The method of claim 15, wherein the modified nucleotide comprises a phosphorothioate.

17. The method of claim 15, wherein the modified nucleotide comprises a phosphorodithioate.

18. The method of claim 15, wherein the one or more thio-modifications is selected from the group consisting of dATP(α

S), dTTP(α

S), dCTP(α

S), dGTP(α

S), rUTP (α

S), rATP(α

S), rCTP(α

S), rGTP(α

S), dATP(α

S₂), dTTP(α

S₂), dCTP(α

S₂), dGTP(α

S₂), rATP(α

S₂), rCTP(α

S₂), rGTP(α

S₂) and rUTP(α

S₂).

19. The method of claim 15, wherein no more than three adjacent phosphate sites are replaced with phosphorothioate groups.

20. The method of claim 15, wherein at least a portion of non-adjacent phosphate sites are replaced with phosphorothioate groups.

21. The method of claim 15, wherein no more than three adjacent phosphate sites are replaced with phosphorodithioate groups.

22. The method of claim 15, wherein at least a portion of non-adjacent phosphate sites are replaced with phosphorodithioate groups.

23. The method of claim 15, wherein the target is a viral protein having RNase H activity.

24. The method of claim 15, wherein the target is HIV RT.

25. A method of identifying a set of aptamers containing an optimal composition of thiophosphate-modified nucleotides such that the aptamers bind with high affinity to a protein having RNase H activity, and have increased resistance to nuclease degradation, said method comprising the steps of:

synthesizing a random partially thiophosphate-modified oligonucleotide combinatorial library wherein at least a portion of the oligonucleotide phosphate groups are thiophosphate-modified nucleotides, and where no more than three of the four different nucleotides are substituted on the 5′

-phosphate positions by 5′

-thiophosphates in each synthesized oligonucleotide are thiophosphate-modified nucleotides;

contacting the amplified library with the protein under conditions favorable for binding of a binding oligonucleotide with said target molecule; and

isolating a subset of binding oligonucleotides from the library, that bind with higher affinity to the protein relative to the original library.

26. The method of claim 25, further comprising the step of:

repeating the selection iteratively, whereby an enriched subset of oligonucleotides binding with higher affinity to the target molecule relative to the original amplified subset, is isolated after each cycle.

27. The method of claim 25, whereby each iteration is performed under conditions of increased stringency in the contacting step until a subset of high affinity binding oligonucleotides is identified.

28. The method of claim 25, whereby synthesis of the combinatorial library is done using constituent oligonucleotides comprising at least a set of 5′

and 3′

PCR primer nucleotide sequences flanking a randomized nucleotide sequence.

29. The method of claim 25, whereby the subset of amplified oligonucleotides is cloned and where individual thiophosphate-modified oligonucleotides that bind to the target are isolated and sequenced.

30. The method of claim 25, whereby the isolated aptamer is screened relative to its respective non-modified oligonucleotide and comprises increased affinity for the protein and increased stability with respect to nuclease degradation.

31. The method of claim 25, whereby the thiophosphate comprises a phosphorothioate, a phosphorodithioate or a combination thereof.

32. A thioaptamer that binds specifically to and inhibits RNase H activity.

33. The thioaptamer of claim 32, wherein the thioaptamer is packed into a capsule, caplet, softgel, gelcap, suppository, film, granule, gum, insert, pastille, pellet, troche, lozenge, disk, poultice or wafer.

34. The thioaptamer of claim 32, wherein thioaptamer is packaged for released within about 60 minutes.

35. The thioaptamer of claim 32, wherein thioaptamer is packaged for release of over 90% within about 60 minutes and 12 hours.

36. The thioaptamer of claim 32, wherein the aptamer is packaged with one or more of the following PVPP, Povidone, a talc and a stearate.

37. The thioaptamer of claim 32, comprising further one or more inactives.

38. The thioaptamer of claim 32, wherein the thioaptamer is lyophilized.

39. The thioaptamer of claim 32, wherein the thioaptamer is suspended for delivery intravenously, intraperitoneally, intramuscularly, subcutaneously, intracutaneously, alveolarly, sublingual or combinations thereof.

40. The thioaptamer of claim 32, having the thioate and sequence substitutions of the oligonucleotides identified by SEQ ID NOS.:

1, 2 and the combination thereof.

41. The thioaptamer of claim 32, having the sequence of the formula:

42. The thioaptamer of claim 32, further comprising one or more pharmaceutically acceptable salts.

43. A pharmaceutical formulation in which a therapeutically effective amount of a thioaptamer that bind specifically to and inhibits RNase H activity is provided to a patient in need thereof.