STRUCTURE BASED AND COMBINATORIALLY SELECTED OLIGONUCLEOSIDE PHOSPHOROTHIOATE AND PHOSPHORODITHIOATE APTAMER TARGETING AP-1 TRANSCRIPTION FACTORS

• US 20110212843A1
• Filed: 01/26/2011
• Published: 09/01/2011
• Est. Priority Date: 05/23/2003
• Status: Active Grant

• 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: Luxon, Bruce A., Leary, James, Gorenstein, David G.
• Granted Patent: US 9,567,579 B2
• Time in Patent Office: Days
• US Class Current: 506/6
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/1048 SELEX
• CPC Class Codes: G01N 33/6818 Sequencing of polypeptides
• CPC Class Codes: G01N 33/6848 Methods of protein analysis involving mass spectrometry
• CPC Class Codes: G01N 33/6851 Methods of protein analysis involving laser desorption ionisation mass spectrometry

Abstract

The present invention includes composition and methods for making and using a combinatorial library to identify modified thioaptamers that bind to, and affect the immune response of a host animal, transcription factors such as IL-6, NF-κB, AP-1 and the like. Composition and methods are also provided for the treatment of viral infections, as well as, vaccines and vaccine adjuvants are provided that modify host immune responses.

Claims

1. A bead-based method for identifying both thioaptamer sequences and binding proteins comprising the steps of:

incubating a thioaptamer library with a sample suspected of comprising one or more proteins;

selecting the one or more thioaptamers of the library to which protein has bound;

identifying the proteins by mass spectrometry; and

identifying the thioaptamer by PCR, cloning and sequencing.

2. The method of claim 1, further comprising the step of separating the protein into fragments prior to separation by liquid chromatography followed by mass spectrometry.

3. The method of claim 1, wherein the steps of identifying the protein by mass spectrometry is preceded by the steps of extracting and separating the proteins by liquid chromatography.

4. The method of claim 1, wherein the steps of identifying the protein using mass spectrometry comprises matrix assisted laser desorption ionization mass spectrometry.

5. The method of claim 1, wherein the one or more thioaptamers are attached to beads.

6. The method of claim 1, wherein the one or more thioaptamers are attached to a substrate.

7. The method of claim 1, wherein the one or more thioaptamers bound to one or more proteins are isolated from a gel.

8. The method of claim 1, wherein the one or more thioaptamers are attached to beads and the one or more proteins that have bound the one or more thioaptamers are labeled and the beads are sorted based on protein binding.

9. The method of claim 1, wherein the one or more thioaptamers are attached to beads and the one or more proteins that have bound the one or more thioaptamers are labeled and the beads are sorted based on fluorescence.

10. The method of claim 1, wherein the one or more thioaptamers are attached to beads and the step of selecting one or more beads to which one or more proteins has bound is defined further as being fluorescent sorting using a flow-cytometer.

11. The method of claim 1, wherein the one or more proteins is further defined as being a cell extract.

12. The method of claim 1, wherein the one or more proteins is further defined as being a cell extract from a virally infected or diseased cell.

13. The method of claim 1, wherein one or more thioaptamers are attached to beads and the beads are substantially protein-free.

14. The method of claim 1, further comprising the step of sequencing the nucleic acid directly.

15. The method of claim 1, wherein the one or more beads comprises an [S]-ODN or [S₂]-ODN combinatorial libraries.

16. The method of claim 1, wherein each of the one or more beads comprises a thio-modified oligonucleotide library.

17. The method of claim 1, wherein the one or more beads comprise a double-stranded nucleic acid library.

18. The method of claim 1, wherein the one or morethioaptamers comprise an oligonucleotide library or library of libraries.

19. The method of claim 1, wherein the library comprises sequence motifs for high affinity with cellular proteins selected from the group consisting essentially of proteins that are members of the AP-1, RBP-Jκ

, NF-κ

B, NF IL-6, CREB and GRE protein families, and combinations thereof.

20. The method of claim 1, further comprising the step of comparing a first and a second incubation of one or more beads to a first and a second sample, respectively, wherein differences in binding are used to detect proteins that expressed differentially.

21. The method of claim 1, further comprising the step of comparing a first and a second incubation of one or more beads to a first and a second sample, respectively, wherein differences in binding are used to detect proteins that expressed differentially and wherein the second sample comprises a diseased cell.

22. The method of claim 1, further comprising the step of

binding the one or more thioaptamers to one or more beads;

incubating the one or more thioaptamer beads with a cell extract from a cell wherein proteins from the cell extract are labeled with a first dye;

incubating the one or more thioaptamers beads with a cell extract from a diseased-cell wherein proteins from the diseased-cell extract are labeled with a second dye;

incubating the one or more thioaptamers beads with a cell extract from a diseased-cell pre-treated with thioaptamers or other drugs, wherein the proteins of the diseased-cell but drug-treated, are labeled with a third dye; and

performing a three-color flow cytometry that measured the relative levels of the first, second and third dyes.

23. The method of claim 1, wherein the diseased-cell comprises a cancer cell.

24. The method of claim 1, wherein the diseased-cell comprises a virally-infected cell.

25. The method of claim 1, wherein the amount of protein of the first, second and third incubations are about the same.

26. A system for identifying proteins that interact with a specific thioaptamer comprising:

a thioaptamer library for binding one or more proteins;

a bead separator wherein the one or more thioaptamer beads of the library to which protein has bound are separated;

a mass spectrometer to identify the proteins that have bound to the one or thioaptamer beads.

27. The system of claim 26, further comprising a nucleic acid sequencer to determine the sequence of the thioaptamer.

28. The system of claim 26, wherein the thioaptamer is PCR amplified and cloned prior to sequencing.

29. The system of claim 26, further comprising a protein separator that separates a bound protein into fragments prior to separation by liquid chromatography followed by mass spectrometry.

30. The system of claim 26, wherein the one or more proteins bound to the thioaptamer are extracted and separated prior to mass spectrometry.

31. The system of claim 26, wherein the one or more proteins bound to the thioaptamer are extracted, separated and identified by liquid chromatography and time of flight (TOF) mass spectrometry (MS) MALDI-TOF.

32. The system of claim 26, wherein the thioaptamers library comprises a one-bead, one thioaptamer library.

33. The system of claim 26, wherein the thioaptamer library is attached to a substrate.

34. The system of claim 26, wherein the one or more proteins is further defined as being a cell extract.

35. The system of claim 26, wherein the one or more proteins is further defined as being a cell extract from a virally infected cell.

36. The system of claim 26, wherein the one or more proteins is further defined as being a cell extract from a diseased cell.

37. The system of claim 26, wherein thioaptamer library is attached to beads and the beads are substantially protein-free.

38. The system of claim 26, wherein the thioaptamer library comprises an [S]-ODN or [S₂]-ODN combinatorial libraries.

39. The system of claim 26, wherein the thioaptamer library is double-stranded.

40. The system of claim 26, wherein the thioaptamer library comprises RNA.

41. The system of claim 26, wherein thioaptamer library comprises a library of libraries.

42. The system of claim 26, wherein the thioaptamer library comprises sequence motifs for high affinity with cellular proteins selected from the group consisting essentially of proteins that are members of the AP-1, RBP-Jκ

, NF-κ

B, NF IL-6, CREB and GRE protein families, and combinations thereof.

43. A bead-based method for identifying one or more proteins that bind to one or more thioaptamers comprising the steps of:

incubating a thioaptamer library with a sample suspected of comprising one or more proteins; and

identifying the proteins by mass spectrometry.

44. A method for identifying one or more targets that bind to one or more thioaptamers comprising the steps of:

incubating a thioaptamer library with a sample suspected of comprising one or more targets; and

identifying the target by mass spectrometry.

45. The composition of claim 44, wherein the thioaptamer comprises a thioaptamer that is concatenated with one or more aptamers.