Structure based and combinatorially selected oligonucleoside phosphorothioate and phosphorodithioate aptamer targeting AP-1 transcription factors

• US 7,910,523 B2
• Filed: 05/20/2004
• Issued: 03/22/2011
• 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.
• Primary Examiner(s): Low; Christopher
• Assistant Examiner(s): Gross; Christopher M.
• Application Number:
  US10/851,864
• Publication Number:
  US 20040265912A1
• Time in Patent Office: 2,497 Days
• Field of Search: None
• US Class Current: 506/31
• 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 thio-modified backbone aptamer sequences and binding proteins comprising the steps of:

A) incubating a partially thio-modified backbone aptamer bead library with a sample suspected of comprising one or more proteins, wherein each bead of the partially thio-modified backbone aptamer bead library comprises more than one copy of an unique aptamer having a unique sequence and backbone modification;

B) selecting one or more beads from the thio-modified backbone aptamer beads library to which the one or more proteins are bound;

C) identifying the one or more proteins by mass spectrometry;

D) identifying the unique sequence of the thio-modified aptamer; and

E) locating the position of a backbone thio-modification of the unique aptamers comparing said sequence to positions encoded during split synthesis.

2. The method of claim 1, further comprising the step of fragmenting the protein and separating the fragments by liquid chromatography followed by mass spectrometry after selection step B).

3. The method of claim 1, wherein the steps of identifying the protein by mass spectrometry, step C) 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 thio-modified aptamers are attached to the beads by a non-cleavable linker.

6. The method of claim 1, wherein the unique one or more thio-modified aptamers bound to one or more proteins are isolated from a gel.

7. The method of claim 1, wherein the unique one or more thio-modified aptamers are attached to beads and the one or more proteins that have bound the one or more unique thio-modified aptamers are labeled and the beads are sorted based on protein binding.

8. The method of claim 1, wherein the one or more unique thio-modified aptamers are attached to beads and the one or more proteins that have bound the unique one or more thio-modified aptamers are labeled and the beads are sorted based on fluorescence.

9. The method of claim 1, wherein 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.

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

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

12. The method of claim 1, wherein one or more thio-modified aptamers are attached to beads and the beads are substantially protein-free.

13. The method of claim 1, further comprising the step of sequencing the aptamer directly.

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

15. The method of claim 1, wherein the one or more beads comprise a double-stranded thio-modified backbone aptamer library.

16. 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 NF-kB protein family.