Bead bound combinatorial oligonucleoside phosphorothioate and phosphorodithioate aptamer libraries

• US 7,338,762 B2
• Filed: 04/21/2004
• Issued: 03/04/2008
• Est. Priority Date: 10/16/2002
• 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: Yang, Xianbin, Luxon, Bruce A., Gorenstein, David G., Herzog, Norbert
• Primary Examiner(s): WESSENDORF, TERESA D
• Application Number:
  US10/828,935
• Publication Number:
  US 20050123939A1
• Time in Patent Office: 1,413 Days
• Field of Search: 435/6, 435/5, 435/4, 536/23.1
• US Class Current: 435/6.11
• CPC Class Codes: B01J 2219/00378 Piezoelectric or ink jet dispensers
• CPC Class Codes: B01J 2219/00387 Applications using probes
• CPC Class Codes: B01J 2219/00497 Features relating to the solid phase supports
• CPC Class Codes: B01J 2219/005 Beads
• CPC Class Codes: B01J 2219/00529 DNA chips
• CPC Class Codes: B01J 2219/00585 Parallel processes
• CPC Class Codes: B01J 2219/0059 Sequential processes
• CPC Class Codes: B01J 2219/00592 Split-and-pool, mix-and-divide processes
• CPC Class Codes: B01J 2219/00596 Solid-phase processes
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/00637 by coating it with another layer
• CPC Class Codes: B01J 2219/00641 the porous medium being continuous, e.g. porous oxide substrates
• CPC Class Codes: B01J 2219/00648 by the use of solid beads
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: B01J 2219/00707 separated from the reactor apparatus
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07B 2200/11 : Compounds covalently bound to a solid support
• 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: C40B 20/04 : Identifying library members by means of a tag, label, or other readable or detectable entity associated with the library members, e.g. decoding processes
• CPC Class Codes: C40B 40/06 : Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/08 : Libraries containing RNA or DNA which encodes proteins, e.g. gene libraries
• CPC Class Codes: C40B 60/14 : for creating libraries
• CPC Class Codes: G01N 33/6803 : General methods of protein analysis not limited to specific proteins or families of proteins
• 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 having two or more beads, wherein attached to each bead is a unique nucleic acid aptamer that have disposed thereon a unique sequence. The library aptamers may be attached covalently to the one or more beads, which may be polystyrene beads. The aptamers may include phosphorothioate, phosphorodithioate and/or methylphosphonate linkages and may be single or double stranded DNA, RNA or even PNAs.

Claims

1. A method for isolation of a target comprising the steps of:

dispersing one or more probe beads comprising a thioaptamer library selected from thioaptamers (S—

ODN) or dithioaptamers (S₂—

ODN) in a thixotropic agent, wherein the thioaptamers comprises one or more but less than all of the linkages selected from the group rATP(α

S₂), rUTP(α

S₂), rGTP(α

S₂), rCTP(α

S₂), rATP(α

S), dTTP(α

S), dGTP(α

S), dCTP(α

S), dATP(α

S₂), dTTP(α

S₂), dGTP(α

S₂) and dCTP(α

S₂);

scanning for probe beads that generate a detectable signal from interaction between the one or more probe beads and the target; and

picking one or more probe beads based on the detectable signal;

extracting the target from the probe bead; and

identifying the target by mass spectrometry after liquid chromatography.

2. The method of claim 1, further comprising the step of identifying the target using mass spectrometry comprises matrix assisted laser desorption ionization mass spectrometry.

3. The method of claim 1, wherein each of the probe beads are further modified to comprise a colorimetric agent.

4. The method of claim 1, wherein each of the probe beads further comprise one or more bases that are attached to a fluorophor.

5. The method of claim 1, wherein each of the probe beads further comprises one or more fluorophors attached to the 5′

end, the 3′

end or internally within the thioaptamers (S—

ODN) or the dithioaptamers (S₂—

ODN).

6. The method of claim 1, wherein the target is labeled with a fluorescent agent.

7. The method of claim 1, wherein the probe bead is acquired by a scanning robotic head and the target is extracted from the probe bead in situ.

8. The method of claim 1, wherein the probe bead is acquired by a scanning robotic head and the target is extracted from the probe bead in situ by proteolysis and transferred to an inlet of an LC-MS or an LC-MS/MS.

9. The method of claim 1, wherein the probe bead is acquired by a scanning robotic head and the target is extracted from the probe bead in situ for MALDI-MS analysis, wherein the MALDI-MS analysis is MALDI-TOF/MS.

10. The method of claim 1, wherein the probe bead is acquired by a scanning robotic head and the target is extracted from the probe bead in situ for LC-MS analysis.

11. The method of claim 1, wherein the probe bead is acquired by a scanning robotic head and the target is extracted from the probe bead in situ for MALDI-MS analysis.

12. The method of claim 1, wherein the probe bead is acquired by a scanning robotic head and the target is extracted from the probe bead in situ for MALDI-MS analysis by SELDI ionization.

13. The method of claim 1, wherein the probe bead is further processed to remove the target bound to the aptamer bead and analyzing the target by binding a second detectable label to the target.

14. The method of claim 1, wherein the thixotrophic agent comprises a polyacrylamide gel.

15. The method of claim 1, wherein picking the one or more probes beads is semi-manually.

16. The method of claim 1, wherein the target is a protein.

17. The method of claim 1, wherein the one or more probe beads are dispersed within the thixotropic agent by molecular printing.

18. The method of claim 1, wherein the one or more probe beads are dispersed within the thixotropic agent using an ink-jet printer.