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Details for Patent: 5,580,737

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Details for Patent: 5,580,737

Title: High-affinity nucleic acid ligands that discriminate between theophylline and caffeine
Abstract:Methods are described for the identification and preparation of nucleic acid ligand ligands to theophylline and caffeine. Included in the invention is a method for identifying nucleic acid ligands with high affinity and selectivity for a target molecule, termed counter-SELEX. One embodiment of counter-SELEX is comprised of the steps of a) preparing a candidate mixture of nucleic acids; b) contacting the candidate mixture with the target, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned form the remainder of the candidate mixture; partitioning the increased affinity nucleic acids from the remainder of the candidate mixture; d) contacting the increased affinity nucleic acids with one or more non-target molecules such that nucleic acid ligands to the non-target molecule(s) are removed; and e) amplifying the nucleic acids with specific affinity to the target molecule to yield a mixture of nucleic acids enriched for nucleic acid sequences with a relatively higher affinity and specificity for binding to the target molecule. Included in the invention are specific nucleic acid ligands to theophylline identified by the method of counter-SELEX.
Inventor(s): Polisky; Barry (Boulder, CO), Jenison; Robert D. (Boulder, CO), Gold; Larry (Boulder, CO)
Assignee: NeXstar Pharmaceuticals, Inc. (Boulder, CO)
Filing Date:May 18, 1995
Application Number:08/443,957
Claims:1. A method for identifying nucleic acid ligands to a target molecule comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting the candidate mixture with the target molecule, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

c) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture;

d) contacting the increased affinity nucleic acids with one or more non-target molecules, wherein nucleic acids with affinity to the non-target molecule(s) are removed; and

e) amplifying the nucleic acids with specific affinity to the target molecule to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding to the target molecule, whereby nucleic acid ligands of the target molecule may be identified.

2. The method of claim 1 further comprising f) repeating steps b), c), d) and e).

3. The method of claim 1 wherein said target molecule is immobilized on a column.

4. The method of claim 1 wherein said candidate mixture of nucleic acids is comprised of single stranded nucleic acids.

5. A purified and isolated non-naturally occurring RNA ligand to theophylline, wherein the nucleotide sequence of said RNA ligand is selected from the group consisting of the nucleotide sequences set forth in FIGS. 2A and 2B (SEQ ID NOS: 3-15).

6. A purified and isolated non-naturally occurring RNA ligand to caffeine, identified by the method of claim 1, wherein the nucleotide sequence of said RNA ligand is selected from the group consisting of the nucleotide sequences set fourth in FIG. 8 (SEQ ID NOS:16-27).

7. Nucleic acid ligands to theophylline identified according to a method for identifying nucleic acid ligands from a candidate mixture comprised of nucleic acids, said method comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting said candidate mixture with theophylline, wherein nucleic acids having an increased affinity to theophylline relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

c) partitioning the increased affinity nucleic acid ligands from the remainder of the candidate mixture; and

d) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acid, whereby nucleic acid ligands of theophylline may be identified.

8. The method of claim 7 further comprising

e) repeating steps b), c) and d).

9. Nucleic acid ligands to caffeine identified according to a method for identifying nucleic acid ligands from a candidate mixture comprised of nucleic acids, said method comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting said candidate mixture with caffeine, wherein nucleic acids having an increased affinity to caffeine relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

c) partitioning the increased affinity nucleic acid ligands from the remainder of the candidate mixture; and

d) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acid, whereby nucleic acid ligands of caffeine may be identified.

10. The method of claim 9 further comprising

e) repeating steps b), c) and d).

11. The method of claim 1 wherein said target molecule is theophylline.

12. A nucleic acid ligand to theophylline, identified according to the method of claim 11.

13. The method of claim 1 wherein said target molecule is caffeine.

14. A nucleic acid ligand to caffeine identified according to the method of claim 13.

15. A non-naturally occurring nucleic acid ligand, having a specific binding affinity for a target molecule, wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule, and wherein said target molecule is selected from the group consisting of theophylline and caffeine.

16. A method for identifying nucleic acid ligands to a target molecule comprising:

(a) preparing a candidate mixture of nucleic acids;

(b) contacting the candidate mixture with one or more non-target molecules, wherein nucleic acids with affinity to the non-target molecule(s) are removed;

(c) contacting the candidate mixture from (b) with the target molecule, wherein nucleic acids having an increased affinity to the target relative to the candidate mixture may be partitioned from the remainder of the candidate mixture;

(d) partitioning the increased affinity nucleic acids from the remainder of the candidate mixture;

(e) amplifying the nucleic acids with specific affinity to the target molecule to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding to the target molecule, whereby nucleic acid ligands of the target molecule may be identified.
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