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Details for Patent: 5,688,935

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Details for Patent: 5,688,935

Title: Nucleic acid ligands of tissue target
Abstract:This invention discloses high-affinity oligonucleotide ligands to complex tissue targets, specifically nucleic acid ligands having the ability to bind to complex tissue targets, and the methods for obtaining such ligands. Tissue targets comprise cells, subcellular components, aggregates or cells, collections of cells, and higher ordered structures. Specifically, nucleic acid ligands to peripheral blood mononuclear cells (PBMC), fibrin clots, and carotid arteries are described.
Inventor(s): Stephens; Andrew (Denver, CO), Schneider; Dan J. (Boulder, CO), Gold; Larry (Boulder, CO), Speck; Ulrich (Berlin, DE)
Assignee: NeXstar Pharmaceuticals, Inc. (Boulder, CO) Schering Aktiengesellschaft (Berlin, DE)
Filing Date:May 03, 1995
Application Number:08/433,126
Claims:1. A nucleic acid ligand to a target selected from the group consisting of peripheral blood mononuclear cells, fibrin clots, and carotid arteries identified according to a method comprising:

a) preparing a candidate mixture of nucleic acids having a mixture of nucleic acid sequences;

b) contacting said candidate mixture of nucleic acids with said target selected from the group consisting of peripheral blood mononuclear cells, fibrin clots, and carotid arteries, 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; and

d) amplifying the increased affinity nucleic acids

e) repeating steps a) through d) as required to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding to said target, whereby a nucleic acid ligand of said target may be identified.

2. A purified and isolated non-naturally occurring nucleic acid ligand to a target selected from the group consisting of peripheral blood mononuclear cells, fibrin clots, and carotid arteries.

3. The purified and isolated nucleic acid ligand of claim 2 wherein said nucleic acid ligand is a non-naturally occurring nucleic acid ligand having a specific binding affinity for said target, such target being a three dimensional chemical structure other than a polynucleotide that binds to said nucleic acid ligand through a mechanism which predominantly depends on Watson/Crick base pairing or triple helix binding, wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target.

4. The nucleic acid ligand of claim 2 which is a deoxyribonucleic acid ligand.

5. The nucleic acid ligand of claim 2 which is a ribonucleic acid ligand.

6. The nucleic acid ligand of claim 2 wherein said target is a peripheral blood mononuclear cell.

7. The nucleic acid ligand to a peripheral blood mononuclear cell of claim 6 wherein said ligand is a DNA ligand selected from the group consisting of the nucleotide sequences set forth in Table 2, SEQ ID NOS: 7-39.

8. The nucleic acid ligand of claim 2 wherein said target is a fibrin clot.

9. The nucleic acid ligand to a fibrin clot of claim 8 wherein said ligand is a RNA ligand selected from the group consisting of the nucleotide sequences set forth in Table 5, SEQ ID NOS: 43-130.

10. The nucleic acid ligand of claim 2 wherein said target is a carotid artery.

11. The nucleic acid ligand to a carotid artery of claim 10 wherein said ligand is a RNA ligand selected from the group consisting of the nucleotide sequences set forth in Table 8, SEQ ID NOS: 141-237.

12. A nucleic acid ligand to a target selected from the group consisting of peripheral blood mononuclear cells, fibrin clots, and carotid arteries identified according to a method comprising:

(a) preparing a candidate mixture of nucleic acids having a mixture of nucleic acid sequences;

(b) contacting the candidate mixture with a first target selected from the group consisting of peripheral blood mononuclear cells, fibrin clots, and carotid arteries, wherein nucleic acids having an increased affinity to the first 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 a second target, wherein nucleic acids with affinity to the second target are removed;

(e) amplifying the nucleic acids with increased affinity to said first target

(f) repeating steps (a) through (e) as required to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding to said target; and

(g) identifying said nucleic acid ligand of said first target.

13. A nucleic acid ligand to a target selected from the group consisting of peripheral blood mononuclear cells, fibrin clots, and carotid arteries identified according to a method comprising:

(a) preparing a candidate mixture of nucleic acids having a mixture of nucleic acid sequences;

(b) contacting the candidate mixture with a first target, wherein nucleic acids with affinity to the first target are removed;

(c) contacting the candidate mixture from (b) with a second target selected from the group consisting of peripheral blood mononuclear cells, fibrin clots, and carotid arteries, wherein nucleic acids having an increased affinity to the second 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 increased affinity to said second target and

(f) repeating steps (a) through (e) as required to yield a mixture of nucleic acids enriched for nucleic acid sequences with relatively higher affinity and specificity for binding to said target; and

(g) identifying said nucleic acid ligand of said second target.
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