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Details for Patent: 5,789,157

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Details for Patent: 5,789,157

Title: Systematic evolution of ligands by exponential enrichment: tissue selex
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 red blood cells ghosts, glioblastomas, and lymphomas are described.
Inventor(s): Jensen; Kirk B. (Boulder, CO), Chen; Hang (Boulder, CO), Morris; Kevin N. (Schwarzach, AT), Stephens; Andrew (Denver, CO), Gold; Larry (Boulder, CO)
Assignee: NeXstar Pharmaceuticals, Inc. (Boulder, CO)
Filing Date:May 03, 1995
Application Number:08/434,425
Claims:1. A method for identifying nucleic acid ligands to a protein component of a biological tissue comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting said candidate mixture of nucleic acids with said biological tissue, wherein nucleic acids having an increased affinity to the biological tissue relative to the candidate mixture may be partitioned from the remainder of the candidate mixture and wherein said nucleic acids have a specific affinity to a protein component of said biological tissue;

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

d) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acids with an increased affinity and an increased specificity for binding to said protein, whereby nucleic acid ligands to a protein component of a biological tissue are identified.

2. The method of claim 1 wherein said nucleic acids of the candidate mixture are comprised of single-stranded nucleic acids.

3. The method of claim 2 wherein said single-stranded nucleic acids are ribonucleic acids.

4. The method of claim 2 wherein said single-stranded nucleic acids are deoxyribonucleic acids.

5. The method of claim 1 wherein said biological tissue is selected from the group consisting of red blood cell ghosts, glioblastoma, and lymphoma.

6. A method for identifying nucleic acid ligands to a protein component of a biological tissue identified according to a method comprising:

(a) preparing a candidate mixture of nucleic acids;

(b) contacting the candidate mixture with a first biological tissue, wherein nucleic acids having an increased affinity to the first biological tissue relative to the candidate mixture may be partitioned from the remainder of the candidate mixture and wherein said nucleic acids have a specific affinity to a protein component of said biological tissue;

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

(d) contacting the increased affinity nucleic acids with a second biological tissue, wherein nucleic acids with affinity to the second biological tissue are removed; and

(e) amplifying the remaining nucleic acids with specific affinity to said protein components to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to said protein components, whereby nucleic acid ligands to a protein component of a biological tissue are identified.

7. A method for identifying nucleic acid ligands to a protein component of a biological tissue identified according to a method comprising:

(a) preparing a candidate mixture of nucleic acids;

(b) contacting the candidate mixture with a first biological tissue, wherein nucleic acids with affinity to the first biological tissue relative to the candidate mixture are removed from the candidate mixture;

(c) contacting the remaining candidate mixture from (b) with a second biological tissue, wherein nucleic acids having an increased affinity to the second biological tissue relative to the candidate mixture may be partitioned from the remainder of the candidate mixture and wherein said nucleic acids have a specific affinity for a protein component of said second biological tissue;

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

(e) amplifying the nucleic acids with specific affinity to said protein component to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to said protein components, whereby nucleic acid ligands to a protein component of a biological tissue are identified.

8. A method for identifying a nucleic acid ligand to a protein component of a biological tissue identified according to a method comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting said candidate mixture of nucleic acids with said biological tissue, wherein nucleic acids having an increased affinity to the biological tissue relative to the candidate mixture may be partitioned from the remainder of the candidate mixture and wherein said nucleic acids have a specific affinity to a protein component of said biological tissue;

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

d) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acids with an increased affinity and an increased specificity for binding to said protein component; and

e) repeating steps b)-d) as necessary, whereby a nucleic acid ligand to protein component of a biological tissue is identified.

9. A method for identifying a nucleic acid ligand to a protein component of a biological tissue identified according to a method comprising:

(a) preparing a candidate mixture of nucleic acids;

(b) contacting the candidate mixture with a first biological tissue, wherein nucleic acids having an increased affinity to the first biological tissue relative to the candidate mixture may be partitioned from the remainder of the candidate mixture and wherein said nucleic acids have a specific affinity to a protein component of said biological tissue;

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

(d) contacting the increased affinity nucleic acids with a second biological tissue, wherein nucleic acids with affinity to the second biological tissue are removed;

(e) amplifying the remaining nucleic acids with specific affinity to said protein component to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to said protein component; and

(f) repeating steps (b)-(e) as necessary, whereby a nucleic acid ligand to a protein component of a biological tissue is identified.

10. A method for identifying a nucleic acid ligand to a protein component of a biological tissue identified according to a method comprising:

(a) preparing a candidate mixture of nucleic acids;

(b) contacting the candidate mixture with a first biological tissue, wherein nucleic acids with affinity to the first biological tissue relative to the candidate mixture are removed from the candidate mixture;

(c) contacting the remaining candidate mixture from (b) with a second biological tissue, wherein nucleic acids having an increased affinity to the second biological tissue relative to the candidate mixture may be partitioned from the remainder of the candidate mixture and wherein said nucleic acids have a specific affinity for protein component of said second biological tissue;

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

(e) amplifying the nucleic acids with specific affinity to said protein component to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to said protein component; and

(f) repeating steps (b)-(e) as necessary, whereby a nucleic acid ligand to a protein component of a biological tissue is identified.

11. A method for identifying a nucleic acid ligand to a protein component of a biological tissue identified according to a method comprising:

a) preparing a candidate mixture of nucleic acids;

b) contacting said candidate mixture of nucleic acids with said biological tissue, wherein nucleic acids having an increased affinity to the biological tissue relative to the candidate mixture may be partitioned from the remainder of the candidate mixture and wherein said nucleic acids have a specific affinity to a protein component of said biological tissue;

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

d) amplifying the increased affinity nucleic acids to yield a mixture of nucleic acids enriched for nucleic acids with an increased affinity and an increased specificity for binding to said protein component;

e) cloning said increased affinity nucleic acids;

f) determining the nucleic acid sequences of said cloned nucleic acids; and

g) determining the specificity of said cloned nucleic acids for binding said protein component of a biological tissue, whereby a nucleic acid ligand to a protein component of a biological tissue is identified.
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