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Generated: August 18, 2017

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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 (Boulder, CO), Chen; Hang (Boulder, CO), Morris; Kevin N. (Schwarzach, AU), Stephens; Andrew (Denver, CO), Gold; Larry (Boulder, CO)
Assignee: NeXstar Pharmaceuticals, Inc. (Boulder, CO)
Filing Date:May 03, 1995
Application Number:08/437,667
Claims:1. 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 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. A purified and isolated non-naturally occurring nucleic acid ligand to a protein component of a biological tissue.

3. The purified nucleic acid ligand of claim 2 which is a non-naturally occurring nucleic acid ligand having a specific binding affinity for a protein component of a biological tissue.

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 biological tissue is a red blood cell ghost.

7. The nucleic acid ligand of claim 6 wherein said ligand is a DNA ligand selected from the group consisting of the nucleotide sequences set forth in Table 1.

8. The nucleic acid ligand of claim 2 wherein said biological tissue is a cell.

9. The nucleic acid ligand of claim 8 wherein said cell is a glioblastoma.

10. The nucleic acid ligand of claim 9 wherein said ligand is a DNA ligand selected from the group consisting of the nucleotide sequences set forth in Table 2.

11. The nucleic acid ligand of claim 8 wherein said cell is a lymphoma.

12. The nucleic acid ligand of claim 1 wherein said candidate mixture is comprised of single-stranded nucleic acids.

13. The nucleic acid ligand of claim 12 wherein said single-stranded nucleic acids are deoxyribonucleic acids.

14. The nucleic acid ligand of claim 12 wherein said single-stranded nucleic acids are ribonucleic acids.

15. The nucleic acid ligand of claim 2 that can modify the function of the protein component.

16. 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 component to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to said protein component, whereby nucleic acid ligands to a protein component of a biological tissue are identified.

17. 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 component, whereby nucleic acid ligands to a protein component of a biological tissue are identified.

18. 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; and

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

19. 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 to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to said protein; and

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

20. 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 a 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 to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to said protein; and

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

21. 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;

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 a protein component of a biological tissue, whereby a nucleic acid ligand to a protein component of a biological tissue is identified.

22. The method of claim 21 wherein said determination of the specificity of said cloned nucleic acid for binding a protein component of a biological tissue comprises:

a) photocrosslinking said cloned nucleic acid to a biological tissue;

b) fractionating the proteins components of said biological tissue; and

c) determining whether said cloned nucleic acid is crosslinked to a protein component of said biological tissue.
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