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Details for Patent: 5,660,985

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Details for Patent: 5,660,985

Title: High affinity nucleic acid ligands containing modified nucleotides
Abstract:Nucleic acid ligands containing modified nucleotides are described as are methods for producing such oligonucleotides. Such ligands enrich the chemical diversity of the candidate mixture for the SELEX process. Specific examples are provided of nucleic acids containing nucleotides modified at the 2'- and 5-position. Specific 2-OH and 2'-NH.sub.2 modified RNA ligands to thrombin are described.
Inventor(s): Pieken; Wolfgang (Longmont, CO), Tasset; Diane (Boulder, CO), Janjic; Nebojsa (Lafayette, CO), Gold; Larry (Boulder, CO), Kirschenheuter; Gary P. (Arvada, CO)
Assignee: NeXstar Pharmaceuticals, Inc. (Boulder, CO)
Filing Date:Apr 27, 1995
Application Number:08/430,709
Claims:1. A method for identifying nucleic acid ligands comprising a modified nucleotide to a target molecule comprising:

a) preparing a candidate mixture of single-stranded nucleic acids wherein each nucleic acid of said candidate mixture comprises a modified nucleotide selected from the group consisting of a 5-position modified pyrimidine, a 2'-position modified pyrimidine, an 8 -position modified purine, and a 2'-position modified purine;

b) contacting the candidate mixture with said target molecule;

c) partitioning the nucleic acids having an increased affinity to the target molecule relative to the candidate mixture from the remainder of the candidate mixture; and

d) amplifying the increased affinity nucleic acids, in vitro, to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands of the target molecule are identified.

2. A method for claim 1 further comprising

e) repeating steps c) and d).

3. The method of claim 1 wherein said single-stranded nucleic acids are selected from the group consisting of single-stranded DNA and single-stranded RNA.

4. The method of claim 1 wherein said modified nucleotide is selected from the group consisting of a 2'-position modified pyrimidine and a 5-position modified pyrimidine.

5. The method of claim 1 wherein said modified nucleotide is selected from the group consisting of a 2'-amino pyrimidine and a 2'-amino purine.

6. The method of claim 1 wherein said modified nueleotide is selected from the group consisting of a 2'-fluoro pyrimidine and a 2'-fluoro purine.

7. The method of claim 4 wherein said 5-position modified pyrimidine is selected from the group consisting of 5-(3-amino alkyl) uridine triphosphate (5-AA-UTP), 5-fluorescein-12-uridine triphosphate (5-F-12-UTP), 5-digoxygenin-11-uridine triphosphate (5-Dig-11-UTP), and 5-bromouridine triphosphate (5-Br-UTP).

8. A nucleic acid ligand to thrombin identified according to the method of claim 1.

9. The nucleic acid ligand of claim 8 wherein said nucleic acid ligand is comprised of 2'-amino pyrimidines, and wherein said ligand is selected from the group consisting of SEQ ID NOS: 20-39.

10. The method of claim 1 wherein said modified nucleotide is selected from the group consisting of a 2'-methoxy pyrimidine and a 2'-methoxy purine.
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