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Details for Patent: 5,496,938

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Details for Patent: 5,496,938

Title: Nucleic acid ligands to HIV-RT and HIV-1 rev
Abstract:Methods for the identification and production of improved nucleic acid ligands are based on the SELEX process. Nucleic acid ligands to HIV-RT and HIV-1 Rev are identified according to the methods described herein.
Inventor(s): Gold; Larry (Boulder, CO), Tuerk; Craig (Morehead, KY)
Assignee: NeXstar Pharmaceuticals, Inc. (Boulder, CO)
Filing Date:Oct 21, 1992
Application Number:07/964,624
Claims:1. A non-naturally occurring nucleic acid ligand to HIV reverse transcriptase (RT) obtained according to a method for identifying a nucleic acid ligand from a candidate mixture of nucleic acids, said method comprising:

a) contacting the candidate mixture with HIV-RT, wherein nucleic acids having an increased affinity to HIV-RT relative to the candidate mixture nucleic acids may be partitioned from the remainder of the candidate mixture;

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

c) amplifying the increased affinity nucleic acids, in vitro, to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands of HIV-RT may be identified and obtained nucleic acids.

2. The nucleic acid ligand to HIV-RT obtained by the method of claim 1 wherein said method further comprises:

d) preparing a modified nucleic acid that is identical to the nucleic acid ligand except for a single residue substitution; and

e) assessing the binding affinity of the modified nucleic acid relative to the nucleic acid ligand whereby nucleic acid ligands to HIV-RT may be obtained.

3. The nucleic acid ligand to HIV-RT may be obtained by the method of claim 1 wherein said method further comprises:

d) preparing a modified nucleic acid that is identical to the nucleic acid ligand except for the absence of one or more terminal residues; and

e) assessing the binding affinity of the modified nucleic acid relative to the nucleic acid ligand whereby nucleic acid ligands to HIV-RT may be obtained.

4. A non-naturally occurring nucleic acid ligand to HIV-RT having the RNA sequence (SEQ ID NO:12): ##STR1## wherein X=any base, and X-X' indicates a preferred base-pair.

5. A non-naturally occurring nucleic acid ligand to HIV-RT obtained according to a method for identifying an extended nucleic acid ligand from a candidate mixture of nucleic acids, said nucleic acid ligand being a ligand of HIV-RT comprising:

a) contacting the candidate mixture with HIV-RT, wherein nucleic acids having an increased affinity to HIV-RT relative to the candidate mixture nucleic acids may be partitioned from the remainder of the candidate mixture;

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

c) amplifying the increased affinity nucleic acids, in vitro, to yield a ligand-enriched mixture of nucleic acids;

d) repeating steps a)-c), as necessary, to identify said nucleic acid ligand to HIV-RT;

e) contacting a second candidate mixture of nucleic acids with HIV-RT, wherein said second candidate mixture is comprised of nucleic acids having a fixed region and a randomized region, said fixed region corresponding to said nucleic acid ligand to HIV-RT identified in d), and wherein nucleic acids having an increased affinity to HIV-RT relative to the candidate mixture nucleic acids may be partitioned from the remainder of the candidate mixture;

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

g) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids; and

h) repeating steps e)-h), as necessary, whereby said nucleic acid ligand to HIV-RT may be obtained.

6. A non-naturally occurring nucleic acid ligand obtained by the method of claim 5 having the RNA sequence (SEQ ID NO: 13): ##STR2## or the corresponding DNA sequence thereof; wherein x is any base, and Z is selected from the group consisting of the sequences set forth in FIG. 9 (SEQ ID NO:14-34).

7. A non-naturally occurring nucleic acid ligand to HIV-1 Rev obtained according to a method for identifying a nucleic acid ligand from a candidate mixture of nucleic acids, said method comprising:

a) contacting the candidate mixture with HIV-1 Rev, wherein nucleic acids having an increased affinity to HIV-1 Rev relative to the candidate mixture nucleic acids may be partitioned from the remainder of the candidate mixture;

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

c) amplifying the increased affinity nucleic acids, in vitro, to yield a ligand-enriched mixture of nucleic acids, whereby nucleic acid ligands to HIV-1 Rev may be identified and obtained.

8. The nucleic acid ligand to HIV-1 Rev obtained by the method of claim 7 wherein said method further comprises:

d) preparing a modified nucleic acid that is identical to the nucleic acid ligand except for a single residue substitution; and

e) assessing the binding affinity of the modified nucleic acid relative to the nucleic acid ligand.

9. The nucleic acid ligand to HIV-1 Rev obtained by the method of claim 7 wherein said method further comprises:

d) preparing a modified nucleic acid that is identical to the nucleic acid ligand except for the absence of one or more terminal residues; and

e) assessing the binding affinity of the modified nucleic acid relative to the nucleic acid ligand whereby nucleic acid ligands to HIV-1 Rev may be obtained.

10. A non-naturally occurring nucleic acid ligand to HIV-1 Rev having a RNA sequence (SEQ ID NO:35): ##STR3## wherein X=any base. f5 whereby nucleic acid ligands to HIV-RT may be obtained
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