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Details for Patent: 5,270,163

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Details for Patent: 5,270,163

Title: Methods for identifying nucleic acid ligands
Abstract:The SELEX (Systematic Evolution of Ligands by EXponential Enrichment) method is disclosed for the identification of nucleic acid ligands. A candidate mixture of single stranded nucleic acids having regions of randomized sequence is contacted with a target compound and those nucleic acids having an increased affinity to the target are partitioned from the remainder of the candidate mixture. The partitioned nucleic acids are amplified to yield a ligand enriched mixture.
Inventor(s): Gold; Larry (Boulder, CO), Tuerk; Craig (Boulder, CO)
Assignee: University Research Corporation (Boulder, CO)
Filing Date:Aug 17, 1992
Application Number:07/931,473
Claims:1. A method for identifying nucleic acid ligands of a target compound from a candidate mixture comprised of single stranded nucleic acids each having a region of randomized sequence, said, method comprising:

a) contacting the candidate mixture with the target, 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;

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 the target compound may be identified.

2. The method of claim 1 wherein said candidate mixture is contacted with the target under conditions favorable for binding, and nucleic acid-target pairs are formed.

3. The method of claim 1 further comprising the step:

d) repeating steps a) through c) using the ligand enriched mixture of each successive repeat as many times as required to yield a desired level of ligand enrichment, whereby nucleic acid ligands of the target compound may be identified.

4. The method of claim 3 wherein said level of ligand enrichment is sufficient to establish a ligand solution to the target.

5. The method of claim 1 wherein said target is a protein.

6. The method of claim 5 wherein said protein is a nucleic acid binding protein.

7. The method of claim 5 wherein said protein is not known to bind nucleic acids.

8. The method of claim 1 wherein said amplification step employs polymerase chain reaction (PCR).

9. The method of claim 1 wherein said partitioning step employs filter binding selection.

10. The method of claim 1 wherein said candidate mixture is comprised of ribonucleic acids.

11. The method of claim 9 wherein said contacting step is performed in the presence of an excess of said target.

12. The method of claim 1 wherein said target is supported on a matrix.

13. The method of claim 12 wherein said matrix-supported target is held in a column.

14. The method of claim 1 wherein said candidate mixture is prepared by synthesis from a template comprising a region of conserved sequence and a region of randomized and/or biased sequence.

15. The method of claim 1 wherein said candidate mixture comprises nucleic acids each comprising a region of conserved sequence and a region of randomized sequence.

16. The method of claim 15 wherein said conserved sequence region of the nucleic acids of said candidate mixture comprises a sequence known to bind to said target.

17. The method of claim 2 wherein said target is a protein and said nucleic acid-target pairs contain Michael adducts.

18. A method for selecting a nucleic acid which affects the function of a target molecule which comprises the additional step of screening nucleic acid ligands identified by the method of claim 1 for increased affinity to said target for their ability to affect the function of said target molecules.

19. The method of claim 4 further comprising the steps of:

preparing a second candidate mixture comprising single stranded nucleic acids, each nucleic acid further comprising a region of conserved sequence derived from said ligand solution; and

f) repeating steps a) through d) using said second candidate mixture, whereby nucleic acid ligands of the target compound may be identified.

20. The method of claim 4 further comprising the steps of:

e) preparing a second candidate mixture comprising single stranded nucleic acids, each nucleic acid further comprising a region of biased sequence derived from said ligand solution; and

f) repeating steps a) through d) using said second candidate mixture, whereby nucleic acid ligands of the target compound may be identified.

21. The method of claim 1 wherein said target is a transition-state analog.
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