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Details for Patent: 5,763,177

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Details for Patent: 5,763,177

Title: Systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands and solution selex
Abstract:A method for identifying nucleic acid ligands to target molecules using the SELEX pocedure wherein the candidate nucleic acids contain photoreactive groups and nucleic acid ligands identified thereby are claimed. The complexes of increased affinity nucleic acids and target molecules formed in the procedure are crosslinked by irradiation to facilitate separation from unbound nucleic acids. In other methods partioning of high and low affinity nucleic acids is facilitated by primer extension steps as shown in the figure in which chain termination nucleotides, digestion resistant nucleotides or nucleotides that allow retention of the cDNA product on an affinity matrix are differentially incorporated into the cDNA products of either the high or low affinity nucleic acids and the cDNA products are treated accordingly to amplification, enzymatic or chemical digestion or by contact with an affinity matrix.
Inventor(s): Gold; Larry (Boulder, CO), Willis; Michael (Louisville, CO), Koch; Tad (Boulder, CO), Ringquist; Steven (Lyons, CO), Jensen; Kirk (Boulder, CO), Atkinson; Brent (Boulder, CO)
Assignee: NeXstar Pharmaceuticals, Inc. (Boulder, CO)
Filing Date:Mar 08, 1996
Application Number:08/612,895
Claims:1. A method for identifying a nucleic acid ligand that photocrosslinks to a protein from a candidate mixture of nucleic acids, wherein each member of said candidate mixture contains a photoreactive group, said method comprising:

a) contacting said candidate mixture with said protein, wherein nucleic acids having an increased affinity to the protein relative to the candidate mixture form nucleic acid-protein complexes with the protein;

b) irradiating said complexes, wherein said nucleic acid-protein photocrosslink;

c) partitioning the photocrosslinked nucleic acid-protein complexes from in the candidate mixture; and

d) identifying a nucleic acid ligand that photocrosslinked to the protein.

2. The method of claim 1 further comprising after step c):

i) repeating steps a) through c); and

ii) amplifying the nucleic acids that photocrosslinked to the protein.

3. The method of claim 1 wherein after step c) the protein is removed from the nucleic acid-protein complex by proteolytic digestion.

4. The method of claim 1 wherein said identified nucleic acid ligand modifies a biological activity of said protein.

5. A method for identifying a photocrosslinking nucleic acid ligand of a protein from a candidate mixture of nucleic acids, said method comprising:

a) contacting said candidate mixture with said protein, wherein nucleic acids having increased affinity to the protein relative to the candidate mixture form nucleic acid-protein complexes with the protein;

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

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

d) incorporating photoreactive groups into said amplified increased affinity nucleic acids;

e) repeating step;

f) irradiating said increased affinity nucleic acids, wherein said nucleic acid-protein complexes photocrosslink;

g) repeating steps c) and d); and

h) identifying a photocrosslinking nucleic acid ligand to the protein.

6. The method of claim 1 for identifying a nucleic acid ligand that photocrosslinks to a protein further comprising the steps:

e) preparing a second candidate mixture of nucleic acids from the nucleic acid ligand identified in step d):

f) contacting said second candidate mixture with said protein wherein nucleic acids having an increased affinity to the protein relative to the second candidate mixture form nucleic acid-protein complexes:

g) partitioning the increased affinity nucleic acids from the remainder of the second candidate mixture, and

h) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids whereby a nucleic acid ligand that photocrosslinks the protein is identified.

7. The method of claim 1 wherein said photoreactive group is selected from the group consisting of 5-bromouracil, 5-iodouracil, 5-bromovinyluracil, 5-iodovinyluracil, 5-azidouracil, 4-thiouracil, 5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine, 5-iodovinylcytosine, 5-azidocytosine, 8-azidoadenine, 8-bromoadenine, 8-iodoadenine, 8-azidoguanine, 8-bromoguanine, 8-iodoguanine, 8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine, 8-azidoxanthine, 8-bromoxanthine, 8-iodoxanthine, 5-bromodeoxyuracil, 8-bromo-2'-deoxyadenine, 5-iodo2'-deoxyuracil, 5-iodo-2'-deoxycytosine, 5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil, 7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine, and 7-deaza-7-bromoguanine.

8. The method of claim 1 wherein the photocrosslinking nucleic acid ligand comprises one or more photoreactive groups, and wherein said photoreactive group is selected from the group consisting of 5-bromouracil, 5-iodouracil, 5-bromovinyluracil, 5-iodovinyluracil, 5-azidouracil, 4-thiouracil, 5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine, 5-iodovinylcytosine, 5-azidocytosine, 8-azidoadenine, 8-bromoadenine, 8-iodoadenine, 8-aziodoguanine, 8-bromoguanine, 8-iodoguanine, 8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine, 8-azidoxanthine, 8-bromoxanthine, 8-iodoxanthine, 5-bromodeoxyuracil, 8-bromo-2'-deoxyadenine, 5-iodo-2'-deoxyuracil, 5-iodo-2'-deoxycytosine, 5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil, 7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine, and 7-deaza-7-bromoguanine.

9. The method of claim 6 wherein the photocrosslinking nucleic acid ligand comprises one or more photoreactive groups, and wherein said photoreactive group is selected from the group consisting of 5-bromouracil, 5-iodouracil, 5-bromovinyluracil, 5-iodovinyluracil, 5-azidouracil, 4-thiouracil, 5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine, 5-iodovinylcytosine, 5-azidocytosine, 8-azidoadenine, 8-bromoadenine, 8-iodoadenine, 8-aziodoguanine, 8-bromoguanine, 8-iodoguanine, 8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine, 8-azidoxanthine, 8-bromoxanthine, 8-iodoxanthine, 5-bromodeoxyuracil, 8-bromo-2'-deoxyadenine, S-iodo-2'-deoxyuracil, 5-iodo-2'-deoxycytosine, 5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil, 7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine, and 7-deaza-7-bromoguanine.

10. The method of claim 6 wherein the photocrosslinking nucleic acid ligand comprises one or more photoreactive groups, and wherein said photoreactive group is selected from the group consisting of 5-bromouracil, 5-iodouracil, 5-bromovinyluracil, 5-iodovinyluracil, 5-azidouracil, 4-thiouracil, 5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine, 5-iodovinylcytosine, 5-azidocytosine, 8-azidoadenine, 8-bromoadenine, 8-iodoadenine, 8-aziodoguanine, 8-bromoguanine, 8-iodoguanine, 8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine, 8-azidoxanthine, 8-bromoxanthine, 8-iodoxanthine, 5-bromodeoxyuracil, 8-bromo-2'-deoxyadenine, 5-iodo-2'-deoxyuracil, 5-iodo-2'-deoxycytosine, 5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil, 7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine, and 7-deaza-7-bromoguanine.

11. A nucleic acid ligand that photocrosslinks to a protein, wherein said nucleic acid ligand is comprised of a non-naturally occurring nucleic acid having a specific binding affinity for a protein, wherein said protein is not a nucleic acid binding protein, and wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the protein, obtained by the process of identifying a nucleic acid ligand of a protein from a candidate mixture of nucleic acids comprised of nucleic acids each having a region of randomized sequence, and wherein each member of said candidate mixture contains a photoreactive group, said method comprising:

a) contacting said candidate mixture with said protein, wherein nucleic acids having an increased affinity to the protein relative to the candidate mixture form nucleic acid-protein complexes with the protein;

b) irradiating said candidate mixture, wherein said nucleic acidprotein complexes photocrosslink;

c) partitioning the photocrosslinked nucleic acid-protein complexes from the candidate mixture; and

d) identifying a nucleic acid ligand that photocrosslinked to the protein.

12. A nucleic acid ligand of claim 11 further comprising one or more of the photoreactive groups selected from the group consisting of 5-bromouracil, 5-iodouracil, 5-bromovinyluracil, 5-iodovinyluracil, 5-azidouracil, 4-thiouracil, 5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine, 5-iodovinylcytosine, 5-azidocytosine, 8-azidoadenine, 8-bromoadenine, 8-iodoadenine, 8-aziodoguanine, 8-bromoguanine, 8-iodoguanine, 8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine, 8-azidoxanthine, 8-bromoxanthine, 8-iodoxanthine, 5-bromodeoxyuracil, 8-bromo-2'-deoxyadenine, 5-iodo-2'-deoxyuracil, 5-iodo-2'-deoxycytosine, 5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil, 7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine, and 7-deaza-7-bromoguanine.

13. A nucleic acid ligand that photocrosslinks to a protein, wherein said nucleic acid ligand is comprised of a non-naturally occurring nucleic acid having a specific binding affinity for a protein, wherein said protein is not a nucleic acid binding protein, and wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the protein, obtained by the process of identifying a nucleic acid ligand of a protein, from a candidate mixture of nucleic acids comprised of nucleic acids each having a region of randomized sequence, and wherein each member of said candidate mixture contains a photoreactive group, said method comprising:

a) contacting said candidate mixture with said protein, wherein nucleic acids having increased affmity to the protein relative to the candidate mixture form nucleic acid-protein complexes with the protein;

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

c) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, whereby a nucleic acid ligand of the protein may be identified;

d) incorporating photoreactive groups into said increased affinity nucleic acids;

e) repeating step a);

f) irradiating said increased affinity nucleic acids, wherein said nucleic acid-protein complexes photocrosslink;

g) repeating step c) and d); and

h) identifying a photocrosslinking nucleic acid ligand to the protein.

14. A nucleic acid ligand of claim 13 further comprising one or more of the photoreactive groups selected from the group consisting of 5-bromouracil, 5-iodouracil, 5-bromovinyluracil, 5-iodovinyluracil, 5-azidouracil, 4-thiouracil, 5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine, 5-iodovinylcytosine, 5-azidocytosine, 8-azidoadenine, 8-bromoadenine, 8-iodoadenine, 8-aziodoguanine, 8-bromoguanine, 8-iodoguanine, 8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine, 8-azidoxanthine, 8-bromoxanthine, 8-iodoxanthine, 5-bromodeoxyuracil, 8-bromo-2'-deoxyadenine, 5-iodo-2'-deoxyuracil, 5-iodo-2'-deoxycytosine, 5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil, 7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine, and 7-deaza-7-bromoguanine.

15. A nucleic acid ligand that photocrosslinks to a protein, wherein said nucleic acid ligand is comprised of a non-naturally occurring nucleic acid having a specific binding affinity for a protein, wherein said protein is not a nucleic acid binding protein and wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the protein, obtained by the process of identifying a nucleic acid ligand of a protein from a candidate mixture of nucleic acids comprised of nucleic acids each having a region of randomized sequence, and wherein each member of said candidate mixture contains a photoreactive group, said method comprising:

a) contacting said candidate mixture with said protein, wherein nucleic acids having an increased affinity to the protein relative to the candidate mixture form nucleic acid-protein complexes with the protein;

b) irradiating said candidate mixture, wherein said nucleic acidprotein complexes photocrosslink;

c) partitioning the photocrosslinked nucleic acid-protein complexes from in the candidate mixture;

d) identifying a nucleic acid ligand that photocrosslinked to the protein;.

e) preparing a second candidate mixture of nucleic acids from those nucleic acid ligands identified in step d);

f) contacting said second candidate mixture with said protein, wherein nucleic acids having an increased affinity to the protein relative to the second candidate mixture form nucleic acid-protein complexes with the protein;

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

h) amplifying the increased affinity nucleic acids to yield a ligandenriched mixture of nucleic acids, whereby a nucleic acid ligand that photocrosslinks the protein is identified.

16. A nucleic acid ligand of claim 15 further comprising one or more of the photoreactive groups selected from the group consisting of 5-bromouracil, 5-iodouracil, 5-bromovinyluracil, 5-iodovinyluracil, 5-azidouracil, 4-thiouracil, 5-bromocytosine, 5-iodocytosine, 5-bromovinylcytosine, 5-iodovinylcytosine, 5-azidocytosine, 8-azidoadenine, 8-bromoadenine, 8-iodoadenine, 8-aziodoguanine, 8-bromoguanine, 8-iodoguanine, 8-azidohypoxanthine, 8-bromohypoxanthine, 8-iodohypoxanthine, 8-azidoxanthine, 8-bromoxanthine, 8-iodoxanthine, 5-bromodeoxyuracil, 8-bromo-2'-deoxyadenine, 5-iodo-2'-deoxyuracil, 5-iodo-2'-deoxycytosine, 5-[(4-azidophenacyl)thio]cytosine, 5-[(4-azidophenacyl)thio]uracil, 7-deaza-7-iodoadenine, 7-deaza-7-iodoguanine, 7-deaza-7-bromoadenine, and 7-deaza-7-bromoguanine.
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