Details for Patent: 6,001,577

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 procedure 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 partitioning 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 arc 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:	Jun 08, 1998
Application Number:	09/093,293
Claims:	1. A method for identifying a nucleic acid ligand to a target molecule that is associated with a disease state in a biological substance, said method comprising: a) identifying a nucleic acid ligand that photocrosslinks to a target molecule from a candidate mixture of nucleic acids, wherein each member of said candidate mixture contains a photoreactive group, said method comprising: i) contacting said candidate mixture of nucleic acids with a first biological substance which contains a target molecule that is associated with said disease state, wherein nucleic acids having an increased affinity to a molecule of said first biological substance relative to the candidate mixture form nucleic acid-molecule complexes with said molecule; ii) irradiating said complexes, wherein said nucleic acid and molecule photocrosslink; iii) partitioning the photocrosslinked nucleic acid-molecule complexes from the remainder of the candidate mixture; and iv) identifying nucleic acid ligands that photocrosslink to said target molecule; b) contacting a second biological substance which does not contain said target molecule that is associated with said disease state with said nucleic acid ligands identified in step iv), wherein the nucleic acids with affinity to a molecule that is not associated with the disease state in the second biological substance is removed; and c) amplifying the remaining nucleic acids with specific affinity to said target molecule that is associated with a disease state to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to said target molecule that is associated with said disease state, whereby a nucleic acid ligand to a target molecule that is associated with a disease state in a biological substance is identified. 2. The method of claim 1 further comprising after step iv); v) repeating steps i) through iii); and vi) amplifying the nucleic acids that photocrosslinked to the target molecule that is associated with a disease state. 3. The method of claim 1 wherein said biological substance is serum. 4. The method of claim 1 wherein said target molecule is selected from the group consisting of a protein, peptide, carbohydrate, polysaccharide, glycoprotien, hormone, receptor, antigen, antibody, virus, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient, and growth factor. 5. The method of claim 4 wherein said target molecule that is associated with a disease state is a protein. 6. The method of claim 4 wherein said protein is alpha-foeto protein. 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-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. 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-azidoguanine, 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. A method for identifying a nucleic acid ligand to a target molecule that is associated with a disease state in a biological substance, said method comprising: a) identifying a nucleic acid ligand that photocrosslinks to a target molecule from a candidate mixture of nucleic acids, said method comprising: i) contacting said candidate mixture of nucleic acids with a first biological substance which contains a target molecule that is associated with said disease state, wherein nucleic acids having an increased affinity to a molecule of said first biological substance relative to the candidate mixture form nucleic acid-molecule complexes with said molecule; ii) partitioning the complexed increased affinity nucleic acids from the remainder of the candidate mixture; iii) amplifying the increased affinity nucleic acids to yield a ligand-enriched mixture of nucleic acids, iv) incorporating photoreactive groups into said amplified increased affinity nucleic acids; v) irradiating said increased affinity nucleic acids, wherein said nucleic acid-molecule complexes photocrosslink; vi) partitioning the photocrosslinked nucleic acid-molecule complexes from the remainder of the candidate mixture; and vii) identifying nucleic acid ligands that photocrosslink to the molecule; b) contacting a second biological substance which does not contain said target molecule that is associated with said disease with said nucleic acid ligands identified in step vii), wherein the nucleic acids with affinity to a molecule that is not associated with said disease is removed; and c) amplifying the remaining nucleic acids with specific affinity to said target molecule that is associated with said disease state to yield a mixture of nucleic acids enriched for nucleic acids with relatively higher affinity and specificity for binding to said target molecule that is associated with said disease state, whereby nucleic acid ligands to a target molecule that is associated with a disease state in a biological substance is identified. 10. The method of claim 9 further comprising: d) repeating step i); and e) repeating steps iii) and iv). 11. The method of claim 9 wherein said biological substance is serum. 12. The method of claim 9 wherein said target molecule that is associated with a disease state is selected from the group consisting of protein, peptide, carbohydrate, polysaccharide, glycoprotien, hormone, receptor, antigen, antibody, virus, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient, and growth factor. 13. The method of claim 12 wherein said target molecule that is associated with a disease state is a protein. 14. The method of claim 13 wherein said protein is alpha-foeto protein. 15. The method of claim 9 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-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. 16. The method of claim 9 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-azidoguanine, 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.