Nexstar Pharmaceuticals, Inc.

Somalogic, Incorporated

University Research Corporation

Steven Ringquist

Larry M Gold

Kirk B Jensen

Tad H Koch

Michael Willis

Brent Atkinson

Penelope J Toothman

Methods to obtain nucleic acid ligands that photocrosslink to target molecules associated with a disease state are provided. The methods presented are variations on the photoSELEX methods for obtaining nucleic acid ligands. In one method, a candidate mixture of photocrosslinkable nucleic acids is contacted with a biological substance obtained from a source associated with a disease state suspected of containing a target molecule to form nucleic acid-target molecule complexes, the complexes are irradiated to form crosslinked complexes, the photocrosslinked complexes are partitioned from the remainder of the candidate mixture; and the nucleic acid ligands that photocrosslink to molecule are retained. These nucleic acids are then contacted with a second biological substance of the same type as the first, but obtained from a source not associated with a disease state. This removes nucleic acids with affinity to molecules that are not associated with the disease state. In another method, photocrosslinkable nucleic acids are not incorporated in the initial candidate mixture, but rather are incorporated in an enriched candidate mixture obtained by contacting a biological substance associated with a disease state containing a target molecule to form nucleic acid-target molecule complexes, partitioning the complexes away from the candidate mixture.

Systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands

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 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.

Systematic evolution of ligands by exponential enrichment: photoselection of nucleic acid ligands and solution selex

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.

Systematic evolution of ligands by exponential enrichment:

Methods are described for the identification and preparation of high-affinity nucleic acid ligands to TGF.beta.. Included in the invention are specific RNA and ssDNA ligands to TGF.beta.1 identified by the SELEX method. Also included are RNA ligands that inhibit the interaction of TGF.beta.1 with its receptor.

High affinity TGF.beta. nucleic acid ligands

High affinity TGF.beta. nucleic acid ligands and inhibitors

Described herein are methods for improved partitioning between high and low affinity nucleic acid ligands identified through the SELEX method, termed solution SELEX. The solution SELEX method achieves partitioning between high and low affinity nucleic acid-target complexes through a number of methods, including (1) primer extension inhibition which results in differentiable cDNA products. Primer extension inhibition is achieved with the use of nucleic acid polymerases, including DNA or RNA polymerases, reverse transcriptase, and Q.beta.-replicase; (2) exonuclease hydrolysis inhibition which results in only the highest affinity ligands amplifying during PCR. This is achieved with the use of any 3'.fwdarw.5' double-stranded exonuclease; (3) linear to circle formation to generate molecules amplifiable during PCR; or (4) PCR amplification of single-stranded nucleic acids. A central theme of the method of the present invention is that the nucleic acid candidate mixture is screened in solution and results in preferential amplification of the highest affinity RNA ligand or catalytic RNA.

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Lyons, CO, United States of America

Steven Ringquist