Patent ID: 9719191
Date: 2017-08-01
CPC Classifications: C12Q,C40B,G01N

Claim:
1. A method for identifying an RNA secondary structure motif which interacts with a ligand, said method comprising: providing a plurality of immobilized small molecule ligands immobilized on a support at two or more different loadings, wherein the two or more different loadings each comprise distinct amounts of immobilized small molecule ligands in the plurality of small molecule ligands, said two or more different loadings being immobilized at separate, discrete locations on the support, incubating the plurality of immobilized small molecule ligands with two or more chase oligonucleotides consisting of the sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10, wherein the chase oligonucleotides are designed to compete with invariant regions of members of an RNA secondary structure motif library for binding to the plurality of immobilized ligands such that members of the RNA secondary structure motif library bind to the immobilized small molecule ligands in the plurality of immobilized small molecule ligands through variant regions, wherein the members of the RNA secondary structure motif library are selected from the group consisting of an internal loop library, a hairpin loop library, a bulge library, a multibranch loop library, a pseudoknot library, and combinations thereof, contacting the plurality of immobilized small molecule ligands with the RNA secondary structure motif library in the presence of the chase oligonucleotides under conditions effective for one or more members of the RNA secondary structure motif library to bind with one or more of the immobilized small molecule ligands, and wherein the RNA secondary structure motif library comprises at least 4 members; and identifying members of the RNA secondary structure motif library that are bound to particular immobilized small molecule ligands in the plurality of immobilized ligands, wherein said identifying the members of the RNA secondary structure motif library that are bound to each particular immobilized small molecule ligand comprises: amplifying and determining the sequence of the members of the RNA secondary structure motif library that are bound at the discrete location on the support corresponding to the particular immobilized small molecule ligand; and using a dose-response curve and a dissociation constant for each small molecule ligand loading to identify members of the RNA secondary structure motif library bound to lower small molecule ligand loading as having greater affinity for the small molecule ligand than members of the RNA secondary structure motif library bound to higher small molecule ligand loading, wherein the higher small molecule ligand loading and lower small molecule ligand loading comprise a serial dilution of the small molecule ligands, wherein the higher small molecule ligand loading comprises up to 100 nanomoles of the small molecule ligand, and wherein the lower ligand loading comprises not less than one femtomole of the small molecule ligand.