Methods for identifying ligands that target nucleic acid molecules and nucleic acid structural motifs

Number: 9719191
Published: 2017-08-01
Filed: 2007-11-29
Assignee: The Research Foundation for The State University of New York
Inventors: Disney; Matthew D. et al.
CPC: C40B30/04; C40B40/08; G01N33/5308; C40B20/08; G01N33/6845; C12Q1/6811; C12Q1/6811; C12Q2565/531; C12Q2525/205
Verdict: Set aside nucleic acid ligand ID (SUNY) - false-positive name match, unrelated Matthew D. Disney
Source: Google Patents · FreePatentsOnline

Abstract

Disclosed are methods for identifying a nucleic acid (e.g., RNA, DNA, etc.) motif which interacts with a ligand. The method includes providing a plurality of ligands immobilized on a support, wherein each particularligand is immobilized at a discrete location on the support; contacting the plurality of immobilized ligands with a nucleic acid motif library under conditions effective for one or more members of the nucleic acid motif library to bind with the immobilized ligands; andidentifying members of the nucleic acid motif library that are bound to a particular immobilized ligand. Also disclosed are methods for selecting, from a plurality of candidate ligands, one or more ligands that have increased likelihood of binding to a nucleic acid molecule comprising a particular nucleic acid motif, as well as methods for identifying a nucleic acid which interacts with a ligand.

Background

BRIEF DESCRIPTION OF THE DRAWINGS(1) FIG. 1A is a drawing showing the structures of small molecule ligands that can be used in the methods of the present invention. 1 is kanamycin A and 2 is 6′-N-5-hexynoate kanamycin A that was used in chemical microarray-based assays to study binding to selected RNA internal loops. FIG. 1B is a reaction scheme showing the synthesis of 6′-N-5-hexynoate kanamycin A (2).(2) FIG. 2 is a drawing showing the sequence and structure of oligonucleotides that can be used in the methods of the present invention. R1 (SEQ ID NO:3) is an RNA motiflibrary displaying a random 3×3 internal loop in which N refers to a random mixture of A,C, G, and U. R2, R3, D1, and D2 are chase oligonucleotides. Structure R2 is a duplex (SEQID NO:4 and SEQ ID NO:5) that is related to the stems of R1; R3 (SEQ ID NO:6) is a short hairpin having the hairpin sequence and closing base pair of R1; D1 (SEQ ID NO:7 and SEQ ID NO:8) and D2 (SEQ ID NO:9 and SEQ ID NO:10) are DNA chase oligonucleotides. The RNA R4 (SEQ ID NO:11) is the cassette into which the library was inserted as shown in R1.(3) FIG. 3 is a drawing showing the secondary structure of the RNA internal loops and the closing base pairs that were selected to bind immobilized kanamycin A using a method according to the present invention. Secondary structures were modeled using free energy minimization using the program RNAStructure. The internal loop library (R1 (SEQ ID NO:3)) is shown at the upper left, and the boxed

Claims

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 structuremotif 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 consistingof 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 theRNA 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 anddetermining the sequence of the members of the RNA secondary structure motif library thatare 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 tohigher 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.