PROFILING RNA-SMALL MOLECULE BINDING SITES WITH OLIGONUCLEOTIDES
Many RNAs cause disease, however RNA is rarely exploited as a small molecule drug target. Disclosed herein are methods for identifying privileged RNA motif-small molecule interactions to enable the rational design of compounds that modulate RNA biology starting from only sequence. A massive, library-versus-library screen was completed that probed over 50 million binding events between RNA motifs and small molecules. The resulting data provide a rich encyclopedia of small molecule-RNA recognition patterns, defining chemotypes and RNA motifs that confer selective, avid binding. The resulting interaction maps were mined against the entire viral genome of hepatitis C virus (HCV). A small molecule was identified that avidly bound RNA motifs present in the HCV3′ untranslated region and inhibited viral replication while having no effect on host cells. Collectively, this investigation represents the first whole genome pattern recognition between small molecules and RNA folds.
1 . A method comprising
contacting a library of RNA sequences, a complementary antisense oligonucleotide, RNase H, and a small molecule candidate RNA-binding compound and determining cleavage of the RNA sequences in the presence of the compound (“presence cleavage”); and
contacting the library of RNA sequences, the complementary antisense oligonucleotide, and RNase H in the absence of the small molecule candidate RNA-binding compound and determining cleavage of the RNA sequences in the absence of the compound (“absence cleavage”);
wherein when cleavage is inhibited (e.g., presence cleavage is lower than absence cleavage), the small molecule candidate RNA-binding compound binds to the RNA sequence.
2 . The method of claim 1 wherein the RNA sequence library comprises a transcriptome.
3 . The method of claim 2 wherein the transcriptome is viral.
4 . The method of claim 2 wherein the transcriptome is mammalian.
5 . The method of claim 2 wherein the transcriptome is bacterial.
6 . The method of claim 1 wherein the RNA sequence library comprises one or more of synthetic, semi-synthetic, or natural RNA.
7 . The method of claim 1 wherein the RNA sequence library comprises the genome of an RNA virus.
8 . The method of claim 1 carried out in vitro.
9 . The method of claim 1 carried out in living cells.
10 . The method of claim 9 wherein the cells are virally- or bacterially-infected cells.
11 . The method of claim 1 wherein a set of complementary antisense oligonucleotides and a set of small molecule candidate RNA-binding compounds are assayed in a 2-dimensional parallel array.