Insights into the development of chemical probes for RNA

Morgan, Brittany S; Forte, Jordan E; Hargrove, Amanda E

In addition to the primary screens, a database of known RNA motif:small molecule interactions, Inforna (39) , was utilized to identify seven small molecules. The database was generated using a library versus library approach named 2-Dimensional Combinatorial Screening (2DCS). In this method, small molecules are immobilized onto a microarray slide and then incubated with libraries of labeled, randomized RNA secondary structures. The bound RNAs are excised, sequenced, and assigned a fitness score using Structure-Activity Relationships Through Sequencing (StARTS). Fitness scores reflect the affinity and selectivity of a given RNA motif:small molecule interaction and are represented on a numerical scale, where a higher score represents greater selectivity. To utilize the Inforna database, a computational or experimental secondary structure of RNA is input, the 2DCS data is searched and lead molecules are proposed. This strategy identified bioactive ligands for five targets: (i) MAPT pre-mRNA, 1-nucleotide bulge; (58) (ii) Pre-miRNA-96, 1 × 1 internal loop (59); (iii) Pre-miRNA-18a, 1-nucleotide bulge (60); (iv) Pre-miRNA-210, 1 × 1 internal loop (16) and (v) Pre-miRNA-544, 1 × 1 internal loop (61) . Other examples of ligands not identified from a primary screening assay included the selection and characterization of four metabolite analogs for riboswitch inhibition (62) (63) (64) (65) .