Insights into the development of chemical probes for RNA

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

The remaining in vitro assays consisted of two activitybased screens and various RNA binding assays [Figure (v) competition dialysis and (vi) indicator displacement. We note that a lack of correlation in small molecule activity between in vitro RNA binding and RNA:protein displacement assays has sometimes been reported, (16, 51, 52) highlighting the importance of multiple assays and/or choosing the most relevant assay for a particular system. We also note that, as in all screens, a lack of correlation can be observed between in vitro activity and cell culture activity. These differences can be attributed to many factors, including small molecule uptake, localization, and metabolism, specificity or off-target effects, and target availability due to binding of other macromolecules or metabolites. Nonetheless, we emphasize the success of the in vitro assays mentioned here in developing RNA bioactives and the valuable insights gained from other ligands discovered by in vitro RNA assays without reported biological activity.