Insights into the development of chemical probes for RNA

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

Compared to the other systems, chemical probes targeting viral RNAs were less characterized in terms of target engagement and specific activity. A select few studies validated probe activity by testing mutant versions of the virus (49, 78) or closely related native viruses (30, 73) . Notably, one report validated probe activity with a second research group to ensure reproducibility of the biological effect (71) . RNAspecific reporter systems were occasionally used to confirm on-target effects, including fusion-induced gene stimulation (79) , heterologous tethering (54) , and a viral protein reporter (48) . A noteworthy example of assessing off-target effects was the use of RNA-Seq at increasing ligand concentrations in the absence of the target RNA (72) . The experiment in HEK293T cells revealed that 53 of the 17 822 transcripts assayed had statistically significant alterations at two concentrations, potentially reflecting a cellular stress response. It is also intriguing that the most biologically potent ligand in this study was the least selective analog in an in vitro tRNA competition assay. This observation underlies potential differences in cellular activity versus in vitro binding selectivity and thus the importance of progressing multiple ligands to biological assays.