Fig. 3. Methods of target validation for small molecules that target RNA.

a | Resistance profiling is applicable when the small molecule exerts enough selective pressure to induce mutations that confer resistance. These mutations, identified by sequencing, reveal the targets of the small molecule. b | The target validation method Chem-CLIP (chemical cross-linking and isolation by pull-down) generates a covalent bond between a small-molecule probe and its targets, which are isolated and purified by bead pull-down. Bona fide targets are those enriched in the pulled-down fractions, as compared with the starting cell lysate. Upon co-treating increasing concentrations of the lead compound with Chem-CLIP probe, a dose-dependent restoration of the RNA target in the pulled-down fractions would indicate target engagement of the lead compound. c | ASO-Bind-Map is based on previous studies that show that structured regions of RNA are protected from antisense oligonucleotide (ASO) hybridization and hence ribonuclease (RNase) H degradation^135,136. Thus, small molecules that bind to and stabilize the structures of RNA targets can elicit a protective effect against ASO-mediated degradation. d | An RNA degrader can cleave its bound RNA target either directly (bleomycin) or by recruiting endogenous RNases (RIBOTAC). Upon co-treating increasing concentrations of the lead compound with degrader probe, a dose-dependent ablation of degradation would indicate target engagement of the lead compound.