Insights into the development of chemical probes for RNA

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

The primary screening assay for each small molecule was categorized as computational, in vitro, or cell-based [ Figure 4A ]. This list contained a wide range of primary screening assays, with limited examples of the same assay being used for multiple targets. The majority of the chemical probes were discovered by in vitro primary screening assays (n = 28) with fewer in cellulo or silico examples. Of those in vitro primary screens, 15 were RNA:protein displacement assays [ Figure 4B ]. These included fluorescence-based assays (Förster resonance energy transfer (FRET) and fluorescence anisotropy) and radiolabel-based methods (mobility shift, scintillation proximity, and filtration assays). One rather unique assay utilized a molecular beacon approach to probe for stabilization of Stem Loop 3 (SL3), a presumptive structural switch located in the -Packing Domain in HIV-1 that is destabilized by binding of the Gag protein prior to packaging of the virus (49) . In this assay, the 5 -and 3 -terminal ends of the SL3 RNA were labeled with a TET fluorophore and a blackhole quencher (BHQ1), respectively. In the presence of Gag protein, the RNA construct became single stranded and the fluorescence was 'turned on'. When a small molecule stabilized the folded hairpin form of SL3 RNA, the Gag-promoted RNA destabilization was reduced and the fluorescence was quenched. The researchers screened a modest sized library (>2500 small molecules) and discovered a ligand that reduced viral production similar to models with a mutated -Packing Domain (p24 50 = 11.3 M).