Disulfide Tethering to Map Small Molecule Binding Sites Transcriptome-Wide

Tether-seq uses s4U metabolic labeling to provide sites for reversible and covalent attachment of small molecule disulfides to the transcriptome. By screening under reducing conditions, we are able to highlight interactions that are stabilized by binding over those driven by the reactivity of the RNA sites. When applied to cellular RNA, Tether-seq with a disulfide analogue of risdiplam (compound 2) revealed a number of potential binding sites. Structure probing by SHAPE-MaP revealed a structured motif and confirmed binding to the lead molecule. Overall design: Tether-seq was performed using RNA from s4U-fed HEK293T cells, extracted under non-denaturing conditions. PNA samples were prepared using trizol-extracted s4U RNA. This involved xyz. Enrichment. TL chemistry. RNA sequencing libraries were prepared for all enrichment samples.