Chemical-guided SHAPE sequencing (cgSHAPE-seq) informs the binding site of RNA-degrading chimeras targeting SARS-CoV-2 5' untranslated region.

One of the hallmarks of RNA viruses is highly structured untranslated regions (UTRs) which are often essential for viral replication, transcription, or translation. In this report, we discovered a series of coumarin derivatives that bind to a four-way RNA helix called SL5 in the 5' UTR of the SARS-CoV-2 RNA genome. To locate the binding site, we developed a sequencing-based method namely cgSHAPE-seq, in which an acylating probe was directed to crosslink with the 2'-OH group of ribose at the binding site to create read-through mutations during reverse transcription. cgSHAPE-seq unambiguously determined a bulged G in SL5 as the primary binding site, which was validated through mutagenesis and in vitro binding experiments. The coumarin derivatives were further used as a warhead in designing RNA-degrading chimeras to reduce viral RNA expression levels. The optimized RNA-degrading chimera C64 inhibited live virus replication in lung epithelial carcinoma cells.