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) in their genomes. These conserved RNA structures are often essential for viral replication, transcription, or translation. In this report, we discovered and optimized a new type of coumarin derivatives, such as and , which 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 novel sequencing-based method namely cgSHAPE-seq, in which the acylating chemical probe was directed to crosslink with the 2'-OH groups of ribose at the ligand binding site. This crosslinked RNA could then create read-through mutations during reverse transcription (i.e., primer extension) at single-nucleotide resolution to uncover the acylation locations. cgSHAPE-seq unambiguously determined that a bulged G in SL5 was the primary binding site of in the SARS-CoV-2 5' UTR, which was validated through mutagenesis and in vitro binding experiments. was further used as a warhead in RNA-degrading chimeras to reduce viral RNA expression levels. We demonstrated that replacing the acylating moiety in the cgSHAPE probe with ribonuclease L recruiter (RLR) moieties yielded RNA degraders active in the in vitro RNase L degradation assay and SARS-CoV-2 5' UTR expressing cells. We further explored another RLR conjugation site on the E ring of and discovered improved RNA degradation activities in vitro and in cells. The optimized RNA-degrading chimera inhibited live virus replication in lung epithelial carcinoma cells.