SHAPE-based chemical probes for studying preQ ₁ -RNA interactions in living bacteria

Interrogating RNA-small molecule interactions inside cells is critical for advancing RNA-targeted drug discovery. In particular, chemical probing technologies that both identify small molecule-bound RNAs and define their binding sites in the complex cellular environment will be key for establishing the on-target activity necessary for successful hit-to-lead campaigns. Using the small molecule metabolite preQ ₁ and its cognate riboswitch RNA as a model, herein we describe a chemical probing strategy for filling this technological gap. Building on well-established RNA acylation chemistry employed by in vivo click selective 2′-hydroxyl acylation analyzed by primer extension (icSHAPE) probes, we developed an icSHAPE-based preQ ₁ probe that retains biological activity in a preQ ₁ riboswitch reporter assay and successfully enriches the preQ ₁ riboswitch from living bacterial cells. Further, we map the preQ ₁ binding site on probe-modified riboswitch RNA by mutational profiling (MaP). As the need for rapid profiling of on- and off-target small molecule interactions continues to grow, this chemical probing strategy offers a method to interrogate cellular RNA-small molecule interactions and support the future development of RNA-targeted therapeutics.