Facile synthesis and evaluation of a dual-functioning furoyl probe for in-cell SHAPE

Recent analysis of transcriptomes has revealed that RNA molecules perform a myriad of functions beyond coding for proteins. RNA molecules can fold into complex secondary and tertiary structures, which are critical for regulating their function. Selective Hydroxyl Acylation analyzed by Primer Extension, or SHAPE is a common method for probing RNA structure in and outside of cells. Recent developments in SHAPE include the design of acyl imidazole acylating electrophiles with alkyl azides to enrich the sites of SHAPE adduct formation. Enrichment is key for next-generation sequencing experiments as it dramatically improves the signal. In a recent comparison of different structures of such reagents, we realized that furoyl acylating reagents form hyper-stable ester adducts with hydroxyls. This prompted us to design, synthesize and test a novel dual-functioning SHAPE probe (FAI-N₃), which has the stable furoyl scaffold and the alkyl azide for enrichment. Herein we present the results that show FAI-N₃ is a suitable probe for RNA structure analysis by SHAPE and that it can be used for enrichment of SHAPE adducts. These results strongly demonstrate that FAI-N₃ is an ideal probe for structure probing in cells and will be very useful for sequencing-based analysis of SHAPE.