A new reagent for in vivo structure probing of RNA G and U residues that improves RNA structure prediction alone and combined with DMS.

Abstract

A key to understanding the roles of RNA in regulating gene expression is knowing their structures in vivo. One way to obtain this information is through probing the structures of RNA with chemicals. To probe RNA structure directly in cells, membrane-permeable reagents that modify the Watson-Crick (WC) face of unpaired nucleotides can be used. Although dimethyl sulfate (DMS) has led to substantial insight into RNA structure, it has limited nucleotide specificity in vivo, with WC face reactivity only at adenine (A) and cytosine (C) at neutral pH. The reagent 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) was recently shown to modify the WC face of guanine (G) and uracil (U). Although useful at lower concentrations in experiments that measure chemical modifications by reverse transcription (RT) stops, at higher concentrations necessary for detection by mutational profiling (MaP), EDC treatment leads to degradation of RNA. Here, we demonstrate EDC-stimulated degradation of RNA in Gram-negative and Gram-positive bacteria. In an attempt to overcome these limitations, we developed a new carbodiimide reagent, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide methiodide (ETC), which we show specifically modifies unpaired Gs and Us in vivo without substantial degradation of RNA. We establish ETC as a probe for MaP and optimize the RT conditions and computational analysis in Escherichia coli Importantly, we demonstrate the utility of ETC as a probe for improving RNA structure prediction both alone and with DMS.