A new reagent for in vivo structure probing of RNA G and U residues that improves RNA structure prediction alone and combined with DMS
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 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. While 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). While useful at lower concentrations in experiments that measure chemical modifications by reverse transcription stops, at higher concentrations necessary for detection by mutational profiling (MaP), EDC treatment leads to degradation of RNA. Indeed, herein 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 reverse transcription 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. Overall design: RNA structure probing MaP-seq libraries were constructed for total RNA from in vivo treated E. coli grown in LB at 37°C. To test initial RT conditions, one replicate of both untreated control RNA and 200mM ETC treated RNA libraries were prepared with TGIRT-III at 65°C, TGIRT-III at 70°C, Marathon, SuperScript-II, and HIV-RT, along with 300 mM ETC and 100 mM DMS detected with TGIRT-III at 65°C. Extension temperature was refined with 3 biological replicates of untreated RNA with TGIRT-III at 50°C, ETC treated RNA with TGIRT-III at 50°C, untreated RNA with TGIRT-III at 55°C, ETC treated RNA with TGIRT-III at 55°C, DMS treated RNA with TGIRT-III at 55°C, untreated RNA with TGIRT-III at 60°C, and ETC treated RNA with TGIRT-III at 60°C.