Talk abstracts
Talk on Friday 01:30-01:45pm submitted by Catherine Douds
A novel reagent for mutational profiling of RNA in vivo
Catherine A. Douds (BMMB Department Pennsylvania State University), Paul Babitzke (BMMB Department Pennsylvania State University), Philip C. Bevilacqua (Chemistry Department Pennsylvania State University, BMMB Department Pennsylvania State University, Plant Biology Department Pennsylvania State University)
Abstract:
A key to understanding the roles that RNAs play in gene regulation is the ability to accurately probe their structure in vivo. To probe RNA structure directly in cells, membrane-permeable reagents that modify the Watson-Crick (WC) face of single stranded RNAs to report on structure can be used. While dimethyl sulfate (DMS) is the most commonly used probe, it has limited nucleotide specificity, reacting only with the WC face of Adenine (A) and Cytosine (C). Thus, N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide (EDC) was developed by our lab and the Simon lab to modify the WC face of Guanine (G) and Uracil (U).1,2 While useful in experiments that measure modifications by reverse transcription stops, at higher concentrations necessary for detection by mutational profiling (MaP), EDC degrades RNA. Here, we demonstrate EDC-stimulated RNA degradation in Gram-negative and Gram-positive bacteria, Escherichia coli and Bacillus subtilis, respectively. In an attempt to overcome these limitations, we developed a new carbodiimide reagent that specifically modifies single stranded G and U in vivo without degrading RNA called 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide methiodide (ETC). We establish ETC as a probe for MaP and optimize the reverse transcription conditions and computational analysis in E. coli. And finally, we demonstrate ETC’s utility as a probe for improving RNA structure prediction alone and together with DMS.
References:
1. Mitchell III, D. et al. In vivo RNA structural probing of uracil and guanine base pairing by 1.ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). RNA 25, 147–157 (2019).
2. Wang, P. Y., Sexton, A. N., Culligan, W. J. & Simon, M. D. Carbodiimide reagents for the chemical probing of RNA structure in cells. RNA 25, 135–146 (2019).
Keywords: structure probing, in vivo, ETC