Guidelines for SHAPE Reagent Choice and Detection Strategy for RNA Structure Probing Studies.

ABSTRACT: Chemical probing is an important tool for characterizing the complex folded structures of RNA molecules, many of which play key cellular roles. Electrophilic SHAPE reagents create adducts at the 2'-hydroxyl position on the RNA backbone of flexible ribonucleotides with relatively little dependence on nucleotide identity. Strategies for adduct detection such as mutational profiling (MaP) allow accurate, automated calculation of relative adduct frequencies for each nucleotide in a given RNA or group of RNAs. A number of alternative reagents and adduct detection strategies have been proposed, especially for use in living cells. Here we evaluate five SHAPE reagents: three previously well-validated reagents 1M7 (1-methyl-7-nitroisatoic anhydride), 1M6 (1-methyl-6-nitroisatoic anhydride), and NMIA ( N-methylisatoic anhydride), one more recently proposed NAI (2-methylnicotinic acid imidazolide), and one novel reagent 5NIA (5-nitroisatoic anhydride). We clarify the importance of carefully designed software in reading out SHAPE experiments using massively parallel sequencing approaches. We examine SHAPE modification in living cells in diverse cell lines, compare MaP and reverse transcription-truncation as SHAPE adduct detection strategies, make recommendations for SHAPE reagent choice, and outline areas for future development.

SUBMITTER: Busan S 

PROVIDER: S-EPMC6712974 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature