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

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.