2A3 is an electrophilic chemical probe that acylates the 2'-OH in the RNA backbone. 2A3 can be used for RNA SHAPE-MaP experiments in cell lines or live bacteria. It labels single-stranded or flexible regions more than folded regions (SHAPE methodology) and enables analysis of RNA secondary structures at single nucleotide resolution. For intracellular experiments, 2A3 has higher signal-to-noise ratio and prediction accuracy than NAI . 2A3 is cell permeable, and can probe both gram-positive and gram-negative bacteria.