High-throughput experimental methods to characterize RNA structure typically involve the treatment of in vitro refolded RNAs with reagents or nucleases that are sensitive to RNA base pairing followed by high-throughput sequencing. To determine how broadly applicable such data is to RNA structure under native conditions in cells, Rouskin et al. used a variation of these methods that used the cell-permeable reagent dimethylsulphate (DMS) in live yeast and human cells. They found that globally, RNAs were substantially more unfolded in vivo than in vitro. This may reflect the presence of RNA helicases and single-stranded RNA-binding proteins in cells.