RNA structure inference through chemical mapping after accidental or intentional mutations

Significance The intricate structures of RNA molecules are crucial to their biological functions but have been difficult to accurately characterize. Multidimensional chemical-mapping methods improve accuracy but have so far involved painstaking experiments and reliance on secondary-structure prediction software. A methodology called M2-seq now lifts these limitations. Mechanistic studies clarify the origin of serendipitous M2-seq–like signals that were recently discovered but not correctly explained and also provide mutational strategies that enable robust M2-seq for new RNA transcripts. The method detects dozens of Watson–Crick helices across diverse RNA folds in vitro and within frog egg extract, with a low false-positive rate (<5%). M2-seq opens a route to unbiased discovery of RNA structures in vitro and beyond.