RESEARCH ARTICLE Rich RNA Structure Landscapes Revealed by Mutate-and-Map Analysis

Landscapes exhibiting multiple secondary structures arise in natural RNA molecules that modulate gene expression, protein synthesis, and viral. We report herein that high-through-put chemical experiments can isolate an RNA’s multiple alternative secondary structures as they are stabilized by systematic mutagenesis (mutate-and-map, M2) and that a computa-tional algorithm, REEFFIT, enables unbiased reconstruction of these states ’ structures and populations. In an in silico benchmark on non-coding RNAs with complex landscapes, M2-REEFFIT recovers 95 % of RNA helices present with at least 25 % population while main-taining a low false discovery rate (10%) and conservative error estimates. In experimental benchmarks, M2-REEFFIT recovers the structure landscapes of a 35-nt MedLoop hairpin, a 110-nt 16S rRNA four-way junction with an excited state, a 25-nt bistable hairpin, and a 112-nt three-state adenine riboswitch with its expression platform, molecules whose char-acterization previously required expert mutational analysis and specialized NMR or chemi-cal mapping experiments. With this validation, M2-REEFFIT enabled tests of whether artificial RNA sequences might exhibit complex landscapes in the absence of explici