RNA structure analyzed by chemical probing and mutational profiling with sequencing (MaPseq)

RNA chemical probing, coupled with mutational profiling read out by sequencing (MaPseq), is a powerful approach for investigating the folding properties of complex RNA molecules. For over half a century, structure probing methods have advanced our understanding of the diverse functions of cellular RNAs. Depending on the analytical goal, these methods can generate models that reflect either the ensemble average or, more recently, deconvoluted structures within heterogeneous folding populations. The integration of chemical probing with next-generation sequencing has transformed the field by greatly accelerating experimental workflows and enabling analysis of complex mixtures of RNA conformations. The latter capability is particularly important, as RNA structural heterogeneity is central to biological function. Here, we present detailed protocols for RNA chemical probing, library preparation, sequencing, and bioinformatic analysis. Using factor VIII pre-mRNA and human telomerase RNA as case studies, we illustrate and compare different experimental strategies. We hope this protocol will provide a flexible framework for researchers seeking to investigate the structural properties of any RNA.