Characterizing RNA structures in vitro and in vivo with selective 2’-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq)

RNA molecules adopt a wide variety of structures that perform many cellular functions, including catalysis, small molecule sensing, and cellular defense, among others. Our ability to characterize, predict, and design RNA structures are key factors for understanding and controlling the biological roles of RNAs. Fortunately, there has been rapid progress in this area, especially with respect to experimental methods that can characterize RNA structures in a high throughput fashion using chemical probing and next-generation sequencing. Here, we describe one such method, selective 2-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq), which measures nucleotide resolution flexibility information for RNAs in vitro and in vivo. We outline the process of designing and performing a SHAPE-Seq experiment and describe methods for using experimental SHAPE-Seq data to restrain computational folding algorithms to generate more accurate predictions of RNA secondary structure. We also provide a number of examples of SHAPE-Seq reactivity spectra obtained in vitro and in vivo and discuss important considerations for performing SHAPE-Seq experiments, both in terms of collecting and analyzing data. Finally we discuss improvements and extensions of these experimental and computational techniques that promise to deepen our knowledge of RNA folding and function.