Use of SHAPE to select 2AP substitution sites for RNA-ligand interactions and dynamics studies.

Most regulatory RNA molecules must adopt a precise secondary fold and tertiary structure to allow their function in cells. A number of experimental approaches, such as the 2-Aminopurine-Based RNA Folding Analysis (2ApFold), have therefore been developed to offer insights into the folding and folding dynamics of RNA. A crucial requirement for this method is the selection of proper 2AP labeling positions. In that regard, we recently discovered that Selective 2'-Hydroxyl Acylation analyzed by Primer Extension (SHAPE) offers a reliable path to identify appropriate nucleotides for 2AP substitution on a target RNA. This chapter describes the straightforward procedure to select 2AP substitution sites in RNA molecules using SHAPE probing. The protocols detail the preparation of the target RNA by transcription, and the SHAPE steps including (1) probing of the RNA, (2) reverse transcription with a radiolabeled primer, (3) sequencing gel, and (4) analysis of the obtained band pattern.