Using 5′ 32P‐labeled Primer and Reverse Transcription to Probe RNA Structure

RNA molecules perform numerous cellular functions necessary for cell viability, some of which can depend on the RNA's structure. Therefore, it is important to study RNA structure and RNA folding to better understand the molecular basis of these functions. RNA chemical mapping strategies to elucidate RNA structural changes involve using chemical reagents that form adducts or cleave RNA. Selective 2′‐hydroxyl acylation analyzed by primer extension (SHAPE) measures RNA flexibility by modification of the 2′ hydroxyl groups of flexible nucleotides. These RNA adducts can be detected using 32P‐labeled primers and reverse transcription (RT) followed by PAGE analysis. This strategy can reveal the base‐paired regions of the RNA and provide insight into tertiary structure and solvent accessibility. This protocol provides a method to interrogate RNA structure using furoyl acylimidazole (FAI). © 2023 Wiley Periodicals LLC. Basic Protocol 1: Reverse transcription (RT) primer labeling with 32P radionuclide Basic Protocol 2: Characterization of RNA structure with radiolabeled primer and reverse transcription (RT)