RNA Folding, Mutation, and Detection

The structure of RNA molecules is absolutely critical to their functions in a biological system. RNA structure is dynamic and changes in response to cellular needs. Within the last few decades, there has been an increased interest in studying the structure of RNA molecules and how they change to support the needs of the cell in different conditions. Selective 2′-hydroxyl acylation-based mutational profiling using high-throughput sequencing is a powerful method to predict the secondary structure of RNA molecules both in vivo and in immunopurified samples. Selective 2′-hydroxyl acylation-based mutational profiling using high-throughput sequencing works by adding bulky groups onto accessible “flexible” bases in an RNA molecule that are not involved in any base-pairing or RNA-protein interactions. When the RNA is reverse transcribed into cDNA, the bulky groups are incorporated as base mutations, which can be compared to an unmodified control to identify the locations of flexible bases. The comparison of sequence data between modified and unmodified samples allows the computer software program (developed to generate reactivity profiles) to generate RNA secondary structure models. These models can be compared in a variety of conditions to determine how specific stimuli influence RNA secondary structures.