RNA G-quadruplex (rG4) structure detection using RTS and SHALiPE assays

RNA G-quadruplexes (rG4s) are non-canonical RNA secondary structures that were first reported several decades ago. Latest studies have suggested that they are widespread in the transcriptomes of diverse species, and they have been demonstrated to have key roles in various fundamental cellular processes. Among the RNA secondary structure probing assays developed recently, Reverse transcriptase stalling (RTS) and selective 2'-hydroxyl acylation analyzed by lithium ion-based primer extension (SHALiPE) enabled the identification and characterization of distinct structural features of an rG4 structure of interest. Herein, we present an experimental protocol describing in detail the procedures involved in the preparation of in vitro transcribed RNAs, buffers, and reagents for RTS and SHALiPE assays, as well as performing RTS and SHALiPE assays, to examine the formation of rG4 and reveal the rG4 structural conformation at nucleotide resolution in vitro. RTS and SHALiPE assays can be performed by an experienced molecular biologist or chemical biologist with a basic understanding of nucleic acids. The duration for the preparation of in vitro transcription and RNA preparation is around 2 days, and the duration for RTS and SHALiPE assays is approximately 5 h.