Profiling RNA G-Quadruplexes In vivo.

RNA G-quadruplexes (RG4s) are specific and complex tertiary structures that form in guanine-rich regions of RNA and can be detected in vitro. A recently developed transcriptome-wide technique, SHALiPE-Seq, enables the assessment of RG4 folding status within living cells in a quantitative manner. This method integrates chemical probing with high-throughput sequencing. SHALiPE-Seq relies on the property of 2-methylnicotinic acid imidazolide (NAI), which preferentially modifies the last guanine in G-tracts when RG4s are folded. To establish reference profiles, in vitro NAI modification patterns are generated under potassium ion (K⁺) conditions, which promote folding, and lithium ion (Li⁺) conditions, which maintain RG4s in an unfolded state. By comparing in vivo SHALiPE-Seq profiles with these in vitro benchmarks, it becomes possible to identify and evaluate the formation of RG4s in living cells. Although this protocol has been applied to Arabidopsis thaliana and rice, SHALiPE-Seq is broadly applicable to other systems and provides a valuable approach for investigating the in vivo dynamics of RG4s and their potential biological functions. © 2025 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: In vitro and in vivo NAI probing of RNA Basic Protocol 2: Construction of the SHALiPE-Seq libraries Basic Protocol 3: Measurement of the RNA G-quadruplex folding status based on SHALiPE-Seq libraries.