Abstract
The intricate structures of RNA molecules facilitate their diverse cellular functions. These structures are shaped by the cellular environment, a context that in silico and in vitro methods typically cannot reconstitute, making it more difficult to study the structure of RNA in cells. In response to these challenges, RNA structure probing using cell-permeable chemicals has emerged as an effective method to capture the RNA structural landscape in its native environment. The integration of these probes with advanced adduct detection techniques, particularly second- and third-generation sequencing, has propelled the field forward, facilitating a deeper understanding of the RNA structurome within its precise functional context, including the examination of RNA structure at the single-molecule and single-cell levels, within specific subcellular compartments, and across various stages of RNA biogenesis and regulation. This Review summarizes the significant advances in the field of RNA structure probing, focusing on the development of novel structural probes, strategies for RNA structure reconstruction, innovative methodologies that offer extended applicability to address unique biological questions, and concludes with an outlook on future directions in the field.