# Recent Advances in Chemical Probing Strategies for RNA Structure Determination In Vivo

## TL;DR

This review discusses how new chemical probing methods and sequencing technologies are improving our understanding of RNA structures inside living cells.

## Contribution

The paper highlights novel structural probes and strategies for RNA structure reconstruction in biological contexts.

## Key findings

- Cell-permeable chemical probes enable RNA structure analysis in native cellular environments.
- Advanced sequencing technologies enhance the resolution of RNA structural studies at single-molecule and single-cell levels.
- New methodologies expand the applicability of RNA structure probing to diverse biological questions.

## 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.

This Review illustrates how structural probes combined with advanced sequencing technologies are revolutionizing our ability to map RNA structures and interactions within living cells. It highlights the development of innovative strategies tailored to probe RNA structures in particular biological contexts.