Detecting and perturbing functional RNA structure ensembles

RNA molecules adopt complex structures in cells that are involved in numerous and varied biological processes. Many RNAs populate heterogeneous ensembles of structures, with states that differentially regulate biological functions. RNA structure ensembles have been challenging to detect by conventional methods for RNA structure determination, limiting our understanding of the prevalence and biological functions of RNA ensembles. To address this limitation, several recent strategies have been developed for deconvoluting RNA structure ensembles by single-molecule correlated chemical probing. In this work I summarize recent strategies for RNA ensemble deconvolution and highlight their key biological findings. I describe the validation of a structure deconvolution method developed in our lab, DANCE-MaP, as well as its application to the 7SK RNA, which revealed an RNA structure-dependent switch that globally regulates transcription. I then apply DANCE-MaP to the human SERPINA1 mRNA, revealing a hidden ensemble of structures in the early coding sequence that collectively regulates mRNA translation. Lastly, I develop an improved method for detecting RNA–ligand interactions by surface plasmon resonance. Altogether, this work demonstrates that structural heterogeneity is an intrinsic and widespread property of RNA, and that structure ensembles play critical roles in numerous biological processes. This deeper understanding of RNA structural heterogeneity might ultimately guide the development of RNA-targeting therapeutic strategies.