Characterization of RNA Structure Dynamics with Trans-Factors Using Single-Molecule Optical Tweezers

RNA folding is a dynamic process that is essential and has a regulatory function in many cellular processes. Additionally, RNA structure and function can be modulated by the binding of trans-acting factors such as translation machinery, RNA-binding proteins, small molecules, or other RNAs. These mechanisms, which add layers of regulation, can exist in many different states that can be difficult to resolve in ensemble methods alone. For this reason, single-molecule methods to monitor RNA folding dynamics in the presence of regulatory factors are crucial in identifying subpopulations, transient intermediates, and understanding the underlying mechanisms of RNA structure, function, and regulation. In this chapter, we describe methods for RNA structure analysis using single-molecule optical tweezers (OTs). This technique can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating RNA-folding pathways or chaperones as they undergo phase separation under changing conditions.