Single-molecule FRET assay to observe the activity of proteins involved in RNA/RNA annealing

Abstract

In recent years, single-molecule fluorescence resonance energy transfer (smFRET) has emerged as a powerful technique to study macromolecular interactions. The chief advantages of smFRET analysis compared to bulk measurements include the possibility to detect sample heterogeneities within a large population of molecules and the facility to measure kinetics without needing the synchronization of intermediate states. As such, the methodology is particularly well adapted to observe and analyze RNA/RNA and RNA/protein interactions involved in small noncoding RNA-mediated gene regulation networks. In this chapter, we describe and discuss protocols that can be used to measure the dynamics of these interactions, with a particular emphasis on the advantages—and experimental pitfalls—of using the smFRET methodology to study sRNA-based biological systems.