Single-Molecule FRET Studies of RNA Structural Rearrangements and RNA-RNA Interactions.

RNA-guided regulation of gene expression is found in all cell types. In this mode of regulation, antisense interactions between the regulatory RNA and its target are typically facilitated by a protein partner. Single-molecule fluorescence microscopy is a powerful tool for dissecting the conformational states and intermediates that contribute to target recognition. This chapter describes protocols for studying target recognition by bacterial small RNAs and their chaperone Hfq on the single-molecule level, using a total internal reflection fluorescence microscope. The sections cover the design of suitable RNA substrates for sRNA-mRNA annealing reactions, preparation of internally labeled mRNA for detecting conformational changes in the target, and key steps of the data analysis. These protocols can be adapted to other RNA-binding proteins that chaperone RNA interactions.