Single-Molecule Fluorescence Methods to Study Protein-RNA Interactions Underlying Biomolecular Condensates

Abstract

Many biomolecular condensates, including nucleoli and stress granules, form via dynamic multivalent protein-protein and protein-RNA interactions. These molecular interactions nucleate liquid-liquid phase separation (LLPS) and determine condensate properties, such as size and fluidity. Here, we outline the experimental procedures for single-molecule fluorescence experiments to probe protein-RNA interactions underlying LLPS. The experiments include single-molecule Förster (Fluorescence) resonance energy transfer (smFRET) to monitor protein-induced conformational changes in the RNA, protein-induced fluorescence enhancement (PIFE) to measure protein-RNA encounters, and single-molecule nucleation experiments to quantify the association and buildup of proteins on a nucleating RNA. Together, these experiments provide complementary approaches to elucidate a molecular view of the protein-RNA interactions that drive ribonucleoprotein condensate formation.

Keywords: Förster resonance energy transfer (FRET); Nucleation; Protein-induced fluorescence enhancement (PIFE); RNA; Ribonucleoprotein (RNP) granule; Single-molecule; Total internal reflection fluorescence (TIRF) microscopy.