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.
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Acknowledgments
We acknowledge funding support from NINDS RF1NS113636, NIGMS 1R01GM115631, and NIA RF1AG071326.
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Ganser, L.R., Ge, Y., Myong, S. (2023). Single-Molecule Fluorescence Methods to Study Protein-RNA Interactions Underlying Biomolecular Condensates. In: Zhou, HX., Spille, JH., Banerjee, P.R. (eds) Phase-Separated Biomolecular Condensates. Methods in Molecular Biology, vol 2563. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2663-4_7
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DOI: https://doi.org/10.1007/978-1-0716-2663-4_7
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