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

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Biomolecular condensates like nucleoli and stress granules form through dynamic protein-protein and protein-RNA interactions.
• These interactions drive liquid-liquid phase separation (LLPS), influencing condensate characteristics such as size and fluidity.

Purpose of the Study:

• To outline experimental procedures for single-molecule fluorescence experiments.
• To probe the specific protein-RNA interactions that underlie LLPS.
• To provide a molecular understanding of ribonucleoprotein condensate formation.

Main Methods:

• Single-molecule Förster resonance energy transfer (smFRET) to detect RNA conformational changes induced by proteins.
• Protein-induced fluorescence enhancement (PIFE) to quantify protein-RNA encounters.
• Single-molecule nucleation assays to measure protein association with RNA.

Main Results:

• Demonstrated smFRET for monitoring RNA dynamics during protein binding.
• Utilized PIFE to quantify direct protein-RNA interaction frequencies.
• Quantified protein assembly kinetics on RNA during nucleation.

Conclusions:

• These complementary single-molecule techniques offer a detailed molecular view of protein-RNA interactions in LLPS.
• The methods elucidate the mechanisms driving the formation of ribonucleoprotein condensates.
• Provides a framework for studying the molecular basis of condensate assembly.