Beyond Droplets: Single-Molecule FRET Illuminates LLPS Dynamics

Abstract

Liquid–liquid phase separation (LLPS) organizes biomolecules into dynamic, membraneless condensates seen in stress granules, nucleoli, and P-bodies, thereby facilitating rapid and reversible cellular compartmentalization related to transcription, RNA processing, and signaling. While LLPS is important for normal physiology, there is mounting evidence that involuntary transitions to solid-like states are involved in neurodegenerative disorders, cancer, and viral pathogenesis. It is difficult to understand the molecular events that regulate LLPS because ensemble techniques blur the inherent heterogeneity and transient interactions. Single-molecule Förster resonance energy transfer (sm-FRET) fills this gap by providing nanometer-scale resolution of conformational dynamics and intermolecular interactions. Recent sm-FRET studies reveal that IDPs and RNA undergo pronounced conformational redistribution during LLPS, including compaction, expansion, and kinetic slowing that correlate with nucleation, maturation, and pathological transitions. Importantly, these conformational changes are inseparable from the reorganization and exchange of water molecules, which govern solvation free energy, osmotic balance, and interfacial transport across condensate boundaries therefore acting as both a thermodynamic driver and a kinetic regulator of condensate assembly and aging. Here, we review recent applications of sm-FRET to LLPS-associated systems, including tau–RNA cophase separation and SARS-CoV-2 nucleocapsid protein assemblies, to illustrate how nanoscale conformational dynamics, hydration energetics, and interfacial transport collectively govern condensate behavior. By integrating sm-FRET-derived observables with phenomenological models of water flux and free energy contributions, we propose a unifying framework that connects molecular insights, hydration-mediated interactions, and disease relevant phase transitions. Together, these insights position sm-FRET as a powerful platform for dissecting the physical chemistry of biomolecular condensates and for guiding the rational development of therapeutic strategies targeting pathological LLPS.