Using Solution NMR to Characterize Biomolecular Condensates Under Biphasic Conditions.

Biomolecular condensates formed through liquid-liquid phase separation (LLPS) organize the intracellular environment and regulate diverse biochemical processes. Despite their importance, probing condensate composition, exchange dynamics, and internal organization remains challenging, particularly without external tags. Nuclear magnetic resonance (NMR) spectroscopy can provide a unique label-free window into these mesoscale assemblies, capturing both molecular motion and environmental heterogeneity. Two complementary NMR methodologies enable a comprehensive characterization of condensates directly within their biphasic state. For condensates which are dynamic enough to be observable by NMR, the diffusion-exchange approach, REstricted DIffusion of INvisible speciEs abbreviated as REDIFINE, utilizes the diffusion contrast with chemical exchange to quantify the fraction of molecules partitioned between condensed and dilute phases, determine droplet size and interface permeability, and extract molecular exchange rates across the phase boundary. For more rigid condensates that are NMR invisible, the water-detected semi-solid magnetization-transfer method, CONdensate DEtectioN by SEmi-solid Magnetization Transfer, or in short CONDENSE-MT, exploits the relaxation contrast and proton exchange between condensed biomolecules and dilute phase solvent to monitor condensates onto the bulk water protons, providing access to relative partitioning, molecular tumbling rates, hydration dynamics, and bound-water content. Together, these approaches deliver a multidimensional, quantitative view of condensate structure and dynamics under near-native biphasic conditions without fluorescent or detection tags. Their integration expands the NMR toolbox for studying biomolecular phase separation and establishes a foundation for connecting condensate physicochemical properties with their biological function and pathological misregulation.