NMR reveals the properties of biomolecular condensates in their biphasic state

Biomolecular condensates formed by liquid–liquid phase separation are inherently biphasic systems in which a condensed phase coexists with a surrounding dilute phase that contains vastly different solute concentrations. While NMR spectroscopy offers unique, quantitative access to molecular structure, interactions and dynamics, its application to phase-separated systems has long been challenged by signal overlap, slow diffusion, and the line broadening in the condensed phase. Recent methodological developments have begun to overcome these limitations by explicitly exploiting the physical properties of biomolecular condensates. In this review, we discuss emerging NMR approaches that enable the characterization of condensates in their native biphasic state. These include the diffusion-based strategy LLPS REDIFINE to disentangle dilute and condensed phase populations and enable quantitative extraction of interphase exchange and droplet properties, the magnetization-transfer-based solvent detection approach CONDENSE-MT, that provides access to NMR-invisible condensed phases, and multiplexed filtering schemes that resolve multiple coexisting components within complex condensates. Together, these methods extend the scope of NMR beyond homogeneous solutions, providing complementary and label-free insights into the structure, dynamics, and phase behavior of multicomponent biomolecular condensates.