The Hidden Geometry of Water: Mapping Nanoscale Confinement and Water Properties Inside Peptide-Nucleotide Biomolecular Condensates.

Biomolecular condensates formed by liquid-liquid phase separation create dynamic, water-rich microenvironments that are essential for cellular organization, yet it is challenging to understand their internal aqueous phase. Here, we introduce Brønsted photoacids and photobases as a new class of environmental fluorescent probes to interrogate the water phase inside biomolecular condensates. Using in vitro condensates formed by intrinsically disordered poly lysine or poly arginine peptides with nucleotides, we combine steady-state and time-resolved fluorescence to resolve the probe's excited-state proton transfer and subsequent proton recombination dynamics. We show that both photoacids and photobases remain largely solvated within poly lysine-based condensates, with only modest slowing of proton transfer relative to bulk water, consistent with a moderately more viscous aqueous environment. We mainly focus on photoacids, which exhibit strongly enhanced geminate proton recombination, attributed to nanometre-scale confinement of water within the condensates, inducing reflective boundaries for the dissociated proton. By modeling the recombination kinetics, we use the photoacid as a molecular ruler to estimate the dimensions of confined aqueous nanocavities, revealing characteristic maximum radii of ∼6 nm. Altering condensate composition systematically modulates these properties, with ATP- and poly arginine-based condensates displaying denser, less hydrated interiors. These findings establish excited-state proton transfer probes as powerful tools for quantifying nanoscale water confinement in biomolecular condensates, with implications for condensate function and molecular sequestration.