Solvent-Isotope Effects in Biomolecular Phase Separation and Fibrillation of Disordered Proteins and Peptides.

Heavy water (D 2 O) is widely used in biomolecular spectroscopy and imaging, often under the assumption that it is an inert replacement for H 2 O. However, D 2 O differs subtly in hydrogen-bonding, viscosity, and dielectric properties, which can alter biomolecular interactions and self-assembly. Here, we test how solvent isotope substitution modulates protein/peptide phase separation and amyloid formation in multiple intrinsically disordered systems. Using turbidity-based phase diagrams and microscopy, we quantify how D 2 O shifts protein-RNA complex coacervation boundaries and alters condensate morphology. Droplet recovery measurements indicate significant solvent-dependent changes in condensate material properties. We further evaluate amyloid formation kinetics, in the presence or absence of a cofactor, supported by orthogonal structural characterization, and assess the functional consequences of tau fibrils using a tau biosensor seeding assay with explicitly defined seed delivery conditions. Together, these results show that D 2 O can systematically bias liquid-liquid phase separation and aggregation readouts and should be treated as an active experimental variable rather than a neutral solvent substitute.