A proline-rich-domain-binding single domain antibody selectively inhibits RNA-induced liquid-liquid phase separation of tau

Liquid-liquid phase separation (LLPS) mediates the formation of biomolecular condensates, which organize cellular processes such as synaptic plasticity and stress response. The neuronal microtubule-associated protein tau undergoes LLPS under specific conditions, regulating synaptic vesicle clustering and microtubule dynamics. In vitro, tau LLPS is induced by cofactors such as polyethylene glycol (PEG) or RNA, mainly via weak multivalent electrostatic interactions. However, the molecular mechanisms governing the formation of tau LLPS, including domain specific contribution, remain unclear. In this study, we used eight single-domain antibodies (VHHs), targeting six distinct short sequences of tau, to explore the mechanisms of tau LLPS in vitro. By combining several biophysical methods, we evaluated the effect of each anti-tau VHH on tau LLPS with two main LLPS inducers, PEG (molecular crowding) and RNA (complex coacervation). With PEG as an inducer, all VHHs targeting tau enhanced tau LLPS formation, regardless of their affinity for tau. With RNA as an inducer, the effect of the VHHs was mixed: VHHs targeting the C-terminal domain promoted condensation, while VHH B1-1, which binds the proline-rich domain (PRD; including residues (221REPKKVAVVRTP232), abolished droplet formation. NMR and surface plasmon resonance confirmed 1 to 1 binding of VHH B1-1 to the PRD, and competition assays with a PRD peptide restored LLPS, demonstrating mechanistic specificity. This result underscores the importance of this region in tau LLPS formation. Our findings provide domain-resolved insights into the regulation of tau LLPS and demonstrate the potential of VHHs as tools to selectively modulate biomolecular condensates in physiological and pathological contexts.