Abstract Tau aggregation is a defining feature of neurodegenerative tauopathies, including Alzheimer’s disease, corticobasal degeneration, and frontotemporal dementia. This aggregation involves the liquid–liquid phase separation (LLPS) of Tau, followed by its sol–gel phase transition, representing a crucial step in aggregate formation both in vitro and in vivo . However, the precise cofactors influencing Tau phase transition and aggregation under physiological conditions (e.g., ion concentration and temperature) remain unclear. In this study, we unveil that nucleic acid secondary structures, specifically RNA G-quadruplexes (rG4s), and calcium ions (Ca 2+ ) synergistically facilitated the sol–gel phase transition of human Tau under mimic intracellular ion conditions (140 mM KCl, 15 mM NaCl, and 10 mM MgCl 2 ) at 37□ in vitro . In the presence of molecular crowding reagents, Tau formed stable liquid droplets through LLPS, maintaining fluidity for 24 h under physiological conditions. Notably, cell-derived RNA promoted Tau sol–gel phase transition, with G4-forming RNA emerging as a crucial factor. Surprisingly, polyanion heparin did not elicit a similar response, indicating a distinct mechanism not rooted in electrostatic interactions. Further exploration underscored the significance of Ca 2+ , which accumulate intracellularly during neurodegeneration, as additional cofactors in promoting Tau phase transition after 24 h. Importantly, our findings demonstrate that rG4s and Ca 2+ synergistically enhance Tau phase transition within 1 h when introduced to Tau droplets. In conclusion, our study illuminates the pivotal roles of rG4s and Ca 2+ in promoting Tau aggregation under physiological conditions in vitro , offering insights into potential triggers for tauopathy.

RNA G-quadruplexes and calcium ions synergistically induce Tau phase transitionin vitro