Protein-Independent Liquid–Liquid Phase Separation of Adenosine Triphosphate under Crowded Conditions

Adenosine triphosphate (ATP), a common constituent of protein-rich biomolecular condensates, itself undergoes liquid–liquid phase separation (LLPS) even in the absence of polypeptides. Using polyethylene glycol as macromolecular crowding agent, we observed robust ATP droplet formation at pH 2–11. Moderate NaCl and lower temperatures promote LLPS by lowering critical ATP and crowder concentrations. Very high salt reverses this trend through anomalous underscreening, but ATP droplets still survive in hypersaline environments. Most importantly, physiological millimolar ATP concentrations are sufficient for phase separation in the presence of millimolar Mg ²⁺ and crowders, mimicking intracellular conditions. pH tunes intermolecular interactions, evidenced by inversion of the adenosine circular dichroism Cotton effect. These results reveal intrinsic, protein-independent LLPS of ATP with potential roles in cellular compartmentalization and pathological phase transitions.