Enzymatic degradation of liquid droplets of DNA is modulated near the phase boundary

Biomolecules can undergo liquid-liquid phase separation (LLPS), forming dense droplets that are increasingly understood to be important for cellular function. Analogous systems are studied as early-life compartmentalization mechanisms, for applications as protocells, or as drug-delivery vehicles. In many of these situ-ations, interactions between the droplet and enzymatic solutes are important to achieve certain functions. To explore this, we carried out experiments in which a model LLPS system, formed from DNA nanostar particles, interacted with a DNA-cleaving restriction enzyme, SmaI, whose activity degraded the droplets, causing them to shrink with time. By controlling adhesion of the DNA droplet to a glass surface, we were able to carry out time resolved imaging of this active dissolution process. We found that the scaling properties of droplet shrinking were sensitive to the proximity to the dissolution (boiling) temperature of the dense liquid: For systems far from the boiling point, enzymes acted only on the droplet surface, while systems poised near the boiling point permitted enzyme penetration. This was corrobo-rated by the observation of enzyme-induced vacuole-formation (bubbling) events, which can only occur through enzyme inter-nalization, and which occurred only in systems poised near the boiling point. Overall, our results demonstrate a mecha-nism through which the phase stability of a liquid affects its enzymatic degradation through modulation of enzyme transport properties.