Light‐Responsive DNA Droplets for Controlling Enzyme Cascade Pathways by Dynamic Phase Separation

Abstract Precise control of enzymatic cascade pathways is essential for deepening our understanding of metabolic processes and designing synthetic biochemical systems. In this study, we present a novel approach to modulate these cascades using light‐responsive DNA droplets. Y‐motif sequences with azobenzene‐modified sticky ends (SE) were designed to form DNA droplets via liquid–liquid phase separation. These droplets enable selective sequestration of enzymes within compartments, allowing dynamic regulation of enzymatic cascades in response to ultraviolet (UV) and visible (vis) light stimuli. As a model system, we employed an incompatible enzymatic cascade, involving glucose oxidase (GOx), horseradish peroxidase (HRP), and catalase (CAT). Under visible light, the DNA droplets co‐localize enzymes within the compartments, effectively regulating metabolic flux without altering the total enzyme concentration. Upon UV exposure, the droplets disassemble, releasing the sequestered enzymes and altering cascade dynamics. The versatility of this approach was further demonstrated using an alternative enzymatic cascade system comprising galactose oxidase (GAO), myeloperoxidase (MPO), and CAT. This system demonstrates that light‐responsive DNA droplets are a powerful tool for regulating enzymatic pathways, with potential applications in synthetic biology, microreactor design, and bioengineering.