Hierarchical control of enzymatic actuators using DNA-based switchable memories

Inspired by signaling networks in living cells, DNA-based programming aims for the engineering of biochemical networks capable of advanced regulatory and computational functions under controlled cell-free conditions. While regulatory circuits in cells control downstream processes through hierarchical layers of signal processing, coupling of enzymatically driven DNA-based networks to downstream processes has rarely been reported. Here, we expand the scope of molecular programming by engineering hierarchical control of enzymatic actuators using feedback-controlled DNA-circuits capable of advanced regulatory dynamics. We developed a translator module that converts signaling molecules from the upstream network to unique DNA strands driving downstream actuators with minimal retroactivity and support these findings with a detailed computational analysis. We show our modular approach by coupling of a previously engineered switchable memories circuit to downstream actuators based on β -lactamase and luciferase. To the best of our knowledge, our work demonstrates one of the most advanced DNA-based circuits regarding complexity and versatility. Naturally evolved regulatory circuits have hierarchical layers of signal generation and processing. Here, the authors emulate these networks using feedback-controlled DNA circuits that convert upstream signaling to downstream enzyme activity in a switchable memories circuit.