A synthetic cell microreactor with two types of interacting dynamic DNA-based pores

Reconstructing cellular complexity is a central challenge in synthetic biology, with profound implications for understanding life and advancing bio-inspired nanotechnologies. A critical step towards this goal is replicating the dynamic interplay among membrane components and their functions. Here we demonstrate a double-necked synthetic cell microreactor (DCM) that incorporates two dynamic, DNA-based pores in the membrane of a giant unilamellar vesicle. The formation of the DCM leverages a signalling pathway mediated by giant unilamellar vesicle membrane dynamics to coordinate interactions between light-responsive small pores and self-arranged sealable large pores. This system enables sequential, on-demand delivery of molecular reactants with high spatiotemporal precision. Using DCMs, we demonstrate confined biochemical reactions, including a glucose oxidase-myoglobin cascade, cytoskeleton-mimetic actin polymerization and bundling, cell-free Spinach RNA transcription and the synthesis of three-dimensional DNA crystals that extend beyond natural systems. By coupling orchestrated multistep signalling with dynamic control of membrane permeability, the DCM establishes a versatile platform for emulating and expanding the functional complexity of natural cellular systems.