Spatiotemporal Regulation of DNA Condensate Formation Through Molecular Reaction Network

Biomolecular condensates formed by liquid–liquid phase separation (LLPS) are involved in diverse cellular functions, motivating efforts to engineer synthetic condensates with programmable behaviors. DNA nanotechnology provides precise control over intermolecular interactions; however, achieving spatiotemporal regulation of condensate formation, an essential feature of cellular LLPS, remains a key challenge. Here, we demonstrate the spatiotemporal regulation of liquid‐like DNA condensate formation by coupling a DNA‐based chemical reaction network (CRN) with a single‐stranded DNA (ssDNA)‐responsive phase‐separation mechanism. The CRN, composed of hybridization and enzyme‐driven reactions, generates a bistable reaction in which either production or degradation of a product ssDNA strand dominates. This product strand hybridizes with a Y‐shaped DNA nanostructure containing two sticky ends, converting it into a three‐armed motif capable of LLPS. We show that CRN‐mediated production of ssDNA induces condensate formation in a time‐programmed manner and that inhibition reactions suppress LLPS depending on inhibitor concentration. Embedding the CRN and the DNA nanostructures in a glass capillary with an inhibitor concentration gradient generates spatially asymmetric, time‐evolving condensate patterns without external control. Our results establish an autonomous framework for controlling when and where DNA condensates are formed, providing a basis for constructing life‐like molecular systems that integrate reaction networks with programmable phase behavior.