Internal Phase Separation in Synthetic DNA Condensates

Abstract Biomolecular condensates regulate cellular biochemistry by organizing enzymes, substrates and metabolites, and often acquire partially de‐mixed states whereby distinct internal domains play functional roles. Despite their physiological relevance, questions remain about the principles underpinning the emergence of multi‐phase condensates. Here, a model system of synthetic DNA nanostructures able to form monophasic or biphasic condensates is presented. Key condensate features, including the degree of interphase mixing and the relative size and spatial arrangement of domains, can be controlled by altering nanostructure stoichiometries. The modular nature of the system facilitates an intuitive understanding of phase behavior, and enables mapping of the experimental phenomenology onto a predictive Flory‐Huggins model. The experimental and theoretical framework introduced is expected to help address open questions on multiphase condensation in biology and aid the design of functional biomolecular condensates in vitro, in synthetic cells, and in living cells.