Controlled Formation of DNA Condensates as Model Nuclei in Monodisperse Giant Vesicles.

Several studies have attempted to replicate the complex hierarchy of eukaryotic cells for the bottom-up construction of artificial cells. Specifically, reconstruction of liquid-liquid phase separation systems as membrane-less organelles is one of the key focuses of this research field, with DNA condensates acting as versatile building blocks whose associative interactions can be precisely controlled via sequence design. However, such control is only possible at the nanoscale as control over the size and morphology of the lipid vesicles and liquid-liquid phase separation systems at the meso-to-microscale is determined by the kinetic aspects of their formation processes. Microfluidics is well-suited for controlling dynamic molecular assemblies at the cellular scale. In this study, we report the controlled condensation of DNA nanostars in mass-produced monodisperse giant vesicles (GVs) generated using a microfluidic device by manipulating the concentrations of DNA and salt associated with the GV volume changes. Our approach facilitates the precise control of the dynamics of DNA condensate formation, final size of condensates, formation of multiple condensates, and reversible formation/dissociation of condensates in GVs serving as a chassis for an artificial cell. Furthermore, our approach eliminates the need for thermal annealing prior to DNA condensation, supporting the coexistence of enzyme-containing biochemical reaction systems, such as gene expression systems.