Molecular Crowding Stabilizes DNA Coacervate Droplets and Generates Self-Organized Characteristic Patterns

Living cells are characterized by highly crowded intracellular environments that are rich in macromolecules. Liquid-like biomolecular condensates play a central role in regulating biochemical reactions in such environments. However, the effects of macromolecular crowding on condensate formation and properties remain inadequately understood, despite their importance for both the fundamental understanding of biomolecular condensates and the development of artificial cells and micromachines. In this study, we systematically investigated the influence of polymeric crowding agentspolyethylene glycol (PEG), dextran, and Ficollon liquid-like DNA condensates (DNA droplets) formed through the sticky-end hybridization of Y-shaped DNA nanostars. We show that PEG, above the molecular weight of several kilograms (k), significantly enhances the thermal stability of DNA droplets compared to dextran and Ficoll, whereas low-molecular-weight PEG (0.6k and 1k) tends to destabilize droplet formation. In contrast, PEG 7.5k robustly promoted droplet formation and enhanced thermal stability. Furthermore, under PEG 7.5k crowding conditions, multiple types of DNA droplets composed of noncomplementary nanostars spontaneously assemble into adjacent and alternating network patterns. The resulting spatial organization is tunable by the PEG concentration, salt concentration, nanostar concentration, and number of droplet species. These findings reveal macromolecular crowding as an effective design parameter for controlling programmable DNA droplets, providing a versatile strategy for constructing functional microstructured systems relevant to micromachines and synthetic-cellular platforms.