DNA-Driven Liquid–Liquid Phase Separation from Cellular Condensates to Programmable Synthetic Systems

Liquid-liquid phase separation (LLPS) compartmentalizes biological systems into dynamic, membraneless condensates that regulate diverse cellular functions. Although protein and RNA-mediated LLPS have dominated research, DNA increasingly emerges as an active driver of phase separation rather than a passive structural scaffold. DNA-driven condensates orchestrate critical nuclear processes, from chromatin organization and transcriptional regulation to genome stability and innate immune responses. Yet LLPS principles extend beyond biology: programmable DNA nanostructures now enable synthetic droplets and hydrogels with tunable mechanical properties, opening pathways to biomaterials, diagnostics, and synthetic cells. Here we synthesize current understanding of DNA-mediated LLPS across biological and synthetic domains, emphasizing five underappreciated topics: (1) DNA's active driving role in LLPS through charge and topology; (2) reversible DNA aggregation and aggregate-to-condensate transitions, distinct from irreversible protein misfolding; (3) non-Fickian transport mechanisms including ballistic wave diffusion triggered by molecular recognition; (4) single-molecule mechanical characterization revealing state-dependent material properties; and (5) the multiscale complexity of cellular DNA condensation shaped by topological constraints and hierarchical organization. We highlight emerging single-molecule technologies, optical tweezers, and scanning probe microscopy that directly measure condensate mechanics and state transitions with unprecedented resolution. This integrated perspective bridges fundamental biophysics of natural DNA condensates with rational engineering principles for programmable synthetic systems, providing a blueprint for therapeutic and biotechnological applications.