Higher-order structure of DNA determines its positioning in cell-size droplets under crowded conditions

Background: It is becoming clearer that living cells use water/water (w/w) phase separation to form membraneless organelles that exhibit various important biological functions. Currently, it is believed that the specific localization of biomacromolecules, including DNA, RNA and proteins in w/w microdroplets is closely related to their bio-activity. Despite the importance of this possible role of micro segregation, our understanding of the underlying physico-chemical mechanism is still unrefined. Further research to unveil the underlying mechanism of the localization of macromolecules in relation to their steric conformation in w/w microdroplets is needed. Principal findings: Single-DNA observation of genome-size DNA (T4 GT7 bacteriophage DNA; 166kbp) by fluorescence microscopy revealed that DNAs are spontaneously incorporated into w/w microdroplets generated in a binary aqueous polymer solution with polyethylene glycol (PEG) and dextran (DEX). Interestingly, DNAs with elongated coil and shrunken conformations exhibit Brownian fluctuation inside the droplet. On the other hand, tightly packed compact globules, as well as assemblies of multiple condensed DNAs, tend to be located near the interface in the droplet. Conclusion and significance: The specific localization of DNA molecules depending on their higher-order structure occurs in w/w microdroplet phase-separation solution under a binary aqueous polymer solution. Such an aqueous solution with polymers mimics the crowded conditions in living cells, where aqueous macromolecules exist at a level of 30–40 weight %. The specific positioning of DNA depending on its higher-order structure in w/w microdroplets is expected to provide novel insights into the mechanism and function of membraneless organelles and micro-segregated particles in living cells.