Cation Accumulation Drives the Preferential Partitioning of DNA in an Aqueous Two-Phase System

Abstract

Mixtures of polyethylene glycol (PEG) and dextran (Dex) represent a widely used class of aqueous two-phase systems (ATPS), with applications ranging from the purification of various biomolecules, such as nucleic acids, to the synthesis of protocells. A key feature underlying these applications is the selective accumulation of biomolecules within Dex-rich droplets in an aqueous PEG phase, but the physical origin of this partitioning remains unclear. Entropic interactions were long assumed to be the primary driving force; however, our systematic experiments using DNA of different lengths indicate that entropy alone cannot fully explain the observed behavior. We identify an additional and previously underappreciated contribution from electrostatic interactions: Dex carries a slightly more negative charge than PEG, which drives preferential cation accumulation in the Dex-rich phase. These counterions facilitate the selective partitioning of DNA inside the Dex-rich droplets. This mechanism explains the dependence of DNA uptake in Dex-rich droplets on the polymer length and salt concentration. Our findings establish that Donnan-type ion partitioning plays a crucial role in the localization of long nucleic acids in Dex-rich droplets, offering a unified explanation for this long-standing phenomenon. They lay the foundation for designing ATPS-based systems and help elucidate the physicochemical principles of biomolecular partition upon phase separation in cells.