Ion accumulation in liquid–liquid phase separation regulates biomolecule localization

Liquid–liquid phase separation is a process in which a uniform solution separates into two coexisting liquid phases. In living cells, it governs the formation of membrane-less compartments that selectively concentrate molecules and regulate biochemical reactions. This phenomenon is also widely used in practical applications, including biomolecular separation and purification.

A long-standing question in phase separation research is why specific molecules preferentially localize in one phase over the other. Conventional explanations have focused on polymer–polymer interactions or entropic effects such as molecular size and shape. However, these frameworks cannot fully explain cases in which similarly charged molecules, including negatively charged biomolecules like DNA, accumulate in the same phase despite electrostatic repulsion.

To address this long-standing question, the researchers investigated a well-known aqueous two-phase system composed of poly(ethylene glycol) (PEG) and dextran (Dex), which has been used both as a model of cellular phase separation and as a separation technology. Although many biomolecules are known to partition preferentially into the Dex-rich phase, the underlying physicochemical mechanism remains unclear.

The international team found that the phase behavior of the PEG/Dex system is strongly affected by salt concentration, pointing to a role for electrostatic effects. Detailed analysis revealed that the Dex-rich phase is slightly more negatively charged than the PEG-rich phase. Using controlled experiments and ion-sensitive fluorescent probes, the researchers demonstrated that positively charged ions selectively accumulate in the Dex-rich phase. This ion partitioning reduces electrostatic repulsion between negatively charged biomolecules, allowing DNA to localize within Dex-rich droplets.

This mechanism represents Donnan-type ion partitioning, previously associated mainly with gels or membranes. The study provides the first direct quantitative evidence that this effect also operates in liquid–liquid phase-separated systems, highlighting ion distribution as a crucial factor in molecular selectivity and advancing our understanding of both cellular organization and biomolecular separation technologies.

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