Depletion of monovalent cations limits the coexistence regime of the DNA nanostar liquid-liquid phase diagram

Liquid-liquid phase separation (LLPS), the physical process by which biomolecular condensates arise from a homogeneous solution, occurs frequently in cells. Due to their programmability, DNA nanostars are increasingly used as a model system for studying LLPS. We study DNA nanostar (NS) LLPS by generating monodisperse water-in-oil emulsions containing different concentrations of NS and measuring the temperature dependence of the volume fraction of NS condensate. We find that in a solution of monovalent salt, the phase diagrams are strongly dependent on the salt concentration. For example, valence-3 NS with 20 bp-long arms in 185 mM NaCl solution will phase separate into a condensate whose [Na+] is roughly 240 mM. Such cation partitioning places a hard upper bound on the allowed volume fraction of condensate. We compare phase diagrams under various salt conditions and identify a “monovalent ion depleted” (MID) regime within each, where LLPS is prohibited. These results contradict current models of NS LLPS, highlight limitations on the applicability of the lever rule to measure phase diagrams, and demonstrate tuning the MID regime using monovalent salt concentration.