Electrostatic bimodality of human cytosolic DNA sensor cGAS-dsDNA condensates revealed by millisecond flashing electrophoretic separation

Electrostatic landscapes within biomolecular condensates are critical regulators of their structural stability and functional plasticity. To quantitatively resolve these elusive electrical signatures, we develop a millisecond Flashing Electrophoretic Separation (msFES) platform enabling nanoliter-scale profiling of biomolecular condensates at near single-droplet resolution—with a focus on zeta potential characterization. Applying msFES to condensates formed by human cytosolic DNA sensor cGAS (hcGAS) and DNA, we unexpectedly discover an intrinsic electrostatic heterogeneity: droplets formed from identical components under identical conditions exhibit two distinct zeta-potential populations. This heterogeneity dictates differential susceptibility to TREX1, an exonuclease that selectively disassembles negatively charged droplets while sparing or amplifying positively charged assemblies, thereby potentially modulating cGAS activation. Pathogenic hcGAS variants (K432T, G303E) disrupt this electrostatic control, rendering hcGAS-DNA droplets hypersensitive to TREX1-mediated DNA degradation. msFES thus uncovers a hidden layer of charge-based regulation in phase-separated systems, potentially linking condensate biophysics to innate immune control. The characterization of LLPS droplets relies on multimodal approaches to probe their structural and dynamic properties, collectively mapping condensate assembly, disassembly, and interaction networks. However, charge-mediated interactions remain poorly resolved, leaving the ζ-potential landscape of LLPS droplets largely unexplored. Here, the authors present a millisecond flashing electrophoretic separation–laserinduced fluorescence (msFES-LIF) platform enabling ζ-potential profiling of fragile biomolecular condensates at pseudo-single-droplet resolution using nanoliter volumes and nanomolar concentrations.