Mechanisms of phase-separation-mediated cGAS activation revealed by dcFCCS

Cyclic GMP–AMP synthase (cGAS), as a DNA sensor, plays an important role in cGAS–STING pathway, which further induces expression of type I interferon as the innate immune response. Previous studies reported that liquid–liquid phase separation (LLPS) driven by cGAS and long DNA is essential to promote catalytic activity of cGAS to produce a second messenger, cyclic GMP–AMP (cGAMP). However, the molecular mechanism of LLPS promoting cGAS activity is still unclear. Here, we applied dual-color fluorescence cross-correlation spectroscopy (dcFCCS), a highly sensitive and quantitative method, to characterize phase separation driven by cGAS and DNA from miscible individual molecule to micronscale. Thus, we captured nanoscale condensates formed by cGAS at close-to-physiological concentration and quantified their sizes, molecular compositions and binding affinities within condensates. Our results pinpointed that interactions between DNA and cGAS at DNA binding sites A, B, and C and the dimerization of cGAS are the fundamental molecular basis to fully activate cGAS in vitro. Due to weak binding constants of these sites, endogenous cGAS cannot form stable interactions at these sites, leading to no activity in the absence of LLPS. Phase separation of cGAS and DNA enriches cGAS and DNA by 2 to 3 orders of magnitude to facilitate these interactions among cGAS and DNA and to promote cGAS activity as an on/off switch. Our discoveries not only shed lights on the molecular mechanisms of phase-separation-mediated cGAS activation, but also guided us to engineer a cGAS fusion, which can be activated by 15 bp short DNA without LLPS.