Fig. 2: Allosteric inhibitors disrupt cGAS-DNA interactions as well as phase separation.

a Electrophoretic mobility shift assays (EMSA) of Cy5-labeled DNA (ISD45, 0.05 μM) incubated with h-cGAS^CD (lanes 1–6: 1, 0.5, 0.25, 0.125, 0.063, and 0.031 μM, respectively) and indicated compound (20 μM). The experiment was repeated three times independently. b Quantified fluorescence intensities of condensates formed by mixing Cy3-labeled h-cGAS^CD at indicated concentration with FAM-labeled DNA (ISD100, 0.25 μM) in buffer containing indicated concentration of NaCl and indicated compound (100 μM) in the FAM channel. Each data point represents the mean intensity from 5 different images. c Representative images of phase separation induced by mixing Cy3-labeled h-cGAS^CD (2 μM) with FAM-labeled DNA (ISD100, 0.25 μM) in buffer containing 150 mM NaCl and different concentrations of indicated compound (left). Green, FAM-labeled DNA (ISD100); Red, Cy3-labeled h-cGAS^CD. Scale bar, 20 μm. Quantification of fluorescence intensities in the images (right). Data represent the mean ± SD from five different images. Statistical significance was determined by an unpaired two-tailed t test. *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001. d Turbidity analysis of h-cGAS^CD at indicated concentration with DNA (ISD100, 0.25 μM) in buffer containing indicated concentration of NaCl and indicated compound (100 μM), where the numbers indicate OD₃₄₀ values of three independent experiments. e EC₅₀ fitting curves and histogram of DNA condensation assays for DNA-h-cGAS^CD interactions. Cy3-labeled DNA (ISD100, 0.15 μM) was incubated with various concentrations of h-cGAS^CD (51–6560 nM, in two-fold increments) in the presence of 50 μM indicated compound. Data are presented as mean ± SD from three independent experiments. Statistical significance was determined by an unpaired two-tailed t test. *p < 0.05, **p < 0.01, ***p < 0.005, and ****p < 0.001. Source data is available with this manuscript as a Source Data file.