Figure 2.

Identification of Svg3 as a potent cGAS agonist by oligonucleotide engineering and screening

(A) Schematic structures of ssDNA oligonucleotides. (B) Elongating dsDNA stem length from 10 to 24 bp elevated IFN-I response in RAW-ISG macrophages, which plateaued at 21 bp in the stem. (C) Optimization of the loop sizes and G numbers in hairpin overhangs for cGAS-mediated IFN-I responses in RAW-ISG macrophages. (D) Svg3 showed a strong binding affinity with human cGAS as measured by MST. (E) The binding of cGAS with Svg3 induced cGAS-Svg3 phase separation to form liquid-like droplets. Alexa Fluor 488-labeled Svg3 was mixed with human cGAS for 30 min. (F) Svg3 elicited strong IFN-I responses in murine bone marrow-derived macrophages (BMDMs) and bone marrow-derived dendritic cells (BMDCs). (G) Svg3 elicited comparably potent IFN-I responses relative to ISD in RAW-ISG macrophages. (H) 2′3′-cGAMP production by RAW 264.7 cells upon treatment with Svg3 or ISD (100 nM) as a control for 4–8 h (n = 3). 2′3′-cGAMP concentration in cell lysis was measured by ELISA. Unless denoted otherwise, 25 nM DNA was transfected into cells by Lipofectamine 2000, followed by incubation for 24 h in cell culture medium before IFN-I measurement. Data: mean ± SD. p values were determined by t test. ns, not significant; p > 0.05, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.