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[Preprint]. 2023 Jul 13:2023.07.13.548237. [Version 1] doi: 10.1101/2023.07.13.548237

Figure 2. Identification of Svg3 as a potent cGAS agonist by oligonucleotide engineering and screening.

Figure 2.

A) Schematic structures of ssDNA oligonucleotides. B) Elongating dsDNA stem length from 10 bp 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 BMDMs and 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. 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 ± S.D. 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).