The study identifies that the cGAS enzyme produces a unique cyclic dinucleotide second messenger, 2'-5'-linked Gp(2'-5')Ap(3'-5'), which activates the STING protein. This molecule is distinct from previously characterized cyclic dinucleotides and is synthesized through a two-step process involving a linear 2'-5'-linked dinucleotide that is then cyclized via a 3'-5' phosphodiester linkage. The structure of this molecule, confirmed by mass spectrometry, NMR, and enzymatic analysis, is different from bacterial cyclic dinucleotides and is structurally and physiochemically distinct. The study also shows that this molecule activates STING in a manner distinct from cyclic di-GMP sensing, as evidenced by the R231A mutation in STING. The cGAS product was shown to be a potent activator of both human and murine STING, with human STING being more responsive to cGAMP(2'-5') than cGAMP(3'-5'). The findings suggest that cGAS produces a novel class of second messenger molecules, extending the family of 2'-5'-linked antiviral biomolecules. The study also highlights the potential for developing specific inhibitors targeting the cGAS-STING axis in autoimmune diseases. The results unify the innate sensing systems of DNA and cyclic dinucleotides, suggesting an evolutionary link between them. The study provides a detailed model of the two-step synthesis of cGAMP(2'-5') and confirms its role as the cGAS-dependent second messenger that activates STING.The study identifies that the cGAS enzyme produces a unique cyclic dinucleotide second messenger, 2'-5'-linked Gp(2'-5')Ap(3'-5'), which activates the STING protein. This molecule is distinct from previously characterized cyclic dinucleotides and is synthesized through a two-step process involving a linear 2'-5'-linked dinucleotide that is then cyclized via a 3'-5' phosphodiester linkage. The structure of this molecule, confirmed by mass spectrometry, NMR, and enzymatic analysis, is different from bacterial cyclic dinucleotides and is structurally and physiochemically distinct. The study also shows that this molecule activates STING in a manner distinct from cyclic di-GMP sensing, as evidenced by the R231A mutation in STING. The cGAS product was shown to be a potent activator of both human and murine STING, with human STING being more responsive to cGAMP(2'-5') than cGAMP(3'-5'). The findings suggest that cGAS produces a novel class of second messenger molecules, extending the family of 2'-5'-linked antiviral biomolecules. The study also highlights the potential for developing specific inhibitors targeting the cGAS-STING axis in autoimmune diseases. The results unify the innate sensing systems of DNA and cyclic dinucleotides, suggesting an evolutionary link between them. The study provides a detailed model of the two-step synthesis of cGAMP(2'-5') and confirms its role as the cGAS-dependent second messenger that activates STING.