Fig. 3: Discovery of 3′2′-cGAMP as a metazoan nucleotide second messenger.

a, High- performance liquid chromatography (HPLC) analysis of the Dm-cGLR1 reaction (orange) and comparison with synthetic standards (black or dashed lines) demonstrates that Dm-cGLR1 synthesizes 3′2′-cGAMP as the major product. A minor Dm-cGLR1 reaction product is 2′3′-c-di-AMP (see also Extended Data Fig. 6a). b, Thin-layer chromatography analysis of mouse cGAS and Dm-cGLR1 reactions labelled with either α-³²P-ATP or α-³²P-GTP (indicated as [α-³²P]NTP) and treated as indicated. Pairwise labelling and nuclease P1 digestion verify that cGAS and Dm-cGLR1 synthesize distinct cGAMP isomers with opposite phosphodiester linkage specificities. Representative of n = 3 independent experiments. High-resolution mass spectrometry confirms the major Diptera cGLR product as 3′2′-cGAMP (see also Extended Data Fig. 6b). c, HPLC quantification of insect cGLR nucleotide products. 3′2′-cGAMP is the dominant product of each identified Diptera cGLR (denoted by a black line), and 2′3′-cGAMP is the dominant product of cGAS and Tc-cGLR. Data are the mean quantified product of n = 3 independent experiments. d, Thermal denaturation assay showing that dSTING selectively recognizes 3′2′-cGAMP (see also Extended Data Fig. 8b, c). Representative of n = 3 independent experiments. e, Crystal structure of the dSTING–3′2′-cGAMP complex reveals a tightly closed homodimer and an ordered β-strand lid, indicating high-affinity engagement of the endogenous Drosophila second messenger 3′2′-cGAMP. f, Alignment and conservation of the stem helix and β-strand lid in human and insect STING proteins. Critical ligand-binding residues (blue dot) and adaptations specific to Diptera (red outline) are denoted. g, Superposition of the dSTING–3′2′-cGAMP (blue–orange) complex and the human STING–2′3′-cGAMP (grey–pink) (PDB: 4KSY)⁵ complex reveals that human STING readout of the 2′–5′ phosphodiester bond by R232 is absent in dSTING (left). Human STING S162 (grey) contacts the free 3′ OH of the guanosine base in 2′3′-cGAMP (pink). dSTING N159 (blue) extends across the ligand-binding pocket to contact the free 3′ OH of the adenosine base in 3′2′-cGAMP (orange) (right).