Extended Data Fig. 6: Synthesis of 3′2′-cGAMP by Diptera cGLRs.
From: cGAS-like receptors sense RNA and control 3′2′-cGAMP signalling in Drosophila
a, HPLC analysis of the nucleotide products of Tc-cGLR, Dm-cGLR1, Ds-cGLR1, Lc-cGLR and Deu-cGLR reactions compared with relevant synthetic controls. Integration of major and minor product peaks in n = 3 independent experiments was used to calculate relative product ratios shown in Fig. 3c. b, The Drosophila cGLR major reaction product was purified from Deu-cGLR reactions and compared with synthetic 3′2′-cGAMP with tandem mass spectrometry analysis. Parent mass extracted ion trace (left) and tandem mass spectra comparison (right) validate the chemical identity of the Drosophila cGLR product as 3′2′-cGAMP. c, Identification of widespread 3′2′-cGAMP synthesis by Diptera cGLRs. The heat map shows the relative concentrations of cGAMP isomers detected by HPLC-MS in bacterial lysates expressing Diptera cGLRs (as described in Extended Data Fig. 2b) (left). In all cases, 3′2′-cGAMP was present as the dominant product with trace amounts of 3′3′-cGAMP and 2′3′-cGAMP detected in some samples as minor species. Right, inset of clade 5 in the Diptera cGLR phylogeny from Fig. 2a annotated to show all enzymes identified to synthesize 3′2′-cGAMP.
