Abstract
Cyclic dinucleotides (CDNs) are key secondary messenger molecules produced by cyclic dinucleotide synthases that trigger various cellular signaling cascades from bacteria to vertebrates. In mammals, cyclic GMP-AMP synthase (cGAS) has been shown to bind to intracellular DNA and catalyze the production of the dinucleotide 2′3′ cGAMP, which signals downstream effectors to regulate immune function, interferon signaling, and the antiviral response. Despite the importance of CDNs, sensitive and accurate methods to measure their levels in vivo are lacking. Here, we report a novel LC-MS/MS method to quantify CDNs in vivo. We characterized the mass spectrometric behavior of four different biologically relevant CDNs (c-di-AMP, c-di-GMP, 3′3′ cGAMP, 2′3′ cGAMP) and provided a means of visually representing fragmentation resulting from collision-induced dissociation at different energies using collision energy breakdown graphs. We then validated the method and quantified CDNs in two in vivo systems, the bacteria Escherichia coli OP50 and the killifish Nothobranchius furzeri. We found that optimization of LC-MS/MS parameters is crucial to sensitivity and accuracy. These technical advances should help illuminate physiological and pathological roles of these CDNs in in vivo settings.Graphical abstract
Highlights
Cyclic dinucleotides (CDNs) are a class of molecules that serve as important secondary messengers conserved across taxa
Bacterial CDNs form through the cyclization of two nucleotides via a phosphodiester bond that links the C3′ of one pentose ring with the C5′ of the other nucleotide, resulting in a 3′5′ cyclic dinucleotide
Recent work suggests that cyclic GMP-AMP synthase (cGAS)/Stimulator of Interferon Genes (STING) signaling is involved in sensing retroviruses, including HIV-1 and HIV-2, as well as the RNA virus COVID-19 [11, 12]
Summary
Cyclic dinucleotides (CDNs) are a class of molecules that serve as important secondary messengers conserved across taxa. Dinucleotide cyclase vibrio (DncV)–like nucleotidyl transferases (CDNTases) catalyze such reactions in prokaryotes and are best known for the synthesis of purine cyclic-bis dimeric GMP and c-di-AMP, heterocyclic 3′3′ cGMP-AMP (cGAMP), but have been recently shown to generate pyrimidine derivatives as well [1]. Vertebrate cyclic GMP-AMP synthase (cGAS) is best known for its role in nucleic acid signaling. It senses both foreign and cytosolically mislocalized selfdouble-stranded (ds)DNA and triggers a defense response [7, 8]. The cGAS/Sting pathway has emerged as central to nucleic acid dynamics, immunity, and aging
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