STING; interferon-β (IFNβ)[1]

There are two different phosphodiester connections in 2',3'-cGAMP sodium (2'-3'-cyclic GMP-AMP sodium): one connects 2'-OH of GMP and 5'-phosphate of AMP, while the other connects 3'-OH of AMP and 5′-phosphate of GMP(1–2).[1]

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2′3′-cGAMP is an endogenous second messenger produced by mammalian cells.
2′3′-cGAMP is a high affinity ligand for STING.
2′3′-cGAMP is a potent inducer of type-I interferons.
2′3′-cGAMP binding induces conformational changes of STING.[1]

Isothermal titration calorimetry (ITC)[1]
Isothermal titration calorimetry (ITC) was employed to measure the binding affinities between STING and cGAMP isomers or c-di-GAMP using a VP-ITC microcalorimeter (GE Healthcare). The protein and the ligand concentrations are shown in Figure 2D. The titrations were performed at 20°C in the buffer containing 25 mM Hepes, pH 7.8, 150 mM NaCl. 32 injections were performed with 4 minutes spacing time. The titration traces were integrated by NITPIC(Keller et al., 2012) and then the curves were fitted by SEDFIT(Houtman et al., 2007). The figures were prepared using GUSSI (http://biophysics.swmed.edu/MBR/software.html).

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Enzymatic Synthesis and Purification of cGAMP[1]
To generate natural cGAMP using the enzyme cGAS, a reaction containing 20mM Tris-Cl, pH7.5, 5mM MgCl2, 10mM CoCl2, 0.01mg/ml herring testis DNA, 1mM ATP, 1mM GTP, and 0.1μM recombinant SUMO-tagged human cGAS (aa147–522) was incubated at 37°C for 1hr. The mixture was fractionated on a Hitrap Q column using a linear 0–0.5M NaCl gradient; a UV peak corresponding to cGAMP was collected and loaded onto a C18 column (201TP510, 1cmX25cm), and eluted with a linear 0–100% methanol gradient.

Preparation of Endogenous cGAMP[1]
Endogenous cGAMP was prepared from DNA transfected L929 and THP-1 cells, respectively. After HT-DNA transfection for 4 hours, about 3× 10~7 cells were lysed in hypotonic buffer [10mM Tris-HCl, pH7.4, 10mM KCl, 1.5mM MgCl2]. The lysates were heated at 95°C for 5 min and centrifuged again at 17,000g for 10 min to remove denatured proteins. The heat-resistant supernatant was fractionated on a C-18 column (Eclipse Plus 4.6×30 mm, 3.5μm, Agilent Technologies) equilibrated with 0.1% formic acid and eluted with a linear gradient of 0–100% methanol. The presence of cGAMP in each fraction was monitored by activity assay (Wu et al., 2013), and the fraction with peak activity was used for further MS and MS/MS analysis.

[1]. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Mol Cell. 2013 Jul 25;51(2):226-35.

The presence of microbial or self DNA in the cytoplasm of mammalian cells is a danger signal detected by the DNA sensor cyclic-GMP-AMP (cGAMP) synthase (cGAS), which catalyzes the production of cGAMP that in turn serves as a second messenger to activate innate immune responses. Here we show that endogenous cGAMP in mammalian cells contains two distinct phosphodiester linkages, one between 2'-OH of GMP and 5'-phosphate of AMP, and the other between 3'-OH of AMP and 5'-phosphate of GMP. This molecule, termed 2'3'-cGAMP, is unique in that it binds to the adaptor protein STING with a much greater affinity than cGAMP molecules containing other combinations of phosphodiester linkages. The crystal structure of STING bound to 2'3'-cGAMP revealed the structural basis of this high-affinity binding and a ligand-induced conformational change in STING that may underlie its activation.[1]

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Although 2′3-cGAMP binds to STING with a much higher affinity than cGAMP isomers containing other phosphodiester linkages, all four cGAMP isomers induced IFNβ with similar EC50 values, which were much lower than that of c-di-GMP. Thus, all cGAMP isoforms are potent inducers of IFNβ, raising the possibility that cGAMP containing distinct phosphodiester linkages might exist in nature, perhaps in some lower organisms. Indeed, Vibrio cholera contains a cyclase that synthesize 3′3′-cGAMP involved in bacterial chemotaxis and colonization (Davieset al., 2012). At present, it is not clear why mammals have evolved to produce 2′3′-cGAMP as the endogenous second messenger to trigger innate immune responses.[1]

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In summary, our results demonstrate that 1) the endogenous second messenger produced in mammalian cells in response to cytosolic DNA stimulation is 2′3′-cGAMP; 2) 2′3′-cGAMP is a high affinity ligand for STING; 3) 2′3′-cGAMP is a potent inducer of IFNβ in mammalian cells; 4) 2′3′-cGAMP induces conformational rearrangements in STING that might underlie its activation; and 5) extensive interactions between 2′3′-cGAMP and STING observed in the crystal structure of the complex explains their specific and high affinity binding.[1]

C20H25N10NAO13P2

698.408995389938

718.063

2734858-36-5

2',3'-cGAMP;1441190-66-4

137120248

White to off-white solid powder

5

19

2

47

1290

8

OC1([H])[C@@]2([H])COP(O[C@@]3([H])[C@@H](O)[C@H](N4C=NC5=C(N=CN=C45)N)O[C@]3([H])COP(O)(=O)O[C@@]1([H])[C@H](N1C=NC3C(N=C(N)NC1=3)=O)O2)(O)=O.[NaH]

CNVCOPPPOWRJAV-DQNSRKNCSA-L

InChI=1S/C20H24N10O13P2.2Na/c21-14-8-15(24-3-23-14)29(4-25-8)18-11(32)12-7(41-18)2-39-45(36,37)43-13-10(31)6(1-38-44(34,35)42-12)40-19(13)30-5-26-9-16(30)27-20(22)28-17(9)33;;/h3-7,10-13,18-19,31-32H,1-2H2,(H,34,35)(H,36,37)(H2,21,23,24)(H3,22,27,28,33);;/q;2*+1/p-2/t6-,7-,10-,11-,12-,13-,18-,19-;;/m1../s1

disodium;2-amino-9-[(1R,6R,8R,9R,10S,15R,17R,18R)-8-(6-aminopurin-9-yl)-9,18-dihydroxy-3,12-dioxido-3,12-dioxo-2,4,7,11,13,16-hexaoxa-3λ5,12λ5-diphosphatricyclo[13.2.1.06,10]octadecan-17-yl]-1H-purin-6-one

2',3'-cGAMP sodium salt; 2734858-36-5; JX6B238JSL; 2'3'-cGAMP (sodium salt); 2'-3'-cyclic GMP-AMP sodium; PD077435; adenylyl-(3'-->5')-2'-guanylic acid, cyclic nucleotide, disodium salt; 2'3'-CYCLIC GUANOSINE MONOPHOSPHATE-ADENOSINE MONOPHOSPHATE DISODIUM SALT

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O: 50 mg/mL (69.60 mM)
DMSO: < 1 mg/mL

Solubility in Formulation 1: 18.33 mg/mL (25.52 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication.

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 (Please use freshly prepared in vivo formulations for optimal results.)