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2',3'-cGAMP (2'-3'-cyclic GMP-AMP) isan endogenous cGAMP molecule found in mammalian cells, it binds to STINGwith high affinity and is a potent inducer of interferon-β(IFNβ). contains two distinct phosphodiester linkages, one between 2′-OHof GMP and 5′-phosphate of AMP, and the other between 3′-OH of AMP and5′-phosphate of GMP. 2',3'-cGAMP is produced in mammalian cells inresponse to DNA in the cytoplasm and binds to STING with a high affinityand is a potent inducer of interferon-β (IFNβ).
| Targets |
STING (stimulator of interferon genes); Kd of cGAS product (endogenous 2',3'-cGAMP sodium) = 4.59 nM; Kd of synthetic 2',3'-cGAMP sodium = 3.79 nM (determined by ITC competition assay).[1]
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| ln Vitro |
2',3'-cGAMP sodium (cGAS product) induced IFNβ in digitonin-permeabilized L929 cells with an EC50 of approximately 20 nM; all cGAMP isomers showed EC50 ranging from 15 to 42 nM, while c-di-GMP had EC50 >500 nM.[1]
The binding affinity (Kd) of 2',3'-cGAMP sodium to STING CTD was 4.59 nM for the cGAS product and 3.79 nM for synthetic 2'3'-cGAMP, which is ~300-fold lower than that of c-di-GMP (Kd 1.21 μM), 3'2'-cGAMP (Kd 1.61 μM), and 3'3'-cGAMP (Kd 1.04 μM), and ~75-fold lower than 2'2'-cGAMP (Kd 287 nM).[1] In STING mutant assays, L929 cells expressing wild-type human STING induced IFNβ in response to DNA or 2',3'-cGAMP sodium, whereas R232A or R232H mutants were defective. HEK293T cells expressing wild-type STING supported IFNβ induction by cGAS, but R232A mutant did not.[1] |
| Enzyme Assay |
Isothermal titration calorimetry (ITC) was used to measure binding affinities between STING CTD (residues 139-379) and 2',3'-cGAMP sodium or other cyclic dinucleotides using a VP-ITC microcalorimeter. Titrations were performed at 20°C in 25 mM Hepes pH 7.8, 150 mM NaCl. For tight binding, compounds were titrated as competitors into the STING-c-di-GMP complex (3.55:1 molar ratio). 32 injections with 4 min spacing; traces integrated by NITPIC and curves fitted by SEDFIT.[1]
High resolution MS and MS/MS analysis was performed on a Q-Exactive mass spectrometer. Full scan mass spectra from m/z 300-700 at resolution 70,000; MS/MS spectra at resolution 35,000 in data-dependent mode; top 5 parent ions fragmented by HCD at normalized collision energy 30.[1] 1H and 31P NMR spectroscopy was performed at 50°C in 30 mM NaH2PO4/Na2HPO4/D2O buffer pH 7.4 to compare the cGAS product with synthetic cGAMP isomers. Anomeric proton (H1) singlet indicated 3'-phosphate; doublet indicated 2'-phosphate.[1] Reverse phase HPLC and circular dichroism (CD) spectroscopy were used to analyze co-elution and confirm ribose configuration.[1] |
| Cell Assay |
Endogenous 2',3'-cGAMP sodium was prepared from L929 mouse cells or THP1 human monocytes transfected with herring testis DNA (HT-DNA). After 4 h, ~3×10^7 cells were lysed in hypotonic buffer (10 mM Tris-HCl pH7.4, 10 mM KCl, 1.5 mM MgCl2). Lysates heated at 95°C for 5 min and centrifuged at 17,000g for 10 min; supernatants fractionated on C-18 column with 0.1% formic acid/methanol gradient; fractions with peak activity analyzed by MS/MS.[1]
For IFNβ induction, different amounts of cGAMP isomers or c-di-GMP were delivered into digitonin-permeabilized L929 cells. After 4 h, IFNβ RNA was measured by qRT-PCR. EC50 values calculated with GraphPad Prism 5.0.[1] For STING functional assays, L929 cells with endogenous STING knocked down by shRNA were stably reconstituted with wild-type, R232A or R232H human STING. Cells were transfected with HT-DNA or treated with 2',3'-cGAMP sodium, then IFNβ RNA measured by qRT-PCR; poly(I:C) was used as control. HEK293T cells (undetectable endogenous STING and cGAS) stably expressing STING-Flag mutants were transfected with human cGAS or MAVS; 24 h later IFNβ RNA was measured. Immunoblotting with anti-STING and anti-β-tubulin confirmed expression.[1] |
| References |
Zhang X, et al. Cyclic GMP-AMP containing mixed phosphodiester linkages is an endogenous high-affinity ligand for STING. Mol Cell. 2013 Jul 25;51(2):226-35. |
| Additional Infomation |
cGAS (cyclic GMP-AMP synthase) is a cytosolic DNA sensor that catalyzes synthesis of 2',3'-cGAMP sodium from ATP and GTP in the presence of DNA. 2',3'-cGAMP sodium binds to STING (endoplasmic reticulum adaptor protein) to activate IKK and TBK1, leading to NF-κB and IRF3 activation and type I interferon production.[1]
The crystal structure of STING CTD (residues 139-379) bound to the cGAS product (2',3'-cGAMP sodium) was solved at 1.88 Å resolution (space group C2). The STING dimer forms a butterfly shape; upon ligand binding, the two protomers undergo inward rotations (arms close by ~20 Å) and a new four-stranded antiparallel β-sheet lid (residues 219-249) forms above the binding pocket. Key interactions: guanine base contacts Glu260, Thr263, and Val239; free 3'-OH of GMP contacts Ser162; α-phosphates contact Arg238 (both protomers) and Arg232 (one protomer). The R232A or R232H mutations severely impair IFNβ induction, highlighting the importance of Arg232 (prevalent human allele) for STING function.[1] |
| Appearance |
White to off-white solid powder
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples
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| Solubility (In Vivo) |
Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.
Injection Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution. Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline) Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil) Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals). View More
Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)] Oral Formulations
Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium) Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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