STING; interferon-β (IFNβ)[1]

2',3'-cGAMP 钠（2'-3'-环 GMP-AMP 钠）中有两种不同的磷酸二酯连接：一种连接 GMP 的 2'-OH 和 AMP 的 5'-磷酸，另一种连接 3 AMP 的 '-OH 和 GMP(1–2) 的 5'-磷酸。

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2’3’-cGAMP是哺乳动物细胞产生的内源性第二信使。 2 ' 3 ' -cGAMP是STING的高亲和力配体。 2 ' 3 ' -cGAMP是一种有效的i型干扰素诱导剂。 2 ' 3 ' -cGAMP结合诱导STING构象变化[1]。

等温滴定量热法（ITC）[1]
等温滴定量热法（ITC）使用VP-ITC微量热计（GE Healthcare）测量STING与cGAMP异构体或c-di-GAMP之间的结合亲和力。蛋白质和配体浓度如图2D所示。滴定在20°C下进行，缓冲液中含有25 mM Hepes， pH 7.8, 150 mM NaCl。注射32次，间隔时间4分钟。通过NITPIC（Keller et al., 2012）对滴定迹线进行积分，然后通过SEDFIT（Houtman et al., 2007）对曲线进行拟合。数据采用GUSSI （http://biophysics.swmed.edu/MBR/software.html）制作。

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酶法合成和纯化cGAMP[1]
为了使用cGAS酶生成天然cGAMP，将含有20mM Tris-Cl， pH7.5, 5mM MgCl2, 10mM CoCl2, 0.01mg/ml鲱鱼睾丸DNA， 1mM ATP， 1mM GTP和0.1μM重组sumo标记的人cGAS （aa147-522）的反应在37℃下孵育1hr。混合物在Hitrap Q柱上以0-0.5M NaCl线性梯度进行分馏；收集cGAMP对应的UV峰，并将其上载到C18色谱柱（201TP510, 1cmX25cm）上，用0-100%的线性甲醇梯度洗脱。

内源性cGAMP的制备[1]
分别从DNA转染的L929和THP-1细胞中制备内源性cGAMP。HT-DNA转染4小时后，约3× 10~7个细胞在低渗缓冲液[10mM Tris-HCl， pH7.4, 10mM KCl， 1.5mM MgCl2]中裂解。裂解液在95°C下加热5分钟，在17000 g下再次离心10分钟，去除变性蛋白。耐热上清液在C-18色谱柱（Eclipse Plus 4.6×30 mm, 3.5μm, Agilent Technologies）上分馏，用0.1%甲酸平衡，用0-100%甲醇线性梯度洗脱。通过活性测定法监测各组分中cGAMP的存在（Wu et al., 2013），并使用活性峰值的组分进行进一步的质谱和质谱/质谱分析。

[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

331

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

注意： 请将本产品存放在密封且受保护的环境中，避免吸湿/受潮。

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

配方 1 中的溶解度: 18.33 mg/mL (25.52 mM) in PBS (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液; 超声助溶。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。