Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Letter
  • Published:

cGAS produces a 2′-5′-linked cyclic dinucleotide second messenger that activates STING


Detection of cytoplasmic DNA represents one of the most fundamental mechanisms of the innate immune system to sense the presence of microbial pathogens1. Moreover, erroneous detection of endogenous DNA by the same sensing mechanisms has an important pathophysiological role in certain sterile inflammatory conditions2,3. The endoplasmic-reticulum-resident protein STING is critically required for the initiation of type I interferon signalling upon detection of cytosolic DNA of both exogenous and endogenous origin4,5,6,7,8. Next to its pivotal role in DNA sensing, STING also serves as a direct receptor for the detection of cyclic dinucleotides, which function as second messenger molecules in bacteria9,10,11,12,13. DNA recognition, however, is triggered in an indirect fashion that depends on a recently characterized cytoplasmic nucleotidyl transferase, termed cGAMP synthase (cGAS), which upon interaction with DNA synthesizes a dinucleotide molecule that in turn binds to and activates STING14,15. We here show in vivo and in vitro that the cGAS-catalysed reaction product is distinct from previously characterized cyclic dinucleotides. Using a combinatorial approach based on mass spectrometry, enzymatic digestion, NMR analysis and chemical synthesis we demonstrate that cGAS produces a cyclic GMP-AMP dinucleotide, which comprises a 2′-5′ and a 3′-5′ phosphodiester linkage >Gp(2′-5′)Ap(3′-5′)>. We found that the presence of this 2′-5′ linkage was required to exert potent activation of human STING. Moreover, we show that cGAS first catalyses the synthesis of a linear 2′-5′-linked dinucleotide, which is then subject to cGAS-dependent cyclization in a second step through a 3′-5′ phosphodiester linkage. This 13-membered ring structure defines a novel class of second messenger molecules, extending the family of 2′-5′-linked antiviral biomolecules.

This is a preview of subscription content, access via your institution

Access options

Buy this article

Prices may be subject to local taxes which are calculated during checkout

Figure 1: The R231A STING mutant uncouples cyclic di-GMP sensing from cGAS-induced activation.
Figure 2: The cGAS reaction product is distinct from cGAMP(3′-5′).
Figure 3: The second messenger produced by cGAS is >Gp(2′-5′)Ap(3′-5′)>.
Figure 4: cGAMP(2′-5′) is a potent activator of human and murine STING.
Figure 5: >Gp(2′-5′)Ap(3′-5′)> is synthesized in a two-step process.

Similar content being viewed by others


  1. Hornung, V. & Latz, E. Intracellular DNA recognition. Nature Rev. Immunol. 10, 123–130 (2010)

    Article  CAS  Google Scholar 

  2. Gall, A. et al. Autoimmunity initiates in nonhematopoietic cells and progresses via lymphocytes in an interferon-dependent autoimmune disease. Immunity 36, 120–131 (2012)

    Article  CAS  Google Scholar 

  3. Ahn, J., Gutman, D., Saijo, S. & Barber, G. N. STING manifests self DNA-dependent inflammatory disease. Proc. Natl Acad. Sci. USA 109, 19386–19391 (2012)

    Article  ADS  CAS  Google Scholar 

  4. Ishikawa, H. & Barber, G. N. STING is an endoplasmic reticulum adaptor that facilitates innate immune signalling. Nature 455, 674–678 (2008)

    Article  ADS  CAS  Google Scholar 

  5. Zhong, B. et al. The adaptor protein MITA links virus-sensing receptors to IRF3 transcription factor activation. Immunity 29, 538–550 (2008)

    Article  CAS  Google Scholar 

  6. Jin, L. et al. MPYS, a novel membrane tetraspanner, is associated with major histocompatibility complex class II and mediates transduction of apoptotic signals. Mol. Cell. Biol. 28, 5014–5026 (2008)

    Article  CAS  Google Scholar 

  7. Sun, W. et al. ERIS, an endoplasmic reticulum IFN stimulator, activates innate immune signaling through dimerization. Proc. Natl Acad. Sci. USA 106, 8653–8658 (2009)

    Article  ADS  CAS  Google Scholar 

  8. Ishikawa, H., Ma, Z. & Barber, G. N. STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature 461, 788–792 (2009)

    Article  ADS  CAS  Google Scholar 

  9. Burdette, D. L. et al. STING is a direct innate immune sensor of cyclic di-GMP. Nature 478, 515–518 (2011)

    Article  ADS  CAS  Google Scholar 

  10. Huang, Y. H., Liu, X. Y., Du, X. X., Jiang, Z. F. & Su, X. D. The structural basis for the sensing and binding of cyclic di-GMP by STING. Nature Struct. Mol. Biol. 19, 728–730 (2012)

    Article  CAS  Google Scholar 

  11. Ouyang, S. et al. Structural analysis of the STING adaptor protein reveals a hydrophobic dimer interface and mode of cyclic di-GMP binding. Immunity 36, 1073–1086 (2012)

    Article  CAS  Google Scholar 

  12. Shang, G. et al. Crystal structures of STING protein reveal basis for recognition of cyclic di-GMP. Nature Struct. Mol. Biol. 19, 725–727 (2012)

    Article  CAS  Google Scholar 

  13. Shu, C., Yi, G., Watts, T., Kao, C. C. & Li, P. Structure of STING bound to cyclic di-GMP reveals the mechanism of cyclic dinucleotide recognition by the immune system. Nature Struct. Mol. Biol. 19, 722–724 (2012)

    Article  CAS  Google Scholar 

  14. Wu, J. et al. Cyclic GMP-AMP is an endogenous second messenger in innate immune signaling by cytosolic DNA. Science 339, 826–830 (2013)

    Article  ADS  CAS  Google Scholar 

  15. Sun, L., Wu, J., Du, F., Chen, X. & Chen, Z. J. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339, 786–791 (2013)

    Article  ADS  CAS  Google Scholar 

  16. Cavlar, T., Deimling, T., Ablasser, A., Hopfner, K. P. & Hornung, V. Species-specific detection of the antiviral small-molecule compound CMA by STING. EMBO J. 32, 1440–1450 (2013)

    Article  CAS  Google Scholar 

  17. Rao, F. et al. Enzymatic synthesis of c-di-GMP using a thermophilic diguanylate cyclase. Anal. Biochem. 389, 138–142 (2009)

    Article  CAS  Google Scholar 

  18. Davies, B. W., Bogard, R. W., Young, T. S. & Mekalanos, J. J. Coordinated regulation of accessory genetic elements produces cyclic di-nucleotides for V. cholerae virulence. Cell 149, 358–370 (2012)

    Article  CAS  Google Scholar 

  19. Conlon, J. et al. Mouse, but not human STING, binds and signals in response to the vascular disrupting agent 5,6-dimethylxanthenone-4-acetic acid. J. Immunol. 190, 5216–5225 (2013)

    Article  CAS  Google Scholar 

  20. Kristiansen, H., Gad, H. H., Eskildsen-Larsen, S., Despres, P. & Hartmann, R. The oligoadenylate synthetase family: an ancient protein family with multiple antiviral activities. J. Interferon Cytokine Res. 31, 41–47 (2011)

    Article  CAS  Google Scholar 

  21. Gao, P. et al. Cyclic [G(2′,5′)pA(3′,5′)p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase. Cell 153, 1094–1107 (2013)

    Article  CAS  Google Scholar 

  22. Diner, E. J. The innate immune DNA sensory cGAS produces a noncanonical cyclic dinucleotide that activates human STING. Cell Rep. 3, 1355–1361 (2013)

    Article  CAS  Google Scholar 

  23. Mizushima, S. & Nagata, S. pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 18, 5322 (1990)

    Article  CAS  Google Scholar 

  24. Gaffney, B. L., Veliath, E., Zhao, J. & Jones, R. A. One-flask syntheses of c-di-GMP and the [Rp,Rp] and [Rp,Sp] thiophosphate analogues. Org. Lett. 12, 3269–3271 (2010)

    Article  CAS  Google Scholar 

Download references


We thank M. Pelegrin for providing us with LL171 cells. K.-P.H. is supported by the National Institutes of Health (U19AI083025), the European Research Council Advanced Grant 322869, and the Center for Integrated Protein Science Munich (CIPSM). A.A. and V.H. are members of the excellence cluster ImmunoSensation. V.H. is supported by grants from the German Research Foundation (SFB670) and the European Research Council (ERC 243046).

Author information

Authors and Affiliations



A.A., M.G., T.C., G.W., T.D., I.R., J.L., K.-P.H. and V.H. designed experiments and analysed the data. A.A., M.G., T.C., G.W., T.D. and I.R. performed experiments. A.A. and V.H. wrote the manuscript. V.H. supervised the project.

Corresponding authors

Correspondence to Andrea Ablasser or Veit Hornung.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Figures 1–15, Supplementary Notes 1–3 and additional references. (PDF 2047 kb)

PowerPoint slides

Rights and permissions

Reprints and permissions

About this article

Cite this article

Ablasser, A., Goldeck, M., Cavlar, T. et al. cGAS produces a 2′-5′-linked cyclic dinucleotide second messenger that activates STING. Nature 498, 380–384 (2013).

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI:

This article is cited by


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing