Published: Vol 8, Iss 20, Oct 20, 2018 DOI: 10.21769/BioProtoc.3055 Views: 5581
Reviewed by: Andrea PuharThomas Alexander PackardSaskia F. Erttmann
Protocol Collections
Comprehensive collections of detailed, peer-reviewed protocols focusing on specific topics
Related protocols
Measurement of the Activity of Wildtype and Disease-Causing ALPK1 Mutants in Transfected Cells With a 96-Well Format NF-κB/AP-1 Reporter Assay
Tom Snelling
Nov 20, 2024 829 Views
Quantitative Measurement of the Kinase Activity of Wildtype ALPK1 and Disease-Causing ALPK1 Mutants Using Cell-Free Radiometric Phosphorylation Assays
Tom Snelling
Nov 20, 2024 780 Views
Fluorescence Polarization-Based High-Throughput Screening Assay for Inhibitors Targeting Cathepsin L
Keyu Guo [...] Shuyi Si
Jul 20, 2025 898 Views
Abstract
Cyclic GMP-AMP synthase (cGAS) is a pattern recognition receptor (PRR) that senses double stranded DNA (dsDNA) in the cytosol and this leads to the activation of stimulator of interferon genes (STING) via the secondary messenger 2’3’-cyclic GMP-AMP (2’3’-cGAMP). STING then recruits TANK binding kinase 1 (TBK-1) and this complex can phosphorylate and activate interferon regulatory factor 3 (IRF3) leading to the induction of type I interferons and other antiviral genes. The cGAS:DNA complex catalyzes the synthesis of 2’3’-cGAMP and the purpose of the protocol presented here is to measure the in vitro activity of purified cGAS in the presence of dsDNA. The protocol was developed to elucidate the relationship between dsDNA length and the level of cGAS activity. The method involves an in vitro reaction with low concentrations of cGAS and dsDNA followed by quantification of the reaction product using anion exchange chromatography. The low concentrations of cGAS and dsDNA and the high sensitivity of this assay is a key advantage when comparing different DNA fragments’ ability to activate cGAS.
Keywords: Cyclic GMP-AMP synthaseBackground
The presence of double stranded DNA within the cytosol of a cell is a potential sign of infection by a DNA or retrovirus. The nucleotidyl transferase cGAS functions as a pattern recognition receptor that senses cytosolic dsDNA. cGAS is allosterically activated by dsDNA and catalyzes the conversion of ATP and GTP into the cyclic dinucleotide 2’3’-cGAMP (or simply cGAMP) (Ablasser et al., 2013; Civril et al., 2013; Diner et al., 2013; Gao et al., 2013; Kranzusch et al., 2013; Sun et al., 2013), which subsequently acts as a secondary messenger that induces an antiviral program in the infected cell. The active site of cGAS contains three acidic residues coordinating two magnesium ions. The role of these ions is to coordinate the triphosphate group of the donor nucleotide and the attacking hydroxyl group of the acceptor nucleotide. cGAS catalyzes the formation of cGAMP in two sequential steps. First, the triphosphate group of ATP is coordinated by the magnesium ions and the 2’-hydroxyl group of GTP makes a nucleophilic attack on the α-phosphate of ATP, which releases the β- and γ-phosphate as pyrophosphate. This leads to the formation of a noncanonical 2’,5’-phosphodiester linkage. The intermediate is then flipped around in the active site and now the triphosphate group of GTP is coordinated by the magnesium ions. This time the 3’-hydroxyl group of the AMP moiety makes the nucleophilic attack on the α-phosphate of GTP forming a 3’,5’-phosphodiester linkage (Civril et al., 2013; Gao et al., 2013; Hornung et al., 2014). Thus, the final product contains both a canonical and noncanonical phosphodiester linkage.
Not all dsDNA is equally efficient at activating cGAS. The minimum DNA length reported to activate cGAS in cells is 12 bp with guanosine overhangs (Herzner et al., 2015). However, the DNA’s ability to activate cGAS is strongly related to the length of the DNA. Increasing the DNA length leads to an increase in its ability to activate cGAS (Andreeva et al., 2017; Luecke et al., 2017). This effect is observed even when increasing the DNA length from 2 kb to 4 kb (Luecke et al., 2017). Furthermore, certain Y-form DNA generated during the reverse transcription of the HIV-1 genome is more potent at activating cGAS compared to conventional dsDNA of similar length (Herzner et al., 2015).
cGAMP acts as a secondary messenger that binds to the adaptor protein STING, and this leads to the induction of antiviral genes (Ablasser et al., 2013; Diner et al., 2013; Li et al., 2013; Sun et al., 2013; Zhang et al., 2013). STING is a transmembrane protein located in the endoplasmic reticulum (ER) membrane with a large C-terminal domain protruding into the cytosol (Ishikawa and Barber, 2008). When STING binds cGAMP, the complex moves to the Golgi apparatus and from there it moves to punctuated foci in the cytoplasm (Saitoh et al., 2009). The STING:cGAMP complex recruits TBK-1, and this leads to the phosphorylation of both STING and TBK-1. This phosphorylated complex can then phosphorylate and thereby activate IRF3, which then translocates to the nucleus where it induces the transcription of antiviral genes including type I interferons (Ishikawa et al., 2009; Tanaka and Chen, 2012). The STING:cGAMP complex will also activate nuclear factor kappa B (NFκB) transcription factors (Abe and Barber, 2014).
The method described in this protocol was used to show that the in vitro activation of recombinant human cGAS truncated to amino acids 155-522 (cGAS [155-522]) is dependent on DNA length. The tested interval of DNA lengths varied from 100 bp to 4,000 bp (Luecke et al., 2017). This method offers an alternative to thin layer chromatography (TLC)-based assays with radiolabeled ATP. Due to poor sensitivity, TLC-based assays normally use concentrations of both dsDNA and cGAS well above physiologically realistic concentrations. The advantage of using the protocol presented here is that no radioactivity or labeling of the substrates are needed and that the high sensitivity of this method makes it possible to use very low concentrations of both cGAS and dsDNA. In this protocol, the concentration of cGAS is ten-fold lower compared to classical TLC assays and we have avoided oversaturating the reaction with DNA. We use 1 ng/μl of dsDNA corresponding to 1.646 x 10-6 M bp. Assuming that one cGAS molecule covers approx. 20 bp (Andreeva et al., 2017), then 1.646 μM bp corresponds to 82.32 nM cGAS binding sites. Under this assumption, there is enough DNA to occupy about 82% of the cGAS used in this protocol. The use of low and approx. equimolar concentrations of cGAS and DNA (measured in cGAS binding sites) is important if you test DNA with small differences in affinity for cGAS. The impact of different affinities might be diminished if for example the DNA concentration is substantial above the saturation point.
This protocol allows for easy and robust quantifications of the cGAS product and compare reaction conditions (such as different buffers, DNA structures, DNA lengths, and cGAS preparations) but it is more time consuming than TLC when running multiple samples. The method described in this protocol was developed from a method designed to measure the activity of the oligoadenylate synthetase (OAS) proteins (Turpaev et al., 1997).
Materials and Reagents
Equipment
Software
Procedure
Note: Gloves should be worn during all steps of this protocol to protect your samples from phosphatase contamination.
Data analysis
Open the Unicorn 5 evaluation window (If you are using Unicorn 7, use the Evaluation Classic application) and open the data you wish to analyze (data from each anion exchange chromatography run can be found in the Result Navigator in the left side of the evaluation window). When the data is open click “Integrate” and choose “Peak Integrate” from the drop down menu.
A new window opens. In this window, there will be two lists on white background. In the left list choose the 254 nm UV for integration (if the program is as described in supplementary Figure 1 the 254 nm UV is the second element from the top and when the list element is highlighted in blue it is chosen). The baseline should be set to “Calculate Baseline” (default). Click “OK” and a peak table appears below the curves. The identified peaks are listed according to retention volume and you can read the area under the curve (AUC) for the cGAMP peak and for any other peak in the chromatogram. Make sure that the calculated baseline looks correct. If the curve has abrupt and discontinuous changes around the peaks due to air in the system or other artifacts, it can give an unreliable baseline and unreliable results. If the curve is discontinuous, it might be necessary to repeat the experiment.
The AUC has the unit mAU•ml and for the cGAMP peak the AUC is a measurement for the amount of cGAMP eluting from ion exchange column. The amount of cGAMP eluting from the column is dependent on the amount of cGAMP produced in a reaction. For this reason, it is possible to use the AUC of the cGAMP peak to represent the activity of cGAS. The AUC can for example be presented in a column bar graph or a column scatter plot.
Note: It is possible to convert the quantification of cGAMP from mAU•ml to nmol. This requires that you make a cGAMP standard curve by running different concentrations of cGAMP on the anion exchange column.
ATP gives a peak at a conductivity of approx. 17.1 mS/cm. There might also be a small ADP peak at a conductivity of approx. 13.6 mS/cm. GTP gives a peak at a conductivity of approx. 18.4 mS/cm. There might also be a small GDP peak at a conductivity of approx. 14.9 mS/cm. 2’3’-cGAMP gives a peak at a conductivity of approx. 9.9 mS/cm.
There can be small variations between runs and between ÄKTApurifier systems. For representative data, see Figure 1 and Luecke et al. (2017).
Figure 1. Examples of chromatograms. A. Chromatograms of 2’3’-cGAMP, ATP, and GTP. B and C. Chromatograms of two different reactions. B) cGAS with a 4 kb PCR fragment. C) cGAS without DNA. The data has previously been published in Luecke et al. (2017).
Notes
The NTP’s are very sensitive to dephosphorylation. It is therefore very important to protect the samples from phosphatases from the environment. That is why gloves should be worn when working with or handling the samples and reagents. The 1.5 ml tubes should be autoclaved and in general care should be taken not to contaminate the samples with phosphatases.
Other reaction conditions suitable for cGAS can also be used in this assay. Avoid chelating agents such as EDTA in the buffers as they interfere with the anion exchange column. In our experience, high salt concentrations can also inhibit the reaction. If you increase the amount of cGAS and DNA, be careful that you do not experience substrate depletion. We recommend that a minimum of 60% of the substrate is remaining after terminating the reaction
Recipes
Acknowledgments
We thank Professor Søren R. Paludan and Stefanie Luecke, Ph.D. for helping with the development of the method described in this protocol. We thank Hans Henrik Gad, Ph.D. for comments on the manuscript.
This work was funded by The Novo Nordisk Foundation (NNF17OC0028184) and the Danish Council for Independent Research, Natural Science (4181-00012B).
Competing interests
The authors declare no conflicts of interest or competing interests.
References
Article Information
Copyright
© 2018 The Authors; exclusive licensee Bio-protocol LLC.
How to cite
Holleufer, A. and Hartmann, R. (2018). A Highly Sensitive Anion Exchange Chromatography Method for Measuring cGAS Activity in vitro. Bio-protocol 8(20): e3055. DOI: 10.21769/BioProtoc.3055.
Category
Immunology > Host defense > General
Biochemistry > Other compound > cGAMP
Biochemistry > Protein > Activity
Do you have any questions about this protocol?
Post your question to gather feedback from the community. We will also invite the authors of this article to respond.
Tips for asking effective questions
+ Description
Write a detailed description. Include all information that will help others answer your question including experimental processes, conditions, and relevant images.
Share
Bluesky
X
Copy link