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Oct 2017

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A Highly Sensitive Anion Exchange Chromatography Method for Measuring cGAS Activity in vitro
一种体外检测cGAS活性的高效阴离子交换色谱法   

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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 synthase (环状GMP-AMP合成酶), cGAS (cGAS), 2'3'-Cyclic GMP-AMP (2'3'-Cyclic GMP-AMP), cGAMP (cGAMP), Nucleotidyl transferase (核苷酸转移酶), Label-free enzyme assay (无标记酶测定)

Background

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

  1. 1 ml single-use syringes (CHIRANA T. Injecta, catalog number: CH03001L )
  2. 100 ml and 500 ml GL45 thread reagent bottles including screw caps (SIMAX, catalog numbers: 1632414321100 and 1632414321500 )
  3. 50 ml tubes (SARSTEDT, catalog number: 62.547.254 )
  4. Autoclaved 1.5 ml tubes (BRAND, catalog number: 780500 )
  5. Cellulose acetate filter membranes 0.22 μm pore size (Frisenette, catalog number: CA047022 )
  6. Disposable nitrile gloves
  7. PCR tubes (VWR, catalog number: 211-0338 )
  8. Pipette tips with barrier (Thermo Fisher Scientific, ARTTM)
  9. Serological pipettes 10 ml (Th. Geyer, Labsolute, catalog number: 7695553 )
  10. dsDNA diluted to a concentration of 5 ng/μl in water or buffer NE (NucleoSpin® Gel and PCR Clean-up) (MACHEREY-NAGEL, catalog number: 740609 )
    Note: If agarose gel purification of the DNA is desired, use NucleoSpin® Gel and PCR Clean-up for extraction of the DNA (MACHEREY-NAGEL, catalog number: 740609 ).
  11. Ice
  12. 100 mM ATP (Thermo Fisher Scientific, catalog number: R0441 )
  13. 100 mM GTP (Thermo Fisher Scientific, catalog number: R0461 )
  14. Concentrated hydrochloric acid (Sigma-Aldrich, catalog number: 30721-1L )
  15. Magnesium chloride hexahydrate (Sigma-Aldrich, catalog number: M2670-1KG )
  16. Sodium hydroxide (VWR, catalog number: 28240.292 )
  17. Sodium chloride (VWR, catalog number: 27810.295 )
  18. Tris (VWR, catalog number: 103156X )
  19. Ultrapure water 18.2 MΩ obtained from PURELAB Chorus 1 (Elga Veolia)
  20. Zinc chloride (VWR, catalog number: 29156.231 )
  21. Glycerol (VWR, catalog number: 24388.295 )
  22. HEPES (VWR, catalog number: 30487.297 )
  23. 2 μM purified cGAS [155-522] stock (see Recipes) 
  24. MgCl2 (200 mM) (see Recipes)
  25. ZnCl2 (10 mM) (see Recipes)
  26. NaOH (5 mM) (see Recipes)
  27. Tris (pH 7.5, 1 M) (see Recipes)
  28. 5x reaction buffer (see Recipes)
  29. Buffer A (see Recipes)
  30. Buffer B (see Recipes)
  31. ATP (10 mM) (see Recipes)
  32. GTP (10 mM) (see Recipes)

Equipment

  1. 2 ml sample loop for ÄKTApurifier 10 (GE Healthcare, catalog number: 18111402 )
  2. ÄKTApurifier 10 (GE Healthcare)
  3. Aluminum cooling block for PCR tubes (e.g., Sigma-Aldrich, catalog number: Z740270-1EA )
  4. -80 °C freezer
  5. Vacuum pump 
  6. Centrifuge for 1.5 ml tubes (Eppendorf, model: MiniSpin® , catalog number: 5452000018)
  7. Injection needle for ÄKTApurifier 10 (GE Healthcare, catalog number: 18180142 )
  8. Laboratory balance with a readability of 0.001 g
  9. Microcentrifuge for PCR tubes (SpectrafugeTM Mini) (Sigma-Aldrich, Labnet International, catalog number: S7816EU-1EA )
  10. pH electrode (VWR, catalog number: 662-1157 )
  11. pH meter (VWR, catalog number: 662-1421 )
  12. Pipetboy
  13. Pipettes (Finnpipette, Thermo Fisher Scientific)
  14. RESOURCE Q 1 ml (GE Healthcare, catalog number: 17117701 )
  15. Thermal Cycler PCR machine (Bio-Rad Laboratories, model: T100TM )
  16. Vacuum filter funnel for GL45 threaded reagent bottles and 47 mm filter membrane diameter, e.g., NalgeneTM Polysulfone Reusable Bottle Top Filter (Thermo Fisher Scientific, catalog number: DS0320-5045 )

Software

  1. Unicorn 5.11 AA or 7 (GE Healthcare, catalog numbers: 28400955 or 29203853)

Procedure

Note: Gloves should be worn during all steps of this protocol to protect your samples from phosphatase contamination.


  1. Preparing control samples for assessing quality and elution profile of ATP, GTP and cGAMP
    Note: Keep everything on ice for this step.
    1. Mix the ATP sample, GTP sample, and cGAMP sample as described below in 1.5 ml tubes.

    2. The samples can be stored at -80 °C and thawed immediately before centrifugation and subsequent analysis on the RESOURCE Q 1 ml column (see Procedure D).

  2. Dilution of DNA fragments and preparation of enzyme master mix
    Notes: 
    1. Procedures B and C in this protocol are performed without pausing. Place the aluminum cooling block on ice for 30 min before starting Procedure B and keep it on ice throughout Procedures B and C.
    2. Keep everything on ice during this step, mix the enzyme master mix on ice, and keep the enzyme master mix on ice.

    1. Dilute the DNA fragments you are testing to a concentration of 5 ng/μl in ultrapure water or buffer NE.
    2. Mix enzyme master mix for the desired number of reactions according to the table below.

      Notes: 
      1. Enzyme master mix can be prepared for any number of reactions simply by upscaling the recipe. It is highly recommended that you make a surplus of the enzyme master mix. For example, if you want to make four reactions you should multiply the recipe for one reaction by five.
      2. The number of reactions that you will make is the number of DNA species you want to test plus a negative control with no DNA.
    3. Mix the master mix well by pipetting carefully up and down several times using a pipette with a suitable volume. Do not introduce air bubbles or make it foam!

  3. In vitro cGAS reaction
    1. Immediately after preparing the enzyme master mix, place one PCR tube per reaction into the aluminum cooling block (remember a tube for the DNA-free negative control).
    2. Pipette 160 μl enzyme master mix into each PCR tube.
    3. Add 40 μl of a given DNA species/fragment (5 ng/μl) into a corresponding PCR tube and pipette up and down ten times to ensure mixing (avoid air bubbles).
    4. For the negative control, add 40 μl of ultrapure water or buffer NE depending on what the DNA fragments are suspended in.
      Note: Addition of the DNA to the PCR tubes should be done swiftly but without forming foam in the samples. The cGAS catalyzed reaction is not occurring to any noticeable extent while the samples are kept on ice.
    5. Centrifuge the PCR tubes for 30-60 s on the small SpectrafugeTM to remove any drops sitting on the side of the tube and to remove any air bubbles.
    6. Place the PCR tubes back in the cooling block.
    7. The T100TM Thermal Cycler PCR machine is programmed to 2 h at 37 °C, 10 min at 95 °C, and 12 °C for indefinite. The lid heating is set to 105 °C and the sample volume is set to maximum (100 μl).
    8. Transfer the PCR tubes to the PCR machine and start the program.
    9. When the 2 h at 37 °C and 10 min at 95 °C has passed, move the PCR tubes to the -80 °C freezer, where they are stored until the analysis (Procedure D).

  4. Analyzing samples on RESOURCE Q 1 ml
    Note: The reactions are analyzed one at a time. The ATP, GTP, and cGAMP samples prepared in Procedure A are analyzed by continuing from Step D3.
    1. Thaw a single PCR tube and transfer all 200 μl to a 1.5 ml tube.
    2. Add 800 μl buffer A to the 1.5 ml tube and mix well.
    3. Centrifuge the now diluted reaction sample at 12,100 x g for 15 min at 4 °C.
    4. Prepare the ÄKTApurifier 10 and RESOURCE Q 1 ml column.
      1. Wash pump A in buffer A and pump B in buffer B.
      2. Wash the entire flow path including the 2 ml sample loop in buffer A with a flow of 1 ml/min until the conductivity is stable and below 2.5 mS/cm.
      3. Connect the RESOURCE Q 1 ml column to the system and equilibrate the column in buffer A.
    5. Fix the injection needle to a 1 ml syringe and draw 900 μl of the diluted reaction sample into the syringe without disturbing the pellet that might have appeared after centrifugation.
    6. Inject 800 μl onto the sample loop.
    7. Use the Unicorn software to program the ÄKTApurifier 10 to do the following (see Table S1 for full variable list):
      Program:
      Wave length 1 = 280 nm
      Wave length 2 = 254 nm
      Wave length 3 = 215 nm (optional)
      Flow rate: 0.5 ml/min
      Equilibrate with 2 column volumes buffer A
      Empty loop with 10 ml
      Linear gradient from 0% to 50% buffer B over 25 column volumes
      Wash column with 100% buffer B over 5 column volumes
      Re-equilibrate with 5 column volumes buffer A
      No fractionation
      Note: The maximal pressure for the column is 1.5 MPa but the pressure generated by the column during this application is usually less than 0.5 MPa. We run the chromatography at 4 °C but it will also work at room temperature.
    8. When the run is finished the next reaction sample can be analyzed by repeating Procedure D.

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

  1. 2 μM purified cGAS [155-522] stock
    2 μM purified human cGAS truncated to amino acids 155-522 suspended in 70 mM NaCl, 10% (v/v) glycerol, 20 mM HEPES, pH 7.5
    Note: For cGAS purification see Luecke et al. (2017). E. coli was transformed with a pET-21a plasmid encoding cGAS [155-522] with an N-terminal Hexa His-MBP tag and a Tobacco Etch Virus (TEV) protease cleavage site. The fusion protein was expressed by the bacterial cells and the MBP tag was cleaved off during purification leaving four residues Gly-Ala-Met-Gly in front of Arg155. Dialysis can be used for changing the buffer of cGAS to 70 mM NaCl, 10% (v/v) glycerol, 20 mM HEPES, pH 7.5 before adjusting the concentration to 2 μM.
  2. MgCl2 (200 mM)
    Dissolve 2.03 g MgCl2•6H2O in 50 ml ultrapure water
  3. ZnCl2 (10 mM)
    Dissolve 0.682 g ZnCl2 in 500 ml ultrapure water
  4. NaOH (5 M)
    Dissolve 100 g NaOH in 500 ml ultrapure water
  5. Tris (pH 7.5 1 M)
    1. Dissolve 60.57 g Tris in 400 ml ultrapure water and adjust the pH to 7.5 at room temperature using concentrated HCl
    2. Adjust the volume to 500 ml using ultrapure water
  6. 5x reaction buffer
    1. Mix 1.46 g NaCl with 12.5 ml 200 mM MgCl2, 0.25 ml 10 mM ZnCl2, 10 ml 1 M Tris pH 7.5 
    2. Adjust the volume to 50 ml using ultrapure water
    3. Filtrate the 5x reaction buffer with a 0.22 μm cellulose acetate filter
    4. Store at -20 °C
    The final composition of the 5x reaction buffer are 500 mM NaCl, 200 mM Tris, 50 mM MgCl2, and 50 μM ZnCl2, pH 7.5
  7. Buffer A
    1. Mix 10 ml 1 M Tris pH 7.5 with 400 ml ultrapure water
    2. Adjust the pH to 7.5 at room temperature using 5 M NaOH
    3. Adjust the volume to 500 ml using ultrapure water
    4. Filtrate the buffer with a 0.22 μm cellulose acetate filter
    5. Prepare the buffer fresh
    The final composition of buffer A is 20 mM Tris pH 7.5
  8. Buffer B
    1. Mix 22 g NaCl and 10 ml 1 M Tris pH 7.5 with 400 ml ultrapure water
    2. Adjust the pH to 7.5 at room temperature using 5 M NaOH
    3. Adjust the volume to 500 ml using ultrapure water
    4. Filtrate the buffer with a 0.22 μm cellulose acetate filter
    5. Prepare the buffer fresh
    The final composition of buffer B is 750 mM NaCl, 20 mM Tris pH 7.5
  9. ATP (10 mM)
    Mix 100 μl 100 mM ATP with 900 μl ultrapure water
    Make aliquots of a size that suits your requirement and store at -80 °C
    Note: Be careful not to contaminate with phosphatases from your hands or the surroundings.
  10. GTP (10 mM)
    Mix 100 μl 100 mM GTP with 900 μl ultrapure water
    Make aliquots of a size that suits your requirement and store at -80 °C
    Note: Be careful not to contaminate with phosphatases from your hands or the surroundings.

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

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简介

环状GMP-AMP合酶(cGAS)是一种模式识别受体(PRR),可以感知胞质溶胶中的双链DNA(dsDNA),并通过第二信使2'3'来激活干扰素基因刺激物(STING)。环GMP-AMP(2'3'-cGAMP)。然后STING募集TANK结合激酶1(TBK-1),该复合物可磷酸化并激活干扰素调节因子3(IRF3),导致I型干扰素和其他抗病毒基因的诱导。 cGAS:DNA复合物催化2'3'-cGAMP的合成,这里提出的方案的目的是测量在dsDNA存在下纯化的cGAS的体外>活性。开发该方案是为了阐明dsDNA长度与cGAS活性水平之间的关系。该方法涉及与低浓度cGAS和dsDNA的体外>反应,然后使用阴离子交换色谱法定量反应产物。当比较不同DNA片段激活cGAS的能力时,低浓度的cGAS和dsDNA以及该测定的高灵敏度是关键优势。

【背景】细胞胞质内存在双链DNA是DNA或逆转录病毒感染的潜在迹象。核苷酸转移酶cGAS作为感知细胞溶质dsDNA的模式识别受体起作用。 cGAS被dsDNA变构激活并催化ATP和GTP转化为环状二核苷酸2'3'-cGAMP(或简称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),后来作为第二信使,在感染细胞中诱导抗病毒程序。 cGAS的活性位点含有三个酸性残基,配位两个镁离子。这些离子的作用是协调供体核苷酸的三磷酸基团和受体核苷酸的攻击羟基。 cGAS在两个连续步骤中催化cGAMP的形成。首先,ATP的三磷酸基团由镁离子配位,GTP的2'-羟基对ATP的α-磷酸进行亲核攻击,其释放β-和γ-磷酸作为焦磷酸盐。这导致形成非经典的2',5'-磷酸二酯键。然后在活性位点翻转中间体,现在GTP的三磷酸酯基团被镁离子配位。这次AMP部分的3'-羟基对GTP的α-磷酸进行亲核攻击,形成3',5'-磷酸二酯键(Civril et al。>,2013; Gao et al。>,2013; Hornung et al。>,2014)。因此,最终产物含有经典和非经典的磷酸二酯键。

并非所有dsDNA在激活cGAS方面都同样有效。报道用于激活细胞中cGAS的最小DNA长度为12bp,具有鸟苷突出端(Herzner et al。>,2015)。然而,DNA激活cGAS的能力与DNA的长度密切相关。增加DNA长度会增加其激活cGAS的能力(Andreeva et al。>,2017; Luecke et al。>,2017)。即使将DNA长度从2kb增加到4kb,也观察到这种效应(Luecke 等,>,2017)。此外,与相似长度的常规dsDNA相比,在HIV-1基因组逆转录期间产生的某些Y型DNA在激活cGAS方面更有效(Herzner et al。>,2015)。cGAMP作为第二信使,与衔接蛋白STING结合,导致抗病毒基因的诱导(Ablasser et al。>,2013; Diner et al。>, 2013; Li et al。>,2013; Sun et al。>,2013; Zhang et al。>,2013)。 STING是位于内质网(ER)膜中的跨膜蛋白,具有突出到胞质溶胶中的大C末端结构域(Ishikawa和Barber,2008)。当STING结合cGAMP时,复合物移动到高尔基体,并从那里移动到细胞质中的间断焦点(Saitoh et al。>,2009)。 STING:cGAMP复合物募集TBK-1,这导致STING和TBK-1的磷酸化。然后,这种磷酸化的复合物可以磷酸化,从而激活IRF3,然后IRF3转移到细胞核,在那里它诱导包括I型干扰素在内的抗病毒基因的转录(Ishikawa et al。>,2009; Tanaka和Chen,2012) 。 STING:cGAMP复合物也将激活核因子κB(NFκB)转录因子(Abe和Barber,2014)。

该方案中描述的方法用于显示截短至氨基酸155-522(cGAS [155-522])的重组人cGAS的体外>活化依赖于DNA长度。测试的DNA长度间隔从100bp到4,000bp不等(Luecke et al。>,2017)。该方法提供了基于薄层色谱(TLC)的放射性标记ATP测定的替代方法。由于灵敏度较差,基于TLC的检测通常使用远低于生理学实际浓度的dsDNA和cGAS浓度。使用本文提出的方案的优点是不需要放射性或基质标记,并且该方法的高灵敏度使得可以使用非常低浓度的cGAS和dsDNA。在该方案中,与经典TLC测定相比,cGAS的浓度低十倍,并且我们避免了与DNA的反应过饱和。我们使用1ng /μl对应于1.646×10 -6 M bp的dsDNA。假设一个cGAS分子覆盖约。 20bp(Andreeva et al。>,2017),然后1.646μMbp对应于82.32nM cGAS结合位点。在这种假设下,有足够的DNA占据该方案中使用的约82%的cGAS。使用低和约。如果您测试对cGAS的亲和力差异很小的DNA,则等摩尔浓度的cGAS和DNA(在cGAS结合位点测量)很重要。如果例如DNA浓度大大高于饱和点,则可能减少不同亲和力的影响。

该协议允许简单和稳健地定量cGAS产品并比较反应条件(例如不同的缓冲液,DNA结构,DNA长度和cGAS制剂),但是当运行多个样品时它比TLC更耗时。该方案中描述的方法是从设计用于测量寡腺苷酸合成酶(OAS)蛋白质活性的方法开发的(Turpaev 等人,>,1997)。

关键字:环状GMP-AMP合成酶, cGAS, 2'3'-Cyclic GMP-AMP, cGAMP, 核苷酸转移酶, 无标记酶测定

材料和试剂

  1. 1毫升一次性注射器(CHIRANA T. Injecta,目录号:CH03001L)
  2. 100毫升和500毫升GL45螺纹试剂瓶,包括螺帽(SIMAX,目录号:1632414321100和1632414321500)
  3. 50毫升管(SARSTEDT,目录号:62.547.254)
  4. 高压灭菌的1.5毫升管(BRAND,目录号:780500)
  5. 醋酸纤维素滤膜0.22μm孔径(Frisenette,目录号:CA047022)
  6. 一次性丁腈手套
  7. PCR管(VWR,目录号:211-0338)
  8. 带屏障的移液器吸头(Thermo Fisher Scientific,ART TM )
  9. 血清移液器10 ml(Th.Geyer,Labsolute,目录号:7695553)
  10. dsDNA在水或缓冲液NE中稀释至浓度为5 ng /μl(NucleoSpin ®凝胶和PCR清理)(MACHEREY-NAGEL,目录号:740609)
    注意:如果需要对DNA进行琼脂糖凝胶纯化,请使用NucleoSpin > ®> 凝胶和PCR纯化来提取DNA (MACHEREY-NAGEL,目录号:740609)。>
  11. 100 mM ATP(赛默飞世尔科技,目录号:R0441)
  12. 100 mM GTP(赛默飞世尔科技,目录号:R0461)
  13. 浓盐酸(Sigma-Aldrich,目录号:30721-1L)
  14. 氯化镁六水合物(Sigma-Aldrich,目录号:M2670-1KG)
  15. 氢氧化钠(VWR,目录号:28240.292)
  16. 氯化钠(VWR,目录号:27810.295)
  17. Tris(VWR,目录号:103156X)
  18. 从PURELAB Chorus 1(Elga Veolia)获得的超纯水18.2MΩ
  19. 氯化锌(VWR,目录号:29156.231)
  20. 甘油(VWR,目录号:24388.295)
  21. HEPES(VWR,目录号:30487.297)
  22. 2μM纯化的cGAS [155-522]库存(参见食谱) 
  23. MgCl 2 (200 mM)(见食谱)
  24. ZnCl 2 (10 mM)(见食谱)
  25. NaOH(5 mM)(见食谱)
  26. Tris(pH 7.5,1 M)(见食谱)
  27. 5x反应缓冲液(见食谱)
  28. 缓冲液A(见食谱)
  29. 缓冲液B(见食谱)
  30. ATP(10 mM)(见食谱)
  31. GTP(10 mM)(见食谱)

设备

  1. 用于ÄKTApurifier10的2 ml样品环(GE Healthcare,目录号:18111402)
  2. ÄKTApurifier10(GE Healthcare)
  3. 用于PCR管的铝冷却块(例如>,Sigma-Aldrich,目录号:Z740270-1EA)
  4. -80°C冰箱
  5. 真空泵 
  6. 离心1.5 ml管(Eppendorf,型号:MiniSpin ®,目录号:5452000018)
  7. ÄKTApurifier10注射针(GE Healthcare,目录号:18180142)
  8. 实验室天平,可读性为0.001克
  9. 用于PCR管的微量离心机(Spectrafuge TM Mini)(Sigma-Aldrich,Labnet International,目录号:S7816EU-1EA)
  10. pH电极(VWR,目录号:662-1157)
  11. pH计(VWR,目录号:662-1421)
  12. Pipetboy
  13. 移液器(Finnpipette,Thermo Fisher Scientific)
  14. 资源Q 1 ml(GE Healthcare,目录号:17117701)
  15. 热循环PCR仪(Bio-Rad Laboratories,型号:T100 TM )
  16. 用于GL45螺纹试剂瓶和47 mm过滤膜直径的真空过滤漏斗,例如>,Nalgene TM 聚砜可重复使用瓶顶过滤器(Thermo Fisher Scientific,目录号:DS0320-5045)

软件

  1. Unicorn 5.11 AA或7(通用电气医疗集团,目录号:28400955或29203853)

程序

注意:在本协议的所有步骤中都应佩戴手套,以保护您的样品免受磷酸酶污染。>


  1. 准备对照样品以评估ATP,GTP和cGAMP的质量和洗脱曲线
    注意:将此步骤保存在冰上。>
    1. 如下所述将ATP样品,GTP样品和cGAMP样品混合在1.5 ml管中。

    2. 样品可以储存在-80°C并在离心之前立即解冻,然后在RESOURCE Q 1 ml色谱柱上进行分析(参见程序D)。

  2. 稀释DNA片段和制备酶主混合物
    注意:  >
    1. 此协议中的过程B和C在不暂停的情况下执行。将铝制冷却块置于冰上30分钟,然后启动程序B,并在整个程序B和C中将其保持在冰上。>
    2. 在此步骤中将所有物质保存在冰上,将酶混合物混合在冰上,并将酶混合物保持在冰上。>

    1. 将您正在测试的DNA片段稀释至浓度为5 ng /μl的超纯水或缓冲液NE中。
    2. 根据下表混合酶主混合物以达到所需数量的反应。

      注意:  >
      1. 只需通过升级配方,就可以为任何数量的反应准备酶主混合物。强烈建议您制备酶主混合物的剩余物。例如,如果你想进行四次反应,你应该将一个反应的配方乘以五。>
      2. 您将要做的反应数量是您要测试的DNA种类数量加上没有DNA的阴性对照。>
    3. 通过使用具有合适体积的移液管小心地上下移液几次,充分混合主混合物。 不要 引入气泡或泡沫!

  3. 体外> cGAS反应
    1. 在制备酶主混合物后,立即将每个反应放置一个PCR管进入铝冷却块(记住用于无DNA阴性对照的管)。
    2. 移取160μl酶主混合物到每个PCR管中。
    3. 将40μl给定的DNA种类/片段(5ng /μl)加入相应的PCR管中,并上下移液10次以确保混合(避免气泡)。
    4. 对于阴性对照,添加40μl超纯水或缓冲液NE,具体取决于DNA片段的悬浮状态。
      注意:应尽快将DNA添加到PCR管中,但样品中不会形成泡沫。当样品保存在冰上时,cGAS催化反应没有发生任何明显的程度。>
    5. 在小型Spectrafuge TM 上将PCR管离心30-60秒,以除去位于管侧面的任何液滴并除去任何气泡。
    6. 将PCR管放回冷却块中。
    7. 将T100 TM 热循环PCR机在37℃,10分钟,95℃和12℃下无限期编程为2小时。盖子加热设定为105℃,样品体积设定为最大值(100μl)。
    8. 将PCR管转移到PCR机器并启动程序。
    9. 当在37℃下2小时和在95℃下10分钟时,将PCR管移至-80℃冰箱,在那里将它们储存直至分析(程序D)。

  4. 分析RESOURCE Q 1 ml的样本
    注意:一次一个地分析反应。通过继续步骤D3分析在程序A中制备的ATP,GTP和cGAMP样品。>
    1. 解冻单个PCR管并将所有200μl转移至1.5ml管中。
    2. 将800μl缓冲液A加入1.5 ml管中并充分混合。
    3. 将现在稀释的反应样品在12,100 x g >下在4℃下离心15分钟。
    4. 准备ÄKTApurifier10和RESOURCE Q 1 ml色谱柱。
      1. 在缓冲液A中清洗泵A,在缓冲液B中清洗泵B.
      2. 清洗整个流路,包括缓冲液A中的2 ml样品环,流速为1 ml / min,直至电导率稳定且低于2.5 mS / cm。
      3. 将RESOURCE Q 1 ml色谱柱连接到系统并平衡缓冲液A中的色谱柱。
    5. 将注射针固定在1 ml注射器上,将900μl稀释的反应样品吸入注射器,不要打扰离心后可能出现的颗粒。
    6. 将800μl注入样品环。
    7. 使用Unicorn软件对ÄKTApurifier10进行编程以执行以下操作(请参阅表S1 为完整变量列表):
      计划:
      波长1 = 280nm
      波长2 = 254nm
      波长3 = 215 nm(可选)
      流速:0.5 ml / min
      用2个柱体积平衡缓冲液A
      空循环用10毫升
      25个柱体积的0%至50%缓冲液B的线性梯度
      用5%柱体积的100%缓冲液B洗涤柱
      用5个柱体积的缓冲液A重新平衡
      没有分馏
      注意:色谱柱的最大压力为1.5 MPa,但在此应用中色谱柱产生的压力通常小于0.5 MPa。我们在4°C下运行色谱,但它也可以在室温下运行。>
    8. 当运行结束时,可以通过重复程序D来分析下一个反应样品。

数据分析

打开Unicorn 5评估窗口(如果您使用的是Unicorn 7,请使用评估经典应用程序)并打开您想要分析的数据(每个阴离子交换层析运行的数据可以在评估左侧的结果导航器中找到窗口)。打开数据后,单击“积分”,然后从下拉菜单中选择“峰积分”。
 将打开一个新窗口。在此窗口中,白色背景上将有两个列表。在左侧列表中选择254 nm UV进行积分(如果程序如补充图1中所述,254 nm UV是顶部的第二个元素,当列表元素以蓝色突出显示时,选择它)。基线应设置为“计算基线”(默认)。单击“确定”,曲线下方将出现一个峰值表。根据保留体积列出鉴定的峰,您可以读取cGAMP峰的曲线下面积(AUC)和色谱图中的任何其他峰。确保计算的基线看起来正确。如果曲线由于系统中的空气或其他伪影而在峰值周围发生突然和不连续的变化,则会产生不可靠的基线和不可靠的结果。如果曲线不连续,可能需要重复实验。
  AUC具有单位mAU•ml,对于cGAMP峰,AUC是从离子交换柱洗脱的cGAMP量的测量值。从柱洗脱的cGAMP的量取决于反应中产生的cGAMP的量。因此,可以使用cGAMP峰的AUC来表示cGAS的活性。例如,AUC可以在列条形图或列散点图中呈现。
注意:可以将cGAMP的定量从mAU•ml转换为nmol。这要求您通过在阴离子交换柱上运行不同浓度的cGAMP来制作cGAMP标准曲线。>
  ATP在电导率约为1时出现峰值。 17.1 mS / cm。电导率约为50时,ADP峰值也可能较小。 13.6 mS / cm。 GTP在电导率约为1时出现峰值。 18.4 mS / cm。电导率约为10时,GDP可能也会出现小峰值。 14.9 mS / cm。 2'3'-cGAMP在电导率约为2时产生峰。 9.9 mS / cm。
在运行之间和ÄKTApurifier系统之间可能存在小的差异。有关代表性数据,请参见图1和Luecke 等人>(2017)。


图1.色谱图示例。 A. 2'3'-cGAMP,ATP和GTP的色谱图。 B和C.两种不同反应的色谱图。 B)具有4kb PCR片段的cGAS。 C)没有DNA的cGAS。该数据先前已在Luecke 等人发表。 >(2017)。

笔记

NTP对去磷酸化非常敏感。因此,保护样品免受环境中的磷酸酶非常重要。这就是为什么在处理或处理样品和试剂时应戴手套的原因。 1.5毫升管应进行高压灭菌,一般应注意不要用磷酸酶污染样品。
 适用于cGAS的其他反应条件也可用于该测定。避免在缓冲液中使用EDTA等螯合剂,因为它们会干扰阴离子交换柱。根据我们的经验,高盐浓度也可以抑制反应。如果增加cGAS和DNA的量,请注意不要遇到底物耗尽。我们建议在终止反应后至少剩余60%的底物

食谱

  1. 2μM纯化的cGAS [155-522]库存
    将2μM纯化的人cGAS截短至氨基酸155-522,悬浮于70 mM NaCl,10%(v / v)甘油,20 mM HEPES,pH 7.5中。 注意:对于cGAS纯化,请参见Luecke等。 (2017年)。用编码cGAS [155-522]的pET-21a质粒转化大肠杆菌,所述质粒具有N-末端Hexa His-MBP标签和烟草蚀刻病毒(TEV)蛋白酶切割位点。融合蛋白由细菌细胞表达,并且在纯化过程中裂解掉MBP标签,在Arg155前面留下四个残基Gly-Ala-Met-Gly。在将浓度调节至2μM之前,透析可用于将cGAS的缓冲液更换为70 mM NaCl,10%(v / v)甘油,20 mM HEPES,pH 7.5。>
  2. MgCl 2 (200 mM)
    将2.03 g MgCl 2 •6H 2 O溶于50 ml超纯水中
  3. ZnCl 2 (10 mM)
    将0.682g ZnCl 2 溶于500ml超纯水中
  4. NaOH(5 M)
    将100g NaOH溶于500ml超纯水中
  5. Tris(pH 7.5 1 M)
    1. 将60.57g Tris溶于400ml超纯水中,并在室温下使用浓HCl调节pH至7.5
    2. 使用超纯水将体积调节至500毫升
  6. 5x反应缓冲液
    1. 将1.46g NaCl与12.5ml 200mM MgCl 2,,0.25ml 10mM ZnCl 2,,10ml 1M Tris pH 7.5混合。
    2. 使用超纯水将体积调节至50毫升
    3. 用0.22μm醋酸纤维素滤器过滤5x反应缓冲液
    4. 储存在-20°C
    5x反应缓冲液的最终组成为500mM NaCl,200mM Tris,50mM MgCl 2,和50μMZnCl 2 ,pH 7.5
  7. 缓冲区A
    1. 将10ml 1M Tris pH 7.5与400ml超纯水混合
    2. 使用5 M NaOH在室温下将pH调节至7.5
    3. 使用超纯水将体积调节至500毫升
    4. 用0.22μm醋酸纤维素过滤器过滤缓冲液
    5. 准备新鲜的缓冲液
    缓冲液A的最终组成为20mM Tris pH 7.5
  8. 缓冲区B
    1. 将22g NaCl和10ml 1M Tris pH 7.5与400ml超纯水混合
    2. 使用5M NaOH在室温下将pH调节至7.5
    3. 使用超纯水将体积调节至500毫升
    4. 用0.22μm醋酸纤维素过滤器过滤缓冲液
    5. 准备新鲜的缓冲液
    缓冲液B的最终组成是750mM NaCl,20mM Tris pH 7.5
  9. ATP(10 mM)
    将100μl100mM ATP与900μl超纯水混合
    制作适合您要求的等分试样,储存在-80°C
    注意:注意不要用手或周围环境中的磷酸酶污染。>
  10. GTP(10 mM)
    将100μl100mM GTP与900μl超纯水混合
    制作适合您要求的等分试样,储存在-80°C
    注意:注意不要用手或周围环境中的磷酸酶污染。>

致谢

我们感谢SørenR。Paludan教授和Stefanie Luecke博士。用于帮助开发本协议中描述的方法。我们感谢Hans Henrik Gad,Ph.D。对手稿的评论。
 这项工作由诺和诺德基金会(NNF17OC0028184)和丹麦独立研究自然科学理事会(4181-00012B)资助。

利益争夺

作者声明没有利益冲突或竞争利益。

参考

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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用: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.
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