This method requires the starting material 3′-amino-3′-deoxy--ATP (15).
| 1. | Activate 7-diethylaminocoumarin-3-carboxylic acid (16.4 mg, 62.8 μmol) by dissolving in dry DMF (1 ml), cooling it on ice, and adding tributylamine (25 μl, 103 μmol) and isobutyl chloroformate (10 μl, 77 μmol). |
| 2. | Leave the reaction mixture on ice for 50 min. |
| 3. | Add 3′-amino-3′-deoxyATP (40 μmol, triethylammonium salt) in water (300 μl) to the activated coumarin and stir at room temperature for 2 h. |
| 4. | Analyze the reaction mixture using HPLC to confirm the formation of deac-aminoATP. Equilibrate a Partisphere SAX column with 0.4 M (NH4)2HPO4 with 5% (v/v) acetonitrile at a flow rate of 1.5 ml/min at room temperature. |
| 5. | Add an aliquot of the reaction mixture (1–10 nmol) to 100 μl of the running buffer. |
| 6. | Inject the solution onto the column. |
| 7. | Follow the absorbance at 254 nm and fluorescence with excitation 435 nm and emission 465 nm. Elution times are approximately 1.6 min for 7-diethylaminocoumarin-3-carboxylic acid, 3.5 min for 3′-amino-3′-deoxyATP, and 13 min for deac--aminoATP (see Note 6). |
| 1. | The reaction mixture was purified on a DEAE-cellulose -column. Equilibrate the column with 10 mM TEAB, pH 7.6 at 1 ml/min at 4°C. |
| 2. | Alter the pH of the reaction mixture to 7.6 using acid or base, reduce the conductivity by dilution in water so it is close to that of 10 mM TEAB and load on to the column. |
| 3. | Wash the column with 10 mM TEAB, pH 7.6 at a flow rate of 1 ml/min for two column volumes. |
| 4. | Elute the nucleotide with a linear gradient of 10–600 mM TEAB (total volume 1 L). Follow the absorbance at 254 nm (see Note 17). Unreacted aminoATP is eluted first followed by deac-aminoATP. |
The product deac-aminoATP is concentrated as described for Cy3-edaATP and stored at ?80°C.
| 1. | Measure the absorbance spectra of deac-aminoATP in 50 mM Tris–HCl, pH 7.5 between 220 and 700 nm. Taking the extinction coefficient for the coumarin to be 46,800 M?1 cm?1 at 429 nm and for adenosine to be 15,200 M?1 cm?1 at 260 nm, calculate the concentrations of the nucleotide (see Note 12). |
| 2. | Characterize deac-aminoATP by HPLC using the same method as above. The major peak should be deac-aminoATP. Determine the purity by integrating the deac-aminoATP peak with any other peaks. |
| 3. | Measure the fluorescence spectra as described for Cy3-edaATP, but using the corresponding excitation and emission peaks for the coumarin (Fig. 2). Fig. 2. Fluorescence change upon binding of deac-aminoADP to myosin S1. Excess -myosin S1 was added to bind all nucleotides. Deac-aminoADP (0.3 μM) was excited at 435 nm and 10 μM S1 was added. Insert shows the titration of myosin S1 into a solution of deac-aminoADP. This highlights the need to saturate the nucleotide to determine the maximum fluorescence change. Myosin S1 was added to a solution of 0.1 μM nucleotide, and the fluorescence was monitored at 480 nm, with excitation at 435 nm. |
| 4. | Follow the same procedure described for the Cy3-edaATP to generate the diphosphate. |
3.3 Assess the Effects of Modifications
The method describes here is for an ATPase or GTPase. This specific example uses a DNA helicase Bacillus stearothermophilus PcrA. The easiest method to provide an overall assessment of the effect of an ATP modification is to measure a steady-state ATPase assay. Should there be a change in the steady-state parameters (greater than 20%), then the individual steps of the ATP cycle could be investigated. It is common for the diphosphate affinity to increase with modifications to the ribose ring (9, 18, 22, 23).
It is also highly recommended that a functional activity assay is performed, such as measuring DNA unwinding by a DNA helicase or an in vitro motility assay with myosin. This is an alternate assessment of the modification effect: the label may interfere with one criterion which may not be noticeable in the other.
| 1. | Prepare a mixture (60 μl) of 2 nM PcrA helicase, 500 nM dT20 oligonucleotide and 10 μM MDCC-PBP (or 6IATR-PBP) in a buffer containing 50 mM Tris–HCl, pH 7.5, 3 mM MgCl2 and 150 mM NaCl. |
| 2. | Record the fluorescence by exciting at 436 nm and emission at 465 nm for MDCC-PBP, or excitation 555 nm and emission 575 nm for 6IATR-PBP. |
| 3. | Add ATP at various concentrations (1 mM to 0.5 μM) (see Note 18). |
| 4. | Repeat the measurements at the same concentrations of deac-aminoATP or Cy3-edaATP (see Note 18). |
| 5. | Perform a calibration of the fluorescence signal using known concentrations of inorganic phosphate. |
| 6. | Compare the V max and K m values for the native and modified nucleotides. |
4 Notes
| 1. | It is also possible to synthesize edaATP (3, 15, 17). |
| 2. | MDCC-PBP is available commercially from Invitrogen, but cannot be used with diethylaminocoumarin-labeled nucleotides because the fluorophores are the same. Similarly, 6IATR-PBP cannot be used if a fluorophore with similar wavelengths is present, such as Cy3 or other rhodamine. |
| 3. | Use an excess of nucleotide over Cy3 NHS-ester due to the expense of the fluorophore. |
| 4. | Filter and degas the (NH4)2HPO4 and then add the HPLC grade methanol. |
| 5. | Alternatively, the reaction can be followed by thin-layer chromatography on silica plates (13). |
| 6. | Using the fluorescence detection is approximately 100-fold more sensitive than absorbance. If necessary, absorbance peaks can be collected, their fluorescence measured in a fluorimeter, and their full absorbance spectrum measured in a spectrometer. |
| 7. | Do not store distilled triethylamine for later use without redistillation: it decomposes on storage. It may be possible to use high-purity triethylamine without distillation, but triethylamine does form impurities on storage. The distillation ensures that only volatile components end up in the TEAB solution. |
| 8. | Triethylamine itself is only partially miscible with water: there will be two layers initially, which becomes a single solution after some CO2 has been absorbed. |
| 9. | Alternatively, follow the fluorescence of Cy3 using an excitation of 550 nm and emission of 570 nm. |
| 10. | Apply and remove the vacuum slowly to prevent the solution splashing and frothing, and so passing into the condenser. |
| 11. | Aliquot the ATP into small amounts before freezing to avoid freeze-thaw cycles. It is advisable to check the purity of the nucleotide by HPLC periodically during long-term storage. |
| 12. | The ratio between the molar amount of the fluorophore and adenosine should be ～1. If not, there is likely to be contaminating fluorophore in the preparation. |
| 13. | Determine the limit of sensitivity for the instrument by injecting known amounts. Typically, 1% contamination should be detected; for example, if 10 nmol is injected, it should be possible to detect 0.1 nmol. It is possible that a greater amount of nucleotide will have to be injected. |
| 14. | Ideally, the addition of excess protein to the nucleotide sample would lead to the maximum potential signal change. However, this is dependent on the affinity between nucleotide and protein. |
| 15. | Unless the protein has a low hydrolysis rate constant, or the protein requires an activator, it is likely that any fluorescence change occurs due to the formation of diphosphate. The diphosphate fluorescence change should be measured independently. |
| 16. | It is also possible to begin the labeling with edaADP and repeat the same protocol as describe above to produce the fluorescent diphosphate. In addition, it is possible to use other ATPases or commercially available glycerol kinase with d-glyceraldehyde (albeit that ribose-modified nucleotides are poor substrates for this kinase) to achieve the hydrolysis of the triphosphate (24). The resulting ADP analog is purified by a desalting column (PD10) or repeating the ion-exchange chromatography. |
| 17. | Alternatively, follow the fluorescence of the diethylaminocoumarin using an excitation of 430 nm and emission of 465 nm. |
| 18. | Avoid diluting the ATP samples to low concentrations. Use 60× concentrated stocks. By adding 1 μl of the ATP to the 60 μl reaction mixture, the correct ATP concentration is achieved. |
Acknowledgments We would like to thank the various coworkers, who have been involved in synthesis and use of fluorescent nucleotides and are coauthors of publications cited here. We thank the Medical Research Council, UK (C.P.T. and M.R.W.) and European Molecular Biology Organization (C.P.T) for financial support.