The protocol allows us to visualize the changes in the second restructure of DNA induced by ATP-dependent chromatin remodeling proteins. The change in the DNA structure can be correlated to transcription regulation. This is an easy and highly-sensitive technique that uses a very small amount of pure DNA to record the structural changes in the oligonucleotide.
This method can provide insight into transcription regulation mediated by ATP-dependent chromatin remodeling proteins. To begin, prepare the working concentrations of buffers and other reaction components freshly and keep them at four degrees Celsius before setting up the reactions, then measure ATPase activity of the protein in the presence of different DNA molecules using an NADH coupled oxidation assay. Mix 0.1 millimolar ADAAD, two millimolar ATP, 10 nanomolar DNA, and 1X REG buffer in a 96-well plate to a final volume of 250 microliters.
Next, incubate the reaction for 30 minutes in a 37 degree Celsius incubator, then measure the amount of NAD+using the software provided along with the microplate reader. To measure the absorbance at 340 nanometers, click on the NADH assay and place the 96-well plate on the plate holder in the instrument, then click on the read plate button to record the absorbance. Collect the CD spectra in high-transparency quartz cuvettes.
Use either rectangular or cylindrical cuvettes. To clean the cuvettes, wash them with water several times, then take a scan of the water or buffer in the cuvette to check whether it is clean. For the reactions, use PAGE-purified DNA oligonucleotides.
For fast cooling, heat the DNA at 94 degrees Celsius for three minutes on the heating block and immediately cool it on ice. For a slow cooling, after heating the DNA at 94 degrees Celsius for three minutes, allow it to cool to room temperature at a rate of one degree Celsius per minute. To record the baseline spectra, set up a total of five control reactions one by one in 1.5 milliliter centrifuge tubes.
Keep the reaction volume at 300 microliters in all the reactions. To record the CD spectra, set up a total of five experimental reactions one by one in 1.5 milliliter centrifuge tubes. To record the scan, turn on the gas and switch on the CD spectrometer.
After 10 to 15 minutes, switch on the lamp, switch on the water bath and set the holder temperature at 37 degrees Celsius. Next, open the CD spectrum software and set the temperature to 37 degrees Celsius, the wavelength range at 180 to 300 nanometers, the time per point to 0.5 seconds, and the scan number to five, then click on the pro data viewer, make a new file, and rename it with the details of the experiment and the date. Next, mix the baseline and experimental reactions by pipetting and carefully transfer the reaction mixes one by one to the cuvette, ensuring that there are no air bubbles.
If performing a time course experiment, incubate the reactions at 37 degrees Celsius for the required time, then take the scan. Add EDTA to the buffer containing the DNA, ATP, magnesium, and protein to stop ATP hydrolysis. To completely inhibit the ATPase activity, increase the EDTA concentration and incubation time.
Subtract the baselines from the corresponding reactions in the software and smoothen the data either in the CD spectrum software or in the data plotting software, then plot a graph of wavelength against mean residue ellipticity and analyze the peaks. M-folds showed that both strands of the MYC DNA could form a stem loop-like structure. The M-fold structures of the forward and the reverse DNA sequence containing the G quadruplex GECE are shown here.
The CD spectra of fast cooled GECE in the absence and presence of ATP and ADAAD showed that ADAAD induces two positive peaks, one at 258 nanometers and the other at 210 nanometers. The addition of EDTA abrogates this conformational change. The CD spectra now have a negative 210 nanometer peak and a broad positive band with peaks at 230 and 250 nanometers.
QGRS mapper and M-fold analysis showed that the promoter regions of DROSHA, DGCR8, and DICER possess the potential to form G quadruplex and stem-like structures. The CD spectra of the DROSHA pair five showed that ADAAD induces a negative peak at 210 nanometers and a positive peak at 260 nanometers. This spectrum is a characteristic of ADNA.
The CD spectra of DGCR8 pair one showed a positive peak at 210 nanometers and a broad negative peak at 260 nanometers. This spectrum is characteristic of B to X transition. For the DGCR8 pair seven, strong positive peaks at 210 and 270 nanometers and a negative peak at 250 nanometers were seen.
This spectrum is characteristic of parallel G quadruplex DNA structures. Lastly, for the DICER pair one, a positive peak at 210 nanometers and two negative peaks, one at 230 nanometers and the other at 260 nanometers were observed. These peaks are characteristic of an A to X DNA transition.
Ensure that the purity of DNA and protein is 99%or more. The cuvette should be clean and the baseline should be less than one millidegree. All the reagents should be pure so that the background is less than one millidegree.
