A robust plate reader assay like ours is very useful for the initial screening of potential methyltransferase inhibitors. The ability to collect data in real time is a big advantage of the continuous endonuclease-coupled assay. Start by preparing 600 microliters of the assay conditions containing 20 micromolar and 50 micromolar compounds separately in the microcentrifuge tubes on ice.
To do so, add double distilled water and 5X methylation buffer to each tube to achieve a final concentration of 1X methylation buffer. Next, add 3.15 microliters of 20 milligrams per milliliter bovine serum albumen, or BSA, to each sample. Then, add two microliters of 3.15 micromolar hairpin DNA substrate and 1.33 microliters of 4.75 micromolar S-adenosylmethionine, or SAM, to each sample.
Add inhibitor to the appropriate samples to achieve a final concentration of 21 micromolar or 52.5 micromolar and an equivalent amount of dimethyl sulfoxide, or DMSO, to a control sample before mixing the solutions by vortexing at 3, 000 rotations per minute for three seconds. Then, spin the samples for a few seconds in a table table mini centrifuge at 1, 200 times G to ensure the solution is collected at the bottom of the tube. Aliquot 95 microliters of each assay solution into six consecutive wells in a black half area 96 well plate.
Prepare 75 microliters of Gla I or DNMT1 plus Gla I enzyme solution in the microcentrifuge tubes as described before to a final concentration of one-time buffer. Next, add 1.2 microliters of 10 units per microliter Gla I to each solution to achieve a final concentration of 0.16 units per microliter. Add 0.6 microliters of five micromolar RFTS-lacking DNMT1 to the DNMT1 plus Gla I solution for a final concentration of 40 nanomolar.
After gentle mixing, spin the samples for a few seconds in a tabletop mini centrifuge at 1, 200 G to ensure that the solution is collected at the bottom of the tube. Then, aliquot 12 microliters of each enzyme solution into six wells in a conical-bottomed 96 well plate. For the methylation assay of the DNA sample, preheat the plate reader to 37 degrees Celsius.
Then, insert the black plate containing the assay solutions in the plate reader. After shaking the plate at 425 CPM, measure the fluorescence with an excitation wavelength of 485 nanometers and an emission wavelength of 528 nanometers. Then, incubate the plate at 37 degrees Celsius for five minutes.
Remove the assay plate from the plate reader and add five microliters of the enzyme solution to each well, followed by mixing the solution by pipetting up and down. Insert the plate into the plate reader and shake the plate before recording the fluorescence every 53 seconds for 30 minutes. Obtain the average fluorescence of the triplicate Gla I control assays for each condition and subtract the average Gla I containing control reaction trays from the triplicate traces obtained in the presence of RFTS-lacking DNMT1.
Then, determine the average and standard error of the mean for the corrected replicates. Plot the average corrected reaction trace and fit the initial linear portion to a line to determine the initial velocity. Determine the percent activity by dividing the velocity observed in the presence of a compound by the velocity observed in the DMSO containing control reaction and multiplying by 100.
The results of a discontinuous endonuclease-coupled assay showed that fully methylated product DNA is protected from cleavage, confirming that RFTS-lacking DNMT1 is active and able to methylate the hemimethylated substrate DNA. In the fluorescence-based assay, in the absence of Gla I, the addition of DNMT1 or buffer alone did not affect the background fluorescence. Subsequent addition of Gla I to all assays resulted in fluorescence generation only in assays that contained DNMT1.
The concurrent addition of RFTS-lacking DNMT1 and Gla I to three assays resulted in robust fluorescence generation. The addition of Gla I alone produced no fluorescence. To screen for potential inhibitors, the DNA methylation activity of RFTS-lacking DNMT1 was examined in the presence of DMSO, compound one, compound two, or compound three at 20 and 50 micromolar concentrations.
The compounds that reduce fluorescence generation in this coupled assay must be further validated. Ensuring the compounds are not inhibiting the coupling enzyme is an important next step.
