病変の特異的分子ビーコンを活用した細胞ライセート中の塩基除去修復活性の定量、リアルタイム分析

The overall goal of the following experiment is to measure rates of DNA repair and cell nuclear extracts in real time using molecular beacons that fluoresce once they're repaired. Basic excision repair molecular beacons are comprised of deoxy oligonucleotides in a stem loop structure containing a single base lesion with six curve oxy fluorescein moieties conjugated to the five prime ends and dil moti conjugated to the three prime ends when folded. The six fam moty is quenched by dab sil in a non fluorescent manner via forester resonance energy transfer.

To assess DNA repair activity, beacons are incubated with cell nuclear extracts, which contain repair enzymes. DNA Glycosylate is recognized and remove the DNA lesion in the beacon. The resulting ABA sites are substrates for AP endonuclease one or ape one Upon recognition APE one cleaves the DNA backbone at the ABA site, allowing the DNA containing the six fam to freely dissociate from the hairpin.

Detachment from the quencher results in an increase of fluorescence that is proportional to the level of DNA repair and can be assessed in real time using a quantitative real-time PCR machine. The capacity to repair DNA damage is crucial to every cell and every organism. Among the multitude of cellular DNA repair pathways, base excision repair targets, damaged bases that may result from oxidative or chemical insults elucidating the role of individual cellular elements that might influence base excision repair is one of the main objectives in our laboratory studies.

As such, measuring DNA repair activity accurately and sensitively has become an interest in our lab. This assay can provide such data. The main advantage of this technique over existing methods like 32 PN labeling and analysis by gel electrophoresis is that our technique does not use radioactive labeled substrates can measure dary repair rates in real time and has a very large dynamic range.

Further, this approach can utilize several different fluoro fours and or base lesions to determine substrate specificity. We have shown that this assay is highly quantitative and very sensitive in different settings. This base excision repair molecular beacon assay is amenable to kinetic analysis.

The high sensitivity and specificity of the assay allows accurate base excision repair quantification, and is therefore useful for repair inhibitor screens or validation. In addition, it is adaptable for measuring dary repair activity and tissues and tumor cell lysates for functional biomarker measurements. When designing molecular beacons that will be used for this assay, consider the following DNA oligonucleotides that are 43 bases in length as shown here.

Work well. The DNA sequence is not unique, but must allow the formation of a hairpin structure. When a kneeled as shown here, specificity is achieved by insertion of specific base lesions such as uracil or tetra hydro furin.

At position X six bases from the five prime end, the GC content should be at least 32%and a GB base should not be placed at the five prime end. Six carboxy fluorescein or six fam should be conjugated to the five prime end and the three prime end should be modified with dab sil. This will be the substrate for the DNA repair proteins to assure binding and activity of the glycosyl releases.

The position of the lesion has been chosen to provide enough space from the hairpin structure and the end of the DNA. The length of the stem or hairpin duplex should be a minimum of 15 base pairs. The recommended loop length is 13 bases.

The control molecular beacon should be identical to the experimental molecular beacon, except that it does not contain a DNA lesion. It should therefore not be recognized by DNA repair enzymes. Once the molecular beacons are in hand, quality assessment should be performed to ensure that the beacons do not fluoresce until heated.

And to determine the optimal melting temperature, all steps during the extraction procedure should be performed on ice or at four degrees Celsius. To ensure protein stability, prepare for the experiment by pre chilling. Two one liter beakers per sample dialysis buffer dialysis chambers, micro centrifuge, tubes, syringes, and needles.

Label 15 milliliter conical tubes and place them on ice. Aspirate the growth medium from the cells and then wash them twice with 7.5 milliliters of cold PBS After the second wash. Add five milliliters of cold PBS to each dish and use a cell lifter to scrape the cells from the plate.

Transfer the cells to a chilled 15 milliliter conical tube, then centrifuge at 500 times G at four degrees Celsius for five minutes. After the spin, remove the supernatant and estimate the packed cell volume by comparing it to known volumes of liquid in similar tubes. Centrifuge the tubes for another minute and then remove the remaining PBS with a pipette resus.

Suspend the cell pellet in 150 microliters of nuke buster extraction reagent one for each 50 microliters of packed cell volume. Then transfer the resuspended cells to a pre chilled 1.5 milliliter tube and vortex for 15 seconds at high speed. After vortexing, incubate the tube on ice for five minutes, then vortex for another 15 seconds and centrifuge at 13, 000 times G for five minutes at four degrees Celsius.

Following the spin, remove and discard the supernatant, which contains the cytoplasmic fraction. Resus suspend the pellet in a mixture of one microliter of resus suspended 100 x protease inhibitor cocktail, one microliter of 100 millimolar DTT and 75 microliters of nuke buster extraction. Reagent two per 50 microliters of packed cell volume.

Vortex's cells for 15 seconds at high speed. Incubate on ice for five minutes and again vortex for 15 seconds at high speed centrifuge at 13, 000 times G for five minutes at four degrees Celsius. Following the spin uses syringe to transfer the supernatant, which contains the nuclear proteins to a slide dialysis cassette with a 7, 000 molecular weight cutoff.

Next, add 500 microliters of one molar DTT to 500 milliliters of pre chilled dialysis buffer and dialyze the lysate for 90 minutes at four degrees Celsius with gentle stirring. After 90 minutes, transfer the dialysis cassette to a second beaker containing 0.5 liters of pre chilled dialysis buffer with DTT and dialyze. The lysate for 90 minutes at four degrees Celsius with gentle stirring following dialysis, the extract may have crystallized due to high protein concentrations.

Next, insert a new syringe into a different port than the one used to inject the cassette and collect the dialyzed nuclear protein solution from the dialysis cassette. Try to include cloudy looking crystals when removing the dialyzed nuclear protein solution. Aliquot the sample to micro centrifuge tubes 20 microliters each quick freeze on dry ice and then store it minus 80 degrees Celsius until needed.

Determine concentration of protein and perform quality control as described in the accompanying document. To perform the molecular beacon assay thaw and dilute the cell lysates to two micrograms per microliter of protein with basic cision repair reaction buffer containing one millimolar of DTT dilute the molecular beacons to 200 micromolar with basic excision repair reaction buffer. Since the quantitative real-time PCR machine is typically not used as a fluorimeter, change the run method on the quantitative real-time PCR machine to collect data every 20 seconds at 37 degrees Celsius for at least 180 cycles.

Then every 20 seconds for 15 cycles at the molecular beacon's maximal fluorescence to obtain data for normalization. Set the reaction volume to 25 microliters. Then set up the plate design on the instrument using the applied biosystem software with standard curve selected as the experiment type, complete the plate layout.

An example is shown in this table, which can also be found in the accompanying document. Do not add anything to the well selected as the standard curve. Place the basic excision repair reaction buffer DTT cell lysate at a concentration of two micrograms per microliter and all molecular beacons being used in the experiment on ice.

Then add the DTT to the basic excision repair reaction buffer to a final concentration of one millimolar pipette. 15 microliters of basic excision repair reaction buffer with DTT into each well of a compatible optical Q-R-T-P-C-R plate on ice. Next, following the plate layout, pipette the beacons into the appropriate wells.

Each combination of lysate and molecular beacon or control should be assayed in triplicate. Finally, pipette five microliters per well of the diluted nuclear lysate into the appropriate wells. It's important to remember to keep all the molecular beacon reagents cold and to pipette the lysates into the plate as the last step.

Remember, the lysates contain active proteins and they'll begin repairing the molecular beacons as soon as they are pipetted into the reaction mixture. Therefore, it's very important to keep all the reagents cold to minimize the amount of beacon repair that occurs before you start taking measurements. Once everything has been added, seal the plate with optical adhesive film and mix by gently tapping the plate.

Then perform a quick ten second spin with a centrifuge to gather solutions at the bottom of the tubes or plate after the spin, insert the plate into the quantitative realtime PCR machine and begin the assay after completion, export the raw data preferably in Excel format and follow the instructions in the accompanying document to analyze the data once the necessary calculations have been made. Plot the normalized fluorescence data as percent free fam against time in seconds in a scatterplot with corresponding errors to assess DNA repair activity and quality of nuclear extracts from the LN 4 28 cells and an MPG overexpression cell line designated as LN 4 28 MPG. The assay described in this video was performed using a molecular beacon containing the APE one substrate tetra furin as a positive control.

As shown in this figure, both cell lines have robust APE one activity as indicated by the steep slope, DNA glycosate activity specific for removal of the MPG substrate. Hypo hypoxanthine indicated as HX was measured in nuclear lysates from both cell lines. Each lysate was analyzed using either a basic excision repair control molecular beacon or a base excision repair HX molecular beacon for the control beacon LN 4 28 is shown in green and LN 4 28 MPG shown in purple for the hypo beacon.

LN 4 28 is shown in blue and LN 4 28 MPG is shown in orange. Results are reported as A the mean fluorescence units and B, the normalized fluorescence. A time dependent increase in fluorescence indicative of HX repair occurred in each cell.

Lysate incubated with the HX molecular beacon as expected. The LN 4 28 MPG cell line indicated by the orange trace had a much higher rate of repair as indicated by the more rapid accumulation of fluorescence than did the parental LN 4 28 cell line, which has minimal MPG protein. Thus the repair rate for specific DNA lesions by specific repair proteins can be measured from cell extracts in real time with minimal background levels of non-specific DNA cleavage.

After watching this video, you should have a good understanding of how to design your molecular beacons and measure realtime DNA repair rates using either purified proteins or cell lysates. With respect to the analysis, it is essential to assess dynamic range and background values do so by the inclusion of control reactions such as with or without the positive and negative control beacons. Considering the well to well variability, it is useful to normalize the data in the individual wells to the input values.

Those are determined by the finer step at melting temperature. This and inclusion of other dyes like a rock die will account for the variability inherent to pipetting errors or differences in a detectors. This technique allowed us to elucidate the influence of individual based excision repair components on the overall repair activity and capacity.

It is sensitive enough to unmask small changes that would have not been detectable with conventional methods.