RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

The overall goal of this procedure is to assay RNA editing using functional omes extracted from Trias Broce for rapid identification of the omes inhibitors. This is accomplished by first isolating mitochondria and the target edito zones from tbr. The second step is to prepare the synthetic RNA editing reporter substrate.

Next compounds are subjected to binding to purified edito zones, along with the RNA editing reporter. In the presence of compounds that block the edito zones function, the RNA reporter will not undergo editing and will remain inactive. The final step is to add the F thread substrate and monitor for the RNA editing reporter's ability to selectively and specifically cleave this substrate.

In the case of an inhibitory compound, RNA editing is blocked and a reduced or no fluorescent signal will be detected. Ultimately, a real-time PCR machine is used to monitor the fluorescence emitted from the fret substrate as a function of time. The main advantage of this technique over conventional in vitro editing assays is that it's a simple and rapid mix and measure fluorescent based reporter assay that can be used for high throughput screening of chemical compounds against RNA editing.

To begin prepare growth medium for tusi pro acyclic form cells as described in the accompanying text protocol. Next, grow 300 milliliters of TBRC 1.7. A wild type pro acyclic form cells at 28 degrees Celsius while shaking them at 70 RPMs for 48 hours.

Once the cells have reached a density of 1.5 times 10 to the seventh cells per milliliter, centrifuge the cells at 6, 000 GS for 10 minutes at four degrees Celsius. Then wash the pellet with 50 milliliters of chilled phosphate, buffer saline, glucose or PBSG buffer and spin down the cells at 10, 000 Gs for 10 minutes at four degrees Celsius. First, we suspend the harvested cells in 30 milliliters of DTE buffer, disrupt the cell membrane using a 40 milliliter pre chilled sterile down homogenizer 10 times on ice.

Next, immediately at 4.3 milliliters of 60%sucrose to the homogenate and centrifuge. The solution at 15, 800 Gs for 10 minutes at four degrees Celsius. To bring down the mitochondria, re suspend the mitochondrial pellet in 4.6 milliliters of STM buffer.

Then add 13.8 microliters of 0.1 molar calcium chloride, and four microliters of RNA free DNAs one, and incubate the mixture for one hour on ice. Next, inactivate the DNAs by adding 4.6 milliliters of STE buffer and centrifuge the mixture at 15, 800 Gs for 10 minutes at four degrees Celsius. Then re suspend the pellet in 400 microliters of lysis buffer and transfer the mixture to a micro fuge tube.

Add 10%Triton X 100 to the tube at a final concentration of 1%and incubate the lysate at four degrees Celsius on a tube rotator for 15 minutes. Then clear the mitochondria lysate by centrifuging the solution two times at 17, 000 Gs for 15 minutes at four degrees Celsius and retain the cleared snat each time. Pour a 10 milliliter, 10%to 30%linear glycerol gradient in an ultracentrifuge tube using two XHHE gradient buffer, and a gradient marker following the instructions in the manual.

Next carefully, remove 500 microliters of solution from the top of the glycerol gradient and gently load 500 microliters of the cleared mitochondrial lysate into the ultracentrifuge tube. Spin the tube at 178, 000 GS for six hours at four degree Celsius. Collect 500 microliter fractions sequentially from the top to the bottom of the gradient at four degrees Celsius.

Then snap, freeze the fractions using liquid nitrogen and store them at minus 80 degrees Celsius. Anil the respective DNA template containing the T seven complementary sequence in a one-to-one molar ratio. Next, transcribe the RNA using an in vitro transcription kit and follow the instruction manual.

Stop the transcription reaction by adding an equal volume of seven molar urea dye. Run on a filter sterilized 9%denaturing poly acrylamide gel. Then use a short wave UV lamp to locate and excise respective RNA.

Place the excised gel piece in a micro fuge tube and add 400 microliters of gel elution buffer elute the RNA overnight at room temperature on a tube rotator. Next, precipitate the eluted RNA by adding one milliliter of cold, 100%ethanol and incubating at minus 20 degrees Celsius for 30 minutes. Centrifuge the mixture at 16, 000 GS for 30 minutes at four degrees Celsius to pellet down the RNA.

Then wash the pellet with one milliliter of 75%ethanol and centrifuge the tube at 16 GS for 20 minutes at four degrees celsius. Next, we suspend the RNA pellet in RNA free water to achieve the desired concentrations using the proportions listed. Start the assay by combining one microliter of pre A six RBZ and one microliter of GA six RBZ in a micro fuge tube.

Incubate the solution at 70 degrees Celsius for three minutes and let it sit at room temperature for 10 minutes. Next, prepare a master mix and add the anil pre A six RBZ and GA six RBZ to the mix. Last, then dispense 18 microliters of the master mix into the wells.

Next, seal the plate with a plate sealer and spin it down to remove any air bubbles. Then incubate the plate at 28 degrees Celsius for four hours after incubation at two microliters of GA six RBZ competitor. To each, well place a fresh seal onto the plate, then spin it down and perform real-time PCR with the following program.

Once the program is completed, add one microliter of fret substrate to each well to a final volume of 23 microliters and seal the plate with a fresh seal. Quickly spin the plate and place it back into the real-time PCR machine. Next program a new experiment with the following steps.

Then set up the plate by selecting all the wells that require reading and choose the FAM filter. Set the input volume as 23 microliters and start the run. Finally, calculate the slope of the values obtained from each sample to obtain a kinetic measurement by plotting the slopes on a bar graph for analysis.

In order to determine the quality of this assay, the signal to noise ratio of different substrates was calculated. Fret substrates with the Tamara Quinter and the Iowa Black dark Quinter are both viable options for use in a high throughput screen. The glycerol gradient fractions were tested for in vitro editing activity using the fluorescence based assay, the most active fractions can be combined when more edit zone is required.

Different control samples were used to analyze the variables in the assay reactions missing any RNA editing components do not. Cleve the substrate cleavage is observed in the presence of all components of the editing reactions in the absence of an inhibitor or in the presence of all editing components and an inactive compound. In contrast, an inhibitory compound can block RNA editing and is used as a positive control While attempting this procedure.

It is important to maintain an RNA free environment and to protect the fret substrate and the light sensitive compounds from light as much as possible.