DNA fiber analysis allows us to understand how nanomaterials impact DNA at single-molecular level. And this information will enable developing strategies that will reduce nanotoxicity. This technique allows us to understand DNA dynamics and how it's impacted when cells or tissues are exposed to DNA damaging agents, either exogenous or endogenous, and we can look at DNA replication, DNA repair, and chromatin dynamics Studying DNA replication dynamics and repair mechanism will assist in developing strategies that will improve the efficacy of therapeutic material or reduce the off-target effect of nanomedicine.
What we really want people to know about this technique is it's the different things that we can or one person can do. One is to understand how novel chemotherapeutic agents can impact the health of a cell, to see if you can kill them faster or more efficiently. The next thing is to think about when you are adding an agent, let's say it's a new therapeutic or pharmaceutical agent, you wanna know if it's gonna hurt a person before you give it to them.
So therefore, you can just give the agent to cells or model systems and see if it hurts the cells or the model system, and if so, how much? The next thing is to kinda look at, we're developing all these new nanomedicines and we wanna make sure that those nanomedicines doesn't harm a person or the place where we're injecting the nanomedicine. The last thing is sometimes you wanna know how a protein impacts DNA replication, DNA repair, or chromatin dynamics and we can use this technique to be able to do that.
This is Shirali Patel, a master's in medical biotechnology research student in my laboratory, will be demonstrating the procedure. Begin the fiber assay preparation by removing the medium from 75%to 80%confluent RAW 264.5 macrophage cells and dividing it into two halves. Reserve one half for the chloro-iododeoxyuridine pulse or chloro-iododeoxyuridine reserve and add five microliters of iododeoxyuridine to the other half, making 50 micromoles as the final concentration.
After mixing, add the iododeoxyuridine-containing medium back to the plates and place the cells in the incubator for 20 minutes. Pre-warm the chloro-iododeoxyuridine reserve medium at 37 degrees Celsius and add chloro-iododeoxyuridine to this medium before the chloro-iododeoxyuridine pulse. After 20 minutes, aspirate the iododeoxyuridine medium mixture from the RAW 264.5 macrophage cell flask.
Gently wash the cells with 500 microliters of PBS, having 7.4 pH, and rotate the plate before discarding the PBS. Treat the cells with various concentrations of graphene oxide nanoparticles in 500 microliters of the medium and incubate for 30 minutes. After incubation, aspirate the treatment-medium mixture and gently wash the cells with 500 microliters of PBS by gently rotating the plate before discarding the PBS.
Next, add five microliters of chloro-iododeoxyuridine to the chloro-iododeoxyuridine reserve medium, and cover the cells with the chloro-iododeoxyuridine reserve medium. For the chloro-iododeoxyuridine pulse, place the cells in the incubator for 20 minutes. After a PBS wash, scrape off the cells for three to four minutes, then add five milliliters of medium to the plate and collect the cells.
Centrifuge the collected cells at 264G for four minutes. Remove the supernatant and re-suspend the cells in one milliliter of ice-cold PBS. To prepare the DNA fibers, label the glass slides with the experimental condition and date, using a pencil.
Aspirate two microliters of cells in a pipette and hold the pipette at an approximately 45 degree angle. Then place the pipette about one centimeter below the slide label and move the pipette horizontally towards the slide label with a slow release of cell solution. Once the solution evaporates and looks sticky and tacky, overlay each line of cells with 15 microliters of lysis buffer without letting the pipette tip touch the slide.
Then tilt the slide at a 25 degree angle and allow the DNA spreads to air dry for a minimum of four hours. On day one, fix the DNA fibers by immersing the slides in methanol acetic acid for two minutes. On day two, place the slides in a glass slide carrier and incubate them at 20 degrees Celsius for a minimum of 24 hours.
To denature the DNA, prepare a humidified chamber and place the slides carefully into a Coplin jar containing deionized, or DI, water, ensuring the coated surfaces do not touch the walls of the jar. After 20 seconds of incubation, pour out the water. Add 2.5 molar hydrochloric acid to the Coplin jar, covering all the slides.
After 80 minutes of incubation, give one wash with PBS plus 0.1%Tween, followed by two washes with PBS for three minutes each at room temperature. For immunostaining, place the slides in the humidified chamber and block them using 5%bovine serum albumin, or BSA, in PBS for 30 minutes at room temperature. Then cover the slides with a transparent plastic sheet.
After blocking, remove the cover slip and add 100 microliters of the primary antibody solution along the slide length. Add a new plastic cover slip and wait for two hours. After two hours, knock off the antibody solution and rinse the slides in a PBS-filled Coplin jar two times.
Block the slides again by adding 200 microliters of 5%BSA along the slide. Add a plastic cover slip and incubate for 15 minutes. After taking off the cover slip and removing excess BSA using a paper towel, add 100 microliters of the secondary antibody solution along the slide length and place a new plastic cover slip.
After one hour, remove the cover slip, knock off the excess BSA on a paper towel, and wash the slides two times in PBS. Add 100 microliters of the tertiary antibody solution along the slide length and place a new plastic cover slip. Again, add 200 microliters of 5%BSA along each slide and incubate for 15 minutes.
After three washes with PBS, remove the excess PBS and allow them to dry off completely. Once dry, add the mounting medium on the slide and place glass cover slips without allowing bubble formation. Visualize the immunostained DNA fibers using an immunofluorescent microscope equipped with a camera, a 60x objective, and appropriate filter sets to detect Alexa Fluor 488 and 594 dyes.
The variation in replication patterns after long-term exposure to the nanomaterials was determined by comparing the replication patterns of cells before and after the iododeoxyuridine and chloro-iododeoxyuridine nanoparticle exposure. DNA fiber analysis and interpretation images revealed a pattern for control macrophage cells and graphene-oxide-nanoparticle-treated macrophage DNA. The variation was observed in the pattern of replication intermediates, showing an increase in new origins on a different region of the same condition.
The conclusive fiber data was obtained by dividing the total area of the slide into five-to-six regions and taking multiple images from each region. Selecting around 500 intermediates from multiple slides or conditions was ideal to investigate the variation in DNA replication dynamics. The analysis also indicated an increase in the red-only tracks, indicating determinations and a decrease in newly fired origins in the graphene-oxide-treated cells Compared to the control.
The quality of the DNA fiber spread depends upon how well the fibers spread out from each other and that they do not overlap. Once the fibers have been spread out and IdU and CldU have been stained, it is extremely important to find the DNA fibers. One of the things you can do after you make the DNA fibers is, one, you can look at how replication dynamics is impacted.
Two, you can look at where DNA damage is located relative to the DNA replication tracks. And the last thing is if you use the FISH probes, you can actually look at specific regions and how they're impacted by that DNA damage. This technique paves the way for development of chemotherapy drugs, drug design, and understanding how DNA replication is impacted by various types of proteins.
