Our work focuses on the development and use of single-molecule methodology to study dynamics in complex biological systems. In particular, we are interested in learning how DNA polymerases behave upon encountering roadblocks, in this case, G-quadruplexes. We have developed a single molecule method based on fluorescence visualization that allows us to see the behavior of individual DNA polymerases upon encountering a G-quadruplex.
With our assay, we have identified a previously uncharacterized exchange pathway for DNA polymerase delta upon hitting G-quadruplexes, in which the polymerase will fall off and a new one will rebind. With our protocol developed, we have finally answered the question of:How do different DNA polymerases behave when they encounter a G-quadruplex, as we can see in real time how the kinetics and mechanics change over time? To begin, remove a functionalized cover slip from a vacuum and place it onto a micro tube rack partially filled with water to create a humid environment.
Pipette 100 microliters of a blocking buffer into a tube, then add 25 microliters NeutrAvidin solution and mix well. Spread the solution onto the surface of the cover slip and allow it to incubate for 15 minutes at room temperature in the humid box. Next, wash the cover slip with water, then dry it using nitrogen gas.
Place a custom-made polydimethylsiloxane block onto the cover slip within a flow cell holder. Insert polyethylene tubes into the holes in the flow cell. Now, heat one-milliliter aliquots of Tween blocking buffer, 500 microliters of wash buffer, and replication buffer to 40 degrees Celsius for 15 minutes to liberate gases from the solutions.
Degas the solutions in a vacuum chamber for an additional 15 minutes at 800 millibars below atmospheric pressure. Apply a drop of oil to the objective. Take a constructed flow cell and place it on the microscope stage.
Then, raise the objective to meet the cover slip. Insert the inlet tube into the degassed Tween blocking buffer and connect the outlet to the syringe pump. Then, pull back on the syringe to draw the Tween blocking buffer through the tubing into the channel.
Flow in 200 microliters of degassed wash buffer into the channel at a rate of 100 microliters per minute to flush out the Tween blocking buffer. Now, dilute the DNA template solutions to 0.5 picomolar in 500 microliters of replication buffer. Let 150 microliters of the solution flow into the channel at a rate of 10 microliters per minute.
Illuminate the sample using a 647-nanometer laser at approximately 900 milliwatts per square centimeter at the sample plane to visualize individual DNA templates. Once a sufficient density of spots is visible, flow in a fresh solution of replication buffer to wash out excess DNA. Move to a new field of view and capture an image of the DNA to determine the degree of colocalization between the labeled polymerase and the DNA substrate.
Once complete, increase the laser power to photobleach the remaining spots. Next, prepare a polymerase solution containing dithiothreitol, dNTPs, and a fluorescent probe in the replication buffer. Flow in 100 microliters of the polymerase solution at a rate of five microliters per minute into the channel.
Once the sample is in focus and the total internal reflection fluorescence angle has been adjusted, set the laser power of the 647-nanometer laser to approximately 900 milliwatts per square centimeter at the sample plane. Begin imaging the field of view for the desired length of time. The G-quadruplex sequence blocked polymerase delta synthesis as indicated by the presence of a 60 nucleotide band in the page gel, while the control substrate without the G-quadruplex produced a 100 nucleotide band.
Single-molecule fluorescence microscopy confirmed that polymerase delta had a shorter dwell time of six plus or minus two seconds on the G-quadruplex substrate, compared to the control substrate, which had a dwell time of 11 plus or minus three seconds. The number of polymerase delta binding events was significantly higher on the G-quadruplex substrate compared to the control, suggesting multiple binding and unbinding cycles due to the synthesis blockade.
