Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

With this protocol, we can directly observe site-specific DNA cleavage by restriction endonucleases at the single molecule level. Our multiplexed approach allows us to measure the time it takes for hundreds of individual REases to complete the catalytic cycle. Multiplexing allows us to generate well-populated dwell time to cleavage distributions that we can fit with the gamma probability distribution to gain insights into the mechanism of DNA cleavage.

Begin by preparing a fresh tube for each resuspended oligonucleotide at 50 microliters of the oligonucleotide and 50 microliters of freshly prepared DTT solution to each tube, pipette up and down to mix and incubate for 30 minutes at room temperature. Pipette the entire volume of each sample mixture gently onto a prepared column and centrifuge at 750G for two minutes. After centrifugation, store any samples that will not be used immediately at 20 degrees Celsius to prevent oxidation of the thiol groups and formation of disulfide bonds.

Pipette one aliquot of 50 microliters of the quantum dot stock into a dialysis device for each construct to be made, making sure not to touch the membrane with the pipette tip. Dialyze against a volume of CHES buffer that is at least 1, 000 times the sample volume with stirring at 100 RPM for 15 minutes. Using a pipetter, carefully retrieve the suspended quantum dots from the dialysis device and transfer them into a fresh tube containing an equal volume of freshly prepared Sulfo-SMCC solution, pipetting up and down to mix.

Incubate for 1 hour at room temperature with shaking at 1, 000 RPM to allow the Sulfo-SMCC to react with the primary amines on the quantum dots. Then, use a pipetter to carefully transfer each sample into a fresh dialysis device. To remove excess Sulfo-SMCC, dialyze against a volume of CHES buffer that is at least 1, 000 times the volume contained in the dialysis device with stirring for 15 minutes.

Exchange the buffer twice and perform dialysis with fresh CHES buffer three times in total, allowing dialysis to proceed for 15 minutes after each buffer exchange. Then repeat the dialysis with PBS. Using a pipetter, carefully recover the solution containing the quantum dots from the dialysis device and transfer each sample into a fresh tube containing an equimolar amount of dilated oligonucleotide diluted in PBS.

Incubate for 2 hours at room temperature with shaking at 1, 000 RPM. Add BSA to each sample for a final concentration of 0.5 milligrams per milliliter. Then use a pipetter to carefully transfer each sample into a fresh dialysis device and dialyze against storage buffer.

After dialysis, carefully recover each sample and place it into a fresh tube. Store the samples at 4 degrees Celsius. Combine a sample of quantum dot labeled oligonucleotides with a tenfold molar excess of biotinylated oligonucleotide.

Heat the mixture in a heat block to 75 degrees Celsius and hold it at that temperature for 5 minutes. Then turn off the heat block and allow the mixture to cool slowly. Store the completed construct at 4 degrees Celsius Using a handheld rotary multi-tool fitted with a tapered diamond point wheel bit, drill two holes in opposite corners of the quartz slide to serve as an inlet and outlet.

Be sure to secure the slide in place, lubricate the bit and slide constantly with water while drilling. After each experiment, recover the prepared quartz slide by soaking the used device in acetone to dissolve the adhesive and epoxy. Discard the remaining components.

Combine 25 microliters of streptavidin solution with 80 microliters of PBS and 20 microliters of blocking buffer. Coat a PEG-treated cover slip with this mixture, and incubate in a humid environment for 30 minutes. During the incubation of the cover slip, prepare the imaging spacer to create a flow channel.

Cut a 1 inch square piece of imaging spacer material, then mark a 2 millimeter wide channel aligned to the ends of the holes drilled in the quartz slide. Cut the channel out of the spacer material with a scalpel. Clean the quartz slide thoroughly with acetone to remove any remnants of adhesive from prior experiments.

Peel one side of the backing from the image spacer and carefully place it onto the quartz slide, taking care not to cover the inlet and outlet holes. Wash the cover slip with water and dry with compressed air. Remove the other side of the backing from the imaging spacer and sandwich it between the quartz slide and the functionalized streptavidin coated cover slip using plastic tweezers to press the assembly together and remove air bubbles from the adhesive.

Cut the end to the tubing at an angle to ensure free flow of solution. Then insert 30 centimeter long polyethylene tubing into each hole in the quartz slide. Hold the tubes in place with a tube rack, then use epoxy to seal the tubes in place and the edges of the assembled device.

Once the epoxy has been set, insert the blunt needle on an empty syringe into the outlet tube of the device and submerge the end of the inlet tube in a container filled with the blocking buffer. Pull back on the syringe plunger to fill the device with blocking buffer. Then leave the device to incubate for at least 30 minutes prior to use.

Attach the microfluidic device to the microscope stage plate with tape. Bring the objective into contact with the bottom of the device and position the objective so that the field of view is within the microfluidic channel. After connecting the outlet tube to the syringe pump, flush the microfluidic channel with fresh blocking buffer by pulling back the syringe plunger.

Check to make sure there are no bubbles trapped in the tubing or in the channel. Dilute 1 microliter of the prepared DNA substrate into 1 milliliter of blocking buffer. Place the inlet tube into the diluted DNA substrate and flow 800 microliters of the substrate solution through the channel at a rate of 200 microliters per minute.

Allow the DNA solution to incubate in the channel for 15 minutes after the flow stops. After the incubation, flow at least 800 microliters of blocking buffer through the channel at a rate of 200 microliters per minute to rinse the unbound DNA out of the channel. Adjust the microscope focus so that the surface-tethered quantum dots are clearly visible.

Flush the microfluidic device with 800 microliters of experimental buffer without magnesium at a flow rate of 200 microliters per minute. Add the REase to an aliquot of experimental buffer without magnesium and mix gently by pipetting. Flow 800 microliters of 400 units per milliliter of EcoRV through the channel at a flow rate of 200 microliters per minute.

Start the experiment by flowing the experimental buffer containing magnesium and fluorescein at a flow rate of 200 microliters per minute. Begin capturing data immediately after starting the syringe pump. Stop data acquisition after flowing 800 microliters of buffer.

After introducing the DNA substrate into the flow cell and washing away excess DNA and quantum dots, there are typically thousands of individual quantum dots in a field of view. They are stably attached to the glass surface and do not undergo noticeable dimming or significant bleaching throughout the experiment. When the REase has flowed through the channel in the absence of magnesium, at least 95%of the quantum dots present at the beginning of the experiment can still be seen at the end of the observation period.

However, when the magnesium-containing buffer is flowed immediately after the REase, up to half of the quantum dots disappear by the end of the observation period, similar to what is observed when REase and magnesium are flowed through the channel together. Quantum dot disappearance happens quickly and results in a sharp decrease in the intensity trajectory, providing a clear indication of the time at which a given DNA molecule is cleaved. This experiment was performed with the well-studied type II P REase EcoRV.

Both non-linear least squares curve fitting and maximum likelihood parameter estimation were used to extract the values of n and beta from the data. The two parameter estimation methods were in agreement. Using the non-linear least squares fit, n was 3.52 and beta was 5.78 seconds.

Using the maximum likelihood estimation, n was 3.41 and beta was 6.06 seconds. This assay can provide evidence of the presence of intermediate steps along the cleavage pathway. Performing this assay under changing reaction conditions can help establish the nature of reaction intermediates that cannot be directly observed.