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

1. General information

1. Oligonucleotide design
   NOTE: The 60 base-pair (bp)-long DNA substrate is formed from a pair of complementary oligonucleotides with a duplex melting temperature of 75 °C in 100 mM NaCl.
   1. Order one oligonucleotide synthesized with a single 5' biotin modification and the other with a 5' thiol modification (with a six-carbon spacer). Place the recognition site in the center of the duplex region.
      NOTE: The oligonucleotide sequences for use with EcoRV are shown below (recognition site in bold).
      5' biotin – AAA ACC GAC ATG TTG ATT TCC TGA AAC GGG ATA TCA TCA AAG CCA TGA ACA AAG CAG CCG – 3'
      5' thiol – CGG CTG CTT TGT TCA TGG CTT TGA TGA TAT CCC GTT TCA GGA AAT CAA CAT GTC GGT TTT – 3'
2. Use ultrapure water with 18 MOhm resistivity for all steps.
3. Protect all solutions containing quantum dots from light to prevent photobleaching.
4. Use a compressed air source to complete this protocol.

2. Preparation of quantum-dot-labeled DNA substrate materials

NOTE: Besides the oligonucleotides described above, see the Table of Materials for other materials and Table 1 for buffers required for the preparation of quantum-dot-labeled DNA substrates.

1. Reduce 5' thiol groups on the oligonucleotide to be coupled to quantum dots.
   1. Resuspend each thiolated oligonucleotide in water at a concentration of 100 µM.
   2. Pipet 650 µL of water into a size-exclusion spin column for each oligonucleotide sample, and vortex for ~15 s. Leave the column to pack for 30 min.
   3. Prepare fresh 100 mM dithiothreitol (DTT) solution immediately prior to each use as DTT degrades in solution rapidly. Carefully open one vial containing 7.7 mg of DTT, and pipet 500 µL of sodium phosphate buffer into the vial; vortex to mix thoroughly.
   4. For each resuspended oligonucleotide, prepare a fresh tube, and add 50 µL of the oligonucleotide and 50 µL of DTT solution to the tube. Pipet up and down to mix. Incubate for 30 min at room temperature to reduce the disulfide bonds between the oxidized thiol groups on the 5' end of the oligonucleotides.
   5. Remove the spin column caps, and place each column into the collection tube. Centrifuge the spin columns at 750 × g for 2 min, and discard the eluate.
   6. Transfer the spin columns into fresh centrifuge tubes. Pipet the entire volume (100 µL) of one sample of DNA/DTT mixture gently onto each prepared column. Centrifuge at 750 × g for 2 min, and measure the absorbance of the eluate at 260 nm to confirm that the concentration is approximately 40 µM.
   7. Store any samples that will not be used immediately at -20 °C to prevent the oxidation of the thiol groups and the formation of disulfide bonds.
2. Coupling of DNA to quantum dots
   1. Pipet one aliquot of 50 µL 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 N-cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer that is at least 1000x the sample volume with stirring at ~100 rpm for 15 min.
   2. Prepare a fresh 6 mM solution of sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) in CHES buffer immediately before each use. Carefully open one vial containing 2 mg of sulfo-SMCC, and pipet 800 µL of CHES buffer into the vial. Vortex to mix thoroughly.
   3. Using a pipettor, carefully retrieve the suspended quantum dots from the dialysis device, and transfer into a fresh tube containing an equal volume of the sulfo-SMCC solution; pipet up and down to mix. Incubate for 1 h at room temperature with shaking at 1000 rpm to allow the sulfo-SMCC to react with the primary amines on the quantum dots.
   4. Using a pipettor, 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 1000x the volume contained in the dialysis device(s), with stirring, for 15 min. Exchange the buffer 2x, and perform dialysis with fresh CHES buffer 3x in total, allowing dialysis to proceed 15 min after each buffer exchange.
   5. To exchange the buffer in preparation for the second reaction, transfer the dialysis devices into a beaker containing a volume of phosphate-buffered saline (PBS) that is at least 1000x the volume contained in the device(s). Dialyze with stirring for 15 min, exchange the buffer 2x, and perform dialysis with fresh PBS 3x in total, allowing dialysis to proceed for 15 min after each buffer exchange.
   6. Using a pipettor, carefully recover the solution containing the quantum dots from the dialysis device(s), and transfer each sample into a fresh tube containing an equimolar amount of thiolated oligonucleotide diluted in PBS. Combine an equal volume of PBS and a 1/10^th the volume of the reduced and purified oligonucleotide sample (approximately 40 µM concentration) as the quantum dot concentration at this point is ~4 µM. Incubate for 2 h at room temperature, with shaking at 1000 rpm.
   7. Add bovine serum albumin (BSA, 10 mg/mL) to each sample to obtain 0.5 mg/mL final BSA concentration. Using a pipettor, carefully transfer each sample into a fresh dialysis device, and dialyze against a volume of storage buffer that is at least 1000x the volume contained in the dialysis device(s). Exchange the buffer 2x, and perform dialysis with fresh storage buffer 3x in total, allowing dialysis to proceed for 15 min after each buffer exchange.
   8. Using a pipettor, carefully recover each sample, place it into a fresh tube, and store at 4 °C.
      NOTE: Do not store at -20 °C as quantum dots are damaged by freezing.
3. Annealing of quantum-dot-labeled oligonucleotide to biotinylated oligonucleotide
   1. Resuspend each biotinylated oligonucleotide in storage buffer at a concentration of 100 µM. Store unused samples at -20 °C.
   2. Combine a sample of quantum dot-labeled oligonucleotide with a 10-fold molar excess of biotinylated oligonucleotide. As the estimated concentration of DNA in the quantum-dot-labeled sample is 2 µM, add 0.2 µL of biotinylated oligonucleotide for every 10 µL of this sample.
   3. Heat the mixture in a heat block to 75 °C (the melting temperature for the complementary region). Hold at that temperature for 5 min. Turn off the heat block, and allow to cool slowly. Store the completed construct at 4 °C; do not freeze.

3. Surface functionalization of coverslips

NOTE: This process has been described previously in other JoVE video protocols^16,17. This protocol describes an adapted version of the procedure with minor changes to accommodate a smaller glass slide. See the Table of Materials for other materials required for the surface functionalization of coverslips.

1. Place 5 coverslips into each coverslip holder. Make sure to skip one space between each pair of coverslips so that they do not stick to one another. Place the coverslips in their holder into a jar with a cover, add ethanol to the jar until the coverslips are covered, and screw the top on securely. Place the entire jar into the water in the bath sonicator, without submerging it completely, and sonicate for 30 min.
2. Fill a clean beaker or jar with ultrapure water. Use metal tweezers to remove the coverslips in their holder from the ethanol, and submerge them in water to rinse. Then, transfer the coverslips and holder into a jar containing a 1 M potassium hydroxide (KOH) solution. Screw the top on securely, place the jar in the sonicator bath, and sonicate for 30 min.
   NOTE: Use care when handling the KOH solution as it is corrosive and an irritant.
3. Repeat the sonication in ethanol and KOH solution, rinsing the coverslips in water as described above between each step.
4. Transfer the coverslips in their holder into a clean jar filled with pure acetone. Finish the cleaning by sonicating the jar as described above for 30 min. Do not rinse with water after this step.
5. Using metal tweezers, transfer the coverslips in their holder into a clean 100 mL beaker containing 80 mL of fresh acetone and a micro-scale stir bar. Place the beaker on a magnetic stir plate set to at least 1,000 rpm. While the acetone is being stirred vigorously, pipet 1.6 mL of 3-aminopropyltriethoxysilane (APTES) into the beaker to make a 2% v/v solution.
   NOTE: Use care when pipetting APTES as it is corrosive.
6. Allow the coverslips to incubate in the solution for 2 min, then use metal tweezers to transfer the coverslips in their holder into a beaker of water to quench the reaction. Rinse the coverslips two more times, replacing the water in the beaker.
7. Cure the silanized glass in the oven at 120 °C for 75 min. If not continuing to the next step immediately, store the coverslips under vacuum for maximum of a few days.
8. Dissolve N-hydroxysuccinimide (NHS) ester-derivatized polyethyleneglycols (PEGs) in 100 mM sodium bicarbonate (pH 8.2). Use a 10:1 ratio of methoxy-terminated PEG to biotin-terminated PEG, with ~100 mg/mL of methoxy-terminated PEG.
9. Pipet 100 µL of the PEG solution onto half of the dry silanized coverslips, and cover each one with a second coverslip. Use small pieces of parafilm placed at each corner as spacers to prevent the coverslips from sticking to each other.
10. Incubate the coverslips in a humid environment for 3.5 h. Separate the coverslip sandwiches. Use a squirt bottle to wash each coverslip with copious water and dry with compressed air.
11. Store functionalized coverslips under vacuum. Be sure to keep the PEG-treated side up because the other side will not capture the DNA substrate.

4. Microfluidic device construction

NOTE: See the Table of Materials for other materials required for the construction of the microfluidic device.

1. Using a handheld rotary multitool 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 for reuse by soaking a used device in acetone to dissolve the adhesive and epoxy; discard the remaining components.
   NOTE: The drilling can be carried out by hand, but the use of a press-style holder for the multitool makes the process easier.
2. Combine 25 µL of streptavidin solution with 80 µL of PBS and 20 µL of blocking buffer. Coat a PEG-treated coverslip with this mixture, and incubate in a humid environment for 30 min.
3. During the incubation of the coverslip, prepare the imaging spacer to create a flow channel. Cut a 1-inch square piece of imaging spacer material, then mark a 2 mm wide channel aligned to the ends of the holes drilled in the quartz slide (Figure 2). Cut the channel out of the spacer material with a scalpel.
4. 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. Be sure to clean the quartz slide thoroughly with acetone to remove any remnants of adhesive from prior experiments.
5. Wash the coverslip with water, and dry with compressed air. Remove the other side of backing from the imaging spacer, and sandwich it between the quartz slide and the functionalized, streptavidin-coated coverslip, using the plastic tweezers to press the assembly together and remove air bubbles from the adhesive.
6. Insert one 30 cm long polyethylene tubing into each hole in the quartz slide. Be sure to cut the ends of the tubing at an angle to ensure free flow of solution. Use a tube rack or other support to hold the tubes in place, and seal the tubes in place and the edges of the assembled device with epoxy.
   NOTE: It works well to build the device on a piece of parafilm, which can easily be peeled off the bottom when the epoxy has set.
7. Once the epoxy has set, insert the blunt needle on the empty syringe into the outlet tube of the device, and submerge the end of the inlet tube in a container filled with blocking buffer. Pull back on the syringe plunger to fill the device with blocking buffer. Leave the device to incubate for at least 30 min prior to use.

5. Surface tethering of quantum-dot-labeled DNA substrate

NOTE: Besides the microfluidic device, DNA substrate, and blocking buffer described above, see the Table of Materials for other materials and Table 1 for buffers required for the surface tethering of quantum-dot-labeled DNA substrates.

1. 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.
2. Flush the microfluidic channel with fresh blocking buffer by pulling back the syringe plunger after connecting the outlet tube to the syringe pump. Check to make sure there are no bubbles trapped in the tubing or in the channel.
   NOTE: From this point on, make sure that the inlet tube is in liquid so that no air bubbles are introduced into the device.
3. Dilute 1 µL of the prepared DNA substrate into 1 mL of blocking buffer. Place the inlet tube into the diluted DNA substrate, and flow 800 µL of the substrate solution through the channel at a rate of 200 µL/min. Allow the DNA solution to incubate in the channel undisturbed for 15 min after the flow stops.
4. Flow at least 800 µL of blocking buffer through the channel at a rate of 200 µL/min to rinse the unbound DNA out of the channel.
5. Adjust the laser power, microscope focus, and TIRF angle so that the surface-tethered quantum dots are clearly visible.
   NOTE: Although quantum dots do not bleach quickly, it is best to keep the power as low as possible to minimize blinking.

6. REase-mediated DNA cleavage

NOTE: See the Table of Materials for materials and Table 1 for buffers required for REase-mediated DNA cleavage.

1. Make sure that the camera is cooled to optimal operating temperature and set up for high-speed streaming with an exposure time of 0.10 s. Be prepared to collect data for ~4 min.
2. Flush the microfluidic device with 800 µL of experimental buffer without magnesium at a flow rate of 200 µL/min.
3. Add the REase to an aliquot (1 mL) of experimental buffer without magnesium, and mix gently by pipetting. Use 4 µL of 100,000 units/mL stock, which corresponds to 400 units/mL of EcoRV. Flow 800 µL of the diluted enzyme through the channel at a flow rate of 200 µL/min.
4. Start the experiment by flowing experimental buffer containing magnesium and fluorescein at a flow rate of 200 µL/min. Begin capturing data immediately after starting the syringe pump. After flowing 800 µL of buffer, stop data acquisition.

7. Data analysis

NOTE: See the Table of Materials for the data analysis software used for this protocol, and make adjustments if using a different analysis platform.

1. Zero time-point determination
   1. Subtract background fluorescence from each frame of the experimental movie using the imtophat function, a built-in morphological top-hat filtering function in the Image Processing Toolbox. Select a disk with a radius of three pixels as the structuring element. Obtain the background intensity by subtracting the filtered image from the original image.
      NOTE: The filter only retains image features brighter than the background and smaller than the structuring element.
   2. Average the subtracted background over each movie frame. Use a trajectory of this value to determine the zero time-point for the experiment (Figure 3A). Determine the start time by finding the first frame at which the rate of increase exceeds 3x the standard deviation of the rate of change during the dead volume flow.
      NOTE: A sharp increase in the rate of change of the average value of the subtracted background for each movie frame indicates the time at which the final experimental buffer containing the tracer dye entered the flow cell (Figure 3B).
2. Quantum dot intensity trajectory calculation and analysis
   1. Calculate a maximum-intensity projection for the set of background-corrected movie frames recorded prior to the arrival of the tracer dye. Determine the locations of quantum dots to one-pixel accuracy in this projection image using a peak-finding function such as pkfnd.
   2. Generate intensity trajectories for individual quantum dots by calculating the average intensity in each movie frame of a square region three pixels per side surrounding the location returned by the peak-finding function.
      NOTE: The background correction described above removes the artefacts introduced by the tracer dye. The resulting intensity trajectories should be relatively flat, but still include naturally occurring intensity fluctuations (Figure 4).
   3. Identify quantum dot disappearance events by statistical analysis of the intensity trajectories. For each quantum dot intensity trajectory, calculate a threshold intensity that is above the minimum value by an appropriate fraction of the difference between the maximum and minimum values for that trajectory, depending on the noise observed in the trajectories.
      NOTE: The intensity fluctuation for the quantum dot is typically much greater than the background fluctuation. An appropriate value can be determined by comparing these values: the threshold selected must be higher than the highest background value, but ideally be lower than the lowest value for quantum dot fluorescence. For the data presented, the threshold used was calculated to be above the minimum value for each trajectory plus one-third of the difference between the maximum and minimum values for that trajectory. A quantum dot can be deemed to disappear at the last time point that the observed intensity at its location was above this threshold.
   4. Confirm putative disappearance events. Only include events in the final analysis if the observed intensity is above the midpoint between the minimum and maximum intensity values for the trajectory for over half of the movie frames prior to the time of disappearance and the standard deviation of the intensity trajectory decreased after the event.
      NOTE: Other statistical tests may be employed to ensure that only valid disappearance events are logged.