Name: single-molecule dwell-time analysis of restriction endonuclease-mediated dna cleavage
Uploaded: 2021-02-07T14:00:00.000+00:00
Duration: 9 min 53 s
Description: Watch this Scientific Journal Video about Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage at JoVE.com

This work was supported by Award Number K12GM074869 to CME from the National Institute of General Medical Sciences. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institute of General Medical Sciences or the National Institutes of Health.

1. General information
<ol>
	<li>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 &#176;C in 100 mM NaCl.
	<ol>
		<li>Order one oligonucleotide synthesized with a single 5&#39; biotin modification and the other with a 5&#39; 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&#39;&#160;biotin - AAA ACC GAC ATG TTG ATT TCC TGA AAC GGG ATA TCA TCA AAG CCA TGA ACA AAG CAG CCG - 3&#39; 
		5&#39;&#160;thiol - CGG CTG CTT TGT TCA TGG CTT TGA TGA TAT CCC GTT TCA GGA AAT CAA CAT GTC GGT TTT - 3&#39;</li>
	</ol>
	</li>
	<li>Use ultrapure water with 18 MOhm resistivity for all steps.</li>
	<li>Protect all solutions containing quantum dots from light to prevent photobleaching.</li>
	<li>Use a compressed air source to complete this protocol.</li>
</ol>
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.
<ol>
	<li>Reduce 5&#39; thiol groups on the oligonucleotide to be coupled to quantum dots.
	<ol>
		<li>Resuspend each thiolated oligonucleotide in water at a concentration of 100 &#181;M.</li>
		<li>Pipet 650 &#181;L 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.</li>
		<li>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 &#181;L of sodium phosphate buffer into the vial; vortex to mix thoroughly.</li>
		<li>For each resuspended oligonucleotide, prepare a fresh tube, and add 50 &#181;L of the oligonucleotide and 50 &#181;L 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&#39; end of the oligonucleotides.</li>
		<li>Remove the spin column caps, and place each column into the collection tube. Centrifuge the spin columns at 750 &#215; g for 2 min, and discard the eluate.</li>
		<li>Transfer the spin columns into fresh centrifuge tubes. Pipet the entire volume (100 &#181;L) of one sample of DNA/DTT mixture gently onto each prepared column. Centrifuge at 750 &#215; g for 2 min, and measure the absorbance of the eluate at 260 nm to confirm that the concentration is approximately 40 &#181;M.</li>
		<li>Store any samples that will not be used immediately at -20 &#176;C to prevent the oxidation of the thiol groups and the formation of disulfide bonds.</li>
	</ol>
	</li>
	<li>Coupling of DNA to quantum dots
	<ol>
		<li>Pipet one aliquot of 50 &#181;L 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.</li>
		<li>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 &#181;L of CHES buffer into the vial. Vortex to mix thoroughly.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
		<li>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 1/10th the volume of the reduced and purified oligonucleotide sample (approximately 40 &#181;M concentration) as the quantum dot concentration at this point is ~4 &#181;M. Incubate for 2 h at room temperature, with shaking at 1000 rpm.</li>
		<li>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.</li>
		<li>Using a pipettor, carefully recover each sample, place it into a fresh tube, and store at 4 &#176;C. 
		NOTE: Do not store at -20 &#176;C as quantum dots are damaged by freezing.</li>
	</ol>
	</li>
	<li>Annealing of quantum-dot-labeled oligonucleotide to biotinylated oligonucleotide
	<ol>
		<li>Resuspend each biotinylated oligonucleotide in storage buffer at a concentration of 100 &#181;M. Store unused samples at -20 &#176;C.</li>
		<li>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 &#181;M, add 0.2 &#181;L of biotinylated oligonucleotide for every 10 &#181;L of this sample.</li>
		<li>Heat the mixture in a heat block to 75 &#176;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 &#176;C; do not freeze.</li>
	</ol>
	</li>
</ol>
3. Surface functionalization of coverslips
NOTE: This process has been described previously in other JoVE video protocols16,17. 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.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Repeat the sonication in ethanol and KOH solution, rinsing the coverslips in water as described above between each step.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Cure the silanized glass in the oven at 120 &#176;C for 75 min. If not continuing to the next step immediately, store the coverslips under vacuum for maximum of a few days.</li>
	<li>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.</li>
	<li>Pipet 100 &#181;L 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.</li>
	<li>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.</li>
	<li>Store functionalized coverslips under vacuum. Be sure to keep the PEG-treated side up because the other side will not capture the DNA substrate.</li>
</ol>
4. Microfluidic device construction
NOTE: See the Table of Materials for other materials required for the construction of the microfluidic device.
<ol>
	<li>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.</li>
	<li>Combine 25 &#181;L of streptavidin solution with 80 &#181;L of PBS and 20 &#181;L of blocking buffer. Coat a PEG-treated coverslip with this mixture, and incubate in a humid environment for 30 min.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
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.
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>Dilute 1 &#181;L of the prepared DNA substrate into 1 mL of blocking buffer. Place the inlet tube into the diluted DNA substrate, and flow 800 &#181;L of the substrate solution through the channel at a rate of 200 &#181;L/min. Allow the DNA solution to incubate in the channel undisturbed for 15 min after the flow stops.</li>
	<li>Flow at least 800 &#181;L of blocking buffer through the channel at a rate of 200 &#181;L/min to rinse the unbound DNA out of the channel.</li>
	<li>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.</li>
</ol>
6. REase-mediated DNA cleavage
NOTE: See the Table of Materials for materials and Table 1 for buffers required for REase-mediated DNA cleavage.
<ol>
	<li>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.</li>
	<li>Flush the microfluidic device with 800 &#181;L of experimental buffer without magnesium at a flow rate of 200 &#181;L/min.</li>
	<li>Add the REase to an aliquot (1 mL) of experimental buffer without magnesium, and mix gently by pipetting. Use 4 &#181;L of 100,000 units/mL stock, which corresponds to 400 units/mL of EcoRV. Flow 800 &#181;L of the diluted enzyme through the channel at a flow rate of 200 &#181;L/min.</li>
	<li>Start the experiment by flowing experimental buffer containing magnesium and fluorescein at a flow rate of 200 &#181;L/min. Begin capturing data immediately after starting the syringe pump. After flowing 800 &#181;L of buffer, stop data acquisition.</li>
</ol>
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.
<ol>
	<li>Zero time-point determination
	<ol>
		<li>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.</li>
		<li>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).</li>
	</ol>
	</li>
	<li>Quantum dot intensity trajectory calculation and analysis
	<ol>
		<li>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.</li>
		<li>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).</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Roberts, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+restriction+enzymes+became+the+workhorses+of+molecular+biology.">How restriction enzymes became the workhorses of molecular biology.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (17), 5905-5908 (2005).</li><li>Loenen, W. A., Dryden, D. T., Raleigh, E. A., Wilson, G. G., Murray, N. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highlights+of+the+DNA+cutters:+a+short+history+of+the+restriction+enzymes.">Highlights of the DNA cutters: a short history of the restriction enzymes.</a> Nucleic Acids Research. 42 (1), 3-19 (2014).</li><li>Roberts, R. J., Vincze, T., Posfai, J., Macelis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=REBASE--a+database+for+DNA+restriction+and+modification:+enzymes,+genes+and+genomes.">REBASE--a database for DNA restriction and modification: enzymes, genes and genomes.</a> Nucleic Acids Research. 43, 298-299 (2015).</li><li>Bonnet, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sliding+and+jumping+of+single+EcoRV+restriction+enzymes+on+non-cognate+DNA.">Sliding and jumping of single EcoRV restriction enzymes on non-cognate DNA.</a> Nucleic Acids Research. 36 (12), 4118-4127 (2008).</li><li>Biebricher, A., Wende, W., Escude, C., Pingoud, A., Desbiolles, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+of+single+quantum+dot+labeled+EcoRV+sliding+along+DNA+manipulated+by+double+optical+tweezers.">Tracking of single quantum dot labeled EcoRV sliding along DNA manipulated by double optical tweezers.</a> Biophysical Journal. 96 (8), 50-52 (2009).</li><li>Blainey, P. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonspecifically+bound+proteins+spin+while+diffusing+along+DNA.">Nonspecifically bound proteins spin while diffusing along DNA.</a> Nature Structural &amp; Molecular Biology. 16 (12), 1224-1229 (2009).</li><li>Huang, C. -. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualization+of+DNA+recognition+by+restriction+endonuclease+EcoRI.">Direct visualization of DNA recognition by restriction endonuclease EcoRI.</a> Journal of Experimental &amp; Clinical Medicine. 5 (1), 25-29 (2013).</li><li>Palma, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+biomolecular+nanoarrays+for+parallel+single-molecule+investigations.">Selective biomolecular nanoarrays for parallel single-molecule investigations.</a> Journal of American Chemical Society. 133 (20), 7656-7659 (2011).</li><li>Reinhard, B. M., Sheikholeslami, S., Mastroianni, A., Alivisatos, A. P., Liphardt, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+plasmon+coupling+to+reveal+the+dynamics+of+DNA+bending+and+cleavage+by+single+EcoRV+restriction+enzymes.">Use of plasmon coupling to reveal the dynamics of DNA bending and cleavage by single EcoRV restriction enzymes.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (8), 2667-2672 (2007).</li><li>van den Broek, B., Noom, M. C., Wuite, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA-tension+dependence+of+restriction+enzyme+activity+reveals+mechanochemical+properties+of+the+reaction+pathway.">DNA-tension dependence of restriction enzyme activity reveals mechanochemical properties of the reaction pathway.</a> Nucleic Acids Research. 33 (8), 2676-2684 (2005).</li><li>Gambino, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+single+molecule+assay+for+measuring+site-specific+DNA+cleavage.">A single molecule assay for measuring site-specific DNA cleavage.</a> Analytical Biochemistry. 495, 3-5 (2016).</li><li>Floyd, D. L., Harrison, S. C., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+kinetic+intermediates+in+single-particle+dwell-time+distributions.">Analysis of kinetic intermediates in single-particle dwell-time distributions.</a> Biophysical Journal. 99 (2), 360-366 (2010).</li><li>Loparo, J. J., van Oijen, A., Hinterdorfer, P., Oijen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+enzymology.">Single-molecule enzymology.</a> Handbook of Single-Molecule Biophysics. , 165-182 (2009).</li><li>Taylor, J. D., Badcoe, I. G., Clarke, A. R., Halford, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EcoRV+restriction+endonuclease+binds+all+DNA+sequences+with+equal+affinity.">EcoRV restriction endonuclease binds all DNA sequences with equal affinity.</a> Biochemistry. 30 (36), 8743-8753 (1991).</li><li>Pingoud, A., Jeltsch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+function+of+type+II+restriction+endonucleases.">Structure and function of type II restriction endonucleases.</a> Nucleic Acids Research. 29 (18), 3705-3727 (2001).</li><li>Tanner, N. A., Loparo, J. J., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+single-molecule+DNA+replication+with+fluorescence+microscopy.">Visualizing single-molecule DNA replication with fluorescence microscopy.</a> Journal of Visualized Experiments: JoVE. (32), e1529 (2009).</li><li>Kulczyk, A. W., Tanner, N. A., Loparo, J. J., Richardson, C. C., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+observation+of+enzymes+replicating+DNA+using+a+single-molecule+DNA+stretching+assay.">Direct observation of enzymes replicating DNA using a single-molecule DNA stretching assay.</a> Journal of Visualized Experiments: JoVE. (37), e1689 (2010).</li><li>Baldwin, G. S., Vipond, I. B., Halford, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+reaction+analysis+of+the+catalytic+cycle+of+the+EcoRV+restriction+endonuclease.">Rapid reaction analysis of the catalytic cycle of the EcoRV restriction endonuclease.</a> Biochemistry. 34 (2), 705-714 (1995).</li><li>Winkler, F. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+crystal-structure+of+Ecorv+endonuclease+and+of+Its+complexes+with+cognate+and+non-cognate+DNA+fragments.">The crystal-structure of Ecorv endonuclease and of Its complexes with cognate and non-cognate DNA fragments.</a> EMBO Journal. 12 (5), 1781-1795 (1993).</li><li>Sioss, J. A., Stoermer, R. L., Sha, M. Y., Keating, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silica-coated,+Au/Ag+striped+nanowires+for+bioanalysis.">Silica-coated, Au/Ag striped nanowires for bioanalysis.</a> Langmuir. 23 (22), 11334-11341 (2007).</li></ol>

The authors have no competing financial interests or other conflicts of interest

The DNA substrate for this assay is labeled with a quantum dot using a two-step reaction scheme using sulfo-SMCC. This bifunctional crosslinker consists of an NHS ester moiety that can react with a primary amine, and a maleimide moiety that can react with a sulfhydryl group20. The thiolated oligonucleotides used to prepare the substrate are shipped in their oxidized form. It is important to reduce and purify them, as described, before proceeding with the coupling procedure, or the efficiency of the coupling reaction will be reduced. It is critical to prepare fresh DTT and sulfo-SMCC solutions immediately before use. These molecules hydrolyze rapidly in aqueous solution. Use care when transferring solutions into and out of the dialysis devices-if the pipette tip touches the membrane, it will cause a rupture, and the sample will be lost. It is ideal to complete all the steps in section 2 without interruption to avoid sample loss.
The coupling reaction used to generate the quantum-dot-labeled oligonucleotide is inefficient. After combining and annealing the quantum-dot-labeled oligonucleotide with the biotinylated oligonucleotide, there will be excess unlabeled DNA present in the prepared substrate sample. Although these species can bind to the functionalized surface, they are not visible under the imaging conditions recommended and do not contribute to the readout of the experiment. To confirm that cleavage of the completed construct results in the release of quantum dots from the surface, carry out the surface tethering as described in section 5 of the protocol, and image a region of the surface to determine how many quantum dots are present. Then, flow 800 &#181;L of a 1 mL aliquot of 10 U/mL of DNase I in DNase buffer (2.5 mM MgCl2; 10 mM Tris-HCl; 0.1 mM CaCl2, pH 7.6) through the microfluidic channel. Allow the DNase I to incubate in the channel for 15 min, wash with 800 &#181;L of DNase buffer (without enzyme), and image the same region of interest. Approximately 90% of the quantum dots should be released from the surface if the construct and surface are properly prepared.
To covalently attach a mixture of methoxy-terminated and biotin-terminated PEG molecules, glass surfaces must be treated to capture the quantum-dot-labeled DNA substrate on the surface for imaging. It is critically important to carry out all cleaning steps rigorously. Multiple cleaning steps are required to remove surface impurities and to expose hydroxyl groups on the glass surface that will react with the alkoxy groups on the silane to form covalent bonds. While it is important to rinse the slides with ultrapure water between the clean steps using ethanol and KOH solutions, no water should be used after the final cleaning with acetone. The presence of excess water during the silanization step will encourage multimerization of the silane in solution at the expense of surface binding. In addition, the silane solution must not be prepared in advance. To prolong its useful life, the silane stock should be stored in a desiccator cabinet.
The single-molecule cleavage assay is designed to be carried out in a microfluidic device (Figure 2). Fabricate a new device prior to each experiment, reusing the drilled quartz slide that forms the top of the device. This procedure has been described previously16,17 and its variations are widely used for TIRF microscopy. The procedure described herein has been adapted to accommodate 1-inch square slides. It is important to work quickly to build the device so that the functionalized surface does not degrade while it is exposed to air. Epoxy is fluorescent, so take care to avoid covering the channel with it. The drilled quartz slide can be recovered for reuse by soaking the used device in acetone and disassembling it.
This assay is designed to be carried out on an inverted microscope that has through-objective total internal reflection illumination capabilities fitted with an electron-multiplying charge-coupled device (EMCCD) camera for data collection. Use a 60x, high numerical aperture objective, and a 488 nm laser excitation source, and use a filter cube that includes a single-bandpass emission filter and single-edge dichroic suitable for the laser, and an emission filter that permits both the fluorescence of the quantum dots (emission peak at 655 nm) and at least some of the fluorescein fluorescence (emission peak at 512 nm) to pass through. For example, the data presented here was collected with a filter cube fitted with a 482/18 nm single bandpass excitation filter, a 488 nm single edge dichroic, and a 675/67 nm single bandpass emission filter. The assay can be carried out by imaging in a single spectral channel, as described in this protocol. If desired, the assay can also be carried out by splitting the fluorescence image into two spectral channels, one for the quantum dots, another for the fluorescein emission. An image splitter fitted with a 640 nm single edge dichroic mirror would be appropriate for this purpose. However, multichannel imaging is not required.
It is important to wash out any untethered DNA substrates from the channel before beginning the experiment. Fluorescein is included at a concentration of 50 &#181;M in the final buffer to signal the arrival of the magnesium into the field of view. Depending on the microscope setup, it may be necessary to adjust the concentration of fluorescein so that its emission does not obscure the quantum dots. Be sure to start acquiring data immediately after starting the flow of the magnesium-containing buffer. Record data during the lag time before the arrival of the magnesium-containing buffer so that the mean and standard deviation of the rate of change in the background intensity can be calculated to facilitate accurate determination of the zero time-point. Note that the most accurate zero time-point is the first frame at which the rate of change of the background fluorescence exceeds the natural fluctuations in this measurement. The background fluorescence intensity continues to increase after this point as fluorescein diffuses across the boundary layer established by the laminar flow in the channel. Magnesium, which is much smaller than fluorescein, diffuses more rapidly, and its concentration can be expected to reach equilibrium much more rapidly.
For the example data presented, a histogram of all 3393 events that occurred after the zero time-point displays a prominent peak within the first 60 s of the experiments. Many bins corresponding to longer lag times contain no events, and those bins that contain events contain only a few, similar to what is observed when no active REase is present. A nonlinear least-squares curve-fit of the gamma probability distribution to all the binned data resulted in parameter estimates of N = 3.06, with a 95% confidence interval of 2.94-3.18, and &#946; = 7.04 s, with a 95% confidence interval of 6.72-7.36 s. However, the maximum-likelihood estimates for the parameters were N = 1.30 (95% confidence interval: 1.24-1.35) and &#946; = 26.7 s (95% confidence interval: 25.3-28.1 s), and by visual inspection, the curve predicted by the maximum-likelihood parameter estimate does not match the data well (Figure 6B). Furthermore, although the curve predicted by the parameters estimated by nonlinear least-squares fitting follows the shape of the histogram of observed event frequencies relatively well, the residuals (difference between observed and predicted event frequencies) become consistently positive for time-points beyond about 60 s (Figure 6B).
As the frequency of events in the later time-points is comparable to the frequency of quantum dot disappearance observed in the absence of active enzyme, most of these later events are likely to be part of a low background level of quantum dot loss that is not caused by REase-mediated DNA cleavage. It is therefore reasonable to exclude those bins from fitting. When all events that occurred more than 60 s after the zero time-point of each experiment were excluded, the two parameter estimation methods were in agreement (Figure 6A). Therefore, to achieve the best parameter estimates, exclude events outside of the activity peak, and confirm that the maximum-likelihood parameter estimate and the nonlinear least-squares curve-fit results have converged.
The experimental approach outlined in this paper permits real-time observations of REase-mediated DNA cleavage at the single-molecule level. Hundreds of events can be observed in each individual experiment, and the results of several experiments can be pooled to obtain the large number of observations required to characterize the distribution of waiting times by fitting the data to the gamma probability distribution. While this assay can only be used as described with REases that bind but do not cleave DNA in the absence of magnesium or another cofactor, many REases fall into this category. It is important to recognize that the exact details of all intermediate steps will not be revealed by this assay. Steps that are significantly faster than the rate-limiting step will not impact the dwell-time distribution and will not figure into the value of N extracted from the data12. Likewise, the value of &#946; that can be extracted from the data is an approximate average of the waiting time for the steps that do impact the dwell-time distribution. The actual waiting time for each step may vary from the value of &#946;12. However, the single-molecule cleavage assay described in this paper can provide evidence of the presence of multiple intermediate steps along the reaction pathway, even if the reaction intermediates themselves cannot be directly observed.

Restriction endonucleases (REases) are enzymes that effect sequence-specific double-strand breaks in DNA. The discovery of REases in the 1970s led to the development of recombinant DNA technology, and these enzymes are now indispensable laboratory tools for genetic modification and manipulation1. Type II REases are the most widely used enzymes in this class as they cleave DNA at a fixed location either within or near their recognition sequence. However, there is a great deal of variation among the Type II REases, and they are divided into several subtypes based on particular enzymatic properties rather than being classified according to their evolutionary relationships. Among each subtype, there are frequent exceptions to the classification scheme, and many enzymes belong to multiple subtypes2. Thousands of Type II REases have been identified, and hundreds of them are commercially available.
However, in spite of the diversity among the Type II REases, very few REases have been studied in detail. According to REBASE, the restriction enzyme database established by Sir Richard Roberts in 19753, published kinetics data are available for fewer than 20 of these enzymes. Furthermore, while some REases have been directly observed at the single-molecule level while diffusing along the DNA prior to encountering and binding to their recognition sequence4,5,6,7, there are very few single-molecule studies of their cleavage reaction kinetics. The existing studies either do not report adequate statistics to undertake detailed analysis of the variation in the times at which single cleavage events take place8,9,10 or are not capable of capturing the full distribution of cleavage times11. This type of analysis can reveal the presence of relatively long-lived kinetic intermediates and could lead to better understanding of the mechanisms of REase-mediated DNA cleavage.
At the single-molecule level, biochemical processes are stochastic-the waiting time for a single instance of the process to occur, &#964;, is variable. However, many measurements of &#964; can be expected to obey a probability distribution, p(&#964;), that is indicative of the type of process taking place. For instance, a single-step process, such as the release of a product molecule from an enzyme, will obey Poisson statistics, and p(&#964;) will take the form of a negative exponential distribution:
<img alt="Equation 1" src="/files/ftp_upload/62112/62112eq1v3.png" />
where &#946; is the mean waiting time. Note that the rate of the process, k, will be equal to 1/&#946;, the inverse of the mean waiting time. For processes that require more than one step, p(&#964;) will be the convolution of the single-exponential distributions for each of the individual steps. A general solution for the convolution of N single-exponential decay functions with identical mean waiting times, &#946;, is the gamma probability distribution:
<img alt="Equation 2" src="/files/ftp_upload/62112/62112eq2v3.png" />
where &#915;(N) is the gamma function, which describes the interpolation of the factorial of N-1 to non-integer values of N. Although this general solution can be used as an approximation when the mean waiting times of individual steps are similar, it must be understood that the presence of relatively fast steps will be masked by steps with significantly longer waiting times. In other words, the value of N represents a lower limit on the number of steps12. With an adequate number of waiting-time measurements, the parameters &#946; and N can be estimated by binning the events and fitting the gamma distribution to the resulting histogram or by using a maximum-likelihood estimation approach. This type of analysis can therefore reveal the presence of kinetic steps that cannot be easily resolved in ensemble assays and requires a large number of observations to estimate parameters accurately12,13.
This paper describes a method to use quantum-dot-labeled DNA and total internal reflection fluorescence (TIRF) microscopy to observe hundreds of individual REase-mediated DNA cleavage events in parallel. The design of the assay makes it possible to pool the results of several experiments and can create dwell-time distributions containing thousands of events. The high photostability and brightness of quantum dots permit a 10 Hz time resolution without sacrificing the ability to observe cleavage events occurring even many minutes after the start of the experiment. Good temporal resolution and a broad dynamic range, combined with the ability to collect a large data set, allow accurate characterization of the dwell-time distributions to uncover the presence of multiple kinetic steps in the cleavage pathways of REases, which have turnover rates in the 1 min-1 range. In the case of EcoRV, three kinetic steps can be resolved, all of which have been identified through other means, confirming that the assay is sensitive to the presence of such steps.
Duplex DNA substrates containing the recognition sequence of interest are produced by annealing a biotinylated oligonucleotide to a complementary strand labeled with a single, covalently attached semiconductor nanocrystal quantum dot. These substrates are introduced into a flow channel built on top of a glass coverslip with a lawn of high molecular weight polyethylene glycol (PEG) molecules covalently attached to its surface. The DNA substrates are captured via a biotin-streptavidin-biotin linkage by a fraction of the PEG molecules that have a biotin at their free end. In TIRF microscopy, an evanescent wave that decays exponentially with distance from the glass-liquid interface provides illumination; the penetration depth is on the order of the wavelength of the light used. Under these conditions, only quantum dots that are tethered to the surface by a DNA molecule that has been captured on the functionalized glass surface will be excited. Quantum dots that are free in solution will not be constrained within the illuminated region and therefore will not luminesce. If the DNA tethering a quantum dot to the surface is cleaved, that quantum dot will be free to diffuse away from the surface, and it will disappear from the fluorescence image.
Although many Type II REases are known to bind DNA in the absence of magnesium14, all require magnesium to mediate DNA cleavage15. These REases can bind the surface-immobilized DNA in the absence of magnesium. When magnesium-containing buffer is flowed through a channel with REase prebound to the DNA, cleavage begins immediately, as indicated by the disappearance of quantum dots. The synchronization achieved by prebinding the REase molecules, and then initiating DNA cleavage by introducing magnesium, facilitates measurement of the lag time to the completion of DNA cleavage independently for each molecule in the population of enzymes observed in an experiment. Fluorescein is included as a tracer dye in the magnesium-containing buffer to indicate the arrival of magnesium into the field of view. As no enzyme is included in the magnesium-containing buffer, the lag time from the arrival of magnesium-containing buffer to the disappearance of each quantum dot indicates the time it takes for an REase that is already bound to the DNA to cleave the DNA and release the quantum dot from the glass surface. 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. The determination of event times is accomplished by mathematical analysis of intensity trajectories, and a typical experiment results in hundreds of identifiable cleavage events. The results of multiple experiments can be pooled to provide adequate statistics to allow estimation of the parameters, N and &#946;, by either nonlinear least-squares or maximum-likelihood analysis.

<table><tbody><tr><td>3-aminopropyltriethoxysilane (APTS)</td><td>Sigma Aldrich</td><td>440140-100ML</td><td>Store in dessicator box</td></tr><tr><td>5 Minute Epoxy</td><td>Devcon</td><td>20845</td><td>use for sealing the microfluidic device</td></tr><tr><td>acetone</td><td>Pharmco</td><td>329000ACS</td><td>use for cleaning coverslips</td></tr><tr><td>bath sonicator</td><td>Fisher Scientific</td><td>CPXH Model 2800</td><td>catalog number 15-337-410</td></tr><tr><td>Beaker, glass, 100 mL</td><td></td><td></td><td></td></tr><tr><td>Benchtop centrifuge</td><td></td><td></td><td></td></tr><tr><td>Biotin-PEG-Succinimidyl Valerate (MW 5,000)</td><td>Laysan Bio</td><td>BIO-SVA-5K</td><td>Succinimidyl valerate has a longer half-life than succinimidyl carbonate</td></tr><tr><td>biotinylated oligonucleotide</td><td>Integrated DNA Technologies</td><td>custom - see protocol for design considerations</td><td>Request 5&#039; Biotin modification and HPLC purification</td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>VWR</td><td>0903-5G</td><td>prepare a 10 mg/mL solution (aq) and heat to 95 &amp;deg;C before using</td></tr><tr><td>Centri-Spin-10 Size Exclusion Spin Columns</td><td>Princeton Separations</td><td>CS-100 or CS-101</td><td>used to purify thiolated oligonucleotides after reducing the disulfide bond</td></tr><tr><td>Centrifuge tubes 1.5. mL</td><td></td><td></td><td></td></tr><tr><td>Coverslips, 1-inch square glass</td><td></td><td></td><td></td></tr><tr><td>coverslip holders</td><td></td><td></td><td></td></tr><tr><td>diamond point wheel</td><td>Dremel</td><td>7134</td><td>use for drilling holes in quartz flow cell topper</td></tr><tr><td>dithiothreitol (DTT)</td><td>Thermo Scientific</td><td>A39255</td><td>No-Weigh Format, 7.7 mg/vial</td></tr><tr><td>drill press rotary tool workstation stand</td><td>Dremel</td><td>220-01</td><td>facilitates quartz drilling</td></tr><tr><td>EcoRV (REase used to generate example data)</td><td>New England Biolabs</td><td>R0195T or R0195M</td><td>Use 100,000 units/mL stock to avoid adding excess glycerol Check REBASE for suppliers of other REases</td></tr><tr><td>ethanol</td><td>various</td><td>CAS 64-17-5</td><td>denatured or 95% are acceptable, use for cleaning coverslips</td></tr><tr><td>Ethylenediaminetetraacetic acid (EDTA)</td><td>Sigma Aldrich</td><td>EDS</td><td>BioUltra, anhydrous, store in dessicator box</td></tr><tr><td>Flea Micro Spinbar</td><td>Fisherbrand</td><td>14-513-65</td><td>3 mm x 10 mm size to fit beneath coverslip rack</td></tr><tr><td>fluorescein</td><td>Acros Organics</td><td>17324</td><td>use to make experimental buffers</td></tr><tr><td>gravity convection oven</td><td>Binder</td><td>9010-0131</td><td></td></tr><tr><td>handheld rotary multitool</td><td>Dremel</td><td>8220</td><td>use for drilling holes in quartz flow cell topper</td></tr><tr><td>ImagEM X2 EM-CCD Camera</td><td>Hamamatsu</td><td>C9100-23B</td><td>air cooling is adequate for this experiment, use HCImage software or similar to control</td></tr><tr><td>Imaging spacer, double-sided, adhesive</td><td></td><td></td><td></td></tr><tr><td>Jar, glass with screw cap, (approximately 50 mm diameter by 50 mm high)</td><td></td><td></td><td></td></tr><tr><td>magnesium chloride hexahydrate</td><td>Fisher Bioreagents</td><td>BP214-500</td><td>use to make experimental buffer with magnesium</td></tr><tr><td>MATLAB software</td><td></td><td></td><td>Data analysis</td></tr><tr><td>metal tweezers</td><td>Fisher Brand</td><td>16-100-110</td><td></td></tr><tr><td>methoxy-PEG-Succinimidyl Valerate (MW 5,000)</td><td>Laysan Bio</td><td>M-SVA-5K</td><td>Both PEGs should have the same NHS ester so that the rate of reaction is consistent</td></tr><tr><td>microcentrifuge</td><td>Eppendorf</td><td>5424</td><td></td></tr><tr><td>multiposition magnetic stirrer</td><td>VWR</td><td>12621-022</td><td></td></tr><tr><td>N-cyclohexyl-2-aminoethanesulfonic acid (CHES)</td><td>Acros Organics</td><td>AC20818</td><td>CAS 103-47-9, use to make CHES buffer</td></tr><tr><td>orbital shaker and heater for microcentrifuge tubes</td><td>Q Instruments</td><td>1808-0506</td><td>with 1808-1061 adaptor for 24 x 2.0 mL or 15 x 0.5 mL tubes</td></tr><tr><td>Parafilm</td><td></td><td></td><td></td></tr><tr><td>PE60 Polyethylene tubing (inner diameter 0.76 mm, outer diameter 1.22 mm)</td><td>Intramedic</td><td>6258917</td><td>22 G blunt needles are a good fit for this tubing size</td></tr><tr><td>Phosphate-Buffered Saline (PBS) 10x</td><td>Sigma Aldrich</td><td>P7059</td><td>Use at 1x strength</td></tr><tr><td>potassium hydroxide</td><td>VWR Chemicals BDH</td><td>BDH9262</td><td>use a 1 M solution to clean coverslips</td></tr><tr><td>Qdot 655 ITK Amino (PEG) Quantum Dots</td><td>Invitrogen</td><td>Q21521MP</td><td></td></tr><tr><td>Quartz Slide, 1 inch square, 1 mm thick</td><td>Electron Microscopy Sciences</td><td>72250-10</td><td>holes must be drilled in the corners for inlet and outlet tubing insertion</td></tr><tr><td>reinforced plastic tweezers</td><td>Rubis</td><td>K35a</td><td>use for handling coverslips and building microfluidic device</td></tr><tr><td>SecureSeal Adhesive Sheets</td><td>Grace Biolabs</td><td>SA-S-1L</td><td>cut to form spacer for microfluidic device</td></tr><tr><td>Single channel syringe pump for microfluidics</td><td>New Era Pump Systems</td><td>NE-1002X-US</td><td>fitted with a 50 mL syringe and a 22 G blunt needle</td></tr><tr><td>Slide-a-Lyzer MINI Dialysis Devices, 10 kDa MWCO, 0.1 mL</td><td>Thermo Scientific</td><td>69570 or 69572</td><td>used for buffer exchange during quantum dot coupling to DNA</td></tr><tr><td>sodium bicarbonate</td><td>EMD Millipore</td><td>SX0320</td><td>use to make buffer for surface functionalization; 100 mM, pH 8</td></tr><tr><td>sodium chloride</td><td>Macron</td><td>7581-12</td><td>use to make experimental buffers</td></tr><tr><td>Sodium phosphate dibasic solution (BioUltra, 0.5 M in water)</td><td>Sigma Aldrich</td><td>94046</td><td>use to make 100 mM sodium phosphate buffer</td></tr><tr><td>Sodium phosphate monobasic solution (BioUltra, 5M in water)</td><td>Sigma Aldrich</td><td>74092</td><td>use to adjust pH of 100 mM sodium phosphate buffer</td></tr><tr><td>Streptavidin from &lt;em&gt;Streptomyces avidinii&lt;/em&gt;</td><td>Sigma Aldrich</td><td>S4762</td><td>dissolve at 1 mg/mL and store 25 mL aliqouts at -20 ?</td></tr><tr><td>Sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC)</td><td>Thermo Scientific</td><td>A39268</td><td>No-Weigh Format, 2 mg/vial</td></tr><tr><td>Syringe fitted with blunt 21 G needle</td><td></td><td></td><td></td></tr><tr><td>Syringe pump</td><td></td><td></td><td></td></tr><tr><td>thiolated oligonucleotide</td><td>Integrated DNA Technologies</td><td>custom - see protocol for design considerations</td><td>Request 5&#039; Thiol Modifier C6 S-S and HPLC purificaiton</td></tr><tr><td>TIRF imaging system with 488 nm laser illumination</td><td>various</td><td></td><td>custom built</td></tr><tr><td>Tris -HCl</td><td>Research Products International</td><td>T60050</td><td>use to make experimental buffers</td></tr><tr><td>Tris base</td><td>JT Baker</td><td>4101</td><td>use to make experimental buffers</td></tr><tr><td>Tween-20</td><td>Sigma</td><td>P7949</td><td>use to make blocking buffer</td></tr><tr><td>Ultrapure water</td><td></td><td></td><td></td></tr><tr><td>vortex mixer</td><td>VWR</td><td>10153-842</td><td></td></tr><tr><td>Wash-N-Dry Coverslip Rack</td><td>Electron Microscopy Sciences</td><td>70366-16</td><td>used for surface functionalization of coverslips</td></tr></tbody></table>

single-molecule dwell-time analysis of restriction endonuclease-mediated dna cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

The flow cell is directly coupled to a high numerical aperture oil-immersion 60x magnification objective on an inverted microscope equipped with laser illumination for through-objective TIRF imaging (Figure 5A). After introducing the DNA substrate and washing away excess DNA and quantum dots, there are typically thousands of individual quantum dots in a field of view (Figure 5B). These quantum dots are stably attached to the glass surface, and they do not undergo noticeable dimming or significant bleaching over the time scale of the experiment. However, if a buffer containing both magnesium and an appropriate REase is flowed through the flow channel, at the end of a typical four-minute observation period, at least 30% of the quantum dots present at the beginning of an experiment will have disappeared from the field of view. Confirming the magnesium requirement, when the REase is flowed through the channel in the absence of magnesium, at least 95% of quantum dots present at the beginning of the experiment can still be seen at the end of the observation period (Figure 5C). However, when magnesium-containing buffer is flowed immediately after allowing the REase to bind the surface-bound DNA in the absence of magnesium, up to half of the quantum dots will have disappeared by the end of the observation period (Figure 5D), similar to what is observed when REase and magnesium were flowed through the channel together. The exact yield of events will depend on the efficiency of the enzyme under the conditions used. When magnesium-containing buffer is flowed without having previously introduced the appropriate REase into the flow channel, less than 5% of quantum dots disappear during the observation period, and there is no discernible peak for a histogram of the events. This result indicates that prebound REase molecules mediate cleavage of the DNA that tethers the quantum dots to the glass surface, and this DNA cleavage is responsible for the vast majority of observed quantum dot disappearance events in these experiments.
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 (Figure 5E). The determination of event times is accomplished by mathematical analysis of intensity trajectories. Single quantum dots generate intensity trajectories with significantly higher variance than the background under the image conditions used, so putative events are confirmed when the variance of the intensity trajectory decreases to a level comparable to the variance of the background after the observed intensity drop. In addition, trajectories that include a large degree of blinking prior to a putative disappearance event are excluded from the final analysis. However, a typical experiment results in hundreds of events that meet these criteria, and the results of multiple experiments can be pooled to provide adequate statistics to allow estimation of the parameters N and &#946; by either nonlinear least-squares curve-fitting or maximum-likelihood parameter estimation.
The representative data presented were collected by performing this experiment with the well-studied Type IIP REase, EcoRV (Video 1). The 60-bp-long duplex DNA substrate is constructed with a biotin molecule on the 5&#39; end of one strand of the duplex and a quantum dot covalently attached to the 5&#39; end of the other strand. The DNA substrate contains a single copy of the recognition sequence, GAT&#8595;ATC, which is cleaved by EcoRV in the middle, as indicated by the downward arrow (&#8595;). EcoRV was prebound to the DNA substrate in the absence of magnesium, and then magnesium-containing buffer was flowed to initiate DNA cleavage. The representative data include the pooled results of five separate experiments, yielding a total of 3451 observed cleavage events. After excluding events that occurred before the zero time-point or outside of the prominent activity peak, 2987 events remained, which were enough to populate a histogram with one-second bins. Both nonlinear least-squares curve-fitting and maximum-likelihood parameter estimation were used to extract the values of N and &#946; from the data. The two parameter estimation methods were in agreement (Figure 6A), with N = 3.52 (95% confidence interval: 3.32-3.71) and &#946; = 5.78 s (95% confidence interval: 5.41-6.15 s) for the nonlinear least-squares fit and N = 3.41 (95% confidence interval: 3.25-3.58) and &#946; = 6.06 s (95% confidence interval: 5.75-6.39 s) for the maximum-likelihood estimation.
The estimate of at least three kinetic steps is reasonable given what is known about the mechanism of EcoRV. This enzyme is known to bind DNA non-specifically in the absence of magnesium14. Bulk solution phase observations of the changes in the intrinsic fluorescence of tryptophan residues in EcoRV under stopped flow conditions suggested that EcoRV that has bound to DNA in the absence of magnesium must undergo a conformational change prior to the entrance of magnesium into the active site18. This observation is corroborated by crystal structure data19. The results of tryptophan fluorescence studies also indicate that DNA cleavage and product release are separate steps that occur with similar rates18. Therefore, a reasonable reaction mechanism for EcoRV-mediated DNA cleavage in this experiment is:
ES &#8594; ES* &#8594; EP &#8594; E+P
In this mechanism, ES, the initial enzyme-substrate complex, is formed when EcoRV is prebound to the DNA in the absence of magnesium. When magnesium enters the flow cell, the enzyme-substrate complex undergoes a conformational change to become ES*, the activated enzyme-substrate complex. This activated complex then cleaves DNA, but does not immediately release the product molecules, becoming an enzyme-product complex (EP). Finally, the product, P, is released in the final step. This mechanism requires three steps, which is in agreement with the data presented. The resulting estimate for the average waiting time for each step is ~6 s, equivalent to a rate of 0.17 s-1 for each step. This calculation is in general agreement with previous estimates of the rates for these processes-on the order of 0.3-0.4 s-1 for DNA cleavage and product release and ~0.5 s-1 for the conformational rearrangement.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/62112/62112fig01.jpg" /> 
Figure 1: Labeled DNA and enzyme reaction schematic. Quantum-dot-labeled DNA molecules are tethered to the functionalized glass surface via biotin-streptavidin-biotin linkage and observed using TIRF microscopy. The DNA molecules contain the recognition site for the REase of interest. When a DNA molecule is cleaved by the REase, the quantum dot is free to diffuse away from the surface and out of the illumination zone. Abbreviations: REase = restriction endonuclease; PEG = polyethylene glycol; TIRF = total internal reflection fluorescence. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62112/62112fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/62112/62112fig02.jpg" /> 
Figure 2: Microfluidic flow cell device. (A) Exploded view showing the three layers used to create the device: the functionalized glass coverslip on the bottom, the quartz slide with inlet and outlet holes on the top, and the double-sided adhesive imaging spacer with a channel cut into it sandwiched in the middle. (B) A completed device with polyethylene tubes sealed in place and with edges coated with epoxy. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62112/62112fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/62112/62112fig03.jpg" /> 
Figure 3: Zero time-point determination. (A) Average background intensity as determined using the morphological top-hat filtering function over each frame of the movie. The background intensity increases markedly when the experimental buffer containing fluorescein enters the field of view. The results from three different experiments are shown here. There is substantial variation in the lag time from experiment to experiment. (B) The sharp increase in the rate of change of the background fluorescence intensity trajectory facilitates accurate determination of the zero time-point. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62112/62112fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/62112/62112fig04.jpg" /> 
Figure 4: Background correction. The increase in the background intensity due to the fluorescein tracer dye indicating the arrival of magnesium can be seen in the raw fluorescence intensity trajectories for individual quantum dots (grey trajectory). After subtracting background using the morphological top-hat filtering function, the artefacts introduced by the tracer dye have been removed from the trajectory (black trajectory). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62112/62112fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/62112/62112fig05.jpg" /> 
Figure 5: Single-molecule TIRF experiment for parallelized observations of DNA cleavage events. (A) A flow cell constructed on a functionalized glass surface designed to capture quantum-dot-labeled DNA is directly coupled to a high numerical aperture 60x oil immersion microscope objective for TIRF imaging. (B) Representative image from the beginning of an experiment. Quantum-dot-labeled DNA has been loaded into the flow cell, and excess unbound quantum dots have been washed away. Individual DNA-tethered quantum dots can be resolved from one another. (C) The same field of view after buffer containing a REase capable of cleaving the DNA tethers has been flowed through the flow channel in the absence of magnesium for four minutes. There has been no significant loss of DNA-tethered quantum dots. (D) The same field of view at the conclusion of an experiment. Magnesium-containing buffer was flowed through the flow channel immediately after flowing REase in a magnesium-free buffer. This image was acquired after approximately four minutes of buffer flow. Prebound REases have cleaved many of the DNA tethers, releasing the quantum dots from the surface. For ease of viewing, only the central quadrant of the objective field of view is shown in each image. Ten movie frames (equivalent to one second of observation time) were averaged to diminish the effects of quantum dot blinking. Brightness and contrast settings are identical for all three images. (E) Representative fluorescence intensity trajectories from image locations where a quantum dot was present at the beginning of the experiment. Trajectories obtained from image locations corresponding to quantum dots that are not released from the surface (grey) may display brief dips to a lower intensity level, but they begin and end at a high intensity level. Trajectories obtained from image locations, corresponding to quantum dots that are released from the surface (black), display a rapid decrease in intensity to a low background level that is instantaneous with respect to the time resolution of the experiment (10 Hz). Released quantum dots do not reappear within the four-minute observation period. Abbreviations: REase = restriction endonuclease; TIRF = total internal reflection fluorescence. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62112/62112fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/62112/62112fig06.jpg" /> 
Figure 6: Dwell-time distribution analysis of EcoRV-mediated DNA cleavage. (A) Histogram and predicted envelopes for the 2987 cleavage events that occur within the main activity peak in a set of five pooled experiments with EcoRV. The two predicted curves are nearly identical, and the fit residuals (below the histogram) do not indicate systematic error in the nonlinear least-squares fit. (B) Histogram of the entire set of 3393 cleavage events that occurred after the zero time-point. The curve predicted by the maximum-likelihood estimation of the parameters (unbroken line, MLE curve) assuming a gamma probability distribution fails to enclose the histogram. The curve predicted by a nonlinear least-squares fit of the formula for the gamma probability distribution to the bin heights (dashed line, NLS curve) is superior, but the residuals of the fit (below the histogram) reveal systematic error. Abbreviations: MLE: maximum-likelihood estimation; NLS = nonlinear least-squares. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62112/62112fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name of Buffer</td>
			<td>Component</td>
			<td>Concentration</td>
			<td>pH at 25 &#176;C</td>
		</tr>
		<tr>
			<td>sodium phosphate buffer</td>
			<td>sodium phosphate</td>
			<td>100 mM</td>
			<td>8.3</td>
		</tr>
		<tr>
			<td>CHES buffer</td>
			<td>N-cyclohexyl-2-aminoethanesulfonic acid (CHES)</td>
			<td>10 mM</td>
			<td>9.0</td>
		</tr>
		<tr>
			<td rowspan="3">PBS</td>
			<td>sodium phospate</td>
			<td>3 mM</td>
			<td rowspan="3">7.2 - 7.6</td>
		</tr>
		<tr>
			<td>sodium chloride</td>
			<td>150 mM</td>
		</tr>
		<tr>
			<td>potassium phosphate</td>
			<td>1.05 mM</td>
		</tr>
		<tr>
			<td rowspan="3">storage buffer</td>
			<td>sodium chloride</td>
			<td>100 mM</td>
			<td rowspan="3">8.0</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td>bovine serum albumin (BSA)</td>
			<td>0.5 mg/mL</td>
		</tr>
		<tr>
			<td>sodium bicarbonate</td>
			<td>sodium bicarbonate</td>
			<td>100 mM</td>
			<td>8.2</td>
		</tr>
		<tr>
			<td rowspan="5">blocking buffer</td>
			<td>Tris-HCl</td>
			<td>20 mM</td>
			<td rowspan="5">7.5</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>2 mM</td>
		</tr>
		<tr>
			<td>sodium chloride</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>0.005% (v/v)</td>
		</tr>
		<tr>
			<td>bovine serum albumin (BSA)</td>
			<td>0.2 mg/mL</td>
		</tr>
		<tr>
			<td rowspan="3">experimental buffer without magnesium</td>
			<td>sodium chloride</td>
			<td>100 mM</td>
			<td rowspan="3">7.9</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td>dithiothreitol (DTT)</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td rowspan="5">experimental buffer with magnesium</td>
			<td>sodium chloride</td>
			<td>100 mM</td>
			<td rowspan="5">7.9</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>50 mM</td>
		</tr>
		<tr>
			<td>magnesium chloride</td>
			<td>10 mM</td>
		</tr>
		<tr>
			<td>dithiothreitol (DTT)&#160;</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>fluorescein</td>
			<td>adjust according to imaging conditions</td>
		</tr>
		<tr>
			<td rowspan="3">DNase buffer</td>
			<td>Magnesium chloride</td>
			<td>2.5 mM</td>
			<td>7.6</td>
		</tr>
		<tr>
			<td>Tris-HCl</td>
			<td>10 mM</td>
			<td></td>
		</tr>
		<tr>
			<td>calcium chloride</td>
			<td>0.1 mM</td>
			<td></td>
		</tr>
	</tbody>
</table>
Table 1: Table of Buffers.
Video 1: Example single-molecule movie. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/62112/example.mp4" target="_blank">Please click here to download&#160;this video.</a>

Watch this Scientific Journal Video about Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage at JoVE.com

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

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

single-molecule-dwell-time-analysis-restriction-endonuclease-mediated

Nanyang Technological University

Research

JoVE Journal

Biochemistry

1.8K Views.  Tufts University. Met dit protocol kunnen we direct plaatsspecifieke DNA-splitsing waarnemen door restrictie-endonucleasen op het niveau van één molecuul. Onze gemultiplexte aanpak stelt ons in staat om de tijd te meten die honderden individuele REases nodig hebben om de katalytische cyclus te voltooien. Multiplexing stelt ons in staat om goed bevolkte verblijftijd tot splitsingsverdelingen te genereren die we kunnen aanpassen aan de gamma-waarschijnlijkheidsverdeling om inzicht te krijgen in het mechanisme ...

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

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

Chemistry

Characterizing DNA Repair Processes at Transient and Long-lasting Double-strand DNA Breaks by Immunofluorescence Microscopy

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair complexes. This process is regulated by a myriad of proteins that promote the association and disassociation of proteins to these lesions. Thanks in large part to the ability to perform functional screens of a vast library of proteins, there is a greater appreciation of the genes necessary for the double-strand DNA break repair. Often knockout or chemical inhibitor screens identify proteins involved in repair processes by using increased toxicity as a marker for a protein that is required for DSB repair. Although useful for identifying novel cellular proteins involved in maintaining genome fidelity, functional analysis requires the determination of whether the protein of interest promotes localization, formation, or resolution of repair complexes. 
The accumulation of repair proteins can be readily detected as distinct nuclear foci by immunofluorescence microscopy. Thus, association and disassociation of these proteins at sites of DNA damage can be accessed by observing these nuclear foci at representative intervals after the induction of double-strand DNA breaks. This approach can also identify mis-localized repair factor proteins, if repair defects do not simultaneously occur with incomplete delays in repair. In this scenario, long-lasting double-strand DNA breaks can be engineered by expressing a rare cutting endonuclease (e.g., I-SceI) in cells where the recognition site for the said enzyme has been integrated into the cellular genome. The resulting lesion is particularly hard to resolve as faithful repair will reintroduce the enzyme's recognition site, prompting another round of cleavage. As a result, differences in the kinetics of repair are eliminated. If repair complexes are not formed, localization has been impeded. This protocol describes the methodology necessary to identify changes in repair kinetics as well as repair protein localization.

Repair of double-strand DNA breaks is a dynamic process, requiring not only formation of repair complexes at the breaks, but also their resolution after the lesion is addressed. Here, we use immunofluorescence microscopy for transient and long-lasting double-stranded breaks as a tool to dissect this genome maintenance mechanism.

The repair of double-stranded breaks (DSBs) in DNA is a highly coordinated process, necessitating the formation and resolution of multi-protein repair ...

Cancer Research

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) and captures double-stranded DNA (dsDNA) non-specifically to interrogate it for a homologous sequence. After encountering homology, Rad51 catalyzes DNA strand exchange to mediate pairing of the ssDNA with the complementary strand of the dsDNA. This reaction is highly regulated by numerous accessary proteins in vivo. Although conventional biochemical assays have been successfully employed to examine the role of such accessory protein in vitro, kinetic analysis of intermediate formation and its progression into a final product has proven challenging due to the unstable and transient nature of the reaction intermediates. To observe these reaction steps directly in solution, fluorescence resonance energy transfer (FRET)-based real-time observation systems of this reaction were established. Kinetic analysis of real-time observations shows that the DNA strand exchange reaction mediated by Rad51 obeys a three-step reaction model involving the formation of a three-strand DNA intermediate, maturation of this intermediate, and the release of ssDNA from the mature intermediate. The Swi5-Sfr1 complex, an accessary protein conserved in eukaryotes, strongly enhances the second and third steps of this reaction. The FRET-based assays presented here enable us to uncover the molecular mechanisms through which recombination accessary proteins stimulate the DNA strand exchange activity of Rad51. The primary goal of this protocol is to enhance the repertoire of techniques available to researchers in the field of homologous recombination, particularly those working with proteins from species other than Schizosaccharomyces pombe, so that the evolutionary conservation of the findings presented herein can be determined.

Fluorescence resonance energy transfer-based real-time observation systems of the DNA strand exchange reaction mediated by Rad51 were developed. Using the protocols presented here, we are able to detect the formation of reaction intermediates and their conversion into products, while also analyzing the enzymatic kinetics of the reaction.

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament ...

In Vitro Directed Evolution of a Restriction Endonuclease with More Stringent Specificity

Restriction endonuclease (REase) specificity engineering is extremely difficult. Here we describe a multistep protocol that helps to produce REase variants that have more stringent specificity than the parental enzyme. The protocol requires the creation of a library of expression selection cassettes (ESCs) for variants of the REase, ideally with variability in positions likely to affect DNA binding. The ESC is flanked on one side by a sequence for the restriction site activity desired and a biotin tag and on the other side by a restriction site for the undesired activity and a primer annealing site. The ESCs are transcribed and translated in a water-in-oil emulsion, in conditions that make the presence of more than one DNA molecule per droplet unlikely. Therefore, the DNA in each cassette molecule is subjected only to the activity of the translated, encoded enzyme. REase variants of the desired specificity remove the biotin tag but not the primer annealing site. After breaking the emulsion, the DNA molecules are subjected to a biotin pulldown, and only those in the supernatant are retained. This step assures that only ESCs for variants that have not lost the desired activity are retained. These DNA molecules are then subjected to a first PCR reaction. Cleavage in the undesired sequence cuts off the primer binding site for one of the primers. Therefore, PCR amplifies only ESCs from droplets without the undesired activity. A second PCR reaction is then carried out to reintroduce the restriction site for the desired specificity and the biotin tag, so that the selection step can be reiterated. Selected open reading frames can be overexpressed in bacterial cells that also express the cognate methyltransferase of the parental REase, because the newly evolved REase targets only a subset of the methyltransferase target sites.

Restriction endonucleases with new sequence specificity can be developed from enzymes recognizing a partially degenerate sequence. Here we provide a detailed protocol that we successfully used to alter the sequence specificity of NlaIV enzyme. Key ingredients of the protocol are the in vitro compartmentalization of the transcription/translation reaction and selection of variants with new sequence specificities.

Restriction endonuclease (REase) specificity engineering is extremely difficult. Here we describe a multistep protocol that helps to produce REase ...

Genetics

DNA Curtains Shed Light on Complex Molecular Systems During Homologous Recombination

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in HR are closely associated with a loss of genome integrity and oncogenic transformation in human cells. HR involves coordinated actions of a complex set of proteins, many of which remain poorly understood. The key aspect of the research described here is a technology called "DNA curtains", a technique which allows for the assembly of aligned DNA molecules on the surface of a microfluidic sample chamber. They can then be visualized by total internal reflection fluorescence microscopy (TIRFM). DNA curtains was pioneered by our laboratory and allows for direct access to spatiotemporal information at millisecond time scales and nanometer scale resolution, which cannot be easily revealed through other methodologies. A major advantage of DNA curtains is that it simplifies the collection of statistically relevant data from single molecule experiments. This research continues to yield new insights into how cells regulate and preserve genome integrity.

DNA curtains present a novel method for visualizing hundreds or even thousands of DNA-binding proteins in real-time as they interact with DNA molecules aligned on the surface of a microfluidic sample chamber.

Homologous recombination (HR) is important for the repair of double-stranded DNA breaks (DSBs) and stalled replication forks in all organisms. Defects in ...

Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. Linearized &lambda; DNA is modified to have a biotin on the end of one strand, and a digoxigenin moiety on the other end of the same strand. The biotinylated end is attached to a functionalized glass coverslip and the digoxigeninated end to a small bead. The assembly of these DNA-bead tethers on the surface of a flow cell allows a laminar flow to be applied to exert a drag force on the bead. As a result, the DNA is stretched close to and parallel to the surface of the coverslip at a force that is determined by the flow rate (Figure 1). The length of the DNA is measured by monitoring the position of the bead. Length differences between single- and double-stranded DNA are utilized to obtain real-time information on the activity of the replication proteins at the fork. Measuring the position of the bead allows precise determination of the rates and processivities of DNA unwinding and polymerization (Figure 2).

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system.

We describe a method for observing real time replication of individual DNA molecules mediated by proteins of the bacteriophage replication system. ...

Biology

CMG-Mediated DNA Unwinding Observed by ssDNA-RPA Tract Growth

Single-Molecule Real-Time Visualization of DNA Unwinding by CMG Helicase

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a dynamic complex of proteins termed the replisome. At the heart of the replisome is the&#160;CDC45-MCM2-7-GINS (CMG) helicase, which separates the two strands of the DNA double helix such that DNA polymerases can copy each strand. During genome duplication, replisomes must overcome a plethora of obstacles and challenges. Each of these threatens genome stability, as failure to replicate DNA completely and accurately can lead to mutations, diseases, or cell death. Therefore, it is of great interest to understand how CMG functions in the replisome during both normal replication and replication stress. Here, we describe a total internal reflection fluorescence (TIRF) microscopy assay using recombinant purified proteins, which allows for real-time visualization of surface-tethered stretched DNA molecules by individual CMG complexes. This assay provides a powerful platform to investigate CMG behavior at the single-molecule level, allowing helicase dynamics to be directly observed with real-time control over reaction conditions.

Author Spotlight: Unraveling the Dynamics of Eukaryotic DNA Replication Through Single-Molecule Visualization

This protocol demonstrates performing a single-molecule assay for live visualization of DNA unwinding by CMG helicase. It describes (1) preparing a DNA substrate, (2) purifying fluorescently labeled Drosophila melanogaster CMG helicase, (3) preparing a microfluidic flow cell for total internal reflection fluorescence (TIRF) microscopy, and (4) the single-molecule DNA unwinding assay.

Faithful genome duplication is essential for preserving the genetic stability of dividing cells. DNA replication is carried out during the S phase by a ...

Quantitative Analysis of RPA-ssDNA Binding Kinetics Using Label-Free Methods

Analyzing DNA-Protein Interactions with Streptavidin-Based Biolayer Interferometry

Protein-DNA interactions underpin essential cellular processes. Understanding these interactions is critical for elucidating the molecular mechanisms of various pathways. Key factors such as the structure, sequence, and length of a DNA molecule can significantly influence protein binding. Bio-layer interferometry (BLI) is a label-free technique that measures binding kinetics between molecules, offering a straightforward and precise approach to quantitatively study protein-DNA interactions. A major advantage of BLI over traditional gel-based methods is its ability to provide real-time data on binding kinetics, enabling accurate measurement of the equilibrium dissociation constant (KD) for dynamic protein-DNA interactions. This article presents a basic protocol for determining the KD value of the interaction between a DNA replication protein, replication protein A (RPA), and a single-stranded DNA (ssDNA) substrate. RPA binds ssDNA with high affinity but must also be easily displaced to facilitate subsequent protein interactions within biological pathways. In the described BLI assay, biotinylated ssDNA is immobilized on a streptavidin-coated biosensor. The binding kinetics (association and dissociation) of RPA to the biosensor-bound DNA are then measured. The resulting data are analyzed to derive precise values for the association rate constant (ka), dissociation rate constant (kd), and equilibrium binding constant (KD) using system software.

This article describes a protocol for studying DNA-protein interactions using a streptavidin-based biolayer interferometry (BLI) system. It outlines the essential steps and considerations for utilizing either basic or advanced binding kinetics to determine the equilibrium binding affinity (KD) of the interaction.

Protein-DNA interactions underpin essential cellular processes. Understanding these interactions is critical for elucidating the molecular mechanisms of ...

A Fluorescence-based Exonuclease Assay to Characterize DmWRNexo, Orthologue of Human Progeroid WRN Exonuclease, and Its Application to Other Nucleases

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. It is likely to play a role in preventing accumulation of recombinogenic intermediates that would otherwise accumulate at transiently stalled replication forks, consistent with a hyper-recombinant phenotype of cells lacking WRN. In humans, the exonuclease domain comprises an N-terminal portion of a much larger protein that also possesses helicase activity, together with additional sites important for DNA and protein interaction. By contrast, in Drosophila, the exonuclease activity of WRN (DmWRNexo) is encoded by a distinct genetic locus from the presumptive helicase, allowing biochemical (and genetic) dissection of the role of the exonuclease activity in genome stability mechanisms. Here, we demonstrate a fluorescent method to determine WRN exonuclease activity using purified recombinant DmWRNexo and end-labeled fluorescent oligonucleotides. This system allows greater reproducibility than radioactive assays as the substrate oligonucleotides remain stable for months, and provides a safer and relatively rapid method for detailed analysis of nuclease activity, permitting determination of nuclease polarity, processivity, and substrate preferences.

Exonucleases play critical roles in ensuring genome stability. Loss of WRN exonuclease function results in premature aging. Studying substrates and other requirements of the nuclease in vitro can help elucidate its role in vivo. Here we demonstrate a rapid and reproducible fluorescence-based assay to measure its nuclease activity.

WRN exonuclease is involved in resolving DNA damage that occurs either during DNA replication or following exposure to endogenous or exogenous genotoxins. ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video