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

Summary

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.

Abstract

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↓ATC (where ↓ 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.

Introduction

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 sequence^4,5,6,7, 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 place^8,9,10 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, τ, is variable. However, many measurements of τ can be expected to obey a probability distribution, p(τ), 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(τ) will take the form of a negative exponential distribution:

where β is the mean waiting time. Note that the rate of the process, k, will be equal to 1/β, the inverse of the mean waiting time. For processes that require more than one step, p(τ) 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, β, is the gamma probability distribution:

where Γ(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 β 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 accurately^12,13.

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 β, by either nonlinear least-squares or maximum-likelihood analysis.

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Protocol

1. General information

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

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

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

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

3. Surface functionalization of coverslips

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

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

4. Microfluidic device construction

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

1. Using a handheld rotary multitool fitted with a tapered diamond point wheel bit, drill two holes in opposite corners of the quartz slide to serve as an inlet and outlet. Be sure to secure the slide in place, lubricate the bit, and slide constantly with water while drilling. After each experiment, recover the prepared quartz slide for reuse by soaking a used device in acetone to dissolve the adhesive and epoxy; discard the remaining components.
   NOTE: The drilling can be carried out by hand, but the use of a press-style holder for the multitool makes the process easier.
2. Combine 25 µL of streptavidin solution with 80 µL of PBS and 20 µL of blocking buffer. Coat a PEG-treated coverslip with this mixture, and incubate in a humid environment for 30 min.
3. During the incubation of the coverslip, prepare the imaging spacer to create a flow channel. Cut a 1-inch square piece of imaging spacer material, then mark a 2 mm wide channel aligned to the ends of the holes drilled in the quartz slide (Figure 2). Cut the channel out of the spacer material with a scalpel.
4. Peel one side of the backing from the image spacer, and carefully place it onto the quartz slide, taking care not to cover the inlet and outlet holes. Be sure to clean the quartz slide thoroughly with acetone to remove any remnants of adhesive from prior experiments.
5. Wash the coverslip with water, and dry with compressed air. Remove the other side of backing from the imaging spacer, and sandwich it between the quartz slide and the functionalized, streptavidin-coated coverslip, using the plastic tweezers to press the assembly together and remove air bubbles from the adhesive.
6. Insert one 30 cm long polyethylene tubing into each hole in the quartz slide. Be sure to cut the ends of the tubing at an angle to ensure free flow of solution. Use a tube rack or other support to hold the tubes in place, and seal the tubes in place and the edges of the assembled device with epoxy.
   NOTE: It works well to build the device on a piece of parafilm, which can easily be peeled off the bottom when the epoxy has set.
7. Once the epoxy has set, insert the blunt needle on the empty syringe into the outlet tube of the device, and submerge the end of the inlet tube in a container filled with blocking buffer. Pull back on the syringe plunger to fill the device with blocking buffer. Leave the device to incubate for at least 30 min prior to use.

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

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

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

6. REase-mediated DNA cleavage

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

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

7. Data analysis

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

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

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Results

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 underg...

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Discussion

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 th...

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Materials

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