Name: single-molecule dwell-time analysis of restriction endonuclease-mediated dna cleavage
Uploaded: 2021-02-07T14:00:00
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 ar...

<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 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...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#39; 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 &#176;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 Streptomyces avidinii</td>
			<td>Sigma Aldrich</td>
			<td>S4762</td>
			<td>dissolve at 1 mg/mL and store 25 mL aliqouts at -20 &#8451;</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&#39; 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 underg...

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 ...

With this protocol, we can directly observe site-specific DNA cleavage by restriction endonucleases at the single molecule level. Our multiplexed approach allows us to measure the time it takes for hundreds of individual REases to complete the catalytic cycle. Multiplexing allows us to generate well-populated dwell time to cleavage distributions that we can fit with the gamma probability distribution to gain insights into the mechanism of DNA cleavage.Begin by preparing a fresh tube for each resuspended oligonucleotide at 50 microliters of the oligonucleotide and 50 microliters of freshly prepared DTT solution to each tube, pipette up and down to mix and incubate for 30 minutes at room temperature. Pipette the entire volume of each sample mixture gently onto a prepared column and centrifuge at 750G for two minutes. After centrifugation, store any samples that will not be used immediately at 20 degrees Celsius to prevent oxidation of the thiol groups and formation of disulfide bonds.Pipette one aliquot of 50 microliters of the quantum dot stock into a dialysis device for each construct to be made, making sure not to touch the membrane with the pipette tip. Dialyze against a volume of CHES buffer that is at least 1, 000 times the sample volume with stirring at 100 RPM for 15 minutes. Using a pipetter, carefully retrieve the suspended quantum dots from the dialysis device and transfer them into a fresh tube containing an equal volume of freshly prepared Sulfo-SMCC solution, pipetting up and down to mix.Incubate for 1 hour at room temperature with shaking at 1, 000 RPM to allow the Sulfo-SMCC to react with the primary amines on the quantum dots. Then, use a pipetter to carefully transfer each sample into a fresh dialysis device. To remove excess Sulfo-SMCC, dialyze against a volume of CHES buffer that is at least 1, 000 times the volume contained in the dialysis device with stirring for 15 minutes.Exchange the buffer twice and perform dialysis with fresh CHES buffer three times in total, allowing dialysis to proceed for 15 minutes after each buffer exchange. Then repeat the dialysis with PBS. Using a pipetter, carefully recover the solution containing the quantum dots from the dialysis device and transfer each sample into a fresh tube containing an equimolar amount of dilated oligonucleotide diluted in PBS.Incubate for 2 hours at room temperature with shaking at 1, 000 RPM. Add BSA to each sample for a final concentration of 0.5 milligrams per milliliter. Then use a pipetter to carefully transfer each sample into a fresh dialysis device and dialyze against storage buffer.After dialysis, carefully recover each sample and place it into a fresh tube. Store the samples at 4 degrees Celsius. Combine a sample of quantum dot labeled oligonucleotides with a tenfold molar excess of biotinylated oligonucleotide.Heat the mixture in a heat block to 75 degrees Celsius and hold it at that temperature for 5 minutes. Then turn off the heat block and allow the mixture to cool slowly. Store the completed construct at 4 degrees Celsius Using a handheld rotary multi-tool fitted with a tapered diamond point wheel bit, drill two holes in opposite corners of the quartz slide to serve as an inlet and outlet.Be sure to secure the slide in place, lubricate the bit and slide constantly with water while drilling. After each experiment, recover the prepared quartz slide by soaking the used device in acetone to dissolve the adhesive and epoxy. Discard the remaining components.Combine 25 microliters of streptavidin solution with 80 microliters of PBS and 20 microliters of blocking buffer. Coat a PEG-treated cover slip with this mixture, and incubate in a humid environment for 30 minutes. During the incubation of the cover slip, prepare the imaging spacer to create a flow channel.Cut a 1 inch square piece of imaging spacer material, then mark a 2 millimeter wide channel aligned to the ends of the holes drilled in the quartz slide. Cut the channel out of the spacer material with a scalpel. Clean the quartz slide thoroughly with acetone to remove any remnants of adhesive from prior experiments.Peel one side of the backing from the image spacer and carefully place it onto the quartz slide, taking care not to cover the inlet and outlet holes. Wash the cover slip with water and dry with compressed air. Remove the other side of the backing from the imaging spacer and sandwich it between the quartz slide and the functionalized streptavidin coated cover slip using plastic tweezers to press the assembly together and remove air bubbles from the adhesive.Cut the end to the tubing at an angle to ensure free flow of solution. Then insert 30 centimeter long polyethylene tubing into each hole in the quartz slide. Hold the tubes in place with a tube rack, then use epoxy to seal the tubes in place and the edges of the assembled device.Once the epoxy has been set, insert the blunt needle on an empty syringe into the outlet tube of the device and submerge the end of the inlet tube in a container filled with the blocking buffer. Pull back on the syringe plunger to fill the device with blocking buffer. Then leave the device to incubate for at least 30 minutes prior to use.Attach the microfluidic device to the microscope stage plate with tape. Bring the objective into contact with the bottom of the device and position the objective so that the field of view is within the microfluidic channel. After connecting the outlet tube to the syringe pump, flush the microfluidic channel with fresh blocking buffer by pulling back the syringe plunger.Check to make sure there are no bubbles trapped in the tubing or in the channel. Dilute 1 microliter of the prepared DNA substrate into 1 milliliter of blocking buffer. Place the inlet tube into the diluted DNA substrate and flow 800 microliters of the substrate solution through the channel at a rate of 200 microliters per minute.Allow the DNA solution to incubate in the channel for 15 minutes after the flow stops. After the incubation, flow at least 800 microliters of blocking buffer through the channel at a rate of 200 microliters per minute to rinse the unbound DNA out of the channel. Adjust the microscope focus so that the surface-tethered quantum dots are clearly visible.Flush the microfluidic device with 800 microliters of experimental buffer without magnesium at a flow rate of 200 microliters per minute. Add the REase to an aliquot of experimental buffer without magnesium and mix gently by pipetting. Flow 800 microliters of 400 units per milliliter of EcoRV through the channel at a flow rate of 200 microliters per minute.Start the experiment by flowing the experimental buffer containing magnesium and fluorescein at a flow rate of 200 microliters per minute. Begin capturing data immediately after starting the syringe pump. Stop data acquisition after flowing 800 microliters of buffer.After introducing the DNA substrate into the flow cell and washing away excess DNA and quantum dots, there are typically thousands of individual quantum dots in a field of view. They are stably attached to the glass surface and do not undergo noticeable dimming or significant bleaching throughout the experiment. When the REase has flowed through the channel in the absence of magnesium, at least 95%of the quantum dots present at the beginning of the experiment can still be seen at the end of the observation period.However, when the magnesium-containing buffer is flowed immediately after the REase, up to half of the quantum dots disappear by the end of the observation period, similar to what is observed when REase and magnesium are flowed through the channel together. Quantum dot disappearance happens quickly and results in a sharp decrease in the intensity trajectory, providing a clear indication of the time at which a given DNA molecule is cleaved. This experiment was performed with the well-studied type II P REase EcoRV.Both non-linear least squares curve fitting and maximum likelihood parameter estimation were used to extract the values of n and beta from the data. The two parameter estimation methods were in agreement. Using the non-linear least squares fit, n was 3.52 and beta was 5.78 seconds.Using the maximum likelihood estimation, n was 3.41 and beta was 6.06 seconds. This assay can provide evidence of the presence of intermediate steps along the cleavage pathway. Performing this assay under changing reaction conditions can help establish the nature of reaction intermediates that cannot be directly observed.

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

Research

JoVE Journal

Biochemistry

A Simple, Robust, and High Throughput Single Molecule Flow Stretching Assay Implementation for Studying Transport of Molecules Along DNA

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, glass coverslips are functionalized in a one-step reaction with silane-PEG-biotin. Flow cells are constructed by sandwiching an adhesive tape with pre-cut channels between a functionalized coverslip and a PDMS slab containing inlet and outlet holes. Multiple channels are integrated into one flow cell and the flow of reagents into each channel can be fully automated, which significantly increases the assay throughput and reduces hands-on time per assay. Inside each channel, biotin-&#955;-DNAs are immobilized on the surface and a laminar flow is applied to flow-stretch the DNAs. The DNA molecules are stretched to &#62;80% of their contour length and serve as spatially extended templates for studying the binding and transport activity of fluorescently labeled molecules. The trajectories of single molecules are tracked by time-lapse Total Internal Reflection Fluorescence (TIRF) imaging. Raw images are analyzed using streamlined custom single particle tracking software to automatically identify trajectories of single molecules diffusing along DNA and estimate their 1D diffusion constants.

This protocol demonstrates a simple, robust and high throughput single molecule flow-stretching assay for studying one-dimensional (1D) diffusion of molecules along DNA.

We describe a simple, robust and high throughput single molecule flow-stretching assay for studying 1D diffusion of molecules along DNA. In this assay, ...

Determining if DNA Stained with a Cyanine Dye Can Be Digested with Restriction Enzymes

Visualization of DNA for fluorescence microscopy utilizes a variety of dyes such as cyanine dyes. These dyes are utilized due to their high affinity and sensitivity for DNA. In order to determine if the DNA molecules are full length after the completion of the experiment, a method is required to determine if the stained molecules are full length by digesting DNA with restriction enzymes. However, stained DNA may inhibit the enzymes, so a method is needed to determine what enzymes one could use for fluorochrome stained DNA. In this method, DNA is stained with a cyanine dye overnight to allow the dye and DNA to equilibrate. Next, stained DNA is digested with a restriction enzyme, loaded into a gel and electrophoresed. The experimental DNA digest bands are compared to an in silico digest to determine the restriction enzyme activity. If there is the same number of bands as expected, then the reaction is complete. More bands than expected indicate partial digestion and less bands indicate incomplete digestion. The advantage of this method is its simplicity and it uses equipment that a scientist would need for a restriction enzyme assay and gel electrophoresis. A limitation of this method is that the enzymes available to most scientists are commercially available enzymes; however, any restriction enzymes could be used.

Staining DNA molecules for fluorescence microscopy allows a scientist to view them during an experiment. In the method presented here, DNA molecules are pre-stained with fluorescent dyes and digested with methylation and non-methylation sensitive restriction enzymes.

Visualization of DNA for fluorescence microscopy utilizes a variety of dyes such as cyanine dyes. These dyes are utilized due to their high affinity and ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Production of Dynein and Kinesin Motor Ensembles on DNA Origami Nanostructures for Single Molecule Observation

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and others. Many of these functions require motors to operate in ensembles. Despite a wealth of knowledge about the mechanisms of individual cytoskeletal motors, comparatively less is known about the mechanisms and emergent behaviors of motor ensembles, examples of which include changes to ensemble processivity and velocity with changing motor number, location, and configuration. Structural DNA nanotechnology, and the specific technique of DNA origami, enables the molecular construction of well-defined architectures of motor ensembles. The shape of cargo structures as well as the type, number and placement of motors on the structure can all be controlled. Here, we provide detailed protocols for producing these ensembles and observing them using total internal reflection fluorescence microscopy. Although these techniques have been specifically applied for cytoskeletal motors, the methods are generalizable to other proteins that assemble in complexes to accomplish their tasks. Overall, the DNA origami method for creating well-defined ensembles of motor proteins provides a powerful tool for dissecting the mechanisms that lead to emergent motile behavior.

The goal of this protocol is to form ensembles of molecular motors on DNA origami nanostructures and observe the ensemble motility using total internal reflection fluorescence microscopy.

Cytoskeletal motors are responsible for a wide variety of functions in eukaryotic cells, including mitosis, cargo transport, cellular motility, and ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved ...

Imaging the Motion of Fully Reconstituted Cdc45/Mcm2-7/GINS (CMG)

Hybrid Ensemble and Single-molecule Assay to Image the Motion of Fully Reconstituted CMG

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication forks. Obtaining a deep mechanistic understanding of the dynamics of CMG is critical to elucidating how cells achieve the enormous task of efficiently and accurately replicating their entire genome once per cell cycle. Single-molecule techniques are uniquely suited to quantify the dynamics of CMG due to their unparalleled temporal and spatial resolution. Nevertheless, single-molecule studies of CMG motion have thus far relied on pre-formed CMG purified from cells as a complex, which precludes the study of the steps leading up to its activation. Here, we describe a hybrid ensemble and single-molecule assay that allowed imaging at the single-molecule level of the motion of fluorescently labeled CMG after fully reconstituting its assembly and activation from 36 different purified S. cerevisiae polypeptides. This assay relies on the double functionalization of the ends of a linear DNA substrate with two orthogonal attachment moieties, and can be adapted to study similarly complex DNA-processing mechanisms at the single-molecule level.

Author Spotlight: Investigating the Motion Dynamics of the Eukaryotic Replisome Components at the Single-Molecule Level

We report a hybrid ensemble and single-molecule assay to directly image and quantify the motion of fluorescently labeled, fully reconstituted Cdc45/Mcm2-7/GINS (CMG) helicase on linear DNA molecules held in place in an optical trap.

Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication ...

Studying TRF1/2 Interactions with Telomeric DNA Using Single-Molecule Assay

Analyzing Telomeric Protein-DNA Interactions Using Single-Molecule Magnetic Tweezers

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper function depends on tightly regulated processes of replication, elongation, and damage response. The shelterin complex, especially Telomere Repeat-binding Factor 1 (TRF1) and TRF2, plays a pivotal role in telomere protection and has emerged as a potential anti-cancer target for drug discovery. These proteins bind to the repetitive telomeric DNA motif TTAGGG, facilitating the formation of protective structures and recruitment of other telomeric proteins. Structural methods and advanced imaging techniques have provided insights into telomeric protein-DNA interactions, but probing the dynamic processes requires single-molecule approaches. Tools like magnetic tweezers, optical tweezers, and atomic force microscopy (AFM) have been employed to study telomeric protein-DNA interactions, revealing important details such as TRF2-dependent DNA distortion and telomerase catalysis. However, the preparation of single-molecule constructs with telomeric repetitive motifs continues to be a challenging task, potentially limiting the breadth of studies utilizing single-molecule mechanical methods. To address this, we developed a method to study interactions using full-length human telomeric DNA with magnetic tweezers. This protocol describes how to express and purify TRF2, prepare telomeric DNA, set up single-molecule mechanical assays, and analyze data. This detailed guide will benefit researchers in telomere biology and telomere-targeted drug discovery.

Author Spotlight: Advanced Single-Molecule Techniques for Investigating Telomeric Protein-DNA Interactions

This protocol demonstrates using single-molecule magnetic tweezers to study interactions between telomeric DNA-binding proteins (Telomere Repeat-binding Factor 1 [TRF1] and TRF2) and long telomeres extracted from human cells. It describes the preparatory steps for telomeres and telomeric repeat-binding factors, the execution of single-molecule experiments, and the data collection and analysis methods.