This work was supported by the Sogang University Research Grant of 201410036.

1. DNA Biotinylation
<ol>
	<li>Biotinylation of blunt ended DNA using Terminal Transferase (TdT) 
	Note: Use T4 DNA (166 kbp), which is a blunt ended DNA.
	<ol>
		<li>Add 5 &#181;l of 2.5 mM CoCl2, 5 &#181;l of 10&#215; reaction buffer, 0.5 &#181;l of 10 mM biotin-11-dUTP, 0.5 &#181;l of terminal transferase (10 units), and 0.5 &#181;l of T4 DNA (0.5 &#181;g/&#181;l) to the reaction mixture. Make to a final volume of 50 &#181;l by adding 38.5 &#181;l of water.</li>
		<li>Incubate the reaction mixture at 37 &#176;C for 1 hr. 
		Note: Extended reaction time may yield double-tethered DNA, i.e., it is possible that biotins are tagged at both ends.</li>
		<li>Stop the reaction by adding 5 &#181;l of 0.5 M EDTA, pH 8.</li>
		<li>Keep the tube at 4&#160;&#176;C.</li>
	</ol>
	</li>
	<li>Biotinylation of sticky ended DNA Using DNA Ligase 
	Note: Use &#955; DNA (48.5 kbp), which is a sticky-end DNA.
	<ol>
		<li>Add 1 &#181;l of T4 DNA ligase, 5 &#181;l of 10&#215; ligation buffer, 1 &#181;l of 25 ng/&#181;l &#955; phage DNA, and add 43 &#181;l of water to make a final volume of 50 &#181;l.</li>
		<li>Keep the tube at 4 &#176;C.</li>
	</ol>
	</li>
</ol>
2. Functionalized Surface Derivatization
Note: In order to tether an end of a DNA molecule on glass surface, a primary amine group is silanized on a coverslip followed by biotin-PEG coating as shown in Figure 1. This PEGylation process is important for single-molecule DNA imaging because it can significantly decrease random noise created by attachment of undesired molecules on the surface.
<ol>
	<li>Piranha Cleaning 
	Note: Piranha solutions react vigorously with organic materials, therefore it should be handled with care, following proper safety guidelines.
	<ol>
		<li>Place coverslips on a polytetrafluoroethylene (PTFE) rack, and hold them by PTFE thread seal tape lengthwise, half on the edge of the surfaces and half on the rack. After wrapping, leave a long piece (~ 5 cm) of tape for handling the rack during the cleaning procedure.</li>
		<li>Fill a 1 L glass beaker with 350 ml of H2SO4 and 150 ml of H2O2 to make piranha solution in a fume hood. Place the rack in piranha solution for 2 hr.</li>
		<li>Empty piranha solution from the beaker, and rinse coverslips thoroughly with deionized water until the pH of water in the beaker reaches neutral. Use pH paper strips.</li>
		<li>Sonicate the racks of cover slips in the beaker containing water, for 30 min. Empty water from the beaker just before amino silanization. Use 75 W of sonication power to clean and derivatize the glass substrates.</li>
	</ol>
	</li>
	<li>Aminosilanization on Glass Surface
	<ol>
		<li>Add 2 ml of N-[3-(trimethoxysilyl)propyl]ethylenediamine and 10 ml of glacial acetic acid to 200 ml of methyl alcohol in a clean polypropylene container for preparing the aminosilanization solution.</li>
		<li>Place cleaned coverslips in the polypropylene container. Shake them at 100 rpm and room temperature for 30 min.</li>
		<li>Sonicate the beaker with derivatized cover slips for 15 min at 75 W. Shake them at 100 rpm and room temperature for at least 30 min.</li>
		<li>Empty the solution from the beaker, and rinse coverslips carefully, once with methyl alcohol, and twice with ethyl alcohol. Store coverslips in ethyl alcohol and use them within two weeks.</li>
	</ol>
	</li>
	<li>PEGylation of the coverslip
	<ol>
		<li>Make 10 ml of 0.1 M sodium bicarbonate (NaHCO3). Filter the solution with a 0.22-&#181;m syringe filter.</li>
		<li>Dissolve 2 mg of biotin-PEG-succinimidyl carbonate (biotin-PEG-SC) and 80 mg of PEG-succinimidyl valerate (mPEG-SVG) in 350 &#181;l of NaHCO3. Use a light protection tube.</li>
		<li>Vortex the tube vigorously for 10 sec, and centrifuge it at 10,000 &#215; g for 1 min to remove bubbles.</li>
		<li>Rinse a glass slide with acetone, followed by rinsing with ethyl alcohol. Allow the slide to air dry completely.</li>
		<li>Place a drop (50 &#181;l) of PEG solution on the clean glass slide. Cover the droplet gently with an amino silanized cover slip, without generating any bubbles.</li>
		<li>Place slides for 3 hr to overnight in a dark, well-leveled humid chamber at room temperature. Use an empty pipette tip holder as the chamber, with water at the bottom. Seal residual PEG solution in the tube and keep it at 4 &#176;C.</li>
		<li>Rinse the PEGylated coverslip thoroughly with deionized water. Store it in a dark and dry place until use.</li>
	</ol>
	</li>
</ol>
3. Assembling a Flow Chamber
<ol>
	<li>Fabricate a punched acrylic holder, as in Figure 2. Ensure that the diameter of the tubing insert is 0.762 mm, and designate one hole is an inlet and the other is an outlet (Figure 2).</li>
	<li>Place double-sided sticky tape strips (width 5-6 mm) on an acrylic holder (Figure 2), aligning perpendicularly to an inlet and outlet hole, not perturbing any channel holes. Use these tape strips as multi-channel walls to make chambers. Scrub the tape using a pipet tip.</li>
	<li>Place a PEGylated coverslip on the top to make flow chambers, with the PEGylated side facedown.</li>
	<li>To prevent any solution from leaking, press the top of a coverslip over the area where double-sided tapes are placed. Add quick-dry epoxy glue to close the edges of the chamber at the top and at the bottom (yellow color in Figure 2).</li>
	<li>Connect a short piece (2.5 cm) of tubing (outer diameter, OD, 0.042&#34;) to a gas-tight syringe and seal the joint with epoxy glue.</li>
	<li>Connect a long flexible tubing (OD: 0.03&#34;) to the tubing linked to the syringe and seal the joint with epoxy glue.</li>
	<li>Fill the tubing linked to the syringe with DI water. Make sure there is no air bubble.</li>
	<li>Insert the tubing into the hole of the flow chamber sealed with epoxy glue.</li>
	<li>Place a yellow tip (200 &#181;l tip) on the other hole as a reservoir.</li>
</ol>
4. Sample Loading into Flow Chamber
Note: Neutravidin can be replaced with other avidin proteins such as streptavidin. All of the reactions can be performed at room temperature, unless it is mentioned. Take sample solution in a yellow tip, and install the tip with the solution onto the hole of acrylic holder (Figure 2b).
<ol>
	<li>Set the flow rate of the syringe pump at 50 &#181;l/min. Load 20 &#181;l of avidin protein (25 &#181;g/ml in T50 solution, Tris 10 mM, NaCl 50 mM, pH 8), and keep it for 10 min.</li>
	<li>Load 20 &#181;l of biotinylated oligodeoxynucleotides (100 &#181;M in 1&#215; TE), and keep it for 10 min. If terminal transferase is used, skip the loading of oligodeoxynucleotides.</li>
	<li>Load 20 &#181;l of DNA solution from Step 1 into the flow chamber at a flow rate of 10 &#181;l/min, and keep it for 30 min.</li>
	<li>Wash the flow chamber with 1&#215; TE, and load 40 &#181;l of FP-DBP10 (~80 nM).</li>
	<li>Observe DNA under a fluorescent microscope with continuous flow of staining molecules in 1&#215; TE on a 60X objective lens. Use 488 nm solid state laser for excitation of FP(eGFP)-DBP. For full stretch of DNA molecules, apply different flow rates according to the DNA lengths used. For example, apply 50 &#181;l/min for 48.5 kbp of &#955; DNA, and 100 &#181;l/min for 166 kbp of T4 DNA.
	<ol>
		<li>For recycling of the acrylic holder, soak assembled flow chambers in a household detergent solution12. Remove tapes and epoxy with a razor blade and rub with hands to completely take them off. Unclog holes with syringe needles. Keep the holders in deionized water until further use.</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Smith, S. B., Finzi, L., Bustamante, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+Mechanical+Measurements+of+the+Elasticity+of+Single+DNA-Molecules+by+Using+Magnetic+Beads.">Direct Mechanical Measurements of the Elasticity of Single DNA-Molecules by Using Magnetic Beads.</a> Science. 258 (5085), 1122-1126 (1992).</li><li>Finkelstein, I. J., Visnapuu, M. -. L., Greene, E. C. <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+imaging+reveals+mechanisms+of+protein+disruption+by+a+DNA+translocase.">Single-molecule imaging reveals mechanisms of protein disruption by a DNA translocase.</a> Nature. 468 (7326), 983-987 (2010).</li><li>Doyle, P. S., Ladoux, B., Viovy, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+a+tethered+polymer+in+shear+flow.">Dynamics of a tethered polymer in shear flow.</a> Phys. Rev. Lett. 84 (20), 4769-4772 (2000).</li><li>Perkins, T. T., Quake, S. R., Smith, D. E., Chu, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Relaxation+of+a+Single+DNA+Molecule+Observed+by+Optical+Microscopy.">Relaxation of a Single DNA Molecule Observed by Optical Microscopy.</a> Science. 264 (5160), 822-826 (1994).</li><li>Cai, W., 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=Ordered+restriction+endonuclease+maps+of+yeast+artificial+chromosomes+created+by+optical+mapping+on+surfaces.">Ordered restriction endonuclease maps of yeast artificial chromosomes created by optical mapping on surfaces.</a> Proc. Natl. Acad. Sci. U.S.A. 92 (11), 5164-5168 (1995).</li><li>Kim, Y., Jo, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutravidin+coated+surfaces+for+single+DNA+molecule+analysis.">Neutravidin coated surfaces for single DNA molecule analysis.</a> Chem. Comm. 47 (22), 6248-6250 (2011).</li><li>Kim, Y., 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=Nanochannel+confinement:+DNA+stretch+approaching+full+contour+length.">Nanochannel confinement: DNA stretch approaching full contour length.</a> Lab on a Chip. 11 (10), 1721-1729 (2011).</li><li>Glazer, A. N., Rye, H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stable+dye-DNA+intercalation+complexes+as+reagents+for+high-sensitivity+fluorescence+detection.">Stable dye-DNA intercalation complexes as reagents for high-sensitivity fluorescence detection.</a> Nature. 359 (6398), 859-861 (1992).</li><li>Graneli, A., Yeykal, C. C., Prasad, T. K., Greene, E. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organized+arrays+of+individual+DNA+molecules+tethered+to+supported+lipid+bilayers.">Organized arrays of individual DNA molecules tethered to supported lipid bilayers.</a> Langmuir. 22 (1), 292-299 (2006).</li><li>Lee, 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=DNA+binding+fluorescent+proteins+for+the+direct+visualization+of+large+DNA+molecules.">DNA binding fluorescent proteins for the direct visualization of large DNA molecules.</a> Nucleic Acids Res. , (2015).</li><li>Roy, R., Hohng, S., Ha, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+guide+to+single-molecule+FRET.">A practical guide to single-molecule FRET.</a> Nat Methods. 5 (6), 507-516 (2008).</li><li>Chandradoss, S. D., 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=Surface+passivation+for+single-molecule+protein+studies.">Surface passivation for single-molecule protein studies.</a> J. Vis. Exp. (86), e50549 (2014).</li></ol>

The authors have nothing to disclose.

Here we present a platform for visualizing long DNA molecules biotinylated for anchoring on surfaces. We have reported an approach for DNA molecules tethered on an avidin protein coated surface with biotinylated bovine serum albumin.6 In the earlier approach, we found a crucial issue of DNA photo-cleavage caused by bis-intercalation dyes that stain DNA molecules tethered on the surface. As these continually excited fluorophores have a high probability to attack a DNA phosphate backbone,9 the excitation light power had to be minimized to avoid photo-cleavage.
In order to overcome this inconvenience, we developed genetically engineered fluorescent proteins with DNA-binding capability (FP-DBP) for staining DNA without photo-cleavage.10 We observed reliable DNA staining on avidin-coated protein surfaces. Nonetheless, this platform had another issue of undesirable aggregation of fluorescent proteins on the protein surface, resulting in increased background noise. It is essential to minimize this random noise for single-molecule DNA analysis to enhance the contrast of DNA and proteins from the background. The use of FP-DBP therefore requires surface passivation to prevent the fluorescent protein from being adsorbed on the surface. PEG could be a powerful candidate for this purpose.12 Hence, we used biotinylated-PEG, which dramatically enhanced signal-to-noise ratio for imaging FP-DBP stained DNA molecules tethered on the surface.
There are several critical steps and notes in this method for higher yields. In order to tether an end of a DNA molecule on a glass surface, a primary amine group is silanized on a coverslip followed by biotin-PEG coating as shown in Figure 1. This surface passivation process is critical for single-molecule DNA imaging because it can significantly decrease noise-generating random adsorption on the surfaces. Therefore, overnight aminosilanization and PEGylation are strongly recommended. In addition, cleaning the glass slide as well as the cover glass with piranha solution can further reduce background noise.
A biotinylated oligonucleotide must have a phosphate group at 5&#39; end in order to link it to 3&#39; hydroxyl group of a large DNA molecule. The sequence of biotinylated oligonucleotide is 5&#39;-p-GGGCGGCGACCT-TEG-Biotin-3&#39; for &#955; DNA tethering. The &#955; DNA solution has to be loaded shortly after the preparation of &#955; DNA solution (described in 1.1), because &#955; DNA often forms concatemers with long-time storage. For blunt-ended DNA such as T4 DNA, terminal transferase can add biotinylated dUTPs at both ends of DNA without any specificity. Thus, terminal transferase reaction time has to be carefully adjusted around an hour, otherwise extended reaction may yield double-labeled DNA (i.e., both ends are labeled with biotin). In addition to viral DNA such as &#955; and T4, any types of DNA can be utilized in this platform. However, it is recommended to use fragments from gigantic genomic DNA after digestion by rare-cutter restriction enzymes such as SwaI or NotI.
Different flow rates are applied for different lengths of DNA. Usually, longer DNA molecules require faster flow rates to fully stretch the DNA molecules, to match the exact contour length. For example, &#955; DNA can stretch up to 16.5 &#181;m, which corresponds to the calculated value of 0.34 nm x 48,502 bp.
It is quite important to freshly prepare all materials, particularly all of proteins and PEGs. For example, Neutravidin readily loses its function of binding biotins if it is stored as a diluted solution. For PEG solutions, pH is critical to conjugate functional groups of a primary amine and n-hydroxysuccinimide. Therefore, it is recommended to freshly prepare all materials at least every week.
Visualization of large DNA molecules tethered on the surface is a versatile platform to be applied for a variety of applications such as genomics, epigenomics, biochemical studies for DNA and protein interaction, and polymer physics studies. However, this approach is currently limited only to model systems using relatively simple and well-known DNA molecules such as the viral genome. To be extended to the human genome and epigenome, it is critically important to establish a robust, reliable, and simple platform for practical use. By overcoming light-induced DNA photo-cleavage and preventing random adsorption of fluorescent protein on the surface, this platform therefore provides clear and high-contrast images for DNA elongated with shear flows.

Visualization of large DNA molecules tethered on glass or bead surfaces has been utilized for investigating DNA-protein interactions, protein dynamics on DNA substrate,1,2 and polymer physics.3,4 A platform for single-tethered large DNA molecules has a few distinct advantages compared to other DNA immobilization methods.5 First, a large DNA molecule tethered on the surface has a natural random-coil conformation without a shear flow, which is critically important for a DNA-binding protein to recognize its binding site. Second, it is very easy to change the chemical environment around DNA molecules for a series of enzymatic reactions in a flow chamber. Third, a microfluidic shear flow induces DNA molecular stretching up to 100% of the full contour length, which is very difficult to achieve using alternative DNA elongation approaches such as surface immobilization6 and nanochannel confinement.7 A fully stretched DNA molecule also provides positional information that can be useful for monitoring enzymatic movements on the genomic map.
Nevertheless, the DNA tethering approach has a critical shortcoming in that the intercalating dye such as YOYO-1 generally causes tethered DNA molecules to be readily broken by fluorescence excitation light. Generally, large DNA molecules have to be stained with a fluorescent dye for visualization under a fluorescent microscope. For this purpose, YOYO-1 or other TOTO series dyes are primarily used because these dyes fluoresce only when they intercalate double-stranded DNA.8 However, it is well-known that bis-intercalating dye causes light-induced DNA photo-cleavage because of the intercalation of fluorophores.9 Further, fluorescently stained DNA molecules tethered on the surface are more fragile since shear flows can exert breaking forces on freely moving DNA molecules. Therefore, we developed FP-DBP as a novel DNA-staining protein dye for imaging large DNA molecules tethered on the surface. The advantage of using FP-DBP is that it does not cause photo-cleavage of the DNA molecules to which it binds.10 In addition, FP-DBP does not increase the contour length of DNA, while bis-intercalating dyes increase the contour length by about 33%.
This video method introduces the experimental approach for tethering large DNA molecules to a PEG-biotin surface. Figure 1 illustrates different approaches of tethering DNA with blunt ends and sticky ends. Thus, this staining method can be applied to any type of DNA molecule. Figure 2 depicts a schematic representation of the flow chamber assembly that can be controlled by a syringe pump to generate shear flows to stretch DNA molecules as well as to load chemical and enzyme solutions. Figure 3 demonstrates micrographs of fully stretched DNA molecules tethered on the PEGylated surface11 and stained with FP-DBP.

<table><tbody><tr><td colspan="4">&lt;strong&gt;1. DNA Biotinylation&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;1.1) Biotinylation of blunt-end DNA using Terminal Transferase (TdT)&lt;/strong&gt;</td></tr><tr><td>Terminal Transferase</td><td>New England Biolabs</td><td>M0315S</td><td>Provided with 10x reaction buffer, 2.5 mM cobalt chloride</td></tr><tr><td>Biotin-11-dUTP</td><td>Invitrogen</td><td>R0081</td><td>Biotin-ddNTP is also available</td></tr><tr><td>T4GT7 Phage DNA</td><td>Nippon Gene</td><td>318-03971</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>Sigma-Aldrich</td><td>E6758</td><td>EDTA</td></tr><tr><td colspan="4">&lt;strong&gt;1.2) Biotinylation of sticky end DNA Using DNA Ligase&lt;/strong&gt;</td></tr><tr><td>T4 DNA Ligase</td><td>New England Biolabs</td><td>M0202S</td><td>Provided with 10x reaction buffer</td></tr><tr><td>Lambda Phage DNA</td><td>Bioneer</td><td>D-2510</td><td>Also available at New England Biolabs</td></tr><tr><td colspan="4">&lt;strong&gt;2. Functionalized Surface Derivatization&lt;/strong&gt;</td></tr><tr><td colspan="4">&lt;strong&gt;2.1) Piranha Cleaning&lt;/strong&gt;</td></tr><tr><td>Coverslip</td><td>Marienfeld-Superior</td><td>0101050</td><td>22 mm x 22 mm, No. 1 Thickness</td></tr><tr><td>Teflon rack</td><td></td><td></td><td>Custom Fabrication</td></tr><tr><td>PTFE Thread Seal Tape</td><td>Han Yang Chemical Co. Ltd.</td><td>3032292</td><td>Teflon&amp;trade; tape</td></tr><tr><td>Sulfuric acid</td><td>Jin Chemical Co. Ltd.</td><td>S280823</td><td>H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;, 95% Purity</td></tr><tr><td>Hydrogen peroxide</td><td>Jin Chemical Co. Ltd.</td><td>H290423</td><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;, 35% in water</td></tr><tr><td>Sonicator</td><td>Daihan Scientific Co. Ltd.</td><td>WUC-A02H</td><td>Table-top Ultrasonic Cleaner</td></tr><tr><td colspan="4">&lt;strong&gt;2.2) Aminosilanization on Glass Surface&lt;/strong&gt;</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-[3-(Trimethoxysilyl)propyl]&lt;br/&gt; ethylenediamine</td><td>Sigma-Aldrich</td><td>104884</td><td></td></tr><tr><td>Glacial Acetic Acid</td><td>Duksan Chemicals</td><td>414</td><td>99% Purity</td></tr><tr><td>Methyl Alcohol</td><td>Jin Chemical Co. Ltd.</td><td>M300318</td><td>99.9% Purity</td></tr><tr><td>Polypropylene Container</td><td>Qorpak</td><td>PLC-04907</td><td></td></tr><tr><td>Ethyl Alcohol</td><td>Jin Chemical Co. Ltd.</td><td>A300202</td><td>99.9% Purity</td></tr><tr><td colspan="4">&lt;strong&gt;2.3) PEGylation of the coverslip&lt;/strong&gt;</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma-Aldrich</td><td>S5761</td><td></td></tr><tr><td>Syringe Filter</td><td>Sartorius</td><td>16534----------K</td><td></td></tr><tr><td>Biotin-PEG-SC</td><td>Laysan Bio</td><td>Biotin-PEG-SC-5000</td><td></td></tr><tr><td>mPEG-SVG</td><td>Laysan Bio</td><td>MPEG-SVA-5000</td><td></td></tr><tr><td>Acetone</td><td>Jin Chemical Co. Ltd.</td><td>A300129</td><td>99% Purity</td></tr><tr><td>Microscope Slides</td><td>Marienfeld-Superior</td><td>1000612</td><td>~76 mm x 26 mm x 1&amp;nbsp;mm</td></tr><tr><td colspan="4">&lt;strong&gt;3. Assembling a Flow Chamber&lt;/strong&gt;</td></tr><tr><td>Acrylic Support</td><td></td><td></td><td>Custom Fabrication</td></tr><tr><td>Double-sided Tape</td><td>3M</td><td></td><td>Transparent type</td></tr><tr><td>Quick-dry Epoxy</td><td>3M</td><td></td><td></td></tr><tr><td>Polyethylene Tubing</td><td>Cole-Parmer</td><td>06417-11, 06417-21</td><td></td></tr><tr><td>Gas Tight 250 &amp;micro;l Syringe</td><td>Hamilton</td><td>81165</td><td></td></tr><tr><td>Syringe Pump</td><td>New Era Pump Systems Inc.</td><td>NE-1000</td><td></td></tr><tr><td colspan="4">&lt;strong&gt;4. Sample Loading into Flow Chamber&lt;/strong&gt;</td></tr><tr><td>Neutravidin</td><td>Thermo Scientific</td><td>31000</td><td></td></tr><tr><td>Tris base</td><td>Sigma-Aldrich</td><td>T1503-5KG</td><td>Trizma base</td></tr><tr><td>Microscope</td><td>Olympus</td><td>IX70</td><td></td></tr><tr><td>EMCCD Camera</td><td>Q Imaging</td><td>Rolera EM-C2</td><td></td></tr><tr><td>Solid-state Laser (488&amp;nbsp;nm)</td><td>Oxxius</td><td>LBX488</td><td></td></tr><tr><td>Alconox</td><td>Alconox Inc.</td><td></td><td></td></tr></tbody></table>

visualization of surface-tethered large dna molecules with a fluorescent protein dna binding peptide

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating interactions between DNA and its binding proteins. Here, we report a method that uses fluorescent microscopy for visualizing large DNA molecules tethered on the surface. First, glass coverslips are biotinylated and passivated by coating with biotinylated polyethylene glycol, which specifically binds biotinylated DNA via avidin protein linkers and significantly reduces undesirable binding from non-specific interactions of proteins or DNA molecules on the surface. Second, the DNA molecules are biotinylated by two different methods depending on their terminals. The blunt ended DNA is tagged with biotinylated dUTP at its 3' hydroxyl terminus, by terminal transferase, while the sticky ended DNA is hybridized with biotinylated complimentary oligonucleotides by DNA ligase. Finally, a microfluidic shear flow makes single DNA molecules stretch to their full contour lengths after being stained with fluorescent protein-DNA binding peptide (FP-DBP).

Figure 1 shows two different DNA tethering methods depending on terminal structures of the DNA molecule. Figure 1a illustrates how the sticky ended DNA molecules are hybridized with complementary biotinylated oligonucleotides, which are immobilized on the avidin-coated PEG surface. Figure 1b shows the addition of biotinylated ddNTP or dNTP to the 3&#39; hydroxyl group of a blunt ended DNA by terminal transferase. We added flexible linkers between DNA molecules and biotin. This increased the ligation and avidin-biotin binding efficiency to provide sufficient space for the reactions. Figure 1c demonstrates the overall schematic representation for DNA tethering on the avidin-coated PEG surface.
Figure 2 depicts the assembly of the flow chamber along with an acrylic holder. Compared to glass/quartz slide glasses, a custom-made acrylic holder simplifies the preparation of the flow chamber for epi-fluorescent microscopy.12 The flow chamber enables instant changing of buffer conditions for enzyme reactions,6 and stretching DNA molecules.
Figure 3 demonstrates single-tethered DNA and T4 &#955;DNA stained with FP-DBP. Microfluidic shear flows elongated single DNA molecules up to full contour lengths such as 16.5 &#956;m (48.5 kb &#955; DNA x 0.34 nm) and 56.4 &#956;m (166 kb T4 DNA x 0.34 nm). We were able to repeat DNA stretching and relaxation multiple times during an extended period (e.g., half-hour), since we utilized FP-DBP that does not cause photo-cleavage.
<img alt="Figure 1" src="/files/ftp_upload/54141/54141fig1.jpg" /> 
Figure 1. Schematic illustration of DNA tethering. a) A sticky ended DNA is hybridized with a biotinylated complementary oligonucleotide. Triethylene glycol is a flexible linker between the oligonucleotide and biotin. b) Biotinylated dNTP is added to a blunt end of double stranded DNA, by terminal transferase. Polycarbon spacer (11-atoms) is a flexible linker between dNTP and biotin. c) Biotinylated DNA is anchored to an avidin protein, which is linked to a biotinylated PEG covalently bonded to the glass surface. The ratio of biotinylated PEG to normal PEG was 0.025 (1:40). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54141/54141fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54141/54141fig2.jpg" /> 
Figure 2. Flow chamber. a) Flow chamber consisting of an acrylic acid resin holder, strips of double-sided tapes and a PEGylated cover slip. The acrylic holder of dimensions 76 mm x 26 mm x 5 mm (L x W x H), was fabricated with laser cutting. b) Side view of the Flow chamber: punched holes are an inlet port on which a yellow tip is installed for a buffer reservoir, and an outlet port that is connected to a tubing controlled by a syringe-pump. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54141/54141fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54141/54141fig3.jpg" /> 
Figure 3. Fluorescent microscopic images of stretched DNA molecules stained with FP-DBP. a) Bacteriophage &#955; DNA (48.5 kbp) tethered on the surface after ligation with biotinylated complementary oligonucleotides. b) Bacteriophage T4 DNA (166 kbp) tethered on the surface after adding biotin with terminal transferase. Indicated arrows show DNA molecules stretched up to their full contour lengths such as 16.5 &#181;m for &#955; DNA and 56.4 &#181;m for T4 DNA. Scale bars: 10 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54141/54141fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide at JoVE.com

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows.

Large DNA molecules tethered on the functionalized glass surface have been utilized in polymer physics and biochemistry particularly for investigating ...

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Nanyang Technological University

Research

JoVE Journal

Biology

10.7K Views.  Sogang University. We present an approach for visualizing fluorescent protein DNA binding peptide (FP-DBP)-stained large DNA molecules tethered on the polyethylene glycol (PEG) and avidin-coated glass surface and stretched with microfluidic shear flows. 

Video: Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Biochemistry

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 ...

Genetics

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome organization are governed by DNA-binding proteins that recognize specific DNA structures or sequences. In vitro experiments have helped to generate detailed models for the function of many types of DNA-binding proteins, yet, the exact mechanisms of these processes and their organization in the complex environment of the living cell remain far less understood. We recently introduced a method for quantifying DNA-repair activities in live Escherichia coli cells using Photoactivated Localization Microscopy (PALM) combined with single-molecule tracking. Our general approach identifies individual DNA-binding events by the change in the mobility of a single protein upon association with the chromosome. The fraction of bound molecules provides a direct quantitative measure for the protein activity and abundance of substrates or binding sites at the single-cell level. Here, we describe the concept of the method and demonstrate sample preparation, data acquisition, and data analysis procedures.

Photoactivated localization microscopy (PALM) combined with single-molecule tracking allows direct observation and quantification of protein-DNA interactions in live Escherichia coli cells.

Protein-DNA interactions are at the heart of many fundamental cellular processes. For example, DNA replication, transcription, repair, and chromosome ...

Immunology and Infection

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular level. To date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements. However, many important processes occur in the living cell at the level of just one or a few molecules; ensemble measurements generally mask the stochastic and heterogeneous nature of these events. Here, using advanced optical microscopy and analytical image analysis tools we demonstrate how to monitor proteins within a single living bacterial cell to a precision of single molecules and how we can observe dynamics within molecular complexes in functioning biological machines. The techniques are directly relevant physiologically. They are minimally-perturbative and non-invasive to the biological sample under study and are fully attuned for investigations in living material, features not readily available to other single-molecule approaches of biophysics. In addition, the biological specimens studied all produce fluorescently-tagged protein at levels which are almost identical to the unmodified cell strains ("genomic encoding"), as opposed to the more common but less ideal approach for generating significantly more protein than would occur naturally ('plasmid expression'). Thus, the actual biological samples which will be investigated are significantly closer to the natural organisms, and therefore the observations more relevant to real physiological processes.

Visualizing Single Molecular Complexes In Vivo Using Advanced Fluorescence Microscopy

Here we demonstrate the protocols for performing single-molecule fluorescence microscopy on living bacterial cells to enable functional molecular complexes to be detected, tracked and quantified.

Full insight into the mechanisms of living cells can be achieved only by investigating the key processes that elicit and direct events at a cellular ...

Functional Surface-immobilization of Genes Using Multistep Strand Displacement Lithography

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic bioreactor system. In contrast to other approaches towards artificial cellular systems, such a setup allows for a continuous supply with gene expression reagents and simultaneous draining of waste products. This facilitates the implementation of cell-free gene expression processes over extended periods of time, which is important for the realization of dynamic gene regulatory feedback systems. Here we provide a detailed protocol for the fabrication of genetic biochips using a simple-to-use lithographic technique based on DNA strand displacement reactions, which exclusively uses commercially available components. We also provide a protocol on the integration of compartmentalized genes with a polydimethylsiloxane (PDMS)-based microfluidic system. Furthermore, we show that the system is compatible with total internal reflection fluorescence (TIRF) microscopy, which can be used for the direct observation of molecular interactions between DNA and molecules contained in the expression mix.

We describe a simple lithographic procedure for the immobilization of gene-length DNA molecules on a surface, which can be used to perform cell-free gene expression experiments on biochips.

Immobilization of genes on lithographically structured surfaces allows the study of compartmentalized gene expression processes in an open microfluidic ...

Bioengineering

In Situ Nucleosome Reconstitution for Single-Molecule Studies

In Situ Nucleosome Assembly for Single-Molecule Correlative Force and Fluorescence Microscopy

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding their biophysical properties and interactions with chromatin-binding proteins. Nucleosome reconstitution on DNA for these studies typically involves a salt dialysis procedure that provides precise control over the placement and number of nucleosomes formed along a DNA tether. However, this protocol is time-consuming and requires a substantial amount of DNA and histone octamers as inputs. To offer an alternative strategy, an in situ nucleosome reconstitution method for single-molecule force and fluorescence microscopy that utilizes the histone chaperone Nap1 is described. This method enables users to assemble nucleosomes on any DNA template without the need for strong nucleosome positioning sequences, adjust nucleosome density on demand, and use fewer reagents. In situ nucleosome formation occurs within seconds, offering a simpler experimental workflow and a convenient transition into single-molecule measurements. Examples of two downstream assays for probing nucleosome mechanics and visualizing the behavior of individual proteins on chromatin are further described.

Author Spotlight: Efficient Nucleosome Reconstitution for Single-Molecule Techniques

This article presents a detailed experimental procedure for reconstituting nucleosome-containing DNA tethers for single-molecule correlative force and fluorescence microscopy. It further describes several downstream experiments that can be conducted to visualize the binding behavior of chromatin-interacting proteins and analyze changes in the physical properties of nucleosomes.

Nucleosomes constitute the primary unit of eukaryotic chromatin and have been the focus of numerous informative single-molecule investigations regarding ...

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 ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified &#955; DNA at the Single Molecule Level

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. In single molecule assays for studying DNA-protein interactions, there are two important factors for consideration: the DNA substrate with enough length for easy observation and labeling a protein with a suitable fluorescent probe. 48.5 kb &#955; DNA is a good candidate for the DNA substrate. Quantum dots (Qdots), as a class of fluorescent probes, allow long-time observation (minutes to hours) and high-quality image acquisition. In this paper, we present a protocol to study DNA-protein interactions at the single-molecule level, which includes preparing a site-specifically modified &#955; DNA and labeling a target protein with streptavidin-coated Qdots. For a proof of concept, we choose ORC (origin recognition complex) in budding yeast as a protein of interest and visualize its interaction with an ARS (autonomously replicating sequence) using TIRFM. Compared with other fluorescent probes, Qdots have obvious advantages in single molecule studies due to its high stability against photobleaching, but it should be noted that this property limits its application in quantitative assays.

Here, we present a protocol to study DNA-protein interactions by total internal reflection fluorescence microscopy (TIRFM) using a site-specifically modified &#955; DNA substrate and a Quantum-dot labeled protein.

The fluorescence microscopy&#160;has made great contributions in dissecting the mechanisms of complex biological processes at the single molecule level. ...

Visualization of Surface-tethered Large DNA Molecules with a Fluorescent Protein DNA Binding Peptide

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