We thank Dr. Hasan Yardimci and Dr.Sevim Yardimci of the Francis Crick Institute for kind help in the single-molecule experiments, Dr. Daniel Duzdevich from Dr. Eric C. Greene&#39;s lab of Columbia University, Dr. Yujie Sun of Peking University and Dr. Chunlai Chen of Tsinghua University for useful discussion. This study was supported by the National Natural Science Foundation of China 31371264, 31401059, CAS Interdisciplinary Innovation Team and the Newton Advanced Fellowship (NA140085) from the Royal Society.

1. Preparation of &#955;-ARS317 DNA substrate
<ol>
	<li>DNA substrate construction and packaging
	<ol>
		<li>Digest native &#955; DNA using XhoI enzyme; amplify a 543 bp DNA fragment bearing ARS317 from the genomic DNA of budding yeast using primers containing 20 bp homologous sequences of upstream and downstream of XhoI enzyme site on lambda DNA. Add 100 ng of XhoI digested &#955; DNA and 10 ng of DNA fragment to 10 &#181;L of homologous recombination reaction system, and incubate the reaction at 37 &#176;C for 30 min.</li>
		<li>To package the recombination &#955; DNA, add 25 &#181;L of lambda packaging extracts into the recombination product, and incubate the reaction at 30 &#176;C for 90 min. Then, add an additional 25 &#181;L of lambda packaging extracts into the reaction tube. Continue to incubate the reaction at 30 &#176;C for 90 min.</li>
		<li>Add 500 &#181;L of sterile dilution buffer (10 mM Tris-HCl (pH 8.3), 100 mM NaCl, 10 mM MgCl2) into the reaction system, and mix gently by turning the tube upside down several times. Add 25 &#181;L of chloroform, mix gently and store at 4 &#176;C.</li>
		<li>Add 100 &#181;L of packaged phage and 100 &#181;L of bacteria LE392MP (cultured at 0.8 - 1.0 using LB medium adding with 10 mM MgSO4) into a new tube. Incubate at 37 &#176;C for 15 minutes.</li>
		<li>Add 200 &#181;L of phage-bacterium mixture into 4 mL oftop agar (LB medium + 0.7% agar + 10 mM MgSO4, cooled to 48 &#176;C). Mix immediately by turning the tube upside down several times and pour it onto a pre-warmed (37 &#176;C) LB plate.</li>
		<li>Incubate the plate at 37 &#176;C overnight and screen the &#955;-ARS317 plaque using PCR and sequencing.</li>
	</ol>
	</li>
	<li>DNA substrate purification from liquid lysates11,18
	<ol>
		<li>Pick one plaque into 200 &#181;L of sterile ddH2O with 10 mM MgCl2 and 10 nM CaCl2. Mix with 200 &#181;L of bacteria LE392MP (cultured overnight in LB medium), and incubate at 37 &#176;C for 15 min.</li>
		<li>Add the phage-bacterium mixture into 100 mL of NZCYM (10 g/L NZ-amine, 5 g/L yeast extract, 5 g/L NaCl, 1 g/L Casamino Hydrolysate, and 2 g/L MgSO4&#183;7H2O) medium and culture for 7 h at 37 &#176;C. Add 250 &#181;L of chloroform into the culture and shake for another 10 min.</li>
		<li>Transfer the culture into a 200 mL flask. Add 5.8 g of NaCl (1 M final concentration), stir to dissolve by hand, and incubate on ice for 30 min.</li>
		<li>Centrifuge at 12,000 x g for 10 min at 4 &#176;C to remove the cell debris. Collect the supernatant into a 200 mL flask and add 10% PEG8000 (m/V). Stir to dissolve by magnetic stirring apparatus and incubate on ice for 30 min or longer.</li>
		<li>Centrifuge at 12,000 x g for 10 min at 4 &#176;C to precipitate the bacteriophage. Remove the supernatant.</li>
		<li>Resuspend the precipitate in 2 mL of phage dilution buffer. Transfer it into a 15 mL tube and add 10 &#181;L of RNase (final concentration is 20 &#181;g/mL) and 40 &#181;L of DNase (final concentration is 5 &#181;g/mL). Incubate at 37 &#176;C for 30 min.</li>
		<li>Add 2 mL of 0.3 M Tris-HCl (pH 9.0), 100 mM EDTA&#160;+ 1.25% SDS, 15 &#181;L proteinase K (final concentration is 10 &#181;g/mL). Incubate at 65 &#176;C for 10 min.</li>
		<li>Add 2 mL of pre-cooled potassium acetate (3 M, pH 4.8), and incubate the tube on ice for 10 min.</li>
		<li>Centrifuge at 8,000 x g for 10 min at 4 &#176;C to remove the insoluble materials.</li>
		<li>Add 0.7x&#160;volume of isopropanol into the supernatant. Mix by turning the tube upside down several times, and incubate for 2 min at room temperature (RT).</li>
		<li>Centrifuge at 8,000 x g for 10 min at RT to precipitate DNA. Remove the supernatant.</li>
		<li>Wash the DNA once with 70% ethanol by centrifugation at 8,000 x g for 10 min. Remove the supernatant.</li>
		<li>Elute the DNA using 500 &#181;L TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) gently, and transfer the DNA into 1.5 mL tube.</li>
		<li>Extract the DNA twice using phenol: chloroform by centrifugation at 8,000 g for 10 min.</li>
		<li>Precipitate with 0.7x&#160;volume isopropanol. Mix by turning the tube upside down gently, and centrifuge at 8,000 x g for 10 min.</li>
		<li>Wash once with 70% ethanol, resuspend in 200 &#181;L of TE. Measure the DNA concentration, and store at -20 &#176;C in 12.5 &#181;g aliquots.</li>
	</ol>
	</li>
</ol>
2. &#955;-ARS317 DNA Biotinylation
<ol>
	<li>To phosphorylate biotinylated oligonucleotides (5&#39;-AGGTCGCCGCC-TEG-Biotin-3&#39;) complementary to the left end of native &#955; DNA, add 1 &#181;L (100 &#181;M) of oligonucleotides, 7.5 &#181;L of ddH2O, 1 &#181;L of 10x&#160;ligase buffer, and 0.5 &#181;L of T4 PNK. Incubate the reaction at 37 &#176;C for 3 h.</li>
	<li>To phosphorylate &#955;-ARS317, add 12.5 &#181;g of &#955;-ARS317, 3 &#181;L of 10x&#160;ligase buffer, 1 &#181;L of T4 PNK, and ddH2O to total volume of 30 &#181;L. Incubate the reaction at 37 &#176;C for 3 h.</li>
	<li>To anneal oligonucleotides with &#955;-ARS317, add 0.5 &#181;L of phosphorylated oligonucleotides, 195 &#181;L of ddH2O, 25 &#181;L of 10&#215;ligase buffer into the phosphorylated &#955;-ARS317 tube. Mix gently by turning the tube upside down and incubate at 65 &#176;C for 5 min. Turn off the block heater, and let the sample cool to below 33 &#176;C in the block.</li>
	<li>To ligate oligonucleotides with &#955;-ARS317, add 1 &#181;L of T4 DNA ligase and 0.63 &#181;L of ATP (200 mM). Mix gently by turning the tube upside down and incubate at room temperature for 2 h or 4 &#176;C overnight. Store at 4 &#176;C for 1 month. 
	NOTE: To avoid breaking up the DNA, all the mixing steps should be done by gently turning the tube upside down, instead of mixing using pipettes.</li>
</ol>
3. Coverslip Cleaning and Functionalization
<ol>
	<li>Coverslip cleaning
	<ol>
		<li>Place 20 coverslips into 4 staining jars (5 coverslips/jar), sonicate for 30 minutes in ethanol, and rinse the coverslips with ultrapure H2O 3 times.</li>
		<li>Sonicate for 30 minutes with 1 M potassium hydroxide (KOH), and rinse the coverslips with ultrapure H2O 3 times. Repeat the ethanol and KOH sonication once.</li>
		<li>Sonicate with acetone for 30 minutes, and then rinse with ultrapure H2O thoroughly (at least 3 times). 
		NOTE: Acetone must be removed thoroughly by rinsing with ultrapure H2O, because it may cause an explosion when organic solvent (such as acetone) mixed with the piranha solution mistakenly14.</li>
		<li>Place the coverslips into piranha solution (3:1 mixture of H2SO4 and 30% H2O2) and incubate at 95 &#176;C for 1 h. 
		NOTE: Piranha solution is highly energetic and erosive, so use with caution. When preparing piranha solution, add 50 mL of H2O2 first into a 500 mL glass beaker, and then add 150 mL of H2SO4 slowly into the beaker.</li>
		<li>Rinse the coverslips with ultrapure H2O 5 times. Then wash each coverslip with ultrapure H2O thoroughly using 3 beakers filled with ultrapure H2O, and dry the coverslip using paper from the edge of the coverslip. Place the coverslip into the staining jar and dry the coverslip thoroughly in a 110 &#176;C oven for 30 minutes.
		<ol>
			<li>Rinse the coverslip with methanol and place the staining jar into the 110 &#176;C oven again to dry the coverslip thoroughly. 
			NOTE: Drying the coverslip thoroughly is very important, because APTES will have lots of possible surface structures once it is in the presence of water19.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Coverslip functionalization
	<ol>
		<li>Add 70 mL of silane solution (93% methanol, 5% acetic, and 2% APTES) into each jar, screw the cap and leave the jar at room temperature overnight.</li>
		<li>Wash and dry the coverslips as described in step 3.1.5, except dry the coverslips thoroughly using nitrogen gas instead of placing them in the oven.</li>
		<li>Dissolve 150 mg of mPEG (methoxy-polyethylene glycol) and 6 mg of biotin-PEG (biotin-polyethylene glycol) into 1 mL of 0.1 M fresh-made NaHCO3 (pH 8.2) thoroughly. Then centrifuge at 17, 000 x g for 1 min to remove insoluble PEGs. 
		NOTE: Freshly made NaHCO3 is ready; there is no need to adjust its pH.</li>
		<li>Place the silanized coverslips in boxes, and put two small coverslips on the top of two ends of the silanized coverslips. Pipette 100 &#181;L of PEG solution on the center of the silanized coverslip, and place another silanized coverslip on the top.</li>
		<li>Add some ultrapure H2O in the box to keep it humid, and incubate the coverslips with PEG solution for at least 3 h in the dark. An overnight incubation can work as well.</li>
		<li>Separate the coverslip pairs and keep the functionalized surface face up, rinse the coverslips using ultrapure H2O extensively, and dry them with nitrogen gas.</li>
		<li>Mark the functionalized side of the coverslips on one of the corners using a marker pen, keep the functionalized side face up, place them in boxes and store the boxes in the vacuum desiccator for 1 month.</li>
		<li>To store the coverslips for a longer&#160;time, place one coverslip into a 50 mL tube drilled with one hole on its cap, then put the tube into a plastic bag, and seal the bag using a vacuum sealer. In this way, the coverslips can be stored at -20 &#176;C about 3 months.</li>
	</ol>
	</li>
</ol>
4. Flow Cell Assembly
<ol>
	<li>Cut a 15 mm &#215; 2 mm channel at the center of a piece of double-sided tape (30 mm &#215; 12 mm) using a puncher.</li>
	<li>Peel off the paper side of the double-sided tape and paste it on the glass slide with two holes. Press to remove air bubbles. Based on our experience, it is easier to remove air bubbles by peeling off the paper side than the plastic side of the double-sided tape.</li>
	<li>Cut a functionalized coverslip (60 mm &#215; 24 mm) into four pieces (30 mm &#215; 12 mm) using a diamond-tipped glass scribe, and remove the debris using nitrogen gas. Remember to keep the functionalized side face up.</li>
	<li>Peel off the plastic side of the double-sided tape, and paste the slide on the functionalized coverslip. Press gently to remove air bubbles between the coverslip and the tape. Removing air bubbles thoroughly can protect the flow cell from leaking while buffers were pumped into it.</li>
	<li>Insert inlet and outlet tubing into small and big holes, respectively. Fix the tubing using epoxy.</li>
	<li>Pump 20 &#181;L of streptavidin (0.2 mg/mL) into flow cell using a syringe manually and incubate at RT for 10 min. Then pump blocking buffer10 into flow cell to replace streptavidin, and keep it at room temperature.</li>
</ol>
5. Single Molecule Visualization
<ol>
	<li>Obtain the focal alignment of red and far-red with A5 on the fluorescence microscope test slide #1 by simultaneous excitation using a 532&#160;nm laser and a 640 nm laser. Produce dual wavelength images with splitting optics (See Table of Materials).</li>
	<li>Place the flow cell on the microscope and connect its outlet tubing to a longer tubing connecting with a 10 mL spring installed on an automated infusion/withdrawal programmable pump. 
	NOTE: Pumping buffers into the flow cell mentioned below means withdrawing buffers from the inlet tubing of the flow cell to the syringe.</li>
	<li>Pump blocking buffer at 500 &#181;L/min into the flow cell to remove air quickly and ensure that the buffer in the flow cell is unblocked. Then pump the blocking buffer at 200 &#181;L/min into the flow cell and flip the outlet tubing to remove air bubbles from the inlet tubing and the flow cell thoroughly. 
	NOTE: All buffers pumped into the flow cell should be degassed in a vacuum desiccator for at least 15 minutes before use.</li>
	<li>Add 0.5 &#181;L of biotinylated &#955;-ARS317 DNA into 80 &#181;L of blocking buffer, and pump it into the flow cell at 25 &#181;L/min for 2 min. Then flush &#955;-ARS317 DNA using 200 &#181;L of blocking buffer at a rate of 50 &#181;L/min.</li>
	<li>Pump 200 &#181;L of binding buffer10 at a rate of 50 &#181;L/min to remove the blocking buffer in the flow cell.</li>
	<li>Add 0.2 &#181;L of streptavidin-coated Qdot705 (1 &#181;M) and 0.2 &#181;L of biotinylated ORC (1.2 &#181;M) into a tube, and incubate at room temperature for 5 min. Then add 20 &#181;L of binding buffer into the tube, and place it on ice. The final concentration of ORC-Qdot705 is about 10 nM.</li>
	<li>Add 2 &#181;L of ORC-Qdot705 (10 nM), 1 &#181;L of DTT (100 mM), 1 &#181;L of ATP (200 mM) into 96 &#181;L of binding buffer. The final concentration of ORC-Qdot705 is 0.2 nM.</li>
	<li>Pump 20 &#181;L of 0.2 nM ORC-Qdot705 at the rate of 10 &#181;L/min into the flow cell. Flush out of excessive ORC-Qdot705 using 200 &#181;L of binding buffer at the rate of 100 &#181;L/min. Then pump binding buffer with 30 nM SYTOX Orange into the flow cell to stain DNA substrates at a rate of 100 &#181;L/min.</li>
	<li>Excite the ORC-Qdot705 signal and SYTOX Orange stained DNA signal using a 405 nm laser and 532 nm laser, respectively. Observe the signals simultaneously with 100 &#181;L/min flow using a Quad-band bandpass filter (FF01-446/510/581/703-25) and record the images by EM-CCD with 100 ms per frame. Collect 20 image stacks from different fields.</li>
</ol>
6. Data Analysis
<ol>
	<li>Crop the images using the Fiji software with a new plugin, which is modified by ourselves based on the Image plugin (OI_cut_RGBmerge) developed by Dr. Ron Vale&#39;s lab at University of California, San Francisco.
	<ol>
		<li>Crop a stack of images (512 &#215; 512 pixels) into two stacks of images (256 &#215; 256 pixels). One is a 532 nm laser exciting result, and the other is a 405 nm laser exciting result.</li>
	</ol>
	</li>
	<li>Process 61 sequential images using Fiji-Image-Stacks-Z project (average intensity).</li>
	<li>Measure the length of DNA (LDNA) and the distance from the site of ORC-Qdot705 (LORC) binding on DNA to the DNA&#39;s tethered end manually.</li>
	<li>Calculate the result of LORC/LDNA using excel, analyze the data using the bootstrap method by R, then create the histogram and fit Gaussian distributions.</li>
</ol>

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The authors have nothing to disclose.

Here, we present a protocol to observe the interaction between the Qdot-labeled protein and the site-specifically modified &#955; DNA using the TIRFM in a flow-cell. The necessary steps include site-specific modification of DNA substrate, DNA biotinylation, coverslip cleaning and functionalization, flow-cell preparation, and single-molecule imaging. There are two key points that should be noted. First, all the steps involved with &#955; DNA should be manipulated gently to decrease any possible damage, e.g., avoi...

Interactions between protein and DNA are essential to many complex biological processes, such as DNA replication, DNA repair, and transcription. Although conventional approaches have shed light on the properties of these processes, many key mechanisms are still unclear. Recently, with the rapidly developing single molecule techniques, some of the mechanisms have been addressed1,2,3.
The application of single-molecule fluorescence microscopy on visualizing protein-DNA interactions in real-time mainly depends on the development of fluorescence detection and fluorescent probes. For a single molecule study, it is important to label the protein of interest with a suitable fluorescent probe since fluorescence detection systems are mostly available commercially.
Fluorescent proteins are commonly used in molecular biology. However, the low fluorescent brightness and stability against photobleaching restrict its application in many single molecule assays. Quantum dots (Qdots) are tiny light-emitting nanoparticles4. Due to their unique optical properties, Qdots are 10 - 20 times brighter and several thousand times more stable than the widely used organic dyes5. Moreover, Qdots have a large Stokes shift (the difference between the position of excitation and emission peaks)5. Thus, Qdots can be used for long-time observation (minutes to hours) and acquisition of images with high signal-to-noise ratios, while they cannot be utilized in the quantitative assays.
To date, there are two approaches to label a target protein with Qdots site-specifically: labeling with the aid of Qdot-conjugated&#160;primary or secondary antibodies6,7,8; or labeling the target protein with Qdots directly, which is based on the strong interaction between biotin and streptavidin9,10,11,12,13. Streptavidin-coated Qdots are commercially available. In our recent study, site-specifically biotinylated proteins in budding yeast with high efficiency were purified by co-overexpression of BirA and Avi-tagged proteins in vivo10. By following and optimizing the single-molecule assays14,15,16,17, we observed the interactions between Qdot-labeled proteins and DNA at the single molecule level using TIRFM10.
Here, we choose the budding yeast origin recognition complex (ORC), which can specifically recognize and bind to the autonomously replicating sequence (ARS), as our protein of interest. The following protocol presents a step-by-step procedure of visualizing the interaction of Qdot-labeled ORC with ARS using TIRFM. The preparation of the site-specifically modified DNA substrate, the DNA biotinylation, the coverslip cleaning and functionalization, the flow-cell assembly, and the single-molecule imaging are described.

<table><tbody><tr><td>Lambda DNA</td><td>New England Biolabs</td><td>N3011</td><td>Store 25 &amp;mu;L aliquots at -20 &amp;ordm;C.</td></tr><tr><td>&lt;em&gt;XhoI&lt;/em&gt; enzyme</td><td>Thermo Fisher Scientific</td><td>FD0694</td><td></td></tr><tr><td>Quick-fusion cloning kit</td><td>Biotool</td><td>B22611</td><td></td></tr><tr><td>MaxPlax Lambda&lt;br/&gt; Packaging Extracts</td><td>Epicentre</td><td>MP5110</td><td>Bacterial strain LE392MP&lt;br/&gt; is included in this package.</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10013092</td><td>Any brand is acceptable.</td></tr><tr><td>Tris</td><td>Amresco</td><td>0497-5KG</td><td></td></tr><tr><td>NaCl</td><td>Beijing Chemical works</td><td>N/A</td><td>Any brand is acceptable.</td></tr><tr><td>MgCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10012818</td><td></td></tr><tr><td>Chloroform</td><td>Beijing Chemical works</td><td>N/A</td><td>Any brand is acceptable.</td></tr><tr><td>NZ-amine</td><td>Amresco</td><td>J853-250G</td><td></td></tr><tr><td>Casamino acids</td><td>Sigma-Aldrich</td><td>22090-500G</td><td></td></tr><tr><td>PEG8000</td><td>Beyotime</td><td>ST483</td><td></td></tr><tr><td>Magnetic stirring apparatus</td><td>IKA</td><td>KMO2 basic</td><td></td></tr><tr><td>15 mL Eppendorf tube</td><td>Eppendorf</td><td>30122151</td><td>15 mL, sterile, bulk, 500pcs</td></tr><tr><td>Rnase</td><td>SIGMA</td><td>R4875-100MG</td><td></td></tr><tr><td>Dnase</td><td>SIGMA</td><td>D5319-500UG</td><td></td></tr><tr><td>Proteinase K</td><td>Amresco</td><td>0706-100MG</td><td></td></tr><tr><td>Biotinylated primers</td><td>Thermo Fisher Scientific</td><td>N/A</td><td></td></tr><tr><td>T4 DNA ligase, T4 DNA Ligase, Reaction Buffer (10x)</td><td>New England Biolabs</td><td>M0202</td><td></td></tr><tr><td>Coverslip</td><td>Thermo Fisher Scientific</td><td>22266882</td><td></td></tr><tr><td>Ethanol</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10009259</td><td></td></tr><tr><td>Potassium hydroxide (KOH)</td><td>Sigma-Aldrich</td><td>306568-100G</td><td></td></tr><tr><td>Acetone</td><td>Thermo Fisher Scientific</td><td>A949-4</td><td></td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;SO&lt;sub&gt;4&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>80120891</td><td>sulfuric acid</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt;</td><td>Sinopharm Chemical Reagent Co.,Ltd</td><td>10011218</td><td>30% Hydrogen peroxide</td></tr><tr><td>Methanol</td><td>Sigma-Aldrich</td><td>322415-2L</td><td></td></tr><tr><td>Acetic acid</td><td>Sigma-Aldrich</td><td>V900798</td><td></td></tr><tr><td>APTES</td><td>Sigma-Aldrich</td><td>A3648</td><td></td></tr><tr><td>mPEG&lt;br/&gt; (methoxy-polyethylene glycol)</td><td>Lysan</td><td>mPEG-SVA-5000</td><td></td></tr><tr><td>biotin-PEG&lt;br/&gt; (biotin-polyethylene glycol)</td><td>Lysan</td><td>Biotin-PEG-SVA-5000</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>31437-500G</td><td></td></tr><tr><td>Vacuum desiccator</td><td>Tianjin Branch Billion Lung Experimental Equipment Co., Ltd.</td><td>IPC250-1</td><td></td></tr><tr><td>Vacuum sealer</td><td>MAGIC SEAL</td><td>WP300</td><td></td></tr><tr><td>Diamond-tipped glass scribe</td><td>ELECTRON MICROSCOPY SCIENCES</td><td>70036</td><td></td></tr><tr><td>Glass slide</td><td>Sail Brand</td><td>7101</td><td></td></tr><tr><td>Inlet tubing</td><td>SCI (Scientific Commodties INC.)</td><td>BB31695-PE/2</td><td>inner diameter 0.38 mm; outer diameter 1.09 mm.</td></tr><tr><td>Outlet tubing</td><td>SCI (Scientific Commodties INC.)</td><td>BB31695-PE/4</td><td>inner diameter 0.76 mm; outer diameter 1.22 mm.</td></tr><tr><td>Double-sided tape</td><td>Sigma-Aldrich</td><td>GBL620001-1EA</td><td></td></tr><tr><td>Epoxy</td><td>LEAFTOP</td><td>9005</td><td>five minutes epoxy</td></tr><tr><td>Streptavidin</td><td>Sigma-Aldrich</td><td>S4762</td><td></td></tr><tr><td>Fluorescence Microscope</td><td>Olympus</td><td>IX71</td><td></td></tr><tr><td>Infusion/withdrawal&lt;br/&gt; programmable pump</td><td>Harvard apparatus</td><td>70-4504</td><td></td></tr><tr><td>532 nm laser</td><td>Coherent</td><td>Sapphire-532-50</td><td></td></tr><tr><td>640 nm laser</td><td>Coherent</td><td>OBIS-640-100</td><td></td></tr><tr><td>EMCCD Camera</td><td>Andor</td><td>DU-897E-CS0-BV</td><td></td></tr><tr><td>W-View Gemini Imaging&lt;br/&gt; splitting optics</td><td>Hamamatsu photonics K.K.</td><td>A12801-01</td><td></td></tr><tr><td>TIRF illumination system</td><td>Olympus</td><td>IX2-RFAEVA2</td><td></td></tr><tr><td>60&amp;times;TIRF objective</td><td>Olympus</td><td>APON60XOTIRF</td><td></td></tr><tr><td>Quad-edge laser dichroic&lt;br/&gt; beamsplitter</td><td>Semrock</td><td>Di01-R405/488/&lt;br/&gt; 532/635-25x36</td><td></td></tr><tr><td>Quad-band bandpass filter</td><td>Semrock</td><td>FF01-446/510/&lt;br/&gt; 581/703-25</td><td></td></tr><tr><td>Dichroic beamsplitter</td><td>Semrock</td><td>FF649-Di01-25x36</td><td></td></tr><tr><td>Emission filter</td><td>Chroma Technology Corp</td><td>ET585/65m</td><td></td></tr><tr><td>Emission filter</td><td>Chroma Technology Corp</td><td>ET665lp</td><td></td></tr><tr><td>FocalCheck fluorescence, microscope test slide #1</td><td>Thermo Fisher Scientific</td><td>F36909</td><td></td></tr><tr><td>SYTOX Orange</td><td>Thermo Fisher Scientific</td><td>S11368</td><td></td></tr><tr><td>Qdot&lt;sub&gt;705 &lt;/sub&gt;Streptavidin Conjugate</td><td>Thermo Fisher Scientific</td><td>Q10163MP</td><td>Store at 4 &amp;ordm;C, do not freeze.</td></tr><tr><td>ATP</td><td>Amresco</td><td>0220-25G</td><td>Prepare 200 mM ATP solution, using ddH&lt;sub&gt;2&lt;/sub&gt;O, adjust pH to 7.0, and store 10 &amp;mu;L aliquots at -20 &amp;ordm;C.</td></tr><tr><td>DTT</td><td>Amresco</td><td>M109-5G</td><td>Prepare 1 M solution using ddH&lt;sub&gt;2&lt;/sub&gt;O, and store 10 &amp;mu;l aliquots at -20 &amp;ordm;C.</td></tr></tbody></table>

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.

To visualize the interaction between Qdot-labeled ORC and the ARS, we first constructed the &#955;-ARS317 DNA substrate. A DNA fragment containing ARS317 was integrated into XhoI site (33.5 kb) of native &#955; DNA by homologous recombination (Figure 1A). The recombination product was packaged using extracts and the packaged phage particles were cultured on LB plates (Figure 1B). The positive phage plaque was screened by...

Watch this Scientific Journal Video about Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified Î» DNA at the Single Molecule Level at JoVE.com

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

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. ...

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Research

JoVE Journal

Biology

7.5K Views.  Chinese Academy of Sciences. Hier presenteren we een protocol om te bestuderen van DNA-eiwit interactie door de totale interne reflectie fluorescentie microscopie (TIRFM) met behulp van een site-specifically gemodificeerde λ DNA substraat en een Quantum-dot label eiwit.

Video: Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified λ DNA at the Single Molecule Level

Detection and Visualization of DNA Damage-induced Protein Complexes in Suspension Cell Cultures Using the Proximity Ligation Assay

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of programmed DNA breaks in lymphocytes. The Ataxia-Telangiectasia Mutated kinase (ATM), ATM- and Rad3-Related kinase (ATR) and the catalytic subunit of DNA-dependent Protein Kinase (DNA-PKcs) are among the first that are activated upon induction of DNA damage, and are central regulators of a network that controls DNA repair, apoptosis and cell survival. As part of a tumor-suppressive pathway, ATM and ATR activate p53 through phosphorylation, thereby regulating the transcriptional activity of p53. DNA damage also results in the formation of so-called ionizing radiation-induced foci (IRIF) that represent complexes of DNA damage sensor and repair proteins that accumulate at the sites of DNA damage, which are visualized by fluorescence microscopy. Co-localization of proteins in IRIFs, however, does not necessarily imply direct protein-protein interactions, as the resolution of fluorescence microscopy is limited. 
In situ Proximity Ligation Assay (PLA) is a novel technique that allows the direct visualization of protein-protein interactions in cells and tissues with unprecedented specificity and sensitivity. This technique is based on the spatial proximity of specific antibodies binding to the proteins of interest. When the interrogated proteins are within ~40 nm an amplification reaction is triggered by oligonucleotides that are conjugated to the antibodies, and the amplification product is visualized by fluorescent labeling, yielding a signal that corresponds to the subcellular location of the interacting proteins. Using the established functional interaction between ATM and p53 as an example, it is demonstrated here how PLA can be used in suspension cell cultures to study the direct interactions between proteins that are integral parts of the DNA damage response.

Here, it is demonstrated how the in situ Proximity Ligation Assay (PLA) can be used to detect and visualize the direct protein-protein interactions between ATM and p53 in suspension cell cultures exposed to genotoxic stress.

The DNA damage response orchestrates the repair of DNA lesions that occur spontaneously, are caused by genotoxic stress, or appear in the context of ...

Biochemistry

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Bioengineering

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 ...

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.

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

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

Single-Molecule 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 ...

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 ...

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).

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 ...

In Situ Monitoring of Transiently Formed Molecular Chaperone Assemblies in Bacteria, Yeast, and Human Cells

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the JDP family could form transient heterocomplexes in eukaryotes to fine-tune substrate selection for the 70 kDa heat shock protein (Hsp70) chaperone-based protein disaggregases. The JDP complexes target acute/chronic stress induced aggregated proteins and presumably help assemble the disaggregases by recruiting multiple Hsp70s to the surface of protein aggregates. The extent of the protein quality control (PQC) network formed by these physically interacting JDPs remains largely uncharacterized in vivo. Here, we describe a microscopy-based in situ protein interaction assay named the proximity ligation assay (PLA), which is able to robustly capture&#160;these transiently formed chaperone complexes in distinct cellular compartments of eukaryotic cells. Our work expands the employment of PLA from human cells to yeast (Saccharomyces cerevisiae) and bacteria (Escherichia coli), thus rendering an important tool to monitor the dynamics of transiently formed protein assemblies in both prokaryotic and eukaryotic cells.

Cognate J-domain proteins cooperate with the Hsp70 chaperone to assist in a myriad of biological processes ranging from protein folding to degradation. Here, we describe an in situ proximity ligation assay, which allows the monitoring of these transiently formed chaperone machineries in bacterial, yeast and human cells.

J-domain proteins (JDPs) form the largest and the most diverse co-chaperone family in eukaryotic cells. Recent findings show that specific members of the ...

Single-Molecule Fluorescence Visualization of DNA Polymerase Dynamics at G-Quadruplexes

The ability of proteins&#160;involved in eukaryotic DNA replication to&#160;overcome obstacles &#8212; such as protein and DNA &#39;roadblocks&#39;&#160;&#8212; is critical for ensuring faithful&#160;genome duplication.&#160;G-quadruplexes are higher-order nucleic acid structures that form in guanine-rich regions of DNA and have been shown to act as obstacles, interfering with genomic maintenance pathways. This study introduces a real-time, fluorescence microscopy-based method to observe DNA polymerase interactions with G-quadruplex structures. Short, primed DNA oligonucleotides containing a G-quadruplex were immobilized on functionalized glass coverslips within a microfluidic flow cell. Fluorescently labeled DNA polymerases were introduced, allowing their behavior and stoichiometry to be monitored over time. This approach enabled the observation of polymerase behavior as it was stalled by a G-quadruplex. Specifically, using fluorescently labeled yeast polymerase &#948;, it was found that upon encountering a G-quadruplex, the polymerase undergoes a continuous cycle of binding and unbinding. This single-molecule assay can be adapted to study interactions between various DNA-maintenance proteins and obstacles on the DNA substrate.

This protocol outlines a fluorescence microscopy-based single-molecule DNA replication assay, enabling real-time visualization of interactions between DNA polymerases and obstacles such as G-quadruplex structures.

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified λ DNA at the Single Molecule Level

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