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 &#956;L aliquots at -20 &#186;C.</td>
		</tr>
		<tr>
			<td>XhoI 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 
			Packaging Extracts</td>
			<td>Epicentre</td>
			<td>MP5110</td>
			<td>Bacterial strain LE392MP 
			is included in this package.</td>
		</tr>
		<tr>
			<td>MgSO4</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>MgCl2</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>H2SO4</td>
			<td>Sinopharm Chemical Reagent Co.,Ltd</td>
			<td>80120891</td>
			<td>sulfuric acid</td>
		</tr>
		<tr>
			<td>H2O2</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 
			(methoxy-polyethylene glycol)</td>
			<td>Lysan</td>
			<td>mPEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>biotin-PEG 
			(biotin-polyethylene glycol)</td>
			<td>Lysan</td>
			<td>Biotin-PEG-SVA-5000</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</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 
			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 
			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&#215;TIRF objective</td>
			<td>Olympus</td>
			<td>APON60XOTIRF</td>
			<td></td>
		</tr>
		<tr>
			<td>Quad-edge laser dichroic 
			beamsplitter</td>
			<td>Semrock</td>
			<td>Di01-R405/488/ 
			532/635-25x36</td>
			<td></td>
		</tr>
		<tr>
			<td>Quad-band bandpass filter</td>
			<td>Semrock</td>
			<td>FF01-446/510/ 
			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>Qdot705 Streptavidin Conjugate</td>
			<td>Thermo Fisher Scientific</td>
			<td>Q10163MP</td>
			<td>Store at 4 &#186;C, do not freeze.</td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Amresco</td>
			<td>0220-25G</td>
			<td>Prepare 200 mM ATP solution, using ddH2O, adjust pH to 7.0, and store 10 &#956;L aliquots at -20 &#186;C.</td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Amresco</td>
			<td>M109-5G</td>
			<td>Prepare 1 M solution using ddH2O, and store 10 &#956;l aliquots at -20 &#186;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. ...

visualizing-interaction-between-qdot-labeled-protein-site

Research

JoVE Journal

Biology

7.5K Views.  Chinese Academy of Sciences. Here, we present a protocol to study DNA-protein interactions by total internal reflection fluorescence microscopy (TIRFM) using a site-specifically modified λ DNA substrate and a Quantum-dot labeled protein.

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

Method for Measurement of Viral Fusion Kinetics at the Single Particle Level

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion events, making it difficult to quantitatively analyze the sequence of the molecular steps involved. We have developed an in vitro, two-color fluorescence assay to monitor kinetics of single virus particles fusing with a target bilayer on an essentially fluid support.
Influenza viral particles are incubated with a green lipophilic fluorophore to stain the membrane and a red hydrophilic fluorophore to stain the viral interior. We deposit a ganglioside-containing lipid bilayer on the dextran-functionilized glass surface of a flow cell, incubate the viral particles on the planar bilayer and image the fluorescence of a 100 x 100 &mu;m2 area, containing several hundreds of particles, on a CCD camera. By imaging both the red and green fluorescence, we can simultaneously monitor the behavior of the membrane dye (green) and the aqueous content (red) of the particles.
Upon lowering the pH to a value below the fusion pH, the particles will fuse with the membrane. Hemifusion, the merging of the outer leaflet of the viral membrane with the outer leaflet of the target membrane, will be visible as a sudden change in the green fluorescence of a particle. Upon the subsequent fusion of the two remaining distal leaflets a pore will be formed and the red-emitting fluorophore in the viral particle will be released under the target membrane. This event will give rise to a decrease of the red fluorescence of individual particles. Finally, the integrated fluorescence from a pH-sensitive fluorophore that is embedded in the target membrane reports on the exact time of the pH drop.
From the three fluorescence-time traces, all the important events (pH drop, lipid mixing upon hemifusion, content mixing upon pore formation) can now be extracted in a straightforward manner and for every particle individually. By collecting the elapsed times for the various transitions for many individual particles in histograms, we can determine the lifetimes of the corresponding intermediates. Even hidden intermediates that do not have a direct fluorescent observable can be visualized directly from these histograms.

We present an in vitro, two-color fluorescence assay to visualize the fusion of single virus particles with a fluid target bilayer. By labeling viral particles with fluorophores that differentially stain the viral membrane and its interior, we are able to monitor the kinetics of hemifusion and pore formation.

Membrane fusion is an essential step during entry of enveloped viruses into cells. Conventional fusion assays typically report on a large number of fusion ...

Visualizing Single-molecule DNA Replication with Fluorescence Microscopy

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA template is attached to a functionalized glass coverslip and replicated extensively after introduction of replication proteins and nucleotides (Figure 1). The growing product double-strand DNA (dsDNA) is extended with laminar flow and visualized by using an intercalating dye. Measuring the position of the growing DNA end in real time allows precise determination of replication rate (Figure 2). Furthermore, the length of completed DNA products reports on the processivity of replication. This experiment can be performed very easily and rapidly and requires only a fluorescence microscope with a reasonably sensitive camera.


This protocol demonstrates a simple single-molecule fluorescence microscopy technique for visualizing DNA replication by individual replisomes in real time.

We describe a simple fluorescence microscopy-based real-time method for observing DNA replication at the single-molecule level. A circular, forked DNA ...

Studying Proteolysis of Cyclin B at the Single Cell Level in Whole Cell Populations

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies during chromosome separation are a hallmark of malignancy and associated with progressive disease 2-4. The spindle assembly checkpoint (SAC) is a mitotic surveillance mechanism that holds back cells at metaphase until every single chromosome has established a stable bipolar attachment to the mitotic spindle1. The SAC exerts its function by interference with the activating APC/C subunit Cdc20 to block proteolysis of securin and cyclin B and thus chromosome separation and mitotic exit. Improper attachment of chromosomes prevents silencing of SAC signaling and causes continued inhibition of APC/CCdc20 until the problem is solved to avoid chromosome missegregation, aneuploidy and malignant growths1.
Most studies that addressed the influence of improper chromosomal attachment on APC/C-dependent proteolysis took advantage of spindle disruption using depolymerizing or microtubule-stabilizing drugs to interfere with chromosomal attachment to microtubules. Since interference with microtubule kinetics can affect the transport and localization of critical regulators, these procedures bear a risk of inducing artificial effects 5.
To study how the SAC interferes with APC/C-dependent proteolysis of cyclin B during mitosis in unperturbed cell populations, we established a histone H2-GFP-based system which allowed the simultaneous monitoring of metaphase alignment of mitotic chromosomes and proteolysis of cyclin B 6.
To depict proteolytic profiles, we generated a chimeric cyclin B reporter molecule with a C-terminal SNAP moiety 6 (Figure 1). In a self-labeling reaction, the SNAP-moiety is able to form covalent bonds with alkylguanine-carriers (SNAP substrate) 7,8 (Figure 1). SNAP substrate molecules are readily available and carry a broad spectrum of different fluorochromes. Chimeric cyclin B-SNAP molecules become labeled upon addition of the membrane-permeable SNAP substrate to the growth medium 7 (Figure 1). Following the labeling reaction, the cyclin B-SNAP fluorescence intensity drops in a pulse-chase reaction-like manner and fluorescence intensities reflect levels of cyclin B degradation 6 (Figure 1). Our system facilitates the monitoring of mitotic APC/C-dependent proteolysis in large numbers of cells (or several cell populations) in parallel. Thereby, the system may be a valuable tool to identify agents/small molecules that are able to interfere with proteolytic activity at the metaphase to anaphase transition. Moreover, as synthesis of cyclin B during mitosis has recently been suggested as an important mechanism in fostering a mitotic block in mice and humans by keeping cyclin B expression levels stable 9,10, this system enabled us to analyze cyclin B proteolysis as one element of a balanced equilibrium 6.

Metaphase to anaphase transition is triggered through anaphase-promoting complex (APC/C)-dependent ubiquitination and subsequent destruction of cyclin B. Here, we established a system which, following pulse-chase labeling, allows monitoring cyclin B proteolysis in entire cell populations and facilitates the detection of interference by the mitotic checkpoint.

Equal distribution of chromosomes between the two daughter cells during cell division is a prerequisite for guaranteeing genetic stability 1. Inaccuracies ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate 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 ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

Analysis of LINE-1 Retrotransposition at the Single Nucleus Level

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are inactive due to 5' truncations, ~80-100 of these elements remain retrotransposition competent and propagate to different locations throughout the genome via RNA intermediates. While older L1s are believed to target AT rich regions of the genome, the chromosomal targets of newer, more active L1s remain poorly defined. Here we describe fluorescence in situ hybridization (FISH) methodology that can be used to track patterns of L1 insertion and rates of ectopic L1 incorporation at the single nucleus level. In these experiments, fluorescein isothiocyanate/cyanine-3 (FITC/CY3) labeled neomycin probes were employed to track L1 retrotransposition in vitro in HepG2 cells stably expressing ectopic L1. This methodology prevents errors in the estimation of rates of retrotransposition posed by toxicity and account for the occurrence of multiple insertions into a single nucleus.

Here we employ FISH methodology to track LINE-1 retrotransposition at the single nuclei level in chromosome spreads of HepG2 cell lines stably expressing synthetic LINE-1.

Long interspersed nuclear element-1 (Line-1 or L1) accounts for approximately 17% of the DNA present in the human genome. While the majority of L1s are ...

CUBIC Protocol Visualizes Protein Expression at Single Cell Resolution in Whole Mount Skin Preparations

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium covering most of our body, and epidermal appendages such as the hair follicles and sweat glands. The epidermis undergoes regeneration throughout life and in response to injury. This is enabled by K14-expressing basal epidermal stem/progenitor cell populations that are tightly regulated by multiple regulatory mechanisms active within the epidermis and between epidermis and dermis. This article describes a simple method to clarify full thickness mouse skin biopsies, and visualize K14 protein expression patterns, Ki67 labeled proliferating cells, Nile Red labeled sebocytes, and DAPI nuclear labeling at single cell resolution in 3D. This method enables accurate assessment and quantification of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines. The CUBIC protocol is the best method available to date to investigate molecular and cellular interactions in full thickness skin biopsies at single cell resolution.

This report describes a CUBIC protocol to clarify full thickness mouse skin biopsies, and visualize protein expression patterns, proliferating cells, and sebocytes at the single cell resolution in 3D. This method enables accurate assessment of skin anatomy and pathology, and of abnormal epidermal phenotypes in genetically modified mouse lines.

The skin is essential for our survival. The outer epidermal layer consists of the interfollicular epidermis, which is a stratified squamous epithelium ...

Advanced Confocal Microscopy Techniques to Study Protein-protein Interactions and Kinetics at DNA Lesions

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological approach to analyzing protein kinetics at DNA lesions over time or protein-protein interactions on locally microirradiated chromatin. We also show how to recognize individual phases of the cell cycle using the Fucci cellular system to study cell-cycle-dependent protein kinetics at DNA lesions. A methodological description of the use of two UV lasers (355 nm and 405 nm) to induce different types of DNA damage is also presented. Only the cells microirradiated by the 405-nm diode laser proceeded through mitosis normally and were devoid of cyclobutane pyrimidine dimers (CPDs). We also show how microirradiated cells can be fixed at a given time point to perform immunodetection of the endogenous proteins of interest. For the DNA repair studies, we additionally describe the use of biophysical methods including FRAP (Fluorescence Recovery After Photobleaching) and FLIM (Fluorescence Lifetime Imaging Microscopy) in cells with spontaneously occurring DNA damage foci. We also show an application of FLIM-FRET (Fluorescence Resonance Energy Transfer) in experimental studies of protein-protein interactions.

Laser microirradiation is a useful tool for studies of DNA repair in living cells. A methodological approach for the use of UVA lasers to induce various DNA lesions is shown. We have optimized a method for local microirradiation that maintains the normal cell cycle; thus, irradiated cells proceed through mitosis.

Local microirradiation with lasers represents a useful tool for studies of DNA-repair-related processes in live cells. Here, we describe a methodological ...

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

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