Name: visualizing the interaction between the qdot-labeled protein and site-specifically modified &#955; dna at the single molecule level
Uploaded: 2018-07-17T14:30:00
Description: 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

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 reacti...

<ol>
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D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+single-molecule+imaging+reveals+a+direct+interaction+between+UvrC+and+UvrB+on+DNA+tightropes.">Real-time single-molecule imaging reveals a direct interaction between UvrC and UvrB on DNA tightropes.</a> Nucleic acids research. 41 (9), 4901-4912 (2013).</li><li>Kad, N. M., Wang, H., Kennedy, G. G., Warshaw, D. M., Van Houten, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collaborative+dynamic+DNA+scanning+by+nucleotide+excision+repair+proteins+investigated+by+single-molecule+imaging+of+quantum-dot-labeled+proteins.">Collaborative dynamic DNA scanning by nucleotide excision repair proteins investigated by single-molecule imaging of quantum-dot-labeled proteins.</a> Molecular cell. 37 (5), 702-713 (2010).</li><li>Chandradoss, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+Passivation+for+Single-molecule+Protein+Studies.">Surface Passivation for Single-molecule Protein Studies.</a> Jove-Journal of Visualized Experiments. (86), (2014).</li><li>Chen, X. Q., Zhao, E. S., Fu, Y. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+single-molecule+approach+to+visualize+the+nucleosome+assembly+in+yeast+nucleoplasmic+extracts.">Using single-molecule approach to visualize the nucleosome assembly in yeast nucleoplasmic extracts.</a> Science Bulletin. 62 (6), 399-404 (2017).</li><li>Yardimci, H., Loveland, A. B., van Oijen, A. M., Walter, J. 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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. ...

This method can help answer the key questions in the field of single molecule imaging of DNA protein interactions such as DNA replication, gene repair and the maintenance of chromosome structure. The main advantage of this technique is that one can obtain modified lambda DNA high collagen and then rapidly cure the labeled proteins. Visual demonstration of this method is critical because the flow cell assembly steps are difficult to learn.To clean the coverslips, place 20 coverslips into four staining jars, pour ethanol into them and sonicate for 30 minutes in ethanol. Then rinse the coverslips with ultrapure water three times. Next, sonicate the coverslips with one molar potassium hydroxide for 30 minutes and rinse the coverslips with ultrapure water three times.Then repeat the ethanol and potassium hydroxide sonication once. Sonicate the coverslips with acetone for 30 minutes and then thoroughly rinse with ultrapure water. Now place the coverslips into Piranha solution and incubate at 95 degrees Celsius for one hour.Following incubation, rinse the coverslips with ultrapure water five times. Then wash each coverslip extensively with ultrapure water. Use paper to dry the coverslip from its edge and place the coverslip into a staining jar.Thoroughly dry the coverslip in a 110 degree Celsius oven. Now rinse the coverslip with methanol and then place the staining jar back into the 110 degree Celsius oven to dry the coverslip. To perform coverslip functionalization, add 70 milliliters of Silane solution into each jar, screw the cap and leave the jar at room temperature overnight.Wash the coverslips using three beakers filled with ultrapure water. Dry the coverslips thoroughly using nitrogen gas. Dissolve 150 milligrams of methoxy-PEG and six milligrams of biotin-PEG into one milliliter of freshly prepared 0.1 molar sodium bicarbonate.Centrifuge at 17, 000 times g for one minute to remove insoluble PEGs. Now pipette 100 microliters of PEG solution on the center of the silanized coverslip and place another silanized coverslip on the top. Incubate the coverslips with PEG solution for at least three hours in the dark.Separate the coverslip pairs and keep the functionalized surface face up. Extensively rinse the coverslips using ultrapure water and dry them with nitrogen gas. Now mark the functionalized side of the coverslips on one of the corners using a marker pen.Place one coverslip into a 50 milliliter tube drilled with one hole on its cap. Place the tube into a plastic bag, seal the bag using a vacuum sealer and store it at minus 20 degrees Celsius. To assemble the flow cell, cut a channel at the center of a piece of double-sided tape using a puncher.Peel off the paper side of the double-sided tape and paste it on a glass side with two holes. It is easier to remove air bubbles by peeling off the paper side than the plastic side of the double-sided tape. Press the tape to remove air bubbles.Then cut a functionalized coverslip into four pieces using a diamond-tipped glass scribe and remove the debris using nitrogen gas keeping the functionalized side up. 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 are pumped into it. Now insert the inlet tubing into the small hole and insert the outlet tubing into the big hole. Fix the tubing using epoxy.Manually pump 20 microliters of 0.2 milligrams per milliliter Streptavidin into the flow cell using a syringe and incubate at room temperature for 10 minutes. Then pump blocking buffer into the flow cell to replace Streptavidin and keep it at room temperature. Obtain the focal alignment of red and far red with A5 on the fluorescence microscope test slide number one by simultaneous excitation using a 532 nanometer laser and a 640 nanometer laser.Produce dual wavelength images with splitting optics. Place the flow cell on the microscope and connect its outlet tubing to a longer tubing that is connected to a 10 milliliter spring installed on an automated infusion withdrawal programmable pump. Remove air bubbles in the flow cell by injecting blocking buffer and flipping the outlet tubing.Add 0.5 microliters of biotinylated lambda-ARS317 DNA into 80 microliters of blocking buffer. Pump the prepared mixture into the flow cell at 25 microliters per minute for two minutes. Then flush the lambda-ARS317 DNA using 200 microliters of blocking buffer at a rate of 50 microliters per minute.Now pump 200 microliters of binding buffer at a rate of 50 microliters per minute to remove the blocking buffer in the flow cell. Next, add two microliters of ORC-Qdot705, one microliter of DTT, and one microliter of ATP into 96 microliters of binding buffer. The final concentration of ORC-Qdot705 is 0.2 nanomolar.After pumping the binding buffer as detailed in the text protocol, pump 20 microliters of the prepared ORC-Qdot705 solution at a rate of 10 microliters per minute into the flow cell. Flush out excessive ORC-Qdot705 using 200 microliters of binding buffer at a rate of 100 microliters per minute. Then pump binding buffer with 30 nanomolar SYTOX Orange into the flow cell to stain DNA substrates at a rate of 100 microliters per minute.Finally, excite the ORC-Qdot705 signal using a 405 nanometer laser and excite the SYTOX Orange stained DNA signal using a 532 nanometer laser. Observe the signals simultaneously with 100 microliters per minute flow using a quad band bandpass filter. Record the images by EMCCD with 100 milliseconds per frame.The illustration of lambda-ARS317 DNA substrate is shown here. Qdot705-labeled ORC was excited by a 405 nanometer laser. SYTOX Orange stained DNA was excited by a 532 nanometer laser.The merged result suggests that Qdot705-labeled ORC binds at ARS317. The result of ORC-binding distribution on lambda-ARS317 DNA shows that ORC binds at ARS317 with a high abundance. Following this procedure, other methods like single-molecule FRET can be performed in order to answer additional questions regarding protein-protein interactions and protein dynamics.After its development, this technique paved the way for researchers in the field of single-molecule fluorescent imaging to explore DNA protein interactions in reconstituted systems of DNA replication and DNA repair in vitro. Don't forget that working with Piranha solution can be extremely hazardous and the precautions such as wearing of protective face masks should always be taken while performing this procedure.

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