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 ÃŽÂ» 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 ÃŽÂ» DNA at the Single Molecule Level at JoVE.com

Visualizing the Interaction Between the Qdot-labeled Protein and Site-specifically Modified ÃƒÆ’Ã…Â½Ãƒâ€šÃ‚Â» 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.

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. ...

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