<meta charset="utf8"></head><body>使用单分子测定法研究 TRF1/2 与端粒 DNA 的相互作用

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Anti-Digoxigenin</td>
			<td>Roche</td>
			<td>11214667001</td>
			<td></td>
		</tr>
		<tr>
			<td>BfaI</td>
			<td>New England Biolab (NEB)</td>
			<td>R0568S</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>V900933</td>
			<td></td>
		</tr>
		<tr>
			<td>CMOS camera&#160;</td>
			<td>Mikrotron</td>
			<td>MC1362</td>
			<td></td>
		</tr>
		<tr>
			<td>CviAII</td>
			<td>New England Biolab (NEB)</td>
			<td>R0640S</td>
			<td></td>
		</tr>
		<tr>
			<td>DIG-11-dUTP</td>
			<td>Jena Bioscience</td>
			<td>NU-803-DIGXL</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA extraction solution</td>
			<td>G-CLONE</td>
			<td>EX0108</td>
			<td></td>
		</tr>
		<tr>
			<td>Dnase I, Rnase-Free, Hc Ea</td>
			<td>Thermo Fisher Scientific</td>
			<td>EN0523</td>
			<td></td>
		</tr>
		<tr>
			<td>dNTP mixture</td>
			<td>Nanjing Vazyme Biotech Co., Ltd (Vazyme)</td>
			<td>P032-02</td>
			<td></td>
		</tr>
		<tr>
			<td>DTT</td>
			<td>Solarbio</td>
			<td>D1070</td>
			<td></td>
		</tr>
		<tr>
			<td>Dynabeads M-270&#160; beads</td>
			<td>Thermo Fisher Scientific</td>
			<td>65305</td>
			<td>Streptavidin beads</td>
		</tr>
		<tr>
			<td>Dynabeads MyOne beads</td>
			<td>Thermo Fisher Scientific</td>
			<td>65001</td>
			<td>Streptavidin beads</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Tianjin No.6 Chemical Reagent Factory</td>
			<td>1083</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Beijing Hwrkchemical Co,. Ltd</td>
			<td>SMG66258-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidazole</td>
			<td>Solarbio</td>
			<td>II0070</td>
			<td></td>
		</tr>
		<tr>
			<td>IPTG</td>
			<td>Solarbio</td>
			<td>I8070</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Tianjin No.6 Chemical Reagent Factory</td>
			<td>A1079</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>EN0523</td>
			<td></td>
		</tr>
		<tr>
			<td>Klenow fragment (3&#8242;-5&#8242; exo-)</td>
			<td>New England Biolab (NEB)</td>
			<td>M0212S</td>
			<td></td>
		</tr>
		<tr>
			<td>LabView</td>
			<td>National Instruments</td>
			<td>https://www.ni.com/en-us/shop/product/labview.html</td>
			<td>Graphical programming software&#160;</td>
		</tr>
		<tr>
			<td>LiCl</td>
			<td>Bide Pharmatech Co., Ltd (bidepharm)</td>
			<td>BD136449</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Solarbio</td>
			<td>L8120-5</td>
			<td></td>
		</tr>
		<tr>
			<td>MseI</td>
			<td>New England Biolab (NEB)</td>
			<td>R0525S</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Shanghai Aladdin</td>
			<td>C111533</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Spectrophotometer</td>
		</tr>
		<tr>
			<td>NdeI</td>
			<td>New England Biolab (NEB)</td>
			<td>R0111S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ni NTA Beads 6FF</td>
			<td>Changzhou Smart-Lifesciences Biotechnology Co.,Ltd</td>
			<td>SA005025</td>
			<td></td>
		</tr>
		<tr>
			<td>Nitrocellulose membrane</td>
			<td>ABclonal</td>
			<td>RM02801</td>
			<td></td>
		</tr>
		<tr>
			<td>PMSF</td>
			<td>Solarbio</td>
			<td>P8340</td>
			<td></td>
		</tr>
		<tr>
			<td>Proteinase K</td>
			<td>Beyotime Biotech Inc (beyotime)</td>
			<td>ST535-500mg</td>
			<td></td>
		</tr>
		<tr>
			<td>rCutSmart Buffer</td>
			<td>New England Biolab (NEB)</td>
			<td>B6004S</td>
			<td></td>
		</tr>
		<tr>
			<td>Rnase A</td>
			<td>Sigma-Aldrich</td>
			<td>R4875</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium acetate</td>
			<td>SERVA Electrophoresis GmbH</td>
			<td>2124902</td>
			<td></td>
		</tr>
		<tr>
			<td>Sumo protease</td>
			<td>Beyotime Biotech Inc (beyotime)</td>
			<td>P2312M</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing telomeric protein-dna interactions using single-molecule magnetic tweezers

Telomeres are protective structures at the ends of chromosomes1,2,3. Telomere erosion during cell division leads to cell senescence and aging, while abnormal elongation of telomeres contributes to cancer4,5. For telomeres to function properly, their replication, elongation, and damage responses must be highly regulated6,7,8. Shelterin, composed of six subunits, plays a central role in telomere protection9,10,11. A deeper understanding of telomeres will provide valuable insights into telomere biology.
TRF1 and TRF2, core subunits of shelterin, are telomeric binding proteins12,13. Both TRF1 and TRF2 bind to the repetitive DNA motif TTAGGG in telomeres via their Myb domains14. They form dimers through their shared TRFH domains, which allow them to encircle telomeric double-stranded DNA and to recruit telomeric proteins15,16,17,18,19. TRF2 is particularly important for the formation of telomeric D-loops and T-loops20,21. Due to their crucial roles in telomere protection, TRF1 and TRF2 have emerged as potential anti-cancer drug targets22,23,24,25.
Significant efforts have been made to investigate the protein-DNA interactions at telomeres. Biochemical methods such as Electrophoretic Mobility Shift Assay (EMSA) and Surface Plasmon Resonance (SPR) have been used to examine binding affinities20,26. Numerous structures of telomeric binding proteins complexed with DNA have been elucidated using cryo-electron microscopy (cryo-EM), X-ray crystallography, and nuclear magnetic resonance (NMR)27,28,29. Super-resolution imaging techniques like stochastic optical reconstruction microscopy (STORM) have revealed TRF2-dependent T-loop formation21. Recently, nanopore sequencing has been developed to profile telomeric sequences4,30,31. These structural insights have greatly enhanced our understanding of telomeric protein-DNA interactions. To further explore the dynamics of telomeric protein-DNA interactions, the development of new technologies is essential.
Single-molecule tools are powerful techniques for exploring protein-DNA interactions at telomere32,33,34. Single-molecule mechanical methods, such as magnetic tweezers, optical tweezers and AFM, have been employed to investigate TRF2-dependent DNA distortion, reveal TRF2-mediated columnar stacking of human telomeric chromatin and observe processive telomerase catalysis, among other applications35,36,37,38,39,40. These methods are particularly useful for probing topological conformations and the kinetics of protein-DNA association and dissociation.
However, the preparation of single-molecule constructs with telomeric repetitive motifs still presents challenges, which limits studies using single-molecule mechanical methods. To address this limitation, we have developed a single-molecule mechanical method to study global protein-DNA interactions on full-length human telomeres41. This method directly extracts telomeric DNA from human cells, circumventing the laborious preparation of artificial telomeric DNA. It facilitates the investigation of kinetic processes on long native telomeres spanning several kilobases.
In this protocol, we provide a detailed description of the steps for probing telomeric protein-DNA interactions using magnetic tweezers, a popular single-molecule mechanical tool42,43,44. We demonstrate how to express and purify telomeric proteins, using TRF2 as an example, and how to prepare telomeric DNA from human cells. Additionally, we show how to set up a single-molecule assay on magnetic tweezers to study telomeric protein-DNA interactions, and we cover the subsequent data analysis of single-molecule experiments. This protocol will benefit researchers in the field of telomere biology and telomere-targeted drug discovery.

<meta charset="utf8"></head><body>作者聚焦：用于研究端粒蛋白-DNA 相互作用的先进单分子技术

使用单分子磁镊分析端粒蛋白-DNA 相互作用

Single-molecule methods have been used to study telomeric protein-DNA interaction. However, preparing single molecule constructs with the telomeric repetitive motifs remains a challenging task. In this protocol, we outline the steps for expressing and purifying TRF2 protein, preparing telomeric DNA, setting up single-molecule mechanical assays, and analyzing the resulting data.Single-molecule tools are powerful techniques for exploring telomeric protein-DNA interactions. Single-molecule mechanical methods, such as magnetic tweezers, optical tweezers, and AFM have been used to investigate TRF2-dependent DNA distortion, reveal TRF2-mediated columnar stacking of human telomeric chromatin, and observe the presence of telomerase catalysis among other applications. We investigated telomeric DNA-protein interactions using single molecular magnetic tweezers, enabling precise measurements of changes in extension and the durations of protein-DNA interactions under applied forces.From these measurements, we derived the dissociation kinetics of the telomeric DNA-protein complexes. Furthermore, the formation of loops within these complexes was revealed. Numerous structures of telomeric-binding proteins in complex with DNA have been resolved using cryo-EM, X-ray crystallography, and NMR.These structural insights have advanced our understanding of telomeric protein-DNA interactions. To further investigate the dynamics of these interactions, we have developed a single-molecule mechanical methods specifically for studying telomeric protein-DNA interactions. Single-molecule tools are powerful techniques for investigating protein-DNA interactions at telomeres.These methods are especially valuable for topological conformations and analyzing the kinetics of protein-DNA association and dissociation.

Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper ...

analyzing-telomeric-protein-dna-interactions-using-single-molecule

Research

JoVE Journal

Biochemistry

Analyzing Telomeric Protein-DNA Interactions Using Single-Molecule Magnetic Tweezers

352 次观看.  Nankai University. 单分子方法已被用于研究端粒蛋白-DNA 相互作用。然而，制备具有端粒重复基序的单分子构建体仍然是一项具有挑战性的任务。在该协议中，我们概述了表达和纯化 TRF2 蛋白、制备端粒 DNA、设置单分子机械测定和分析所得数据的步骤。单分子工具是探索端粒蛋白质-DNA 相互作用的强大技术。单分子力学方法，如磁镊、光镊和 AFM，已被用于研究 TR...