This work was supported by the National Natural Science Foundation of China [Grant 32071227 to Z.Y.], Tianjin Municipal Natural Science Foundation of China (22JCYBJC01070 to Z.Y.), and State Key Laboratory of Precision Measuring Technology and Instruments (Tianjin University) [Grant pilab2210 to Z.Y.].

1. General materials and methods
<ol>
	<li>Refer to the Table of Materials file for the salts, chemicals, antibiotics, enzymes, antibodies, and resin materials used in this protocol.</li>
	<li>Prepare Luria-Bertani (LB) liquid medium, LB agar plates, HEPES buffer, SDS polyacrylamide gel electrophoresis (SDS-PAGE), Phosphate-buffered saline (PBS), Tris-EDTA (TE) buffer according to the recipes from cold spring harbor protocols45,46</s...

Studying TRF1/2 Interactions with Telomeric DNA Using Single-Molecule Assay

The authors declare no competing financial interests or other conflicts of interest.

This protocol employs magnetic tweezers for the manipulation of TRFs at the single-molecule level57,58,59. We utilize magnetic beads to separate TRFs from genomic DNA fragments. Following restriction digestion, TRFs bind to the magnetic beads, enabling their easy separation from genomic DNA fragments. This approach allows for manipulation using magnetic tweezers, which can effectively trap magnetic beads, unlike optical 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.

<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, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper function depends on tightly regulated processes of replication, elongation, and damage response. The shelterin complex, especially Telomere Repeat-binding Factor 1 (TRF1) and TRF2, plays a pivotal role in telomere protection and has emerged as a potential anti-cancer target for drug discovery. These proteins bind to the repetitive telomeric DNA motif TTAGGG, facilitating the formation of protective structures and recruitment of other telomeric proteins. Structural methods and advanced imaging techniques have provided insights into telomeric protein-DNA interactions, but probing the dynamic processes requires single-molecule approaches. Tools like magnetic tweezers, optical tweezers, and atomic force microscopy (AFM) have been employed to study telomeric protein-DNA interactions, revealing important details such as TRF2-dependent DNA distortion and telomerase catalysis. However, the preparation of single-molecule constructs with telomeric repetitive motifs continues to be a challenging task, potentially limiting the breadth of studies utilizing single-molecule mechanical methods. To address this, we developed a method to study interactions using full-length human telomeric DNA with magnetic tweezers. This protocol describes how to express and purify TRF2, prepare telomeric DNA, set up single-molecule mechanical assays, and analyze data. This detailed guide will benefit researchers in telomere biology and telomere-targeted drug discovery.

Author Spotlight: Advanced Single-Molecule Techniques for Investigating Telomeric Protein-DNA Interactions

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

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.

Figure 1A illustrates the schematic domains and structures of TRF1 and TRF2, consisting of 439 and 542 amino acids, respectively, which can be expressed in prokaryotic cells. The preparation of TRF1 has been previously described in the literature41. Here, we provide a comprehensive description and representative results of the preparation of TRF2. Figure 1B shows the plasmid map used for expressing TRF2 in E. coli. We evaluated T...

This protocol demonstrates using single-molecule magnetic tweezers to study interactions between telomeric DNA-binding proteins (Telomere Repeat-binding Factor 1 [TRF1] and TRF2) and long telomeres extracted from human cells. It describes the preparatory steps for telomeres and telomeric repeat-binding factors, the execution of single-molecule experiments, and the data collection and analysis methods.

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

Expression and Purification of Telomeric DNA-Binding Protein TRF2 Using SUMO Fusion Tag

To begin the transformation of Escherichia coli BL21 DE3 cells, thaw 50 microliter aliquot of competent cell suspension on ice. Add one microliter of DNA of pET 28a Sumo TRF2 plasmid to the competent cell suspension, and gently swirl the tube to mix the suspension. After transformation, spread 100 microliters of the cell suspension on a luria bertani, or LB, agar plate containing 50 micrograms per milliliter kanamycin.Incubate the cells overnight at 37 degrees Celsius. The next day, pick a colony of the transformed cells, and culture it in five milliliters of LB medium, supplemented with 50 micrograms per milliliter kanamycin. Incubate for 18 hours at 37 degrees Celsius, with shaking at 220 RPM.After incubation, induce the culture with one millimolar IPTG as a final concentration. Incubate at 20 degrees Celsius for 17 hours to promote protein expression. Load the crude protein extracts, along with the loading buffer, onto an SDS-PAGE gel.Run the samples initially at 100 volts for 30 minutes, followed by 120 volts for 50 minutes in a tris-glycine buffer. Then, stain with Coomassie blue to visualize protein expression. For protein purification, centrifuge the crude protein extract at 38, 000 G for 40 minutes at four degrees Celsius, and filter the supernatant through a 0.22 micrometer syringe filter.Load the filtered supernatant onto the pre-equilibrated nickel column in the binding buffer. Wash the column with binding buffer and elute the protein with the prepared imidazole gradient, collecting 12 fractions of one milliliter each. Add glycerol to a final concentration of 50%to the concentrated and buffer exchanged protein for storage.The expression of TRF2 in Escherichia coli was successfully demonstrated, as shown by SDS-PAGE, where distinct bands corresponding to TRF2 appeared before and after induction.

Preparation of Human Telomeric Restriction Fragments and Measurement of Telomeric DNA Using Single-Molecule Magnetic Tweezers

To begin, centrifuge one times 10 to the power of seven HeLa cell lines containing telomeric restriction fragments DNA at 1, 000g for three minutes. Discard the supernatant and resuspend the pellet in 200 microliters of PBS. After SDS proteinase K and sodium chloride treatment, centrifuge the cell suspension at 16, 900g for 10 minutes.Transfer the supernatant to a new centrifuge tube and add an equal volume of isopropanol, avoiding floating lipids or sediment. Centrifuge at high speed, and wash the pellet with 500 microliters of 70%ethanol. After drying the DNA pellet, carefully resuspend it in 475 microliters of TE buffer and gently mix by tapping the bottom of the tube.Now, add 25 microliters of 10 milligrams per milliliter RNAsay and tap the tube until the pellet is completely dissolved. Next, add 1/10 volume of three-molar sodium acetate and two volumes of cold 100%ethanol and place the tube at minus 80 degrees Celsius for two to three hours. After centrifugation and 70%ethanol washes, add 100 microliters of TE buffer to the DNA pellet, tap the tube to mix, and place it at four degrees Celsius for two hours to fully dissolve.For DNA digestion, combine four micrograms of genomic DNA with one microliter of CviAII, two microliters of 10X digestion buffer, and water to reach a total volume of 20 microliters. Incubate the mixture at 25 degrees Celsius for 12 hours. In a thermal cycler, heat the mixture at 75 degrees Celsius for three minutes.Then, decrease the temperature by 0.1 degrees Celsius every 30 seconds until reaching 25 degrees Celsius. After cleaning and drying, coat the bottom cover slips with 20 microliters of 0.1%nitrocellulose and add reference beads. Bake the cover slips at 100 degrees Celsius for four minutes.Assemble the flow cell sandwich. And after heating at 85 degrees Celsius, use two swabs to massage the assembly until the Parafilm seals the channel. Wash 10 microliters of M-270 beads five times with 50 microliters of working buffer using a magnet.After washing, add the beads on top of the digested genomic DNA into a 1.5-milliliter centrifuge tube. Gently flick the tube a few times to mix the beads and DNA sample. Leave the mixture on ice for one hour.After incubation, wash the mixture with 500 microliters of working buffer three times using a magnet to pull the beads down with 10-minute intervals between washes. Resuspend the sample in a working buffer, and load 30 microliters of the mixture into the flow cell. Flush the unbound magnetic beads after 30 minutes.After placing the flow cell on top of the objective lens, select a pair of five-millimeter cubic magnets arranged in a vertical configuration, and align the magnet holder with the x-axis of the magnetic tweezers'light path for imaging. Launch the graphical programming software and connect the controllers for the magnetic tweezers. Adjust the field of view to locate a reference bead at the bottom of the flow cell, and adjust the objective lens slightly so that the reference bead shows clear defraction rings.Write a script in MATLAB to control motor movements for force ramp assays. Import the script into graphical programming software to test the single-molecule experiments. At a slow flow rate, load 200 microliters of 10-nanomolar TRF1 into the flow cell.After 30 minutes of binding, choose a script for a force ramp experiment with a force loading rate of plus/minus one piconewton per second. Name the data files and run the experiment. Genomic DNA integrity was confirmed using agarose gel electrophoresis, with the resulting TRS displaying consistent lengths across various human cell lines.Telomeric repeat sequences in TRS were detected via southern blotting, showing clear hybridization signals. Force extension curves from the force ramp assay showed zigzag patterns during stretching, indicating the breaking of protein DNA interactions. The dissociation kinetics of telomeric DNA protein complexes showed a linear relationship between force and dissociation rate.Moreover, the length heterogeneity of telomeric DNA from human cells investigates the loop formation mechanism in telomeres of various lengths.

300 Views.  Nankai University.  This protocol demonstrates using single-molecule magnetic tweezers to study interactions between telomeric DNA-binding proteins (Telomere Repeat-binding Factor 1 [TRF1] and TRF2) and long telomeres extracted from human cells. It describes the preparatory steps for telomeres and telomeric repeat-binding factors, the execution of single-molecule experiments, and the data collection and analysis methods.

Studying TRF1/2 Interactions with Telomeric DNA Using Single-Molecule Assay

Summary