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.].

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 that are limited by transparency issues. Moreover, while AFM can be used for single-molecule manipulation, additional steps are required to dissociate TRFs from magnetic beads and immobilize them between a mica surface and a cantilever tip. In contrast, magnetic tweezers offer a more straightforward and efficient method for single-molecule manipulation of TRFs, eliminating the need for these extra steps41.
Maintaining the integrity of genomic DNA and TRFs is crucial, as single-molecule manipulation depends on intact TRFs. Using magnetic tweezers, we stretch and relax TRFs by holding their two ends to probe protein-DNA interactions. Accurate force measurement on magnetic beads is fundamental for single-molecule mechanical assays54. Thus, cubic-shaped magnets should be aligned parallel or orthogonal to the camera&#39;s light path in magnetic tweezers (NOTE below step 4.3.3), ensuring precise three-dimensional bead position recording per established equations54,60. Magnet offset is crucial for determining their positions and the resulting forces on magnetic beads. We use #2 cover glass with a thickness of approximately 0.2 mm for consistency or adjust the offset in the equation for different cover glasses54. Frame rate is vital for capturing the dynamics of protein-DNA interactions, necessitating a shutter dead time of zero for complete bead movement data. The motor movements, encoded by a script, are central to designing single-molecule mechanical assays. Critical parameters such as force loading rate, forces, and durations should be set and optimized iteratively. Protein concentrations used in single-molecule assays require extensive titration to determine the optimal level for studying protein-DNA interactions. Initial force testing can be performed using force-ramp assays with force spectroscopy, while force-jump assays evaluate forces of interest at a constant level.
One may encounter a few challenges when adapting this protocol. If TRF1 or TRF2 is not expressed in E. coli, start with a small-scale trial, lyse cells, and analyze proteins via SDS-PAGE and staining, confirming with Western Blot using tag-specific antibodies. One should verify the construct and plasmid to ensure correct cloning without mutations, frameshifts, or premature stop codons, and check promoter suitability for E. coli. The expression should be optimized by testing different E. coli strains (e.g., BL21(DE3), Rosetta), adjusting growth temperature (15-25 &#176;C), induction time (2-16 h), and IPTG concentration (0.1-1 mM). Inclusion bodies should be addressed by analyzing cell pellets before and after sonication. One should tackle codon bias with strains providing rare tRNAs or codon optimization. Try different growth media (LB, TB, auto-induction) and improve aeration by adjusting shaking speed and culture volume. For toxicity issues, use tightly regulated promoters or lower-copy-number vectors and add glucose (0.2%) to repress leaky lac promoter expression. Maintain plasmid integrity with proper antibiotic selection and ensure complete protein release by optimizing the cell lysis method61,62.
If no TRF tethers are found in single-molecule assays, one possibility is that not enough initial genomic DNA was used. Try using 500 ng of genomic DNA equivalent of TRFs to set up a single-molecule mechanical assay. Persistent issues with finding TRF tethers in magnetic tweezers might be due to excessively long digestion times of genomic DNA. Keep the digestion procedure under 12 h to avoid star activity. Additionally, the formation of G-quadruplexes at the single-stranded overhang of telomeric DNA could inhibit the annealing of biotin-labeled oligonucleotides for binding to magnetic beads. To prevent this, maintain potassium or sodium ion concentrations below 1 mM to avoid G-quadruplex formation and increase the likelihood of forming TRF tethers in the single-molecule setup. Additionally, increasing the heating temperature during the annealing step (step 3.2.5) helps to destabilize G-quadruplexes and facilitates the binding of biotin-labeled oligonucleotides to TRFs.
In the presence of TRF1 or TRF2 proteins, TRF DNA tethers could become tightly compacted and too difficult to stretch in magnetic tweezers due to the abundance of binding sites on the telomere for these proteins. Titrating the protein concentrations can help identify optimal levels for single-molecule mechanical assays. The protein-DNA binding affinity can be assessed using EMSA or SPR assays to determine the dissociation constant (Kd), which can then inform suitable concentrations for single-molecule assays.
The limited amount of TRFs generated from genomic DNA could hold back the efficiency of single-molecule assays. Consequently, collecting single-molecule data may take longer time compared to using artificially synthesized telomeric DNA constructs. However, using telomeres sourced directly from the human genome provides several advantages that artificial telomeric DNA, commonly prepared by plasmid in bacteria or PCR methods, cannot offer. One advantage is that human TRFs are heterogeneous in length, allowing the examination of telomere length dependency in protein-DNA interactions. In contrast, artificial telomeric DNA is usually uniform in length, and generating artificial telomeric DNA of various lengths would be time-consuming and labor-intensive. Additionally, human TRFs contain original sequences from cells, including natural markers such as modifications, mutations, and damages. This allows for the investigation of protein-DNA interactions under physiological or pathological conditions, which is not possible with artificial telomeric DNA.
This protocol is essential for studies in the field of telomere biology at the single-molecule level and can potentially benefit telomere-targeting drug discovery.

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.

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 TRF2 expression before and after induction in E. coli, as depicted in Figure 1B. The purification and tag cleavage processes were also analyzed using SDS-PAGE (Figure 1C). Following this protocol, both telomeric binding proteins, TRF1 and TRF2, were successfully expressed in E. coli.
Telomeric DNA, in the form of terminal restriction fragments (TRFs), is generated from human genomes using a combination of four restriction enzymes. Following the flowchart shown in Figure 2A, we extracted genomes from human cells. The enzymes selectively digest genomic DNA while leaving the full-length telomeric DNA intact, thus providing material for single-molecule assays. We have extensively tested the TRF DNA preparation using various human cell lines. The integrity of the genomic DNA was examined using agarose gel electrophoresis, as shown in Figure 2B. The TRFs were subsequently analyzed by Southern blotting, employing fluorescently labeled oligonucleotides complementary to the telomeric repeat sequence TTAGGG (Figure 2C), following a method described elsewhere41,56. On average, the length of human TRFs is around a few kilobases.
Single-molecule assays take place in a flow cell assembled as described in Figure 3A. A nitrocellulose coating covers the negatively charged glass surface, providing a hydrophilic matrix for antibody adsorption. BSA passivation blocks areas of the nitrocellulose matrix not bound by anti-digoxigenin antibodies. Polystyrene beads, which do not contain iron nanoparticles, serve as reference beads. TRF tethers are formed through affinity interactions between the digoxigenin-antibody on the nitrocellulose matrix and biotin-streptavidin on the magnetic bead surface (Figure 3B). TRF1 or TRF2 are introduced into the flow cell, binding to the TRF DNA tethers via free diffusion. Magnetic tweezers are used to modulate the magnetic field, generating forces on the magnetic beads to stretch and relax the TRF tethers, thereby enabling the probing of protein-DNA interactions.
A single-molecule mechanical assay is designed by developing a script to control the motions of the magnetic tweezers&#39; motors. A flowchart provides a structured approach to creating this script in MatLab for a single-molecule assay using magnetic tweezers (Figure 4A). The magnets move along the z-axis within a range of 0-25 mm. Forces are calculated based on the magnet positions, according to the magnet configuration, using equations described in the literature54. For a specific force-loading rate, the magnet positions and movement speeds are designed and programmed in MatLab to achieve the desired force manipulation profile, as demonstrated in the force ramp assay example (Figure 4B-D).
Using TRF1 as an example, we probed the telomeric DNA-protein interactions with single-molecule magnetic tweezers. The experimental setup can be configured as a force-ramp assay, as shown in Figure 5A, where the force-extension curves exhibit zigzag features during stretching due to the breaking of protein-DNA interactions and smooth traces during relaxation, reflecting the DNA fiber without protein-mediated loops. The setup can also be configured as a force-jump assay, as shown in Figure 5B,C, allowing us to measure changes in extension and the durations of protein-DNA interactions under specific forces. The dissociation kinetics of the telomeric DNA-protein complexes can be derived from these measurements (Figure 5D). Additionally, the formation of loops in the DNA-protein complexes can be revealed by changes in extension (Figure 5E). Furthermore, the protein concentration can be titrated within this telomeric DNA experimental setup. Moreover, the length heterogeneity of telomeric DNA from human cells allows us to investigate the loop formation mechanism in telomeres of various lengths (Figure 6).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/67251/67251fig01v2.jpg" /> 
Figure 1: The expression and purification of TRF1/2. (A) The domain architecture of TRF1 and TRF2. (B) The plasmid map of pET28a-SUMO-TRF2 for expressing TRF2 in E. coli. (C) SDS-PAGE analysis of the samples obtained at various stages of expression and purification. Lane 1: Uninduced by IPTG. Lane 2: IPTG-induced precipitate obtained by cell lysis. Lane 3: IPTG-induced supernatant obtained after cell lysis. Lane 4: Flow through Ni column. Lane 5: Wash with low imidazole buffer to remove impurity proteins. Lane 6: Elution of 6xHis-SUMO-TRF2 with buffer containing 300 mM imidazole. Lane 7: Digestion of SUMO tags by SUMO protease enzyme. Lane 8: Repurification with Ni column and elution of TRF2 (6xHis and SUMO tags removed) with buffer containing 20 mM imidazole. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67251/67251fig01largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/67251/67251fig02.jpg" /> 
Figure 2: Preparation of terminal restriction fragments (TRF). (A) Flowchart for the preparation of TRFs from human cells. (B) Examination of genomic DNA integrity using a 1% agarose gel for 5 human cell lines. (C) TRF analysis performed by Southern blotting for 5 human cell lines. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67251/67251fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/67251/67251fig03.jpg" /> 
Figure 3: Preparation of single-molecule mechanical assays. (A) Flowchart depicting the preparation of a flow cell for single-molecule mechanical assays. (B) Schematic representation of single-molecule mechanical assays using magnetic tweezers. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67251/67251fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/67251/67251fig04.jpg" /> 
Figure 4: Design of a single-molecule mechanical assay. (A) Flowchart for generating a script to execute a force-ramp assay. (B) The movement profile of magnets along the z-axis. (C) The force profile corresponding to magnet movement. (D) The correlation between the magnet positions and the resulting forces. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67251/67251fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/67251/67251fig05.jpg" /> 
Figure 5: Telomeric DNA-protein interactions probed by single-molecule magnetic tweezers. (A) TRF1-telomere interactions were probed in force-ramp assays (N = 10) in a buffer containing 20 mM HEPES (pH 7.5), 1 mM EDTA, and 100 mM NaCl at 23 &#176;C. The concentration of TRF1 was 10 nM, with a force loading rate of &#177;1 pN/s. (B) TRF tether was stretched and relaxed in force-jump assays. The applied force protocol was Frest = 0 pN, Ftest = 2 pN - 8 pN, Fhigh = 10 pN, and Fmax = 20 pN. Buffer and temperature conditions were the same as in (A). Frame rate = 200 Hz. (C) TRF1-telomere interactions were probed in force-jump assays with a TRF1 concentration of 10 nM. (D) Dissociation kinetics of TRF1-telomere complexes. The logarithm of the dissociation rate at tested forces (Mean &#177; SD, n = 206) follows the Kramer-Bell-Evans model (red curve and legend equation). The inset shows the average dissociation time (&#60;&#964;&#62;) at Ftest. (E) Distribution of changes in extension (&#916;L) upon rupture events of TRF1-telomere complexes. Black and red curves represent Gaussian fittings. This figure has been modified with permission from Li et al.41. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67251/67251fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/67251/67251fig06.jpg" /> 
Figure 6: The number and sizes of DNA loops formed by TRF1 in a telomere. (A) Telomere length dependency of DNA loop sizes (&#916;L) at [TRF1] = 20 nM. Curves represent Gaussian fittings. (B) Telomere length dependency of &#916;L at [TRF1] = 40 nM. (C) Correlation between &#916;L and the number of rupture events per telomere, N. The zero-order correlation is r = -0.20, with p &#60; 0.001 (sample size = 1496). (D) A cartoon illustrating a possible mechanism explaining the negative correlation between &#916;L and N suggesting that TRF1 can compact a single telomere with primary loop domains into a high-order topology. This figure has been modified with permission from Li et al.41. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67251/67251fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary File 1: MatLab code for generating scripts.m <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67251/JoVE67251-coding files-MatLab code for generating scripts.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 2: Script for constant force assay.txt. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67251/JoVE67251-coding files-Script for constant force assay.zip" target="_blank">Please click here to download this File.</a>
Supplementary File 3: Script for force ramp assay.txt. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67251/JoVE67251-coding files-Script for force ramp assay.zip" target="_blank">Please click here to download this File.</a>
Supplementary Table 1: Recipes <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/67251/Supplementary Table 1.zip" target="_blank">Please click here to download this File.</a>

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

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.

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Biochemistry

575 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.

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

Structural Information from Single-molecule FRET Experiments Using the Fast Nano-positioning System

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. Thereby, multiple smFRET measurements are used to localize an unknown dye position inside a protein complex by means of trilateration. In order to obtain quantitative information, the Nano-Positioning System (NPS) uses probabilistic data analysis to combine structural information from X-ray crystallography with single-molecule fluorescence data to calculate not only the most probable position but the complete three-dimensional probability distribution, termed posterior, which indicates the experimental uncertainty. The concept was generalized for the analysis of smFRET networks containing numerous dye molecules. The latest version of NPS, Fast-NPS, features a new algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering that allows for the analysis of large smFRET networks in a comparably short time. Moreover, Fast-NPS allows the calculation of the posterior by choosing one of five different models for each dye, that account for the different spatial and orientational behavior exhibited by the dye molecules due to their local environment.
Here we present a detailed protocol for obtaining smFRET data and applying the Fast-NPS. We provide detailed instructions for the acquisition of the three input parameters of Fast-NPS: the smFRET values, as well as the quantum yield and anisotropy of the dye molecules. Recently, the NPS has been used to elucidate the architecture of an archaeal open promotor complex. This data is used to demonstrate the influence of the five different dye models on the posterior distribution.

We present the setup and experimental procedure to obtain smFRET data from large donor-acceptor networks with a TIRF microscope. The step-by-step analysis of these measurements with the Bayesian inference software Fast-NPS yields high-resolved structural information via the application of adapted dye models.

Single-molecule F&#246;rster Resonance Energy Transfer (smFRET) can be used to obtain structural information on biomolecular complexes in real-time. ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA processing, and telomere elongation. During these processes, various proteins bind and resolve G4 structures to perform their function. As the function of G4 often depends on the stability of its folded structure, it is important to investigate how G4 binding proteins regulate the stability of G4. This work presents a method to manipulate single G4 molecules using magnetic tweezers, which enables studies of the regulation of G4 binding proteins on a single G4 molecule in real time. In general, this method is suitable for a wide scope of applications in studies for proteins/ligands interactions and regulations on various DNA or RNA secondary structures.

A single-molecule magnetic tweezers platform to manipulate G-quadruplexes is reported, which allows for the study of G4 stability and regulation by various proteins.

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For many molecular machines in a cell proteins have to act together&#160;with interaction partners in a functional cycle to fulfill their task. The extension of two-color to multi-color smFRET makes it possible to simultaneously probe more than one interaction or conformational change. This not only adds a new dimension to smFRET experiments but it also offers the unique possibility to directly study the sequence of events and to detect correlated interactions when using an immobilized sample and a total internal reflection fluorescence microscope (TIRFM). Therefore, multi-color smFRET is a versatile tool for studying biomolecular complexes in a quantitative manner and in a previously unachievable detail.
Here, we demonstrate how to overcome the special challenges of multi-color smFRET experiments on proteins. We present detailed protocols for obtaining the data and for extracting kinetic information. This includes trace selection criteria, state separation, and the recovery of state trajectories from the noisy data using a 3D ensemble Hidden Markov Model (HMM). Compared to other methods, the kinetic information is not recovered from dwell time histograms but directly from the HMM. The maximum likelihood framework allows us to critically evaluate the kinetic model and to provide meaningful uncertainties for the rates.
By applying our method to the heat shock protein 90 (Hsp90), we are able to disentangle the nucleotide binding and the global conformational changes of the protein. This allows us to directly observe the cooperativity between the two nucleotide binding pockets of the Hsp90 dimer.

Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...

Optical Tweezers to Study RNA-Protein Interactions in Translation Regulation

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the structure and function of an RNA can be modulated by various trans-acting factors, such as proteins, metabolites or other RNAs. Frameshifting RNA molecules, for instance, are regulatory RNAs located in coding regions, which direct translating ribosomes into an alternative open reading frame, and thereby act as gene switches. They may also adopt different folds after binding to proteins or other trans-factors. To dissect the role of RNA-binding proteins in translation and how they modulate RNA structure and stability, it is crucial to study the interplay and mechanical features of these RNA-protein complexes simultaneously.&#160;This work illustrates how to employ single-molecule-fluorescence-coupled optical tweezers to explore the conformational and thermodynamic landscape of RNA-protein complexes at a high resolution. As an example, the interaction of the SARS-CoV-2 programmed ribosomal frameshifting element with the trans-acting factor short isoform of zinc-finger antiviral protein is elaborated. In addition, fluorescence-labeled ribosomes were monitored using the confocal unit, which would ultimately enable the study of translation elongation. The fluorescence coupled OT assay can be widely applied to explore diverse RNA-protein complexes or trans-acting factors regulating translation and could facilitate studies of RNA-based gene regulation.

This protocol presents a complete experimental workflow for studying RNA-protein interactions using optical tweezers. Several possible experimental setups are outlined including the combination of optical tweezers with confocal microscopy.

RNA adopts diverse structural folds, which are essential for its functions and thereby can impact diverse processes in the cell. In addition, the ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately 20 nm to accommodate large tRNA substrates at the A-, P- and E-sites. Consequently, the ribosome dynamics are naturally de-phased quickly. Single molecule method can detect each ribosome separately and distinguish inhomogeneous populations, which is essential to reveal the complicated mechanisms of multi-component systems. We report the details of a smFRET method based on the Nikon Ti2 inverted microscope to probe the ribosome dynamics between the ribosomal protein L27 and tRNAs. The L27 is labeled at its unique Cys 53 position and reconstituted into a ribosome that is engineered to lack L27. The tRNA is labeled at its elbow region. As the tRNA moves to different locations inside the ribosome during the elongation cycle, such as pre- and post- translocation, the FRET efficiencies and dynamics exhibit differences, which have suggested multiple subpopulations. These subpopulations are not detectable by ensemble methods. The TIRF-based smFRET microscope is built on a manual or motorized inverted microscope, with home-built laser illumination. The ribosome samples are purified by ultracentrifugation, loaded into a home-built multi-channel sample cell and then illuminated via an evanescent laser field. The reflection laser spot can be used to achieve feedback control of perfect focus. The fluorescence signals are separated by a motorized filter-turret and collected by two digital CMOS cameras. The intensities are retrieved via the NIS-Elements software.

Single molecule fluorescence energy transfer is a method that tracks the tRNA dynamics during ribosomal protein synthesis. By tracking individual ribosomes, inhomogeneous populations are identified, which shed light on mechanisms. This method can be used to track biological conformational changes in general to reveal dynamic-function relationships in many other complexed biosystems. Single molecule methods can observe non-rate limiting steps and low-populated key intermediates, which are not accessible by conventional ensemble methods due to the average effect.

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 (FET) family of fusion oncoproteins, is one of the most frequently involved fusion genes in Ewing sarcoma. These FET family fusion proteins typically harbor a low-complexity domain (LCD) of FET protein at their N-terminus and a DNA-binding domain (DBD) at their C-terminus. EWS-FLI1 has been confirmed to form biomolecular condensates at its target binding loci due to LCD-LCD and LCD-DBD interactions, and these condensates can recruit RNA polymerase II to enhance gene transcription. However, how these condensates are assembled at their binding sites remains unclear. Recently, a single-molecule biophysics method-DNA Curtains-was applied to visualize these assembling processes of EWS-FLI1 condensates. Here, the detailed experimental protocol and data analysis approaches are discussed for the application of DNA Curtains in studying the biomolecular condensates assembling on target DNA.

Here, we describe the use of the single-molecule imaging method, DNA Curtains, to study the biophysical mechanism of EWS-FLI1 condensates assembling on DNA.

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...

Single-Molecule Analysis of Sf9 Purified Superprocessive Kinesin-3 Family Motors

A complex cellular environment poses challenges for single-molecule motility analysis. However, advancement in imaging techniques have improved single-molecule studies and has gained immense popularity in detecting and understanding the dynamic behavior of fluorescent-tagged molecules. Here, we describe a detailed method for&#160;in vitro single-molecule studies of kinesin-3 family motors using Total Internal Reflection Fluorescence (TIRF) microscopy. Kinesin-3 is a large family that plays critical roles in cellular and physiological functions ranging from intracellular cargo transport to cell division to development. We have shown previously that constitutively active dimeric kinesin-3 motors exhibit fast and superprocessive motility with high microtubule affinity at the single-molecule level using cell lysates prepared by expressing motor in mammalian cells. Our lab studies kinesin-3 motors and their regulatory mechanisms using cellular, biochemical and biophysical approaches, and such studies demand purified proteins at a large scale. Expression and purification of these motors using mammalian cells would be expensive and time-consuming, whereas expression in a prokaryotic expression system resulted in significantly aggregated and&#160;inactive protein. To overcome the limitations posed by bacterial purification systems and mammalian cell lysate, we have established a robust Sf9-baculovirus expression system to express and purify these motors. The kinesin-3 motors are C-terminally tagged with 3-tandem fluorescent proteins (3xmCitirine or 3xmCit) that provide enhanced signals and decreased photobleaching. In vitro single-molecule and multi-motor gliding analysis of Sf9 purified proteins demonstrate that kinesin-3 motors are fast and superprocessive akin to our previous studies using mammalian cell lysates. Other applications using these assays include detailed knowledge of oligomer conditions of motors, specific binding partners paralleling biochemical studies, and their kinetic state.

This study details purification of KIF1A(1-393LZ), a member of kinesin-3 family, using Sf9-baculovirus expression system. In vitro single-molecule and multi-motor gliding analysis of these purified motors exhibited robust motility properties comparable to motors from mammalian cell lysate. Thus, Sf9-baculovirus system is amenable to express and purify motor protein of interest.