Name: analyzing telomeric protein-dna interactions using single-molecule magnetic tweezers
Uploaded: 2024-08-30T14:20:00.000+00:00
Duration: 11 min 21 s
Description: Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper ...

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><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&amp;nbsp;</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&amp;nbsp; 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&amp;prime;-5&amp;prime; 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&amp;nbsp;</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/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/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/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/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/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/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/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/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/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/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

607 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. ...

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

Bioengineering

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 ...

Magnetic Tweezers for the Measurement of Twist and Torque

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques include so-called force spectroscopy approaches such as atomic force microscopy, optical tweezers, flow stretching, and magnetic tweezers. Amongst these approaches, magnetic tweezers have distinguished themselves by their ability to apply torque while maintaining a constant stretching force. Here, it is illustrated how such a &#8220;conventional&#8221; magnetic tweezers experimental configuration can, through a straightforward modification of its field configuration to minimize the magnitude of the transverse field, be adapted to measure the degree of twist in a biological molecule. The resulting configuration is termed the freely-orbiting magnetic tweezers. Additionally, it is shown how further modification of the field configuration can yield a transverse field with a magnitude intermediate between that of the &#8220;conventional&#8221; magnetic tweezers and the freely-orbiting magnetic tweezers, which makes it possible to directly measure the torque stored in a biological molecule. This configuration is termed the magnetic torque tweezers. The accompanying video explains in detail how the conversion of conventional magnetic tweezers into freely-orbiting magnetic tweezers and magnetic torque tweezers can be accomplished, and demonstrates the use of these techniques. These adaptations maintain all the strengths of conventional magnetic tweezers while greatly expanding the versatility of this powerful instrument.

Magnetic tweezers, a powerful single-molecule manipulation technique, can be adapted for the direct measurements of the twist (using a configuration called freely-orbiting magnetic tweezers) and torque (using a configuration termed magnetic torque tweezers) in biological macromolecules. Guidelines for performing such measurements are given, including applications to the study of DNA and associated nucleo-protein filaments.

Single-molecule techniques make it possible to investigate the behavior of individual biological molecules in solution in real time. These techniques ...

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk population methods (Northern blot, PCR, and RNAseq) that measure mRNA levels lack the ability to provide information on cell-to-cell variation in responses. Precise single cell and allelic visualization and quantification is possible via single molecule RNA fluorescence in situ hybridization (smFISH). RNA-FISH is performed by hybridizing target RNAs with labeled oligonucleotide probes. These can be imaged in medium/high throughput modalities, and, through image analysis pipelines, provide quantitative data on both mature and nascent RNAs, all at the single cell level. The fixation, permeabilization, hybridization and imaging steps have been optimized in the lab over many years using the model system described herein, which results in successful and robust single cell analysis of smFISH labeling. The main goal with sample preparation and processing is to produce high quality images characterized by a high signal-to-noise ratio to reduce false positives and provide data that are more accurate. Here, we present a protocol describing the pipeline from sample preparation to data analysis in conjunction with suggestions and optimization steps to tailor to specific samples.&#160;

Single molecule RNA fluorescence in situ hybridization (smFISH) is a method to accurately quantify levels and localization of specific RNAs at the single cell level. Here, we report our validated lab protocols for wet-bench processing, imaging and image analysis for single cell quantification of specific RNAs.

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Magnetic Tweezers in a Microplate Format

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these approaches are limited single-molecule methods, which restricts their availability. To address this need, a microplate assay was developed that enables mechanical manipulation while performing standard biochemical assays. This is achieved using magnets incorporated into a microplate lid to create multiple magnetic tweezers. In this format, force is exerted across biomolecules connected to paramagnetic beads, equivalent to a typical magnetic tweezer. The study demonstrates the application of this tool with FRET-based assays to monitor protein conformations. However, this approach is widely applicable to different biological systems ranging from measuring enzymatic activity through to the activation of signaling pathways in live cells.

Here, we describe the use of a novel microplate assay to enable mechanical manipulation of biomolecules while performing ensemble biochemical assays. This is achieved using a microplate lid modified with magnets to create multiple static magnetic tweezers across the microplate.

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these ...

High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are therefore poised to be useful in the field of mechanobiology. Since the method commonly relies on image-based tracking of magnetic beads, the speed limit in recording and analyzing images, as well as the thermal fluctuations of the beads, has long hampered its application in observing small and fast structural changes in target molecules. This article describes detailed methods for the construction and operation of a high-resolution MT setup that can resolve nanoscale, millisecond dynamics of biomolecules and their complexes. As application examples, experiments with DNA hairpins and SNARE complexes (membrane-fusion machinery) are demonstrated, focusing on how their transient states and transitions can be detected in the presence of piconewton-scale forces. We expect that high-speed MTs will continue to enable high-precision nanomechanical measurements on molecules that sense, transmit, and generate forces in cells, and thereby deepen our molecular-level understanding of mechanobiology.

Here, we describe a high-speed magnetic tweezer setup that performs nanomechanical measurements on force-sensitive biomolecules at the maximum rate of 1.2 kHz. We introduce its application to DNA hairpins and SNARE complexes as model systems, but it will be also applicable to other molecules involved in mechanobiological events.

Single-molecule magnetic tweezers (MTs) have served as powerful tools to forcefully interrogate biomolecules, such as nucleic acids and proteins, and are ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...