This protocol demonstrates using single-molecule magnetic tweezers to study interactions between telomeric DNA-binding proteins (Telomere Repeat-binding Factor 1 [TRF1] and TRF2) and long telomeres extracted from human cells. It describes the preparatory steps for telomeres and telomeric repeat-binding factors, the execution of single-molecule experiments, and the data collection and analysis methods.
Telomeres, the protective structures at the ends of chromosomes, are crucial for maintaining cellular longevity and genome stability. Their proper 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.
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.
1. General materials and methods
2. Protein expression and purification of telomeric DNA-binding proteins
3. Preparation of human telomeric restriction fragments
4. Setting up a flow cell for telomeric DNA sample on magnetic tweezers
5. Measurements of a telomere using single-molecule magnetic tweezers
6. Measurements of TRF1/2 on a telomere using magnetic tweezers
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' 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).
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 °C. The concentration of TRF1 was 10 nM, with a force loading rate of ±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 ± SD, n = 206) follows the Kramer-Bell-Evans model (red curve and legend equation). The inset shows the average dissociation time (<τ>) at Ftest. (E) Distribution of changes in extension (ΔL) 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. Please click here to view a larger version of this figure.
Figure 6: The number and sizes of DNA loops formed by TRF1 in a telomere. (A) Telomere length dependency of DNA loop sizes (ΔL) at [TRF1] = 20 nM. Curves represent Gaussian fittings. (B) Telomere length dependency of ΔL at [TRF1] = 40 nM. (C) Correlation between ΔL and the number of rupture events per telomere, N. The zero-order correlation is r = -0.20, with p < 0.001 (sample size = 1496). (D) A cartoon illustrating a possible mechanism explaining the negative correlation between ΔL 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. Please click here to view a larger version of this figure.
Supplementary File 1: MatLab code for generating scripts.m Please click here to download this File.
Supplementary File 2: Script for constant force assay.txt. Please click here to download this File.
Supplementary File 3: Script for force ramp assay.txt. Please click here to download this File.
Supplementary Table 1: Recipes Please click here to download this File.
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'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 °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.
The authors have nothing to disclose.
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.].
Anti-Digoxigenin | Roche | 11214667001 | |
BfaI | New England Biolab (NEB) | R0568S | |
BSA | Sigma-Aldrich | V900933 | |
CMOS camera | Mikrotron | MC1362 | |
CviAII | New England Biolab (NEB) | R0640S | |
DIG-11-dUTP | Jena Bioscience | NU-803-DIGXL | |
DNA extraction solution | G-CLONE | EX0108 | |
Dnase I, Rnase-Free, Hc Ea | Thermo Fisher Scientific | EN0523 | |
dNTP mixture | Nanjing Vazyme Biotech Co., Ltd (Vazyme) | P032-02 | |
DTT | Solarbio | D1070 | |
Dynabeads M-270 beads | Thermo Fisher Scientific | 65305 | Streptavidin beads |
Dynabeads MyOne beads | Thermo Fisher Scientific | 65001 | Streptavidin beads |
Ethanol | Tianjin No.6 Chemical Reagent Factory | 1083 | |
Glycerol | Beijing Hwrkchemical Co,. Ltd | SMG66258-1 | |
Imidazole | Solarbio | II0070 | |
IPTG | Solarbio | I8070 | |
Isopropanol | Tianjin No.6 Chemical Reagent Factory | A1079 | |
Kanamycin | Thermo Fisher Scientific | EN0523 | |
Klenow fragment (3′-5′ exo-) | New England Biolab (NEB) | M0212S | |
LabView | National Instruments | https://www.ni.com/en-us/shop/product/labview.html | Graphical programming software |
LiCl | Bide Pharmatech Co., Ltd (bidepharm) | BD136449 | |
Lysozyme | Solarbio | L8120-5 | |
MseI | New England Biolab (NEB) | R0525S | |
NaCl | Shanghai Aladdin | C111533 | |
NanoDrop | Thermo Fisher Scientific | Spectrophotometer | |
NdeI | New England Biolab (NEB) | R0111S | |
Ni NTA Beads 6FF | Changzhou Smart-Lifesciences Biotechnology Co.,Ltd | SA005025 | |
Nitrocellulose membrane | ABclonal | RM02801 | |
PMSF | Solarbio | P8340 | |
Proteinase K | Beyotime Biotech Inc (beyotime) | ST535-500mg | |
rCutSmart Buffer | New England Biolab (NEB) | B6004S | |
Rnase A | Sigma-Aldrich | R4875 | |
Sodium acetate | SERVA Electrophoresis GmbH | 2124902 | |
Sumo protease | Beyotime Biotech Inc (beyotime) | P2312M |
.