Magnetic Tweezers for the Measurement of Twist and Torque
May 19th, 2014
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.
The overall goal of this experiment is to directly measure torsional strains or changes in the twist of double stranded DNA molecules at the single molecule level. This is accomplished using two assays in the first assay called freely orbiting magnetic tweezers or thumped. A single functionalized DNA molecule is tethered between a magnetic bead and a glass surface.
While a cylindrically shaped magnet exerts a force stretching the DNA In this configuration, the beads angular position is only constrained by the tethered DNA, not by the magnet, allowing bead rotation as shown by the red arrow to report on changes in the twist of the DNA rotational thermal fluctuations of the bead cause. Its XY position to lie on A circular annulus or donut. Conversion of this XY position to rotation angle makes it possible to monitor changes in the twist of the tethered DNA in a second related assay called magnetic torque tweezers, or MTTA side magnet is added to the main cylindrical magnet to constrain the beads angular motion.
With this magnet configuration, external torques can be applied to the tethered DNA molecule through simple rotation of the magnet assembly, measurements of the deviation of the beads angular position after applying a number of turns compared to its initial torsionally relaxed configuration, together with calibration of stiffness of the magnetic trap that confines the beads, angular motion makes it possible to quantify the buildup of torque in the DNA. The main advantage of using font and MTT over conventional magnetic tweezers is that we can directly measure torque and changes in the twist of nucleic acids. This method can help answer key questions about the mechanics of DNA and RNA by allowing us to map out their responses to external forces and torques.
The implications of this technique extend to probing the interactions of DNA with proteins. For example, proteins responsible for DNA repair storage, or transcription Visual demonstration of this method illustrates how easily a conventional magnetic tweezer set up can be modified to give it new capabilities. The setup used for the following experiments is based on a conventional magnetic tweezers setup.
At its center is a flow cell illuminated from the top by an LED and imaged through a microscope objective and CCD camera from below. Above the flow cell is a magnet head that can be moved up and down and rotated using computer controlled motors. The images from the CCD camera are analyzed in real time by custom lab view software to determine the X, Y, and Z position of DNA tethered beads.
The custom lab view software is available from the authors upon request. After preparing a flow cell with DNA tethered magnetic beads, mounting it on the conventional magnetic tweezers and selecting both a surface immobilized to reference bead and a bead to which an individual DNA molecule of the proper length is tethered. The setup may be converted to the font mode.
Begin by manually unscrewing the complete magnet head that holds the magnet for the conventional tweezers configuration. Replace it with the magnet head that holds a cylindrical magnet for font. While placing the cylindrical magnet used for F in the magnet head, be sure to keep the selected DNA tether within the field of view.
The most difficult aspect of this procedure is to correctly align the magnets for the form geometry. A good alignment is achieved by systematically moving the magnets and testing the alignment after every step, which we will demonstrate. Now, Perform a course alignment of the magnet in the font using the position stages to manually move the magnets in the lab view software.
Click on the record button to measure the XY position fluctuations or excursions. The recorded traces are displayed on the screen in real time and saved as text files that contain the X, Y, Z position information. If the XY excursions follow an arc as shown here, the cylindrical magnet is not properly aligned, continue moving the cylindrical magnet in the appropriate direction and taking measurements until the XY fluctuations trace out a complete circular pattern, which indicates that the course alignment is achieved.Next.
If required for further experiments, perform a fine alignment in the font by using a high resolution automated stage to move the flow cell, aligning the cylindrical magnet within about 10 microns of the bead. Then as before, record the XY excursions. Continue moving the stage and recording excursions until the fluctuations on the circular annulus are nearly uniform.
To verify the final alignment, use a MATLAB script which is available from the authors upon request to plot the fluctuations in a histogram or a thermogram and inspect it for uniformity. To take measurements of DNA torque, remove the cylindrical magnet that is used for font and replace it with a cylindrical magnet plus a permanent side magnet. For the MTT, ensure that the selected DNA tether remains within the field of view.
Enter the number and rate of magnetic turns in the corresponding panel of the controlling lab view software.Here. The number of turns is set to five and the rate is set to 0.1 hertz. This will cause the magnets to rotate slowly during the measurement.
Next in matlab, use an angular tracking script based on monitoring the XY position, which is available from the author upon request. A plot that displays the angular fluctuations as a function of time, theta T will appear. Once everything is set up in lab view, click the record button.
The traces will appear in real time as before. In matlab, use the MATLAB script to produce plots of angle theta T and bead height Z of T trace on screen with a Gaussian fit to the angle signal to determine the standard deviation of the angular fluctuations. Sigma theta.
This script directly determines the stiffness of the torsional trap from the variance of the angular fluctuations. Sigma theta squared in radians. Using the formula shown here, Note that it is typical in the MTT to achieve rotational trap stiffness of 10 to 1000 pico newton nanometer per radian, which is much lower than in the conventional magnetic tweezers.
Here, for example, we've determined the rotational trap stiffness to be about 52 pig nanometer per radian. The rotational trap stiffness of the magnetic torque tweezers compared to the conventional magnetic tweezers renders it suitable for measurements of single molecule torque, but also means that the maximum torque that can be exerted is reduced. This implies that the MTT cannot counterbalance drag torques caused by rapid rotation.
Care must therefore be taken not to rotate too fast. We typically rotate at rates of about 0.1 hertz. Next, the DNA tether is overwhelmed by slowly rotating the magnets, a set number of turns, and by recording another trace of angular fluctuations, the number and rate of magnet turns is again entered in the corresponding panel of the controlling lab view software.
Here the number of turns is set to 40 and the rate is set to 0.1 hertz. This will cause the magnets to rotate slowly during the measurement to determine the torque accumulated in the nucleic acid tether. After N turns, we use the formula shown here where the angled brackets denote the average and theta zero.
And theta N are the angle at zero turns corresponding to a torsionally relaxed tether and turns respectively. Repeat the steps which the magnets are rotated and record a plateau of angular fluctuations as necessary in order to fully determine a molecule's toque response in a single measurement run. To measure changes in the twist of the DNA induced by the repair protein RAD 51 whose binding to double stranded DNA, both lengthens and unwind.
DNA rad 51 was added to a DNA molecule tethered in the font. As shown here, the bead traces out a spiraling trajectory. This motion can be decoupled into components that describe how the DNA lengthens and unwinds over time.
To measure the torque stored in DNA using the MTT, the molecule with systematically over and under wound and the angular fluctuations were measured for each number of applied turns. The standard deviation of the angular fluctuations, which reports on the angular trap stiffness should be independent of the number of applied turns. Here, the standard deviation is about nine degrees as shown here.
The mean of the angular positions systematically changes with the number of applied turns using the constant angular trap stiffness. The changes in the mean angle are converted to torque, yielding the torque stored in the DNA versus applied turns. The simultaneous recording of the beads Z position yields the length of the DNA versus applied turns.
Together these two curves yield the full mechanical response of DNA to over and under winding. After watching this video, you should have a good understanding of how to measure, twist and torque on biological molecules using the freely orbiting magnetic tweezers and the magnetic torque tweezers to novel single molecule assays to which conventional magnetic tweezers can readily be adapted. The development of these techniques paves the way for research in the field of biophysics, for example, to study the torsional properties of DNA or RNA and to observe processes such as DNA compaction and repair The font and MTT techniques can be enhanced by other methods like fluorescence detection in order to answer additional questions such as determining the particular location of a protein on a tethered DNA.
Overview
This article discusses the use of magnetic tweezers to measure the twist and torque in biological macromolecules, specifically focusing on DNA. The techniques described include freely-orbiting magnetic tweezers and magnetic torque tweezers, which allow for direct measurements at the single-molecule level.
Key Study Components
Area of Science
- Biophysics
- Molecular Biology
- Single-Molecule Techniques
Background
- Magnetic tweezers are a powerful tool for manipulating single molecules.
- They can measure torsional strains and changes in twist of DNA.
- Understanding DNA mechanics is crucial for insights into biological processes.
- The methods can probe interactions between DNA and proteins.
Purpose of Study
- To measure torsional strains in double-stranded DNA at the single-molecule level.
- To provide guidelines for using magnetic tweezers in biological research.
- To illustrate the modifications needed for conventional magnetic tweezers to measure torque.
Methods Used
- Freely-orbiting magnetic tweezers for measuring twist.
- Magnetic torque tweezers for applying and measuring torque.
- Real-time analysis of bead position using custom software.
- Calibration of the magnetic trap to quantify torque buildup.
Main Results
- Successful measurement of DNA twist and torque using modified setups.
- Demonstrated the ability to map DNA responses to external forces.
- Provided a visual demonstration of the experimental setup.
- Showed the importance of proper alignment in the experimental procedure.
Conclusions
- Magnetic tweezers can be adapted for direct measurements of DNA mechanics.
- This technique enhances our understanding of DNA-protein interactions.
- It opens avenues for further research in molecular biology.
Frequently Asked Questions
Executive Industry Relevance
Measuring torsional strain and torque in nucleic acids at the single-molecule level enables mechanistic de-risking of nucleic acid-targeting therapeutics by revealing how DNA and RNA respond to external forces. This capability supports target validation by clarifying the functional consequences of protein-DNA interactions, such as those involving repair or transcription factors. The method provides quantitative, real-time readouts that improve predictive confidence in early discovery stages where understanding molecular mechanics informs lead identification and preclinical model selection.
Strategic Applications in Biopharma R&D
Early Discovery & Target Validation
- Scientific Value: Interrogates therapeutic hypotheses by measuring how proteins like RAD 51 alter DNA twist and length, clarifying functional mechanisms.
- Operational Value: Enables direct observation of torque and twist changes, reducing ambiguity in target engagement assays.
- Predictive Value: Supports portfolio triage by quantifying the mechanical impact of nucleic acid-binding compounds.
Screening & Assay Development
- Assay Readiness: Produces standardized, quantifiable outputs (angular fluctuations, bead height) for reproducible compound screening.
- Scalability: Adapted magnetic tweezers configurations allow reuse across different nucleic acid targets with minimal retooling.
- Quantitative Output: Delivers torque and twist measurements that enable dose-response profiling of molecular interactions.
Translational & Preclinical Research
- Disease Relevance: Probes nucleic acid mechanics in contexts relevant to genetic disorders, cancer, and infectious disease mechanisms.
- Translational Continuity: Bridges discovery and preclinical work by providing mechanistic insights into DNA-protein interactions that inform biomarker selection.
- Risk-Adjusted Advancement: Enables go/no-go decisions based on torsional response profiles, reducing late-stage failure risk.
Pipeline & Workflow Integration
The method fits within the discovery continuum from target validation through lead identification to preclinical evaluation, where understanding nucleic acid mechanics informs compound optimization and safety assessment.
- Discovery Biology: Supports hypothesis testing by quantifying how ligands or proteins alter DNA topology and mechanical stability.
- Screening: Enables assay readiness through real-time, label-free detection of torsional changes in tethered nucleic acids.
- Analytics: Provides angular and positional readouts (theta, Z) that allow teams to correlate mechanical responses with biochemical activity.
- Translational Research: Connects to preclinical work by revealing how disease-associated mutations or modifications alter nucleic acid mechanics.
- Enterprise Reuse: Configurations (freely-orbiting and torque modes) are interchangeable on a common platform, supporting broad target applicability.
Operational & Enterprise Impact
- Scientific Value: Delivers predictive confidence in target validation by reducing mechanistic ambiguity in nucleic acid-protein interactions.
- Operational Value: Ensures standardization and reproducibility through calibrated torque measurements and real-time tracking.
- Strategic Value: Improves capital efficiency by enabling early de-risking of targets with unclear mechanical mechanisms of action.
- Portfolio Impact: Supports risk-adjusted prioritization by quantifying the torsional response of lead compounds to nucleic acid targets.
Implementation Considerations
- Requires expertise in single-molecule biophysics and magnetic tweezers operation.
- Depends on precise magnetic field alignment and stabilization for accurate torque and twist measurements.
- Necessitates cross-team standardization of calibration protocols (e.g., rotational trap stiffness) for reproducible results across labs.
- Involves adaptation considerations when applying the technique to different nucleic acid lengths, sequences, or protein complexes.
- Limited by maximum applicable torque due to low torsional trap stiffness, constraining use in high-torque scenarios.
