Conventional Magnetic Tweezers Research Articles

Magnetic tweezers with magnetic flux density feedback control.

In this work, we present a single-pole magnetic tweezers (MT) device designed for integration with substrate deformation tracking microscopy and/or traction force microscopy experiments intended to explore extracellular matrix rheology and human epidermal keratinocyte mechanobiology. Assembled from commercially available off-the-shelf electronics hardware and software, the MT device is amenable to replication in the basic biology laboratory. In contrast to conventional solenoid current-controlled MT devices, operation of this instrument is based on real-time feedback control of the magnetic flux density emanating from the blunt end of the needle core using a cascade control scheme and a digital proportional-integral-derivative (PID) controller. Algorithms that compensate for a spatially non-uniform remnant magnetization of the needle core that develops during actuation are implemented into the feedback control scheme. Through optimization of PID gain scheduling, the MT device exhibits magnetization and demagnetization response times of less than 100ms without overshoot over a wide range of magnetic flux density setpoints. Compared to current-based control, magnetic flux density-based control allows for more accurate and precise magnetic actuation forces by compensating for temperature increases within the needle core due to heat generated by the applied solenoid currents. Near field calibrations validate the ability of the MT device to actuate 4.5 μm-diameter superparamagnetic beads with forces up to 25 nN with maximum relative uncertainties of ±30% for beads positioned between 2.5 and 40 µm from the needle tip.

Force determination in lateral magnetic tweezers combined with TIRF microscopy.

Combining single-molecule techniques with fluorescence microscopy has attracted much interest because it allows the correlation of mechanical measurements with directly visualized DNA : protein interactions. In particular, its combination with total internal reflection fluorescence microscopy (TIRF) is advantageous because of the high signal-to-noise ratio this technique achieves. This, however, requires stretching long DNA molecules across the surface of a flow cell to maximize polymer exposure to the excitation light. In this work, we develop a module to laterally stretch DNA molecules at a constant force, which can be easily implemented in regular or combined magnetic tweezers (MT)-TIRF setups. The pulling module is further characterized in standard flow cells of different thicknesses and glass capillaries, using two types of micrometer size superparamagnetic beads, long DNA molecules, and a home-built device to rotate capillaries with mrad precision. The force range achieved by the magnetic pulling module was between 0.1 and 30 pN. A formalism for estimating forces in flow-stretched tethered beads is also proposed, and the results compared with those of lateral MT, demonstrating that lateral MT achieve higher forces with lower dispersion. Finally, we show the compatibility with TIRF microscopy and the parallelization of measurements by characterizing DNA binding by the centromere-binding protein ParB from Bacillus subtilis. Simultaneous MT pulling and fluorescence imaging demonstrate the non-specific binding of BsParB on DNA under conditions restrictive to condensation.

Measuring Single-Molecule Twist and Torque in Multiplexed Magnetic Tweezers.

Magnetic tweezers permit application of precisely calibrated stretching forces to nucleic acid molecules tethered between a surface and superparamagnetic beads. In addition, magnetic tweezers can control the tethers' twist. Here, we focus on recent extensions of the technique that expand the capabilities of conventional magnetic tweezers by enabling direct measurements of single-molecule torque and twist. Magnetic torque tweezers (MTT) still control the DNA or RNA tether's twist, but directly measure molecular torque by monitoring changes in the equilibrium rotation angle upon overwinding and underwinding of the tether. In freely orbiting magnetic tweezers (FOMT), one end of the tether is allowed to rotate freely, while still applying stretching forces and monitoring rotation angle. Both MTT and FOMT have provided unique insights into the mechanical properties, structural transitions, and interactions of DNA and RNA. Here, we provide step-by-step protocols to carry out FOMT and MTT measurements. In particular, we focus on multiplexed measurements, i.e., measurements that record data for multiple nucleic acid tethers at the same time, to improve statistics and to facilitate the observation of rare events.

Probing the mechanical properties, conformational changes, and interactions of nucleic acids with magnetic tweezers.

Nucleic acids are central to the storage and transmission of genetic information. Mechanical properties, along with their sequence, both enable and fundamentally constrain the biological functions of DNA and RNA. For small deformations from the equilibrium conformations, nucleic acids are well described by an isotropic elastic rod model. However, external forces and torsional strains can induce conformational changes, giving rise to a complex force-torque phase diagram. This review focuses on magnetic tweezers as a powerful tool to precisely determine both the elastic parameters and conformational transitions of nucleic acids under external forces and torques at the single-molecule level. We review several variations of magnetic tweezers, in particular conventional magnetic tweezers, freely orbiting magnetic tweezers and magnetic torque tweezers, and discuss their characteristic capabilities. We then describe the elastic rod model for DNA and RNA and discuss conformational changes induced by mechanical stress. The focus lies on the responses to torque and twist, which are crucial in the mechanics and interactions of nucleic acids and can directly be measured using magnetic tweezers. We conclude by highlighting several recent studies of nucleic acid-protein and nucleic acid-small-molecule interactions as further applications of magnetic tweezers and give an outlook of some exciting developments to come.

Combined Magneto-Optical Tweezers and Supperesolution Fluorescence Imaging for Probing Dynamic Single-Molecule Topology of DNA, and Protein Machines that Manipulate DNA Topology

Conventional magnetic tweezers (MT) designs suffer from a few limitations: (i) it is not possible to independently control torque while maintaining the desired force magnitude. The applied torque is orders of magnitude higher than biologically relevant values, precluding direct measurement of torque. (ii) They do not image the biological molecule. Rather, they image the magnetic beads attached to the molecules and infer the transformation of the molecules. This limits experiments to only high-torque processes that result in measurable change of molecular geometry, and even in these experiments the location of events on the molecule cannot be known. (iii) Due to the video-imaging based position detection of the magnetic bead, as opposed to interferometry based tracking typically used in optical tweezers, the low spatial and temporal resolutions prevent the observation of many molecular activities.The above limitations have been overcome individually but there has not been a design that overcomes all the drawbacks. Here we propose magneto-optical tweezers (MOT) to independently apply torques and forces at biologically relevant values while also arranging the DNA molecules in a transverse orientation to allow fluorescence microscopy to be performed and utilising back-focal-plane interferometry for high resolution tracking [1, 2]. We also present preliminary data on DNA topological response to MOT manipulation.1. Miller, H., Zhou, Z., Wollman, A.J., and Leake, M.C. Superresolution imaging of single DNA molecules using stochastic photoblinking of minor groove and intercalating dyes. Methods 2015.2. Zhou, Z., Miller, H., Wollman, A., and Leake, M. Developing a New Biophysical Tool to Combine Magneto-Optical Tweezers with Super-Resolution Fluorescence Microscopy. Photonics 2015. 2, 758.

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 “conventional” 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 “conventional” 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 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 “conventional” 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 “conventional” 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.

Magnetization Properties of Superparamagnetic Beads

Magnetic beads are frequently used in molecular biology and biophysics. In particular, nanometer- to micrometer-sized magnetic beads are a key component in magnetic tweezers and enable the application of precisely calibrated forces and torques to individual molecules of interest. Interestingly, despite the extensive use of magnetic beads, the origin of some of their apparent magnetic properties remains controversial. While the forces exerted on the particles in magnetic tweezers are well understood and have been quantitatively modeled from first principles (Lipfert et al., Biophys J., 2009), the torques are less well understood. Application of torque in magnetic tweezers requires the magnetization of the beads to exhibit some amount of anisotropy, the exact mechanism, however, remains poorly understood. Two models have been put forward: one relies on a small ferromagnetic component in addition to a larger paramagnetic component, while the other model postulates an ‘easy’ magnetization axis. We have performed two separate experiments to address this issue. First, we used direct angle tracking (Lipfert et al., Rev. Sci. Instrum., 2011) of DNA-tethered beads held in conventional magnetic tweezers to determine the magnetic field dependence of the rotational trap stiffness. The results are well described by a model proposed by Pavone and coworkers (Normanno et al., PRA, 2004) and argue in favor of a weak axis model. Second, we probed beads tethered to rotating flagellar motors of fixed E. coli cells to determine the field-dependent angular speed. Again, the studies suggest that the beads possess an ‘easy’ magnetization axis. Further insight and understanding of the intrinsic magnetization properties is desirable to improve the sensitivity of single-molecule magnetic tweezers and will be important for a quantitative understanding and optimization of the torque exerted in magnetic tweezers.

A pair of high resolution magnetic tweezers with illumination of total reflection evanescent field and its application in the study of DNA helicases

The resolution of conventional magnetic tweezers is limited by the Brown motion of magnetic beads. When the force is lower than ～10 pN, the resolution of magnetic tweezers decreases significantly because of the increased Brown motion. To improve the resolution of magnetic tweezers under low forces, we combine the total internal reflection fluorescence techniques with magnetic tweezers, and design a novel single molecule connection: magnetic bead-DNA linker-fluorescent bead-single molecule. With the improved magnetic tweezers, we study the folding dynamics of a DNA hairpin. The results reveal that a nanometer-scale resolution is obtained. By analyzing these results, we calibrate the penetration depth of the total internal reflection field. Finally, we investigate the unwinding dynamics of a BLM helicase core protein. Some preliminary results of the helicase unwinding experiments confirm the practicability of the improved magnetic tweezers in the field of single molecular research.

A method to track rotational motion for use in single-molecule biophysics

The double helical nature of DNA links many cellular processes such as DNA replication, transcription, and repair to rotational motion and the accumulation of torsional strain. Magnetic tweezers (MTs) are a single-molecule technique that enables the application of precisely calibrated stretching forces to nucleic acid tethers and to control their rotational motion. However, conventional magnetic tweezers do not directly monitor rotation or measure torque. Here, we describe a method to directly measure rotational motion of particles in MT. The method relies on attaching small, non-magnetic beads to the magnetic beads to act as fiducial markers for rotational tracking. CCD images of the beads are analyzed with a tracking algorithm specifically designed to minimize crosstalk between translational and rotational motion: first, the in-plane center position of the magnetic bead is determined with a kernel-based tracker, while subsequently the height and rotation angle of the bead are determined via correlation-based algorithms. Evaluation of the tracking algorithm using both simulated images and recorded images of surface-immobilized beads demonstrates a rotational resolution of 0.1°, while maintaining a translational resolution of 1-2 nm. Example traces of the rotational fluctuations exhibited by DNA-tethered beads confined in magnetic potentials of varying stiffness demonstrate the robustness of the method and the potential for simultaneous tracking of multiple beads. Our rotation tracking algorithm enables the extension of MTs to magnetic torque tweezers (MTT) to directly measure the torque in single molecules. In addition, we envision uses of the algorithm in a range of biophysical measurements, including further extensions of MT, tethered particle motion, and optical trapping measurements.

Freely orbiting magnetic tweezers to directly monitor changes in the twist of nucleic acids

The double-stranded nature of DNA links its replication, transcription and repair to rotational motion and torsional strain. Magnetic tweezers (MT) are a powerful single-molecule technique to apply both forces and torques to individual DNA or RNA molecules. However, conventional MT do not track rotational motion directly and constrain the free rotation of the nucleic acid tether. Here we present freely orbiting MT (FOMT) that allow the measurement of equilibrium fluctuations and changes in the twist of tethered nucleic acid molecules. Using a precisely aligned vertically oriented magnetic field, FOMT enable tracking of the rotation angle from straight forward (x,y)-position tracking and permits the application of calibrated stretching forces, without biasing the tether's free rotation. We utilize FOMT to measure the force-dependent torsional stiffness of DNA from equilibrium rotational fluctuations and to follow the assembly of recombination protein A filaments on DNA.

Magnetic Torque Tweezers and Their Application in Probing the Torsional Properties of DNA, RNA, and DNA Filaments

The double-stranded nature of DNA links processes such as replication, transcription, and repair, to rotational motion and torsional strains. Magnetic tweezers (MT) are a powerful single-molecule technique to apply both forces and torques to individual molecules. However, while the forces applied in conventional MT can be calibrated from thermal fluctuations or computed from first principles [1], direct measurement of the torque is challenging.Here we present the magnetic torque tweezers (MTT), which enable the direct measurement of torque [2] based on a tracking protocol that monitors x, y, z, and angle and on a redesigned magnet configuration. We have applied the MTT to dsDNA, dsRNA, and RecA-DNA heteroduplex filaments. Our measurements of the effective torsional stiffness C of dsDNA indicate a significant force dependence, with C ≈ 40 nm at low forces up to C ≈ 100 nm at high forces, reconciling previous partially conflicting measurements. Torque measurements on RecA-DNA heteroduplex filaments reveal an initial torsional stiffness about two-fold higher than that of DNA. However, at relatively moderate torques further build-up of torsional strain is prevented by partial RecA disassembly and structural transitions in the filament. Preliminary results on the torsional properties of fully dsRNA indicate static properties overall similar to dsDNA, but significantly different dynamics of supercoil formation.Finally, we compare the MTT configuration with recently developed strategies to measure torque using optical tweezers [3] and with a related MT approach that allows straight-forward measurements of free rotation termed freely-orbiting magnetic tweezers [4].[1] Lipfert, Hao & Dekker, Biophys. J. (2009).[2] Lipfert, Kerssemakers, Jager & Dekker, Nature Methods, in press (2010).[3] Forth, et al. Phys. Rev. Lett. (2008).[4] Lipfert, Wiggin, Kerssemakers & Dekker, submitted (2010).