This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MSIT) (NRF-2022R1C1C1012176, NRF-2021R1A4A1031754, and NRF- 2021R1A6A1A10042944). S.-H.R. was supported by the NRF grant (2021R1C1C2009717).

All materials and equipment described in this protocol are listed in the Table of Materials. LabVIEW software to operate the high-speed MT setup described below, as well as the MATLAB scripts to analyze sample data, are deposited on GitHub (https://github.com/ShonLab/Magnetic-Tweezers) and publicly available.
1. Construction of apparatus
NOTE: The general principle of the high-speed MT construction is similar to standard, conventional...

In this work, we introduced a single-molecule force spectroscopy setup that can observe structural changes of biomolecules at high spatiotemporal precision. The high-speed CMOS camera used acquires 1,200 frames s&#8722;1 at 1,280 x 1,024 resolution, enabling 1.2 kHz bead tracking. However, the speed of measurements is currently limited by the bead tracking software, so the ROI is typically reduced to smaller areas in high-speed measurements. The high power of the SLD provides a bright illumination that is crit...

Cells actively sense and respond to mechanical stimuli. In doing so, many biomolecules display force-dependent properties that enable dynamic structural changes. Well-appreciated examples include mechanosensitive ion channels and cytoskeletal elements that provide the cells with key mechanical information from their surrounding environment.
In addition, molecules that show a unique force-bearing nature can also be considered mechanosensitive in a broader sense. For example, local formation and melting of nucleic acid duplexes, as well as higher-order structures such as G-quadruplexes, play crucial roles in replication, transcription, recombination, and more recently, genome editing. Moreover, some neuronal proteins involved in synaptic communications perform their functions by generating physical forces that exceed the levels of typical intermolecular interactions. No matter which example one studies, investigating nanomechanics of the involved biomolecules with high spatiotemporal precision will prove highly useful in revealing molecular mechanisms of the associated mechanobiological processes1,2,3.
Single-molecule force spectroscopy methods have served as powerful tools to examine the mechanical properties of biomolecules2,4,5,6. They can monitor structural changes in nucleic acids and proteins concurrently with the application of force, thereby examining force-dependent properties. Two well-known setups are optical tweezers and magnetic tweezers (MTs), which employ micron-sized beads to manipulate molecules5,6,7,8. In these platforms, polystyrene (for optical tweezers) or magnetic beads (for MTs) are tethered to target molecules (e.g., nucleic acids and proteins) via molecular &#34;handles&#34;, typically made of short fragments of double-stranded DNA (dsDNA). The beads are then moved to exert force and imaged to track their locations that report on structural changes in target molecules. Optical and magnetic tweezers are largely interchangeable in their applications, but there exist important differences in their approaches to controlling force. Optical tweezers are intrinsically position-clamp instruments that trap beads in position, because of which the applied force fluctuates when a target construct undergoes shape changes; extension increase, such as from unfolding, loosens the tether and reduces tension, and vice versa. Although active feedback can be implemented to control the force in optical tweezers, MTs in contrast naturally operate as a force-clamp device, taking advantage of stable, far-field magnetic forces by permanent magnets, which can also withstand environmental perturbation.
Despite their long history and simple design, MTs have lagged behind optical tweezers in their applications to high-precision measurements, largely because of technical challenges in fast bead tracking. Recently, however, several groups have jointly led a multi-faceted improvement of both hardware and software for MT instruments2,9,10,11,12,13,14,15,16,17,18,19. In this work, we introduce an example of such a setup running at 1.2 kHz and describe how to use it to perform nanomechanical measurements on force-sensitive biomolecules. As model systems, we employ DNA hairpins and neuronal SNARE complexes and examine their fast, structural changes in the piconewton regime. DNA hairpins exhibit simple two-state transitions in a well-defined force range20,21, and therefore serve as toy models to verify the performance of a tweezer setup. As the SNARE proteins assemble into a force-sensitive complex that drives membrane fusion22, they have also been extensively studied by single-molecule force spectroscopy14,23,24,25. Standard approaches to analyzing data and extracting useful information on thermodynamics and kinetics are presented. We hope this article can facilitate the adoption of high-precision MTs in mechanobiological studies and motivate readers to explore their own force-sensitive systems of interest.

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.

Force calibration 
The results from the two force measurement methods (beads&#39; lateral displacement variance and power spectrum analysis) differed by 0-2 pN (Figure 2G). According to the results in Figure 2F, we can reliably reach up to 30 pN with regular neodymium magnets.
Two-state transitions of an 8 bp DNA hairpin 
We first investigated the nanomechanics of a short DNA hairpin (<stron...

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

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 ...

Biomolecules undergo small and rapid structural changes often in response to force. High resolution magnetic tweezers can explore these dynamics under physiologically relevant forces. As the method makes nanoscale measurements with millisecond precision, it is possible to monitor subtle changes in nucleic acids and proteins in real time.Malfunctions in first bearing and mechanosensitive proteins can potentially lead to cardiovascular and musculoskeletal disorders. Magnetic tweezers can provide insight on the working mechanisms of these proteins. The set must be properly aligned and calibrated to obtain good resolutions.Also, care must be taken during the preparation and identification of molecular constructs for measurements. Begin by setting up an inverted microscope on an anti-vibration optical table. Next, install a high speed CMOS camera and a frame grabber.Vertically mount a motorized linear stage with more than 20 millimeter travel length on a manual XY stage to build a translation stage for magnet manipulation in 3D. Next, install a rotary stepper motor and a belt and pulley system for magnet rotation. Mount the magnets on an acrylic holder in which the identical parallel magnets are spaced one millimeter apart.Adjust the vertical position of the translation stage so that the magnet's bottom surface aligns with the sample plane at the lowest stage position. Using a low magnification objective lens, align the magnets to the center of the field of view. Check the magnet rotation to ensure that the displacement of the center of the magnet pair is limited.Install a super luminescent diode for bead illumination. Pass the beam through the magnet gap ensuring the beam is properly collimated to fit in the gap and the illumination is not shadowed by the magnets. Now install a Piezo lens scanner on the nose piece and mount 100X oil immersion objective lens with a 1.5 numerical aperture for bead tracking.Ensure that illumination is uniform with magnet movement to avoid potential artifacts in bead tracking. Finally, adjust the light to maximum brightness without pixel saturation. Begin by taking two glass coverslips for the top and bottom of the cell.Clean the coverslips by sonicating them in one molar potassium hydroxide for 30 minutes. Next, rinse the coverslips in distilled water and keep them immersed in water. Pegylate the bottom coverslip and store it at minus 20 degrees Celsius until further use.On the day of the experiment, blow dry the pegylated coverslips with a nitrogen gun. To make the simple channels, prepare approximately two millimeter wide strips of double-sided tape and lay down four strips parallel to each other five millimeters apart on the bottom coverslip. Now place a top coverslip in the center of the bottom coverslip leaving about five millimeter space on the short edges for channel inlets and outlets.Using tweezers, gently press the back of the top coverslip to firmly seal the channels. Trim the edge of a 200 microliter pipette tip and cut out about 10 millimeters from the wider opening to allow it to hold more than 200 microliters of solution. Prepare three syringe needles that fit the tubing for the syringe pump.Connect the needle on the flow cell channel exit to the syringe pump with polyethylene tubing. Equilibrate the channels with PBS. Resuspend bead aggregates by vortexing the bead solutions.Then sequentially introduce the required solutions into the channel by suctioning with the pump. Wash away any unbound beads while applying 0.1 piconewtons of force. On the flow cell channel surface, identify the magnetic beads tethered to single molecules of the DNA construct.Locate a reference bead nearby. Rotate the candidate bead to check if it swivels freely. Rotate the beads for a few more turns and estimate the radius of rotation, choosing a bead with a smaller rotational radius.Increase the force from zero to five piconewtons to identify good single tethered beads by looking for a large change in the bead diffraction pattern resulting from a five kilobase pair tether stretch. Using the PCR, prepare five kilobase pair of double-stranded DNA fragments labeled with biotin on one end and azide on the other end. After preparing a flow cell with the PCR product, record the X and Y coordinates of the tethered bead at 1.2 kilohertz and the magnets in the resting position.Move the magnets closer to the flow cell and repeat the bead position measurements until the magnets barely touch the top of the flow cell. Calculate the force at each magnet position D using either of the alternative methods. Repeat the force measurements for a few more DNA constructs probing three to five beads to average out the force variability between the magnetic beads.Once a proper magnetic bead is located together with a reference bead, click on the calibrate button to start preparing for bead tracking. Click on the beads in the image to define the locations of the beads. For tracking in the Z direction, the software will step the objective lens with a Piezo scanner in equidistant steps and record fluctuation averaged bead images at each position to generate a lookup table.Enable tracking and autofocusing and click on the acquire button to record the bead positions. Force application to a DNA hairpin construct resulted in the worm-like chain model of force-induced extension. At six piconewtons, the construct displayed fluctuations in extension associated with reversible unzipping, which disappeared at eight piconewtons and snapped onto a new model curve.Hairpin tracking experiments at 100 hertz showed an increase in extension, but the histograms did not resolve distinct populations. At 1.2 kilohertz, the median filtered trajectories revealed two distinct populations. The separation between the two populations remained the same in the unzipping force regime.The upper open state became gradually dominant with increased force. Transition rates varied exponentially with applied force favoring unzipping and inhibiting rezipping. In the intermediate force regime, an Allan deviation of two to three nanometers was obtained at the maximum speed.The DNA handles of a SNARE complex construct behaved as worm-like chain polymers. When the force was relaxed from a higher regime, the extension returned to the original curve or followed a new model. At 14 piconewtons, the SNARE complex failed to transition to the unzipped state, whereas an increased force allowed the transition.The complete unfolding of the complex at higher forces was confirmed by refolding observed at two piconewtons. Identification of properly formed sample bead tethers is the most important thing to remember when attempting our procedure. This step enables accurate selection and thus analysis of the target structure.Magnetic tweezers can further be used to study DNA super coiling by applying torque. Also, it could be coupled with fluorescence imaging to observe highly complex protein interactions or unfolding.

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

Abstract