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).

The authors have no conflicts of interest to declare.

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...

Watch this Scientific Journal Video about High-Speed Magnetic Tweezers for Nanomechanical Measurements on Force-Sensitive Elements at JoVE.com

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 ...

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Research

JoVE Journal

Biochemistry

2.0K Views.  Pohang University of Science and Technology (POSTECH). Biomoleculen ondergaan kleine en snelle structurele veranderingen, vaak als reactie op geweld. Hoge resolutie magnetische pincetten kunnen deze dynamiek onderzoeken onder fysiologisch relevante krachten. Omdat de methode metingen op nanoschaal doet met milliseconde precisie, is het mogelijk om subtiele veranderingen in nucleïnezuren en eiwitten in realtime te volgen.Storingen in eerste dragende en mechanosensitieve eiwitten kunnen mogelijk leiden tot cardiovasculaire en musculoskeleta...

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

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

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 ...

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is subject to chemical and mechanical signals, and the mechanisms by which it senses and responds to mechanical signals are poorly understood. Elucidating the role of forces in cell maturation will enable the design of scaffolds that can promote cell adhesion and cytoskeletal coupling to the substrate, and therefore improve the capacity of different neuronal types to regenerate after injury.
Here, we describe a method to apply simultaneous force spectroscopy measurements during laser induced cell lesion. We measure tension release in the partially lesioned axon by simultaneous interferometric tracking of an optically trapped probe adhered to the membrane of the axon. Our experimental protocol detects the tension release with piconewton sensitivity, and the dynamic of the tension release at millisecond time resolution. Therefore, it offers a high-resolution method to study how the mechanical coupling between cells and substrates can be modulated by pharmacological treatment and/or by distinct mechanical properties of the substrate.

We measured the tension release in an axon that was partially lesioned with a laser dissector by simultaneous force spectroscopy measurement performed on an optically-trapped probe adhered to the membrane of the axon. The developed experimental protocol evaluates the axon adhesion to the culture substrate.

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is ...

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 ...

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 ...

Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.

Visual, single-molecule biochemistry studied through microfluidic chambers is greatly facilitated using glass barrel, gas-tight syringes, stable connections of tubing to flow cells, and elimination of bubbles by placing switching valves between the syringes and tubing. The protocol describes dual optical traps that enable visualization of DNA transactions and intermolecular interactions.

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the ...

Direct Force Measurements of Subcellular Mechanics in Confinement using Optical Tweezers

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue morphogenesis goes in hand with molecular and structural changes at the single cell level that result in variations of subcellular mechanical properties. As a consequence, even within the same cell, different organelles and compartments resist differently to mechanical stresses; and mechanotransduction pathways can actively regulate their mechanical properties. The ability of a cell to adapt to the microenvironment of the tissue niche thus is in part due to the ability to sense and respond to mechanical stresses. We recently proposed a new mechanosensation paradigm in which nuclear deformation and positioning enables a cell to gauge the physical 3D environment and endows the cell with a sense of proprioception to decode changes in cell shape. In this article, we describe a new method to measure the forces and material properties that shape the cell nucleus inside living cells, exemplified on adherent cells and mechanically confined cells. The measurements can be performed non-invasively with optical traps inside cells, and the forces are directly accessible through calibration-free detection of light momentum. This allows measuring the mechanics of the nucleus independently from cell surface deformations and allowing dissection of exteroceptive and interoceptive mechanotransduction pathways. Importantly, the trapping experiment can be combined with optical microscopy to investigate the cellular response and subcellular dynamics using fluorescence imaging of the cytoskeleton, calcium ions, or nuclear morphology. The presented method is straightforward to apply, compatible with commercial solutions for force measurements, and can easily be extended to investigate the mechanics of other subcellular compartments, e.g., mitochondria, stress-fibers, and endosomes.

Here, we present a protocol to investigate the intracellular mechanical properties of isolated embryonic zebrafish cells in three-dimensional confinement with direct force measurement by an optical trap.

During the development of a multicellular organism, a single fertilized cell divides and gives rise to multiple tissues with diverse functions. Tissue ...

Developmental Biology

Studying TRF1/2 Interactions with Telomeric DNA Using Single-Molecule Assay

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.

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.

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

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