Fig 1 - uploaded by Keir C Neuman
Content may be subject to copyright.
Magnetic tweezers instrument. ( a ) Photograph and ( b ) diagram of magnetic tweezers. The magnet assembly and the illumination LED are mounted on a structural rail. The flow-cell holder sits on the piezoelectric stages, which are mounted on the platform. The objective is mounted on a piezoelectric focusing element attached to the underside of the platform. The illumination ( heavy dashed line ) is collected by the objective and directed to the CCD camera via a mirror. The detection laser light ( light dashed lines ) is introduced into the objective via a dichroic mirror that sits in a cage-cube below the objective. The front view illustrates how the PIFOC adaptor is mounted on the platform with two right angle brackets. ( c ) Backscattered laser detection setup. The expanded laser beam is reflected by the turning mirror and passes through a half wave plate, polarizing beam splitter (PBS), a pair of lenses, and a quarter wave plate before being focused by the objective. Light backscattered by the bead is collected by the objective and follows the reverse path, until the PBS, where it is reflected onto a position sensitive detector.
Source publication
Magnetic tweezers provide a versatile tool enabling the application of force and torque on individual biomolecules. Magnetic tweezers are uniquely suited to the study of DNA topology and protein-DNA interactions that modify DNA topology. Perhaps due to its presumed simplicity, magnetic tweezers instrumentation has been described in less detail than...
Context in source publication
Context 1
... have found that it is more efficient to build magnetic tweezers instrumentation from components, rather than modifying a com- mercial microscope. The design and construction of a microscope with the modest imaging capability required for magnetic tweezers are relatively straightforward. We provide a brief overview of the microscope assembly and details on specific topics essential for magnetic tweezers (11, 12). First, design considerations for the magnet geometry are discussed, followed by the design and implementation of illumination, imaging, and tracking. Our magnetic tweezers apparatus (Fig. 1) is assembled on a vibration isolation workstation (VH-FR3036, Newport). A 95- mm structural rail (XT95-750, Thorlabs) supports the magnet assembly and the illumination source. A 10 00 Â 12 00 aluminum breadboard on four 1.5 00 solid stainless steel posts (P10, Thorlabs) supports a fine and a course scale piezoelectric stage to which the flow-cell sample holder is affixed. The illumination system (LED and two lenses) is attached by an angle bracket to a manual stage on the structural rail. The linear motorized stage to control the axial position of the magnets is mounted on a ...
Similar publications
Histone H1 binds to linker DNA between nucleosomes, but the dynamics and biological ramifications of this interaction remain poorly understood. We have performed single molecule experiments using magnetic tweezers to determine the effects of H1 on naked DNA in buffer or during chromatin assembly in Xenopus egg extracts. In buffer, nanomolar concent...
Mutations in SARS-CoV-2 have shown effective evasion of population immunity and increased affinity to the cellular receptor angiotensin-converting enzyme 2 (ACE2). However, in the dynamic environment of the respiratory tract, forces act on the binding partners, which raises the question of whether not only affinity but also force stability of the S...
Transcription in RNA viruses is highly dynamic, with a variety of pauses interrupting nucleotide addition by RNA-dependent
RNA polymerase (RdRp). For example, rare but lengthy pauses (>20 s) have been linked to backtracking for viral single-subunit
RdRps. However, while such backtracking has been well characterized for multi-subunit RNA polymerases...
Single-molecule biophysics has transformed our understanding of the fundamental molecular processes involved in living biological systems, but also of the fascinating physics of life. Far more exotic than a collection of exemplars of soft matter behaviour, active biological matter lives far from thermal equilibrium, and typically covers multiple le...
Ring-shaped replicative helicases are hexameric and play a key role in cellular DNA replication. Despite their importance, our understanding of the unwinding mechanism of replicative helicases is far from perfect. Bovine papillomavirus E1 is one of the best-known model systems for replicative helicases. E1 is a multifunctional initiator that senses...
Citations
... The DNA substrate of interest was then incubated at concentrations ranging from 1.5 pM to 10 pM for 15 min, followed by magnetic beads at a concentration of 20 µg/ml for 15 min. The buffer in the sample chamber was then exchanged for the topo reaction buffer.Magnetic tweezersAll experiments were carried out on a custom-built magnetic tweezers (MT) setup based on previous designs27,66,67,68 . In brief, a pair of 0.25" cube neodymium dipole magnets (K&J Magnetics B444) with a separation gap of 0.5 mm maintained by aluminum spacers was used to generate the magnetic field over the stage of a microscope. ...
Type IIA topoisomerases are essential DNA processing enzymes that must robustly and reliably relax DNA torsional stress in vivo. While cellular processes constantly create different degrees of torsional stress, how this stress feeds back to control type IIA topoisomerase function remains obscure. Using a suite of single-molecule approaches, we examined the torsional impact on supercoiling relaxation of both naked DNA and chromatin by eukaryotic topoisomerase II (topo II). We observed that topo II was at least ~ 50-fold more processive on plectonemic DNA than previously estimated, capable of relaxing > 6000 turns. We further discovered that topo II could relax supercoiled DNA prior to plectoneme formation, but with a ~100-fold reduction in processivity; strikingly, the relaxation rate in this regime decreased with diminishing torsion in a manner consistent with the capture of transient DNA loops by topo II. Chromatinization preserved the high processivity of the enzyme under high torsional stress. Interestingly, topo II was still highly processive (~ 1000 turns) even under low torsional stress, consistent with the predisposition of chromatin to readily form DNA crossings. This work establishes that chromatin is a major stimulant of topo II function, capable of enhancing function even under low torsional stress.
... However, it is not straightforward to insert specific sequences or structures at arbitrary locations since nicking endonucleases are limited to certain sites along the DNA. For the generation of shorter constructs with a wide variety of structural features and labels, a range of methods based on PCR digestionligation and single-stranded annealing have been described (17)(18)(19)(20)(21)(22)(23). However, these methods typically involve multiple rounds of digestion, purification and ligation which is time consuming and can limit the synthesis yield. ...
Single-molecule techniques such as optical tweezers and fluorescence imaging are powerful tools for probing the biophysics of DNA and DNA-protein interactions. The application of these methods requires efficient approaches for creating designed DNA structures with labels for binding to a surface or microscopic beads. In this paper, we develop a simple and fast technique for making a diverse range of such DNA constructs by combining PCR amplicons and synthetic oligonucleotides using golden gate assembly rules. We demonstrate high yield fabrication of torsionally-constrained duplex DNA up to 10 kbp in length and a variety of DNA hairpin structures. We also show how tethering to a cross-linked antibody substrate significantly enhances measurement lifetime under high force. This rapid and adaptable fabrication method streamlines the assembly of DNA constructs for single molecule biophysics.
... Magnetic beads attached to polymers, such as nucleic acids or proteins, are used in order to exert forces or torques and probe their conformations at the single-molecule level (magnetic tweezers) [8][9][10][11][12]. In addition to their use in single-molecule experiments, magnetic beads are used to analyse the elasticity of cells or extracellular matrices [13,14]. ...
Semiflexible nunchucks are block copolymers, which consist of two long blocks of high bending stiffness jointed together by a short block of low bending stiffness. Semiflexible nunchucks that consist of two DNA nanorods jointed by a short segment of double-stranded (ds) DNA and confined in two dimensions have been used in recent experiments by Fygenson and coworkers as a tool to magnify the bending fluctuations of the linking dsDNA, which in turn are used to deduce the persistence length of dsDNA. In a recent theoretical analysis, we showed that in a semiflexible nunchuck with one end grafted, the fluctuations of the position of the free end that is transverse to the grafting direction exhibit a pronounced bimodality, provided that the bending stiffness of the hinge is not very large. In this article, we theoretically analyse a grafted semiflexible nunchuck with a magnetic bead attached to its free end. We show that a transverse magnetic field induces an asymmetry in the bimodal distribution of the transverse fluctuations of the free end. This asymmetry is very sensitive to interactions with a magnetic field and, in principle, could be used in magnetometry (the measurement of a magnetic field or the magnetic moment of the bead). We also investigate how the response of the bimodal distribution of the transverse fluctuations of the free end to a magnetic field depends on the bending stiffness of the nunchuck hinge. In addition, we analyse the closely related systems of a single filament and two filaments jointed at a kink point with one end grafted and the other end attached to a magnetic bead.
... Topo VI is a slow, chirally selective and highly distributive DNA relaxase To begin exploring the single-molecule behaviour of M. mazei topo VI, a magnetic tweezers supercoiling assay was employed, described in detail elsewhere (Seol and Neuman, 2011a;Seol and Neuman, 2011b). As opposed to agarose gel analysis of DNA topology, the magnetic tweezers facilitates control over the precise level and chirality of DNA supercoiling, as well as the real-time detection of supercoil relaxation by topo VI with the ability to capture single strand-passage events. ...
... The magnetic tweezers instrumentation used here has been detailed extensively elsewhere (Charvin et al., 2003;Dittmore et al., 2017;Seol and Neuman, 2011a;Seol and Neuman, 2011b). Here, both DNA braids and supercoils have been utilised. ...
DNA topoisomerase VI (topo VI) is a type IIB DNA topoisomerase found predominantly in archaea and some bacteria, but also in plants and algae. Since its discovery, topo VI has been proposed to be a DNA decatenase, however robust evidence and a mechanism for its preferential decatenation activity was lacking. Using single-molecule magnetic tweezers measurements and supporting ensemble biochemistry, we demonstrate that Methanosarcina mazei topo VI preferentially unlinks, or decatenates DNA crossings, in comparison to relaxing supercoils, through a preference for certain DNA crossing geometries. In addition, topo VI demonstrates a significant increase in ATPase activity, DNA binding and rate of strand passage, with increasing DNA writhe, providing further evidence that topo VI is a DNA crossing sensor. Our study strongly suggests that topo VI has evolved an intrinsic preference for the unknotting and decatenation of interlinked chromosomes by sensing and preferentially unlinking DNA crossings with geometries close to 90°.
... Using this calibration method, vertical displacements of the bead may be determined with a precision of up to 10 nm [24,25]. Further refinements have come through instrument designs that compensate for sample-cell drift [26][27][28][29][30]. These advancements, however, have increased the complexity of vertical MTs and necessitated the use of sophisticated calibration procedures, for instance, for determining the previously-mentioned relationship between diffraction rings and DNA extension. ...
... The commonly used attachments are biotin-streptavidin and digoxigenin-antidigoxigenin. To increase the resiliency of the tethered DNA at higher forces, multiple biotins and digoxigenin molecules are attached to each end of the DNA molecule (details can be found in the review paper by Seol and Neuman [29]). This is also necessary to torsionally constrain the DNA. ...
The use of magnetic tweezers for single-molecule micromanipulation has evolved rapidly since its introduction approximately 30 years ago. Magnetic tweezers have provided important insights into the dynamic activity of DNA-processing enzymes, as well as detailed, high-resolution information on the mechanical properties of DNA. These successes have been enabled by major advancements in the hardware and software components of these devices. These developments now allow for a much richer mechanistic understanding of the functions and mechanisms of DNA-binding enzymes. In this review, the authors briefly discuss the fundamental principles of magnetic tweezers and describe the advancements that have made it a superlative tool for investigating, at the single-molecule level, DNA and its interactions with DNA-binding proteins.
... The magnetic tweezer setup and assays conducted with it were performed as previously 744 described (Seol and Neuman, 2011;Seol et al., 2016). 745 ...
ParABS partition systems, comprising the centromere-like DNA sequence parS, the parS-binding ParB-CTPase and the nucleoid-binding ParA-ATPase, ensure faithful segregation of bacterial chromosomes and low-copy-number plasmids. F-plasmid partition complexes containing ParBF and parSF move by generating and following a local concentration gradient of nucleoid-bound ParAF. However, the process through which ParBF activates ParAF-ATPase has not been defined. We studied CTP- and parSF-modulated ParAF-ParBF complex assembly, in which DNA-bound ParAF-ATP dimers are activated for ATP hydrolysis by interacting with two ParBF N-terminal domains. CTP or parSF enhances the ATPase rate without significantly accelerating ParAF-ParBF complex assembly. Together, parSF and CTP accelerate ParAF-ParBF assembly without further significant increase in ATPase rate. Magnetic-tweezers experiments showed that CTP promotes multiple ParBF loading onto parSF-containing DNA, generating condensed partition complex-like assemblies. We propose that ParBF in the partition complex adopts a conformation that enhances ParBF-ParBF and ParAF-ParBF interactions promoting efficient partitioning.
... To begin exploring the single-molecule behaviour of M. mazei topo VI, a magnetic tweezers supercoiling assay was employed, described in detail elsewhere (39,40). As opposed to agarose gel analysis of DNA topology, the magnetic tweezers facilitates control over the precise level and chirality of DNA supercoiling, as well as the real-time detection of supercoil relaxation by topo VI with the ability to capture single strand-passage events. ...
... The magnetic tweezers instrumentation used here has been detailed extensively elsewhere (39,40,46,70). Here, both DNA braids and supercoils have been utilised. ...
DNA topoisomerase VI (topo VI) is a type IIB DNA topoisomerase found predominantly in archaea and some bacteria, but also in plants and algae. Since its discovery, topo VI has been proposed to be a DNA decatenase, however robust evidence and a mechanism for its preferential decatenation activity was lacking. Using single-molecule magnetic tweezers measurements and supporting ensemble biochemistry, we demonstrate that Methanosarcina mazei topo VI preferentially unlinks, or decatenates, DNA crossings, in comparison to relaxing supercoils, through a preference for certain DNA crossing geometries. In addition, topo VI demonstrates a dramatic increase in ATPase activity, DNA binding and rate of strand passage, with increasing DNA writhe, providing further evidence that topo VI is a DNA crossing sensor. Our study strongly suggests that topo VI has evolved an intrinsic preference for the unknotting and decatenation of interlinked chromosomes by sensing and preferentially unlinking DNA crossings with geometries close to 90°.
... The general design of the TIRF microscopy setup was essentially as previously described (Ivanov 586 The magnetic tweezer setup and assays conducted with it were performed as previously described 655 (Seol and Neuman, 2011;Seol et al., 2016). 656 . ...
ParABS partition systems, comprising the centromere-like DNA sequence parS, the parS-binding ParB-CTPase and the nucleoid-binding ParA-ATPase, ensure faithful segregation of bacterial chromosomes and low-copy-number plasmids. F-plasmid partition complexes containing ParBF and parSF move by generating and following a local concentration gradient of nucleoid-bound ParAF. However, the process of ParBF activation of ParAF-ATPase has not been defined. We studied CTP- and parSF-modulated ParAF—ParBF complex assembly leading to ParAF-ATPase activation. Activation of DNA-bound ParAF-ATP dimers for ATP hydrolysis requires binding of two ParBF N-terminal domains. CTP or parSF enhances the ATPase rate without increasing ParAF—ParBF assembly kinetics. Together, parSF and CTP accelerate ParAF—ParBF assembly without further increasing the ATPase rate. Magnetic-tweezers experiments showed that CTP promotes multiple ParBF loading onto parSF-containing DNA, generating condensed partition complex-like assemblies. We propose that ParBF in the partition complex adopts a conformation that enhances ParBF—ParBF and ParAF—ParBF interactions promoting efficient partitioning.
... Most of these observations have been performed on instruments in which only one molecule can be studied at a time (i.e., optical tweezers), impeding the rapid collection of statistically significant datasets. Several approaches have been developed that provide greater multiplexing [6][7][8][9] including magnetic tweezers (MT) 4,10,11 , acoustic force spectroscopy 7 (AFS), and centrifuge force microscopy 8 (CFM). However, all of these methods depend on high axial resolution to observe length changes. ...
The application of forces and torques on the single molecule level has transformed our understanding of the dynamic properties of biomolecules, but rare intermediates have remained difficult to characterize due to limited throughput. Here, we describe a method that provides a 100-fold improvement in the throughput of force spectroscopy measurements with topological control, which enables routine imaging of 50,000 single molecules and a 100 million reaction cycles in parallel. This improvement enables detection of rare events in the life cycle of the cell. As a demonstration, we characterize the supercoiling dynamics and drug-induced DNA break intermediates of topoisomerases. To rapidly quantify distinct classes of dynamic behaviors and rare events, we developed a software platform with an automated feature classification pipeline. The method and software can be readily adapted for studies of a broad range of complex, multistep enzymatic pathways in which rare intermediates have escaped classification due to limited throughput.
... While scanning techniques such as confocal and light-sheet microscopy suffice for imaging stationary or slow-moving systems (such as fixed tissue samples), highly dynamic systems require frame rates only accessible with wide-field microscopy. In particular, force spectroscopy applications such as Optical Traps (OT), Magnetic Tweezers (MT), or Acoustic Force Spectroscopy (AFS) often require sampling rates as high as 1 kHz, so that high-speed wide-field or non-image-based tracking techniques are the only viable options [6][7][8][9]. Moreover, studies employing those techniques typically require nanometer-scale localization accuracy. ...
... The data presented here was acquired using a Physik-Instrumente P-721.CDQ objective scanner and E-665 piezo controller (Physik Instrumente, Auburn, MA). Other scanners [6,15] have been used in similar implementations. [12]. ...
... For an un-weighted two-variable least-squares fit of N data points, the average residual of the estimate, x in Eq. (2), is given by (6) and the standard error of x is given by the matrix ...
Volumetric imaging and 3D particle tracking are becoming increasingly common and have a variety of microscopy applications including in situ fluorescent imaging, in-vitro single-molecule characterization, and analysis of colloidal systems. While recent interest has generated discussion of optimal schemes for localizing diffraction-limited fluorescent puncta, there have been relatively few published routines for tracking particles imaged with bright-field illumination. To address this, we outline a simple, look-up-table based 3D tracking strategy, which can be adapted to most commercially available wide-field microscopes, and present two image processing algorithms that together yield high-precision localization and return estimates of statistical accuracy. Under bright-field illumination, a particle’s depth can be determined based on the size and shape of its diffractive pattern due to Mie scattering. Contrary to typical “super-resolution” fluorescence tracking routines, which typically fit a diffraction-limited spot to a model point-spread-function, the lateral (XY) tracking routine relies on symmetry to locate a particle without prior knowledge of the form of the particle. At low noise levels (signal:noise > 1000), the symmetry routine estimates particle positions with accuracy better than 0.01 pixel. Depth localization is accomplished by matching images of particles to those in a pre-recorded look-up-table. The routine presented here optimally interpolates between LUT entries with better than 0.05 step accuracy. Both routines are tolerant of high levels of image noise, yielding sub-pixel/step accuracy with signal-to-noise ratios as small as 1, and, by design, return confidence intervals indicating the expected accuracy of each calculated position. The included implementations operate extremely quickly and are amenable to real-time analysis at frame rates exceeding several hundred frames per second.
