Magnetic tweezers

UvrD, a model for non-hexameric Superfamily 1 helicases, utilizes ATP hydrolysis to translocate stepwise along single-stranded DNA and unwind the duplex. Previous estimates of its step size have been indirect, and a consensus on its stepping mechanism is lacking. To dissect the mechanism underlying DNA unwinding, we use optical tweezers to measure directly the stepping behavior of UvrD as it processes a DNA hairpin and show that UvrD exhibits a variable step size averaging~3 base pairs. Analyzing stepping kinetics across ATP reveals the type and number of catalytic events that occur with different step sizes. These single-molecule data reveal a mechanism in which UvrD moves one base pair at a time but sequesters the nascent single strands, releasing them non-uniformly after a variable number of catalytic cycles. Molecular dynamics simulations point to a structural basis for this behavior, identifying the protein-DNA interactions responsible for strand sequestration. Based on structural and sequence alignment data, we propose that this stepping mechanism may be conserved among other non-hexameric helicases.

ABSTRACTMost biomolecular systems are dependent on a complex interplay of forces. Modern force spectroscopy techniques provide means of interrogating these forces. These techniques, however, are not optimized for studies in constrained or crowded environments as they typically require micron-scale beads in the case of magnetic or optical tweezers, or direct attachment to a cantilever in the case of atomic force microscopy. We implement a nanoscale force-sensing device using a DNA origami which is highly customizable in geometry, functionalization, and mechanical properties. The device, referred to as the NanoDyn, functions as a binary (open or closed) force sensor that undergoes a structural transition under an external force. The transition force is tuned with minor alterations of 1 to 3 DNA oligonucleotides and spans tens of picoNewtons (pN). This actuation of the NanoDyn is reversible and the design parameters strongly influence the efficiency of resetting the initial state, with...

In recent years, techniques have been developed to study and manipulate single molecules of DNA and other biopolymers. In one such technique, the magnetic trap, a single DNA molecule is bound at one end to a glass surface and at the other to a magnetic microbead. Small magnets, whose position and rotation can be controlled, pull on and rotate the microbead. This provides a simple method to stretch and twist the molecule. The system allows one to apply and measure forces ranging from 10(-3) to >100 pN. In contrast to other techniques, the force measurement is absolute and does not require calibration of the sensor. In this article, we describe the principle of the magnetic trap, as well as its use in the measurement of the elastic properties of DNA and the study of DNA-protein interactions.

Controlled transport of multiple individual nanostructures is crucial for nanoassembly and nanodelivery but is challenging because of small particle size. Although atomic force microscopy and optical and magnetic tweezers can control single particles, it is extremely difficult to scale these technologies for multiple structures. Here, we demonstrate a "nano-conveyer-belt" technology that permits simultaneous transport and tracking of multiple individual nanospecies in a selected direction. The technology consists of two components: nanocontainers, which encapsulate the nanomaterials transported, and nanoconveyer arrays, which use magnetic force to manipulate individual or aggregate nanocontainers. This technology is extremely versatile. For example, nanocontainers encapsulate quantum dots or rods and superparamagnetic iron oxide nanoparticles in <100 nm nanocontainers, the smallest magnetic composites to have been simultaneously moved and optically tracked. Similarly, the nanoconveyers consist of patterned microdisks or zigzag nanowires, whose dimensions can be controlled through micropatterning. The nanoconveyer belt technology could impact multiple fields, including nanoassembly, biomechanics, nanomedicine, and nanofluidics.

Detailed mechanisms of DNA clamps in prokaryotic and eukaryotic systems were investigated by probing their mechanics with single-molecule force spectroscopy. Specifically, the mechanical forces required for the Escherichia coli and Saccharomyces cerevisiae clamp opening were measured at the single-molecule level by optical tweezers. Steered molecular dynamics simulations further examined the forces involved in DNA clamp opening from the perspective of the interface binding energies associated with the clamp opening processes. In combination with additional molecular dynamics simulations, we identified the contact networks between the clamp subunits that contribute significantly to the interface stability of the S.cerevisiae and E. coli clamps. These studies provide a vivid picture of the mechanics and energy landscape of clamp opening and reveal how the prokaryotic and eukaryotic clamps function through different mechanisms.

This study evaluates the influence of vinculin in closed conformation on the mechanical properties of cells. We demonstrate that MEFvin À/À cells transfected with the eGFP-vinculin mutant A50I (talin-binding-deficient-vinculin in a constitutively closed conformation) show 2-fold lower stiffness and focal adhesion density compared to MEFvin +/+ and MEF Rescue cells. MEF A50I cells are as stiff as MEFvin À/À cells with similar focal adhesion density. Further, 2D traction microscopy indicates that MEF A50I and MEFvin À/ À cells generate 3-to 4-fold less strain energy than MEFvin +/+ and MEF Rescue cells. These results demonstrate that vinculin's mechano-coupling function is dependent on its conformational state.

Arthur Ashkin was awarded the 2018 Nobel prize in physics for the invention of optical tweezers. Since the first publication in 1986 Optical Tweezers have been used as a tool to measure forces and rheological properties of microscopic systems. For the calibration of these measurements, the knowledge of the forces is fundamental. However, it is still common to deduce the optical forces from assumptions based on the particle size with respect to the trapping laser wavelength. This show the necessity to develop a complete and accurate electromagnetic model that does not depend on early approximations of the force model. Furthermore, the model we developed has several advantages, such as morphology-dependent resonances, size dependence for large spheres, and multipole effects from smaller particles, just to name a few. In this tutorial, we review and discuss the physical modeling of optical forces in optical tweezers, which are the resultant forces exerted by a trapping beam on a sphere of any size and composition.

Background: DNA bridging promoted by the H-NS protein, combined with the compaction induced by cellular crowding, plays a major role in the structuring of the E. coli genome. However, only few studies consider the effects of the physical interplay of these two factors in a controlled environment. Methods: We apply a single molecule technique (Magnetic Tweezers) to study the nanomechanics of compaction and folding kinetics of a 6 kb DNA fragment, induced by H-NS bridging and/or PEG crowding. Results: In the presence of H-NS alone, the DNA shows a step-wise collapse driven by the formation of multiple bridges, and little variations in the H-NS concentration-dependent unfolding force. Conversely, the DNA collapse force observed with PEG was highly dependent on the volume fraction of the crowding agent. The two limit cases were interpreted considering the models of loop formation in a pulled chain and pulling of an equilibrium globule respectively. Conclusions: We observed an evident cooperative effect between H-NS activity and the depletion of forces induced by PEG. General Significance: Our data suggest a double role for H-NS in enhancing compaction while forming specific loops, which could be crucial in vivo for defining specific mesoscale domains in chromosomal regions in response to environmental changes.

Single molecule experiments have demonstrated a progressive transition from a B-to an L-form helix as DNA is gently stretched and progressively unwound. Since the particular sequence of a DNA segment influences both base stacking and hydrogen bonding, the conformational dynamics of B-to-L transitions should be tunable. To test this idea, DNA with diaminopurine replacing adenine was synthesized to produce linear fragments with triply hydrogen-bonded A:T base pairs. Triple hydrogen bonding stiffened the DNA by 30% flexurally. In addition, DAP-substituted DNA formed plectonemes with larger gyres for both Band L-form helices. Both unmodified and DAP-substituted DNA transitioned from a B-to an L-helix under physiological conditions of mild tension and unwinding. This transition avoids writhing by DNA stretched and unwound by enzymatic activity. The intramolecular nature and ease of this transition likely prevent cumbersome topological rearrangements in genomic DNA that would require topoisomerase activity to resolve. L-DNA displayed about tenfold lower persistence length indicating it is much more contractile and prone to sharp bends and kinks. However, left-handed DAP DNA was twice as stiff as unmodified L-DNA. Thus, significantly doubly and triply hydrogen bonded segments have very distinct mechanical dynamics at physiological levels of negative supercoiling and tension.

G-quadruplexes (G4s) are tetrahelical DNA structures stabilized by four guanines paired via Hoogsteen hydrogen bonds into quartets. While their presence within eukaryotic DNA is known to play a key role in regulatory processes, their functional mechanisms are still under investigation. In the present work, we analysed the nanomechanical properties of three G4s present within the promoter of the KIT proto-oncogene from a single-molecule point of view through the use of magnetic tweezers (MTs). The study of DNA extension fluctuations under negative supercoiling allowed us to identify a characteristic fingerprint of G4 folding. We further analysed the energetic contribution of G4 to the double-strand denaturation process in the presence of negative supercoiling, and we observed a reduction in the energy required for strands separation.

Platinum-containing molecules are widely used as anticancer drugs. These molecules exert cytotoxic effects by binding to DNA through various mechanisms. The binding between DNA and platinum-based drugs hinders the opening of DNA, and therefore, DNA duplication and transcription are severely hampered. Overall, impeding the above-mentioned important DNA mechanisms results in irreversible DNA damage and the induction of apoptosis. Several molecules, including multinuclear platinum compounds, belong to the family of platinum drugs, and there is a body of research devoted to developing more efficient and less toxic versions of these compounds. In this study, we combined different biophysical methods, including single-molecule assays (magnetic tweezers) and bulk experiments (ultraviolet absorption for thermal denaturation) to analyze the differential stability of double-stranded DNA in complex with either cisplatin or multinuclear platinum agents. Specifically, we analyzed how the binding of BBR3005 and BBR3464, two representative multinuclear platinum-based compounds, to DNA affects its stability as compared with cisplatin binding. Our results suggest that single-molecule approaches can provide insights into the drug-DNA interactions that underlie drug potency and provide information that is complementary to that generated from bulk analysis; thus, single-molecule approaches have the potential to facilitate the selection and design of optimized drug compounds. In particular, relevant differences in DNA stability at the single-molecule level are demonstrated by analyzing nanomechanically induced DNA denaturation. On the basis of the comparison between the single-molecule and bulk analyses, we suggest that transplatinated drugs are able to locally destabilize small portions of the DNA chain, whereas other regions are stabilized.

Controlled transport of multiple individual nanostructures is crucial for nanoassembly and nanodelivery but is challenging because of small particle size. Although atomic force microscopy and optical and magnetic tweezers can control single particles, it is extremely difficult to scale these technologies for multiple structures. Here, we demonstrate a "nano-conveyer-belt" technology that permits simultaneous transport and tracking of multiple individual nanospecies in a selected direction. The technology consists of two components: nanocontainers, which encapsulate the nanomaterials transported, and nanoconveyer arrays, which use magnetic force to manipulate individual or aggregate nanocontainers. This technology is extremely versatile. For example, nanocontainers encapsulate quantum dots or rods and superparamagnetic iron oxide nanoparticles in <100 nm nanocontainers, the smallest magnetic composites to have been simultaneously moved and optically tracked. Similarly, the nanoconveyers consist of patterned microdisks or zigzag nanowires, whose dimensions can be controlled through micropatterning. The nanoconveyer belt technology could impact multiple fields, including nanoassembly, biomechanics, nanomedicine, and nanofluidics.

Torsional stress in linear biopolymers such as DNA and chromatin has important consequences for nanoscale biological processes. We have developed a new method to directly measure torque on single molecules. Using a cylindrical magnet, we manipulate a novel probe consisting of a nanorod with a 0.1 μm ferromagnetic segment coupled to a magnetic bead. We achieve controlled introduction of turns into the molecule and precise measurement of torque and molecule extension as a function of the number of turns at low pulling force. We show torque measurement of single DNA molecules and demonstrate for the first time measurements of single chromatin fibers.

Movie S1-PROBE ROTATION. Real time movie of the rotation of the nanorod-bead probe by translation of the stage. The probe was tethered to the glass surface of the capillary tube by a 3 µm DNA molecule and lifted using the cylindrical magnet. Movement of the stage in a path around the projection of the center of the cylindrical magnet allows the nanorod-bead probe to be rotated in a controlled manner. Movie S2-NANOROD-BEAD PROBE. Fluctuations of the nanorod-bead probe under a vertical magnetic field created by a cylindrical magnet. Nanorod-bead probe was tethered to the glass surface by a 3µm DNA molecule. The vertical magnetic field lifts the probe while providing a weak horizontal angular trap. The horizontal angular fluctuations in this case spanned ≈100°. The left side is a bright field movie. The right side is the same movie as a binary image. The red line is the computed orientation of the probe based on the center of the bead and the center of the rod. Images were analyzed at 21 Hz. Movie S3-VERTICAL ANGLE. Fluctuations of the vertical angle (α) between the nanorod and the horizontal plane. Top left: bright field movie of the fluctuating nanorod-bead probe tethered by a DNA molecule and subject to the magnetic field created by a cylindrical magnet. Blue and red lines show where the intensity profiles were taken. Note that the diffraction pattern remains homogeneous along the rod which indicates that α is small and relatively constant. Top right: intensity profiles for the red and blue lines in bright field movie. Bottom: plot of the vertical angle as a function of movie frame. We obtain this angle from the heights of the two positions along the nanorod (indicated by the blue and red lines) and the distance between them (660 nm). See Figure S7 caption for details.

Fragile X syndrome and other triplet repeat diseases are characterized by an elongation of a repeating DNA triplet. The ensemble-averaged lambda exonuclease digestion rate of different substrates, including one with an elongated FMR1 gene containing 120 CGG repeats, was measured using absorption and fluorescence spectroscopy. Using magnetic tweezers sequencedependent digestion rates and pausing was measured for individual lambda exonucleases. Within the triplet repeats a lower average and narrower distribution of rates and a higher frequency of pausing was observed.

Through its capability to transiently pack and unpack our genome, chromatin is a key player in the regulation of gene expression. Single‐molecule approaches have recently complemented conventional biochemical and biophysical techniques to decipher the complex mechanisms ruling chromatin dynamics. Micromanipulations with tweezers (magnetic or optical) and imaging with molecular microscopy (electron or atomic force) have indeed provided opportunities to handle and visualize single molecules, and to measure the forces and torques produced by molecular motors, along with their effects on DNA or nucleosomal templates. By giving access to dynamic events that tend to be blurred in traditional biochemical bulk experiments, these techniques provide critical information regarding the mechanisms underlying the regulation of gene activation and deactivation by nucleosome and chromatin structural changes. This minireview describes some single‐molecule approaches to the study of ATP‐consuming mol...

Retroviruses must integrate their linear viral cDNA into the host genome for a productive infection. Integration is catalysed by the retrovirus-encoded integrase (IN), which forms a tetramer or octamer complex with the viral cDNA long terminal repeat (LTR) ends termed an intasome. IN removes two 3&#39;-nucleotides from both LTR ends and catalyses strand transfer of the recessed 3&#39;-hydroxyls into the target DNA separated by 4-6 bp. Host DNA repair restores the resulting 5&#39;-Flap and single-stranded DNA (ssDNA) gap. Here we have used multiple single molecule imaging tools to determine that the prototype foamy virus (PFV) retroviral intasome searches for an integration site by one-dimensional (1D) rotation-coupled diffusion along DNA. Once a target site is identified, the time between PFV strand transfer events is 470 ms. The majority of PFV intasome search events were non-productive. These observations identify new dynamic IN functions and suggest that target site-selection lim...

Background: Despite the profound current knowledge of the architecture and dynamics of nucleosomes, little is known about the structures generated by the interaction of histones with single-stranded DNA (ssDNA), which is widely present during replication and transcription. Methods: Non-denaturing gel electrophoresis, transmission electron microscopy, atomic force microscopy, magnetic tweezers. Results: Histones have a high affinity for ssDNA at physiological salt concentrations, with an apparent binding constant similar to that calculated for their association with double-stranded DNA (dsDNA). The length of DNA (number of nucleotides in ssDNA or base pairs in dsDNA) associated with a fixed core histone mass is the same for both ssDNA and dsDNA. Whereas histone-ssDNA complexes show a high tendency to aggregate in 0.2 M NaCl, at lower ionic strength nucleosome-like structures are formed. Core histones are able to protect ssDNA from digestion by micrococcal nuclease, and a shortening of ssDNA occurs upon its interaction with histones. The purified (+) strand of a cloned DNA fragment of nucleosomal origin has a higher affinity for histones than the purified complementary (-) strand. Conclusions: At physiological ionic strength histones have high affinity for ssDNA, possibly associating with it into nucleosome-like structures. General Significance: In the cell nucleus histones may spontaneously interact with ssDNA to facilitate their participation in the replication and transcription of chromatin.

A new kind of nanodevice that acts like tweezers through remote actuation by an external magnetic field is designed. Such device is meant to mechanically grab micrometric objects. The nanotweezers are built by using a top-down approach and are made of two parallelepipedic microelements, at least one of them being magnetic, bound by a flexible nanohinge. The presence of an external magnetic field induces a torque on the magnetic elements that competes with the elastic torque provided by the nanohinge. A model is established in order to evaluate the values of the balanced torques as a function of the tweezers opening angles. The results of the calculations are confronted to the expected values and validate the overall working principle of the magnetic nanotweezers. Several applications in life science and biotechnology require tools for manipulating and exerting forces on micro or nanometric objects. In that regard, biocompatible magnetic nanoparticles have widely been developed and used 1-3 , owing to their ability to be actuated by external magnetic fields. The advantage of such method is that magnetic fields can penetrate human tissues in a noninvasive way. Bottom-up approaches are an efficient way to chemically produce functionalized superparamagnetic iron oxide nanoparticles in ligand matrices, and their superparamagnetic nature allows them to not agglomerate in the absence of magnetic field 4. Functionalized magnetic nanoparticles can target specific biological entities for drug delivery, hyperthermia treatment or mechanical actuation 1. Other methods for manipulating micro or nanoscale objects have also been explored. Notable examples are the concepts of magnetic tweezers 5-13 and optical tweezers 14. The former concept consists in binding a molecule to a substrate at one end and to a magnetic particle at the other end. By applying a magnetic field gradient, forces can be exerted on biomolecules such as DNA or cells to test their mechanical properties. Optical tweezers trap accelerated particles in an optical potential well. While the optical tweezers are a kind of "contactless" tweezers, past works have also established designs for "contact" tweezers 15, 16 that are controlled by magnetic fields for grasping objects physically as well as for moving in liquid environments. They succeeded in demonstrating the possibility of transporting cargoes in the submillimeter scale through pick-and-place experiments. Devices based on the use of magnetic particles or elements along with elastic materials are also being actively developed to make programmable actuators 17. In this article, we present a new design of magnetic tweezers prepared by a top-down approach. An advantage of such fabrication process is the fact that a vast number of nanotweezers can be produced at the same time. Moreover, with sizes between the nanometric and micrometric scales the tweezers will ultimately be able to interact with objects of comparable size such as cells, bacteria, or long chains of macromolecules. Being made of two actuable jaws, the concept of magnetic tweezer we propose is quite similar to that of real-life tweezers except that their ultimate role is to interact with micrometric species in fluids. They can potentially be an efficient way to trap or deliver molecules to targeted biological zones. Like for the nanoparticles developed in the past, self-agglomeration needs to be avoided by a proper choice of materials for the tweezers, e.g. synthetic antiferromagnetic 18, 19 or vortex 20, 21 particles. Due to their in-plane magnetic shape anisotropy, they can be remotely controlled by magnetic fields rather than magnetic field gradients, as seen in our previous works 22, 23 .

We have determined the change in the number of proteins bound non-specifically to DNA as a function of applied force on tethered DNA. Using magnetic tweezers, single molecules of λ DNA were stretched in the absence and presence of λ repressor protein (CI). The force versus extension data was analyzed using a model for quantitative determination of protein binding. The results indicate that the non-specific binding of CI changes the forceextension relation significantly in comparison to that of naked DNA. We demonstrate that the force-extension relation provides an effective approach for estimating the number of proteins bound non-specifically to DNA.

Proton-pumping NADH-ubiquinone oxidoreductase (complex I) is a very large membrane protein complex that is found in many bacteria and almost all eukaryotes (Brandt 2006). In the mitochondrial respiratory chain it couples the transfer of two electrons from NADH to ubiquinone to the vectorial translocation of four protons across the inner membrane of the organelle and thereby generates up to 40% of the driving force for ATP synthesis by ATP synthase. Complex I is a source of reactive oxygen species and is involved in the pathogenesis of encephalomyopathies and neurodegenerative diseases.

A novel platform for microfluidic manipulation of magnetic particles is discussed. The particles are confined by an array of magnetic spin valves with bistable ferromagnetic ''ON'' and antiferromagnetic ''OFF'' net magnetization states. The switchable fringing fields near the spin-valve traps can be used to selectively confine or release particles for transport or sorting. Spin-valve traps may be potentially used as magnetic molecular tweezers or adapted to a low-power magnetic random access memory (MRAM) switching architecture for massively parallel particle sorting applications.

The viral RNA-dependent RNA polymerase (RdRp) is a well-established target for development of broad-spectrum antiviral therapeutics. Incorporation of ribonucleotide analogues by the RdRp will either cause termination of RNA synthesis or mutagenesis of the RNA product. We demonstrated recently that incorporation of a pyrazine-carboxamide ribonucleotide into nascent RNA leads to pausing and backtracking of the elongating RdRp. Here, we provide evidence for the single-stranded RNA product of backtracking serving as an intermediate in RdRp-catalyzed, template-switching reactions. This intermediate is used for both intramolecular template-switching (copy-back RNA synthesis) and intermolecular template-switching (homologous RNA recombination). The use of a magnetic-tweezers platform to monitor RdRp elongation dynamics permitted direct observation of copy-back synthesis and illuminated properties of the RdRp that promote copy-back synthesis, including stability of the RdRp-nascent-RNA comp...

In recent years, techniques have been developed to study and manipulate single molecules of DNA and other biopolymers. In one such technique, the magnetic trap, a single DNA molecule is bound at one end to a glass surface and at the other to a magnetic microbead. Small magnets, whose position and rotation can be controlled, pull on and rotate the microbead. This provides a simple method to stretch and twist the molecule. The system allows one to apply and measure forces ranging from 10(-3) to &gt;100 pN. In contrast to other techniques, the force measurement is absolute and does not require calibration of the sensor. In this article, we describe the principle of the magnetic trap, as well as its use in the measurement of the elastic properties of DNA and the study of DNA-protein interactions.

The condensin SMC protein complex organizes chromosomal structure by extruding loops of DNA. Its ATP-dependent motor mechanism remains unclear but likely involves steps associated with large conformational changes within the ∼50 nm protein complex. Here, using high-resolution magnetic tweezers, we resolve single steps in the loop extrusion process by individual yeast condensins. The measured median step sizes range between 20-40 nm at forces of 1.0-0.2 pN, respectively, comparable with the holocomplex size. These large steps show that, strikingly, condensin typically reels in DNA in very sizeable amounts with ∼200 bp on average per single extrusion step at low force, and occasionally even much larger, exceeding 500 bp per step. Using Molecular Dynamics simulations, we demonstrate that this is due to the structural flexibility of the DNA polymer at these low forces. Using ATP-binding-impaired and ATP-hydrolysis-deficient mutants, we find that ATP binding is the primary step-generating stage underlying DNA loop extrusion. We discuss our findings in terms of a scrunching model where a stepwise DNA loop extrusion is generated by an ATP-bindinginduced engagement of the hinge and the globular domain of the SMC complex.

Bacillus subtilis bacteriophage AR9 employs two strategies for efficient host takeover control and host antiviral defense evasion – it encodes two unique DNA-dependent RNA polymerases (RNAPs) that function at different stages of virus morphogenesis in the cell, and its double stranded (ds) DNA genome contains uracils instead of thymines throughout1,2. Unlike any known RNAP, the AR9 non-virion RNAP (nvRNAP), which transcribes late phage genes, recognizes promoters in the template strand of dsDNA and efficiently differentiates obligatory uracils from thymines in its promoters3. Here, using structural data obtained by cryo-electron microscopy and X-ray crystallography on the AR9 nvRNAP core, holoenzyme, and a promoter complex, and a variety of in vitro transcription assays, we elucidate a unique mode of uracil-specific, template strand-dependent promoter recognition. It is achieved by a tripartite interaction between the promoter specificity subunit, the core enzyme, and DNA adopting a...

Single molecule force spectroscopy is an emerging technique that can be used to measure the biophysical properties of single macromolecules such as nucleic acids and proteins. In particular, single DNA molecule stretching experiments are used to measure the elastic properties of these molecules and to induce structural transitions. We have demonstrated that double‒stranded DNA molecules undergo a force‒induced melting transition at high forces. Force–extension measurements of single DNA molecules using optical tweezers allow us to measure the stability of DNA under a variety of solution conditions and in the presence of DNA binding proteins. Here we review the evidence of DNA melting in these experiments and discuss the example of DNA force‒induced melting in the presence of the single‒stranded DNA binding protein T4 gene 32. We show that this force spectroscopy technique is a useful probe of DNA–protein interactions, which allows us to obtain binding rates and binding free energies...

Mechanical forces influence many aspects of cell behavior. Forces are detected and transduced into biochemical signals by force bearing molecular elements located at the cell surface, in adhesion complexes or in cytoskeletal structures 1. The nucleus is physically connected to the cell surface through the cytoskeleton and the linker of nucleoskeleton and cytoskeleton (LINC) complex, allowing rapid mechanical stress transmission from adhesions to the nucleus 2. Whereas it has been demonstrated that nuclei experience force 3 , the direct effect of force on the nucleus is not known. Here we show that isolated nuclei are able to respond to force by adjusting their stiffness to resist the applied tension. Using magnetic tweezers, we found that applying force on nesprin-1 triggers nuclear stiffening that does not involve chromatin or nuclear actin, but requires an intact nuclear lamina and emerin, a protein of the inner nuclear membrane. Emerin becomes tyrosine phosphorylated in response to force and mediates the nuclear mechanical response to tension. Our results demonstrate that mechanotransduction is not restricted to cell surface receptors and adhesions but can occur within the nucleus. To mimic the transmission of mechanical stress from the cytoskeleton to the nucleus, we applied force directly on isolated nuclei via the LINC complex component nesprin-1 (Figure 1a). We used magnetic tweezers to stimulate magnetic beads coated with anti nesprin-1 antibody and we measured bead displacements due to a known force induced by a magnetic field. Application of successive pulses of constant force triggered an increase in nuclear stiffness, resulting in decreased bead displacement (Figure 1b, Supplementary Figure 1a and Supplementary figure 2). The "relative bead displacement" was calculated by normalizing the displacement for pulses 2, 3, 4, 5 and 6 to that observed during the first pulse. The AUTHOR CONTRIBUTIONS: C.G designed and performed experiments.

Single molecule force spectroscopy is an emerging technique that can be used to measure the biophysical properties of single macromolecules such as nucleic acids and proteins. In particular, single DNA molecule stretching experiments are used to measure the elastic properties of these molecules and to induce structural transitions. We have demonstrated that doublestranded DNA molecules undergo a force-induced melting transition at high forces. Force-extension measurements of single DNA molecules using optical tweezers allow us to measure the stability of DNA under a variety of solution conditions and in the presence of DNA binding proteins. Here we review the evidence of DNA melting in these experiments and discuss the example of DNA force-induced melting in the presence of the single-stranded DNA binding protein T4 gene 32. We show that this force spectroscopy technique is a useful probe of DNA-protein interactions, which allows us to obtain binding rates and binding free energies for these interactions.

A compact single beam optical tweezers system for force measurements and manipulation of individual double-stranded deoxyribonucleic acid ͑DNA͒ molecules was integrated into a commercial inverted optical microscope. A maximal force of 150 pN combined with a force sensitivity of less than 0.5 pN allows measurements of elastic properties of single molecules which complements and overlaps the force regime accessible with atomic force microscopy ͑AFM͒. The manipulation and measurement performance of this system was tested with individual-DNA molecules and renders new aspects of dynamic forces phenomena with higher precision in contrast to AFM studies. An integrated liquid handling system with a fluid cell allows investigation of the force response of individual DNA molecules in the presence of DNA binding agents. Comparison of YOYO-1-, ethidium bromide intercalated DNA, and distamycin-A complexed DNA revealed accurate and reproducible differences in the force response to an external load. This opens the possibility to use it as a single molecule biosensor to investigate DNA binding agents and even to identify molecular binding mechanisms.

Fluorescent DNA dyes are broadly used in many biotechnological applications for detecting and imaging DNA in cells and gels. Their binding alters the structural and nanomechanical properties of DNA and affects the biological processes that are associated with it. Although interaction modes like intercalation and minor groove binding already have been identified, associated mechanic effects like local elongation, unwinding, and softening of the DNA often remain in question. We used magnetic tweezers to quantitatively investigate the impact of three DNA-binding dyes (YOYO-1, DAPI, and DRAQ5) in a concentration-dependent manner. By extending and overwinding individual, torsionally constrained, nickfree dsDNA molecules, we measured the contour lengths and molecular forces that allow estimation of thermodynamic and nanomechanical binding parameters. Whereas for YOYO-1 and DAPI the binding mechanisms could be assigned to bis-intercalation and minor groove binding, respectively, DRAQ5 exhibited both binding modes in a concentration-dependent manner.

DNA-binding agents are broadly used in many biotechnological or biomedical applications for detecting DNA in cells and gels or as drugs in cytotoxic oncologic cancer treatments. Their binding alters the structural and nanomechanical properties of DNA and affects the associated biological processes. Although interaction modes like intercalation and minor grove binding already have been identified, associated mechanic effects like DNA strand elongation as well as unwinding and softening of the dsDNA strand often remain obscure. We used single-molecule magnetic tweezers force experiments to quantitatively investigate the impact of four DNA-dyes (YOYO-1, DAPI, DRAQ5 and PicoGreen) as well as the anti-cancer drug mitoxantrone at room temperature in a concentration dependent manner. By extending and overwinding individual, torsionally constrained, nick-free dsDNA molecules, we determined the contour lengths, persistence lengths and molecular forces, which allow estimation of several thermodynamic and nanomechanical binding parameters. Whereas for YOYO-1 and DAPI the binding mechanisms can be assigned to bisintercalation and minor groove binding, respectively, DRAQ5 and Pico-Green exhibit both binding modes in a concentration dependent manner. Similarly, the chemotherapeutic drug mitoxantrone is found to interact with dsDNA depending on the concentration as a groove binder and an intercalator. Our experiments show that single-molecule nanomechanical experiments can be used for elucidating the binding mechanics of DNA binders and can contribute to the registration process in drug regulation.

Fluorescent DNA dyes are broadly used in many biotechnological applications for detecting and imaging DNA in cells and gels. Their binding alters the structural and nanomechanical properties of DNA and affects the biological processes that are associated with it. Although interaction modes like intercalation and minor groove binding already have been identified, associated mechanic effects like local elongation, unwinding, and softening of the DNA often remain in question. We used magnetic tweezers to quantitatively investigate the impact of three DNA-binding dyes (YOYO-1, DAPI, and DRAQ5) in a concentration-dependent manner. By extending and overwinding individual, torsionally constrained, nick-free dsDNA molecules, we measured the contour lengths and molecular forces that allow estimation of thermodynamic and nanomechanical binding parameters. Whereas for YOYO-1 and DAPI the binding mechanisms could be assigned to bis-intercalation and minor groove binding, respectively, DRAQ5 exh...

A precise characterization of cell elastic properties is crucial for understanding the mechanisms by which cells sense mechanical stimuli and how these factors alter cellular functions. Optical and magnetic tweezers are micromanipulation techniques which are widely used for quantifying the stiffness of adherent cells from their response to an external force applied on a bead partially embedded within the cell cortex. However, the relationships between imposed external force and resulting bead translation or rotation obtained from these experimental techniques only characterize the apparent cell stiffness. Indeed, the value of the estimated apparent cell stiffness integrates the effect of different geometrical parameters, the most important being the bead embedding angle 2␥, bead radius R, and cell height h. In this paper, a three-dimensional finite element analysis was used to compute the cell mechanical response to applied force in tweezer experiments and to explicit the correcting functions which have to be used in order to infer the intrinsic cell Young's modulus from the apparent elasticity modulus. Our analysis, performed for an extensive set of values of ␥, h, and R, shows that the most relevant parameters for computing the correcting functions are the embedding half angle ␥ and the ratio h u /2R, where h u is the under bead cell thickness. This paper provides original analytical expressions of these correcting functions as well as the critical values of the cell thickness below which corrections of the apparent modulus are necessary to get an accurate value of cell Young's modulus. Moreover, considering these results and taking benefit of previous results obtained on the estimation of cell Young's modulus of adherent cells probed by magnetic twisting cytometry (MTC) (Ohayon, J., and Tracqui, P., 2005, Ann. Biomed. Eng., 33, pp. 131-141), we were able to clarify and to solve the still unexplained discrepancies reported between estimations of elasticity modulus performed on the same cell type and probed with MTC and optical tweezers (OT). More generally, this study may strengthen the applicability of optical and magnetic tweezers techniques by insuring a more precise estimation of the intrinsic cell Young's modulus (CYM).

A precise characterization of cell elastic properties is crucial for understanding the mechanisms by which cells sense mechanical stimuli and how these factors alter cellular functions. Optical and magnetic tweezers are micromanipulation techniques which are widely used for quantifying the stiffness of adherent cells from their response to an external force applied on a bead partially embedded within the cell cortex. However, the relationships between imposed external force and resulting bead translation or rotation obtained from these experimental techniques only characterize the apparent cell stiffness. Indeed, the value of the estimated apparent cell stiffness integrates the effect of different geometrical parameters, the most important being the bead embedding angle 2γ, bead radius R, and cell height h. In this paper, a three-dimensional finite element analysis was used to compute the cell mechanical response to applied force in tweezer experiments and to explicit the correcting...

Optical tweezers are a powerful tool for micromanipulation and measurement of picoNewton sized forces. However, conventional interfaces present difficulties as the user cannot feel the forces involved. We present an interface to optical tweezers, based around a low-cost commercial force feedback device. The different dynamics of the micro-world make intuitive force feedback a challenge. We propose a coupling method using an existing optical tweezers system and discuss stability and transparency. Our system allows the user to perceive real Brownian motion and viscosity, as well as forces exerted during manipulation of objects by a trapped bead.