and Z.B. The bench FTY reproduces the real joint behavior, testing the tool in the same real condition of use. Shelby Township, MI 48315 - USA. Our fit values for ΔG0 and J are close to bulk measurements for the B-Z transition in GC repeats (Table 1). These systems were limited to frequency responses around the 1000 Hz in addition to being cumbersome and difficult to set their hoop antennas. 1D and Table S1). Red and yellow lines show transition model fits. The high-resolution torque measurements techniques we have established may be adapted to mechanochemical studies of DNA-associated enzymes and are broadly applicable to mechanical investigations of multistranded biopolymers. 2), consistent with bulk kinetics: at a constant supercoiling density of σ = -0.05, the half time for the B-Z transition of d(GC)16 was measured to be approximately 20 min (38). Z-DNA formation can have specific effects on both the initiation (36) and elongation (37) phases of transcription, and Z-forming sequences may also act as efficient buffers to absorb changes in linking number without changing torque, avoiding perturbations of biological processes. Unlike angular optical trap measurements (23), the torque measurements reported here do not include simultaneous high-resolution extension measurements, which may be incorporated in future improvements to the assays. (A) Experimental setup for dynamic torque measurements. (A) Templates containing 50 bp (yellow, Z50) or 22 bp (green, Z22) GC repeat SOIs were assayed in PBS under 1.6 pN. The force applied to the end of the lever arm is measured using a force transducer. The disadvantage of its correct solution by experimental methods is substantially limited by the need … technique. Strand separation is required for the initiation of both transcription and replication, and it is favored by negative superhelical stress (1). The difference in compliance between B- and Z-states is also smaller than seen at lower ionic strength. Using a U2B force tran… Inhibiting a signaling pathway protects microgravity-exposed mice from losing muscle and bone mass, a study finds. As it was inherently limited by lower speeds and plagued by brush-ring wear, frequent maintenance was required. Measuring the power of the electric motor in the drive train is the indirect method of torque measurement. The torque measurement system can be implemented as an independent system or be a constituent part of the measurement system based on the digital modules of ZETSENSOR series. Some of these inherent limitations were; antennas were limited to a few select sizes, cables from the signal conditioning receiver to data acquisition systems were also limited to certain lengths, proximity to steel structures affect readings accuracy, and emitted emi (electro-magnetic interference) were sometimes more than allowed by government. Copyright © 2021 National Academy of Sciences. L-DNA has been proposed to be partially or entirely composed of strand-separated DNA, and thermodynamic models of supercoiling-induced strand separation depend on knowledge of the mechanical properties of strand-separated DNA. 1) and also display reduced hysteresis (Fig. Localized sequence-dependent structural transitions are important for the regulation of biological processes such as DNA replication and transcription. Unless stated otherwise, all such curves are obtained from four independent twist clamp experiments, and unwinding curves are superimposable with rewinding. Neither assay relies on a micron-sized rotational probe (12, 21, 22), allowing torque resolutions sufficient for routine sequence-dependent torque spectroscopy. Manufacturers making use of these developments in digital telemetry introduced several look-alikes that were almost identical in form and in inherent limitations as well. We measure Δθ0 = -1.11 ± 0.02 rad/bp, indistinguishable from previously measured values of -1.12 rad/bp, and the coexistence plateau occurs at a very small negative torque of only -0.4 pNnm (Fig. Extrapolating from this region, we obtain a difference in helicity between B-DNA and L-DNA at zero torque, Δθ0,L = 1 rad/bp. Campaign information and peer influence give rise to partisan echo chambers. We have measured torque-twist curves for constructs containing 100 bp (Bub100) or 50 bp (Bub50) of mismatched DNA. For any torque measurement method, resolution is ultimately limited by the Brownian torque noise (4kBTγrB)1/2, where γr is the rotational drag coefficient of the torque probe and B is the bandwidth of the measurement (22). The sequence-dependent torsional response of paired and mispaired duplexes is also of direct relevance to understanding and designing the mechanical behavior of DNA nanostructures. We fit the linear region of L-DNA elasticity to obtain the torsional rigidity of L-DNA, which is 10–15 times softer than B-DNA (Fig. load from the fuel rack. 2 A and B and SI Text) to verify our physical understanding and extract structural, mechanical, and thermodynamic parameters for alternative structural forms. Measuring torque while installing a bolt prevents the overtightening of bolts. The importance of this problem is increased by an order in terms of unsteady loads (random, impulsive, alternating, etc.). S4). Our theory and methods establish a framework for understanding the torsional response of arbitrary sequences, which ultimately should be predictable from reductionist measurements on simple sequences such as those described here. Required accuracy 5. Comparison between "damp-like" torque and "field-like" torque. Saturation of the underwinding transition leads to a net left-handed twist (14⇓–16), which may reflect an average property of multiple contributing states. GACT bubbles seem to adopt a B-DNA-like structure (Fig. Complete transformation of kilobase-scale DNA segments is unlikely to occur in biology, but knowledge of the overall helicity and rigidity of the resulting “L-DNA” places important constraints on any detailed model for sequence-dependent responses to negative supercoiling. While untainted by slip ring technology shortcomings, they had some of their own. Image credit: Shutterstock/Sketchy Bytes. We have further discovered ionic strength effects (Fig. In a plot of torque as a function of twist (Fig. In single stretched DNA molecules, B-DNA ceases to display linear torsional elasticity at a critical torque of approximately -10 pNnm, entering an apparent cooperative structural transition to an underwound form (11, 12). 1D) is substantially larger than previous estimates of a few bp for strand-separated DNA (27, 28). 3A), notably yielding a lower value for the boundary penalty J than is generally obtained for denaturation of matched duplexes (2, 27, 32), and lower than we obtained for B-Z transitions (Table 1 and Table S2). DNA nanotechnology has already benefited from mechanical models parameterized using previous single molecule measurements (47). In an additional tradeoff with competing methods, our assays based on rotor bead tracking (11) require specialized molecular constructs with three distinct attachment chemistries. FCC had to stop one such model and fine its manufacturer. The use of homogeneous simple repeat sequences facilitates meaningful comparisons of experiment with theory and allows us to extract thermodynamic and structural parameters for sequence-dependent transitions. The secret is now out. Torque is determined indirectly through measurement of certain auxiliaryquantities in the drive train. Averaged traces are each based on three independent measurements. All methods employ at least one microparticle as a handle for applying pN-scale tensions to the DNA using optical or magnetic forces. Torque Measurement Techniques – A Timeline, Real-Time Wireless Conveyor Diagnostic System, Wireless Coupled Flange Torque Transducer. Torque-twist plots of Z50 and Z22 collected at 1.6 pN of tension show clear transitions to a second state upon application of negative torque (Fig. In contrast to dynamic torque measurements based on the angular velocity of the rotor, this second approach uses a static experimental setup in which torque is quantified by measuring the SOI against a calibrated transducer DNA segment. The torque magnetometry technique has been successfully used to detect anisotropy changes in strongly correlated magnetic systems and resolve quantum oscillations for mapping out … Mechanical perturbations can be used to obtain detailed sequence-dependent structural, thermodynamic, and kinetic properties of nucleic acids (17). (B) Torque-twist diagram for 4.6-kb control DNA tethers. (B) Response of 100 bp (dark green, Bub100) and 50 bp (light green, Bub50) d(pGpApCpT)n•d(pGpApCpT)n SOIs assayed in PBS under 10 pN of tension. This article addresses the many techniques and tradeoff of torque measurement techniques. Torque basics, Hooke's law Torque is force around a point or axis — a twisting force — and is calculated by multiplying an applied force by the distance through which the force works. Junctions may also contribute additional compliance to the Z-state, explaining unexpected posttransition softening for Z50 (Fig. Data analysis, models for fitting and statistics are described in SI Text. Plumbing a variety of historical data could offer important insights into trends in insect declines. Our measurements of mechanics and thermodynamics of short homogeneous sequences have begun to describe the sequence-dependent folding landscape for superhelical double-stranded DNA. We do not capture any email address. Fits to a cooperative transition model (see SI Text) are displayed (inset) along with the corresponding experimental data (violet, Z50; red, Z22). Although it offers many advantages, the technique had fallen out of fashion but is now making a comeback due to new developments in inductive angle sensors. Torque is described as a force producing rotation about an axis. These measurements are all consistent with a previous proposal (11, 16) in which L-DNA consists of a mixture of strand-separated DNA and noncanonical structures such as Z-DNA. The base pair geometry of GACT mismatch bubbles, in which pyrimidine bases oppose purines, may help conserve a Watson–Crick-like structure with a reduced number of hydrogen bonds in comparison with complementary B-DNA. The dynamic implementation relies on the viscous drag of the rotor bead and allows a direct torque measurement with similar performance to assays employing an angular optical trap and nanofabricated quartz cylinders (23, 24): Using approximately 400 nm rotor beads, we measure torque to within approximately 1 pNnm by integrating over 3 s of data. The rotor bead tracking assay was the first of a number of methods introduced over the past decade (11, 12, 21 ⇓ ⇓ –24) for measuring torque on single stretched DNA molecules. Calculation can be done along with measurement of rotational speed. Parts may not be assembled securely enough for the unit to function properly or threads may be stripped because the torque was too high, causing the unit to fail. Duplexes with extended mismatch regions are intermediates in homologous DNA recombination (39, 40) and other hybridization-driven processes. This guide will help you develop your own torque measurement program. However, the plateau and overshoot motifs are seen in both the unwinding and rewinding curves. This latter effect of salt is in the opposite direction from the expected result if the B-L transition consisted solely of strand separation. L-DNA has a torsional persistence length significantly greater than either our estimates or previous estimates for strand-separated DNA, is stabilized rather than destabilized by high ionic strength, and is confirmed to be highly helical. Accurate quantitative models of sequence-dependent structural transitions are needed to explain sophisticated cellular functions such as supercoiling-dependent regulation of gene expression profiles (5, 6). The torque measuring device comprises or consists of the complete set of measuring instr ents carry out torque measurem ll components of the torque measuring device (including cables for electrical connection) shall individually and uniquely identified (for example by the manufacturer’s name, the type, four or six conductor circuit or similar, and the serial number). 1 B and C). In this model, each basepair within the SOI can exist in one of two states with differences in free energy (ΔG0) and twist (Δθ0) as well as a change in compliance associated with the transition, accounting for differing twist persistence lengths. The example vehicle is a four wheel drive (4WD) agricultural tractor - but it is proposed that the same measurement techniques … (D) The torsional response of the DNA handles has been subtracted from averaged rewinding curves in order to show the inferred response of the SOI alone, plotted as torque vs. twist/bp. Our measurements of the helicity and torsional rigidity of L-DNA are in good agreement with another study (46) that appeared while our manuscript was under review. 3C). Compromises between the conflicting requirements of torque probes and force handles have been a factor in the poor torque resolution of some previous methods, necessitating hours or even days (22) of noise-averaging data collection to determine torque-twist relationships. Hybrid destabilization due to individual basepair mismatches has been extensively characterized (34), but little is known about the structure of long mismatched stretches or the cooperativity of mismatched strand–strand interactions. L-DNA thus has the external properties of a left-handed structure, with an average extrapolated unstressed helical pitch of approximately 15 bp. The end results are measurement sensors and transducers you can trust, and we can be proud of. (A) Fits are shown for Z50, Z22, Bub100, and T100, demonstrating the range of behaviors explained by the model described in SI Text. From model fits, the change in free energy per base pair ΔG0 for disrupting GACT mismatches is approximately 0.55 kcal/mol, comparable to previous measurements based on van’t Hoff analysis of single base pair mismatches in oligonucleotides (34). To mitigate the effect of B/Z junctions, we created new constructs incorporating 9-atom-long spacers within the phosphate backbone at all 3′ and 5′ ends of the Z22 GC tract (Fig. By the late 80’s microprocessor and surface mount technologies has already made tremendous strides leading to the miniaturization of circuitries and the development of several families of micro-chips. The main selection criteria are: 1. (D) L-DNA helicity and elasticity vary with tension. Z-DNA formation is cooperative (9, 10, 30), which may be explained by a high nucleation penalty due to the formation of unfavorable conformations at junctions between Z-DNA and B-DNA (10, 30, 31). Torsional responses of mismatch bubbles. 4B). Individual unwinding curves (fine lines) were variable and differed from the consistent behavior of rewinding curves (averaged, bold lines). Modern measurement and test gear makes it straightforward to establish rotational speed and electric power in electrical machines. This method allows the torque sensor to be placed as close as possible to the torque of interest and avoid possible errors in the … analyzed data; and F.C.O. Ensure that torque is set between every measurement in the settable torque wrench to simulate actual usage between repeated readings. For long random sequences, the single “B-DNA to L-DNA” plateau at -10 pNnm may obscure a complex underlying response, in which specific sequences preferentially undergo differing structural transitions. Let us do the same for you. Torque measurements reveal sequence-specific cooperative transitions in supercoiled DNA. The thermodynamic parameters of Z-DNA formation calculated from our measurements are in good agreement with previous measurements based on gel electrophoresis (10) (Table 1). For the static implementation (Fig. This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Base-base mismatches. 2 A and B). A critical review, Crystal structure of a junction between B-DNA and Z-DNA reveals two extruded bases, Determination of DNA cooperativity factor, Synthetic DNA replication bubbles bound and unwound with twofold symmetry by a simian virus 40 T-antigen double hexamer. 2 A and B) and explain the torsional response of GC tracts: As negative twists are added to the DNA, negative torque initially accumulates linearly until a nucleus of Z-DNA is formed, upon which torque is partially relieved and the molecule enters a B-Z coexistence torque plateau, followed by a further linear accumulation of torque once the entire GC tract has been converted to Z-DNA (Fig. Freely available online through the PNAS open access option. For B-Z transitions under moderate or high salt conditions, we observe hysteretic effects reflecting kinetic barriers to Z-DNA formation (Fig. From the automotive to medical, aerospace, defense, and the pharmaceutical industries. A possible explanation for this discrepancy is the contribution of helical distortions at B/Z junctions, which are believed to be formed within the GC-repeat region and have been estimated to occupy approximately 3 bp (30). The noise floor of a typical balanced bridge resistance measurement is less than 5mΩ/500Ω. 2 and Table S2). Finally, our analysis enables compliance measurements for soft high-energy states such as strand-separated DNA, providing an important (2, 27) but often poorly quantified parameter needed for predicting mechanical transitions. On dual fuel engines, running on gas mode, and common rail engines. In addition to quantifying base pair stabilities, measurements of torque as a function of imposed twist allow sensitive determination of the cooperativity parameter J that arises from junctions between differing states (Fig. SensorData Torque sensors are designed with attention to the ever demanding testing requirements of today. Piezoelectric reaction torque sensors offer convincing features such as exceptional overload … High salt: 1.8 M NaCl, 0.5 mg/mL BSA, 50 mM Tris, pH 7.4. Image credit: José Francisco Salgado (artist). (A) Torque-twist curve for d(pT)100•d(pT)100 SOI (T100) and negative control DNA (gray tones) in PBS, together with fits to the cooperative transition model (light blue and green lines). 3A). 1 C and D). Online ISSN 1091-6490. The coexistence plateau occurs at τZ = -3 pNnm in PBS. Figure 2. At 1.6 pN, the twist persistence length of this state is approximately 7 bp (Fig. Hydrogen bonds within dT•dT base pairs have been observed in single dT•dT mismatches with NMR spectroscopy (41) and predicted based on a X-ray crystal structure of two stemloop DNAs (42). The research presented in this paper focuses on the use of novel, non-contact torque sensors to measure the driveline torque distribution. This transition has been proposed to represent strand separation (13), but it has been noted that other noncanonical structures such as Z-DNA may also be simultaneously populated in random sequences under negative torques (14, 15). NOTE: We only request your email address so that the person you are recommending the page to knows that you wanted them to see it, and that it is not junk mail. Under high salt conditions (Lower) the B-Z coexistence plateau occurs at lower absolute torques, and unwinding (bold dark red) and rewinding (fine light red) traces show hysteretic behavior. How to Measure Torque On an Existing Shaft. When the molecules are overwound to a critical torque of approximately 8 pNnm, a reversible transition occurs to a second structural state. Measuring torque with angle sensors. Traceability techniques. All of the transitions we have investigated are well described by a single statistical mechanical model for cooperative structural transitions in polymers. B-Z transitions for Z50 show a negative torque overshoot followed by a plateau. 1 C and D). 50207 Hayes Road To investigate Z-DNA formation, we used SOIs containing either a 50 bp or a 22 bp GC repeat. How to Use Torque Analyzers and Sensors. 12 December 2017. Once a nucleus of Z-DNA has been formed in a GC tract, it may be extended readily due to the small free energy difference between B-form and Z-form for d(pGpC). Torque measurement system can also be used for the control of rotary oscillations attributed to changes in the dynamics of torque value in time domain and to the fluctuations in the resistance level. PBS: 137 mM NaCl, 2.7 mM KCl, 0.5 mg/mL BSA, 10 mM Na2HPO4, 2 mM KH2PO4, pH 7.4. Low salt: 0.5 mg/mL BSA, 10 mM Tris, pH 7.4. These measurements suggest a low-stability B-DNA-like structure for the mismatch inserts. Cost. We fit the measured torque-twist relationships to a model for cooperative structural transitions in polymers (Fig. The angular position ϕ of the MB is controlled by rotating the magnets, and the angle ψ of the RB is measured using videomicroscopy. Our direct measurements of B-Z transitions underscore the ease with which high-propensity sequences can form Z-DNA in preference to strand separation, and we have measured properties of L-DNA that are difficult to reconcile with a model in which L-DNA consists of strand-separated DNA alone. In three divergent examples, we found that the behaviors of short regions of homogenous sequences were well described by a model for cooperative structural transitions in polymers, allowing the extraction of thermodynamic parameters relevant to sequence-dependent torsional responses (Fig. For the shorter Z22 construct, the plateau is absent and the torque-twist diagram resembles the two-state behavior seen in RNA hairpin unzipping experiments (17). Measuring torque is a very important aspect of the electric machine industry. The newest kid on the block was the digital telemetry system. 1A). In biological contexts, trans-acting factors can bias the equilibrium between available structural transitions, and it should be emphasized that stress-induced destabilization of the duplex (1) is important even when spontaneous strand separation is absent: Linking number deficits may be absorbed by stabilizing a bubble upon protein binding (44) or invasion of a homologous strand to form a D loop (45), without ever forming an open bubble as a stable intermediate in this transaction. A signal processing module tethered to the rotating sensor by no more than 0.25” also acted as the radio receiver. A dramatic change in behavior is observed for B-Z transitions under high salt conditions of 1.8 M NaCl. The wrong torque can result in the assembly failing due to a number of problems. In contrast to poly(dT) bubbles, torsionally relaxed GACT mismatch bubbles are not detectably softer than B-DNA. By saturating the transition, the extrapolated change in helicity per base pair Δθ0,L can be obtained. Characteristic curves show a plateau at a critical torque τcrit, a posttransition torsional spring constant κpost, and a change in helicity Δθ0 for the transition. ; by a Morgridge Family Stanford Graduate Fellowship to L.E.F. 2B). S1 B and C and Table S1), including a decrease in the critical torque for the B-L transition by approximately 15% at high ionic strength (Fig. At low negative torques, Bub50 and Bub100 exhibit a reversible plateau corresponding to a structural transition (Fig. Our methods based on the rotor bead tracking assay (11) avoid compromises by segregating the force handle from the torque probe. The rotor bead tracking assay (11) was the first of a number of methods introduced over the past decade (11, 12, 21⇓⇓–24) for measuring torque on single stretched DNA molecules. To investigate these properties, we tested the torsional response of DNA mismatch bubbles. DNA molecules, beads, and flow cells were prepared using similar procedures to previous work (11). Understanding the type of torque to be measured, as well as the different types of torque sensors that are available, will have a profound impact on the accuracy of the resulting data, as well as the cost of the measurement. Static vs. Although Yokogawa analyzers can interface with most speed and torque sensors, it's still wise to confirm compatibility in each case. Bulk supercoiling and single molecule twisting experiments have been used to characterize the response of DNA to negative superhelical stress (4, 8, 9, 11). Negative control DNA (black line) under identical conditions serves as a reference. Ensure the number of repeated reading and replicated readings are the same for all appraisers. Measure the residual torque by the breakaway method implies a possible over tightening of the joint under test (even if, in general, the residual torque is less than the tightening torque, so it is likely that the test of residual torque does not exceed the nominal value). Detailed methods are described in SI Text. Extended mismatches have been assumed to function as open bubbles in helicase loading experiments (33). The constant-torque plateau (τL) at approximately -10 pNnm reflects the structural transition from B-form to L-form (Fig. This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1113532109/-/DCSupplemental. We have also introduced an experimental strategy that rigorously challenges transition theory by changing the boundary conditions: Atomic spacers can be introduced at the edge of an SOI to create a boundary that thermodynamically approximates a free end but still permits the application of a torsional constraint. Comparison of unwinding (fine lines) with averaged rewinding curves (bold lines) shows reduced hysteresis in Z22sp. The twist persistence length of L-DNA Pt,eff = 20–30 bp (Fig. To directly probe the effect of sequence on structural transitions driven by torque, we have measured the torsional response of a panel of DNA sequences using single molecule assays that employ nanosphere rotational probes to achieve high torque resolution. The methods used to measure torque can be further divided into two more categories, either reaction or in-line. Here, we use our torque-measurement techniques to investi-gate specific states that can contribute to localized sequence-dependent responses to negative supercoiling. The task of measuring, monitoring and recording of the torque, mechanical stress and capacity is relevant for most transmissions of the process and transport machines. Torque wrenches are the most commonly used measures for this torque. These shortcomings opened the door for the development of rotary transformers. The rotor will spin to relieve this twist, but a fixed twist condition can be imposed by rotating the magnets to counteract this relaxation, using a “twist clamp” feedback algorithm. A specific “sequence of interest” (SOI, red) is placed between the MB and the RB attachment of the DNA molecule. Torque wrenches must be tested on a routine basis using torque measurement devices, including torque analyzers and sensors. To measure the sequence-averaged mechanical properties of structures that form at the critical torque, the DNA molecule can be unwound through the entire torque plateau, so that no B-DNA remains (Fig. In addition to strand separation, superhelical tension may also be released by the formation of Z-DNA, a left-handed double-helical structure (7, 8). Twist was ramped at 0.05 turns/s, torque was obtained from rotor angular velocities averaged over a 3-s window, and data were subsequently binned along the twist axis and averaged (red line). (B) Visualization of the effect of cooperativity in our model, shown by a series of calculated torque-twist curves, using all the parameters from Z50 except for the domain wall penalty J, which was varied from 0.5 to 12 kcal/mol. Modern strain gage technology allows for sensors to be smaller, stiffer and faster than ever before, the very attributes SensorData capitalizes on. 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