Quantitative Guidelines for Force Calibration through Spectral Analysis of Magnetic Tweezers Data

Single-molecule techniques are powerful tools that can be used to study the kinetics and mechanics of a variety of enzymes and their complexes. Force spectroscopy, for example, can be used to control the force applied to a single molecule and thereby facilitate the investigation of real-time nucleic acid-protein interactions. In magnetic tweezers, which offer straightforward control and compatibility with fluorescence measurements or parallel tracking modes, force-measurement typically relies on the analysis of positional fluctuations through video microscopy. Significant errors in force estimates, however, may arise from incorrect spectral analysis of the Brownian motion in the magnetic tweezers. Here we investigated physical and analytical optimization procedures that can be used to improve the range over which forces can be reliably measured. To systematically probe the limitations of magnetic tweezers spectral analysis, we have developed a magnetic tweezers simulator, whose outcome was validated with experimental data. Using this simulator, we evaluate methods to correctly perform force experiments and provide guidelines for correct force calibration under configurations that can be encountered in typical magnetic tweezers experiments

Gold rotor bead tracking for high-speed measurements of DNA twist, torque and extension

Simultaneous measurements of DNA twist and extension have been used to measure physical properties of the double helix and to characterize structural dynamics and mechanochemistry in nucleoprotein complexes. However, the spatiotemporal resolution of twist measurements has been limited by the use of angular probes with large rotational drags, preventing the detection of short-lived intermediates or small angular steps. Here we introduce AuRBT, demonstrating a >100X improvement in time resolution over previous techniques. AuRBT employs gold nanoparticles as bright low-drag rotational and extensional probes, relying on instrumentation that combines magnetic tweezers with objective-side evanescent darkfield microscopy. In an initial application to molecular motor mechanism, we have examined the high-speed structural dynamics of DNA gyrase, revealing an unanticipated transient intermediate. AuRBT also enables direct measurements of DNA torque with >50X shorter integration times than previous techniques; here we demonstrate high-resolution torque spectroscopy by mapping the conformational landscape of a Z-forming DNA sequence