Magnetic Torque Tweezers and Their Application in Probing the Torsional Properties of DNA, RNA, and DNA Filaments

Abstract

The double-stranded nature of DNA links processes such as replication, transcription, and repair, to rotational motion and torsional strains. Magnetic tweezers (MT) are a powerful single-molecule technique to apply both forces and torques to individual molecules. However, while the forces applied in conventional MT can be calibrated from thermal fluctuations or computed from first principles [1], direct measurement of the torque is challenging.Here we present the magnetic torque tweezers (MTT), which enable the direct measurement of torque [2] based on a tracking protocol that monitors x, y, z, and angle and on a redesigned magnet configuration. We have applied the MTT to dsDNA, dsRNA, and RecA-DNA heteroduplex filaments. Our measurements of the effective torsional stiffness C of dsDNA indicate a significant force dependence, with C ≈ 40 nm at low forces up to C ≈ 100 nm at high forces, reconciling previous partially conflicting measurements. Torque measurements on RecA-DNA heteroduplex filaments reveal an initial torsional stiffness about two-fold higher than that of DNA. However, at relatively moderate torques further build-up of torsional strain is prevented by partial RecA disassembly and structural transitions in the filament. Preliminary results on the torsional properties of fully dsRNA indicate static properties overall similar to dsDNA, but significantly different dynamics of supercoil formation.Finally, we compare the MTT configuration with recently developed strategies to measure torque using optical tweezers [3] and with a related MT approach that allows straight-forward measurements of free rotation termed freely-orbiting magnetic tweezers [4].[1] Lipfert, Hao & Dekker, Biophys. J. (2009).[2] Lipfert, Kerssemakers, Jager & Dekker, Nature Methods, in press (2010).[3] Forth, et al. Phys. Rev. Lett. (2008).[4] Lipfert, Wiggin, Kerssemakers & Dekker, submitted (2010).