Measuring Single-Molecule Twist and Torque in Multiplexed Magnetic Tweezers.

Abstract

Magnetic tweezers permit application of precisely calibrated stretching forces to nucleic acid molecules tethered between a surface and superparamagnetic beads. In addition, magnetic tweezers can control the tethers' twist. Here, we focus on recent extensions of the technique that expand the capabilities of conventional magnetic tweezers by enabling direct measurements of single-molecule torque and twist. Magnetic torque tweezers (MTT) still control the DNA or RNA tether's twist, but directly measure molecular torque by monitoring changes in the equilibrium rotation angle upon overwinding and underwinding of the tether. In freely orbiting magnetic tweezers (FOMT), one end of the tether is allowed to rotate freely, while still applying stretching forces and monitoring rotation angle. Both MTT and FOMT have provided unique insights into the mechanical properties, structural transitions, and interactions of DNA and RNA. Here, we provide step-by-step protocols to carry out FOMT and MTT measurements. In particular, we focus on multiplexed measurements, i.e., measurements that record data for multiple nucleic acid tethers at the same time, to improve statistics and to facilitate the observation of rare events.