Rotation tracking of genome-processing enzymes using DNA origami rotors

Many genome-processing reactions, including transcription, replication and repair, generate DNA rotation. Methods that directly measure DNA rotation, such as rotor bead tracking^1-3, angular optical trapping⁴ and magnetic tweezers⁵, have helped to unravel the action mechanisms of a range of genome-processing enzymes that includes RNA polymerase (RNAP)⁶, gyrase², a viral DNA packaging motor⁷ and DNA recombination enzymes⁸. Despite the potential of rotation measurements to transform our understanding of genome-processing reactions, measuring DNA rotation remains a difficult task. The time resolution of existing methods is insufficient for tracking the rotation induced by many enzymes under physiological conditions, and the measurement throughput is typically low. Here we introduce origami-rotor-based imaging and tracking (ORBIT), a method that uses fluorescently labelled DNA origami rotors to track DNA rotation at the single-molecule level with a time resolution of milliseconds. We used ORBIT to track the DNA rotations that result from unwinding by the RecBCD complex, a helicase that is involved in DNA repair⁹, as well as from transcription by RNAP. We characterized a series of events that occur during RecBCD-induced DNA unwinding-including initiation, processive translocation, pausing and backtracking-and revealed an initiation mechanism that involves reversible ATP-independent DNA unwinding and engagement of the RecB motor. During transcription by RNAP, we directly observed rotational steps that correspond to the unwinding of single base pairs. We envisage that ORBIT will enable studies of a wide range of interactions between proteins and DNA.