Rotation tracking of genome-processing enzymes using DNA origami rotors

Abstract

Many genome-processing reactions, such as transcription, replication and repair, generate DNA rotation. Methods that directly measure DNA rotation, including rotor bead tracking 1-3 , angular optical trap 4 , and magnetic tweezers 5 have helped unravel the action mechanisms of a range of genome-processing enzymes, such as RNA polymerase (RNAP) 6 , gyrase 2 , viral DNA packaging motor 7 , and DNA recombination enzymes 8 . However, 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 to track 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 labeled DNA origami rotors to track DNA rotation at the single-molecule level with millisecond time resolution. We used ORBIT to track DNA rotation resulted from unwinding by RecBCD, a helicase involved in DNA repair 9 , and transcription by RNAP. We characterized a series of events 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 corresponding to single-base-pair unwinding. We envision ORBIT will enable studies of a wide range of protein-DNA interactions.

Intermediate states of molecular self-assembly from liquid-cell electron microscopy

“…Tentatively, we attribute in-plane rotations and enhanced translational mobility during hybridization to release of torsion energy (24). Model systems show that when the DNA helix is perturbed, overwound, or underwound, this generates torque (38), and the same is reasonable to expect as strands progressively twist failed encounter times for the three base-pair sequences studied.…”

“…A conundrum for single-molecule imaging-it has been called "molecular individuality" regarding single-molecule fluorescence imaging (22)(23)(24)-is kinetic heterogeneity. Microenvironments of individual molecules can differ significantly; they do not average out as would be the case for larger, colloidal-sized particles (25).…”

DNA-Scaffolded Proximity Assembly and Confinement of Multienzyme Reactions

“…As shown in Fig. 13c, two research groups, namely those of Zhuang and coworkers and Yin and coworkers, collaborated to develop a DNA origami rotor that was driven by the unwinding function of a helicase (RecBCD complex) during the transcription process [131]. These authors introduced a method of origami-rotor-based imaging and tracking (ORBIT) to track DNA rotation at the single-molecule level with a time resolution of milliseconds.…”

“…c DNA origami rotor driven by a motor protein (left), atomic force microscopy images of a DNA rotor (middle) and origami-rotor-based imaging and tracking (ORBIT) for tracking DNA rotation (right). Reproduced from Kosuri et al [131], with permission, copyright 2019, Springer Nature Tinnefeld and coworkers were the first to report a multi-fluorophore array on a DNA origami tile; these researchers controlled the spacing distance and the position of each fluorophore to direct the energy transfer [133]. Yan and coworkers constructed an artificial light-harvesting antenna by assembling multiple donor/acceptor pairs on a seven-helix DNA bundle (Fig.…”

Adenita: Interactive 3D modeling and visualization of DNA Nanostructures

“…With the aforementioned applications, the field of DNA nanotechnology advances rapidly and the involved DNA nanostructures are ever increasing in size and complexity [16]. With the recent developments in hybrid DNA-protein systems [17,18], the need for sophisticated modeling and visualization tools becomes apparent. We aim to facilitate the combination of DNA nanostructures with other molecules, such as aptamers, proteins or nanoparticles, in a feasible manner that does not require a large pipeline of tools or the inspection of nanostructures at the atomic scale.…”