Scientists at the Salk Institute have extended the observation window for tracking molecular rotation during gene transcription from seconds to hours by combining a DNA origami rotor with a dye-cycling strategy that continuously replenishes fluorescent probes. The method, called dye-cycling ORBIT, was published in Cell Reports Methods and builds on an earlier technique that used a self-assembling DNA nanostructure to amplify the tiny rotational movements of RNA polymerase as it reads DNA.
RNA polymerase must rotate along DNA's helical structure to transcribe genetic information into RNA, but the rotation's diameter is roughly 100 times smaller than the wavelength of visible light, making direct observation impossible with standard fluorescence microscopy. The ORBIT rotor attaches a corkscrew-shaped stem to the polymerase and a large, fluorescent X-shaped handle that magnifies the rotation into a detectable signal, achieving single base-pair resolution.
A persistent limitation of fluorescence-based tracking is photobleaching, where fluorescent tags permanently lose brightness after emitting light, typically restricting measurements to a few seconds. The dye-cycling innovation solves this by washing in fresh fluorescent probes during the experiment, effectively refueling the rotor mid-flight and sustaining the signal.
In the reported experiments, dye-cycling ORBIT maintained tracking for 10 minutes, and the lab has since recorded continuous rotation data for hours. This duration covers thousands of base pairs and multiple transcriptional events, providing a mechanical view of gene expression that was previously inaccessible.
The DNA origami structures self-assemble from designed strands, enabling near-atomic precision without top-down nanofabrication. Researchers emphasize that the approach is rapid to iterate, inexpensive, and biodegradable, making it broadly adaptable for studying other DNA-interacting proteins whose rotational mechanics remain unknown.
Senior author Pallav Kosuri describes the mechanical side of molecular biology as a "Wild West" compared to the well-cataloged chemical reactions, and argues that visualizing physical movements is essential for understanding, manipulating, and altering molecular function. The method opens the door to systematic study of the rotational dynamics underlying transcription, replication, and repair.
The work was led by first author Amanda Wacker, who completed her Ph.D. in Kosuri's lab, and supported by the Salk Institute. The peer-reviewed publication includes a graphical abstract and detailed methods, and the DOI is 10.1016/j.crmeth.2026.101550.
Tiny DNA rotor lets microscopes track gene transcription one base pair at a time
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