Dye-cycling DNA origami rotors for long-term tracking of transcription at base-pair resolution

Protein-DNA interactions often result in rotational movements. These movements underlie many genetic processes such as DNA transcription by RNA polymerases (RNAPs). To illuminate these movements, we previously developed origami-rotor-based imaging and tracking (ORBIT), a fluorescence-based method that enables high-speed tracking of DNA rotation. When used to track DNA rotation during transcription by E. coli RNAP, ORBIT enabled the detection of single base-pair steps. However, the duration of ORBIT experiments was limited due to fluorescence photobleaching. To overcome this limitation and extend the total possible observation time, we here introduce dye-cycling ORBIT (dcORBIT), a method that enables observation of protein-DNA interactions for over 10 min at 20 Hz while maintaining single-base-pair precision. dcORBIT thereby opens new possibilities to study the fundamental rotational movements underlying gene expression.