UC San Diego

Twisting, pulling, bending, winding, and unwinding are intrinsic consequences of protein interactions with DNA. These physical movements are critical to genetic processes such as DNA repair, transcription, gene editing, and epigenetic regulation. Single-molecule methods have enabled discoveries of discrete protein-DNA interaction patterns that can be obscured in bulk datasets. One powerful branch of single-molecule methods is fluorescent microscopy-based methods that involve tagging a protein of interest and observing its interactions with DNA over time. However, the fluorescent dyes used to tag molecules of interest can only emit a finite number of photons before photobleaching, limiting observation time. To extend total observation time, researchers must sacrifice other aspects of measurements such as temporal resolution or localization precision. Here, I introduce a dye-cycling labeling strategy for the fluorescence-based Origami rotor-based imaging and tracking (ORBIT) method that enables long-term observation of protein-DNA interactions without sacrificing temporal resolution or localization precision. I apply this dye-cycling ORBIT method to uncover interactions of E. coli RNA polymerase with DNA, including aspects of backstepping. I also explore how the dye-cycling ORBIT method expands the possibilities for investigating diverse protein-DNA dynamics.