Fig. 6.

Single-molecule microscopy to visualize Apo3G scanning on ssDNA. a Total internal reflectance fluorescence (TIRF) microscope set up for analyzing Apo3G motion along ssDNA. ssDNA (92 nt) is annealed to a Cy3-labeled (D donor), surface immobilized anchor DNA. Binding and scanning of Cy5-labeled (A acceptor) Apo3G results in FRET changes. b, c The representative smFRET Scanning Trajectories for poly dT substrate with a 5′ hot CCC motif (pdT 5′ hot) or poly dT without C (pdT) show FRET traces (black) with a hidden Markov Model fit (red). Apo3G motion toward 5′- and 3′-directions is observed as increases and decreases in FRET, respectively. The transition density plot (TDP) showing the relative frequency of FRET transitions observed between initial and final FRET states (low frequency = blue, high frequency = yellow). Movements toward the 5′- and 3′-ends appear as peaks above and below the diagonal, respectively. The transitions are symmetric in both directions, indicating that Apo3G scans ssDNA without directional preference. The presence of bright yellow spots in the TDP plot for the pdT 5′ hot motif, but not for pdT, arise from a large excess of FRET transitions caused by quasi-localized scanning, i.e., “hovering”, of Apo3G in the vicinity of the 5′ hot motif [176]