FIGURE 1. Single-molecule FRET assay for probing the orientational dynamics of RT.

a, The structure of HIV-1 RT bound to a DNA–DNA substrate²¹. Labelling sites for Cy3 on RT are highlighted by green stars. b, Nucleic-acid substrates consisted of a 19–21-nt primer strand annealed to a 50-nt template strand containing a Cy5 label (red star). Cy5 was either 3 nt from the 5' end (circle) or 4–6 nt from the 3' end (arrow) of the primer. c, Single-molecule detection of Cy3 (green star or sphere)-labelled RT binding to and dissociating from the surface-immobilized nucleic-acid substrates labelled with Cy5 (red star or sphere). The stars and spheres indicate dyes that do and do not emit fluorescence, respectively. d, FRET analysis for RT binding to a single primer–template complex. Top: fluorescence time traces from Cy3 (green) and Cy5 (red) under excitation at 532 nm and that from Cy5 (pink) under excitation at 635 nm. Middle: FRET value calculated over the duration of the binding events (yellow shaded regions). Bottom: FRET distribution histogram created for the binding events.