Population distributions of FRET efficiencies for Cy3–Cy5-labeled IRES junctions studied as single molecules. Except for the different fluorophores, equivalent junction species as used in the steady-state fluorescence experiments (Fig. 4) were studied. In addition, a Cy3–Cy5-labeled duplex was prepared by hybridization of the junction e strand to its complement, to provide a model for coaxially stacked C and E arms. The different species were encapsulated in phospholipid vesicles in 10 mM Tris-HCl (pH 8.1), 50 mM NaCl, 10 mM MgCl2, and imaging buffer (see Materials and Methods) and studied by total internal reflection fluorescence microscopy. Hundreds of single molecules were studied for each species, and fluorescent intensities at Cy3 and Cy5 emission wavelengths recorded for a number of minutes with a 100-msec resolution. Molecules with aberrant spectral properties were rejected and FRET efficiencies calculated from the remainder. These were plotted as the histograms shown, fitted to Gaussian distributions. Histograms are shown for the CD junction, the CE junction, and the CE duplex. The measured mean EFRET and half-widths for each distribution are presented in Table 1.
