Fig. 1.
Structural characterization of d(TGGAA)n using single-molecule FRET. (A) Illustrations of the single-molecule assay used in this experiment. The green and red stars represent the donor (Cy3) and acceptor (Cy5) labeling sites, respectively. (B) EFRET histograms of d(TGGAA)3–6 (colored) and the assay used as a caliper of the end-to-end alignment (cartoon in Bottom). The EFRET histogram of secondary structure-free dT15 is overlaid as a gray histogram for comparison. (C) Representative EFRET time traces for d(TGGAA)3 and d(TGGAA)4. The green and red curves are intensities of the Cy3 and Cy5 fluorescence signals, respectively. The gray curves represent the EFRET (unitless). The vast majority of the traces, including those not shown here, are virtually stable at a single EFRET state. I, intensity; A.U., arbitrary unit. (D) EFRET histograms of d(TGGAA)4 and mutation assays for simulating the hairpin with overhang structures. N denotes the number of molecules used for building the histogram.