CueR-specific Holliday junction, HJC2. (A) Design of HJC2 with the dyad-symmetric sequence in arms M and N, Cy3 and Cy5 labels on strands a and d, and BiotinTEG on strand c. (Inset) Absorption spectrum of the Cy3- and Cy5-labeled HJC2. (B) Exemplary single-molecule fluorescence (top) and EFRET (bottom) trajectories of HJC2, showing two-state fluctuations. EFRET is approximated as ICy5/(ICy3 + ICy5) (I: fluorescence intensity). The values τI and τII are the waiting times on the EFRET states of conf-I and conf-II, respectively. Note that a.u. = arbitrary units.