Nucleic Acids Research, 2012, Vol. 40, No. 13 6203 Figure 2. sm-FRET recordings of hairpin substrates in the presence of eIF4AI. (A) Schematic diagram illustrating DNA/RNA hairpin sub- strate harboring a 12-nt loop and 12 nt duplex region, labeled with a FRET pair (Cy3–Cy5). The substrate is immobilized to a glass surface via the 30-end biotin. (B) sm-FRET of the immobilized RNA/DNA hairpin in the presence of eIF4AI, displaying episodes consisting of decreased FRET signals followed by return to original values. (C) FRET distribution from individual sm traces (N = 38 out of 1000) showing two distinct peaks at 0.9 and 0.6. Figure 3. sm-FRET recordings of hairpin substrates in the presence of eIF4AI/eIF4H. (A) A representative sm time trace of an RNA/DNA hairpin structure (Schematic Illustration in Figure 2A). The hairpin molecule is immobilized to the cover-slip surface using a biotin moiety. eIF4AI/eIF4H are introduced at a concentration of 200 nM and ATP concentration is 0.1 mM. Donor (ID) and acceptor (IA) intensities (green and red, respectively) are measured and the corres- ponding FRET trace is calculated by: IA=ðIA+IDÞ (blue). (B) Histogram of FRET levels obtained from N > 2000 individual molecules under the same conditions as in A, showing two prominent peaks at 0.9 and 0.4. The oscillating FRET behavior is observed in over 50% of the sm events and is ATP dependent. (C) Dwell times of the low- and high-FRET states are used to construct the time histograms of the unwinding time and waiting time intervals (blue and green, respect- ively). Single exponential models are used to ﬁt the data (black lines), yielding the characteristic timescales U and W.