6202 Nucleic Acids Research, 2012, Vol. 40, No. 13 Figure 1. Bulk FRET assay displays an eIF4AI-dependent decrease in the steady-state FRET level. (A) A schematic diagram showing the substrate molecules for bulk FRET assay. An 18 base pair RNA/DNA duplex labeled with a FRET pair introduced at a very low concentration (1 nM) is unwound only in the presence of ATP and eIF4AI, reaching a lower steady-state FRET level. (B) Results from bulk FRET assays monitoring eIF4AI helicase activity at the indicated eIF4AI concentrations and 1 mM ATP. We next sought to characterize eIF4AI helicase activity using sm-FRET. Initial attempts to use hybrid duplexes of the type shown in Figure 1A as substrates were found to be ineffective for sm-FRET since following unwinding of the duplexes by eIF4AI, non-biotinylated strands (carrying either the donor or the acceptor dye) diffused away from the coverslip surface resulting in loss of FRET signal. Hence, we developed a series of RNA/DNA hybrid hairpin structures in which we designed loops of various sizes, ranging from 3 to 12 nt long (a hairpin molecule with a 12-nt long loop is shown schematically in Figure 2A). All the hairpin substrate molecules used in this study have G values within the range of 16 to 15 kcal/mol (Supplementary Figure S1), which according to Figure 1 and previous reports (30), should be completely unwound by eIF4AI. The loop structure facilitated multiple observations of repeated unzipping/re-annealing kinetics for the same molecule. Figure 2B displays a sm time-trace of the donor (Cy3 green line, ID) and acceptor (Cy5 red line, IA) intensities and their corresponding FRET value (IA/(ID+IA), blue line) as a function of time. When both ATP (1 mM) and eIF4AI (2 mM) were added, rare but distinct episodes of FRET transitions were observed, characteristically moving from an initial FRET value of 0.90 (correspond- ing to a closed hairpin structure) down to a value of 0.60, then returning to 0.90. Statistical analysis was performed on a set of 1000 individual molecules, all of which ex- hibited stable donor and acceptor emission for a minimum of 2 min. We found that traces containing at least one FRET transition of any magnitude composed only 3.8% of all traces. This subset of events was used to construct a histogram of FRET values that displays two clear peaks at FRET levels of 0.9 and 0.6, correspond- ing to two well-deﬁned FRET states that can be approximated by a double Gaussian ﬁt (black line; Figure 2C). Photophysical effects could be ruled out as the source of the FRET transitions for two reasons: (i) Control experiments showed that in the absence of either ATP or eIF4AI no FRET transitions were observed (Supplementary Figure S3). (ii) It is well estab- lished that ﬂuorophore ‘blinking’ involves complete ‘switching off’ of one of the dyes, rather than a transition to an intermediate FRET level (31,32). We observed that even at low FRET values we still detect substantial photon emissions from both dyes (Figure 2), making it unlikely that these transitions are caused by photophysical effects. Repetitive unwinding is induced in the presence of eIF4H Next, we wished to assess the consequences of eIF4H on the unwinding parameters of eIF4AI, given that previous bulk studies have reported elevated eIF4AI ATPase activity in the presence of eIF4H (20,33), as well as a stimulation of helicase activity (20,34) (Supplementary Figure S4). The addition of 1 mM eIF4H to eIF4AI (at a 1:1 ratio) into the sm assay produced a striking change in the FRET dynamics. The FRET levels repeat- edly and rapidly varied between two discrete values (0.9 and 0.4) for periods lasting several minutes (Figure 3A). Moreover, in sharp contrast to the isolated FRET transi- tions caused by eIF4AI alone (Figure 2B), the oscillating two-level transition pattern for the 12-base hairpin loop accounted for >50% of all sm traces in a population of more than 2000 molecules (Figure 3B). Since the transi- tions in FRET value from 0.9 to 0.4 reﬂect roughly a 3 nm change in donor–acceptor distance (approximately the length of the eIF4AI dumbbell conﬁguration at its widest point) (35), and given that the FRET transitions are ATP-dependent, the results are consistent with previous results that eIF4H, which does not possess inherent helicase activity, stimulates the unwinding activity of eIF4AI. The eIF4AI/eIF4H complex leads to reversible unwinding of structured RNA, while simultan- eously preventing fast reannealing of the RNA template. Each oscillation of the FRET trace between the high and low level represents a single unwinding cycle. A Hidden– Markov Model (HMM) analysis of the observed two-state ﬂuctuations (36) was used to extract the dynamics of this process, characterized by ‘waiting time’ (high FRET level)