confocal setup as previously described (27). Cy3 was excited by a 514.5-nm line of Ar+ laser (Spectra-Physics, Santa Clara, CA, USA). Emissions from donor and acceptor channel were discriminated using dichroic mirrors (Chroma Technology, Bellows Falls, VT, USA) and measured by two avalanche photodiodes (Perkin-Elmer, Wellesley, MA, USA). The exposure time is 1 s and the sampling rate is 2 Hz. In all bulk experi- ments, 1 nM of duplex substrate molecules were used to minimize reannealing of the strands upon unwinding. sm-FRET assays Sm measurements were performed using a homemade microﬂuidic chamber equipped with inlet and outlet ﬂow tubing as described in Di Fiori and Meller (28). The quartz coverslip (Esco Products, Oak Ridge, NJ, USA) of the cell was ﬁrst coated with biotinylated BSA (Sigma, St Louis, MO, USA) and then with streptavidin (Thermo, Rockford, IL, USA). Biotinylated samples were immobilized on the biotinylated BSA-anchored streptavidin as previously described (28). In a typical experiment, a 10 pM RNA/DNA hybrid hairpin solution was ﬂowed through the cell, immobilizing roughly 100 molecules in a single ﬁeld of view of the microscope. In order to remove unattached hybrids, the cell was washed 10 times with imaging buffer, comple- mented with an oxygen scavenging system [50 mM Tris (pH 7.5), 50 mM NaCl, saturated Trolox (>2 mM) (Sigma), 5 mM Protocatechusate (Sigma) and 0.15 mM Protocatechuate 3,4-Dioxygenase(Sigma)] (29). eIF4AI or eIF4AI/eIF4H (1:1) complex (1 mM) was incubated with the immobilized hybrids for 10 min at 22 C in imaging buffer containing 1 mM ATP (Roche Applied Science, Indianapolis, IN, USA) and 1 mM MgCl2. sm data acquisition and analysis The FRET donor (Cy3) and acceptor (Cy5) signals were collected using a modiﬁed objective-type total internal reﬂection (TIRF) microscope (IX71, Olympus, Japan). Cy3 was excited by a 532-nm laser (Millennia V, Spectra- Physics, Santa Clara, CA, USA). Emissions from donor and acceptor were discriminated using dichroic mirrors (Chroma Technology, Bellows Falls, VT, USA) and imaged by an EMCCD camera (iXon DV 897, Andor Technology, Belfast, Ireland) at 10 frames/s, except where otherwise noted. A custom image acquisition program written using LabVIEW (National Instruments, Austin, TX, USA) was used to acquire movies and stream data to the computer’s hard-drive. sm traces were extracted and analyzed using programs written in Matlab (Mathworks, Natick, MA, USA). FRET values were calculated as IA/(IA+ID), where IA and ID are the ﬂuorescence intensity of the acceptor and donor channels, respectively, following correction of back- ground noise. The two-state FRET oscillations were analyzed using Hidden Markov Modeling (HAMMY). Electrophoretic mobility shift assays Electrophoretic mobility shift assays (EMSA) reactions were performed in a binding buffer (20 mM Tris–Cl, pH 7.4, 80 mM KCl, 2.5 mM MgCl2, 1 mM DTT and Nucleic Acids Research, 2012, Vol. 40, No. 13 6201 1% glycerol). Reactions containing the indicated proteins, ﬂuorophore-labeled RNA, BSA, PEG and ATP were performed at RT over the course of 30 min. RNA/DNA hairpin: substrates included RNA/DNA hybrid hairpins carrying 3 - to 12-nt long loops, with a 12-bp stem region. The single-stranded overhang region consisted of 23 nt. A Cy3 ﬂuorophore was attached internally to the DNA sequence. Samples were loaded onto 10% non-denaturing polyacrylamide gel and following electrophoresis, gels were scanned for Cy3 emission using a Pharos FX Scanner (BioRad, Hercules, CA, USA). ATPase activity Phosphate (Pi) release from eIF4AI (and eIF4AI/eIF4H complex) upon addition of ATP (10–100 mM) was measured using a ﬂuorescently labeled mutant of phos- phate-binding protein (MDCC-PiBP, Invitrogen PV4407). Background Pi was removed from the reaction buffer [20 mM Tris–Cl (pH 7.5), 80 mM KCl, 2.5 mM MgCl2] using a MOP solution containing 7-methylguanosine (200 mM) and purine nucleoside phosphorylase (Sigma- Aldrich). RESULTS Bulk and sm characterization of eIF4AI unwinding activity using FRET Before undertaking sm-FRET measurements, we veriﬁed that FRET could be used to assess eIF4AI helicase activity in bulk. As previously reported (8), the helicase activity of eIF4A was observed using a radiometric-based gel assay (Supplementary Figure S2). We then developed a bulk FRET assay for eIF4AI, using an RNA/DNA hybrid duplexes since our plan was to immobilize RNA sub- strates to solid support matrices using DNA for sm-FRET experiments (see below) and eIF4AI is able to unwind such substrates (30). Substrates consisting of 18-mer RNA/DNA hybrid duplexes (Tm = 51.1 C, G = 21.4 kcal/mol) labeled using a FRET pair (Cy3–Cy5) located on opposite strands displayed a stable, high FRET level (Figure 1). Upon addition of 1 mM ATP and increasing concentrations of eIF4AI, we observed a gradual decrease in FRET signal, followed by a steady lower FRET state determined by eIF4AI’s concentration (Figure 1). The characteristic half-life for FRET kinetics was 3 min. In this assay, we used an extremely low RNA/DNA duplex concentration (1 nM) to avoid re-annealing of the two nucleic acids strands within the experimental timeframe. No FRET change was observed in the absence of ATP and the FRET signal was eIF4AI concentration dependent (Figure 1). Gel electrophoresis analysis of radio-labeled RNA duplexes showed that in the presence of ATP, eIF4AI unwinds RNA duplexes, in agreement with the FRET measurements, and consistent with previous studies (19). These experiments allow us to conclude that increasing eIF4AI concentrations lead to an increase in unwound RNA:DNA duplexes, although we can make no inferences on the linearity of the response from these experiments.