Competitive ARGONAUTE2 loading in the DICER-AGO2 enzyme complex correlates with efficacy in cell-based RNAi assays.

<p>Kinetic assays of enzymatic AGO loading were performed by applying DICER+AGO2 (30 nM each) to mixtures containing a fixed concentration of fluorogenic AGO substrate (siRNA BoPsi664, 80 nM) and variable concentrations (0, 25, 100, 400 nM shown) of unlabeled competing DICER substrate D03 (<b>A</b>, panel above) or DICER substrate D10 (<b>A</b>, panel below). “No enzyme” control wells contain BoPsi664 without DICER or AGO2. The apparent initial rate of fluorogenic AGO loading was dependent upon the concentration of unlabeled competing DICER substrates D03, D10 and D11 (<b>B</b>, colored symbols) or unlabeled competing AGO substrate (<b>B</b>, AllStars siRNA) compared to control (<b>B</b>, “Ctrl”). The potency of competitive AGO loading of DICER substrates by the DICER-AGO2 complex (<i>IC</i>₅₀) was correlated to <i>HIF1A</i> mRNA in Huh-7.5 cells as detected by branched DNA (bDNA) assay (<b>C</b>). Dotted lines indicate the 90% confidence interval, and error bars show SEM. “n.d.,” not detectable.