DICER+AGO2 enzymes cleave fluorogenic substrates into duplex products that are unstable at T>25°C.

<p>Enzymatic activity (37°C) is shown for the dsRNA DICER substrates (BoGD664AS * GD664S, <b>A</b>; BoGD664AS-dAdG * GD664S, <b>C</b>; BoPD664S * PD664AS, <b>E</b>; BoPD664S-dAdG * PD664AS, <b>G</b>) and fluorogenic siRNA BoPsi664 (BoPsi664S * Psi664AS; <b>I</b>) by AGO2 (120 nM) + DICER (30 nM) but not by <i>E</i>. <i>coli</i> RNase H (0.24 U/mL). Melting analysis shows that only products of DICER+AGO2 cleavage are already melted at <i>T</i> ≥ 25°C, whereas control reactions containing either no enzyme or RNAse H instead of DICER+AGO2 have fluorimetric dsRNA stability (as measured by <i>T</i>_m) that is indistinguishable from <i>T</i>_m of the substrates (<b>B, D, F, H, J</b>). EDTA inhibits enzymatic cleavage of substrates and gives the characteristic decrease in <i>T</i>_m compared to Assay Buffer containing divalent cations (<b>B, D, F, H, J</b>). First derivatives are displayed using dashed lines.