Fig 1: “Quencherless” fluorogenic substrates allow real-time monitoring of enzymatic activity of DICER and reconstituted RISC complex for enzyme kinetics assays.Recombinant human RNAi proteins were expressed in insect cells, purified and analyzed by SDS-PAGE (2.5 μg/lane) with Coomassie Blue staining (A). Lanes: SeeBlue Plus2 pre-stained markers (M), DICER (1), TRBP (2) and AGO2 (3). Continuous enzymatic assays of DICER using fluorogenic substrates (250 nM) bearing asymmetric overhangs (3’-dinucleotide overhang on anti-sense strand and long sense overhang) are shown (B), and linearity with enzyme concentration is shown (C). Continuous assays of reconstituted human RISC (combinations of purified enzymes) using DICER substrate BoGD664 (250 nM; D-E) or fluorogenic siRNA (AGO-loading substrate BoPsi664; 250 nM; F). AGO2 increases enzymatic activity of reconstituted RISC: (DICER+AGO2 > DICER alone (E-F). AGO2 or TRBP alone show no activity (E-F). *, p<0.05.
Fig 2: Binding interactions in the RISC complex are functionally determined using enzyme kinetics.DICER-AGO2 binding interaction was assessed by enzymatic activity assays modeled using the Morrison equation [17]. Titration of DICER (25 nM) with AGO2 enhances enzymatic activity for cleavage of either fluorogenic DICER substrate (250 nM; A) and the fluorogenic siRNA (AGO-loading substrate; 250 nM; B) apparently via high-affinity binding interaction. AGO loading is dependent on AGO2 concentration and requires DICER (B). Michaelis-Menten kinetics were observed for DICER and minimal reconstituted RISC using both DICER substrates (C-D). AGO-loading siRNA exhibited kinetics consistent with a substrate inhibition model (E), whereas DICER or DICER+TRBP demonstrate minimal enzymatic activity. Lesser apparent fluorogenic activity was observed in combination with the dsRNA-binding protein TRBP.
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