Skip to main content
. 2015 Mar 20;10(3):e0120614. doi: 10.1371/journal.pone.0120614

Fig 3. “Quencherless” fluorogenic substrates allow real-time monitoring of enzymatic activity of DICER and reconstituted RISC complex for enzyme kinetics assays.

Fig 3

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.