Fig. 2: RNA cleavage assay by Siwi-piRISC immobilized on the surface.
From: Heterogeneous dissociation process of truncated RNAs by oligomerized Vasa helicase
a RNA cleavage assay of target RNA. In the presence of Siwi, Vasa and ATP, target RNA was cleaved and dissociated from Siwi-piRISC in bulk. Black and red arrows indicate the target RNA (64 nt) and cleaved RNA (31 nt), respectively. Full gel image is shown in Fig. S6a. b Schematic representation of dissociation of cleaved target RNA loaded on Siwi-piRISC and Vasa. c The relative intensity decay of target RNA after adding 0 (blue) or 1 mM (black) ATP with Vasa in a certain (27.6 × 27.6 µm2) region. The red curve shows the exponential decay with the time constant of 46.5 ± 0.2 s (R2 = 0.998) in addition of 1 mM ATP. With dN-Vasa, the intensity was shown in green. The intensity of fluorescent-labelled RNA was constant coloured in grey. d The example time-trace intensity of single target RNA after adding 1 mM or 10 µM ATP. Red lines show the analysed stepwise intensity. e The distribution of dwell time before cleaved RNA detached in 1 mM ATP with the time constant of 49.8 ± 2.9 s (R2 = 0.994) or in 10 µM ATP. 71.6% of RNA remained over 400 s in 10 µM ATP. Insets are the corresponding histograms fitted with a single exponential curve.
