Figure 5
Confirmation of cleavage activities for representative hRzs. (A and B) Maps showing the in vitro transcripts generated from the linearized pET11a vectors for the hRz # 2 (A) and hRz # 7 (B) target substrates. Positions of the T7 promoter (T7-Pro), hRz-T7 cleavage site (hRz7), T7 terminator (T7 Term), and Cla I site used for linearization (Cla I) are indicated. T1 and T2 show the extent of two transcripts that are generated in the in vitro transcription reaction for each substrate. C1 shows the extent of the single 5' cleavage product from both transcripts from each substrate, while C2 and C3 show the extent of the two 3' cleavage products generated from the two different transcripts produced from each substrate. (C) Agarose gel of in vitro cleavage reaction products. In vitro transcribed targets and their respective hRz # 2 and hRz # 7 were incubated for 30 min at 37°C. Cleavage products were separated in 2% agarose gels stained with ethidium bromide. Lane M: Millenium™ RNA Marker. Lanes 1–3: In vitro transcribed DENV-ENV region target without MgCl2 (lane 1), hRz # 2 and without MgCl2 (lane 2), and hRz # 2 with MgCl2 (lane 3). Lanes 4–6: In vitro transcribed 3'NCR region target without MgCl2 (lane 4), hRz # 7 without MgCl2 (lane 5), and hRz # 7 with MgCl2 (lane 6). Arrows in lanes 3 and 6 show the expected hRz # 2 and hRz#7 cleavage products, respectively.