Figure 5. Site specific cleavage of a truncated target affects of alternative substrate structure

A: anti-V230 hRzs (Rz2A and Rz2B) were tested against a truncated WT opsin mRNA containing 450 nt of the full length mRNA assymetrically embracing the V230 GUC` cleavage site. The truncated S is 510 nt long due to vector sequences read during in vitro transcription. Cleavage at V230 GUC` is predicted to yield products P₁ (5'=198 nt) and P₂ (3'=312 nt). Rz2A and Rz2B were reacted with truncated WT S at 37 °C and 50 °C. The E:S ratio was 6:1 (S at 650 nM). Lane 1 is an RNA ladder. Lane 2 is open. Lane 3 and 4 are short WT S at 37 °C and 50 °C, respectively but without hRz and Mg²⁺. Lanes 5 and 6 are short WT S with Rz2A and Mg²⁺ at 37 °C and 50 °C, respectively. Lanes 7 and 8 are short WT S with Rz2B, 50 °C and 37 °C, respectively but without Mg²⁺. Lanes 9 and 10 are WT-short with Rz2B and 50 °C and 37 °C, respectively, with Mg²⁺. B: Short WT and short opsin AS constructs were transcribed (in duplicate) with Rz2B ([E]:[S] template ratio 1:6) for 1 h at 37 °C. The label in the header indicates the S used and the lanes with ladders.