FIGURE 5.

Effect of the “anticodon stem design” on the in vitro trans-splicing efficiency with Azoarcus ribozymes. (A) Schematic for the secondary structure, with a focus on interactions between substrate mRNA (red), ribozyme 5′ extension (green), and ribozyme 3′ exon (blue). The secondary structure is based on the model describing the Azoarcus ribozyme crystal structure (Adams et al. 2004a). The positions of the P1 helix (P1), P1 extension (P1ex), and anticodon stem are indicated. (B) Autoradiogram of 5′ radiolabeled trans-splicing products separated by denaturing polyacrylamide gel electrophoresis. The targeted splice site and the designed number of base pairs in the P1 extension (P1ex) are indicated for each ribozyme variant. Size markers in the first and last lane indicate the position of full-length CAT mRNA substrate (Sub), the expected trans-splicing product (Prod), and the 5′ fragments produced by the first step of splicing (5′-Frag) at splice sites 97, 177, and 258. The length of the trans-splicing products is the same for all splice sites because the length of the ribozyme 3′ exon was covaried with the splice sites. The quantitations of trans-splicing efficiencies are shown below the autoradiogram, as percentage of substrate converted to product. The scale is the same as in Figures 3 and 4 to facilitate comparison. Error bars denote standard deviations from three experiments.