C-to-U editing-specific cleavage of ribozyme against the APOB mRNA fragment. (A) Sequences of ribozymes and substrates of the synthetic APOB mRNA fragment. HR-APOB-edit was generated by introducing A as the recognition base for C-to-U editing recognition. The recognition base of the ribozyme is marked by a circle with a dotted outline, and the sequence of the catalytic core is the same as that of the HR-HTR2C. The cleavage site is indicated by a white arrowhead, and the target C-to-U editing site of C is underlined in the substrate sequence. Unedited and edited substrates are denoted as APOB-cyt and APOB-uri, respectively. (B) Analysis of editing-specific cleavage of HR-APOB-edit using denaturing PAGE (15%). The cleavage reaction was performed with APOB-cyt (left) and APOB-uri (right) in the presence of excess ribozyme at 37°C for 1 h. (C) Time course of cleavage reaction and kinetic analysis for HR-APOB-edit against unedited and edited APOB RNA fragments under single-turnover conditions. The diagram represents the cleavage ratio as a function of reaction time and the sets of ribozyme/substrate are shown as triangles (HR-APOB-edit/APOB-cyt) and circles (HR-APOB-edit/APOB-uri). Data were obtained from two independent experiments. Fitting curves were generated using a single-exponential equation. Kinetic parameters obtained from this experiment are summarized in Table 1.
