Time course of cleavage reaction and kinetic analysis of selected HHRs for the UAA (HTR2C-wt) and UIA (HTR2C-Eino) substrates under single-turnover conditions. (A) Denaturing PAGE (15%) showing the time course of cleavage of HTR2C-wt (left) and HTR2C-Eino (right) by the selected HHRs. (B) Graphs representing the cleavage ratio as a function of reaction time. Each data point for individual sets of ribozyme and substrate (ribozyme/substrate) is represented as circles (HHR/HTR2C-wt) and triangles (HHR/HTR2C-Eino). The cleavage ratio at different time points was determined using quantitative scanning of the corresponding gel bands and calculated using the expression P/(S + P), in which S and P represent the intensities of the substrate band and cleavage product band, respectively. All data were obtained from two independent experiments for reproducibility. Fitting curves were generated using a single-exponential equation. The kinetic parameters obtained from this experiment are summarized in Table 1.
