Substrate-sequence and Mg2+-dependent cleavage activity of the selected HHR. (A) The nucleotide sequences of the HHR based on the HR-F39 framework targeting the A-to-I editing site on NEIL1 mRNA (HR-F39-NEIL1) and the synthetic NEIL1 RNA fragment (NEIL1 RNA). The sequence of the catalytic core was used for the HR-F39 sequence. The adenosine editing sites on the HTR2C RNA are indicated with black arrowheads. (B) Analysis of A-to-I unedited-specific cleavage by HR-F39-NEIL1 using denaturing PAGE (15%). The cleavage reaction was performed with NEIL1 RNA in which the editing site was adenosine (NEIL1-ade) or inosine (NEIL1-ino) in the presence of excess HR-F39-NEIL1 at 37°C for different durations. The positions of the resulting 5′-cleavage products (P) are indicated. (C) 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 (HR-F39-NEIL1/NEIL1-ade) and triangles (HR-F39-NEIL1/NEIL1-ino). (D) Cleavage activity of HR-F39 against HTR2C-wt RNA under the low Mg2+ concentrations. Data points at the various Mg2+ concentrations are as follows: (circles) 10 mM; (triangles) 5 mM; (squares) 2 mM; (inverted triangles) 1 mM. The cleavage ratios at different time points were determined by quantitation of the corresponding gel bands and calculated by P/(S + P), where S and P represent the intensity of the substrate band and cleavage product band, respectively. All data were obtained from two independent experiments to ensure reproducibility. Best-fit curves were generated using a single-exponential equation. The kinetic parameters obtained from this experiment are summarized in Table 1.
