A-to-I editing-specific cleavage of ribozyme against HTR2C mRNA fragment. (A) Sequences of ribozymes and substrates of synthetic HTR2C mRNA fragment. Parental ribozyme HR-HTR2C was designed to cleave 5′ to the editing site. HR-HTR2C-edit was generated from HR-HTR2C by altering the recognition base to C for A-to-I editing recognition. The recognition base of the ribozyme is marked by a circle with a dotted outline. Sequence of a catalytic core for all ribozyme constructs is 5′-CUGAUGAGGCCGAAAGGCCGAA-3′. The cleavage site is indicated by a white arrowhead, and target A-to-I editing site of A is underlined in the substrate sequence. Unedited and edited substrates are denoted as HTR2C-ade and HTR2C-ino, respectively. (B) Analysis of A-to-I-editing-specific cleavage by ribozyme using denaturing PAGE (15%). The cleavage reaction was performed with HTR2C-ade (left) and HTR2C-ino (right) in the presence of an excess of ribozyme at 37°C for 1 h. Positions of the 5′-labeled substrates (S) and of the resulting 5′-cleavage products (P) are indicated. (C) Summary of the cleavage ratios obtained from experiment B for HR-HTR2C and HR-HTR2C-edit against HTR2C-ade and HTR2C-ino. Intensities of the substrate band (S) and cleavage product band (P) were quantified, and the cleavage ratios were calculated using the expression P/(S+P).
