Construction of the trans-acting HHR designed utilizing the NHH preference for specific target cleavage depending on the site-specific A-to-I unedited state. (A) Schematic representation of minimal-HHR (minHHR) with NHH cleavage preference. HHR and substrate RNA are shown as black and gray lines, respectively, and dashed lines indicate the base-pair interactions. A minHHR has three stems: Stem I and stem III are in the hybridizing arms for recognizing the target sequence with Watson-Crick base-pairing, and stem–loop II is in the catalytic core. On the substrate RNA, black and gray circles denote the first and second H positions, respectively. The nucleotide denoted as D (A or G or U, except for C) forms a base pair with the first H. (B) HHR design utilizing the first H position of NHH specificity for specific HTR2C RNA cleavage when the E site is unedited. Sequences of ribozyme (HR-HTR2C-Eedit) and a part of the synthetic HTR2C RNA fragment are shown. The nucleotides corresponding to the NHH triplet are indicated in gray. The editing sites of adenosines on the HTR2C RNA are indicated as black arrowheads, in which only the E site is represented as X (A or I). HR-HTR2C-Eedit was designed to set the E site to the second H position for unedited-specific cleavage. The sequence of the catalytic core is 5′-CUGAUGAGGCCGAAAGGCCGAA-3′. Substrate RNAs that are unedited (X = A) and edited (X = I) at the E site are denoted as HTR2C-wt and HTR2C-Eino, respectively. (C) Analysis of A-to-I unedited-specific cleavage by HR-HTR2C-Eedit using denaturing PAGE (15%). The cleavage reaction was performed with HTR2C-wt (upper) and HTR2C-Eino (lower) in the presence of an excess of HR-HTR2C-Eedit at 37°C for different times. The positions of the resulting 5′-cleavage products (P) are indicated.
