Construction of the extended-HHR (HHR) for effective unedited-specific cleavage by an in vitro selection method with a partially randomized HHR library. (A) Schematic illustration (upper) and the nucleotide sequences (lower) of the partially randomized HHR library for selecting exHHR cleaving UAA substrate. In this picture, the positions of target triplet nucleotides are indicated as black circles and each stem region (stem III, stem Ia, and stem Ib) is represented. Fixed and randomized nucleotides are represented as white and gray circles, respectively. The white arrowhead indicates the target cleavage site. Based on minHR-HTR2C-Eedit, four randomized nucleotides were introduced into stem I as an artificial loop 1 that has a potential to form a tertiary stabilizing motif with loop II. The catalytic core region including the stem II–loop II and the nucleotide paired with the E site were substituted with 21 randomized nucleotides. In order to select the HHR cleaving at the target site, the consensus sequence of 5′-CUGA-3′ was fixed. The region of helix Ia, Ib, and III was designed to form base-pairing with the target RNA, and the lengths of each of these regions were 6, 11, and 5 bp, respectively. The target RNA based on the HTR2C RNA sequence was 5′-biotinylated for subsequent selection. (B) Schematic representation of in vitro selection method for obtaining active exHHRs. First, the RNA library was annealed with 5′-biotinylated target RNA, and then the resulting complex was immobilized on streptavidin-coated magnetic beads. After performing an on-beads cleavage reaction, the supernatant containing the ribozyme candidates that dissociated from the beads due to their cleavage activity was collected. Next, RT-PCR was performed with specific primers for the amplification of cDNA from the selected ribozyme, and then the RNA library was regenerated with amplified dsDNA as a template in an in vitro transcription reaction. (C) Nucleotide sequences of selected HHR candidates after eight rounds of in vitro selection. The sequence of HHR library and the selected RNAs were indicated. The corresponding regions for stem Ia, loop I, stem Ib, catalytic core including stem–loop II, and stem III are denoted in parentheses above the sequences: (N) random nucleotide. Canonical HHR consensus sequences (5′-CUGANGA-GAA-3′) are underlined. Nucleotides that were predicted to form stem II are represented in gray, and the nucleotide paired with E site is denoted in italics.
