Schematic representation of the precleaved RNA editing assays. (A) Trimolecular hybrids are involved in the PC-deletion, -insertion, and -ligation assays. Three U residues were designed for removal from the 5′Del fragment in PC-deletion; one missing U residue was designed for addition to the 3′Ins fragment in PC-insertion; no requirement for U-deletion or -insertion in PC-ligation. The Watson–Crick and G·U wobble base pairs are depicted by solid and dashed lines. The critical nucleotides of the catalytic site of the HHR are indicated in bold and highlighted in yellow and orange (B) RNA editing results in the formation of an active HHR. (C) Enzymatic cleavage activity of the active HHR on the FRET substrate (containing a fluorophore and a quencher). An arrow indicates the cleavage site. The inset depicts the usage of nonconventional termini in the RNA substrates to reduce/eliminate aberrant editing products. Guide RNA gHHRc (gHHR with cytidine in the editing site) was used for efficient RNA editing (Supplemental Fig. S2).
