Site-specific cleavage of RNA encoding garlic latent virus coat protein by a hairpin type ribozyme

To test the possibility of producing virus resistant garlic plants, we have designed a hairpin type ribozyme to cleave at the middle of the RNA encoding the coat protein of garlic latent virus (GLV). It is known that the hairpin ribozyme derived from the negative strand of satellite RNA of tobacco ringspot virus [(-)sTRSV] cleaves RNA at 5′-phosphodiester bond of the non-base paired -N^↓GUC- bulge flanked by the base-paired region next to the hairpin domain. The -N^↓GUC- containing target site was selected from the region which was predicted open by the secondary structure analysis. The ribozyme cleaved two different sizes of RNA substrates, V9S (148 nucleotides) and V9 RNA (1,361 nucleotides) in vitro at the predicted site. The cleavage reaction was optimal in 4 mM MgCl₂, 2 mM spermidine and 40 mM Tris-HCI (pH 8.0), at 50°C. Magnesium and calcium ions enhanced RNA cleavage whereas limited cleavage was observed with other divalent cationas such as Mn²⁺, Zn²⁺ and Ni²⁺. The k_cat/K_M value with V9S was 0.22 μM^-1min^-1, which is lower than that of the Hampel and Tritz (1989) system of 70 μM^-1min^-1, probably due to the size difference of the substrate.