A hairpin ribozyme from a satellite RNA of chicory mottle virus : cleavage efficiency, substrate selectivity and targeting HIV1
The catalytic capacity, substrate selectivity and application in HIV1 treatment of a hairpin ribozyme derived from sCYMVl were studied. Based on the sequence comparisons of hairpin ribozymes derived from sTRSV (GUC substrate) and sCYMVl (GUA substrate), the helix 1 length of the sCYMVl ribozyme was extended from 5bp to 6bp, and the mutagenesis of the sCYMVl ribozyme was performed in C7, G20 and U46 of loops 1, 2 and 4. Futhermore, helix 4 and loop 3 were replaced by a tetraloop and altered tetraloop. Cleavage and kinetics studies have shown that the C7 position is critical for the GUA/GUC substrate selection; a mutant sCYMVl ribozyme (CIA) with a 6 bp helix 1 increased the cleavage activity 4-fold, mainly by decreasing the Km values. The thermostable tetraloop and altered tetraloop have improved the catalytic activity 1.4x to 1.7x. The possible mechanisms of these results and the experimental methodology are discussed. sCYMVl ribozyme has, for the first time, been shown to successfully cleave HIV1 GUA sequences in this thesis. Nef/LTR of 9218 position and Tat/Vpr of 5839 position were the two target sites selected. The ribozyme targeted to the Nef/LTR site has a cleavage efficiency (kcat/Km) of 5.1x10^5 M^(-1)min^(-1), which is very close to that of the native sCYMVl ribozyme (6.3x10^5 M^(-1)min^(-1)). The ribozyme cleaving the Tat/Vpr site has a cleavage efficiency of 16x10^4 M^(-1) min^(-1). These results have demonstrated that the sCYMVl ribozyme efficiently cleaves the native GUA substrate and a heterologous GUA substrate. Therefore, it expands the repertoire of hairpin ribozymes by including GUA containing sequences as potential target sites for gene therapy.