The hammerhead ribozyme, which was first found in plant viroid, can be modified to bind and cleave any target RNAs of choice in a sequence-specific manner. Consequently, they could be used to suppress the expression of a target gene. In this study, suppression of the lacZ' and HBV X genes by hammerhead ribozymes was investigated both in vitro and in vivo.
The activity of a ribozyme, which was designed to suppress the expression of the lacZ' gene, was investigated in a cis-manner in vitro and in E. coli. Over 90% of the transcripts were cleaved at 37 and 50℃ in vitro. The plaques became colorless by the introduction of a plasmid (pMP18-Rz) which carried the ribozyme in the correct orientation into E. coli, whereas in control experiments plaques were blue in the introduction of a plasmid (pMP19-Rz) which carried the same ribozyme in the reverse orientation. The ribozyme was found to reduce the expression of lacZ' gene by 90%, which was determined by the β-galactosidase assay. We show in this study not only intracellular cleavage product but also the exact site where the cleavage took place by primer extension analysis.
The in vitro cleavage reactions of five different ribozymes which targeted the HBV X gene were studied in trans. Rz4 and Rz3 showed significant cleavage activity while the GUU-targeting Rz5 showed to be considerably less active. Rz1 and Rz2 showed little or almost no activity (Rz4＞Rz3≫Rz5≫Rz1, Rz2). For Rz3 and Rz4, the influences of temperature, formamide concentration and incubation time on cleavage activity were investigated. The difference in activity between Rz3 and Rz4 was thought to result from their binding ability to their respected target RNA sequences.
Rz3 and Rz4 were also introduced into E. coli to investigate their in vivo activities. Although the ribozymes were successfully produced, suppression of the target gene was not observed, and cleavage products could not be detected in the RNA level.
To evaluate the activity of Rz3 and Rz4 in animal cell line, the X gene and the synthetic oligomers encoding both ribozymes plus its null mutants were cloned into an expression vector pRC/RSV separately and cotransfected into Huh7 cells. Intracellular cleavage of X mRNA was confirmed by the primer extension analysis. The cleavage events occurred in Rz3 and Rz4, whereas the mutated Rz3 and Rz4 did not show any cleavage products.
From this study, we suggest that the activity of ribozyme is determined according to the secondary structure of the target RNA region. Furthermore, we found that the exhibition of a ribozyme's catalytic activity also depends on the nature of the cell type (i.e E. coli vs animal cells).