M Oketani1, Y Asahina, C H Wu, G Y Wu. 1. Department of Medicine, University of Connecticut Health Center, Farmington 06030, USA.
Abstract
BACKGROUND/AIMS: The aim of this study was to determine whether DNA analogs of ribozymes could be prepared to inhibit hepatitis C virus (HCV) gene expression. METHODS: Two DNA ribonucleases, Dz2 and Dz4, were designed with varying arm lengths, to cleave at the 5'-noncoding region (NCR) just upstream from the translation start site, and core region of HCV genome, respectively. A reporter vector was prepared to contain target HCV regulatory sequences controlling a downstream luciferase gene. DNA ribonucleases with normal phosphodiester, as well as with terminal phosphorothioate linkages, were administered to Huh7 cells, and luciferase activity was measured. RESULTS: DNA ribonucleases were highly active in cleaving HCV RNA targets. Enzymes with longer arms had consistently higher cleavage activity compared to enzymes with shorter arms under cell-free conditions. Furthermore, in Huh7 cells, terminal phosphorothioate derivatives, Dz2 and Dz4, significantly suppressed HCV-luciferase fusion gene expression up to 45% and 67% of controls, respectively. Interestingly, phosphorothioate-modified DNA ribonucleases had greater inhibitory effects on target gene expression than their unmodified counterparts. In contrast, DNA ribonucleases with point mutations in the catalytic domain had significantly lower inhibitory effects compared to wild-type DNA ribonucleases. However, activity was not eliminated, suggesting that some antisense contribution was present. CONCLUSIONS: DNA ribonucleases directed against the HCV genome can specifically cleave target HCV RNA. Modifications of the extreme 3'- and 5'-termini protect against nuclease degradation without appreciable reduction in inhibitory activity against viral gene expression under intracellular conditions.
BACKGROUND/AIMS: The aim of this study was to determine whether DNA analogs of ribozymes could be prepared to inhibit hepatitis C virus (HCV) gene expression. METHODS: Two DNA ribonucleases, Dz2 and Dz4, were designed with varying arm lengths, to cleave at the 5'-noncoding region (NCR) just upstream from the translation start site, and core region of HCV genome, respectively. A reporter vector was prepared to contain target HCV regulatory sequences controlling a downstream luciferase gene. DNA ribonucleases with normal phosphodiester, as well as with terminal phosphorothioate linkages, were administered to Huh7 cells, and luciferase activity was measured. RESULTS: DNA ribonucleases were highly active in cleaving HCV RNA targets. Enzymes with longer arms had consistently higher cleavage activity compared to enzymes with shorter arms under cell-free conditions. Furthermore, in Huh7 cells, terminal phosphorothioate derivatives, Dz2 and Dz4, significantly suppressed HCV-luciferase fusion gene expression up to 45% and 67% of controls, respectively. Interestingly, phosphorothioate-modified DNA ribonucleases had greater inhibitory effects on target gene expression than their unmodified counterparts. In contrast, DNA ribonucleases with point mutations in the catalytic domain had significantly lower inhibitory effects compared to wild-type DNA ribonucleases. However, activity was not eliminated, suggesting that some antisense contribution was present. CONCLUSIONS: DNA ribonucleases directed against the HCV genome can specifically cleave target HCV RNA. Modifications of the extreme 3'- and 5'-termini protect against nuclease degradation without appreciable reduction in inhibitory activity against viral gene expression under intracellular conditions.