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Novel antiviral approaches are needed to control Hepatitis B virus infection worldwide. X protein of this virus activates various promoters and is strongly associated with hepatocellular carcinoma. Although several groups, including ours, reported sequence-specific cleavage of X RNA by either ribozymes (Rzs) or DNA-enzymes (Dzs) earlier, but none of these studies reported 100% in vitro cleavage of the full-length X RNA. We reasoned that by melting the secondary structures near the Rz/Dz cleavage site with specific antisense DNA oligonucleotides (ODNs) or 10-23 Dz, it may be pos- sible to achieve this objective. Hammerhead motif containing Rz-170 specific for X RNA was constructed by recombinant techniques and Dz-237 was synthesized using the 10-23 catalytic motif. When specific ODNs or 10-23 Dzs were included in the cleavage reaction with either Rz-170 or Dz-237, increased cleavage was observed in a dose-dependent manner which often resulted in almost complete in vitro cleavage of the target RNA. Rz-170 in combination with specific ODNs caused potent intracellular reduction of HBx RNA. Thus, the cleavage activity of catalytic nucleic acids (Rzs or Dzs) can be increased significantly by specific ODNs or Dzs and this treatment also results in potent intracellular target RNA re- duction. These findings have important therapeutic implications.

the open biotechnology journal

Potent Intracellular Knock Down of Hepatitis B Virus X RNA by Catalytic Hammerhead Ribozymes or DNA-Enzymes with Antisense DNA-Oligonucleotides or 10-23 DNA-Enzymes that Powerfully Augment In Vitro Sequence-Specific Cleavage Activities