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
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 possible
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 reduction.
These findings have important therapeutic implications.