Synthetic ribozyme suppressing HBV X gene expression

Abstract

Ribozymes are potentially very powerful agents for perturbing intracellular gene expression. Hammerhead structure ribozymes were designed to cleave the mRNA of hepatitis B virus X gene. Its cleavage was designed to result in the suppressed expression of lacZ fused to a 5$^\prime$462-bp fragment of X gene. The hammerhead ribozymes consist of a core region for cleavage activity and two variable regions for binding to a target RNA sequenec. A target region of X mRNA was chosen among 12 GUC-containing regions, because it is predicted not to be base-paired by a computer algorthm of RNA secondary structure prediction. Each target-binding, variable region was designed to contain 4 matched bases near the core sequence and 4 2-base wobbles (matched and mismatched) in the rest. Among 256 possible sequences 12 sequences have so far been identified to yield suppressed expression of the fused lacZ. The $\beta$-galactosidase activities were reduced at least 10-fold. Also corresponding polypeptedes were not produced as revealed by SDS-polyacrylamide gel electrophoresis.