Hammerhead Ribozyme-Mediated Cleavage of Hepatitis B Virus RNA

Hammerhead ribozymes are short catalytic RNA molecules possessing endoribonuclease activity. The enzyme’s specificity is mediated by variable flanking sequences complementary to the RNA target substrate (1 ,2 ). Cleavage occurs via transesterification 3′ to a nucleotide triplet NUX where N may be any base and X any base except G (3 ,4 ). Hepadnaviruses are partially double stranded DNA viruses that share a common replication mechanism involving the reverse transcription of a pregenomic RNA intermediate (5 ). Cleavage of the pregenomic RNA, therefore, should interrupt the life cycle of the hepadnaviruses. In the present chapter, protocols for construction of ribozyme expression vectors and ribozyme-mediated cleavage of hepadnaviral RNA in vitro are provided. Furthermore, possible reasons for inefficient cleavage in vivo are discussed.