Construction of a novel artificial-ribozyme-releasing plasmid

Abstract

A novel ‘active-ribozyme-releasing system’ was constructed, taking advantage of the consensus sequence of a new class of ribozyme. An active ribozyme sequence, targeted for the SFWl gene (a yeast suppressor gene for flocculation) was fused just downstream of the T7 promoter. The 3′ terminus of the Arst ribozyme was designed to be trimmed by the second ribozyme connected to the downstream of the first active ribozyme. In vitro experiments revealed that the active ribozyme targeted to SFLl was successfully released by the action of the second rhzyme, subsequently cleaving the SFL1 mRNA at the predetermined site. Since the first active ribozyme with a defined 3′-terminus can be produced even when a circular DNA is used as a template, this kind of construct has a potential to release an ‘active ribozyme’ tailored to destroy a target gene (RNA) in vivo. Moreover, the second ribozyme in this construct can be utilized as a universal pseudo-terminator for generation of any RNA transcripts inserted in place of the cassette portion of the first ribozyme.