Identification of genes by hybrid ribozymes that couple cleavage activity with the unwinding activity of an endogenous RNA helicase.

Abstract

Novel ribozymes that couple the cleavage activity of hammerhead ribozymes with the unwinding activity of RNA helicase eIF4AI were constructed. This leads to extremely efficient cleavage of the target mRNA, regardless of the secondary structure of the RNA, and eliminates one of the major problems: many target sites on the RNA were previously inaccessible to cleavage due to secondary and/or tertiary structure formation. Moreover, libraries of hybrid ribozymes with randomized binding arms were introduced into cells. This procedure made it possible to readily identify the relevant genes associated with phenotype. Specifically, four genes known to be in the Fas-mediated apoptosis pathway were identified along with additional genes. This application of a randomized library of hybrid ribozymes represents a simple, powerful method for the identification of genes associated with specific phenotypes in the post-genome era.