A hammerhead ribozyme was demonstrated to be a metalloenzyme. By controlling the metal-binding ability of the hammerhead ribozyme in the presence or absence of a specific sequence of interest, we engineered an allosterically controllable ribozyme, designated the maxizyme. Hybrid ribozymes were then constructed by coupling the site-specific cleavage activity of a hammerhead ribozyme with the unwinding activity of an endogenous RNA helicase. This leads to extremely efficient cleavage of target mRNA, not only in vitro, but also in vivo, and eliminates one of the major problems arising in the application of ribozymes for cleavage of mRNA in vivo: that many target sites on the RNA were previously inaccessible to cleavage owing to secondary and/or tertiary structure formation. Since hybrid ribozymes can efficiently attack target sites within mRNA, libraries were made of hybrid ribozymes with randomized binding arms, which were then introduced into cells. This procedure made it possible to readily identify the relevant genes associated with a specific phenotype, such as in apoptosis and cancer metastasis pathways. This application of a randomized library of hybrid ribozymes represents a simple, yet powerful, method for the identification of genes associated with specific phenotypes in the post-genome era. Moreover, vector-based siRNA (short-interfering RNA for RNA interference, RNAi) can also be used for the creation of the libraries and for the subsequent confirmation of the identified genes, relevant in the examined phenotype.
Mechanism of action of hammerhead ribozymes and their applications in vivo: rapid identification of functional genes in the post-genome era by novel hybrid ribozyme libraries
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Y. Takagi, E. Suyama, H. Kawasaki, M. Miyagishi, K. Taira; Mechanism of action of hammerhead ribozymes and their applications in vivo: rapid identification of functional genes in the post-genome era by novel hybrid ribozyme libraries. Biochem Soc Trans 1 November 2002; 30 (6): 1145–1149. doi: https://doi.org/10.1042/bst0301145
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