Target Discovery and Validation Reviews and Protocols

8
Hammerhead Ribozyme-Based Target Discovery
Masayuki Sano and Kazunari Taira
Summary
With the accumulation of vast amounts of data as a result of the sequencing of the human genome,
it is necessary to identify human genes that are involved in various cellular, developmental, and
disease-related processes and to clarify their functions and potential utility as targets in the treatment
of disease. Identification methods based on the use of hammerhead and hairpin ribozymes
have received increasing attention as possible tools for the rapid identification of key genes
involved in biological processes. This chapter describes the method known as gene-discovery by
a hammerhead ribozyme library for elucidation of the gene function. Use of this technology has
already revealed new insights into several important biological phenomena.
1. Introduction
A hammerhead ribozyme is a small RNA molecule with catalytic activity
and it can be engineered to optimize its activity in the intracellular environment.
The hammerhead ribozyme recognizes a target gene via interactions between
the binding arms of the ribozyme and the gene transcript, and it can cleave any
RNA substrate that contains an NUH triple, where N is any nucleotide and H is
A, C, or U. To date, numerous studies have been directed towards applications
of ribozymes in vivo, and many successful experiments suppressing genes of
interest have been reported (1,2).
In our laboratory, successful inactivation of genes by hammerhead ribozymes
in living cells has been achieved by using an appropriate expression cassette
(Fig. 1A). The use of the human tRNAVal promoter ensures a high level of
expression of the ribozymes, and an appropriate design of the expression
cassette leads to the intracellular stability and cytoplasmic transport of the
ribozyme transcripts (3–5). These features would apparently be advantageous
for the exploitation of ribozymes in vivo.
From: Methods in Molecular Biology, vol. 360, Target Discovery and Validation Reviews and Protocols
Volume I, Emerging Strategies for Targets and Biomarker Discovery
Edited by: M. Sioud © Humana Press Inc., Totowa, NJ
143