RULES FOR RIBOZYMES

The selective inactivation of genes, in a tissue-specific or temporall y controled manner, is now an important requirement for the analysis o f nervous system development and function. Hammerhead ribozymes-cataly tic RNA enzymes that specifically bind to and then cleave target RNAs- may provide a way to meet this requirement, particularly for organisms in which gene inactivation by homologous recombination is not feasibl e. However, ribozyme application has to some extent been hampered by l imited knowledge as to the base-pairing accessibility of RNA target si tes in vivo. In an attempt to circumvent this limitation, we have used a computer program based on free energy minimization to predict secon dary structures for 128 RNA sequences for which corresponding ribozyme s or antisense oligonucleotides have been synthesized, tested, and rep orted in the literature. A comparative analysis of the predicted struc tures of these targets with the reported efficacy of their correspondi ng antisense reagents has allowed us to formulate a set of rules for t he rational choice of hammerhead ribozyme flanking arms and cleavage s ites.