A method for prediction of accessible sites on an mRNA sequence for target selection of hammerhead ribozymes

Hammerhead ribozymes are short RNA molecules endowed with endoribonucleolytic activity. Their use as molecular tools for specific inhibition of gene translation is affected by many factors including the target accessibility. A method for the prediction of accessible target sites for hammerhead ribozymes within a given RNA sequence is described. This method maps all putative NUH cleavage sites (N = A, C, G, U and H = A, C, U) and picks out short flanking regions as the binding domain for the corresponding ribozyme. The probabilistic level of unfolding, accessibility score (AS), is then calculated for each target region on the basis of a comparison of all folding structures obtained for the target RNA and arranged according to the Boltzmann's distribution. At the end, a series of imposed limits gives the best target sequences endowed with highly probable accessibility and with a potentially active catalytic structure of the hammerhead sequence. A successive experimental approach to verify the effective accessibility of selected targets was used. For that, antisense oligonucleotides addressed to the coding region of bcl2 mRNA were synthesized and administered to the MCF7 human cell line. The inhibition of gene expression, as measured by western analysis of the BCL2 protein, demonstrated that all target sites selected on the basis of their putative accessibility were actually sensitive to antisense treatments while the inaccessible ones were not. The application of this target discovery method to ribozyme design is proposed in order to satisfy a crucial condition.