Secondary structure prediction and in vitro accessibility of mRNA as toolsin the selection of target sites for ribozymes

We have investigated the relative merits of two commonly used methods for t arget site selection for ribozymes: secondary structure prediction (MFold p rogram) and in vitro accessibility assays. A total of eight methylated ribo zymes with DNA arms were synthesized and analyzed in a transient co-transfe ction assay in HeLa cells. Residual expression levels ranging from 23 to 72 % were obtained with anti-PSKH1 ribozymes compared to cells transfected wit h an irrelevant control ribozyme, Ribozyme efficacy depended on both ribozy me concentration and the steady state expression levels of the target mRNA. Allylated ribozymes against a subset of the target sites generally display ed poorer efficacy than their methylated counterparts. This effect appeared to be influenced by in vivo accessibility of the target site. Ribozymes de signed on the basis of either selection method displayed a wide range of ef ficacies with no significant differences in the average activities of the t wo groups of ribozymes. While in vitro accessibility assays had limited pre dictive power, there was a significant correlation between certain features of the predicted secondary structure of the target sequence and the effica cy of the corresponding ribozyme, Specifically, ribozyme efficacy appeared to be positively correlated with the presence of short stem regions and hel ices of low stability within their target sequences. There were no correlat ions with predicted free energy or loop length.