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Nucleic Acids Research, 2000, Vol. 28, No. 21 4113-4124
© 2000 Oxford University Press

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

Mohammed Amarzguioui, Gaute Brede, Eshrat Babaie, Morten Grøtli1, Brian Sproat2 and Hans Prydz*

The Biotechnology Centre of Oslo, University of Oslo, Gaustadalleen 21, 0349 Oslo, Norway, 1Department of Chemistry, Carlsberg Laboratory, Gamle Carlsberg Vej 10, DK-2500 Valby, Denmark and 2Institut für Organische Chemie, Universität Göttingen, Tammann strasse 2, 37077 Göttingen, Germany

We have investigated the relative merits of two commonly used methods for target site selection for ribozymes: secondary structure prediction (MFold program) and in vitro accessibility assays. A total of eight methylated ribozymes with DNA arms were synthesized and analyzed in a transient co-transfection assay in HeLa cells. Residual expression levels ranging from 23 to 72% were obtained with anti-PSKH1 ribozymes compared to cells transfected with an irrelevant control ribozyme. Ribozyme efficacy depended on both ribozyme concentration and the steady state expression levels of the target mRNA. Allylated ribozymes against a subset of the target sites generally displayed poorer efficacy than their methylated counterparts. This effect appeared to be influenced by in vivo accessibility of the target site. Ribozymes designed on the basis of either selection method displayed a wide range of efficacies with no significant differences in the average activities of the two groups of ribozymes. While in vitro accessibility assays had limited predictive power, there was a significant correlation between certain features of the predicted secondary structure of the target sequence and the efficacy of the corresponding ribozyme. Specifically, ribozyme efficacy appeared to be positively correlated with the presence of short stem regions and helices of low stability within their target sequences. There were no correlations with predicted free energy or loop length.

* To whom correspondence should be addressed. Tel: +47 2295 8755; Fax: +47 2269 4130; Email: hans.prydz@biotek.uio.no


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