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Open Access Research article

A structural analysis of in vitro catalytic activities of hammerhead ribozymes

Yu Shao, Susan Wu, Chi Yu Chan, Jessie R Klapper, Erasmus Schneider and Ye Ding*

Author Affiliations

Wadsworth Center, New York State Department of Health, 150 New Scotland Avenue, Albany, NY 12208, USA

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BMC Bioinformatics 2007, 8:469  doi:10.1186/1471-2105-8-469

Published: 30 November 2007



Ribozymes are small catalytic RNAs that possess the dual functions of sequence-specific RNA recognition and site-specific cleavage. Trans-cleaving ribozymes can inhibit translation of genes at the messenger RNA (mRNA) level in both eukaryotic and prokaryotic systems and are thus useful tools for studies of gene function. However, identification of target sites for efficient cleavage poses a challenge. Here, we have considered a number of structural and thermodynamic parameters that can affect the efficiency of target cleavage, in an attempt to identify rules for the selection of functional ribozymes.


We employed the Sfold program for RNA secondary structure prediction, to account for the likely population of target structures that co-exist in dynamic equilibrium for a specific mRNA molecule. We designed and prepared 15 hammerhead ribozymes to target GUC cleavage sites in the mRNA of the breast cancer resistance protein (BCRP). These ribozymes were tested, and their catalytic activities were measured in vitro. We found that target disruption energy owing to the alteration of the local target structure necessary for ribozyme binding, and the total energy change of the ribozyme-target hybridization, are two significant parameters for prediction of ribozyme activity. Importantly, target disruption energy is the major contributor to the predictability of ribozyme activity by the total energy change. Furthermore, for a target-site specific ribozyme, incorrect folding of the catalytic core, or interactions involving the two binding arms and the end sequences of the catalytic core, can have detrimental effects on ribozyme activity.


The findings from this study suggest rules for structure-based rational design of trans-cleaving hammerhead ribozymes in gene knockdown studies. Tools implementing these rules are available from the Sribo module and the Srna module of the Sfold program available through Web server at webcite.