Abstract
BackgroundRibozymes 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.ResultsWe 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.ConclusionThe 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 .
Highlights
Ribozymes are small catalytic RNAs that possess the dual functions of sequencespecific RNA recognition and site-specific cleavage
Measurement of ribozyme activity Traditionally, ribozyme activity is determined through in vitro cleavage followed by gel electrophoresis; the latter most often uses a radiolabeled substrate RNA combined with autoradiography [39], non-radioactive detection by ethidium bromide staining has been employed [40]
Ribozyme GUC7 was incubated for varying lengths of time with the appropriate substrate RNA, and the remaining substrate was analyzed either by agarose gel electrophoresis followed by densitometry, or else by real time RT-PCR on the LightCycler
Summary
Ribozymes are small catalytic RNAs that possess the dual functions of sequencespecific RNA recognition and site-specific cleavage. Ribozymes are short catalytic RNAs that possess the dual functions of sequence-specific RNA recognition and sitespecific cleavage. The catalytic core of the ribozyme contains helix II and largely conserved nucleotides. For inhibition of the expression of a gene through targeting of the gene's mRNA, trans-cleaving hammerhead ribozymes can be engineered with binding arms whose sequences are complementary to the target mRNA sequences flanking a cleavage triplet NUH, where N is any nucleotide and H is any nucleotide except. Among all possible NUH combinations, cleavage at GUC (see Figure 1A) has been reported to be the most effective [2]
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