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A structural analysis of in vitro catalytic activities of hammerhead ribozymes.

Shao Y, Wu S, Chan CY, Klapper JR, Schneider E, Ding Y - BMC Bioinformatics (2007)

Bottom Line: These ribozymes were tested, and their catalytic activities were measured in vitro.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wadsworth Center, New York State Department of Health, 150 New Scotland Avenue, Albany, NY 12208, USA. yushao04@yahoo.com

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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 http://sfold.wadsworth.org.

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Structural analysis of GUC11, the other outlier in Figure 3. For a substantial portion of the structure sample generated by Sfold for GUC11, the predicted structure has a misfolded catalytic core. (A) Ribozyme in "active" binding conformation with correctly folded core (i.e., helix II and single stranded end sequences of the core including 9 conserved nucleotides, as shown in Figure 1A), representing 66.5% of the predicted structures. (B) A conformation with a misfolded core, representing 28% of the predicted structures. (C) Another conformation with a misfolded core, representing 5.5% of the predicted structures. The sequence for the ribozyme 5' binding arm ends at A9, and the sequence for the ribozyme 3' binding arm starts at A32.
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Figure 5: Structural analysis of GUC11, the other outlier in Figure 3. For a substantial portion of the structure sample generated by Sfold for GUC11, the predicted structure has a misfolded catalytic core. (A) Ribozyme in "active" binding conformation with correctly folded core (i.e., helix II and single stranded end sequences of the core including 9 conserved nucleotides, as shown in Figure 1A), representing 66.5% of the predicted structures. (B) A conformation with a misfolded core, representing 28% of the predicted structures. (C) Another conformation with a misfolded core, representing 5.5% of the predicted structures. The sequence for the ribozyme 5' binding arm ends at A9, and the sequence for the ribozyme 3' binding arm starts at A32.

Mentions: To understand the behaviors of the two outliers, ribozymes GUC3 and GUC11, we examined the structures predicted by Sfold for each of the ribozymes. In the case of ribozyme GUC3, we found that, for 79.1% of the structures, there are at least four base pairs formed by nucleotides in the two binding arms and the ends of the catalytic core sequence (Figure 4). In the "active" ribozyme binding conformation (Figure 1B), all of not only the binding arms but also the end sequences of the catalytic core are single-stranded. Thus, substantial intramolecular structure involving these regions can hinder target binding by the ribozyme, despite the correct formation of helix II (Fig 1A). For GUC11, we found that 33.5% of sampled structures have the catalytic core misfolded so that it lacks a correctly formed helix II (Figure 5). This could explain the observation that GUC11 was the least effective for target cleavage (Table 1, Figure 3), despite a moderately accessible target site as indicated by ΔGdisruption (Table 1). In contrast, for each of the other 14 ribozymes in our study, the percentage of the sampled structures having a misfolded core is less than 1%.


A structural analysis of in vitro catalytic activities of hammerhead ribozymes.

Shao Y, Wu S, Chan CY, Klapper JR, Schneider E, Ding Y - BMC Bioinformatics (2007)

Structural analysis of GUC11, the other outlier in Figure 3. For a substantial portion of the structure sample generated by Sfold for GUC11, the predicted structure has a misfolded catalytic core. (A) Ribozyme in "active" binding conformation with correctly folded core (i.e., helix II and single stranded end sequences of the core including 9 conserved nucleotides, as shown in Figure 1A), representing 66.5% of the predicted structures. (B) A conformation with a misfolded core, representing 28% of the predicted structures. (C) Another conformation with a misfolded core, representing 5.5% of the predicted structures. The sequence for the ribozyme 5' binding arm ends at A9, and the sequence for the ribozyme 3' binding arm starts at A32.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2238771&req=5

Figure 5: Structural analysis of GUC11, the other outlier in Figure 3. For a substantial portion of the structure sample generated by Sfold for GUC11, the predicted structure has a misfolded catalytic core. (A) Ribozyme in "active" binding conformation with correctly folded core (i.e., helix II and single stranded end sequences of the core including 9 conserved nucleotides, as shown in Figure 1A), representing 66.5% of the predicted structures. (B) A conformation with a misfolded core, representing 28% of the predicted structures. (C) Another conformation with a misfolded core, representing 5.5% of the predicted structures. The sequence for the ribozyme 5' binding arm ends at A9, and the sequence for the ribozyme 3' binding arm starts at A32.
Mentions: To understand the behaviors of the two outliers, ribozymes GUC3 and GUC11, we examined the structures predicted by Sfold for each of the ribozymes. In the case of ribozyme GUC3, we found that, for 79.1% of the structures, there are at least four base pairs formed by nucleotides in the two binding arms and the ends of the catalytic core sequence (Figure 4). In the "active" ribozyme binding conformation (Figure 1B), all of not only the binding arms but also the end sequences of the catalytic core are single-stranded. Thus, substantial intramolecular structure involving these regions can hinder target binding by the ribozyme, despite the correct formation of helix II (Fig 1A). For GUC11, we found that 33.5% of sampled structures have the catalytic core misfolded so that it lacks a correctly formed helix II (Figure 5). This could explain the observation that GUC11 was the least effective for target cleavage (Table 1, Figure 3), despite a moderately accessible target site as indicated by ΔGdisruption (Table 1). In contrast, for each of the other 14 ribozymes in our study, the percentage of the sampled structures having a misfolded core is less than 1%.

Bottom Line: These ribozymes were tested, and their catalytic activities were measured in vitro.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Wadsworth Center, New York State Department of Health, 150 New Scotland Avenue, Albany, NY 12208, USA. yushao04@yahoo.com

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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 http://sfold.wadsworth.org.

Show MeSH
Related in: MedlinePlus