BACKGROUND
ribozymes are short catalytic rnas that possess the dual functions of sequence-specific rna recognition and site-specific cleavage.
for the self-cleaving  hammerhead ribozyme discovered by haseloff and gerlach  <cit> , the binding arms at the 5' and 3' ends of the ribozyme form two helices, termed helix i and helix iii, with the substrate.
the catalytic core of the ribozyme contains helix ii and largely conserved nucleotides.
these structure and sequence features are illustrated in figure 1a for the hammerhead conformation of a specific mrna-targeting ribozyme studied here.
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 g. among all possible nuh combinations, cleavage at guc  has been reported to be the most effective  <cit> .
in recent years, gene silencing by rna interference   <cit>  has emerged as a powerful tool for gene knockdown studies.
however, applications of rnai are limited to eukaryotes, whereas ribozymes can be applied in both eukaryotic and prokaryotic systems  <cit> .
furthermore, off-target effects by rnai have been well noted  <cit> .
ribozymes, in contrast, generally possess high target specificity, owing to the dual requirements for cleavage, i.e., complementarity for the binding arms and a cleavage triplet.
on the other hand, the knockdown effects by rnai are stronger than those for ribozymes, suggesting that the two technologies can play complementary roles in functional genomics  <cit> .
ribozyme libraries constructed with randomized binding arms have been employed for successful identification of novel functional genes in mammalian cells  <cit> .
the activities of trans-cleaving ribozymes can vary greatly for different sites on the same target mrna.
such variability is considered to be largely due to differences in the accessibility of the target sites  <cit> .
there is compelling evidence that, to a large extent, the secondary structure of an mrna molecule determines the accessibility of the mrna for numerous gene regulatory mechanisms that require complementary base-pairing for target recognition, including translational inhibition by antisense oligonucleotides  <cit> , target cleavage by ribozymes  <cit>  and sirnas  <cit> , and, more recently, repression of translation by micrornas  <cit> .
experimental approaches to the identification of accessible target sites are tedious and time-consuming.
the design of effective ribozymes presents a challenge and has largely been based on trial and error.
several computational methods  <cit>  make accessibility predictions through structures predicted by the free energy minimization approach  <cit> .
however, this method is not well suited to characterization of the likely population of structures that can exist in dynamic equilibrium in vivo for a specific mrna molecule  <cit> .
in recent years, an alternative sampling approach to rna secondary structure prediction has been developed  <cit>  and has been implemented as the sfold program  <cit> .
the structure sampling algorithm generates a statistically representative sample from the boltzmann-weighted ensemble of rna secondary structures for the rna.
in comparison to the minimum free energy method, this approach has been shown to better represent the likely population of mrna structures  <cit> , and to make improved predictions for structural rnas  <cit> .
not surprisingly, predictions by sfold significantly correlate with experimental results in gene down-regulation studies using antisense oligonucleotides  <cit> , rnai  <cit> , or micrornas  <cit> ; in contrast, a lack of significant correlation was found in these applications for predictions based on free energy minimization.
here, we explore the potential value of using sfold to predicting activities of hammerhead ribozymes.
in this study, a set of hammerhead ribozymes targeted to the transcript of the human abcg <dig> gene encoding the breast cancer resistance protein  were designed and analyzed in vitro .
we considered a number of structural and thermodynamic parameters that can affect the activity of a hammerhead ribozyme.
several of these parameters were computed with structures predicted by sfold for the target mrna and for the hammerhead ribozyme.
one of the parameters measures the target accessibility, and is termed the target disruption energy; it represents the energy cost expended in altering the local target structure so as to allow ribozyme binding .
we found that the target disruption energy and the total energy change of the ribozyme-target hybridization are two significant parameters for prediction of ribozyme activity.
the target disruption energy is the major contributor to the predictability by the total energy change.
in addition, for the ribozyme itself, 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.
these findings suggest rules for a structure-based rational design of trans-cleaving hammerhead ribozymes.
a ribozymes guc <dig>  guc <dig>  guc <dig> and guc <dig> were not tested.
b position of g of the guc cleavage site in the abcg <dig> cdna relative to the a of the atg start codon .
RESULTS
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  <cit> , although non-radioactive detection by ethidium bromide staining has also been employed  <cit> .
for both methods, quantification then requires densitometry of the cleavage products on the gel.
here, we wished to evaluate whether ribozyme cleavage activity can also be measured via quantitative rt-pcr.
accordingly, ribozyme guc <dig> 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.
as shown by figure  <dig>  the results from these two methods are in good agreement.
we concluded that quantitative rt-pcr is a valid method by which to determine ribozyme activity in vitro; thus, all activity measurements for our study were therefore made by this method.
statistical analyses
for the  <dig> ribozymes tested in vitro, we performed both correlation and regression analyses, using the ribozyme catalytic activity measured by , and each of the computational parameters .
first, we observed that there were two outliers, namely, ribozymes guc <dig> and guc <dig>  which behaved differently from the other  <dig> ribozymes.
we thus initially focused on the analyses for the  <dig> "normally behaving" ribozymes, and then investigated possible explanations for the two outliers.
for the  <dig> well behaved ribozymes, we found, among the structural and thermodynamic parameters, that Δgdisruption and Δgtotal are significantly correlated with ribozyme activity .
the correlation coefficient for Δgdisruption is  <dig>  with a p-value of  <dig> , and the correlation coefficient for Δgtotal is - <dig>  with a p-value of  <dig> .
ribozyme activity, however, was not significantly correlated with Δgswitch, or Δghybrid.
because Δgtotal is computed from Δgdisruption, Δgswitch, and Δghybrid , Δgtotal and Δgdisruption are significantly correlated .
thus, the significance of the correlation with the ribozyme activity for Δgtotal is mainly due to Δgdisruption.
from linear regression analysis, either Δgdisruption or Δgtotal is significantly predictive of the ribozyme activity .
furthermore, in a comparison of the r <dig> values for Δgdisruption and Δgtotal, a relative improvement of about  <dig> % is observed for Δgtotal.
this suggests that, although Δgswitch and Δghybrid are insignificant as individual predictors, they do contribute to the improved predictability by Δgtotal.
the r <dig> for Δgtotal indicates that over 60% of the variability in the ribozyme cleavage activity can be attributed to Δgtotal.
a two outliers, guc <dig> and guc <dig>  were excluded in these analyses
to understand the behaviors of the two outliers, ribozymes guc <dig> and guc <dig>  we examined the structures predicted by sfold for each of the ribozymes.
in the case of ribozyme guc <dig>  we found that, for  <dig> % 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 .
in the "active" ribozyme binding conformation , 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 .
for guc <dig>  we found that  <dig> % of sampled structures have the catalytic core misfolded so that it lacks a correctly formed helix ii .
this could explain the observation that guc <dig> was the least effective for target cleavage , despite a moderately accessible target site as indicated by Δgdisruption .
in contrast, for each of the other  <dig> ribozymes in our study, the percentage of the sampled structures having a misfolded core is less than 1%.
discussion and
CONCLUSIONS
in this work, we have considered a number of structural and thermodynamic parameters and have assessed their effects on the in vitro activities of trans-cleaving hammerhead ribozymes.
we found that Δgdisruption, a measure of accessibility at the target site, and Δgtotal, a measure of the total energy change for the ribozyme-target hybridization process, are two significant parameters for predicting ribozyme activity, and that Δgdisruption is the major contributor to the predictability by Δgtotal.
in this analysis, the stability of the ribozyme-target hybrid as measured by Δghybrid had no impact on ribozyme cleavage activity.
furthermore, we found that incorrect folding of the ribozyme catalytic core or interactions involving the two binding arms and the end sequences of the catalytic core can have detrimental effects on ribozyme activity.
for the selection of functional ribozymes for gene knock-down studies, it is advisable to choose highly accessible target sites  and favorable Δgtotal.
in addition, a ribozyme with either a misfolded core or structures that are substantially different from the "active" binding conformation  should be avoided.
our analyses are limited to modeling of rna secondary structures.
the r <dig> of  <dig> % for Δgtotal indicates that the remaining  <dig> % of the variability in the ribozyme cleavage activity must be attributed to other factors that likely include rna tertiary interactions  <cit> .
we adopted a population approach to modeling of mrna secondary structure, by employing the sfold program.
in an antisense application, predictions by sfold were significantly correlated with activity of antisense oligonucleotides, whereas there was a lack of correlation for the minimum free energy  predictions  <cit> .
in an rnai application, the sfold predictions were significantly predictive of rnai efficacy  <cit> .
in contrast, the predictive significance was either lacking or poor in terms of r <dig> for predictions by mfe structures, by heuristic suboptimal foldings  <cit> , or by complete suboptimal foldings  <cit> .
furthermore, for all of these rna folding programs, only sfold predictions were found to fully explain sensitivity of lin- <dig> mutants to microrna repression by let- <dig>  <cit> .
in the current application, if we employ the minimum free energy predictions by mfold  <cit>  in the calculation of Δgdisruption and Δgswitch, we observe an insignificant correlation with the ribozyme activity for Δgdisruption , and substantially reduced significance and predictability for Δgtotal .
this finding further validates the sampling approach to characterization of the likely population of mrna structures.
in the calculation of Δgdisruption, we assumed that the binding of target mrna by a ribozyme induces only a local structural alteration at the target site.
it is likely that in some, if not all, cases, nucleotides outside the target site will also contribute to the energy change due to ribozyme binding.
an alternative to the local disruption model is a global disruption model, which assumes that the rest of the target mrna molecule can refold after ribozyme binding.
for this model, Δgdisruption can be re-calculated by constraining the target site to be single-stranded and refolding the rest of the target mrna.
surprisingly, we observed insignificant correlation and poor predictability either for Δgdisruption , or for Δgtotal .
these results suggest that target cleavage occurs rapidly after the completion of ribozyme-target hybridization such that global refolding of the target before cleavage is unlikely.
while partial refolding is a possibility, it is highly uncertain what region of the target will be involved in refolding.
thus, it is difficult to construct a computational model that can represent a reasonable compromise between the local model and the global model.
when the standard deviations for the measured activities are available, an alternative to the standard linear regression analysis is the weighted regression analysis.
in a weighted least-squares regression, the square term in the sum of squares for a data point is multiplied by a weight  <cit> .
in general, a larger weight is assigned to a data point with a higher precision as indicated by a smaller standard deviation.
specifically, with the standard deviation of cleavage activity available for every ribozyme , 1/ <dig>  that is, 1/variance, can be used as the weight  <cit> .
the weighted regression yielded results that are highly similar to those from the un-weighted regression analysis.
for example, for Δgtotal, the r <dig> is  <dig> , with a p-value of  <dig> .
while we focused on guc cleavage sites in the present study, we have also tested a small set of non-guc nuh sites.
we observed a similar level of correlation between ribozyme activity and Δgdisruption , suggesting that the critical parameters identified here are generally relevant for the prediction of the activity of hammerhead ribozymes.
however, the levels of activities for non-guc sites were generally lower than those for the guc sites, consistent with a previous report that guc is the most effective target site  <cit> .
to generalize our findings for guc target sites, further in vitro testing using other targets, as well as in vivo testing in cultured cells, will be required.
the latter is currently in progress for the ribozymes described here.

 ribozymes are short catalytic rnas that possess the dual functions of sequence-specific rna recognition and site-specific cleavage. for the self-cleaving hammerhead ribozyme discovered by haseloff and gerlach, the binding arms at the 5' and 3' ends of the ribozyme form two helices, termed helix i and helix iii, with the substrate. the catalytic core of the ribozyme contains helix ii and largely conserved nucleotides. these structure and sequence features are illustrated in figure 1a for the hammerhead conformation of a specific mrna-targeting ribozyme studied here. 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 g. among all possible nuh combinations, cleavage at guc has been reported to be the most effective. in recent years, gene silencing by rna interference has emerged as a powerful tool for gene knockdown studies. however, applications of rnai are limited to eukaryotes, whereas ribozymes can be applied in both eukaryotic and prokaryotic systems. furthermore, off-target effects by rnai have been well noted. ribozymes, in contrast, generally possess high target specificity, owing to the dual requirements for cleavage, i.e., complementarity for the binding arms and a cleavage triplet. on the other hand, the knockdown effects by rnai are stronger than those for ribozymes, suggesting that the two technologies can play complementary roles in functional genomics. ribozyme libraries constructed with randomized binding arms have been employed for successful identification of novel functional genes in mammalian cells. the activities of trans-cleaving ribozymes can vary greatly for different sites on the same target mrna. such variability is considered to be largely due to differences in the accessibility of the target sites. there is compelling evidence that, to a large extent, the secondary structure of an mrna molecule determines the accessibility of the mrna for numerous gene regulatory mechanisms that require complementary base-pairing for target recognition, including translational inhibition by antisense oligonucleotides, target cleavage by ribozymes and sirnas, and, more recently, repression of translation by micrornas. experimental approaches to the identification of accessible target sites are tedious and time-consuming. the design of effective ribozymes presents a challenge and has largely been based on trial and error. several computational methods make accessibility predictions through structures predicted by the free energy minimization approach. however, this method is not well suited to characterization of the likely population of structures that can exist in dynamic equilibrium in vivo for a specific mrna molecule. in recent years, an alternative sampling approach to rna secondary structure prediction has been developed and has been implemented as the sfold program. the structure sampling algorithm generates a statistically representative sample from the boltzmann-weighted ensemble of rna secondary structures for the rna. in comparison to the minimum free energy method, this approach has been shown to better represent the likely population of mrna structures, and to make improved predictions for structural rnas. not surprisingly, predictions by sfold significantly correlate with experimental results in gene down-regulation studies using antisense oligonucleotides, rnai, or micrornas; in contrast, a lack of significant correlation was found in these applications for predictions based on free energy minimization. here, we explore the potential value of using sfold to predicting activities of hammerhead ribozymes. in this study, a set of hammerhead ribozymes targeted to the transcript of the human abcg gene encoding the breast cancer resistance protein were designed and analyzed in vitro. we considered a number of structural and thermodynamic parameters that can affect the activity of a hammerhead ribozyme. several of these parameters were computed with structures predicted by sfold for the target mrna and for the hammerhead ribozyme. one of the parameters measures the target accessibility, and is termed the target disruption energy; it represents the energy cost expended in altering the local target structure so as to allow ribozyme binding. we found that the target disruption energy and the total energy change of the ribozyme-target hybridization are two significant parameters for prediction of ribozyme activity. the target disruption energy is the major contributor to the predictability by the total energy change. in addition, for the ribozyme itself, 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. these findings suggest rules for a structure-based rational design of trans-cleaving hammerhead ribozymes. a ribozymes guc guc guc and guc were not tested. b position of g of the guc cleavage site in the abcg cdna relative to the a of the atg start codon.  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, although non-radioactive detection by ethidium bromide staining has also been employed. for both methods, quantification then requires densitometry of the cleavage products on the gel. here, we wished to evaluate whether ribozyme cleavage activity can also be measured via quantitative rt-pcr. accordingly, ribozyme guc 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. as shown by figure the results from these two methods are in good agreement. we concluded that quantitative rt-pcr is a valid method by which to determine ribozyme activity in vitro; thus, all activity measurements for our study were therefore made by this method. statistical analyses for the ribozymes tested in vitro, we performed both correlation and regression analyses, using the ribozyme catalytic activity measured by, and each of the computational parameters. first, we observed that there were two outliers, namely, ribozymes guc and guc which behaved differently from the other ribozymes. we thus initially focused on the analyses for the "normally behaving" ribozymes, and then investigated possible explanations for the two outliers. for the well behaved ribozymes, we found, among the structural and thermodynamic parameters, that Δgdisruption and Δgtotal are significantly correlated with ribozyme activity. the correlation coefficient for Δgdisruption is with a p-value of, and the correlation coefficient for Δgtotal is - with a p-value of. ribozyme activity, however, was not significantly correlated with Δgswitch, or Δghybrid. because Δgtotal is computed from Δgdisruption, Δgswitch, and Δghybrid, Δgtotal and Δgdisruption are significantly correlated. thus, the significance of the correlation with the ribozyme activity for Δgtotal is mainly due to Δgdisruption. from linear regression analysis, either Δgdisruption or Δgtotal is significantly predictive of the ribozyme activity. furthermore, in a comparison of the r values for Δgdisruption and Δgtotal, a relative improvement of about % is observed for Δgtotal. this suggests that, although Δgswitch and Δghybrid are insignificant as individual predictors, they do contribute to the improved predictability by Δgtotal. the r for Δgtotal indicates that over 60% of the variability in the ribozyme cleavage activity can be attributed to Δgtotal. a two outliers, guc and guc were excluded in these analyses to understand the behaviors of the two outliers, ribozymes guc and guc we examined the structures predicted by sfold for each of the ribozymes. in the case of ribozyme guc we found that, for % 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. in the "active" ribozyme binding conformation, 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. for guc we found that % of sampled structures have the catalytic core misfolded so that it lacks a correctly formed helix ii. this could explain the observation that guc was the least effective for target cleavage, despite a moderately accessible target site as indicated by Δgdisruption. in contrast, for each of the other ribozymes in our study, the percentage of the sampled structures having a misfolded core is less than 1%. discussion and  in this work, we have considered a number of structural and thermodynamic parameters and have assessed their effects on the in vitro activities of trans-cleaving hammerhead ribozymes. we found that Δgdisruption, a measure of accessibility at the target site, and Δgtotal, a measure of the total energy change for the ribozyme-target hybridization process, are two significant parameters for predicting ribozyme activity, and that Δgdisruption is the major contributor to the predictability by Δgtotal. in this analysis, the stability of the ribozyme-target hybrid as measured by Δghybrid had no impact on ribozyme cleavage activity. furthermore, we found that incorrect folding of the ribozyme catalytic core or interactions involving the two binding arms and the end sequences of the catalytic core can have detrimental effects on ribozyme activity. for the selection of functional ribozymes for gene knock-down studies, it is advisable to choose highly accessible target sites and favorable Δgtotal. in addition, a ribozyme with either a misfolded core or structures that are substantially different from the "active" binding conformation should be avoided. our analyses are limited to modeling of rna secondary structures. the r of % for Δgtotal indicates that the remaining % of the variability in the ribozyme cleavage activity must be attributed to other factors that likely include rna tertiary interactions. we adopted a population approach to modeling of mrna secondary structure, by employing the sfold program. in an antisense application, predictions by sfold were significantly correlated with activity of antisense oligonucleotides, whereas there was a lack of correlation for the minimum free energy predictions. in an rnai application, the sfold predictions were significantly predictive of rnai efficacy. in contrast, the predictive significance was either lacking or poor in terms of r for predictions by mfe structures, by heuristic suboptimal foldings, or by complete suboptimal foldings. furthermore, for all of these rna folding programs, only sfold predictions were found to fully explain sensitivity of lin- mutants to microrna repression by let-. in the current application, if we employ the minimum free energy predictions by mfold in the calculation of Δgdisruption and Δgswitch, we observe an insignificant correlation with the ribozyme activity for Δgdisruption, and substantially reduced significance and predictability for Δgtotal. this finding further validates the sampling approach to characterization of the likely population of mrna structures. in the calculation of Δgdisruption, we assumed that the binding of target mrna by a ribozyme induces only a local structural alteration at the target site. it is likely that in some, if not all, cases, nucleotides outside the target site will also contribute to the energy change due to ribozyme binding. an alternative to the local disruption model is a global disruption model, which assumes that the rest of the target mrna molecule can refold after ribozyme binding. for this model, Δgdisruption can be re-calculated by constraining the target site to be single-stranded and refolding the rest of the target mrna. surprisingly, we observed insignificant correlation and poor predictability either for Δgdisruption, or for Δgtotal. these results suggest that target cleavage occurs rapidly after the completion of ribozyme-target hybridization such that global refolding of the target before cleavage is unlikely. while partial refolding is a possibility, it is highly uncertain what region of the target will be involved in refolding. thus, it is difficult to construct a computational model that can represent a reasonable compromise between the local model and the global model. when the standard deviations for the measured activities are available, an alternative to the standard linear regression analysis is the weighted regression analysis. in a weighted least-squares regression, the square term in the sum of squares for a data point is multiplied by a weight. in general, a larger weight is assigned to a data point with a higher precision as indicated by a smaller standard deviation. specifically, with the standard deviation of cleavage activity available for every ribozyme, 1/ that is, 1/variance, can be used as the weight. the weighted regression yielded results that are highly similar to those from the un-weighted regression analysis. for example, for Δgtotal, the r is, with a p-value of. while we focused on guc cleavage sites in the present study, we have also tested a small set of non-guc nuh sites. we observed a similar level of correlation between ribozyme activity and Δgdisruption, suggesting that the critical parameters identified here are generally relevant for the prediction of the activity of hammerhead ribozymes. however, the levels of activities for non-guc sites were generally lower than those for the guc sites, consistent with a previous report that guc is the most effective target site. to generalize our findings for guc target sites, further in vitro testing using other targets, as well as in vivo testing in cultured cells, will be required. the latter is currently in progress for the ribozymes described here. 
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. ribozymes are small catalytic rnas that possess the dual functions of sequence-specific rna recognition and site-specific cleavage. 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. the findings from this study suggest rules for structure-based rational design of trans-cleaving hammerhead ribozymes in gene knockdown studies. we designed and prepared hammerhead ribozymes to target guc cleavage sites in the mrna of the breast cancer resistance protein. tools implementing these rules are available from the sribo module and the srna module of the sfold program available through web server at.
 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 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 hammerhead ribozymes to target guc cleavage sites in the mrna of the breast cancer resistance protein. 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. 