BACKGROUND
rna interference   <cit>  is a sequence-specific gene silencing mechanism that can be mediated either by small interfering rnas  of about  <dig> nt with two-nucleotide 3' overhang  <cit> , or by stably expressed short hairpin rnas , which are processed by dicer into sirnas  <cit> .
the antisense  strand guides argonaute <dig> , the catalytic component of the rna-induced silencing complex , to cleave mrna by base-pairing with the complementary site in the target.
large variation in the efficiency of sirnas has been commonly observed  <cit> .
usually, only a small proportion of randomly selected sirnas are potent.
thus, there has been a great interest in determining rules for improvement of rnai design.
it has been commonly observed that high gc content negatively correlates with rnai activity.
thus, a low gc-content is among a number of empirical rules on sirna duplex features that have been proposed  <cit> .
in addition, the importance of target secondary structure and accessibility has been supported by numerous studies  <cit> .
it is tempting to seek a mechanistic interpretation for the effect of gc-content on rnai efficiency.
because high gc can give rise to stable rna secondary structure, one possible interpretation is the proposal that self-structure of the sirna guide strand can be detrimental to rnai activity  <cit> .
we here investigate this issue by considering the secondary structures for both the target messenger rna  and the sirna guide strand.
from analyses of a unique homogeneous data set of  <dig> shrnas targeted to  <dig> endogenous human genes, the results support a target-structure-based interpretation for the effect of gc-content.
RESULTS
for the shrna dataset, we first computed pearson's correlation coefficient and the significance of the correlation for the rnai activity and a structural parameter or gc%.
these calculations were performed by using the r statistical package  <cit> , and the results were summarized in table  <dig>  we find that, with the highest and significant correlation, target site accessibility is more important than gc-content.
we also observe an appreciable negative correlation of - <dig>  between gc-content and rnai activity, albeit with a p-value of  <dig> .
surprisingly, for the predicted optimal structure for the sirna guide strand, there is a lack of correlation between rnai activity and either the stability  or the number of free dangling nucleotides at an end of the structure.
because the effect of gc-content cannot be explained by the structure of the sirna guide strand, we hypothesized that, to some extent, gc-content is indicative of target site accessibility.
indeed, there is a highly significant correlation between target site accessibility and gc-content .
furthermore, for the representative set of structural rnas,  <dig> % of paired bases are gc, significantly higher than  <dig> % for unpaired bases .
thus, for a structured rna, a region with higher gc content is likely to have more stable secondary structure.
to further investigate the relationships among gc-content, target accessibility and rnai activity, we performed partial correlation analysis.
partial correlation measures the degree of association between two random variables, with the effect of a set of controlling random variables removed.
for the three variables of our interest here, the calculation was performed with a published r script for three variables  <cit> .
by partial correlation analysis, we found that when the effect of target accessibility is controlled , the correlation between gc-content and rnai activity is  <dig> , with a p-value of  <dig> .
this near complete diminishment of correlation supports the hypothesis that the negative correlation typically observed between gc-content and rnai efficiency is mainly due to structural inaccessibility often associated with high gc-content of the target site.
because the protein complexes involved in gene regulation are similar for micrornas  <cit>  and sirnas, we folded all of  <dig> worm micrornas from microrna registry  <cit> .
we found that for  <dig>  of the micrornas, the predicted optimal structure has a stability  under  <dig>  kcal/mol, typically with two to five consecutive base pairs.
furthermore, for  <dig>  of the micrornas, either the 5' end or the 3' end is completely involved in intramolecular base-pairing.
these suggest that some intramolecular structures can be tolerated for the regulatory functions by animal micrornas, and these structures are likely to be weakened or completely abolished upon interaction with the risc.
discussion
the results of the analyses suggest that, to a large extent, the effect of gc-content on rnai is due to the target structure and site accessibility rather than the structure of the sirna guide strand.
however, for the purpose of the rational design of rnai experiments, gc-content cannot be a substitute for predicted target site accessibility, owning to its substantially lower correlation .
similar observations were made in a previous analysis of other rnai datasets based on alternative structural calculations  <cit> .
the common findings from this study and the previous study support a target-structure-based interpretation for the effect of gc-content on the rnai efficiency.
contrasting with the conclusion from a published study  <cit> , we did not observe any effect of potential folding of sirna guide strand on rnai efficiency.
it is likely that the ability for the sirna guide strand to fold may be negatively affected by the enzymatic activity by the rnai machinery including duplex unwinding by helicase.
in addition, constrained by risc, the sirna guide strand is unlikely to fold freely into a stable structure, regardless of gc-content.
based on nine sirnas with  <dig> common nts for a region of a single target, it was reported that the rnai activity was strongly correlated with the number of free  dangling nts at the ends of the structure predicted for the sirna guide strand, and there was a poor correlation for other sequence features and target accessibility  <cit> .
with our much larger and more representative dataset, we did not observe any correlation for the number of free dangling nts.
for the negative finding on target accessibility, there may be two reasons.
first, the accessibility was calculated with probabilities of unpaired individual bases in the boltzmann ensemble of rna structures  <cit> .
although this represents a major improvement over the use of a single structure such as the optimal fold, the accessibility can be arguably better assessed by consideration of free energy changes for sirna-target hybridization  <cit> .
second, the assessment of the effect of target accessibility on target recognition and rnai activity requires controlling for the upstream effect of sirna duplex asymmetry  <cit> .
the small data set of nine sirnas is inadequate.
the analysis of  <dig> worm micrornas suggests that some intramolecular structures can be tolerated for the regulatory functions by micrornas, and these structures are likely to be weakened or completely abolished upon interaction with the risc.
target cleavage by rnai machinery and translation repression by microrna pathway may have different effects on the structure of the target and the small rna:target duplex.
it is possible that the effects of intramolecular structure can be different between sirnas and micrornas, so that the lack of negative effect of structure for sirnas cannot be simply reasoned by the lack of self-folding effect for micrornas.
nevertheless, the results of our analyses on the shrna data do not support the previous conclusion on the significance of the effects of structures of the sirna guide strands  <cit> .
CONCLUSIONS
target accessibility as primarily determined by target secondary structure is an important determinant for rnai potency.
the commonly observed negative effect of high sirna gc-content on rnai potency is due to generally poor target accessibility for a high gc target site, rather than the likelihood that the high gc sirna guide strand may form stable intramolecular secondary structure.
these findings provide a target-structure-based interpretation and mechanistic insight for the effect of sirna gc-content on rnai efficiency.

 rna interference is a sequence-specific gene silencing mechanism that can be mediated either by small interfering rnas of about nt with two-nucleotide 3' overhang, or by stably expressed short hairpin rnas, which are processed by dicer into sirnas. the antisense strand guides argonaute, the catalytic component of the rna-induced silencing complex, to cleave mrna by base-pairing with the complementary site in the target. large variation in the efficiency of sirnas has been commonly observed. usually, only a small proportion of randomly selected sirnas are potent. thus, there has been a great interest in determining rules for improvement of rnai design. it has been commonly observed that high gc content negatively correlates with rnai activity. thus, a low gc-content is among a number of empirical rules on sirna duplex features that have been proposed. in addition, the importance of target secondary structure and accessibility has been supported by numerous studies. it is tempting to seek a mechanistic interpretation for the effect of gc-content on rnai efficiency. because high gc can give rise to stable rna secondary structure, one possible interpretation is the proposal that self-structure of the sirna guide strand can be detrimental to rnai activity. we here investigate this issue by considering the secondary structures for both the target messenger rna and the sirna guide strand. from analyses of a unique homogeneous data set of shrnas targeted to endogenous human genes, the results support a target-structure-based interpretation for the effect of gc-content.  for the shrna dataset, we first computed pearson's correlation coefficient and the significance of the correlation for the rnai activity and a structural parameter or gc%. these calculations were performed by using the r statistical package, and the results were summarized in table we find that, with the highest and significant correlation, target site accessibility is more important than gc-content. we also observe an appreciable negative correlation of - between gc-content and rnai activity, albeit with a p-value of. surprisingly, for the predicted optimal structure for the sirna guide strand, there is a lack of correlation between rnai activity and either the stability or the number of free dangling nucleotides at an end of the structure. because the effect of gc-content cannot be explained by the structure of the sirna guide strand, we hypothesized that, to some extent, gc-content is indicative of target site accessibility. indeed, there is a highly significant correlation between target site accessibility and gc-content. furthermore, for the representative set of structural rnas, % of paired bases are gc, significantly higher than % for unpaired bases. thus, for a structured rna, a region with higher gc content is likely to have more stable secondary structure. to further investigate the relationships among gc-content, target accessibility and rnai activity, we performed partial correlation analysis. partial correlation measures the degree of association between two random variables, with the effect of a set of controlling random variables removed. for the three variables of our interest here, the calculation was performed with a published r script for three variables. by partial correlation analysis, we found that when the effect of target accessibility is controlled, the correlation between gc-content and rnai activity is, with a p-value of. this near complete diminishment of correlation supports the hypothesis that the negative correlation typically observed between gc-content and rnai efficiency is mainly due to structural inaccessibility often associated with high gc-content of the target site. because the protein complexes involved in gene regulation are similar for micrornas and sirnas, we folded all of worm micrornas from microrna registry. we found that for of the micrornas, the predicted optimal structure has a stability under kcal/mol, typically with two to five consecutive base pairs. furthermore, for of the micrornas, either the 5' end or the 3' end is completely involved in intramolecular base-pairing. these suggest that some intramolecular structures can be tolerated for the regulatory functions by animal micrornas, and these structures are likely to be weakened or completely abolished upon interaction with the risc. discussion the results of the analyses suggest that, to a large extent, the effect of gc-content on rnai is due to the target structure and site accessibility rather than the structure of the sirna guide strand. however, for the purpose of the rational design of rnai experiments, gc-content cannot be a substitute for predicted target site accessibility, owning to its substantially lower correlation. similar observations were made in a previous analysis of other rnai datasets based on alternative structural calculations. the common findings from this study and the previous study support a target-structure-based interpretation for the effect of gc-content on the rnai efficiency. contrasting with the conclusion from a published study, we did not observe any effect of potential folding of sirna guide strand on rnai efficiency. it is likely that the ability for the sirna guide strand to fold may be negatively affected by the enzymatic activity by the rnai machinery including duplex unwinding by helicase. in addition, constrained by risc, the sirna guide strand is unlikely to fold freely into a stable structure, regardless of gc-content. based on nine sirnas with common nts for a region of a single target, it was reported that the rnai activity was strongly correlated with the number of free dangling nts at the ends of the structure predicted for the sirna guide strand, and there was a poor correlation for other sequence features and target accessibility. with our much larger and more representative dataset, we did not observe any correlation for the number of free dangling nts. for the negative finding on target accessibility, there may be two reasons. first, the accessibility was calculated with probabilities of unpaired individual bases in the boltzmann ensemble of rna structures. although this represents a major improvement over the use of a single structure such as the optimal fold, the accessibility can be arguably better assessed by consideration of free energy changes for sirna-target hybridization. second, the assessment of the effect of target accessibility on target recognition and rnai activity requires controlling for the upstream effect of sirna duplex asymmetry. the small data set of nine sirnas is inadequate. the analysis of worm micrornas suggests that some intramolecular structures can be tolerated for the regulatory functions by micrornas, and these structures are likely to be weakened or completely abolished upon interaction with the risc. target cleavage by rnai machinery and translation repression by microrna pathway may have different effects on the structure of the target and the small rna:target duplex. it is possible that the effects of intramolecular structure can be different between sirnas and micrornas, so that the lack of negative effect of structure for sirnas cannot be simply reasoned by the lack of self-folding effect for micrornas. nevertheless, the results of our analyses on the shrna data do not support the previous conclusion on the significance of the effects of structures of the sirna guide strands.  target accessibility as primarily determined by target secondary structure is an important determinant for rnai potency. the commonly observed negative effect of high sirna gc-content on rnai potency is due to generally poor target accessibility for a high gc target site, rather than the likelihood that the high gc sirna guide strand may form stable intramolecular secondary structure. these findings provide a target-structure-based interpretation and mechanistic insight for the effect of sirna gc-content on rnai efficiency. 
sirna gc-content negatively correlates with rnai efficiency, and it is of interest to have a convincing mechanistic interpretation of this observation. for a set of representative structural rnas, the gc content of % for paired bases is significantly higher than the gc content of % for unpaired bases. thus, for a structured rna, a region with higher gc content is likely to have more stable secondary structure. furthermore, by partial correlation analysis, the correlation for gc-content is almost completely diminished, when the effect of target accessibility is controlled. these findings provide a target-structure-based interpretation and mechanistic insight for the effect of gc-content on rnai efficiency. by analyzing a unique homogeneous data set of shrnas targeted to endogenous human genes, we find that: 1) target site accessibility is more important than gc-content for efficient rnai; 2) there is an appreciable negative correlation between gc-content and rnai activity; 3) for the predicted structure of the sirna guide strand, there is a lack of correlation between rnai activity and either the stability or the number of free dangling nucleotides at an end of the structure; 4) there is a high correlation between target site accessibility and gc-content. rna interference mediated by small interfering rnas or short hairpin rnas has become a powerful technique for eukaryotic gene knockdown. we here examine this issue by considering the secondary structures for both the target messenger rna and the sirna guide strand.
 rna interference mediated by small interfering rnas or short hairpin rnas has become a powerful technique for eukaryotic gene knockdown. sirna gc-content negatively correlates with rnai efficiency, and it is of interest to have a convincing mechanistic interpretation of this observation. we here examine this issue by considering the secondary structures for both the target messenger rna and the sirna guide strand.  by analyzing a unique homogeneous data set of shrnas targeted to endogenous human genes, we find that: 1) target site accessibility is more important than gc-content for efficient rnai; 2) there is an appreciable negative correlation between gc-content and rnai activity; 3) for the predicted structure of the sirna guide strand, there is a lack of correlation between rnai activity and either the stability or the number of free dangling nucleotides at an end of the structure; 4) there is a high correlation between target site accessibility and gc-content. for a set of representative structural rnas, the gc content of % for paired bases is significantly higher than the gc content of % for unpaired bases. thus, for a structured rna, a region with higher gc content is likely to have more stable secondary structure. furthermore, by partial correlation analysis, the correlation for gc-content is almost completely diminished, when the effect of target accessibility is controlled.  these findings provide a target-structure-based interpretation and mechanistic insight for the effect of gc-content on rnai efficiency. 13– january the seventh asia pacific bioinformatics conference beijing, china 