A Structural Interpretation of the Effect of GC-Content on Efficiency of RNA Interference

Chi Yu Chan, Wadsworth Center, New York State Department of Health
C Steven Carmack
Dang D. Long
Anil Maliyekkel
Yu Shao
Igor Roninson, Department of Drug Discovery and Biomedical Sciences, University of South Carolina
Ye Ding

© 2009 Chan et al; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


BACKGROUND: RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) 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 (mRNA) and the siRNA guide strand. RESULTS: By analyzing a unique homogeneous data set of 101 shRNAs targeted to 100 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 62.6% for paired bases is significantly higher than the GC content of 38.7% 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. CONCLUSION: These findings provide a target-structure-based interpretation and mechanistic insight for the effect of GC-content on RNAi efficiency.