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Improved siRNA/shRNA functionality by mismatched duplex.

ABSTRACT: siRNA (small interfering RNA) and shRNA (small hairpin RNA) are powerful and commonly used tools in biomedical research. Currently, siRNAs are generally designed as two 21 nt strands of RNA that include a 19 nt completely complementary part and a 2 nt overhang. However, since the si/shRNAs use the endogenous miRNA machinery for gene silencing and the miRNAs are generally 22 nt in length and contain multiple internal mismatches, we tested if the functionality can be increased by designing the si/shRNAs to mimic a miRNA structure. We systematically investigated the effect of single or multiple mismatches introduced in the passenger strand at different positions on siRNA functionality. Mismatches at certain positions could significantly increase the functionality of siRNAs and also, in some cases decreased the unwanted passenger strand functionality. The same strategy could also be used to design shRNAs. Finally, we showed that both si and miRNA structured oligos (siRNA with or without mismatches in the passenger strand) can repress targets in all individual Ago containing cells, suggesting that the Ago proteins do not differentiate between si/miRNA-based structure for silencing activity.

SUBMITTER: Wu H

PROVIDER: S-EPMC3235145 | biostudies-literature | 2011

REPOSITORIES: biostudies-literature

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Improved siRNA/shRNA functionality by mismatched duplex.

Wu Haoquan H Ma Hongming H Ye Chunting C Ramirez Danielle D Chen Shuiping S Montoya Jessica J Shankar Premlata P Wang Xiaozhong A XA Manjunath N N

PloS one 20111209 12

siRNA (small interfering RNA) and shRNA (small hairpin RNA) are powerful and commonly used tools in biomedical research. Currently, siRNAs are generally designed as two 21 nt strands of RNA that include a 19 nt completely complementary part and a 2 nt overhang. However, since the si/shRNAs use the endogenous miRNA machinery for gene silencing and the miRNAs are generally 22 nt in length and contain multiple internal mismatches, we tested if the functionality can be increased by designing the si/ ...[more]

PMID: 22174840

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Project description:Prediction of efficient oligonucleotides for RNA interference presents a serious challenge, especially for the development of genome-wide RNAi libraries which encounter difficulties and limitations due to ambiguities in the results and the requirement for significant computational resources. Here we present a fast and practical algorithm for shRNA design based on the thermodynamic parameters. In order to identify shRNA and siRNA features universally associated with high silencing efficiency, we analyzed structure-activity relationships in thousands of individual RNAi experiments from publicly available databases (ftp://ftp.ncbi.nlm.nih.gov/pub/shabalin/siRNA/si_shRNA_selector/). Using this statistical analysis, we found free energy ranges for the terminal duplex asymmetry and for fully paired duplex stability, such that shRNAs or siRNAs falling in both ranges have a high probability of being efficient. When combined, these two parameters yield a approximately 72% success rate on shRNAs from the siRecords database, with the target RNA levels reduced to below 20% of the control. Two other parameters correlate well with silencing efficiency: the stability of target RNA and the antisense strand secondary structure. Both parameters also correlate with the short RNA duplex stability; as a consequence, adding these parameters to our prediction scheme did not substantially improve classification accuracy. To test the validity of our predictions, we designed 83 shRNAs with optimal terminal asymmetry, and experimentally verified that small shifts in duplex stability strongly affected silencing efficiency. We showed that shRNAs with short fully paired stems could be successfully selected by optimizing only two parameters: terminal duplex asymmetry and duplex stability of the hypothetical cleavage product, which also relates to the specificity of mRNA target recognition. Our approach performs at the level of the best currently utilized algorithms that take into account prediction of the secondary structure of the target and antisense RNAs, but at significantly lower computational costs. Based on this study, we created the si-shRNA Selector program that predicts both highly efficient shRNAs and functional siRNAs (ftp://ftp.ncbi.nlm.nih.gov/pub/shabalin/siRNA/si_shRNA_selector/).

Dataset Information

Improved siRNA/shRNA functionality by mismatched duplex.

Publications

Improved siRNA/shRNA functionality by mismatched duplex.

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