{"title":"Functional shRNA expression system with reduced off-target effects","authors":"Y. Naito, K. Nishi, Aya Juni, K. Ui-Tei","doi":"10.1109/MHS.2009.5351912","DOIUrl":null,"url":null,"abstract":"The capability to induce off-target effects by small interfering RNA (siRNA) is highly correlated with the calculated melting temperature (Tm) of the protein-free seed-target duplex, indicating that its thermodynamic stability is a major factor in determining the degree of the off-target effect. In this study, we analyzed changes in microarray expression profiles following transfection of short hairpin RNAs (shRNAs). In a similar manner to siRNA, no or little off-target effects were observed by shRNA with a low Tm value in the seed-target duplex, whereas a high degree of off-target effect was induced by shRNA with a high Tm. Furthermore, shRNAs were often cleaved by Drosha and/or Dicer at the shifted sites in their primary transcripts, resulting in unexpected siRNA products with shifted sequences. Based on these results, the following conditions should be satisfied to ensure a functional shRNA with reduced off-target effects: the presence of A/U residues at nucleo tide positions 1 and −1 from the 5' end of the guide strand, three to six A/Us in nucleotide positions 2–7, G/C at positions 19 and 18, and no GC stretch of more than nine nucleotides. In addition, the Tm values of the seed regions in both strands should be low.","PeriodicalId":344667,"journal":{"name":"2009 International Symposium on Micro-NanoMechatronics and Human Science","volume":"43 1","pages":"0"},"PeriodicalIF":0.0000,"publicationDate":"2009-12-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"1","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"2009 International Symposium on Micro-NanoMechatronics and Human Science","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1109/MHS.2009.5351912","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 1
Abstract
The capability to induce off-target effects by small interfering RNA (siRNA) is highly correlated with the calculated melting temperature (Tm) of the protein-free seed-target duplex, indicating that its thermodynamic stability is a major factor in determining the degree of the off-target effect. In this study, we analyzed changes in microarray expression profiles following transfection of short hairpin RNAs (shRNAs). In a similar manner to siRNA, no or little off-target effects were observed by shRNA with a low Tm value in the seed-target duplex, whereas a high degree of off-target effect was induced by shRNA with a high Tm. Furthermore, shRNAs were often cleaved by Drosha and/or Dicer at the shifted sites in their primary transcripts, resulting in unexpected siRNA products with shifted sequences. Based on these results, the following conditions should be satisfied to ensure a functional shRNA with reduced off-target effects: the presence of A/U residues at nucleo tide positions 1 and −1 from the 5' end of the guide strand, three to six A/Us in nucleotide positions 2–7, G/C at positions 19 and 18, and no GC stretch of more than nine nucleotides. In addition, the Tm values of the seed regions in both strands should be low.