TAILIEUCHUNG - Báo cáo khoa học: Effect of siRNA terminal mismatches on TRBP and Dicer binding and silencing efficacy

To enhance silencing and avoid off-target effects, siRNAs are often designed with an intentional bias to ensure that the end of the siRNA that contains the guide strand 5¢ end is less stably hybridized relative to the end containing the passenger strand 5¢ end. One means by which this is accom-plished is to introduce a terminal mismatch, typically by changing the passenger strand sequence to impair its hybridization with the guide strand 5¢ end. | Effect of siRNA terminal mismatches on TRBP and Dicer binding and silencing efficacy Hemant K. Kini and S. P. Walton Applied Biomolecular Engineering Laboratory Cellular and Biomolecular Laboratory Department of ChemicalEngineering and Materials Science Michigan State University East Lansing MI USA Keywords Dicer mismatches RNA interference short interfering RNA TRBP Correspondence S. P Walton Applied Biomolecular Engineering Laboratory Cellular and Biomolecular Laboratory Department of ChemicalEngineering and Materials Science Michigan State University 3249 Engineering Building East Lansing MI 48824-1226 USA Fax 1 517 432 1105 Tel 1 517 432 8733 E-mail spwalton@ Website http abel Received 2 July 2009 revised 28 August 2009 accepted 7 September 2009 doi To enhance silencing and avoid off-target effects siRNAs are often designed with an intentional bias to ensure that the end of the siRNA that contains the guide strand 5 end is less stably hybridized relative to the end containing the passenger strand 5 end. One means by which this is accomplished is to introduce a terminal mismatch typically by changing the passenger strand sequence to impair its hybridization with the guide strand 5 end. However there are conflicting reports about the influence of terminal mismatches on the silencing efficacy of siRNAs. Here the silencing efficiency of siRNAs with a terminal mismatch generated either by altering the guide strand at the 5 end nucleotide 1 or the passenger strand nucleotide 19 from the 5 end was examined. Subsequently we studied the relationship between the silencing efficiency of the siRNAs and their binding to the RNA-induced silencing complex loading complex proteins HIV transactivating response RNA-binding protein and Dicer in H1299 cytoplasmic extracts. Binding of siRNA and the transactivating response RNA-binding protein was significantly reduced by terminal mismatches which largely agrees with the .

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