Document Detail


The design, preparation, and evaluation of asymmetric small interfering RNA for specific gene silencing in Mammalian cells.
MedLine Citation:
PMID:  23027049     Owner:  NLM     Status:  In-Data-Review    
Abstract/OtherAbstract:
RNA interference (RNAi) is a highly efficient endogenous gene silencing mechanism mediated by short double-stranded RNAs termed small interfering RNAs (siRNAs). The current standard siRNA structure, which is used by most researchers to trigger sequence-specific target gene silencing, consists of a double strand region of 19 bp with 2 nt 3'-overhangs at both ends. However, in addition to the desired target gene silencing, this conventional siRNA structure also exhibits several unintended effects that constitute obstacles to the use of siRNA in gene function studies and therapeutics development. Here, we provide protocols for designing and preparing an alternative structure for RNAi trigger, termed asymmetric shorter-duplex RNA (asiRNA). The asiRNA structure has a duplex region shorter than 19 bp and has an asymmetric 3'-overhang structure. Importantly, the asiRNA structure not only triggers efficient target gene silencing comparable to that of the 19 bp standard siRNA structure but also significantly reduces nonspecific effects triggered by 19 bp siRNAs such as sense-strand-mediated off-target silencing and the saturation of RNAi machinery. Procedures are described for verifying that asiRNA activates gene silencing through an Ago2-dependent pathway and for assessing the miRNA pathway competition potency and specific and nonspecific silencing abilities of asiRNAs. We propose that asiRNA, an improved RNAi trigger that can overcome the nonspecific effects evoked by standard siRNA structures, can be developed as a precise and effective tool for both functional genomics and therapeutic applications.
Authors:
Chanil Chang; Sun Woo Hong; Pooja Dua; Soyoun Kim; Dong-Ki Lee
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  Methods in molecular biology (Clifton, N.J.)     Volume:  942     ISSN:  1940-6029     ISO Abbreviation:  Methods Mol. Biol.     Publication Date:  2013  
Date Detail:
Created Date:  2012-10-02     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  9214969     Medline TA:  Methods Mol Biol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  135-52     Citation Subset:  IM    
Affiliation:
Global Research Laboratory for RNAi Medicine, Department of Chemistry, BK21 School of Chemical Materials Science, Sungkyunkwan University, Suwon, South Korea.
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