title: 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.
authors.affiliation: Center of Excellence in Infectious Disease Research, Department of Biomedical Sciences, Paul L Foster School of Medicine, Texas Tech University Health Sciences Center, El Paso, Texas, United States of America. haoquan.wu@ttuhsc.edu
authors.forename: Haoquan
authors.initials: H
authors.lastname: Wu
authors.forename: Hongming
authors.initials: H
authors.lastname: Ma
authors.forename: Chunting
authors.initials: C
authors.lastname: Ye
authors.forename: Danielle
authors.initials: D
authors.lastname: Ramirez
authors.forename: Shuiping
authors.initials: S
authors.lastname: Chen
authors.forename: Jessica
authors.initials: J
authors.lastname: Montoya
authors.forename: Premlata
authors.initials: P
authors.lastname: Shankar
authors.forename: Xiaozhong A
authors.initials: XA
authors.lastname: Wang
authors.forename: N
authors.initials: N
authors.lastname: Manjunath
mesh_terms: D060565:Argonaute Proteins / Q000378:metabolism; D020137:Base Pair Mismatch / Q000235:genetics*; D001483:Base Sequence; D017124:Conserved Sequence; D057809:HEK293 Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D034741:RNA, Small Interfering / Q000737:chemistry / Q000235:genetics*; D034743:RNA-Induced Silencing Complex / Q000378:metabolism; D013816:Thermodynamics
chemical_list: D060565:Argonaute Proteins; D034741:RNA, Small Interfering; D034743:RNA-Induced Silencing Complex