title: Analysis of siRNA specificity on targets with double-nucleotide mismatches.
abstract: Although RNA interference as a tool for gene knockdown is a great promise for future applications, the specificity of small interfering RNA (siRNA)-mediated gene silencing needs to be thoroughly investigated. Most research regarding siRNA specificity has involved analysis of affected off-target genes instead of exploring the specificity of the siRNA itself. In this study we have developed an efficient method for generating a siRNA target library by combining a siRNA target validation vector with a nucleotide oligomix. We have used this library to perform an analysis of the silencing effects of a functional siRNA towards its target site with double-nucleotide mismatches. The results indicated that not only the positions of the mismatched base pair have an impact on silencing efficiency but also the identity of the mismatched nucleotide. Our data strengthen earlier observations of widespread siRNA off-target effects and shows that approximately 35% of the double-mutated target sites still causes knockdown efficiency of >50%. We also provide evidence that there may be substantial differences in knockdown efficiency depending on whether the mutations are positioned within the siRNA itself or in the corresponding target site.
authors.affiliation: Department of Molecular Medicine and Surgery, Karolinska Institutet, CMM L8:01, 171 76 Stockholm, Sweden. dahlgren.cecilia@gmail.com
authors.forename: Cecilia
authors.initials: C
authors.lastname: Dahlgren
authors.forename: Hong-Yan
authors.initials: HY
authors.lastname: Zhang
authors.forename: Quan
authors.initials: Q
authors.lastname: Du
authors.forename: Maria
authors.initials: M
authors.lastname: Grahn
authors.forename: Gunnar
authors.initials: G
authors.lastname: Norstedt
authors.forename: Claes
authors.initials: C
authors.lastname: Wahlestedt
authors.forename: Zicai
authors.initials: Z
authors.lastname: Liang
mesh_terms: D020137:Base Pair Mismatch; D002460:Cell Line; D015723:Gene Library; D006801:Humans; D051916:Membrane Cofactor Protein / Q000235:genetics; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry*
chemical_list: D051916:Membrane Cofactor Protein; D009711:Nucleotides; D034741:RNA, Small Interfering