title: Abasic pivot substitution harnesses target specificity of RNA interference.
abstract: Gene silencing via RNA interference inadvertently represses hundreds of off-target transcripts. Because small interfering RNAs (siRNAs) can function as microRNAs, avoiding miRNA-like off-target repression is a major challenge. Functional miRNA-target interactions are known to pre-require transitional nucleation, base pairs from position 2 to the pivot (position 6). Here, by substituting nucleotide in pivot with abasic spacers, which prevent base pairing and alleviate steric hindrance, we eliminate miRNA-like off-target repression while preserving on-target activity at ∼ 80-100%. Specifically, miR-124 containing dSpacer pivot substitution (6pi) loses seed-mediated transcriptome-wide target interactions, repression activity and biological function, whereas other conventional modifications are ineffective. Application of 6pi allows PCSK9 siRNA to efficiently lower plasma cholesterol concentration in vivo, and abolish potentially deleterious off-target phenotypes. The smallest spacer, C3, also shows the same improvement in target specificity. Abasic pivot substitution serves as a general means to harness the specificity of siRNA experiments and therapeutic applications.
authors.affiliation: Division of Life Sciences, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea.
authors.forename: Hye-Sook
authors.initials: HS
authors.lastname: Lee
authors.affiliation: Division of Life Sciences, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea.
authors.forename: Heeyoung
authors.initials: H
authors.lastname: Seok
authors.affiliation: Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul 135-710, Korea.
authors.forename: Dong Ha
authors.initials: DH
authors.lastname: Lee
authors.affiliation: Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul 135-710, Korea.
authors.forename: Juyoung
authors.initials: J
authors.lastname: Ham
authors.affiliation: Division of Life Sciences, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea.
authors.forename: Wooje
authors.initials: W
authors.lastname: Lee
authors.affiliation: Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul 135-710, Korea.
authors.forename: Emilia Moonkyung
authors.initials: EM
authors.lastname: Youm
authors.affiliation: Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul 135-710, Korea.
authors.forename: Jin Seon
authors.initials: JS
authors.lastname: Yoo
authors.affiliation: EncodeGEN Co. Ltd., Seoul 135-994, Korea.
authors.forename: Yong-Seung
authors.initials: YS
authors.lastname: Lee
authors.affiliation: Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul 135-710, Korea.|EncodeGEN Co. Ltd., Seoul 135-994, Korea.
authors.forename: Eun-Sook
authors.initials: ES
authors.lastname: Jang
authors.affiliation: Division of Life Sciences, College of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea.|Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul 135-710, Korea.|Samsung Research Institute for Future Medicine, Samsung Medical Center, Seoul 135-710, Korea.
authors.forename: Sung Wook
authors.initials: SW
authors.lastname: Chi
mesh_terms: D000818:Animals; D060565:Argonaute Proteins / Q000378:metabolism; D020029:Base Pairing; D015152:Blotting, Northern; D002460:Cell Line; D045744:Cell Line, Tumor; D018390:Gene Targeting / Q000379:methods*; D006367:HeLa Cells; D056945:Hep G2 Cells; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D035683:MicroRNAs / Q000235:genetics*; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics*; D000067492:RNAi Therapeutics / Q000379:methods; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000235:genetics
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; C509207:MIRN124 microRNA, human; D035683:MicroRNAs; D034741:RNA, Small Interfering; C472125:PCSK9 protein, human; D000071449:Proprotein Convertase 9; D043484:Proprotein Convertases; D012697:Serine Endopeptidases