title: RNA interference tolerates 2'-fluoro modifications at the Argonaute2 cleavage site.
abstract: Short interfering RNA (siRNA) molecules with good gene-silencing properties are needed for drug development based on RNA interference (RNAi). An initial step in RNAi is the activation of the RNA-induced silencing complex RISC, which requires degradation of the sense strand of the siRNA duplex. Although various chemical modifications have been introduced to the antisense strand, modifications to the Argonaute2 (Ago2) cleavage site in the sense strand have, so far, not been described in detail. In this work, novel 2'-F-purine modifications were introduced to siRNAs, and their biological efficacies were tested in cells stably expressing human tartrate-resistant acid phosphatase (TRACP). A validated siRNA that contains both purine and pyrimidine nucleotides at the putative Ago2 cleavage site was chemically modified to contain all possible combinations of 2'-fluorinated 2'-deoxypurines and/or 2'-deoxypyrimidines in the antisense and/or sense strands. The capacity of 2'-F-modified siRNAs to knock down their target mRNA and protein was studied, together with monitoring siRNA toxicity. All 2'-F-modified siRNAs resulted in target knockdown at nanomolar concentrations, despite their high thermal stability. These experiments provide the first evidence that RISC activation not only allows 2'-F modifications at the sense-strand cleavage site, but also increase the biological efficacy of modified siRNAs in vitro.
authors.affiliation: Bone Biology Research Consortium, Institute of Biomedicine, Department of Anatomy, University of Turku.
authors.forename: Pirkko
authors.initials: P
authors.lastname: Muhonen
authors.forename: Tuula
authors.initials: T
authors.lastname: Tennilä
authors.forename: Elena
authors.initials: E
authors.lastname: Azhayeva
authors.forename: Ranga N
authors.initials: RN
authors.lastname: Parthasarathy
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Janckila
authors.forename: H Kalervo
authors.initials: HK
authors.lastname: Väänänen
authors.forename: Alex
authors.initials: A
authors.lastname: Azhayev
authors.forename: Tiina
authors.initials: T
authors.lastname: Laitala-Leinonen
mesh_terms: D000135:Acid Phosphatase / Q000037:antagonists & inhibitors* / Q000737:chemistry; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D005461:Fluorine / Q000737:chemistry*; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors* / Q000737:chemistry; D010448:Peptide Initiation Factors / Q000737:chemistry* / Q000378:metabolism; D034622:RNA Interference; D020871:RNA Stability; D012333:RNA, Messenger / Q000187:drug effects; D034741:RNA, Small Interfering / Q000737:chemistry / Q000494:pharmacology*; D000071681:Tartrate-Resistant Acid Phosphatase
chemical_list: C466422:AGO2 protein, human; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D007527:Isoenzymes; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D005461:Fluorine; D000135:Acid Phosphatase; D000071681:Tartrate-Resistant Acid Phosphatase