title: A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects.
abstract: Small interfering RNAs (siRNAs) are now established as the preferred tool to inhibit gene function in mammalian cells yet trigger unintended gene silencing due to their inherent miRNA-like behavior. Such off-target effects are primarily mediated by the sequence-specific interaction between the siRNA seed regions (position 2-8 of either siRNA strand counting from the 5'-end) and complementary sequences in the 3'UTR of (off-) targets. It was previously shown that chemical modification of siRNAs can reduce off-targeting but only very few modifications have been tested leaving more to be identified. Here we developed a luciferase reporter-based assay suitable to monitor siRNA off-targeting in a high throughput manner using stable cell lines. We investigated the impact of chemically modifying single nucleotide positions within the siRNA seed on siRNA function and off-targeting using 10 different types of chemical modifications, three different target sequences and three siRNA concentrations. We found several differently modified siRNAs to exercise reduced off-targeting yet incorporation of the strongly destabilizing unlocked nucleic acid (UNA) modification into position 7 of the siRNA most potently reduced off-targeting for all tested sequences. Notably, such position-specific destabilization of siRNA-target interactions did not significantly reduce siRNA potency and is therefore well suited for future siRNA designs especially for applications in vivo where siRNA concentrations, expectedly, will be low.
authors.affiliation: Department of Molecular Biology, University of Aarhus, Arhus, Nucleic Acid Center, Department of Physics and Chemistry, University of Southern Denmark, Odense, Denmark. jebb@mb.au.dk
authors.forename: Jesper B
authors.initials: JB
authors.lastname: Bramsen
authors.forename: Malgorzata M
authors.initials: MM
authors.lastname: Pakula
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Hansen
authors.forename: Claus
authors.initials: C
authors.lastname: Bus
authors.forename: Niels
authors.initials: N
authors.lastname: Langkjær
authors.forename: Dalibor
authors.initials: D
authors.lastname: Odadzic
authors.forename: Romualdas
authors.initials: R
authors.lastname: Smicius
authors.forename: Suzy L
authors.initials: SL
authors.lastname: Wengel
authors.forename: Jyoti
authors.initials: J
authors.lastname: Chattopadhyaya
authors.forename: Joachim W
authors.initials: JW
authors.lastname: Engels
authors.forename: Piet
authors.initials: P
authors.lastname: Herdewijn
authors.forename: Jesper
authors.initials: J
authors.lastname: Wengel
authors.forename: Jørgen
authors.initials: J
authors.lastname: Kjems
mesh_terms: D045744:Cell Line, Tumor; D017930:Genes, Reporter; D006801:Humans; D009696:Nucleic Acids / Q000737:chemistry; D034622:RNA Interference; D016372:RNA, Antisense / Q000737:chemistry; D034741:RNA, Small Interfering / Q000737:chemistry*
chemical_list: D009696:Nucleic Acids; D016372:RNA, Antisense; D034741:RNA, Small Interfering
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