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Nucleic Acids Res. 2010 Sep;38(17):5761-73. doi: 10.1093/nar/gkq341. Epub 2010 May 7.

A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects.

Author information

1
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

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.

PMID:
20453030
PMCID:
PMC2943616
DOI:
10.1093/nar/gkq341
[Indexed for MEDLINE]
Free PMC Article

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