A screen of chemical modifications identifies position-specific modification by UNA to most potently reduce siRNA off-target effects.
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.
Bramsen
Jesper B
JB
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
Pakula
Malgorzata M
MM
Hansen
Thomas B
TB
Bus
Claus
C
Langkjær
Niels
N
Odadzic
Dalibor
D
Smicius
Romualdas
R
Wengel
Suzy L
SL
Chattopadhyaya
Jyoti
J
Engels
Joachim W
JW
Herdewijn
Piet
P
Wengel
Jesper
J
Kjems
Jørgen
J
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*
D009696:Nucleic Acids; D016372:RNA, Antisense; D034741:RNA, Small Interfering
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