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.

Increased siRNA duplex stability correlates with reduced off-target and elevated on-target effects

“…In addition, it could be very interesting combining our approach of siRNA passenger strand modification to generate thermodynamically stabilized siRNA duplexes with guide strand modifications. Very interesting combinations could be siRNA guide strand modifications in the seed region, which have been described to cause reduced siRNA off-target activity, for example, 29-O-methyl nucleotides (Jackson et al 2006a) or UNA nucleotides (Bramsen et al 2010;Laursen et al 2010;Vaish et al 2010). Since siRNAs are being used as drugs our siRNA selection parameters may also have an impact on siRNA-based therapy.…”

Screening siRNAs targeting a novel gene (HA117) and the development of a derivative recombinant adenovirus delivery system

“…Even one mispairing of base can reduce the silence effects greatly. 9 It is very crucial to select both efficient suppression characteristic and conservative siRNA target sites. But even if the siRNAs are strictly followed the principle of siRNA design, 10,11 there is only 60-70% of them can have a gene silence effects.…”

“…Different softwaredesigned siRNAs targeting the same gene may have different interference efficiencies. [9][10][11] If the most efficient siRNA can be quickly and easily screened from among many siRNAs, this would greatly facilitate successful silencing of target gene expression with RNAi. This capability would be especially helpful in the study of novel gene function.…”

Construction, screening and evaluation of a derivative recombinant adenovirus for the optimal siRNA targeting of a novel gene (HA117)

“…Currently, siRNAs are designed based on software analysis or literature study. However, different software-designed siRNAs targeting the same gene may have different interference efficiencies (Bramsen et al, 2010;Strapps et al, 2010;Tiemann, 2009). Therefore, quick and easy screening of the most efficient siRNA from among many siRNAs would greatly facilitate subsequent successful silencing of target gene expression with RNAi; this capability would be especially helpful in the study of novel gene function.…”