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

TLDR

siRNAs are a preferred tool for gene silencing but can trigger unintended off‑target effects through seed‑region interactions, and although chemical modifications can mitigate this, only a few have been explored. The study aimed to develop a high‑throughput luciferase reporter assay to monitor siRNA off‑targeting in stable cell lines. The authors tested 10 chemical modifications at individual seed positions across three target sequences and three concentrations to assess effects on siRNA function and off‑targeting. UNA modification at position 7 markedly reduced off‑targeting across all sequences without compromising potency, making it a promising strategy for low‑concentration in‑vivo siRNA applications.

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.