# Taming the Achilles’ Heel: A Chemical and Structural Design to Address Off-Target Effects in siRNA Therapeutics

## TL;DR

This paper introduces a new chemical and structural design for siRNA to reduce off-target effects while preserving its effectiveness in RNA interference.

## Contribution

The novel 2′-diol modification and extended 3′-overhang design reduce off-target interactions in siRNA therapeutics.

## Key findings

- 2′-diol modifications in the seed-region reduced melting temperature and imposed thermodynamic asymmetry.
- The US-siRNA design with a 3′-overhang improved antisense strand loading into RISC.
- The combined strategy minimizes off-target effects without compromising on-target RNAi activity.

## Abstract

Off-target effects represent one of the major bottlenecks
for RNA
interference (RNAi) technology. To address this issue, we present
a novel strategy by combining seed-region chemical modification with
an extended 3′-overhang on the sense strand (SS) to mitigate
SS-mediated and miRNA-like nontargeted interactions. To modify the
seed-region, we developed a novel 2′-diol modification that
was selectively installed at different positions within the seed-region
of siRNA. For this purpose, we synthesized universal 2′-diacetate
phosphoramidites that yielded a free 2′-diol functionality
after standard deprotection of oligonucleotides. The 2′-diol
moieties with single (positions 3–7) and dual (6 + 7) insertions
in the seed-region decreased the melting temperature (T
m) by ca. −1 to −4.0 °C, imposing thermodynamic
asymmetry. To improve the end-asymmetry of siRNA, we developed a structurally
unsymmetrical siRNA (US-siRNA) design (five-nucleotides at the 3′-overhang
region of SS), which together with seed-region modifications significantly
increased the relative RISC loading of antisense strand (AS) with
respect to their canonical sense variants. Overall, our rational design
of chemical modifications of the seed-region with a 2′-diol
moiety, in concert with the US-siRNA design, furnishes a simple, modular
strategy to minimize off-target effects while maintaining the on-target
RNAi activity.