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Updated: Jan 7, 2026
Probing RNA Structure with Dimethyl Sulfate Mutational Profiling with Sequencing In Vitro and in Cells
Published on: December 9, 2022
An siRMSD parameter of structural distortion induced by chemical modification is predictive of the off-target effect
Seongjin An1, Kohei Nomura2, Yoshiaki Kobayashi3
1Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8561, Japan.
Abstract:
We developed siRMSD, a predictive parameter for off-target effects induced by chemical modifications, to optimize siRNA therapeutics. In RNA interference, small interfering RNA (siRNA) suppresses gene function by degrading mRNA with perfect sequence complementarity, providing therapeutic potential through the targeted inhibition of disease-related genes. However, off-target effects on unintended mRNAs pose a significant challenge to clinical application. While chemical modifications improve nuclease stability and reduce off-target effects, the underlying mechanisms remain unclear. Here, we show that structural distortions caused by chemical modifications determine off-target effects. Modifications, including 2'-O-methoxyethyl, 2'-O-methyl, and 2'-formamido, at positions 2-5 disrupted the A-form RNA duplex on argonaute 2, preventing stable binding to target mRNA. In contrast, modifications at positions 6-8 had minimal impact on off-target effect resulting from changes in thermodynamic stability.
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