Abstract
Small interfering RNA (siRNA) has been recognized as a powerful gene-silencing tool. For therapeutic application, chemical modification is often required to improve the properties of siRNA, including its nuclease resistance, activity, off-target effects, and tissue distribution. Careful siRNA guide strand selection in the RNA-induced silencing complex (RISC) is important to increase the RNA interference (RNAi) activity as well as to reduce off-target effects. The passenger strand-mediated off-target activity was previously reduced and on-target activity was enhanced by substitution with acyclic artificial nucleic acid, namely serinol nucleic acid (SNA). In the present study, the reduction of off-target activity caused by the passenger strand was investigated by modifying siRNAs with SNA. The interactions of SNA-substituted mononucleotides, dinucleotides, and (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO)-labeled double-stranded RNA (dsRNA) with the MID domain of the Argonaute 2 (AGO2) protein, which plays a pivotal role in strand selection by accommodation of the 5’-terminus of siRNA, were comprehensively analyzed. The obtained nuclear magnetic resonance (NMR) data revealed that AGO2-MID selectively bound to the guide strand of siRNA due to the inhibitory effect of the SNA backbone located at the 5’ end of the passenger strand.
Highlights
Small interfering RNAs are duplexes of approximately 23 base pairs composed of passenger strands and guide strands
Translational inhibition mediated by Small interfering RNA (siRNA) is executed by the RNA-induced silencing complex (RISC), which comprises a siRNA guide strand and the Argonaute 2 (AGO2) protein
As base pairing between the strand loaded into RISC and the messenger RNA (mRNA) target determine the selectivity of gene silencing
Summary
Small interfering RNAs (siRNAs) are duplexes of approximately 23 base pairs composed of passenger strands and guide strands. The control of the guide strand selection only by using RNA nucleotides is insufficient; it is necessary to develop methods that would result in a reduction of off-target activity mediated by the passenger strand-incorporated RISC. The 5’-terminal phosphate and nucleotide are not involved in pairing with the target mRNA These moieties are located in a narrow binding pocket on the MID domain of AGO2 and exhibit stacking interactions and extensive hydrogen bonds with the amino acid residues of MID and PIWI. We expect that the SNA-substituted siRNA design can be applied in the development of nucleic acid drugs to knock down any disease-related genes and supplement miRNA To make this design versatile, it is necessary to elucidate the mechanisms by which the SNA substitution at the terminal positions of siRNA reduces the off-target effects. We determined that AGO2-MID asymmetrically binds to the guide strand of the SNA-modified siRNA, in which the passenger strand contains an SNA moiety at the 5’ end
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