RNA interference activity of single-stranded oligonucleotides linked between the passenger strand and the guide strand with an aryl phosphate linker

Abstract

Recently, we demonstrated that asymmetrical 18 base-paired double-strand oligonucleotides comprised of alternately combined 2’-O-methyl RNA and DNA, termed MED-siRNAs, show high RNase resistance, efficient cleavage of target mRNA, and the subsequent reduction of target protein expression. The 5’-terminal phosphate group and the 3’-overhang of the guide strand were required to fully activate the RNAi activity of MED-siRNAs. Here, we evaluated MED-siRNAs modified with aryl phosphate groups at the 5’-end of the guide strand. The 5’-aryl phosphorylated MED-siRNAs showed highly efficient reduction of target protein expression comparable to 5’-phosphorylated MED-siRNAs. Moreover, 5’-aryl phosphorylated MED-siRNAs linked between the aryl phosphate group at the 5’-end of the guide strand and the hydroxyl group at the 3’-end of the passenger strand with alkyl amide linkers or peptides (e.g., DL-Ser-L-Ala-L-Tyr), resulted in single-stranded MED-siRNAs with a highly efficient cleavage activity of target mRNA with binding to Argonaute 2 via an RNA interference mechanism. These linker techniques could also be used to create siRNAs composed of naturally-occurring molecules such as amino acids. These findings suggest the possibility of using these single-stranded MED-siRNAs as siRNA reagents.

Supplemental data for this article is available online at https://doi.org/10.1080/15257770.2021.1927077 .

Keywords:

• Aryl phosphate
• 2’-O-methyl RNA
• siRNA
• linkage
• chemical modification

Acknowledgements

We gratefully acknowledge Dr. Tatsuya Kawaguchi of Daiichi Sankyo RD Novare for his assistance of quantification of protein levels.

Disclosure statement

All authors are employees of Daiichi Sankyo. Co. Ltd.