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 .

摘要

最近，我们证明了由交替组合的 2'- O组成的不对称 18 个碱基配对双链寡核苷酸- 甲基 RNA 和 DNA，称为 MED-siRNA，显示出高 RNase 抗性、目标 mRNA 的有效切割以及随后目标蛋白表达的减少。5'-末端磷酸基团和引导链的 3'-突出端是完全激活 MED-siRNAs 的 RNAi 活性所必需的。在这里，我们评估了在引导链的 5' 末端用芳基磷酸基团修饰的 MED-siRNA。5'-芳基磷酸化 MED-siRNAs 显示出与 5'-磷酸化 MED-siRNAs 相当的靶蛋白表达的高效降低。此外，5'-芳基磷酸化 MED-siRNAs 连接在引导链 5'-末端的芳基磷酸基团和过客链 3'-末端的羟基之间，带有烷基酰胺接头或肽（例如，DL -Ser-L-Ala-L-Tyr), 导致单链 MED-siRNAs 具有高效的靶 mRNA 切割活性，并通过 RNA 干扰机制与 Argonaute 2 结合。这些接头技术也可用于创建由天然存在的分子（如氨基酸）组成的 siRNA。这些发现表明使用这些单链 MED-siRNA 作为 siRNA 试剂的可能性。

本文的补充数据可在 https://doi.org/10.1080/15257770.2021.1927077 在线获得。