Specificity, duplex degradation and subcellular localization of antagomirs

TLDR

MicroRNAs are abundant 20–23‑nt regulators of gene expression, and studying their function and therapeutic potential in mice relies on modified oligonucleotides. The study aims to further characterize the properties and function of antagomirs in mice. Antagomirs are degraded in mouse liver by a mechanism independent of RNAi, as evidenced by the degradation of chemically protected duplexes and their localization to a cytosolic compartment distinct from P‑bodies. Antagomirs with optimized phosphorothioate modifications and lengths >19 nt efficiently silence miRNAs, discriminate single‑nucleotide mismatches, localize to a cytosolic compartment, are ineffective systemically in the CNS but effective when injected locally into cortex, thereby validating their in‑vivo utility.

Abstract

MicroRNAs (miRNAs) are an abundant class of 20–23-nt long regulators of gene expression. The study of miRNA function in mice and potential therapeutic approaches largely depend on modified oligonucleotides. We recently demonstrated silencing miRNA function in mice using chemically modified and cholesterol-conjugated RNAs termed ‘antagomirs’. Here, we further characterize the properties and function of antagomirs in mice. We demonstrate that antagomirs harbor optimized phosphorothioate modifications, require >19-nt length for highest efficiency and can discriminate between single nucleotide mismatches of the targeted miRNA. Degradation of different chemically protected miRNA/antagomir duplexes in mouse livers and localization of antagomirs in a cytosolic compartment that is distinct from processing (P)-bodies indicates a degradation mechanism independent of the RNA interference (RNAi) pathway. Finally, we show that antagomirs, although incapable of silencing miRNAs in the central nervous system (CNS) when injected systemically, efficiently target miRNAs when injected locally into the mouse cortex. Our data further validate the effectiveness of antagomirs in vivo and should facilitate future studies to silence miRNAs for functional analysis and in clinically relevant settings.