Specificity, duplex degradation and subcellular localization of antagomirs

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.

Chemical modification and design of anti-miRNA oligonucleotides

“…The next advance in AMO technology came from Krützfeldt et al 21,22 who employed 2¢OMe oligonucleotides with several PS bonds between the internucleotide linkages at each end of the molecule to defend against exonuclease attack (Table 1). A cholesterol group was attached to the 3¢-end to assist with delivery in vivo; this specific design of AMO was called an 'antagomir' .…”

A microRNA component of the hypoxic response

“…Although the traditional drug targets have been protein products, the recent development of microRNA derivatives with increased stability and binding efficiency, such as AMOs (antimicroRNA oligonucleotides) and LNAs (locked nucleic acids) represent potentially important developments for such purpose. [36][37][38] For example, targeting an HRM that plays a survival role in hypoxia could provide a new angle in targeting a notoriously refractory fraction of tumor cells. Moreover, manipulation of select microRNAs could synergize with conventional therapies.…”

Role of MicroRNA29b in Blood–Brain Barrier Dysfunction during Hyperhomocysteinemia: An Epigenetic Mechanism

“…To strengthen our hypothesis, we administered locked nucleic acidanti-miR29b injection in vivo to conclusively show miR29b regulation in BBB permeability. We administered scramble miR and anti-miR29b into CBS þ / À mice through intracerebral route, as explained by Krutzfeldt et al 40 The author described that local administration of antagomiR-16 is effective as compared with systemic infusions, most likely because of inability of oligonucleotides to cross the blood-brain barrier. 40 As compared to CBS þ / À þ scramble miR mice, CBS þ / À þ anti-miR29b mice showed significant decrease in miR29b levels along with considerable decrease in FITC-BSA leakage through brain pial venules (Supplementary Figure 2).…”

“…We administered scramble miR and anti-miR29b into CBS þ / À mice through intracerebral route, as explained by Krutzfeldt et al 40 The author described that local administration of antagomiR-16 is effective as compared with systemic infusions, most likely because of inability of oligonucleotides to cross the blood-brain barrier. 40 As compared to CBS þ / À þ scramble miR mice, CBS þ / À þ anti-miR29b mice showed significant decrease in miR29b levels along with considerable decrease in FITC-BSA leakage through brain pial venules (Supplementary Figure 2). In agreement to our finding, Zhou et al 41 also found miR29 family member 'miR29a' involvement in the regulation of intestinal membrane permeability in patients with irritable bowel disease.…”

“…Homocysteine-induced BBB disruption model in bEnd.3 cells was confirmed by analyzing paracellular and transcellular transport systems using Lucifer yellow and BSA-647 fluorescence probes leakage at different time points (10,20,40,60, 120 minutes) in four treatment groups. In Hcy-treated cells, significant endothelial cell permeability was observed, as evident through increased leakage of the Lucifer yellow (at 40 minutes and 60 minutes; Figure 1A) and BSA-647 (at 40 minutes to 120 minutes; Figure 1B) fluorescent dyes, which suggested that both paracellular and transcellular transport systems are affected during endothelial disruption.…”