In this study, we characterized the inhibition of miRNAs with antagomirs in vivo. A detailed understanding of this process is needed to investigate miRNA function in mice and exploit their therapeutic potential. Our study provides a unique platform since its major read-out is based on the dose-and time-dependent regulation of several endogenous and validated targets of miR-122.
Specificity of drug-like oligonucleotides is important to minimize off-target effects and to discriminate between related miRNAs that sometimes differ by only a single nucleotide. Previous studies have demonstrated that locked nucleic acid (LNA) are sensitive to single mismatch in in-situ
staining protocols of zebrafish embryos (11
). Tissue-culture-based luciferase assays indicated that 2′-O
-methoxyethyl oligonucleotides might have a similar specificity (12
). In line with these data we show that the antagomir chemistry enables discrimination of a single nucleotide. Intriguingly, this effect depends on the position of the mismatch within the antagomir sequence. Nucleotide exchanges at the very 5′-end of the antagomir or in the center did not prevent downregulation of miR-122 levels in northern blots and upregulation of miR-122 targets. This observation is in agreement with the work published by Davis et al
., who reported a similar result in luciferase-based assays (12
). Asymmetry of a single nucleotide mismatch may therefore be more detrimental for targeting miRNAs than symmetric changes. These data are important for the design of antagomirs that target specific members of miRNA families or when off-target effects have to be considered.
The mechanism of oligonucleotide-mediated miRNA silencing is still unknown. Previous data from our group and others suggested that this process involves degradation of the miRNA in vitro
) and in vivo
). Northern blots of tissue samples treated with antagomirs fail to detect fragments of the targeted miRNA. This could be explained by cellular RNase activity that readily degrades them. We have previously demonstrated that increasing the cellular amount of endogenous miRNA by introducing duplexes of miRNA/antagomir leads to detectable degradation products (7
). In this study, we used this approach to ask whether antagomir-mediated silencing of miRNA involves a RNA-induced silencing complex (RISC)-dependent cleavage mechanism. In the RNAi pathway, the siRNA duplex of passenger strand and guide strand is integrated into the RISC complex and the argonaute-2 (Ago2) protein subsequently cleaves the passenger strand across from the guide strand's phosphate bond between position 10 and 11 (13
). This cleavage was inhibited by a single 2′-O
-methylation of the passenger strand corresponding to nucleotide 11 of the guide strand (13
). Hypothetically, antagomirs could cleave miRNAs within RISC with the antagomir acting as the guide strand. To test this, we injected miRNA/antagomir-duplexes into mice that harbored a 2′-O
-methyl endonuclease protection of the microRNA corresponding to nucleotide 10 and 11 of the antagomir. However, endonuclease protection between nucleotides 10 and 11 did not prevent the degradation of the miRNA as demonstrated by abundant miRNA fragments in northern analysis, nor did it prevent the upregulation of miR-122 targets. Thus Ago2-mediated cleavage is unlikely to mediate this process. Similar results were obtained when the miRNA was protected at the outside positions using phosphorothioates, indicating that the miRNA targeting does not dependent on exonuclease activity either. However, the fact that miRNA/antagomir-duplexes regulate miRNA targets suggests antagomir recycling. The appearance of miRNA fragments of decreased length suggests that degradation is involved in this recycling process. Further elucidation of the mechanism of degradation will be important for optimizing miRNA silencing.
To address the subcellular compartment where interaction of miRNA and antagomir occurs we engineered flurophore-labeled antagomirs. Flurophore labeling of siRNA has previously been used to evaluate cellular uptake of siRNA (15
). Q570-labeled antagomirs were cleared from the plasma at a t1/2
min (data not shown), which is considerably faster than the plasma-clearance of cholesterol-conjugated siRNA of ~90
). A striking overlap of the subcellular localization profiles of antagomirs and miRNAs by sucrose gradient ultracentrifugation analysis of liver homogenates indicates that they might share subcellular compartments. Antagomir localization within hepatocytes was strictly limited to the cytosol. This could explain why antagomirs did not influence steady-state levels of the nuclear precursors of miRNAs (7
). We asked whether antagomirs could localize to P-bodies, since this compartment has been linked to the miRNA pathway. P-bodies are enriched in Ago 2 as well as mRNA that is targeted by miRNAs. It is, however, still unknown to which degree endogenous miRNAs localize to P-bodies. In any case, we did not observe any co-localization of antagomirs and P-bodies and conclude that the interaction of antagomirs and miRNAs occurs upstream of this compartment.
In this study, we also extended our previous analysis of the influence of different chemical modifications on antagomirs. Phosphorothioate modifications provide protection against RNase activity and their lipophilicity contributes to enhanced tissue uptake. On the other hand, phosphorothioates decrease the melting temperature of RNA duplexes (12
) and have been shown to be general inhibitors of cellular RNAse activity (18
). Indeed our results indicate a critical balance of the number of phosphorothioates within the antagomir chemistry. While a significant number of phosphorothioates increases efficiency, complete phosphorothioate modification decreased efficiency. Our data also demonstrate that antagomirs require >19-nt length for optimal function.
Lastly, we demonstrate that antagomirs can efficiently decrease miR-16 levels in mouse brain when injected locally. Systemic infusions of antagomir-16 do not change brain levels of miR-16, most likely because of an inability to cross the blood-brain barrier (7
). Local injections of small amounts of antagomir-16 efficiently reduced expression of this miR-16 in the cortex. This inhibition was specific since the expression of other miRNAs was not affected and no alteration in miR-16 levels were measured in the contralateral hemisphere that was injected with PBS. These results suggest that miRNA-inhibitors could facilitate the elucidation of miRNA function in the CNS.
Different chemical modifications have been described to block miRNA function in vitro
) or in vivo
). However, detailed characterizations of these inhibitors are either lacking or based on heterologous miRNA target reporter assays (12
). In this study, we used the expression levels of endogenous miR-122 targets as a read-out. We show that antagomirs can be used in a time- and dose-dependent fashion to study miRNA targets. Furthermore, we characterized antagomirs with regard to specificity, functional minimal-length requirements and effectiveness in the CNS following direct application. Our data support the conclusion that antagomirs will be powerful tools for further studies on miRNA function in mice and possibly non-rodent models and enhance the validation of miRNA inhibitors as potential therapeutics.