Inhibiting miRNAs using antisense oligonucleotides

MicroRNAs (miRNAs) are important modulators of gene expression, and their dysregulation is implicated in numerous diseases. Synthetic oligonucleotides that alter expression of functional miRNAs are being used to identify biological targets, and can be used therapeutically when miRNA dysregulation contributes to pathophysiology.

Mature miRNAs, with a typical length of 19–24 nt, can be inhibited by steric-blocking using IDT^® miRNA Inhibitors. Also known as anti-miRNA oligonucleotides (AMOs), miRNA Inhibitors are often chemically modified to improve functional potency (by increasing their binding affinity to target miRNAs) and to provide protection against nuclease degradation. An ideal modification should be non-toxic and should not increase binding affinity to the extent that specificity is compromised. IDT has developed a ZEN™ non-nucleotide modifier that confers favorable properties to 2′-O-methyl RNA miRNA Inhibitors. These ZEN miRNA Inhibitors are highly stable in cell culture and provide high potency, high specificity, and low toxicity.

The effects of IDT miRNA Inhibitors on miRNA activity were studied in HeLa cells transfected with a modified psiCHECK™-2 vector (Promega) containing a miR-21 binding site cloned downstream of the translational stop codon for the Renilla luciferase gene. The miR-21 binding site allows miR-21 to cleave and degrade Renilla luciferase mRNA (Figure 1).

Figure 1. Modulating miRNA function. A miR-21 binding site was inserted into the multiple cloning site of the psiCHECK-2 vector (Promega) at the 3′ end of the Renilla luciferase gene. Upon transfection into HeLa cells, miR-21 binds to its binding site within the psiCHECK-miR-21 plasmid, causing degradation of the Renilla luciferase mRNA and, thereby, preventing luminescence. Subsequent transfection with IDT miRNA Inhibitors targeting miR-21 (labeled here as anti-miRNA oligonucleotides or AMOs) leads to miRNA Inhibitor binding of miR-21, allowing translation of the Renilla luciferase mRNA and observed luminescence.

The decrease in Renilla luciferase activity is observed as reduced luminescence in the Dual-Luciferase^® Assay (Promega). Addition of miRNA Inhibitors prevented miRNA binding to the miR-21 binding site and restored Renilla luciferase activity (Figures 2 and 3).

Figure 2. IDT miRNA Inhibitors with ZEN modifications are potent inhibitors of miRNA function. The psiCHECK-miR-21 plasmid was transfected into HeLa cells, followed by miRNA Inhibitor transfection for 24 hr. Residual mRNA was measured as luciferase activity, using the Dual-Luciferase^® Assay (Promega), and Renilla luciferase was normalized to the internal firefly luciferase control. Insertion of the non-nucleotide modifying group, ZEN, between the terminal and adjacent bases at the flanking ends of a non-toxic 2′-O-methyl miRNA Inhibitor targeting miR-21 increased its potency to a level comparable to existing highly potent miRNA Inhibitors (circled). Reagent only control is set to 1. (HP: hairpin; RC: reverse complement; PS: phosphorothioate; PO: standard phosphate linkage; C3: C3 spacer).

Figure 3. IDT miRNA Inhibitors with ZEN modifications have increased specificity. The psiCHECK-miR-21 plasmid was transfected into HeLa cells, followed by miRNA Inhibitor transfection for 24 hr. Residual mRNA was measured as luciferase activity, using the Dual-Luciferase® Assay (Promega), and Renilla luciferase was normalized to the internal firefly luciferase control. The modified miRNA Inhibitors were mutated at 1, 2, or 3 positions and analyzed for off-target effects. ZEN-modified miRNA Inhibitors showed the highest specificity of the 4 modified miRNA Inhibitors tested (boxed). Reagent only control is set to 1. (HP: hairpin; RC: reverse complement; PS: phosphorothioate; PO: standard phosphate linkage; C3: C3 spacer).

Cytotoxicity levels were measured using the MultiTox-Glo Multiplex Cytotoxicity Assay (Promega) (Figure 4).

Figure 4. IDT miRNA Inhibitors with ZEN modifications are non-toxic. Modified miRNA Inhibitors comprising a non-immunostimulatory sequence independent of known human miRNAs, were used to compare toxicity in HeLa cells. The cells were incubated for 24 hr and cell viability was measured. Staurosporine (1 mM) was used as a positive control for cytotoxicity. ZEN-containing miRNA Inhibitors were shown to be the least toxic modified miRNA Inhibitors tested at 50 and 100 nM (circled). (HP: hairpin; RC: reverse complement; PS: phosphorothioate; PO: standard phosphate linkage; C3: C3 spacer).