Anti-miRNA Oligonucleotides (AMOs)
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, of typical length 19–24 nt, can be inhibited by steric-blocking anti-miRNA oligonucleotides (AMOs). AMOs are often chemicallymodified to improve functional potency (by increasing their binding affinity to target miRNAs) and 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 developeda ZEN™ non-nucleotide modifier that confers favorable properties to 2’-O-methyl RNA AMOs. These ZEN AMOs are highly stable in cell culture and provide high potency high specificity, and low toxicity.
The effects of AMOs 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). The decrease in Renilla luciferase activity is observed as reduced luminescence in the Dual Luciferase Assay (Promega). Addition of AMOs prevented miRNA binding to the miR-21 binding site and restored Renilla luciferase activity (Figures 2 and 3). Cytotoxicity levels were measured using the MultiTox-Glo Multiplex Cytotoxicity Assay (Promega) (Figure 4).
Figure 1. Modulating miRNA Function. A miR-21 binding site was inserted into the multiple cloning site of the PsiCHECK™-2 vector, 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 gene and, thereby, preventing luminescence. Subsequent transfection with anti-miRNA oligonucleotides (AMOs) targeted to miR-21 leads to AMO binding of miR-21, allowing translation of the Renilla luciferase gene and observed luminescence.
Figure 2. ZEN™ Anti-miRNA Oligonucleotides are Potent Inhibitors. The PsiCHECK-miR-21 plasmid was transfected into HeLa cells, followed by Anti-miRNA Oligonucleotides (AMO) 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 AMO targeting miR-21 increased its potency to a level comparable to existing highly potent AMOs (circled). Reagent only control is set to 1. (HP: hairpin; RC: reverse complement; PS: phosphorothioate; PO: standard phosphate linkage; C3: C3 spacer)
Figure 3. ZEN™ Anti-miRNA Oligonucleotides Have Increased Specificity. The PsiCHECK-miR-21 plasmid was transfected into HeLa cells, followed by AMO 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. Modified AMOs were mutated at 1, 2, or 3 positions and analyzed for off-target effects. ZEN AMOs showed the highest specificity of the 4 modified AMOs tested (boxed). Reagent only control is set to 1. (HP: hairpin; RC: reverse complement; PS: phosphorothioate; PO: standard phosphate linkage; C3: C3 spacer)
Figure 4. ZEN™ Anti-miRNA Oligonucleotides are Non-Toxic. Modified AMOs 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 AMOs were shown to be the least toxic modified AMOs tested at 50 and 100 nM (circled). (HP: hairpin; RC: reverse complement; PS: phosphorothioate; PO: standard phosphate linkage; C3: C3 spacer)