miRNA Inhibitor oligonucleotides functions as non-toxic, potent inhibitors of miRNAs
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 by 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). 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). 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 (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.
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)
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)
Product focus—Small RNAs for functional genomics
miRNA Inhibitors are steric blocking oligonucleotides that hybridize to mature miRNAs, inhibiting their function. IDT® miRNA Inhibitors are oligonucleotides comprised of 2'-O-methyl residues with ZEN™ modifications at or near the ends. Incorporating 2'-O-methyl residues confers resistance to endonuclease degradation and increases binding affinity to RNA targets, while the ZEN modification blocks exonuclease degradation and further increases binding affinity.
Find out more about IDT miRNA Inhibitors.
Antisense Oligonucleotides (ASOs)
Antisense Oligonucleotides are short, synthetic 15–25 nt oligonucleotides that localize to the nucleus. They are ordered as desalted DNA oligos, and include phosphorothioate (PS) linkages that confer nuclease resistance, thus enhancing intracellular stability. 2′-O-methyl RNA bases can be added in addition to the PS linkages to decrease toxicity and increase ASO efficacy.
Learn more about IDT Antisense Oligonucleotides.
Dicer-Substrate RNAs (DsiRNAs) and the TriFECTa® RNAi Kit
DsiRNAs are chemically synthesized 27mer duplex RNAs that have increased potency in RNAi experiments compared to traditional siRNAs. The TriFECTa RNAi Kit contains 3 Dicer-substrate 27mer duplexes, targeting a specific gene, that are selected from a predesigned set of duplexes from the RefSeq collection of human, mouse, and rat genes in Genbank.
Learn more about DsiRNAs and the TriFECTa RNAi Kit.
Secondary structure prediction software
The Unafold tool is free, online, secondary structure prediction software. The program calculates the probability of single-stranded RNA or DNA folding, or hybridization between 2 single strands.
Acces the UNAFold tool.
Author: Nicola Brookman-Amissah is a senior scientific writer at IDT.
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