The success of miRNA inhibition experiments is dependent on several factors, the most important of which are potency, specificity, stability, and toxicity. High potency ensures that only small doses are required to produce the desired phenotype, also reducing toxicity of the administered compound. miRNA inhibitors that are specific to their target reduce the incidence of off-target effects, ensuring that any observed phenotypes are the result of the effect on the target under investigation. Stability and low toxicity are essential for in vivo experiments.
Figures 1–4 demonstrate the high potency, specificity, stability, and extremely low toxicity of IDT miRNA Inhibitors compared to other modified oligonucleotides.
|IDT miRNA Inhibitors||UzC A A C A U C A G U C U G A U A A G C U Az|
|DNA||t c a a c a t c a g t c t g a t a a g c t a|
|2’OMe||U C A A C A U C A G U C U G A U A A G C U A|
|2’OMe 3PS Ends||U*C*A*A C A U C A G U C U G A U A A G*C*U*A|
|2’OMe 3’inZEN||U C A A C A U C A G U C U G A U A A G C UzA|
|2’OMe 5’inZEN||UzC A A C A U C A G U C U G A U A A G C U A|
|2’OMe 3’inC3||U C A A C A U C A G U C U G A U A A G C U+A|
|2’OMe 5’inC3||U+C A A C A U C A G U C U G A U A A G C U A|
|2’OMe 5’inC3, 3’inC3 (2’OMe 2XC3)||U+C A A C A U C A G U C U G A U A A G C U+A|
High potency of IDT miRNA Inhibitors
Figure 1. IDT miRNA Inhibitors exhibit high potency. Oligonucleotides designed to target miR-21 were transfected at 0.3–30 nM in HeLa cells expressing the psiCHECK-miR-21 plasmid using Lipofectamine® RNAiMAX transfection reagent. The cells were lysed after 24 hr and analyzed for luciferase activity. Results were normalized with the internal firefly luciferase (FLuc) control and are shown as fold change in Renilla luciferase (RLuc) compared with the lipid reagent control, which was set at 1. Tm values for the various oligos are shown above the respective profiles.
High specificity of IDT miRNA Inhibitors
miRNA inhibitors with various modifications were tested against wild-type miR-21 and 3 "mutant" versions containing 1, 2, or 3 mismatches (Table 1). IDT miRNA Inhibitors demonstrated high specificity at all concentrations tested (Figure 2).
Table 1. Sequences of wild-type and "mutant" miR-21 used to test specificity of miRNA inhibitors.
|Mutant type||miR-21 IDT miRNA Inhibitor sequences (5′ to 3′)*|
|Wild type (0 MUT)||C A A C A U C A G U C U G A U A A G C U|
|1 MUT||C A A C A U C A G U C A G A U A A G C U|
|2 MUT||C A A C C U C A G U C A G
A U A A G C U|
|3 MUT||C A A C C U C A G U C A G
A U A A C C U|
Figure 2. IDT miRNA Inhibitors are highly specific. Different miR-21 inhibitors (n=5) were synthesized, complementary to wild-type miR-21 or containing 1, 2, or 3 mismatched—0 MUT, 1 MUT, 2 MUT, and 3 MUT, respectively. The inhibitors were transfected into HeLa cells expressing the psiCHECK-miR-21 plasmid. After 24 hr, the cells were lysed and analyzed for luciferase activity. Values were normalized with internal firefly luciferase (FLuc) control and reported as a fold change in Renilla luciferase (RLuc) compared with lipid reagent control, which was set at 1.
High nuclease stability of IDT miRNA Inhibitors
Figure 3. IDT miRNA Inhibitors are resistant to nucleases. 1.1 nmol of each oligonucleotide was incubated in (A) 10% FBS, high exonuclease environment; or (B) 20% mouse liver protein extract, high endonuclease environment, for the indicated lengths of time. Each reaction was analyzed on a denaturing polyacrylamide gel stained with methylene blue.
Low cellular toxicity of IDT miRNA Inhibitors
Figure 4. IDT miRNA Inhibitors exhibit low toxicity to cells. Modified oligonucleotides corresponding to a nontargeting negative control sequence, NC1, unrelated to any known human miRNA were transfected into HeLa cells at concentrations of 10, 30, and 100 nM to investigate and compare toxicity. The cells were visualized by phase contrast microscopy (10X magnification).