Each dot represents an individual human miRNA inhibitor in which the Tm is shown as a function of the GC content of the miRNA target. Blue dots correspond to full-length inhibitors with classical nucleotide chemistry. The red dots correspond to the new generation of miRCURY LNA miRNA inhibitors. The affinity of traditional full-length miRNA inhibitors is highly influenced by the GC content and Tm values spanning >40°C. In contrast, the Tm of miRCURY LNA miRNA inhibitors are all focused within a 10°C interval around an optimal high temperature.
HeLa cells were transfected with a plasmid containing Renilla luciferase for the transfection efficiency control and the miRNA target sequence of a firefly luciferase reporter gene in the 3’ UTR. Firefly luciferase expression is suppressed by the corresponding endogenous miRNA level in the cell. One day later, the cell cultures were transfected with various concentrations of the corresponding regular miRCURY LNA miRNA Inhibitor. Reporter gene expression was measured with a dual luciferase assay 24 H after transfection. Ratios of firefly and Renilla luciferase activity were calculated and normalized to values obtained with a firefly luciferase reporter with no miR target sequence (pLuc). The results illustrate that our regular miRCURY LNA miRNA Inhibitors display sub-nanomolar potency under optimal transfection conditions.
HeLa cells were transfected with a plasmid with a Renilla luciferase (transfection efficiency control) and an hsa-miR-21-5p target sequence in the 3'UTR of a Firefly luciferase reporter gene. Firefly luciferase expression is therefore suppressed by the corresponding endogenous miR-21-5p level in the cell. The next day, the cell cultures were transfected with different concentrations of regular and Power miRCURY LNA hsa-miR-21-5p inhibitors and negative controls. Reporter gene expression was measured with a dual Luciferase assay 24 H after transfection. Ratios of firefly and Renilla luciferase activity were calculated and normalized to values obtained with a firefly luciferase reporter with no miR target sequence (pLuc). The results illustrate that our miRNA inhibitors display sub-nanomolar potency under optimal transfection conditions, and the Power inhibitors display superior activity compared with the regular inhibitors.
HepG2, HeLa and HEK293 cells were transfected with a plasmid encoding Renilla luciferase (transfection efficiency control) and a firefly luciferase reporter gene with either an miR-21-5p target site or an miR-27a-3p target site in the 3’-UTR (pmiR-21-5p or pmiR-27a-3p). 24 H after removal of the transfection reagent, corresponding miRCURY LNA miRNA Power Inhibitors were added directly to culture medium in different concentrations. Reporter gene expression was measured with a dual luciferase assay 48 H (HepG2) and 72 H (HeLa and HEK293) after addition of the inhibitors. Ratios of firefly and Renilla luciferase activity were calculated and normalized to values obtained with a firefly luciferase reporter with no miR target sequence (pLuc) in each of the three cell lines. The results illustrate that efficient miRNA inhibition can be achieved by adding high concentrations of Power Inhibitor directly to the culture medium. However, uptake kinetics with gymnosis is slower than delivery of the inhibitors using transfection reagents. When using transfection reagents, we normally observe strong inhibition after just 24 H. With unassisted uptake, we observe activity with some cell lines and certain inhibitors after one day, but it typically peaks between 48–72 H after addition of the inhibitors. Normal inhibitors with an unmodified, normal phosphodiester backbone are ineffective with gymnotic delivery, probably due to insufficient stability.