Custom miRCURY LNA miRNA Inhibitors efficiently inhibit target miRNA activity, enabling you to perform miRNA functional studies on sequences not in miRBase. These antisense oligonucleotides have perfect sequence complementary to their targets, and when introduced into cells, they sequester the target miRNA in highly stable heteroduplexes that prevent interaction with the normal cellular partners.
Custom miRCURY LNA Inhibitors and Power Inhibitors are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
Overall features of the third-generation miRNA inhibitor design.
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.
Examples of miRNA silencing with miRCURY LNA miRNA inhibitors.
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.
Enhanced potency of miRCURY LNA miRNA Power Inhibitors.
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.
miRNA silencing via direct uptake (gymnosis) of Power 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.
Tm-normalized miRCURY LNA miRNA Inhibitors have unmatched potency against all miRNAs, regardless of their GC content (see figure Examples of miRNA silencing with miRCURY LNA miRNA Inhibitors). The Power Inhibitors are so potent that they can be added directly to cell cultures without the need for transfection reagents (see figure miRNA silencing via direct uptake (gymnosis) of Power Inhibitors).
miRCURY LNA miRNA Inhibitors are antisense oligonucleotides with perfect sequence complementary to their targets. When introduced into cells, they sequester the target miRNA in highly stable heteroduplexes, effectively preventing the miRNA from hybridizing with its normal cellular interaction partners.
The potency of miRNA antisense inhibitors is determined by their affinity for their miRNA target and their biological stability. The affinity of normal inhibitors with conventional nucleotide chemistry is a function of the GC content of the miRNA target. Since the GC content of miRNAs can vary from 5–95%, potencies of such inhibitors will vary greatly according to the miRNA sequence, with essentially no potency with AT-rich miRNAs.
We have exploited the high affinity properties of LNA chemistry to create Tm-normalized miRCURY LNA miRNA Inhibitors and miRCURY LNA miRNA Power Inhibitors. Varying the numbers and positions of LNA nucleotides in these DNA/LNA mixmer inhibitors and carefully choosing the target sequences normalizes the melting temperatures of the oligonucleotides within a narrow window around an empirically determined optimal high temperature (see figure Overall features of the third-generation miRNA inhibitor design). These design features ensure that miRCURY LNA miRNA Inhibitors have the same high efficacy, regardless of the GC content of their miRNA targets.
The combination of LNA and phosphorothioate modifications dramatically improves the stability of miRCURY LNA miRNA Power Inhibitors against enzymatic degradation, so the efficacy of Power Inhibitors is significantly better than the regular inhibitors (see figure Enhanced potency of miRCURY LNA miRNA Power Inhibitors). In fact, they are so stable and potent that they can be added directly to serum-containing culture medium without the need for transfection reagents, providing efficient miRNA inhibition via unassisted "naked" delivery, also known as gymnosis. This allows you to assess the consequences of miRNA silencing without worrying about confounding side effects from the transfection reagents.
Unassisted delivery does require significantly higher inhibitor concentrations, and uptake kinetics are generally slower than when using transfection reagents (see figure miRNA silencing via direct uptake (gymnosis) of Power Inhibitors). Furthermore, the ability to take up oligonucleotides directly from the culture medium varies significantly among cell lines.
Power Inhibitors are especially useful for complex applications, such as those involving difficult-to-transfect cells, highly expressed miRNA targets, long-duration experiments and when normal transfection procedures result in unacceptable phenotypic consequences.
miRCURY LNA miRNA Power Inhibitors are not recommended for use with cells or cell lines derived from muscle or the central nervous system (CNS). These cell types are known to be particularly sensitive to phosphorothioate-modified oligonucleotides, which may give misleading phenotypes that are unrelated to antisense activity.
The high potency of miRCURY LNA miRNA Inhibitors and miRCURY LNA miRNA Power Inhibitors allows them to be used at low concentrations, minimizing the risk of undesired secondary effects unrelated to the antisense activity.
DNA/RNA duplexes are a substrate for RNase H, which degrades the RNA strand, and we use this catalytic activity in our GapmeRs for RNA silencing. To ensure that our miRNA inhibitors do not degrade mRNA and long non-coding RNAs that happen to contain a complementary sequence, we have positioned the LNA nucleotides such that they are never distanced by more than a few bases. LNA effectively inhibits RNase H activity, ensuring that the LNA inhibitor/RNA duplexes are not recognized as substrates by RNase H. As a result, off-target effects on mRNA and lncRNA stability are minimized. Finally, ribosomes have strong helicase activity that can effectively remove even LNA-enhanced, high-affinity, short oligonucleotides. Therefore, our inhibitors also have minimal effects on translation of mRNAs that have complementary sequences in the open reading frame.
Predesigned miRCURY LNA miRNA Inhibitors and miRCURY LNA miRNA Power Inhibitors have been designed for all known human, mouse and rat miRNAs. Since many miRNAs are phylogenetically conserved, this set of inhibitors covers a large proportion of vertebrate and invertebrate miRNAs. These inhibitors are also available with fluorescein (6-FAM) labels. All miRNA inhibitors are desalted and delivered dried down in tubes in 1 nmol, 5 nmol and 15 nmol quantities.
We currently offer four predesigned negative controls, two for use with miRCURY LNA miRNA Inhibitors and two for use with miRCURY LNA miRNA Power Inhibitors. The miRCURY LNA miRNA Power Inhibitor Controls have phosphorothioate-modified backbones to match the miRCURY LNA miRNA Power Inhibitors.
Negative Control A: no hits of >70% homology to any sequence in any organism in the NCBI and miRBase databases.
miRCURY LNA miRNA Inhibitors are desalted and delivered dried down in tubes. Following resuspension, regular inhibitors can be delivered to cells with a transfection reagent or by electroporation. Alternatively, Power Inhibitors can be added directly to the cell culture medium for unassisted uptake via gymnosis. Phenotypic effects of the miRNA inhibitor are normally assessed 24–72 hours after delivery. For some applications, such as cell differentiation assays, the phenotypic readout may take place 7–10 days after transfection.
miRCURY LNA miRNA Inhibitors are primarily used for miRNA loss-of-function studies by assessing the biological consequences of inhibiting miRNA activity. These effects can be observed in a variety of ways, including using cellular assays to monitor cell proliferation, cell differentiation or apoptosis. Changes to gene expression of putative miRNA targets can also be measured at the mRNA or protein level.