Antisense LNA GapmeRs
For highly effective knockdown of mRNA and lncRNA using LNA-enhanced antisense oligonucleotides
Antisense LNA GapmeRs are highly potent, single-stranded antisense oligonucleotides (ASO) for silencing of lncRNA and mRNA in cell cultures and even in animal models. Antisense LNA GapmeR Positive Controls enable optimization of conditions for lipid-based transfection, electroporation or unassisted delivery. The GapmeRs are enhanced with LNA technology and are designed using sophisticated and empirically developed algorithms and offer excellent performance and high success rates.
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Potent knockdown of mRNA or lncRNAThe efficacy of mRNA knockdown using Antisense LNA GapmeRs rivals that of siRNA-based methods (see figure Antisense LNA GapmeRs have higher success rate and potency than siRNAs), providing an excellent alternative for researchers looking for a technique that works independently of RISC and has no miRNA-like, off-target effects.
Tool-of-choice for silencing of lncRNAlncRNA loss-of-function studies can be particularly challenging for several reasons. Many lncRNAs are involved in transcriptional regulation by attracting chromatin-modifying enzymes to certain DNA targets. Since they are confined to the nuclear compartment, these lncRNAs are inefficiently targeted by siRNA. In contrast, RNAs retained in the nucleus are particularly sensitive to Antisense LNA GapmeRs, because they share the nuclear compartment with RNase H, the endonuclease responsible for Antisense LNA GapmeR activity (see figure Silencing of mRNA and long non-coding RNA using Antisense LNA GapmeRs). In addition, lncRNAs often derive from transcriptionally complex loci with overlapping sense and antisense transcripts. Strand-specific knockdown is therefore crucial, and this is guaranteed with Antisense LNA GapmeRs, because they are single stranded. Antisense LNA GapmeRs provide effective knockdown of various lncRNAs, regardless of their intracellular localization (see figure Efficient knockdown with Antisense LNA GapmeRs, regardless of RNA target type and subcellular localization).
No transfection reagent neededAntisense LNA GapmeRs are efficiently taken up by cells directly from the culture medium due to their small size and exceptional potency and stability. This makes it possible to achieve potent knockdown of target RNA in many cell lines with unassisted delivery (see figure LNA GapmeRs can be used without a transfection agent), avoiding the cytotoxic effects associated with transfection reagents. Non-assisted uptake does require higher concentrations of the Antisense LNA GapmeR than would be needed with lipid-based transfection, and the knockdown kinetics are slower. Usually, knockdown is observed after only 48 H of culture in the presence of the Antisense LNA GapmeR.
Potent positive controls with optimal specificityAntisense LNA GapmeR Positive Controls are experimentally validated and feature very potent activity against different types of RNA targets expressed in a broad range of cell types. The controls are available for different types of RNA with different subcellular localization (see figure Performance of Antisense LNA GapmeR Positive Controls), making it possible to identify an appropriate control for most applications. Every Antisense LNA GapmeR Positive Control was designed for optimal specificity and was selected based on experiments demonstrating highly potent activity against its intended target.
Study RNA function in live animal modelsExcellent pharmacokinetic and pharmacodynamic properties of Antisense LNA GapmeRs have been demonstrated in many different tissues and organs. These LNA antisense oligonucleotides are well tolerated and show low toxicity in vivo. In addition, short, high-affinity Antisense LNA GapmeRs are active at lower concentrations than other antisense oligonucleotides. The incorporation of LNA also increases the serum stability of the ASO.
Antisense LNA GapmeRs have high potential to penetrate the cell membrane barrier and successfully interact with intracellular and even nuclear-retained targets. They also provide effective and long-lasting knockdown of mRNA and lncRNA in a broad range of tissues in live animal models. Plus, the workflow is easier, because specific formulation using liposomes or cationic complexes, for example, is not required for efficient in vivo delivery. See figure Efficient in vivo knockdown with LNA GapmeRs in a broad spectrum of tissues for an example of in vivo knockdown of a highly abundant, nuclear-retained lncRNA.
Efficient silencing of mRNA and lncRNA with fewer off-target effectsAntisense LNA GapmeRs are powerful tools for protein, mRNA and lncRNA loss-of-function studies. These single-stranded, antisense oligonucleotides (ASOs) catalyze RNase H-dependent degradation of complementary RNA targets. The Antisense LNA GapmeRs are 16 nucleotides long and are enriched with LNA in the flanking regions and DNA in an LNA-free central gap, hence the name "GapmeR" (see figure A unique short, single-stranded antisense design). The LNA-containing flanking regions confer nuclease resistance to the antisense oligo, while also increasing target binding affinity, regardless of the GC content. The central DNA "gap" activates RNase H cleavage of the target RNA upon binding. Antisense LNA GapmeRs have fully modified phosphorothioate (PS) backbones, which ensure exceptional resistance to enzymatic degradation.
Sophisticated design parametersAntisense LNA GapmeRs are designed using our empirically derived design tool that incorporates more than 20 years of experience with LNA design. For each RNA target, the tool evaluates thousands of possible designs against more than 30 design parameters to identify the Antisense LNA GapmeRs most likely to provide potent and specific target knockdown.
The primary design parameters include the following:
CoverageAntisense LNA GapmeRs can be designed for any RNA target >80 nucleotides and are available in several different grades, depending on your application:
Highly purified Antisense LNA GapmeRs for in vivo studiesWe also offer higher-purity Antisense LNA GapmeRs for use in in vivo studies. These Antisense LNA GapmeRs have excellent pharmacokinetic and pharmacodynamic properties and offer effective and long-lasting RNA silencing in a broad range of tissues. They are well-tolerated and show low toxicity in vivo, due to the following unique features:
Antisense LNA GapmeRs are primarily used to study the functions of mRNA or lncRNA by assessing the biological consequences of inhibiting their expression. The effect of silencing an mRNA or lncRNA can be studied in numerous ways, such as using cellular assays to monitor cell proliferation, cell differentiation, or apoptosis. The effect on gene expression can also be measured at the level of RNA or putative protein targets. Following resuspension, Antisense LNA GapmeRs are introduced into cells using a transfection reagent or via unassisted uptake (gymnosis), and phenotypic effects are assessed at an appropriate time afterwards.
The in vivo grade of Antisense LNA GapmeRs is available with fluorescein or other custom modifications. They are delivered dried down in Na salt, ready to be dissolved in PBS. If needed, they can be delivered in the quality required and with the necessary documentation for preclinical toxicity studies and human trials. After resuspension, these GapmeRs can be directly injected systemically with no need for expensive formulation in nanoparticles or liposomes. LNA GapmeRs can be administered via various routes (subcutaneous, intravenous, intraperitoneal, etc.). For more information, refer to the in vivo guidelines.
Antisense LNA GapmeRs are powerful tools for loss-of-function studies of proteins, mRNA and lncRNAs.
When delivered effectively to cell cultures, Antisense LNA GapmeR Positive Controls are certain to provide very significant knockdown of their RNA targets, so they are very useful for optimizing conditions for lipid-based transfection or electroporation. They can also be used to test the susceptibility of a particular cell line to unassisted uptake.
RNA function in animal models can be inferred by studying the biological consequences of RNA silencing with Antisense LNA GapmeRs. Such studies have revealed the important role played by lncRNAs in several human diseases.
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