Antisense LNA GapmeR in vivo LS (B)

Potent knockdown of mRNA or lncRNA

The 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 lncRNA

lncRNA 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 needed

Antisense 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 specificity

Antisense 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 models

Excellent 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 effects

Antisense 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 parameters

Antisense 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:

• Optimal target sequence accessibility to ensure high potency. The design tool selects target sequences based on local secondary structure prediction.
• Antisense off-target evaluation. GapmeR sequences are aligned against ENSEMBL to enable selection of the most specific Antisense LNA GapmeRs with minimal off-targets in the spliced and unspliced transcriptomes.
• Optimal oligo