Antisense LNA GapmeR Custom Plate

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.

Antisense LNA GapmeR Custom Plates for RNA functional analysis screening projects

Antisense LNA GapmeRs are available in a convenient 96-well plate format that is useful for loss-of-function screening of multiple RNAs in parallel (see figure Custom plates with Antisense LNA GapmeRs for RNA functional analysis screening). The custom plates can also be used for identifying super-potent LNA GapmeRs through screening multiple LNA GapmeRs per RNA target.

Antisense LNA GapmeR Custom plate details:

• Antisense LNA GapmeRs with custom-defined purity and amount, delivered dried-down in 96-well plates
• Minimum order is 18 Antisense LNA GapmeRs
• Fully flexible plate layout
• Cost-effective solution

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 oligonucleotide design, including length, T_m, gap size, self-complementarity, LNA positions, etc.

Antisense LNA GapmeRs are antisense oligonucleotides with perfect sequence complementary to their RNA target. When introduced into cells, they sequester their target RNA in highly stable DNA:RNA heteroduplexes, leading to RNase H-mediated target degradation. The sequences of the oligonucleotides and their LNA spiking patterns have been carefully designed to achieve high target affinity with excellent sequence specificity and biological stability, while keeping the self-annealing properties to a minimum.

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.

Antisense LNA GapmeRs are potent antisense oligonucleotides 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.