text.skipToContent text.skipToNavigation

Antisense LNA GapmeR Custom Plate

For RNA functional analysis screening projects using custom 96-well plates of Antisense LNA GapmeRs
  • Custom plates of Antisense LNA GapmeRs for RNA functional analysis screening projects
  • Function by RNase H-dependent degradation of complementary RNA targets
  • Provide strand-specific knockdown with no RISC-associated, off-target activity
  • Excellent alternative to siRNA for knockdown of mRNA and lncRNA
  • Taken up by cells without need for transfection reagents

Customize your own 96-well plates of Antisense LNA GapmeRs for use in RNA functional analysis screening projects. Antisense LNA GapmeRs are highly potent, single-stranded antisense oligonucleotides (ASO) for silencing of lncRNA and mRNA. The LNA-enhanced GapmeRs are designed using sophisticated and empirically developed algorithms and offer excellent performance and high success rates.

产品 Product no. 货号 目录价:
 
 
Show details
    varies
Can't order online?
To place an order via phone, email or for requesting a quote, please provide the product’s name, number and catalog number.
产品 Product no. 货号 目录价:
Antisense LNA GapmeR Custom Plate (5 nmol)
5 nmol Custom Antisense LNA GapmeRs, option of in vitro standard grade or in vivo ready with different labels, provided in plate
339530 Varies Create and order

Antisense LNA GapmeR Custom Plate适用于分子生物学应用。该产品不适用于疾病的诊断、预防或治疗。


0
Silencing of mRNA and long non-coding RNA using Antisense LNA GapmeRs.
Antisense LNA GapmeRs are single-stranded LNA oligonucleotides that hybridize and catalyze degradation of their target RNA. We exploit the fact that DNA:RNA duplexes are recognized by RNase H1 in the nucleus, creating an endonucleolytic cleavage in the RNA strand. The two generated fragments are then degraded by exonucleases. The Antisense LNA GapmeR is released and will continue on, guiding degradation of another RNA strand.
1
Antisense LNA GapmeRs have higher success rate and potency than siRNAs.
The potency of different designs of Antisense LNA GapmeRs and siRNAs from a leading provider for mRNA targets X and Y was compared. All of the Antisense LNA GapmeRs demonstrated the most potent effect in reduction of the two mRNA targets and with excellent dose-response. For each of the two targets X and Y, five different LNA GapmeRs and four different siRNA designs were tested in a dose-response setup using three oligonucleotide concentrations: 1 nM, 5 nM and 25 nM. A negative control (mock or scrambled control) was included in each study. The effect of the knockdown was determined by qPCR and reported as CT of a control (actin)/CT of the target. (A–B) Antisense LNA GapmeRs and siRNAs targeting protein X were transfected into BT474 cells. (C–D) Antisense LNA GapmeRs and siRNAs targeting protein Y were transfected into HN5 cells.
2
Efficient knockdown with Antisense LNA GapmeRs, regardless of RNA target type and subcellular localization.
Antisense LNA GapmeRs were designed for different RNA targets with our online design tool. The targets included mRNA (A–B) and lncRNAs (C–E) with different subcellular localizations. The five highest-ranking LNA GapmeRs for each target were tested for efficacy. In all cases, at least two out of the five LNA GapmeRs tested provided highly potent knockdown of their RNA target in concentrations as low as 1–10 nM and irrespective of their subcellular localization. HeLa cells were transfected. Cells were harvested 48 H after transfection and analyzed for target RNA content by qPCR. Data were normalized to GAPDH, and the relative target RNA content was compared to that from untreated cells.




3
LNA GapmeRs can be used without a transfection agent.
Antisense LNA GapmeRs can be delivered to cell lines by unassisted uptake or gymnosis, when they are added directly to the culture medium without transfection reagents. This is useful for difficult-to-transfect cell lines and for avoiding experimental artifacts introduced by transfection reagents. Malat1 is a highly abundant lncRNA localized in nuclear speckles. HeLa cells were transfected with a Malat1 LNA GapmeR or a negative LNA GapmeR control, or the GapmeRs were added directly to HeLa cell cultures without transfection reagents for unassisted uptake. Cells were harvested 48 H after transfection or direct additon of the LNA GapmeR and were analyzed for Malat1 content by qPCR. Data were normalized to GAPDH, and the relative Malat1 content was compared with untreated cells. The results demonstrate almost complete elimination of Malat1 both by classical transfection and by unassisted uptake.
4
Efficient in vivo knockdown with LNA GapmeRs in a broad spectrum of tissues.
The Antisense LNA GapmeR for knockdown of Malat1 in mice was injected subcutaneously over a period of 5 weeks. Animals received 2 x 10 mg the first week and 2 x 15 mg/week for another 4 weeks. Tissue samples from the mice were collected (A) 2 days after the final LNA GapmeR administration or (B) for up to 15 weeks after the final LNA GapmeR administration. Total RNA was extracted from tissue samples and analyzed for Malat1 content by qPCR. The results were normalized with Actin and are an average from groups of six animals. The results show highly efficient knockdown of Malat1 in a broad range of different tissues. The knockdown effect was still highly efficient in most tissues even 5 weeks after the last dose. The duration of the knockdown varied between tissues, from long to medium to short, reflecting differences in the pharmacodynamics of the Antisense LNA GapmeRs across tissue types.
5
A unique short, single-stranded antisense design.
(A) Antisense LNA GapmeRs are single-stranded, short 16mer oligonucleotides containing a DNA portion flanked by LNA. (B) The LNA parts increase the affinity for the target and confer nuclease resistance, regardless of GC content. Generally, duplexes of DNA hybridized to RNA catalyze RNase H-dependent degradation of the RNA strand. However, LNA inhibits RNase H activity. For this reason, Antisense LNA GapmeRs are designed with a central LNA-free gap containing traditional DNA nucleotides only, a crucial feature for efficient endonucleolytic cleavage of the target RNA. (C) RNase H is activated by the DNA part of the antisense oligonucleotide
6
Custom plates with Antisense LNA GapmeRs for RNA functional analysis screening.
Antisense LNA GapmeRs are available in a convenient 96-well plate format useful for various screening applications, including identifying super-potent LNA GapmeRs by screening multiple GapmeRs per RNA target and loss-of-function screening of multiple RNAs in parallel. Antisense LNA GapmeRs are transfected into cell lines, and relevant functional phenotypic assays are used to identify genes involved in biological processes of interest. The GapmeRs may also be screened for potency (measurement of target RNA knockdown efficiency) using RT-qPCR, for example.

Custom plates contain 0.2 nmol Antisense LNA GapmeRs in vitro Standard delivered dried down in 96-well plates, with a minimum 18 GapmeRs per plate. The plate layout is fully flexible. This example includes positive and negative control oligonucleotides with empty wells at the edge of the plate to avoid evaporation edge effects.
7
Performance of Antisense LNA GapmeR Positive Controls.
HeLa cells were transfected with Antisense LNA GapmeRs against the indicated RNA targets. Cells were harvested 48 H after transfection and were analyzed for target RNA content by qPCR. (A) MALAT-1: HeLa cells were either transfected with a MALAT1 LNA GapmeR and an LNA GapmeR negative control, or LNA GapmeRs were added directly to HeLa cell cultures without transfection reagent (unassisted uptake). qPCR data were normalized to GAPDH, and the relative MALAT1 content was compared with untreated cells. The results demonstrate almost complete elimination of MALAT1 both by classical transfection and by unassisted uptake. (B–C) PTEN and mTOR: qPCR data were normalized to GAPDH, and the relative target RNA content was compared with untreated cells. (D) Hotair: qPCR data were normalized to GAPDH, and the relative target RNA content was compared with untreated cells. (E) GAPDH: qPCR data were normalized with a normalization factor based on HPRT, ACTB and MALAT1 RNA, and the relative GAPDH content was compared with untreated cells.

Performance
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.
Principle
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, Tm, gap size, self-complementarity, LNA positions, etc.
Procedure
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.
Applications
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.

您无权限查看此资源

fragment fix placeholder