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.
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.
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.
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.
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.
(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
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.
