Abstract
There have been many attempts to unveil the therapeutic potential of antisense molecules during the last decade. Due to its specific role in canonical Wnt signalling, β-catenin is a potential target for an antisense-based antitumour therapy. In order to establish such a strategy with peptide nucleic acids, we developed a reporter assay for quantification of antisense effects. The luciferase-based assay detects splice blocking with high sensitivity. Using this assay, we show that the splice donor of exon 13 of β-catenin is particularly suitable for an antisense strategy, as it results in a truncated protein which lacks transactivating functions. Since the truncated proteins retain the interactions with Tcf/Lef proteins, they act in a dominant negative fashion competing with wild-type proteins and thus blocking the transcriptional activity of β-catenin. Furthermore, we show that the truncation does not interfere with binding of cadherin and α-catenin, both essential for its function in cell adhesion. Therefore, the antisense strategy blocks Wnt signalling with high efficiency but retains other important functions of β-catenin.
Highlights
In 1978 a synthetic antisense oligonucleotide was successfully used by Zamecnik and Stephensen for targeting of Rous sarcoma virus RNA within chick embryo fibroblasts [1]
In order to quantify the efficiency of the antisense molecules, a splice-dependant reporter assay was established
On the contrary, blocking of splicing by antisense molecules generates an mRNA including the non-spliced intron, which, contains an open reading frame directly combining those of exon 1 and 2, and resulting in a functional luciferase fusion protein
Summary
In 1978 a synthetic antisense oligonucleotide was successfully used by Zamecnik and Stephensen for targeting of Rous sarcoma virus RNA within chick embryo fibroblasts [1]. The successful clinical application demonstrated the potential of antisense reagents and motivated scientists to overcome issues with solubility, degradation or cellular delivery [6]. Due to their good affinity for complementary RNA sequences, lack of interactions with natural proteins and excellent water solubility, morpholino oligos became the gold standard for antisense knock-down experiments [7, 8]. They show a characteristic structure with a 6-membered morpholine ring replacing ring sugars present within naturally occurring nucleic acids. Negatively charged phosphate linkages of DNA and RNA have been replaced by phosphorodiamidate intersubunit linkages [7]
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