AROs targeted to TBK1 knock down RNA expression across multiple locations on exon 14.
To evaluate whether AROs could knock down the expression of other genes, we designed AROs against the TBK1 gene (Figure 3A). We targeted exon 14 because it contained multiple ASF/SF2 sites according to ESE finder (Cartegni et al., 2003) and because its moderate size (122 bp) allowed us to evaluate multiple AROs targeting the same exon, as diagrammed in Figure 3A. To investigate whether AROs act in a location-specific manner, we designed 5 different AROs targeting exon 14 of TBK1 in 20 base pair segments and tested them in HaCat cells. All 5 AROs significantly knocked down TBK1 expression (P =< 0.01) when compared to the mock transfection carrier control. The largest knockdown was seen in ARL-TBK1-O3-ARL (51%) and the smallest in TBK1-O5-ARL (37%). These results indicate that all AROs targeting exon 14 of TKB1 were effective at gene knockdown.
As a negative control, we included the ALR-Oligo-ARL construct targeting the KRT14 gene from the previous experiment. As expected, the KRT14-targeting ARO did not decrease TBK1 transcript levels. Instead, we observed a significant (P <= 0.01) increase in TBK1 gene expression when KRT14 is knocked down. We hypothesize that the increase in TBK1 expression is a downstream result of perturbation of KRT14 expression levels.
Our experiments do not seem to indicate any preference for targeting oligo location along the length of the exon as all of the oligonucleotides that targeted exon 14 of TBK1 showed similar knockdown effects.