AROs can be delivered directly via lipofection as synthesized oligonucleotides.
A major appeal of AROs as a potential therapeutic is their small size and single-stranded nature. Smaller molecules are easier to deliver and single-stranded RNA is not targeted by cellular mechanisms that actively seek out double-stranded RNA for degradation. To determine if AROs could be delivered directly as single-stranded RNA oligonucleotides, we ordered synthesized AROs targeting KRT14 and tested them in HaCat cells. Each molecule was designed to contain phosphorothioate bonds between the first two and last two bases to inhibit exonuclease degradation. The targeting domain was the same sequence used in the previous plasmid based knockdown experiment (Figure 1). We tested a single 5’ ARL (ARL-Oligo) as well as ARLs fused to each end of the targeting oligo (ARL-Oligo-ARL) and compared them to a previously validated siRNA targeting KRT14 at varying concentrations (Figure 4A).
We observed significant knockdown effects at all concentrations of ARL-Oligo and anti-KRT14 siRNA conditions, with diminishing returns at above 100nM. Interestingly, we did not observe significant knockdown with ARL-Oligo-ARL oligos, despite this configuration being the most effective in plasmid experiments, until concentrations of 1 µM. One possibility for this may be that the ARL-Oligo-ARL may have had difficulty making it to the nucleus due to its increased size. Another possibility is that interactions between complementary regions in the two ARLs on the oligo caused the targeting domain to be less available or degraded more quickly than its single ARL counterpart.