Superior Silencing by 2’,4’-BNANC-based Short Antisense Oligonucleotides Compared to 2’,4’-BNA/LNA-based Apolipoprotein B-Antisense-Inhibitors

Tsuyoshi Yamamoto, et al.
The duplex stability with target mRNA and the gene silencing potential of a novel bridged nucleic acid analogue are described. The analogue, 2,4-BNANC antisense oligonucleotides (AONs) ranging from 10- to 20-nt-long, targeted apolipoprotein B. 2,4- BNANC was directly compared to its conventional bridged (or locked) nucleic acid (2,4-BNA/LNA)-based counterparts. Melting temperatures of duplexes formed between 2,4-BNANC-based antisense oligonucleotides and the target mRNA surpassed those of 2,4-BNA/LNA-based counterparts at all lengths. An in vitro transfection study revealed that when compared to the identical length 2,4-BNA/LNA-based counterpart, the corresponding 2,4-BNANC-based antisense oligonucleotide showed significantly stronger inhibitory activity. This inhibitory activity was more pronounced in shorter (13-, 14-, and 16-mer) oligonucleotides. On the other hand, the 2,4-BNANC-based 20-mer AON exhibited the highest affinity but the worst IC50 value, indicating that very high affinity may undermine antisense potency. These results suggest that the potency of AONs requires a balance between reward term and penalty term. Balance of these two parameters would depend on affinity, length, and the specific chemistry of the AON, and fine-tuning of this balance could lead to improved potency. We demonstrate that 2,4-BNANC may be a better alternative to conventional 2,4-BNA/LNA, even for &ldquo;short&rdquo; antisense oligonucleotides, which are attractive in terms of drug-likeness and cost-effective bulk production.<br />