The duplex stability with target mRNA and the gene silencing potential of a novel bridged nucleic acid analogue are described. The analogue, , - antisense oligonucleotides (AONs) ranging from 10- to 20-nt-long, targeted apolipoprotein B. , - was directly compared to its conventional bridged (or locked) nucleic acid ( , -BNA/LNA)-based counterparts. Melting temperatures of duplexes formed between , - -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 , -BNA/LNA-based counterpart, the corresponding , - -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 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 , - may be a better alternative to conventional , -BNA/LNA, even for “short” antisense oligonucleotides, which are attractive in terms of drug-likeness and cost-effective bulk production. 1. Introduction Recently designed and synthesized high-performance modified-nucleic-acids (HiPerNAs) such as 2′-O-methyl RNA (2′-OMe), 2′-O-methoxyethyl RNA (MOE), and 2′,4′-bridged nucleic acid/locked nucleic acid (2′,4′-BNA/LNA) have improved performance compared to phosphorothioate antisense oligonucleotides (AONs). HiPerNAs overcome the systemic antisense effects of these earlier antisense oligonucleotides and show promise as antisense therapeutics for the treatment of a variety of diseases [1–5]. However, more potent and less toxic AONs are required, since several clinical trials of AON drugs carrying HiPerNAs have been recently terminated due to the lack of efficacy or because of safety concerns. In addition, toxicity and delivery problems remain [6–8]. We previously described a unique modified nucleic acid, 2′,4′-bridged nucleic acid (2′,4′-BNA; also known as LNA) [9, 10]. Its high therapeutic efficacy is based on the extraordinarily high target binding of the original 2′,4′-BNA/LNA-based AON. 2′,4′-BNA/LNA-based AON is widely accepted as one of the most promising antisense drugs,
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