Exploring the Potential of Phosphoramide N‑Benzimidazole Oligonucleotides (PABAOs) as Antisense Agents.

This study provides the first assessment of the therapeutic potential of phosphoramide benzoazole oligonucleotides (PABAOs) bearing N-benzimidazole moieties. Two series of antisense oligonucleotides (ASOs) targeting distinct regions of enhanced green fluorescent protein (eGFP) mRNA were synthesized via automated solid-phase phosphoramidite chemistry. Three N-benzimidazole modification patterns for each ASO were systematically evaluated. Incorporation of modification pairs proximal to both the 3' and 5' termini conferred enhanced ASO's resistance to degradation in fetal bovine serum (FBS). Although N-benzimidazole incorporation reduced hybridization affinity, all modified ASOs retained the ability to form stable complexes with structured RNA targets under conditions close to physiological. Notably, one of the two series of modified ASOs preserved RNase H-mediated cleavage activity at levels comparable to its unmodified counterpart. Cytotoxicity assessments revealed sequence-specific effects, with modified ASOs demonstrating negligible toxicity in HEK293-eGFP cells at concentrations up to 10 μM. Among the constructs, the one ASO bearing paired N-benzimidazole modifications proximal to both the 3' and 5' termini induced substantial eGFP knockdown, whereas the other ASOs and controls, including scrambled and phosphorothioate-containing variants, were inactive. In addition, oligonucleotide accumulation in cells increases with the number of modifications in the ASOs. These results identify N-benzimidazole-modified PABAOs as RNase H-competent antisense oligomers with tunable nuclease resistance and support further optimization of benzoazole backbones for therapeutic ASO design.