Abstract

Antisense oligonucleotides (ASOs) have emerged as a powerful therapeutic modality for targeting disease‑associated RNAs. However, most clinical ASOs rely on chemical modifications to enhance stability and pharmacokinetic properties. In this study, we developed a series of pH‑responsive hairpin‑structured ASO prodrugs based on the i‑motif. By systematically varying loop size, loop position, and stem length, we obtained prodrugs with distinct structural stability and release kinetics. Notably, the R-series ASO prodrugs demonstrated the highest stability among all designed sequences, a property attributable to their largest loop structure. Among these, the construct with a 5‑base‑pair stem exhibited the optimal balance between stability and acid‑triggered release efficiency. In SK-BE (2) cells, these ASO prodrugs, particularly the R3‑5 and R5‑5 sequences, effectively silenced MYCN expression and induced apoptosis. This work provides a rational structure‑activity design strategy for tumor‑microenvironment‑responsive nucleic acid therapeutics and establishes a reference for further structural optimization.