Electrochemical Sensing of Charge-Neutral Antisense Oligonucleotides Using Hairpin Probes: A Case Study with Viltolarsen

Antisense oligonucleotides (ASOs) are synthetic, single-stranded oligonucleotides gaining traction in clinical therapeutics. Although pharmacokinetic analyses of ASOs have progressed, in vitro platforms integrating sensors─essential for improving analytical efficiency─are still under development. Electrochemical sensors are well-suited for the quantitative monitoring of various compounds, yet their application to ASOs─with their unique backbone linkages and sugar rings─remains unexplored. This study presents the first electrochemical sensing approach for ASOs, using Viltolarsen, a clinically approved charge-neutral ASO, as a representative case study. Hairpin-structured oligonucleotide probes complementary to the target sequence were engineered, immobilized on nanostructured electrodes, and hybridization events were monitored electrochemically. Compared with DNA analogs, sensing of Viltolarsen produced smaller signal changes but achieved hybridization more rapidly, likely due to reduced electrostatic repulsion between duplexes on the electrode surface. Furthermore, once hybridized, the hairpin probe did not readily revert to its initial conformation. This behavior arises from the high thermodynamic stability of the probe–Viltolarsen duplex, which results from minimal electrostatic repulsion between strands. These observed behaviors differ from those typically observed with DNA targets. Sensors composed of either DNA or RNA probes exhibited limits of detection of 150 and 50 nM, respectively. These findings provide new insights for designing electrochemical sensors specifically tailored to charge-neutral ASOs, advancing the development of pharmacokinetic analytical platforms.