Abstract
Discriminating single-nucleotide variations at the terminal regions of short nucleic acids remains a major challenge for hybridization-based electrochemical biosensors (E-biosensors), as terminal mismatches often produce insufficient destabilization for selective recognition. This limitation is particularly relevant for microRNAs and isomiRNAs, which frequently differ by only one or a few nucleotides at the 3' or 5' termini. Here, we investigate the electrochemical behavior of the OWL DNA architecture following its integration into a surface-confined sensing platform. Beyond demonstrating electrochemical target recognition, we examine how structural rigidity influences electron-transfer dynamics, signal generation, and terminal single-nucleotide polymorphism (SNP) discrimination relative to conventional duplex probes. Electrochemical characterization revealed a signaling mechanism distinct from duplex sensors: whereas duplex controls displayed frequency-dependent signal inversion, the OWL architecture produced consistent signal suppression over the explored frequency range. Additionally, targets containing single mismatches at either the 3' or 5' termini produced significantly lower responses, demonstrating effective terminal SNP discrimination. Preliminary matrix studies showed preserved functionality in diluted artificial urine and artificial saliva, whereas serum introduced significant signal variations. These findings establish rigid multistrand DNA architectures as promising recognition elements for electrochemical nucleic acid sensing and provide mechanistic insight into electron transfer within surface-confined DNA assemblies.