Triplex-forming oligonucleotides as programmable recognition interfaces for biosensing and translational diagnostics

Triplex-forming oligonucleotides (TFOs) are programmable nucleic acid ligands that recognize duplex DNA through Hoogsteen or reverse Hoogsteen interactions, providing a unique molecular recognition strategy beyond conventional Watson-Crick hybridization. Recent advances in nucleic acid engineering, chemical modification, nanotechnology, and signal amplification have transformed TFOs from classical antigene agents into versatile platforms for biosensing and bioanalysis. In this review, we highlight the chemical principles governing triplex formation and discuss recent developments in TFO-enabled sensing architectures, including optical, electrochemical, and multimodal platforms. Emerging applications in molecular diagnostics, intracellular analysis, and translational bioanalysis are critically evaluated, together with the integration of nanomaterials, microfluidics, and CRISPR-based technologies. We further discuss key challenges, including triplex stability, target accessibility, delivery, and scalability, and provide perspectives on the future of programmable triplex-based interfaces for intelligent biosensing and precision diagnostics.