Isothermal Detection of Single-Nucleotide Variants via pH-Mediated Toehold-Controlled Ligation on Double-Stranded DNA

Single-nucleotide variants (SNVs) are essential biomarkers in molecular diagnostics, yet their rapid and precise detection remains difficult due to the dependence on thermally driven DNA denaturation and instrument-intensive workflows. Here, we introduce a simple and cost-effective physicochemical strategy that exploits the strong pH sensitivity of DNA duplex stability to enable direct SNV detection on double-stranded DNA using a toehold-gated probe. Exposure to extreme pH conditions disrupts hydrogen bonding within the DNA duplex, inducing rapid strand unwinding. During the subsequent neutralization, the toehold-gated probe efficiently assembles at the target SNV site. Leveraging this principle, we developed a pH-mediated, toehold-controlled SNV detection method that eliminates the need for thermal denaturation. Acid-base treatment replaces conventional thermal denaturation, enabling rapid dsDNA unwinding and precise probe assembly on the target sequence, which selectively recognizes mutant alleles through toehold-mediated strand displacement and designed ligation. The resulting ligation product triggers a highly efficient in vitro transcription reaction, producing a visually distinguishable signal within minutes. The entire workflow comprises only two operational steps and achieves detection and readout within 45 min. This platform provides a low-cost, portable, and instrument-free solution for precise SNV identification, offering strong potential for rapid on-site molecular diagnostics.