Plug-and-Play Competitive Hairpin Conversion Module Enables Enzyme-Free High-Resolution Discrimination of Single Nucleotide Polymorphisms

The accuracy of single-nucleotide polymorphism (SNP) detection in long sequences is fundamentally constrained by the minuscule thermodynamic differences arising from single-base mismatches, wherein the overall probe binding energy frequently masks single-base-discrimination signals. Here, we develop a plug-and-play competitive hairpin conversion module (CHCM) based on strand-competitive hybridization and toehold-mediated strand displacement. CHCM incorporates two structured hairpin probes (H1 and H2) that specifically recognize wild-type and mutant alleles, respectively. Through thermodynamic competition triggered by single-base variations, the system precisely modulates probe-target binding stability, directing the formation of differential DNA assemblies that convert single-nucleotide information into detectable sequence signals. Critically, CHCM discriminates heterozygous samples with 0.1% mutation abundance in long-sequence backgrounds using only conventional fluorescent probes without enzymatic assistance. Furthermore, CHCM exhibits exceptional technical compatibility: it seamlessly integrates with upstream PCR amplification or downstream catalytic hairpin assembly (CHA) signal amplification while sustaining robust performance. Successful SNP genotyping in soybean leaf genomic DNA validates its practical utility. This work delivers a high-resolution, cost-effective solution for SNP detection in complex matrices and establishes a scalable modular framework for dynamic nucleic acid probe design.