Programmable DNAzyme switch integrated with low-background cross-reaction for sensitive and selective SNP identification

Single-nucleotide polymorphism (SNP) detection plays a critical role in early disease screening, personalized medicine, and crop genetic improvement. In recent years, DNAzymes have attracted widespread attention in molecular recognition and catalytic diagnostics because of the sequence programmability and strong strand-cleavage activity. However, current DNAzyme-based systems still face significant challenges, such as limited sequence selectivity and high nonspecific reactivity, which constrain their broader application in high-precision genotyping, particularly in SNP discrimination. To overcome these limitations, we rationally redesigned the catalytic core of the DNAzyme to construct a competitive molecular switch governed by an “activation–silencing” mechanism, thereby addressing the bottleneck of single-base specificity in DNAzyme systems. This protein-enzyme-free strategy for SNP recognition also breaks the strict stoichiometric paradigm of conventional enzyme-free strand displacement reactions, resulting in a significant enhancement of target selectivity. To tackle the common issue where improved selectivity often compromises detection sensitivity, we innovatively introduced a solid-liquid phase cross-reaction mechanism and developed a cascade system based on electrochemical biosensing to improve analytical sensitivity. Our strategy enables sensitive detection of SNPs with a detection limit as low as 11.3 aM, representing a marked improvement over sensors with conventionally vertical amplification (370 aM). Furthermore, it demonstrates high consistency in genotyping representative soybean variants. Beyond theoretical model for improved single-base recognition and signal transduction, this work provides an innovative, enzyme-free, and scalable platform for SNP sensing and signal regulation, offering new concepts for precision genotyping and molecular diagnostics.