Turning DNAzyme into a Stitch-to-Activate Biosensor for Single-Base-Resolution Molecular Diagnostics

Abstract The catalytic core of DNAzymes is highly sensitive to local structural perturbations. Conventional DNAzyme-based SNV sensing mainly relies on differences in hybridization stability between matched and mismatched targets. Here, we position the SNV immediately adjacent to the catalytic-core reconstitution junction of a split DNAzyme, allowing a single-base mismatch to directly affect the catalytic core reconstitution and subsequent substrate cleavage. Systematic analysis of insertions, deletions, and substitutions within the catalytic core further confirmed the strong dependence of DNAzyme activity on local core structure. To enhance serum stability and sustained catalytic performance, we anchored the split probes onto cholesterol-modified micelles (DZM). Micellar confinement protects against nuclease degradation and promotes efficient repeated catalytic turnover, resulting in enhanced signal accumulation. DZM achieves a 100 fM detection limit and a two-fold higher signal-to-noise ratio than the free split DNAzyme, and discriminates KRAS G12D at a mutant fraction as low as 0.1%. Clinical validation on serum samples yields an AUC of 0.9940. The stitch-to-activate design also enables multiplexed detection by incorporating distinct recognition arms at the same junction, allowing simultaneous interrogation of multiple variants in one pot. This modular architecture is reprogrammable and compatible with DNA logic gates, offering a cost-effective liquid biopsy tool.