Hairpin-Mediated TMSDR-CHA Signal Conversion for Highly Specific Single-Base Discrimination of EGFR L858R Mutation

The detection of EGFR L858R mutations (a key driver of non-small-cell lung cancer (NSCLC)) has become critical for precision medicine, driven by rapid advancements in molecular diagnostics. Signal-converting nucleic acid technology simplifies assays and reduces costs in high-throughput screening, but suffers from insufficient single-base discrimination and severe nonspecific background signal─two long-standing bottlenecks in low-abundance mutation detection. To address these issues, this study proposes a synergistic hairpin-mediated system by integrating the toehold-mediated strand displacement reaction (TMSDR) with catalytic hairpin assembly (CHA), where TMSDR enables specific signal conversion via toehold-triggered strand displacement, and the designed hairpin structure provides intrinsic anti-interference capability through internal complementary sequences (thus avoiding nonspecific binding with wild-type (WT) sequences). Compared with conventional linear nucleic acid converters, the proposed system demonstrates up to a 100-fold increase in specificity for single-base mutation discrimination, accompanied by a significant 58% reduction in nonspecific background signal. Notably, the TMSDR-CHA synergy achieves ultrasensitive detection of L858R mutations at an allele frequency as low as 0.5%, providing a novel technical platform for low-abundance genetic variant analysis.