Photo-Programmable Temporal Uncoupling Enables One-Pot Isothermal Enrichment and Detection of Low-Abundance Single-Nucleotide Variants

Sensitive detection of low-abundance single-nucleotide variants (SNVs) remains challenging because rare mutant alleles are frequently masked by overwhelming wild-type DNA backgrounds. Herein, we develop a photo-programmable temporally uncoupled one-pot isothermal sensing platform (PTU-One-Pot) for sensitive enrichment and detection of the EGFR L858R mutation. The platform integrates restriction endonuclease-mediated wild-type DNA digestion with recombinase polymerase amplification (RPA) in a closed tube. To resolve the intrinsic kinetic conflict between relatively slow wild-type DNA digestion and rapid RPA amplification, RPA primers are modified with a photocleavable 6-nitropiperonyloxymethyl (NPOM) group. During the initial phase, NPOM-caged primers suppress RPA amplification, allowing MlsI restriction endonuclease to selectively digest wild-type templates. Subsequent brief ultraviolet irradiation removes the NPOM protection and activates RPA amplification of remaining mutant-type templates. Mechanistic studies confirm RPA photo-programmable trigger and sequence-selective wild-type template cleavage. Droplet digital PCR, high-resolution melting, Sanger sequencing verify that PTU-One-Pot can effectively eliminate wild-type templates and convert the final products from wild-type-dominated to mutant-enriched. Under optimized conditions, fluorescence readout achieves a limit of detection of 0.05% variant allele frequency, showing high specificity to EGFR L858R mutation. Lateral flow assay integration enables low-instrument visual detection at 1% variant allele frequency. Evaluation using 23 formalin-fixed paraffin-embedded patient samples demonstrates that both fluorescence and lateral flow readouts are consistent with next-generation sequencing and droplet digital PCR reference results. Overall, this PTU-One-Pot strategy provides a rapid and sensitive isothermal platform for low-abundance mutation enrichment and analysis.