Dual‐Stage Mismatch Interrogation (DMI) for High‐Fidelity G–T Discrimination by a DNA Nanodevice

ABSTRACT Precise discrimination of single‐nucleotide variants (SNVs) under physiological conditions remains a significant challenge for nucleic acid technologies, particularly in the case of G>A mutations, where relatively stable G–T mismatches substantially reduce recognition selectivity. Here, we report a modular DNA nanodevice, termed CAPDV (Cut and Paste nanoDevice), that achieves high‐fidelity SNV recognition through a novel concept referred to as dual‐stage mismatch interrogation (DMI). CAPDV incorporates two functional modules. Module 1 selectively excises an RNA fragment containing the mutation using paired DNAzymes. The excised fragment is subsequently transferred to Module 2, where a second round of mutation interrogation occurs. This step is followed by excised fragment‐dependent cleavage of specific RNA sequence by RNase H. While each module individually exhibits limited selectivity, their integration within a CAPDV enables nearly perfect 94% differentiation of G–T mismatch. The DMI mechanism enhances overall recognition selectivity and may serve as a novel strategy for improving selectivity in challenging cases of SNV recognition.