Generation and Single-Molecule Characterization of a Sequence-Selective Covalent Cross-Link Mediated by Mechlorethamine at a C–C Mismatch in Duplex DNA for Discrimination of a Disease-Relevant Single Nucleotide Polymorphism

Many strategies for the detection of nucleic acid sequence rely upon Watson–Crick hybridization of a probe strand to the target strand, but the reversible nature of nucleic acid hybridization presents an inherent challenge: short probes that provide high target specificity have relatively low target affinity resulting in signal losses. Sequence-specific covalent cross-linking reactions have the potential to provide both selective target capture and durable signal. We explore a novel approach involving sequence-specific covalent cross-linking of a probe to target DNA combined with single-molecule nanopore detection of the cross-linked DNA. Here, we exploited the selective reaction of mechlorethamine at a C–C mismatch for covalent capture of a target DNA sequence corresponding to a cancer-driving mutation at position 1799 of the human BRAF kinase gene. We then demonstrated that the α-hemolysin protein nanopore can be employed for the unambiguous, single-molecule detection of the cross-linked probe-target co...