Selective Covalent Capture of a DNA Sequence Corresponding to a Cancer-driving C>G Mutation in the Gene by a Chemically Reactive Probe: Optimizing a Cross-linking Reaction with Non-canonical Duplex Structures

Covalent reactions are used in the detection of various biological analytes ranging from low molecular weight metabolites to protein-protein complexes. The detection of specific nucleic acid sequences is important in molecular biology and medicine but covalent approaches are less common in this field, in part, due to a deficit of simple and reliable reactions for the covalent capture of target sequences. Covalent anchoring can prevent the denaturation (melting) of probe-target complexes and causes signal degradation in typical hybridization-based assays. Here, we used chemically reactive nucleic acid probes that hybridize with, and covalently capture, a target sequence corresponding to a cancer-driving variant of the human gene. Our approach exploits a reductive amination reaction to generate a stable covalent attachment between an abasic site in the probe strand and a guanine mutation at position 35 in the gene sequence. Importantly, systematic variation of the probe sequence in a manner that formally introduces non-canonical structures such as bulges and mispairs into the probe-target duplex led to probes with dramatically improved cross-linking properties. An optimized abasic site-containing probe enabled simultaneous quantitative detection of both mutant and wild-type sequences in mixtures.