SCHEDULED MAINTENANCE
Selective covalent capture of a DNA sequence corresponding to a cancer-driving C>G mutation in the KRAS gene by a chemically reactive probe: optimizing a cross-linking reaction with non-canonical duplex structures†
c
and
Kent S.
Gates
*ab
Abstract
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 KRAS 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 KRAS 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 KRAS sequences in mixtures.
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