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.
Xu GuoMaryam Imani NejadLi-Qun GuKent S GatesPublished in: RSC advances (2019)
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.
Keyphrases
- nucleic acid
- wild type
- living cells
- single molecule
- quantum dots
- high resolution
- fluorescent probe
- small molecule
- protein protein
- copy number
- papillary thyroid
- genome wide
- label free
- real time pcr
- squamous cell carcinoma
- amino acid
- childhood cancer
- induced pluripotent stem cells
- transcription factor
- high throughput
- photodynamic therapy
- genome wide analysis