Unlabeled hairpin DNA probe for electrochemical detection of single-nucleotide mismatches based on MutS-DNA interactions

The paper described a label-free assay for the detection of single-nucleotide mismatches in which an unlabeled hairpin DNA probe and a MutS protein conjugate (His6-MutS-linker peptide-streptavidin binding peptide (HMLS)) are exploited for the detection of mismatches by electrochemical impedance spectroscopy (EIS). We demonstrate this method for eight single-nucleotide mismatches. Upon hybridization of the target strand with the hairpin DNA probe, the stem-loop structure is opened forming a duplex DNA. In duplexes containing a single nucleotide mismatch, the mismatch is present at the solvent exposed side, enabling more effective HMLS recognition and binding. The binding event is evaluated by EIS and analyzed with the help of Randles' equivalent circuits. The differences in the charge transfer resistance DeltaR(CT) before and after protein binding to the duplex DNA allows the unequivocal detection of all eight single-nucleotide mismatches. DeltaR(CT) allows the discrimination of a C-A mismatch with the concentration of the target strand as low as 100 pM.