Characterization of Mismatched DNA Hybridization via a Redox-Active Diviologen Bound in the PNA-DNA Minor Groove

Diviologen molecules of the general formula CH3(CH2)(11)V2+(CH2)(6)V2+(CH2)(11)CH3 (C12VC6VC12, V2+ = 4,4'-bipyridinium or viologen) were employed to electrochemically assay DNA hybridization to PNA probes immobilized at Au electrodes. Immobilized 15-mer PNA probes were exposed to 25-mer DNA oligonucleotides containing either complementary or single base mismatched sequences. In the presence of complementary PNA-DNA hybrids, the V2+/+ redox couple of C12VC6VC12 exhibited a unique double-wave cyclic voltammogram, with a formal potential shifted -100 mV from the E-f in the presence of single base mismatched DNA hybrids or PNA probes alone. Integration of the CVs demonstrated that C12VC6VC12 exhibited binding cooperativity to the complementary PNA-DNA hybrids and saturated at a ratio of 2:1 (C12VC6VC12:hybrid). Reduced C12VC6VC12 (V+) absorption spectra showed a significant lambda(max) blue shift (22 nm) in the presence of complementary hybrids compared to the lambda(max) in the presence of PNA or mismatched DNA hybrids. Chronocoulometry was employed to assay surface populations and obtain thermodynamics for C12VC6VC12 binding. These data are consistent with C12VC6VC12 bound in the minor groove of complementary hybrids as face-to-face pi-dimers. This approach to distinguishing complementary hybrids from mismatched hybrids is novel, with potential applications involving detection of DNA damage or single nucleotide polymorphism (SNP) analysis.