Characterization of mismatched DNA hybridization via a redox-active diviologen bound in the PNA-DNA minor groove

Diviologen molecules of the general formula CH ₃(CH ₂) ₁₁V ²⁺(CH ₂) ₁₁V ²⁺(CH ₂) ₁₁CH ₃ (C ₁₂VC ₆VC ₁₂, V ²⁺ = 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 y ^2+/+ redox couple C ₁₂VC ₆VC ₁₂ 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 C ₁₂VC ₆VC ₁₂ exhibited binding cooperativity to the complementary PNA-DNA hybrids and saturated at a ratio of 2:1 (C ₁₂VC ₆VC ₁₂:hybrid). Reduced C ₁₂VC ₆VC ₁₂ (V ⁺) absorption spectra showed a significant λ _max blue shift (22 nm) in the presence of complementary hybrids compared to the AmaK in the presence of PNA or mismatched DNA hybrids. Chronocoulometry was employed to assay surface populations and obtain thermodynamics for C ₁₂VC ₆VC ₁₂ binding. These data are consistent with C ₁₂VC ₆VC ₁₂ bound in the minor groove of complementary hybrids as face-to-face π-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.