Analysis of 5-Methyl Cytosine Containing Oligomers Via Reduction of Tetracationic Di-Viologen Derivatives Using Square Wave Voltammetry

Abstract

5-Methylcytosine (5 mC) is an epigenetic modification that plays several important roles ranging from development to disease progression. Local cytosine methylation is still detected and quantified utilizing bisulfite cytosine to uracil conversion and methylated base counting following PCR. Electrochemical approaches may offer more rapid means to provide this output. Here, small oligomeric DNA sequences from the CD8+ T-cell reporter gene featuring between 0 and 4 5 mC sites were immobilized on Au electrodes via self-assembly. 5 mC content was assayed via square wave voltametric reduction of a di-viologen molecule (C₁₂H₂₅V²⁺C₆H₁₂V²⁺C₁₂H₂₅, V²⁺=4,4’-bipyridyl or viologen) bound to the DNA. 5 mC presence induced subtle structural changes in the oligomers, which could be detected monitoring di-viologen reduction current changes at −0.39 V vs. SCE after exposure to higher ionic strength conditions. Solution phase CD spectroscopy was employed to validate the DNA structural changes occurring at the electrode surface and provide insight into the structural perturbations resulting from 5 mC presence. Overall, this method provides a relatively simple electrochemical 5 mC monitoring approach for small gene sequences based on helical structure.