Square Wave Voltammetry Detection and CD Spectroscopy Structural Characterization of 5- Methyl Cytosine-Containing Oligomeric DNA Sequences

DNA methylation, primarily at the 5-carbon in cytosine (5-methyl cytosine) plays a\nsignificant role in key processes and functions of the body, such as development of the brain and\nin neuronal cell differentiation. Lack of DNA methylation control has been linked to cancer,\ncardiovascular diseases, autoimmune diseases, and imprinting disorders. Due to its role in\nneuronal development, DNA methylation has also been implicated in the development of\nAlzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, Huntington’s disease\nand multiple sclerosis (MS). Because aberrant methylation can arise at such an early stage in\ndisease progression, detection of hypermethylated DNA sequences, or those sequences\nexhibiting elevated levels of cytosine methylation, may allow for earlier detection of these\ndiseases, resulting in more optimal treatment options.\nHere, we describe an electrochemical assay designed to detect cytosine methylation\nlevels in DNA sequences from CD8+ T cells. Methylation in this gene has been linked to\nmultiple sclerosis. Short, defined oligomeric sequences from this gene were immobilized on gold\nelectrodes via thiol linkages and exposed to target, complementary sequences featuring varying\nnumbers (0-4) 5-methyl cytosine modifications forming double stranded DNA hybrids on the\nelectrode. The DNA was then exposed to high ionic strength (0.1 M MgCl2) and a redox active\ndiviologen molecule of the form C12H25V2+C6H12V2+C12H25 (where V2+ = viologen, 4-4’-\nbipyridyl, C12Viologen). Detection of the DNA took place using square wave voltammetry via\nreduction of C12Viologen, which has been previously shown to bind to DNA in a structure\nspecific manner. MgCl2 induced structural changes in the DNA, which were related to the\namount of cytosine methylation featured in the oligomers. This could be detected via the\nemergence of a significant reduction current appearing at ~-0.37 V vs. SCE in non-methylated\noligomers that decreased in magnitude with DNA featuring increasing methylation content.\nStatistically significant differences in this current were detected between non-methylated\noligomers and all oligomers featuring any amount of cytosine methylation.\nThe DNA oligomers were characterized using circular dichroism (CD) spectroscopy and\nUV-Vis thermal melting studies. CD spectra showed that the oligomers adopted typical\nconformations based on their high GC content (likely favoring the A-form of DNA), and upon\nexposure to MgCl2, the strong positive bands decreased slightly, with the most change occurring\nin non-methylated oligomers. This data showed that the structural changes to either nonmethylated\nor methylated DNA were subtle, and likely resulted in a A or BII form where the\nbases were slightly altered from the initial structure upon exposure to MgCl2. Overall, these\nchanges were consistent with the electrochemical findings, showing that very small structural\nchanges related to DNA methylation could be detected using electrochemical methods, leading\nto the possibility that this approach could be used as a diagnostic tool to detect cytosine\nmethylation in similar short gene segments