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

Abstract

DNA methylation, primarily at the 5-carbon in cytosine (5-methyl cytosine) plays a significant role in key processes and functions of the body, such as development of the brain and in neuronal cell differentiation. Lack of DNA methylation control has been linked to cancer, cardiovascular diseases, autoimmune diseases, and imprinting disorders. Due to its role in neuronal development, DNA methylation has also been implicated in the development of Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, Huntington’s disease and multiple sclerosis (MS). Because aberrant methylation can arise at such an early stage in disease progression, detection of hypermethylated DNA sequences, or those sequences exhibiting elevated levels of cytosine methylation, may allow for earlier detection of these diseases, resulting in more optimal treatment options. Here, we describe an electrochemical assay designed to detect cytosine methylation levels in DNA sequences from CD8+ T cells. Methylation in this gene has been linked to multiple sclerosis. Short, defined oligomeric sequences from this gene were immobilized on gold electrodes via thiol linkages and exposed to target, complementary sequences featuring varying numbers (0-4) 5-methyl cytosine modifications forming double stranded DNA hybrids on the electrode. The DNA was then exposed to high ionic strength (0.1 M MgCl2) and a redox active diviologen molecule of the form C12H25V2+C6H12V2+C12H25 (where V2+ = viologen, 4-4’- bipyridyl, C12Viologen). Detection of the DNA took place using square wave voltammetry via reduction of C12Viologen, which has been previously shown to bind to DNA in a structure specific manner. MgCl2 induced structural changes in the DNA, which were related to the amount of cytosine methylation featured in the oligomers. This could be detected via the emergence of a significant reduction current appearing at ~-0.37 V vs. SCE in non-methylated oligomers that decreased in magnitude with DNA featuring increasing methylation content. Statistically significant differences in this current were detected between non-methylated oligomers and all oligomers featuring any amount of cytosine methylation. The DNA oligomers were characterized using circular dichroism (CD) spectroscopy and UV-Vis thermal melting studies. CD spectra showed that the oligomers adopted typical conformations based on their high GC content (likely favoring the A-form of DNA), and upon exposure to MgCl2, the strong positive bands decreased slightly, with the most change occurring in non-methylated oligomers. This data showed that the structural changes to either nonmethylated or methylated DNA were subtle, and likely resulted in a A or BII form where the bases were slightly altered from the initial structure upon exposure to MgCl2. Overall, these changes were consistent with the electrochemical findings, showing that very small structural changes related to DNA methylation could be detected using electrochemical methods, leading to the possibility that this approach could be used as a diagnostic tool to detect cytosine methylation in similar short gene segments.