Square Wave Voltammetry Detection and CD Spectroscopy Structural Characterization of 5- Methyl Cytosine-Containing Oligomeric DNA Sequences
Author
Preston, Victoria
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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.
Date
2023-01-04
Citation:
APA:
Preston, Victoria.
(January 2023).
Square Wave Voltammetry Detection and CD Spectroscopy Structural Characterization of 5- Methyl Cytosine-Containing Oligomeric DNA Sequences
(Master's Thesis, East Carolina University). Retrieved from the Scholarship.
(http://hdl.handle.net/10342/12247.)
MLA:
Preston, Victoria.
Square Wave Voltammetry Detection and CD Spectroscopy Structural Characterization of 5- Methyl Cytosine-Containing Oligomeric DNA Sequences.
Master's Thesis. East Carolina University,
January 2023. The Scholarship.
http://hdl.handle.net/10342/12247.
April 18, 2024.
Chicago:
Preston, Victoria,
“Square Wave Voltammetry Detection and CD Spectroscopy Structural Characterization of 5- Methyl Cytosine-Containing Oligomeric DNA Sequences”
(Master's Thesis., East Carolina University,
January 2023).
AMA:
Preston, Victoria.
Square Wave Voltammetry Detection and CD Spectroscopy Structural Characterization of 5- Methyl Cytosine-Containing Oligomeric DNA Sequences
[Master's Thesis]. Greenville, NC: East Carolina University;
January 2023.
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Publisher
East Carolina University