Nuclear Magnetic Resonance (NMR) Spectroscopy is a crucial tool for determining the structures of biological molecules. This technique can also be used to extract thermodynamic parameters of these molecules, enhancing our understanding of their biological roles. DNA is analyzed through NMR Spectroscopy in order to identify the effect of sequence on expressivity. DNA predominantly resides in BI orientation, but a second conformation, BII, also exists. DNA can switch between BI and BII backbone conformations and the likelihood of this switching is dependent upon the energetic barrier between these two sub-states. The secondary structure of DNA, and thus its adoption of BI and BII conformation, is sequence-dependent. Therefore, the identity and neighboring base pairs of a segment of DNA have a large effect on the flexibility of the backbone. Methylation also affects backbone structure. The methyl group has been shown to promote either stabilization and/or destabilization on proximate bases. This thesis uses variable temperature NMR and Mathematica modeling to determine the backbone conformations, rate of inter-conversion between BI/BII conformations, and the energetic barrier of this fluctuation for each nucleotide step in DNA dodecamers containing the CRE binding sequence. This has been a long-term goal of the Hatcher-Skeers lab, and the data from this thesis would have been added to years of flanked CRE DNA information to reveal any patterns. In this experiment, 5’-TTTC-3’ CRE DNA dodecamers underwent NMR analyses to extract backbone flexibility parameters. Additionally, the effect of methylation was studied in scans with methylated cytosine in the central CRE sequence. The TRX scale was used to predict the BII character of these sequences. Due to technical errors, the experimental results were not able to accurately represent the specific dynamics of each backbone step. However, general trends were identified, such as adherence to and veracity of the TRX scale and the effect of methylation. It was found that the %BII of the native DNA closely resembled the TRX predictions, whilst the methylated sequence did not. The largest changes in activation energy due to methylation occurred in the central CRE sequence, suggesting methylation is a localized effect. The results reflected several trends from past CRE experiments, but the data cannot be explicitly analyzed due to the technical errors.
| Identifer | oai:union.ndltd.org:CLAREMONT/oai:scholarship.claremont.edu:cmc_theses-2750 |
| Date | 01 January 2017 |
| Creators | Johnson, Amy |
| Publisher | Scholarship @ Claremont |
| Source Sets | Claremont Colleges |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | CMC Senior Theses |
| Rights | © 2017 Amy D Johnson, default |
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