Date of Award
Bachelor of Science
Many important enzymatic and physical processes occur specifically at the ends of DNA molecules. The goal of this project was to develop a molecule that would be an effective probe of the chemistry a. the ends of DNA. To do this, ethidium bromide, a well-known intercalation (a molecule that binds between DNA base pairs), was blocked to intercalation with bulky t-butyl groups. We proposed that this new molecule would preferentially bind to blunt-ended DNA, where there is much less steric hindrance. The synthesized molecule was characterized by NMR, Fluorescence, UV/Vis, and Mass Spectroscopy. Supporting data were acquired through fluorescence lifetime measurements, which reflect the chemical environment of the fluorophore. Preliminary data indicated that the lifetime of our target molecule lengthened from 3.8 ns to 13.8 ns when the molecule was exposed to short DNA with blunt ends. This effect could not have been due to intercalation, because there was no observed change in the lifer>me of the molecule when it was exposed to long DNA with few blunt ends. Unfortunately, we have not yet been able to duplicate the data supporting blunt-end binding, partially because the association between our target molecule and blunt-ended DNA is extremely small. An interesting aside is that the target molecule converts to another form at low pH and low ionic strength. We believe that it might be stacking on itself to form multimers, but the current evidence is inconclusive. In addition to the fluorescence studies computer modeling was done using Macromodel and Spartan on an SGI workstation. After minimizing the energies of ethidium bromide and the target molecule, several docking studies were done to predict the ability of each molecule to intercalate DNA. Modeling will be used to help select future blunt-end binding target molecules.
Farwell, Michael D., "Molecular recognition of blunt-ended DNA" (1997). Honors Theses. 2061.