Masters Theses

Date of Award

8-1996

Degree Type

Thesis

Degree Name

Master of Science

Major

Physics

Major Professor

Solon Georghiou

Committee Members

Dan Roberts, Cynthia Peterson

Abstract

Thymine appears to be the major fluorophore in DNA. In the present study, the photophysical properties of thymidine have been studied by varying the solvent viscosity and polarity (dielectric constant ε and polarizability α), as well as the pH of the aqueous environment. The fluorescence quantum yield q is found to decrease linearly with ε and nonlinearly with α. For diethyl ether, which is the solvent with the lowest ε and α value of α (10.2 Å3) very similar to that of thymidine, in which thymidine is soluble, q decreases by 60% relative to its value in aqueous solution, pH 7. This decrease suggests that both electrostatic and polarizability effects are important in the deactivation processes of the excited state. The shifts of the fluorescence and absorption spectra, however, are too small to allow a quantitative comparison with theory. At pH 0.5 and pH 11.5, the photophysical properties have been found to be similar with those at pH 7; this suggests that the formation of hydrogen bonds at positions N3 and O4 with the solvent does not have a significant effect on these properties. Upon increasing the viscosity to 59 cP (by addition of 77% sucrose by volume) and to 159 cP (by addition of 77% sucrose by volume at 5°C), q exhibits a large enhancement, by about 570% and 710%, respectively. An attempt has been made to simulate the fluorescence properties of thymine in DNA on the basis of these results for free thymidine. Since there is evidence that stacking interactions stem from polarizability effects, and the a for thymidine is very similar to that for diethyl ether, this solvent may be considered to simulate best both the electrostatic and dipole-induced dipole interactions which thymine undergoes with its environment in DNA. The rigidity of DNA appears to be responsible for the large enhancement of the fluorescence quantum yield of its thymine residue relative to its value when free in water, and for the shift of the fluorescence spectral peak to shorter wavelengths. By noting that steady-state fluorescence measurements report mainly on the long-decay components from DNA, the results of the present study suggest that these components have a relatively rigid environment Their high fluorescence quantum yield implies a long lifetime, however, and this would facilitate photodimer formation, a process that necessitates considerable conformational rearrangements. In another series of experiments, the fluorescence emission anisotropies of poly(dA)*poly(dT), (dA)20*(dT)20, poly(dT), and (dT)20 were compared. It was found that the values for the double-stranded systems are lower than those for the corresponding single-stranded systems. This suggests that in double-stranded systems there are cooperative interactions which enhance their flexibility. This inference is in agreement with the results of a very recent time-resolved fluorescence anisotropy study which reported on the flexibility of DNA in the picosecond-nanosecond time ranges (Georghiou et al., 1996). Differences in the spectrofluorometric properties of poly(dT) and (dT)20 cast doubt on the assumption made in the literature that the structure of the dinucleotide can be used to deduce that of the polymer.

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