Masters Theses

Shen Zhu

Date of Award

12-1990

Degree Type

Thesis

Degree Name

Master of Science

Major

Physics

Major Professor

Solon Georghiou

Abstract

In this thesis I discuss the properties of the excited electronic states in mehtylated DNA and its bases. In the first part I describe steady-state measurements of fluorescence anisotropy in order to resolve the long-wavelength absoption spectrum of 7-methyl GMP in pH 5 buffer at room temperature into component spectra that correspond to the electronic transitions I and II present in that spectral region. We have chosen this derivative of guanine because its fluorescence quantum yield is much greater than that of GMP. It is found that the data are adequately described by a model that involves emission exclusively from state I, with state II converting to it with 100% efficiency. The shape of the absorption spectrum of state II is virtually independent of the angle θ between the absorption transition dipole moments of states I and II, whereas that of state I is dependent on θ. We analyze the data on the basis of the premise that in the short-wavelength region state II is the predominantly absorbing state. This premise is based on studies of single-crystal polarized reflection and linear dichroism from stretched films. The spectral maxima for the two states are found to be at about 290 nm and 260 nm, respectively. There is also a weak band which is centered at about 245 nm. The oscillator strengths are found to be 0.07, 0.21 and ≈ 0.04, for states I, II and that associated with the weak band, respectively. The importance of these findings with regard to the photophysical properties of nucleic acids and calculations of their GD spectra is discussed. In the second part, I report the results of a study in which an irreversible electronic energy trap has been formed in calf thymus DNA by methylating about 75% of its G bases at position N-7. This has allowed us to measure for the first time the efficiency of transfer of energy along the helix of a double-stranded nucleic acid at room temperature. It is found that about one out of every three photons absorbed by the other bases is trapped. We have also simulated the data with a stochastic model that uses the dipole-dipole interaction to calculate the efficiency of transfer. In order to approximate the experimental results, the model requires that: (i) the fluorescence quantum yield of T, C, and G in DNA be about 2 x 10-3, which is about two orders of magnitude larger that the value of the fluorescence quantum yield reported for DNA; and (ii) the fluorescence quantum yield of A in DNA be negligibly small. Requirement (i) is consistent with energy transfer taking place before a very efficient fluorescence quenching process sets in, which could be formation of excited-state complexes (excimers) that do not fluoresce appreciably. Requirement (ii) implies a very short fluorescence lifetime for A, which is consistent with the reported absence of a significant number of photoproducts formed by A in DNA. The simulations find that, on the average, the excitation energy takes about 1.2 steps to reach the trap; that is to say, bases that are nearest and next nearest neighbors of the trap are, in effect, the only energy donors. Both intra- as well as interstrand energy transfer ( the latter only for the C-trap base pair) make significant contributions. The values of the efficiency for pairwise base-base intrastrand transfer are 90% and 80%, respectively. The corresponding values for the rate constant of transfer are 2 x 1011, 1 x 1012, and 4 X 1011 s-1 Transfer is inefficient when A is the donor or the acceptor. In addition to the dipole-dipole term, the only other significant term in the expansion of the interaction potential is the dipole-quadrupole term which, however, makes only a small contribution to the overall transfer efficiency. The electron exchange interaction appears to be much less efficient than the coulombic interaction.

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