Excimer formation in DNA as a probe of its dynamics and stability

Author

Steven Michael Kubala

Date of Award

5-1996

Degree Type

Thesis

Degree Name

Master of Science

Major

Physics

Major Professor

Solon Georghiou

Committee Members

Daniel Roberts, Cynthia B. Peterson

Abstract

Excimer formation in DNA is of extreme interest as it is thought to be involved in the formation of photodimers. This work has investigated the effects of ionic strength, viscosity η, and presence of organic solvent on excimer formation in the alternating polynucleotide poly(dA-dT) •poly(dA-dT). For the ionic strength measurements, in all cases the buffer consisted of 0.05 M sodium cacodylate and was pH 7.0 in triply distilled water, but the amount of NaCl was varied from 0.01 M to 2.0 M. This increase of NaCl resulted in little change of the excimer-to-monomer ratio, which suggests that base motions have not changed greatly even though the thermal melting transition temperature increases from 38.2°C to 79.2°C. Thus, the transition temperature cannot be used as a reliable indicator of the stability of the helix. Viscosity was increased through the addition of sucrose, as well as through temperature reduction. The excimer peak was found to be shifted from 415 nm in buffer to 383 nm with η=14 cP and 367 nm with η=59 cP. This 3000 cm^-1 shift suggests that the mobility of the bases is greatly reduced since the most favored sandwich conformation excimer is not achieved. Methanol and acetonitrile were used to study their effects on excimer formation. For both solvents the excimer-to-monomer fluorescence ratio was found to decrease as the solvent concentration was increased, for those concentrations for which no hyperchromic changes were observed in the absorption spectrum. However, upon denaturation, the solvents differed in that methanol caused a huge increase in the excimer band and acetonitrile caused aggregation. This aggregation is likely a result of the difference of the basicity of the two solvents, with acetonitrile displacing some water from the phosphate groups and causing strand separation, due to enhanced electrostatic repulsion. This emphasizes the importance of hydration in maintaining the double helical structure. The changes observed in the absorption and fluorescence spectra were found to be reversible upon the reduction of solvent concentration through the addition of buffer, provided the solvent concentration was below that which causes denaturation or aggregation. By varying the parameters of ionic strength, viscosity, and presence of solvent, this work has been able to investigate subtle changes in the stability of the helix that affect excimer formation.

Recommended Citation

Kubala, Steven Michael, "Excimer formation in DNA as a probe of its dynamics and stability. " Master's Thesis, University of Tennessee, 1996.
https://trace.tennessee.edu/utk_gradthes/10870