Macrosolute effects on nucleic acids

Obando, Sarai.

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Obando, Sarai.

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Thesis (Ph.D.)--Georgetown University, 2008.; Includes bibliographical references. The intracellular environment contains a variety of solutes that cumulatively occupy a significant volume of the cell (20-30%). The high volume occupancy generates a system which is macromolecularly crowded. This crowding, also known as the excluded volume effect, can lead to an increase in the chemical activity of solutes and influence thermodynamic and kinetic values as compared to a dilute system. Synthetic, inert cosolutes were used to provide a simplified mimic of the intracellular environment, in which the DNA structures were studied. Various macrosolutes, cosolutes of molecular weight greater than 1000, and an osmolyte, a cosolute of molecular weight less than 1000, were used to created crowded conditions. In the presence of macrosolute, differential stabilization of a complementary DNA duplex over duplexes containing a single mismatched base pair was observed. Hence, crowding effectively increased the specificity of the hybridization reaction. In contrast, a study of single bulge base duplexes demonstrated the differential stabilization of a bulge duplex over all other duplexes when in the presence of a macrosolute. Crowding studies on unimolecular systems demonstrated marginal stabilization of stem-loop structures as compared to duplexes. However, in systems with molecularities ranging from one to four, as the molecularity of a system increased, the crowding effects also increased. For all systems studied, crowding enhanced the rate constant of hybridization. As the activation energies of the systems studied increased, the crowding effects also increased. This work contributes to our understanding of the effects that the complex intracellular environment has on biochemical interactions and processes. These results are also of potential use to bioanalytical techniques which employ nucleic acid hybridization. Additionally, these results could be used to design molecular systems, which can switch structure based on the volume occupancy of the surrounding medium.