Molecular standards for analytical ultracentrifugation: investigating the suitability of double-stranded DNA

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Ranasinghe, Maduni Charuni
University of Lethbridge. Faculty of Arts and Science
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Lethbridge, Alta. : University of Lethbridge, Dept. of Chemistry and Biochemistry
This thesis focuses on the development of molecular standards to validate analytical ultracentrifugation (AUC) instruments, alongside an exploration of multiple applications of AUC. The goal of the main study is to address the critical need to develop a robust molecular standard for validating AUC instruments, with a specific emphasis on evaluating double-stranded DNA molecules as a potential candidate. By extensive investigation into the hydrodynamic properties of different topologies of double-stranded DNA at a wide range of temperatures, this study reveals the potential of linear and nicked double-stranded DNA as a reliable standard for AUC, contributing to the accurate characterization of macromolecules in solution. Supplementary research findings, as detailed in the attached publications presented in the attached appendix, further illustrate the versatility of AUC in various scientific domains. In one study, we employed AUC as a powerful technique to measure the sedimentation and diffusion coefficients of DNA minicircles to validate the elastic theory results obtained from our other collaborator. Also, it explores the impact of DNA supercoiling-induced shapes on minicircle hydrodynamics. In another collaborative effort, we study the oligomerization behavior of a de novo designed metalloprotein for photocatalytic hydrogen evolution, leveraging AUC for comprehensive characterization. These collective studies highlight the indispensable role of AUC in the characterization of macromolecules in solution, with applications ranging from DNA dynamics to functional protein characterization, and they demonstrate the crucial importance of reliable molecular standards in enhancing the accuracy of AUC measurements. Together, they contribute to the advancement of analytical science and its applications across diverse research domains.
analytical ultracentrifugation , molecular standards , double-stranded DNA , DNA minicircle , hydrodynamics , global analysis