|Title||Single-Molecule Kinetic and Thermodynamic Studies of Cosolute-Influenced Nucleic Acid Conformational Transitions|
|Year of Publication||2014|
|Academic Department||Chemistry and Biochemistry|
|Number of Pages||285|
|University||University of Colorado|
Over the last 40 years the number of biochemical functionalities attributed to nucleic acids has increased tremendously. This diverse array of chemical functionality is intimately coupled to the spatial arrangement of atoms associated with these molecules. The three-dimensional structures and functions of nucleic acids are known to be dependent on the concentration and identity of solutes in solution. These nucleic acid cosolutes can be as simple and universal as atomic metal cations that favorably interact with the negatively charged phosphate backbone of nucleic acids and resulting in stabilization of electronegatively dense conformations. Alternatively, they may be complex organic molecules that are able to promote conformational transitions in certain RNA sequences responsible for regulating gene expression. Understanding the biophysical principles responsible for these cosolutes-influenced conformational transitions represents the primary objective of this work.
Single-molecule fluorescence resonance energy transfer (smFRET) is a technique that is well suited for this goal because it provides the ability to watch individual nucleic acids undergo cosolute-influenced conformational transitions in real-time. These experimental results provide direct access to rate and equilibrium constants for any nucleic acid of interest. The temperature dependence of these smFRET experiments can be used to partition the free energy change associated with these conformational transitions into the enthalpic and entropic components. The kinetic and thermodynamic information acquired from these experiments, in conjunction with existing structural models, is used to describe the folding pathways and free energy landscapes associated with these nucleic acids.
The types of nucleic acid conformational transitions described in this thesis are as follows: the GAAA Tetraloop Receptor interaction, a 21 bp DNA duplex, and 8bp DNA duplex, a minimal version of the human Telomerase RNA pseudoknot, and a vitamin-B12 riboswitch. The cosolutes investigated for these nucleic acid constructs include: MgCl2, KCl, NaCl, urea, trimethylamine oxide, and vitamin-B12. Lastly, this thesis documents the development of an advanced heating technique used to conduct many of the temperature-dependent experiments associated with the aforementioned nucleic acids.