| The massive charge of nucleic acids creates strong intramolecular repulsions that attract and in return are modulated by a group of surrounding positive counterions, referred as the "ion atmosphere". These counterions inherent to any nucleic acid profoundly influence its physical properties and biological functions but are difficult to quantitate and understand.; This dissertation pursues quantitative investigations of the thermodynamic nature of the ion atmosphere. An analytical assay was developed and used to fully dissect the ionic constituents around simple, well-defined DNA duplexes. This quantitative and comprehensive thermodynamic description of the ion atmosphere founded the subsequent energetic investigation of counterions.; To isolate the counterion-induced electrostatic forces from complications of tertiary interactions or the specific ion-binding, a model system that consists of two DNA duplexes connected by a neutral flexible polyethylene glycol tether was employed. The model system simulates a fundamental structural component of large RNAs and thus captures the essential aspects of the electrostatically-mediated folding of structured RNAs. Experiments investigated the counterion-mediated electrostatic potential between the tethered duplexes, helping distinguish hypotheses regarding to the driving force of RNA folding. In addition, structural measurements in conjunction with the theoretical modeling of the tethered duplexes revealed the ensemble nature of the unfolded state of RNA that is critical to understand the often complex and multi-dimensional folding landscapes. A further extension of the study utilized the tethered duplexes as a common scaffold to probe how the junctions modulate the conformational distribution of the unfolded ensemble. It provided an initial investigation of the thermodynamic role of junctions in RNA folding.; Together, the presented work highlights the success of applying well-defined model systems to gain quantitative and comprehensive understanding of the physical principles that drive the behaviors of nucleic acids. These quantitative measurements of the ion atmosphere further enable a rigorous examination of current understandings of the nucleic acid electrostatics and guide the development of improved theories. |
