Because RNA carries a high negative charge, its folding into secondary and tertiary structures can be exquisitely sensitive to salt concentration. Folding of RNA tertiary structures is particularly affected by the concentration of divalent ions and the types of mono- and divalent ions presents. Investigation ion association with an RNA is a difficult problem, as binding in the sample molecule may vary from entirely delocalized interactions with the RNA electrostatic field to chelation of partially dehydrated ions at specific sites. How one views the role of ions in directing RNA folding depends on the relative importance assigned to such non-specific and specific binding modes. The purpose of the proposed work is to systematically examine ion-RNA interactions in a series of RNAs of known and increasingly complex non- canonical and tertiary structures. Approaches that will be used are calorimetric and UV melting experiments (which report the differences in the extent of ion association between folded and unfolded states, and provide a direct measure of ion-induced stabilization), and methods that detect the extent of monovalent or divalent ion association with the folded RNA structure (equilibrium dialysis or titrations using fluorescent indicators of free ion concentrations).
Specific aims of the work include (i) Investigation of divalent ion interactions at specific RNA sites, including the affinity, monovalent ion salt dependence of binding, and selectivity for different alkaline earth ions; (ii) Determination of what RNA structures develop selectivity for monovalent ions (alkali metal and ammonium), and whether selectivity can be attributed to one or a few specific coordination sites; (iii) Initial attempts to compare calculated and experimentally measured electrostatic potentials in an RNA. These experiments should provide a picture of how ions are distributed around each RNA and the degree to which different classes of associated ions contribute to thermodynamic stability of RNA structures. This information will fill in a crucial aspect of non-canonical and tertiary RNA folding energetics.
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