Calorimetric Studies of Site-Specific Ligand Binding By RNA

Open Access
- Author:
- Sokoloski, Joshua Edward
- Graduate Program:
- Chemistry
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- August 12, 2011
- Committee Members:
- Philip C. Bevilacqua, Dissertation Advisor/Co-Advisor
Philip C. Bevilacqua, Committee Chair/Co-Chair
Scott A Showalter, Committee Member
David Lawrence Allara, Committee Member
Song Tan, Committee Member - Keywords:
- Isothermal Titration Calorimetry
RNA - Abstract:
- RNA is a biopolymer capable of carrying genetic information and performing diverse chemistry, where thermodynamics of ligand binding to RNA reveal crucial insight into its molecular structure and function. Isothermal titration calorimetry (ITC) provides a direct, label-free method to measure ligand-macromolecule interactions in biological systems. This dissertation details projects exploring structure/function relationships in RNA, particularly focusing on ITC to elucidate the molecular underpinnings of site-specific ligand interactions with RNA. Nucleotides with the syn orientation were compiled into a database, and their relevance to RNA function was analyzed. It was concluded that syn bases are low-occurring, but not uncommon, and that they cluster in binding pockets and active sites of functional RNA. One such functional RNA with several syn bases, the malachite green aptamer (MGA), was chosen for thermodynamic characterization by ITC. It was shown that the binding of two related ligands, malachite green (MG) and tetramethylrosamine (TMR), features a favorable binding enthalpy, an unfavorable binding entropy, and a negative heat capacity, with MG binding having the greater amount for each one of these quantities. The temperature dependent ITC data also indicates that MG binding has two binding regimes: one low temperature regime where the stoichiometry is n = 0.4 and one high temperature regime where the stoichiometry goes to n = 1. TMR binding lacked this feature. Preliminary results for manipulating MGA activity by incorporation of conformationally restricted nucleotides at syn positions are presented. Site-specific metal ion binding was also investigated with ITC. These results show that metal ion binding to RNA is driven by entropy, with an unfavorable enthalpy, and that they can be detected and quantified via ITC. The negative heat capacity for site-specific magnesium ion-RNA interactions compared to a positive heat capacity for nonspecific interactions indicate that the heat capacity may be a general thermodynamic marker for RNA specificity in its binding behavior.