Open Access
Brown, Trevor S.
Graduate Program:
Integrative Biosciences
Doctor of Philosophy
Document Type:
Date of Defense:
July 15, 2005
Committee Members:
  • Philip C. Bevilacqua, Committee Chair
  • Juliette T J Lecomte, Committee Chair
  • Anne Milasincic Andrews, Committee Member
  • Stephen James Benkovic, Committee Member
  • Craig Eugene Cameron, Committee Member
  • 31P NMR
  • hepatitis delta virus (HDV) ribozyme
  • Leadzyme
  • RNA folding
  • RNA structure
  • CD spectroscopy
  • UV spectroscopy and fluorescence spectroscopy
Nucleobase-pKa shifting is important in expanding the structural and catalytic range in RNA ribozymes. RNA is built from only four similar heterocyclic nucleotides with pKas of < 4.5 and > 9. Moreover, nucleotide pKa values shift further from neutrality in typical base pairs. Thus, nucleotides seem to lend themselves neither to electrostatic nor general acid-base catalysis of biologically relevant reactions. However, seven naturally occurring ribozymes have been discovered and several artificial ribozymes synthesized that are capable of catalyzing phosphodiester-bond synthesis and cleavage, aminoacylation of RNA, glycosidic and amide bond formation, hydroxyl phosphorylation, alkylation, acyl transfer, and Diels¡VAlder reactions. To achieve these functions, nucleobase pKas are shifted by attaining complex structures with specific microenvironments. The HDV and leadzyme ribozymes serve as models throughout this dissertation highlighting the importance and complexity of understanding nucleic acid catalysis. These ribozymes have in vivo significance in that HDV is a human pathogen and the essential sequence of leadzyme has been found throughout eukaryotic genomes leading to implications of its function in lead toxicity. A number of methodologies are used in this dissertation. A novel application of chip electrophoresis with capillary sample introduction is developed to measure ribozyme kinetics. Additionally, rational mutant design, enzymatic structure mapping, pH-dependent PAGE, and CD, UV-absorbance, and fluorescence spectroscopy are used to investigate ribozyme folding pathways and nucleobase pKa perturbation. To obtain a suitable construct for biophysical studies, the G11C/U27ƒ´ HDV ribozyme double mutant was prepared in an effort to destabilize known alternative pairings¡XAlt 1, Alt 2, Alt 3, and Alt P1¡Xwhile leaving the catalytic core intact (Chapter 2.1). However, biphasic kinetics and antisense oligonucleotide response trends opposing those of the well¡Vstudied G11C mutant were observed, suggesting that new alternative pairings with multiple registers, termed ¡¥Alt X¡¦ and ¡¥Alt Y¡¦, had been created. Inclusion of biologically relevant concentrations of Na+ realized the goal of monophasic, fast¡Vfolding kinetics (kobs „l 60 min¡V1). A model is developed wherein Na+, which destabilizes secondary and tertiary structures in the presence of Mg2+, facilitates native folding by destabilizing multiple alternate secondary structures with a higher¡Vorder dependence. Thus, design of a highly reactive HDV ribozyme sequence uncovered facilitation of RNA folding by alternate pairings and near physiological ionic strength. In Chapter 4.1, continuous analysis of a fluorescene-labeled leadzyme reaction is demonstrated using completely automated capillary sample introduction onto a microfabricated chip with an array of separation lanes, laser-induced fluorescence detection and differing lead concentrations. The automated results are in agreement with rates determined by conventional, more labor intensive, manual sample collection and PAGE analysis. This work demonstrates the potential of this method to provide valuable kinetic information for other, more complex, biologically relevant RNA and protein enzymes. Specific measurements of critical ribozyme features, such as nucleobase pKa, are of even greater interest than the global molecular characteristics. In Chapter 3.1, a simple method for determining nucleobase pKa values by indirect labeling is developed and used to demonstrate a pKa of neutrality in dsDNA using optimal nearest-neighbor partners and lower temperature. Such pKa values expand the catalytic repertoire of nucleic acids at physiological pH. C75 of the HDV ribozyme has been proposed to be involved in both the general acid-base mechanism and coupled to conformational changes in the folded ribozyme. Because of this, pH-dependent structural changes are followed by CD, UV-absorbance, and pyrene fluorescence spectroscopy (Chapter 5.1). In addition, non-denaturing PAGE is employed over a broad pH range to further elucidate linked structure formation. Unfortunately, the spectra of each method were complicated by the presence of the ¡¥non-targeted¡¦ nucleotides. In conclusion, these methods are not effective reporters of subtle changes associated with this single protonation. These efforts demonstrate that changes in structure coupled to the protonation state of C75 in the pre-cleaved HDV ribozyme are subtle. This thesis highlights the importance of rational mutant design, describes the use of Na+ as a denaturant, and demonstrates combining these two points to achieve a homogeneous, pre-cleaved, folded ribozyme population suitable for biophysical studies, which uncovered only a subtle structural change upon protonation of C75 and suggested that the large rearrangements previously reported may have been the result of stable misfolded structures.