Author: Leung, Edward Ki-Yun
The following studies have been focused on studying two catalytic DNA molecules, the 8-17 and bipartite deoxyribozymes; both of which cleave single-strand RNA molecules. A new technique was developed to study the 8-17 by correlating photo-induced oxidative damage to its structure and structure folding transitions. A detailed mechanistic study was performed on the bipartite deoxyribozyme. Detailed structural information of the bipartite deoxyribozyme was obtained by photo-induced crosslinking and detailed mutation analyses. The charge flow patterns within an intricately folded DNA complex, the 8-17 bound to a DNA pseudosubstrate, incorporating three helical elements and two catalytically relevant loops were extensively studied. The stacking preferences of the three constituent helices were studied and provided evidence for significant transitions within the complex’s global geometry. The patterns further suggested varying levels of solvent exposure of the complex’s constituent parts, and revealed that a catalytically relevant cytosine within the folded complex exists in an unusual structural/electronic environment. The bipartite deoxyribozyme was found to have a mechanism of significant complexity. A dissection of metal usage indicated the involvement of two catalytically relevant magnesium ions for optimal activity. The deoxyribozyme was able to utilize manganese(II) as well as magnesium; however titration with hexaamminecobalt(III) chloride inhibited the activity of the bipartite; this suggests that it is a metalloenzyme that utilizes metal hydroxide as a general base. Overall, the bipartite deoxyribozyme appeared to be kinetically distinct not only from the self-cleaving ribozymes but also from other in vitro selected, RNA-cleaving deoxyribozymes. The catalytic core of the bipartite deoxyribozyme was studied by mutagenesis and photo-induced crosslinking. Mutation analyses of the catalytic core revealed that a stem structure is important for the catalytic activity of the deoxyribozyme and that four bases within both loop regions are possible candidates for the direct co-ordination with the catalytically relevant divalent metal ions. Thio-modified nucleotides were substituted throughout the bipartite bound to a DNA pseudosubstrate. The mapping of the crosslinked species and the mutagenesis data suggested the catalytic core of the bipartite folded in a way that positions a stem region of a hairpin close to active site of the enzyme.
Copyright is held by the author.
The author has not granted permission for the file to be printed nor for the text to be copied and pasted. If you would like a printable copy of this thesis, please contact email@example.com.
Member of collection