Year of Publication
Doctor of Philosophy (PhD)
Arts and Sciences
Dr. Stephen M. Testa
Group I intron-derived ribozymes are catalytic RNAs that have been engineered to catalyze a variety of different reactions, in addition to the native self-splicing reaction. One such ribozyme, derived from a group I intron of Pneumocystis carinii, can modify RNA transcripts through either the excision or insertion of RNA sequences. These reactions are mediated through the trans excision-splicing (TES) or trans insertionsplicing (TIS) reaction pathways. To increase our current understanding of these reactions, as well as their general applicability, a mechanistic and kinetic framework for the TES reaction was established. Furthermore, additional ribozymes were investigated for their ability to catalyze the TES reaction. Lastly, the development of the TIS reaction into a viable strategy for the manipulation of RNA transcripts was investigated.
The TES reaction proceeds through two reaction steps: substrate cleavage followed by exon ligation. Mechanistic studies revealed that substrate cleavage is catalyzed by the 3’ terminal guanosine of the Pneumocystis ribozyme. Moreover, kinetic studies suggest that a conformational change exists between the individual reaction steps. Intron-derived ribozymes from Tetrahymena thermophila and Candida albicans were also investigated for their propensity to catalyze the TES reaction. The results showed that each ribozyme could catalyze the TES reaction; however, Pneumocystis carinii is the most effective using the model constructs.
Investigations of the TIS reaction focused on developing a new strategy for the insertion of modified oligonucleotides into an RNA substrate. These studies used oligonucleotides with modifications to the sugar, base, and backbone positions. Each of the modified oligonucleotides was shown to be an effective TIS substrate. These results demonstrate that TIS is a viable strategy for the incorporation of modified oligonucleotides, of varying composition, into an intended RNA target.
The results from these studies show that group I introns are highly adaptable for catalyzing non-native reactions, including the TES and TIS reactions. Furthermore, group I introns are capable of catalyzing these unique reactions through distinct reaction pathways. Overall, these results demonstrate that group I introns are multi-faceted catalysts.
Dotson, Perry Patrick II, "MECHANISTIC INVESTIGATIONS OF THE TRANS EXCISION-SPLICING AND TRANS INSERTION-SPLICING REACTION" (2008). University of Kentucky Doctoral Dissertations. 605.