University of Kentucky UKnowledge University of Kentucky Doctoral Dissertations Graduate School 2008 MECHANISTIC INVESTIGATIONS OF THE TRANS EXCISION- SPLICING AND TRANS INSERTION-SPLICING REACTION Perry Patrick Dotson II University of Kentucky, [email protected] Right click to open a feedback form in a new tab to let us know how this document benefits ou.y Recommended Citation Dotson, Perry Patrick II, "MECHANISTIC INVESTIGATIONS OF THE TRANS EXCISION-SPLICING AND TRANS INSERTION-SPLICING REACTION" (2008). University of Kentucky Doctoral Dissertations. 605. https://uknowledge.uky.edu/gradschool_diss/605 This Dissertation is brought to you for free and open access by the Graduate School at UKnowledge. It has been accepted for inclusion in University of Kentucky Doctoral Dissertations by an authorized administrator of UKnowledge. For more information, please contact [email protected]. ABSTRACT OF DISSERTATION Perry Patrick Dotson II The Graduate School University of Kentucky 2008 iii MECHANISTIC INVESTIGATIONS OF THE TRANS EXCISION-SPLICING AND TRANS INSERTION-SPLICING REACTION ABSTRACT OF DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Arts and Sciences at the University of Kentucky By Perry Patrick Dotson II Lexington, Kentucky Director: Dr. Stephen M. Testa, Associate Professor of Chemistry Lexington, Kentucky 2008 Copyright © Perry Patrick Dotson II 2008 iv ABSTRACT OF DISSERTATION MECHANISTIC INVESTIGATIONS OF THE TRANS EXCISION-SPLICING AND TRANS INSERTION-SPLICING REACTION 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 insertion- splicing (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. v KEYWORDS: RNA, Group I intron, ribozyme, trans excision-splicing reaction, trans insertion-splicing reaction Perry Patrick Dotson II May 01, 2008 vi MECHANISTIC INVESTIGATIONS OF THE TRANS EXCISION-SPLICING AND TRANS INSERTION-SPLICING REACTION By Perry Patrick Dotson II Stephen M. Testa, Ph.D. Director of Dissertation Robert B. Grossman, Ph.D. Director of Graduate Studies May 01, 2007 vii RULES FOR THE USE OF DISSERTATIONS Unpublished dissertations submitted for the Doctor's degree and deposited in the University of Kentucky Library are as a rule open for inspection, but are to be used only with due regard to the rights of the authors. Bibliographical references may be noted, but quotations or summaries of parts may be published only with the permission of the author, and with the usual scholarly acknowledgments. Extensive copying or publication of the dissertation in whole or in part also requires the consent of the Dean of the Graduate School of the University of Kentucky A library that borrows this dissertation for use by its patrons is expected to secure the signature of each user. Name Date viii DISSERTATION Perry Patrick Dotson II The Graduate School University of Kentucky 2008 ix MECHANISTIC INVESTIGATIONS OF THE TRANS EXCISION-SPLICING AND TRANS INSERTION-SPLICING REACTION DISSERTATION A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the College of Arts and Sciences at the University of Kentucky By Perry Patrick Dotson II Lexington, Kentucky Director: Dr. Stephen M. Testa, Associate Professor of Chemistry Lexington, Kentucky 2008 Copyright © Perry Patrick Dotson II 2008 x ACKNOWLEDGEMENTS There are many people that I would like to thank who have given their time and support during my graduate career. I would like to thank my research advisor, Dr. Stephen Testa. Dr. Testa has challenged me throughout my graduate career, and has made me a more efficient scientist. I would also like to thank the members of my doctoral committee; Dr. DeMoll, Dr. Lovell, Dr. Peterson, and my outside examiner, Dr. Hunt, for their time and help throughout my graduate career. I would also like to thank the members of the Testa lab, including Dr. Rashada Alexander, Dr. Ashley Johnson, Dr. Dana Baum, Dr. Joy Sinha, Nick Tzouanakis, and Dustin Lafferty. I would like to thank Dr. Rashada Alexander and Dr. Dana Baum for showing me the techniques that I would need to get started in the lab. I would especially like to thank Dr. Joy Sinha and Nick Tzouanakis for always making things in the lab interesting. I would like to take this opportunity to thank the people who have supported me the most. I would like to thank my parents, Perry Patrick and Lorena Lynn Dotson. I appreciate everything that they have done for me throughout my entire life. They have always been supportive of my decisions, and I am eternally grateful to have them as my parents. Without them, I would not be where I am today. I would also like to thank my grandparents, including Michael Louis and Margaret Curry Dotson, and Walter Glenn and Lorena May Davis for their support. Although both of my grandfathers and one of my grandmothers are gone, they are not forgotten. In addition, I would like to thank all of my family members, including the Dotson’s, Davis’, Donohue’s, Cipoletti’s, and Potter’s. Lastly, I would like to thank Larry and Erin White for being good friends. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS...............................................................................................iii TABLE OF CONTENTS................................................................................................... iv LIST OF TABLES............................................................................................................. ix LIST OF FIGURES ............................................................................................................ x CHAPTER ONE-SUMMARY ........................................................................................... 1 Investigation of the Reaction Mechanism for the Trans Excision-Splicing Reaction..... 2 Kinetic Characterization of the First Step of the TES Reaction ..................................... 2 The Trans Excision-Splicing Reaction Can be Catalyzed by Group I Intron-Derived Ribozymes from Tetrahymena thermophila and Candida albicans................................ 3 Insertion of Modified Oligonucleotides using the Trans Insertion-Splicing Reaction ... 4 CHAPTER TWO-BACKGROUND................................................................................... 5 The Central Dogma of Molecular Biology. .................................................................... 5 Nucleic Acids .................................................................................................................. 5 Deoxyribonucleic Acid................................................................................................ 6 Ribonucleic Acid ......................................................................................................... 6 Catalytic RNA ................................................................................................................. 7 RNA Splicing................................................................................................................... 7 Group I Introns ........................................................................................................... 8 The Self-Splicing Reaction.......................................................................................... 9 Group I Intron-Derived Ribozymes .........................................................................