Louisiana State University LSU Digital Commons LSU Historical Dissertations and Theses Graduate School 1999 The hC aracterization of Oligonucleotides and Nucleic Acids Using Ribonuclease H and Mass Spectrometry. Lenore Marie Polo Louisiana State University and Agricultural & Mechanical College Follow this and additional works at: https://digitalcommons.lsu.edu/gradschool_disstheses Recommended Citation Polo, Lenore Marie, "The hC aracterization of Oligonucleotides and Nucleic Acids Using Ribonuclease H and Mass Spectrometry." (1999). LSU Historical Dissertations and Theses. 6923. https://digitalcommons.lsu.edu/gradschool_disstheses/6923 This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Historical Dissertations and Theses by an authorized administrator of LSU Digital Commons. For more information, please contact [email protected]. INFORMATION TO USERS This manuscript has been reproduced from the microfilm master. UMI films the text directly from the original or copy submitted. Thus, some thesis and dissertation copies are in typewriter free, while others may be from any type of computer printer. The quality of this reproduction is dependent upon the quality of the copy submitted. Broken or indistinct print, colored or poor quality illustrations and photographs, print bleedthrough, substandard margins, and improper alignment can adversely affect reproduction. In the unlikely event that the author did not send UMI a complete manuscript and there are missing pages, these will be noted. Also, if unauthorized copyright material had to be removed, a note will indicate the deletion. Oversize materials (e.g., maps, drawings, charts) are reproduced by sectioning the original, beginning at the upper left-hand corner and continuing from left to right in equal sections with small overlaps. Each original is also photographed in one exposure and is included in reduced form at the back of the book. Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6” x 9” black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. UMI A Bell & Howell Information Company 300 North Zceb Road, Ann Arbor MI 48106-1346 USA 313/761-4700 800/521-0600 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. THE CHARACTERIZATION OF OLIGONUCLEOTIDES AND NUCLEIC ACIDS USING RIBONUCLEASE H AND MASS SPECTROMETRY A Dissertation Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy in The Department of Chemistry by Lenore Marie Polo B.S., Barry University, 1994 May, 1999 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. UMI Number: 9926424 Copyright 1999 by Polo, Lenore Marie All rights reserved. UMI Microform 9926424 Copyright 1999, by UMI Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. UMI 300 North Zeeb Road Ann Arbor, MI 48103 Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Copyright © 1999 Lenore M. Polo All rights reserved ii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. For Sergio iii Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. ACKNOWLEDGMENTS First and foremost, I would like to thank God for granting me the patience, perseverance, and talent that have allowed me to attain this goal. I would like to thank my undergraduate professors, Dr. Fisher, Dr. Jungbauer, Sr. O’Donnell, and Dr. Boulos, for encouraging me to pursue my graduate career. I would like to thank Dr. Hammer, Dr. Morden, Kyle Waite, Dr. T. McCarley, and Dr. DiMario for their helpful discussions and their invaluable assistance in various portions of this work. I would also like to thank Dr. Carlton at the LSU Medical Center for allowing me the use of his laboratory’s DE-MALDI-TOF-MS and ESI-MS, and Anthony Haag for his assistance in operating the instruments mentioned. I would like to thank in a special way all my fellow group members, especially Kari Green-Church, Victor Vandell, and Tracey Simmons, for all their support during my time here at LSU. I especially thank my adviser, Dr. Limbach, for his help, patience, and dedication to my success as a graduate student, as well as for his critical review of this dissertation. I would also like to thank my entire family, but especially my parents, for all their encouragement and support throughout all my years in school—I owe my success to their dedication. I also thank in a special way, my fiance, Sergio, to whom this work is dedicated for his love and support which helped carry me through the most trying stages of my career. I also thank him for waiting so patiently for me for so long. Lastly, I would like to thank my grandfather who passed away before the completion of this work. He is the reason I am a chemist today—this would have been one of his proudest moments. iv Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. TABLE OF CONTENTS Acknowledgments ............... .iv List of Tables.......................... vii List of Figures..........................................................................................................viii List of Schemes........................................................................................................... x A bstract...................................................................................................................... xi Chapter 1. Current methods for the determination of posttranscriptional modifications in ribosomal RNA, and proposed method for indirect mass spectrometric sequencing of selected regions of ribosomal RNA ....................... 1 1.1 Significance ...............................................................................................1 1.2 Posttranscriptional modifications in rRNA ............................................2 13 Current methods for the determination of pseudouridine in ribosomal RNA...................................................................................................................8 1.4 Mass spectrometric techniques for the analysis of nucleic acids and the determination of posttranscriptional modifications .....................................12 1.5 Protocol for the determination of pseudouridine in selected regions of rRNA using mass spectrometry .................................................................... 18 Chapter 2. The analysis of CMC derivatives of nucleic acids and oligonucleotides using mass spectrometry ................................... 23 2.1 Introduction ............................................................................................ 23 2.2 Experimental ........................................................................................... 26 2 3 Results and discussion ........................................................................... 29 2.4 Conclusions ............................................................................................ 46 Chapter 3. The analysis of ribonuclease H cleavage products of oligonucleotides using mass spectrometry ......................................................................................... 49 3.1 Introduction ............................................................................................ 49 33 Experimental ............................................................................................50 3 3 Results and discussion ........................................................................... 55 3.4 Conclusions..............................................................................................62 Chapter 4. Cleavage of 16S ribosomal RNA using ribonuclease H ............... 63 4.1 Introduction ............................................................................................ 63 4 3 Experimental............................................................................................64 4 3 Results and discussion ........................................................................... 72 4.4 Conclusions ............................................................................................ 91 v Reproduced with permission of the copyright owner. Further reproduction prohibited without permission. Chapters. Conclusion .............................................................................................93 5.1 Summary ..................................................................................................93 5.2 Future experiments ................................................................................. 96 References ................................................................................................................. 101 Appendix A. List of abbreviations ...................................................................... 108 Appendix B. Copyright consent form .................................................................110 V ita.............................................................................................................................I