A Chemical Biology Toolbox for Studying A-to-I RNA Editing Open Access
Knutson, Steven (Spring 2021)
Abstract
RNA carries and transports information within cells to significantly influence their function and behavior. After transcription, RNA is also extensively modified by a number of enzymes to further alter this information. Adenosine-to-inosine (A-to-I) editing is one of the most widespread and impactful RNA modifications, and is catalyzed by adenosine deaminases acting on RNA (ADARs). Editing is essential for a number of biological processes, and dysfunctional editing is directly linked to several disease pathologies. Despite this major importance, our overall understanding of A-to-I editing is quite limited. In particular, existing technical challenges obscure the true prevalence and landscape of A-to-I editing in the human transcriptome, and it is unclear why certain sites are edited over others and what precise function they each serve. While it is known that dysregulated editing is linked with numerous diseases, the exact molecular and cellular mechanisms responsible for this relationship are also poorly understood. We also have indirect evidence that different cell types and tissues display vastly different editing patterns, but there are no approaches to measure these differences. As a result, we have little understanding of how this heterogeneity affects overall tissue and organ function which might contribute to disease. Given these limitations, the primary goal of my research has been to develop improved tools to better probe A-to-I RNA editing and characterize its biological functions. This thesis aims to highlight these efforts, and in Chapter 1 I will first provide an overview of the biological roles of A-to-I editing and summarize current methodology for studying and harnessing this modification. In Chapters 2 and 3, I then describe explorations of inosine labeling using acrylamide derivatives to enable affinity enrichment and chemical profiling of A-to-I edited transcripts. In Chapters 4 and 5, I develop a protein-based platform for binding and detecting inosine in RNA using Endonuclease V (EndoV), and highlight the powerful applications that have been leveraged from these investigations. In Chapter 6, I describe our use of the covalent denaturant glyoxal as a synthetic nucleic-acid modification which enables thermoreversible control over the structure and activity of a variety of biomolecular constructs. Finally, in Chapter 7 I summarize the implications of these present studies, discuss opportunities for advancing each technology platform, and describe future perspectives and challenges for studying A-to-I RNA editing.
Table of Contents
Chapter 1: Introduction
1.1 RNA Modifications ...........................................................................................................1
1.2 A-to-I RNA Editing...........................................................................................................3
1.3 Biological Functions and Disease Mechanisms ...................................................................6
1.4 Detecting A-to-I RNA Editing...........................................................................................10
1.5 Summary and Conclusions of this Dissertation ..................................................................14
1.6 References ......................................................................................................................16
Chapter 2: Chemical Labeling and Affinity Capture of Inosine-Containing RNAs Using Acrylamidofluorescein
2.1 Abstract .........................................................................................................................28
2.2 Introduction...................................................................................................................29
2.3 Results and Discussion....................................................................................................31
2.4 Conclusion .....................................................................................................................36
2.5 Materials and Methods....................................................................................................38
2.6 References .....................................................................................................................41
Chapter 3: Chemical Profiling of A-to-I RNA Editing Using a Click-Compatible Phenylacrylamide
3.1 Abstract .........................................................................................................................44
3.2 Introduction...................................................................................................................45
3.3 Results and Discussion....................................................................................................47
3.4 Conclusions ...................................................................................................................54
3.5 Materials and Methods....................................................................................................55
3.6 References .....................................................................................................................61
Chapter 4: Selective Enrichment of A-to-I Edited Transcripts from Cellular RNA Using Endonuclease V
4.1 Abstract .........................................................................................................................66
4.2 Introduction...................................................................................................................67
4.3 Results and Discussion....................................................................................................70
4.4 Conclusions ...................................................................................................................81
4.5 Materials and Methods....................................................................................................83
4.6 References .....................................................................................................................92
Chapter 5: Direct Immunodetecton of Global A-to-I RNA Editing Activity with a Chemiluminescent Bioassay
5.1 Abstract:........................................................................................................................97
5.2 Introduction:..................................................................................................................98
5.3 Results and Discussion:..................................................................................................101
5.4 Conclusions: .................................................................................................................116
5.5 Materials and Methods: .................................................................................................118
5.6 References ....................................................................................................................128
Chapter 6: Thermoreversible Control of Nucleic Acid Structure and Function with Glyoxal Caging
6.1 Abstract ....................................................................................................................... 138
6.2 Introduction.................................................................................................................139
6.3 Results and Discussion..................................................................................................142
6.4 Conclusions .................................................................................................................167
6.5 Materials and Methods..................................................................................................168
6.6 References ...................................................................................................................192
Chapter 7: Conclusions and Future Perspectives
7.1 Chemical Profiling of ADAR Mechanisms and Substrate Preferences ................................205
7.2 Engineering EndoV for Enhanced Inosine Recognition....................................................206
7.3 Deep RNA sequencing using an Optimized EndoVIPER Workflow.....................................208
7.4 Elucidating the Natural Function of EndoV in Humans....................................................209
7.5 Implementation of the EndoVLISA Bioassay ..................................................................210
7.6 Visualizing Global A-to-I Editing Patterns with EndoV Immunostaining...........................211
7.7 Parallel Study of A-to-I Editing and Subcellular RNA Localization....................................212
7.8 Directed Evolution of Deaminase Ribozymes for Site-Directed RNA Editing......................214
7.9 Single-cell Profiling of A-to-I Editing ........................................................................... 216
7.10 Improved Control over Glyoxal Caging and Decaging....................................................220
7.11 Glyoxal Caging of Anti-Viral Drugs..............................................................................222
7.12 References.................................................................................................................224
Appendix A: Omitted Data from Chapter 2..........................................................................235
Appendix B: Omitted Data from Chapter 3..........................................................................248
Appendix C: Omitted Data from Chapter 4..........................................................................261
Appendix D: Omitted Data from Chapter 5..........................................................................277
Appendix E: Omitted Data from Chapter 6..........................................................................306
About this Dissertation
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