Small-Molecule Tools to Probe and Modulate RNA Function Restricted; Files Only
Karloff, Diane (Fall 2023)
Abstract
Once considered a mere messenger, RNA is now known to perform diverse regulatory roles in the cell, from maintaining genome stability and modulating gene expression to scaffolding protein:protein interactions and enabling cell polarization. Aberrant RNA processing is linked to a wide array of neurodegenerative diseases and cancers, and RNA also serves as the genetic material for dozens of devastating human viruses. As small molecules are well-established tools to interrogate and disrupt biological systems, the elucidation of principles that drive RNA:small molecule binding interactions facilitates basic science discovery and therapeutic drug development. This dissertation highlights opportunities that small molecules present toward exploring and perturbing cellular RNA function. In Chapter 1, we discuss four key themes relating to the clinical importance of the transcriptome and its amenability to small molecule-based probing. In Chapter 2, we describe a photoaffinity labeling method to improve the robustness of cellular RNA imaging. Visualization of RNA dynamics is critical to unraveling effects on cell signaling. Further, reducing the molecular weight of the cargo required for RNA imaging reduces impacts on native behavior. Chapter 3 explores photoaffinity labeling as a means to evaluate RNA:drug target engagement. We find that 10 chemical probes from diverse structural classes display unique binding profiles with breast cancer-associated messenger RNAs. Chapter 4 investigates the small molecule glyoxal as a thermoreversible regulator of nucleoside antiviral bioactivity. This strategy has the potential to improve patient adherence during antiretroviral therapy and enhance the poor pharmacokinetic profile associated with this compound class. Beyond the scope of RNA:small molecule interactions, Chapter 5 elaborates on the vital role that researchers play in shaping scientific literacy and accessibility in society. This role can be realized within departments, via social media, or through engagement in global organizations. In Chapter 6, we summarize the implications of the work described herein and present exciting future opportunities at the interface of RNA:ligand binding.
Table of Contents
Chapter 1: Introduction ................................................................................................................................... 1
1.1 RNA Function in Cells ................................................................................................................................ 1
1.2 RNA:Small Molecule Interactions ............................................................................................................... 4
1.3 Photoaffinity Labeling ............................................................................................................................... 6
1.4 RNA SELEX ............................................................................................................................................... 8
1.5 Summary and Conclusions of this Dissertation ............................................................................................ 9
1.6 References ............................................................................................................................................... 11
Chapter 2: Imaging and Tracking RNA in Live Mammalian Cells via Fluorogenic Photoaffinity Labeling .............. 18
2.1 Abstract .................................................................................................................................................. 18
2.2 Introduction ............................................................................................................................................ 19
2.3 Results and Discussion ............................................................................................................................. 23
2.4 Conclusion .............................................................................................................................................. 30
2.5 Materials and Methods ............................................................................................................................. 33
2.6 References ............................................................................................................................................... 43
Chapter 3: Profiling the Ligandability of the Human mRNAome with Small-Molecule PAL Probes ....................... 50
3.1 Abstract .................................................................................................................................................. 50
3.2 Introduction ............................................................................................................................................ 51
3.3 Results and Discussion ............................................................................................................................. 54
3.4 Conclusion .............................................................................................................................................. 72
3.5 Materials and Methods ............................................................................................................................. 76
3.6 References ............................................................................................................................................... 79
Chapter 4: Glyoxal-Caged Nucleoside Antivirals as Extended-Release Prodrugs ................................................. 88
4.1 Abstract .................................................................................................................................................. 88
4.2 Introduction ............................................................................................................................................ 89
4.3 Results and Discussion ............................................................................................................................. 90
4.4 Conclusion .............................................................................................................................................. 96
4.5 Materials and Methods ............................................................................................................................. 97
4.6 References ............................................................................................................................................. 103
Chapter 5: Expanding STEM Accessibility and Inclusivity ............................................................................... 108
5.1 Engagement in Departmental, Local, National, and International Initiatives Promoting Scientific Literacy ... 108
5.2 Recruit and Retain a Diverse Workforce .................................................................................................... 112
5.3 References ............................................................................................................................................. 118
5.4 Related Links ......................................................................................................................................... 119
Chapter 6: Conclusions and Future Perspectives ............................................................................................ 121
6.1 References ............................................................................................................................................. 126
Appendix A: Data Omitted from Chapter 2 .................................................................................................... 131
Appendix B: Data Omitted from Chapter 3 .................................................................................................... 143
Appendix C: Data Omitted from Chapter 4 .................................................................................................... 146
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File download under embargo until 10 January 2026 | 2023-11-01 10:55:55 -0400 | File download under embargo until 10 January 2026 |
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