The central dogma originally postulated that RNA solely played the role of genetic information transfer from DNA to protein. But that has been proven to be an inadequate depiction of this genetic material. RNA plays an integral role in the cell beyond just translation alone. RNA is capable of regulating every step of the central dogma. Because of this realization, the development of the RNA toolkit has been an ever-growing field. This toolkit consists of techniques to study RNA structure, localization, associated binding proteins, and nascent transcription. Herein, my studies aimed to enhance the field of RNA structure probing, as well as the field of labeling nascent RNA with bioorthogonal nucleosides. To that aim, I have synthesized a new probe with increased stability for icSHAPE structure probing of RNA. Additionally, I have synthesized the first azide containing bioorthogonal nucleosides for RNA labeling. Lastly, I extended the PGA toolkit to include pyrimidine nucleosides so that the cell-specific RNA labeling can be biased for either RNA or polyadenylated RNA, based on the nucleoside being used.

RNA is a highly regulatory biomolecule within the cell. RNA has been identified in many biological functions that involve growth and development, and dysregulation of RNA has been demonstrated to be critical in the onset of cancer and neurodegenerative diseases. Toward the goal of elucidating novel RNA functions chemical methods have been developed. These include chemical probing of RNA structures and metabolic incorporation of modified nucleosides into nascent transcripts. These approaches add precision to current methods through subcellular profiling, enzyme-substrate pairs for cell-type specific labeling, multifunctional nucleosides for profiling RNA-protein interactions, and multicomponent labeling for RNA lifetimes with determination of sites of incorporation. Combining these chemical techniques with modern sequencing methods offers a holistic view of RNA biology in real time. Here, is presented the work toward advancing chemical methods for studying RNA biology.

From epigenetic regulation to protein regulation, RNAs structure is integral to a wide variety of cellular processes. Many functions performed by RNAs are now just beginning to be discovered and explored. An important step forward is developing methods that can relate the structure of a given RNA molecule to its function. Chemical probing is commonly the method of choice for interrogating RNA structure; however, the field is limited to select chemistries available for studying RNA structure. Herein, my studies provide avenues to improve upon, as well as invent, chemical approaches necessary to add to the current toolbox of chemical methods for measuring RNA structure inside living cells. Furthermore, these tools are adapted to next-generation sequencing methodologies which can provide a convenient access to analyzing the structures of thousands of RNAs in parallel. The merger of chemistry and transcriptomics is poised to provide novel inroads to exploring how groups of RNAs are regulated by common structural motifs that impart function.

The transcriptional landscape has revealed itself to be highly complex, wherein RNA serves both coding and non-coding functions that are essential for cell differentiation, development and survival. Because the role of RNA has continued to expand, there is increasing need for biochemical methods to label and track RNA transcription, post-transcriptional regulation, and other RNA-based mechanisms. Herein, my doctoral work to expand the scope of metabolic reporters for RNA synthesis details a systematic expansion of chemistries and modifications for nucleoside analogs. This work has also served as a foundation for the development of novel, cell-specific RNA tagging approaches, both chemical and enzymatic. Throughout these studies, I have characterized the limitations of functional group tolerance and in many cases interrogated their effects on cell uptake and metabolism. Furthermore, I have worked to characterize the limitations of current cell-specific methods, with the goal of developing more stringent and robust approaches. These efforts are poised to provide greater context and engineering opportunities for methods to tag, visualize and study RNA gene expression.

A cancer gene therapy involves the expression of a foreign gene in cancer cells. However, the current methods to control the transgene expression are imperfect, with leaky expression usually found in normal cells. This leakiness can lead to harmful side effects in patients, especially if the transgene encodes a toxic protein. Herein, my studies aim to invent, as well as improve upon regulatory tools to control transgene expression in cancer gene therapies. Compared to the traditional promoter-based or cell surface marker-based approaches, my approach targets an intracellular protein defect caused by several mutations that are prevalent in cancers. Additionally, I improved the inducible gene expression system, further eliminating its background leakiness. Together, my studies offer novel cell-specific targeting and temporal control options, expanding the current gene therapy toolbox.

The development of new tools for RNA metabolic labeling is critical for analyzing nascent transcription in cells and animal models to elucidate specific regulatory mechanisms of RNA. These approaches have recently been demonstrated with commercially available methods, such as thiol (SH)-linked alkylation for the metabolic sequencing of RNA (SLAM-seq), yet there remain many opportunities to refine RNA metabolic labeling for cell-specific approaches. In the following dissertation, I will highlight the important work that has already been developed towards these efforts. I will also introduce a novel cell-specific RNA metabolic labeling method using a new enzyme-analog pair. For this approach, I will demonstrate how it can be used in vivo to capture nascent RNA within a metastatic breast cancer mouse model. Lastly, I will discuss a new chemo-selective reagent pair for vinyl nucleosides that can be used to analyze two RNA populations simultaneously in cells. Throughout the following document, I will emphasize the chemical and biochemical challenges and advantages of these labeling methods and discuss opportunities for future development. These tools are dedicated to improving biological insight to study transcription with greater physiological specificity.