Discovery and Application of Natural and Synthetic RNA Switches
Date of Award
Spring 2023
Document Type
Dissertation
Degree Name
Doctor of Philosophy (PhD)
Department
Molecular, Cellular, and Developmental Biology
First Advisor
Breaker, Ronald
Abstract
RNA biology has become one of the most talked about fields of research in both the biological and biomedical sciences, and understanding the role of RNAs, both coding and noncoding, has been tremendously helpful to researchers in advancing molecular biology and therapeutics. With the ongoing COVID-19 pandemic, RNA related research has been at the forefront of groundbreaking scientific developments at the basic sciences and applied sciences level, i.e., combating infections from a single-stranded RNA virus with an mRNA vaccine. My thesis work, similarly, spans the spectrum between understanding the fundamental biology of small, structured, noncoding RNAs (riboswitches) to using these RNAs in drug discovery and development. In Chapter One, I discuss the progress made in riboswitch discovery and application. Riboswitches are structured, noncoding RNAs typically found in the 5′ UTR of bacterial mRNA that bind a ligand to regulate gene expression. The aptamer domain of the riboswitch recognizes and binds the ligand, inducing a conformational change in the adjacent expression platform. Given that approximately 90% of bacterial genomes are coding regions, these noncoding RNAs must play a crucial role in managing important biosynthetic and metabolic processes in the cell. Understanding riboswitch biology has a provider deeper insight into these important cellular pathways. In addition to their role in the basic sciences, riboswitches have also been used in innumerable ways in the applied sciences, as drug targets and as biosensors. Moreover, riboswitches have also served as a model for developing synthetic switches that operate similarly to regulate gene expression. In Chapter Two, I discuss the work done to identify a novel class of riboswitches that regulates genes related to guanidine biology. The guanidyl moiety is a part of many key metabolites, including arginine, guanine, and creatine. However, the source nor the exact function of free guanidine is unknown. Given the numerous genes known to alleviate guanidine toxicity, guanidine is biologically relevant compound. The validation of this fourth riboswitch class furthers the mystery behind this intriguing compound. Additionally, this chapter focuses on the efforts to uncover a variant of the SAM-I riboswitch class, thought to bind spermidine, providing the first evidence of spermidine-sensing riboswitch class. Lastly, this chapter presents ongoing efforts in validating other riboswitch candidates. In Chapter Three, I describe high-throughput screening campaigns (HTS) using riboswitch reporters to identify compounds that perturb essential biological pathways regulated by the riboswitches. The first HTS uses an SAH riboswitch to detect small molecule inhibitors of the SAH nucleosidase enzyme. This enzyme is an attractive target for antibiotic development as it plays a crucial role in several bacterial processes, including recycling toxic levels of SAH. Through screening efforts, we identified a compound capable of permeating E. coli, increasing reporter expression, and inhibiting SAH nucleosidase. A similar HTS was done with a TPP riboswitch to identify compounds that disturb thiamin homeostasis. However, challenges faced in this screen give insights on how to better develop such campaigns using the riboswitch reporter systems in the future. In Chapter Four, I discuss the work done to engineer synthetic RNA switches. In using a natural guanine aptamer as a scaffold, we identified novel aptamer classes that bind quinine and caffeine through selection methods. To determine whether these aptamers function in vivo, they were tested in the context of the original guanine riboswitch’s expression platform in B. subtilis and shown to regulate gene expression upon binding by quinine or caffeine. This chapter also explores the development of novel expression platforms for use in eukaryotic systems. Many groups have used self-cleaving ribozymes such as hammerhead, twister, and pistol for designing their synthetic switches; however, dynamic range in living systems has proved to be an issue. I describe my work in using the twister sister self-cleaving ribozyme to address this problem and the potential pitfalls in developing a selection for an allosteric ribozyme. Lastly, this chapter focuses on ongoing efforts in the lab to develop synthetic switches for drug compounds to be used for gene therapy.
Recommended Citation
Balaji, Aparaajita, "Discovery and Application of Natural and Synthetic RNA Switches" (2023). Yale Graduate School of Arts and Sciences Dissertations. 916.
https://elischolar.library.yale.edu/gsas_dissertations/916
