Characterization and Targeting of G-quadruplex Structures


Student thesis: Doctoral Thesis

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Award date31 Aug 2020


G-quadruplexes (G4s) are exceptional nucleic acid structures resulting from the stacking of G-tetrads in a stretched G-rich nucleic acid sequence. Studies have shown that G4s participate in diverse cellular activities in both macro- and micro-organisms. Efforts have been made to understand the mechanisms of G4 folding and targeting both in vivo and in vitro. However, our current understanding of the dynamics and potential novel applications of G4s remains very limited. There are two main reasons for this limitation. The first (problem I) is the lack of an optimised biophysical and biochemical assay for effective G4 characterisation across different systems. a) Most studies on G4s have relied on the use of canonical (i.e., natural) G4s; non-canonical G4s (which also exist in cells) have been significantly understudied. b) The presence and functions of G4s in small noncoding RNAs, such as micro-RNAs (miRNAs), also remain elusive. c) Likewise, most studies on G4s have focused on eukaryotes, providing little or no information on the existence and roles of G4s in prokaryotes (specifically bacteria). The second reason for our limited understanding of the dynamics and applications of G4s (problem II) relates to the lack of development and applications of G4-specific tools for targeting and potentially manipulating cellular processes (e.g., G4–protein interactions). To address these problems, the objective of my PhD research was to investigate the chemical and biophysical dynamics of G4 structures, the prevalence of G4s across several species and the biological roles of these structures in cellular events. This research was also conducted to develop a novel and highly selective tool for and approach to G4 targeting (e.g., aptamers) and to explore the potential use of this tool to inhibit RNA G4 (rG4)–protein interactions.

To solve problem I, I developed an optimised biophysical and biochemical method that was more sensitive, robust and time- and cost-effective than previous methods. I also demonstrated the applicability of this method across different systems through comparisons with existing approaches. Using this new approach, I successfully addressed problems a)–c). As reported in Chapter Two, I used intrinsic fluorescence and other spectroscopic methods to explore and uncover the effect of a single bulge position and bulge identity on a G4. Prior to this study, little was known about the implications and biophysical features of non-canonical G4s. Notably, my mentee and I collaboratively reported substantial variation in the spectroscopic features of bulged DNA and RNA G4s and established the usefulness of intrinsic fluorescence for identifying the formation of both canonical and non-canonical G4s. As reported in Chapter Three, I extended this work by exploring rG4 candidates from miRNAs that are conserved in mammalian species. My collaborators and I discovered the formation of rG4s in miRNAs and the roles of these structures in miRNA-mediated post-transcriptional regulation. To showcase the utility of this newly developed method across different systems and to tackle problem c), Chapter Four of my thesis explained the presence of rG4 in prokaryotes and the collaborative discovery that rG4s are predominantly RNA structural motifs across a broad range of bacterial species. Using a state-of-the-art rG4 sequencing platform (rG4-seq), we uncovered many rG4 sites in E. coli and other bacterial strains and the roles of these structures in cellular regulation.

The roles of G4s in cellular activities are mostly controlled by their associations with other biomolecules, such as G4-binding proteins. Over the years, many classes of G4-sensitive and -specific apparatus have been developed to interfere with G4–protein interactions. Nonetheless, no aptamer tool had been developed for this purpose prior to this study. Therefore, as reported in Chapter Five of my thesis, in response to problem II, I developed a novel L-RNA aptamer (L-Apt.4-1c) that could bind highly selectively to the rG4 D-hTERC and inhibit its interactions with protein. Chapter Five also explains that L-RNA aptamer-mediated interference with G4–protein interactions is equivalent and even superior to interference mediated by existing G4-specific tools. This observation showcases the potential of our approach and results not only to broaden the existing G4 toolbox, but also to open a new window for diverse applications.