Studies Towards Identification and Targeting of Functional G-quadruplexes

Abstract

Nucleic acids, including DNA and RNA sequences, can fold into diverse secondary and tertiary structures to perform specific biological functions. Among them, a non-canonical four-stranded structure, named Guanine (G)-quadruplex (G4), formed by stacked G-tetrads in G-rich DNA or RNA sequences has been reported to be widely dispersed in eukaryotic genes and transcripts, as well as bacteria and virus. DNA and RNA G4s play significant regulatory roles in various cellular processes and are involved in many diseases and cancers, making them potential targets for drug development and anti-cancer therapeutics. The identification and targeting of G4s can help to understand their different biological roles and potential applications in diagnosis and therapy. Despite the methodological development allowing the detection of G4s in recent years, the understanding of their biological significance is still limited owing to the structure complexity of G4 and the limitations of current detection methods. In this thesis, besides using computational methods to predict the potential formation of G4, I also performed biophysical and biochemical methods to identify and characterize G4, as well as developed L-RNA aptamers as new tools to target G4 and regulate G4-mediated gene activity.

The pandemic of coronavirus disease 2019 (COVID-19) caused by the SARS-CoV-2 virus has become a global public health emergency. Understanding the structure information of SARS-CoV-2 RNA genome and its regulatory mechanism is important for effective drugs development. Considering the importance of G4 structure in virus pathogenesis, it is interesting to investigate the potential G4 formation in SARS-CoV-2 RNA genome. I analyzed SARS-CoV-2 genome using bioinformatic tool QGRS-Mapper and found 25 putative G4-forming sequences (PQSs) with four contiguous GG runs (G₂NxG₂NyG₂NzG₂). The identified PQSs are located in the open reading frames of ORF1 ab, spike (S), ORF3a, membrane (M) and nucleocapsid (N) genes of SARS-CoV-2 genome and found in other Coronaviridae family as well. The PQSs at positions 13385 and 24268 were analyzed to have the highest G-score and were confirmed to fold into G-quadruplex structure in vitro by using multiple biophysical assays including circular dichroism (CD), ultraviolet (UV) melting and fluorescence assays. Microscale thermophoresis (MST) assays showed the binding interactions between PQSs and viral helicase (nsp13). Molecular docking analysis indicated that nsp13 can unfold G4 structures by rearrangement of guanine bases. These results suggest that targeting viral helicase and G4 structures represents a potential approach for inhibiting SARS-CoV-2 virus.

Identification and characterization of G4 enable the understanding of their biological importance, providing new insights into therapeutics. However, challenges remain in distinguishing different G4 conformations. As a special nucleic acid structure with rich conformational polymorphisms, specific targeting of G4 conformations and regulating their biological functions provide promising therapeutic intervention. Despite the large repertoire of G4-binding tools, only limited of them can specifically target particular G4 conformation. To solve this problem, I developed the first L-RNA aptamer, L-Apt12-6, with high binding affinity and selectivity to parallel G4 over other nucleic acid structures. L-RNA aptamer is composed of unnatural L-RNA nucleotides and resistant to nuclease degradation, making it well-suited for biological and biomedical applications. Using parallel dG4 c-kit 1 as an example, I demonstrated the strong binding affinity between L-Apt12-6 and c-kit 1, and notably reported the potential applications of L-Apt12-6 in controlling DNA replication and gene activity. My results and findings suggested that L-Apt12-6 will be a valuable tool for determination of G-quadruplex conformation and regulating of G4-mediated biological processes both in vitro and in vivo.

Despite increasing L-RNA aptamers have been developing for different targets, their binding properties are not satisfactory and limit their applications. To improve the binding performance, I developed a simple and robust method to synthesize circular L-RNA aptamer. Using our recently reported L-Apt.4-1c as an example, I generated the first circular L-RNA aptamer, cycL-Apt.4-1c. The 5’ and 3’ terminals of L-Apt.4-1c were ligated through intramolecular click chemistry reaction with high efficiency. The cyclized L-RNA aptamer exhibits stronger binding affinity, improved specificity, and higher conformational stability than linear L-Apt.4-1c. Competitive binding assay with protein, telomerase repeated amplification protocol (TRAP) assay and dual luciferase reporter gene assay further validated the promising applications of cycL-Apt.4-1c in controlling RNA-protein interaction, regulating telomerase activity and gene expression. My important findings demonstrate that cyclization can be an effective way to strengthen the function of L-RNA aptamers. Moreover, my approach will be applicable to any L-RNA aptamers and open up a new avenue for diverse applications.

In summary, the extensive works in this thesis are devoted to the investigation of functional G4 structure, including G4 identification, characterization, and G4-targeting L-RNA aptamers development and modification. These works serve to reveal the regulatory role of G4 in important cellular processes, and generate new insights into the understanding of G4 structure and function. In addition, the novel L-RNA aptamers developed in this thesis significantly improved the current G4-targeting strategies, with great potential to be applied for biological and biotechnological applications, such as diagnosis, therapeutics, and biosensing.

Date of Award	5 May 2023
Original language	English
Awarding Institution	City University of Hong Kong
Supervisor	Chun Kit KWOK (Supervisor)