DOI
https://doi.org/10.25772/P3VA-KC94
Author ORCID Identifier
https://orcid.org/0000-0002-2264-3715
Defense Date
2024
Document Type
Dissertation
Degree Name
Doctor of Philosophy
Department
Chemistry
First Advisor
Dr. Soma Dhakal
Abstract
DNA is inherently dynamic and topologically diverse and can fold into many different structures. Besides the canonical Watson-Crick structure, other higher-order structures such as G-quadruplexes (G4), i-motifs (iM), and four-way DNA junctions are possible. Although these high-order DNA structures are known to form transiently, they are important due to the crucial roles they play in many cellular processes including DNA replication, recombination, and repair. Among these DNA structures, 4-way junctions (also known as Holliday junctions, HJ) which are formed during the repair of double-strand DNA breaks (DSBs) and interact with proteins have garnered significant attention due to their central role in DNA damage repair. Therefore, dissecting the role of HJs, their topological properties, and their interactions with the HJ-binding proteins and small-molecule ligands is not only critical for understanding the mechanism of repair but also for deciphering the therapeutic potential of HJs.
Besides the inherent conformational dynamics, the HJs can migrate along the DNA axis through progressive base-pair rearrangements between the homologous DNA strands, called branch migration (BM). BM is conserved among organisms and is essential to stabilize recombination intermediates, which occurs by avoiding the reversal of strand exchange leading to the faithful repair of DSBs; however, molecular insights into the BM process including kinetics, the effects of microenvironments, and the role of HJ-binding proteins are poorly understood. Along this line, we have investigated the effects of cell-mimic solvent compositions and molecular crowding on the BM process by varying the concentration of cosolutes, namely dimethyl sulfoxide (DMSO) and polyethylene glycol (PEG). Using a single-molecule technique called fluorescence resonance energy transfer (FRET) and a mobile HJ analog capable of migrating, we have demonstrated that BM is significantly affected by cosolutes. The kinetic analyses of the FRET data showed that the BM is enhanced under crowded environments. Furthermore, due to the importance of HJ in maintaining the genomic integrity of highly replicating cancer cells, there is a growing interest in exploiting HJ for cancer therapy via drug targeting. Therefore, we have expanded this project to investigate the binding of a small drug-like molecule to the HJ. Using both immobile and mobile junctions, we determined that the ligand called “VE-822” alters HJ conformations and dynamics, suggesting a potential application in therapeutics.
On the therapeutic side, we are also pursuing the engineering of cell surfaces with biomolecules for targeted applications. My project dealt with cytokines such as tumor necrosis factor-alpha (TNFα) for potential islet transplant therapy in the treatment of type 1 diabetes (T1D). Diabetes is one of many diseases that are life-threatening, affecting millions of individuals at different ages. T1D is characterized by a lack of insulin production due to the destruction of insulin-producing beta cells by the innate immune system. One novel therapeutic approach that has recently emerged as an alternative to exogenous insulin administration is islet cell transplantation (ICT). However, one major challenge in ICT is a considerable loss (50-80%) of the transplanted tissue mass due to inflammation and islet cell death post transplantation. To protect islets from inflammation, we used DNA aptamers to block TNFα and improve islet function. Using an aptamer-based single-molecule FRET (smFRET) coupled with in-vitro functional assays, we have evaluated the anti-inflammatory potential of a group of DNA aptamers by probing their binding interaction with TNFα and their ability to alleviate cytokine-induced islet cell death. Our analyses demonstrated the high potential of aptamers as anti-inflammatory agents in islet transplant therapy.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
4-22-2024