MSc in Molecular and Macromolecular Sciences 

Presenter:  Daniel Kimani

Title of the Thesis: “A New Method to Manipulate DNA Topology Using Poly(B5AMA) Biomaterials”

Deoxyribonucleic acid (DNA) topology plays a critical role in regulating vital biological processes such as replication, transcription, and recombination. Plasmid deoxyribonucleic acid (pDNA) can exist in various structural configurations such as supercoiled DNA, open circular DNA, and linear DNA, each exhibiting unique functional properties, rendering them indispensable in various applications. Supercoiled DNA, the prevalent form harvested from Escherichia coli bacteria, serves as the primary research material. However, different DNA topologies offer advantages for specific therapeutic interventions, such as targeted gene delivery or expression regulation. Open circular DNA presents enhanced accessibility for gene expression studies, while linear DNA facilitates precise gene editing and integration. Natural enzymes such are topoisomerases and restriction endonucleases are used to convert the topology from supercoil to open circular (nicked) or linear DNA. Enzymatic DNA topology manipulation poses limitations due to their specificity, instability and storage challenges associated with these enzymes. While natural enzymes have traditionally been employed to conduct this transformation, the use of synthetic biomaterials as alternative tools for DNA topology conversion remains largely unexplored. Recent studies have highlighted the potential of ozone treatment and alcohol precipitation, such as isopropanol, in converting supercoiled DNA to open circular or linear forms. However, these methods are complex procedures and exhibit lower efficiencies.

This study demonstrates a more efficient method by employing poly vitamin B5 analogous methacrylamide (poly(B5AMA)n) materials such as hydrogels and nanogel to convert supercoiled plasmid DNA to other DNA topologies as a function of temperature. Hydrogels and nanogels were synthesized through free radical polymerization and different molecular weights and chirality of (poly(B5AMA)n) were synthesized through reversible addition-fragmentation chain transfer (RAFT) polymerization and were all evaluated for their effect on DNA topology. The findings were consistent with previously reported methods involving enzymatic or chemical treatments but demonstrate a novel, enzyme-free approach using biocompatible materials. This novel method holds promise to streamline plasmid DNA topology conversion process, offering potential advancements in therapeutic applications and research endeavours. This study also provides new insights into the application of synthetic biomaterials for DNA topology manipulation and opens avenues for their use in gene therapy.

April 29, 2025, 9:00 am in AVC 286A

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