Harnessing Supercoiling to Regulate DNA Activity

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ABSTRACT This MIRA proposal presents my vision for how my research will evolve over the next five years and culminates from our long-term, rigorous studies of the diverse structures and properties of supercoiled DNA and its interaction with topoisomerases. Within cells, DNA is supercoiled and often constrained into small DNA loops that can be experimentally recapitulated with supercoiled DNA minicircles small enough for use in a wide range of biophysical and biochemical assays. The methods we have developed and extensive knowledge acquired thus far will be invaluable for our proposed studies of DNA topoisomerases, actions of important antimicrobial and anticancer agents that target them, the utility of engineered DNA minicircles as gene therapy vectors, and supercoiling-induced noncanonical DNA structures that are implicated in human disease. We will first utilize state-of-the-art electron cryo-microscopy and cryo-tomography to determine the 3-D structure of topoisomerases bound to physiologically relevant DNA substrates. This approach will be coupled with comprehensive quantitative assays using electrophoretic and fluorescence techniques and analytical ultracentrifugation to characterize how DNA supercoiling so strongly affects topoisomerase-drug interactions. Many topoisomerases, particularly those that are important drug targets, preferentially act on positively supercoiled DNA. Consequently, corresponding anti-topoisomerase drugs interact with positively supercoiled DNA as well, although research of chemotherapeutics that target topoisomerases has largely disregarded the effect of supercoiled DNA on drug action. We plan to identify new inhibitors of validated drug targets by screening, for the first time, active topoisomerase bound to positively supercoiled DNA against a library of over 5 billion diverse compounds. We will next apply our innovative tools and compelling data of how supercoiling, curvature, and sequence dictate DNA conformation to design and construct DNA nanoparticles with specific, desired shapes that are ideal for cellular uptake needed in a variety of clinical applications. Existing nanoparticles, such as those composed of gold or monosaccharides, are inert; therefore, we propose utilizing DNA minicircles, as both the vehicle and cargo in one, for gene therapy to overcome many of the barriers to effective gene delivery. Finally, we will employ DNA minicircles to investigate how supercoiling promotes the formation of non-B-DNA structures, which are known to impact DNA replication, repair, transcription, yet their in vivo frequency is controversial. This work is transformative, as our novel DNA minicircles, advanced imaging tools, and quantitative analyses will enable us to achieve unprecedented and previously unattainable insights into the structure and function of supercoiled DNA. Our fundamental research will continue to challenge the paradigm that DNA is passively acted upon by topoisomerases but instead drives numerous critical cellular processes. Moreover, this project has substantial human therapeutic applications related to anti-topoisomerase drug efficacy, improved gene therapy delivery, and mitigating genomic instability caused by non-B-DNA forms.