Programming DNA topology: from folding DNA minicircles to revealing the spatial organization of bacterial genomes

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While rapid DNA sequencing has led to significant increases in the amount of genetic information available, we are still far from a comprehensive understanding of how DNA operates. Recent experiments have shown that DNA looping and folding are essential mechanisms in the switching of genes between their on and off states and that different patterns of gene expression are strongly influenced by genomic spatial organisation. This has led to the idea that genetic information may also be encoded through DNA topology and highlights the importance of studying the physical properties of DNA and its interacting molecules. An understanding of DNA topology will provide us with the capacity to further control genetic information and to design genomes optimal for utilisation in synthetic biology. In this fellowship, I aim to obtain the ability to program and predict DNA topology on a broad range of length scales: from DNA minicircles around the kilo-bp (kbp) scale to bacterial genomes containing several mega-bps (Mbps).To tackle this issue, I propose to develop a physics-based computational methodology that will range from establishing protocols and models for atomistic and coarse-grained simulations to the development of a statistical-mechanics algorithm for the fast prediction of the topology of DNA. These theoretical methods will be complementary and will be supported by an appropriate set of experiments performed using a range of single molecule techniques, including atomic force microscopy (AFM) . Firstly, I plan to quantify the capacity of DNA to encode its own topology and its interdependence with DNA-recognising proteins by means of the design and modelling of artificially folded DNA minicircles. I will then transfer the acquired structural information towards developing an efficient prediction algorithm that will be converted into a "genome-wide DNA loop locator". As the apical part of a supercoiled DNA loop is determined by a single helical turn (approximately), an extraction of the fluctuations at the base-pair level will be sufficient to deal with sequences at the genomic scale. A selection of proof-of-concept systems will be used to elucidate the governing rules of DNA topology and to test the novel computational methodology. The gained technology could then be easily applied to a multitude of interesting cases soon after. The capability to program DNA minicircles with a specific conformation will have consequences on gene therapy because these tiny DNA molecules are being recognised as highly efficient agents for the introduction of genetic material into cells without negative side effects. In parallel, the "genome-wide DNA loop locator" will be used to predict the architecture of bacterial genomes and, as a consequence, will help in the design of genetically stable microorganisms with constitutively-expressed synthetic metabolic routes. Thus, if the proposed research is successful, its impact could be broad as it would lead to advances in the fields of healthcare and synthetic biology. It would benefit society in the longer term, through the development of new effective diagnoses and medicines and through increasing its capacity to tackle important socioeconomic challenges, including the supply of renewable energy, clean water and safe food.