Targeting Twist: Single-molecule insights into supercoiled DNA-topoisomerase interactions for drug discovery

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Topoisomerases are enzymes, present in all organisms from humans to bacteria, which are essential for life due to their ability to untangle knotted and twisted DNA. The knotting and twisting of our DNA occurs as 2 metres of our DNA is folded into the cell nucleus; much narrower than the width of a human hair. This is exacerbated by the molecular machinery in our cells which travel along our DNA, pulling it apart and manipulating it, in a compact environment. Without topoisomerases, our DNA becomes irreversibly knotted and twisted, and the cell will die. For this reason, key therapeutics such as anticancer and antibiotic drugs target human and bacterial topoisomerases respectively either killing cancerous or bacterial cells.The pharmaceutical pipeline is continually working both to improve the therapeutics that we have, and to create novel therapeutics. To improve existing therapeutics, we require better understanding of how they work. There remains much room for development in this field. Anti-cancer therapeutics that target topoisomerases currently suffer from a lack of selectivity. Further to this, there remain a number of unexplored antibacterial targets, which have significant potential, but are as yet untargeted. I propose to use single molecule techniques such as Atomic Force Microscopy, where a needle moves up and down as it is scanned over a surface, building up a picture of what the surface looks like, line by line, (much like a record player reads out a record by feeling the contours of its surface). These will be complemented by faster single molecule techniques, such as magnetic tweezers where a piece of DNA is tethered to a magnetic ball at one end, and a glass surface at the other. The DNA is twisted into a more complex state, by rotating the magnetic ball. The length of DNA is determined by measuring the height of the ball. This gives a very fast readout of how topoisomerases are able to untwist DNA, and how this process can be disrupted by drugs such as anti-cancer therapeutics and antibiotics. The aim of the project is to improve our understanding of how topoisomerases untwist DNA, and how this is prevented by topoisomerase inhibitors, to aid in the development of new or improved anti-cancer drugs and antibiotics.