Periodic Report Summary - SMDNA (Novel approaches to the study of enzymatic diffusion on single DNA molecules) The overall aim of this project was to 'investigate the binding and diffusion of sequence-specific DNA binding enzymes on single DNA molecules'. Furthermore, the project aims to 'advance current understanding in the field by applying novel methodologies at the single molecule level'. The project has generated internationally outstanding results, which are discussed in more detail in relation to the specific project objectives below. Enzymatic diffusion at the single-molecule level: The role of DNA topology A key aspect of this part of the proposal is to sequence-specifically label DNA using a DNA methyltransferase enzyme. This was carried out in collaboration with Prof. Saulius Klimasauskas (Vilnius University). As a proof-of-principle study, the lambda-phage genome was labelled using the HhaI DNA methyltransferase enzyme at sites reading 5'-GCGC-3'. By depositing this DNA onto a surface, its sequence was directly visualized. This work has resulted in a patent application, two publications and a feature article that covered the wok in the Royal Society of Chemistry news magazine 'Highlights in Chemical Biology'. This work was deemed of such importance that the focus of the project was diverted in order to pursue it in preference to the objectives originally proposed. An investigation of the mechanism of diffusion along linear DNA: the effect of target site and half-site binding on diffusion of the structurally well-characterised BamHI and BstYI enzymes A collaboration with scientists at New England Biolabs examined the effect of heterodimerisation on the diffusional behaviour behaviour of the BtsI restriction enzyme. Preliminary work shows promise. The BtsI B subunit has been successfully visualized at the single molecule level and preliminary analysis of its diffusional behaviour shows that it displays behaviour consistent with other, previously studied enzymes. This work will continue in the future. The fellow has developed a good understanding of fluorescence microscopy and is now capable of building and optimising a microscope for single-molecule imaging. Furthermore, new skills in the fabrication of microfluidic devices and the techniques fundamental to preparing samples for single-molecule experiments will be invaluable in his future career. The fellow attended the Gordon Research conference on the application of single-molecule imaging to biology in Lucca, Italy in June 2010 and the VIB course on Entrepreneurship in the biosciences in 2011. The fellow wrote and adopted the patent covering his DNA mapping work. Hence, the fellow has developed an extensive knowledge of the patent application process. The fellow collaborated extensively with staff at Leuven Research and Development throughout this process and continues to do so as plans to proceed with the commercialisation of the projects output progress. The fellow has also acquired an extensive knowledge of the process of valorisation, which will be invaluable in the future.
