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Publication

Synthetic approaches towards sequence-specific DNA labelling

Book - Dissertation

DNA, often referred to as the blueprint of life, encodes all the genetic information that is required for an organism. Since the elucidation of the general structure of DNA nearly 70 years ago, scientists have become increasingly interested in the study of this polynucleotide. One of the issues with this study is the visualization of DNA. Unmodified DNA lacks an appropriate observable emission and cannot be studied by the naked eye. Thus, over the past decades, various techniques have been developed to introduce fluorescent properties onto the double helix in order to study the DNA substrate.These techniques often make use of the functionalization of DNA targets, and utilise specifically designed fluorescent molecules. Unfortunately, the described synthetic pathways towards these molecules are often challenging and faster or more cost efficient pathways are generally desired. Thus, the main objective of the research is finding new and readily accessible DNA labelling approaches and components, suitable for optical mapping.The first part of the research focuses on the development of new cysteine-based S-adenosyl-L-methionine (SAM) analogues for sequence-specific DNA labelling. This chemo-enzymatic approach to DNA labelling has been extensively studied, but requires non-natural cofactors which come with a high synthetic workload. These new analogues, comprising cysteine rather than the natural homolog, can be obtained in near quantitative conversions from readily available starting materials without relying on using an excess amount of labor-intensive molecules. The synthetic strategy was used to generate fluorescent cofactors with colors spanning the whole visible spectrum, and the stability of these analogues under various conditions was examined.In the second part, a synthetic pathway towards small molecules based on netropsin and distamycin with an appended fluorescent dye is described. These analogues can be used to specifically target AT rich regions within the genome, providing an alternative approach for DNA optical mapping. Reactive warheads are introduced to ensure covalent labelling of the analogues to DNA.Finally, the DNA labelling capacity of the newly developed molecules is evaluated using several biological screening assays. The compatibility between the cysteine-based cofactors and different methyltransferase enzymes is examined, and after positive results the cofactors are used in genomic mapping experiments. The small molecules are subjected to an experiment similar to the competitive binding assay.
Publication year:2020