| Popis: |
In this thesis, we aimed to create specific binders for 1-methyl adenine (1mA) in methylation-damaged DNA. Such binders may have potential as biosensor of DNA methylation lesions as the current experimental state-of-the- art does not allow for in vivo detection of methylated DNA. To that end, we initiated the molecular evolution of the E. coli AlkB (EcAlkB) DNA repair protein, a prototypical and promiscuous demethylase known to preferentially act on 1mA DNA. We aimed at improving specificity and affinity interaction with this particular DNA modification. To do so, we randomised six well- chosen amino acids in EcAlkB. The obtained library served as input in phage display experiments under catalytically inactive conditions, in order to prevent enzymatic release from the binding site. Next, the evolving library composition was thoroughly evaluated by Illumina sequencing and Nanopore sequencing. This analysis revealed several mutated sequence patterns. Most promising mutants were constructed and biochemically characterised. This yielded a higher affinity variant with a single amino acid mutation (AlkB W69F). Other variants still bound 1mA-modified oligonucleotides, albeit with a lower affinity. Furthermore, a serendipitous 3-amino acid insertion mutant appeared to maintain binding properties, showcasing the mutational tolerability of the AlkB scaffold. In parallel to the directed evolution experiments, a rational approach was followed where a 10-amino acid loop from a human dsDNA specific homologue was grafted into AlkB in order to tune binding selectivity for modified ssDNA towards dsDNA. Lastly, we show an explorative study to assess the feasibility of fluorescently tagged AlkB as in vivo biosensor. status: published |
