Ribonucleic acids are essential to many processes of life and, to fulfil their functions, they have numerous chemical modifications. Most of such modifications are found in tRNA, where they affect metabolism, structure, stability, localisation and transport. Modified purine base derivatives are critical in various biological process regulation across both prokaryotic and eukaryotic organisms. These modifications affect gene expression, the stability of RNA, and protein interactions, which are crucial for cellular differentiation, development, and reactions to environmental factors. In order to use modified purine base as drugs or as other medicval tools, it is crucial to know and understand their metabolic pathways. This is why investigation of enzymes that play part in modified purine base metabolism are highly significant for basic research and translational applications [1].
In this study, modified purine heterocyclic bases, such as N2-methylguanine, N6-methylguanine, N6-methyladenine, N6,N6-dimethyladenine etc., were synthesized and purified by flash column chromatography. The structure and the purity of synthesized derivatives were proven by UV/Vis, NMR spectroscopy, TLC and HPLC-MS analysis. The synthesized methylated compounds and some other commercially available modified purines were used as substrates to select enzymes involved in the catabolism of the modified heterocyclic bases. For this purpose, purine auxotrophy-based selection systems, similar to previously described [2], were used. These systems allow the selection of enzymes that convert modified respective purines into unmodified counterparts. Also, new metagenomic libraries, created by isolating DNA from the environmental samples, sharing and cloning into the multicopy cloning vectors, were used for selection of genes involved in metabolism of modified heterocyclic bases. Several candidate DNA fragments were obtained during the screening procedure and are currently under the investigation.