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Self-labelling protein tags (SLPs) are resourceful tools that revolutionized sensor imaging, having the versatile ability of being genetically fused with any protein of interest and undergoing activation with alternative probes specifically designed for each variant (namely, SNAP

We present a new class of DNA-based nanoswitches that, upon enzymatic repair, could undergo a conformational change mechanism leading to a change in fluorescent signal. Such folding-upon-repair DNA nanoswitches are synthetic DNA sequences containing O(6)-methyl-guanine (O(6)-MeG) nucleobases and labelled with a fluorophore/quencher optical pair. The nanoswitches are rationally designed so that only upon enzymatic demethylation of the O(6)-MeG nucleobases they can form stable intramolecular Hoogsteen interactions and fold into an optically active triplex DNA structure. We have first characterized the folding mechanism induced by the enzymatic repair activity through fluorescent experiments and Molecular Dynamics simulations. We then demonstrated that the folding-upon-repair DNA nanoswitches are suitable and specific substrates for different methyltransferase enzymes including the human homologue (hMGMT) and they allow the screening of novel potential methyltransferase inhibitors.

In the past decade, a powerful biotechnological tool for specific in vivo and in vitro labeling of proteins of interest, the SNAP-tag technology, has been proposed as a valid alternative to the classic protein-tags. This self-labeling protein-tag is an engineered variant of the human O6-alkylguanine-DNA-alkyltransferases (hMGMT) enzyme, which specifically reacts with benzyl-guanine (BG) derivatives, maintaining a part of these substrates in the catalytic site, with a one-step reaction. However, given the mesophilic nature of this protein-tag, the general use of this new approach is restricted to mesophilic model systems and mild reaction conditions. To overcome this limitation, it is necessary the exploitation of homologous activities from extremophilic sources, to apply this technology also to (hyper)thermophilic organisms and harsher reaction conditions. To this purpose, it has been first characterized an archaeal alkylguanine-DNA-alkyltransferases (AGT or OGT) from Saccharolobus solfataricus (SsOGT), which presented an exceptional in vitro stability at high temperatures, physical and chemical denaturing agents, making it suitable not only for the in vivo heterologous expression in Escherichia coli, but also in the thermophilic Thermus thermophilus and Sulfolobus islandicus organisms. Furthermore, the growing demand to apply this biotechnological tool to extreme conditions led to look for new protein-tags from hot sources. Recently, new AGTs from Thermotoga neapolitana (TnOGT) and Pyrococcus furiosus (PfuOGT) resulted different from SsOGT in terms of activity and stability, proposing them as potential tool for the in vivo analysis and function of enzymes of interest in these hyperthermophilic model systems.

Alkylguanine-DNA-alkyltransferases (AGTs) have a natural role in the protection of DNA from mutations caused by alkylating agents. Their peculiar irreversible self-alkylation reaction led to the development as new tools in the modern biotechnology. SNAP-tag® is a powerful enzyme for the specific labelling of protein/enzymes by using benzyl-guanine (BG) derivatives as substrates. This technology has some limitations, as the mesophilic nature of the tag (an engineered variant of the human enzyme) and the needs of purify each substrate. Recently, the SNAP-tag® technology was successfully implemented by the employment of thermostable “SNAP-tag-like” variants from (hyper)thermophilic sources, and by the utilization of BG-substrates containing an azide group to be combined with DBCO-derivatives by azide-alkyne Huisgen cycloaddition. The introduction of these new actors on the scene made possible the expansion of the methodology to in vivo and in vitro harsh reaction conditions, as well as the utilization of more chemical groups in the overall reaction enzyme labelling.

The specific labelling of proteins in recent years has made use of self-labelling proteins, such as the SNAP-tag(®) and the Halotag(®). These enzymes, by their nature or suitably engineered, have the ability to specifically react with their respective substrates, but covalently retaining a part of them in the catalytic site upon reaction. This led to the synthesis of substrates conjugated with, e.g., fluorophores (proposing them as alternatives to fluorescent proteins), but also with others chemical groups, for numerous biotechnological applications. Recently, a mutant of the OGT from Saccharolobus solfataricus (H(5)) very stable to high temperatures and in the presence of physical and chemical denaturing agents has been proposed as a thermostable SNAP-tag(®) for in vivo and in vitro harsh reaction conditions. Here, we show two new thermostable OGTs from Thermotoga neapolitana and Pyrococcus furiosus, which, respectively, display a higher catalytic activity and thermostability respect to H(5), proposing them as alternatives for in vivo studies in these extreme model organisms.