RNA chemical and synthetic biology:
RNA switches can be designed and constructed by combining ligand-binding RNA domains (so-called aptamers) with sequences that affect gene expression (so-called expression platforms). Traditionally, we have utilized small self-cleaving ribozymes as expression platforms. In these applications the designed RNA switch is inserted into the mRNA of a desired transgene. We have shown that they are versatile tools for controlling gene expression in a variety of organisms such as bacteria (M. Felletti, J. Stifel, L. A. Wurmthaler, S. Geiger, J. S. Hartig: Twister ribozymes as highly versatile expression platforms for artificial riboswitches. Nature Communications, 2016, 12834), yeast (B. Klauser, J. Atanasov, L. Siewert, J.S. Hartig: Ribozyme-based aminoglycoside switches of gene expression engineered by genetic selection in S. cerevisiae. ACS Synthetic Biology, 2014, 4, 516), nematodes (L. Wurmthaler, M. Sack, K. Gense, J. S. Hartig, and M. Gamerdinger: A tetracycline-dependent ribozyme switch allows conditional induction of gene expression in Caenorhabditis elegans. Nature Communications, 2019, 10, 491), plants (N. Shanidze, F. Lenkeit, J. S. Hartig, and D. Funck: A theophylline-responsive riboswitch regulates expression of nuclear-encoded genes in Arabidopsis. Plant Physiology, 2019, doi: 10.1104/pp.19.00625) and human cell lines (B. Strobel, M. Spöring, H. Klein, S. Sayols Puig, J. S. Hartig, and S. Kreuz, High-throughput identification of synthetic riboswitches by barcode-free amplicon-sequencing in human cells. Nature Communications, 2020, 11, 714; S. Ausländer , P. Stücheli , C. Rehm , D. Ausländer , J. S. Hartig, M. Fussenegger: A general design strategy for protein-responsive riboswitches in mammalian cells. Nature Methods, 2014, 11, 1154). Since these control elements have a small genetic footprint and are considered to be non-immunogenic, they are considered to be of high interest for controlling transgene expression in future gene-therapeutic applications (B. Strobel, M. J. Düchs, D. Blazevic, P. Rechtsteiner, C. Braun, K.S. Baum-Kroker, B. Schmid, T. Ciossek, D. Gottschling, J. S. Hartig, and S. Kreuz: A Small-Molecule-Responsive Riboswitch Enables Conditional Induction of Viral Vector-Mediated Gene Expression in Mice. ACS Synthetic Biology, 2020, 9, 1292; P. Ketzer, J. K. Kaufmann, S. Engelhardt, S. Bossow, C. von Kalle, J. S. Hartig, G. Ungerechts, D. M. Nettelbeck: Artificial riboswitches for gene expression and replication control of DNA and RNA viruses. Proceedings of the National Academy of Sciences, 2014, 111, E554).
In general, such RNA switches are very modular with respect to their application and can be utilized to control further RNA species such as rRNA (M. Wieland, B. Berschneider, M. D. Erlacher, J. S. Hartig: Aptazyme-mediated regulation of 16S ribosomal RNA. Chemistry & Biology, 2010, 17, 236) and tRNAs (A. Saragliadis, J. S. Hartig: Ribozyme-based tRNA switches for post-transcriptional control of amino acid identity in protein synthesis. Journal of the American Chemical Society, 2013, 135, 8222). Lately, we have broadened the available synthetic riboswitch-toolbox by exploring alternative expression platforms such as controlling polyadenylation (M. Spöring, R. Boneberg, and J. S. Hartig: Aptamer-mediated control of polyadenylation for gene expression regulation in mammalian cells. ACS Synthetic Biology, 2020, 9, 3008) as well as splicing processes (M. Finke, D. Brecht, J. Stifel, K. Gense, M. Gamerdinger, and J. S. Hartig: Efficient splicing-based RNA regulators for tetracycline-inducible gene expression in human cell culture and C. elegans. Nucleic Acids Research, 2021, 49, e71). These concepts are characterized by improved applicability and performances and, together with ribozyme-dependent switches, are currently further developed by us in order to be maximally functional in future applications.
Discovery of novel enzymes and pathways:
Starting with the discovery of novel guanidine-responsive riboswitches (F. Lenkeit, I. Eckert, J. S. Hartig, and Z. Weinberg: Discovery and Characterization of a Fourth Class of Guanidine Riboswitches. Nucleic Acids Research, 2020, 16, 12889), we are currently aiming at clarifying the metabolism of free guanidine in nature. Natural riboswitches represent interesting starting points for such an endeavor since they deliver a list of genes under control of and hence functionally connected to guanidine. Many of the functions of guanidine riboswitch-associated genes are currently unknown or not well characterized and their functional study will enable us to shed light on still unknown metabolism and functions of guanidine in nature.
So-far we have clarified the degradation of guanidine for the purpose of nitrogen assimilation. The necessary enzymatic activities are special since they need to overcome a high degree of resonance stabilization of guanidine. One system utilizes carboxylation and subsequent hydrolysis for degradation (M. Sinn, F. Hauth, F. Lenkeit, Z. Weinberg, and J. S. Hartig: Widespread bacterial utilization of guanidine as nitrogen source. Molecular Microbiology, 2021, doi: 10.1111/mmi.14702). In analogy to urea degradation where both carboxylation as well as direct hydrolysis activities via the Ni-dependent enzyme urease exist, we searched for an enzyme capable of hydrolyzing guanidine to urea. We discovered a highly specific guanidine hydrolase enzyme from the arginase family that surprisingly also depends on Ni (D. Funck, M. Sinn, J. Fleming, M. Stanoppi, J. Dietrich, R. López-Igual, O. Mayans, and J. S. Hartig: Discovery of a Ni2+-dependent guanidine hydrolase in bacteria. Nature, 2022, 603, 515).
In addition to its degradation, we are currently investigating biotic sources of guanidine. For example, we have identified a novel bacterium able to utilize the toxic guanidine-containing plant metabolite canavanine as sole N- and C-source (F. Hauth, H. Buck, and J. S. Hartig: Pseudomonas canavaninivorans sp. nov. isolated from bean rhizosphere. Int. J. Syst. Evol. Microbiol., 2022, doi: 10.1099/ijsem.0.005203). We clarified the responsible degradation pathway with a novel PLP-dependent enzyme termed canavanine-g-lyase as key activity producing hydroxyguanidine (F. Hauth, H. Buck, M. Stanoppi and J. S. Hartig: Canavanine utilization via homoserine and hydroxyguanidine by a PLP-dependent g-lyase in Pseudomonadaceae and Rhizobiales. RSC Chemical Biology, 2022, DOI: 10.1039/D2CB00128D). Hydroxyguanidine is excreted as waste product and is able to trigger guanidine riboswitches. Results in ongoing projects regarding further riboswitch-associated genes point at additional functional connections with canavanine metabolism. The characterization of additional sources of guanidine and its physiological functions are among current topics in our group.