Publikation: Catalytic RNAs as a target and a tool for the control of gene expression in mammalian cells and bacteria
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RNAs with catalytic functions were discovered over 40 years ago. Since then, major advances were made investigating the biological function of catalytic RNAs and exploiting them as tools for artificial systems. Especially, the self-cleaving ribozymes are a suitable element for synthetic riboswitch design. Generally, riboswitches consist of a ligand-binding aptamer domain that is fused to an expression platform via a communication module. Upon ligand-binding, due to a conformational change of the expression platform, the information is transferred and gene expression can be regulated. Over the years, the interest in controlling transgene expression increased dramatically and artificial riboswitches are useful tools in versatile applications ranging from synthetic biology for basic research interests to potential therapeutic approaches. Therapeutic contexts involve for instance the controlled expression or induction of cytokines like interleukins or interferon-gamma. Therefore, synthetic riboswitches require to be transferrable between different contexts maintaining their dynamic range, while regulating the expression of the respective gene of interest. Moreover, the system has to be sensitive and highly selective to the chosen ligand. Thereby, side effects from unspecific binding events or leaking background expression in the uninduced state can be prevented. Common therapeutic approaches involve viral vectors, such as Adeno-associated virus or Measles virus, as carriers to bring the synthetic riboswitch directly into the target cells. This poses limits to the switching system in order to be combinable with the viral context, like the small coding space. Moreover, for instance, transcription-dependent RNA-switches are difficult to apply to negative-sense single-stranded RNA-viruses, as Measles virus vectors. Therefore, it is important to develop always new switching systems to exploit them for different therapeutic strategies. Riboswitches differ a lot in size and complexity. The simplest design utilizes an aptamer as cis-regulatory element to act as a roadblock for the ribosome or to control the accessibility of essential sequence elements within the mRNA upon ligand addition. Combining multiple elements, the dynamic range increases and the background expression in the OFF-state can be reduced. One common strategy is to fuse an aptamer to small self-cleaving ribozymes to create allosteric ribozymes. These so-called aptazymes can be inserted in either of the untranslated regions (UTR) of an mRNA to control its stability in a ligand-dependent manner. By now, several systems with high dynamic ranges exploiting aptazymes were designed. However, their context-dependent performance poses an obstacle to use them in therapeutic approaches. In this study, we implemented a comprehensive and comparative survey of the effect of inserting different self-cleaving ribozymes into mRNAs in human cell culture to expand the toolbox of ribozyme platforms. Furthermore, we used the obtained results to create new 10 tetracycline (Tet)-dependent aptazymes, based on the hepatitis delta virus-ribozyme (HDV). Although their induction levels upon ligand addition were lower than expected, we found strategies to optimize the system. Following different approaches, we were able to generate a set of ON-switching HDV-based aptazymes. Moreover, we could show their great combinability with other synthetic riboswitches. By developing a Tet-dependent splice-based system, which is residing in the 5’-UTR of the mRNA of the gene of interest, we could develop combinations with aptazymes showing increased dynamic ranges. The splice-based system controls the presence of an upstream open reading frame (uORF) within the mRNA, which is effectively downregulating gene expression. By investigating different uORF sequences, aptamer structures and splice site properties, we were able to create a set of ON-switches. These show Tet-dependent switching on the protein level as well as on the RNA level. Additionally, we could show that this system is easily transferrable to another gene context maintaining its switching ability. The systems presented in this thesis add up to the already existing toolbox of synthetic riboswitches for human cell culture and our data can be used to improve future aptazyme designs. However, we did not only examine self-cleaving ribozymes on their potential as a tool for controlling gene expression. We were also investigating the glmS ribozyme as it exploits another molecule to enhance its biological function. It uses glucosamine-6-phosphate (GlcN6P) as a coenzyme for self-cleavage. The presence of the glmS ribozyme almost exclusively in gram-positive bacteria makes it a convenient target for antibiotic drug development. Using artificial mimics of the natural ligand, which are not metabolized but efficiently trigger the self-cleavage of the ribozyme, we were able to interfere with cell metabolism and inhibited the growth of multiple different species, including human pathogens such as S. aureus. Thus, within this thesis we were able to develop genetic devices important for therapeutic strategies in human cell culture as well as to study the pathogenicity of different gram-positive bacteria using GlcN6P mimics.
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KLÄGE, Dennis, 2024. Catalytic RNAs as a target and a tool for the control of gene expression in mammalian cells and bacteria [Dissertation]. Konstanz: Universität KonstanzBibTex
@phdthesis{Klage2024Catal-70615, year={2024}, title={Catalytic RNAs as a target and a tool for the control of gene expression in mammalian cells and bacteria}, author={Kläge, Dennis}, address={Konstanz}, school={Universität Konstanz} }
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Therefore, synthetic riboswitches require to be transferrable between different contexts maintaining their dynamic range, while regulating the expression of the respective gene of interest. Moreover, the system has to be sensitive and highly selective to the chosen ligand. Thereby, side effects from unspecific binding events or leaking background expression in the uninduced state can be prevented. Common therapeutic approaches involve viral vectors, such as Adeno-associated virus or Measles virus, as carriers to bring the synthetic riboswitch directly into the target cells. This poses limits to the switching system in order to be combinable with the viral context, like the small coding space. Moreover, for instance, transcription-dependent RNA-switches are difficult to apply to negative-sense single-stranded RNA-viruses, as Measles virus vectors. Therefore, it is important to develop always new switching systems to exploit them for different therapeutic strategies. Riboswitches differ a lot in size and complexity. The simplest design utilizes an aptamer as cis-regulatory element to act as a roadblock for the ribosome or to control the accessibility of essential sequence elements within the mRNA upon ligand addition. Combining multiple elements, the dynamic range increases and the background expression in the OFF-state can be reduced. One common strategy is to fuse an aptamer to small self-cleaving ribozymes to create allosteric ribozymes. These so-called aptazymes can be inserted in either of the untranslated regions (UTR) of an mRNA to control its stability in a ligand-dependent manner. By now, several systems with high dynamic ranges exploiting aptazymes were designed. However, their context-dependent performance poses an obstacle to use them in therapeutic approaches. In this study, we implemented a comprehensive and comparative survey of the effect of inserting different self-cleaving ribozymes into mRNAs in human cell culture to expand the toolbox of ribozyme platforms. Furthermore, we used the obtained results to create new 10 tetracycline (Tet)-dependent aptazymes, based on the hepatitis delta virus-ribozyme (HDV). Although their induction levels upon ligand addition were lower than expected, we found strategies to optimize the system. Following different approaches, we were able to generate a set of ON-switching HDV-based aptazymes. Moreover, we could show their great combinability with other synthetic riboswitches. By developing a Tet-dependent splice-based system, which is residing in the 5’-UTR of the mRNA of the gene of interest, we could develop combinations with aptazymes showing increased dynamic ranges. The splice-based system controls the presence of an upstream open reading frame (uORF) within the mRNA, which is effectively downregulating gene expression. By investigating different uORF sequences, aptamer structures and splice site properties, we were able to create a set of ON-switches. These show Tet-dependent switching on the protein level as well as on the RNA level. Additionally, we could show that this system is easily transferrable to another gene context maintaining its switching ability. The systems presented in this thesis add up to the already existing toolbox of synthetic riboswitches for human cell culture and our data can be used to improve future aptazyme designs. However, we did not only examine self-cleaving ribozymes on their potential as a tool for controlling gene expression. We were also investigating the glmS ribozyme as it exploits another molecule to enhance its biological function. It uses glucosamine-6-phosphate (GlcN6P) as a coenzyme for self-cleavage. The presence of the glmS ribozyme almost exclusively in gram-positive bacteria makes it a convenient target for antibiotic drug development. Using artificial mimics of the natural ligand, which are not metabolized but efficiently trigger the self-cleavage of the ribozyme, we were able to interfere with cell metabolism and inhibited the growth of multiple different species, including human pathogens such as S. aureus. Thus, within this thesis we were able to develop genetic devices important for therapeutic strategies in human cell culture as well as to study the pathogenicity of different gram-positive bacteria using GlcN6P mimics.</dcterms:abstract> <dcterms:issued>2024</dcterms:issued> <dspace:hasBitstream rdf:resource="https://kops.uni-konstanz.de/bitstream/123456789/70615/4/Klaege-2-195vvd50ffu7e0.pdf"/> <void:sparqlEndpoint rdf:resource="http://localhost/fuseki/dspace/sparql"/> </rdf:Description> </rdf:RDF>