TetR-binding aptamer as a versatile regulatory element

Abstract

Synthetic biology explores the means of redesign and fabrication of existing biological systems or the de novo design and generation of biological components that are entirely new to nature. The main focus of the relatively new and dynamic discipline is to develop programmable genetic regulatory systems. Precise, reversible and temporary control of gene expression as well as in-depth understanding of fundamental genetics are crucial for the programming of new genetic circuits. RNA represents one of the most powerful substrates in the engineering of biological systems, as it is versatile, designable and easily characterizable. Among the diverse functions of RNA molecules, their role as natural riboswitches has predominantly inspired researchers to design synthetic RNA-based regulators. Most of RNA devices contain a sensor element, an aptamer domain, which recognizes small molecules or protein ligands with high specificity and affinity, and an expression platform, controlling gene expression via various mechanisms. Generally, binding of a specific ligand to the aptamer domain stabilizes the RNA molecule or causes conformational changes in its structure; these further regulate transcription, translation and mRNA processing and degradation. Engineered RNA-based devices have already demonstrated multiple applications in synthetic biology. However, their implementation was mostly validated in bacteria and yeast, while mammalian synthetic biology has lagged behind. Splicing of pre-mRNAs is an essential process in human cells that generates a diverse proteome through networks of coordinated splicing events and offers an additional layer of control for synthetic RNA devices. The reprogrammed removal of intronic sequences could provide a novel approach for the development of gene therapies to tackle disease phenotypes. For this purpose, it is necessary to design tools that allow precise and timely control of the splicing mechanisms. In the first research project described in the presented doctoral thesis, a versatile and highly efficient splicing device enabling control of gene expression in human cells and making use of an RNA aptamer recognized by the TetR was designed. Further, the portability of the splicing device was shown through its functionality in various reporter systems and the endogenous gene context. In the course of the thesis, the first inducible model for alternative 3’ splice site recognition with the tetracycline repressor (TetR) aptamer leading to production of splice variants with different subcellular localization was generated. The applicability of the system was corroborated in experiments aimed at controlling nuclear import in human cells. The proposed approach may prove valuable in phenotypic studies of essential genes and provide an alternative in the development of therapeutic strategies, as the nucleo-cytoplasmic transport is vital for the maintenance of balanced cell physiology, with aberrant spatiotemporal localization of proteins leading to the development of various disorders and cancer. Finally, the third research project undertaken in the course of my doctoral studies focused on the development and optimization of the TetR aptamer dependent translational control system in human cells. Translational regulation constitutes an important point of post-transcriptional control of gene expression, enabling the cell to rapidly change the level of a specific gene product. Up to now, no efficient aptamer-ligand based translational regulatory system has been demonstrated in mammalian cells.