Spatially Confining Translation to Enable Optimized Genetic Code Expansion in Eukaryotes
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Abstract
The genetic code is the operating system of every living cell. It is executed through the central dogma of molecular biology and defines how the ribosome translates genetic information into a polypeptide.
The genetic code is conserved throughout all domains of life and encodes three stop codons as well as the incorporation of the canonical amino acids. The naturally occurring amino acids can be classified in four different chemical functionalities (nonpolar, polar, basic and acidic). Despite this simplicity the genetic code gives rise to the entire diversity seen across all kingdoms of life and it can only be imagined how life might look like if the ribosome could also incorporate noncanonical amino acids (ncAAs), covering the full spectrum of chemical functionalities, into proteins.
Genetic code expansion (GCE) is a powerful method to site-specifically incorporate ncAAs into proteins in vivo. To this end, typically an orthogonal aminoacyl-tRNA-synthetase/tRNA (RS/tRNA) suppressor pair is used to reassign a rare stop codon to be read as a sense codon. During the last decades of research GCE has been established to permit the genetic incorporation of a plethora of ncAAs, which have for example been used to enable site-specific protein labeling for super-resolution microscopy.
However, for imaging applications as well as for applications aiming to synthesize fully artificial polymers in eukaryotes, the current technology has at least three major limitations. First, GCE is codon specific but it cannot distinguish the mRNA of the protein of interest from endogenous mRNAs, leading to recoding of untargeted codons in the transcriptome. Due to this activity, GCE can have adverse side effects or even be toxic. Second, only a few orthogonal RS/tRNA suppressor pairs have been established for eukaryotic systems and third, only two different stop codons can be suppressed at a time.
In this cumulative thesis I address the first problem, by developing synthetic membraneless organelles that allow to selectively translate only selected mRNAs with an expanded genetic code (Chapters2&3 and Appendix II). An alternative development using inducible expression systems, allowing to regulate the GCE components, is presented in Chapter 2 and Appendix IV. I further develop multiple mutually orthogonally translating organelles to equip cells with multiple genetic codes, which represents a new way to obtain orthogonal RS/tRNA suppressor pairs and enables to multiple times reassign the same stop codon, thus solving the remaining two major contemporary limitation of GCE (Chapter 5).