Synthesis of designer proteins by manipulating translation
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Abstract
Proteins, one of the primary workhorses of a living cell, are composed of 22 different amino acids. The impressive diversity in protein structure and function that has been achieved by only 22 building blocks draws attention to the untapped potential of expanding the repertoire of canonical amino acids. Genetic code expansion (GCE) is an in vivo protein modification technology that allows the incorporation of non-canonical amino acids (ncAAs) in a protein of interest (POI). Most commonly GCE utilizes a special pair of aminoacyl tRNA synthetase (aaRS) and its cognate tRNA to repurpose a stop codon to incorporate an ncAA instead of signaling the termination of translation. These GCE-specific aaRS/tRNA pairs need to be orthogonal to the endogenous aaRS/tRNA pairs to minimize unspecific modification of the host proteome.
Decades of research towards perfecting the GCE technology has facilitated the incorporation of over 500 ncAAs of diverse chemical properties into POIs. These ncAAs can potentially have most desired chemical handle and can render new functionalities to the POIs, for example, post-translational modifications (PTMs), ability to bio-orthogonally react with probes including synthetic dyes, to name a few. Despite being an extremely powerful technology, GCE is not devoid of limitations. Some of the pressing challenges include lack of mRNA specificity leading to unspecific ncAA incorporation in the host proteome, limited availability of codons for reassignment and generation of truncated POI due to failure of GCE. To mitigate the first problem in mammalian cells Reinkemeier et al. developed membraneless orthogonally translating organelles (OTOs) to confine the GCE process. mRNA of the POI was selectively recruited to the OTOs thereby minimizing unspecific ncAA incorporation in the host proteome.
In order to truly harness the potential of GCE it is necessary to expand beyond the three available stop codons. This thesis explores the possibility of reassigning sense codons to perform mRNA specific GCE in a POI in mammalian cells. Sense codons have been reassigned both in site-specific and residue-specific manner. In site-specific sense codon reassignment, the codon to be reassigned occurs only at pre-determined locations in the gene of the POI. On the other hand, for residue-specific reassignment, a particular sense codon that naturally occurs in the gene of the POI is chosen and all occurrences of the selected codon are attempted to be reassigned. Although proteome wide residue specific sense codon reassignment has been performed before, this technology has not been applied in a mRNA-specific manner.
A set of 8 sense codons could be successfully reassigned site-specifically in EGFP-based reporters in HEK293T cells. The highest fold change selectivity of 14fold for site-specific sense codon reassignment was achieved for the CTA codon with an OTO as compared to the non-selective, cytoplasmic GCE machinery. For residue-specific sense codon reassignment, 61 sense codons were individually reassigned in two different reporters to select the suitable codon for reassignment. mRNA selective residue-specific sense codon reassignment by OTOs was successfully demonstrated for multiple sense codons in vimentin::mcerulean in HEK293T cells. Both mRNA selective site- and residue-specific sense codon reassignment technologies could be used for labelling POIs for confocal microscopy applications.
The success of selective residue-specific sense codon reassignment is the key step in the development of a novel protein labelling technology that would allow in vivo visualization of protein shapes when combined with super resolution microscopy, thereby elucidating yet invisible sub-cellular structures. Besides fluorescent microscopy applications, both site- and residue-specific sense codon reassignment hold the potential of facilitating in vivo synthesis of artificial biopolymers of potentially any functionality and the work of this thesis brings us one step closer to achieving greater feats than billions of years of evolution.