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Thesis advisor: Abhishek Chatterjee / Genetic code expansion (GCE) is an enabling technology whereby noncanonical amino acids (ncAAs) can be site-specifically incorporated into proteins of interest, allowing for vast applications and an improved understanding of structure-function relationships in biology. GCE stands out as a versatile platform due to the use of a variety of engineered aminoacyl-tRNA synthetase (aaRS)/transfer RNA (tRNA) pairs, and it has endowed proteins with over 200 distinct ncAAs in both prokaryotic and eukaryotic systems. My dissertation outlines endeavors aimed at broadening the chemical diversity of α-amino side chains and substrates beyond α-amino acids in both prokaryotic and eukaryotic organisms through the utilization of GCE technology. This was achieved by creating universal GCE platforms called altered translational machinery (ATM) strains, which eliminate the limitations of orthogonality for the evolution of aaRS/tRNA pairs. This expansion enables the use of the same aaRS/tRNA pair for ncAA incorporation functionalities into multiple domains of life. Moreover, the diversity of ncAAs that can be genetically encoded in eukaryotic cells was enhanced by evolving the E. coli leucyl-tRNA synthetase (EcLeuRS)/tRNA pair using a yeast-based selection system. This advancement facilitated the incorporation of novel ncAAs into proteins within mammalian cells. Additionally, I worked toward developing a platform for introducing monomers into the genetic code beyond α-amino acids. This involved developing an aaRS evolution platform that doesn't rely on translation as a selectable readout. Finally, I worked towards the creation of polyester-polyamide oligomers with sequence control as a step towards the goal of generating sequence-defined biopolymers with new-to-nature backbone chemistries. / Thesis (PhD) — Boston College, 2024. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Production of mutant biological molecules for understanding biological principles or as therapeutic agents has gained considerable interest recently. Synthetic genes are today being widely used for production of such molecules due to the substantial decrease in the costs associated with gene synthesis technology. Along one such line, we have engineered tRNA genes in order to dissect the effects of G:U base-pairs on the accuracy of the protein translation machinery. Our results provide greater detail into the thermodynamic interactions between tRNA molecules and an Elongation Factor protein (termed EF-Tu in bacteria and eEF1A in eukaryotes) and how these interactions influence the delivery of aminoacylated tRNAs to the ribosome. We anticipate that our studies not only shed light on the basic mechanisms of molecular machines but may also help us to develop therapeutic or novel proteins that contain unnatural amino acids. Further, the manipulation of the translation machinery holds promise for the development of new methods to understand the origins of life. Along another line, we have used the power of synthetic biology to experimentally validate an evolutionary model. We exploited the functional diversity contained within the EF-Tu/eEF1A gene family to experimentally validate the model of evolution termed ‘heterotachy’. Heterotachy refers to a switch in a site’s mutational rate class. For instance, a site in a protein sequence may be invariant across all bacterial homologs while that same site may be highly variable across eukaryotic homologs. Such patterns imply that the selective constraints acting on this site differs between bacteria and eukaryotes. Despite intense efforts and large interest in understanding these patterns, no studies have experimentally validated these concepts until now. In the present study, we analyzed EF-Tu/eEF1A gene family members between bacteria and eukaryotes to identify heterotachous patterns (also called Type-I functional divergence). We applied statistical tests to identify sites possibly responsible for biomolecular functional divergence between EF-Tu and eEF1A. We then synthesized protein variants in the laboratory to validate our computational predictions. The results demonstrate for the first time that the identification of heterotachous sites can be specifically implicated in functional divergence among homologous proteins. In total, this work supports an evolutionary synthetic biology paradigm that in one direction uses synthetic molecules to better understand the mechanisms and constraints governing biomolecular behavior while in another direction uses principles of molecular sequence evolution to generate novel biomolecules that have utility for industry and/or biomedicine.