Title: Reprogramming protein synthesis for cell engineering
Description: Synthetic biology, which aims to enable the design and assembly of customized biological systems, holds great promise for delivering solutions to numerous modern day challenges in agriculture, sustainable energy production, and medicine. However, at its current stage, synthetic biology is not yet equipped with the necessary tools and understanding to reprogram the immensely complex molecular environment of the cell beyond simple proof of concept demonstrations. One current objective within synthetic biology is to create robust tools that can be used to manipulate biological systems in a predictable and reliable manner. While many transcription-based control devices have been reported, little consideration has been given to the eukaryotic protein translation apparatus as a target for engineering gene-regulatory tools. In this work, we explore the potential for reprogramming the protein synthesis machinery for cell engineering. We begin in Chapter 1 by reviewing canonical protein synthesis and survey the assortment of translation reprogramming mechanisms that exist in nature, focusing on the role of RNA in these processes.

We then cover previous efforts to engineer the protein synthesis machinery and discuss their methodological approaches. Lastly, we examine potential opportunities for engineering protein synthesis that have not yet been explored. RNA’s prominent role in protein synthesis and its amenability to high-throughput in vitro selection approaches raises the possibility that the translation apparatus could be engineered through in vitro directed evolution of its RNA components. In Chapter 2, we develop an experimental framework for identifying mRNA sequence elements that reprogram protein synthesis, focusing on stop codon readthrough. By adapting a previously developed in vitro selection technology called mRNA display, we demonstrate that molecules of RNA derived from expansive libraries of random sequences can be enriched as a result of their translation reprogramming activity. We then analyze these stop codon readthrough signals and propose the use of these sequences for enhanced unnatural amino acid incorporation technologies.

In Chapter 3, we apply this very same selection principle for the in vitro directed evolution of RNA sequences that stimulate -1 programmed ribosomal frameshifting. Then, using previously reported RNA aptamers, we rationally engineer RNA switches that regulate translation reading frame in response to small molecule inputs. To further optimize switch performance, an in vivo directed evolution platform was established. We explore the utility of these RNA switches, particularly their ability to regulate multi-protein stoichiometry, for performing cellular logic operations and controlling cell fate. A major focus of translation engineering has been the incorporation of unnatural amino acids for fluorescent labeling of proteins in living cells. The successful achievement of this goal will require small molecule fluorophores with desirable biological properties, as well as robust synthetic methods for their production. In Chapter 4, we present a scalable approach to oxazine and xanthene fluorophores that utilizes a general diaryl ether synthetic intermediate.

Finally, in Chapter 5, we describe a photoactivatable oxazine fluorophore and demonstrate its utility as a live-cell imaging reagent with applicability to advanced microscopy techniques.