Thesis advisor: Abhishek Chatterjee / Directed evolution is a powerful technique to expand chemical space in biological systems. In particular, this method has been used to develop cellular machinery to enable genetic code expansion (GCE), the incorporation of unnatural amino acids (UAAs) into proteins during the translation process. GCE relies on evolving an aminoacyl tRNA synthetase (aaRS) and tRNA pair from a different domain of life to incorporate a UAA into proteins in their new host, as these evolutionarily distant pairs are less likely to be cross-reactive with host pairs. The aaRS and tRNA must meet a number of conditions to be useful for GCE: the pair must be orthogonal (non-cross-reactive) to the host’s native aaRS/tRNA pairs in order to ensure site-specific UAA incorporation; the aaRS must have an active site suited to accept the shape of the UAA; and the tRNA must cooperate with the host ribosome, elongation and release factors, and other translational machinery to efficiently incorporate the UAA into the protein. Numerous aaRS/tRNA pairs have been evolved to allow incorporation of diverse UAAs in bacteria due to the tractable nature of these organisms for directed evolution experiments. While an aaRS evolved in bacteria to charge a novel UAA can be used in eukaryotes, tRNAs cannot be evolved for GCE in bacteria and then used in eukaryotes because they will not have evolved in the presence of the correct translational machinery. It is necessary to evolve tRNAs directly in their host cells. Unfortunately for researchers working on GCE in mammalian cells, it is difficult to perform directed evolution on small gene products in these hosts. Transformation efficiency in mammalian cells is poor, and transient transfection yields heterogeneous DNA distribution to target cells, making selection based on performance of individual library members impossible. Viruses are an ideal DNA delivery vector for mammalian cells, as production of recombinant viruses allows control over library member generation, and viruses can be delivered with exquisite copy number control. The Chatterjee lab recently developed a platform, Virus-Assisted Directed Evolution of tRNAs (VADER), using adeno-associated virus (AAV) to evolve tRNAs for GCE directly in mammalian cells.
While VADER is the first directed evolution platform that allows the evolution of small gene products in mammalian cells, its efficiency is limited by its continued reliance on transient transfection to deliver non-library DNA that is necessary for the production of rAAV. To overcome this limitation, baculovirus delivery vectors were developed to boost DNA delivery and AAV capsid production to improve virus production efficiency during selections. VADER allows the evolution of tRNAs to incorporate certain UAAs, but the technique relies on installing a UAA into the AAV capsid, which is sensitive to disruption caused by slight modifications in structure. To expand the scope of VADER to evolve tRNAs for UAAs that cannot be incorporated into the AAV capsid, an alternate selection handle (Assembly Activating Protein, or AAP) was deleted from the genome and provided in trans to incorporate 5-hydroxytryptophan (5HTP). Incorporating the UAA into this flexible protein allows UAA-dependent production of AAV and expands the scope of tRNAs that can be evolved in mammalian cells. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.
| Identifer | oai:union.ndltd.org:BOSTON/oai:dlib.bc.edu:bc-ir_109192 |
| Date | January 2021 |
| Creators | Levinson, Samantha D. |
| Publisher | Boston College |
| Source Sets | Boston College |
| Language | English |
| Detected Language | English |
| Type | Text, thesis |
| Format | electronic, application/pdf |
| Rights | Copyright is held by the author, with all rights reserved, unless otherwise noted. |
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