Thesis advisor: Abhishek Chatterjee / Thesis advisor: Eranthie Weerapana / In nature, just twenty canonical amino acids are responsible for the creation of nearly all proteins. Genetic code expansion (GCE), or the incorporation of noncanonical amino acids (ncAAs) into living cells, is a powerful tool that expands the studies we are capable of performing using proteins. This technology relies on engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pairs that are orthogonal to the host cells’ endogenous aaRS/tRNA pairs, and one of the main limitations of GCE arises from the inefficiency of these suppressor tRNAs when expressed in a foreign host cell. To address this limitation, we have previously reported a strategy for the virus-assisted directed evolution of tRNAs (VADER) which is uniquely capable of addressing the specific needs of tRNA evolution. In order to advance the capabilities of VADER, we made a number of modifications to the VADER selection scheme. First, we designed and executed a modified VADER selection that enabled the evolution of a new class of tRNAs, and with this VADER selection, we were able to generate a first-generation E. coli tyrosyl tRNA (tRNATyr) variant that was three times as active as its wild-type equivalent. Next, we introduced a number of refinements to the VADER strategy to generate VADER 2.0, an improved workflow capable of screening larger libraries and libraries encoding more active variants. Using VADER 2.0, we created second-generation tRNAPyl and tRNATyr mutants that achieved incorporation efficiencies that were greater than five-fold higher than their wild-type equivalents across a wide variety of substrates, enabling exciting GCE experiments that would not be possible otherwise. / Thesis (PhD) — Boston College, 2023. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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Thesis advisor: Abhishek Chatterjee / In the last two decades, unnatural amino acid (UAA) mutagenesis has emerged as a powerful new method to probe and engineer protein structure and function. This technology enables precise incorporation of a rapidly expanding repertoire of UAAs into predefined sites of a target protein expressed in living cells. Owing to the small footprint of these genetically encoded UAAs and the large variety of enabling functionalities they offer, this technology has tremendous potential for deciphering the delicate and complex biology of the mammalian cells. We describe the application of this technology to the modification of adeno-associated virus (AAV) for the first time, enabling the generation of vectors with precisely re-engineered cell-targeting for gene therapy. Our UAA-AAV production platform enables the incorporation of UAAs bearing bio-orthogonal reactive handles into multiple specific sites on the virus capsid and their subsequent functionalization with various labeling molecules. Incorporation of an azido-UAA enabled site-specific attachment of a cyclic-RGD peptide onto the capsid, retargeting the virus to the αv β3 integrin receptors, which are overexpressed in tumor vasculature. This work provides a general chemical approach to introduce various receptor binding agents onto the AAV capsid with site selectivity to generate optimized vectors with engineered infectivity. Next, we used our unique UAA-AAV vector as a tool for the directed evolution of more active UAA incorporation machinery in mammalian cells. It is well known that the efficiency of unnatural amino acid mutagenesis in mammalian cells is limited by the suboptimal activity of the suppressor tRNAs currently in use. The ability to improve their performance through directed evolution can address this limitation, but no suitable selection system was previously available to achieve this. We have developed a novel platform for virus-assisted directed evolution of enhanced suppressor tRNAs (VADER) in live mammalian cells. Our system applies selective pressure for tRNA activity via the nonsense suppression-dependent production of UAA-AAV, and selectivity for the specific incorporation of interest comes from a novel virus purification strategy based on the unique chemistry of the UAA. We demonstrated > 10,000-fold selectivity for active tRNAs out of mock libraries and used this system to evolve libraries generated from the commonly used archaeal pyrrolysyl suppressor tRNA, ultimately identifying a variant which is three times as active as the original tRNA. Finally, we used next-generation sequencing to analyze the fate of every library member over the course of the selection and found that our VADER selection scheme is indeed selective for the enrichment of more active tRNA variants. This work provides a general blueprint for the evolution of better orthogonal suppressor tRNAs in mammalian cells. / Thesis (PhD) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

Thesis advisor: Abhishek Chatterjee / Thesis advisor: Eranthie Weerapana / Over the past twenty years, the site-specific incorporation of unnatural amino acids (UAAs) into a target protein through genetic code expansion (GCE) has emerged as one of the foremost technologies to selectively modify proteins in their native cellular context. This technology relies on engineered aminoacyl-tRNA synthetase (aaRS)/tRNA pairs that are orthogonal to the host cells’ endogenous aaRS/tRNA pairs. Traditionally, scientists look towards evolutionarily distant domains of life to identify orthogonal aaRS/tRNA pairs that can be further engineered for GCE applications in the host system. For example, bacterial aaRS/tRNA pairs are used for GCE in eukaryotes. The directed evolution of orthogonal aaRS/tRNA pairs for eukaryotic GCE has been less fortuitous due to the cumbersome nature of established yeast-based selection platforms. Recently, our lab circumvented this platform-based limitation by developing “altered translational machinery” (ATM) Escherichia coli strains that enabled the directed evolution of bacterial aaRS/tRNA pairs for eukaryotic GCE applications. In the ATM-tyrosyl (ATMY) E. coli strain, reintroduction of the E. coli tyrosyl-tRNA (tRNAEcTyrCUA) as a nonsense suppressor led to cross-reactivity with the endogenous E. coli glutaminyl-tRNA synthetase (EcGlnRS), restricting the activity range of aaRSs that could be selected, ultimately diminishing the scope of incorporable UAAs. To recover the dynamic range of this platform, cross-reactivity of the tRNAEcTyrCUA was eliminated through directed evolution of the tRNA acceptor stem. This new, orthogonal tRNA revealed weak mutant aaRSs whose suppression efficiencies were boosted through additional rounds of directed evolution. Improved aaRS mutants exhibited higher solubility, thermal stability, and suppression efficiency than their predecessor. While the newly engineered, orthogonal tRNAEcTyrCUA gave access to novel aaRS/tRNA pairs for eukaryotic GCE, some notable UAAs were still missing that could be incorporated with the archaeal Methanococcus jannaschii tyrosyl-tRNA synthetase (MjTyrRS)/tRNA pair in bacteria. Following a systematic investigation into the discrepancy between the E. coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNA and MjTyrRS/tRNA pairs, we found that it can be partially attributed to the low structural robustness of the EcTyrRS. This limitation was overcome by rationally designing chimeric TyrRSs composed of EcTyrRS and a structural homologue from the thermophilic bacterium Geobacillus stearothermophilus. The chimeric scaffolds demonstrated enhanced stability, activity, and resilience to destabilizing active site mutations, offering a potentially more attractive scaffold for GCE. / Thesis (PhD) — Boston College, 2021. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

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.

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The genetic code of all known organisms is comprised of the 20 proteinogenic amino acids that serve as building blocks on a peptide chain to form a vast array of proteins. Proteins are responsible for virtually every biological process in all organisms; however, the 20 amino acids contain a limited number of functional groups that often leaves much to be desired. The lack of diversity addresses the need to increase the genetic repertoire of living cells to include a variety of amino acids with novel structural, chemical, and physical properties not found in the common 20 amino acids. In order to expand the chemical scope of the genetic code beyond the functionalities that can be directly genetically encoded, unnatural amino acids must be added to the proteome. The ability to incorporate unnatural amino acids (UAAs) into proteins at defined sites has a direct impact on the ability of scientists to study biological processes that are difficult or impossible to address by more classical methods. The UUAs of interest are acylated lysine derivatives (isovaleryl, isobutyryl, and β-hydroxybutyryl) and photocaged histidine. Acylation of histone lysine has been linked to epigenetic regulation of metabolism.1 A means to site-specifically incorporate each acylated lysine derivative would help study the effect of acylated lysine in epigenetic regulation. Likewise, in order to elucidate the role of histidine in specific protein functions, one can replace a critical histidine with a photocaged histidine. Photocaged amino acids are those that possess a photo-cleavable, aromatic caged group. Light-induced protein activation allows for the biological activity of the protein to be spatiotemporally regulated under non-invasive external control.2 The site-specific in vivo incorporation of unnatural amino acids is made possible by amber codon suppression by an orthogonal suppressor aminoacyl-tRNA synthetase (aaRS)/tRNA pair.3 In amber codon suppression the amber stop codon is decoded for an UAA by a suppressor aaRS/tRNA pair. To accept the UAA, the aaRS must be evolved to achieve orthogonal activity with specific UUAs. The pyrrolysyl aaRS/tRNA (PylRS/PylT) pair from M. barkeri and M. mazei was used to construct multiple, large-scale aaRS mutant libraries where critical residues within the active site of PylRS are mutated via site-saturated mutagenesis.4 The libraries were subjected to directed evolution through a series of positive and negative selections to enrich aaRS variants that exclusively bind to acylated lysine derivatives and photocaged histidine as substrates.5 The PylRS selection survivors were screened for UAA activity and identified successful clones underwent a fluorescent activity assay. The active aaRS were used for amber codon suppression to express the respective UAA in ubiquitin and green fluorescent protein constructs.

Catechol 1,2-Dioxygenase from Rhodococcus opacus is a type of intradiol dioxygenase enzyme that catalyzes the conversion of catechol to cis, cis muconic acid. This enzymatic conversion has the potential to be useful in a number of different applications such as treating wastewater contaminated with aromatic compounds to creating a greener method to produce cis, cis muconic acid which can be used to make a number of industrially important base chemicals. However, for enzymes to be used in industrial conditions, they must be highly stable. The experimental chapters in this thesis explore whether this enzyme can be stabilized to meet industrial requirements while minimizing any loss in catalytic activity. Through the studies described in Chapter 2, a mutant enzyme was generated through disulfide bond engineering with significantly improved thermostability. However overall catalytic activity was reduced. Toward addressing this loss of catalytic activity, in Chapter 3, attempts were made to implement state-of-the-art genetic code expansion strategies to increase catalytic activity of the enzymes. However, these attempts were unsuccessful. Finally, Chapter 4 describes how future stability engineering could be optimized using design pipelines similar to the one developed in this study. Additionally, it describes possible additional optimizations toward making the application of these enzymes cost effective in the near future.

Over the past years, small RNAs (smRNAs) have been identified as important molecular regulators of gene expression and specifically eukaryotic messenger RNAs (mRNAs). Small RNAs including small-interfering RNAs (siRNAs) and microRNAs (miRNAs) take part in the RNA silencing pathway and regulate various pathways in the cell including transcription, genome integrity, chromatin structure, mRNA stability, and translation. siRNAs are usually from exogenously derived molecules, while miRNAs are expressed endogenously by the genome. The RNA silencing pathway is highly conserved between organisms and plays a critical part in maintaining homeostasis, host-pathogen interaction, and disease progression. Thus, a better understanding of the RNA silencing pathway and probing of the molecules involved in the process is instrumental in developing tools that can better regulate the expression of specific genes. The viral suppressor of RNA silencing (VSRS) p19, is a 19 kDa protein that is expressed by tombusviruses and exhibits the highest reported affinity to small RNAs, including siRNA and miRNA. Further engineering of this protein acts as an interesting means to control the RNA silencing pathway and provides a platform to design novel tools to further modulate the activity of smRNAs in living systems. The ability to incorporate new and useful chemical functionality into proteins within living organisms has been greatly enhanced by technologies that expand the genetic code. These usually involve bioorthogonal transfer RNA (tRNA) /aminoacyl-tRNA synthetase (aaRS) pairs that can selectively incorporate an unnatural amino acid (UAA) site specifically into ribosomally synthesized proteins. Site-specificity is coded for by using a rare codon such as the amber stop codon. In Chapter 2, we demonstrate the engineering of p19 for the development of a Förster resonance energy transfer (FRET) reporter system for the visualization of RNA delivery and release in cells using UAAs and bioorthogonal click chemistry, which was done by incorporating azidophenylalanine (AzF). In Chapter 3, by incorporating UAAs into p19’s binding pocket, we were able to enhance its smRNA suppressing activity by covalently trapping the bound substrates. We have demonstrated the engineering of a molecular switch that contains photo-crosslinking groups that covalently trap smRNAs. In Chapter 4, incorporating a metal-ion chelating UAA (2,2′-bipyridin-5-yl) alanine (BpyAla) into p19’s binding pocket has successfully led to site-specific cleavage of small RNAs including siRNAs and endogenous miRNAs. The genetic introduction of BpyAla provides a unique method of introducing catalytic activity into proteins of interest. The developed unnatural enzyme provides a new tool for catalytic suppression of the RNA silencing pathway. These results demonstrate the power of adding new chemistries to proteins using UAAs to achieve possible, diverse applications in therapy and biotechnology.

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