Hepatitis C virus (HCV) infects an estimated 170 million people worldwide and is a major cause of chronic hepatitis and hepatocellular carcinoma. As there are limited treatment options, the elucidation of novel host-viral interactions during HCV pathogenesis will be critical for the development of new therapeutics. My thesis work has identified cell death-inducing DFF45-like effector B (CIDEB) as a host factor that is disregulated during HCV infection, and has delineated the relevance of CIDEB’s dual roles in apoptosis and lipid metabolism in the context of the HCV lifecycle. Moreover, additional host factors necessary for the HCV lifecycle were investigated using unnatural amino acid (UAA) technology. With this technique, the photo-cross-linking UAA p-azido-phenlyalanine (AZF) and 3’-azibutyl-N-carbamoyl-lysine (Abk) were incorporated into viral proteins by expanding the genetic code of the host organism. This conferred diverse physicochemical and biological properties to these proteins that were exploited to investigate protein structure and function in vitro and in vivo. In summary, gaining insight into the numerous host-viral interactions that take place during HCV infection will both advance our understanding of HCV pathogenesis and uncover potential therapeutic targets.

Thesis advisor: Eranthie Weerapana / Adeno-Associated Virus (AAV) has been developed into a powerful therapeutic tool - in the last ten years it has acted as a gene-delivery vehicle in several approved therapeutics and many more therapeutics on trial. Despite extensive research, gaps in our understanding of AAV’s infectious cycle still exist, and further development is needed for the creation of improved gene therapy vectors. Technology to incorporate Unnatural Amino Acids (UAAs) into the AAV capsid has recently been developed, and could aid in both furthering our understanding of AAV’s biology and in the therapeutic advancement of AAV. In this work, we demonstrate how the functionalization of the AAV capsid using UAA incorporation can advance our control over the AAV capsid and aid in probing and manipulating AAV biology. We describe our use UAA incorporation to place a bio-orthogonal reactive handle into AAV’s capsid followed by functionalization with a targeting moiety and demonstrate the unprecedented amount of control that UAA incorporation provides in the creation of a functional virus conjugate. We are able to control both the precise placement and the stoichiometry of the targeting moiety on the AAV capsid, providing a platform that, for the first time, can undergo rigorous optimization analogous to that which medicinal chemists put small molecules through. We also describe the creation of a new platform to site-specifically modify the AAV capsid using cysteine incorporation, a technique that retains the ability to site-specifically modify the capsid as UAA incorporation does, but does not require the excess machinery that UAA incorporation requires. Next we discuss the incorporation of a photocaging amino acid, NBK, into the AAV capsid. Using NBK, we were able to effectively block AAV’s primary binding interaction with Heparan Sulfate Proteoglycan (HSPG) and control the timing of AAV infection using light to chemically remove the photo-protecting group. While photocaging the HSPG interaction is only a proof of concept, it demonstrates the remarkable amount of control that UAA incorporation affords, and lends insight to what could be accomplished using the functionalities that can be placed on the AAV capsid with UAAs. / Thesis (PhD) — Boston College, 2020. / 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.

Genetic code expansion allows the incorporation of non-canonical amino acids with a variety of new functional groups: fluorescent amino acids,1-3 azides,4-6 alkynes,5-10 and photocrosslinkers.4,11,12 This incorporation requires the evolution of new tRNA/aminoacyl tRNA sythetase pairs. Traditionally screenings of novel tRNA/aminoacyl tRNA synthetase pairs have been done in vivo. While these in vivo screenings have proven robust, they are limited in multiple ways: non-canonical amino acids (ncAAs) must be nontoxic and bioavailable. Furthermore, library size is limited by transformation efficiency. Lastly, in vivo screenings require substantial amounts of the target ncAA, which is often not available in large masses. In vitro screenings bypass these limitations: toxicity and bioavailibilty are no longer concerns. Library size can be expanded by several orders of magnitude as we are no longer limited by transformation efficiency. Lastly, because in vitro transcription/translation reactions are routinely conducted on the μL scale, ncAA usage can be minimized. We set out to use in vitro compartmentalization to further expand the code. In an in vitro compartmentalization screening, the water droplets in a water-in-oil emulsion serve as separate reaction chambers in which individual library members are transcribed and translated. Here we report optimization of S30 transcription/translation reactions. Optimizations include cell lysis method, reaction temperature, template amount, and T7 RNA polymerase amounts. Yields remained low and we transistioned into the use of PURExpress. Fluorohistidines are isosteric with histidine, but not isoelectronic.13 This change in environment results in a reduction of pKa. We set out to synthesize 4-fluorohistidine to use as a pH probe in several target proteins. A synthesis of 4-fluorohistidine was published in 1973.14,15 We were able to improve upon this synthesis by reducing cost and improving yield of a key step in the reaction. Next, small peptides with polyhistidine tags were translated in vitro using our 4-fluorohistidine. We are calling this polyhistidine tag incorporating 4-fluorohistidine our “hexafluorohistag.” Because of the reduced pKa of the 4-fluorohistidine, the hexafluorohistag showed affinity to Nickel-NTA resin even at reduced pH. This allowed for the purification of hexafluorohistagged peptides in the presence of traditional polyhistidine-tagged peptides.

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.

Mn-Mediated radical addition has been developed within the Friestad laboratory as a versatile method toward addition to C=N bonds. N-Acylhydrazones generated by condensation between an aldehyde and an N-acylamine serves as the substrate toward radical addition. A bulky directed group attached with the N-acyl moiety and restricted rotation around N-N bond due to a three point chelation with a Lewis acid differentiates the faces of the C=N bond of the N-acylhydrazones. Radical generation initiated by photolysis of Mn2(CO)10 causing homolysis of C-X bond in alkyl halide serves as the radical donor to the N-acylhydrazones. Radical addition thereafter occurs stereoselectively from the less hindered face of the C=N bond of the N-acylhydrazones. The product N-acylhydrazines can be effectively transformed to α-chiral amines. In this thesis, a new protocol toward generation of non-proteogenic γ-amino esters using Mn-mediated radical addition has been described. Moreover, the utility of the Mn-mediated radical addition has been demonstrated through studies toward synthesis of tubulysin U and V. Chapter 3 describes a new strategy for asymmetric synthesis of γ-amino esters starting from non-amino precursors. The α-substituted γ-amino esters are prevalent in drugs, drug candidates, and in peptidomimetics. As a part of progressing the Mn-mediated radical addition reaction, highly stereoselective reactions were devised for addition to N-acylhydrazonoesters in absence of Lewis acid. Spectroscopic investigations were carried out to decipher the Lewis acid chelation of N-acylhydrazones. Finally, a novel microwave mediated trifluoroacylation of N-acylhydrazinoesters facilitated the cleavage of N-N bond to liberate γ-aminoester. Chapter 4 describes application of Mn-mediated radical addition toward synthesis of tubulysin natural products. Tubulysins are natural products, isolated from myxobacteria, that have exhibited potent cytotoxicity toward cancer cells in the picomolar regime. The Mn-mediated radical addition was used to prepare two chiral amine subunits in highly diastereoselective fashion. The subunits were then assembled after required manipulations into the tetrapeptide structure characteristic of tubulysins. This strategy to synthesize tubulysins is the most stereoselective of all efforts toward the synthesis of this molecule. Synthesis toward tubulysin was achieved in 18 steps as the longest linear sequence with a 31% overall yield to tubulysin V in benzyl protected form. Chapter 5 describes a new strategy toward installation of N-hydroxymethyl unit into a peptide chain. N,O-Acetals are acid-base labile species that is present in some tubulysin natural analogs. This new approach exploits Fleming-Tamao oxidation and hence introduce the hydroxymethyl unit of the N,O-acetal in a masked form. Following peptide construction the masked hydroxy group is released to liberate the N-hydroxymethyl moiety. Acylation of the free hydroxy group furnishes the N,O-acetal moiety in a strategy that is potentially applicable toward synthesis of tubulysin D.

There are multiple mechanisms by which DNA can become damaged. Such damage must be repaired for the cell to avoid ill-health consequences. Homologous recombination (HR) is a means of repairing one specific type of damage, a double-strand break (DSB). This complex pathway includes the Rad51-DNA nucleoprotein filament as its primary machinery. Current methodology for studying HR proteins includes the use of fluorescently labeled DNA to probe for HR dynamics. This technique limits the number of proteins that can be involved in experimentation, and often only works as an end reporter. The work here aims at improving upon standard techniques by creating two fluorescent protein probes. The first probe was developed by directly attaching a fluorophore to Saccharomyces cerevisiae Rad51 with the use of click chemistry and the incorporation of unnatural amino acids. This probe could function as a primary reporter on the formation and dissociation of the Rad51-DNA filament in the presence of pro- and anti- HR mediator proteins. The second probe was created by labeling the exterior cysteine residues of Plasmodium falciparum single strand DNA binding protein (SSB) with a fluorophore via maleimide chemistry. This probe acts as a secondary reporter for HR dynamics by signaling for when free single stranded DNA (ssDNA) is available.

This project report analyzes the emergence of categories of sexual deviancy as they appear in selected medical treatises from the eighteenth century. Terms such as homosexual or lesbian were not yet available in medical or public discourse but the early modern writers did use a variety of other references to establish the existence of such categories. For instance, one might label deviants as hermaphrodites, eunuchs, sodomites, or monsters to describe what were perceived as unnatural forms of sexual expression which ostensibly posed a threat to the social order largely because they were not procreative, but also because of the fear that they might produce children of the same ilk. Moreover, the sudden explosion in scientific and medical knowledge during the Enlightenment created a need for the organization and classification of such knowledge, as well as a fascination with anomalies and how they might be cured. My argument is that four of these deviant categoriesthe chronic masturbator, tribades or hermaphrodites, mollies (or effeminate male homosexuals), and eunuchswere considered unnatural because they fell outside normative prescriptions of acceptable sexual conduct that was based primarily on pro-natal and pro-nutpial ideologies. I rely on experts in eighteenth-century scholarship, such as Rictor Norton, Randolph Trumbach, Thomas Laqueur, Robert Darby, Thomas A. King, and George Rousseau, to inform my discussion of writings from this period. Although contemporary scholars in this field have made significant contributions to our knowledge of early modern understandings of sexual deviancy, relatively few of them seem to have investigated how medical treatises on generation provided a scientific basis for the marginalization of specific types of people. By identifying these types under the larger category of generation, I argue that these medical texts and popular sexologies function as vehicles of social control by emphasizing that the only legitimate form of sexual expression was within the context of marriage and that its sole purpose was for reproduction.

This project report analyzes the emergence of categories of sexual deviancy as they appear in selected medical treatises from the eighteenth century. Terms such as homosexual or lesbian were not yet available in medical or public discourse but the early modern writers did use a variety of other references to establish the existence of such categories. For instance, one might label deviants as hermaphrodites, eunuchs, sodomites, or monsters to describe what were perceived as unnatural forms of sexual expression which ostensibly posed a threat to the social order largely because they were not procreative, but also because of the fear that they might produce children of the same ilk. Moreover, the sudden explosion in scientific and medical knowledge during the Enlightenment created a need for the organization and classification of such knowledge, as well as a fascination with anomalies and how they might be cured. My argument is that four of these deviant categoriesthe chronic masturbator, tribades or hermaphrodites, mollies (or effeminate male homosexuals), and eunuchswere considered unnatural because they fell outside normative prescriptions of acceptable sexual conduct that was based primarily on pro-natal and pro-nutpial ideologies. I rely on experts in eighteenth-century scholarship, such as Rictor Norton, Randolph Trumbach, Thomas Laqueur, Robert Darby, Thomas A. King, and George Rousseau, to inform my discussion of writings from this period. Although contemporary scholars in this field have made significant contributions to our knowledge of early modern understandings of sexual deviancy, relatively few of them seem to have investigated how medical treatises on generation provided a scientific basis for the marginalization of specific types of people. By identifying these types under the larger category of generation, I argue that these medical texts and popular sexologies function as vehicles of social control by emphasizing that the only legitimate form of sexual expression was within the context of marriage and that its sole purpose was for reproduction.

The field of protein engineering has been greatly augmented by the expansion of the genetic code using unnatural amino acids as well as the development of cell-free synthesis systems with high protein yield. Cell-free synthesis systems have improved considerably since they were first described almost 40 years ago. Residue specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an amino acid depleted cell-free protein synthesis system that can be used to study residue specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines high protein expression yields with a high level of analog substitution in the target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid-depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo format. We use this amino acid depleted cell-free synthesis system for the directed evolution of streptavidin, a protein that finds wide application in molecular biology and biotechnology. We evolve streptavidin using in vitro compartmentalization in emulsions to bind to desthiobiotin and find, at the conclusion of our experiment, that our evolved streptavidin variants are capable of binding to both biotin and desthiobiotin equally well. We also discover a set of mutations for streptavidin that are potentially powerful stabilizing mutations that we believe will be of great use to the greater research community. / text