This dissertation will first explore methods to selectively direct proteins to phospholipid membranes. The natural mechanisms that tether proteins to membranes are typically complex, requiring multiple steps and accessory components. It would be advantageous to develop simplified methods to direct proteins of interest to phospholipid membranes in a single step. We have developed a modular method for membrane localization of proteins by using chemically modified phospholipid anchors capable of covalent attachment to O6-methylguanine DNA methyltransferase (SNAP-tag) fusion proteins. We demonstrate that photocaged lipid precursors enable light-triggered spatial and temporal control over protein localization. The anchoring system is compatible with cell-free expression, allowing for genetic targeting of proteins to lipid membranes of giant unilamellar vesicles. This technique can be used to control membrane curvature effects, similar to what has been previously observed with certain membrane-bound proteins. This work addresses a current need in synthetic biology for simplified and robust methods to control membrane localization of expressed proteins and shows promise as a general tool for protein targeting to lipid vesicles and cellular membranes.

In the second investigation, this dissertation will discuss a new method for the nonenzymatic remodeling of lipid membranes. Cell membranes have a vast repertoire of phospholipid species whose structures can be dynamically modified by enzymatic remodeling of acyl chains and polar head groups. Lipid remodeling plays important roles in membrane biology and dysregulation can lead to disease. Although there have been tremendous advances in creating artificial membranes to model the properties of native membranes, a major obstacle has been developing straightforward methods to mimic lipid membrane remodeling. Stable liposomes are typically kinetically trapped and are not prone to exchanging diacylphospholipids. An approach relies on transthioesterification/acyl shift reactions that occur spontaneously and reversibly between tertiary amides and thioesters. We demonstrate exchange and remodeling of both lipid acyl chains and head groups. Using our synthetic model system we demonstrate the ability of spontaneous phospholipid remodeling to trigger changes in vesicle spatial organization, composition, and morphology as well as recruit proteins that can affect vesicle curvature. Membranes capable of chemically exchanging lipid fragments could be used to help further understand the specific roles of lipid structure remodeling in biological membranes.

In the third section of this dissertation, a novel approach for the in situ synthesis of natural lipids in living cells will be presented. Mammalian cells synthesize thousands of distinct lipids, yet the function of many of these lipid species is unknown. Ceramides, a class of sphingolipid, are implicated in several cell-signaling pathways but poor cell permeability and lack of selectivity in endogenous synthesis pathways have hampered direct study of their effects. Here we report a strategy that overcomes the inherent biological limitations of ceramide delivery by chemoselectively ligating lipid precursors in vivo to yield natural ceramides in a traceless manner. Using this method, we uncovered the apoptotic effects of several ceramide species and observed differences in their apoptotic activity based on acyl-chain saturation. Additionally, we demonstrate spatiotemporally controlled ceramide synthesis in live cells through photoinitiated lipid ligation. Our in situ lipid ligation approach addresses the long-standing problem of lipid-specific delivery and enables the direct study of unique ceramide species in live cells.

Finally, this dissertation will discuss the development of small molecules for the depalmitoylation of proteins in vivo. Post-translational S-palmitoylation plays a central role in protein localization, trafficking, stability, aggregation, and cell signaling. Dysregulation of palmitoylation pathways in cells can alter protein function and is the cause of several diseases. Considering the biological and clinical importance of S-palmitoylation, tools for direct, in vivo modulation of this lipid modification would be extremely valuable. Here, we describe a method for the cleavage of native S-palmitoyl groups from proteins in living cells. Using a cell permeable, cysteine-functionalized amphiphile, we demonstrate the direct depalmitoylation of cellular proteins. We show that amphiphile-mediated depalmitoylation (AMD) can effectively cleave S-palmitoyl groups from the native GTPase HRas and successfully depalmitoylate mislocalized proteins in an infantile neuronal ceroid lipofuscinosis (INCL) disease model. AMD enables direct and facile depalmitoylation of proteins in live cells and has potential therapeutic applications for diseases such as INCL, where native protein thioesterase activity is deficient.

Protein S-palmitoylation is an essential post-translational modification that regulates protein structure and function. It consists of the attachment of a palmitic acid to one or more cysteine residues through a thioester linkage. In cellular proteins, S-palmitoylation plays an important role in membrane localization, trafficking, lipid raft association, and cell signaling. Various viral proteins are also modified with S-palmitoylation; however, their functional role differs from that of cellular proteins. One of the main differences is that S-palmitoylation is a reversible process in cellular proteins, which allows for recycling and regulation of protein function, whereas in viral proteins, there is no functional benefit to recycle and reuse proteins after viral entry and infection has occurred. The unique nature of S-palmitoylation on protein function makes it an interesting lipid post-translational modification to further characterize and potentially target for therapeutic purposes. There are not many studies that have focused on S-palmitoylation as a targeted therapeutic approach, which makes it even more intriguing to fully understand its effects on cellular and viral proteins. In this dissertation, I examined the effects of directly targeting S-palmitoylation of the NRAS protein, which is highly mutated in multiple cancers including melanoma, using a novel amphiphile-mediated depalmitoylation approach. I discovered that this chemoselective strategy causes delocalization of NRAS from the plasma membrane, inhibition of downstream oncogenic signaling pathways in NRAS-mutated melanoma cells, and reduction of tumor growth in NRAS-mutated melanoma xenograft mice. Additionally, I focused on understanding the role of S-palmitoylation on the SARS-CoV-2 spike glycoprotein. I determined the essential sites of palmitoylation within the C-terminal cysteine-rich region as well as the palmitoylation-dependence of membrane fusion between the spike protein and the host ACE2 receptor. Taken together, these studies highlight the significance of S-palmitoylation on cellular proteins, such as the effect on NRAS function and downstream signaling, and viral proteins, specifically the role on SARS-CoV-2 spike protein-mediated virus-cell membrane fusion, that are involved in various diseases.

It is of great fundamental interest to develop experimental model systems to reconstruct design principles underlying formation of living cells and shed light on how life emerged on Earth and possibly elsewhere. An ambitious strategy is the bottom-up approach, which aims to systematically control the assembly of basic building blocks with defined functionality to construct a life-like entity. This dissertation will explore the roles of miscellaneous lipid assemblies as building blocks of artificial cellular compartments.

The first part (Chapters 2 and 3) of this dissertation shows how lipid environments facilitate chemoselective reactions between amphiphilic partners through physical partitioning. Through examples of histidine ligation, and acyl phosphate chemistry we show functionalized micelle-forming amphiphiles react to form amidophospholipids which self-assemble into micron-sized vesicles.

The second part (Chapter 3) of the dissertation seeks to understand the origins of cellular phospholipid synthesis pathways by repurposing soluble fatty acid activating enzymes (fatty acyl adenylate ligase and fatty acyl CoA ligase) to synthesize phospholipids. Such simplified biochemical pathways may provide a hint at how lipids were likely synthesized in a minimal protocell without the necessity of transmembrane protein enzymes.

The third part (Chapters 4 and 5) of the dissertation discusses the vesicle formation from a novel class of single-chain amphiphiles derived from galactopyranosyl head groups and unsaturated fatty acid tails. The geometric parameters and thermodynamic properties of these amphiphilic assemblies are characterized by a host of physical techniques. The vesicles are further shown to sustain model biochemical reactions.

The final part (Chapters 6) of the dissertation describes the formation of sponge phase droplets from fatty acyl galactopyranosylamides and non-ionic detergents. The droplets contain a dense bicontinuous network of bilayers and nanometric aqueous channels, which facilitates molecules to partition into them based on their size, polarity, and specific binding motifs. The sequestration of biomolecules can be programmed by doping the droplets with suitably functionalized amphiphiles. The droplets can harbor functional soluble and transmembrane proteins, allowing for the co-localization and concentration of enzymes and substrates to enhance reaction rates. Droplets protect bound proteins from proteases, and these interactions can be engineered to be reversible and optically controlled.

The bottom-up generation of lipid membranes from minimal precursors is a key objective in synthetic biology research. In living cells, the synthesis of lipid assemblies is ubiquitously coordinated by several membrane-bound enzymes. However, these pathways are tough to replicate, owing to the difficulty of reconstituting the enzymes in vitro. Lipid membrane synthesis from simple metabolic building blocks remains challenging. We demonstrate a chemoenzymatic schematic for lipid membrane generation, utilizing a bacterial fatty acid synthase (cgFAS I) to synthesize palmitoyl-CoA in situ from acetyl-CoA and malonyl-CoA as precursors. Palmitoyl-CoA spontaneously reacts with a cysteine-modified lysophospholipid via native chemical ligation (NCL), generating noncanonical amidophospholipids that self-assemble into micron-sized membranes. The results demonstrate that combining the specificity and efficiency of a type I fatty acid synthase with a selective bioconjugation reaction provides a biomimetic route for the de novo formation of membrane-bound vesicles. Utilizing this route, we further investigate the application of non-amphiphilic, minimal precursor pathway to generate lipid membranes. We explore the use of acetate as the carbon feedstock for the chemoenzymatic reaction strategy, generation palmitoyl-CoA in situ and coupling it with small molecule head group such as cysteine to form phospholipid analogs. Ubiquitous enzymes acetyl-CoA synthetase (ACS) from E. coli was used to generate acetyl-CoA from acetate and human acetyl-CoA carboxylase (ACC) was used to then synthesize malonyl-CoA from acetyl-CoA. Additionally, cgFAS catalyzed the in situ generation of palmitoyl-CoA from acetyl-CoA and malonyl-CoA. Finally, we explore the formation of a unique analog of sphingolipid membranes from water-soluble precursors that have markedly different biophysical properties to their natural counterparts. We show that numerous transition metal ions, particularly Cu(II), catalyze the selective O-acylation of the biologically occurring single-chain amphiphile sphingosylphosphorylcholine using fatty acyl phosphates or thioesters as acyl donors under mild aqueous conditions. This work demonstrates the value of de novo membrane generation strategies starting from minimal, water-soluble precursors. Utilizing such approaches, we can control both the chemical structures of the lipid analogs as well as their biophysical properties in aqueous media. This effort contributes towards understanding the fundamental requirements for bottom-up generation of lipid membranes, providing alternative strategies to those previously shown.

This dissertation will explore two different methods of generating enzymatically-amplified signals in response to cell surface features. The first method uses a variation on traditional catalyzed reporter deposition employing the enzyme laccase to covalently deposit a fluorescent ferulic acid derivative. This enzyme has distinct properties that make it more suitable for use with catalyzed reporter deposition in living systems. The second portion of this thesis focuses on the development of xanthine methylating enzymes as a reporter protein. It demonstrates how synthetic juxtacrine signaling receptors can be used to induce expression of enzymes capable of methylating a variety of xanthines to produce a detectable, biocompatible small molecule reporter. This approach required heterologous expression of many xanthine methyltransferase enzymes in human cells for the first time so that their properties could be evaluated. In addition to being potentially useful as a reporter protein, the use of xanthine methylating enzymes in mammalian cells has potential connections to synthetic biology and the development of synthetic paracrine signaling pathways using small molecules.

This dissertation will first explore the use of methylcyclopropene as an activated dienophile in the [4 + 2] inverse Diels–Alder cycloaddition with a tetrazine coupling partner for applications in bioimaging. Recently, bioorthogonal chemistries to label and track biomolecules in their native environment has received considerable interest from biochemists and chemical biologists. The tetrazine ligation is one such example, exhibiting robust kinetics and is mutually exclusive to other bioorthogonal reactions

making it feasible to carryout multiple-color labeling experiments. However, the classical coupling partners of tetrazine are bulky dienophiles, like norbornene and trans-cyclooctene. This potentially limits live-cell applications requiring sterically small labeling probes. In contrast, methylcyclopropene is the smallest cyclic alkene and as a tetrazine coupling partner it provides minimal steric impact that is often desired in intracellular investigations. In addition to the fast kinetics (k 13 M−1s−1), fluorophore conjugated tetrazines can also exhibit a fluorogenic “turn-on” upon cycloaddition with methylcyclopropene, making them well suited for live-cell imaging probes.

In the second investigation, this dissertation will explore two fundamental features of a phospholipid bilayer; their ability to encapsulate macromolecules and reconstitute transmembrane proteins. Phospholipid liposomes are akin to micron-sized flasks that can function as a delivery system and/or a bioreactor. In both of these applications high encapsulation efficiency is greatly desired or even necessary, but there are few liposomal methodologies that can achieve this. In order to integrate genetic circuits in liposomes we employed the inverted emulsion technique to make giant unilamellar vesicles that can be visualized by light microscopy. In addition, this method achieves greater than 90% encapsulation efficiency for polar macromolecules. Building off this technique we show it is possible to encapsulate live bacteria and yeast at high densities, which was previously only possible via microfluidics.

An alternative methodology of encapsulation can be accomplished with synthetic lipids that are composed of two clickable precursors, comprising of an alkyl chain and a lysophospholipid. Initial we demonstrated how this could work between an oleoyl azide and an alkyne lysophospholipid to form a triazole phospholipid, but due to the low solubility of the azide oil in aqueous solutions we pioneered vesicle formation by native chemical ligation (NCL). In this system both precursors are water soluble allowing for higher encapsulation efficiency and similarly to the Cu(I)-catalyzed azide–alkyne cycloaddition, the NCL system can also spontaneously reconstitute active transmembrane proteins during membrane growth.

This dissertation first explores the use of photo-activated thiol-yne click chemistry methodology to gain access to phospho- and glycolipid analogs. Phospholipids and glycolipids constitute an essential part of biological membranes, and are of tremendous fundamental and practical interest. Unfortunately, the preparation of functional phospholipids, or synthetic analogs, is often synthetically challenging. We utilized thiol-yne click chemistry methodology to assemble the alkynyl hydrophilic head groups with numerous thiol modified lipid tails to yield the appropriate dithioether phospho- or glycolipids. The resulting structures closely resemble the structure and function of native diacylglycerolipids. Dithioether phosphatidylcholines (PCs) are suitable for forming giant unilamellar vesicles (GUV), which can be used as vessels for cell-free expression systems. The unnatural thioether linkages render the lipids resistant to phospholipase A2 hydrolysis. We utilized the improved stability of these lipids to control the shrinkage of GUVs composed of a mixture of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and dioleyl-dithioether PC, and concentrating encapsulated nanoparticles. We imagine that these readily accessible lipids could find a number of applications as natural lipid substitutes.

In the second investigation, this dissertation reports the development of a wash-free cellular mRNA imaging technology utilizing RNA-TAG (transglycosylation at guanine) methodology. The many roles of RNA in cellular regulation and function increasingly demand for tools to explore RNA tracking and localization within cells. Our recently reported RNA-TAG approach uses an RNA-modifying enzyme, tRNA-guanine transglycosylase, to accomplish covalent labeling of an RNA of interest with fluorescent tracking agents in a highly selective and efficient manner. Unfortunately, labeling by this method suffers from a high nonspecific fluorescent background and is unsuitable for imaging RNA within complex cellular environments. We designed and synthesized novel fluorogenic thiazole orange probes that significantly lower nonspecific binding and background fluorescence and, as a result, provide up to a 100-fold fluorescence intensity increase after labeling. Using these fluorogenic labeling agents, we were able to image mRNA expressed in Chinese Hamster Ovary cells in a wash-free manner.

RNA-TAG methodology was also applied to the modification of therapeutically relevant modified mRNA (mod-mRNA). Mod-mRNA has recently been widely studied as the form of RNA useful for therapeutic applications due to its high stability and lowered immune response. As a proof of concept, we covalently attached a fluorescent probe to mCherry encoding mod-mRNA transcripts bearing 5-methylcytidine and/or pseudouridine substitutions with high labeling efficiencies. To provide a versatile labeling methodology with a wide range of possible applications, we employed a two-step strategy for functionalization of the mod-mRNA to highlight the therapeutic potential of this new methodology. We envision that this novel and facile labeling methodology of mod-RNA will have great potential in decorating both coding and noncoding therapeutic RNAs with a variety of diagnostic and functional moieties.

The importance of nucleic acids has expended past their role in encoding life and into a space of scientific utility and discovery. Developing new, robust methods to study and manipulate them in their natural environments and harness their programmability for fundamental research tools and biomedical applications has become a critical goal for researchers. This work presents an enzymatic tool for functional modification of single stranded DNA oligonucleotides as well a strategy to assemble functional nucleic acid protein conjugates.

Due to its greater stability and ease of synthesis, DNA, rather than RNA, is the nucleic acid of choice for many functional applications. Often, these applications require the DNA oligonucleotide to be modified in some way with an affinity tag or reporter. There are a limited number of methods to label DNA oligonucleotides, all of which have their shortcomings, including being cost prohibitive, inefficient, or a limited small molecule scope. In this dissertation I develop an enzymatic single stranded DNA modification method, expanding on the powerful RNA transglycosylation at guanine (RNA-TAG) modification tool developed in our lab. DNA-TAG offers researchers with an unprecedented tool to generate modified oligonucleotides in their lab. This work describes the rational and iterative design of DNA-TAG and demonstrates its utility through fluorescent northern blot and RNA fluorescent in situ hybridization (FISH).

Combining the programmability of nucleic acids with the functions of proteins has the potential to enable powerful assemblies with unique and critical functions, both natural and synthetic. Developing novel strategies to bring these classes of biomolecules together in a robust and specific manner continues to facilitate new applications. Here I present a fully enzymatic method to covalently assemble a functional RNA-Protein conjugate using a novel bifunctional small molecule probe and demonstrate he utility of the system by recruitment of an endonuclease to an RNA of interest to induce degradation.

With the herein presented advancements, we expand the application space of nucleic acid modification. Nucleic acid transglycosylation at guanine (NA-TAG) technologies provide researchers with a single tool capable of modifying nearly any single stranded nucleic acid substrate with a small molecule of their choice for downstream applications.