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Synthesis and Characterization of Biomimetic Compartments
Full text linkThis dissertation will first explore methods for the structural characterization of artificial phospholipid membrane assemblies. We employed confocal microscopy, negative stain transmission electron microscopy (TEM), cryo-electron microscopy (cryo-EM), and small angle x-ray scattering (SAXS) to analyze the structure of two artificial lipid systems from the millimeter to atomic scale. These biophysical techniques reveal their necessity as a general toolkit for the structural characterization of artificial lipid systems in a range of spatial resolutions.Next, this dissertation will discuss the biochemical and biophysical characterization of lipid sponge droplets that have potential application as an artificial organelle. The spatial organization afforded by organelles can possibly expand the functions of synthetic reaction systems, especially within artificial cells. Here, we describe highly stable nonlamellar, galactolipid-based sponge phase droplets as programmable synthetic organelles. We use various biochemical and biophysical techniques to structurally characterize the dense network of lipid bilayers and nanometric aqueous channels of the sponge phase. In the third section of this dissertation, a novel approach for the semisynthesis and reconstitution of transmembrane (TM) proteins in giant unilamellar vesicles (GUVs) will be presented. Herein, we test an alternative solution to detergent-based TM protein reconstitution strategies involving the in vitro assembly of TM proteins from synthetic TM domains and expressed soluble domains using chemoselective peptide ligation. We developed an intein mediated ligation strategy to semisynthesize single-pass TM proteins in synthetic GUV membranes by covalently attaching soluble protein domains to a synthetic TM polypeptide, avoiding the requirement for detergent. Finally, this dissertation will discuss the total synthesis of protein-based compartments as drug delivery vehicles. Common vehicles are empty viral capsids and bacterial microcompartments comprised of protein subunits. Usually, these compartments are expressed in E. coli, purified, disassembled, and reassembled with a cargo (e.g. a therapeutic) of interest. The total synthesis of protein-based compartments would increase the versatility of the protein sequence and enable the incorporation of noncanonical amino acids, fluorescent or radioactive labels, and chemical tags. Herein, we describe the total synthesis and characterization of the 129-mer bacteriophage MS2 coat protein for its future application as a versatile and robust drug delivery vehicle
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Enzymatic Methods for the Study of RNA in Mammalian Cells
Full text linkTechnologies for the labelling, detection, and manipulation of biomolecules have drastically improved our understanding of cell biology. As the myriad of functional roles for RNA in the cell are increasingly recognized, such tools to enable further investigation of RNA are the subject of much interest.We demonstrate the site-specific incorporation of nucleobase derivatives bearing fluorophores or affinity labels into a short RNA stem loop recognition motif by exchange of a guanine residue. The RNA-TAG (transglycosylation at guanosine) is carried out by a bacterial (E. coli) tRNA guanine transglycosylase (TGT), whose natural substrate is the nitrogenous base PreQ1. Remarkably, we have successfully incorporated large functional groups including biotin, BODIPY, thiazole orange, and Cy7 through a linker attached to the exocyclic amine of PreQ1. Larger RNAs, such as mRNA transcripts, can be site-specifically labeled if they possess the 17-nucleotide hairpin recognition motif. The RNA-TAG methodology could facilitate the detection and manipulation of RNA molecules by enabling the direct incorporation of functional artificial nucleobases using a simple hairpin recognition element.Leveraging the ability to site-specifically and covalently label an RNA of interest using E. Coli TGT and unnatural nucleobase substrate, we establish the identification of RNA-protein interactions and the selective enrichment of cellular RNA in mammalian systems. We demonstrate the utility of this approach through the identification of known binding partners of 7SK snRNA via mass spectrometry. Through a minimal 4-nucleotide mutation of the long noncoding RNA HOTAIR, enzymatic biotinylation enables identification putative HOTAIR binding partners in MCF7 breast cancer cells that suggest new potential pathways for oncogenic function. Furthermore, using RNA sequencing and qPCR, we establish that an engineered enzyme variant achieves high levels of labeling selectivity against the human transcriptome allowing for 145-fold enrichment of cellular RNA directly from mammalian cell lysates.Finally, we examine the use of RNA-TAG labeling in live cells, exploring design principles of the nucleobase substrate for use in applications in RNA imaging and beyond. The flexibility and breadth of this approach suggests that this system could be routinely applied to the functional characterization of RNA, greatly expanding the toolbox available for studying mammalian RNA biology
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Use of Enzymes for Amplified Signal Generation
Full text linkThis 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
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Utilization of Bioorthogonal Chemistry for Live-Cell Labeling of Glycoconjugates and the Liposomal-Based Engineering of Dynamic Artificial Cellular Models
Full text linkThis 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
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Self-Assembling Lipopeptide Nanocarriers for Targeted Cellular Uptake
Full text linkSelf-assembling lipopeptide nanocarriers have emerged as one of the most powerful delivery vehicles, providing unique opportunities to transport therapeutic drugs. In efforts to improve target specificity, such carriers are functionalized with integrin-binding motifs (such as RGD peptide sequences), enhancing cellular uptake via ligand-receptor interactions. However, common approaches for the preparation of the desired structures require complex synthetic routes to covalently conjugate the peptide ligands to the lipid fragments. It would be thus valuable to develop a straightforward methodology to construct biocompatible vehicles that can efficiently deliver functional cargos to live cells. Herein, we describe a new class of nanocarrier systems constituted by self-assembling lipopeptides conjugated with specific integrin-binding motifs. The approach takes advantage of chemoselective and non-enzymatic methods to synthesize functional lipopeptides, which spontaneously self-assemble into micron-sized vesicles. The straightforward construction of our model systems, jointly with their robustness, biocompatibility and simplicity, highlights their relevant use as carriers for the enhanced cellular uptake of fluorescently labeled biomacromolecules (Texas Red-Dextran) and therapeutic agents (doxorubicin) via receptor-mediated endocytosis pathway
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Chemical and Enzymatic Methods to Regulate Phospholipid Membrane Formation and Manipulate Cellular mRNA
Full text linkThis 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
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Development of hybrid lipid membranes: From construction to application of novel lipid-based artificial compartments
Full text linkMembrane-forming phospholipids provide essential functions to living organisms, rendering them as one of the ideal candidates to explore the evolutionary origins of compartmentalization. However, current model systems to generate membranes, have been mainly focused on working with long chain amphiphiles containing phospho-head groups, which require a biochemically complex machinery unlikely to have been achieved in the early stages of life. Therefore, it highlights the significance of novel systems of membrane synthesis prior to the development of integral membrane proteins in early protocells. To better understand the plausible mechanisms of early lipid synthesis and membrane compartmentalization, strategies of in situ formation membranes may be developed using simple molecular building blocks, such as soluble proteins, simple peptides, and short acyl chains. This summary includes construction of amphiphilic lipids by using a chemoselective reaction to demonstrate how artificial cells may potentially mimic the properties of natural biological membranes and thus, spontaneously create biocompatible membranes
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Applications of Lipid Assemblies in Artificial Cell Development
Full text linkIt 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
Chemoenzymatic Generation of Lipid Membranes
Full text linkThe 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
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