Synthetic organic chemistry studies toward designer lipids and aptamer ligand systems with controlled functionalities
Creators: name: Gutierrez, Bryan
Publisher: name: No Publisher Supplied
Year: 2023

description: This thesis describes two areas of research: the design of lipid-based sensors and biomimetic protocells; and the implementation of activity-based aptamer systems using modified small molecule ligands which target specific stimuli in diseased cells and bacteria. Liposomal delivery of fluorescent lipid probes is a dynamic and customizable platform that will elucidate the trafficking, localization, and interplay between the major cellular compartments and their complicated biochemical equilibrium. Chapter 1 is comprised of three separate lipid projects 1) Peroxynitrite (ONOO–), a biologically relevant redox-active species generated under nitrosative stress conditions. Notably, ONOO– promotes oxidative damage of lipids. Herein, we report the development and multifaceted utilization of a novel, activity-based phospholipid probe, 1.1, for imaging lipid environments that are targeted by ONOO– in biomimetic and biological systems. Using 1.1 and the natural lipid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, we built synthetic protocell membranes in the form of giant vesicles (GVs). These GVs can respond to ONOO– by lighting up at the membrane and displayed excellent specificity for ONOO– when compared to other redox-active species, introducing an unprecedented function to biomimetic membrane architectures. Live HeLa cells containing 1.1 were investigated under cellular nitrosative stress induced by endogenous stimulation with interferon-y / lipopolysaccharide / phorbol myristate acetate. Both conditions enhanced the coumarin fluorescence within cells, providing insight into intracellular lipid environments potentially prone to peroxynitrite-mediated oxidative damage. This work offers a chemical reactivity-dependent function for synthetic vesicles and opens a possibility for investigating nitrosative stress at subcellular levels. 2) Similar to 1), but here two novel activity-based phospholipids 1.12 and 1.14 were synthesized for sensing hydrogen sulfide and hydrogen peroxide. Lastly, 3) is a short synopsis of a collaboration involving the regulatory pathway of phosphatidate (PA) phosphatase. PA is an enzyme involving the conversion of diacylglycerol to triacylglycerol and Cytidine diphosphate (CDP)-diacylglycerol for lipid synthesis. PA is regulated by kinase-mediated phosphorylation and phosphatase complex Nem1-Spo7 for dephosphorylation on the membrane of the endoplasmic reticulum. Here, our collaborator developed a proteoliposome model system that mimics the Nem1-Spo7 phosphatase activity similar to that in cells for studying PA regulation. For chapter 2, I will introduce a fundamental concept for functional aptamer-ligand pairs that enable activity-based sensing, which can easily be configured to sense a new biomarker, thus eliminating the need for in vitro selection. This system will allow us to visualize the complex phenomena of RNA biology and its consequences in diagnostics, therapeutics, and biotechnology. In addition, the activity-based sensing platform will allow for the investigations of RNA dynamics and localization in living cells under specific redox physiochemical imbalances such as ROS.