Chemical and Enzymatic Methods to Regulate Phospholipid Membrane Formation and Manipulate Cellular mRNA
- Author(s): Zhou, Eric Cunyu
- Advisor(s): Devaraj, Neal K
- et al.
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.