Abstract
Over the past few decades, remarkable developments in chemical probes and microscopy have been significantly advancing bioimaging technology which plays a key role in modern biomedical research and practices. GFPs provided a great tool for labeling proteins for visualizing their dynamics such as interactions with other proteins and nucleic acids. However, the relatively large size of GFPs limits its application as it can interrupt the functionality, localization, and interactions of the targeted proteins. Proteins can also be labeled/modified through residue specific chemical reactions such as biotin ligase-based reactions, Staudinger ligation, 3+2 cycloaddition reactions, click and photo-click reactions, cross-metathesis reactions. However, these post-translational modifications pose some toxicity issues, background signals, solubility, interference with the native forms of proteins, or being limited to in vitro labeling only. Small molecule-based fluorescent probes such as fluorescent unnatural α-amino acids (FAA) can be suitable candidates for incorporation to target proteins without perturbing their native forms. Despite some availability, most of them absorb at short wavelength (UV-region) thus undesirable for bio-imaging. Harnessing the Suzuki-Cross Coupling reaction, a novel fluorescent α-amino acid 4-phenanthracen-9-yl-L-phenylalanine (Phen-AA) has been synthesized via the coupling of an anthracene ring to the benzene ring of L-phenylalanine, creating a novel fluorescent α-amino acid that emits blue fluorescence (ChemComm 2020). Subsequently, a cyan-emitting fluorescent α-amino acid DBT-FAA has been synthesized with a sulfur-containing a thiophene ring introduced to the side chain of the phenanthracenyl amino acid to bring novel photophysical and physiochemical properties. Both the FAAs showed excellent photophysical properties and biocompatibility and bioimaging capability with living human cells. However, their size and relative short optical wavelength could potentially disrupt the structure and limit their application in site-specific incorporation into proteins for bioimaging. To improve the photophysical properties, a bright cyan emitting fluorescent α-amino acid Coum-PheAA was successfully developed with much enhanced photophysical properties and bioimaging capability in Hela cells and live-zebra fish embryos. Coum-PheAA was then chosen as the fluorescent α-amino acid for site-specific incorporation into amber suppressed proteins in E. coli using an orthogonal aminoacyl-tRNA synthetase (aaRS)/ tRNA pair. The aaRS was rationally designed first via amino acid scanning and docking simulations. The promising candidates were subsequently created via site-directed mutagenesis. The engineered synthetase variants showed substantial improvement in recognition and incorporation of Coum-PheAA in an amber suppressed target protein and successfully imaged in live E. coli cells. In parallel, a compartmentalized partnered replication based directed evolution methodology was attempted to develop aaRS variants with high efficiency and fidelity for incorporating Coum-PheAA with high selectivity. A pool of randomized aaRS gene library was linked to the production of Taq DNA polymerase and enriched in emulsion-based compartments using PCR. Overall, this dissertation study was dedicated in designing new FAAs and aaRS as well as their utilization for site-specific incorporation into proteins and their bio-imaging in live-cells and other living systems. These novel chemical tools will pave the way for a better understanding of cell molecular biology and protein dynamics, localization, and protein imaging which may find broad applications in biomedical research and the pharmaceutical industry.