The Design, Synthesis and Characterization of Bright Fluorescent Probes for Biomolecule Detection

The ability to observe and quantify interactions within a cellular environment has allowed scientist to develop a detailed understanding of biomolecular interactions, in addition to identifying and diagnosing disease states. Advancements in fluorescent probes and imaging
techniques have proven to be instrumental for peering deeper into these molecular interactions in real time. Although fluorescent small molecules are well-established and versatile tools, fine tuning the spectral properties of either the probe or the small molecule is necessary in order to
obtain a clear and accurate picture of the desired molecular environment. In this thesis, we develop and explore new fluorescent probes for adaptable, efficient, and sensitive detection assays. The first portion of this thesis looks at manipulating the scaffold of a fluorogenic dye in order to develop a versatile fluorophore to stain non-specifically DNA as well as bind to a promiscuous protein. Fluorogenic dyes are a special class of fluorophores that show distinctly
different emission intensities depending on their local environment. When free in a solution, the dye can exploit non-radiative twisting pathways about the central methine bridge; but when in a viscous media or when bound by a biomolecule, this pathway is eliminated and fluorescence
increases. The fluoromodule spectral range is dependent upon the properties of the chromophore that is bound. In this thesis, we present a series of thiazole orange analogs with
varied substituents and electronic properties, and investigate their spectroscopic properties and their fluorogenic behavior in different solvents and biomolecular environments (e.g. DNA and scFv). It was determined that a 40nm spectral shift could be obtained allowing for a brighter image at common laser wavelengths. The second portion of the thesis focuses on improving hybridization probes targeting nucleic acids. Traditional strategies lack the ability to distinguish bound from unbound probes. In order to minimize background fluorescence from unbound probes we developed a modified
binary probe used to label DNA telomeric repeats in fixed cells and tissue. By utilizing γ- modified Peptide Nucleic Acids (γ PNAs), we were able to shorten and simplify the binary probe design with two terminally-labeled γPNA 9-mer probes designed to hybridize in tandem allowing for a Förster Resonance Energy Transfer (FRET) signal to indicate hybridization to the target. First, the γPNA probes were analyzed for affinity, structure, and fluorescence properties. Next, the γPNA probes were applied for the analysis of telomeric DNA in genomic DNA, as well as
cellular imaging using fluorescence in-situ hybridization (FISH). The final portion of the thesis focuses on enhancing the brightness of hybridization probes. Traditional PNA probes are designed to be complementary to the target and a single dye is covalently attached at the terminus. In this strategy, sensitivity of the probe is dependent on the fluorescent properties of a single dye. In order to enhance the brightness of the probe, we proposed an internal labeling strategy to improve the local concentration of dye per probe.
We developed PNA probes with multiple internal fluorescent dyes using the Huisgen-Meldal Sharpless “Click” reaction between an azide-modified fluorescent dye and alkynyl-uracilcontaining PNA. We have synthesized a dye-modified PNA monomer and have incorporated it into a series of oligomers. It was determined that the PNA:DNA duplex stability was not affected by the presence of the dye and only a marginal decrease in fluorescence due to quenching was observed, even when the two dyes were placed in adjacent positions. We have demonstrated the feasibility of this approach, which suggests that several fluorescent dyes can be introduced into a PNA probe at internal positions in order to enhance the brightness of DNA/RNA-targeting
PNA probes. We have improved the efficacy of fluorescent probes by altering the structure of a fluorogenic dye, and designing new strategies for nucleic acid hybridization probes. Versatile, brighter, and more accurate probes will ultimately give biologist better tools to diagnose,
characterize, and ultimately understand biological interactions at a molecular level.