Design, Synthesis and Characterization of Fluorescent Probes for Biomolecule Detection

Observing individual components and processes that occur between DNA, RNA and proteins is not only fundamental to advancing our basic understanding of biology, but it is also critical for identifying and diagnosing disease states. Fluorescent probes and advancements in optical techniques have proven to be instrumental for peering deeper into these molecular orchestrations in real-time. In this post-genomic era, efficiently labeling and detecting these proteins and their interactions with other biomolecules has gained considerable attention.;The first portion of this thesis covers the development of new probes, dye-protein pairs termed 'fluoromodules', based on unsymmetrical cyanine dyes to create genetically encodable fluorogenic protein labels. Bridge substitution on a small family of trimethine dyes is shown to reduce dye background fluorescence, which ultimately improves the signal to noise ratio. Theoretical calculations corroborate this finding and establish that the reduced fluorescence signal is due to a lower energy requirement associated with torsional rotation in the excited state which provides a non-radiative relaxation pathway. Bridge substitutions with a cyano group at the alpha position in the bridge were also used to enhance the chemo and photostability of mono and trimethine dyes alone or with a cognate protein binding partner.;Induced steric crowding in a series of cyano substituted monomethine dyes resulted in significant biological ramifications. Importantly, non-specific biomolecule interactions and staining of cellular components was eliminated. Crystal structures suggest that this reduced affinity of the substituted dyes is due to twisting of the dye from planarity, which hinders favorable dye-biomolecule interactions. Selection of new binding proteins for one of these dyes lead to the identification of new fluoromodules; most of which are highly selective towards the target dye but a promiscuous binder was also identified.;In the final section of this thesis, multiple chromophores were organized on a PNA-DNA scaffold to create constructs with quantum yields and over-all brightness greater than that of the unmodified dye. The covalently attached dyes also improve the thermal stability of the duplex and are able to participate in Forster energy transfer with an acceptor dye attached at the terminal end of the duplex.