Combining biomolecules and fluorogenic cyanine dyes into novel fluorescent labels

Fluorescence technology is a pivotal element of modern science. Advances in a variety of fields from medical diagnostics to nanotechnology are highly interrelated to advances in new fluorescence detection systems, or fluorescent labels that will be employed. This dissertation describes our efforts to contribute to the progress in fluorescence technology by developing novel fluorescent labels. We developed two different types of fluorescent labels with different superiorities and thus different application areas. Despite being developed based on different rationales, the approach we took was common, that is the utilization of biomolecules and fluorogenic cyanine dyes and their unique binding interactions.;Chapters 3 and 4 describe novel genetically encodable fluorescent labels that promise low signal-to-noise ratios, and spectral versatility for in-vivo applications. Magnetic and fluorescence cell sorting was applied to isolate single chain variable fragment (scFv) proteins from a yeast-surface displayed library for their ability to activate the fluorescence of the fluorogenic dye Dimethyl Indole Red (DIR). Isolated scFvs exhibited strong binding affinities for DIR by enhancing its fluorescence more than 100-fold. One of the isolated scFvs (K7) demonstrated promiscuity, binding to several other fluorogenic cyanine dyes with strong affinity and high quantum yields. K7 was further characterized in terms of the origins for the promiscuity, employing fluorescence melting and intrinsic fluorescence studies. In an attempt to demonstrate the potential of these fluoromodules for being engineered according to various different needs, we employed directed molecular evolution on one of the DIR activating scFvs. We used error-prone PCR mutagenesis and fluorescence activated cell sorting to manipulate the affinity and ligand specificity of the parent scFv. The fluorescence spectroscopic analysis of selected scFvs revealed several fold increase in DIR affinity. Furthermore, the affinity of the scFvs for structurally similar fluorogenic cyanine dyes was suppressed which improved the DIR specificity of matured scFvs.;Chapter 2 describes the design, preparation and characterization of fluorescent DNA nanotags that are brighter than their competitors and have great potential for in-vitro labeling assays. DNA naotags are intercalator dye arrays on a compact 3-dimensional DNA-Tetrahedron (TH) nanostructure. The template tolerates the structural distortions introduced by intercalation and allows concentration of multiple fluorophores within a small volume, resulting in brightly fluorescent nanotags with effective extinction coefficients in the order of 106 M-1 cm-1 . Forster resonance energy transfer from intercalated donor dyes to covalently attached acceptor dyes is efficient in the TH-nanotags leading to wavelength tunability. The compact nature of the TH also provides a protective medium for the fluorophores, leading to improved photostability and enhanced resistance to nuclease digestion, relative to one- or two-dimensional nanotags described previously.