Biomolecular labeling with combinatorial oligodeoxyfluorosides [electronic resource]

This thesis describes the combinatorial synthesis of libraries of "oligodeoxyfluorosides" and their applications in quenching probes, as FRET donors and in live cell imaging. Oligodeoxyfluorosides (ODFs) are a multichromophoric system based on the assembly of fluorescent deoxyriboside monomers ("fluorosides") into DNA-like phosphodiester oligomers. Hydrocarbon and heterocyclic aromatic fluorophores replace nucleobases and are covalently conjugated to the anomeric position of deoxyribose. When arrayed in an oligomeric form, these fluorophores are well stacked on one another and interact in both the ground state and the excited state, forming excimers and exciplexes. These DNA-based ODFs were conjugated to short oligonucleotides and used as fluorophores in DNA hybridization probes. In particular, their quenching and FRET properties were studied in the DNA hybridization context. Hybridization of an ODF-labeled probe to a dabcyl quencher-labeled complement resulted in efficient quenching in 85% of the cases tested. High efficiency of quenching was also established by their large Stern-Volmer constants of between 2.1 x 104 M-1 and 4.3 x 105 M-1. These orders of magnitude in Stern-Volmer constants were only previously observed in large conjugated polymers and have not been observed in discrete molecules. The excited state interactions such as exciplexes and excimers formed in ODFs were also found to act as donors for energy transfer by the Förster mechanism. Although such delocalized excited states have been widely studied, we know of no prior report of their use in FRET. We tested the ability of the twenty ODFs to donate energy to Cy5 and TAMRA dyes conjugated to a complementary strand of DNA, with these acceptors oriented either at the near or far end of the ODF-conjugated probes. Results showed that a number of the ODF fluorophores exhibited relatively efficient energy transfer characteristic of the Förster mechanism, as judged by drops in donor emission quantum yield and fluorescence lifetime, accompanied by increases in intensity of acceptor emission bands. Excimer/exciplex bands in the donors were selectively quenched while shorter-wavelength monomer emission stayed relatively constant, consistent with the notion that the delocalized excited states, rather than individual fluorophores, are the donors. Next, a set of 23 ODFs spanning the entire visible spectrum was identified from a combinatorial library of 4096 tetramer ODFs constructed from eight monomeric fluorosides. Interestingly, all 23 ODFs can be simultaneously excited by long-wavelength UV excitation at 354 nm. Experiments with human tumor (HeLa) cells, observing ODFs by laser confocal microscopy, showed that they could penetrate the outer cellular membrane upon simple incubation, yielding cytoplasmic localization. Further experiments showed that ODFs were taken up through an energy-dependent endocytosis process and were localized in endosomes and lysosomes in the cells. The results suggest that ODF dyes may be broadly useful as labels in biological systems, allowing the simultaneous tracking of multiple species by color, and allowing visualization in moving systems.

Keywords: Chemistry
Advisor: Kool Eric T. primary advisor. Thesis advisor
Huestis Wray advisor. Thesis advisor
Khosla Chaitan