This proposal is aimed at development of a new class of fluorescent reporters for biomolecules. We have adopted a strategy of replacement of DNA bases with fluorescent hydrocarbons and heterocycles, and we use the DNA backbone to assemble these into multi-fluor composite molecules. Because the DNA backbone has evolved for intimate interaction between stacked aromatic rings, it encourages close physical, electronic, and photophysical interaction between individual fluorescent species. This results in """"""""polyfluor"""""""" molecules having properties that are often quite different than the individual fluors making them up, leading to more intense fluorescence, to unusual modes of energy transfer, and to labels with tunable excitation and emission. ? ? In preliminary work we have synthesized several fluorescent nucleosides (""""""""fluorosides"""""""") as candidate monomers for incorporation into polyfluor strings. The preparation of the first libraries of polyfluor molecules has allowed us to generate hundreds of varied fluorescent species. These have been evaluated on beads under an epifluorescence microscope for emission intensity and hue at varied excitation wavelengths. Picking and decoding of individually interesting polyfluors has made it possible to find useful combinations of fluorosides that give especially intense emission and a wide spectrum of emission wavelengths at specified excitation wavelength. We have already identified a number of different forms of multiple energy transfer in such polyfluors. This combinatorial approach allows us to discover such complex photophysical interactions where they would otherwise be difficult or impossible to predict. ? ? We expect that this approach will lead to the development of exceptionally bright and tunable fluorescent markers for biomolecules, having widely varied excitation and emission characteristics that common single dyes do not have. In the term covered by this proposal, our aims are to (1) synthesize a new set of fluorosides having broader photophysical characteristics; (2) develop a polyfluor library with excitation at 410-440 nm (for eventual blue laser excitation) and a wide variety of emission wavelengths; (3) prepare a library of """"""""antenna"""""""" sensitizers for common commercial dyes; (4) develop and test high-efficiency quenchers of polyfluors; and (5) develop methods for conjugating polyfluors to peptides, antibodies, and small molecules. ? ?
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