The need for improved fluorescent labels increases as The Genome Project progresses and expands to include genomes of other organisms relevant to human health. Better fluorescent labels are also required for gene-profiling experiments and other applications in proteomics. This proposal outlines why resolution and fluorescence intensities are difficult to reconcile in multiplexing. Briefly, dyes that emit close to the excitation wavelength are not well resolved, and ones that emit far from the excitation wavelength do not absorb it strongly hence do not fluoresce intensely. Even the state-of-the-art through-space energy transfer cassettes (eg Perkin-Elmer """"""""Big Dyes"""""""") are restricted by these constraints since the fluorescence spectrum of the absorbing dye (the donor) must overlap with the absorption spectrum of the emitting dye (the acceptor). The central hypothesis of this proposal is that relatively small dye-cassettes could be designed to exploit through-bond, rather than through-space, energy transfer and that such cassettes would not be restricted by the constraints described above. Proof-of- concept for this hypothesis was obtained in the first funding cycle. Novel, functionalized BODIPY derivatives that fluoresce at different wavelengths were made, coupled to donor molecules via conjugated linkers, and spectroscopic studies of these materials were performed. Some of these cassettes were shown to absorb radiation at 270 nm and emit it between 515 and 676 nm, corresponding to pseudo-Stokes shifts of up to 408 nm! The next step in this project is to prepare hydrophilic cassettes that can be easily coupled to DNA. This is not a trivial thing to do but, since the last submission, progress has been made in this direction such that we are within a few steps of the first water soluble systems. This proposal is to develop the syntheses and design of water-soluble dye-cassettes that maximize through- bond energy transfer. Unique acceptor fragments that fluoresce below 515 nm above 676 nm will be prepared to take advantage of the enormous resolution without loss of signal intensity that becomes possible via through-bond energy transfer. Experimental and computational studies to characterize the mechanism of the energy transfer are proposed. Other experiments are described for correction of mobility shifts and testing/application of the dyes in DNA sequencing.
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