This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The central hypothesis of this work is that when a donor and acceptor systems are connected via a conjugated linker that does not allow them to become planar then rapid energy transfer from the donor to the acceptor may occur through bonds. Through-bond energy transfer is mechanistically different to the F?rster basis for FRET, and there is no known requirement for overlap of the emission of the donor fragment with the absorption of the acceptor part. Thus, appropriately designed through-bond energy transfer cassettes could absorb photons via a donor part, or parts, at a convenient wavelength (eg 488 nm: excitation from an Ar-laser), transfer the energy rapidly through the conjugated linker to the acceptor fragment that emits at a far longer wavelength. There is no constraint on the difference between the donor absorption and the acceptor emission wavelengths in this scheme. It therefore is possible to design dyes that absorb strongly at a short wavelength and emit brightly with very similar intensities at several wavelengths (governed by the chemical nature of the acceptor) that are many wavenumbers apart, ie with excellent resolution. Coupling more than one donor in a conjugated system with an acceptor facilitates absorption of more light thereby increasing the intensity of the emission. In summary, through bond energy transfer cassettes have the potential to increase both the resolution and fluorescence intensities obtained from several probes excited by a laser source operating at a single wavelength. Proteins generally cannot enter cells by passive diffusion, but require active transport. While some proteins can also be transported into cells by microinjection, entrapped in liposomes, viral vectors, and electroporation, such methods are laborious, time consuming, and often have low efficiencies. A recently developed method involving a peptide called """"""""Chariot"""""""" (Active Motif, Carlsbad, CA) overcomes these problems. Chariot non-covalently complexes with proteins to peptides and facilitates their transport into cells. The Chariot peptide is non-cytotoxic, and crosses plasma membranes independent of transporters or specific receptors, thus avoiding the lysosomal degradative pathway. The Chariot peptide has high transport efficiency (65-95%) and has already been shown to rapidly co-transport large fluorescent proteins. Once internalized, the fluorescent protein-Chariot peptide complex rapidly dissociates, thereby allowing the fluorescent-tagged protein to proceed to its intracellular target while the Chariot peptide is rapidly degraded. Use of the Pep1 peptide (and other carrier systems) to transfer protein/through-bond cassette conjugates into living cells opens new vistas of research. It is not yet evident that proteins imported into cells using the Chariot system are free in the cytosol;they could be encapsulated in intracellular vesicles. One of the objectives of our research is to elucidate this with single molecule studies performed at the center.
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