Through-bond Energy Transfer Cassettes for Intracellular Imaging

Equipment routinely used for fluorescence detection of several biomolecules each labeled with a fluorescent dye typically has only one laser source that emits at a single wavelength (eg typically 488 nm). The four dyes must each emit at wavelengths that are different enough to allow them to be spectroscopically distinguished, and they must do so with high quantum yields when irradiated at 488 nm (or at a wavelength corresponding to some other laser source). This is called multiplexing. Ideally, dyes should emit very strongly when irradiated at the source wavelength, and each one should do so at different wavelengths that are far removed from that of the laser source.

To prepare better dyes for multiplexing we conceived and patented a new approach to design of fluorescent dyes. This involved organic syntheses of molecules with donor and acceptor parts linked together in one molecule (Figure 4).

Figure 1

a Through-bond energy transfer cassettes for the production of fluorescent dyes with large Stoke’s shifts. 

b Typical design of a through-bond energy transfer cassette. 

c Dyes in multiplexing should be well resolved and equally fluorescent. 

d In fact, the dyes actually used in tend to fluoresce at too similar wavelengths and their fluorescence tapers off at long wavelengths.

 

Initially, uor interests were focused on dyes for DNA sequencing in The Human Genome Project; multiplexing with fluorescent dyes are critical to this effort. More recently the emphesis of our efforts is on intracellular imaging. Specifically, we would like to use a combination of through-space energy transfer (FRET) and through-bond energy transfer to monitor protein-protein interactions in live cells, as outlined in Figure 5.

Labeled proteins in this scheme are imported into live cells via a non-covalently bound cell-penetrating peptide called Chariot. We are interested in the ways Chariot achieves this, and the possibilities for making more readily accessible Chariot analogs that perform the same task.

In the medium term, this project is inteded to converge with our interest in small molecules that interfere with protein-protein interactions. The small molecules and/or the proteins can be labeled, imported into cells, then imaged ex vivo.

 

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