Design, synthesis, and spectroscopic properties of peptide-bridged fluorescence energy-transfer cassettes

A general partial solid-phase synthetic scheme was developed for the synthesis of energy-transfer cassettes with the donor and acceptor dyes bridged by a peptide. In these cassettes, 6-carboxyfluorescein (Fam) served as a donor. For the second dye, 6-carboxy-X-rhodamine (Rox) was used as a fluorescent acceptor or erythrosin B as a quencher. Different peptides bearing Rox at the amino terminus and Fam linked through different diamines to the carboxyl terminus were synthesized to examine the effects of the chain length and rigidity on energy-transfer efficiency. The ratio of emission intensities at 605 nm of the acceptor dye (ROX) in the cassette Rox-GPPPEPPP-p-xylylenediamine-Fam versus free ROX with 488 nm excitation was approximately 14 and is similar to that obtained for optimized oligonucleotide primers bearing the same dyes [Ju, J., Ruan, C., Fuller, C. W., Glazer, A. N., and Mathies, R. A. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 4347-4351].