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

A general partial solid-phase synthetic scheme was developed for the synthe sis of energy-transfer cassettes with the donor and acceptor dyes bridged b y a peptide. In these cassettes, 6-carboxyfluorescein (Fam) served as a don or. For the second dye, 6-carboxy-X-rhodamine (Rox) was used as a fluoresce nt acceptor or erythrosin B as a quencher. Different peptides bearing Rox a t the amino terminus and Fam linked through different diamines to the carbo xyl terminus were synthesized to examine the effects of the chain length an d rigidity on energy-transfer efficiency. The ratio of emission intensities at 605 nm of the acceptor dye (ROX) in the cassette Rox-GPPPEPPP-p-xylylen ediamine-Fam versus free ROX with 488 nm excitation was similar to 14 and i s similar to that obtained for optimized oligonucleotide primers bearing th e 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].