HETERODIMERIC DNA-BINDING DYES DESIGNED FOR ENERGY-TRANSFER - SYNTHESIS AND SPECTROSCOPIC PROPERTIES

Heterodimeric dyes are described which bind tightly to double-stranded (dsDNA) with large fluorescence enhancements. These dyes are designed to exploit energy transfer between donor and acceptor chromophores to tune the separation between excitation and emission wavelengths. The dyes described here absorb strongly at the 488 nm argon ion line, but emit at different wavelengths, and can be applied to multiplex detecti on of various targets. The chromophores in these dyes, a thiazole oran ge-thiazole blue heterodimer (TOTAB), two different thiazole orange-et hidium heterodimers (TOED1 and TOED2), and a fluorescein-ethidium hete rodimer (FED), are in each case linked through polymethylene-amine lin kers. The emission maxima of the DNA-bound dyes lie at 662 (TOTAB), 61 4 (TOED 2), and 610 nm (FED). The dyes showed a > 100 fold enhancement of the acceptor chromophore fluorescence on binding to dsDNA and no s equence selectivity. In comparison with direct 488 nm excitation of th e constituent monomeric dyes, in the heterodimers the fluorescence of the acceptor chromophores was greatly enhanced and the emission of the donor chromophores quenched by over 90%. The acceptor emission per DN A-bound dye molecule was constant from 100 DNA bp:dye to 20 bp:dye and decreased sharply at higher dye:DNA ratios.