Fluorescence-quenching phenomenon by photoinduced electron transfer between a fluorescent dye and a nucleotide base

Fluorescently labeled oligonucleotide probes have been widely used in biote chnology, and fluorescence quenching by the interaction between the dyes an d a nucleobase has been pointed out. This quenching causes big problem in a nalytical methods, but is useful in some other cases. Therefore, it is nece ssary to estimate the fluorescence quenching intensity under various condit ions. We focused on the redox properties of some commercially available flu orescent dyes, and investigated dye-nucleotide interactions between a free dye and a nucleotide in aqueous solution by electrochemical and spectroscop ic techniques. Our results suggested that the quenching was accompanied by photoinduced electron transfer between a thermodynamically quenchable excit ed dye and a specific base. Several kinds of fluorescent dyes labeled to th e 5'-end of oligonucleotide C10T6 Were prepared, and their quenching ratios compared upon hybridization with the complementary oligonucleotide A(6)G(1 0) The quenching was completely reversible and their efficiencies depended on the attached fluorophore types. The fluorescence of 5-FAM, BODIPY FL or TAMRA-modified probe was strongly quenched by hybridization.