Excited-state quenching of dye-linked oligonucleotides

摘要

Fluorescence energy transfer (FET) can be used to obtain structural and dynamic information on duplex and branched DNA molecules suitably labeled with donor and acceptor dyes. However, dye-DNA interaction cause excited-state quenching of the dyes and complicate the analysis of FET data. We have investigated the excited-state quenching of two common FET dyes, fluorescein and tetramethyl rhodamine, covalently linked to synthetic oligonucleotides. The rate of quenching is shown to depend on the base sequence and association state of the oligonucleotide, and also the length of the linker chain. Furthermore, the dyes can adopt more than one configuration with respect to the oligonucleotide strand and the degree of quenching is different in each configuration. The implications of these findings for FET measurements in nucleic acids are discussed.