Mechanism of Ethidium Bromide Fluorescence Enhancement on

The mechanism of the enhancement of the fluorescence of ethidium bromide on binding to double helical RNA and DNA has been investigated. From an examination of the effect of different solvents on the fluorescence lifetime, quenching of fluorescence by proton acceptors, and the substantial lengthening of lifetime observed upon deuteration of the amino protons, regardless of the medium, we conclude that proton transfer from the excited singlet state is the process primarily responsible for the low fluorescence yield in most polar solvents. Enhancement of fluorescence upon intercalation E t h i d i u m bromide, a cationic dye (structure I ) which interacts strongly and specifically with double helical RNAs and DNAs, is widely used in spectrofluorimetric studies because of the striking fluorescence enhancement it displays upon binding (Waring, 1965; LePecq and Paoletti, 1967; Bittman, 1969; Burns, 1969, 1971; Tao et al., 1970; LePecq, 1971; Angerer et al., 1974; Bontemps and Fredericq, 1974; Genest et al., 1974; Gatti et al., 1975). It is generally agreed that strong fluorescence enhancement accompanies intercalation of the dye into the double helix conformation of the nucleic acid but there is also evidence for additional nonintercalative, less fluorescence-enhanced sites which are presumed to involve electrostatic binding (Waring, 1965; LePecq and Paoletti, 1967; Bittman, 1969). Recently, ethidium bromide has been used to probe tRNA structure (Bittman, 1969; Urbanke et al., 1973), 5s RNA (Gray and Saunders, 1971; Feunteun et al., 1975), circular DNA (Hudson et a]., 1969), chromatin structure (Ide and Baserga, 1976), ribosomal RNA (Lawrence and Daune, 1976), synthetic DNA (Aktipis and Martz, 1974), tRNA protein interactions (Rigler et al., 1971), and to determine the molecular weight of DNA (Weissman et al., 1976). Ethidium bromide also elicits a wide range of biochemical effects (Lurquin and Buchet-Mahieu, 1971; Avadhani et al., 1973; Kramer et al., 1974; Criddle et al., 1976). Despite extensive studies of ethidium bromide complexes with polynucleotides, a satisfactory mechanism explaining the high degree of fluorescence enhancement upon binding has not From the Department of Chemistry, Revelle College, University 01 California, San Diego, La Jolla, California 92093. Received October 17, 1976; revised manuscript received February 10, 1977. This work was supported by The American Cancer Society. is attributed to a reduction in the rate of excited state proton transfer to solvent molecules. The proposed mechanism accounts for the -3.5-fold increase in the lifetime of free ethidium bromide in going from H2O to D2O; the fact that addition of small amounts of water to nonaqueous solvents decreases the fluorescence whereas addition of small amounts of D2O enhances the fluorescence; and the enhancement of the ethidium bromide triplet state yield on binding to DNA. Other proposed mechanisms are shown to be inconsistent with our