Stable fluorescent complexes of double-stranded DNA with bis-intercalating asymmetric cyanine dyes: properties and applications

Abstract

The synthesis, proof of structure, and the absorption and fluorescence properties of two new unsymmetrical cyanine dyes, thiazole orange dimer (TOTO; 1,1′-(4,4,7,7-tetramethyl-4,7-diazaundecamethylene)-bis-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methyl-idenel-quinolinium tetraiodide) and oxazole yellow dimer (YOYO; an analogue of TOTO with a benzo-1,3 oxazole in place of the benzo-1,3-thiazole) are reported. TOTO and YOYO are virtually non-fluorescent in solution, but form highly fluorescent complexes with double-stranded DNA (dsDNA), up to a maximum dye to DNA bp ratio of 1:4, with>1000-fold fluorescence enhancement. The dsDNA-TOTO (λ _max 513 nm; $λmaxF$ 532 nm) and dsDNA-YOYO (λ _max 489 nm; $λmaxF$ 509 nm) complexes are completely stable to electrophoresis agarose and acrylamide gels. Mixtures of restriction fragments pre-labeled with ethidium dimer (EthD; $λmaxF$ 616 nm) and those prelabeled with either TOTO or YOYO were separated by electrophoresis. Laser excitation at 488 nm and simultaneous confocal fluorescence detection at 620–750 nm (dsDNA-EthD emission) and 500–565 nm (dsDNA-TOTO or dsDNA-YOYO emission) allowed sensitive detection, quantitation, and accurate sizing of restriction fragments ranging from 600 to 24,000 bp. The limit of detection of dsDNA-TOTO and YOYO complexes with a laser-excited confocal fluorescence gel scanner for a band 5-mm wide on a 1-mm thick agarose gel was 4 picograms, about 500-fold lower than attainable by conventional staining with ethidium bromide.