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-thiaz ole)-2- methylidene]-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 greater than 1000-fold fluorescence enhancement. The dsDNA-TOTO (lambda max 513 nm; lambda maxF 532 nm) and dsDNA-YOYO (lambda max 489 nm; lambda maxF 509 nm) complexes are completely stable to electrophoresis on agarose and acrylamide gels. Mixtures of restriction fragments pre-labeled with ethidium dimer (EthD; lambda maxF 616 nm) and those pre-labeled 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.

Development and validation of flow cytometric measurement for parasitemia in cultures of P. falciparum vitally stained with YOYO‐1

“…Jacobberger et al (9) used DiOC1, a membrane potential respon-sive dye and Hoechst 33342 to evaluate parasitemia levels in mice (7). Recently, YOYO-1, a dimeric cyanine nucleic acid dye, is among the highest sensitivity fluorescent probes available for nucleic acid staining and has been added to this list (10,11). YOYO-1 has an extremely high affinity for DNA, and it can be excited at 488 nm, which is the excitation wavelength available from most lasers employed in FCM.…”

Design and characterization of a compact dual channel virus counter

“…POPO-3 has an excitation maximum at 534 nm and an emission maximum at 570 nm. Although POPO-3 has a high affinity for DNA, it is essentially nonfluorescent in the absence of nucleic acids and exhibits 100-to 1,000-fold fluorescence enhancements upon binding to DNA (16,17). Because POPO-3 is cell impermeant, a detergent, sodium dodecylsulfate (SDS), was required to allow the stain access to the DNA.…”

Microwave synthesis and fluorescence properties of homo- and heterodimeric monomethine cyanine dyes TOTO and their precursors

“…Interest in nonradioactive DNA stains that are stable under gel electrophoretic conditions has led to the synthesis and characterization of a family of homo-and heterodimeric DNA-binding dyes (1)(2)(3). Organic fluorescent probes (FPs) are, as a rule, polycyclic aromatic cations with the planar structure capable of incorporating (intercalating) between the planes of the DNA bases, pulling apart these planes and changing the structure of nucleic acids.…”