Specific High-Affinity Binding of Thiazole Orange to Triplex and G-Quadruplex DNA

Abstract

Interaction of Thiazole Orange (TO) with double-, triple-, and quadruple-stranded forms of DNA was studied. We have demonstrated by UV-vis absorption, circular dichroism (CD), and fluorescence spectroscopy that TO binds with much higher affinity to triplex and G-quadruplex DNA structures compared to double-stranded (ds) DNA. Complexes of the dye with DNA triplexes and G-quadruplexes are very stable and do not dissociate during chromatography and gel electrophoresis. TO binding to either triple- or quadruple-stranded DNA structures results in a >1000-fold increase in dye fluorescence. The fluorescence titration data showed that TO to triad and tetrad ratios, in tight complexes with the triplex and the G-quadruplex, are equal to 0.5 and 1, respectively. Preferential binding of TO to triplexes and G-quadruplexes enables selective detection of only these DNA forms in gels in the absence of free TO in electrophoresis running buffer. We have also demonstrated that incubation of U2OS cells with submicromolar concentrations of TO results in preferential staining of certain areas in the nucleus in contrast to DAPI which binds to dsDNA and efficiently stains regions that are unstained with TO. We suggest that TO staining may be useful for the detection of noncanonical structural motifs in genomic DNA.

Interaction of hemin with quadruplex DNA

“…Thiazole orange (TO) is generally used as a DNA-bound intercalator. Initial studies have demonstrated that thiazole orange binds to the quadruplex-forming oligonucleotide with high affinity [23,24]. In this investigation, TO was used for fluorescent intercalator displacement assay.…”

Fluorescent triplex-forming DNA oligonucleotides labeled with a thiazole orange dimer unit

“…[10][11][12][13][14][15] Pyrimidine TFOs labeled with thiazole orange (TO) exhibited comparably larger changes in fluorescence intensities before and after triplex formation 14,15 because the fluorescence intensity of TO molecules is low in the presence of poly-pyrimidine singlestranded DNA 16 and high in the presence of DNA triplexes. 15,17 To suppress the fluorescence of the TO molecules covalently attached to DNA oligonucleotides, the utilization of excitonic interaction between two molecules of TO is effective. 18,19 Exciton theory explains that the fluorescence of two dye molecules forming an H-dimer is strongly suppressed.…”

“…[21][22][23][24][25][26][27][28][29][30][31][32] ECHO probes emitting low background fluorescence were expected to be also applicable to detect double-stranded DNA as TO molecules emit intense fluorescence in the presence of DNA triplexes. 15,17 However, DNA triplexes formed by ECHO probes have not been reported and the photophysical properties are unknown. Besides the photophysical properties, the effects on the thermal stability and kinetic parameters of the DNA triplexes formed by the ECHO probes, which are caused by the interaction of the two TO molecules with the DNA triplexes, are also important, because DNA triplexes intrinsically have a low thermal stability and slow formation rate.…”

Parallel Polyadenine Duplex Formation at Low pH Facilitates DNA Conjugation onto Gold Nanoparticles

“…Poly-G DNA was not considered here, since they tend to form G-quadruplex and emit very strongly with TO. 43 TO is a useful dye for staining the A-motif if G-quadruplex can be excluded. We then tested the response of TO with DNA 1-4.…”