A theoretical and experimental study of two thiazole orange derivatives with single- and double-stranded oligonucleotides, polydeoxyribonucleotides and DNA.
ABSTRACT The effect of interaction with DNA and oligonucleotides on the photophysical properties of two thiazole orange (TO) derivatives, with different side chains (-(CH2)3-N+(CH3)3 and -(CH2)6-I)) linked to the nitrogen of the quinoline ring of the thiazole orange, is presented here. The first one called TO-PRO1 is a commercially available dye, whereas the second one called TO-MET has been specially synthesized for further covalent binding to oligonucleotides with the aim of being used for specific in situ detection of biomolecular interactions. Both photophysical measurements and molecular calculations have been done to assess their possible mode of interaction with DNA. When dissolved in buffered aqueous solutions both derivatives exhibit very low fluorescence quantum yields of 8 x 10(-5) and 2 x 10(-4), respectively. However, upon binding to double-stranded DNA, large spectroscopic changes result and the quantum yield of fluorescence is enhanced by four orders of magnitude, reaching values up to phi F = 0.2 and 0.3, respectively, as a result of an intercalation mechanism between DNA base pairs. A modulation of the quantum yield is observed as a function of the base sequence. The two derivatives also bind with single-stranded oligonucleotides, but the fluorescence quantum yield is not so great as that when bound to double-stranded samples. Typical fluorescence quantum yields of 7 x 10(-3) to 3 x 10(-2) are observed when the dyes interact with short oligonucleotides, whereas the fluorescence quantum yield remains below 10(-2) when interacting with single-stranded oligonucleotides. This slight but significant quantum-yield increase is interpreted as a folding of the single strand around the dye, which reduces the internal rotation of the two heterocycles around the central methine bridge that links the two moieties of the dye. From these properties, it is proposed to link monomer covalently to oligonucleotides for the subsequent detection of target sequences within cells.
- SourceAvailable from: Richard Paul Haugland[show abstract] [hide abstract]
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.Nucleic Acids Research 07/1992; 20(11):2803-12. · 8.28 Impact Factor
- Journal of The American Chemical Society - J AM CHEM SOC. 04/2002; 117(48).
- [show abstract] [hide abstract]
ABSTRACT: One- and two-dimensional 1H NMR spectroscopy were used to characterize the binding of a homodimeric thiazole orange dye, 1,1'-(4,4,8,8-tetramethyl-4,8-diazaundecamethylene)-bis-4-(3 -methyl-2,3-dihydro-(benzo- 1,3-thiazole)-2-methylidene)-quinolinium tetraiodide (TOTO), to various double-stranded DNA oligonucleotides containing symmetric (5'-pyr-pyr-pu-pu-3')2 or (5'-pu-pu-pyr-pyr-3')2 sequences. It was found that TOTO binds preferentially to oligonucleotides containing a (5'-CTAG-3')2 or a (5'-CCGG-3')2 sequence. Binding to the (5'-CCGG-3')2 sequence is less favored than to the (5'-CTAG-3')2 sequence. The complexes of TOTO with d(CGCTAGCGCTAGCG)2 (10) and d(CGCTAGCCGGCG):d(CGCCGGCTAGCG) (11) oligonucleotides, each containing two preferential binding sites, was also examined. In both cases TOTO forms mixtures of 1:1 and 1:2 dsDNA-TOTO complexes in ratios dependent on the relative amount of TOTO and the oligonucleotides in the sample. Binding of TOTO to the two oligonucleotides is sequence selective at the (5'-CTAG-3')2 and (5'-CCGG-3')2 sites. The 1H NMR spectra of both the 1:2 complexes and the three different 1:1 complexes have been assigned. A slight negative cooperativity is observed in formation of the 1:2 complexes. The ratio between the two different 1:1 complexes formed with oligonucleotide 11 is 2.4 in favor of binding to the (5'-CTAG-3')2 site. This is very similar to results obtained when the two sites are in different oligonucleotides. Thus the distribution of TOTO among the (5'-CTAG-3')2 and (5'-CCGG-3')2 sites is independent of whether the two sites are in the same or two different oligonucleotides.Nucleic Acids Research 04/1996; 24(5):859-67. · 8.28 Impact Factor