Fluorescence lifetime distributions of DNA-4',6-diamidino-2-phenylindole complex.
ABSTRACT Time-resolved fluorescence of 4',6-diamidino-2-phenylindole (DAPI) complexes show that for a homogeneous polymer (polyd(AT) or polyd(A).polyd(T)) at high P/D (phosphate/dye) ratio, a single exponential component adequately describes the fluorescence decay. For the AT polymers at low P/D ratio or for native DNA, the decay cannot be described by a single-exponential term. A continuous distribution of lifetime values of Gaussian shape gives a good fit to the decay data. We propose that the lifetime distribution method for the analysis of the fluorescence decay of DNA-DAPI complexes provides a useful method of characterizing the microheterogeneity of site binding.
Article: DNA mediated resonance energy transfer from 4',6-diamidino-2-phenylindole to [Ru(1,10-phenanthroline)2L]2+.[show abstract] [hide abstract]
ABSTRACT: The binding site of Delta- and Lambda-[Ru(phenanthroline)2L]2+ (L being phenanthroline (phen), dipyrido[3,2-a:2'3'-c]phenazine (DPPZ), and benzodipyrido[3,2-a:2'3'-c]phenazine (benzoDPPZ)), bound to poly[d(A-T)2] in the presence and absence of 4',6-diamidino-2-phenylindole (DAPI) was investigated by circular dichroism and fluorescence techniques. DAPI binds at the minor groove of poly[d(A-T)2] and blocks the groove. The circular dichroism spectrum of all Ru(II) complexes are essentially unaffected whether the minor groove of poly[d(A-T)2] is blocked by DAPI or not, indicating that the Ru(II) complexes are intercalated from the major groove. When DAPI and Ru(II) complexes simultaneously bound to poly[d(A-T)2], the fluorescence intensity of DAPI decreases upon increasing Ru(II) complex concentrations. The energy of DAPI at excited state transfers to Ru(II) complexes across the DNA via the Förster type resonance energy transfer. The efficiency of the energy transfer is similar for both [Ru(phen)2DPPZ]2+ and [Ru(phen)2benzoDPPZ]2+ complexes, whereas that of [Ru(phen)3]2+ is significantly lower. The distance between DAPI and [Ru(phen)3]2+ is estimated as 0.38 and 0.64 Förster distance, respectively, for the Delta- and Lambda-isomer.Biophysical Journal 01/2004; 85(6):3865-71. · 3.65 Impact Factor
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ABSTRACT: The fluorescence properties of thiazole orange, linked via a (1) hydrophobic alkyl or a (2) hydrophilic ethylene glycol chain to the central internucleotidic phosphate group of a pentadeca-2'-deoxyriboadenylate (dA15), are evaluated. Linkage at the phosphate group yields two stereoisomers, S-isomer of the phosphorus chiral center (Sp) and R-isomer of the phosphorus chiral center (Rp); these are studied separately. The character of the linkage chain and the chirality of the internucleotidic phosphate linkage site influence the fluorescent properties of these thiazole orange-oligonucleotide conjugates (TO-probes). Quantum yields of fluorescence (phifl) of between 0.04 and 0.07 were determined for the single-stranded conjugates. The fluorescence yield increased by up to five times upon hybridization with the complementary sequence (d5'[CACT15CAC3']); (phifl values of between 0.06-0.35 were determined for the double-stranded conjugates. The phifl value (0.17) of thiazole orange, 1-(N,N'-trimethylaminopropyl)-4-[3-methyl-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene]-quinolinium iodide (TO-Pro 1) in the presence of the oligonucleotide duplex (TO-Pro 1: dA15.d5'[CACT15CAC3'] (1:1)) is much less than that for some of the hybrids of the conjugates. Our studies, using steady-state and time-resolved fluorescence experiments, show that a number of discrete fluorescent association species between the thiazole orange and the helix are formed. Time-resolved studies on the four double-stranded TO-probes revealed that the fluorescent oligonucleotide-thiazole orange complexes are common, only the distribution of the species varies with the character of the chain and the chirality at the internucleotidic phosphate site. Those TO-probes in which the isomeric structure of the phosphate-chain linkage is Rp, and therefore such that the fluorophore is directed toward the minor groove, have higher phifl values than the Sp isomer. Of the systems studied, thiazole orange linked by an alkyl chain to the internucleotidic phosphate (Rp isomer) has the highest phifl and the greatest fraction of the longest-lived fluorescent thiazole orange species (in the hybrid form).Photochemistry and Photobiology 04/2002; 75(3):201-10. · 2.41 Impact Factor