Abstract
Fluorescence decay studies, obtained by multifrequency phase-modulation fluorometry, have been performed on DAPI in solution and complexed with natural and synthetic polydeoxynucleotides. DAPI decay at pH 7 was decomposed using two exponential components of 2.8 and 0.2 ns of lifetime values, respectively. The double exponential character of the decay was maintained over a large pH range. Phase- and modulation-resolved spectra, collected between 420 and 550 nm, have indicated at least two spectral components associated with the two lifetime values. This, plus the observation of the dependence of the emission spectrum on the excitation wavelength, suggests a lifetime heterogeneity originating from ground-state molecular conformers, partially affected by pH changes. DAPI complexed with natural polydeoxynucleotides retained most of the features of DAPI decay in solution, except for the value of the long lifetime component that was longer (approximately 4 ns) and the relative fractional fluorescence intensities of the two components that were inverted. AT polymers/DAPI complexes show single exponential decay. Solvent shielding when DAPI is bound to DNA changes the indole ring solvation and stabilizes the longer lifetime decay component. For poly(GC)/DAPI complex, the decay was similar to that of free DAPI in solution, proving the dependence on the polydeoxynucleotides sequence the different types of binding and the reliability of the fluorescence method to solve them.
Highlights
Fluorescence decay studies, obtained by multifrequency phase-modulation fluorometry, have been performed on DAPI in solution and complexed with natural and synthetic polydeoxynucleotides
Phase- and modulationresolved spectra were virtually identical, confirming that only two major spectral components associated with the two lifetime values were present: the long one, with an emission maximum near 445 nm and the short one, with a broad maximum centered at 482 nm and with a long red tail (Fig. 2)
The analysis of the DAPI fluorescence decay collected over the large pH range from pH 2 to pH 10 using two exponential components (Table 1) showed a progressive lengthening of both lifetime values from 2.0 to 3.3 nsec for the long component and from 0.06 to 1.40 nsec for the short one
Summary
Fluorescence decay studies, obtained by multifrequency phase-modulation fluorometry, have been performed on DAPI in solution and complexed with natural and synthetic polydeoxynucleotides. Previous studies have shown that only the binding between AT, AU, and IC clusters is responsible for the fluorescent complex formation [4] Because of this property, DAPI is currently used in a number of biochemical and cytochemical investigations, including a staining procedure for the selective visualization of some huma.n chromosomes, as well as to probe the molecular environment of restriction endonuclease cleavage sites, the effectiveness in inhibiting initiation at the Zac UV5 promoter by E.coli RNA polymerase, and to act as a possible modulator Iof a genetic expression [5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20]. The strong fluorescence enhancement following DNA binding has been interpreted in terms of two different modes of binding: [1] a highly energetic (-8 Kcal/mol of DAPI) and intercalative type of interaction [1,4], characterized by a high value of the affinity constant and a high specificity for AT, AU, IC clusters; and [2] a weaker one, nonspecific, with a lower affinity constant and no larger fluorescence enhancement [2,3,5,6]
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