Time-resolved fluorescence of DAPI in solution and bound to polydeoxynucleotides

Fluorescence decay studies, obtained by multifrequency phase-modulation fluorometry,

For polyd(GC)/DAPI complex, the decay was similar to that of free DAPI in solution, proving the dependence on the polydeoxynucleotides sequence of the different types of binding and the reliability of the fluorescence method to solve them.for data acquisition and analysis.
The light source was a HeCd laser emitting at 325 nm.Phase and modulation data were analyzed using a sum of exponentials by a nonlinear least-squares routine (24).Phase-resolved spectra were obtained using the software method described by Gratton and Jameson (25).A solution of POPOP in ethyl alcohol with a lifetime value of 1.35 ns was used as a reference.
The integration time was 2 set per point and data were collected each 5 nm.

Results
The fluorescence decay of an aqueous solution of DAPI at pH 7 was The solid lines correspond to the best fit using two exponential decays.

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 (-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.
l-31, see Figure 1.Previous studies have shown that only the binding between AT, AU, and IC clusters 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 strong binding and suggested a model of interaction in which there is formation in the narrow helical groove of two hydrogen bonds between donor groups of the drug and N3 and 02 atoms of adjacent adenine and thymine rings, with electrostatic interactions.This model is also supported by the molecular structure of DAPI and by the lack of coplanarity of the rings in the molecule (7,9).In this study, we investigated the fluorescence decay of DAPI in pure solvents and bound to natural and synthetic purine-pyrimidine alternating polydeoxynucleotides using multifrequency phase fluorometry, which is particularly suitable to resolve multiexponential decays rapidly and accurately.Furthermore, the technique of phase-and modulation-resolved spectra (21) allowed the direct detection of various components of the emission spectra.Materials and Methods Calf thymus DNA, Co1 El plasmid DNA, poly d(AT), poly d(A)*poly d(T), and poly d(GC) were obtained from Boehringer Mannheim Biochemicals.DNA molecules were checked for protein contamination either by the value of the optical density ratio, A260lA280 > 1.8, or by Cs Cl-Eb buoyant density gradient centrifugation.The two fractions obtained by centrifugation, lower and upper band, corresponding to cc and linear DNA molecules, respectively, were collected and the intercalator removed (22).DAPI was purchased from Serva Biochemicals.The solution concentration was determined using the following molar extinction coefficients: calf thymus DNA (6600); Co1 El plasmid DNA (6600); poly d(AT) (6600); poly d(A)*poly d(T) (6000) and poly d(GC) (8400).A molar extinction coefficient of 23000 M-1 cm-l at 342 nm was used to determine the concentration of DAPI solutions.Depending on the final concentration of the polydeoxynucleotides, different ratios as moles of phosphate (P) to dye (D) were obtained.In the measurements pertaining to solvent and to pH variation effects, typically a small amount of a concentrated DAPI aqueous solution was diluted to the with an ISSlADC interface (ISS, Inc., Champaign, Illinois)

FiP
of the long decay component.F1 = fractional fluorescence intensity of the long lifetime component.T2 = lifetime of the short decay component.theemission maximum of about 10 nm, with respect to the free DALI.The poly d(GC)/DAPI complex, instead, did not exhibit a significant fluorescence enhancement, even at a high phosphate/dye ratio (P/D).The fluorescence decay of linear (1) or covalently closed (cc) DNA and polydeoxynucleotide/API complexes at high P/D ratios is shown in Fig.3aand Table2.The value of the longer component was about 3.6 nsec and gave the major contribution to the decay.The short lifetime component had a larger value than that found for DAPI in aqueous solution and its fractional contribution was strongly reduced.At low P/D ratio (about 1.0, data not shown), the decay was adequately described using two exponentials and both lifetimes had lower values

( 2 .
photophysic.alorigin of the spectroscopic properties of DAPI bound to nucleic acids and can be used to discriminate among the different types of binding.