Abstract
The flexibility of calf thymus DNA and several polynucleotides was measured using the anisotropy decay of DAPI bound to DNA, a minor groove probe. DNA torsional dynamics were analyzed using the Schurr model [Allison, S. A., & Schurr, J. M. (1979) Chem. Phys. 41, 35-44] in the infinite polymer length approximation. Time-resolved fluorescence depolarization was measured using a frequency-double mode-locked dye laser and frequency-domain acquisition methods. At very high P/D ratios, the anisotropy decay is dominated by DNA torsional dynamics. The recovered values of the torsional elastic constant were in good agreement with literature values obtained using other DNA probes. The exact knowledge of the angle between the probe emission dipole transition moment and the helix axis is critical for the determination of the polymer elastic constant. At low P/D ratios, energy transfer between dye molecules strongly contributes to the anisotropy decay. We have developed a statistical model that describes the anisotropy decay, when the correct geometrical factors are included. At low P/D ratios the anisotropy decay is dominated by fluorescence homotransfer. In this regime, it is possible to determine the orientation of the dye molecule with respect to the polymer with accuracy. The values obtained for the distance and orientation of the DAPI molecules in solution using the fluorescence measurements are in excellent agreement with those from the crystal structure of the oligonucleotides-DAPI complex by Dickerson's group [Larsen T.A., Goodsell, D. S., Cascio, D., Grzeskowiak, K., & Dickerson, R. E. (1989) J. Biomol. Struct. Dyn. 7, 477-491].
Highlights
Man, 1983a,b; Gratton et al, 1984a,b; Beechem & Gratton, 1988) have offered new possibilities for a better understanding of DNA dynamics
The model used by Schurr assumes that the DNA molecule can be treated as a flexible rod with hydrodynamic properties
In this article we report a time-resolved fluorescence depolarization study of DAPI bound to DNA to determine the potential use of such a minor groove binding ligand for probing nucleic acid structure and dynamics
Summary
4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) was obtained from Boehringer Mannheim and checked for purity by thin layer chromatography. Steady-state fluorescence spectra of DAPI were performed at 460 and 340 nm, for the emission and the excitation, respectively, on the ISS (ISS Inc., Champaign, IL) photon counting spectrofluorometer, at the Laboratory for Fluorescence Dynamics at the University of Illinois at Urbana-Champaign. Steady-state polarization measurements of DAPI complexed with DNA and with polydeoxynucleotides are previously reported (Barcellona & Gratton, 1993). A set of 10-20 different modulation frequencies were employed in the range 10-200 MHz. The emission polarizer was automatically alternated between vertical and horizontal orientations for the measurement of anisotropy decays. Solution concentrations were determined spectrophotometrically using the following molar extinction coefficients: DAPI in buffer solution ) 23 000 M-1 cm-1 at 342 nm, for the polydeoxynucleotides in homopolymers and alternating copolymer sequences were 6000 and 6600 M-1 cm-1, respectively. All experiments each sample was allowed to equilibrate at room temperature for at least 5 min before measurement
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