Torsional dynamics and orientation of DNA--DAPI complexes.
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].
Barcellona
M L
ML
Istituto di Chimica Biologica, Università di Catania, Italy.
Gratton
E
E
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D004247:DNA / Q000737:chemistry*; D004548:Elasticity; D004735:Energy Transfer; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes; D007211:Indoles / Q000737:chemistry*; D008433:Mathematics; D008961:Models, Structural; D008962:Models, Theoretical; D009690:Nucleic Acid Conformation; D011336:Probability; D013050:Spectrometry, Fluorescence; D013950:Thymus Gland
D005456:Fluorescent Dyes; D007211:Indoles; C007293:DAPI; D004247:DNA