TORSIONAL DYNAMICS AND ORIENTATION OF DNA-DAPI COMPLEXES

The flexibility of calf thymus DNA and several polynucleotides was mea sured using the anisotropy decay of DAPI bound to DNA, a minor groove probe. DNA torsional dynamics were analyzed using the Schurr model [Al lison, S. A., & Schurr, J. M. (1979) Chem. Phys. 41, 35-44] in the inf inite polymer length approximation. Time-resolved fluorescence depolar ization was measured using a frequency-doubled mode-locked dye laser a nd 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 elasti c constant. At low P/D ratios, energy transfer between dye molecules s trongly contributes to the anisotropy decay. We have developed a stati stical model that describes the anisotropy decay, when the correct geo metrical factors are included. At low P/D ratios the anisotropy decay is dominated by fluorescence homotransfer. In this regime, it is possi ble to determine the orientation of the dye molecule with respect to t he polymer with accuracy. The values obtained for the distance and ori entation of the DAPI molecules in solution using the fluorescence meas urements are in excellent agreement with those from the crystal struct ure 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].