Denaturation and condensation of intracellular nucleic acids monitored by fluorescence depolarization of intercalating dyes in individual cells.

The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society
W Beisker, W G Eisert

Abstract

The intercalating binding of planar aromatic dye molecules to nucleic acids can be analyzed using fluorescence depolarization measurements of the dye molecules excited by linearly polarized light. In this study, we investigated the conformational changes of the intracellular DNA-dye complex in single cells. Flow cytometry, combined with a newly developed double-beam autocompensation technique, permitted rapid high-precision fluorescence depolarization measurements on a large number of individual cells. The dyes ethidium bromide (EB), propidium iodide (PI), and acridine orange (AO) were used in this study. Depending on the dye-to-phosphate ratio of the nuclear acid-dye complex, as well as on the spatial dye structure itself, internal and external binding sites can be monitored by fluorescence depolarization analysis. Both energy transfer and rotation and vibration of the dye molecules cause depolarization of the fluorescence emission. Differences in the concentration-dependent dye fluorescence depolarization values between PI and EB on one side and AO on the other side can be interpreted as a denaturation and condensation of double-stranded DNA regions by AO. We further show that the fluorescence polarization measurement techniq...Continue Reading

Citations

May 11, 2000·Cytometry·C L AsburyG van den Engh
Apr 1, 1992·Biophysical Journal·M LudwigR J Hartzman
Jun 6, 2000·Advanced Drug Delivery Reviews·M B BallyD L Reimer
Sep 29, 2009·Expert Opinion on Drug Delivery·Yan DuanDefu Zhi

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