Spectral analysis of the DNA targeting bisalkylaminoanthraquinone DRAQ5 in intact living cells

Abstract

Background: We report on the potential DNA binding modes and spectral characteristics of the cell-permeant far red fluorescent DNA dye, DRAQ5, in solution and bound within intact cells. Our aim was to determine the constraints for its use in flow cytometry and bioimaging. Methods: Solution characteristics and quantum yields were determined by spectroscopy. DRAQ5 binding to nuclear DNA was analyzed using fluorescence quenching of Hoechst 33342 dye, emission profiling by flow cytometry, and spectral confocal laser scanning microscopy of the complex DRAQ5 emission spectrum. Cell cycle profiling utilized an EGFP-cyclin B1 reporter as an independent marker of cell age. Molecular modeling was used to explore the modes of DNA binding. Results: DRAQ5 showed a low quantum yield in solution and a spectral shift upon DNA binding, but no significant fluorescence enhancement. DRAQ5 caused a reduction in the fluorescence intensity of Hoechst 33342 in live cells prelabeled with the UV excitable dye, consistent with molecular modeling that suggests AT preference and an en-

Modified Annexin V/Propidium Iodide Apoptosis Assay For Accurate Assessment of Cell Death

“…True positive events display the expected co-localization between PI and DRAQ5 within the nuclear compartment (yellow areas depict colocalization between PI and DRAQ5). DRAQ5 is highly membrane permeable dye that accurately stains nuclear compartment in both live and dead cells 10,11,12 . For easier visual determination of colocalization DRAQ5 stain was given a green pseudocolour.…”

Nanomechanics of Fluorescent DNA Dyes on DNA Investigated by Magnetic Tweezers

“…Yet, the binding mode of DRAQ5 to DNA is still debated. Japardize et al (15) and Martin et al (17) suggested DNA intercalation as the dominating binding mechanism whereas Njoh et al (18) concluded in their molecular docking studies that the binding mode can adequately be described by AT preferred engagement in the DNA minor groove. A more competitive bi-mechanistic interaction was suggested by Islam et al (19) in their theoretical modeling experiments, however, without experimentally investigating further associated nanomechanical properties.…”

Reliability of confocal microscopy spectral imaging systems: Use of multispectral beads

“…This equipment has the capacity to obtain unique fluorescence spectra of specific dye molecules that will increase our understanding of biological fluorescence emission process (9)(10)(11)(12)(13)(14)(15)(16). The popularity of this new field in biology has been described in a special issue of Cytometry that includes both review articles and specific application articles (17)(18)(19)(20)(21)(22)(23)(24)(25)(26). To insure that this equipment is functioning properly, new and improved tests have been developed to evaluate spectral performance on these instruments (1,2,5,6).…”