Differential effects of deuterium oxide on the fluorescence lifetimes and intensities of dyes with different modes of binding to DNA.

Deuterium oxide (D2O) increases both the fluorescence lifetime and the fluorescence intensity of the intercalating dyes propidium iodide (PI) and ethidium bromide (EB) when bound to nucleic acid structures. We have used spectroscopic analysis coupled with conventional and phase-sensitive flow cytometry to compare the alterations in intensity and lifetime of various DNA-binding fluorochromes bound to DNA and Chinese hamster ovary (CHO) cells in the presence of D2O vs phosphate-buffered saline (PBS). Spectroscopic and flow cytometric studies showed a differential enhancement of intensity and lifetime based on the mode of fluorochrome-DNA interaction. The fluorescence properties of intercalating probes, such as 7-aminoactinomycin D (7.AAD) and ethidium homodimer II (EthD II) were enhanced to the greatest degree, followed by the probes TOTO and YOYO, and the non-intercalating probes Hoechst 33342 (HO) and 4,6-diamidino-2-phenylindole (DAPI). The non-intercalating probe mithramycin (MI) gave unexpected results, showing a great enhancement of fluorescence intensity and lifetime in D2O, indicating that when staining is performed in PBS, much of the MI fluorescence is quenched by the solvent environment. Apoptotic subpopulations of HL-60 cells had a shorter lifetime compared to non-apoptotic subpopulations when stained with EthD II. These results indicate that accessibility of the dye molecules to the solvent environment once bound to DNA, leads to the differential enhancement effects of D2O on fluorescence intensity and lifetime of these probes.