Differential Effects of Deuterium Oxide on the Fluorescence Lifetimes and Intensities of Dyes with Different Modes of Binding to DNA

Abstract

SUMMARY Deuterium oxide (D 2 O) 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 D 2 O 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 Ј ,6-diamidino-2-phenylindole (DAPI). The non-intercalating probe mithramycin (MI) gave unexpected results, showing a great enhancement of fluorescence intensity and lifetime in D 2 O, 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 nonapoptotic 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 D 2 O on fluorescence intensity and lifetime of these probes. D na-binding fluorochromes have been used extensively in many applications, including flow cytometry and gel and capillary electrophoresis. Fluorochromes utilized in these applications must satisfy several criteria. They must bind specifically to nucleic acids, there must be a low quantum yield as free dye in solution, and there must be enhancement of quantum yield on binding to nucleic acid structures. Several classes of DNA-specific fluorochromes with different modes of base pair ( bp ) binding are available: (a) the DNA intercalators propidium iodide (PI) and ethidium bromide (EB), which lack appreciable bp specificity; (b) DNA intercalators with A-T bp preference, such as ethidium homodimer II (EthD II) or with G-C preference, such as 7-amino actinomycin D (7-AAD); and (c) non-intercalating probes with either A-T bp specificity, such as Hoechst 33342 (HO) or 4 Ј ,6-diamidino-2-phenylindole (DAPI) or with G-C specificity, such as mithramycin (MI). Two high quantum yield cyanine fluorochromes, TOTO and YOYO, are dimers of the dyes thiazole orange and oxazole yellow, respectively (Lee et al. 1986). These fluorochromes are believed to bind to DNA by intercalation, without base specificity (Haugland 1992) but with some base sequence preference (Netzel et al. 1995).Enhancement of the fluorescence intensity of DNAbinding probes improves the signal-to-noise ratio, allowing the use of lower dye concentrations and thereby reducing potential self-quenching between d...

A Fluorescence Study of the Structure and Accessibility of Plasmid DNA Condensed with Cationic Gene Delivery Vehicles

“…Since H 2 O and D 2 O are known to differentially quench HOC, 38 differences in spectral responses during titrations performed in the two solvents would confirm a role for solvation changes in the observed spectral changes in HOC-DNA upon the binding of cations. When HOC-DNA is dissolved in deuterated buffer, a 30% increase in fluorescence and a substantial red shift in the wavelength of emission maximum are observed.…”

“…These results are in agreement with previous reports. 38 Titrations of DOTAP into HOC-DNA were performed in both solvents and the wavelength of the emission maximum was monitored as a function of charge ratio (Fig. 8).…”

A protocol to effectively create single cell suspensions of adherent cells for multiparameter high-throughput flow cytometry

“…680 nm. The fluorescence lifetimes of these and most organic fluorophores are less than 20 ns (Watt and Voss 1979;Araki and Yamada 1985;Sailer et al 1997;Buschmann et al 2003). Therefore, fluorophores with similar emission, but different excitation profiles, can be analyzed on the same system for the same event by utilizing temporal discrimination.…”

Apoptosis induced with different cycle-perturbing agents produces differential changes in the fluorescence lifetime of DNA-bound ethidium bromide

“…However, the position of the apoptotic peak relative to the nonapoptotic G 0 /G 1 peak was different depending on the fluorochrome utilized. For example, when labeled with the nonspecific intercalating fluorochrome PI, the ratio of the apoptotic peak mean to the G 0 /G 1 peak mean was 6.5, while staining with nonintercalating fluorochromes, such as A-T-specific Hoechst, yielded a ratio of 2.4, and G-C specific mithramycin provided a ratio of 2.1 (23). Although all fluorochromes tested so far demonstrate the sub-G 0 /G 1 apoptotic subpopulation in the DNA content distribution, only some fluorochromes such as PI, EB, or EthD II, but not necessarily all of the dyes which also bind by DNA intercalation, demonstrate lifetime alterations when bound to the chromatin remaining within apoptotic cells (22,23).…”