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 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 D2O on fluorescence intensity and lifetime of these probes.

[1]  H. Crissman,et al.  Staining of DNA in live and fixed cells. , 1994, Methods in cell biology.

[2]  T. Jovin,et al.  Actinomycin D and 7-aminoactinomycin D binding to single-stranded DNA. , 1991, Biochemistry.

[3]  I Schmid,et al.  Dead cell discrimination with 7-amino-actinomycin D in combination with dual color immunofluorescence in single laser flow cytometry. , 1992, Cytometry.

[4]  L. Masotti,et al.  EXCITED STATE pKa BEHAVIOUR OF DAPI. A RATIONALIZATION OF THE FLUORESCENCE ENHANCEMENT OF DAPI IN DAPI‐NUCLEIC ACID COMPLEXES * , 1986 .

[5]  Joseph R. Lakowicz,et al.  Time-resolved laser spectroscopy in biochemistry II , 1990 .

[6]  Robert C. Habbersett,et al.  Improved multilaser/multiparameter flow cytometer for analysis and sorting of cells and particles , 1991 .

[7]  Z. Darżynkiewicz,et al.  A selective procedure for DNA extraction from apoptotic cells applicable for gel electrophoresis and flow cytometry. , 1994, Analytical biochemistry.

[8]  Z. Darżynkiewicz,et al.  Features of apoptotic cells measured by flow cytometry. , 1992, Cytometry.

[9]  David P. Millar,et al.  Fluorescence self-quenching of ethidium bromide intercalated in DNA , 1992, Photonics West - Lasers and Applications in Science and Engineering.

[10]  T. Dewey,et al.  Biophysical and Biochemical Aspects of Fluorescence Spectroscopy , 2013, Springer US.

[11]  F. Dörr.  Mechanisms of Energy Transfer , 1983 .

[12]  J A Steinkamp,et al.  Interactions of intercalating fluorochromes with DNA analyzed by conventional and fluorescence lifetime flow cytometry utilizing deuterium oxide. , 1996, Cytometry.

[13]  J. Olmsted,et al.  Mechanism of ethidium bromide fluorescence enhancement on binding to nucleic acids. , 1977, Biochemistry.

[14]  J. Steinkamp,et al.  Three-color fluorescence measurements on single cells excited at three laser wavelengths. , 2005, Cytometry.

[15]  C. Greenstock,et al.  Fluorescence lifetime analysis of DNA intercalated ethidium bromide and quenching by free dye. , 1994, Biophysical chemistry.

[16]  Richard P. Haugland,et al.  Handbook of fluorescent probes and research chemicals , 1996 .

[17]  J A Steinkamp,et al.  Fluorescence lifetime measurements in a flow cytometer by amplitude demodulation using digital data acquisition technique. , 1994, Cytometry.

[18]  M. V. Lancker,et al.  A Comparison of Four Frequently Used Assays for Quantitative Determination of DNA , 1986 .

[19]  D. Ward,et al.  Base Specificity in the Interaction of Polynucleotides with Antibiotic Drugs , 1965, Science.

[20]  T. Netzel,et al.  Base-Content Dependence of Emission Enhancements, Quantum Yields, and Lifetimes for Cyanine Dyes Bound to Double-Strand DNA: Photophysical Properties of Monomeric and Bichromomphoric DNA Stains , 1995 .

[21]  R. Humphrey,et al.  Comparison of ethidium bromide and 4'6-diamidino-2-phenylindole as quantitative fluorescent stains for DNA in agarose gels. , 1982, Journal of Biochemical and Biophysical Methods.

[22]  J A Steinkamp,et al.  Resolution of fluorescence signals from cells labeled with fluorochromes having different lifetimes by phase-sensitive flow cytometry. , 1993, Cytometry.

[23]  J. Lakowicz,et al.  Phase-resolved fluorescence lifetime measurements for flow cytometry. , 1993, Cytometry.

[24]  H. Crissman,et al.  TOTO and YOYO: new very bright fluorochromes for DNA content analyses by flow cytometry. , 1994, Cytometry.

[25]  E. Gratton,et al.  Time-resolved fluorescence of DAPI in solution and bound to polydeoxynucleotides. , 1990, Biochemical and biophysical research communications.

[26]  Z. Darżynkiewicz,et al.  Changes in accessibility of DNA to various fluorochromes during spermatogenesis. , 1986, Cytometry.

[27]  M. Melamed,et al.  Accessibility of DNA in situ to various fluorochromes: relationship to chromatin changes during erythroid differentiation of Friend leukemia cells. , 1984, Cytometry.

[28]  J. Kapuscinski,et al.  Interactions of 4', 6-diamidine-2-phenylindole with synthetic polynucleotides. , 1979, Nucleic acids research.

[29]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[30]  L. G. Lee,et al.  Thiazole orange: a new dye for reticulocyte analysis. , 1986, Cytometry.

[31]  R A Mathies,et al.  A stable double-stranded DNA-ethidium homodimer complex: application to picogram fluorescence detection of DNA in agarose gels. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[32]  John A. Steinkamp,et al.  Flow cytometer for resolving signals from heterogeneous fluorescence emissions and quantifying lifetime in fluorochrome-labeled cells/particles by phase-sensitive detection , 1993 .

[33]  G. Mazzini,et al.  A flow cytometric study of the propidium iodide staining kinetics of human leukocytes and its relationship with chromatin structure. , 1983, Cytometry.