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

Netzel, Thomas L.; Nafisi, Kambiz; Zhao, Min; Lenhard, J. R.; Johnson, Iain

doi:10.1021/j100051a019

Cited by 203 publications

(279 citation statements)

References 20 publications

(29 reference statements)

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“…These alterations are postulated to indicate different binding modes of the dyes and changes in the accessibility of the dye molecules to the solvent environment (Heller and Greenstock 1994). Phase-sensitive flow cytometric analysis of the DNAbinding fluorochromes provided fluorescence lifetime values in accord with previously published results (Netzel et al 1995;Hochstrasser and Millar 1992;Wadkins and Jovin 1991;Olmsted and Kearns 1977). These results indicate that our flow cytometric measurements of fluorescence lifetimes are accurate, and provide a valuable new parameter for single-cell analyses.…”

Section: Discussionsupporting

confidence: 83%

“…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 dye molecules bound in close proximity. Deuterium oxide (D 2 O; heavy water) increases EB and PI fluorescence Correspondence to: Harry A. Crissman, Life Sciences Div., Los Alamos National Laboratory, LS-4 M888, Los Alamos, NM 87545.…”

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

Sailer

Nastasi

Valdez

et al. 1997

J Histochem Cytochem.

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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...

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Section: Discussionsupporting

confidence: 83%

mentioning

confidence: 99%

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

Sailer

Nastasi

Valdez

et al. 1997

J Histochem Cytochem.

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“…POPO-3 has an excitation maximum at 534 nm and an emission maximum at 570 nm. Although POPO-3 has a high affinity for DNA, it is essentially nonfluorescent in the absence of nucleic acids and exhibits 100-to 1,000-fold fluorescence enhancements upon binding to DNA (16,17). Because POPO-3 is cell impermeant, a detergent, sodium dodecylsulfate (SDS), was required to allow the stain access to the DNA.…”

Section: Materials and Methods Two-dye Systemmentioning

confidence: 99%

Design and characterization of a compact dual channel virus counter

2005

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Background: Although there is a growing need in the field of biotechnology to rapidly and accurately quantify viruses, time-consuming techniques such as the plaque titer method remain the ''gold standard.'' Flow cytometric methods for virus quantification offer the advantages of rapid analysis and statistical treatment. The technique presented in this work represents the first demonstration of a flow cytometric determination of a viral count that is directly related to the count obtained by plaque titer. Methods: A flow cytometric instrument for rapid quantification of virus particles was designed, constructed, and thoroughly characterized. A two-color method, which involved staining the viral genome and the protein coat for baculoviruses, was developed in addition to an algorithm to identify simultaneous events on the DNA and protein channels.

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“…The probability of this situation (with fewer than 1 reversal) occurring is ~1 in 40. More exactly, this probability is (1 -p) 30 + 30 p (1 -p) 29 = 0.023. This difference may signal the requirement for more sophisticated analysis, or indicates the presence of a potentially useful physical effect.…”

Section: -H--hr5kb_mentioning

confidence: 98%