Nucleic Acid Stains—Section 8.1

Page Contents

• Properties of Cyanine Dyes
• Premier Cyanine Dyes for Ultrasensitive Nucleic Acid Detection and Quantitation
• Cell-Impermeant Cyanine Dimers: The TOTO Family of Dyes
• Cell-Impermeant Cyanine Monomers: The TO-PRO Family of Dyes
• Cell-Impermeant SYTOX Dyes for Dead-Cell Staining
• Cell-Permeant Cyanine Dyes: The SYTO Nucleic Acid Stains
• Amine-Reactive Cyanine Dye
• Phenanthridines and Acridines: Classic Intercalating Dyes
• Indoles and Imidazoles: Classic Minor Groove–Binding Dyes
• Other Nucleic Acid Stains
• Data Table
• Ordering Information

We offer an extensive assortment of nucleic acid stains, many of which have been developed in our research laboratories. This section discusses the physical properties of the various classes of dyes listed below. The sections that follow describe applications of these nucleic acid stains for genomics research.

The four classes of Molecular Probes® cyanine dyes include:

• Premier dyes for ultrasensitive nucleic acid quantitation and gel staining (Specialty nucleic acid reagents for molecular biology—Table 8.1)
• Cell-impermeant TOTO, TO-PRO and SYTOX families of dyes (Cell membrane–impermeant cyanine nucleic acid stains—Table 8.2)
• Cell-permeant SYTO family of dyes (Cell-permeant cyanine nucleic acid stains—Table 8.3)
• Amine-reactive SYBR dye that can be used to form bioconjugates

The three classes of classic nucleic acid stains (Properties of classic nucleic acid stains—Table 8.4) include:

• Intercalating dyes (Figure 8.1.1), such as ethidium bromide and propidium iodide
• Minor-groove binders (Figure 8.1.1), such as DAPI and the Hoechst dyes
• Other nucleic acid stains, including acridine orange, 7-AAD, LDS 751 and hydroxystilbamidine

Figure 8.1.1 Schematic diagram showing the different binding modes of dyes (and other ligands) to DNA.

Molecular Probes® nucleic acid–binding cyanine dyes share several important spectroscopic and physical properties:

• High molar absorptivity, with extinction coefficients typically greater than 50,000 cm^-1M^-1 at visible wavelengths
• Very low intrinsic fluorescence, with quantum yields usually less than 0.01 when not bound to nucleic acids
• Large fluorescence enhancements (often over 1000-fold) upon binding to nucleic acids, with increases in quantum yields to as high as 0.9
• Moderate to very high affinity for nucleic acids, with little or no staining of other biopolymers

Their fluorescence absorption and emission spectra span the visible-light spectrum from blue to near-infrared (Figure 8.1.2) with additional absorption peaks in the UV, making them compatible with many different types of instrumentation. The cyanine dyes also show important differences in some physical characteristics—particularly in cell membrane permeability and nucleic acid specificity—that allow their distribution into distinct classes, and these classes are discussed in detail below and in the following sections of this chapter.

Several of our cyanine dyes provide exceptional sensitivity in specific nucleic acid assays (Specialty nucleic acid reagents for molecular biology—Table 8.1). For these dyes, we have developed detailed and extensively tested protocols to facilitate reproducible, high-sensitivity results in these assays.

• PicoGreen, OliGreen and RiboGreen quantitation reagents and their Quant-iT reagent counterparts in Nucleic Acid Quantitation in Solution—Section 8.3 set a benchmark for the detection and quantitation of DNA, oligonucleotides and RNA in solution. These reagents offer extremely simple and rapid protocols as well as linear ranges that span up to four orders of magnitude in nucleic acid concentration.
• SYBR Gold, SYBR Green I and SYBR Green II nucleic acid gel stains in Nucleic Acid Detection on Gels, Blots and Arrays—Section 8.4 are ultrasensitive gel stains that surpass the sensitivity of ethidium bromide by more than an order of magnitude in nucleic acid detection.
• SYBR Safe DNA gel stain (Nucleic Acid Detection on Gels, Blots and Arrays—Section 8.4) is not only significantly less mutagenic than ethidium bromide, but SYBR Safe stain's detection sensitivity is comparable to that of ethidium bromide. SYBR Safe stain showed no or very low mutagenic activity when tested by an independent, licensed testing laboratory, and it is not classified as hazardous waste under U.S. Federal regulations.
• CyQUANT GR dye (C7026), discussed in Assays for Cell Enumeration, Cell Proliferation and Cell Cycle—Section 15.4, is a cyanine dye designed to quantitate cell proliferation and can reliably detect the nucleic acids in as few as 50 cells.

The cyanine dimer dyes listed in Cell membrane–impermeant cyanine nucleic acid stains—Table 8.2—sometimes referred to as the TOTO family of dyes—are symmetric dimers of cyanine dyes with exceptional sensitivity for nucleic acids. This sensitivity is due to a high affinity for nucleic acids, in combination with a very high fluorescence enhancement and quantum yield upon binding. The unique physical characteristics of these dyes and some illustrative applications are discussed below; specific applications are discussed in later sections of this chapter.

Each of the cyanine dimer dyes is available separately. For researchers designing new applications, the Nucleic Acid Stains Dimer Sampler Kit (N7565) provides samples of eight spectrally distinct analogs of the dimeric cyanine dyes for testing (Cell membrane–impermeant cyanine nucleic acid stains—Table 8.2).

High Affinity for Nucleic Acids

Appropriately designed dimers of nucleic acid–binding dyes have nucleic acid–binding affinities that are several orders of magnitude greater than those of their parent monomer dyes. For example, the intrinsic DNA binding affinity constants of ethidium bromide and ethidium homodimer-1 (E1169) are reported to be 1.5 × 10⁵ and 2 × 10⁸ M^-1, respectively, in 0.2 M Na⁺. As a result, the dimeric cyanine dyes are among the highest-affinity fluorescent probes available for nucleic acid staining.

For example, in the TOTO-1 dimeric cyanine dye (T3600), the positively charged side chains of the TO-PRO-1 monomeric cyanine dye (T3602, ) are covalently linked to form the TOTO-1 molecule, with four positive charges (). This linkage gives TOTO-1 dye a greatly enhanced affinity for nucleic acids—more than 100 times greater than that of the TO-PRO-1 monomer. TOTO-1 dye exhibits a higher affinity for double-stranded DNA (dsDNA) than even the ethidium homodimers and also binds to both single-stranded DNA (ssDNA) and RNA. The extraordinary stability of TOTO-1–nucleic acid complexes allows the dye–DNA association to remain stable, even during electrophoresis (); thus, samples can be stained with nanomolar dye concentrations prior to electrophoresis, thereby reducing the hazards inherent in handling large volumes of ethidium bromide staining solutions. In contrast, the binding of thiazole orange—the parent compound of TOTO-1 and TO-PRO-1—is rapidly reversible, limiting the dye's sensitivity and rendering its nucleic acid complex unstable to electrophoresis.

High Fluorescence Enhancements and High Quantum Yields upon Binding to Nucleic Acids

In addition to their superior binding properties, TOTO-1 dye and the other cyanine dimers are essentially nonfluorescent in the absence of nucleic acids and exhibit fluorescence enhancements upon DNA binding of 100- to 1000-fold, which compares favorably with the fluorescence enhancement of thiazole orange upon DNA binding (~3000-fold). Furthermore, the fluorescence quantum yields of the cyanine dimers bound to DNA are high (generally between 0.2 and 0.6), and their extinction coefficients are an order of magnitude greater than those of the ethidium homodimers. This sensitivity is sufficient for detecting single molecules of labeled nucleic acids by optical imaging () and flow cytometry and for tracking dye-labeled virus particles in microbial communities and aquatic systems by fluorescence microscopy. These dyes are generally considered to be cell impermeant, although their use to stain reticulocytes permeabilized by 5% DMSO has been reported.

Modifying the Dimers Creates Compounds with Different Spectral Characteristics

Simply by changing the aromatic rings and the number of carbon atoms linking the cyanine monomers, we were able to synthesize an extended series of these dyes with different spectral characteristics (Cell membrane–impermeant cyanine nucleic acid stains—Table 8.2). Chemical modifications produce dramatic shifts in the absorption and emission spectra and reduce the quantum yields of the bound dyes but cause little or no change in their high affinity for DNA. The names of the dyes reflect their basic structure and spectral characteristics. For example, YOYO-1 iodide (491/509) has one carbon atom bridging the aromatic rings of the oxacyanine dye and exhibits absorption/emission maxima of 491/509 nm when bound to dsDNA. YOYO-3 dye (612/631)—which differs from YOYO-1 dye only in the number of bridging carbon atoms—has absorption/emission maxima of 612/631 nm when bound to dsDNA. Fluorescence spectra for the POPO, BOBO, YOYO, TOTO, JOJO and LOLO dyes bound to dsDNA are shown in Figure 8.1.2. The spectra of these dyes at dye:base ratios of less than 1:1 are essentially the same for the corresponding dye–ssDNA and dye–RNA complexes. At higher dye:base ratios, however, ssDNA and RNA complexes of all of the monomethine ("-1") dyes of the TOTO series and TO-PRO series have red-shifted emissions, whereas corresponding complexes of the trimethine ("-3") analogs do not. Thus, the cyanine dimer family provides dyes with a broad range of spectral characteristics to match the output of almost any available excitation source.

Figure 8.1.2 Normalized fluorescence emission spectra of DNA-bound cyanine dimers, identified by the color key on the sidebar.

Binding Modes of the Cyanine Dimers

The studies on cyanine dimer binding modes have focused on the YOYO-1 and TOTO-1 dyes. YOYO-1 dye was found to exhibit at least two distinct binding modes. At low dye:base pair ratios, the binding mode appears to consist primarily of bis-intercalation (Figure 8.1.1). Each monomer unit intercalates between bases, with the benzazolium ring system sandwiched between the pyrimidines and the quinolinium ring between the purine rings, causing the helix to unwind. The distortion in the local DNA structure caused by YOYO-1 bis-intercalation has been observed by two-dimensional NMR spectroscopy. At high dye:base pair ratios, a second, less well characterized mode of external binding begins to contribute. Circular dichroism measurements also indicate a possible difference in the binding modes of YOYO-1 dye to ssDNA and dsDNA. These data are consistent with our own results, including the observation that the fluorescence emission of the YOYO-1 dye complex with nucleic acids shifts to longer wavelengths at high dye:base ratios upon binding to single-stranded nucleic acids and that the salt, ethanol and sodium dodecyl sulfate (SDS) sensitivity of YOYO-1 dye binding to DNA is a function of the dye:base pair ratio.

TOTO-1 dye is also capable of bis-intercalation, although it reportedly interacts with dsDNA and ssDNA with similarly high affinity. NMR studies of TOTO-1 dye interactions with a double-stranded 8-mer indicate that TOTO-1 dye is a bis-intercalator, with the fluorophores intercalating between the bases and the linker region having interactions in the minor groove (Figure 8.1.3). Binding of the dye partially unwinds the DNA, distorting and elongating the helix. However, another study using fluorescence polarization measurements suggests that an external binding mode, where the dipole of the dye molecule is aligned with the DNA grooves, may be more important. TOTO-1 dye reportedly exhibits some sequence selectivity for the site 5'-CTAG-3', although it will bind to almost any sequence in dsDNA. TOTO-1 dye does not exhibit cooperative binding to DNA, suggesting that it may be a suitable dye for detecting nucleic acids in gels.

The binding modes of the other members of the TOTO dye series have also been partially characterized. Electrophoresis and fluorescence lifetime measurements have shown that YOYO-3 dye also appears to intercalate into DNA. During application development, we have determined that staining of nucleic acids by BOBO-1 and POPO-1 dyes is much faster (occurring within minutes) than staining by YOYO-1 or TOTO-1 dyes (which can take several hours to reach equilibrium under the same experimental conditions), indicating possible differences in their binding mechanisms. Fluorescence yield and lifetime measurements have been used to assess the base selectivity of an extensive series of these dyes. Circular dichroism measurements have shown that bis-intercalation is the predominant binding mode for the POPO-1 dye.

Figure 8.1.3 NMR solution structure of the TOTO-1 dye (T3600) bound to DNA; the image was derived from data submitted to the Protein Data Bank (number PDB 108D, www.rcsb.org/pdb/ ). The NMR structure shows that TOTO-1 binds to DNA through bis-intercalation.

Working with Cyanine Dimers

All of the dyes in the TOTO series (Cell membrane–impermeant cyanine nucleic acid stains—Table 8.2) are supplied as 1 mM solutions in dimethylsulfoxide (DMSO), except for POPO-3 (P3584), which is supplied as a 1 mM solution in dimethylformamide (DMF). These cationic dyes appear to be readily adsorbed out of aqueous solutions onto surfaces (particularly glass) but are very stable once complexed to nucleic acids.

Our TO-PRO family of dyes, all of which are listed in Cell membrane–impermeant cyanine nucleic acid stains—Table 8.2, each comprise a single cyanine dye and a cationic side chain (). The monomeric dyes in the TO-PRO series are spectrally analogous to the corresponding dimeric cyanine dyes; however, with only two positive charges and one intercalating unit, the TO-PRO dyes exhibit somewhat reduced affinity for nucleic acids relative to the dyes in the TOTO series. Like their dimeric counterparts, these monomeric cyanine dyes are typically impermeant to cells, although YO-PRO-1 (Y3603) dye has been shown to be permeant to apoptotic cells, providing a convenient indicator of apoptosis (Assays for Apoptosis—Section 15.5). YO-PRO-1 has also been observed to pass through P2X₇ receptor channels of live cells.

Spectral Characteristics of the Cyanine Dye Monomers

The TO-PRO family of dyes retains all of the exceptional spectral properties of the dimeric cyanine dyes discussed above. The absorption and emission spectra of these monomeric cyanine dyes cover the visible and near-infrared spectrum (Cell membrane–impermeant cyanine nucleic acid stains—Table 8.2). They also have relatively narrow emission bandwidths, thus facilitating multicolor applications in imaging and flow cytometry. YO-PRO-1 (491/509) and TO-PRO-1 (515/531) dyes are optimally excited by the 488 nm and 514 nm spectral lines of the argon-ion laser, respectively. In flow cytometric analysis, the TO-PRO-3 (642/661) complex with nucleic acids has been excited directly by the red He-Ne laser and indirectly by the argon-ion laser by using fluorescence resonance energy transfer (FRET) from co-bound propidium iodide (Fluorescence Resonance Energy Transfer (FRET)—Note 1.2). The TO-PRO-3 complex with nucleic acids has also been detected in a flow cytometer equipped with an inexpensive 3 mW visible-wavelength diode laser that provides excitation at 635 nm. Although the DNA-induced fluorescence enhancement of TO-PRO-5 dye (T7596) is not as large as that observed with our other cyanine dyes, its spectral characteristics (excitation/emission maxima ~745/770 nm) provide a unique alternative for multicolor applications.

Working with Cyanine Monomers

The binding affinity of the TO-PRO series of dyes to dsDNA is lower than that of the TOTO series of dyes but is still very high, with dissociation constants in the micromolar range. TO-PRO dyes also bind to RNA and ssDNA, although typically with somewhat lower fluorescence quantum yields. Fluorescence polarization studies indicate that TO-PRO-1 and PO-PRO-1 dyes bind by intercalation, with unwinding angles of 2° and 31°, respectively. Binding of these dyes to dsDNA is not sequence selective. All dyes of the TO-PRO series (Cell membrane–impermeant cyanine nucleic acid stains—Table 8.2) are supplied as 1 mM solutions in DMSO.

SYTOX Green Stain

Our SYTOX nucleic acid stains (Cell membrane–impermeant cyanine nucleic acid stains—Table 8.2) are cell-impermeant cyanine dyes that are particularly useful as dead-cell stains. SYTOX Green nucleic acid stain (S7020, S34860) is a high-affinity nucleic acid stain that easily penetrates cells with compromised plasma membranes and yet will not cross the membranes of live cells. It is especially useful for staining both gram-positive and gram-negative bacteria, in which an exceptionally bright signal is required. Following brief incubation with the SYTOX Green stain, dead cells fluoresce bright green when excited with the 488 nm spectral line of the argon-ion laser or with any other 450–500 nm source (). Because all of the SYTOX dyes are essentially nonfluorescent in aqueous medium, no wash steps are required. Unlike the DAPI or Hoechst dyes, the SYTOX Green nucleic acid stain shows little base selectivity. These properties, combined with its ~1000-fold fluorescence enhancement upon nucleic acid binding and high quantum yield, make our SYTOX Green stain a simple and quantitative single-step dead-cell indicator for use with epifluorescence and confocal laser-scanning microscopes, fluorometers, fluorescence microplate readers and flow cytometers (Figure 8.1.4).

The SYTOX Green nucleic acid stain can be used with blue- and red-fluorescent labels for multiparameter analyses (). It is also possible to combine the SYTOX Green nucleic acid stain with the SYTO 17 red-fluorescent nucleic acid stain (S7579) for two-color visualization of dead and live cells. Because the SYTOX Green nucleic acid stain is an excellent DNA counterstain for chromosome labeling and for fixed cells and tissues (), we have incorporated it into our SelectFX Nuclear Labeling Kit (S33025), which is discussed in Probes for the Nucleus—Section 12.5.

Figure 8.1.4 Quantitative flow cytometric analysis of Escherichia coli viability using the SYTOX Green nucleic acid stain (S7020). A bacterial suspension containing an equal number of live and isopropyl alcohol–killed E. coli was stained with SYTOX Green and analyzed using excitation at 488 nm. A bivariate frequency distribution for forward light scatter versus log fluorescence intensity (collected with a 510 nm longpass optical filter) shows two clearly distinct populations. When live and dead bacteria were mixed in varying proportions, a linear relationship between the population numbers and the actual percentage of live cells in the sample was obtained (see inset).

SYTOX Blue Stain

SYTOX Blue stain (S11348, S34857) is a high-affinity nucleic acid stain that typically penetrates only cells with compromised plasma membranes (). The SYTOX Blue stain labels both DNA and RNA with extremely bright fluorescence centered near 470 nm (). The absorption maximum of the nucleic acid–bound SYTOX Blue stain (~445 nm) permits very efficient fluorescence excitation by the 436 nm spectral line of the mercury-arc lamp. Unlike many blue-fluorescent dyes, the SYTOX Blue stain is also efficiently excited by tungsten–halogen lamps and other sources that have relatively poor emission in the UV portion of the spectrum. The brightness of the SYTOX Blue stain allows sensitive detection with fluorometers, microplate readers, arc-lamp–equipped flow cytometers and epifluorescence microscopes, including those not equipped with UV-pass optics.

In a side-by-side comparison with the SYTOX Green stain, the SYTOX Blue stain yielded identical results when quantitating membrane-compromised bacterial cells. Furthermore, like the SYTOX Green stain, the SYTOX Blue stain does not interfere with bacterial cell growth. Because their emission spectra overlap somewhat, we have found that it is not ideal to use the SYTOX Blue stain and green-fluorescent dyes together; however, fluorescence emission of the SYTOX Blue stain permits clear discrimination from orange- or red-fluorescent probes, facilitating the development of multicolor assays with minimal spectral overlap between signals.

SYTOX Orange Stain

SYTOX Orange nucleic acid stain (S11368, S34861) is designed to clearly distinguish dead bacteria, yeast or mammalian cells from live cells. As compared with propidium iodide, SYTOX Orange stain has shorter-wavelength emission and its spectra more closely matches the rhodamine filter set (). In addition, the SYTOX Orange stain has a much higher molar absorptivity (extinction coefficient) than propidium iodide and a far greater fluorescence enhancement upon binding DNA, suggesting that it may have a higher sensitivity as a dead-cell stain or as a nuclear counterstain. The SYTOX Orange stain was shown to be extremely useful for DNA fragment sizing by single-molecule flow cytometry when using a Nd:YAG excitation source, with a 450-fold enhancement upon binding to dsDNA.

SYTO Nucleic Acid Stains for DNA and RNA

The SYTO dyes are somewhat lower-affinity nucleic acid stains that passively diffuse through the membranes of most cells. These UV- or visible light–excitable dyes can be used to stain RNA and DNA in both live and dead eukaryotic cells, as well as in gram-positive and gram-negative bacteria. We have synthesized a large number of SYTO dyes (Cell-permeant cyanine nucleic acid stains—Table 8.3) that share several important characteristics:

• Permeability to virtually all cell membranes, including mammalian cells and bacteria (Assays for Cell Viability, Proliferation and Function—Chapter 15)
• High molar absorptivity, with extinction coefficients greater than 50,000 cm^-1M^-1 at visible absorption maxima
• Extremely low intrinsic fluorescence, with quantum yields typically less than 0.01 when not bound to nucleic acids
• Quantum yields typically greater than 0.4 when bound to nucleic acids

Available as blue-, green-, orange- or red-fluorescent dyes, these novel SYTO stains provide researchers with visible light–excitable dyes for labeling DNA and RNA in live cells (). SYTO dyes differ from each other in one or more characteristics, including cell permeability, fluorescence enhancement upon binding nucleic acids, excitation and emission spectra (Cell-permeant cyanine nucleic acid stains—Table 8.3), DNA/RNA selectivity and binding affinity. The SYTO dyes are compatible with a variety of fluorescence-based instruments that use either laser excitation or a conventional broadband illumination source (e.g., mercury- and xenon-arc lamps).

The SYTO dyes can stain both DNA and RNA. In most cases, the fluorescence wavelengths and emission intensities are similar for solution measurements of DNA or RNA binding. Exceptions include the SYTO 12 and SYTO 14 dyes, which are about twice as fluorescent when complexed with RNA as with DNA, and SYTO 16, which is about twice as fluorescent on DNA than RNA. Consequently, the SYTO dyes do not act exclusively as nuclear stains in live cells and should not be equated in this regard with DNA-selective compounds such as DAPI or the Hoechst 33258 and Hoechst 33342 dyes, which readily stain cell nuclei at low concentrations in most cells. SYTO dye–stained eukaryotic cells will generally show diffuse cytoplasmic staining, as well as nuclear staining. The SYTO 14 dye (S7576) has been used to visualize the translocation of endogenous RNA found in polyribosome complexes in living cells. Particularly intense staining of intranuclear bodies is frequently observed. Because these dyes are generally cell permeant and most of the SYTO dyes contain a net positive charge at neutral pH, they may also stain mitochondria. In addition, the SYTO dyes will stain most gram-positive and gram-negative bacterial cells. Dead yeast cells are brightly stained with the SYTO dyes, and live yeast cells typically exhibit staining of both the mitochondria and the nucleus. Some of the SYTO dyes have been reported to be useful for detecting apoptosis (Assays for Apoptosis—Section 15.5), and dyes structurally similar to the SYTO dyes have been used to detect multidrug-resistant cells (Probes for Cell Adhesion, Chemotaxis, Multidrug Resistance and Glutathione—Section 15.6). The red-fluorescent SYTO dyes are proving useful as counterstains (Probes for the Nucleus—Section 12.5) when combined with green-fluorescent antibodies, lectins or the cell-impermeant SYTOX Green nucleic acid stain.

All of the SYTO dyes are available separately (Cell-permeant cyanine nucleic acid stains—Table 8.3), and several SYTO dyes are included in our LIVE/DEAD Viability Kits (Viability and Cytotoxicity Assay Kits for Diverse Cell Types—Section 15.3, Molecular Probes assay kits for cell viability, cell counting and bacterial gram staining—Table 15.2). The green-fluorescent SYBR 14 dye, a component of our LIVE/DEAD Sperm Viability Kit (L7011, Viability and Cytotoxicity Assay Kits for Diverse Cell Types—Section 15.3) is also in the SYTO family of dyes. To facilitate testing the SYTO dyes in new applications, we offer several sampler kits containing sample sizes of SYTO dyes in each color set (Cell-permeant cyanine nucleic acid stains—Table 8.3). The recommended dye concentration for cell staining depends on the assay and may vary widely but is typically 1–20 µM for bacteria, 1–100 µM for yeast and 10 nM–5 µM for other eukaryotes.

SYTO RNASelect Green-Fluorescent Cell Stain

SYTO RNASelect green-fluorescent cell stain (S32703, Viability and Cytotoxicity Assay Reagents—Section 15.2) is a cell-permeant nucleic acid stain that selectively stains RNA (Figure 8.1.5, Figure 8.1.6). Although virtually nonfluorescent in the absence of nucleic acids, the SYTO RNASelect stain exhibits bright green fluorescence when bound to RNA (absorption/emission maxima ~490/530 nm), but only a weak fluorescent signal when bound to DNA (Figure 8.1.5). Filter sets that are suitable for imaging cells labeled with fluorescein (FITC) will work well for imaging cells stained with SYTO RNASelect stain ().

Figure 8.1.5 Relative absorption (A) and fluorescence emission (B) spectra of SYTO RNASelect green-fluorescent cell stain (S32703) in the presence of Escherichia coli DNA or in buffer alone.

Figure 8.1.6 Methanol-fixed bovine pulmonary artery endothelial cells treated with RNase, DNase or both, and then labeled with SYTO RNASelect Green cell stain (S32703). Removal of RNA with RNase prevented nucleolar labeling and greatly decreased nuclear and cytoplasmic labeling. Use of DNase resulted in less of a loss of label intensity in these cell compartments, reflecting the RNA-selective nature of this dye.

The amine-reactive succinimidyl ester of SYBR 101 dye (S21500) can be conjugated to peptides, proteins, drugs, polymeric matrices and biomolecules with primary amine groups. The conjugates are expected to be essentially nonfluorescent until they complex with nucleic acids, resulting in strong green fluorescence. Thus, they may be useful for studies of nucleic acid binding to various biomolecules, such as DNA-binding proteins. It is also possible that the fluorescence enhancement upon nucleic acid binding of SYBR 101 dye conjugates will be useful for monitoring their transport into the nucleus. SYBR 101 dye conjugates of solid or semisolid matrices (such as microspheres, magnetic particles or various resins) may be useful for detection or affinity isolation of nucleic acids.

The reactive SYBR 101 dye may also be conjugated to amine-modified nucleic acids. Although it is possible that the SYBR 101 dye may show some fluorescence when conjugated to amine groups on nucleic acids, they may be useful for developing homogeneous hybridization assays in which a specific sequence can be quantitated in solution without the need to separate bound and free probes. For example, a similar reactive nucleic acid stain has been used to label peptide–nucleic acid conjugates (PNA) for use as probes in real-time PCR. The labeled PNA probes exhibited a fluorescence increase upon hybridization to their complementary sequence and have been used to identify a single-base mismatch in a 10-base target sequence.

Cell-Impermeant Ethidium Bromide and Propidium Iodide

Ethidium bromide (EtBr, ) and propidium iodide (PI, P1304MP; P3566, P21493; ) are structurally similar phenanthridinium intercalators. PI is more soluble in water and less membrane-permeant than EtBr, although both dyes are generally excluded from viable cells. EtBr and PI can be excited with mercury- or xenon-arc lamps or with the argon-ion laser, making them suitable for fluorescence microscopy, confocal laser-scanning microscopy (), flow cytometry and fluorometry. These dyes bind with little or no sequence preference at a stoichiometry of one dye per 4–5 base pairs of DNA. Excitation of the EtBr–DNA complex may result in photobleaching of the dye and single-strand breaks. Both EtBr and PI also bind to RNA, necessitating treatment with nucleases to distinguish between RNA and DNA. Once these dyes are bound to nucleic acids, their fluorescence is enhanced ~10-fold, their excitation maxima are shifted ~30–40 nm to the red and their emission maxima are shifted ~15 nm to the blue (Figure 8.1.7, Properties of classic nucleic acid stains—Table 8.4). Although their molar absorptivities (extinction coefficients) are relatively low, EtBr and PI exhibit sufficiently large Stokes shifts to allow simultaneous detection of nuclear DNA and fluorescein-labeled antibodies, provided that the proper optical filters are used.

PI is commonly used as a nuclear or chromosome counterstain (Probes for the Nucleus—Section 12.5, ) and as a stain for dead cells (Viability and Cytotoxicity Assay Reagents—Section 15.2, ). EtBr is the conventional dye used for nucleic acid gel staining (Nucleic Acid Detection on Gels, Blots and Arrays—Section 8.4). However, our SYBR Gold and SYBR Green nucleic acid gel stains are far more sensitive than EtBr, and the SYBR Green I stain has been shown to be significantly less mutagenic than EtBr by Ames testing (Nucleic Acid Detection on Gels, Blots and Arrays—Section 8.4). Furthermore, our SYBR Safe DNA gel stain, which is as sensitive as EtBr and less mutagenic in the standard Ames test, has tested negative in three mammalian cell–based assays for genotoxicity and is not classified as hazardous waste under U.S. Federal regulations (Nucleic Acid Detection on Gels, Blots and Arrays—Section 8.4).

EtBr and PI are potent mutagens and must be handled with extreme care. Solutions containing EtBr or PI can be decontaminated by filtration through activated charcoal, which is then incinerated, thus providing an economical decontamination procedure. Alternatively, the dyes can be completely degraded in buffer by reaction with sodium nitrite and hypophosphorous acid. PI is offered as a solid (P1304MP, P21493), and both EtBr and PI are available as aqueous solutions (15585-011, P3566).

Figure 8.1.7 Normalized fluorescence emission spectra of DNA-bound 1) Hoechst 33258 (H1398, H3569, H21491), 2) acridine orange (A1301, A3568), 3) ethidium bromide (E1305, E3565) and 4) 7-aminoactinomycin D (A1310).

Cell-Permeant Hexidium Iodide

Hexidium iodide (H7593) is a moderately lipophilic phenanthridinium dye () that is permeant to mammalian cells and selectively stains almost all gram-positive bacteria in the presence of gram-negative bacteria. Our LIVE BacLight Bacterial Gram Stain Kit and ViaGram Red⁺ Bacterial Gram Stain and Viability Kit (L7005, V7023; Viability and Cytotoxicity Assay Kits for Diverse Cell Types—Section 15.3) use hexidium iodide for the discrimination of bacterial gram sign (). Hexidium iodide yields slightly shorter-wavelength spectra upon DNA binding than our ethidium or propidium dyes. Generally, both the cytoplasm and nuclei of eukaryotic cells show staining with hexidium iodide; however, mitochondria and nucleoli may also be stained.

Cell-Permeant Dihydroethidium (Hydroethidine)

Dihydroethidium (also known as hydroethidine) is a chemically reduced ethidium derivative () that is permeant to live cells and exhibits blue fluorescence in the cytoplasm. Many viable cells oxidize the probe to ethidium, which then fluoresces red upon DNA intercalation (). Dihydroethidium, which is somewhat air sensitive, is available in a 25 mg vial (D1168) or specially packaged in 10 vials of 1 mg each (D11347); the special packaging is strongly recommended when small quantities of the dye will be used at a time. Dihydroethidium is also available as a 5 mM stabilized solution in dimethylsulfoxide (D23107).

High-Affinity Ethidium Homodimers

Ethidium homodimer-1 (EthD-1, E1169; ) and ethidium homodimer-2 (EthD-2, E3599; ) strongly bind to dsDNA, ssDNA, RNA and oligonucleotides with a significant fluorescence enhancement (>40-fold). EthD-1 also binds with high affinity to triplex nucleic acid structures. One molecule of EthD-1 binds per four base pairs in dsDNA, and the dye's intercalation is not sequence selective. It was originally reported that only one of the two phenanthridinium rings of EthD-1 is bound at a time; subsequent reports indicate that bis-intercalation appears to be involved in staining both double-stranded and triplex nucleic acids.

The spectra and other properties of the EthD-1 and EthD-2 dimers are almost identical (). However, the DNA affinity of EthD-2 is about twice that of EthD-1. EthD-2 is also about twice as fluorescent bound to dsDNA than to RNA. Because both EthD-1 and EthD-2 can be excited with UV light or by the 488 nm spectral line of the argon-ion laser, either dye can be used in combination with the TOTO-1, YOYO-1 or SYTOX Green nucleic acid stains for multicolor experiments (Figure 8.1.8). The ethidium homodimer dyes are impermeant to cells with intact membranes, a property that makes EthD-1 useful as a dead-cell indicator in our LIVE/DEAD Viability/Cytotoxicity Kit (L3224, Viability and Cytotoxicity Assay Kits for Diverse Cell Types—Section 15.3, ) and EthD-2 (as DEAD Red nucleic acid stain) a suitable dead-cell indicator in our LIVE/DEAD Reduced Biohazard Cell Viability Kit #1 (L7013, Viability and Cytotoxicity Assay Kits for Diverse Cell Types—Section 15.3, ). These dyes have also been used to detect DNA in solution, although they are not as sensitive or as easy to use as our Quant-iT PicoGreen dsDNA reagent (Nucleic Acid Quantitation in Solution—Section 8.3).

Figure 8.1.8 Normalized fluorescence emission spectra of DNA-bound SYTOX Green nucleic acid stain (S7020) and ethidium homodimer-1 (EthD-1, E1169). Both spectra were obtained using excitation at 488 nm.

Ethidium Monoazide: A Photocrosslinking Reagent

Nucleic acids can be covalently photolabeled by various DNA intercalators. Ethidium monoazide (E1374, ) is a fluorescent photoaffinity label that, after photolysis, binds covalently to nucleic acids both in solution and in cells that have compromised membranes. The quantum yield for covalent photolabeling by ethidium monoazide is unusually high (>0.4).

The membrane-impermeant ethidium monoazide is reported to only label dead cells and is therefore particularly useful for assaying the viability of pathogenic cells (Viability and Cytotoxicity Assay Reagents—Section 15.2). A mixed population of live and dead cells incubated with this reagent can be illuminated with a visible-light source, washed, fixed and then analyzed in order to determine the viability of the cells at the time of photolysis. This method not only reduces some of the hazards inherent in working with pathogenic cells, but also is compatible with immunocytochemical analyses requiring fixation. We have developed alternative assays for determining the original viability of fixed samples and provide these in the LIVE/DEAD Reduced Biohazard Cell Viability Kit #1 (L7013) and the LIVE/DEAD Fixable Dead Cell Stain Kits, which are described in Viability and Cytotoxicity Assay Kits for Diverse Cell Types—Section 15.3.

In addition to its utility as a viability indicator, ethidium monoazide has been used to irreversibly label the DNA of Candida albicans in order to investigate phagocytic capacity of leukocytes. Ethidium monoazide has also been employed to "footprint" drug-binding sites on DNA, to probe for ethidium-binding sites in DNA and transfer RNA (tRNA) and to selectively photoinactivate the expression of genes in vertebrate cells.

Acridine Orange: A Dual-Fluorescence Nucleic Acid Stain

We offer highly purified, flow cytometry–grade acridine orange, a dye that interacts with DNA and RNA by intercalation or electrostatic attractions. In condensed chromatin, however, the bulk of DNA is packed in a way that does not allow efficient acridine orange intercalation. This cationic dye () has green fluorescence with an emission maximum at 525 nm when bound to DNA. Upon association with RNA, its emission is shifted to ~650 nm (red fluorescence). Acridine orange is available as a solid (A1301) and, for ease of handling, as a 10 mg/mL aqueous solution (A3568).

AT-Selective Acridine Homodimer

The water-soluble acridine homodimer bis-(6-chloro-2-methoxy-9-acridinyl)spermine (A666, ) is one of several acridine dimers that have been described in the literature. This dye has extremely high affinity for AT-rich regions of nucleic acids, making it particularly useful for chromosome banding. Acridine homodimer emits a blue-green fluorescence when bound to DNA, yielding fluorescence that is proportional to the fourth power of the AT base-pair content. Because of its greater brightness and photostability, acridine homodimer has been recommended as an alternative to quinacrine for Q banding.

AT-Selective ACMA

ACMA (9-amino-6-chloro-2-methoxyacridine, A1324, ) is a DNA intercalator that selectively binds to poly(d(AT)) with a binding affinity constant of 2 × 10⁵ M^-1 at pH 7.4. Excitation of the ACMA–DNA complex (excitation/emission maxima ~419/483 nm) is possible with most UV-light sources, making it compatible for use with both shorter- and longer-wavelength dyes. ACMA also apparently binds to membranes in the energized state and becomes quenched if a pH gradient forms. It has been extensively employed to follow cation and anion movement across membranes and to study the proton-pumping activity of various membrane-bound ATPases (Probes Useful at Acidic pH—Section 20.3).

DNA-Selective Hoechst Dyes

The bisbenzimide dyes—Hoechst 33258 (), Hoechst 33342 () and Hoechst 34580—are cell membrane–permeant, minor groove–binding DNA stains (Figure 8.1.1) that fluoresce bright blue upon binding to DNA. Hoechst 33342 has slightly higher membrane permeability than Hoechst 33258, but both dyes are quite soluble in water (up to 2% solutions can be prepared) and relatively nontoxic. These Hoechst dyes, which can be excited with the UV spectral lines of the argon-ion laser and by most conventional fluorescence excitation sources, exhibit relatively large Stokes shifts () (excitation/emission maxima ~350/460 nm), making them suitable for multicolor labeling experiments. Hoechst 34580 has somewhat longer-wavelength spectra than the other Hoechst dyes when bound to nucleic acids.

The Hoechst 33258 and Hoechst 33342 dyes have complex, pH-dependent spectra when not bound to nucleic acids, with a much higher fluorescence quantum yield at pH 5 than at pH 8. Their fluorescence is also enhanced by surfactants such as sodium dodecyl sulfate (SDS). These dyes appear to show a wide spectrum of sequence-dependent DNA affinities and bind with sufficient strength to poly(d(AT)) sequences that they can displace several known DNA intercalators. They also exhibit multiple binding modes and distinct fluorescence emission spectra that are dependent on dye:base pair ratios. Hoechst dyes are used in many cellular applications, including cell-cycle and apoptosis studies (Assays for Cell Enumeration, Cell Proliferation and Cell Cycle—Section 15.4, Assays for Apoptosis—Section 15.5), and they are common nuclear counterstains (Probes for the Nucleus—Section 12.5). Hoechst 33258, which is selectively toxic to malaria parasites, is also useful for flow cytometric screening of blood samples for malaria parasites and for assessing their susceptibility to drugs.

The Hoechst 33258 and Hoechst 33342 dyes are available as solids (H1398, H1399), as high-purity solids (H21491, H21492) and, for ease of handling, as 10 mg/mL aqueous solutions (H3569, H3570). The Hoechst 34580 dye is available as a solid (H21486).

AT-Selective DAPI

DAPI (4',6-diamidino-2-phenylindole; D1306, D3571, D21490; ) shows blue fluorescence () upon binding DNA and can be excited with a mercury-arc lamp or with the UV lines of the argon-ion laser. Like the Hoechst dyes, the blue-fluorescent DAPI stain apparently associates with the minor groove of dsDNA (Figure 8.1.9), preferentially binding to AT clusters; there is evidence that DAPI also binds to DNA sequences that contain as few as two consecutive AT base pairs, perhaps employing a different binding mode. DAPI is thought to employ an intercalating binding mode with RNA that is AU selective.

The selectivity of DAPI for DNA over RNA is reported to be greater than that displayed by ethidium bromide and propidium iodide. Furthermore, the DAPI–RNA complex exhibits a longer-wavelength fluorescence emission maximum than the DAPI–dsDNA complex (~500 nm versus ~460 nm) but a quantum yield that is only about 20% as high.

Binding of DAPI to dsDNA produces an ~20-fold fluorescence enhancement, apparently due to the displacement of water molecules from both DAPI and the minor groove. Although the Hoechst dyes may be somewhat brighter in some applications, their photostability when bound to dsDNA is less than that of DAPI. In the presence of appropriate salt concentrations, DAPI usually does not exhibit fluorescence enhancement upon binding to ssDNA or GC base pairs. However, the fluorescence of DAPI does increase significantly upon binding to detergents, dextran sulfate, polyphosphates and other polyanions. A review by Kapuscinski discusses the mechanisms of DAPI binding to nucleic acids, its spectral properties and its uses in flow cytometry and for chromosome staining. DAPI is an excellent nuclear counterstain, showing a distinct banding pattern in chromosomes (), and we have included it in our SelectFX Nuclear Labeling Kit (S33025, Probes for the Nucleus—Section 12.5). DAPI is quite soluble in water but has limited solubility in phosphate-buffered saline.

We also offer DAPI premixed with our ProLong Gold and SlowFade Gold antifade reagents (P36931, P36935, S36938, S36939; Fluorescence Microscopy Accessories and Reference Standards—Section 23.1). This combination of nucleic acid dye and antifade reagent permits simultaneous staining and protection of the stained sample from photobleaching.

Figure 8.1.9 X-ray crystal structure of DAPI (D1306, D3571, D21490) bound to DNA; the image was derived from data submitted to the Protein Data Bank (number PDB 1D30, www.rcsb.org/pdb ). X-ray crystallography shows that DAPI binds to DNA in the minor groove.

7-Aminoactinomycin D and Actinomycin D: Fluorescent Intercalators

7-AAD (7-aminoactinomycin D, A1310; ) is a fluorescent intercalator that undergoes a spectral shift upon association with DNA. 7-AAD–DNA complexes can be excited by the argon-ion laser and emit beyond 610 nm (Properties of classic nucleic acid stains—Table 8.4, Figure 8.1.7, ), making this nucleic acid stain useful for multicolor fluorescence microscopy (), confocal laser-scanning microscopy and immunophenotyping by flow cytometry. 7-AAD appears to be generally excluded from live cells, although it has been reported to label the nuclear region of live cultured mouse L cells and salivary gland polytene chromosomes of Chironomus thummi larvae. 7-AAD binds selectively to GC regions of DNA, yielding a distinct banding pattern in polytene chromosomes and chromatin. This sequence selectivity has been exploited for chromosome banding studies.

Actinomycin D (A7592) is a nonfluorescent intercalator that exhibits high GC selectivity and causes distortion at its binding site. Binding of the nonfluorescent actinomycin D to nucleic acids changes the absorbance of the dye. Like 7-AAD, actinomycin D has been used for chromosome banding studies. Binding of actinomycin D to ssDNA is reported to inhibit reverse transcriptase and other polymerases.

Hydroxystilbamidine

The trypanocidal drug hydroxystilbamidine (H22845, ) is an interesting probe of nucleic acid conformation; its nucleic acid staining properties were first described in 1973. Hydroxystilbamidine, a nonintercalating dye, exhibits AT-selective binding that is reported to favor regions of nucleic acids that have secondary structure. The interaction between hydroxystilbamidine and DNA has been investigated using binding isotherms and temperature-jump relaxation studies.

Hydroxystilbamidine has some unique spectral properties upon binding nucleic acids. At pH 5, the free dye exhibits UV excitation maxima at ~330 nm and ~390 nm, with dual emission at ~450 nm and ~600 nm (Figure 8.1.10). Although the red-fluorescent component remains present when bound to DNA, it is never observed when the dye is bound to RNA, permitting potential discrimination to be made between these two types of nucleic acids. The enhancement of its metachromatic fluorescence upon binding to DNA is proportional to the square of the AT base-pair content. Hydroxystilbamidine is reported to exhibit red fluorescence when bound to calf thymus DNA and T5 DNA, orange fluorescence with Micrococcus lysodeikticus DNA and blue-violet fluorescence on poly(d(AT)). It has been used for the treatment of myeloma, binding selectively to myeloma cells in the bone marrow.

Murgatroyd described the use of hydroxystilbamidine's metachromatic fluorescence properties for the selective permanent staining of DNA (with yellow fluorescence), mucosubstances and elastic fibers in paraffin sections. He also reported that hydroxystilbamidine (as its isethionate salt, which we do not currently offer) is nonmutagenic in Salmonella typhimurium by the Ames test.

Figure 8.1.10 Fluorescence spectra of hydroxystilbamidine bound to different forms of DNA. Hydroxystilbamidine (H22845) was incubated with either calf thymus DNA (red) or a hybrid of poly(d(A)) and poly(d(T)) homopolymers (blue) in 50 mM sodium acetate, pH 5.0. The fluorescence emission spectra changes when the dye is bound to AT-rich DNA versus calf-thymus genomic DNA.

Long-Wavelength LDS 751 Dye

LDS 751 (L7595, ) is a cell-permeant nucleic acid stain that has been used to discriminate intact nucleated cells from nonnucleated and damaged nucleated cells, as well as to identify distinct cell types in mixed populations of neutrophils, leukocytes and monocytes by flow cytometry. LDS 751, which has its peak excitation at ~543 nm on dsDNA, can be excited by the argon-ion laser at 488 nm and is particularly useful in multicolor analyses due to its long-wavelength emission maximum (~712 nm). Binding of LDS 751 to dsDNA results in an ~20-fold fluorescence enhancement. When LDS 751 binds to RNA, we have observed a significant red shift in its excitation maximum to 590 nm and blue shift in its emission maxima to 607 nm, which may permit its use to discriminate DNA and RNA in cells. A report has ascribed the name LDS 751 to a dye called styryl 8; however, their chemical structures are not the same.

NeuroTrace Fluorescent Nissl Stains

The Nissl substance, described by Franz Nissl more than 100 years ago, is unique to neuronal cells. Composed of an extraordinary amount of rough endoplasmic reticulum, the Nissl substance reflects the unusually high protein synthesis capacity of neuronal cells. Various fluorescent or chromophoric "Nissl stains" have been used for this counterstaining, including acridine orange, ethidium bromide, neutral red (N3246, Viability and Cytotoxicity Assay Reagents—Section 15.2), cresyl violet, methylene blue, safranin-O and toluidine blue-O. We have developed five fluorescent Nissl stains (Fluorescence characteristics of NeuroTrace fluorescent Nissl stains—Table 14.2) that not only provide a wide spectrum of fluorescent colors for staining neurons, but also are far more sensitive than the conventional dyes:

• NeuroTrace 435/455 blue-fluorescent Nissl stain (N21479)
• NeuroTrace 500/525 green-fluorescent Nissl stain (N21480)
• NeuroTrace 515/535 yellow-fluorescent Nissl stain (N21481)
• NeuroTrace 530/615 red-fluorescent Nissl stain (N21482)
• NeuroTrace 640/660 deep red–fluorescent Nissl stain (N21483)

In addition, the Nissl substance redistributes within the cell body in injured or regenerating neurons. Therefore, these Nissl stains can also act as markers for physically or chemically induced neurostructural damage. Staining by the Nissl stains is completely eliminated by pretreatment of tissue specimens with RNase; however, these dyes are not specific stains for RNA in solutions. The strong fluorescence (emission maximum ~515–520 nm) of NeuroTrace 500/525 green-fluorescent Nissl stain (N21480) makes it a preferred dye for use as a counterstain in combination with orange- or red-fluorescent neuroanatomical tracers such as DiI (D282, D3911, V22885; Tracers for Membrane Labeling—Section 14.4).

For Research Use Only. Not for human or animal therapeutic or diagnostic use.