Imaging of Nucleic acids

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Nucleic acid stains are usually cationic dyes that bind with the polyanionic nucleic acid, thus showing specificity for DNA and RNA. Furthermore, hydrophilic/hydrophobic interactions play an important role in (a) providing some dyes with the ability to intercalate between the nucleic acid bases and (b) providing other dyes with the ability to bind with double-stranded nucleic acids in grooves. In double-stranded DNA, the alignment of the strands is antiparallel and asymmetric along the axis. This asymmetry creates two different grooves on opposite sides of the base pairs, called major and minor grooves. Examples of intercalating dyes are phenanthridines (ethidium bromide, propidium iodide), acridines (acridine orange, ACMA), and numerous cyanine dyes (TO, YO, PO, JO, BO, LO, and their derivatives; SYTO and SYTOX dye families). The chemical conjugation of intercalating fluorophore and a linker carrying additional cationic charge provides both intercalating ability and the electrostatic mode of binding. The dimeric probes obtained in this way have extremely high binding efficiencies and sensitivities to nucleic acids. These features of the dimeric probes have led to their usage in a variety of DNA studies, including imaging at a single molecule level. Imaging of a single chain of 39-mm-long DNA molecules, stained with the dimeric dye YOYO-1 and attached to a 1-mm-diameter polystyrene bead, has been used to study the relaxation of a single DNA molecule stretched by a laser tweezer. Laser tweezer action is discussed in detail in Chapter 14. An example of this single DNA molecule imaging is shown in Figure 14.17.

Some of the nucleic acid stains, such as Hoechst dyes and DAPI, bind with double-stranded DNA in minor grooves. For these dyes, binding in the DNA minor grooves determines their selectivity not only to the double-stranded DNA (ds DNA)but also to the A-T sequences of the DNA molecule because of the narrow minor grooves of the A-T sequences in comparison with the wider minor grooves of the G-C sequences. Narrower minor grooves of the

Flow Cytometry

Figure 8.16. Top panel shows the two-photon fluorescence image of breast cancer cell treated with AN-152:C625 (drug labeled with dye C625) and [d-Lys6]LH-RH:TPR (the peptide carrier labeled with dye TPR). The bottom panel shows the localized spectra obtained from different parts of the cell as indicated by the arrows in the image.

Figure 8.16. Top panel shows the two-photon fluorescence image of breast cancer cell treated with AN-152:C625 (drug labeled with dye C625) and [d-Lys6]LH-RH:TPR (the peptide carrier labeled with dye TPR). The bottom panel shows the localized spectra obtained from different parts of the cell as indicated by the arrows in the image.

A-T sequences provide a snag fit for the ribbon-like molecule of a groove-binding stain.

Nucleic acid stains can be used for staining nucleic acids inside cells, where the selection of the probe is determined by its ability to permeate through the membrane of the live cell. Cell-membrane-impermeant dyes (e.g., ethidium

Figure 8.17. Two-photon laser scanning microscopic images of a KB cell stained with Hoechst 33342 and SYTO 43. (A) Transmission image; (B) SYTO 43 fluorescence image (excitation with 860 nm). (C) Hoechst 33342 fluorescence image (excitation with 750nm). (D) Merged image of fluorescence and tramission images (Blue transmission, Green-Hoechst fluorescence, and Red-Syto 43 fluorescence) Hoechst stains exclusively the dsDNA sites; SYTO 43 labels both DNA and RNA. Arrow shows nucleolus, the major repository of RNA in the nucleus. (See color figure.)

Figure 8.17. Two-photon laser scanning microscopic images of a KB cell stained with Hoechst 33342 and SYTO 43. (A) Transmission image; (B) SYTO 43 fluorescence image (excitation with 860 nm). (C) Hoechst 33342 fluorescence image (excitation with 750nm). (D) Merged image of fluorescence and tramission images (Blue transmission, Green-Hoechst fluorescence, and Red-Syto 43 fluorescence) Hoechst stains exclusively the dsDNA sites; SYTO 43 labels both DNA and RNA. Arrow shows nucleolus, the major repository of RNA in the nucleus. (See color figure.)

bromide, SYTOX dyes) are involved in various studies of the cell apoptosis because of their increased permeability into apoptotic cells, caused by the compromised membranes in apoptotic cells. Consequently, the dyes are used as simple, single-step dead cell indicators.

In contrast, cell-membrane-permeant probes, such as SYTO dyes, are nucleic acid stains that passively diffuse through the membranes of live cells. These dyes are used to stain RNA and DNA in both live and dead eukaryotic cells, as well as in gram-positive and gram-negative bacteria. Hence, in terms of cell organelles, the SYTO dyes do not exclusively stain nuclei in live cells. In contrast, dyes like DAPI or Hoechst readily stain nuclei in live cells due to their selectivity to DNA concentrated in cell nucleus. These features of selective staining are demonstrated in Figure 8.17, which shows two-photon scanning microscopic images of KB cells (human oral epidermoid carcinoma cell line) stained with Hoechst 33342 and SYTO 43, obtained at our Institute. Hoechst 33342 is a cell-membrane-permeant, minor groove-binding DNA stain, showing a sequence-dependent DNA affinity as it binds to the A-T sequences.

The SYTO dyes do not exclusively stain nuclei in live cells. In contrast, dyes like DAPI or the Hoechst dyes at low concentrations readily stain nuclei in live cells and do not affect cell viability because of their high specificity to dsDNA.

Determination of the relative contents of DNA and RNA at definite sites inside a cell nucleus is still a challenging task for biologists. One approach to this problem is double staining with DNA- and RNA-selective stains. Though no true RNA-specific stains are commercially available, there are many specific fluorescent stains for double-stranded nucleic acids. A common approach, therefore, is to use a fluorescent stain, selective to the double-stranded DNA (e.g., Hoechst and DAPI), and another nucleic acid stain that does not show sufficient discrimination between double-stranded DNA and single-stranded RNA, existing in a cell nucleus.

Figure 8.17 shows a confocal image of a KB cell, stained with SYTO 43 and Hoechst, where this approach has been used. The observed difference between the SYTO 43 and the Hoechst fluorescence pattern reveals the distribution of RNA in the cell.

Another approach to differentiating DNA and RNA uses a single dye that shows a shift in its fluorescence spectra, when bound to the double-stranded DNA (dsDNA) and to single-stranded RNA. The cell-membrane-permeant intercalative dye acridine orange exhibits green fluorescence (530-nm peak), when staining dsDNA. However, when binding with the single-stranded RNA, it shows red fluorescence (640-nm peak), which is apparently associated with the formation of a dye aggregate on the RNA molecule. The image of KB cell stained with acridine orange is shown in Figure 8.18. In this case, two-photon excitation of acridine orange was used for imaging and localized spectroscopy (see Figure 7.22). Localized spectroscopy allowed ratiometric profiling of the "green" and the "red" components (indicating dsDNA and RNA, in this case) to estimate the DNA/RNA content in certain locations inside the nucleus (Figure 8.18).

It is important to note that the different conditions for the nucleic acids outside a cell and inside the cell (which includes an intracellular organization of RNA and DNA) can cause a difference in the binding efficiency of a probe to DNA and RNA in the two cases. For instance, a monomethine cyanine dye Cyan 40 (4-((1-methylbenzothiazolyliliden-2)methyl)-1,2,6-trimethylpyri-dinium perchlorate) does not show any significant preference in RNA staining versus DNA staining outside a cellular environment, but, as shown in Figure 8.19, it does show an apparent preferential binding with RNA inside a living cell (Ohulchanskyy et al., 2003).

An important technique enabling the detection and determination of spatial distribution of specific DNA or RNA sequences in the cytoplasm, nucleus, and chromosomes as well as in other organelles is that of fluorescence in situ hybridization (FISH) (Pinkel, 1999). Hence the imaging utilizing this technique is also referred to as FISH imaging (Kozubek, 2002). FISH imaging has proved to be of value in the analysis of the structures and functions of chromosomes and genomes. It has been used for the determination of the spatial and temporal expression of genes.

Hybridization, in the context of DNA and RNA, refers to nucleotide base pairing of two single-stranded nucleic acid chains (DNA or RNA). The FISH technique involves in situ hybridization of nucleic acids in the target cells or chromosomes, to be detected or imaged, with fluorescently labeled, single-stranded probe nucleic acids. This method is illustrated in Figure 8.20. First, the nucleic acid (DNA) in the target cell is made single-stranded, for example, by heating. Next, the cell is incubated with fluorescently labeled single-stranded probe nucleic acid molecules (DNA or RNA). In situ hybridization occurs between the target and the probe under the conditions of the matching of their base sequence for pairing (conditions of complementary base sequences). After hybridization, fluorescence imaging can be used to deter-

Flow Cytometry Acridine Orange Lin
Figure 8.18. Top panel shows two-photon excited imaging of a KB cell with DNA (green pseudocolor) and RNA (red pseudocolor) staining with acridine orange. Bottom panel shows the fluorescence spectra obtained, using localized spectroscopy, from different locations in the cell. (See color figure.)

mine the number, intensity, and spatial distribution of each of the different-colored hybridization signals, probes, or segments fluorescing at different wavelengths.

Fluorescence labeling of the probe nucleic acid can involve two types of approaches, also shown in Figure 8.20. In one approach, called the direct method, the fluorescent label (fluorophore, represented by letter F in the figure) is directly attached to the end of the nucleic acid probe. In the other method, called the indirect method, the probe is modified chemically with molecules of biotin or digoxigenin (represented by the letter H in the figure).

Figure 8.19. KB cell stained simultaneously with Hoechst 33342 and Cyan 40. (I) Light transmission image. (II) Fluorescence image with Hoechst excitation (1ex = 750nm). (III) Fluorescence image with Cyan 40 excitation (1ex = 860 nm). (IV) Image generated by overlapping of I, II, and III images. Green pseudocolor marks Hoechst fluorescence, red pseudocolor represents Cyan 40, and blue pseudocolor means transmission. Scanning for both excitation wavelengths was performed in one focal plane. (Reproduced with permission from Ohulchansky et al., 2002.) (See color figure.)

Figure 8.19. KB cell stained simultaneously with Hoechst 33342 and Cyan 40. (I) Light transmission image. (II) Fluorescence image with Hoechst excitation (1ex = 750nm). (III) Fluorescence image with Cyan 40 excitation (1ex = 860 nm). (IV) Image generated by overlapping of I, II, and III images. Green pseudocolor marks Hoechst fluorescence, red pseudocolor represents Cyan 40, and blue pseudocolor means transmission. Scanning for both excitation wavelengths was performed in one focal plane. (Reproduced with permission from Ohulchansky et al., 2002.) (See color figure.)

Figure 8.20. FISH Schematic to target DNA. (a) Double-stranded DNA (bold lines) is denatured to make them single-stranded. (b) The target is incubated with denatured probes (bold lines) that are labeled with fluorochromes. (c) Hapten-labeled probes need to be rendered visible using affinity reagents such as avidin or antibodies (bold lines) that carry fluorochromes. (Reproduced with permission from Pinkel, 1999.)

After hybridization with the target nucleic acid, fluorescently labeled affinity reagents are used to couple to the H group to render it fluorescent. Because the fluorophore number densities can significantly be increased using the indirect method, enhancement of sensitivity can be accomplished. The ideal length of the probe is 100-300bp (base pairs), a shorter length resulting in lower stability of hybridization. On the other hand, larger-size probes pose difficulties

Figure 8.20. FISH Schematic to target DNA. (a) Double-stranded DNA (bold lines) is denatured to make them single-stranded. (b) The target is incubated with denatured probes (bold lines) that are labeled with fluorochromes. (c) Hapten-labeled probes need to be rendered visible using affinity reagents such as avidin or antibodies (bold lines) that carry fluorochromes. (Reproduced with permission from Pinkel, 1999.)

in penetrating into the cell structure. The probes can be produced by cloning (recombinant DNA technique whereby DNA is inserted into a vector and amplified together inside an appropriate host cell). Synthetic oligonuclectides have also been used as probes. Multicolor FISH utilizes several types of probes simultaneously, which have been labeled with different fluorophores.

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  • TIBLETS
    Where on the dna molecule does syto bind?
    8 years ago
  • nasih
    Why acridine orange gives red and green electrostatics?
    8 years ago

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