Ethidium bromide structure

Several complexes that involve intercalation of an acridine in a portion of a nucleic acid have been studied by X-ray crystallographic techniques. These include complexes of dinucleoside phosphates with ethidium bromide, 9-aminoacridine, acridine orange, proflavine and ellipticine (65-69). A representation of the geometry of an intercalated proflavine molecule is illustrated in Figure 6 (b) this is a view of the crystal structure of proflavine intercalated in a dinucleoside phosphate, cytidylyl- -S ) guano-sine (CpG) (70, TV). For comparison an example of the situation before such intercalation is also illustrated in Figure 6 (a) by three adjacent base pairs found in the crystal structure of a polynucleotide (72, 73). In this latter structure the vertical distance (parallel to the helix axis) between the bases is approximately... 

Intercalating agents are hydrophobic, planar structures that can fit between the DNA base pairs in the center of the DNA double helix. These compounds (ethidium bromide and actinomycin D are often-used examples) take up space in the helix and cause the helix to unwind a little bit by increasing the pitch. The pitch is a measure of the distance between successive base pairs. 

In the absence of conclusive data on the role of a positive supercoiling wave, static positive supercoiling elicited by nucleosome reconstitution on relaxed or slightly positively-supercoiled plasmids [51] or by ethidium bromide intercalation in the loop of mononucleosomes on DNA minicircles [52] did not succeed either in releasing dimers. Moreover, circular dichroism, histone chemical modi-flcation and H3-thiol accessibility failed to detect an even slight alteration in the structure of such torsionally-stressed nucleosomes [51]. The reason was later found to lie in the ability of nucleosome entry/exit DNAs to form a positive crossing [52]. 

The scatchard technique consists of studying the difficulties encountered by a fluorescent dye, ethidium bromide (Fig. 19), with respect to its intercalation between the base pairs of DNA when the secondary structure of the macromolecule is modified by binding (or intercalation) of a drug. 

Anyhow, a combination of the Scatchard technique and Raman spectroscopy shows (i) that SOAz actually interacts with DNA at the level of ribose backbones and (ii) that this kind of interaction does not drastically modify the DNA secondary structure, ethidium bromide encountering no more difficulty to intercalate between DNA plates SOAz being grafted or not on the nucleic acid. Thus, the behaviour of MYKO 63 and of SOAz appears quite different with respect to their mode of interaction with DNA despite their close chemical and molecular structure. This surprising observation may be of interest for understanding why SOAz does not induce any cumulative toxicity in vivo in contrast with MYKO 63. 

Figure 5.13 shows the structures of extrinsic fluors that have been of value in studying biochemical systems. ANS, dansyl chloride, and fluorescein are used for protein studies, whereas ethidium, proflavine, and various acridines are useful for nucleic acid characterization. Ethidium bromide has the unique characteristic of enhanced fluorescence when bound to double-stranded DNA but not to single-stranded DNA. Aminomethyl coumarin (AMC) is of value as a fluorogenic leaving group in measuring peptidase activity. 

Fluorescence assays are considered among the most convenient, sensitive, and versatile of all laboratory techniques. However, the purine and pyrimidine bases yield only weak fluorescence spectra. Le Pecq and Paoletti (1967) showed that the fluorescence of a dye, ethidium bromide, is enhanced about 25-fold when it interacts with DNA. Ethidium bromide, which is a relatively small planar molecule (Figure El3.4), binds to DNA by insertion between stacked base pairs (intercalation). The process of intercalation is especially significant for aromatic dyes, antibiotics, and other drugs. Some dyes, when intercalated into DNA, show an enhanced fluorescence that can be used to detect DNA molecules after gel electrophoresis measurements (see Chapter 4 and Experiments 14 and 15) and to characterize the physical structure of DNA. Two analyses of DNA will be completed in this experiment ... 

B 9. Compare the structure of ethidium bromide (Figure E13.4) with those of the polyamines (Figure E13.5). Would you expect the binding of spermine or spermidine to DNA to be competitive with the binding of ethidium Explain. 

A Lehmnger, D Nelson, and M Cox, Principles of Biochemistry, 3rd ed (1999), Worth Publishers (New York), pp 907-930 DNA structure and function J Le Pecq and C Paoletti,/ Mol. Biol. 27, 87 (1967) A Fluorescent Complex Be tween Ethidium Bromide and Nucleic Acids ... 

As an alternative to radiation, a stain such as ethidium bromide is used to visualize DNA. The ethidium may be incorporated into the structure of DNA either before or after electrophoresis. The gel is then visualized under a fluorescent lamp. 

Matsuzawa, Y. and Yoshikawa, K. (1994) Change of the higher-order structure in a giant DNA induced by 4 ,6-diamidino-2-phenylindole as a minor groove binder and ethidium bromide as an intercalator. Nucleosides Nucleotides, 13, 1415-1423. 

Perhaps the most well-recognized fluorescent dye for detection of DNA hybridization is ethidium bromide (EtBr). EtBr is a cationic phenanthridinium compound that can bind to DNA by intercalation. This dye has an excitation maxima at 518 nm when bound to double-stranded DNA (dsDNA). Excitation of EtBr is often done by use of an argon ion laser, making this fluorophore a viable choice for applications in optical sensors as well as confocal scanning laser microscopy and fluorometry [41]. The structure of ethidium bromide is shown in Fig. 6. 

Intercalators associate with dsDNA by insertion between the stacked base pairs of DNA [52], EtBr binds to dsDNA with little to no sequence specificity, with one dye molecule inserting for every 4-5 base pairs [53]. It also binds weakly via a non-intercalative binding mechanism only after the intercalative sites have been saturated [54], Propidium iodide (PRO) is structurally similar to ethidium bromide, and both dyes show a fluorescence enhancement of approximately 20-30 fold upon binding to dsDNA [41]. As well, their excitation maxima shift 30-40 nm upon binding due to the environment change associated with intercalation into the more rigid and hydrophobic interior of the double-stranded nucleic acid structure relative to aqueous solution [41]. 

The increase in fluorescence intensity of TO upon binding to dsDNA is due to the restriction of rotation around the monomethine bridge upon intercalation of the dye into the double helical structure as the benzothiazole and quinolinium rings adapt to the propeller twist of the base pairs [49]. The monomethine bridge has a low energy barrier to rotation and hence is free to rotate in solution, allowing for the electronically excited dye to relax by non-radiative decay [49]. The quantum yield of free TO in solution has been reported to be 2 x 10-4 at 25 °C [43]. The binding constant for TO is 106 M 1 while that of ethidium bromide is 1.5 x 105 M 1 [59]. 

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