Between Ethidium Bromide and DNA

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. 

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]. 

Finally, one can see that upon addition of DNA to the ethidium bromide solution, the orange color of the fluorophore turns to pink (Figure 12.4). This color modification is the result of the interaction between the two molecules and mainly binding of ethidium bromide to DNA. 

Schiff bases and their complexes with Cu ", Zn ", Ni and Sn " as potential antitumor agents, were used in the fluorimetric method for preliminary screening of antitumor agents (Lee etal. 1993). The method was based on the consistency of the in-vivo and in-vitro interactions of drugs with DNA. Studies of Schiff bases-metal complex interaction with DNA using a fluorescence probe [ethidium bromide (EthBr)-DNA system] identified the parallelism between binding constants and antineoplastic... 

Cw-platin diacetate binds to ethidium bromide and yields a thermochromic 1 1 complex. The transition at 490 nm (orange) shifts to 640 nm (blue) as the temperature is increased and a proton transfer occurs between the coordinated amino group of the dye and the acetate counterion (Fig. 8.6.19). Ternary ethid-ium-cisplatin-DNA complexes are also known (Ren et al., 1993). 

The development and application of fluorescent dyes, which are incorporated into the PCR product, and fluorochromes for labelling of oligonucleotids opened the possibility for real-time monitoring of the product formation cycle by cycle. The fluorescence signal increases in proportion to the amount of amplicon. Dyes, such as ethidium bromide and SYBR Green I, that intercalate into the DNA are the simplest way to follow at real time the increase of amplicon. The disadvantage of this method is that distinguishing between different PCR products is not possible. 

It should be pointed out that when using ethidium bromide the sensitivity of the assays varies depending on the physical state of the nucleic acids (see Table I). Ethidium does not discriminate between RNA and DNA, although dyes are available which bind DNA exclusively, so the relative amounts of each may be determined by taking two sets of measurements. Alternatively, nucleases (DNA-ase or RNA-ase) can be used to exclusively remove one or the other in a mixture. Nucleic acids from different sources (see Table II) also show a variation in sensitivity, and the fluorescence assay lacks the selectivity of the hybridization technique. Nevertheless, for rapid screening or quality-control applications the fluorescence assay is still the method of choice. 

Ethidium bromide (1) is a widely used guest molecule (Scheme 1). The property that makes 1 a good probe for DNA binding is that its fluorescence quantum yield is very low in water and increases significantly when 1 intercalates between the base pairs of DNA.139... 

Chracteristics of B. m terium s plasmid system have been summarized by Carlton (3p. The plasmids are typical in that they band as covalently closed circular DNA in ethidium bromide-cesium chloride gradients and they are resistant to irreversible alkaline denaturation (17). However, B. megaterium plasmids are atypical in that they exist in approximately 10 size classes and as many copies per cell ( ). In fact, for the smaller plasmids there are hundreds of copies per cell so that plasmid DNA may represent up to 40% of the total extractable DNA (31). This is unusual since for most plasmids there are usually no more than a few copies per cell. Also, hybridization studies suggest that there is extensive homology between three B. megaterium plasmids of different sizes and between these plasmids and the chromosomal DNA (31,33). Carlton (31) concludes that the most likely explanation of the origin of B. megaterium plasmids is that they are molecular hybrids between one or more plasmid elements and various portions of the chromosomal DNA. 

When the stretched DNA-lipid film was soaked in an aqueous solution of ethidium bromide (itmax = 480 nm) for a day at room temperature, the transparent film turned red (itmax = 520 nm) and the aqueous solution became clear (Fig. 9a). Thus, the ethidium intercalated completely between base pairs of the DNA film. When the film was moved into the new aqueous buffer solution, the intercalated dye molecules were hardly removed from the film at least for a day. Similar intercalation behavior into the film was observed for other dyes such as proflavine, acridine orange, and safranine T [14-17]. 

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. 

---