Nucleic acids ethidium bromide binding

Haag, D. Tschahargane, C. Goerttler, K. Does ethidium bromide bind selectively and stoichiometrically to nucleic acids in histological tissues. Histochemie 1971,27,119-124. 

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. 

Much attention has been focussed lately on the family of asymmetric cyanine dyes for use in fluorescence detection of nucleic acids. These dyes show a significant enhancement in fluorescence intensity (100- to 1000-fold) upon binding to double-stranded DNA as compared to that from the fluorophore in solution. Use of cyanine fluorophores may be advantageous for use in assay design and sensor applications with respect to some of the more commonplace dyes, such as ethidium bromide and Hoechst 33342, as these latter dyes exhibit significant fluorescence intensity as background when in solution and have significantly lower enhancement in emission intensity [42]. 

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]. 

Lower nucleic acid concentrations are best determined fluorimetrically. These methods generally depend on the fact that certain dyes can bind to nucleic acids by intercalating between successive base pairs, and this binding is accompanied by marked increases in the fluorescence quantum yield. Ethidium bromide fluorescence (Aex 260-360 nm Af.rr] 560 nm), which is commonly used to visualise nucleic acids in gel electrophoresis, can also be used to quantitate double stranded DNA and RNA with a sensitivity of about 10 ng (Karsten and Wollenberger 1977). The dye 4,6-diamidino-2-phenylindole (DAPI) (Aex 360 nm 2f.m 450 nm) can be used to quantitate DNA specifically with a detection limit of about lng (Brunk et al. 1979). 

DNA accessibility can be determined by how well the DNA binds fluorescent intercalating dyes. TO-PRO-1 is a cyanine dye that fluoresces only when bound to nucleic acid (82). It is more sensitive for fluorescence detection than ethidium bromide and binds stably to the DNA. The relative degree of protection of the DNA can be quantified. 

Some of these, like ANS and ethidium bromide, will bind non-covalently to particular regions of proteins and nucleic acids, with large changes in their fluorescent properties. ANS lends to bind to hydrophobic patches on proteins and partially unfolded polypeptides, with a blue shift and increase in fluorescence intensity. Ethidium bromide molecules intercalate between the base pairs of double-stranded DNA, resulting in a large increase in fluorescence that is used routinely for detecting and visualizing bands of nucleic acids in gel electrophoresis, for example. 

The representation of fluctuations connected with fluorescence correlation spectroscopy has been adopted for the study of the reversible binding of ethidium bromide to DNA. The reaction has biological significance in connection with the discovery of the transcriptive mechanism of the genetic code, since ethidium bromide inhibits nucleic acid synthesis. The DNA-ethidium bromide complex is strongly fluorescent, its fluorescent quantum... 

The recognition of the limitations of short radiative fluorescence lifetime of some covalently bound labels used in studies of nucleic acids prompted Saavedra and Picozza to bind terbium to DNA via inunobilized DTPA [47]. Instead of the nanosecond lifetimes achieved with stains such as ethidium bromide, a radiative lifetime of 1.5 msec was obtained for terbium-labeled DNA. In addition, this material is stable under the conditions frequently encountered in polyacrylamide gel electrophoresis. This labeling technique could also be used for RNA. There can be no doubt that the requirements of genetic manipulation will bring about many similar procedures for analyzing nucleic acids. 

Macromolecules may or may not fluoresce. Those that do are considered to contain intrinsic fluors. The common intrinsic fluors for proteins are tryptophan, tyrosine, and phenylalanine (the same three groups that absorb UV radiation). Macromolecules that have no intrinsic fluors can be made fluorescent by adding an extrinsic fluor to them. This is done by the process of chemical coupling or sample binding. The most common extrinsic fluors for proteins are l-aniline-8-naphthalene sulfonate, l-dimethylaminonaphthalene-5-sulfonate, dansyl chloride, 2-p-toluidyl-naphthalene-6-sulfonate, rhodamine, and fluorescein. The most common extrinsic fluor for nucleic acids are various acridienes (acridine orange, proflavin, acriflavin) and ethidium bromide. 

The intercalation process requires (1) planarity and a certain size of the ligand, (2) a base-paired helical secondary structure of the nucleic acid. Proflavine and ethidium bromide have been shown to bind strongly to RNA, and to synthetic polymers in an amount proportional to their degree of double-helical conformation. Single-stranded or denaturated nucleic acids have very flexible structures, which allow the stacking of an amount of dye greater than what is allowed by more rigid double-stranded molecules. 

Other fluorogenic dyes, such as DAPI, ethidium bromide and the cyanine dyes, bind to DNA and/or RNA and undergo changes in fluorescence intensity of up to 1000-fold. Depending on the probe, these compounds can be used to image fixed cells or track mitotic events. Combinations of probes with different specificities for single-stranded, double-stranded and ribosomal RNA have been used to characterize the quantity and conformation of nucleic acids throughout the cell cycle. 

Of the few relaxation studies that have been made, binding of the interca-lator ethidium bromide to nucleic acids has been of interest in most... 

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