Ethidium Fluorescence

Since tRNA is more varied structurally than DNA, ethidium could reside in pockets as well as intercalate into double-strand regions. The fluorescence decay provides information about the type, or types, of binding sites occupied by ethidium. It is currently believed that the excited state of ethidium is quenched by proton transfer to the solvent0 86-1 and that its lifetime is reduced with increasing solvent exposure. If ethidium occupies two or more kinds of sites with different degrees of exposure to solvent, then its fluorescence decay is expected to be multiexponential. 

Satisfactory fits of the fluorescence decays for ethidium bound to yeast tRNAPhe and E. coli tRNA al require (at least) two exponentials in the sum response S(t) [cf. Eq. (4.56)] under all conditions studied.0 870 88) The normalized amplitudes and lifetimes for tRNA 1 (extrapolated to zero concentration) are S° = 0.917, r, = 25.6ns and S02 = 0.082, t2 = 5.6 ns.(187) The results for tRNAPhe are similar.(188) This requirement for two (or more) exponentials is unequivocal evidence for at least two ethidium binding sites. The dominant component has a lifetime similar to, but slightly longer than, that of ethidium intercalated in DNA and is taken to represent ethidium 

If the tRNA can be treated as a rigid ellipsoid of revolution, then its anisotropy is expressed by Eq. (4.24) with I given by Eqs. (4.28a-c), Cn(0 = exp(- 2I iit), and F (/) = exp[-(6- 2)D ], where DM and DL are the rotational diffusion coefficients around the symmetry and transverse axes, respectively. In general, a multiexponential decay is expected. However, the data for tRNAPh and tRNA in the presence of endogenous Mg2+are well fitted by the single-exponential function r(t) = r0 exp( — t/xR) +, l87,, 88 The 

The rotational relaxation time zK can be combined with time-dependent nuclear Overhauser effect (NOE) measurements to determine interproton 

Removal of endogeneous Mg2+ by rigorous treatment (heating to 80 °C in the presence of 10 mM EDTA) introduces a fundamental difference between yeast tRNAPhe and E. coli tRNA]781, although their xR values remain 

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The reader is referred to other reviews for detailed discussions of the electronic states and luminescence of nucleic acids and their constituents/0 fluorescence correlation spectroscopy/2) spectroscopy of dye/DNA complexes/0 and ethidium fluorescence assays/4,0 A brief review of early work on DNA dynamics as well as a review of tRNA kinetics and dynamics have also appeared. The diverse and voluminous literature on the use of fluorescence techniques to assay the binding of proteins and antitumor drugs to nucleic acids and on the use of fluorescent DNA/dye complexes in cytometry and cytochemistry lies entirely outside the scope of this chapter. 

Figure 4.15. Effect of chloroquine on linear pBR322 DNA. Torsion constant (top) and fluorescence amplitude ratio of the slow (intercalated) and fast (free) components in the decay of the ethidium fluorescence intensity (hottom) versus ln(added chl/bp). The DNA is present in 0.1 M NaCl, 10 mM Tris, 10 mM EDTA, pH 8.5, at 20°C, and ethidium is present at 1 dye per 300 base pairs. The points at the left of the figure apply for zero added chloroquine. The best-fit...

At binding ratios r > 0.27, both linear and supercoiled DNAs show evidence of a marked structural change. A component with intermediate lifetime (t 5 ns) appears in the ethidium fluorescence decay, which may represent a partially intercalated species. The apparent torsion constants become highly nonuniform and exhibit considerably altered values. The long-range torsion constant increases appreciably for the linear DNA, but decreases for the supercoiled DNAs, which are substantially positively supercoiled at that point.(53)... 

In most studies published after the initial reports on the existence of M-DNA (67, 68), the formation of M-DNA for a variety of DNA duplexes (Table III) was demonstrated using an ethidium fluorescence assay (70, 71, 73-77). This assay is based on the fact that ethidium bromide intercalates in B-DNA and consequently fluoresces, but it cannot intercalate in M-DNA because of electrostatic repulsion between ethidium and the metal ions within the duplex. 

ScHiMERLiK, M. L, U. Quast, and M. A. Raftery Ligand-induced changes in membrane-bound acetylcholine receptor observed by ethidium fluorescence. 3. Stopped-flow studies with histrionicotoxin. Biochemistry 18, 1902—1906 (1979). 

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