Rotating couette

Reference axis = hydrodynamic field vector

Fig. 2.5.5. Schematic illustration of the orientation of a cylindrical Couette flow cell relative to the linearly polarized light.

(a) the generation of an electric field (''electric LD'') which forces the molecules to arrange in this field depending on their intrinsic dipole moment; and (b) the implementation of a hydrodynamic field in a rotating couette (''flow LD'', Figure 2.5.5), in which molecules can align along the flow field. The latter method is only useful for macromolecules such as peptides or DNA, because small molecules cannot be oriented along the flow lines of the field. Nevertheless, for the determination of the binding mode of a dye-DNA complex flow-LD spectroscopy has been shown to be useful. In a hydrodynamic field most of the DNA molecules are partially arranged along the flow lines (with the flow lines as reference axis, a a 90°), so that the DNA bases afford a clear negative LD signal (Figure 2.5.6A, solid line). Consequently, an intercalator should also give a negative LD signal, because the transition moment is almost coplanar to those of the nucleic acid bases. In contrast, for a groove binder the angle a is 45° to the helix axis and thus to the flow lines. Consequently, a groove binder should give a positive signal, which is relatively weak compared with that of an intercalator.

The flow LD spectrum of aminoacridizinium 5a in the presence of st DNA at a molar dye:DNA ratio of 0.025 contains a negative LD signal in the long-wavelength absorption region of the acridisinium salt (Figure 2.5.6A, dashed line). The negative sign of the LD signals in this region is indicative of an intercalative binding mode between 5a and DNA. The LDr spectrum also provides information about the average orientation of the molecular plane of the aromatic dye relative to those of the DNA bases (Eq. 7). Typically, LDr bands are of constant signal intensity, except for the region of overlap between different polarizations. A LDr band with varying signal intensity usually results from heterogeneous binding. For the 9-amino derivative 5a, a nearly constant LDr value over the range 350-500 nm was

Fig. 2.5.6.

Wavelength j Linear-flow-dichroism (A), and reduced linear-flow-dichroism (B) spectra of acridizinium salt 5a in buffer solution (1 mM EDTA, 10 mM Tris buffer, 10 mM NaCl, pH 7.0); solid line, DNA without dye; dashed line, [5c]/[DNA] = 0.025.

observed (Figure 2.5.6B); this indicates that its orientation properties are fully consistent with intercalation into the DNA.

Further information can be gained from inspection of DNA base absorption. In the absence of acridizinium 5a the DNA bases give a negative LD band in the absorption region 230-300 nm (Figure 2.5.6A, solid line). Most notably, a significant increase of this LD absorption occurs on addition of the 9-aminoacridizinium salt 5a (Figure 2.5.6A, dashed line); this indicates better orientation of the macro-molecule within the hydrodynamic field. It can this be concluded that the alignment of the DNA becomes more pronounced because of stiffening of the helix on intercalation of the ligand.

In summary, it has been demonstrated that complex formation between dyes and DNA may be conveniently monitored by absorption and emission spectroscopy and that these methods provide useful data for discussion of the binding strength and binding mode.

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