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. 2003 Nov 1;31(21):6235-42.
doi: 10.1093/nar/gkg822.

Groove-binding unsymmetrical cyanine dyes for staining of DNA: dissociation rates in free solution and electrophoresis gels

Maja Eriksson  1 , H Jonas Karlsson, Gunnar Westman, Björn Akerman
Affiliations

Groove-binding unsymmetrical cyanine dyes for staining of DNA: dissociation rates in free solution and electrophoresis gels

Maja Eriksson et al. Nucleic Acids Res. .

Abstract

The rates of dissociation of three non-intercalative unsymmetrical cyanine dyes, BEBO, BETO and BOXTO from mixed-sequence DNA have been studied with the DNA either free in solution or in confining porous agarose gels. The properties of the new dyes were compared to the related intercalating dyes BO, BO-PRO, TO-PRO and YO-PRO. With DNA in solution, BEBO dissociates more slowly than the monovalent BO and interestingly also more slowly than the divalent dye BO-PRO. Similarly, both BETO and BOXTO exhibit considerably slower dissociation than TO-PRO. The new dyes show biexponential dissociation kinetics in mixed-sequence DNA. The average rate of dissociation increases with increasing ionic strength, but the salt dependence of the dissociation is weaker than for the corresponding intercalating dye. The rate of dye-dissociation decreases by a factor of about 10(5) in the gel. The rates for the dyes generally follow the pattern that we observe with the DNA in free solution, however a more accentuated stabilization was seen for intercalators than for groove-bound dyes. The results show that, in particular, BOXTO is a promising candidate as a preferentially groove-bound DNA-stain with a large enhancement of the fluorescence quantum yield upon binding to DNA, and which exhibits slow and salt-insensitive dissociation compared to corresponding intercalative dyes.

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Figures

Figure 1
Figure 1
The structures of three new dyes BEBO, BETO and BOXTO and the dyes compared in the study BO, BO-PRO, TO-PRO and YO-PRO.
Figure 2
Figure 2
The kinetics of dissociation of BEBO from ctDNA (at 26 mM NaCl), after 1:1 dilution by an aqueous solution of SDS in NaCl (26 mM) (measured by stopped-flow technique). The emission intensity is normalized to the (constant) intensity measured after dilution of the same sample with the 26 mM NaCl solution without SDS. The dissociation data were analyzed with two exponential decay (light grey line) (Residual, grey square).
Figure 3
Figure 3
(A) The salt dependence of the two rate constants for the dissociation of BEBO from ctDNA (k1, filled square, k2 filled diamond) and for the single rate constant for the dissociation of BEBO from poly(dA-dT)2 (open triangle). (B) The salt dependence of the rate constants for the dissociation of BO-PRO from ctDNA (biexponential decay, k1, filled square, k2, open diamond), of BO-PRO from poly(dA-dT)2 (single exponential decay, filled triangle) and of BO from ctDNA (single exponential decay, k, open triangle).
Figure 4
Figure 4
The relative amplitude of the rate constant for the fast component of the dissociation of BEBO (filled up-triangle), BO-PRO (filled star), BETO (open diamond), BOXTO (filled down-triangle) and TO-PRO (open circle) from ctDNA (the lines in the figure are only guides to the eye).
Figure 5
Figure 5
(A) The salt dependence of the two rate constants for the dissociation of BOXTO from ctDNA (biexponential decay, k1, filled square, k2, filled triangle). (B) The salt dependence of the two rate constants for the dissociation of TO-PRO from ctDNA (biexponential decay, k1, filled square, k2, filled diamond), and for the single rate constant for dissociation from poly(dA-dT)2 (open triangle).
Figure 6
Figure 6
The average rate constant (see equation 1) versus concentration of NaCl for the dissociation from DNA. (A) BO (filled triangle, single exponential decay), BO-PRO (filled square), and BEBO (open diamond) from ctDNA. (B) TO-PRO (open square), BETO (filled down-triangle, the line is a guide to the eye), and BOXTO (open down-triangle) from ctDNA. (C) BO (filled triangle), BO-PRO (filled square), EB (open circle), PI (filled circle), TO-PRO (open square) from ctDNA and DAPI from poly(dA-dT) (filled diamond) and poly(dG-dC) (open diamond). Data for EB, PI and DAPI are from Wilson et al. (14).
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