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

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.

Time-resolved electrophoretic analysis of mobility shifts for dissociating DNA ligands

“…1a) could be obtained, however, because substantial dissociation occurred before electrophoretic separation of the two DNA forms was achieved (data not shown). This interpretation is consistent with the fact that in this case the measured dissociation time of 7 min [7] is short compared to the typical experimental time (40 min in Fig. 1a).…”

“…2c) shows that dissociation of BOXTO does not influence the unwinding assay, and in particular supports that the lack of minimum is not due to dissociation of the dye. A dissociation halftime of 147 min was obtained from the decrease in fluorescence intensity, in agreement with earlier determinations during electrophoresis under approximately same conditions [7]. This comparatively slow dissociation is consistent with the linear behavior in Fig.…”

“…Regarding the stability of the DNA-dye complex during gel electrophoresis, we have earlier found that dyes of the types studied here dissociate in agreement with a first order process [7] r(t) = r(0) ? e…”

“…The position of native (nonstained) DNA was obtained by loading the same DNA sample in parallel lanes on the same gel but after different durations of electrophoresis, and the position of the zones vs. electrophoresis time was determined by scanning the fluorescence of the ethidium bromide post-stained gel. The dissociation kinetics of the dyes were obtained from the integrated zone intensity measured after various durations of electrophoresis, as described in [7]. The intensity decays were monoexponetial and the reported halftimes are t 1/2 = ln2/k where k is the fitted rate constant.…”

“…Generally the complex is assumed to be stable for the duration of the electrophoretic analysis, for instance due to a strong interaction, such as between the thiolmodified DNA and the gold particles [6]. However, some complexes do dissociate, which sometimes can be utilized to study dissociation kinetics, for example, when ligands dissociate from DNA [7]. Under other conditions dissociation is a disadvantage, for instance, when it is caused by the migration itself, as is the case for complexes between DNA and the Escherichia coli recombination protein RecA [5].…”