Groove-binding Unsymmetrical Cyanine Dyes for DNA Applications
Doctoral thesis, 2006
This work focuses on interactions between DNA and a group of newly developed unsymmetrical cyanine dyes. The aim of the work was to discover fluorescent dyes that can be used in biophysical studies of natural DNA where minimum perturbation of the DNA-helix is essential, where a slow dissociation rate of the dyes is needed and where the ionic strength is an important parameter. One long-term goal is to be suitable for use in fluorescence based techniques to study ejection of pre-stained DNA from bacteriophages. Since bacteriophage DNA is naturally densely packed in a restricted volume inside the phage capsid, the dyes used for staining the DNA should preferably bind in such way that the DNA conformation is altered as little as possible.
The new dyes are structurally very similar to the cyanine dyes TO and BO, but are extended with different heterocyclic groups. In our newly developed time-resolved electrophoretic mobility shift assay, it was found that none of the new dyes is able to unwind supercoiled DNA, and we therefore concluded that the new dyes do not intercalate DNA. Instead, the new dyes were found to bind to the DNA grooves. The crescent shape and the enhanced hydrophobic properties of the new dyes seem essential for groove-binding. In free solution, the new dyes dissociate more slowly from DNA and, in contrast to predictions, demonstrate a reduced sensitivity to the ionic strength compared to the corresponding intercalating dyes. Since BOXTO exhibits the highest increase in fluorescence quantum yield upon DNA-binding among the new dyes and has the slowest rate of dissociation from DNA, we suggest that BOXTO is the most promising candidate for future DNA applications.
Finally, we discovered that both intercalating and groove-binding cyanine dyes are able to bind to densely packed phage DNA and that the association rates are inversely proportional to the size of the dyes. Counterintuitively, the phages seem less affected by binding of intercalators, despite intercalation perturbing the DNA-helical structure more than groove-binding.
cryo-transmission electron microscopy
dynamic light scattering