Chemistry Reference
In-Depth Information
but they should be able to bind with higher affi nity. No conclusive reason was pro-
posed for why D
D - [{Ru(phen)
2
}
2
( m - bb7)]
4 +
bound more strongly than the corre-
sponding complexes containing two, fi ve or ten methylene groups. It was considered
likely that the bb2-bridged complex contains insuffi cient degrees of freedom to bind
tightly, whereas the preference of the bb7-bridged complex over the bb5- and bb-10
bridged complexes may rely on the differential binding potential of the DNA
sequence where the second ruthenium centre binds.
11.3.7 Fluorescent DAPI - Displacement Assay
While NMR spectroscopy provides the most detailed information on the nature of
the binding of the ruthenium complexes with any of the DNA or RNA structures,
it is a relatively time-consuming and expensive technique (in terms of the cost of
the oligonucleotides). A suitable method is required to screen the relative binding
affi nity of a large number of ruthenium complexes, in terms of variable ligands and
stereoisomers, against a range of DNA and RNA structures.
Boger
et al.
120
developed a now widely used fl uorescent intercalator displace-
ment (FID) assay to screen organic compounds that bind, by intercalation or groove
association, with oligonucleotides. The assay is based upon the loss of fl uorescence
resulting from the displacement of an intercalating dye such as ethidium bromide
(EthBr) or thiazole orange (TO) from a DNA sequence by the molecule of interest.
While the assay appears to be widely applicable, some anomalies were observed
with dinuclear ruthenium complexes between the results obtained with the FID
method and observations on the same oligonucleotide-metal complex combinations
using other techniques. As the disparity may have been due to differences in binding
modes between the intercalating dyes and the minor groove-binding metal com-
plexes, a modifi ed fl uorescence assay was proposed that used the minor groove-
binding compound DAPI. It was suggested that there may be a direct displacement
of the fl uorescent DAPI dye by the minor groove-binding ruthenium complexes.
121
In a systematic evaluation of the DNA-binding affi nities of a range of dinuclear
ruthenium complexes with various oligonucleotides, some notable inconsistencies
were observed between the results of the FID fl uorescence assay using an intercalat-
ing dye, compared with DAPI. The metal complex-oligonucleotide combinations
that gave inconsistent results in the fl uorescent displacement assays were further
examined by other techniques, including NMR, affi nity chromatography and equi-
librium dialysis. The results from the DAPI assay were totally consistent with the
relative metal complex-oligonucleotide affi nities determined by the other tech-
niques. It was concluded that the difference in the binding mode between the inter-
calating dye and the groove-binding ruthenium complexes were responsible for the
discrepancies.
121
11.3.8 Affi nity Chromatography
The interactions of DNA with metal complexes may be utilized in the 'reverse'
sense to develop a remarkably effi cient affi nity chromatographic technique for the
