Minor-Groove Binding Drugs: Where Is the Second Hoechst
33258 Molecule?
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Abstract
Hoechst 33258 binds with high affinity
into the minor groove of
AT-rich sequences of double-helical DNA. Despite extensive studies
of this and analogous DNA binding molecules, there still remains uncertainty
concerning the interactions when multiple ligand molecules are accommodated
within close distance. Albeit not of direct concern for most biomedical
applications, which are at low drug concentrations, interaction studies
for higher drug binding are important as they can give fundamental
insight into binding mechanisms and specificity, including drug self-stacking
interactions that can provide base-sequence specificity. Using circular
dichroism (CD), isothermal titration calorimetry (ITC), and proton
nuclear magnetic resonance (<sup>1</sup>H NMR), we examine the binding
of Hoechst 33258 to three oligonucleotide duplexes containing AT regions
of different lengths: [d(CGCGAATTCGCG)]<sub>2</sub> (A<sub>2</sub>T<sub>2</sub>), [d(CGCAAATTTGCG)]<sub>2</sub> (A<sub>3</sub>T<sub>3</sub>), and [d(CGAAAATTTTCG)]<sub>2</sub> (A<sub>4</sub>T<sub>4</sub>). We find similar binding geometries in the minor groove for all
oligonucleotides when the ligand-to-duplex ratio is less than 1:1.
At higher ratios, a second ligand can be accommodated in the minor
groove of A<sub>4</sub>T<sub>4</sub> but not A<sub>2</sub>T<sub>2</sub> or A<sub>3</sub>T<sub>3</sub>. We conclude that the binding of the
second Hoechst to A<sub>4</sub>T<sub>4</sub> is not cooperative and
that the molecules are sitting with a small separation apart, one
after the other, and not in a sandwich structure as previously proposed
