Use of an 125I-labelled DNA ligand to probe DNA structure

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Abstract
It no longer seems likely that DNA molecules in situ have a uniform conformation, represented by the classical B-form helix. For example, recent structural studies have shown that in certain conditions DNA can have a left-handed (so-called Z-form) helix1, and have revealed extensive sequence-dependent variations of B-DNA helical parameters2. Such sequence-dependent variations in DNA structure can be investigated in solution with reagents that bind to DNA in a conformation-dependent manner, and cut one or both strands of the double-helix at the site of binding, as, for example, has been shown for the endonuclease DNase I3. We describe here a simple way to endow a DNA-binding ligand with the ability to cleave DNA—labelling with 125I. The radiochemical damage associated with 125I decay induces a double-stranded DNA break. Using this technique we have shown that a sequence of four consecutive A·T base pairs is a necessary, but not sufficient, condition for strong binding to DNA of the bis-benzamide Hoechst 33258—presumably the other important factor is the conformation of the double-helix at the site of the (A/T)4 sequence. We suggest 125I-Hoechst 33258 may be a useful new probe of DNA structure.

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