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

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.