Interaction of three-way DNA junctions with steroids

DNA aptamers that bind to cholic acid were previously isolated by an in vit ro selection method. Secondary structural prediction and deletion-mutant ex periments suggested that the cholic-acid binding regions of 19 sequenced cl ones could form three-way-junction structures. In this article, the seconda ry structures of the sequenced clones and the structural requirements for b inding to cholic acid were evaluated. A course of mutational-analysis and c hemical-modification experiments provided strong support for the predicted secondary structure and also indicated that the binding site is located at the branching point of the three-way junction. Sequence analysis revealed t hat the sequences of the three base pairs flanking the junction of the thre e stems are highly conserved among selected clones. The evaluation of the r elative binding of several bile acids and structurally related steroids wit h the aptamer was also carried out. The results revealed a broad range of s electivity and preference for hydrophobic steroids rather than for cholic a cid upon binding, indicating that the binding is driven by a hydrophobic in teraction. The experimental results reported here allowed us to propose a s tructural model of a binding site formed by three Watson-Crick base pairs.