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Nucleic Acids Research, 2000, Vol. 28, No. 9 1963-1968
© 2000 Oxford University Press

Interaction of three-way DNA junctions with steroids

Teru Kato, Kazuyoshi Yano, Kazunori Ikebukuro and Isao Karube*

Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan

DNA aptamers that bind to cholic acid were previously isolated by an in vitro selection method. Secondary structural prediction and deletion-mutant experiments suggested that the cholic-acid binding regions of 19 sequenced clones could form three-way-junction structures. In this article, the secondary structures of the sequenced clones and the structural requirements for binding to cholic acid were evaluated. A course of mutational-analysis and chemical-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 that the sequences of the three base pairs flanking the junction of the three stems are highly conserved among selected clones. The evaluation of the relative binding of several bile acids and structurally related steroids with the aptamer was also carried out. The results revealed a broad range of selectivity and preference for hydrophobic steroids rather than for cholic acid upon binding, indicating that the binding is driven by a hydrophobic interaction. The experimental results reported here allowed us to propose a structural model of a binding site formed by three Watson–Crick base pairs.

* To whom correspondence should be addressed. Tel: +81 3 5452 5220; Fax: +81 3 5452 5227; Email: karube@bio.rcast.u-tokyo.ac.jp


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L. S. Shlyakhtenko, V. N. Potaman, R. R. Sinden, A. A. Gall, and Y. L. Lyubchenko
Structure and dynamics of three-way DNA junctions: atomic force microscopy studies
Nucleic Acids Res., September 15, 2000; 28(18): 3472 - 3477.
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