PMID: 9539736May 16, 1998Paper

Simultaneous determination of helical unwinding angles and intrinsic association constants in ligand-DNA complexes: the interaction between DNA and calichearubicin B

Proceedings of the National Academy of Sciences of the United States of America
S M ZemanD M Crothers

Abstract

We present a helical unwinding assay for reversibly binding DNA ligands that uses closed circular DNA, topoisomerase I (Topo I), and two-dimensional agarose gel electrophoresis. Serially diluted Topo I relaxation reactions at constant DNA/ligand ratio are performed, and the resulting apparent unwinding of the closed circular DNA is used to calculate both ligand unwinding angle (phi) and intrinsic association constant (Ka). Mathematical treatment of apparent unwinding is formally analogous to that of apparent extinction coefficient data for optical binding titrations. Extrapolation to infinite DNA concentration yields the true unwinding angle of a given ligand and its association constant under Topo I relaxation conditions. Thus this assay delivers simultaneous structural and thermodynamic information describing the ligand-DNA complex. The utility of this assay has been demonstrated by using calichearubicin B (CRB), a synthetic hybrid molecule containing the anthraquinone chromophore of (DA) and the carbohydrate domain of calicheamicin gamma1I. The unwinding angle for CRB calculated by this method is -5. 3 +/- 0.5 degrees. Its Ka value is 0.20 x 10(6) M-1. For comparison, the unwinding angles of ethidium bromide and DA have been...Continue Reading

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Citations

Feb 11, 2004·Journal of Biomolecular Structure & Dynamics·Michael TriebKlaus R Liedl
Jul 6, 2010·Nucleic Acids Research·Domenico SalernoFrancesco Mantegazza
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Jun 3, 2021·Methods and Protocols·Ruixin LiJong Hyun Choi
Sep 10, 2010·The Journal of Physical Chemistry. B·Huan-Quan SunQi Liao

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