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Published August 26, 1992 | public
Journal Article

Thermodynamics of oligodeoxyribonucleotide-directed triple helix formation: an analysis using quantitative affinity cleavage titration

Abstract

The free energy for oligodeoxyribonucleotide-directed triple helix formation at a single site on a DNA plasmid fragment has been analyzed using quantitative affinity cleavage titration. Measurement of the amount of site-specific cleavage of a 339-bp radiolabeled DNA duplex produced at 24-degrees-C (100 mM Na+, 1 mM spermine-4HCl, 50 mM Tris-acetate, pH 7.0) over a concentration change of four orders of magnitude for oligodeoxyribonucleotide-EDTA.Fe 1.Fe, (5'-T*TTTTCTCTCTCTCT-3') yields an equilibrium binding constant, K(T) = 3.7 +/- 1.1 x 10(6) M-1 (DELTA-G(T) = -9.0 +/- 0.2 kcal.mol-1). Quantitative affinity cleavage titration affords association constants that are identical within experimental uncertainty with those obtained from quantitative DNase I footprint titration of the same oligonucleotide with and without EDTA.In (1.In and 6, respectively). Removal of one thymidine and one cytidine residue from the 3' end of 1.Fe reduces the free energy of binding by 0.5 kcal.mol-1, and removal of two thymidine and two cytidine residues from its 3' terminus decreases the binding free energy by 1.1 kcal.mol-1 at pH 7.0. Single internal base triplet mismatches result in a destabilization of the local triple-helical structure by 2.5-3.0 kcal.mol-1. Quantitative affinity cleavage titration is a general method which should allow for the measurement of equilibrium constants for the association of many DNA-binding molecules to single sites on relatively large DNA under a broad range of solution conditions.

Additional Information

© 1992 American Chemical Society. Received February 20, 1992. We thank Warren Wade and Jumi Shin for helpful discussions. We are grateful for generous support from the National Institutes of Health (GM-35724) and for National Science Foundation and Ralph M. Parsons Foundation predoctoral fellowships to S.F.S.

Additional details

Created:
August 20, 2023
Modified:
October 18, 2023