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

Singleton, Scott F. and Dervan, Peter B. (1992) Thermodynamics of oligodeoxyribonucleotide-directed triple helix formation: an analysis using quantitative affinity cleavage titration. Journal of the American Chemical Society, 114 (18). pp. 6957-6965. ISSN 0002-7863. doi:10.1021/ja00044a002. https://resolver.caltech.edu/CaltechAUTHORS:20160510-141441505

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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.


Item Type:Article
Related URLs:
URLURL TypeDescription
http://dx.doi.org/10.1021/ja00044a002DOIArticle
http://pubs.acs.org/doi/abs/10.1021/ja00044a002PublisherArticle
ORCID:
AuthorORCID
Dervan, Peter B.0000-0001-8852-7306
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.
Funders:
Funding AgencyGrant Number
NIHGM-35724
NSF Predoctoral FellowshipUNSPECIFIED
Ralph M. Parsons FoundationUNSPECIFIED
Issue or Number:18
DOI:10.1021/ja00044a002
Record Number:CaltechAUTHORS:20160510-141441505
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20160510-141441505
Official Citation:Thermodynamics of oligodeoxyribonucleotide-directed triple helix formation: an analysis using quantitative affinity cleavage titration Scott F. Singleton and Peter B. Dervan Journal of the American Chemical Society 1992 114 (18), 6957-6965 DOI: 10.1021/ja00044a002
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:66936
Collection:CaltechAUTHORS
Deposited By: Victoria Brennan
Deposited On:19 May 2016 18:03
Last Modified:11 Nov 2021 00:24

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