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Footprinting methods for analysis of pyrrole-imidazole polyamide/DNA complexes

Trauger, John W. and Dervan, Peter B. (2001) Footprinting methods for analysis of pyrrole-imidazole polyamide/DNA complexes. In: Drug-Nucleic Acid Interactions. Methods in Enzymology. No.340. Academic Press , San Diego, CA, pp. 450-466. ISBN 9780121822415.

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This chapter describes three complementary footprinting methods and the protocols used for analysis of polyamide : DNA complexes: (1) MPE–Fe(II) footprinting, (2) affinity cleavage, and (3) quantitative DNase I footprint titration. Footprinting with methidiumpropyl–EDTA–Fe(II) [MPE–Fe(II)] is used to identify high-affinity polyamide-binding sites to near nucleotide resolution. Affinity cleavage is used to determine the orientation of the bound polyamide in the minor groove of DNA. Quantitative DNase I footprinting is used to determine equilibrium association constants (K_a) for polyamide–DNA complexes at previously identified match and mismatch sites. A pivotal step in the discovery—evaluation process of new polyamide motifs is the characterization of the affinity and specificity of next-generation molecules following the design-sythesis phase. In the design phase, it is often the case that the sequence preference, as well as the energetics of any new molecule binding at each potential site, is not perfectly understood and it is crucial to scan “libraries” of many potential DNA-binding sites in order to identify true high-affinity binding sites.

Item Type:Book Section
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Dervan, Peter B.0000-0001-8852-7306
Additional Information:© 2001 Academic Press. We are grateful to the National Institutes of Health (General Medical) and the National Foundation for Cancer Research for research support, and to the National Science Foundation and the Ralph M. Parsons Foundation for predoctoral fellowships to J.W.T.
Funding AgencyGrant Number
National Foundation for Cancer ResearchUNSPECIFIED
Ralph M. Parsons FoundationUNSPECIFIED
Series Name:Methods in Enzymology
Issue or Number:340
Record Number:CaltechAUTHORS:20160510-082727863
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Official Citation:John W Trauger, Peter B Dvan, Footprinting methods for analysis of pyrrole-imidazole polyamide/DNA complexes, Methods in Enzymology, Academic Press, 2001, Volume 340, Pages 450-466, ISSN 0076-6879, ISBN 9780121822415, (
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:66824
Deposited By: Victoria Brennan
Deposited On:18 May 2016 19:12
Last Modified:11 Nov 2021 00:03

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