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Nucleic Acids Research Advance Access originally published online on June 22, 2007
Nucleic Acids Research 2007 35(12):e89; doi:10.1093/nar/gkm467
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Nucleic Acids Research, 2007, Vol. 35, No. 12 e89
© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

A 96-well DNase I footprinting screen for drug–DNA interactions

Tom Ellis1, David A. Evans1, Christopher R. H. Martin1 and John A. Hartley1,2,*

1Spirogen Ltd, London Bioscience Innovation Centre, 2 Royal College Street, London, NW1 0NH and 2Cancer Research UK Drug-DNA Interactions Research Group, Department of Oncology, University College London, 91 Riding House Street, London W1W 7BS, UK

*To whom correspondence should be addressed. Tel: +44 (0)20 7679 9326; Fax: +44 (0)20 7436 2956; Email: john.hartley{at}ucl.ac.uk

Received March 21, 2007. Revised May 17, 2007. Accepted May 28, 2007.

The established protocol for DNase I footprinting has been modified to allow multiple parallel reactions to be rapidly performed in 96-well microtitre plates. By scrutinizing every aspect of the traditional method and making appropriate modifications it has been possible to considerably reduce the time, risk of sample loss and complexity of footprinting, whilst dramatically increasing the yield of data (30-fold). A semi-automated analysis system has also been developed to present footprinting data as an estimate of the binding affinity of each tested compound to any base pair in the assessed DNA sequence, giving an intuitive ‘one compound–one line’ scheme. Here, we demonstrate the screening capabilities of the 96-well assay and the subsequent data analysis using a series of six pyrrolobenzodiazepine-polypyrrole compounds and human Topoisomerase II alpha promoter DNA. The dramatic increase in throughput, quantified data and decreased handling time allow, for the first time, DNase I footprinting to be used as a screening tool to assess DNA-binding agents.


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