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  • Title: Relative DNA binding affinity of helix 3 homeodomain analogues, major groove binders, can be rapidly screened by displacement of prebound ethidium bromide. A comparative study.
    Author: Shim YH, Arimondo PB, Laigle A, Garbesi A, Lavielle S.
    Journal: Org Biomol Chem; 2004 Mar 21; 2(6):915-21. PubMed ID: 15007422.
    Abstract:
    The binding affinity for a 12-bp dsDNA of Antennapedia helix 3 analogues, major groove binders, has been measured by displacement of prebound ethidium bromide, a fluorescent displacement assay proposed for minor groove binders by Boger et al.(J. Am. Chem. Soc., 2000, 122, 6382-6394). Relative binding affinities determined by this method were compared to those obtained by gel mobility shift and footprinting assays for the 12-bp dsDNA and a 178-bp DNA fragment. The present work demonstrates that the fluorescence displacement assay is suitable for rapid screening of major groove binders, even though about 60 to 70% of the prebound ethidium bromide is displaced by these peptides. Total (100%) displacement of ethidium bromide was serendipitously achieved by addition in the peptide sequence, at the N-terminus, of a S-3-nitro-2-pyridinesulfenyl-N-acetyl-cysteine residue. S-3-nitro-2-pyridinesulfenylcysteine was shown to (i) bind to dsDNA with a micromolar affinity and (ii) direct within DNA grooves a peptide with no affinity for dsDNA.
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