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Effects of a hairpin polyamide on DNA melting: comparison with distamycin and Hoechst 33258

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Author

  • PL James
  • L Le Strat
  • Ulf Ellervik
  • C Bratwall
  • B Norden
  • T Brown
  • KR Fox

Summary, in English

We have used DNase I footprinting and fluorescence melting studies to study the interaction of the hairpin polyamide Im-Py-Py-Py-(R)(H2N)gamma-Im`-PY-PY-PY-beta-Dp with its preferred binding sites (5'-WGWWCW; W=A or T) and other sequences. DNase I footprinting confirmed that the ligand binds to the sequence AGAACA at nanomolar concentrations and that changing the terminal A to G causes a dramatic decrease in affinity, while there was no. interaction with the reverse sequence WCWWGW Fluorescence melting studies with I I mer duplexes showed that the polyamide had very different effects on the forward (TGWWCT) and reverse (TCTAGT) sequences. At low concentrations, the polyamide produced biphasic melting curves with TGATCT, TGTACT and TGAACT, suggesting a strong interaction. In contrast, the melting profiles with TCTAGT were always monophasic and showed much smaller concentration dependent changes in T-m. The polyamide also showed weak binding to the sequence TGATCT when one of the central AT pairs was replaced with an AC mismatch. These melting profiles were compared with those produced by the AT-selective minor groove binding agents distamycin and Hoechst 33258 at the same site's and at similar sequences containing A(5) and (AT)(3), which are expected to bind distamycin in the 1:1 and 2:1 modes, respectively. These ligands produced simple monophasic melting curves in which the T-m steadily increased as the ligand concentration was raised. (C) 2004 Elsevier B.V. All rights reserved.

Publishing year

2004

Language

English

Pages

205-212

Publication/Series

Biophysical Chemistry

Volume

111

Issue

3

Document type

Journal article

Publisher

Elsevier

Topic

  • Organic Chemistry

Keywords

  • Polyamide
  • Distamycin
  • Hoechst 33258
  • Molecular beacons
  • DNA melting

Status

Published

ISBN/ISSN/Other

  • ISSN: 1873-4200