A Fluorescent Intercalator Displacement Assay for Establishing DNA Binding Selectivity and Affinity

Winston C. Tse1, Dale L. Boger2

1 Gilead Sciences, Foster City, California, 2 The Scripps Research Institute, La Jolla, California
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 8.5
DOI:  10.1002/0471142700.nc0805s20
Online Posting Date:  April, 2005
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A protocol for a fluorescent intercalator displacement (FID) assay useful for establishing DNA binding selectivity, affinity, stoichiometry, and binding site size, and for distinguishing modes of DNA binding is presented.

Keywords: DNA binding assay; DNA sequence selectivity; intercalator; fluorescence; FID

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Table of Contents

  • Strategic Planning
  • Basic Protocol 1: 96‐Well Single‐Point Assay
  • Basic Protocol 2: Fid Tiration Assay
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: 96‐Well Single‐Point Assay

  • 8.57 µM ethidium bromide in Tris buffer
  • Tris buffer: 0.1 M Tris⋅Cl, pH 8.0 ( appendix 2A), with 0.1 M NaCl
  • 15 µM solutions of each DNA hairpin oligodeoxyribonucleotide in H 2O (100 µL each)
  • Agent blank solution: Tris buffer containing 10% dimethylsulfoxide (DMSO)
  • 7.5 µM and 10.0 µM compound of interest in Tris buffer containing 10% DMSO
  • 96‐well assay plates: tissue culture (TC)‐treated, black, flat‐bottom, polystyrene microtiter plates (e.g., Costar 3916)
  • Top‐reading fluorescence plate reader (e.g., Molecular Devices Spectra Max Gemini)
NOTE: To benefit measurement accuracy, ensure that the same percentage of DMSO is in the agent blank solution and each agent solution (typically 10% DMSO in stock solutions).

Basic Protocol 2: Fid Tiration Assay

  • Ethidium bromide in Tris buffer (see protocol 1)
  • Hairpin oligodeoxyribonucleotide of interest
  • Compound of interest at 0.05 M in Tris buffer containing 10% DMSO
  • 3‐mL quartz cuvette
  • Fluorospectrophotometer (e.g., JY Horiba FluoroMax‐3 with Hamilton autotitration apparatus)
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Literature Cited

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