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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Abstract

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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Materials

Basic Protocol 1: 96‐Well Single‐Point Assay

  Materials
  • 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

  Materials
  • 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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Figures

Videos

Literature Cited

Literature Cited
   Blackwell, T.K. and Weintraub, H. 1990. Differences and similarities in DNA‐binding preferences of MyoD and E2A protein complexes revealed by binding site selection. Science 250:1104‐1110.
   Blackwell, T.K., Kretzner, L., Blackwood, E.M., Eisenman, R.N., and Weintraub, H. 1990. Sequence‐specific DNA binding by the c‐Myc protein. Science 250:1149‐1151.
   Boger, D.L. and Johnson, D.S. 1996. CC‐1065 and the duocarmycins: Understanding their biological function through mechanistic studies. Angew. Chem. Int. Ed. Engl. 35:1438‐1474.
   Boger, D.L. and Tse, W.C. 2001. Thiazole orange as the fluorescent intercalator in a high resolution FID assay for determining DNA binding affinity and sequence selectivity of small molecules. Bioorg. Med. Chem. 9:2511‐2518.
   Boger, D.L., Dechantsreiter, M.A., Ishii, T., Fink, B.E., and Hedrick, M.P. 2000a. Assessment of solution‐phase positional scanning libraries based on distamycin A for the discovery of new DNA binding agents. Bioorg. Med. Chem. 8:2049‐2057.
   Boger, D.L., Fink, B.E., and Hedrick, M.P. 2000b. Total synthesis of distamycin A and 2640 analogs: A solution‐phase combinatorial approach to the discovery of new, bioactive DNA binding agents and development of a rapid, high‐throughput screen for determining relative DNA binding affinity or DNA binding sequence selectivity. J. Am. Chem. Soc. 122:6382‐6394.
   Boger, D.L., Fink, B.E., Brunette, S.R., Tse, W.C., and Hedrick, M.P. 2001. A simple, high‐resolution method for establishing DNA binding affinity and sequence selectivity. J. Am. Chem. Soc. 123:5878‐5891.
   Broude, N.E. 2002. Stem‐loop oligonucleotides: A robust tool for molecular biology and biotechnology. Trends Biotechnol. 20:249‐256.
   Browne, M.J. and Thurlbey, P.L. 1996. Genomes, Molecular Biology and Drug Discovery. Academic Press, London.
   Browne, K.A., He, G.X., and Bruice, T.C. 1993. Microgonotropens and their interactions with DNA. 2. Quantitative evaluation of equilibrium constants for 1:1 and 2:1 binding of dien‐microgonotropen‐a, ‐b, and ‐c as well as distamycin and Hoechst‐33258 to d(GGCGCAAATTTGGCGG)/d(CCGCCAAATTTGCGCC). J. Am. Chem. Soc. 115:7072‐7079.
   Chaltin, P., Borgions, F., Van Aerschot, A., and Herdewijn, P. 2003. Comparison of library screening techniques used in the development of dsDNA ligands. Bioorg. Med. Chem. Lett. 13:47‐50.
   Chiang, S.Y., Azizkhan, J.C., and Beerman, T.A. 1998. A comparison of DNA‐binding drugs as inhibitors of E2F1‐ and Sp1‐DNA complexes and associated gene expression. Biochemistry 37:3109‐3115.
   Chodosh, L.A., Carthew, R.W., and Sharp, P.A. 1986. A single polypeptide possesses the binding and transcription activities of the adenovirus major late transcription factor. Mol. Cell Biol. 6:4723‐4733.
   Choo, Y., Sanchez‐Garcia, I., and Klug, A. 1994. In vivo repression by a site‐specific DNA‐binding protein designed against an oncogenic sequence. Nature 372:642‐645.
   Dervan, P.B. 1986. Design of sequence‐specific DNA‐binding molecules. Science 232:464‐471.
   Galas, D.J. and Schmitz, A. 1978. DNase footprinting: A simple method for the detection of protein‐DNA binding specificity. Nucl. Acids Res. 5:3157‐3170.
   Gottesfeld, J.M., Neely, L., Trauger, J.W., Baird, E.E., and Dervan, P.B. 1997. Regulation of gene expression by small molecules. Nature 387:202‐205.
   Guelev, V.M., Harting, M.T., Lokey, R.S., and Iverson, B.L. 2000. Altered sequence specificity identified from a library of DNA‐binding small molecules. Chem. Biol. 7:1‐8.
   Ham, Y.W., Tse, W.C., and Boger, D.L. 2003. High‐resolution assessment of protein DNA binding affinity and selectivity utilizing a fluorescent intercalator displacement (FID) assay. Biorg. Med. Chem. Lett. 13:3805‐3807.
   Hamy, F., Albrecht, G., Florsheimer, A., and Bailly, C. 2000. An ARE‐selective DNA minor groove binder from a combinatorial approach. Biochem. Biophys. Res. Commun. 270:393‐399.
   Hardenbol, P., Wang, J.C., and Van Dyke, M.W. 1997. Identification of preferred distamycin‐DNA binding sites by the combinatorial method REPSA. Bioconjugate Chem. 8:617‐620.
   Hayashibara, K.C. and Verdine, G.L. 1992. Template‐directed interference footprinting of cytosine contacts in a protein‐DNA complex: Potent interference by 5‐aza‐2′‐deoxycytidine. Biochemistry 31:11265‐11273.
   Knudsen, H. and Nielsen, P.E. 1996. Antisense properties of duplex‐ and triplex‐forming PNAs. Nucl. Acids Res. 24:494‐500.
   Kuwabara, M.D. and Sigman, D.S. 1987. Footprinting DNA‐protein complexes in situ following gel retardation assays using 1,10‐phenanthroline‐copper ion: Escherichia coli RNA polymerase‐lac promoter complexes. Biochemistry 26:7234‐7238.
   Larson, C.J. and Verdine, G.L. 1996. Bioorganic Chemistry: Nucleic Acids (S.M. Hecht, ed.) pp.  324–346. Oxford University Press, Oxford.
   Lee, L.G., Chen, C.H., and Chiu, L.A. 1986. Thiazole orange: A new dye for reticulocyte analysis. Cytometry 7:508‐517.
   LePecq, J.B. and Paoletti, C. 1967. A fluorescent complex between ethidium bromide and nucleic acids. Physical‐chemical characterization. J. Mol. Biol. 27:87‐106.
   Matteucci, M.D. and Wagner, R.W. 1996. In pursuit of antisense. Nature 384:20‐22.
   Neidle, S. 1997. Recent developments in triple‐helix regulation of gene expression. Anticancer Drug Des. 12:433‐442.
   Neidle, S. and Thurston, D.E. 1994. New Targets for Cancer Chemotherapy (D.J. Kerr and P. Workman, eds.). CRC Press, Boca Raton, Fla.
   Nygren, J., Svanvik, N., and Kubista, M. 1998. The interactions between the fluorescent dye thiazole orange and DNA. Biopolymers 46:39‐51.
   Rajski, S.R. and Williams, R.M. 1998. DNA cross‐linking agents as antitumor drugs. Chem. Rev. 98:2723‐2796.
   Royer‐Pokora, B., Gordon, L.K., and Haseltine, W.A. 1981. Use of exonuclease III to determine the site of stable lesions in defined sequences of DNA: The cyclobutane pyrimidine dimer and cis and trans dichlorodiammine platinum II examples. Nucl. Acids Res. 9:4595‐4609.
   Satz, A.L. and Bruice, T.C. 2000. Synthesis of fluorescent microgonotropens (FMGTs) and their interactions with dsDNA. Bioorg. Med. Chem. 8:1871‐1880.
   Scatchard, G. 1949. The attractions of proteins for small molecules and ions. Ann. N.Y. Acad. Sci. 51:660‐672.
   Tao, Z.F., Saito, I., and Sugiyama, H. 2000. Highly cooperative DNA dialkylation by the homodimer of imidazole‐pyrrole diamide‐CPI conjugate with vinyl linker. J. Am. Chem. Soc. 122:1602‐1608.
   Taylor, J.S., Schultz, P.G., and Dervan, P.B. 1984. DNA affinity cleaving. Sequence specific cleavage of DNA by distamycin‐EDTA‐iron(II) and EDTA‐distamycin‐iron(II). Tetrahedron 40:457‐465.
   Thiesen, H.J. and Bach, C. 1990. Target detection assay (TDA): A versatile procedure to determine DNA binding sites as demonstrated on SP1 protein. Nucl. Acids Res. 18:3203‐3209.
   Thurston, D.E. 1999. Nucleic acid targeting: Therapeutic strategies for the 21st century. Br. J. Cancer 80(Suppl 1):65–85.
   Trauger, J.W., Baird, E.E., and Dervan, P.B. 1996. Recognition of DNA by designed ligands at subnanomolar concentrations. Nature 382:559‐561.
   Tse, W.C. and Boger, D.L. 2004. A fluorescent intercalator displacement assay for establishing DNA binding selectivity and affinty. Acc. Chem. Res. 37:61‐69.
   Tse, W.C., Ishii, T., and Boger, D.L. 2003. Comprehensive high‐resolution analysis of hairpin polyamides utilizing a fluorescent intercalator displacement (FID) assay. Biorg. Med. Chem. 11:4479‐4486.
   Tullius, T.D., Dombroski, B.A., Churchill, M.E., and Kam, L. 1987. Hydroxyl radical footprinting: A high‐resolution method for mapping protein‐DNA contacts. Methods Enzymol. 155:537‐558.
   Van Dyke, M.W., Hertzberg, R.P., and Dervan, P.B. 1982. Map of distamycin, netropsin, and actinomycin binding sites on heterogeneous DNA: DNA cleavage‐inhibition patterns with methidiumpropyl‐EDTA‐Fe(II). Proc. Natl. Acad. Sci. U.S.A. 79:5470‐5474.
   Werstuck, G. and Green, M.R. 1998. Controlling gene expression in living cells through small molecule‐RNA interactions. Science 282:296‐298.
   Woods, C.R., Ishii, T., Wu, B., Bair, K.W., and Boger, D.L. 2002a. Hairpin versus extended DNA binding of a substituted β‐alanine linked polyamide. J. Am. Chem. Soc. 124:2148‐2152.
   Woods, C.R., Ishii, T., and Boger, D.L. 2002b. Synthesis and DNA binding properties of iminodiacetic acid‐linked polyamides: Characterization of cooperative extended 2:1 side‐by‐side parallel binding. J. Am. Chem. Soc. 124:10676‐10682.
   Yeung, B.K.S., Tse, W.C., and Boger, D.L. 2003. Determination of binding affinities of triplex forming oligonucleotides using a fluorescent intercalator displacement (FID) assay. Biorg. Med. Chem. Lett. 13:3801‐3804.
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