A Competition Dialysis Assay for the Study of Structure‐Selective Ligand Binding to Nucleic Acids

Jonathan B. Chaires1

1 University of Mississippi Medical Center, Jackson, Mississippi
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 8.3
DOI:  10.1002/0471142700.nc0803s11
Online Posting Date:  February, 2003
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Abstract

Unique DNA structures represent potential targets for small molecules, and provide a promising new avenue for drug development. However, attempts to rationally design small molecules that bind selectively to a particular DNA structure have been hampered by the lack of a rapid and convenient assay for structural selectivity. Determination of structure‚Äźselective ligand binding using competition dialysis is described in this unit. The competition dialysis assay is simple, straightforward, and rapid once stock solutions of the nucleic acid structures of interest have been prepared as described.

     
 
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Table of Contents

  • Basic Protocol 1: Determination of Structure‐Selective Ligand Binding by Competition Dialysis
  • Support Protocol 1: Preparation of Nucleic Acid Solutions
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Determination of Structure‐Selective Ligand Binding by Competition Dialysis

  Materials
  • Dialysate solution: 1 µM test ligand in BPES (see recipe for BPES)
  • Nucleic acid samples (see protocol 2)
  • 10% (w/v) SDS
  • Slide‐a‐Lyzer MINI dialysis units with appropriate molecular weight cutoff (e.g., 3500 Da; Pierce), and floatation device
  • Submicro spectrophotometer cell (Starna Cells) with 1‐cm path length and 160‐µL geometric volume
  • Graphics software (e.g., Microcal Origin)

Support Protocol 1: Preparation of Nucleic Acid Solutions

  Materials
  • Natural DNA samples from Clostridium perfringens, calf thymus, and Micrococcus lysodeikticus (Sigma)
  • BPES (see recipe)
  • Synthetic polydeoxyribonucteotides (Amersham Pharmacia Biotech): poly(dA), poly(dT), poly(dC), poly(dA):poly(dT), poly(dAdT), and poly(dGdC)
  • Synthetic polyribonucleotides (Sigma): poly(rA) and poly(rA):poly(rU)
  • Custom synthetic deoxyoligonucleotides 5′T 2G 20T 2 and 5′AG 3(T 2AG 3) 3 (Research Genetics or Oligos Etc.)
  • Additional reagents and equipment for preparing genomic DNA fragments for viscometry (unit 8.1), and for UV absorbance (unit 7.3) and circular dichroism (unit 7.12) spectroscopy
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Figures

Videos

Literature Cited

Literature Cited
   Alberti, P., Ren, J., Teulade‐Fichou, M.P., Guittat, L., Riou, J.F., Chaires, J., Helene, C., Vigneron, J.P., Lehn, J.M., and Mergny, J.L. 2001. Interaction of an acridine dimer with DNA quadruplex structures. J. Biomol. Struct. Dyn. 19:505‐513.
   Becker, M.M. and Dervan, P.B. 1979. Molecular recognition of nucleic acids by small molecules. Binding affinity and structural specificity of bis(methdium)spermine. J. Am. Chem. Soc. 101:3664‐3666.
   Chaires, J.B. 1985. Long‐range allosteric effects on the B to Z equilibrium by daunomycin. Biochemistry 24:7479‐7486.
   Chaires, J.B. 1992. Application of equilibrium binding methods to elucidate the sequence specificity of antibiotic binding to DNA. In Advances in DNA Sequence Specific Agents (L.H. Hurley, ed.) pp. 3‐23. JAI Press, Greenwich, Conn.
   Haq, I., Ladbury, J.E., Chowdhry, B.Z., and Jenkins, T.C. 1996. Molecular anchoring of duplex and triplex DNA by disubstituted anthracene‐9,10‐diones: Calorimetric, UV melting and competition dialysis studies. J. Am. Chem. Soc. 118:10693‐10701.
   Herrera, J.E. and Chaires, J.B. 1989. A premelting conformational transition in poly(dA)‐poly(dT) coupled to daunomycin binding. Biochemistry 28:1993‐2000.
   Lisgarten, J.N., Coll, M., Portugal, J., Wright, C.W., and Aymami, J. 2002. The antimalarial and cytotoxic drug cryptolepine intercalates into DNA at cytosine‐cytosine sites. Nat. Struct. Biol. 9:57‐60.
   Müller, W. and Crothers, D.M. 1975. Interactions of heteroaromatic compounds with nucleic acids. 1. The influence of heteroatoms and polarizability on the base specificity of intercalating ligands. Eur. J. Biochem. 54:267‐277.
   Perry, P.J. and Jenkins, T.C. 1999. Recent advances in the development of telomerase inhibitors for the treatment of cancer. Exp. Opin. Invest. Drugs. 8:1981‐2008.
   Ren, J. and Chaires, J.B. 1999. Sequence and structural selectivity of nucleic acid binding ligands. Biochemistry 38:16067‐16075.
   Ren, J. and Chaires, J.B. 2000. Preferential binding of 3, 3′‐diethyloxadicarbocyanine to triplex DNA. J. Am. Chem. Soc. 122:424‐425.
   Ren, J. and Chaires, J.B. 2001. Rapid screening of structurally selective ligand binding to nucleic acids. Methods Enzymol. 340:99‐108.
   Ren, J., Bailly, C., and Chaires, J.B. 2000. NB‐506, an indolocarbazole topoisomerase I inhibitor, binds preferentially to triplex DNA. FEBS Lett. 470:355‐359.
   Ren, J., Qu, X., Dattagupta, N., and Chaires, J.B. 2001. Molecular recognition of a RNA:DNA hybrid structure. J. Am. Chem. Soc. 123:6742‐6743.
   Satyanarayana, S., Dabrowiak, J.C., and Chaires, J.B. 1992. Neither delta‐ nor lambda‐tris(phenanthroline)ruthenium(II) binds to DNA by classical intercalation. Biochemistry. 31:9319‐9324.
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