Determination of Binding Mode: Intercalation

Peter C. Dedon1

1 Massachusetts Institute of Technology, Cambridge, Massachusetts
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 8.1
DOI:  10.1002/0471142700.nc0801s00
Online Posting Date:  May, 2001
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Abstract

A small molecule can be assumed to bind to DNA by intercalation between base pairs if it causes lengthening and unwinding of the DNA helix and undergoes changes in its spectral properties, such as DNA‐induced hypochromism and quenching of its UV absorbance. DNA lengthening and unwinding can be determined from the change in viscosity of a solution of linear or plasmid DNA, respectively. Intercalation of a ligand can also be seen as a reduction in the UV/visible absorbance of the intercalator, as well as a shift in the absorbance maximum.

     
 
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Table of Contents

  • Basic Protocol 1: Determination of DNA Lengthening by Viscometry
  • Alternate Protocol 1: Determination of DNA Lengthening by Viscometry for Ligands Soluble in Organic Solvents
  • Basic Protocol 2: Assessment of DNA Unwinding by Plasmid Viscometry
  • Alternate Protocol 2: Assessment of DNA Unwinding by Plasmid Viscometry for Ligands Soluble in Organic Solvents
  • Basic Protocol 3: Assessment of Changes in the Optical Properties of Ligands Upon Binding to DNA
  • Support Protocol 1: Preparation of Genomic DNA Fragments for Viscometry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Determination of DNA Lengthening by Viscometry

  Materials
  • 10% HNO 3
  • 10 mM buffer (Tris⋅Cl, HEPES, or other buffer) containing 1 mM EDTA, pH 7 (for TE buffer see appendix 2A)
  • 0.8 to 2.5 mM (base pairs) sonicated S1 nuclease‐treated calf thymus DNA of ∼200‐bp average length, in above buffer (see protocol 6)
  • Ligand of interest dissolved in above buffer
  • Semi‐micro capillary viscometer (Cannon‐Ubehold or Cannon‐Manning semi‐micro type 75, see Fig. )
  • 0.4‐µm membrane filtration apparatus (e.g., Millipore Ultrafree MC or Centricon filters)
  • Constant‐temperature water bath, 25° ± 0.1°C
  • Stopwatch (± 0.01 sec) or Wescan fiber‐optic detection unit and timer (Wescan Instruments)
  • Micropipettor with extension or extended microsyringe assembly (Hamilton or Stoelting)
NOTE: All solutions should be passed through a 0.4‐µm membrane filter to remove particulate matter that could clog the viscometer capillary and thus produce aberrant flow times.

Alternate Protocol 1: Determination of DNA Lengthening by Viscometry for Ligands Soluble in Organic Solvents

  • Ligand of interest dissolved in an organic solvent that is miscible with water
  • Organic solvent
  • Microcentrifuge tubes
  • Small vacuum desiccator with water aspirator
  • Micro stir bars to fit in microcentrifuge tubes
  • Magnetic stirring plate

Basic Protocol 2: Assessment of DNA Unwinding by Plasmid Viscometry

  Materials
  • DNA: Phage DNA M13mp, PM2, or ∅X174; plasmids COL E1 or pBR325; or any similarly sized plasmid (5 to 7 kbp); each prepared by standard alkaline lysis (e.g., CPMB UNIT ) or similar procedures, and suspended at 1 mg/mL in TE ( appendix 2A) or other buffer
  • 10 mM buffer (Tris⋅Cl, HEPES, or other buffer) containing 1 mM EDTA, pH 7 (for TE buffer see appendix 2A)
  • Ligand of interest dissolved in above buffer (∼1 mM)
  • 1 to 2 mM ethidium bromide solution (see recipe)
  • Semi‐micro capillary viscometer (Cannon‐Ubehold or Cannon‐Manning semi‐micro type 75, see Fig. )
  • 0.4‐µm membrane filtration apparatus (e.g., Millipore Ultrafree MC or Centricon filters)
  • Constant‐temperature water bath, 25° ± 0.1°C
  • Stopwatch (± 0.01 sec) or Wescan fiber‐optic detection unit and timer (Wescan Instruments)
  • Micropipettor with extension or extended microsyringe assembly (Hamilton or Stoelting)
CAUTION: Ethidium bromide is a mutagen and an environmental hazard. It should be handled carefully with gloves and disposed of properly. Methods of disposal may vary between institutions. Consult with the institution's environmental safety office for the preferred means of storage and disposal of ethidium bromide.

Alternate Protocol 2: Assessment of DNA Unwinding by Plasmid Viscometry for Ligands Soluble in Organic Solvents

  Materials
  • Sonicated, S1 nuclease–treated calf thymus DNA (see protocol 6)
  • 10 mM buffer (Tris⋅Cl, HEPES, or other buffer) containing 1 mM EDTA, pH 7 (for TE buffer see appendix 2A)
  • Ligand of interest dissolved in above buffer
  • Digital or double‐beam UV/visible spectrophotometer capable of recording absorbance spectra

Basic Protocol 3: Assessment of Changes in the Optical Properties of Ligands Upon Binding to DNA

  Materials
  • Calf thymus DNA (sodium salt)
  • Phosphate/EDTA buffer: 50 mM sodium phosphate with 1 mM EDTA, pH 7
  • Nitrogen gas
  • Gel loading buffer ( appendix 2A)
  • 1% (w/v) agarose gel in TBE buffer
  • TBE buffer ( appendix 2A)
  • DNA size markers, 100 to 1000 bp (e.g., HaeIII‐digested ∅X174 DNA)
  • 10 mM sodium acetate, pH 5/100 mM NaCl/30 µM ZnCl 2
  • S1 nuclease
  • Buffered phenol or 1:1 (v/v) phenol/chloroform ( appendix 2A)
  • 3 M sodium acetate, pH 7
  • 100%, 95%, and 70% (v/v) ethanol
  • TE buffer, pH 7.5 ( appendix 2A)
  • 10 mg/mL DNase‐free RNase A (see recipe)
  • 20 mg/mL proteinase K in water (store in single‐use aliquots at −20°C)
  • 24:1 (v/v) chloroform/isoamyl alcohol
  • 1 µg/mL ethidium bromide solution (see recipe)
  • Sonicator (e.g., Branson model 450)
  • 3000‐ to 10,000‐MWCO dialysis tubing
  • 3 × 60–cm column of Sepharose 4B or Sephacryl C‐500‐HR
  • UV transilluminator
  • Additional reagents and equipment for agarose gel electrophoresis (e.g., CPMB UNIT ) and phenol/chloroform extraction ( appendix 2A)
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Figures

Videos

Literature Cited

Literature Cited
   Cohen, G. and Eisenberg, H. 1969. Viscosity and sedimentation study of sonicated DNA‐proflavine complexes. Biopolymers 8:45‐55.
   Cohen, G.L., Bauer, W.R., Barton, J.K., and Lippard, S.J. 1979. Binding of cis‐ and trans‐dichlorodiammineplatinum(II) to DNA: Evidence for unwinding and shortening of the double helix. Science 203:1014‐1016.
   Lerman, L.S. 1961. Structural considerations in the interaction of DNA and acridines. J. Mol. Biol. 3:18‐30.
   Révet, B.M., Schmir, M., and Vinograd, J. 1971. Direct determination of the superhelix density of closed circular DNA by viscometric titration. Nature 229:10‐13.
   Satyanarayana, S., Dabrowiak, J.C., and Chaires, J.B. 1993. Tris(phenanthroline)ruthenium(II) enantiomer interactions with DNA: Mode and specificity of binding. Biochemistry 32:2573‐2584.
   Saucier, J.‐M. 1977. Physicochemical studies on the interaction of irehdiamine A with bihelical DNA. Biochemistry 16:5879‐5889.
   Waring, M. 1970. Variation in the supercoils in closed circular DNA by binding of antibiotics and drugs: Evidence for molecular models involving intercalation. J. Mol. Biol. 54:247‐279.
   Wilson, W.D. and Jones, R.L. 1982. Intercalation in biological systems. In Intercalation Chemistry (M.S. Whittingham and A.J. Jacobson, eds.) pp. 445‐501. Academic Press, New York.
   Yu, L., Golik, J., Harrison, R., and Dedon, P. 1994. The deoxyfucose‐anthranilate of esperamicin A1 confers intercalative DNA binding and causes a switch in the chemistry of bistranded DNA lesions. J. Am. Chem. Soc. 116:9733‐9738.
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