Use of Electrophilic Substitution to Form Site‐Specific Cross‐Links in DNA

Richard M. Pires1, Robert S. Coleman1

1 The Ohio State University, Columbus, Ohio
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 5.5
DOI:  10.1002/0471142700.nc0505s06
Online Posting Date:  November, 2001
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Abstract

This unit describes the synthesis, purification, and analysis of modified oligodeoxynucleotides containing 4‐thio‐2′‐deoxyuridine, and the use of these modified oligodeoxynucleotides in conjunction with bis‐electrophilic tethers to form site‐specific cross‐links with a complementary DNA target. These modified oligodeoxynucleotides provide a flexible tool for studies directed at biophysical investigation of oligonucleotide structure, as well as investigation of DNA hybridization, stability, and sequence‐dependent base nucleophilicity.

     
 
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Table of Contents

  • Basic Protocol 1: Formation of Site‐Specific Cross‐Links in DNA
  • Support Protocol 1: Synthesis of N,N′‐Bis‐Bromoacetyl‐1,2‐Diaminobenzene
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Formation of Site‐Specific Cross‐Links in DNA

  Materials
  • S‐(2‐Cyanoethyl)‐protected 4‐thio‐2′‐deoxyuridine (Glen Research)
  • Dry solvent‐grade acetonitrile
  • 1.0 M DBU solution (see recipe)
  • 50 mM NaSH/NH 4OH solution (see recipe)
  • Nitrogen gas (optional)
  • HPLC buffer A (see recipe)
  • HPLC buffer B: HPLC‐grade acetonitrile
  • 80% (v/v) distilled glacial acetic acid
  • Dry ice
  • Double‐deionized water
  • 2 mg/mL (1.5 U/mg) snake venom phosphodiesterase (SVP) from Crotalus durissus
  • 1 U/µL alkaline phosphatase (AP) from calf intestine
  • 0.1 M potassium phosphate buffer, pH 8.0 ( appendix 2A)
  • Electrophile: N,N′‐bis‐bromoacetyl‐1,2‐diaminobenzene (see protocol 2)
  • Dimethylformamide (DMF)
  • n‐Butanol
  • Radiolabeled complimentary DNA solution (1 to 5 OD/mL)
  • DNA synthesizer with bottle (and septum) that fits a phosphoramidite inlet port
  • Vacuum pump
  • Desiccator
  • Centrifugal evaporator
  • Binary high‐performance liquid chromatograph (HPLC) equipped with UV detector and recorder
  • Polystyrene reversed‐phase (PRP‐1) HPLC column (Hamilton)
  • Spectrophotometer
  • Small spin column containing 0.3 ml Sephadex G‐25 equilibrated with double‐deionized water
  • Additional reagents and equipment for DNA synthesis ( appendix 3C), SVP/AP digestion (unit 10.6), and denaturing polyacrylamide gel electrophoresis (PAGE; appendix 3B)

Support Protocol 1: Synthesis of N,N′‐Bis‐Bromoacetyl‐1,2‐Diaminobenzene

  Materials
  • 1,2‐Diaminobenzene
  • Nitrogen gas
  • Tetrahydrofuran (THF), freshly distilled
  • Triethylamine (TEA), freshly distilled
  • Bromoacetyl bromide
  • Saturated aqueous NaHCO 3
  • Ethyl acetate
  • Saturated NaCl
  • 5% (v/v) HCl
  • MgSO 4
  • Silica
  • 9:1 (v/v) dichloromethane/methanol (CH 2Cl 2/CH 3OH)
  • Flame‐dried 100‐mL flask
  • 3 × 15–cm glass column
  • Additional reagents and equipment for flash column chromatography ( appendix 3E)
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Figures

Videos

Literature Cited

Literature Cited
   Coleman, R.S. and Kesicki, E.A. 1994. Synthesis and postsynthetic modification of oligodeoxynucleotides containing 4‐thio‐2′‐deoxyuridine (dS4U). J. Am. Chem. Soc. 116:11636‐11642.
   Coleman, R.S. and Kesicki, E.A. 1995. Template‐directed cross‐linking of oligonucleotides: Site specific covalent modification of dG‐N7 within duplex DNA. J. Org. Chem. 60:6252‐6253.
   Coleman, R.S. and Pires, R.M. 1997. Covalent cross‐linking of duplex DNA using 4‐thio‐2′‐deoxyuridine as a readily modifiable platform for introduction of reactive functionalities into oligonucleotides. Nucl. Acids Res. 25:4771‐4777.
   Coleman, R.S. and Siedlecki, J.M. 1991. Synthesis and stability of S‐cyanoethyl protected thiouridine and 2′‐deoxy‐4‐thiouridine. Tetrahedron Lett. 32:3033‐3034.
   Skinner, W.A., Cory, M., Shellenberger, T.E., and Degraw, J.I. 1967. Effect of organic compounds on reproductive processes. V: Alkylating agents derived from aryl‐, aralkyl‐, and cyclohexylmethylenediamines. J. Med. Chem. 10:120‐121.
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