Use of maleimide-thiol coupling chemistry for efficient syntheses of oligonucleotide-enzyme conjugate hybridization probes

Bioconjug Chem. 1990 Jan-Feb;1(1):71-6. doi: 10.1021/bc00001a009.


Two general methods which exploit the reactivity of sulfhydryl groups toward maleimides are described for the synthesis of oligonucleotide-enzyme conjugates for use as nonradioisotopic hybridization probes. In the first approach, 6-maleimidohexanoic acid succinimido ester was used to couple 5'-thiolated oligonucleotide to calf intestine alkaline phosphatase to provide a 1:1 conjugate in 80-85% yield. The second strategy employed N,N'-1,2-phenylenedimaleimide to cross-link thiolated horseradish peroxidase or beta-galactosidase with a 5'-thiolated oligonucleotide in 58% and 65% yields, respectively. The oligonucleotide-alkaline phosphatase conjugate was able to detect 6 amol of target DNA in 4 h, while the horseradish peroxidase conjugate was found to be 40-fold lower in its sensitivity of detection by using dye precipitation assays.

MeSH terms

  • Alkaline Phosphatase*
  • Animals
  • Base Sequence
  • Cattle
  • Cross-Linking Reagents*
  • DNA / analysis
  • DNA / genetics*
  • Horseradish Peroxidase*
  • Indicators and Reagents
  • Maleimides*
  • Molecular Sequence Data
  • Nucleic Acid Hybridization*
  • Oligonucleotide Probes / chemical synthesis*
  • Plasmids
  • Protein Binding
  • Sulfhydryl Compounds
  • beta-Galactosidase / genetics*


  • Cross-Linking Reagents
  • Indicators and Reagents
  • Maleimides
  • Oligonucleotide Probes
  • Sulfhydryl Compounds
  • DNA
  • Horseradish Peroxidase
  • Alkaline Phosphatase
  • beta-Galactosidase
  • N,N'-2-phenylenedimaleimide