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

• DOI: 10.1021/bc00001a009
• Publication Date: 2005-03-08T00:43:06Z
• Authors: Soumitra S. Ghosh; Philip M. Kao; Ann W. McCue; Hugh L. Chappelle
• Keywords: Conjugate; Horseradish peroxidase; Maleimide; Thiol; Molecular beacon; Reactivity

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