Use of maleimide-thiol coupling chemistry for efficient syntheses of oligonucleotide-enzyme conjugate hybridization probes.
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.
Ghosh
S S
SS
SISKA Diagnostics, Inc., La Jolla, California.
Kao
P M
PM
McCue
A W
AW
Chappelle
H L
HL
D000469:Alkaline Phosphatase; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D003432:Cross-Linking Reagents; D004247:DNA / Q000032:analysis / Q000235:genetics*; D006735:Horseradish Peroxidase; D007202:Indicators and Reagents; D008301:Maleimides; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015345:Oligonucleotide Probes / Q000138:chemical synthesis*; D010957:Plasmids; D011485:Protein Binding; D013438:Sulfhydryl Compounds; D001616:beta-Galactosidase / Q000235:genetics*
D003432:Cross-Linking Reagents; D007202:Indicators and Reagents; D008301:Maleimides; D015345:Oligonucleotide Probes; D013438:Sulfhydryl Compounds; D004247:DNA; D006735:Horseradish Peroxidase; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase; C023860:N,N'-2-phenylenedimaleimide