Use of Maleimide-Thiol Coupling Chemistry for Efficient Syntheses of Oligonucleotide-Enzyme Conjugate Hybridization Probes
Use of Maleimide-Thiol Coupling Chemistry for Efficient Syntheses of Oligonucleotide-Enzyme Conjugate Hybridization Probes
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DOI：
10.1021/bc00001a009
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发表时间：
1990-01-01
影响因子：
4.7
通讯作者：
Chappelle, Hugh L.
中科院分区：
文献类型：
--
作者：
Ghosh, Sournitra S.;Kao, Philip M.;Chappelle, Hugh L.
本文描述了两种利用巯基对马来酰亚胺的反应性来合成用作非放射性同位素杂交探针的寡核苷酸-酶偶联物的一般方法。第一种方法采用6-马来胺己酸琥珀酰亚胺酯将5′-硫代寡核苷酸偶联至犊牛肠碱性磷酸酶，偶联率为1:1，产率为80-85%。第二种方法采用N，N'-1,2-苯二马来酰亚胺与5'-硫代寡核苷酸交联硫代辣根过氧化物酶或β -半乳糖苷酶，产率分别为58%和65%。寡核苷酸-碱性磷酸酶偶联物可在4 h内检测到6个mol的目标DNA，而用染料沉淀法检测辣根过氧化物酶偶联物的灵敏度要低40倍。
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.