Chen C H, Sigman D S
Proc Natl Acad Sci U S A. 1986 Oct;83(19):7147-51. doi: 10.1073/pnas.83.19.7147.
The nuclease activity of 1,10-phenanthroline-copper ion can be targeted to specific DNA sequences by attachment of the ligand to the 5' end of complementary deoxyoligonucleotides via a phosphoramidate linkage. To synthesize the adduct, the phosphorimidazolide of the deoxyoligonucleotide is prepared using a water-soluble carbodiimide and is then coupled to 5-glycylamido-1,10-phenanthroline. After hybridization to the target DNA, sequence-specific cleavage is observed upon the addition of cupric ion and 3-mercaptopropionic acid. Two methods of assaying the cutting of the operator sequence of the lac operon have been employed using the oligonucleotide 5'-AATTGTTATCCGCTCACAATT-3' representing sequence positions 21-1 of the template strand. In the first, the single-stranded DNA of the phage M13mp8 was the target, and cuts were detected using a primer-extension assay. In the second, the substrate was an EcoRI fragment 3' labeled in the nontemplate strand. After denaturation and reannealing to the oligonucleotide-1,10-phenanthroline adduct, cupric ion and 3-mercaptopropionic acid were added, and the products were analyzed directly on a sequencing gel. With the phenanthroline moiety attached to position 21 of the oligonucleotide carrier, cutting was observed at positions 20-25 using both assays.
通过磷酰胺酯键将配体连接到互补脱氧寡核苷酸的5'端，可使1,10 - 菲咯啉 - 铜离子的核酸酶活性靶向特定的DNA序列。为了合成加合物，使用水溶性碳二亚胺制备脱氧寡核苷酸的磷酰亚胺唑化物，然后将其与5 - 甘氨酰胺基 - 1,10 - 菲咯啉偶联。与靶DNA杂交后，加入铜离子和3 - 巯基丙酸时观察到序列特异性切割。使用代表模板链第21 - 1位序列的寡核苷酸5'-AATTGTTATCCGCTCACAATT-3'，采用了两种测定乳糖操纵子操纵序列切割的方法。第一种方法中，噬菌体M13mp8的单链DNA作为靶标，使用引物延伸测定法检测切割情况。第二种方法中，底物是在非模板链上3'端标记的EcoRI片段。变性并与寡核苷酸 - 1,10 - 菲咯啉加合物重退火后，加入铜离子和3 - 巯基丙酸，产物直接在测序凝胶上进行分析。当菲咯啉部分连接到寡核苷酸载体的第21位时，两种测定方法均在第20 - 25位观察到切割。
