Nuclease activity of 1,10-phenanthroline-copper: sequence-specific targeting.
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.
D001483:Base Sequence; D002384:Catalysis; D004262:DNA Restriction Enzymes; D003851:Deoxyribonucleases; D006861:Hydrogen Peroxide; D007763:Lac Operon; D009841:Oligonucleotides; D010618:Phenanthrolines; D013379:Substrate Specificity
D009841:Oligonucleotides; D010618:Phenanthrolines; C014741:bis(1,10-phenanthroline)copper(2+) ion; D006861:Hydrogen Peroxide; D003851:Deoxyribonucleases; D004262:DNA Restriction Enzymes