Nuclease activity of 1,10-phenanthroline-copper: sequence-specific targeting.
Chen, C H
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.
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- Sequence-specific scission of DNA by the chemical nuclease activity of 1,10-phenanthroline-copper(I) targeted by RNA.
- Secondary structure specificity of the nuclease activity of the 1,10-phenanthroline-copper complex.
- Scission of RNA by the chemical nuclease of 1,10-phenanthroline-copper ion: preference for single-stranded loops.
- Sequence-specific recognition and cleavage of duplex DNA via triple-helix formation by oligonucleotides covalently linked to a phenanthroline-copper chelate.
- A versatile in vivo footprinting technique using 1,10-phenanthroline–copper complex to study important cellular processes