Optimizing the Targeted Chemical Nuclease Activity of 1,10-Phenanthroline−Copper by Ligand Modification

Abstract

Our interest in improving the efficiency of targeted scission reagents has prompted us to study the influence of ring substituents on the nuclease activity of 1,10-phenanthroline-copper conjugated to oligonucleotides and DNA-binding proteins. Since methyl substitution at all but the 2 and 9 positions enhances the copper-dependent chemical nuclease activity of 1,10-phenanthroline, we have compared the activity of conjugates prepared from 5-(aminomethyl)-1,10-phenanthroline (MOP) to those of conjugates prepared from 5-amino-1,10-phenanthroline (amino-OP). Tethering MOP derivatives to the Escherichia coli Fis protein enhances DNA scission several-fold at the weaker cleavage sites initially observed with conjugates prepared from amino-OP. However, scission efficiency is not increased at the stronger cleavage sites, or when scission is targeted to single-stranded DNA by a complementary oligonucleotide. These results are consistent with a change in the rate-determining step for cleavage associated with the differential accessibility of the DNA-bound coordination complex to solvent and reductant. Although the free bis cuprous complex of 2,9-dimethyl-1,10-phenanthroline (neocuproine) is redox-inactive, an oligonucleotide tethered to neocuproine through C5 of the phenanthroline ring efficiently cleaves a complementary DNA sequence. These results establish that the nucleolytic species in targeted scission is the 1:1 cuprous complex and suggest that the oxidative reaction proceeds through a copper-oxo intermediate rather than a metal-coordinated peroxy species. However, substituents at the 2 and 9 positions of the ligand will often hinder close approach of the phenanthroline-copper moiety to the oxidatively sensitive ribose as shown by the preference of the oligonucleotide-targeted chimera for cleavage of single-stranded regions and the failure of neocuproine-DNA-binding protein chimeras and a C2-tethered chimera to cleave DNA.

Bidirectional binding of the TATA box binding protein to the TATA box

“…The cleavage patterns generated by E188C-OP and K97C-OP each span two turns of the DNA helix and as such are considerably broader than those observed for most protein-OP conjugates (26). Because DNA cleavage by tethered OP derivatives is mediated by a nondiffusible copper-oxo species, the unusually broad patterns cannot result from cleavage by a diffusible cleavage agent (27). Two explanations exist for the spread in the cleavage patterns.…”

Variable Structures of Fis-DNA Complexes Determined by Flanking DNA – Protein Contacts

“…The OP moieties iodoacetamido-OP (Sigman et al, 1991), iodomethylacetamido-OP (Gallagher et al, 1996), iodoacetylglycylamino-OP, and iodoacetyl-b-alanylamino-OP (Pan et al, 1995) were coupled to Fis mutants containing single cysteine residues as described previously . The Fis-OP conjugates were typically incubated with approximately 50 fmol endlabeled DNA fragments in a 20 ml solution containing 20 mM Tris-HCl (pH 7.5), 80 mM NaCl, 50 mM CuSO4 and up to 50 mg/ml sonicated herring testes DNA for ten minutes.…”

Copper(II) complexes of 1,10-phenanthroline-derived ligands: Studies on DNA binding properties and nuclease activity

“…There are also some reports of copper-phen complexes cleaving DNA hydrolytically [11]. Several copper complexes containing phen moiety have been synthesized and studied as sequence specific DNA binding or cleaving reagents [12][13][14][15][16].…”