Optimizing the targeted chemical nuclease activity of 1,10-phenanthroline-copper by ligand modification.
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.
Gallagher
J
J
Department of Biological Chemistry, School of Medicine, University of California, Los Angeles 90095-1570, USA.
Chen
C H
CH
Pan
C Q
CQ
Perrin
D M
DM
Cho
Y M
YM
Sigman
D S
DS
D026921:Amino Acid Transport Systems, Neutral; D001483:Base Sequence; D002614:Chelating Agents / Q000737:chemistry; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism*; D026901:Membrane Transport Proteins / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010618:Phenanthrolines / Q000737:chemistry* / Q000378:metabolism; D011401:Promoter Regions, Genetic
D026921:Amino Acid Transport Systems, Neutral; D002614:Chelating Agents; D026901:Membrane Transport Proteins; D010618:Phenanthrolines; C038556:bis(1,10-phenanthroline)copper(1+) ion; C002701:neocuproine; C033082:proline transporter; D012321:DNA-Directed RNA Polymerases; D003851:Deoxyribonucleases