Genome Engineering with Synthetic Copper Nucleases

Abstract

Oxidative chemical nucleases represent an important class of synthetic compound that mediate nucleic acid recognition and strand scission. These agents have found utility as footprinting probes to map drug-DNA and protein-DNA interactions and also as clinical cytotoxins of human cancer. Herein, we highlight a recent contribution from our group on the application of the copper(II) 1,10-phenanthroline (OP) complex Cu(OP) 2 as a novel DNA-shuffling agent capable of generating highly diverse mutant protein antibody libraries. By employing a recombinant antibody gene fragment specific for prostate-specific antigen (PSA), we compared variation and PSA-binding specificity within Taq DNA polymerase I reconstructed Cu(OP) 2 oxidative digest fragments with hydrolytic restriction digests generated by DNase I. Results show the Cu(OP) 2 mutant library contained highly PSA-specific binding antibodies thus indicating that oxidative chemical nucleases may be applied to generate novel antibody clones not accessible through the use of the traditional DNA-shuffling enzyme DNase I. Key words copper(II), nuclease, nucleic acids, radical oxidation, H-atom abstraction, strand scissionIn the field of DNA manipulation, the design of artificial DNA cutters has become an area of considerable research interest. Synthetic copper chemical nucleases are a class of DNA cutter and can be categorised in two ways: i) those that mimic natural restriction endonucleases by catalysing hydrolysis of the phosphodiester backbone, 1 and ii) compounds that induce direct strand scission by oxidative Hatom abstraction from the pentose ring of DNA. 2 The biochemistry of bovine pancreatic deoxyribonuclease (DNase I) -a calcium-and magnesium-dependent hydrolytic enzyme that nonspecifically degrades both single-and double-stranded DNA -is described in Scheme 1 (A). Here, glutamate (Glu75) and histidine (His131) residues combine to catalyse hydroxide anion formation in proximity to the phosphate backbone. 3 The resulting 5′-phosphorylated and 3′-hydroxylated ends produced by hydrolytic nucleases (e.g., DNase I or site-specific restriction endonucleases) can then be further manipulated, for example, in DNA recombi-