Protein engineering with artificial chemical nucleases

Abstract

Herein we report the application of oxidative artificial chemical nucleases as novel agents for protein engineering. The complex ion [Cu(Phen)2(H2O)](2+) (CuPhen; Phen = 1,10-phenanthroline) was applied under Fenton-type conditions against a recombinant antibody fragment specific for prostate-specific antigen (PSA) and compared against traditional DNA shuffling using DNase I for the generation of recombinant mutagenesis libraries. We show that digestion and re-annealment of single chain variable fragment (scFv) coding DNA is possible using CuPhen. Results indicate recombinant library generation in this manner may generate novel clones—not accessible through the use of DNase I—with CuPhen producing highly PSA-specific binding antibodies identified by surface plasmon resonance.

Polypyridyl‐Based Copper Phenanthrene Complexes: A New Type of Stabilized Artificial Chemical Nuclease

“…TPMA-DPPZ, all produced OC-DNA at lower concentrations (1.0-7.5 mm), with linear (L) conformationsf orming with Cu-TPMA-Phena nd Cu-TPMA-DPQ complexes ( Figure 6A,l anes [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. The overall trend in chemical nuclease activity was Cu-TPMA-Phen > Cu-TPMA-DPQ > Cu-TPMA-DPPZ @ Cu-TPMA, which indicates that the ancillary N,N' phenanthrene group is critical to ROS catalysis at the DNA interface.…”

“…Additionally to hydrolysis, AMNs provide another pathway for cutting DNA. In this case, cleavage occurs by DNA oxidation, mediated by reactive oxygen species (ROS), and contributes towards therapeutic utility by damaging the genome of cancer cells to impede faithful cell replication . DNA oxidation by metallo‐nucleases is generally irreversible and relies on the binding of the activated AMN to its requisite target site through groove binding, insertion or intercalation interactions .…”

“…Additionally to hydrolysis, AMNs provide anotherp athway for cutting DNA.I nt his case, cleavage occurs by DNA oxidation, mediated by reactive oxygen species (ROS), and contributes towards therapeutic utility by damaging the genome of cancer cells to impede faithful cell replication. [4][5][6] DNA oxidation by metallo-nucleases is generally irreversible and relies on the binding of the activated AMN to its requisite targets ite through groove binding, insertion or intercalation interac-tions. [7] Considering that these interactions dependo nt he shape and chargeo ft he complex, the synthesis of selective and robust inorganic scaffolds as well as the building of effective biomimetic enzymes [8][9][10][11] are important researchg oals for assembling bioactive materials and in exploring the requirementsf or their biological use.…”

Genome Engineering with Synthetic Copper Nucleases

“…In our recent contribution, we utilised the oxidative nuclease complex, [Cu(OP) 2 (H 2 O)]·2NO 3 , to generate a highly diverse recombinant library of PSA-specific antibodies through DNA polymerase I shuffling (Figure 1,A). 5 Although Cu(OP) x complexes have been unambiguously shown to inhibit the activity of a variety of DNA polymerase I enzymes, 4,11 it has not yet been shown if purified oxidatively sheared fragments are suitable for polymerase 'reprocessing'. The complex (Scheme 1,C) was synthesised and characterised by single X-ray crystallography before exposure to a dsDNA gene fragment identified to produce PSA-specific antibodies by the ELISA.…”

“…4 Our group actively explore the biochemical and therapeutic applications of Cu(OP) 2 -based systems and have recently applied this oxidative nuclease to genetically engineer a library of high-affinity prostate-specific antigen (PSA) binding antibodies through a process called DNA shuffling. 5 In this work, the selectivity and diversity of the Cu(OP) 2 recombinant antibody library was then compared directly to a library of mutant PSA-specific antibodies generated by DNase I.…”