Stabilised and targeted copper(II) polypyridyl oxidative chemical nucleases

Gene editing has emerged as an important tool for altering the genomes of living cells with unprecedented ease. By targeting underlying genes of interest, this field has the potential to transform the treatment of human disease. Enzymatic nucleases—in particular CRISPRCas9—epitomize the current state of art in gene editing technology but are often limited by large size and complex design. The development of small molecule bio-mimetics of enzymatic nucleases has recently sparked efforts for potential applications in cancer therapy, DNA footprinting and protein engineering. One such example is represented by the artificial metallo-nuclease (AMN) [Cu(1,10-phenanthroline)2]+ (Cu-Phen) which, in the reduced Cu(I) form, semi-intercalates duplex DNA, promoting reactive oxygen species (ROS) production and DNA oxidative cleavage. Cu-Phen type systems, however, exhibit limitations including: (i) speciation, (ii) promiscuous binding and off-target damage leading to general toxicity. The first part of this thesis reports a new family of stabilized Cu(II)-AMNs where a caging polypyridyl ligand scaffold (tris-(2-pyridylmethyl)amine, TPMA) was combined with Cu(II)-phenathrene chemical-nuclease cores (with phenanthrene being either 2,2'-bipyridine or phenazines such as Phen, 1,10-phenanthroline-5,6-dione (PD), dipyridoquinoxaline (DPQ) and dipyridophenazine (DPPZ)). EPR studies identified these complexes to have high solution stability with phenazine intercalators enhancing DNA recognition and binding compared to Cu-TPMA alone. Nuclease analysis then identified a distinctive oxidative profile compared to classical Sigman and Fenton-type reagents. Although the TPMA ligand afforded increased solution stability, its steric hindrance prevented the high DNA binding observed for [Cu(Phen)(Phenazine)]2+-type complexes. A new series of Cu(II)-DPA derivatives (where DPA is di-(2-pyridylmethyl)amine) was therefore developed. By removing one pyridine from the TPMA scaffold we sought to enhance intercalation while maintaining suitable solution stability. Furthermore, pentacoordination around the Cu(II) center exposed a free coordination site for the inner-sphere production of ROS. Finally, caged Cu(II)-AMNs were coupled with triplex forming oligonucleotides (TFOs) for GFP-gene targeted knockdown. To achieve this, azide-alkyne cycloaddition was employed to ‘click’ azide-modified AMN scaffolds to various alkyne-TFO derivatives. Gene-directing properties were then studied using a variety of techniques including UV melting, polyacrylamide and agarose gel electrophoresis and quantitative polymerase chain reaction (qPCR). The results of the AMN-TFO hybrids were compared to state-of-the-art nucleases such as CRISPR-Cpf1 and type II restriction endonucleases