To date, DNA cleavage, caused by cleavage agents, has been monitored mainly by gel and capillary electrophoresis. However, these techniques are time-consuming, non-quantitative and require gel stains. In this work, a novel, simple and, importantly, a quantitative method for monitoring the DNA nuclease activity of potential anti-cancer drugs, at a DNA electrochemical sensor, is presented. The DNA sensors were prepared using thiol-modified oligonucleotides that self-assembled to create DNA monolayers at gold electrode surfaces. The quantification of DNA double-strand breaks is based on calculating the DNA surface coverage, before and after exposure to DNA cleavage agents, using a method developed by a Tarlov group. The nuclease properties of a model DNA cleavage agent, copper bisphenanthroline ([Cu II (phen) 2 ] 2+), that cleaves DNA in a Fenton-type reaction, were quantified electrochemically. The DNA surface coverage decreased on average by 21 % after immersing the DNA sensor in a nuclease assay containing [Cu II (phen) 2 ] 2+ , a reductant and an oxidant. This percentage indicates that 6 base pairs were cleaved in the nuclease assay from the immobilised 30 base pair strands. The DNA cleavage can be also induced electrochemically in the absence of a chemical reductant. [Cu II (phen) 2 ] 2+ , intercalates between DNA base pairs and, on application of a suitable potential, can be reduced to [Cu I (phen) 2 ] + , with solution oxygen acting as the required oxidant. This reduction process is facilitated through DNA strands via long-range electron transfer, resulting in DNA cleavage of 23 %. The control measurements for both chemically and electrochemically induced cleavage revealed that DNA strand breaks did not occur under experimental conditions in the absence of [Cu II (phen) 2 ] 2+ .
We report a series of copper(II) artificial metallo‐nucleases (AMNs) and demonstrate their DNA damaging properties and in‐vitro cytotoxicity against human‐derived pancreatic cancer cells. The compounds combine a tris‐chelating polypyridyl ligand, di‐(2‐pycolyl)amine (DPA), and a DNA intercalating phenanthrene unit. Their general formula is Cu‐DPA‐N,N' (where N,N'=1,10‐phenanthroline (Phen), dipyridoquinoxaline (DPQ) or dipyridophenazine (DPPZ)). Characterisation was achieved by X‐ray crystallography and continuous‐wave EPR (cw‐EPR), hyperfine sublevel correlation (HYSCORE) and Davies electron‐nuclear double resonance (ENDOR) spectroscopies. The presence of the DPA ligand enhances solution stability and facilitates enhanced DNA recognition with apparent binding constants (Kapp) rising from 105 to 107 m−1 with increasing extent of planar phenanthrene. Cu‐DPA‐DPPZ, the complex with greatest DNA binding and intercalation effects, recognises the minor groove of guanine–cytosine (G‐C) rich sequences. Oxidative DNA damage also occurs in the minor groove and can be inhibited by superoxide and hydroxyl radical trapping agents. The complexes, particularly Cu‐DPA‐DPPZ, display promising anticancer activity against human pancreatic tumour cells with in‐vitro results surpassing the clinical platinum(II) drug oxaliplatin.
The technique chosen to immobilise DNA onto electrodes can determine the density and stability of the resultant immobilised layer. DNA-modified electrodes were prepared using four common DNA immobilisation methods and characterised using ferrocyanide. The negatively charged DNA strands should repel ferrocyanide anions. The DNA layers created using adsorption at glassy carbon electrodes were unstable, while those created through chemisorption of thiol-modified DNA onto gold electrodes were repeatable and stable, and returned, on average, 94 % repulsion of the probe. The presented results show how the immobilisation protocol, and DNA type, affects the stability, repeatability, and integrity of resultant DNA layers.
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