DNA-Mediated Oxidation of p53

Transcription factor p53 is the most commonly altered gene in human cancer. As a redox-active protein in direct contact with DNA, p53 can directly sense oxidative stress through DNA-mediated charge transport. Electron hole transport occurs over long distances through the π-stacked bases and leads to the oxidative dissociation of p53. The extent of protein dissociation depends upon the redox potential of the DNA in direct contact with each p53 monomer. The DNA sequence dependence of p53 oxidative dissociation was examined by electrophoretic mobility shift assays using oligonucleotides containing both synthetic and human p53 consensus sequences with an appended photooxidant, anthraquinone. Greater p53 dissociation is observed from sequences containing low-redox potential purine regions, particularly guanine triplets. Using denaturing polyacrylamide gel electrophoresis of irradiated anthraquinone-modified DNA, the DNA damage sites corresponding to sites of preferred electron hole localization were determined. The resulting DNA damage preferentially localizes to guanine doublets and triplets. Oxidative DNA damage is inhibited in the presence of p53, but only at sites in direct contact with p53. From these data, predictions about the sensitivity of human p53-binding sites to oxidative stress as well as possible biological implications have been made. On the basis of our data, the guanine pattern within the purine region of each p53-binding site determines the response of p53 to DNA oxidation, yielding for some sequences the oxidative dissociation of p53 from a distance and thereby providing another potential role for DNA charge transport chemistry within the cell

A Family of Rhodium Complexes with Selective Toxicity toward Mismatch Repair-Deficient Cancers

Rhodium metalloinsertors are a unique set of metal complexes that bind specifically to DNA base pair mismatches <i>in vitro</i> and kill mismatch repair (MMR)-deficient cells at lower concentrations than their MMR-proficient counterparts. A family of metalloinsertors containing rhodium–oxygen ligand coordination, termed “<b>Rh–O</b>” metalloinsertors, has been prepared and shown to have a significant increase in both overall potency and selectivity toward MMR-deficient cells regardless of structural changes in the ancillary ligands. Here we describe DNA-binding and cellular studies with the second generation of <b>Rh–O</b> metalloinsertors in which an ancillary ligand is varied in both steric bulk and lipophilicity. These complexes, of the form [Rh­(L)­(chrysi)­(PPO)]²⁺, all include the O-containing PPO ligand (PPO = 2-(pyridine-2-yl)­propan-2-ol) and the aromatic inserting ligand chrysi (5,6-chrysene quinone diimine) but differ in the identity of their ancillary ligand L, where L is a phenanthroline or bipyridyl derivative. The <b>Rh–O</b> metalloinsertors in this family all show micromolar binding affinities for a 29-mer DNA hairpin containing a single CC mismatch. The complexes display comparable lipophilic tendencies and p<i>K</i>_a values of 8.1–9.1 for dissociation of an imine proton on the chrysi ligand. In cellular proliferation and cytotoxicity assays with MMR-deficient cells (HCT116O) and MMR-proficient cells (HCT116N), the complexes containing the phenanthroline-derived ligands show highly selective cytotoxic preference for the MMR-deficient cells at nanomolar concentrations. Using mass spectral analyses, it is shown that the complexes are taken into cells through a passive mechanism and exhibit low accumulation in mitochondria, an off-target organelle that, when targeted by parent metalloinsertors, can lead to nonselective cytotoxicity. Overall, these <b>Rh–O</b> metalloinsertors have distinct and improved behavior compared to previous generations of parent metalloinsertors, making them ideal candidates for further therapeutic assessment

An Unusual Ligand Coordination Gives Rise to a New Family of Rhodium Metalloinsertors with Improved Selectivity and Potency

Rhodium metalloinsertors are octahedral complexes that bind DNA mismatches with high affinity and specificity and exhibit unique cell-selective cytotoxicity, targeting mismatch repair (MMR)-deficient cells over MMR-proficient cells. Here we describe a new generation of metalloinsertors with enhanced biological potency and selectivity, in which the complexes show Rh–O coordination. In particular, it has been found that both Δ- and Λ-[Rh­(chrysi)­(phen)­(DPE)]²⁺ (where chrysi =5,6 chrysenequinone diimmine, phen =1,10-phenanthroline, and DPE = 1,1-di­(pyridine-2-yl)­ethan-1-ol) bind to DNA containing a single CC mismatch with similar affinities and without racemization. This is in direct contrast with previous metalloinsertors and suggests a possible different binding disposition for these complexes in the mismatch site. We ascribe this difference to the higher p<i>K</i>_a of the coordinated immine of the chrysi ligand in these complexes, so that the complexes must insert into the DNA helix with the inserting ligand in a buckled orientation; spectroscopic studies in the presence and absence of DNA along with the crystal structure of the complex without DNA support this assignment. Remarkably, all members of this new family of compounds have significantly increased potency in a range of cellular assays; indeed, all are more potent than cisplatin and <i>N</i>-methyl-<i>N</i>′-nitro-nitrosoguanidine (MNNG, a common DNA-alkylating chemotherapeutic agent). Moreover, the activities of the new metalloinsertors are coupled with high levels of selective cytotoxicity for MMR-deficient versus proficient colorectal cancer cells

DNA Protection by the Bacterial Ferritin Dps via DNA Charge Transport

Dps proteins, bacterial mini-ferritins that protect DNA from oxidative stress, are implicated in the survival and virulence of pathogenic bacteria. Here we examine the mechanism of <i>E. coli</i> Dps protection of DNA, specifically whether this DNA-binding protein can utilize DNA charge transport through the base pair π-stack to protect the genome from a distance. An intercalating ruthenium photooxidant was employed to generate DNA damage localized to guanine repeats, the sites of lowest potential in DNA. We find that Dps loaded with ferrous iron, in contrast to Apo-Dps and ferric iron-loaded Dps, significantly attenuates the yield of oxidative DNA damage. These data demonstrate that ferrous iron-loaded Dps is selectively oxidized to fill guanine radical holes, thereby restoring the integrity of the DNA. Luminescence studies indicate no direct interaction between the ruthenium photooxidant and Dps, supporting the DNA-mediated oxidation of ferrous iron-loaded Dps. Thus DNA charge transport may be a mechanism by which Dps efficiently protects the genome of pathogenic bacteria from a distance

[Ru(Me₄phen)₂dppz]²⁺, a Light Switch for DNA Mismatches

[Ru­(Me₄phen)₂dppz]²⁺ serves as a luminescent “light switch” for single base mismatches in DNA. The preferential luminescence enhancement observed with mismatches results from two factors: (i) the complex possesses a 26-fold higher binding affinity toward the mismatch compared to well-matched base pairs, and (ii) the excited state emission lifetime of the ruthenium bound to the DNA mismatch is 160 ns versus 35 ns when bound to a matched site. Results indicate that the complex binds to the mismatch through a metalloinsertion binding mode. Cu­(phen)₂²⁺ quenching experiments show that the complex binds to the mismatch from the minor groove, characteristic of metalloinsertion. Additionally, the luminescence intensity of the complex with DNA containing single base mismatches correlates with the thermodynamic destabilization of the mismatch, also consistent with binding through metalloinsertion. This complex represents a potentially new early cancer diagnostic for detecting deficiencies in mismatch repair

A Rhodium-Cyanine Fluorescent Probe: Detection and Signaling of Mismatches in DNA

We report a bifunctional fluorescent probe that combines a rhodium metalloinsertor with a cyanine dye as the fluorescent reporter. The conjugate shows weak luminescence when free in solution or with well matched DNA but exhibits a significant luminescence increase in the presence of a 27-mer DNA duplex containing a central CC mismatch. DNA photocleavage experiments demonstrate that, upon photoactivation, the conjugate cleaves the DNA backbone specifically near the mismatch site on a 27-mer fragment, consistent with mismatch targeting. Fluorescence titrations with the 27-mer duplex containing the CC mismatch reveal a DNA binding affinity of 3.1 × 10⁶ M^–1, similar to that of other rhodium metalloinsertors. Fluorescence titrations using genomic DNA extracted from various cell lines demonstrate a clear discrimination in fluorescence between those cell lines that are proficient or deficient in mismatch repair. This differential luminescence reflects the sensitive detection of the mismatchrepair-deficient phenotype

Luminescence of [Ru(bpy)₂(dppz)]²⁺ Bound to RNA Mismatches

The luminescence of <i>rac</i>-[Ru­(bpy)₂(dppz)]²⁺ (bpy = 2,2′-bipyridine and dppz = dipyrido­[3,2-<i>a</i>:2′,3′-<i>c</i>]­phenazine) was explored in the presence of RNA oligonucleotides containing a single RNA mismatch (CA and GG) in order to develop a probe for RNA mismatches. While there is minimal luminescence of [Ru­(bpy)₂(dppz)]²⁺ in the presence of matched RNA due to weak binding, the luminescence is significantly enhanced in the presence of a single CA mismatch. The luminescence differential between CA mismatched and matched RNA is substantially higher compared to the DNA analogue, and therefore, [Ru(bpy)₂(dppz)]²⁺ appears to be also a sensitive light switch probe for a CA mismatch in duplex RNA. Although the luminescence intensity is lower in the presence of RNA than DNA, Förster resonance energy transfer (FRET) between the donor ruthenium complex and FRET acceptor SYTO 61 is successfully exploited to amplify the luminescence in the presence of the mismatch. Luminescence and quenching studies with sodium iodide suggest that [Ru­(bpy)₂(dppz)]²⁺ binds to these mismatches via metalloinsertion from the minor groove. This work provides further evidence that metalloinsertion is a general binding mode of octahedral metal complexes to thermodynamically destabilized mismatches not only in DNA but also in RNA

DNA-Modified Electrodes Fabricated Using Copper-Free Click Chemistry for Enhanced Protein Detection

A method of DNA monolayer formation has been developed using copper-free click chemistry that yields enhanced surface homogeneity and enables variation in the amount of DNA assembled; extremely low-density DNA monolayers, with as little as 5% of the monolayer being DNA, have been formed. These DNA-modified electrodes (DMEs) were characterized visually, with AFM, and electrochemically, and were found to facilitate DNA-mediated reduction of a distally bound redox probe. These low-density monolayers were found to be more homogeneous than traditional thiol-modified DNA monolayers, with greater helix accessibility through an increased surface area-to-volume ratio. Protein binding efficiency of the transcriptional activator TATA-binding protein (TBP) was also investigated on these surfaces and compared to that on DNA monolayers formed with standard thiol-modified DNA. Our low-density monolayers were found to be extremely sensitive to TBP binding, with a signal decrease in excess of 75% for 150 nM protein. This protein was detectable at 4 nM, on the order of its dissociation constant, with our low-density monolayers. The improved DNA helix accessibility and sensitivity of our low-density DNA monolayers to TBP binding reflects the general utility of this method of DNA monolayer formation for DNA-based electrochemical sensor development

Electrochemical Patterning and Detection of DNA Arrays on a Two-Electrode Platform

We report a novel method of DNA array formation that is electrochemically formed and addressed with a two-electrode platform. Electrochemical activation of a copper catalyst, patterned with one electrode, enables precise placement of multiple sequences of DNA onto a second electrode surface. The two-electrode patterning and detection platform allows for both spatial resolution of the patterned DNA array and optimization of detection through DNA-mediated charge transport with electrocatalysis. This two-electrode platform has been used to form arrays that enable differentiation between well-matched and mismatched sequences, the detection of TATA-binding protein, and sequence-selective DNA hybridization

Multiplexed Electrochemistry of DNA-Bound Metalloproteins

Here we describe a multiplexed electrochemical characterization of DNA-bound proteins containing [4Fe-4S] clusters. DNA-modified electrodes have become an essential tool for the characterization of the redox chemistry of DNA repair proteins containing redox cofactors, and multiplexing offers a means to probe different complex samples and substrates in parallel to elucidate this chemistry. Multiplexed analysis of endonuclease III (EndoIII), a DNA repair protein containing a [4Fe-4S] cluster known to be accessible via DNA-mediated charge transport, shows subtle differences in the electrochemical behavior as a function of DNA morphology. The peak splitting, signal broadness, sensitivity to π-stack perturbations, and kinetics were all characterized for the DNA-bound reduction of EndoIII on both closely and loosely packed DNA films. DNA-bound EndoIII is seen to have two different electron transfer pathways for reduction, either through the DNA base stack or through direct surface reduction; closely packed DNA films, where the protein has limited surface accessibility, produce electrochemical signals reflecting electron transfer that is DNA-mediated. Multiplexing furthermore permits the comparison of the electrochemistry of EndoIII mutants, including a new family of mutations altering the electrostatics surrounding the [4Fe-4S] cluster. While little change in the midpoint potential was found for this family of mutants, significant variations in the efficiency of DNA-mediated electron transfer were apparent. On the basis of the stability of these proteins, examined by circular dichroism, we propose that the electron transfer pathway can be perturbed not only by the removal of aromatic residues but also through changes in solvation near the cluster