Jacqueline K. Barton

Electrochemical DNA sensors

Electrochemistry-based sensors offer sensitivity, selectivity and low cost for the detection of selected DNA sequences or mutated genes associated with human disease. DNA-based electrochemical sensors exploit a range of different chemistries, but all take advantage of nanoscale interactions between the target in solution, the recognition layer and a solid electrode surface. Numerous approaches to electrochemical detection have been developed, including direct electrochemistry of DNA, electrochemistry at polymer-modified electrodes, electrochemistry of DNA-specific redox reporters, electrochemical amplifications with nanoparticles, and electrochemical devices based on DNA-mediated charge transport chemistry.

Mutation detection by electrocatalysis at DNA-modified electrodes

Detection of mutations and damaged DNA bases is important for the early diagnosis of genetic disease. Here we describe an electrocatalytic method for the detection of single-base mismatches as well as DNA base lesions in fully hybridized duplexes, based on charge transport through DNA films. Gold electrodes modified with preassembled DNA duplexes are used to monitor the electrocatalytic signal of methylene blue, a redox-active DNA intercalator, coupled to [Fe(CN)6]3−. The presence of mismatched or damaged DNA bases substantially diminishes the electrocatalytic signal. Because this assay is not a measure of differential hybridization, all single-base mismatches, including thermodynamically stable GT and GA mismatches, can be detected without stringent hybridization conditions. Furthermore, many common DNA lesions and “hot spot” mutations in the human p53 genome can be distinguished from perfect duplexes. Finally, we have demonstrated the application of this technology in a chip-based format. This system provides a sensitive method for probing the integrity of DNA sequences and a completely new approach to single-base mismatch detection.

Long‐Range Electron Transfer through DNA Films

Regardless of its position within the DNA film, cross-linked daunomycin (DM) is efficiently reduced electrochemically, indicating that the electron transfer exhibits a shallow distance dependence. Upon the introduction of an intervening cytosine-adenine (CA) mismatch, the electrochemical response is dramatically attenuated (shown schematically). Therefore, the DNA double helix can facilitate long-range electron transfer, but only in the presence of a well-stacked pathway.

Rates of DNA-Mediated Electron Transfer Between Metallointercalators

Ultrafast emission and absorption spectroscopies were used to measure the kinetics of DNA-mediated electron transfer reactions between metal complexes intercalated into DNA. In the presence of rhodium(III) acceptor, a substantial fraction of photoexcited donor exhibits fast oxidative quenching (>3 × 1010 per second). Transient-absorption experiments indicate that, for a series of donors, the majority of back electron transfer is also very fast (∼1010 per second). This rate is independent of the loading of acceptors on the helix, but is sensitive to sequence and π stacking. The cooperative binding of donor and acceptor is considered unlikely on the basis of structural models and DNA photocleavage studies of binding. These data show that the DNA double helix differs significantly from proteins as a bridge for electron transfer. On-Line References and Notes

Mechanism of Cellular Uptake of a Ruthenium Polypyridyl Complex

Transition metal complexes provide a promising avenue for the design of therapeutic and diagnostic agents, but the limited understanding of their cellular uptake is a roadblock to their effective application. Here, we examine the mechanism of cellular entry of a luminescent ruthenium(II) polypyridyl complex, Ru(DIP) 2dppz (2+) (where DIP = 4,7-diphenyl-1,10-phenanthroline and dppz = dipyridophenazine), into HeLa cells, with the extent of uptake measured by flow cytometry. No diminution of cellular uptake is observed under metabolic inhibition with deoxyglucose and oligomycin, indicating an energy-independent mode of entry. The presence of organic cation transporter inhibitors also does not significantly alter uptake. However, the cellular internalization of Ru(DIP) 2dppz (2+) is sensitive to the membrane potential. Uptake decreases when cells are depolarized with high potassium buffer and increases when cells are hyperpolarized with valinomycin. These results support passive diffusion of Ru(DIP) 2dppz (2+) into the cell.

DNA charge transport over 34 nm

Molecular wires show promise in nanoscale electronics, but the synthesis of uniform, long conductive molecules is a significant challenge. Deoxyribonucleic acid (DNA) of precise length, by contrast, is synthesized easily, but its conductivity over the distances required for nanoscale devices has not been explored. Here we demonstrate DNA charge transport (CT) over 34 nm in 100-mer monolayers on gold. Multiplexed gold electrodes modified with 100-mer DNA yield sizable electrochemical signals from a distal, covalent Nile Blue redox probe. Significant signal attenuation upon incorporation of a single base-pair mismatch demonstrates that CT is DNA-mediated. Efficient cleavage of these 100-mers by a restriction enzyme indicates that the DNA adopts a native conformation accessible to protein binding. Similar electron-transfer rates measured through 100-mer and 17-mer monolayers are consistent with rate-limiting electron tunnelling through the saturated carbon linker. This DNA-mediated CT distance of 34 nm surpasses that of most reports of molecular wires.

An electrical probe of protein–DNA interactions on DNA-modified surfaces

DNA charge transport chemistry is found to provide a sensitive method for probing protein-dependent changes in DNA structure and enzymatic reactions. Here we describe the development of an electrochemical assay of protein binding to DNA-modified electrodes based upon the detection of associated perturbations in DNA base stacking. Gold electrode surfaces that were modified with loosely packed DNA duplexes, covalently crosslinked to a redox-active intercalator and containing the binding site of the test protein, were constructed. Charge transport through DNA as a function of protein binding was then assayed. Substantial attenuation in current is seen in the presence of the base-flipping enzymes HhaI methylase and uracil DNA glycosylase, as well as with TATA-binding protein. When restriction endonuclease PvuII (R.PvuII) binds to its methylated target, little base-stacking perturbation occurs and little diminution in current flow is observed. Importantly, the kinetics of restriction by R.PvuII of its nonmethylated target is also easily monitored electrochemically. This approach should be generally applicable to assaying protein–DNA interactions and reactions on surfaces.

Fluorescein Redirects a Ruthenium-Octaarginine Conjugate to the Nucleus

The cellular uptake and localization of a Ru-octaarginine conjugate with and without an appended fluorescein are compared. The inherent luminescence of the Ru(II) dipyridophenazine complex allows observation of its uptake without the addition of a fluorophore. Ru-octaarginine-fluorescein stains the cytosol, nuclei, and nucleoli of HeLa cells under conditions where the Ru-octaarginine conjugate without fluorescein shows only punctate cytoplasmic labeling. At higher concentrations, however, Ru-octaarginine without the fluorescein tag does exhibit cytoplasmic, nuclear, and nucleolar staining. Attaching fluorescein to Ru-octaarginine lowers the threshold concentration required for diffuse cytoplasmic labeling and nuclear entry. Hence, the localization of the fluorophore-bound peptide cannot serve as a proxy for that of the free peptide.

Crystal structure of Δ-[Ru(bpy)2dppz]2+ bound to mismatched DNA reveals side-by-side metalloinsertion and intercalation

DNA mismatches represent a novel target in the development of diagnostics and therapeutics for cancer, because deficiencies in DNA mismatch repair are implicated in cancers, and cells that are repair-deficient show a high frequency of mismatches. Metal complexes with bulky intercalating ligands serve as probes for DNA mismatches. Here, we report the high-resolution (0.92 Å) crystal structure of the ruthenium ‘light switch’ complex Δ-[Ru(bpy)2dppz]2+ (bpy = 2,2′-bipyridine and dppz = dipyridophenazine), which is known to show luminescence on binding to duplex DNA, bound to both mismatched and well-matched sites in the oligonucleotide 5′-(dCGGAAATTACCG)2-3′ (underline denotes AA mismatches). Two crystallographically independent views reveal that the complex binds mismatches through metalloinsertion, ejecting both mispaired adenosines. Additional ruthenium complexes are intercalated at well-matched sites, creating an array of complexes in the minor groove stabilized by stacking interactions between bpy ligands and extruded adenosines. This structure attests to the generality of metalloinsertion and metallointercalation as DNA binding modes. A ‘light switch’ ruthenium complex is known to show enhanced luminescence in the presence of DNA mismatches — emerging targets for cancer diagnostics and therapeutics — but the way it interacts with DNA has remained unclear. Now, metalloinsertion into and metallointercalation at the minor groove of the double helix have been unambiguously observed in a high-resolution crystal structure.

Luminescence of ruthenium(II) polypyridyls: evidence for intercalative binding to Z-DNA

Photophysical studies have been undertaken to characterize the binding interactions of enantiomers of Ru(phen)3(2+), Ru(DIP)3(2+), and racemic Ru(bpy)2dppz2+ (where phen = 1,10-phenanthroline, DIP = 4,7-diphenylphenanthroline, and dppz = dipyridophenazine) with Z-form poly d(GC). Parallel enhancements in steady state luminescent intensity and a lengthening of luminescent lifetimes are seen for ruthenium enantiomers with Z-DNA as for B-DNA but with enantioselectivities reversed. Greater enhancements are seen for delta-isomers with the right-handed helix but for lambda-isomers with the left-handed helix. Ru(bpy)2dppz2+, an avid intercalator in B-DNA, displays no luminescence free in aqueous solution, but luminesces brightly bound to either B- or Z-poly d(GC). Stern-Volmer quenching studies also support the enantioselective preference in binding to B-DNA by delta-isomers and a reversal with binding to Z-DNA preferentially by the lambda-isomers. Steady state polarization studies indicate a rigid association of the complexes with both B- and Z-DNA on the time-scale of their emission and again with symmetrical enantioselectivities for the left and right-handed helices. Given the well characterized intercalative association of the complexes with B-DNA, the parallel results seen here with Z-DNA point strongly to a comparable intercalative association with the Z-form helix. That molecules may interact with Z-DNA through intercalation has not been demonstrated previously and now requires consideration in describing the range of interactions of small molecules and proteins with Z-DNA.