Showing papers by "Jacqueline K. Barton published in 2008"
TL;DR: Certain DNA sequences oriented within this gap are substrates for Alu I, a blunt end restriction enzyme, supporting the supposition that the DNA is in its native conformation when bridging the ends of the single-walled carbon nanotubes.
Abstract: We describe a general method to integrate DNA strands between single-walled carbon nanotube electrodes and to measure their electrical properties. We modified DNA sequences with amines on either the 5' terminus or both the 3' and 5' termini and coupled these to the single-walled carbon nanotube electrodes through amide linkages, enabling the electrical properties of complementary and mismatched strands to be measured. Well-matched duplex DNA in the gap between the electrodes exhibits a resistance on the order of 1 M(Omega). A single GT or CA mismatch in a DNA 15-mer increases the resistance of the duplex approximately 300-fold relative to a well-matched one. Certain DNA sequences oriented within this gap are substrates for Alu I, a blunt end restriction enzyme. This enzyme cuts the DNA and eliminates the conductive path, supporting the supposition that the DNA is in its native conformation when bridging the ends of the single-walled carbon nanotubes.
313 citations
TL;DR: In this paper, the authors examined the mechanism of cellular entry of a luminescent ruthenium(II) polypyridyl complex, Ru(DIP)2dppz2+ (where DIP = 4,7-diphenyl-1,10-phenanthroline and dppz = dipyridophenazine), into HeLa cells, with the extent of uptake measured by flow cytometry.
Abstract: 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)2dppz2+ (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)2dppz2+ 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)2dppz2+ into the cell.
289 citations
TL;DR: The characteristic features, applications, and advantages of DNA-mediated electrochemistry are discussed, including reliable electrochemical assays based on DNA charge transport at self-assembled DNA monolayers.
152 citations
TL;DR: Observations highlight a means through which oxidative stress may be chemically signaled in the genome over long distances through CT from guanine radicals to DNA-bound proteins.
113 citations
TL;DR: The scheme outlined provides a basis for the sensitive electrical detection of numerous proteins on a single DNA chip.
Abstract: A simple method for the electrochemical detection of TATA-binding protein is demonstrated at DNA-modified microelectrodes. The assay is general and based on the interruption of DNA-mediated charge transport to Nile Blue, a redox-active probe covalently attached to the DNA base pair stack. Nanomolar quantities of TATA binding protein can be detected on the microelectrodes even in the presence of micromolar amounts of bovine serum albumin, EndonucleaseIII, or Bam HI methyltransferase. The scheme outlined provides a basis for the sensitive electrical detection of numerous proteins on a single DNA chip.
82 citations
TL;DR: Analysis of cleavage products and the correlation of affinities with helix destabilization suggest that Rh(bpy)2(chrysi)3+ binds both lesions via metalloinsertion, as observed for Rh binding at mismatched sites.
Abstract: The site-specific recognition of abasic sites and single base bulges in duplex DNA by sterically expansive rhodium metalloinsertors has been investigated. Through DNA photocleavage experiments, Rh(bpy)_2(chrysi)^(3+) is shown to bind both abasic sites and single base bulges site-specifically and, upon irradiation, to cleave the backbone of the defect-containing DNA. Photocleavage titrations reveal that the metal complex binds DNA containing an abasic site with high affinity (2.6(5) × 10^6 M^(−1)), comparably to the metalloinsertor and a CC mismatch. The complex binds single base bulge sites with lower affinity (∼10^5 M^(−1)). Analysis of cleavage products and the correlation of affinities with helix destabilization suggest that Rh(bpy)_2(chrysi)^(3+) binds both lesions via metalloinsertion, as observed for Rh binding at mismatched sites, a binding mode in which the mismatched or unpaired bases are extruded from the helix and replaced in the base stack by the sterically expansive ligand of the metalloinsertor.
72 citations
TL;DR: Cyclometalated Ir(III) complexes tethered to 18-mer oligonucleotides through a functionalized dipyridophenazine ligand have been used to study the distance dependence profile of hole and electron transport along DNA.
Abstract: Cyclometalated Ir(III) complexes tethered to 18-mer oligonucleotides through a functionalized dipyridophenazine ligand have been used to study the distance dependence profile of hole and electron transport along DNA. These DNA assemblies allow a direct comparison of hole and electron transport with a single donor coupled into the base stack. Interestingly, both processes, monitored with modified bases as hole or electron kinetic traps incorporated in the strands, appear to have similarly shallow dependences in their reactions with distance. As with hole transport, perturbations to the base stack also attenuate electron transport.
71 citations
TL;DR: The results more generally underscore the importance of using DNA electrochemistry to determine DNA-bound potentials for redox-sensitive transcription factors because such binding can dramatically affect this key protein property.
Abstract: Electrochemistry measurements on DNA-modified electrodes are used to probe the effects of binding to DNA on the redox potential of SoxR, a transcription factor that contains a [2Fe-2S] cluster and is activated through oxidation. A DNA-bound potential of +200 mV versus NHE (normal hydrogen electrode) is found for SoxR isolated from Escherichia coli and Pseudomonas aeruginosa. This potential value corresponds to a dramatic shift of +490 mV versus values found in the absence of DNA. Using Redmond red as a covalently bound redox reporter affixed above the SoxR binding site, we also see, associated with SoxR binding, an attenuation in the Redmond red signal compared with that for Redmond red attached below the SoxR binding site. This observation is consistent with a SoxR-binding-induced structural distortion in the DNA base stack that inhibits DNA-mediated charge transport to the Redmond red probe. The dramatic shift in potential for DNA-bound SoxR compared with the free form is thus reconciled based on a high-energy conformational change in the SoxR–DNA complex. The substantial positive shift in potential for DNA-bound SoxR furthermore indicates that, in the reducing intracellular environment, DNA-bound SoxR is primarily in the reduced form; the activation of DNA-bound SoxR would then be limited to strong oxidants, making SoxR an effective sensor for oxidative stress. These results more generally underscore the importance of using DNA electrochemistry to determine DNA-bound potentials for redox-sensitive transcription factors because such binding can dramatically affect this key protein property.
64 citations
TL;DR: In this article, the authors review recent experiments in their laboratory aimed at the design and study of octahedral metal complexes that bind DNA non-covalently and target reactions to specific sites.
Abstract: Since the elucidation of the structure of double helical DNA, the construction of small molecules that recognize and react at specific DNA sites has been an area of considerable interest. In particular, the study of transition metal complexes that bind DNA with specificity has been a burgeoning field. This growth has been due in large part to the useful properties of metal complexes, which possess a wide array of photophysical attributes and allow for the modular assembly of an ensemble of recognition elements. Here we review recent experiments in our laboratory aimed at the design and study of octahedral metal complexes that bind DNA non-covalently and target reactions to specific sites. Emphasis is placed both on the variety of methods employed to confer site-specificity and upon the many applications for these complexes. Particular attention is given to the family of complexes recently designed that target single base mismatches in duplex DNA through metallo-insertion.
47 citations
TL;DR: In this paper, a ping-pong electron transfer (PIET) system was proposed to promote the reduction of pyrimidine bases from a distance without the presence of an external quencher.
Abstract: Herein we describe a novel Ir system that is able to promote the reduction of pyrimidine bases from a distance without the presence of an external quencher. Instead, DNA-mediated ET is triggered by DNA-mediated HT. Thus, photoactivation of these Ir assemblies results in both a forward and a reverse pattern for charge migration, which we term ping-pong electron transfer through DNA (Scheme 1).
46 citations
TL;DR: In this paper, the authors used scanning electrochemical microscopy (SECM) to probe long-range charge transport (CT) through DNA monolayers containing the redox-active Nile Blue (NB) intercalator covalently affixed at a specific location in the DNA film.
Abstract: Scanning electrochemical microscopy (SECM) is used to probe long-range charge transport (CT) through DNA monolayers containing the redox-active Nile Blue (NB) intercalator covalently affixed at a specific location in the DNA film. At substrate potentials negative of the formal potential of covalently attached NB, the electrocatalytic reduction of Fe(CN)63− generated at the SECM tip is observed only when NB is located at the DNA/solution interface; for DNA films containing NB in close proximity to the DNA/electrode interface, the electrocatalytic effect is absent. This behavior is consistent with both rapid DNA-mediated CT between the NB intercalator and the gold electrode as well as a rate-limiting electron transfer between NB and the solution phase Fe(CN)63−. The DNA-mediated nature of the catalytic cycle is confirmed through sequence-specific and localized detection of attomoles of TATA-binding protein, a transcription factor that severely distorts DNA upon binding. Importantly, the strategy outlined here is general and allows for the local investigation of the surface characteristics of DNA monolayers both in the absence and in the presence of DNA binding proteins. These experiments highlight the utility of DNA-modified electrodes as versatile platforms for SECM detection schemes that take advantage of CT mediated by the DNA base pair stack.
TL;DR: Redmond Red, a fluoropore containing a redox-active phenoxazine core, has been explored as a new electrochemical probe for the detection of abasic sites in double-stranded DNA.
TL;DR: The periodicity of DNA-mediated charge transport is controlled by engineering the extent of BET across the bridge, which is consistent with a model where multiples of four to five base pairs form an ideal CT-active length of a bridging adenine domain.
Abstract: DNA-mediated charge transport (CT) is exquisitely sensitive to the integrity of the bridging π-stack and is characterized by a shallow distance dependence. These properties are obscured by poor coupling between the donor/acceptor pair and the DNA bridge, or by convolution with other processes. Previously, we found a surprising periodic length dependence for the rate of DNA-mediated CT across adenine tracts monitored by 2-aminopurine fluorescence. Here we report a similar periodicity by monitoring N2-cyclopropylguanosine decomposition by rhodium and anthraquinone photooxidants. Furthermore, we find that this periodicity is attenuated by consequent back-electron transfer (BET), as observed by direct comparison between sequences that allow and suppress BET. Thus, the periodicity can be controlled by engineering the extent of BET across the bridge. The periodic length dependence is not consistent with a periodicity predicted by molecular wire theory but is consistent with a model where multiples of four to five base pairs form an ideal CT-active length of a bridging adenine domain.
TL;DR: It is shown that a primer extension reaction can be used to monitor oxidative DNA damage directly in functioning mitochondria after photoreaction with a rhodium intercalator that penetrates the intact mitochondrial membrane.
Abstract: Sites of oxidative DNA damage in functioning mitochondria have been identified using a rhodium photooxidant as a probe. Here we show that a primer extension reaction can be used to monitor oxidative DNA damage directly in functioning mitochondria after photoreaction with a rhodium intercalator that penetrates the intact mitochondrial membrane. The complex [Rh(phi)_2bpy]Cl_3 (phi = 9,10-phenanthrenequinonediimine) binds to DNA within the mitochondria and, upon irradiation, initiates DNA oxidation reactions. Significantly, piperidine treatment of the mitochondria leads to protein-dependent primer extension stops spaced every ∼20 base pairs. Hence, within the mitochondria, the DNA is well covered and packaged by proteins. Photolysis of the mitochondria containing [Rh(phi)_2bpy]^(3+) leads to oxidative DNA damage at positions 260 and 298; both are mutational hot spots associated with cancers. The latter position is the 5‘-nucleotide of conserved sequence block II and is critical to replication of the mitochondrial DNA. The oxidative damage is found to be DNA-mediated, utilizing a charge transport mechanism, as the Rh binding sites are spatially separated from the oxidation-prone regions. This long-range DNA-mediated oxidation occurs despite protein association. Indeed, the oxidation of the mitochondrial DNA leads not only to specific oxidative lesions, but also to a corresponding change in the protein-induced stops in the primer extension. Mitochondrial DNA damage promotes specific changes in protein−DNA contacts and is thus sensed by the mitochondrial protein machinery.
TL;DR: The size and shape of the eilatin ligand allow stacking with both well-matched and mismatched DNA, and displays lower selectivity for the mismatch than does a metalloinsertor containing the smaller, though still bulky, chrysene-5,6-quinonediimine ligand.
Abstract: The DNA-binding properties of Ru(bpy)_2(eilatin)^(2+) have been investigated to determine if the sterically expansive eilatin ligand confers specificity for destabilized single-base mismatches in DNA. Competitive DNA photocleavage experiments employing a sequence-neutral metallointercalator, Rh(bpy)_2(phi)^(3+)(phi = 9,10-phenanthrenequinonediimine), and a mismatch-specific metalloinsertor, Rh(bpy)_2(chrysi)^(3+) (chrysi = chrysene-5,6-quinonediimine), reveal that the eilatin complex binds to a CC mismatched site with an apparent binding constant of 2.2(2) × 10^6 M^(−1). Nonetheless, the selectivity in binding mismatched DNA is not high: competitive titrations with Rh(bpy)_2(phi)^(3+) show that the complex binds also to well-matched B-form sites. Thus, Ru(bpy)_2(eilatin)^(2+), despite containing the extremely expansive eilatin ligand, displays lower selectivity for the mismatch than does Rh(bpy)_2(chrysi)^(3+), a metalloinsertor containing the smaller, though still bulky, chrysene-5,6-quinonediimine ligand. In summary, the size and shape of the eilatin ligand allow stacking with both well-matched and mismatched DNA.
15 May 2008
TL;DR: The metallointercalators may serve as models for DNA-binding proteins, not only in binding DNA sites with high specificity, but also in carrying out electron transfer chemistry mediated by the DNA base-pair stack, as powerful probes of this chemistry.
Abstract: Here we describe studies of metallointercalators bound to DNA. These octahedral transition metal complexes primarily bind noncovalently by stacking within the DNA helix. Given the rich photophysics and photochemistry of the ruthenium and rhodium complexes we employ, we have used a variety of biophysical studies to characterize their interactions with DNA. X-ray crystallography has also provided atomic resolution detail as to their binding to the duplex. Complexes have been designed that target DNA with high specificity. We have, for example, designed metal complexes that bind specifically to mismatched sites in the DNA duplex, and these have found application in the detection of single nucleotide polymorphisms and studies of mismatch repair deficiency. The photophysical properties of the metal complexes along with their intercalative stacking have been useful in particular as tools to characterize long-range charge transport in DNA. Using metallointercalators tethered to the duplex, oxidative damage to DNA from a distance has been demonstrated. The metallointercalators may serve as models for DNA-binding proteins, not only in binding DNA sites with high specificity, but also in carrying out electron transfer chemistry mediated by the DNA base-pair stack. Certainly these metallointercalators have proven to be powerful probes of this chemistry.