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J K Barton

Publications and source records attributed to J K Barton.

At least 37 records · Page 2Linked to original sources
PubMed2000

Luminescence quenching by DNA-bound viologens: effect of reactant identity on efficiency and dynamics of electron transfer in DNA.

Photoinduced electron transfer from two intercalating photoactive donors, Ru(phen)2dppz2+ and ethidium, to intercalating viologen acceptors of the N,N'-dialkyl-6-(2'-pyridiniumyl)phenanthridinium family has been investigated through steady-state and time-resolved luminescence quenching measurements. Efficient quenching of the emission from these donors bound to DNA is observed at low concentrations of acceptor (1-10 eq.), and in time-resolved emission experiments it is determined that electron transfer occurs on the nanosecond time scale. Furthermore, transient absorption measurements confirm that the quenching is the result of a charge-transfer process; upon photoreaction of intercalated Ru(phen)2dppz2+ with a viologen acceptor, an intermediate with spectral properties resembling the expected charge-separated pair is observed. The quenching yields and kinetics obtained with this quencher are in marked contrast to those observed with these same donors paired with Rh(phi)2bpy3+ as an acceptor. The differing efficiencies of electron transfer for these donor/acceptor pairs bound to DNA as compared to others previously described are discussed qualitatively in terms of the structural and electronic properties of the different reactants.

DNA↗
PubMed2000

Robust charge transport in DNA double crossover assemblies.

BACKGROUND: Multiple-stranded DNA assemblies, encoded by sequence, have been constructed in an effort to self-assemble nanodevices of defined molecular architecture. Double-helical DNA has been probed also as a molecular medium for charge transport. Conductivity studies suggest that DNA displays semiconductor properties, whereas biochemical studies have shown that oxidative damage to B-DNA at the 5'-G of a 5'-GG-3' doublet can occur by charge transport through DNA up to 20 nm from a photo-excited metallointercalator. The possible application of DNA assemblies, in particular double crossover (DX) molecules, in electrical nanodevices prompted the design of a DNA DX assembly with oxidatively sensitive guanine moieties and a tethered rhodium photo-oxidant strategically placed to probe charge transport. RESULTS: DX assemblies support long-range charge transport selectively down the base stack bearing the intercalated photo-oxidant. Despite tight packing, no electron transfer (ET) crossover to the adjacent base stack is observed. Moreover, the base stack of a DX assembly is well-coupled and less susceptible than duplex DNA to stacking perturbations. Introducing a double mismatch along the path for charge transport entirely disrupts long-range ET in duplex DNA, but only marginally decreases it in the analogous stack within DX molecules. CONCLUSIONS: The path for charge transport in a DX DNA assembly is determined directly by base stacking. As a result, the two closely packed stacks within this assembly are electronically insulated from one another. Therefore, DX DNA assemblies may serve as robust, insulated conduits for charge transport in nanoscale devices.

Base Pair Mismatch↗
PubMed2000

Probing DNA charge transport with metallointercalators.

A wide range of experiments have emerged recently regarding charge transport through DNA, including spectroscopic studies of rates of DNA-mediated electron transfer and biochemical studies of DNA base oxidation over long distances. These experiments have, in turn, led to new proposals about the way in which charge moves through DNA and have prompted the consideration of physiological roles for DNA electron transfer. Importantly, metallointercalators have been key players in many of these experiments. Metallointercalators provide critical probes to examine the migration of charge through the DNA base stack.

DNA↗
PubMed2000

Structure of a photoactive rhodium complex intercalated into DNA.

Intercalating complexes of rhodium(III) are strong photo-oxidants that promote DNA strand cleavage or electron transfer through the double helix. The 1.2 A resolution crystal structure of a sequence-specific rhodium intercalator bound to a DNA helix provides a rationale for the sequence specificity of rhodium intercalators. It also explains how intercalation in the center of an oligonucleotide modifies DNA conformation. The rhodium complex intercalates via the major groove where specific contacts are formed with the edges of the bases at the target site. The phi ligand is deeply inserted into the DNA base pair stack. The primary conformational change of the DNA is a doubling of the rise per residue, with no change in sugar pucker from B-form DNA. Based upon the five crystallographically independent views of an intercalated DNA helix observed in this structure, the intercalator may be considered as an additional base pair with specific functional groups positioned in the major groove.

Base Sequence↗
PubMed2000

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.

Base Pair Mismatch↗
PubMed1999

Single-base mismatch detection based on charge transduction through DNA.

High-throughput DNA sensors capable of detecting single-base mismatches are required for the routine screening of genetic mutations and disease. A new strategy for the electrochemical detection of single-base mismatches in DNA has been developed based upon charge transport through DNA films. Double-helical DNA films on gold surfaces have been prepared and used to detect DNA mismatches electrochemically. The signals obtained from redox-active intercalators bound to DNA-modified gold surfaces display a marked sensitivity to the presence of base mismatches within the immobilized duplexes. Differential mismatch detection was accomplished irrespective of DNA sequence composition and mismatch identity. Single-base changes in sequences hybridized at the electrode surface are also detected accurately. Coupling the redox reactions of intercalated species to electrocatalytic processes in solution considerably increases the sensitivity of this assay. Reporting on the electronic structure of DNA, as opposed to the hybridization energetics of single-stranded oligonucleotides, electrochemical sensors based on charge transport may offer fundamental advantages in both scope and sensitivity.

Base Pair Mismatch↗
PubMed1999

Perturbing the DNA sequence selectivity of metallointercalator-peptide conjugates by single amino acid modification.

Metallointercalator-peptide conjugates that provide small molecular mimics to explore peptide-nucleic acid recognition have been prepared. Specifically, a family of peptide conjugates of [Rh(phi)(2)(phen')](3+) [where phi = 9,10-phenanthrenequinone diimine and phen' = 5-(amidoglutaryl)-1,10-phenanthroline] has been synthesized and their DNA-binding characteristics examined. Single amino acid modifications were made from the parent metallointercalator-peptide conjugate [Rh(phi)(2)(phen')](3+)-AANVAIAAWERAA-CONH(2), which targets 5'-CCA-3' site-specifically. Moving the glutamate at position 10 in the sequence of the appended peptide to position 6 {[Rh(phi)(2)(phen')](3+)-AANVAEAAWARAA-CONH(2)} changed the sequence preference of the metallointercalator-peptide conjugate to 5'-ACA-3'. Subsequent mutation of the glutamate at position 6 to arginine {[Rh(phi)(2)(phen')](3+)-AANVARAAWARAA-CONH(2)} caused more complex changes in DNA recognition. Thermodynamic dissociation constants were determined for these metallointercalator-peptide conjugates by photoactivated DNA cleavage assays with the rhodium intercalators. At 55 degrees C in the presence of 5 mM MnCl(2), [Rh(phi)(2)(phen')](3+)-AANVAIAAWERAA-CONH(2) binds to a 5'-CCA-3' site with K(d) = 5.7 x 10(-)(8) M, whereas [Rh(phi)(2)(phen')](3+)-AANVAEAAWARAA-CONH(2) binds to its target 5'-ACA-3' site with K(d) = 9.9 x 10(-8) M. The dissociation constant for [Rh(phi)(2)(phen')](3+) with random-sequence DNA is 7.0 x 10(-7) M. Structural models have been developed and refined to account for the observed sequence specificities. As with much larger DNA-binding proteins, with these metal-peptide conjugate mimics, single amino acid changes can lead to single or multiple base changes in the DNA site targeted.

Amino Acid Sequence↗
PubMed1999

Femtosecond dynamics of DNA-mediated electron transfer.

Diverse biophysical and biochemical studies have sought to understand electron transfer (ET) in DNA in part because of its importance to DNA damage and its repair. However, the dynamics and mechanisms of the elementary processes of ET in this medium are not fully understood and have been heavily debated. Two fundamental issues are the distance over which charge is transported and the time-scale on which the transport through the pi-stack of the DNA base pairs may occur. With femtosecond resolution, we report direct observation in DNA of ultrafast ET, initiated by excitation of tethered ethidium (E), the intercalated electron acceptor (A); the electron donor (D) is 7-deazaguanine (Z), a modified base, placed at different, fixed distances from A. The ultrafast ET between these reactants in DNA has been observed with time constants of 5 ps and 75 ps and was found to be essentially independent of the D-A separation (10-17 A). However, the ET efficiency does depend on the D-A distance. The 5-ps decay corresponds to direct ET observed from 7-deazaguanine but not guanine to E. From measurements of orientation anisotropies, we conclude that the slower 75-ps process requires the reorientation of E before ET, similar to E/nucleotide complexes in water. These results reveal the nature of ultrafast ET and its mechanism: in DNA, ET cannot be described as in proteins simply by a phenomenological parameter, beta. Instead, the involvement of the base pairs controls the time scale and the degree of coherent transport.

Base Sequence↗
PubMed1999

Site-specific inhibition of transcription factor binding to DNA by a metallointercalator.

The metallointercalator Lambda-1-Rh(MGP)2phi5+ binds tightly and specifically to the site 5'-CATATG-3' in the major groove of double helical DNA by a combination of direct readout and shape selection. To examine competitive interactions between this small metal complex and a DNA-binding transcription factor, the preferred binding site for Lambda-1-Rh(MGP)2phi5+ was engineered into the AP-1 recognition element (ARE) of the major-groove binding bZIP transcription factor yAP-1, the yeast analogue of mammalian AP-1. Binding experiments confirmed that the modified ARE retained normal yAP-1 binding affinity. Photocleavage experiments demonstrated that the modified ARE contained a high-affinity binding site for Lambda-1-Rh(MGP)2phi5+, whereas the native ARE showed no interaction. Competition experiments using gel shift mobility assays demonstrated that Lambda-1-Rh(MGP)2phi5+ at 120 nM competes 50% of yAP-1 binding to the 5'-CATATG-3' containing oligonucleotide. In contrast, competitive disruption of protein binding to the native ARE requires 3 microM Lambda-1-Rh(MGP)2phi5+. Metallointercalator derivatives, including geometric isomers of Lambda-1-Rh(MGP)2phi5+, show no specific binding to the target site and show no inhibition of yAP-1/DNA complexes at concentrations as high as 20 microM. Thus, metallointercalators can be tuned to show selectivity for major groove sites on DNA comparable to transcription factors and indeed can inhibit transcription factor binding site selectively.

Binding Sites↗
PubMed1999

A versatile mismatch recognition agent: specific cleavage of a plasmid DNA at a single base mispair.

[Rh(bpy)2(chrysi)]3+ is a novel, sterically bulky DNA intercalator that has been designed to bind specifically in the destabilized regions near DNA base mismatches and, upon photoactivation, to cleave the DNA backbone. Here the molecule is shown to be both a general and remarkably specific mismatch recognition agent. Specific DNA cleavage is observed at over 80% of mismatch sites in all the possible single base pair sequence contexts around the mispaired bases. Moreover, the complex is highly site-specific; it is shown to recognize and photocleave at a single base mismatch in a 2725 base pair linearized plasmid heteroduplex. Sterically demanding intercalators such as [Rh(bpy)2(chrysi)]3+ may have application both in mutation detection systems and as mismatch-specific chemotherapeutic agents.

Base Pair Mismatch↗
PubMed1999

Femtosecond dynamics of the DNA intercalator ethidium and electron transfer with mononucleotides in water.

Ethidium (E) is a powerful probe of DNA dynamics and DNA-mediated electron transfer (ET). Molecular dynamical processes, such as solvation and orientation, are important on the time scale of ET. Here, we report studies of the femtosecond and picosecond time-resolved dynamics of E, E with 2'deoxyguanosine triphosphate (GTP) in water, and E with 7-deaza-2'-deoxyguanosine triphosphate (ZTP) in water; E undergoes ET with ZTP but not GTP. These studies elucidate the critical role of relative orientational motions of the donor-acceptor complex on ET processes in solution. For ET from ZTP to E, such motions are in fact the rate-determining step. Our results indicate that these complexes reorient before ET. The time scale for the solvation of E in water is 1 ps, and the orientational relaxation time of E is 70 ps. The impact of orientational and solvation effects on ET between E and mononucleotides must be considered in the application of E as a probe of DNA ET.

DNA↗
PubMed1999

Electron transfer between bases in double helical DNA.

Fluorescent analogs of adenine that selectively oxidize guanine were used to investigate photoinduced electron transfer through the DNA pi-stack as a function of reactant stacking and energetics. Small variations in these factors led to profound changes in the kinetics and distance dependences of DNA-mediated electron-transfer reactions. Values of beta, a parameter reflecting the dependence of electron transfer on distance, ranged from 0.1 to 1.0 per angstrom. Strong stacking interactions result in the fastest electron-transfer kinetics. Electrons are thus transported preferentially through an intrastrand rather than interstrand pathway. Reactant energetics also modulate the distance dependence of DNA-mediated charge transport. These studies may resolve the range of disparate results previously reported, and paradigms must now be developed to describe these properties of the DNA pi-stack, which can range from insulator- to "wire"-like.

2-Aminopurine↗
PubMed1999

Use of an agent to reduce scattering in skin.

BACKGROUND AND OBJECTIVE: A method to increase light transport deeply into target areas of tissue would enhance both therapeutic and diagnostic laser applications. The effects of a hyperosmotic agent on the scattering properties of rat and hamster skin were investigated. STUDY DESIGN/MATERIALS AND METHODS: A hyperosmotic agent, glycerol, was applied in vitro and in vivo to rat and hamster skin to assess the changes in tissue optical properties. Changes in the reduced scattering coefficient after application of the agent in vitro to rat skin and after the skin has been rehydrated were assessed to evaluate the effect of the agent on tissue. RESULTS: Experimental results showed a transient change in the optical properties of in vitro rat skin. A 50% increase in transmittance and decrease in diffuse reflectance occurred within 5-10 min after the introduction of anhydrous glycerol. In addition, reduction of light scattering with this technique increased depth of visibility with optical coherence tomography. Injection of glycerol under the skin allowed in vivo visualization of blood vessels. CONCLUSIONS: The application of the agent reduces the amount of refractive mismatch found in the tissue and markedly reduces random scattering, thereby making the skin less turbid for visible wavelengths for a controlled period of time.

Administration, Topical↗
PubMed1999

Modeling laser treatment of port wine stains with a computer-reconstructed biopsy.

BACKGROUND AND OBJECTIVE: The efficacy of laser treatment of port wine stains (PWS) has been shown to be highly dependent on patient-specific vasculature. The effect of tissue structure on optical and thermal mechanisms was investigated for different pulse durations by using a novel theoretical model that incorporates tissue morphology reconstructed tomographically from a PWS biopsy. STUDY DESIGN/MATERIALS AND METHODS: An optical-thermal numerical model capable of simulating arbitrarily complex, three-dimensional tissue geometries was developed. The model is comprised of (1) a voxel-based Monte Carlo optical model, (2) a finite difference thermal model, and (3) an Arrhenius rate process calculation to predict the distribution of thermal damage. Simulations based on previous computer-based reconstruction of a series of 6 microm sections from a PWS biopsy were performed for laser pulse durations (taup) of 0.5, 5.0, and 10.0 ms at a wavelength of 585 nm. RESULTS: Energy deposition rate in the blood vessels was primarily a function of vessel depth in skin, although shading effects were evident. Thermal confinement and selectivity of damage were seen to be inversely proportional to pulse duration. The model predicted blood-specific damage for taup = 0.5 ms, vascular and perivascular damage for taup = 5 ms, and widespread damage in superficial regions for taup = 10 ms. The effect of energy deposition in the epidermis was most pronounced for longer pulse durations, resulting in increased temperature and extent of damage. CONCLUSION: Pulse durations between 0.5 and 5 ms are likely optimal for the PWS analyzed. The incorporation of a tomographically reconstructed PWS biopsy into an optical-thermal model represents a significant advance in numerical modeling of laser-tissue interaction.

Adult↗
PubMed1999

Simultaneous irradiation and imaging of blood vessels during pulsed laser delivery.

BACKGROUND AND OBJECTIVE: Simultaneous irradiation and viewing of 10-120 microm cutaneous blood vessels were performed to investigate the effects of 2-micros 577-nm dye laser pulses. STUDY DESIGN/MATERIALS AND METHODS: A modified scanning laser confocal microscope recorded vessel response to different radiant exposures (J/cm2). Probit analysis determined the 50% probability ("threshold") radiant exposure necessary to cause embolized or partly occluding coagula, coagula causing complete blood flow stoppage, and hemorrhage. RESULTS: A statistically significant difference in the threshold radiant exposure existed for each damage category for blood vessels 10-30 microm in diameter, but not for larger vessels. For vessels over 60 microm, complete flow stoppage was unattainable; increasing laser pulse energy produced hemorrhage. In larger vessels, coagula often were attached to the superficial vessel wall while blood flowed underneath. Monte Carlo optical and finite difference thermal modeling confirmed experimental results. CONCLUSION: These results provide insight into the role of pulse duration and vessel diameter in the outcome of pulsed dye laser irradiation.

Angiography↗
PubMed1999

Long-range oxidative damage to DNA: effects of distance and sequence.

INTRODUCTION: Oxidative damage to DNA in vivo can lead to mutations and cancer. DNA damage and repair studies have not yet revealed whether permanent oxidative lesions are generated by charges migrating over long distances. Both photoexcited *Rh(III) and ground-state Ru(III) intercalators were previously shown to oxidize guanine bases from a remote site in oligonucleotide duplexes by DNA-mediated electron transfer. Here we examine much longer charge-transport distances and explore the sensitivity of the reaction to intervening sequences. RESULTS: Oxidative damage was examined in a series of DNA duplexes containing a pendant intercalating photooxidant. These studies revealed a shallow dependence on distance and no dependence on the phasing orientation of the oxidant relative to the site of damage, 5'-GG-3'. The intervening DNA sequence has a significant effect on the yield of guanine oxidation, however. Oxidation through multiple 5'-TA-3' steps is substantially diminished compared to through other base steps. We observed intraduplex guanine oxidation by tethered *Rh(III) and Ru(III) over a distance of 200 A. The distribution of oxidized guanine varied as a function of temperature between 5 and 35 degrees C, with an increase in the proportion of long-range damage (> 100 A) occurring at higher temperatures. CONCLUSIONS: Guanines are oxidized as a result of DNA-mediated charge transport over significant distances (e.g. 200 A). Although long-range charge transfer is dependent on distance, it appears to be modulated by intervening sequence and sequence-dependent dynamics. These discoveries hold important implications with respect to DNA damage in vivo.

DNA↗