Biomedical subjects

Jacqueline K Barton

Publications and source records attributed to Jacqueline K Barton.

At least 37 records · Page 2Linked to original sources

PubMed2004

Electron transfer rates in DNA films as a function of tether length.

A homologous series of DNA-modified electrodes has been investigated in which the molecular tether length varies. Using intercalated, covalently bound daunomycin as a redox probe, an exponential dependence of electron transfer rates on the number of intervening methylene groups in the sigma-bonded tether is observed. In contrast, variation in DM position within DNA yields no detectable change in rate. These data confirm that overall electron transfer rates in DNA films are limited by the tether, not the DNA.

DNA↗

PubMed2004

Tuning the DNA reactivity of cis-platinum: conjugation to a mismatch-specific metallointercalator.

A novel bimetallic conjugate combining a rhodium intercalator that selectively binds DNA mismatches and a reactive cis-platinum analogue that targets DNA by coordination has been prepared. The site-selectivity of the bimetallic complex in forming coordination adducts is examined using mismatched and well-matched oligonucleotides of different sequences. The results indicate that through the bifunctional complex, the platinum center can be targeted near mismatched sites. Interestingly, with mismatched, DNA both intrastrand and the less common interstrand cross-linked adducts are formed. The recognition of a DNA mismatch by the bulky Rh intercalator appears to direct the Pt unit, depending upon steric contraints, to react preferentially with mismatched DNA at a site that may or may not be the preferred site of Pt coordination. Thus, the presence of a permanent link to a site-specific intercalator is able to tune the reactivity of the cis-platinum analogue.

Base Pair Mismatch↗

PubMed2004

DNA-mediated charge transport requires conformational motion of the DNA bases: elimination of charge transport in rigid glasses at 77 K.

We have proposed that DNA-mediated charge transport (CT) is gated by base motions, with only certain base conformations being CT-active; a CT-active conformation can be described as a domain, a transiently extended pi-orbital defined dynamically by DNA sequence. Here, to explore these CT-active conformations, we examine the yield of base-base CT between photoexcited 2-aminopurine (Ap*) and guanine in DNA in rigid LiCl glasses at 77 K, where conformational rearrangement is effectively eliminated. Duplex DNA assemblies (35-mers) were constructed containing adenine bridges Ap(A)nG (n = 0-4). The yield of CT was monitored through fluorescence quenching of Ap* by G. We find, first, that the emission intensity of Ap* in all DNA duplexes increases dramatically upon cooling and becomes comparable to free Ap*. This indicates that all quenching of Ap* in duplex DNA is a dynamic process that requires conformational motion of the DNA bases. Second, DNA-mediated CT between Ap* and G is not observed at 77 K; rather than hindering the ability of DNA to transport charge, conformational motion is required. Moreover, the lack of DNA-mediated CT at 77 K, even through the shortest bridge, suggests that the static structures adopted upon cooling do not represent optimum CT-active conformations. These observations are consistent with our model of conformationally gated CT. Through conformational motion of the DNA bases, CT-active domains form and break-up transiently, both facilitating and limiting CT.

2-Aminopurine↗

PubMed2004

DNA charge transport: conformationally gated hopping through stacked domains.

The role of base motions in delocalization and propagation of charge through double helical DNA must be established experimentally and incorporated into mechanistic descriptions of DNA-mediated charge transport (CT). Here, we address these fundamental issues by examining the temperature dependence of the yield of CT between photoexcited 2-aminopurine (Ap) and G through DNA bridges of varied length and sequence. DNA assemblies (35-mers) were constructed containing adenine bridges Ap(A)(n)()G (n = 0-9, 3.4-34 A) and mixed bridges, ApAAIAG and ApATATG. CT was monitored through fluorescence quenching of Ap by G and through HPLC analysis of photolyzed DNA assemblies containing Ap and the modified guanine, N(2)-cyclopropylguanosine ((CP)G); upon oxidation, the (CP)G radical cation undergoes rapid ring opening. First, we find that below the duplex melting temperature ( approximately 60 degrees C), the yield of CT through duplex DNA increases with increasing temperature governed by the length and sequence of the DNA bridge. Second, the distance dependence of CT is regulated by temperature; enhanced DNA base fluctuations within duplex DNA extend CT to significantly longer distances, here up to 34 A in <10 ns. Third, at all temperatures the yield of CT does not exhibit a simple distance dependence; an oscillatory component, with a period of approximately 4-5 base pairs, is evident. These data cannot be rationalized by superexchange, hopping of a localized charge injected into the DNA bridge, a temperature-induced transition from superexchange to thermally induced hopping, or by phonon-assisted polaron hopping. Instead, we propose that CT occurs within DNA assemblies possessing specific, well-coupled conformations of the DNA bases, CT-active domains, accessed through base motion. CT through DNA is described as conformationally gated hopping among stacked domains. Enhanced DNA base motions lead to longer range CT with a complex distance dependence that reflects the roles of coherent dynamics and charge delocalization through transient domains. Consequently, DNA CT is not a simple function of distance but is intimately related to the dynamical structure of the DNA bridge.

2-Aminopurine↗

PubMed2004

Single-nucleotide polymorphism discovery by targeted DNA photocleavage.

Single-nucleotide polymorphisms are the largest source of genetic variation in humans. We report a method for the discovery of single-nucleotide polymorphisms within genomic DNA. Pooled genomic samples are amplified, denatured, and annealed to generate mismatches at polymorphic DNA sites. Upon photoactivation, these DNA mismatches are then cleaved site-specifically by using a small molecular probe, a bulky metallointercalator, Rhchrysi or Rhphzi. Fluorescent labeling of the cleaved products and separation by capillary electrophoresis permits rapid identification with single-base resolution of the single-nucleotide polymorphism site. This method is remarkably sensitive and minor allele frequencies as low as 5% can be readily detected.

Animals↗

PubMed2004

[Ru(bpy)2(L)]Cl2: luminescent metal complexes that bind DNA base mismatches.

Here we report the synthesis of luminescent ruthenium complexes that bind DNA base pair mismatches. [Ru(bpy)2(tpqp)]Cl2 (tpqp = 7,8,13,14-tetrahydro-6-phenylquino[8,7-k][1,8]phenanthroline), [Ru(bpy)2(pqp)]Cl2 (pqp = 6-phenylquino[8,7-k][1,8]phenanthroline), and [Ru(bpy)2(tactp)]Cl2 [tactp = 4,5,9,18-tetraazachryseno[9,10-b]triphenylene] have been synthesized, and their spectroscopic properties in the absence and presence of DNA have been examined. While [Ru(bpy)2(pqp)]2+ shows no detectable luminescence, [Ru(bpy)2(tpqp)]2+ is luminescent in the absence and presence of DNA with an excited-state lifetime of 10 ns and a quantum yield of 0.002. Although no increase in emission intensity is associated with binding to mismatch-containing DNA, luminescence quenching experiments and measurements of steady-state fluorescence polarization provide evidence for preferential binding to oligonucleotides containing a CC mismatch. Furthermore, by marking the site of binding through singlet oxygen sensitized damage, the complex has been shown to target a CC mismatch site directly with a specific binding affinity, Kb = 4 x 10(6) M(-1). [Ru(bpy)2(tactp)]2+, an analogue of [Ru(bpy)2(dppz)]2+ containing a bulky intercalating ligand, is luminescent in aqueous solution at micromolar concentrations and exhibits a 12-fold enhancement in luminescence in the presence of DNA. The complex, however, tends to aggregate in aqueous solution; we find a dimerization constant of 9.8 x 10(5) M(-1). Again, by singlet oxygen sensitization it is apparent that [Ru(bpy)2(tactp)]2+ binds preferentially to a CC mismatch; using a DNase I footprinting assay, a binding constant to a CC mismatch of 8 x 10(5) M(-1) is found. Hence results with these novel luminescent complexes support the concept of using a structurally demanding ligand to obtain selectivity in targeting single base mismatches in DNA. The challenge is coupling the differential binding we can obtain to differential luminescence.

Base Pair Mismatch↗

PubMed2004

Bifunctional rhodium intercalator conjugates as mismatch-directing DNA alkylating agents.

A conjugate of a DNA mismatch-specific rhodium intercalator, containing the bulky chrysenediimine ligand, and an aniline mustard has been prepared, and targeting of mismatches in DNA by this conjugate has been examined. The preferential alkylation of mismatched over fully matched DNA is found by a mobility shift assay at concentrations where untethered organic mustards show little reaction. The binding site of the Rh intercalator was determined by DNA photocleavage, and the position of covalent modification was established on the basis of the enhanced depurination associated with N-alkylation. The site-selective alkylation at mismatched DNA renders these conjugates useful tools for the covalent tagging of DNA base pair mismatches and new chemotherapeutic design.

2,2'-Dipyridyl↗

PubMed2004

Charge equilibration between two distinct sites in double helical DNA.

DNA assemblies containing a pendant dipyridophenazine complex of Ru(II) along with two oxidative traps, a site containing the nucleoside analog methylindole (5'-GMG-3') and a 5'-GGG-3' site, have been constructed to explore long-range charge transport through the base pair stack. With these chemically well defined assemblies, in combination with the flash/quench technique, formation of the methylindole cation radical and the neutral guanine radical is monitored directly by using transient absorption spectroscopy, and yields of oxidative damage are quantitated biochemically by gel electrophoresis. In these assemblies the base radicals form with a rate of > or =10(7) s(-1). The rate of base radical formation does not change upon the addition of a second radical trap, the 5'-GGG-3' site; however, the yield of methylindole oxidation is significantly lower. This observation indicates that the 5'-GGG-3' site is effective in competing for the migrating charge and provides a second trapping site. Switching the orientation of the two trapping sites does not affect the yield of oxidized products at either site. Therefore, in DNA both forward and reverse charge transport occur so as to provide equilibration across the duplex on a timescale that is fast compared with trapping at a particular site. Further evidence of charge equilibration results from incorporating an intervening base-stacking perturbation and monitoring the fate of the injected charge. These experiments underscore the dynamic nature of DNA charge transport and reveal the importance of considering radical propagation in both directions along the DNA duplex.

Base Sequence↗

PubMed2004

Effects of the photooxidant on DNA-mediated charge transport.

A direct comparison of DNA charge transport (CT) with different photooxidants has been made. Photooxidants tested include the two metallointercalators, Rh(phi)(2)(bpy')(3+) and Ru(phen)(bpy')(dppz)(2+), and three organic intercalators, ethidium (Et), thionine (Th), and anthraquinone (AQ). CT has been examined through a DNA duplex containing an A(6)-tract intervening between two 5'-CGGC-3' sites with each of the photooxidants covalently tethered to one end of the DNA duplex. CT is assayed both through determination of the yield of oxidative guanine damage and, in derivative DNA assemblies, by analysis of the yield of a faster oxidative trapping reaction, ring opening of N(2)-cyclopropylguanine (d(CP)G) within the DNA duplex. We find clear differences in oxidative damage ratios at the distal versus proximal 5'-CGGC-3' sites depending upon the photooxidant employed. Importantly, nondenaturing gel electrophoresis data demonstrate the absence of any DNA aggregation by the DNA-bound intercalators. Hence, differences seen with assemblies containing various photooxidants cannot be attributed to differential aggregation. Comparisons in assemblies using different photooxidants thus reveal characteristics of the photooxidant as well as characteristics of the DNA assembly. In the series examined, the lowest distal/proximal DNA damage ratios are obtained with Ru and AQ, while, for both Rh and Et, high distal/proximal damage ratios are found. The oxidative damage yields vary in the order Ru > AQ > Rh > Et, and photooxidants that produce higher distal/proximal damage ratios have lower yields. While no oxidative DNA damage is detected using thionine as a photooxidant, oxidation is evident using the faster cyclopropylguanosine trap; here, a complex distance dependence is found. Differences observed among photooxidants as well as the complex distance dependence are attributed to differences in rates of back electron transfer (BET). Such differences are important to consider in developing mechanistic models for DNA CT.

Base Sequence↗

PubMed2004

Direct chemical evidence for charge transfer between photoexcited 2-aminopurine and guanine in duplex DNA.

Photoexcited 2-aminopurine (Ap*) is extensively exploited as a fluorescent base analogue in the study of DNA structure and dynamics. Quenching of Ap* in DNA is often attributed to stacking interactions between Ap* and DNA bases, despite compelling evidence indicating that charge transfer (CT) between Ap* and DNA bases contributes to quenching. Here we present direct chemical evidence that Ap* undergoes CT with guanine residues in duplex DNA, generating oxidative damage at a distance. Irradiation of Ap in DNA containing the modified guanine, cyclopropylguanosine (CPG), initiates hole transfer from Ap* followed by rapid ring opening of the CPG radical cation. Ring opening accelerates hole trapping to a much shorter time regime than for guanine radicals in DNA; consequently, trapping effectively competes with back electron transfer (BET) leading to permanent CT chemistry. Significantly, BET remains competitive, even with this much faster trapping reaction, consistent with measured kinetics of DNA-mediated CT. The distance dependence of BET is sharper than that of forward CT, leading to an inverted dependence of product yield on distance; at short distances product yield is inhibited by BET, while at longer distances trapping dominates, leading to permanent products. The distance dependence of product yield is distinct from forward CT, or charge injection. As with photoinduced charge transfer in other chemical and biological systems, rapid kinetics for charge injection into DNA need not be associated with a high yield of DNA damage products.

2-Aminopurine↗

PubMed2003

1H NMR studies of nickel(II) complexes bound to oligonucleotides: a novel technique for distinguishing the binding locations of metal complexes in DNA.

The selective paramagnetic relaxation of oligonucleotide proton resonances of d(GTCGAC)(2) and d(GTGCAC)(2) by Ni(phen)(2)(L)(2+) where L = dipyridophenazine (dppz), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), and phenanthrenequinone (phi) has been examined to obtain structural insight into the noncovalent binding of these metal complexes to DNA. In the oligonucleotide d(GTCGAC)(2), preferential broadening of the G1H8, G4H8, T2H6, and C3H6 proton resonances was observed with Ni(phen)(2)(dppz)(2+), Ni(phen)(2)(dpq)(2+), and Ni(phen)(2)(phi)(2+). In the case of the sequence d(GTGCAC)(2), where the central two bases are juxtaposed from the previous one, preferential broadening was observed instead for the A5H2 proton resonance. Thus, a subtle change in the sequence of the oligonucleotide can cause significant change in the binding location of the metal complex in the oligonucleotide. Owing to comparable changes for all metal complexes and sequences in broadening of the thymine methyl proton resonances, we attribute the switch in preferential broadening to a change in site location within the oligomer rather than to an alteration of groove location. Therefore, even for DNA-binding complexes of low sequence-specificity, distinct variations in binding as a function of sequence are apparent.

Cations, Divalent↗

PubMed2003

In situ scanning tunneling microscopy of DNA-modified gold surfaces: bias and mismatch dependence.

In situ scanning tunneling microscopy has been performed on DNA-modified gold surfaces under physiological conditions. The STM images of DNA-modified gold surfaces are strongly dependent on the applied potential and percentage of DNA duplexes containing a single base mismatch. At negative surface potentials we observe reproducible features that are attributed to DNA agglomerates where the DNA duplexes are in the upright orientation; at positive potentials, when DNA molecules lie down on the surface, the film is transparent, and only the gold surface is distinguishable. These observations indicate that DNA possesses a non-negligible local density of states which can be probed when the DNA duplex is in the upright orientation. By varying the percentage of DNA duplexes containing a single base mismatch, we have observed a dramatic change in the image contrast as a result of the perturbation induced by the mismatch on the electronic pathway inside the DNA. These results emphasize the central role of the integrity of the pi-stack for DNA charge transport. Duplex DNA is a promising candidate in molecular electronics, but only in arrangements where the orbitals can efficiently overlap with the electronic states of the electrodes and the environment does not constrain the DNA in non-native, poorly stacked conformations.

DNA↗

PubMed2003

Charge transport in DNA duplex/quadruplex conjugates.

DNA conjugates containing adjacent duplex and guanine quadruplex assemblies have been designed to explore charge transport into quadruplex architectures. The quadruplex assemblies have been characterized structurally using circular dichroism and by assaying for chemical protection. Using an intercalating rhodium photooxidant, noncovalently bound or tethered to the duplex end, oxidizing radicals are found to be trapped in the folded quadruplex. Damage is observed almost exclusively at the external tetrads of the quadruplex. Little damage of the center tetrad is observed, due most likely to lowered efficiency of radical trapping within the quadruplex core. This pattern of damage is distinct from that observed for repetitive G sequences within duplex DNA. The data indicate, furthermore, that in the conjugates examined, the guanine quadruplex provides a more effective trap than a 5'-GG-3' guanine doublet within duplex DNA. Within these assemblies, sufficient base-base overlap must exist at the duplex/quadruplex junction to allow for charge migration. This funneling of damage to the quadruplex, as well as the unique pattern of damage within the quadruplex, requires consideration with respect to the analysis of oxidative DNA damage within the cell.

Circular Dichroism↗

PubMed2003

DNA-mediated charge transport for DNA repair.

MutY, like many DNA base excision repair enzymes, contains a [4Fe4S]2+ cluster of undetermined function. Electrochemical studies of MutY bound to a DNA-modified gold electrode demonstrate that the [4Fe4S] cluster of MutY can be accessed in a DNA-mediated redox reaction. Although not detectable without DNA, the redox potential of DNA-bound MutY is approximately 275 mV versus NHE, which is characteristic of HiPiP iron proteins. Binding to DNA is thus associated with a change in [4Fe4S]3+/2+ potential, activating the cluster toward oxidation. Given that DNA charge transport chemistry is exquisitely sensitive to perturbations in base pair structure, such as mismatches, we propose that this redox process of MutY bound to DNA exploits DNA charge transport and provides a DNA signaling mechanism to scan for mismatches and lesions in vivo.

Amides↗

PubMed2003

Long-range DNA charge transport.

The stack of base pairs within double helical DNA has been shown to mediate charge transport reactions. Charge transport through DNA can result in chemistry at a distance, yielding oxidative DNA damage at a site remote from the bound oxidant. Since DNA charge transport chemistry depends on coupling within the stacked base pair array, this chemistry is remarkably sensitive to sequence-dependent DNA structure and dynamics. Here, we discuss different features of DNA charge transport chemistry, including applications as well as possible biological consequences and opportunities.

DNA↗

PubMed2003

Fast back electron transfer prevents guanine damage by photoexcited thionine bound to DNA.

The phenothiazinium dye thionine has a high excited state reduction potential and is quenched by guanine on the femtosecond time scale. Here, we show by gel electrophoresis that irradiation of thionine with 599 nm light in the presence of an oligonucleotide duplex does not produce permanent DNA damage. Upon photoexcitation of thionine weakly associated with guanosine-5'-monophosphate, the reduced protonated thionine radical and neutral guanine radical are detected by transient absorption spectroscopy, indicating that the quenching of thionine by guanine occurs via an electron-transfer mechanism. The observation of radical formation without permanent guanine damage indicates that fast back electron transfer plays a critical role in governing the yield of damage by DNA-binding molecules.

DNA↗

PubMed2003

Rapid radical formation by DNA charge transport through sequences lacking intervening guanines.

Using the flash-quench technique to probe DNA charge transport in assemblies containing a tethered ruthenium intercalator, the kinetics and yield of methylindole radical formation as a function of DNA sequence were studied by laser spectroscopy and biochemical methods. In these assemblies, the methylindole moiety serves as an artificial base of low oxidation potential. Hole injection and subsequent formation of the methylindole radical cation were observed at a distance of over 30 A at rates >/=107 s-1 in assemblies containing no guanine bases intervening the ruthenium intercalator and GMG oxidation site. Radical yield was, however, strikingly sensitive to an intervening base mismatch; no significant methylindole radical formation was evident with an intervening AA mismatch. Also critical is the sequence at the injection site; this sequence determines initial hole localization and hence the probability of hole propagation. With guanine rather than inosine near the site of hole injection, decreased yields of radicals and long-range oxidative damage are observed. The presence of the low-energy guanine site in this case serves to localize the hole and therefore diminish charge transport through the base pair stack.

Base Sequence↗

PubMed2003

A rhodium(III) complex for high-affinity DNA base-pair mismatch recognition.

A rhodium(III) complex, rac-[Rh(bpy)(2)phzi](3+) (bpy, 2,2'-bipyridine; phzi, benzo[a]phenazine-5,6-quinone diimine) has been designed as a sterically demanding intercalator targeted to destabilized mismatched sites in double-helical DNA. The complex is readily synthesized by condensation of the phenazine quinone with the corresponding diammine complex. Upon photoactivation, the complex promotes direct strand scission at single-base mismatch sites within the DNA duplex. As with the parent mismatch-specific reagent, [Rh(bpy)(2)(chrysi)](3+) [chrysene-5,6-quinone diimine (chrysi)], mismatch selectivity depends on the helix destabilization associated with mispairing. Unlike the parent chrysi complex, the phzi analogue binds and cleaves with high affinity and efficiency. The specific binding constants for CA, CC, and CT mismatches within a 31-mer oligonucleotide duplex are 0.3, 1, and 6 x 10(7) M(-1), respectively; site-specific photocleavage is evident at nanomolar concentrations. Moreover, the specificity, defined as the ratio in binding affinities for mispaired vs. well paired sites, is maintained. The increase in affinity is attributed to greater stability in the mismatched site associated with stacking by the heterocyclic aromatic ligand. The high-affinity complex is also applied in the differential cleavage of DNA obtained from cell lines deficient in mismatch repair vs. those proficient in mismatch repair. Agreement is found between photocleavage by the mismatch-specific probes and deficiency in mismatch repair. This mismatch-specific targeting, therefore, offers a potential strategy for new chemotherapeutic design.

Antineoplastic Agents↗