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The DNA duplex is an exquisite macromolecular array that stores genetic information to encode proteins and regulate pathways. Its unique structure also imparts chemical function that allows it also to mediate charge transport (CT). We have utilized diverse platforms to probe DNA CT, using spectroscopic, electrochemical, and even genetic methods. These studies have established powerful features of DNA CT chemistry. DNA CT can occur over long molecular distances as long as the bases are well stacked. The perturbations in base stacking that arise with single base mismatches, DNA lesions, and the binding of some proteins that kink the DNA all inhibit DNA CT. Significantly, single molecule studies of DNA CT show that ground state CT can occur over 34 nm if the duplex is well stacked; one single base mismatch inhibits CT. The DNA duplex is an effective sensor for the integrity of the base pair stack. Moreover, the efficiency of DNA CT is what one would expect for a stack of graphite sheets: equivalent to the stack of DNA base pairs and independent of the sugar-phosphate backbone. Since DNA CT offers a means to carry out redox chemistry from a distance, we have considered how this chemistry might be used for long range biological signaling. We have taken advantage of our chemical probes and platforms to characterize DNA CT in the context of the cell. CT can occur over long distances, perhaps funneling damage to particular sites and insulating others from oxidative stress. Significantly, transcription factors that activate the genome to respond to oxidative stress can also be activated from a distance through DNA CT. Numerous proteins maintain the integrity of the genome and an increasing number of them contain [4Fe-4S] clusters that do not appear to carry out either structural or enzymatic roles. Using electrochemical methods, we find that DNA binding shifts the redox potentials of the clusters, activating them towards oxidation at physiological potentials. We have proposed a model that describes how repair proteins may utilize DNA CT to efficiently search the genome for lesions. Importantly, many of these proteins occur in low copy numbers within the cell, and thus a processive mechanism does not provide a sufficient explanation of how they find and repair lesions before the cell divides. Using atomic force microscopy and genetic assays, we show that repair proteins proficient at DNA CT can relocalize in the vicinity of DNA lesions and can cooperate in finding lesions within the cell. Conversely, proteins defective in DNA CT cannot relocalize in the vicinity of lesions and do not assist other proteins involved in repair within the cell. Moreover such genetic defects are associated with disease in human protein analogues. As we continue to unravel this chemistry and discover more proteins with redox cofactors involved in genome maintenance, we are learning more regarding opportunities for long range signaling and sensing, and more examples of DNA CT chemistry that may provide critical functions within the cell.

This study creates a device where the DNA is electronically integrated to serve as both the biological target and electrical transducer in a CNT–DNA–CNT device. We detect DNA binding and methylation by the methyltransferase M.SssI at the single molecule level. We demonstrate sequence-specific, reversible binding of M.SssI and protein-catalyzed methylation that alters the protein-binding affinity of the device. This device, which relies on the exquisite electrical sensitivity of DNA, represents a unique route for the specific, single molecule detection of enzymatic activity.

Metallointercalating photooxidants interact intimately with the base stack of double-stranded DNA and exhibit rich photophysical and electrochemical properties, making them ideal probes for the study of DNA-mediated charge transport (CT). The complexes [Rh(phi)(2)(bpy')](3+) (phi = 9,10-phenanthrenequinone diimine; bpy' = 4-methyl-4'-(butyric acid)-2,2'-bipyridine), [Ir(ppy)(2)(dppz')](+) (ppy = 2-phenylpyridine; dppz' = 6-(dipyrido[3,2-a:2',3'-c]phenazin-11-yl)hex-5-ynoic acid), and [Re(CO)(3)(dppz)(py')](+) (dppz = dipyrido[2,3-a:2',3'-c]phenazine; py' = 3-(pyridin-4-yl)-propanoic acid) were each covalently tethered to DNA to compare their photooxidation efficiencies. Biochemical studies show that upon irradiation, the three complexes oxidize guanine by long-range DNA-mediated CT with the efficiency: Rh > Re > Ir. Comparison of spectra obtained by spectroelectrochemistry after bulk reduction of the free metal complexes with those obtained by transient absorption (TA) spectroscopy of the conjugates suggests that the reduced metal states form following excitation of the conjugates at 355 nm. Electrochemical experiments and kinetic analysis of the TA decays indicate that the thermodynamic driving force for CT, variations in the efficiency of back electron transfer, and coupling to DNA are the primary factors responsible for the trend observed in the guanine oxidation yields of the three complexes.

The complex [Re(CO)(3)(dppz)(py'-OR)](+) (dppz = dipyrido[3,2-a:2',3'-c]phenazine; py'-OR = 4-functionalized pyridine) offers IR sensitivity and can oxidize DNA directly from the excited state, making it a promising probe for the study of DNA-mediated charge transport (CT). The behavior of several covalent and noncovalent Re-DNA constructs was monitored by time-resolved IR (TRIR) and UV/visible spectroscopies, as well as biochemical methods, confirming the long-range oxidation of DNA by the excited complex. Optical excitation of the complex leads to population of MLCT and at least two distinct intraligand states. Experimental observations that are consistent with charge injection from these excited states include similarity between long-time TRIR spectra and the reduced state spectrum observed by spectroelectrochemistry, the appearance of a guanine radical signal in TRIR spectra, and the eventual formation of permanent guanine oxidation products. The majority of reactivity occurs on the ultrafast time scale, although processes dependent on slower conformational motions of DNA, such as the accumulation of oxidative damage at guanine, are also observed. The ability to measure events on such disparate time scales, its superior selectivity in comparison to other spectroscopic techniques, and the ability to simultaneously monitor carbonyl ligand and DNA IR absorption bands make TRIR a valuable tool for the study of CT in DNA.

Quantum yields for charge transport across adenine tracts of increasing length have been measured by monitoring hole transport in synthetic oligonucleotides between photoexcited 2-aminopurine, a fluorescent analogue of adenine, and N2-cyclopropyl guanine. Using fluorescence quenching, a measure of hole injection, and hole trapping by the cyclopropyl guanine derivative, we separate the individual contributions of single- and multi-step channels to DNA charge transport, and find that with 7 or 8 intervening adenines the charge transport is a coherent, single-step process. Moreover, a transition occurs from multi-step to single-step charge transport with increasing donor/acceptor separation, opposite to that generally observed in molecular wires. These results establish that coherent transport through DNA occurs preferentially across 10 base pairs, favored by delocalization over a full turn of the helix.

In an effort to develop octahedral metal complexes as chemotherapeutic and diagnostic agents targeted to DNA, it is critical to optimize the properties of their cellular uptake. Appending d-octaarginine has been found to improve both the uptake and nuclear localization efficiency of these complexes, but the increased positive charge interferes with selective DNA binding and hence activity. Herein, we evaluate the nuclear entry of a series of luminescent ruthenium peptide conjugates of shorter sequence and lower charge. As is the case for the d-octaarginine conjugate (Ru-D-R8), the tetrapeptide RrRK (where r = d-arginine) facilitates nuclear localization of the ruthenium complex above a threshold concentration, though the threshold is higher for this conjugate (Ru-RrRK) than for Ru-D-R8. Furthermore, appended fluorescein, which lowers the threshold concentration for Ru-D-R8, does not improve nuclear entry of Ru-RrRK, indicating that fluorescein conjugation is not a general strategy for modulating the distribution of cell-penetrating peptides. Similarly, the concentration required for nuclear entry of Ru-RrRK is much higher than has been reported for a thiazole orange RrRK conjugate, demonstrating the influence of payload on the efficiency of uptake and localization of cell-penetrating peptides.

We report the use of silicon chips with 16 DNA-modified electrodes (DME chips) utilizing DNA-mediated charge transport for multiplexed detection of DNA and DNA-binding protein targets. Four DNA sequences were simultaneously distinguished on a single DME chip with 4-fold redundancy, including one incorporating a single base mismatch. These chips also enabled investigation of the sequence-specific activity of the restriction enzyme Alu1. DME chips supported dense DNA monolayer formation with high reproducibility, as confirmed by statistical comparison to commercially available rod electrodes. The working electrode areas on the chips were reduced to 10 microm in diameter, revealing microelectrode behavior that is beneficial for high sensitivity and rapid kinetic analysis. These results illustrate how DME chips facilitate sensitive and selective detection of DNA and DNA-binding protein targets in a robust and internally standardized multiplexed format.

The transport of charge through the DNA base pair stack offers a route to carry out redox chemistry at a distance. Here we describe characteristics of this chemistry that have been elucidated and how this chemistry may be utilized within the cell. The shallow distance dependence associated with these redox reactions permits DNA-mediated signaling over long molecular distances in the genome and facilitates the activation of redox-sensitive transcription factors globally in response to oxidative stress. The long-range funneling of oxidative damage to sites of low oxidation potential in the genome also may provide a means of protection within the cell. Furthermore, the sensitivity of DNA charge transport to perturbations in base pair stacking, as may arise with base lesions and mismatches, may be used as a route to scan the genome for damage as a first step in DNA repair. Thus, the ability of double helical DNA, in mediating redox chemistry at a distance, provides a natural mechanism for redox sensing and signaling in the genome.

Deficiencies in mismatch repair (MMR) are associated with carcinogenesis. Rhodium metalloinsertors bind to DNA base mismatches with high specificity and inhibit cellular proliferation preferentially in MMR-deficient cells versus MMR-proficient cells. A family of chrysenequinone diimine complexes of rhodium with varying ancillary ligands that serve as DNA metalloinsertors has been synthesized, and both DNA mismatch binding affinities and antiproliferative activities against the human colorectal carcinoma cell lines HCT116N and HCT116O, an isogenic model system for MMR deficiency, have been determined. DNA photocleavage experiments reveal that all complexes bind to the mismatch sites with high specificities; DNA binding affinities to oligonucleotides containing single base CA and CC mismatches, obtained through photocleavage titration or competition, vary from 10(4) to 10(8) M(-1) for the series of complexes. Significantly, binding affinities are found to be inversely related to ancillary ligand size and directly related to differential inhibition of the HCT116 cell lines. The observed trend in binding affinity is consistent with the metalloinsertion mode where the complex binds from the minor groove with ejection of mismatched base pairs. The correlation between binding affinity and targeting of the MMR-deficient cell line suggests that rhodium metalloinsertors exert their selective biological effects on MMR-deficient cells through mismatch binding in vivo.

The base pair stack of DNA has been demonstrated as a medium for long-range charge transport chemistry both in solution and at DNA-modified surfaces. This chemistry is exquisitely sensitive to structural perturbations in the base pair stack as occur with lesions, single base mismatches, and protein binding. We have exploited this sensitivity for the development of reliable electrochemical assays based on DNA charge transport at self-assembled DNA monolayers. Here, we discuss the characteristic features, applications, and advantages of DNA-mediated electrochemistry.

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.

De novo initiation of DNA synthesis by DNA polymerases requires formation of an initial “primer” on the template. In eukaryotes, this function is provided by a distinct DNA primase. Primase, a DNA-dependent RNA polymerase, generates short (8-12 nt) RNA primers on ssDNA templates. These initial RNA primers are handed off to DNA polymerase α (pol α), which extends the primer by ∼20 nts. This primed RNA/DNA substrate is then handed off to processive polymerases e or δ, which synthesize the vast majority of new DNA on the leading and lagging strands. X-ray crystal structures of human primase have been used to propose models for the catalytic mechanism. However, the molecular basis for primer counting and handoff remains unclear. Primase contains a 4Fe-4S cluster in the p58C domain critical to pol-prim loading and activity, but its specific function is not known. We are investigating the hypothesis that the function of the cluster is to drive primer length counting. Well-stacked duplex DNA has been shown capable of transporting charge over long distances, and it has been proposed this phenomenon enables communication between Fe-S cluster-containing proteins. In this study, we show the 4Fe-4S cluster in wild-type p58C is capable of mediating charge transport (CT) with DNA. DNA-modified electrodes were used to characterize the electrochemistry of p58C. Based on previously determined WT structures, we identified a possible pathway from the cluster to the DNA binding site in p58C and designed mutants of key residues in this pathway. Mutation of Tyr345 to Phe greatly diminishes DNA CT. Primase activity assays with WT and mutant proteins revealed substantial differences in product length distribution. These data suggest charge transport is involved in primer length counting and may signal for primer template handoff to pol α.

Charge transport (CT) through the DNA base pairs provides a means to promote redox reactions at a remote site and potentially to effect signaling between molecules bound to DNA. Here we describe the oxidation of a cell-cycle regulatory protein, p53, from a distance through DNA-mediated CT. A consensus p53 binding site as well as three DNA promoters regulated by p53 were synthesized containing a tethered DNA photooxidant, anthraquinone. Photoinduced oxidation of the protein occurs from a distance; introduction of an intervening CA mismatch, which inhibits DNA-mediated CT, prevents oxidation of p53. DNA-mediated oxidation is shown to promote dissociation of p53 from only some promoters, and this sequence-selectivity in oxidative dissociation correlates with the biological regulation of p53. Under severe oxidative stress, effected here through oxidation at long range, p53 dissociates from a promoter that activates DNA repair as well as the promoter for the negative regulator of p53, Mdm2, but not from a promoter activating cell-cycle arrest. Mass spectrometry results are consistent with disulfide bond formation in p53 upon DNA-mediated oxidation. Furthermore, DNA-bound p53 oxidation is shown in vivo by up-regulation of p53 and subsequent irradiation in the presence of a rhodium photooxidant to give a new p53 adduct that can be reversed with thiol treatment. This DNA-mediated oxidation of p53 parallels that seen by treating cells with hydrogen peroxide. These results indicate a unique mechanism using DNA-mediated CT chemistry by which p53 activity on different promoters may be controlled globally under conditions of oxidative stress.

The DNA double helix has captured the imagination of many, bringing it to the forefront of biological research. DNA has unique features that extend our interest into areas of chemistry, physics, material science, and engineering. Our laboratory has focused on studies of DNA charge transport (CT), wherein charges can efficiently travel long molecular distances through the DNA helix while maintaining an exquisite sensitivity to base pair π-stacking. Because DNA CT chemistry reports on the integrity of the DNA duplex, this property may be exploited to develop electrochemical devices to detect DNA lesions and DNA-binding proteins. Furthermore, studies now indicate that DNA CT may also be used in the cell by, for example, DNA repair proteins, as a cellular diagnostic, in order to scan the genome to localize efficiently to damage sites. In this review, we describe this evolution of DNA CT chemistry from the discovery of fundamental chemical principles to applications in diagnostic strategies and possible roles in biology.

We report the synthesis and characterization of a bimetallic complex derived from a new family of potent and selective metalloinsertors containing an unusual Rh-O axial coordination. This complex incorporates a monofunctional platinum center containing only one labile site for coordination to DNA, rather than two, and coordinates DNA nonclassically through adduct formation in the minor groove. This conjugate displays bifunctional, interdependent binding of mismatched DNA via metalloinsertion at a mismatch as well as covalent platinum binding. DNA sequencing experiments revealed that the preferred site of platinum coordination is not the traditional N7-guanine site in the major groove, but rather N3-adenine in the minor groove. The complex also displays enhanced cytotoxicity in mismatch repair-deficient and mismatch repair-proficient human colorectal carcinoma cell lines compared to the chemotherapeutic cisplatin, and it triggers cell death via an apoptotic pathway, rather than the necrotic pathway induced by rhodium metalloinsertors.

DNMT1, the most abundant human methyltransferase, is responsible for translating the correct methylation pattern during DNA replication, and aberrant methylation by DNMT1 has been linked to tumorigenesis. We have developed a sensitive signal-on electrochemical assay for the measurement of DNMT1 activity in crude tissue lysates. We have further analyzed ten tumor sets and found a direct correlation between DNMT1 hyperactivity and tumorous tissue. In the majority of samples analyzed, the tumorous tissue has significantly higher DNMT1 activity than the healthy adjacent tissue. No such correlation is observed in measurements of DNMT1 expression by quantitative PCR, DNMT1 protein abundance by Western blotting, or DNMT1 activity using a radiometric DNA labeling assay. DNMT1 hyperactivity can result from both protein overexpression and/or enzyme hyperactivity. DNMT1 activity measured electrochemically provides a direct measure of activity in cell lysates and, as a result, provides a sensitive and early indication of cancerous transformation.

We have developed a thin layer, multiplexed biosensing platform that features two working-electrode arrays for detecting small molecules, nucleic acid sequences, and DNA-binding proteins. DNA duplexes are patterned onto the primary electrode array, while a secondary electrode array is used both to initiate DNA monolayer formation and for electrochemical readout via DNA-mediated charge transport (DNA CT) chemistry. Electrochemical reduction of Cu(phendione)_2^(2+) (phendione is 1,10-phenanthroline-5,6-dione) at the secondary electrodes induces covalent attachment via click chemistry of ethynyl-labeled DNA probe duplexes onto the primary electrodes that have been treated with azide-terminated alkylthiols. Electrochemical impedance spectroscopy and cyclic voltammetry confirm that catalyst activation at the secondary electrode is essential to maintain the integrity of the DNA monolayer. Electrochemical readout of DNA CT processes that occur at the primary electrode is accomplished also at the secondary electrode. The two-electrode system enables the platform to function as a collector–generator using either ferrocyanide or ferricyanide as mediators with methylene blue and DNA charge transport. Electrochemical measurements at the secondary electrode eliminate the need for large background corrections. The resulting sensitivity of this platform enables the reliable and simultaneous detection of femtomoles of the transcription factors TATA-binding protein and CopG on a single multiplexed device.

Mismatch repair (MMR) is critical to maintaining the integrity of the genome, and deficiencies in MMR are correlated with cancerous transformations. Bulky rhodium intercalators target DNA base mismatches with high specificity. Here we describe the application of bulky rhodium intercalators to inhibit cellular proliferation differentially in MMR-deficient cells compared with cells that are MMR-proficient. Preferential inhibition by the rhodium complexes associated with MMR deficiency is seen both in a human colon cancer cell line and in normal mouse fibroblast cells; the inhibition of cellular proliferation depends strictly on the MMR deficiency of the cell. Furthermore, our assay of cellular proliferation is found to correlate with DNA mismatch targeting by the bulky metallointercalators. It is the Δ-isomer that is active both in targeting base mismatches and in inhibiting DNA synthesis. Additionally, the rhodium intercalators promote strand cleavage at the mismatch site with photoactivation, and we observe that the cellular response is enhanced with photoactivation. Targeting DNA mismatches may therefore provide a cell-selective strategy for chemotherapeutic design.

Duplex DNA functionalized with pyrene has been utilized to fabricate DNA-modified electrodes on highly oriented pyrolytic graphite (HOPG). Films have been characterized using AFM and radioactive labeling as well as electrochemically. The data obtained are consistent with a close-packed structure in the film with helices oriented in a nearly upright orientation, as seen earlier with the fabrication of thiol-tethered duplexes on gold. Also as on gold, we observe the reduction of DNA-bound intercalators in a DNA-mediated reaction. The reduction of the intercalator is attenuated in the presence of the single-base mismatches, CA and GT, independent of the sequence composition of the oligonucleotide. This sensitivity to single-base mismatches is enhanced when methylene blue reduction is coupled in an electrocatalytic cycle with ferricyanide. The extended potential range afforded by the HOPG surface has allowed us also to investigate the electrochemistry of previously inaccessible metallointercalators, Ru(bpy)_2dppz^(2+) and Os(phen)_2dppz^(2+), at the DNA-modified HOPG surface. These results support the application of DNA-modified HOPG as a convenient and reproducible surface for electrochemical DNA sensors using DNA-mediated charge transport.

Several photoactive platinum alpha-diimine intercalators have been prepared to develop new probes of DNA oxidation and reduction chemistry. Five water-soluble bis(mes')Pt(II) complexes (mes' = N,N,N,3,5-pentamethylaniline) with various aromatic alpha-diimine ligands (dppz = dipyridophenazine, np = naphtha[2,3-f][1,omega]phenanthroline, CN-np = naphtho[2,3-f][1,10]phenanthroline-9-carbonitrile, CN(2)-np = naphtho[2,3-f][1,10]phenanthroline-9,14-dicarbonitrile, and bp = benzo-[f][1,10]phenanthroline) were synthesized. The complex [(np)Pt(mes')(2)]Cl(2) was also characterized by X-ray crystallography, and the crystal structure shows that the ortho-methyl groups of the mes' ligands conveniently block substitution at the vacant sites of platinum without overlapping with the intercalating alpha-diimine ligand. The Pt(II) complexes were found to have excited-state oxidation and reduction potentials of -0.6 to -1.0 and 1.0 to 1.5 V versus NHE, respectively, making them potent photoreductants as well as photooxidants. Many of the complexes are found to promote the photooxidation of N(2)-cyclopropyldeoxyguanosine (d(Cp)G). Photoexcited [(dppz)Pt(mes')(2)](2+) is found to be most efficient in this photooxidation, as well as in the photoreduction of N(4)-cyclopropylcytidine ((Cp)C); these modified nucleosides rapidly decompose in a ring-opening reaction upon oxidation or reduction. Photoexcited [(dppz)Pt(mes')(2)]Cl(2), upon intercalation into the DNA pi stack, is found, in addition, to promote reductive and oxidative damage within the DNA duplex, as is also probed using the kinetically fast electron and hole traps, (Cp)C and (Cp)G. These Pt complexes may therefore offer useful reactive tools to compare and contrast directly reductive and oxidative chemistry in double helical DNA.

Cyclobutane pyrimidine dimers are the major DNA photoproducts produced upon exposure to UV radiation. If left unrepaired, these lesions can lead to replication errors, mutation, and cell death. Photolyase is a light-activated flavoenzyme that binds to pyrimidine dimers in DNA and repairs them in a reaction triggered by electron transfer from the photoexcited flavin cofactor to the dimer. Using gold electrodes modified with DNA duplexes containing a cyclobutane thymine dimer (T<>T), here we probe the electrochemistry of the flavin cofactor in Escherichia coli photolyase. Cyclic and square-wave voltammograms of photolyase deposited on these electrodes show a redox signal at 40 mV versus normal hydrogen electrode, consistent with electron transfer to and from the flavin in the DNA-bound protein. This signal is dramatically attenuated on surfaces where the π-stacking of the DNA bases is perturbed by the presence of an abasic site below the T<>T, an indication that the redox pathway is DNA-mediated. DNA repair can, moreover, be monitored electrically. Exposure of photolyase on T<>T-damaged DNA films to near-UV/blue light leads to changes in the flavin signal consistent with repair, as confirmed by parallel HPLC experiments. These results demonstrate the exquisite sensitivity of DNA electrochemistry to perturbations in base pair stacking and the applicability of this chemistry to probe reactions of proteins with DNA.

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.

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 in10 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.

Here we report the synthesis of luminescent ruthenium complexes that bind DNA base pair mismatches. [Ru(bpy)_2(tpqp)]Cl_2 (tpqp = 7,8,13,14-tetrahydro-6-phenylquino[8,7-k][1,8]phenanthroline), [Ru(bpy)_2(pqp)]Cl_2 (pqp = 6-phenylquino[8,7-k][1,8]phenanthroline), and [Ru(bpy)_2(tactp)]Cl_2 [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, K_b = 4 × 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 × 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 × 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.

The ultrafast dynamics of electron transfer (ET) between bases in DNA have been determined to elucidate the critical role of base-pair gating on the timescale of the electron transport and the temperature dependence of the rates. ET occurs only through DNA duplexes that adopt a specific, well-coupled alignment of bases (ET-active; see picture). Descriptions of ET dynamics must include fluctuations of DNA bases.

In electrochemistry experiments on DNA-modified electrodes, features of the redox probe that determine efficient charge transport through DNA-modified surfaces have been explored using methylene blue (MB^+) and Ru(NH_3)_6^(3+) as DNA-binding redox probes. The electrochemistry of these molecules is studied as a function of ionic strength to determine the necessity of tight binding to DNA and the number of electrons involved in the redox reaction; on the DNA surface, MB^+ displays 2e^-/1H^+ electrochemistry (pH 7) and Ru(NH^3)_6^(3+) displays 1e^- electrochemistry. We examine also the effect of electrode surface passivation and the effect of the mode (intercalation or electrostatic) of MB^+ and Ru(NH_3)_6^(3+) binding to DNA to highlight the importance of intercalation for reduction by a DNA-mediated charge-transport pathway. Furthermore, in experiments in which MB^+ is covalently linked to the DNA through a σ-bonded tether and the ionic strength is varied, it is demonstrated that intercalative stacking rather than covalent σ-bonding is essential for efficient reduction of MB^+. The results presented here therefore establish that efficient charge transport to the DNA-binding moiety in DNA films requires intercalative stacking and is mediated by the DNA base pair array.

The electrocatalytic reduction of ferricyanide mediated by methylene blue (MB) at a DNA-modified electrode was investigated by linear-sweep voltammetry at a rotating-gold-disk electrode (RDE). Electrodes were modified with thiol-terminated double-stranded oligonucleotides (sequence: 5‘-SH-AGTACAGTCATCCGC-3‘) to form densely packed DNA films that blocked the electrochemical reduction of millimolar solutions of ferricyanide at overpotentials greater than 0.5 V. Addition of micromolar concentrations of MB to these solutions, however, resulted in the rapid appearance of catalytic currents as long as the potential was held negative of the formal potential of MB within the DNA film (−0.30 V vs SCE). MB binds reversibly to these DNA-modified surfaces, and the adsorption kinetics of MB were determined by a coulometric assay. These data fit a simple Langmuir model with k_1 (adsorption) = 7.5(6) × 10^5 M^(-1) s^(-1) and k_(-1) (desorption) = 0.043(8) s^(-1). Interestingly, linear-sweep RDE voltammograms recorded in the presence of MB revealed “peaked”-shaped i−V traces, which took several seconds to reach their steady-state values. Moreover, the steady-state current density for reduction of Fe(CN)_6^(3-) was found to depend on the bulk concentration of MB in solution. A model is proposed to account for these data in which Fe(CN)_6^(3-) turnover is limited ultimately by the kinetics of MB crossing into and out of the film, and the chemical reaction between Fe(CN)_6^(3-) and reduced MB occurs at the film/solution interface.

The opportunistic pathogen Pseudomonas aeruginosa forms metabolically stratified biofilms. Redox-active phenazine metabolites shuttle metabolic electrons from the oxygen depleted biofilm interior to the oxygen saturated exterior, facilitating anoxic survival. We are testing the hypothesis that phenazine retention and/or charge transfer through the biofilm matrix are mediated by extracellular DNA (eDNA), which is the most abundant polymer in the P. aeruginosa matrix and is known to provide structural integrity to the biofilm. Specifically, we are growing biofilms on electrode arrays and performing in vitro studies of phenazine interactions with DNA. Biofilm experiments show that conductivity through the matrix is mediated by sequential electron transfer between phenazine molecules. Transferring electrode grown biofilms to fresh liquid medium demonstrates that some phenazines are retained tightly in the biofilm matrix, while others rapidly diffuse away. The retention of different phenazines corresponds to their affinity for DNA in vitro, consistent with the idea that some phenazines might bind the eDNA in the biofilm. We are testing whether phenazines bound to DNA can also mediate charge transfer. Collectively, these experiments are helping us better understand how P. aeruginosa utilizes its self-produced extracellular electron shuttles to survive biofilm oxidant limitation, and are opening up new perspectives on the role(s) that eDNA may play in the biofilm matrix.

The unique characteristics of DNA charge transport (CT) have prompted an examination of roles for this chemistry within a biological context. Not only can DNA CT facilitate long-range oxidative damage of DNA, but redox-active proteins can couple to the DNA base stack and participate in long-range redox reactions using DNA CT. DNA transcription factors with redox-active moieties such as SoxR and p53 can use DNA CT as a form of redox sensing. DNA CT chemistry also provides a means to monitor the integrity of the DNA, given the sensitivity of DNA CT to perturbations in base stacking as arise with mismatches and lesions. Enzymes that utilize this chemistry include an interesting and ever-growing class of DNA-processing enzymes involved in DNA repair, replication, and transcription that have been found to contain 4Fe-4S clusters. DNA repair enzymes containing 4Fe-4S clusters, that include endonuclease III (EndoIII), MutY, and DinG from bacteria, as well as XPD from archaea, have been shown to be redox-active when bound to DNA, share a DNA-bound redox potential, and can be reduced and oxidized at long-range via DNA CT. Interactions between DNA and these proteins in solution, in addition to genetics experiments within Escherichia coli, suggest that DNA-mediated CT can be used as a means of cooperative signaling among DNA repair proteins that contain 4Fe-4S clusters as a first step in finding DNA damage, even within cells. On the basis of these data, we can consider also how DNA-mediated CT may be used as a means of signaling to coordinate DNA processing across the genome.

Mechanistic models of charge transfer (CT) in macromolecules often focus on CT energetics and distance as the chief parameters governing CT rates and efficiencies. However, in DNA, features unique to the DNA molecule, in particular, the structure and dynamics of the DNA base stack, also have a dramatic impact on CT. Here we probe the influence of subtle structural variations on base–base CT within a DNA duplex by examining photoinduced quenching of 2-aminopurine (Ap) as a result of hole transfer (HT) to guanine (G). Photoexcited Ap is used as a dual reporter of variations in base stacking and CT efficiency. Significantly, the unique features of DNA, including the strandedness and directional asymmetry of the double helix, play a defining role in CT efficiency. For an (AT) n bridge, the orientation of the base pairs is critical; the yield of intrastrand HT is markedly higher through (A) n compared with (T) n bridges, whereas HT via intrastrand pathways is more efficient than through interstrand pathways. Remarkably, for reactions through the same DNA bridge, over the same distance, and with the same driving force, HT from photoexcited Ap to G in the 5′ to 3′ direction is more efficient and less dependent on distance than HT from 3′ to 5′. We attribute these differences in HT efficiency to variations in base–base coupling within the DNA assemblies. Thus base–base coupling is a critical parameter in DNA CT and strongly depends on subtle structural nuances of duplex DNA.

Double helical DNA, containing a π-stacked array of base pairs within its interior, can be considered as a molecular analogue of solid state π-stacked arrays. Like the solid state materials, the DNA base pair stack provides a medium to facilitate charge transport. However, owing to the dynamical motions of the base pairs within the molecular stack, as well as sequence-dependent inhomogeneities in energetics and base–base couplings, DNA charge transport differs considerably from that in solid state π-stacked materials. Here we review some of the experimental techniques and chemical assemblies used to probe charge transport in DNA. We focus on those parameters that distinguish charge transport within the molecular base pair stack and highlight the sensitivity of DNA charge transport to dynamical variations in base stacking and couplings. Exploiting the sensitivity of DNA charge transport to these sequence- and structure-dependent variations in stacking provides a route to the design of DNA-based nanoscale sensors. Certainly the application of DNA in molecular electronic devices must take into consideration those factors that promote and inhibit DNA-mediated charge transport.

The base pair stack within double helical DNA provides an effective medium for charge transport. The DNA pi-stack mediates oxidative DNA damage over long molecular distances in a reaction that is exquisitely sensitive to the sequence-dependent conformation and dynamics of DNA. A mixture of tunneling and hopping mechanisms have been proposed to account for this long-range chemistry, which is gated by dynamical variations within the stack. Electrochemical sensors have also been developed, based upon the sensitivity of DNA charge transport to base pair stacking, and these sensors provide a completely new approach to diagnosing single base mismatches in DNA and monitoring protein-DNA interactions electrically. DNA charge transport, furthermore, may play a role within the cell and, indeed, oxidative damage to DNA from a distance has been demonstrated in the cell nucleus. As a result, the biological consequences of and opportunities for DNA-mediated charge transport now require consideration.

Interactions with DNA by a family of ruthenium(II) complexes bearing the dppz (dppz = dipyridophenazine) ligand or its derivatives have been examined. The complexes include Ru(bpy)(2)(dppx)(2+) (dppx = 7,8-dimethyldipyridophenazine), Ru(bpy)(2)(dpq)(2+) (dpq = dipyridoquinoxaline), and Ru(bpy)(2)(dpqC)(2+) (dpqC = dipyrido-6,7,8,9-tetrahydrophenazine). Their ground and excited state oxidation/reduction potentials have been determined using cyclic voltammetry and fluorescence spectroscopy. An intercalative binding mode has been established on the basis of luminescence enhancements in the presence of DNA, excited state quenching, fluorescence polarization values, and enantioselectivity. Oxidative damage to DNA by these complexes using the flash/quench method has been examined. A direct correlation between the amount of guanine oxidation obtained via DNA charge transport and the strength of intercalative binding was observed. Oxidative damage to DNA through DNA-mediated charge transport was also compared directly for two DNA-tethered ruthenium complexes. One contains the dppz ligand that binds avidly by intercalation, and the other contains only bpy ligands, that, while bound covalently, can only associate with the base pairs through groove binding. Long range oxidative damage was observed only with the tethered, intercalating complex. These results, taken together, all support the importance of close association and intercalation for DNA-mediated charge transport. Electronic access to the DNA base pairs, provided by intercalation of the oxidant, is a prerequisite for efficient charge transport through the DNA pi-stack.

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.

The role of abnormal DNA methyltransferase activity in the development and progression of cancer is an essential and rapidly growing area of research, both for improved diagnosis and treatment. However, current technologies for the assessment of methyltransferase activity, particularly from crude tumor samples, limit this work because they rely on radioactivity or fluorescence and require bulky instrumentation. Here, we report an electrochemical platform that overcomes these limitations for the label-free detection of human DNA(cytosine-5)-methyltransferase1 (DNMT1) methyltransferase activity, enabling measurements from crude cultured colorectal cancer cell lysates (HCT116) and biopsied tumor tissues. Our multiplexed detection system involving patterning and detection from a secondary electrode array combines low-density DNA monolayer patterning and electrocatalytically amplified DNA charge transport chemistry to measure selectively and sensitively DNMT1 activity within these complex and congested cellular samples. Based on differences in DNMT1 activity measured with this assay, we distinguish colorectal tumor tissue from healthy adjacent tissue, illustrating the effectiveness of this two-electrode platform for clinical applications.

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 pKa 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 N-methyl-N'-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.

We report the synthesis and characterization of a bimetallic conjugate (RhPt) in which an oxaliplatin derivative is tethered to a rhodium metalloinsertor through an aminomalonate leaving group ligand. The complex interacts with DNA through metalloinsertion at a base pair mismatch followed by formation of a covalent Pt–DNA adduct. Characterization of RhPt in mismatch repair-deficient HCT116O cells reveals increased cytotoxicity compared to cisplatin and oxaliplatin as well as relative to the unconjugated rhodium and platinum counterparts. Caspase and poly-ADP ribose polymerase inhibition assays indicate that RhPt induces apoptotic cell death. Inductively coupled plasma mass spectrometry (ICP-MS) experiments reveal that RhPt exhibits enhanced cellular uptake properties that contribute to its increased efficacy., We report the synthesis and characterization of a bifunctional DNA metalloinsertor conjugate (“RhPt”). The bimetallic complex contains a rhodium mismatch recognition component tethered to a platinum anticancer agent and exhibits dual binding to DNA involving both metalloinsertion at a base pair mismatch and covalent cross-linking of platinum. In mismatch repair-deficient cells, RhPt exhibits enhanced cellular uptake and cytotoxicity over traditional platinum therapeutics.

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.

Classical chemotherapeutics, such as cisplatin and its analogues, have been highly successful in the clinic, yet improvements can certainly be made, given the significant side effects associated with the killing of healthy cells. Recent advances in the field of chemotherapy include the development of targeted anticancer agents, compounds that are directed towards a specific biomarker of cancer, with the hopes that such targeted therapies might have reduced side effects given their greater selectivity. Here we discuss several transition metal complexes that are tailored towards various biomolecules associated with cancer. Most notably, the success of rhodium metalloinsertors, which specifically bind to nucleic acid base mismatches in DNA, highlight the enormous potential of this exciting new strategy.

Iron–sulfur clusters have increasingly been found to be associated with enzymes involved in DNA processing. Here we describe a role for these redox clusters in DNA-mediated charge-transport signaling in E. coli between DNA repair proteins from distinct pathways. DNA-modified electrochemistry shows that the 4Fe–4S cluster of DNA-bound DinG, an ATP-dependent helicase that repairs R-loops, is redox-active at cellular potentials and ATP hydrolysis increases DNA-mediated redox signaling. Atomic force microscopy experiments demonstrate that DinG and Endonuclease III (EndoIII), a base excision repair enzyme, cooperate at long-range using DNA charge transport to redistribute to regions of DNA damage. Genetics experiments, moreover, reveal that this DNA-mediated signaling among proteins also occurs within the cell and, remarkably, is required for cellular viability under conditions of stress. Silencing the gene encoding EndoIII in a strain of E. coli where repair by DinG is essential results in a significant growth defect that is rescued by complementation with EndoIII but not with an EndoIII mutant that is enzymatically active but unable to carry out DNA charge transport. This work thus elucidates a fundamental mechanism to coordinate the activities of DNA repair enzymes across the genome.

With the goal of developing artificial nucleases for DNA hydrolysis, metal-coordinating peptides have been tethered to a DNA-intercalating rhodium complex to deliver metal ions to the sugar-phosphate backbone. The intercalator, [Rh(phi)(2)bpy']Cl(3) [phi = 9,10-phenanthrenequinone diimine; bpy' = 4-(butyric acid)-4'-methyl-2,2'-bipyridine], provides DNA binding affinity, and a metal-binding peptide contributes reactivity. This strategy for DNA hydrolysis is a general one, and zinc(II)-promoted cleavage has been demonstrated for two widely different tethered metallopeptides. An intercalator coupled with a de novo-designed alpha helix containing two histidine residues has been demonstrated to cleave both supercoiled plasmid and linear DNA substrates. Mutation of this peptide confirms that the two histidine residues are essential for Zn(2+) binding and cleavage. Zinc(II)-promoted cleavage of supercoiled plasmid has also been demonstrated with an intercalator-peptide conjugate containing acidic residues and modeled after the active site of the BamHI endonuclease. Other redox-active metals, such as copper, have been delivered to DNA with our intercalator-peptide conjugates to effect oxidative chemistry. Copper cleavage experiments and photocleavage experiments with [Rh(phi)(2)bpy'](3+) complement the hydrolysis studies and provide structural information about the interactions between the tethered metallopeptides and DNA. Variation of the rhodium intercalator was also explored, but with a mismatch-specific intercalator, no site-specific hydrolysis was found. These experiments, in which the peptide, the metal cation, and the intercalator components of the conjugate are each varied, illustrate some of the issues involved in creating an artificial nuclease with DNA intercalators and metallopeptides.

Oxidative damage to DNA bases in isolated HeLa nuclei occurs upon treatment with rhodium intercalators and photoactivation. Oxidation occurs preferentially at the 5'-guanine of 5'-GG-3' sites, indicative of base damage by DNA-mediated charge transfer chemistry. Moreover, oxidative damage occurs at protein-bound sites which are inaccessible to rhodium. Thus, on transcriptionally active DNA within the cell nucleus, DNA-mediated charge transport leads to base damage from a distance, and direct interaction of an oxidant is not necessary to generate a base lesion at a specific site. These observations require consideration in designing new chemotherapeutics and in understanding cellular mechanisms for DNA damage and repair.

A tris(heteroleptic) phenanthrenequinone diimine (phi) complex of Ir(III), Ir(bpy)(phen)(phi)(3+), was synthesized through the stepwise introduction of three different bidentate ligands, and the Lambda- and Delta-enantiomers were resolved and characterized by CD spectroscopy. Like other phi complexes, this tris(heteroleptic) iridium complex binds avidly to DNA by intercalation. Electrochemical studies show that Ir(bpy)(phen)(phi)(3+) undergoes a reversible one-electron reduction at E(0) = -0.025 V in 0.1 M TBAH/DMF (versus Ag/AgCl), and spectroelectrochemical studies indicate that this reduction is centered on the phi ligand. The EPR spectrum of electrochemically generated Ir(bpy)(phen)(phi)(2+) is consistent with a phi-based radical. The electrochemistry of Ir(bpy)(phen)(phi)(3+) was also probed at a DNA-modified electrode, where a DNA binding affinity of K = 1.1 x 10(6) M(-1) was measured. In contrast to Ir(bpy)(phen)(phi)(3+) free in solution, the complex bound to DNA undergoes a concerted two-electron reduction, to form a diradical species. On the basis of UV-visible and EPR spectroscopies, it is found that disproportionation of electrochemically generated Ir(bpy)(phen)(phi)(2+) occurs upon DNA binding. These results underscore the rich redox chemistry associated with metallointercalators bound to DNA.

The long-range film structure of 15 base pair, thiol-derivatized duplexes tethered through single 3‘ and 5‘ linkages to a Au(111) surface is measured via scanning probe microscopy. Film morphology of the 3‘ linked duplexes differs from that of its 5‘ counterpart as observed by scanning probe microscopy in contact mode under buffer solution. No structural features are distinguished in a film formed with the 5‘ linked duplexes as measured with either a bare silicon nitride or chemically modified probe. However, a distinct pattern in the film structure of the 3‘ linked duplexes is measured with a chemically modified, hydrophobic tip. Small changes in linker orientation can greatly affect DNA film structure, and a hypothesis for the self-assembly mechanism of the 3‘ linked duplexes is presented. Factors such as linker placement and composition must be considered in comparing DNA microarrays formed with different tethering methods.

A systematic investigation of the efficiency of oxidative damage at guanine residues through long-range charge transport was carried out as a function of intervening base mismatches. A series of DNA oligonucleotides were synthesized that incorporate a ruthenium intercalator linked covalently to the 5' terminus of one strand and containing two 5'-GG-3' sites in the complementary strand. Single base mismatches were introduced between the two guanine doublet steps, and the efficiency of transport through the mismatches was determined through measurements of the ratio of oxidative damage at the guanine doublets distal versus proximal to the intercalated ruthenium oxidant. Differing relative extents of guanine oxidation were observed for the different mismatches. The damage ratio of oxidation at the distal versus proximal site for the duplexes containing different mismatches varies in the order GC approximately GG approximately GT approximately GAAACC approximately TT approximately CA approximately CT. For all assemblies, damage found with the Delta-Ru diastereomer was found to be greater than with the Lambda-diastereomer. The extent of distal/proximal guanine oxidation in different mismatch-containing duplexes was compared with the helical stability of the duplexes, electrochemical data for intercalator reduction on different mismatch-containing DNA films, and base-pair lifetimes for oligomers containing the different mismatches derived from 1H NMR measurements of the imino proton exchange rates. While a clear correlation is evident both with helix stability and electrochemical data monitoring reduction of an intercalator through DNA films, damage ratios correlate most closely with base-pair lifetimes. Competitive hole trapping at the mismatch site does not appear to be a key factor governing the efficiency of transport through the mismatch. These results underscore the importance of base dynamics in modulating long-range charge transport through the DNA base-pair stack.

Oxidative damage as a result of DNA-mediated long-range charge transport occurs readily and at high yield in duplex DNA, and it is of interest whether similar damage can occur in duplex oligonucleotides that include both ribo- and deoxyribonucleotides. Assemblies containing RNA and mixed RNA.DNA strands were constructed containing tethered ethidium as a photooxidant. In photooxidation experiments, long-range oxidative damage to the ribose-containing strand of the oligonucleotide duplexes was examined. Hole injection by photoexcited ethidium followed by radical migration to oxidatively susceptible guanines afforded significant damage on ribose-containing strands at long range ( approximately 35 A). This damage does not differ substantially in yield and location from that found in B-DNA duplexes. No oxidative damage was found upon photooxidation of DNA/RNA duplexes containing tethered metallointercalator, despite the ability of the rhodium complex to promote oxidative damage at a distance in DNA duplexes. This result is attributed to the poor coupling of the rhodium complex into the A-like RNA/DNA duplex. The ability for long-range charge transport to occur in double-stranded nucleic acids of different comformations is considered in light of modeling studies that show interstrand base-base overlap between the opposing, complementary strands that make up RNA/DNA hybrid duplexes. Thus, the possibility of long-range radical migration to effect oxidative damage or signaling may be considered also in the context of transcriptional events.

Charge migration through the DNA base stack has been probed both spectroscopically, to observe the formation of radical intermediates, and biochemically, to assess irreversible oxidative DNA damage. Charge transport and radical trapping were examined in DNA assemblies in the presence of a site-specifically bound methyltransferase HhaI mutant and an intercalating ruthenium photooxidant using the flash-quench technique. The methyltransferase mutant, which can flip out a base and insert a tryptophan side chain within the DNA cavity, is found to activate long-range hole transfer through the base pair stack. Protein-dependent DNA charge transport is observed over 50 A with guanine radicals formed10(6) s(-1); hole transport through DNA over this distance is not rate-limiting. Given the time scale and distance regime, such protein-dependent DNA charge transport chemistry requires consideration physiologically.