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Here the effect on DNA-mediated charge transport of binding by a variety of proteins is examined. DNA assemblies were constructed that contain a tethered rhodium intercalator, as photooxidant, as well as two 5'-GG-3' sites flanking the DNA-binding site for the different proteins. By monitoring the ratio of oxidative damage promoted at the guanine doublet situated distal to the protein-binding site versus that at the proximal site as a function of protein binding, the effects of binding the proteins on DNA-mediated charge transport were determined. Proteins examined included both the wild-type and mutant methyltransferase, M.HhaI, which are base-flipping enzymes, the restriction endonuclease R.PvuII, a TATA-binding protein, which kinks the DNA, and the transcription factor Antennapedia homeodomain protein, which binds DNA through a helix-turn-helix motif. In general, it was observed that yields of long-range oxidative damage correlate with protein-dependent alterations in DNA base stacking. Interactions that disturb the DNA pi-stack inhibit DNA charge transport. Alternatively, interactions that promote no helix distortion but, as a result of tight packing, may rigidify the pi-stack, serve instead to enhance the ability of the DNA base pairs to serve as a conduit for charge transport. Thus, protein binding to DNA modulates long-range charge transport both negatively and positively, depending upon the specific protein/DNA interactions in play. Long-range DNA charge transport and this modulation by protein binding may be important to consider physiologically.

Two cyclometalated complexes of Rh(III), rac-[Rh(ppy)_2chrysi]^+ and rac-[Rh(ppy)_2phi]^+, have been synthesized and characterized with respect to their binding to DNA. The structure of rac-[Rh(ppy)_2phi]Cl·H_2O·CH_2Cl_2 has been determined by X-ray diffraction (monoclinic, P2_1/c, Z = 4, a =18.447(3) Å, b = 9.770(1) Å, c = 17.661(3) Å, β = 94.821(11)°, V = 3172.0(8) Å^3) and reveals that the complex is a distorted octahedron with nearly planar ligands, similar in structure to the DNA mismatch recognition agent [Rh(bpy)_2chrysi]^(3+). The 2-phenylpyridyl nitrogen atoms are shown to be in the axial positions, as a result of trans-directing effects. This tendency simplifies the synthesis and purification of such complexes by limiting the number of possible isomers generated. The abilities of [Rh(ppy)_2chrysi]^+ and [Rh(ppy)_2phi]^+ to bind and, with photoactivation, to cleave DNA have been demonstrated in assays on duplex DNA in the absence and presence of a single CC mismatch. [Rh(ppy)_2chrysi]^+ was shown upon photoactivation to cleave DNA selectively at the base pair mismatch whereas [Rh(ppy)_2phi]^+ cleaves B-DNA nonspecifically. The reactivity of [Rh(ppy)_2chrysi]^+ was also compared to that of the known mismatch recognition agent [Rh(bpy)_2chrysi]^(3+). Competitive photocleavage studies revealed that a 14-fold excess of [Rh(ppy)_2chrysi]^+ was required to achieve the same level of binding as that of [Rh(bpy)_2chrysi]^(3+). However, the ratio of damage induced by [Rh(bpy)_2chrysi]^(3+) to that induced by [Rh(ppy)_2chrysi]^+ is considerably greater than this value, indicating that decreased photoefficiency for the cyclometalated complex must contribute to its significantly attenuated photoreactivity. These cyclometalated intercalators provide the starting points for the design of a new family of metal complexes targeted to DNA.

A thymine cyclobutane dimer, site-specifically incorporated in a DNA duplex, is shown to be repaired upon photoexcitation (at 380 nm) of a naphthalene diimide intercalator (NDI), either bound noncovalently to the duplex or covalently appended to the C4 amine of a methylated cytosine base well separated from the thymine dimer. The repair of the thymine dimer is triggered by photooxidation either directly or by DNA-mediated charge transport over a distance of ∼22 A, the separation between NDI and the cyclobutane ring. Photooxidative repair with covalently and noncovalently bound NDI is demonstrated using HPLC under denaturing conditions, where the loss of the thymine dimer-containing strand and the formation of the repaired strand are monitored directly, as well as using a novel gel electrophoretic assay. In this assay, two strands of oligonucleotides containing 5‘- and 3‘-terminal thymidines are first ligated photochemically to yield thymine dimers, and repair is then assayed by monitoring the reversal of the photoligation by intercalators bound either noncovalently or at a distance. Although both NDI and a rhodium intercalator were seen to reverse the photoligation, several anthraquinones and ethidium were unable to promote repair upon irradiation at 350 nm. This photoligation reversal assay provides a rapid screen for thymine dimer repair. The oxidative repair of thymine dimers in a DNA duplex from a distance appears now to be a general phenomenon and requires consideration in developing mechanisms for DNA-mediated charge transport.

Continuous-wave X-band EPR spectroscopy has been employed in examining the guanine radical within a DNA duplex at ambient temperature using the flash-quench technique. Guanine was selectively oxidized by DNA-bound [Ru(phen)_2dppz]^(3+) (dppz = dipyridophenazine, phen = 1,10-phenanthroline) generated in situ by photolysis in the presence of [Co(NH_3)_5Cl]^(2+) as the oxidative quencher. An EPR signal centered at g_(iso) = 2.0048 is observed in experiments with poly(dG-dC) as substrate. Comparable signals are also detected with a 13-mer oligonucleotide duplex containing only one guanine base and with calf thymus DNA, but no signal is observed with poly(dA-dT) or poly(dI-dC). These observations reflect the base selectivity of the reaction in forming the guanine radical. With ruthenium hexaammine as oxidative quencher, no signal is observed, while, with methyl viologen, a strong signal with hyperfine pattern is seen, characteristic of the reduced viologen radical and indicating that [Ru(phen)_2dppz]^(3+) was generated. The guanine radical signal, once formed upon continuous irradiation in argon-saturated aqueous buffer solution (pH 7), decays with a half-life of 30 s, but vanishes instantaneously in the dark or upon introduction of oxygen. Spin trapping experiments with N-tert-butyl-α-phenylnitrone substantiate the selectivity in generating the guanine radical; in the presence of poly(dG-dC), calf thymus DNA, the 13-mer oligonucleotide but not with poly(dA-dT) and poly(dI-dC), the detected nitroxide EPR signals are the same with g_(iso) = 2.0059, 〈a_N〉 = 15.05 G, and 〈a_H〉 = 3.11 G. Upon titration of the ruthenium intercalator into poly(dG-dC), the signal intensity increases smoothly as the [base pair]/[intercalator] ratio decreases from 100 to 25, at which point the signal intensity decreases markedly; this result may be an indication of an antiferromagnetic exchange interaction between guanine radicals. Indeed, using the flash-quench technique, EPR spectroscopy of guanine radicals within DNA now will permit the evaluation of how radicals within the DNA base stack may be coupled under biological conditions.

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.

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.

Using luminescence spectroscopy and single photon counting, photoinduced electron transfer (ET) reactions between photoexcited [M(phen)_2dppz ^(2+) (phen = 1,10-phenanthroline, dppz=dipyridophenazine, M=Ru or Os) and the electron acceptors Rh(phi)_2bpy^(3+) (phi=9,10-phenanthrenequinone diimine, bpy=2,2′-bipyridine) and Ru(NH_3)_6^(3+) were studied as a function of DNA sequence in long DNA polymers. In addition, the thermal back reactions between M(III) and reduced acceptor were also followed by transient absorption spectroscopy. The comparison of ET reactions of the isostructural donors Os and Ru with an intercalated acceptor, Rh(phi)_2bpy^(3+), and an externally bound acceptor, Ru(NH_3)_6^(3+), helps to elucidate which factors are important for electron transfer between DNA-bound intercalators. Ru(phen)_2dppz^(2+) and Os(phen)_2dppz^(2+) show nearly identical quenching by Rh(phi)_2bpy^(3+) for a given DNA polymer, with an efficient quenching process that occurs on a time scale much faster than the excited state lifetime. We find that Rh(phi)_2bpy^(3+) and Ru(NH_3)_6^(3+) show opposite trends for quenching of DNA-bound M(phen)_2dppz^(2+). Quenching by intercalated Rh(phi)_2bpy^(3+) is most efficient in AT-only DNA polymers and less efficient in GC-only polymers, whereas for groove-bound Ru(NH_3)_6^(3+), the reverse is observed. The intrinsic excited state lifetime of Ru(phen)_2dppz^(2+) bound to DNA and the luminescence quenching efficiency by Ru(NH_3)_6^(3+) provide indicators of the solvent accessibility of the DNA-bound dppz donor. On this basis, we attribute the difference in ET reactivity among the various DNA polymers to differences in how well M(phen)_2dppz^(2+) stacks in DNA.

A flash-quench method has been employed to probe electron-transfer reactions from peptides to DNA. The photoexcited intercalators [Ru(phen)2(dppz)]2+ (phen = 1,10-phenanthroline; dppz = dipyridophenazine) and [Ru(phen)(bpy‘)(dppz)]2+ (bpy‘ = 4-(4‘-methyl-2,2‘-bipyridyl)valerate) are quenched by a nonintercalating and weakly bound electron-transfer quencher to generate the corresponding DNA-bound Ru(III) complexes in situ. Both Ru(III) complexes are powerful ground-state oxidants, capable of oxidizing guanine in DNA or DNA-bound tryptophan of the intercalating peptide, Lys-Trp-Lys. In mixed-sequence oligonucleotide duplexes containing [Ru(phen)(bpy‘)(dppz)]2+ tethered at one end, damage to distant guanines is observed by gel electrophoresis, consistent with the mobility of the electron through the DNA duplex. This damage at guanines is observed in both the presence and absence of Lys-Trp-Lys, but the presence of the peptide affects the distribution. In flash-quench experiments using mixed-sequence oligonuc...

Metallointercalator-DNA conjugates were prepared by amide bond formation between active esters on the nonintercalating ligands of transition metal complexes and primary amines presented at the 5' or the 3' termini of oligonucleotides attached to solid supports. The conjugates were liberated from the support by aminolysis and purified by HPLC on C18 or C4 stationary phases, which separates the two diastereomeric forms of the conjugates containing either the Lambda or the Delta enantiomer of the octahedral metal complex. The coupling reaction proceeds with approximately 75% conversion of the amino-terminated oligonucleotide into the conjugate; the isolated yield is approximately 200 nmol for syntheses initiated on DNA-synthesis columns with a loading of 2 micromol. The conjugates were characterized by ultraviolet-visible and circular dichorism absorption spectroscopy, electrospray ionization mass spectrometry, enzymatic digestion, and polyacrylamide gel electrophoresis (PAGE). Oligonucleotides bearing [Rh(phi)(2)(bpy')](3+) (phi = 9, 10-phenanthrene quinone diimine; bpy' = 4-butyric acid-4'-methyl bipyridyl) form 1:1 duplexes with the complementary strand, and the electrophoretic mobility under nondenaturating PAGE of duplexes containing Delta-Rh is notably different from duplexes containing Lambda-Rh. High-resolution PAGE of DNA photocleavage reactions initiated by irradiation of the tethered Rh complexes reveal intercalation of the complex only near the tethered end of the duplex. Analogous DNA-binding properties were observed with [Rh(phi)(2)(bpy')](3+) tethered to the 3' terminus. By combining the 3' and 5' modification strategies, a mixed-metal DNA conjugate containing both [Os(phen)(bpy')(Me(2)-dppz)](2+) (Me(2)-dppz = 7, 8-dimethyldipyridophenazine) on the 3' terminus and [Rh(phi)(2)(bpy')](3+) on the 5' terminus was prepared and isolated. Taken together, these strategies for preparing metallointercalator-DNA conjugates offer a useful approach to generate chemical assemblies to probe long-range DNA-mediated charge transfer where the redox initiator is confined to and intercalated in a well-defined binding site.

The design of small complexes that bind and react at specific sequences of DNA becomes important as we begin to delineate, on a molecular level, how genetic information is expressed. A more complete understanding of how to target DNA sites with specificity will lead not only to novel chemotherapeutics but also to a greatly expanded ability for chemists to probe DNA and to develop highly sensitive diagnostic agents.

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 π-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 Å). 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, β. Instead, the involvement of the base pairs controls the time scale and the degree of coherent transport.

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

Unabhangig von der Position im DNA-Film wird kovalent an die DNA angeknupftes Daunomycin (DM) elektrochemisch effizient reduziert – ein Hinweis auf eine nur geringe Abhangigkeit des Elektronentransfers von der Entfernung. Nach Einfuhrung einer C-A-Fehlpaarung geht das elektrochemische Ansprechen drastisch zuruck (schematisch dargestellt). Die DNA-Doppelhelix kann demnach weitreichenden Elektronentransfer erleichtern, aber nur bei Vorliegen eines Ubertragungsweges durch einen geordneten π-Basenstapel.

Our laboratory has proposed redox signaling among DNA repair proteins containing 4Fe-4S clusters through DNA charge transport (DNA CT) as a first step in lesion detection. Recently, we have explored whether UvrC, the endonuclease in Nucleotide Excision Repair (NER), may also contain a 4Fe-4S cluster. A new purification route was developed to express an MBP-UvrC fusion protein, and evidence of a 4Fe-4S cluster was seen by UV-Visible (UV-Vis) and Electron Paramagnetic Resonance (EPR) spectroscopy. Like other 4Fe-4S repair proteins studied in our lab, MBP-UvrC can participate in DNA CT chemistry, as evidenced by its electrochemical activity on DNA-modified gold electrodes. Complementing in vivo genetic assays have been developed and indicate that DNA-mediated signaling between UvrC and other DNA-processing enzymes containing 4Fe-4S clusters is occurring. Taken together, these results have suggested that UvrC is part of a network of 4Fe-4S proteins that communicate using DNA CT to find lesions and maintain genomic integrity. Additional in vitro and in vivo characterization is underway to understand further the biological implications of the newly-discovered, DNA-mediated redox chemistry of UvrC.

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.

Reagents for recognition and efficient cleavage of mismatched DNA without photoactivation were designed. They contain a combination of a mismatch-directing metalloinsertor, [Rh(bpy)_2(chrysi)]^(3+) (bpy = 2,2‘-bipyridyl, chrysi = 5,6-chrysenequinone diimine), and an oxidative cleavage functionality, [Cu(phen)_2]^+ (Cu). Both unconjugated (Rh+Cu) and conjugated (Rh−Cu) frameworks of the Rh insertor and Cu were prepared. Compared to Cu, both constructs Rh+Cu and Rh−Cu exhibit efficient site-specific DNA scission only with mismatched DNA, confirmed by experiments with ^(32)P-labeled oligonucleotides. Furthermore, these studies indicate that DNA cleavage occurs near the mismatch in the minor groove and on both strands. Interestingly, the order of reactivity of the three systems with a CC mismatch is Rh+Cu > Rh−Cu ≫ Cu. Rh binding appears to direct Cu reactivity with or without tethering. These results illustrate advantages and disadvantages in bifunctional conjugation.

Charging along: DNA-mediated charge transport across adenine tracts is monitored by using a probe interior to the bridge (N^6-cyclopropyladenine (^(CP)A), shown in red). This trap was incorporated serially across the bridge and could be oxidized by a distal rhodium photooxidant without significant attenuation in yield over a distance of 5 nm. These results are consistent with complete delocalization of charge across the DNA bridge.

Gold surfaces modified with thiol-derivatized DNA duplexes have been investigated as a function of applied electrochemical potential via atomic force microscopy (EC-AFM). At open circuit, monolayers of well-packed DNA helices form with a film depth of 45(3) A. On the basis of the anisotropic dimensions of these 15 base pair duplexes (20 A in diameter versus 50 A in length), this corresponds to an average ∼45° orientation of the helical axis with respect to the gold surface. Under potential control, the monolayer thickness (and therefore the orientation of the helices) changes dramatically with applied potential. At potentials negative of ∼0.45 V (versus a Ag wire quasi-reference electrode) film thicknesses of ∼55 A are observed, whereas at more positive potentials the monolayer thickness drops to a limiting value of ∼20 A. These results are consistent with a morphology change in which the helices either stand straight up or lie flat down on the metal surface, depending on the electrode potential relative to the potential of zero charge (pzc). This voltage-induced morphology change is reversible and effectively constitutes a nanoscale mechanical “switch”.

The solution structure of the complex of the metallointercalator Δ-α-[Rh[(R,R)-Me2trien]phi]3+ ((R,R)-Me2trien = 2R,9R-diamino-4,7-diazadecane; phi = 9,10-phenanthrenequinone diimine) bound to d(GAGTGCACTC)2 at 4 °C has been determined using 1H NMR spectroscopy coupled with restrained molecular dynamics calculations. Δ-α-[Rh[(R,R)-Me2trien]phi]3+ binds specifically to its designed site, 5‘-TGCA−3‘, with slow exchange kinetics. Using interproton distance restraints (including 70 intermolecular restraints) derived from NOESY spectra in both D2O and H2O and torsional restraints derived from ECOSY data, 20 disparate starting structures converged to a set of final structures with an average RMSD (between structures) of 1.0 A. As shown previously, binding occurs by deep intercalation of the phi ligand at the central 5‘-GC-3‘ step of the decamer, positioning the ancillary Me2trien ligand in the major groove. C2 symmetry is maintained with no kinking or bending of the DNA double helix. The binding site is deforme...

RNA pur•pur-pyr and pyr•pur-pyr (pur = purine, pyr = pyrimidine) triple helices consisting of a Watson−Crick base-paired 28mer hairpin duplex and a Hoogsteen base-paired purine or pyrimidine 12mer are targeted with photoactivated cleavage by the metal complex Rh(phen)_2phi^(3+) (phen = phenanthroline, phi = 9,10-phenanthrenequinone diimine). The metal complex interacts with these triple helices in a structure-specific manner. Different cleavage patterns are seen with the pyr•pur-pyr and pur•pur-pyr motifs. Cleavage is seen on both of the Watson−Crick strands of the former motif and primarily on the purine Watson−Crick strand of the latter motif. Little cleavage is seen on the Hoogsteen strand for either motif. Importantly, the metal complex shows no detectable cleavage on the A-form RNA duplex in the absence of the third Hoogsteen strand. The cleavage patterns are consistent with an intercalated model for the metal complex in the triple helix. Similar cleavage is seen on DNA triple helices, but over a background of duplex cleavage. Targeting of synthetic RNA triple helices, but not duplex regions, by Rh(phen)_2phi^(3+) provides a basis for the chemical probing of triply bonded sites in folded RNA molecules.

New paradigms and new truths in science are not usually adopted because opponents are eventually convinced of their persuasive character, but, more often, because opponents gradually die; the new paradigms are readily adopted by the new generation of students as the laws of nature, just as the dead opponents accepted the old paradigm M. Planck [2]

The DNA double helix, containing a π-stacked array of base pairs in its core, represents a unique and efficient medium for long-range charge transport. DNA assemblies have been constructed containing tethered metallointercalators, and these provide chemically well-defined systems through which to probe the DNA π-stack. Using both spectroscopy and chemical assays of reactivity, we find electron transfer reactions mediated by the DNA base pairs to occur over long molecular distances. The structure of DNA facilitates chemistry at a distance. Importantly, these long-range reactions depend sensitively upon base pair stacking, and hence are modulated by and report on the characteristic stacking within the double helix.

Seit dem ersten Strukturvorschlag fur die DNA-Doppelhelix besteht eine Diskussion daruber, ob die Stapelung der DNA-Basen eine effiziente, weitreichende Ladungsubertragung ermoglicht. Die Konsequenzen einer solchen Radikalstellenwanderung durch die DNA sollten fur das Verstandnis von Carcinogenese und Mutagenese in Betracht gezogen werden. Die DNA zeichnet sich im Innern der Helix durch eine stapelformige Anordnung von heterocyclischen, aromatischen Basenpaaren aus; sie ist ein einmaliges System zur Erforschung des Elektronentransfers. In unserer Arbeitsgruppe wurden Metallkomplexe entwickelt, die zwischen die Basenpaare der DNA-Helix intercalieren und die es ermoglichen, DNA-vermittelten Elektronentransfer zu untersuchen. In diesem Aufsatz beschreiben wir einige von uns durchgefuhrte Elektronentransferreaktionen, die durch den DNA-Basenstapel vermittelt werden. In einigen dient die DNA als Brucke, und mit spektroskopischen Verfahren konnen wir untersuchen, wie der Basenstapel DNA-gebundene Donoren und Acceptoren koppelt. Die Untersuchungen deuten auf eine hohe Empfindlichkeit der Kopplung gegenuber der DNA-Intercalation hin. Wenn der DNA-Basenstapel Donoren und Acceptoren effektiv miteinander verbindet, kann er auch als ausgezeichneter Elektronentransferkanal der DNA selbst dienen, allerdings in Reaktionen, die von entfernten Positionen ausgelost wurden. Wir haben auch viele Versuche zur oxidativen DNA-Schadigung durchgefuhrt, die von einem in groserer Entfernung an der Helix fixierten Oxidationsmittel initiiert wird. Die Ladungsubertragungen hangen sehr von der DNA-Intercalation und von der Integritat des Basenstapels ab, aber nicht von der Entfernung. Auch die als DNA-Schadigung physiologisch bedeutsame Bildung von Thymindimeren kann in einer durch Elektronentransfer initiierten Reaktion ruckgangig gemacht werden. Uber grosere Entfernungen vermittelte chemische Reaktionen werden also durch die DNA-Doppelhelix ermoglicht.

[Ru(phen)2dppz]2+ (phen = 1,10-phenanthroline, dppz = dipyridophenazine) and closely related complexes have previously been observed to have an undetectably small quantum yield of photoluminescence in water but a moderate emission yield when bound to DNA. This so-called “light-switch” effect is a critical factor in the utility of these complexes as spectroscopic probes for DNA. Here we describe a detailed investigation of the photophysics of [Ru(phen)2dppz]2+ in aqueous solution, and in mixtures of acetonitrile and water, by time-resolved absorption and emission spectroscopies. The emission of the complex in water has been measured for the first time. A prompt initial emission, derived from a metal-to-ligand charge-transfer (MLCT) excited state typical for polypyridyl−ruthenium complexes, is observed along with a delayed emission attributed to a novel MLCT species. The small quantum yield of photoluminescence for [Ru(phen)2dppz]2+ in water, and in water/acetonitrile, depends upon efficient formation of a ...

Long-range photoinduced electron transfer has been systematically examined in a series of small DNA duplexes covalently modified with ethidium and Rh(phi)2bpy3+ through time-resolved and steady-state measurements of fluorescence quenching. Fast fluorescence quenching (k ≳ 1010 s-1) is observed for this donor/acceptor pair noncovalently bound to DNA, and transient absorption studies allow the assignment of this quenching to an electron-transfer mechanism. In the duplexes modified with tethered intercalators, intrahelix fluorescence quenching attributed to electron transfer occurs at distances up to 30 A. Over a donor/acceptor separation of ∼20 A, approximately 30% of the ethidium fluorescence is quenched, while at a separation of ∼30 A, approximately 10% quenching is observed. Time-resolved measurements indicate that this quenching is primarily static. Fluorescence polarization and melting studies indicate that the intercalators are rigidly bound, enhance helix stability, and the duplex populations are str...

Resonance Raman (rR) spectroscopy was utilized to gain insight into the electronic structure of dppz (dppz = dipyrido[3,2-a:2‘,3‘-c]phenazine) complexes of Ru(II) and Os(II). The time-resolved resonance Raman (TR3) spectrum of Ru(phen)2(dppz)2+ (phen = 1,10-phenanthroline) collected under 355 nm (fwhm ≈ 10 ns) excitation is consistent with the population of a ligand-centered (LC) 3ππ* state of the dppz ligand. Since emission from the Ru(II) → (dppz) metal-to-ligand charge transfer (MLCT) excited state is observed, both states must possess similar energies. Lowering of the MLCT state energy in Ru(phen)2(F2-dppz)2+ and Os(phen)2(dppz)2+ with respect to that of the 3ππ* state results in the disappearance of the LC 3ππ* excited-state peaks in the rR spectrum, indicative of its fast deactivation to the lower MLCT state in these complexes. Addition of DNA to the samples containing Ru(phen)2(dppz)2+ and Ru(phen)2(F2-dppz)2+ leads to a decrease in intensity of the peaks associated with the intercalating phenazine...

The Tat-binding site of the bovine immunodeficiency virus TAR RNA hairpin has been targeted by Rh(phen)_2phi^(3+) (phen = phenanthroline, phi = 9,10-phenanthrenequinone diimine), a photochemical probe of RNA tertiary structure. The primary site cleaved by the rhodium complex, upon photoactivation, is U24, a base which participates in the novel base triple (with bases A13 and U10) characteristic of this folded RNA. Δ-Rh(phen)_2phi^(3+) binds to this site with an affinity of 2 × 10^6M^(−1). Upon mutation of U24 and A13 to A24 and U13, respectively, so that the RNA oligomer is unable to form the base triple, site-specific cleavage by the rhodium complex is abolished. Moreover, as determined through rhodium photocleavage, at a concentration of 20 μM, Rh(phen)_2phi^(3+) inhibits specific binding of BIV-Tat peptide (2 μM) to its target site. Thus the rhodium complex, in matching its shape to the opened major groove of the properly folded RNA, specifically targets its site and is able to compete for its target with the BIV-Tat peptide.

A flash-quench method has been developed to probe oxidative damage to DNA. A photoexcited Ru(II) intercalator is quenched in DNA by a weakly bound, electron-transfer quencher to generate Ru(III), a powerful ground-state oxidant. Once generated, Δ-Ru(phen)2(dppz)3+ bound to poly(dG-dC) rapidly oxidizes guanine within the DNA duplex. Transient absorption spectroscopy indicates rapid formation of the neutral guanine radical within the DNA duplex. Permanent damage resulting from the flash-quench experiment is monitored by gel electrophoresis of synthetic oligonucleotide duplexes. Oxidative damage, visualized by treatment with piperidine, occurs selectively at the 5‘-G of 5‘-GG-3‘ sites and at the 5‘- and central G of 5‘-GGG-3‘ triplets; enzymatic digestion in the absence of piperidine treatment shows formation of 8-oxo-2‘-deoxyguanosine with Ru(NH3)63+ as quencher. The yield of base damage is, furthermore, modulated by the choice of electron-transfer quencher. Quantum yields for damage vary in the order Ru(NH...

Whether the DNA base pair stack might serve as a medium for efficient, long-range charge transfer has been debated almost since the first proposal of the double-helical structure of DNA. The consequences of long-range radical migration through DNA are important with respect to understanding carcinogenesis and mutagenesis. Double-helical DNA has in its core a stacked array of aromatic heterocyclic base pairs, and this molecular π stack represents a unique system in which to explore the chemistry of electron transfer. We designed a family of metal complexes which bind to DNA by intercalative stacking within the helix; these metallointercalators may be usefully applied in probing DNA-mediated electron transfer. Here we describe a range of electron transfer reactions we carried out which are mediated by the DNA base paired stack. In some cases, DNA serves as a bridge, and spectroscopic analyses permit us to probe how the π stack couples DNA-bound donors and acceptors. These studies point to the sensitivity of coupling to DNA intercalation. However, if the DNA π stack effectively bridges donors and acceptors, the base-pair stack itself might serve not only as a conduit for electron transfer in DNA, but also in reactions initiated from a remote position. We carried out a series of reactions involving oxidative damage to DNA arising from the remotely positioned oxidant on the helix. The implications of long-range charge migration through DNA to effect damage are substantial. As in other DNA-mediated charge transfers, these reactions are highly dependent on DNA intercalation and the integrity of the intervening base-pair stack, but not on molecular distance. Furthermore, a physiologically important DNA lesion, the thymine dimers, can be reversed in a reaction initiated by electron transfer. This repair reaction can also be promoted from a distance as a result of long-range charge migration through the DNA base pair stack.

1H and 31P NMR spectroscopies have been applied in the structural characterization of the enantioselective interactions between Λ- and Δ-[Ru(phen)2dppz]2+ (dppz = dipyridophenazine) and the hexamer oligonucleotide d(GTCGAC)2. Issues of intercalation, exchange rate, sequence specificity, enantioselectivity, and the distribution of binding geometries have been explored. Several forms of evidence support intercalation by both isomers: (i) upfield changes in 1H chemical shift for protons of the dppz ligand; (ii) characteristic downfield changes in 31P chemical shifts for the duplex bound by the metal complex; (iii) increases in duplex melting temperature in the presence of both isomers. Slow exchange is achieved near 0 °C, thus permitting the observation of individual binding events. While both isomers intercalate into the helix, enantioselective differences in intercalation are evident. Differences in intercalative geometries are clearly manifested through chiral shifts in racemic mixtures and distinct reso...

A method of DNA monolayer formation has been developed using copper-free click chemistry that yields enhanced surface homogeneity and enables variation in the amount of DNA assembled; extremely low-density DNA monolayers, with as little as 5% of the monolayer being DNA, have been formed. These DNA-modified electrodes (DMEs) were characterized visually, with AFM, and electrochemically, and were found to facilitate DNA-mediated reduction of a distally bound redox probe. These low-density monolayers were found to be more homogeneous than traditional thiol-modified DNA monolayers, with greater helix accessibility through an increased surface area-to-volume ratio. Protein binding efficiency of the transcriptional activator TATA-binding protein (TBP) was also investigated on these surfaces and compared to that on DNA monolayers formed with standard thiol-modified DNA. Our low-density monolayers were found to be extremely sensitive to TBP binding, with a signal decrease in excess of 75% for 150 nM protein. This protein was detectable at 4 nM, on the order of its dissociation constant, with our low-density monolayers. The improved DNA helix accessibility and sensitivity of our low-density DNA monolayers to TBP binding reflects the general utility of this method of DNA monolayer formation for DNA-based electrochemical sensor development.

We report a novel method of DNA array formation that is electrochemically formed and addressed with a two-electrode platform. Electrochemical activation of a copper catalyst, patterned with one electrode, enables precise placement of multiple sequences of DNA onto a second electrode surface. The two-electrode patterning and detection platform allows for both spatial resolution of the patterned DNA array and optimization of detection through DNA-mediated charge transport with electrocatalysis. This two-electrode platform has been used to form arrays that enable differentiation between well-matched and mismatched sequences, the detection of TATA-binding protein, and sequence-selective DNA hybridization.

The DNA mismatch repair system (MMR) maintains genome stability through recognition and repair of single-base mismatches and small insertion-deletion loops. Inactivation of the MMR pathway causes microsatellite instability and the accumulation of genomic mutations that can cause or contribute to cancer. In fact, 10-20% of certain solid and hematologic cancers are MMR-deficient. MMR-deficient cancers do not respond to some standard of care chemotherapeutics because of presumed increased tolerance of DNA damage, highlighting the need for novel therapeutic drugs. Toward this goal, we generated isogenic cancer cell lines for direct comparison of MMR-proficient and MMR-deficient cells. We engineered NCI-H23 lung adenocarcinoma cells to contain a doxycycline-inducible shRNA designed to suppress the expression of the mismatch repair gene MLH1, and compared single cell subclones that were uninduced (MLH1-proficient) versus induced for the MLH1 shRNA (MLH1-deficient). Here we present the characterization of these MMR-inducible cell lines and validate a novel class of rhodium metalloinsertor compounds that differentially inhibit the proliferation of MMR-deficient cancer cells.

Dps proteins, bacterial mini-ferritins that protect DNA from oxidative stress, are implicated in the survival and virulence of pathogenic bacteria. Here we examine the mechanism of E. coli Dps protection of DNA, specifically whether this DNA-binding protein can utilize DNA charge transport through the base pair π-stack to protect the genome from a distance. An intercalating ruthenium photooxidant was employed to generate DNA damage localized to guanine repeats, the sites of lowest potential in DNA. We find that Dps loaded with ferrous iron, in contrast to Apo-Dps and ferric iron-loaded Dps, significantly attenuates the yield of oxidative DNA damage. These data demonstrate that ferrous iron-loaded Dps is selectively oxidized to fill guanine radical holes, thereby restoring the integrity of the DNA. Luminescence studies indicate no direct interaction between the ruthenium photooxidant and Dps, supporting the DNA-mediated oxidation of ferrous iron-loaded Dps. Thus DNA charge transport may be a mechanism by which Dps efficiently protects the genome of pathogenic bacteria from a distance.

Intraduplex DNA-mediated reduction is established as a general mechanism for the reduction of distally bound stacked redox-active species covalently tethered to DNA through flexible alkane linkages. Methylene Blue (MB), Nile Blue (NB), and Anthraquinone (AQ) were covalently tethered to DNA with three different covalent linkages. Using these reporters DNA electrochemistry was shown to be both DNA-mediated and intra-, rather than inter-, duplex. Significantly, the charge transport pathway occurring through the DNA π-stack is established by using an intervening AC mismatch to break this path. The fact that the DNA-mediated reduction of MB occurs primarily via intraduplex intercalation is established through varying the proximity and integrity of the neighboring duplex DNA.

Here we describe a multiplexed electrochemical characterization of DNA-bound proteins containing [4Fe-4S] clusters. DNA-modified electrodes have become an essential tool for the characterization of the redox chemistry of DNA repair proteins containing redox cofactors, and multiplexing offers a means to probe different complex samples and substrates in parallel to elucidate this chemistry. Multiplexed analysis of endonuclease III (EndoIII), a DNA repair protein containing a [4Fe-4S] cluster known to be accessible via DNA-mediated charge transport, shows subtle differences in the electrochemical behavior as a function of DNA morphology. The peak splitting, signal broadness, sensitivity to π-stack perturbations, and kinetics were all characterized for the DNA-bound reduction of EndoIII on both closely and loosely packed DNA films. DNA-bound EndoIII is seen to have two different electron transfer pathways for reduction, either through the DNA base stack or through direct surface reduction; closely packed DNA films, where the protein has limited surface accessibility, produce electrochemical signals reflecting electron transfer that is DNA-mediated. Multiplexing furthermore permits the comparison of the electrochemistry of EndoIII mutants, including a new family of mutations altering the electrostatics surrounding the [4Fe-4S] cluster. While little change in the midpoint potential was found for this family of mutants, significant variations in the efficiency of DNA-mediated electron transfer were apparent. On the basis of the stability of these proteins, examined by circular dichroism, we propose that the electron transfer pathway can be perturbed not only by the removal of aromatic residues but also through changes in solvation near the cluster.

The discovery of cisplatin as a therapeutic agent stimulated a new era in the application of transition metal complexes for therapeutic design. Here we describe recent results on a variety of transition metal complexes targeted to DNA to illustrate many of the issues involved in new therapeutic design. We describe first structural studies of complexes bound covalently and non-covalently to DNA to identify potential lesions within the cell. We then review the biological fates of these complexes, illustrating the key elements in obtaining potent activity, the importance of uptake and subcellular localization of the complexes, as well as the techniques used to delineate these characteristics. Genomic DNA provides a challenging but valuable target for new transition metal-based therapeutics.

The driving force dependence for the photoinduced electron transfer (ET) from ∗ Ru(phen) 3 2+ (phen = 1.10-phenanthroline) to several cationic M(phen) 3 ″+ complexes (MCo, Ni, Rh, Cr; n = 2, 3) has been measured in water and in the presence of polyanionic microheterogeneous environments. The anionic host utilized in this study are poly(styrenesulfonate) (PSS) and calf-thymus DNA. In water the driving force dependence of the observed rates, plot of log( k q ) versus ΔG (Marcus plot), reaches a plateau corresponding to the rate of diffusion of the reactants. Qualitatively, the Marcus plot is shallower in the case of PSS compared to that measured in DNA and water. This behavior is explained in terms of differences in bimolecular ET kinetics between the DNA and PSS systems.

Ultrafast emission and absorption spectroscopies were used to measure the kinetics of DNA-mediated electron transfer reactions between metal complexes intercalated into DNA. In the presence of rhodium(III) acceptor, a substantial fraction of photoexcited donor exhibits fast oxidative quenching (3 x 10(10) per second). Transient-absorption experiments indicate that, for a series of donors, the majority of back electron transfer is also very fast (approximately 10(10) per second). This rate is independent of the loading of acceptors on the helix, but is sensitive to sequence and pi stacking. The cooperative binding of donor and acceptor is considered unlikely on the basis of structural models and DNA photocleavage studies of binding. These data show that the DNA double helix differs significantly from proteins as a bridge for electron transfer.

The photoinduced electron transfer chemistry between Os(phen)2dppz2+ and Rh(phi)2bpy3+ bound to DNA has been characterized. Os(phen)2dppz2+ serves as an isostructural analogue for Ru(phen)2dppz2+ with a red-shifted emission spectrum, access to a 3+ oxidation state which is stabilized by ∼500 mV relative to the ruthenium complex, and excited-state lifetimes below 10 ns in the presence of DNA. Emission from Δ-Os(phen)2dppz2+ bound to calf thymus DNA is efficiently quenched by Δ-Rh(phi)2bpy3+, and a lower limit for the quenching constant is set at 7 × 109 s-1. The quenching profile over a range of quencher concentrations is found to be remarkably similar to that of the ruthenium analogue, despite an increase of ∼200 mV in ΔG for the photoinduced, forward electron transfer reaction. Such an observation may indicate the importance of the HOMO energy in the donor excited state, which is similar for both donors. Owing to the lack of spectral overlap between Os(phen)2dppz2+ emission and Rh(phi)2bpy3+ absorption, ...

Strategies for the site-specific recognition of DNA are described through the design of a family of octahedral metallointercalating complexes. Coordination complexes, which mimic DNA-binding proteins with regard to their affinity and specificity for DNA sites, may be prepared by the specific functionalization of chiral metal complexes so as to achieve the ensemble of non-covalent contacts in the DNA major groove anchored through intercalation.

The excited state properties and reactivity of Rh(phi) 2 (phen) 3+ (phi = 9,10-phenanthrenequinone diimine, phen = 1,10-phenanthroline) were investigated and compared to those of related rhodium(III) and zinc(II) complexes. At 77 K Rh(phi) 2 (phen) 3+ exhibits LC 3 ππ ∗ dual emission from both phi and phen ligands, whose energies are 2.8 eV and 3.0 eV, respectively, with a biexponential decay that can be fit to 5 ns (98%) and 60 ns (2%) lifetime components. A longer-lived transient is observed by transient absorption both at 77 K and 298 K, which decays monoexponentially with a time constant of 900 ns and 235 ns at each temperature, respectively, in ethanol. The transient can be attained with visible excitation (420 nm to 532 nm) and it is assigned to be intra-ligand charge transfer (ILCT) in nature. It was determined from transient absorption experiments that the ILCT excited state of Rh(phi) 2 (phen) 3+ lies approximately 2.0 eV above the ground state and that it is a powerful oxidizing agent with E 1 2 ([ Rh ] 3+∗/2+ )≈2.0 V versus NHE. The transient absorption experiments with electron donors reveal that the one-electron reduced Rh(phi) 2 (phen) 2+ absorbs at 540 nm with e ≈ 3300 M −1 cm −1 .

Photoinduced electron transfer reactions between photoexcited Ru(phen)2dppz2+ (phen = 1,10-phenanthroline, dppz = dipyridophenazine) and acceptors Rh(phi)2bpy3+ and Rh(phen)2phi3+ (phi = 9,10-phena...

The enantioselective recognition of d(GTGCAC)_2 by Δ- and Λ-Rh(en)_2phi^(3+) (en= ethylenediarnine; phi = 9, 10-phenanthrenequinone diimine) has been examined in a series of one-dimensional (ID) and two-dimensional (2D) 500 MHz ^1H NMR experiments both to extend our understanding of the basis for the enantioselective DNA binding and to gain structural information concerning intercalation by the octahedral metal complexes. Δ-Rh(en)_2phi^(3+) forms a symmetric 1:1 complex with d(GTGCAC)_2, and the metal complex is in slow exchange with the oligodeoxynucleotide bound form at 295 K. The strong upfield shifts of the phi ligand's aromatic protons (0.6-1.3 ppm) are consistent with full intercalation of the phi ligand into the DNA base stack. 2D-NOESY experiments reveal a loss in internucleotide connectivity between G_3 and C_4 bases, while new NOE cross peaks are observed between the phi ligand and the G_3 deoxyribose sugar. In contrast to binding by Δ-Rh(en)_2phi^(3+), the 1:1 Δ-Rh(en)_2phi^(3+)-d(GTGAC)_2 complex shows much broader resonances, and both metal complex and DNA protons appear to be in the intermediate exchange regime. The loss of C_2 symmetry in the 1:1 complex is consistent with binding by Λ-Rh(en)_2phi^(3+) at the T_2G_3 step. Although the enantiomeric metal complexes display different sequence selectivities and exchange characteristics, Λ- and Δ-Rh(en)_2phi^(3+) interact with the oligonucleotide duplex in a fundamentally similar manner, through the full intercalation of the phi ligand. Upfield movements in chemical shifts of phi protons are nearly identical for the two enantiomers, and both Λ- and Δ-Rh(en)_2phi^(3+) stabilize the duplex to melting by 5-10 °C. Given the common binding mode of the two enantiomers, the differences in their binding characteristics emanate from interactions with the ancillary nonintercalating ligands. Thus, as a general strategy, intercalation may provide an anchor for sequence-selective interactions of octahedral metal complexes in the groove of duplex DNA.

The chemical construction of small molecules targeted to DNA depends upon the sequence-dependent structure of the double helix. Here we describe a new structural element to be considered in the sequence-specific recognition of DNA, sequence-dependent DNA twistability. The importance of sequence-dependent DNA twistability is demonstrated in the DNA recognition properties of a novel synthetic rhodium intercalator, lambda-1-Rh(MGP)2phi5+. This metallointercalator, containing pendant guanidinium groups, binds in the major groove of DNA at subnanomolar concentrations to the 6 base pair sequence 5'-CATATG-3' with enantiospecificity. An essential feature of this recognition is the sequence-specific unwinding of the DNA helix, which permits direct contacts between guanidinium functionalities on the metal complex and guanine residues. Through an assay developed to test for sequence-specific DNA unwinding, a 70 +/- 10 degrees unwinding of the sequence 5'-CATATG-3' is established with specific binding by the metal complex. This sequence-dependent twistability may be an essential feature of the recognition of sequences by DNA-binding proteins and may be exploited in future design.

A new family of ruthenium(II) complexes with sterically expansive ligands for targeting DNA defects was prepared, and their luminescent responses to base pair mismatches and/or abasic sites were investigated. The design of the complexes sought to combine the mismatch specificity of sterically expansive metalloinsertors, such as [Rh(bpy)2(chrysi)]3+ (chrysi = chrysene-5,6-quinone diimine), and the light switch behavior of [Ru(bpy)2(dppz)]2+ (dppz = dipyrido[3,2-a:2′,3′-c]phenazine). In one approach, complexes bearing analogues of chrysi incorporating hydrogen-bonding functionality similar to dppz were synthesized. While the complexes show luminescence only at low temperatures (77 K), competition experiments with [Ru(bpy)2(dppz)]2+ at ambient temperatures reveal that the chrysi derivatives preferentially bind DNA mismatches. In another approach, various substituents were introduced onto the dppz ligand to increase its steric bulk for mismatch binding while maintaining planarity. Steady state luminescence and luminescence lifetime measurements reveal that these dppz derivative complexes behave as DNA “light switches,” but that the selectivity in binding and in luminescence with mismatched/abasic versus well-matched DNA is not high. In all cases, the luminescence depends sensitively upon structural perturbations to the dppz ligand.

The structural core of DNA, a continuous stack of aromatic heterocycles, the base pairs, which extends down the helical axis, gives rise to the fascinating electronic properties of this molecule that is so critical for life. Our laboratory and others have developed diverse experimental platforms to investigate the capacity of DNA to conduct charge, termed DNA-mediated charge transport (DNA CT). Here, we present an overview of DNA CT experiments in solution, on surfaces, and with single molecules that collectively provide a broad and consistent perspective on the essential characteristics of this chemistry. DNA CT can proceed over long molecular distances but is remarkably sensitive to perturbations in base pair stacking. We discuss how this foundation, built with data from diverse platforms, can be used both to inform a mechanistic description of DNA CT and to inspire the next platforms for its study: living organisms and molecular electronics.

Electrocatalysis offers a means of electrochemical signal amplification, yet in DNA-based sensors, electrocatalysis has required high-density DNA films and strict assembly and passivation conditions. Here, we describe the use of hemoglobin as a robust and effective electron sink for electrocatalysis in DNA sensing on low-density DNA films. Protein shielding of the heme redox center minimizes direct reduction at the electrode surface and permits assays on low-density DNA films. Electrocatalysis with methylene blue that is covalently tethered to the DNA by a flexible alkyl chain linkage allows for efficient interactions with both the base stack and hemoglobin. Consistent suppression of the redox signal upon incorporation of a single cytosine-adenine (CA) mismatch in the DNA oligomer demonstrates that both the unamplified and the electrocatalytically amplified redox signals are generated through DNA-mediated charge transport. Electrocatalysis with hemoglobin is robust: It is stable to pH and temperature variations. The utility and applicability of electrocatalysis with hemoglobin is demonstrated through restriction enzyme detection, and an enhancement in sensitivity permits femtomole DNA sampling.