Complexation and (templated) synthesis of rhenium complexes with cyclodextrins and cyclodextrin dimers in water

Several small, lipophilic rhenium complexes form inclusion complexes with native beta-cyclodextrin (beta-CD) and beta-CD dimers. Association constants larger than 10(9)M(-1) were obtained using dimers. The use of beta-CD also enabled the synthesis of these rhenium complexes in water, in excellent yields, through complexation of the otherwise insoluble corresponding ligands. The influence of the reaction time and temperature on the configuration of the reaction products has been investigated in depth for one of these complexes. Using a beta-CD dimer, it proved possible to specifically template the formation of one configuration. The strength of the complexes of the rhenium complexes in cyclodextrin dimers may allow radiolabeling of biomolecules.     

Photocontrolled DNA Origami Assembly by Using Two Photoswitches

Stimuli-responsive switching molecules have been widely investigated for the purpose of the mechanical control of biomolecules. Recently developed arylazopyrazole (AAP) shows photoisomerization activity, displaying a faster response to light-induced conformational changes and unique absorption spectral properties compared with those of conventionally used azobenzene. Herein, it is demonstrated that AAP can be used as a photoswitching molecule to control photoinduced assembly and disassembly of DNA origami nanostructures. An AAP-modified DNA origami has been designed and constructed. It is observed that the repeated assembly and disassembly of AAP-modified X-shaped DNA origami and hexagonal origami with complementary strands can be achieved by alternating UV and visible-light irradiation. Closed and linear assemblies of AAP-modified X-shaped origami were successfully formed by photoirradiation, and more than 1 μm linear assemblies were formed. Finally, it is shown that the two photoswitches, AAP and azobenzene, can be used in tandem to independently control different assembly configurations by using different irradiation wavelengths. AAP can extend the variety of available wavelengths of photoswitches and stably result in the assembly and disassembly of various DNA origami nanostructures.     

Regioselectivity control of radiation-induced reaction: electron beam-induced fries rearrangement of sulfonamide within a beta-cyclodextrin inclusion complex

EB (electron beam) irradiation of sulfonamide within a beta-cyclodextrin (beta-CD) inclusion complex in the solid state induced the solvent-free Fries rearrangement, which proceeded at a shorter reaction time with reversed regioselectivity by inclusion into the beta-CD, compared with that of sulfonamide crystals; the beta-CD as a restricted nanospace had a large effect on the reactivity and regioselectivity of the solvent-free EB-Fries rearrangement.     

Atomic force microscopic study of low temperature induced disassembly of RecA-dsDNA filaments

The assembly and disassembly of RecA-DNA nucleoprotein filaments on double-stranded DNA (dsDNA) or single-stranded DNA (ssDNA) are important steps for homologous recombination and DNA repair. The assembly and disassembly of the nucleoprotein filaments are sensitive to the reaction conditions. In this work, we investigated different morphologies of the formed nucleoprotein filaments at low temperature under different solution conditions by atomic force microscopy (AFM). We found that low temperature and long keeping time could induce the incomplete disassembly of the formed nucleoprotein filaments. In addition, when the formed filaments were kept at -20 degrees C for 20 h with 1,4-dithiothreitol (DTT), the integrated filaments disassembled. It was similar to the case under the same condition without anything added. However, when glycerol was used as a substitute for DTT, there was no obvious disassembly at the same condition. Oppositely, when the formed filaments were kept at 4 degrees C for 20 h, the disassembly with additional DTT was not as obvious as the case at -20 degrees C for 20 h, whereas the case with additional glycerol disassembled. The experiments indicated the effect of cold denaturation on the interaction of DNA and RecA. Meanwhile, the study of these phenomena can supply guidelines for the property and stability of RecA as well as the relevant roles of influencing factors to RecA and DNA in further theoretical studies.     

Inclusion kinetics of a nucleotide into a cyclodextrin cavity by means of ultrasonic relaxation

To examine a dynamic interaction between nucleotide and cyclic oligosaccharide, ultrasonic absorption measurements were carried out in aqueous solution containing beta-cyclodextrin (beta-CD) and adenosine 5'-monophosphate (AMP) in the frequency range of 0.8-95 MHz. A relaxational absorption was observed in the solution, although it was not found in the individual solution of beta-CD or AMP. From the concentration dependences of AMP on the relaxation time and the maximum absorption per wavelength, the cause of the relaxation was attributed to a perturbation of a chemical equilibrium associated with a complex formation between beta-CD (host) and AMP (guest). The rate constants for the formation and breakup processes of the complex were determined. Also, a standard volume change of the reaction was obtained. From comparisons of the obtained rate and thermodynamic parameters with those for beta-CD and various guests, it has been concluded that the adenine moiety is included in the beta-CD cavity and that the hydrogen bonds may play a role in the complex formation.     

Exploiting Complex Fluorophore Interactions to Monitor Virus Capsid Disassembly

Supramolecular protein complexes are the corner stone of biological processes; they are essential for many biological functions. Unraveling the interactions responsible for the (dis)assembly of these complexes is required to understand nature and to exploit such systems in future applications. Virus capsids are well-defined assemblies of hundreds of proteins and form the outer shell of non-enveloped viruses. Due to their potential as a drug carriers or nano-reactors and the need for virus inactivation strategies, assessing the intactness of virus capsids is of great interest. Current methods to evaluate the (dis)assembly of these protein assemblies are experimentally demanding in terms of instrumentation, expertise and time. Here we investigate a new strategy to monitor the disassembly of fluorescently labeled virus capsids. To monitor surfactant-induced capsid disassembly, we exploit the complex photophysical interplay between multiple fluorophores conjugated to capsid proteins. The disassembly of the capsid changes the photophysical interactions between the fluorophores, and this can be spectrally monitored. The presented data show that this low complexity method can be used to study and monitor the disassembly of supramolecular protein complexes like virus capsids. However, the range of labeling densities that is suitable for this assay is surprisingly narrow.     

Dynamic study of interaction between beta-cyclodextrin and aspirin by the ultrasonic relaxation method

A single ultrasonic relaxational phenomenon was observed in aqueous solutions containing both beta-cyclodextrin (beta-CD) as host and nonionized or ionized acetylsalicylic acid (aspirin) as guest. The observed relaxation was responsible for a dynamic complexation reaction between beta-CD and aspirin molecules, concomitant with a volume change during the reaction. The kinetic and equilibrium constants for the complexation in the acid (nonionized) form of the aspirin system were derived from the guest concentration dependence of the relaxation frequency. The equilibrium constant for the carboxylate (ionized) form of aspirin was determined from the concentration dependence of a maximum absorption per wavelength, and the rate constants were calculated by using the determined equilibrium constant and the observed relaxation frequencies, which remained nearly almost constant over the concentration range studied. The results showed that the effect of charge on the aspirin molecule was reflected only in the dissociation process from the beta-CD cavity, while no remarkable change was seen in the association process whose rate was diffusion controlled. The results could be explained on the basis of the difference of the hydrophobic moieties in the two guests that were included in the host cavity. The results of the standard volume change for the complexation reaction were closely related to the number of expelled water molecules originally located in the beta-CD cavity and the volume of the aspirin molecule incorporated into the beta-CD cavity.     

Water‐Dependent Optical Activity Inversion of Chiral DNA–Silica Assemblies

Chirality is widely found in nature and is expressed hierarchically in many organic–inorganic hybrid materials. Optical activity (OA) is the most fundamental attribute of these chiral materials. In this study, we found that the OA of impeller‐like chiral DNA–silica assemblies (CDSAs) was inverted with the addition of water. The state of DNA under dry and wet conditions, and the dual chirality of chiral DNA layers and twisted helical arrays of opposite handedness in CDSAs were considered to exert predominant effects on the OAs. The circular dichroism (CD) responses for the dry CDSAs were mostly attributed to the chiral arrangement of DNA layers, whereas the opposite CD responses for the wet CDSAs primarily originated from twisted helical arrays of DNA molecules. The observed CD signals were a super‐position of the two opposing OA responses. The increase in the longitudinal relation of DNA molecules due to the recovery of a double‐helical structure of DNA in the presence of water was considered to be the reason for the increase in intensity of the CD signals that originated from the twisted helical array, which led to the inversion of OA of the CDSAs. The inversion of the plasmon‐resonance‐based OAs for the chiral‐arranged achiral Ag nanoparticles (NPs) located in the channels of the CDSAs in dry and wet states further confirmed the dual chirality of DNA packing. Such research on DNA assemblies and metal NPs with dual, opposite chirality assists in the understanding of DNA hierarchical chirality in living systems and the creation of macroscopic ordered helical materials and biosensors.     

DNA-programmed modular assembly of cyclic and linear nanoarrays for the synthesis of two-dimensional conducting polymers

Nanometer-scale arrays of conducting polymers were prepared on scaffolds of self-assembling DNA modules. A series of DNA oligomers was prepared, each containing six 2,5-bis(2-thienyl)pyrrole (SNS) monomer units linked covalently to N4 atoms of alternating cytosines placed between leading and trailing 12-nucleobase recognition sequences. These DNA modules were encoded so the recognition sequences would uniquely associate through Watson-Crick assembly to form closed-cycle or linear arrays of aligned SNS monomers. The melting behavior and electrophoretic migration of these assemblies showed cooperative formation of multicomponent arrays containing two to five DNA modules (i.e., 12-30 SNS monomers). The treatment of these arrays with horseradish peroxidase and H(2)O(2) resulted in oxidative polymerization of the SNS monomers with concomitant ligation of the DNA modules. The resulting cyclic and linear arrays exhibited chemical and optical properties typical of conducting thiophene-like polymers, with a red-end absorption beyond 1250 nm. AFM images of the cyclic array containing 18 SNS units revealed highly regular 10 nm diameter objects.     

Hybridization chain reaction triggered controllable one-dimensional assembly of gold nanoparticles

Assembling and ordering nanomaterials into desirable patterns are considerably significant, since the properties of nanomaterials depend not only on the size and shape, but also on the spatial arrangement among the collective building blocks. In this work, the DNA self-assembly technology of hybridization chain reaction (HCR) provided a convenient method to yield long double-strand DNA (dsDNA) to install gold nanoparticles (AuNPs) into one dimensional assembly along the skeleton of dsDNA. Interestingly, the tunable length of AuNPs assemblies along dsDNA chain could be achieved by adjusting the reaction time of HCR, which is based on the formation of covalent bond between Au and the -SH group of DNA. Compared with weak light scattering of single AuNP, these AuNPs assemblies could be clearly imaged under the dark field microscopy, indicating that the light scattering was greatly improved after assembling.     

Redox-controlled interaction of biferrocenyl-terminated dendrimers with beta-cyclodextrin molecular printboards

This paper describes the synthesis and electrochemistry of biferrocenyl-terminated dendrimers and their beta-cyclodextrin (beta-CD) inclusion complexes in aqueous solution and at surfaces. Three generations of poly(propylene imine) (PPI) dendrimers, decorated with 4, 8, and 16 biferrocenyl (BFc) units, respectively, were synthesized. A water-soluble BFc derivative forms stable inclusion complexes with beta-CD. The intrinsic binding constant is K(i)=2.5 x 10(4) M(-1). The BFc dendrimers were solubilized in water by complexation of the end groups with beta-CD, resulting in large water-soluble supramolecular assemblies. Cyclic voltammetry (CV) and differential pulse voltammetry (DPV) showed that all the end groups are complexed to beta-CD. Adsorption of the dendrimers at self-assembled monolayers (SAMs) of heptathioether-functionalized beta-CD on gold ("molecular printboards") resulted in stable monolayers of the dendrimers due to the formation of multivalent host-guest interactions between the BFc end groups of the dendrimers and the immobilized beta-CD molecules. The number of interacting end groups is 3, 4, and 4 for dendrimer generations 1, 2, and 3, respectively. The complexation of BFc to beta-CD is sensitive to the oxidation state of the BFc unit. Oxidation of neutral BFc-Fe(2) ((II,II)) to the cationic, mixed-valence biferrocenium BFc-Fe(2) ((II,III)+) resulted in dissociation of the host-guest complexes. Scan-rate-dependent CV and DPV analyses of the dendrimer-beta-CD assemblies immobilized at the beta-CD host surface and in solution revealed that the dendrimers are oxidized in three steps. First, the surface-beta-CD-bound BFc moieties are oxidized to the mixed-valence state, Fe(2) ((II,III)+), followed by the oxidation of the non-surface-interacting BFc groups to the Fe(2) ((II,III)+) state. The third step involves the oxidation of all the BFc moieties to the Fe(2) ((III,III)2+) state.     

Ester hydrolysis and enol nitrosation reactions of ethyl cyclohexanone-2-carboxylate inhibited by beta-cyclodextrin

Both the ester hydrolysis and the nitrosation reactions of the enol tautomer of ethyl cyclohexanone-2-carboxylate (ECHC) are investigated in the absence and presence of beta-cyclodextrin (beta-CD). The ester hydrolysis reaction is studied in dilute H2O and D2O solutions of hydrochloric acid and in aqueous buffered solutions of carboxylic acids (acetic acid and its chloro derivatives). The pseudo-first-order rate constant increases with both the [H+] and the total buffer concentration, indicating that the hydrolysis is subject to acid and general base catalysis. Substantial solvent isotope effects in the normal direction (kH/kD > 1) for the acid-catalyzed hydrolysis was observed. Addition of beta-CD strongly slows the hydrolysis reaction. The variation of the observed rate constant (k(o)) with [beta-CD] exhibits saturation behavior, consistent with 1:1 binding between the enol of ECHC and beta-CD. The binding is quite strong, and bound ECHC-enol is unreactive. The nitrosation reaction of ECHC in aqueous acid medium, using sodium nitrite in great excess over the concentration of ECHC, yields perfect first-order kinetics, indicating that the slow step is the nitrosation of the enol tautomer. This finding suggests that a great percentage of the total ECHC concentration must exist in the enol form. The nitrosation reaction is of first order in [nitrite] and is catalyzed by the presence of Cl-, Br-, or SCN- ions, which indicates that the attack of the nitrosating agent is the slow step. The nitrosation reaction is also strongly inhibited by the presence of beta-CD because of the formation of unreactive inclusion complexes between the host, beta-CD, and the guest, the enol of ECHC. In alkaline medium, the formation of the enolate ion is observed, which absorbs at higher wavelengths (lambda(max) = 256 nm in acid medium shifts to lambda(max) = 288 nm in alkaline medium). This anion also undergoes ester hydrolysis spontaneously, but shows neither specific basic catalysis nor appreciable effect by the presence of beta-CD. From kinetic and spectroscopic measurements the pKa of the enol of ECHC has been determined as 12.35.     

Monitoring dediazoniation product formation by high-performance liquid chromatography after derivatization

A derivatization protocol that exploits the rapid reaction between arenediazonium ions and a suitable coupling agent followed by high-performance liquid chromatography analyses of the reaction mixture was employed to determine the product distribution, the rate constants for product formation and the association constant of 4-nitrobenzenediazonium, PNBD, ion with beta-cyclodextrin, beta-CD. The derivatization of PNBD with the coupling agent leads to the formation of a stable azo dye that prevents by-side reactions of PNBD with the solvents of the mobile phase, including water, or the metallic parts of the chromatographic system that would eventually lead to erroneous identification and quantification of dediazoniation products. The results show that in the presence of beta-CD, nitrobenzene is formed at the expense of 4-nitrophenol, which is the major product in its absence. The observed rate constants for the interaction between PNBD and beta-CD increase upon increasing [beta-CD] showing a saturation profile indicative of the formation of an inclusion complex between PNBD and beta-CD. By fitting the experimental data to a simplified Lineaweaver-Burk equation, the corresponding association constant and the maximum acceleration rate of beta-CD towards PNBD were estimated. The protocol is applicable under a variety of experimental conditions provided that the rate of the coupling reaction is much faster than that of dediazoniation.     

Kinetic study for the inclusion complex of carboxylic acids with cyclodextrin by the ultrasonic relaxation method

Ultrasonic absorption coefficients in the frequency range of 0.8-95 MHz were measured in aqueous solutions containing both beta-cyclodextrin (beta-CD) (host) and butanoic acid (in its dissociated form and undissociated one) (guest). A single relaxational phenomenon was observed only when the solutes were coexisting, although no relaxation was found in the beta-CD solution or in the acid solutions. The absorption was also measured in a solution of pentanoic acid (dissociated form) with beta-CD, and single relaxation was detected. The ultrasonic relaxation observed in these solutions was due to a perturbation of a chemical equilibrium related to a reaction of an inclusion complex formed by the host and guest. The equilibrium constant was obtained from the dependence of the maximum absorption per wavelength on the guest concentration. The rate constant for the inclusion process of the guest into a cavity of beta-CD and that for the leaving process from the cavity were determined from the obtained relaxation frequency and the equilibrium constant. The standard volume change of the reaction was also computed from the maximum absorption per wavelength. These results were compared with those in solutions containing both beta-CD and different guest molecules. It was found that the hydrophobicity of guest molecules played an important role in the formation of the inclusion complex and also that the charge on the carboxylic group had a considerable effect on the kinetic characteristics of the complexation reaction.     

Heterodimerization of dye-modified cyclodextrins with native cyclodextrins

The heterodimerization behavior of dye-modified beta-cyclodextrins (1-6) with native cyclodextrins (CDs) was investigated by means of absorption and induced circular dichroism spectroscopy in an aqueous solution. Three types of azo dye-modified beta-CDs (1-3) show different association behaviors, depending on the positional difference and the electronic character of substituent connected to the CD unit in the dye moiety. p-Methyl red-modified beta-CD (1), which has a 4-(dimethylamino)azobenzene moiety connected to the CD unit at the 4' position by an amido linkage, forms an intramolecular self-complex, inserting the dye moiety in its beta-CD cavity. It also associates with the native alpha-CD by inserting the moiety of 1 into the alpha-CD cavity. The association constants for such heterodimerization are 198 M(-1) at pH 1.00 and 305 M(-1) at pH 6.59, which are larger than the association constant of 1 for beta-CD (43 M(-1) at pH 1.00). Methyl red-modified 2, which has the same dye moiety as that for 1 although its substituent position is different from that of 1, does not associate even with alpha-CD due to the stable self-intramolecular complex, in which the dye moiety is deeply included in its own cavity of beta-CD. Alizarin yellow-modified CD (3), which has an azo dye moiety different from that of 1 and 2, caused a slight spectral variation upon addition of alpha-CD, suggesting that the interaction between 3 and alpha-CD is weak. On the other hand, phenolphthalein-modified beta-CD (4), which forms an intermolecular association complex in its higher concentrations, binds with beta-CD with an association constant of 787 M(-1) at pH 10.80, where 4 exists as the dianion monomer in the absence of beta-CD. p-Nitorophenol-modified beta-CDs (5 and 6), each having p-nitorophenol moieties with a different connecting part with an amido and amidophenyl group, respectively, associated with alpha-CD with association constants of 66 and 16 M(-1) for 5 and 6, respectively. The phenyl unit in the connecting part of 6 may prevent the smooth binding with alpha-CD. All these results suggest that the dye-modified CDs, in which the dye part is not tightly included in its CD cavity, associate with the native CD to form heterodimer composed of two different CD units by inserting the dye moiety into the native CD unit. The resulting heterodimers have a cavity that can bind another appending moiety of host molecules. On this basis, more ordered molecular arrays or the supramolecular hereropolymers can be constructed.     

Inhibition of the beta-cyclodextrin catalyzed dediazoniation of 4-nitrobenzenediazonium tetrafluoroborate. blocking effect of sodium dodecyl sulfate

We have investigated the effects of sodium dodecyl sulfate, SDS, on the reaction between 4-nitrobenzenediazonium, 4NBD, ions and beta-cyclodextrin, beta-CD, under acidic conditions at T = 60 degrees C by employing a combination of spectrophotometric, chromatographic, and conductometric techniques. Previous studies under acidic conditions indicate that the secondary -OH groups of beta-CD solvate 4NBD ions, which are included in the beta-CD cavity, leading to the formation of a highly unstable transient diazo ether complex that undergoes homolytic fragmentation with an observed rate constant about 1700 times higher than that in pure aqueous acid solution (t(1/2) = 6 h at T = 60 degrees C) when [beta-CD]/[4NBD] = 40. Addition of SDS to a 4NBD/beta-CD system makes the k(obs) values decrease up to its value in a SDS micellar solution, which is similar to that in aqueous acid solution. Dediazoniation product distribution is significantly affected; the reaction between 4NBD and beta-CD ([beta-CD]/[4NBD] = 40), in the absence of SDS, proceeds exclusively through a homolytic mechanism leading to the quantitative formation of nitrobenzene, ArH, but addition of SDS turns over the mechanism by promoting the heterolytic mechanism. In addition, mixtures of 4-nitrophenol, ArOH, and ArH dediazoniation products are formed; their relative yields depend on the amount of added SDS so that at very high [SDS(T)], the heterolytic mechanism becomes the predominant one. Results are consistent with conductometric measurements showing that addition of beta-CD to an aqueous surfactant solution inhibits micelle formation and elevates CMC(app) values because CD encapsulation of surfactant monomers competes with the micellization process and are interpreted in terms of SDS monomers blocking the beta-CD cavity by forming a nonreactive complex, releasing 4NBD to the bulk solution.     

DNA charge transport leading to disulfide bond formation

Here, we show that DNA-mediated charge transport (CT) can lead to the oxidation of thiols to form disulfide bonds in DNA. DNA assemblies were prepared possessing anthraquinone (AQ) as a photooxidant spatially separated on the duplex from two SH groups incorporated into the DNA backbone. Upon AQ irradiation, HPLC analysis reveals DNA ligated through a disulfide. The reaction efficiency is seen to vary in assemblies containing intervening DNA mismatches, confirming that the reaction is DNA-mediated. Interestingly, one intervening mismatch near the thiols promotes an increase in efficiency, which we attribute to increased base dynamics. Hence, here, where the reaction is on the backbone rather than within the base stack, stacking perturbations do not necessarily lead to an inhibitory effect on DNA CT.     

Controlling chemical self-assembly by solvent-dependent dynamics

The influence of the ratio between poor and good solvent on the stability and dynamics of supramolecular polymers is studied via a combination of experiments and simulations. Step-wise addition of good solvent to supramolecular polymers assembled via a cooperative (nucleated) growth mechanism results in complete disassembly at a critical good/poor solvent ratio. In contrast, gradual disassembly profiles upon addition of good solvent are observed for isodesmic (non-nucleated) systems. Due to the weak association of good solvent molecules to monomers, the solvent-dependent aggregate stability can be described by a linear free-energy relationship. With respect to dynamics, the depolymerization of π-conjugated oligo(p-phenylene vinylene) (OPV) assemblies in methylcyclohexane (MCH) upon addition of chloroform as a good solvent is shown to proceed with a minimum rate around a critical chloroform/MCH solvent ratio. This minimum disassembly rate bears an intriguing resemblance to phenomena observed in protein unfolding, where minimum rates are observed at the thermodynamic midpoint of a protein denaturation experiment. A kinetic nucleation-elongation model in which the rate constants explicitly depend on the good solvent fraction is developed to rationalize the kinetic traces and further extend the insights by simulation. It is shown that cooperativity, i.e., the nucleation of new aggregates, plays a key role in the minimum polymerization and depolymerization rate at the critical solvent composition. Importantly, this shows that the mixing protocol by which one-dimensional aggregates are prepared via solution-based processing using good/poor solvent mixtures is of major influence on self-assembly dynamics.     

Multivalent host-guest interactions between beta-cyclodextrin self-assembled monolayers and poly(isobutene-alt-maleic acid)s modified with hydrophobic guest moieties

Poly(isobutene-alt-maleic acid)s modified with p-tert-butylphenyl or adamantyl groups interact with beta-cyclodextrin self-assembled monolayers (beta-CD SAMs) by inclusion of the hydrophobic substituents in the beta-cyclodextrin cavities. The adsorption was shown to be strong, specific, and irreversible. Even with a monovalent competitor in solution, adsorption to the beta-CD SAMs was observed, and desorption proved impossible. The adsorbed polymer layer was very thin as evidenced by surface plasmon resonance spectroscopy and AFM. Apparently, all or most hydrophobic groups of the polymers were employed efficiently in multivalent binding, as was further supported by the absence of specific binding of beta-CD-modified gold nanoparticles to the polymer surface assemblies. Supramolecular microcontact printing of the polymers onto the beta-CD SAMs led to assembly formation in the targeted areas of the substrates.     

DNA mediated charge transport: characterization of a DNA radical localized at an artificial nucleic acid base

DNA assemblies containing 4-methylindole incorporated as an artificial base provide a chemically well-defined system in which to explore the oxidative charge transport process in DNA. Using this artificial base, we have combined transient absorption and EPR spectroscopies as well as biochemical methods to test experimentally current mechanisms for DNA charge transport. The 4-methylindole radical cation intermediate has been identified using both EPR and transient absorption spectroscopies in oxidative flash-quench studies using a dipyridophenazine complex of ruthenium as the intercalating oxidant. The 4-methylindole radical cation intermediate is particularly amenable to study given its strong absorptivity at 600 nm and EPR signal measured at 77 K with g = 2.0065. Both transient absorption and EPR spectroscopies show that the 4-methylindole is well incorporated in the duplex; the data also indicate no evidence of guanine radicals, given the low oxidation potential of 4-methylindole relative to the nucleic acid bases. Biochemical studies further support the irreversible oxidation of the indole moiety and allow the determination of yields of irreversible product formation. The construction of these assemblies containing 4-methylindole as an artificial base is also applied in examining long-range charge transport mediated by the DNA base pair stack as a function of intervening distance and sequence. The rate of formation of the indole radical cation is >/=10(7) s(-)(1) for different assemblies with the ruthenium positioned 17-37 A away from the methylindole and with intervening A-T base pairs primarily composing the bridge. In these assemblies, methylindole radical formation at a distance is essentially coincident with quenching of the ruthenium excited state to form the Ru(III) oxidant; charge transport is not rate limiting over this distance regime. The measurements here of rates of radical cation formation establish that a model of G-hopping and AT-tunneling is not sufficient to account for DNA charge transport. Instead, these data are viewed mechanistically as charge transport through the DNA duplex primarily through hopping among well stacked domains of the helix defined by DNA sequence and dynamics.