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.     

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.     

Direct observation of radical intermediates in protein-dependent DNA charge transport.

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 formed >10(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.     

寡聚酰胺为探针的电化学DNA生物传感器

DNA分子中的碱基对可以长程传递电荷, DNA分子中的碱基π堆积结构为电荷的长程传递提供了良好的通道. 电荷在DNA分子中的传递受碱基序列的影响, 利用这种性质可以构建DNA碱基错配检测的电化学传感器. 寡聚酰胺能和DNA以小沟绑定方式高亲和力地结合, 并且具有序列识别功能, 本文以带有硝基官能团的寡聚酰胺分子为电化学探针, 设计了电化学DNA生物传感器. 结果显示, 寡聚酰胺与DNA修饰电极作用后, 电化学响应显著增强, 并且可以作为检测DNA碱基错配的电化学探针分子.     

The effect of varied ion distributions on long-range DNA charge transport

Long-range oxidative damage to DNA was utilized as a probe to delineate the effects of different ion distributions on DNA charge transport. DNA assemblies were constructed, containing a tethered rhodium intercalating photooxidant, spatially separated from two 5'-GG-3' sites of oxidative damage, with either an A6-tract or a mixed DNA sequence intervening between the guanine doublets; the extent of charge transport was assessed through measurements of the ratio of yields of damage at the guanine doublet distal versus that proximal to the metal binding site. The distal/proximal damage ratios were compared after photooxidation of otherwise identical Rh-tethered assemblies, except for 32P-labeling either at the 5'- or 3'-end; this labeling difference corresponds, in the absence of charge neutralization by condensed counterions, to a shift in negative charge from one end of the duplex to the other. Both with assemblies containing the mixed sequence and the A6-tract, we observed that moving the negative charges to the proximal end of the duplex significantly decreased hole transport to the distal end. We propose that these results reflect variations in the thermodynamic potential at the proximal and distal guanine sites because of the change in charges at the termini of the oligomer. High values for the internal dielectric constant of the stacked base pairs are suggested by these data. Hence, the longitudinal polarizability of DNA may be important to consider in mechanisms for long-range DNA charge transport.     

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

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

Charge Transfer through the DNA Base Stack

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.     

Sequence dependence of charge transport through DNA domains

Here we examine the photooxidation of two kinetically fast electron hole traps, N4-cyclopropylcytosine (CPC) and N2-cyclopropylamine-guanosine (CPG), incorporated in DNA duplexes of various sequence using different photooxidants. DNA oxidation studies are carried out either with noncovalently bound [Ru(phen)(dppz)(bpy')]3+ (dppz = dipyridophenazine) and [Rh(phi)2(bpy)]3+ (phi = phenanthrenequinone diimine) or with anthraquinone tethered to DNA. Because the cyclopropylamine-substituted bases decompose rapidly upon oxidation, their efficiency of decomposition provides a measure of relative hole localization. Consistent with a higher oxidation potential for CPC versus CPG in DNA, CPC decomposes with photooxidation by [Rh(phi)2(bpy)]3+, while CPG undergoes ring-opening both with photoexcited [Rh(phi)2(bpy)]3+ and with [Ru(phen)(dppz)(bpy')]3+. Anthraquinone-modified DNA assemblies of identical base composition but different base sequence are also probed. Single and double base substitutions within adenine tracts modulate CPC decomposition. In fact, the entire sequence within the DNA assembly is seen to govern CPC oxidation, not simply the bases intervening between CPC and the tethered photooxidant. These data are reconciled in the context of a mechanistic model of conformationally gated charge transport through delocalized DNA domains. Photooxidations of anthraquinone-modified DNA assemblies containing both CPC and CPG, but with varied distances separating the modified bases, point to a domain size of at least three bases. Our model for DNA charge transport is distinguished from polaron models. In our model, delocalized domains within the base pair stack form transiently based upon sequence-dependent DNA structure and dynamics. Given these results, DNA charge transport is indeed remarkably sensitive to DNA sequence and structure.     

Two-dimensional LNA/DNA arrays: estimating the helicity of LNA/DNA hybrid duplex

We measured the helical repeats of a non-natural nucleic acid, locked nucleic acid (LNA), by incorporating LNA strands into the outer arms of a DNA double crossover (DX) molecule; atomic force microscopy (AFM) imaging of the two-dimensional (2D) arrays self-assembled from these DX molecules allows us to derive the helical repeat of the LNA/DNA hetero-duplex to be 13.2 +/- 0.9 base pairs per turn.     

Long-range oxidation of guanine by Ru(III) in duplex DNA

Background: Theoretical and experimental studies have demonstrated that 5′-GG-3′ sequences in DNA are ‘hot spots’ for oxidative damage, but few studies have definitively addressed whether oxidative damage to DNA may arise from a distance via long-range charge migration. Towards this end, we have prepared tethered ruthenium (Ru)-oligonucleotide duplexes and used a flash—quench strategy to demonstrate long-range charge transport through the DNA double helix.Results: DNA assemblies containing a tethered Ru(II) intercalator have been synthesized. Ru(III), generated in situ in the presence of externally bound electron-transfer quenchers, promotes base damage selectively at the 5′-G of a 5′-GG-3′ doublet located ∼ 37 Å from the binding site of the oxidant. In the absence of a guanine doublet, oxidative damage occurs equally at all guanine bases in the strand. Oxidative damage is also observed at long range for guanine in a G·A mismatch but not in a G·T mismatch.Conclusions: The present study expands the scope of long-range electron-transfer chemistry in terms of experiments, applications, and possible reactions within the cell. Here we demonstrate oxidative damage to DNA occurring with a high quantum yield over a distance of ∼37 Å using a ground-state oxidant. These results point to the equilibration of the radical across the DNA duplex to the sites of lowest energy. In addition, this charge migration is sensitive to the intervening π-stack formed by DNA base pairs and hence may be useful for the detection of mismatches.     

Oxidative charge transport through DNA in nucleosome core particles

Eukaryotic DNA is packaged into nucleosomes, made up of 146 bp of DNA wrapped around a core of histone proteins. We used photoexcited rhodium intercalators to explore DNA charge transport within these assemblies. Although histone proteins inhibit intercalation of the rhodium complex within the core particle, they do not prevent 5'-GG-3' oxidation, the signature of oxidative charge transport through DNA. Moreover, using rhodium intercalators tethered to the 5' terminus of the DNA, we found that guanine bases within the nucleosome can be oxidized from a distance of 24 bp. Histone binding did not affect the pattern and extent of this oxidation. Therefore, although the structure of the nucleosome core particle generally protects DNA from damage by solution-borne molecules, packaging within the nucleosome does not protect DNA from charge transfer damage through the base pair stack.     

Influence of intervening mismatches on long-range guanine oxidation in DNA duplexes

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 GA > AA > CC 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.     

Long-range DNA charge transport

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

Long-range electron and hole transport through DNA with tethered cyclometalated iridium(III) complexes

A cyclometalated complex of Ir(III) is covalently tethered to DNA oligonucleotides and serves as both a photooxidant and photoreductant in the study of DNA-mediated hole transport (HT) and electron transport (ET). Spectroscopic and melting temperature studies support intercalation of the tethered complex into the DNA duplex through the functionalized dppz ligand. Using these tethered assemblies, ET and HT is initiated in DNA by the same photoredox probe. Cyclopropylamine substituted bases, N4-cyclopropylcytosine (CPC) and N2-cyclopropylguanine (CPG) are used as kinetically fast electron and hole traps to probe the resulting electron migration processes after direct irradiation of the tethered Ir assembly. Oxidation of CPG and CPC is promoted efficiently by HT from photoexcited Ir(III) when the modified bases are positioned in the purine strands of the A-tract. In contrast, when CPC is embedded in a pyrimidine tract, ET to yield reductive decomposition is observed. Thus, the Ir(III)-tethered DNA assembly containing cyclopropyl-modified bases provides a unique model system to explore the two DNA-mediated electron migration processes using the same photoredox probe and the same DNA bridge.     

Watson–Crick Base Pairing Controls Excited‐State Decay in Natural DNA

Excited‐state dynamics are essential to understanding the formation of DNA lesions induced by UV light. By using femtosecond IR spectroscopy, it was possible to determine the lifetimes of the excited states of all four bases in the double‐stranded environment of natural DNA. After UV excitation of the DNA duplex, we detected a concerted decay of base pairs connected by Watson–Crick hydrogen bonds. A comparison of single‐ and double‐stranded DNA showed that the reactive charge‐transfer states formed in the single strands are suppressed by base pairing in the duplex. The strong influence of the Watson–Crick hydrogen bonds indicates that proton transfer opens an efficient decay path in the duplex that prohibits the formation or reduces the lifetime of reactive charge‐transfer states.     

Pseudohexagonal 2D DNA crystals from double crossover cohesion

Two-dimensional pseudohexagonal trigonal arrays have been constructed by self-assembly from DNA. The motif used is a bulged-junction DNA triangle whose edges and extensions are DNA double crossover (DX) molecules, rather than conventional DNA double helices. Experiments were performed to establish whether the success of this system results from the added stiffness of DX molecules or the presence of two sticky ends at the terminus of each edge. Removal of one sticky end precludes lattice formation, suggesting that it is the double sticky end that is the primary factor enabling lattice formation.     

Two dimensional PNA/DNA arrays: estimating the helicity of unusual nucleic acid polymers

We extend the generality of nucleic acid-based structural nanotechnology by incorporating non-natural nucleic acids into a DNA double crossover (DX) molecule; visualizing two-dimensional arrays of these DX molecules by Atomic Force Microscopy (AFM) enables us to measure the helical repeat of any heteroduplex sequence capable of forming the outer arms of a DX.     

DNA electrochemistry through the base pairs not the sugar-phosphate backbone

Using intercalated, covalently bound daunomycin as a redox probe, ground state charge transport in DNA films with a perturbation in base pair stacking was examined in comparison with breaks in the sugar-phosphate backbone. While the introduction of one or even two nicks in the sugar-phosphate backbone yields no detectable effect on electron transfer, a CA mismatch significantly attenuates the electron transfer yield. These results confirm that the base pair stack is the pathway for DNA-mediated charge transfer, not the sugar-phosphate backbone.     

Hole polarons in poly(G)-poly(C) and poly(A)-poly(T) DNA molecules

The polaron might play an important role in the process of charge migration through duplex DNA stack. In the present work, we investigate properties of hole polarons in DNA molecules containing identical base pairs, such as poly(G)-poly(C) and poly(A)-poly(T), An extended tight-binding model (extended Su-Schrieffer-Heeger model), which involves the effect of an electric field in the direction of DNA stack, will be introduced. The transfer integral and electron-phonon coupling parameters in this model are obtained according to ab initio calculation for different base pair dimers. Calculations reveal that the polaron in poly(A)-poly(T) has a wider shape and a higher mobility under a specific electric field than that in poly(G)-poly(C) DNA stack.