Photoelectrochemical evaluation of pH effect on hole transport through triplex-forming DNA immobilized on a gold electrode

We characterized pH effect on hole transport through DNA duplexes possessing a partial triplex-forming region. Direct electrochemical measurement of the current response of photosensitizer-tethered DNA immobilized on a gold electrode revealed that the partial triplex formation under acidic conditions suppressed photocurrent due to hole transport, while dissociation of the triplex into the duplex as occurred upon increasing pH values recovered the photocurrent efficiency. Reversible conversion between duplex and triplex induced upon cyclic alternation of pH values resulted in a rise and fall of photocurrent responses, indicating that pH change may feature in the switching function of hole transport in DNA. These electrochemical behaviors could be correlated to the results obtained in long-range photo-oxidative DNA cleavage experiments, in which DNA cleavage at the hole trapping site beyond the triplex region was significantly suppressed under triplex-forming acidic conditions.     

Photocurrent response after enzymatic treatment of DNA duplexes immobilized on gold electrodes: electrochemical discrimination of 5-methylcytosine modification in DNA

We demonstrate a photoelectrochemical approach to the detection of the methylation status of cytosine bases in DNA. We prepared anthraquinone (AQ) photosensitizer-tethered oligodeoxynucleotide (ODN) duplexes bearing 5-methylcytosine (mC) or the corresponding cytosine (C) at a restriction site of the ODN strand immobilized on gold electrodes, and measured their photocurrent responses arising from hole transport after enzymatic digestion. Treatment with HapII or HhaI of the duplexes bearing normal C led to strand cleavage, and the photosensitizer unit was eliminated from the ODN strand immobilized on the gold electrode, exclusively reducing the photocurrent density. With a similar treatment, the duplexes bearing mC showed higher photocurrent responses arising from hole transport through the duplex. This significant difference in the photocurrent response between mC and normal C residues in DNA on the gold electrodes is potentially applicable to the detection of mC modification in DNA.     

Photostimulated hole transport through a DNA duplex immobilized on a gold electrode

Photostimulated hole transport through DNA duplexes immobilized on gold electrodes has been investigated. By modifying a gold electrode with a DNA duplex containing a photosensitizer, we have observed a sequence-dependent cathodic photocurrent. DNA acts as a good mediator for cathodic photocurrent when appropriate sequences are selected.     

Long-distance radical cation migration in duplex DNA: the effect of contiguous A.A and T.T mismatches on efficiency and mechanism

A series of DNA oligomers was prepared. Each oligomer contained an anthraquinone group (AQ, sensitizer) covalently linked at a 5'-end and two GG steps that surrounded a variable region. The variable region was composed of A.T base pairs or A.A or T.T mismatches. Irradiation of the AQ injected a radical cation (hole) into the DNA that migrated through the duplex, being trapped by reaction with H2O of O2 at the GG steps. The effect of substituting A.A or T.T mismatches for Watson-Crick base pairs was examined. For A.A mispairs, charge transfer through the mismatch region was as efficient as through normal DNA. For the T.T mismatches, radical cation transport was strongly distance-dependent. These findings suggest that A.A mismatches form a zipper-like motif, and charge transport proceeds by a hopping mechanism. In contrast, charge transport through the T.T mismatches (where there are no purines) may proceed by quantum mechanical tunneling.     

Guanine of the third strand of C.G*G triplex serves as an effective hole trap

We have examined the structural and electronic effects of the one-electron oxidation of the C.GG triplex, where G is located in a quite different environment from the G of duplex DNA. Upon photoirradiation of an external photosensitizer (riboflavin) with the C.GG triplex, oxidative DNA cleavage occurred exclusively at guanine repeat sequences in the third strand of triple helix DNA. Hole transport through the C.GG triplex also occurred, resulting in selective cleavage at G in the third strand. Thus, the hole generated in the duplex can migrate to GGG in the third strand and is trapped exclusively at Gs in the third strand. These experimental results, together with molecular orbital calculations, suggest that the origin of the selective strand cleavage can be explained as follows: (i) guanine repeat sequences in the third strand are more easily oxidized than in duplex DNA and (ii) in their radical cation states, G of the third strand rapidly deprotonates and reacts with oxygen and/or water, leading to strand cleavage. These results indicate that the oxidative damage preferentially occurred at Gs of the third strand owing to thermodynamic and kinetic features of the one-electron oxidation of the C.GG triplex.     

DNA logic gates

A conceptually new logic gate based on DNA has been devised. Methoxybenzodeazaadenine ((MD)A), an artificial nucleobase which we recently developed for efficient hole transport through DNA, formed stable base pairs with T and C. However, a reasonable hole-transport efficiency was observed in the reaction for the duplex containing an (MD)A/T base pair, whereas the hole transport was strongly suppressed in the reaction using a duplex where the base opposite (MD)A was replaced by C. The influence of complementary pyrimidines on the efficiency of hole transport through (MD)A was quite contrary to the selectivity observed for hole transport through G. The orthogonality of the modulation of these hole-transport properties by complementary pyrimidine bases is promising for the design of a new molecular logic gate. The logic gate system was executed by hole transport through short DNA duplexes, which consisted of the "logic gate strand", containing hole-transporting nucleobases, and the "input strand", containing pyrimidines which modulate the hole-transport efficiency of logic bases. A logic gate strand containing multiple (MD)A bases in series provided the basis for a sharp AND logic action. On the other hand, for OR logic and combinational logic, conversion of Boolean expressions to standard sum-of-product (SOP) expressions was indispensable. Three logic gate strands were designed for OR logic according to each product term in the standard SOP expression of OR logic. The hole-transport efficiency observed for the mixed sample of logic gate strands exhibited an OR logic behavior. This approach is generally applicable to the design of other complicated combinational logic circuits such as the full-adder.     

Base sequence effects in radical cation migration in duplex DNA: support for the polaron-like hopping model

A series of anthraquinone-linked (AQ) duplex DNA oligomers were prepared and investigated. Irradiation of the AQ injects a radical cation into the DNA. The radical cation migrates through the DNA and reacts selectively at GG steps, which leads to strand cleavage after treatment with piperidine. The oligomers investigated in this work were selected to assess the effect on long-distance charge transport of placing a T base (or bases) in a strand of repeating purine bases. With notable exceptions, the amount of strand scission decreases with the distance between the AQ and the GG step. The results are consistent only with models for long-distance transport, such as thermally activated polaron-like hopping, that incorporate radical cation delocalization over two or more adjacent bases.     

Long-range oxidative damage to DNA: protection of guanines by a nonspecifically bound disulfide

One-electron oxidation of DNA generates a base radical cation ("hole") that migrates through the duplex and causes damage at guanines. Unrepaired damage may lead to mutations. It has been suggested that "sacrificial guanines" in intron regions of DNA might serve to protect genes from damage. We have investigated the ability of a noncovalently bound sacrificial reagent to protect DNA from damage. Irradiation of an anthraquinone (AQ)-linked DNA duplex injects a radical cation into the DNA that causes reactions at GG steps close to and farther from the AQ. Bis[2-(3-(aminopropyl)amino)ethyl]disulfide, an analogue of spermine, binds to duplex DNA. Irradiation of the AQ-linked DNA in the presence of this disulfide suppresses the reaction at both GG steps and protects the DNA from damage. It is suggested that evolutionary pressure for the preservation of genomic integrity would yield disulfide-containing compounds optimized to bind to DNA and neutralize base radical cations.     

Ligand binding mode to duplex and triplex dna assessed by combining electrospray tandem mass spectrometry and molecular modeling

In this paper, we report the analysis of seven benzopyridoindole and benzopyridoquinoxaline drugs binding to different duplex DNA and triple helical DNA, using an approach combining electrospray ionization mass spectrometry (ESI-MS), tandem mass spectrometry (MS/MS), and molecular modeling. The ligands were ranked according to the collision energy (CE(50)) necessary to dissociate 50% of the complex with the duplex or the triplex in tandem MS. To determine the probable ligand binding site and binding mode, molecular modeling was used to calculate relative ligand binding energies in different binding sites and binding modes. For duplex DNA binding, the ligand-DNA interaction energies are roughly correlated with the experimental CE(50), with the two benzopyridoindole ligands more tightly bound than the benzopyridoquinoxaline ligands. There is, however, no marked AT versus GC base preference in binding, as supported both by the ESI-MS and the calculated ligand binding energies. Product ion spectra of the complexes with triplex DNA show only loss of neutral ligand for the benzopyridoquinoxalines, and loss of the third strand for the benzopyridoindoles, the ligand remaining on the duplex part. This indicates a higher binding energy of the benzopyridoindoles, and also shows that the ligands interact with the triplex via the duplex. The ranking of the ligand interaction energies compared with the CE(50) values obtained by MS/MS on the complexes with the triplex clearly indicates that the ligands intercalate via the minor groove of the Watson-Crick duplex. Regarding triplex versus duplex selectivity, our experiments have demonstrated that the most selective drugs for triplex share the same heteroaromatic core.     

Unmediated by DNA electron transfer in redox-labeled DNA duplexes end-tethered to gold electrodes

Electron transfer (ET) between gold electrodes and redox-labeled DNA duplexes, immobilized onto the electrodes through the alkanethiol linker at the 3'-end and having internal either methylene blue (MB) or anthraquinone (AQ) redox labels, was shown to depend on the redox label charge and the way the redox label is linked to DNA. For loosely packed DNA monolayers, the conjugation of the positively charged MB to DNA through the long and flexible alkane linker provided ET whose kinetics was formally governed by the diffusion of the redox label to the negatively charged electrode surface. For the uncharged AQ label no ET signal was detected. The conjugation of AQ to DNA through the short and more conductive acetylene linker did not provide the anticipated DNA-mediated ET to the AQ-moiety: ET appeared to be low-efficient if any in the studied system, for which no intercalation of AQ within the DNA duplex occurred. The ET communication between the electrode and AQ, built in DNA through the acetylene linker, was achieved only when Ru(NH(3))(6)(3+) molecules were electrostatically attached to the DNA duplex, thus forming the electronic wire. These results are of particular importance both for the fundamental understanding of the interfacial behavior of the redox labeled DNA on electrodes and for the design of biosensors exploiting a variation of ET properties of DNA in the course of hybridization.     

DNA hole transport on an electrode: application to effective photoelectrochemical SNP typing

A useful feature of DNA is that long-range hole transport through DNA is readily achieved. Photostimulated long-range hole transport through DNA has prospective use in the development of a conceptually new electrochemical single-nucleotide polymorphism (SNP) typing method for use as a versatile platform for gene diagnostics and pharmacogenetics. We have applied artificial DNAs designed for photostimulated long-range hole transport through DNA to SNP typing. By hybridizing photosensitizer-equipped DNA probes, immobilized on gold working electrodes, with a target DNA strand containing an SNP site, we observed a cathodic photocurrent, which markedly changed depending on the nature of the base at the specific site. The use of a combination of hole-transporting bases constitutes a very powerful method for a single-step electrochemical assay applicable to SNP typing of all types of sequences.     

Effects of the photooxidant on DNA-mediated charge transport

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

Charge carrier transport properties in polymer liquid crystals containing oxadiazole and amine moieties in the same side chain

Steady-state and transient photocurrent measurements were carried out to study the charge carrier transport properties of polymer liquid crystal (LC) containing oxadiazole (OXD) and amine moieties in the same side chain. The steady-state photocurrent measurement with asymmetric electrodes of ITO and Al and a short penetration depth of the illumination light indicated that both electrons and holes can be transported in this film. The transient hole photocurrent observed by time-of-flight (TOF) experiments was dispersive at room temperature. The hole drift mobility significantly depended on temperature and electric field and was determined to be 6.1 x 10(-8) cm2/Vs at a field of 9.1 x 10(5) V/cm. According to the disorder formalism, the Gaussian width of the density of states was determined to be 170 meV for holes. Despite the indication of possible electron transport in this film, we could not determine the electron mobility by TOF experiments due to strong dispersive photocurrent. We discuss the present charge transport properties of the film in relation to a large dipole attributed to an electrical push-pull structure of p-dimethylaminophenyl-substitited OXD moiety in polymer LC and its electroluminescent properties.     

A gold nanoparticle based approach for screening triplex DNA binders

Nanoparticle assemblies interconnected with DNA triple helixes can be used to colorimetrically screen for triplex DNA binding molecules and simultaneously determine their relative binding affinities based on melting temperatures. Nanoparticles assemble only when DNA triple helixes form between DNA from two different particles and a third strand of free DNA. In addition, the triple helix structure is unstable at room temperature and only forms in the presence of triplex DNA binding molecules which stabilize the triple helix. The resulting melting transition of the nanoparticle assembly is much sharper and at a significantly higher Tm than the analogous triplex structure without nanoparticles. Upon nanoparticle assembly, a concomitant red-to-blue color change occurs. The assembly process and color change do not occur in the presence of duplex DNA binders and therefore provide a significantly better screening process for triplex DNA binding molecules compared to standard methods.     

long-range charge transport in duplex DNA: anthraquinone sensitization results are independent of terminal ionic distribution

Irradiation of an anthraquinone (AQ) derivative linked to a 5'-terminus of duplex DNA results in the formation of a base radical cation ("hole") that can migrate through the DNA. Reaction of the radical cation occurs primarily at the 5'-G of GG sequences. This reaction results in the formation of strand breaks when the irradiated DNA is treated with piperidine. The strand breaks are detected by polyacrylamide gel electrophoresis of samples that are labeled at the 3'- or 5'-terminus with (32)P. In contrast to a previous report in which a linked rhodium metallointercalator is used as the sensitizer to oxidize the DNA (Williams, T. T.; Barton, J. K. J. Am. Chem. Soc. 2002, 124, 1840-1841), we find that the position of the label does not affect the relative reactivity of the GG steps when AQ is the sensitizer.     

Rupture force between the third strand and the double strand within a triplex DNA

The rupture force to separate the third strand and the duplex within a triplex DNA was measured by means of atomic force spectroscopy. The tip and the sample surfaces were functionalized by oligodeoxyribonucleotides 5'-TTCTTCTTTCTTTTCCTTTTCTTTCTTCTTACTTCTCTCTCTC TCTCTCT-SH-3'. The sample surface was hybridized with 5'-AAGAAGAAAGAAAAGGAAAAGAAAGAAGAA-3' to form a double strand DNA on the surface prior to the force measurements. These sequences form triple helices with 30 base pairs under a pH of 5.8 and in the presence of 2.0 mM spermine. Signals of rupture of single and multiple triplex DNA were observed in the force distance curves. Rupture force histograms revealed a force of 42.6 +/- 1.9 pN from 24 independent measurements at a tip velocity of 400 nm/s to separate the third strand from duplex DNA. The velocity dependence of the rupture force quantum indicates a thermal dissociation process similar to that of rupturing a ds-DNA. The number of rupture events was controlled by adding oligonucleotides 5'-AAGAAGAAAGAAAAGGAAAAGAAAGAAGAA-3' either to reduce or to initiate triplex formation.     

Photoregulation of DNA triplex formation by azobenzene

Formation and dissociation of DNA triplex are reversibly photoregulated by cis <--> trans isomerization of the azobenzene tethered to the third strand. When the azobenzene takes the trans from, a stable triplex is formed. Upon the isomerization of trans-azobenzene to its cis form by UV light irradiation (300 < lambda < 400 nm), however, the modified oligonucleotide is removed from the target duplex. The triplex is re-formed on photoinduced cis --> trans isomerization (lambda > 400 nm). The photoregulating activity significantly depends on the position of azobenzene in the third strand, as well as on the geometric position (meta or para) of its amido substituent. For m-amidoazobenzene, the photoregulation is the most effective when it is tethered to the 5'-end of the third strand. However, p-amidoazobenzene should be introduced into the middle of the strand for effective regulation. In the optimal cases, the change of T(m) of the triplex, caused by the cis <--> trans isomerization of azobenzene, is greater than 30 degrees C. UV-visible and CD spectroscopy, as well as computer modeling studies, clearly demonstrate that the trans-azobenzene intercalates between the base pairs in the target duplex and thus stabilizes the triplex by stacking interactions. On the other hand, nonplanar cis-azobenzene destabilizes the triplex due to its steric hindrance against the adjacent base pairs.     

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.     

Fluorescently Sensing of DNA Triplex Assembly Using an Isoquinoline Alkaloid as Selector,Stabilizer, Inducer,and Switch‐On Emitter

DNA triplex assembly has attracted a variety of interest in the regulation of genetic expression, drug screening, molecular switches, and sensors. However, these achievements are essentially dependent on the formation and stability of the triplex assembly. Herein, the recognition of DNA triplex assembly with various isoquinoline alkaloids was investigated. We found that natural chelerythrine (CHE) exhibits the highest selectivity in recognizing the triplex structure. The DNA triplex stability is substantially increased upon CHE binding, as opposed to the invariance in the stability of the duplex counterpart. CHE also favors the assembly of the triplex‐forming oligonucleotide (TFO) with its duplex counterpart. The triplex binding switches CHE to a strong fluorescent emitter, which suggests CHE as a useful probe in following triplex assembly. As a unique triplex selector, inducer, and emitter, CHE successfully reports the wide pH‐ and metal‐ion‐dependent tunability of the triplex nanoswitch in a label‐free manner.     

Rational design of a DNA wire possessing an extremely high hole transport ability

DNA is a promising conductive biopolymer. However, there are problems that need to be solved to realize real DNA wires. These include the low efficiency of hole transport and the serious oxidative damage that can occur during hole transport. We have demonstrated a protocol for the design of a DNA wire that can effectively mediate hole transport that is not adversely affected by oxidation during hole transport through the DNA duplex. We have synthesized a stable and effective DNA wire by incorporating a designer nucleobase, benzodeazaadenine derivatives, which have lower oxidation potentials and wider stacking areas but are not decomposed during hole transport.