One-electron oxidation of DNA oligomers that lack guanine: reaction and strand cleavage at remote thymines by long-distance radical cation hopping

The anthraquinone (AQ) photosensitized one-electron oxidation of DNA introduces a radical cation (electron "hole") that migrates through the duplex by hopping. The radical cation normally is trapped irreversibly by reaction at guanine. We constructed AQ-linked DNA oligomers composed exclusively of A/T base pairs. Their irradiation led to reaction and strand cleavage primarily at thymines. Long-distance radical cation hopping to distant thymines was demonstrated by the distance dependence of the process and by experiments with DNA oligomers that contain a single remote GG step. The reaction of the radical cation at thymine was shown to involve its 5-methyl group by the replacement of selected thymines with uracils. These findings show that the reactivity of radical cations in DNA cannot be explained simply by exclusive reliance on the relative oxidation potential of the nucleobases. Instead, the site of reaction is determined in accord with the Curtin-Hammett principle for reactive species in rapid equilibrium.     

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.     

Emergent functionality of nucleobase radical cations in duplex DNA: prediction of reactivity using qualitative potential energy landscapes

The one-electron oxidation of a series of DNA oligonucleotides was examined. Each oligomer contains a covalently linked anthraquinone (AQ) group. Irradiation of the AQ group with near-UV light results in a one-electron oxidation of the DNA that generates a radical cation (electron "hole"). The radical cation migrates through the DNA by a hopping mechanism and is trapped by reaction with water or molecular oxygen, which results in chemical reaction at particular nucleobases. This reaction is revealed as strand cleavage when the irradiated oligonucleotide is treated with piperidine. The specific oligomers examined reveal the existence of three categories of nucleobase sequences: charge shuttles, charge traps, and barriers to charge migration. The characterization of a sequence is not independent of the identity of other sequences in the oligonucleotide, and for this reason, the function of a particular sequence emerges from an analysis of the entire structure. Qualitative potential energy landscapes are introduced as a tool to assist in the rationalization and prediction of the reactions of nucleobases in oxidized DNA.     

Charge-transfer and spin dynamics in DNA hairpin conjugates with perylenediimide as a base-pair surrogate

A perylenediimide chromophore (P) was incorporated into DNA hairpins as a base-pair surrogate to prevent the self-aggregation of P that is typical when it is used as the hairpin linker. The photoinduced charge-transfer and spin dynamics of these hairpins were studied using femtosecond transient absorption spectroscopy and time-resolved EPR spectroscopy (TREPR). P is a photooxidant that is sufficiently powerful to quantitatively inject holes into adjacent adenine (A) and guanine (G) nucleobases. The charge-transfer dynamics observed following hole injection from P into the A-tract of the DNA hairpins is consistent with formation of a polaron involving an estimated 3-4 A bases. Trapping of the (A 3-4) (+*) polaron by a G base at the opposite end of the A-tract from P is competitive with charge recombination of the polaron and P (-*) only at short P-G distances. In a hairpin having 3 A-T base pairs between P and G ( 4G), the radical ion pair that results from trapping of the hole by G is spin-correlated and displays TREPR spectra at 295 and 85 K that are consistent with its formation from (1*)P by the radical-pair intersystem crossing mechanism. Charge recombination is spin-selective and produces (3*)P, which at 85 K exhibits a spin-polarized TREPR spectrum that is diagnostic of its origin from the spin-correlated radical ion pair. Interestingly, in a hairpin having no G bases ( 0G), TREPR spectra at 85 K revealed a spin-correlated radical pair with a dipolar interaction identical to that of 4G, implying that the A-base in the fourth A-T base pair away from the P chromophore serves as a hole trap. Our data suggest that hole injection and transport in these hairpins is completely dominated by polaron generation and movement to a trap site rather than by superexchange. On the other hand, the barrier for charge injection from G (+*) back onto the A-T base pairs is strongly activated, so charge recombination from G (or even A trap sites at 85 K) most likely proceeds by a superexchange mechanism.     

Mechanism for radical cation transport in duplex DNA oligonucleotides

We investigated the photoinduced one-electron oxidation of a series of DNA oligomers having a covalently linked anthraquinone group (AQ) and containing [(A)(n)GG](m) or [(T)(n)GG](m) segments. These oligomers have m GG steps, where m = 4 or 6, separated by (A)(n) or (T)(n) segments, where n = 1-7 for the (A)(n) set and 1-5 for the (T)(n) set. Irradiation with UV light that is absorbed by the AQ causes injection of a radical cation into the DNA. The radical cation migrates through the DNA, causing chemical reaction, primarily at GG steps, that leads to strand cleavage after piperidine treatment. The uniform, systematic structure of the DNA oligonucleotides investigated permits the numerical solution of a kinetic scheme that models these reactions. This analysis yields two rate constants, k(hop), for hopping of the radical cation from one site to adjacent sites, and k(trap), for irreversible reaction of the radical cation with H(2)O or O(2). Analysis of these findings indicates that radical cation hopping in these duplex DNA oligomers is a process that occurs on a microsecond time scale. The value of k(hop) depends on the number of base pairs in the (A)(n) and (T)(n) segments in a systematic way. We interpret these results in terms of a thermally activated adiabatic mechanism for radical cation hopping that we identify as phonon-assisted polaron hopping.     

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.     

Elucidation of the mechanism of redox grafting of diazotated anthraquinone

Redox grafting of aryldiazonium salts containing redox units may be used to form exceptionally thick covalently attached conducting films, even in the micrometers range, in a controlled manner on glassy carbon and gold substrates. With the objective to investigate the mechanism of this process in detail, 1-anthraquinone (AQ) redox units were immobilized on these substrates by electroreduction of 9,10-dioxo-9,10-dihydroanthracene-1-diazonium tetrafluoroborate. Electrochemical quartz crystal microbalance was employed to follow the grafting process during a cyclic voltammetric sweep by recording the frequency change. The redox grafting is shown to have two mass gain regions/phases: an irreversible one due to the addition of AQ units to the substrate/film and a reversible one due to the association of cations from the supporting electrolyte with the AQ radical anions formed during the sweeping process. Scanning electrochemical microscopy was used to study the relationship between the conductivity of the film and the charging level of the AQ redox units in the grafted film. For that purpose, approach curves were recorded at a platinum ultramicroelectrode for AQ-containing films on gold and glassy carbon surfaces using the ferro/ferricyanide redox system as redox probe. It is concluded that the film growth has its origin in electron transfer processes occurring through the layer mediated by the redox moieties embedded in the organic film.     

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.     

Tethered DNA hairpins facilitate electrochemical detection of DNA ligation

A novel electrochemical assay for DNA ligase activity is described. The assay exploits the properties of DNA hairpins tethered at one terminus to a gold electrode and labelled at the other with a ferrocene group for rapid characterisation of DNA status by cyclic voltammetry. Successful ligation of 'nicked' DNA hairpins is indicated by retention of the ferrocene couple when exposure to DNA ligase is followed by conditions that denature the hairpin. The results demonstrate the simplicity of integrating electrochemical detection with hairpin based biosensors and illustrate a new approach to the assay of DNA ligases, of which the NAD(+)-dependent enzymes represent a potential broad spectrum antibacterial drug target.     

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.     

Syntheses of anthraquinone capped hairpin DNAs and electrochemical redox responses from their self-assembled monolayers on gold electrode

An anthraquinone (AQ) based DNA linker and hairpin-forming DNAs linked by the AQ linker with variable A-T base pairs were synthesized for the investigation of electron transfer through double helical DNA (DNA-ET) in self-assembled monolayers (SAMs). The spectroscopic analysis of absorption spectra indicated that the AQ of the hairpin DNA stacked with adjacent A-T base pair. Electrochemical redox response due to the AQ was observed from the hairpin DNA immobilized on gold electrode, thus the hairpin DNA is suitable for the investigation of DNA-ET in SAMs.     

Near-UV induced interstrand cross-links in anthraquinone-DNA duplexes

Anthraquinone (AQ) has been extensively used as a photosensitizer to study charge transfer in DNA. Near-UV photolysis of AQ induces electron abstraction in oligonucleotides leading to AQ radical anions and base radical cations. In general, this reaction is followed by the transport of base radical cations to sites of low oxidation potential, that is, GG, and conversion of G radical cations to DNA breaks. Here, we show that AQ also produces interstrand cross-links in DNA duplexes. About half of the cross-links collapse to single strands in hot piperidine treatment. The structure of stable interstrand cross-links was deduced by MS, NMR, and sequence substitution. The cross-links consist of a covalent link between the methyl group of T on one strand with either C6 or C7 of AQ on the other strand. The formation of interstrand cross-links decreased in O2 compared to deoxygenated solutions. In the presence of O2, the yield of breaks at GG doublets was 10-fold greater than that of cross-links for end tethered AQ, while cross-links exceeded breaks for centrally located AQ. The formation of stable cross-links can be explained by initial charge transfer from T to excited AQ, deprotonation of T radical cations, and condensation of the latter species with AQ radicals. These studies reveal a novel pathway of damage in the photolysis of AQ-DNA duplexes.     

Anthraquinone-sensitized photooxidation of 5-methylcytosine in DNA leading to piperidine-induced efficient strand cleavage

One-electron photooxidations of 5-methyl-2'-deoxycytidine (d(m)C) and 5-trideuteriomethyl-2'-deoxycytidine ([D(3)]d(m)C) by sensitization with anthraquinone (AQ) derivatives were investigated. Photoirradiation of an aerated aqueous solution containing d(m)C and anthraquinone 2-sulfonate (AQS) afforded 5-formyl-2'-deoxycytidine (d(f)C) and 5-hydroxymethyl-2'-deoxycytidine (d(hm)C) in good yield through an initial one-electron oxidation process. The deuterium isotope effect on the AQS-sensitized photooxidation of d(m)C suggests that the rate-determining step in the photosensitized oxidation of d(m)C involves internal transfer of the C5-hydrogen atom of a d(m)C-tetroxide intermediate to produce d(f)C and d(hm)C. In the case of a 5-methylcytosine ((m)C)-containing duplex DNA with an AQ chromophore that is incorporated into the backbone of the DNA strand so as to be immobilized at a specific position, (m)C underwent efficient direct one-electron oxidation by the photoexcited AQ, which resulted in an exclusive DNA strand cleavage at the target (m)C site upon hot piperidine treatment. In accordance with the suppression of the strand cleavage at 5-trideuterio-methylcytosine observed in a similar AQ photosensitization, it is suggested that deprotonation at the C5-methyl group of an intermediate (m)C radical cation may occur as a key elementary reaction in the photooxidative strand cleavage at the (m)C site. Incorporation of an AQ sensitizer into the interior of a strand of the duplex enhanced the one-electron photooxidation of (m)C, presumably because of an increased intersystem crossing efficiency that may lead to efficient piperidine-induced strand cleavage at an (m)C site in a DNA duplex.     

An ultrasensitive fluorescent aptasensor based on truncated aptamer and AGET ATRP for the detection of bisphenol A

Given the gigantic harmfulness of bisphenol A (BPA), a novel and ultrasensitive aptasensor, which employs the truncated BPA aptamer, click chemistry, and activators generated by electron transfer for atom transfer radical polymerization (AGET ATRP), was developed herein for the quantitative determination of BPA. Firstly, hairpin DNAs (hairpins) with a thiol at the 5′ end and an azide group at the 3′ end were conjugated with aminated magnetic beads (MBs) through heterobifunctional cross-linkers. BPA truncated aptamer (ssDNA-A) hybridizes with its complementary single-stranded DNA (ssDNA-B) to form double-stranded DNA. In the presence of BPA, ssDNA-A specifically captures BPA, and then ssDNA-B is released. Subsequently, the ssDNA-B hybridizes with hairpins to expose the azide group near the surface of the MBs. Then, propargyl-2-bromoisobutyrate (PBIB), the initiator of AGET ATRP containing alkynyl group, was conjugated with azide group of hairpins via the Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC). Consequently, a large number of fluorescein-o-acrylate (FA) were introduced to the MBs through AGET ATRP, resulting in that the fluorescence intensity was increased dramatically. Obviously, the fluorescence intensity was especially sensitive to the change of BPA concentration, and this method can be used in quantitative determination of BPA. Under optimal conditions, a broad liner range from 100 fM to 100 nM and a low limit of detection (LOD) of 6.6 fM were obtained. Moreover, the method exhibits not only excellent specificity for BPA detection over BPA analogues but high anti-interference ability in real water sample detection, indicating that it has huge application prospect in food safety and environment monitoring.

     

DNA confined in nanochannels: hairpin tightening by entropic depletion

A theory is presented of the elongation of double-stranded DNA confined in a nanochannel based on a study of the formation of hairpins. A hairpin becomes constrained as it approaches the wall of a channel which leads to an entropic force causing the hairpin to tighten. The DNA in the hairpin remains double-stranded. The free energy of the hairpin is significantly larger than what one would expect if this entropic effect were unimportant. As a result, the distance between hairpins or the global persistence length is often tens of micrometer long and may even reach millimeter sizes for 10 nm thin channels. The hairpin shape and size and the DNA elongation are computed for nanoslits and circular and square nanochannels. A comparison with experiment is given.     

Sensitivity and specificity of metal surface-immobilized "molecular beacon" biosensors

The separate developments of microarray patterning of DNA oligonucleotides, and of DNA hairpins as sensitive probes for oligonucleotide identification in solution, have had a tremendous impact on basic biological research and clinical applications. We have combined these two approaches to develop arrayable and label-free biological sensors based on fluorescence unquenching of DNA hairpins immobilized on metal surfaces. The thermodynamic and kinetic response of these sensors, and the factors important in hybridization efficiency, were investigated. Hybridization efficiency was found to be sensitive to hairpin secondary structure, as well as to the surface distribution of DNA hairpins on the substrate. The identity of the bases used in the hairpin stem as well as the overall loop length significantly affected sensitivity and selectivity. Surface-immobilized hairpins discriminated between two sequences with a single base-pair mismatch with high sensitivity (over an order of magnitude difference in signal) under identical assay conditions (no change in stringency). This represents a significant improvement over other microarray-based techniques.     

Effect of secondary structure on the thermodynamics and kinetics of PNA hybridization to DNA hairpins.

The binding of a series of PNA and DNA probes to a group of unusually stable DNA hairpins of the tetraloop motif has been observed using absorbance hypochromicity (ABS), circular dichroism (CD), and a colorimetric assay for PNA/DNA duplex detection. These results indicate that both stable PNA-DNA and DNA-DNA duplexes can be formed with these target hairpins, even when the melting temperatures for the resulting duplexes are up to 50 degrees C lower than that of the hairpin target. Both hairpin/single-stranded and hairpin/hairpin interactions are considered in the scope of these studies. Secondary structures in both target and probe molecules are shown to depress the melting temperatures and free energies of the probe-target duplexes. Kinetic analysis of hybridization yields reaction rates that are up to 160-fold slower than hybridization between two unstructured strands. The thermodynamic and kinetic obstacles to hybridization imposed by both target and probe secondary structure are significant concerns for the continued development of antisense agents and especially diagnostic probes.     

Selective photocleavage of DNA and RNA by anthraquinone derivatives: targeting the single-strand region of hairpin structures

A tetracationic anthraquinone derivative (27AQS2) binds to hairpin DNA and RNA. Ultraviolet irradiation of the bound quinone causes cleavage in the loop region of both oligonucleotides and at guanines in the stem region of the DNA hairpin. The absence of observable strand cleavage at guanines in the RNA hairpin suggests that either aniline treatment does not cause cleavage at damaged guanines in RNA or that radical cation migration does not occur readily in RNA duplexes. The ability to target the single-stranded regions of DNA and RNA structures is an important property of this photonuclease.     

Folding and unfolding kinetics of DNA hairpins in flowing solution by multiparameter fluorescence correlation spectroscopy

Dynamic equilibrium between the folded and unfolded conformations of single stranded DNA hairpin molecules containing polythymine hairpin loops was investigated using simultaneous two-beam fluorescence cross-correlation spectroscopy and single beam autocorrelation spectroscopy. The hairpins were end-labeled with a fluorescent dye and a quencher, such that folding and unfolding of the DNA hairpin primary structure caused the dye fluorescence to fluctuate on the same characteristic time scale as the folding and unfolding reaction. These fluctuations were observed as the molecules flowed sequentially between two spatially offset, microscopic detection volumes. Cross-correlation analysis of fluorescence from the two detection volumes revealed the translational diffusion and flow properties of the hairpins, as well as the average molecular occupancy of the two volumes. Autocorrelation analysis of the fluorescence from the individual detection volumes revealed the kinetics of hairpin folding and unfolding, with the parameters relating to diffusion, flow, and molecular occupancy constrained to the values determined from the cross-correlation analysis. This allowed unambiguous characterization of the folding and unfolding kinetics, without the need to determine the hydrodynamic properties by analyzing a separate control sample. The analysis revealed nonexponential relaxation kinetics and DNA size-dependent folding times characteristic of dynamic heterogeneity in the DNA hairpin-forming mechanism.     

Selective one-electron oxidation of duplex DNA oligomers: reaction at thymines

The one-electron oxidation of duplex DNA generates a nucleobase radical cation (electron "hole") that migrates long distances by a hopping mechanism. The radical cation reacts irreversibly with H2O or O2 to form oxidation products (damaged bases). In normal DNA (containing the four common DNA bases), reaction occurs most frequently at guanine. However, in DNA duplexes that do not contain guanine (i.e., those comprised exclusively of A/T base pairs), we discovered that reaction occurs primarily at thymine and gives products resulting from oxidation of the T-C5 methyl group and from addition to its C5-C6 double bond. This surprising result shows that it is the relative reactivity, not the stability, of a nucleobase radical cation that determines the nature of the products formed from oxidation of DNA. A mechanism for reaction is proposed whereby a thymine radical cation may either lose a proton from its methyl group or H2O/O2 may add across its double bond. In the latter case, addition may initiate a tandem reaction that converts both thymines of a TT step to oxidation products.