Gamma and ultraviolet radiation cause DNA crosslinking in the presence of metal ions at high pH.

M-DNA is a novel duplex conformation in which metal ions such as Co2+, Ni2+ or Zn2+ replace the imino protons of every base pair. An ethidium fluorescence assay was used to estimate lesions in M-DNA induced by gamma- and UV radiation. General damage to DNA was assessed from the loss of ethidium fluorescence after irradiation of calf thymus DNA. Crosslinks were measured from the return of ethidium fluorescence after a heating and cooling step. Strand breaks were estimated from the loss of fluorescence in covalently closed circular plasmid DNA after a heating and cooling step. For the Co2+ form of M-DNA, gamma-radiation caused the very efficient formation of crosslinks which was not observed with B-DNA nor with the Ni2+ or Zn2+ forms of M-DNA. The crosslinks occurred in both A-T and G-C base pairs but did not form in the presence of a free radical scavenger. Crosslinks induced by UV radiation also formed at a faster rate in the Co2+, Ni2+ and Zn2+ forms of M-DNA compared to B-DNA; crosslinking occurred in all DNA but was more prominent in AT-rich sequences and was not inhibited by a free radical scavenger. Therefore, the presence of certain metal ions may lead to large increases in the formation of radiation-induced crosslinks in DNA.     

Scanning electrochemical microscopy. 51. Studies of self-assembled monolayers of DNA in the absence and presence of metal ions

Scanning electrochemical microscopy was used to examine electron transfer across a self-assembled monolayer of thiol-modified DNA duplexes on a gold electrode. The apparent rate constant for heterogeneous ET from a solution redox probe, Fe(CN)6(3-/4-), to the gold surface through ds-DNA was 4.6 (+/-0.2) x 10(-7) cm/s. With the addition of Zn2+, which resulted in the formation of a metalated DNA (M-DNA) monolayer, the rate constant increased to 5.0 (+/-0.3) x 10(-6) cm/s. Upon treating M-DNA with EDTA, the zinc ions were released from the monolayer and the original rate constant for the DNA duplexes was restored. The enhanced ET rate was also observed at a DNA monolayer treated with Ca2+ or Mg2+, which does not complex by the DNA bases to form M-DNA. The binding of these cations facilitated the monolayer penetration by the probe mediator Fe(CN)6(3-/4-) and accordingly caused an increased redox signal of the mediator at the ds-DNA-modified electrode. Cationic or neutral mediators were not blocked by the ds-DNA monolayer. These results suggest that although the increased electron transport through M-DNA could partially be ascribed to the intrinsic enhancement of electric conductivity of M-DNA, which has been confirmed by photochemical studies, the change in the surface charge of DNA monolayers on the electrode caused by the binding of metal ions to DNA molecules may play a more important role in the enhancement of current with M-DNA.     

Hexachloroiridate(IV) as a redox probe for the electrochemical discrimination of B-DNA and M-DNA monolayers on gold

This paper demonstrates the effectiveness of using the redox couple to investigate DNA monolayers, and compares the potential advantages of this system to the standard redox couple. B-DNA monolayers were converted to M-DNA by incubation in buffer containing 0.4 mM Zn²⁺ at pH 8.6 and studied by cyclic voltammetry (CV), impedance spectroscopy (IS) and chronoamperometry (CA) with . Compared to B-DNA, M-DNA showed significant changes in CV, IS and CA spectra. However, only small changes were observed when the monolayers were incubated in Mg²⁺ at pH 8.6 or in Zn²⁺ at pH 6.0. The heterorgeneous electron-transfer rate (k_ET) between the redox probe and the surface of a bare gold electrode was determined to be 5.7 × 10⁻³ cm/s. For a B-DNA modified electrode, the k_ET through the monolayer was too slow to be measured. However, under M-DNA conditions, a k_ET of 1.5 × 10⁻³ cm/s was reached. As well, the percent change in resistance to charge transfer, measured by IS, was used to illustrate the dependence of M-DNA formation on pH. This result is consistent with Zn²⁺ ions replacing the imino protons on thymine and guanine residues. The redox couple was also effective in differentiating between single-stranded and double-stranded DNA during de-hybridization and rehybridization experiments.     

Structure of the alpha-homo-DNA:RNA duplex and the function of twist and slide to catalogue nucleic acid duplexes

High-resolution NMR studies of an alpha-homo-DNA:RNA duplex reveal the formation of a right-handed parallel-oriented helix. It differs significantly from a standard A- or B-type helix by a small twist value (26.2 degrees ), which leads to a helical pitch of 13.7 base pairs per helical turn, a negative inclination (-1.78 Angstrom) and a large x displacement (5.90 Angstrom). The rise (3.4 Angstrom) is similar to that found in B-DNA. The solution of this new helix structure has stimulated us to develop a mathematical and geometrical model based on slide and twist parameters to describe nucleic acid duplexes. All existing duplexes can be positioned within this landscape, which can be used to understand the helicalization process.     

Electronic structure of xDNA

xDNA is an artificial duplex made of natural and benzo-homologated bases. The latter can be seen as a fusion between benzene and a natural base. We have used two different ab initio techniques, one based on B3LYP and a Gaussian expansion of the wave functions, and the other on GGA and plane-waves, to investigate the electronic properties of an xDNA duplex and a natural one with an analogous sequence. The calculations were performed in dry conditions, i.e., H atoms were used to neutralize the charge. It is found that the HOMO-LUMO gap of xDNA is about 0.5 eV smaller than that of B-DNA, independent of the technique used. The pi-pi* gap of xDNA is 1.3 or 1.0 eV smaller than that of B-DNA, depending on whether one uses B3LYP/6-31G or GGA/plane-waves, respectively. An analysis of how saturation changes the electronic properties of the nucleotide pairs that make up these duplexes suggests that different saturation schemes significantly affect the HOMO-LUMO gap value of xDNA and B-DNA. The same is not true for the pi-pi* gap. That xDNA has a smaller pi-pi* gap than B-DNA suggests that xDNA could be a plausible candidate for molecular-wire applications.     

Probing the B-to-Z-DNA duplex transition using terminally stacking ethynyl pyrene-modified adenosine and uridine bases

Pyrene-modified adenosine and uridine bases located in the dangling positions of G,C-alternating oligodeoxynucleotides undergo pi-stacking in their B-DNA duplexes, but not in their Z-DNA duplexes; fluorescence quenching in the former, through photoinduced electron transfer, but not in the latter, allows the state of the B-to-Z-DNA transition to be characterized visually.     

2-Aminopurine: a probe of structural dynamics and charge transfer in DNA and DNA:RNA hybrids

Spectroscopic techniques are employed to probe relationships between structural dynamics and charge transfer (CT) efficiency in DNA duplexes and DNA:RNA hybrids containing photoexcited 2-aminopurine (Ap). To better understand the variety of interactions and reactions, including CT, between Ap and DNA, the fluorescence behavior of Ap is investigated in a full series of redox-inactive as well as redox-active assemblies. Thus, Ap is developed as a dual reporter of structural dynamics and base-base CT reactions in nucleic acid duplexes. CD, NMR, and thermal denaturation profiles are consistent with the family of DNA duplexes adopting a distinct conformation versus the DNA:RNA hybrids. Fluorescence measurements establish that the d(A)-r(U) tract of the DNA:RNA hybrid exhibits enhanced structural flexibility relative to that of the d(A)-d(T) tract of the DNA duplexes. The yield of CT from either G or 7-deazaguanine (Z) to Ap in the assemblies was determined by comparing Ap emission in redox-active G- or Z-containing duplexes to otherwise identical duplexes in which the G or Z is replaced by inosine (I), the redox-inactive nucleoside analogue. Investigations of CT not only demonstrate efficient intrastrand base-base CT in the DNA:RNA hybrids but also reveal a distance dependence of CT yield that is more shallow through the d(A)-r(U) bridge of the A-form DNA:RNA hybrids than through the d(A)-d(T) bridge of the B-form DNA duplexes. The shallow distance dependence of intrastrand CT in DNA:RNA hybrids correlates with the increased conformational flexibility of bases within the hybrid duplexes. Measurements of interstrand base-base CT provide another means to distinguish between the A- and B-form helices. Significantly, in the A-form DNA:RNA hybrids, a similar distance dependence is obtained for inter- and intrastrand reactions, while, in B-DNA, a more shallow distance dependence is evident with interstrand CT reactions. These observations are consistent with evaluations of intra- and interstrand base overlap in A- versus B-form duplexes. Overall, these data underscore the sensitivity of CT chemistry to nucleic acid structure and structural dynamics.     

B-DNA Helix Stability in a Solvent-Free Environment

B-DNA is the most common DNA helix conformation under physiological conditions. However, when the amount of water in a DNA solution is decreased, B-to-A helix transitions have been observed. To understand what type of helix conformations exist in a solvent-free environment, a series of poly d(CG)(n) and mixed sequence DNA duplexes from 18 to 30 bp were examined with circular dichroism (CD), ESI-MS, ion mobility, and molecular dynamics. From the CD spectra, it was observed that all sequences had B-form helices in solution. However, the solvent-free results were more complex. For the poly d(CG)(n) series, the 18 bp duplex had an A-form helix conformation, both A- and B-helices were present for the 22 bp duplex, and only B-helices were observed for the 26 and 30 bp duplexes. Since these sequences were all present as B-DNA in solution, the observed solvent-free structures illustrate that smaller helices with fewer base pairs convert to A-DNA more easily than larger helices in the absence of solvent. A similar trend was observed for the mixed sequence duplexes where both an A- and B-helix were present for the 18 bp duplex, while only B-helices occur for the larger 22, 26, and 30 bp duplexes. Since the solvent-free B-helices appear at smaller sizes for the mixed sequences than for the pure d(CG)(n) duplexes, the pure d(CG)(n) duplexes have a greater A-philicity.     

Sharp melting transitions in DNA hybrids without aggregate dissolution: proof of neighboring-duplex cooperativity

Similar to DNA-modified gold nanoparticles, comb polymer-DNA hybrids exhibit very sharp melting transitions that can be utilized in highly selective DNA detection systems. Current theories suggest that such sharp melting results from either a phase transition caused by the macroscopic dissolution of the aggregate or neighboring-duplex interactions in the close-packed environment between adjacent DNA duplexes. To delineate the contributions of each of these effects, an aggregate system based on polymer-DNA hybrids was designed to include both polymer-linked and partially untethered duplexes. When this hybridized system was subjected to thermal analysis, both types of duplexes exhibited sharp melting transitions. The very sharp melting transition displayed by the partially untethered DNA duplexes offers proof that neighboring-duplex interactions can indeed induce cooperativity. Contributions of this neighboring-duplex effect, as well as the enhanced stabilization observed in polymer-DNA:polymer-DNA aggregates, can be quantitatively assessed using a simple thermodynamic model. While neighboring-duplex interactions alone can lead to cooperative melting, the enhanced stabilization observed in polymer-DNA aggregates is a function of both neighboring-duplex interactions and multivalent or aggregate properties.     

Oligonucleotides containing 6-aza-2'-deoxyuridine: synthesis, nucleobase protection, pH-dependent duplex stability, and metal-DNA formation

Oligodeoxyribonucleotides containing the nucleoside 6-aza-2'-deoxyuridine z6Ud (1a) were prepared by using solid-phase synthesis. As the pKa value of this nucleoside is 6.8, unwanted side reactions are observed. Consequently, nitrogen-3 was protected (o-anisoyl protection). The phosphoramidite of 1a prepared on this route was as efficient as the building blocks of canonical nucleosides in allowing multiple incorporations into oligonucleotides. Oligonucleotide duplexes containing 1a show a pH dependence of the Tm value. This is caused by nucleobase deprotonation occurring on compound 1a already under neutral conditions. Metal (M)-DNA formation was studied in the presence of Zn+2 ions. It is demonstrated that 6-azauracil-modified duplexes form M-DNA already in neutral medium while alkaline conditions (above pH 8.5) are required for natural DNA. The conformational analysis of the sugar moiety of the nucleoside 1a and its anisoyl derivative 5a shows a preferred N-conformation in solution while an S-conformation for compound 1a was obtained in the solid state (single-crystal X-ray analysis).     

pH‐Dependent Assembly of DNA–Gold Nanoparticles Based on the i‐Motif: A Switchable Device with the Potential of a Nanomachine

The pH‐dependent self‐assembling of gold nanoparticles is described. Oligonucleotides containing four or six consecutive dC residues are immobilized on 15‐nm gold nanoparticles. Their assembly is based on the formation of a DNA i‐motif as determined by the color change from red to blue between pH 5.5 and 6.5. The process occurs within a narrow pH range and is reversible. The i‐motif is formed by the antiparallel intercalation of two parallel duplexes provided by two different gold nanoparticles. This assembly process can be utilized to generate novel systems for colorimetric sensing, applications in medical imaging and therapy, and for the construction of a proton‐driven nanomachine.     

Bicyclo[3.2.1]amide-DNA: a chiral,nonchiroselective base-pairing system

The design, synthesis, and base-pairing properties of bicyclo[3.2.1]amide-DNA (bca-DNA), a novel phosphodiester-based DNA analogue, are reported. This analogue consists of a conformationally constrained backbone entity, which emulates a B-DNA geometry, to which the nucleo-bases were attached through an extended, acyclic amide linker. Homobasic adenine-containing bca decamers form duplexes with complementary oligonucleotides containing bca, DNA, RNA, and, surprisingly, also L-RNA backbones. UV and CD spectroscopic investigations revealed the duplexes with D- or L-complements to be of similar stability and enantiomorphic in structure. Bca oligonucleotides that contain all four bases form strictly antiparallel, left-handed complementary duplexes with themselves and with complementary DNA, but not with RNA. Base-mismatch discrimination is comparable to that of DNA, while the overall thermal stabilities of bca-oligonucleotide duplexes are inferior to those of DNA or RNA. A detailed molecular modeling study of left- and right-handed bca-DNA-containing duplexes showed only minor changes in the backbone structure and revealed a structural switch around the base-linker unit to be responsible for the generation of enantiomorphic duplex structures. The obtained data are discussed with respect to the structural and energetic role of the ribofuranose entities in DNA and RNA association.     

B-DNA Structure and Stability: The Role of Nucleotide Composition and Order

We have quantum chemically analyzed the influence of nucleotide composition and sequence (that is, order) on the stability of double-stranded B-DNA triplets in aqueous solution. To this end, we have investigated the structure and bonding of all 32 possible DNA duplexes with Watson–Crick base pairing, using dispersion-corrected DFT at the BLYP-D3(BJ)/TZ2P level and COSMO for simulating aqueous solvation. We find enhanced stabilities for duplexes possessing a higher GC base pair content. Our activation strain analyses unexpectedly identify the loss of stacking interactions within individual strands as a destabilizing factor in the duplex formation, in addition to the better-known effects of partial desolvation. Furthermore, we show that the sequence-dependent differences in the interaction energy for duplexes of the same overall base pair composition result from the so-called “diagonal interactions” or “cross terms”. Whether cross terms are stabilizing or destabilizing depends on the nature of the electrostatic interaction between polar functional groups in the pertinent nucleobases.     

In situ scanning tunneling microscopy of DNA-modified gold surfaces: bias and mismatch dependence

In situ scanning tunneling microscopy has been performed on DNA-modified gold surfaces under physiological conditions. The STM images of DNA-modified gold surfaces are strongly dependent on the applied potential and percentage of DNA duplexes containing a single base mismatch. At negative surface potentials we observe reproducible features that are attributed to DNA agglomerates where the DNA duplexes are in the upright orientation; at positive potentials, when DNA molecules lie down on the surface, the film is transparent, and only the gold surface is distinguishable. These observations indicate that DNA possesses a non-negligible local density of states which can be probed when the DNA duplex is in the upright orientation. By varying the percentage of DNA duplexes containing a single base mismatch, we have observed a dramatic change in the image contrast as a result of the perturbation induced by the mismatch on the electronic pathway inside the DNA. These results emphasize the central role of the integrity of the pi-stack for DNA charge transport. Duplex DNA is a promising candidate in molecular electronics, but only in arrangements where the orbitals can efficiently overlap with the electronic states of the electrodes and the environment does not constrain the DNA in non-native, poorly stacked conformations.     

Bicyclo-DNA: a Hoogsteen-selective pairing system

Background: The natural nucleic acids (DNA and RNA) can adopt a variety of structures besides the antiparallel double helix described by Watson and Crick, depending on base sequence and solvent conditions. Specifically base-paired DNA structures with regular backbone units include left-handed and parallel duplexes and triple and quadruple helical arrangements. Given the base-pairing pattern of the natural bases, preferences for how single strands associate are determined by the structure and flexibility of the sugar-phosphate backbone. We set out to determine the role of the backbone in complex formation by designing DNA analogs with well defined modifications in backbone structure.Results: We recently developed a DNA analog (bicyclo-DNA) in which one (γ) of the six torsion angles (a-ζ) describing the DNA-backbone conformation is fixed in an orientation that deviates from that observed in B-DNA duplexes by about +100°, a shift from the synclinal to the antiperiplanar range. Upon duplex formation between homopurine and homopyrimidine sequences, this analog preferentially selects the Hoogsteen and reversed Hoogsteen mode, forming A-T and G-C⁺ base pairs. Base-pair formation is highly selective, but degeneracy is observed with respect to strand orientation in the duplex.Conclusions: The flexibility and orientation of the DNA backbone can influence the preferences of the natural bases for base-pairing modes, and can alter the relative stability of duplexes and triplexes.     

Hydration changes accompanying nucleic acid intercalation reactions:volumetric characterizations

We use high precision ultrasonic velocimetric and densimetric techniques to determine at 25 degrees C the changes in volume, deltaV, and adiabatic compressibility, deltaK(S), that accompany the binding of ethidium to the poly(rA)poly(rU), poly(dAdT)poly(dAdT), poly(dGdC)poly(dGdC), and poly(dIdC)poly(dIdC) duplexes, as well as to the poly(rU)poly(rA)poly(rU) triplex. The binding of ethidium to each of the duplexes and the triplex is accompanied by negative changes in volume, deltaV, and adiabatic compressibility, deltaK(S). We discuss the basis for relating macroscopic and microscopic properties, particularly, emphasizing how measured changes in volume and compressibility can be quantitatively interpreted in terms of the differential hydration properties of DNA and RNA structures in their ligand-free and ligand-bound states. We also estimate the entropic cost of intercalation-induced changes in hydration of each of the nucleic acid structures and the drug. In general, our results emphasize the vital role of hydration in modulating the energetics of drug-DNA binding, while also underscoring the fact that hydration must be carefully taken into account in analysis and prediction of the energetics of nucleic acid recognition.     

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.     

Preferentially linear connection of gold nanoparticles in derivatization with phosphorothioate oligonucleotides

A gold nanosphere in water is considered to attain special stability in derivatization like an artificial atom when the octet rule is satisfied by forming four covalent bonds with two 5'-phosphorothioate-modified oligonucleotide molecules. Owing to this, the hybridization of two mutually complementary gold-bound oligonucleotides makes gold nanospheres preferentially connected linearly by duplexes to produce strands like linear artificial molecules. We have then fixated the linear strands of DNA-linked gold nanospheres by reducing Ag(+) ion clusters immobilized around duplexes to show the absorption spectrum of silver-coated artificial-molecular nanorods.     

Detection of non-cross-linking interaction between DNA-modified gold nanoparticles and a DNA-modified flat gold surface using surface plasmon resonance imaging on a microchip

Gold nanoparticles (GNPs) with fully matched DNA duplexes on their surfaces aggregate together without molecular cross-linking at high salt concentrations. The mechanism of this non-cross-linking (NCL) interaction has been elusive. In this paper, NCL interaction between duplex-modified GNPs and a duplex-modified flat gold surface is presented for the first time. This new experimental platform has enabled us to study the NCL interaction between duplexes with different sequences. We immobilized 15-base single-stranded (ss) DNA onto the surfaces of GNPs with a diameter of 40nm and onto a flat gold substrate. The GNPs were hybridized with 15-base ssDNA at a low salt concentration. A microfluidic device was used for simultaneous delivery of the following three components onto the gold substrate: the duplex-modified GNPs, 15-base ssDNA to be hybridized onto the substrate, and NaCl at a high concentration. Adsorption of the GNPs onto the substrate was monitored using surface plasmon resonance imaging. When the GNPs and the substrate had an identical sequence, the adsorption behavior was analogous to the aggregation behavior of GNPs in test tubes. Furthermore, we investigated 12 cases in which the GNPs and the substrate had completely different sequences, and obtained results suggesting that the NCL attraction force primarily depends on the terminal base pairs of the duplexes. This means that the main mechanism of the NCL interaction is likely to be inter-duplex base stacking rather than formation of Holliday junctions.