On the stability of single- and double-stranded DNA helices: The application of the PPT-MCF method on large fragments of DNA

The pseudo-polarization tensor mutually consistent field (PPT -MCF ) method recently introduced [1] has been applied to study the stacking interactions between the nucleotide bases in large periodic B-DNA fragments. The effects on the global and local binding properties caused by replacing one base in the periodic sequence by another base are investigated. The increase in the stability for comparable fragments owing to this base substitution is further enforced in the case of periodic alternating helices. The most important results are that the stacking interaction between two bases is slowly converging with the interbase distance and that the average contribution per base to the binding energy is repulsive. Furthermore, the energetical properties of double helix models in B- and Z-DNA configurations, respectively, consisting of up to five base pairs have been compared. It turns out that the G C G C sequence in Z-DNA is significantly more stable than either in periodic or periodic alternating B-DNA. In these cases the average energy contribution of a single Watson–Crick-type base pair is predicted also to be positive. From the calculations it follows that the double helix is not stabilized owing to the hydrogen bonding between the bases belonging to both strands, in contradiction to most other investigations.     

A theoretical study on the hydration of B- and Z-DNA double helices

A theoretical study on the hydration of B- and Z-DNA double helices has been carried out using empirical potential energy functions. The interaction energy between water and the model compounds has been computed considering only the first hydration shell.The results show the number of binding water molecules to be thirty-six and twenty-five in B- and in Z-DNA, respectively. The water molecules in the first hydration shell of B-DNA are very well ordered along the phosphate groups of the backbone whereas those of Z-DNA are more disordered than in B-DNA and are more strongly bound. The water molecules near the first hydration shell of Z-DNA are thought to move more freely than those of B-DNA.     

Structure of a copper-mediated base pair in DNA.

Stable and selective DNA base pairing by metal coordination was recently demonstrated with nucleotides containing complementary pyridine-2,6-dicarboxylate (Dipic) and pyridine (Py) bases (Meggers, E.; Holland, P. L.; Tolman; W. B.; Romesberg, F. E.; Schultz, P. G. J. Am. Chem. Soc. 2000, 122, 10714-10715). To understand the structural consequences of introducing this novel base pair into DNA we have solved the crystal structure of a duplex containing the metallo-base pair. The structure shows that the bases pair as designed, but in a Z-DNA conformation. The structure also provides a structural explanation for the B- to Z-DNA transition in this duplex. Further solution studies demonstrate that the metallo-base pair is compatible with Z- or B-DNA conformations, depending on the duplex sequence.     

(P)-helicene displays chiral selection in binding to Z-DNA

Enantiomeric helicenes of (P)-A and (M)-A were synthesized. The binding of the helicenes to B- and Z-DNA was studied quantitatively by CD, equilibrium dialysis, and fluorescence spectroscopy. Enantiomeric (P)-A not only bound selectively to Z-DNA but also effectively converted the B-DNA conformation to Z-DNA. The enantioselectivity of the helicenes offers a new route for the rational design of inhibitors of biological functions that may depend on Z-DNA.     

Fragmentation of electrospray-produced oligodeoxynucleotide ions adducted to metal ions

In this article, we describe the unique fragmentations of oligodeoxynucleotides (ODNs) whose phosphate groups are completely depleted of protons and replaced with metal ions. The production of the ubiquitous [a(n) - base] ions still occurs, but no longer by transfer of an acidic phosphate proton to an adjoining 3' base. Nor is the extent of the reaction determined by the proton affinity of that base. Rather, the reaction now occurs via a cleavage 3' to both pyrimidines and purines; cleavage 3' to pyrimidine is more favorable than that 3' to purine. We also demonstrate that an ODN is more stable in the gas phase when its phosphate groups are bound to metal ions than when its phosphate groups are attached to hydrogens. This study also provides further evidence for the ODN fragmentation mechanism that involves H transfer to a nucleobase. To establish the structural utility of this new fragmentation, we applied it to distinguishing small ODNs containing a photomodified cis,syn-cyclobutane pyrimidine dimer from the parent ODNs, a system that cannot be distinguished by collisional activation of precursor species that do not contain metal ions.     

Sequence-specific B-DNA flexibility modulates Z-DNA formation

Conversion of right-handed B-DNA into left-handed Z-DNA is one of the largest structural transitions in biology that plays fundamental roles in gene expression and regulation. Z-DNA segments must form within genomes surrounded by a sea of B-DNA and require creation of energetically costly B/Z junctions. Here, we show using a combination of natural abundance NMR R(1ρ) carbon relaxation measurements and CD spectroscopy that sequence-specific B-DNA flexibility modulates the thermodynamic propensity to form Z-DNA and the location of B/Z junctions. We observe sequence-specific flexibility in B-DNA spanning fast (ps-ns) and slow (μs-ms) time scales localized at the site of B/Z junction formation. Further, our studies show that CG-repeats play an active role tuning this intrinsic B-DNA flexibility. Taken together, our results suggest that sequence-specific B-DNA flexibility may provide a mechanism for defining the length and location of Z-DNA in genomes.     

Probing the microenvironments in the grooves of Z-DNA using dan-modified oligonucleotides

The environment-sensitive fluorophore dan (6-dimethylamino-2-acyl-naphthalene)- modified dC or dG bases were introduced into the Z-DNA forming sequence. It was demonstrated that both grooves of Z-DNA are more hydrated than those of B-DNA. Dan will be useful for probing the microenvironments in the grooves among the DNA polymorphs.     

Synthesis,structure, and photochemistry of exceptionally stable synthetic DNA hairpins with stilbene diether linkers

The structure and properties of 18 hairpin-forming bis(oligonucleotide) conjugates possessing stilbene diether linkers are reported. Conjugates possessing bis(2-hydroxyethyl)stilbene 4,4'-diether linkers form the most stable DNA hairpins reported to date. Hairpins with as few as two T:A base pairs or four noncanonical G:G base pairs are stable at room temperature. Increasing the length of the hydroxyalkyl groups results in a decrease in hairpin thermal stability. On the basis of the investigation of their circular dichroism spectra, all of the hairpins investigated adopt B-DNA structures, except for a hairpin with a short poly(G:C) stem which forms a Z-DNA structure. Both the strong fluorescence of the stilbene diether linkers and their trans-cis photoisomerization are totally quenched in hairpins possessing neighboring T:A and G:C base pairs. Quenching is attributed to an electron-transfer mechanism in which the singlet stilbene serves as an electron donor and T or C serves as an electron acceptor. In contrast, in denatured hairpins and hairpins possessing neighboring G:G base pairs the stilbene diether linkers undergo efficient photoisomerization.     

DNA中的B-Z"链接"

DNA能够通过改变自身的构象来完成它在生命过程中的一些重要作用。在进行转录和其它一些生理过程中,B-DNA的局部区域会转变为Z-DNA,形成具有两个B-Z接合点的奇特的B-DNA-Z-DNA-B-DNA结构,在每个接合点处有一对碱基的氢键发生断裂且碱基被挤出而连接在双螺旋的外侧。探讨这种结构的形成机制对于理解DNA的结构与功能的关系具有重要意义,也将有助于更深刻地认识Z-DNA所扮演的角色。     

Effect of base sequence and deprotonation of Guanine cation radical in DNA

The deprotonation of guanine cation radical (G+*) in oligonucleotides (ODNs) was measured spectroscopically by nanosecond pulse radiolysis. The G+* in ODN, produced by oxidation with SO4-*, deprotonates to form the neutral G radical (G(-H)*). In experiments using 5-substituted cytosine-modified ODN, substitution of the cytosine C5 hydrogen by a methyl group increased the rate constant of deprotonation, whereas replacement by bromine decreased the rate constant. Kinetic solvent isotope effects on the kinetics of deoxyguanosine (dG) and ODN duplexes were examined in H2O and D2O. The rate constant of formation of G(-H)* in dG was 1.7-fold larger in H2O than D2O, whereas the rate constant in the ODN duplex was 3.8-fold larger in H2O than D2O. These results suggest that the formation of G(-H)* from G+* in the ODN corresponds to the deprotonation of the oxidized hydrogen-bridged (G+*-C) base pair by a water molecule. The characteristic absorption maxima of G+* around 400 nm were shifted to a longer wavelength in the order of G相似文献   

Exclusion of aromatic radical cations from cyclodextrin nanocavity studied by pulse radiolysis

The influence of hydroxypropyl-beta-cyclodextrin (HP-beta-CD) on the one-electron oxidation reaction of aromatic sulfides (S) with Br2*- and the decay process of the S radical cation (S*+) was investigated by pulse radiolysis. The dissociation kinetics of S*+ from the CD cavity was examined in terms of the apparent equilibrium constants (Kapp) for the formation and decay processes of S*+. Inhibition of the one-electron oxidation reaction of S by Br2*- was clearly observed in the presence of HP-beta-CD. On the basis of a comparison between the determined Kapp values, it was found that the binding ability of S*+ with HP-beta-CD is much lower than that of S, because of the hydrophobic nature of the cavity. The formation process of the dimer radical cation of 4-(methylthio)phenylmethanol ((MTPM)2*+), which is generated between MTPM(*+) and neutral MTPM in solution, was also inhibited by the addition of HP-beta-CD.     

Proton-coupled hole hopping in nucleosomal and free DNA initiated by site-specific hole injection

Nucleosomes were reconstituted from recombinant histones and a 147-mer DNA sequence containing the damage reporter sequence 5'-…d([2AP]T[GGG](1)TT[GGG](2)TTT[GGG](3)TAT)… with 2-aminopurine (2AP) at position 27 from the dyad axis. Footprinting studies with ˙OH radicals reflect the usual effects of "in" and "out" rotational settings, while, interestingly, the guanine oxidizing one-electron oxidant CO(3)(˙-) radical does not. Site-specific hole injection was achieved by 308 nm excimer laser pulses to produce 2AP(˙+) cations, and superoxide via the trapping of hydrated electrons. Rapid deprotonation (~100 ns) and proton coupled electron transfer generates neutral guanine radicals, G(-H)˙ and hole hopping between the three groups of [GGG] on micro- to millisecond time scales. Hole transfer competes with hole trapping that involves the combination of O(2)(˙-) with G(-H)˙ radicals to yield predominantly 2,5-diamino-4H-imidazolone (Iz) and minor 8-oxo-7,8-dihydroguanine (8-oxoG) end-products in free DNA (Misiaszek et al., J. Biol. Chem. 2004, 279, 32106). Hole migration is less efficient in nucleosomal than in the identical protein-free DNA by a factor of 1.2-1.5. The Fpg/piperidine strand cleavage ratio is ~1.0 in free DNA at all three GGG sequences and at the "in" rotational settings [GGG](1,3) facing the histone core, and ~2.3 at the "out" setting at [GGG](2) facing away from the histone core. These results are interpreted in terms of competitive reaction pathways of O(2)(˙-) with G(-H)˙ radicals at the C5 (yielding Iz) and C8 (yielding 8-oxoG) positions. These differences in product distributions are attributed to variations in the local nucleosomal B-DNA base pair structural parameters that are a function of surrounding sequence context and rotational setting.     

New base pairing motifs. The synthesis and thermal stability of oligodeoxynucleotides containing imidazopyridopyrimidine nucleosides with the ability to form four hydrogen bonds

The synthesis and thermal stability of oligodeoxynucleotides (ODNs) containing imidazo[5',4':4,5]pyrido[2,3-d]pyrimidine nucleosides 1-4 (N(N), O(O), N(O), and O(N), respectively) with the aim of developing two sets of new base pairing motifs consisting of four hydrogen bonds (H-bonds) is described. The proposed four tricyclic nucleosides 1-4 were synthesized through the Stille coupling reaction of a 5-iodoimidazole nucleoside with an appropriate 5-stannylpyrimidine derivative, followed by an intramolecular cyclization. These nucleosides were incorporated into ODNs to investigate the H-bonding ability. When one molecule of the tricyclic nucleosides was incorporated into the center of each ODN (ODN I and II, each 17mer), no apparent specificity of base pairing was observed, and all duplexes were less stable than the duplexes containing natural G:C and A:T pairs. On the other hand, when three molecules of the tricyclic nucleosides were consecutively incorporated into the center of each ODN (ODN III and IV, each 17mer), thermal and thermodynamic stabilization of the duplexes due to the specific base pairings was observed. The melting temperature (T(m)) of the duplex containing the N(O):O(N) pairs showed the highest T(m) of 84.0 degrees C, which was 18.2 and 23.5 degrees C higher than that of the duplexes containing G:C and A:T pairs, respectively. This result implies that N(O)and O(N) form base pairs with four H-bonds when they are incorporated into ODNs. The duplex containing N(O):O(N) pairs was markedly stabilized by the assistance of the stacking ability of the imidazopyridopyrimidine bases. Thus, we developed a thermally stable new base pairing motif, which should be useful for the stabilization and regulation of a variety of DNA structures.     

Fluorescence properties of 2-aminopurine-cytidine-7-deazaguanine (5'-ApCdzG-3') trimer in B- and Z-DNA

The electron transfer quenching of 2-aminopurine by guanine and 7-deazaguanine was investigated in B- and Z-DNA, and an increase in the fluorescence intensity of 2-aminopurine upon B- to Z-DNA transition was demonstrated.     

Site-selective electron transfer from purines to electrocatalysts: voltammetric detection of a biologically relevant deletion in hybridized DNA duplexes.

BACKGROUND: The one-electron oxidation of guanine nucleobases is of interest for understanding the mechanisms of mutagenesis, probing electron-transfer reactions in DNA, and developing sensing schemes for nucleic acids. The electron-transfer rates for oxidation of guanine by exogenous redox catalysts depend on the base paired to the guanine. An important goal in developing the mismatch sensitivity is to identify a means for monitoring the current resulting from electron transfer at a single base in the presence of native oligonucleotides that contain all four bases. RESULTS: The nucleobase 8-oxo-guanine (8G) is selectively oxidized by the redox catalyst Os(bpy)(3)(3+/2+) (bpy = 2,2'-bipyridine) in the presence of native guanine. Cyclic voltammograms of Os(bpy)(3)(2+) show current enhancements indicative of nucleobase oxidation upon addition of oligonucleotides that contain 8G, but not in the presence of native guanine. As expected, similar experiments with Ru(bpy)(3)(2+) show enhancement with both guanine and 8G. The current enhancements for the 8G/Os(III) reaction increase in the order 8G-C approximately 8G.T < 8G.G < 8G.A < 8G, the same order as that observed for guanine/Ru(III). This site-selective mismatch sensitivity can be applied to detection of a TTT deletion, which is important in cystic fibrosis. CONCLUSIONS: The base 8G can be effectively used in conjunction with a low-potential redox catalyst as a probe for selective electron transfer at a single site. Because of the high selectivity for 8G, rate constants can be obtained that reflect the oxidation of only one base. The mismatch sensitivity can be used to detect biologically relevant abnormalities in DNA.     

One-electron reductive template-directed ligation of oligodeoxynucleotides possessing a disulfide bond

Hypoxic X-radiolysis of diluted aqueous solutions was performed to generate hydrated electrons that induced one-electron reduction of oligodeoxynucleotides (ODNs) possessing a disulfide bond. Upon hypoxic irradiation of dinucleotides, two forms of dinucleotides were produced via intermolecular exchange of the disulfides and ligation that proceeded with a multiple turnover. In contrast to the efficient reaction induced by hypoxic irradiation, the reaction efficiency was dramatically decreased when irradiation was performed under aerobic conditions, presumably due to capturing reactive hydrated electrons by molecular oxygen. We subsequently applied these unique reaction characteristics to template-directed ligation. In the presence of a complementary template ODN, two ODNs possessing a disulfide bond produced a prescribed ODN with high regioselectivity via interstrand crossing upon hypoxic irradiation.     

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.     

Microanalysis of oligodeoxynucleotides by cathodic stripping voltammetry at amalgam-alloy surfaces in the presence of copper ions

The application of gold amalgam-alloy electrode (AuAE) for a sensitive voltammetric detection of different oligodeoxynucleotides (ODNs) containing the purine units within the ODN-chains in the presence of copper is described. The detection of ODNs is based on the following procedure: (i) the first step includes an acidic hydrolysis of the ODN (ahODN) samples performing the release of the purine bases from ODN-chain; (ii) the second step includes an electrochemical accumulation of the complex of the purine base residues released from ODN-chain with copper ions Cu(I) (ahODN-Cu(I) complex) at the potential of reduction of copper ions Cu(II) on the amalgam-alloy electrode surfaces; (iii) finally followed the cathodic stripping of the electrochemically accumulated ahODN-Cu(I) complex from the electrode surface. The proposed electrochemical method was used for: (a) detection of different ODN lengths containing only adenine units (the number of adenine units within the ODN-chains was changed from 10 to 80), and (b) determination of the number of purine units within the 30-mer ODNs containing a random sequence segments involving both the purine and pyrimidine units. The intensity of the cathodic stripping current density peak (j_CSP) of the electrochemically accumulated ahODN-Cu(I) complex increased linearly with the increasing number of purine units within the ODN-chains. We observed a good correlation between the percentage content of purine units to the whole length of different 30-mer ODNs and the percentage content of the intensity of the j_CSP of the electrochemically accumulated 30-mer ahODN-Cu(I) complexes. The detection of acid hydrolysed 80-mer (A₈₀) in the bulk solution and in a 20-μl volume is possible down to 200 pM and 2 nM at the AuAE, respectively. For the shortest 10-mer (A₁₀) a detectable value of 5 nM in the bulk solution on the AuAE was observed. The sensitive detection of different ODNs containing the purine units in their chains in the presence of copper can be also performed at the platinum amalgam-alloy (PtAE) and copper amalgam-alloy (CuAE) contrary to a lower sensitivity at the silver amalgam-alloy (AgAE) electrode.     

Unique charge transfer properties of the four-base pi-stacks in Z-DNA

To investigate the photoreactions of BrU in Z-DNA, the photoirradiation of 5'-d(C1G2C3G4BrU5G6C7G8)-3'/5'-d(C9mG10C11A12C13mG14C15G16)-3'(ODN 1-2) was investigated. In accord with previous observations, B-form ODN 1-2 with the 5'-GBrU sequence showed very weak photoreactivity. However, Z-form ODN 1-2 in 2 M NaCl underwent photoreaction to afford 5'-d(CGC)rGd(UGCG)-3' together with the formation of imidazolone (Iz) contained 5'-d(CIzCACmGCG)-3'. The results clearly indicate that structural changes caused by the B-Z transition dramatically increased the photoreactivity of ODN 1-2. Inspection of the molecular structure of Z-DNA suggests that there is unique four-base pi-stacks at the G4-BrU5-C11-mG10 in ODN 1-2. These results suggest that the intriguing possibility that the mG10 in a complementary strand located at the end of the four-base pi-stacks may act as an electron donor. To test the hypothesis of interstrand charge transfer from mG10 to BrU5 within the four-base pi-stacks in Z-DNA, ODN 1-3 samples in which the putative donor G10 residue was replaced with 8-methoxyguanine (moG) were prepared, since moG is known to trap cation radicals to yield Iz moieties in DNA. Photoirradiation of ODN 1-3 efficiently produced 5'-d(CGC)rGd(UGCG)-3' together with formation of 5'-d(CIzCACmGCG)-3'. These results clearly indicate that the interstrand charge transfer from mG10 to BrU5 initiates the photoreaction. In clear contrast, other replacements of G with moG did not enhance the photoreactivity. The present study revealed the presence of unique four-base pi-stacks in Z-DNA and photoirradition of BrU in Z-DNA causes efficient electron transfer from G within this cluster.