Identification of novel ligands for the Z-DNA binding protein by structure-based virtual screening

We describe the first discovery of small molecules that bind to the Z-DNA binding domain of human ADAR1 (Adenosine Deaminase Acting on RNA 1) by structure-based virtual screening of chemical database. These molecules bind to Z-DNA binding domain to inhibit the interaction with the Z-DNA. Many viruses have Z-DNA binding proteins, which are structurally similar to Z-DNA binding domain of human ADAR1, and the ability of Z-DNA binding protein to bind the Z-DNA is essential for their pathogenicity. Therefore, the molecules identified in this study may serve as novel leads for the design of agents that inhibit biological functions of those pathogenic viruses.     

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.     

One-electron attachment reaction of B- and Z-DNA modified by 8-bromo-2'-deoxyguanosine

The one-electron attachment reaction of 8-bromo-2'-deoxyguanosine ((Br)G) in DNA was studied by comparing that in B- and Z-DNA. Oligodeoxynucleotides (ODNs) modified by (Br)G were synthesized as Z-DNA in which the syn-conformation deoxyguanosine is stabilized by steric interference between the 8-bromo group of (Br)G and the sugar moiety. Debromination from the (Br)G-modified ODNs occurred from the one-electron attachment during the gamma-radiolysis. The structural dependence of B- and Z-DNA was observed for the one-electron attachment reaction. The conversion of (Br)G was higher in Z-DNA than in B-DNA. Because the solvent-accessible surface of the purine base in Z-DNA is greater than that in B-DNA, it is demonstrated that the reactivity of purine base C8 is enhanced in Z-DNA compared to that in B-DNA.     

(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.     

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.     

NMR study on the B-Z junction formation of DNA duplexes induced by Z-DNA binding domain of human ADAR1

Z-DNA is produced in a long genomic DNA by Z-DNA binding proteins, through formation of two B-Z junctions with the extrusion of one base pair from each junction. To answer the question of how Z-DNA binding proteins induce B-Z transitions in CG-rich segments while maintaining the B-conformation of surrounding segments, we investigated the kinetics and thermodynamics of base-pair openings of a 13-bp DNA in complex with the Z-DNA binding protein, Zα(ADAR1). We also studied perturbations in the backbone of Zα(ADAR1) upon binding to DNA. Our study demonstrates the initial contact conformation as an intermediate structure during B-Z junction formation induced by Zα(ADAR1), in which the Zα(ADAR1) protein displays unique backbone conformational changes, but the 13-bp DNA duplex maintains the B-form helix. We also found the unique structural features of the 13-bp DNA duplex in the initial contact conformation: (i) instability of the AT-rich region II and (ii) longer lifetime for the opening state of the CG-rich region I. Our findings suggest a three-step mechanism of B-Z junction formation: (i) Zα(ADAR1) specifically interacts with a CG-rich DNA segment maintaining B-form helix via a unique conformation; (ii) the neighboring AT-rich region becomes very unstable, and the CG-rich DNA segment is easily converted to Z-DNA; and (iii) the AT-rich regions are base-paired again, and the B-Z junction structure is formed.     

Construction of ssDNA-Attached LR-Chimera Involving Z-DNA for ZBP1 Binding Analysis

The binding of proteins to Z-DNA is hard to analyze, especially for short non-modified DNA, because it is easily transferred to B-DNA. Here, by the hybridization of a larger circular single-stranded DNA (ssDNA) with a smaller one, an LR-chimera (involving a left-handed part and a right-handed one) with an ssDNA loop is produced. The circular ssDNAs are prepared by the hybridization of two ssDNA fragments to form two nicks, followed by nick sealing with T4 DNA ligase. No splint (a scaffold DNA for circularizing ssDNA) is required, and no polymeric byproducts are produced. The ssDNA loop on the LR-chimera can be used to attach it with other molecules by hybridization with another ssDNA. The gel shift binding assay with Z-DNA specific binding antibody (Z22) or Z-DNA binding protein 1 (ZBP1) shows that stable Z-DNA can form under physiological ionic conditions even when the extra ssDNA part is present. Concretely, a 5′-terminal biotin-modified DNA oligonucleotide complementary to the ssDNA loop on the LR-chimera is used to attach it on the surface of a biosensor inlaid with streptavidin molecules, and the binding constant of ZBP1 with Z-DNA is analyzed by BLI (bio-layer interferometry). This approach is convenient for quantitatively analyzing the binding dynamics of Z-DNA with other molecules.     

Computer simulation of the ionic atmosphere around Z-DNA

We describe a coarse-grained model for Z-DNA that mimics the DNA shape with a relatively small number of repulsive interaction sites. In addition, negative charges are placed at the phosphate positions. The ionic atmosphere around this grooved Z-DNA model is then investigated with Monte Carlo simulation. Cylindrically averaged concentration profiles as well as the spatial distribution of ions have been calculated. The results are compared to those for other DNA models differing in the repulsive core. This allows the examination of the effect of the DNA shape in the ionic distribution. It is seen that the penetrability of the ions to the DNA groove plays an important role in the ionic distribution. The results are also compared with those reported for B-DNA. In both conformers the ions are structured in alternating layers of positive and negative charge. In Z-DNA the layers are more or less concentric to the molecular axis. Besides, no coions enter into the single groove of this conformer. On the contrary, the alternating layers of B-DNA are also structured along the axial coordinate with some coions penetrating into the major groove. In both cases we have found five preferred locations of the counterions and two for the coions. The concentration of counterions reaches its absolute maximum at the narrow Z-DNA groove and at the minor groove of B-DNA, the value of the maximum being higher in the Z conformer.     

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.     

To be dinuclear or not: Z-DNA induction by nickel complexes

The left-handed Z-DNA has been identified as a gene regulating element. Therefore the generation of Z-DNA through metal complexes might be an innovative way for the regulation of gene expression. Use of the new dinuclear complex N,N,N',N'-tetrakis-[2-(3,5-dimethylpyrazol-1-yl)ethyl]-1,3-propylenediamine-bis(nickel(II) dinitrate) (2) reversibly induced Z-DNA formation. However, when a 1:1 ratio of metal/dinucleating ligand was used as a control, the midpoint of the B- to Z-DNA transition was at the same nickel concentration as in case of the dinuclear complex. The novel mononuclear analogue, N-methyl-N,N-bis-[2-(3,5-dimethylpyrazol-1-yl)ethyl]amine-nickel(II)-dinitrate (3) was inducing the Z-DNA at a similar ratio versus nucleotides as free nickel(II) itself. For the first time, proton and nickel binding constants for the bis-[2-(pyrazol-1-yl)ethyl]amine ligand system are reported and discussed. Both nickel complexes 2 and 3 were structurally characterized by single crystal analysis. Furthermore, the synthesis of the two new ligands, N,N,N',N'-tetrakis-[2-(3,5-dimethylpyrazol-1-yl)ethyl]-1,2-propylenediamine (4) and N-methyl-N,N-bis-[2-(3,5-dimethylpyrazol-1-yl)ethyl]amine (5) is described. The two major synthetic pathways leading to polypyrazoyl amines in general are critically discussed with respect to yield, reproducibility and handling of the intermediates.     

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.     

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。     

Porphyrins conjugated to DNA as CD reporters of the salt-induced B to Z-DNA transition

The porphyrin chromophore incorporated at the 5'-position of an oligonucleotide allows the simultaneous detection of the B- to Z-DNA transition via the porphyrin Soret band circular dichroism exciton couplet signal around 420 nm and the oligonucleotide CD region below 300 nm, at micromolar concentrations.     

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.     

Engraving the Surface of Electrospun Microfibers with Nanoscale Grooves Promotes the Outgrowth of Neurites and the Migration of Schwann Cells

We report a simple method based upon coaxial electrospinning for the fabrication of aligned microfibers engraved with nanoscale grooves to promote neurite outgrowth and cell migration. The success of this method relies on the immiscibility between poly(?‐caprolactone) (PCL) and poly(vinyl pyrrolidone) (PVP) in 2,2,2‐trifluoroethanol (TFE) for the generation of PVP/TFE pockets on the surface of a PCL jet. The pockets are stretched and elongated along with the jet, eventually resulting in the formation of nanoscale grooves upon the removal of PVP. The presence of nanoscale grooves greatly enhances the outgrowth of neurites from both PC12 cells and chick embryonic dorsal root ganglia (DRG) bodies, as well as the migration of Schwann cells. The enhancements can be maximized by optimizing the dimensions of the grooves for potential use in applications involving neurite extension and wound closure.     

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.     

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.     

Effect of fluid-substrate attraction and pore geometry on fluid adsorption

We employ grand canonical ensemble Monte Carlo simulations to investigate the impact of substrate curvature on the phase behavior of an adjacent fluid. The substrates consist of a periodic sequence of grooves in the x direction; the grooves are infinitely long in the y direction. The shape of the grooves is controlled by a parameter eta. For eta = 0 the substrates are planar. If eta = 1, the grooves are wedge shaped. If eta > 1 the grooves become concave and in the limit eta = infinity rectangular. The fluid-substrate potential representing a groove consists of two contributions, namely, that of the homogeneous substrate base corresponding to a semi-infinite solid and that of a finite piece of solid with nonplanar surfaces. Whereas the former contribution can be calculated analytically, the latter needs to be evaluated numerically. For very large values of eta, that is in (almost) rectangular grooves, we observe capillary condensation of that portion of fluid located inside the grooves. As eta decreases capillary condensation gives way to continuous filling. In all cases, a nearly planar film-gas interface eventually forms in the direction normal to the surface of the substrate base and outside the grooves if one increases the chemical potential sufficiently.     

The elastic distortion and stability of biaxial nematic liquid crystal on the surface grooves

Fukuda et al. reexamined the Berreman's model which attributes the surface anchoring to the elastic distortion of the uniaxial nematic liquid crystal induced by the grooves of a surface. They showed that at the variance with the assumption made in the original approach of Berreman, the azimuthal distortion of the director cannot be considered as negligibly small. Now this method is generalized to the biaxial nematic liquid crystals, with some approximations for the elastic constants. We obtain an additional term in the elastic distortion energy per unit area which depends on the second power of the cosine of the angle made between the main director n at infinity and the direction of the surface grooves. This additional term describes the distortion energy of the minor director m induced by the surface grooves when the n director is anchored exactly along the grooves. We have studied the stability of the n director around the grooves, and in one-constant model for each director the stability condition is that the elastic constant of the n director is the maximum.     

Anions or cations: who is in charge of inhibiting the nickel(II) promoted B- to Z-DNA transition?

Various weakly binding cations and anions were studied at a concentration of 10 mM to ascertain their interaction with the nickel(II) promoted B- to Z-DNA transition of poly d(GC). These salts were ranked according to the decreasing amounts of nickel needed for the B- to Z-DNA transition and provided the following order: NaCl approximately Me4NCl > LiCl > MgCl2 > no salt > NaBF4 approximately NaNO3 approximately NaClO4. Remarkably, it was found that going from sodium nitrate to sodium chloride increased the necessary amount of nickel to induce the transition to the left-handed helix of poly d(GC) by a factor of 10. This dramatic effect cannot be explained by the binding constant of nickel(II) to chloride to form the monocationic complex. We believe that this is the first reported example of the role of chloride anions, which appear to modulate the interaction of nickel(II) ions with the polyanionic DNA.