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Even in high-quality X-ray crystal structures of oligonucleotides determined at a resolution of 1 Å or higher, the orientations of first-shell water molecules remain unclear. We used cryo neutron crystallography to gain insight into the H-bonding patterns of water molecules around the left-handed Z-DNA duplex [d(CGCGCG)]2. The neutron density visualized at 1.5 Å resolution for the first time allows us to pinpoint the orientations of most of the water molecules directly contacting the DNA and of many second-shell waters. In particular, H-bond acceptor and donor patterns for water participating in prominent hydration motifs inside the minor groove, on the convex surface or bridging nucleobase and phosphate oxygen atoms are finally revealed. Several water molecules display entirely unexpected orientations. For example, a water molecule located at H-bonding distance from O6 keto oxygen atoms of two adjacent guanines directs both its deuterium atoms away from the keto groups. Exocyclic amino groups of guanine (N2) and cytosine (N4) unexpectedly stabilize waters H-bonded to O2 keto oxygens from adjacent cytosines and O6 keto oxygens from adjacent guanines, respectively. Our structure offers the most detailed view to date of DNA solvation in the solid-state undistorted by metal ions or polyamines.

The binding mode of cationic porphyrin (trans-BMPyP) with poly[d(G-C)2] and poly[d(A-T)2] was examined according to the site of the periphery cationic methyl pyridine ion of the cationic porphyrin (o-, m-, p-) as well as the possibility of a B-Z transition depending on the binding modes by measuring the absorption spectrum and circular dichroism (CD). The negative band found in the soret region showed the intercalation mode of m- and p-trans-BMPyP-poly[d(G-C)2] to the DNA base pairs, but no B-Z transition was induced. On the other hand, the distinctive bisignate band found in the soret region of the CD spectrum for m- and p-trans-BMPyP-poly[d(A-T)2] suggests that m- and p-trans-BMPyP have an effective extensive stacking-based binding mode along with the skeleton of poly[d(A-T)2], wherein the B-Z transition was induced through extensive stacking. The difference in binding mode was attributed to the difference in the molecular structure depending on the site of the periphery cationic methyl pyridine ion in the cationic porphyrin. In other words, o-trans-BMPyP is nonplanar because of the steric hindrance of the cationic methyl pyridine ion at the o-site. In contrast, m- and p-trans-BMPyP are planar, but not all porphyrins with a planar structure undergo the B-Z transition. In conclusion, a B-Z transition is induced if the structure of a porphyrin is planar and the binding mode allows the porphyrins to be stacked effectively along the DNA skeleton, not in a binding mode where the porphyrin is intercalated to the DNA.Communicated by Ramaswamy H. Sarma.

The DNA in the left-handed conformation (Z-conformation) was first discovered by A. Rich, who revealed the crystalline structure of a DNA oligomer d(GC)3 by X-ray diffraction method. Later it was also found that DNA molecules change their conformations from typical right-handed form (B-DNA) to the left-handed form (Z-DNA) under specific conditions (B–Z transition). Furthermore, the detailed structures of the interface between B- and Z-DNAs, B-Z junction, was also determined with an atomic resolution. Recently it was found that some proteins have the Z-DNA binding domains, but the biological functions of Z-DNA are not well understood yet. Therefore the investigation of Z-DNA under physiological conditions is highly essential. In this study, we demonstrated the high-resolution real-space imaging of DNA molecules having the Z- and B-form conformations by frequency-modulation atomic force microscopy (FM-AFM), that has made a great progress in recent years, in an aqueous solution. The major and minor grooves of both DNA conformations were clearly visualized. Furthermore, the surface charge density was measured by three-dimensional (3D) force mapping method. We found that Z-form region was less negatively charged than the B-form region., 左巻きDNAの2重らせん構造の直接可視化に成功 --液中原子間力顕微鏡によるDNA高分解能観察とその電荷分布計測--. 京都大学プレスリリース. 2019-05-17.

Acta crystallographica / B 77(3), 331-338 (2021). doi:10.1107/S2052520621002663, The X-ray crystal structure of the d(CGCGCG)$_2$/putrescine(2+)/K$^+$ com��plex has been determined at 0.60 �� resolution. Stereochemical restraints were used only for the putrescinium dication, and 23 bonds and 18 angles of the Z-DNA nucleotides with dual conformation. The N atoms of the putrescine(2+) dication form three direct hydrogen bonds with the N7_G atoms of three different Z-DNA molecules, plus three water-mediated hydrogen bonds with cytosine, guanine and phosphate acceptors. A unique potassium cation was also unambiguously identified in the structure, albeit at a ���0.5 occupation site shared with a water molecule, providing the first example of such a com��plex with Z-DNA. The K$^+$ cation has coordination number of eight and an irregular coordination sphere, formed by four water molecules and four O atoms from two phosphate groups of the Z-DNA, including ligands present at fractional occupancy. The structural disorder of the Z-DNA duplex is manifested by the presence of alternate conformations along the DNA backbone. Comparison of the position and interactions of putrescine(2+) in the present structure with other ultra-high-resolution structures of Z-DNA in com��plexes with Mn$^{2+}$ and Zn$^{2+}$ ions shows that the dicationic putrescinium moiety can effectively substitute these metal ions for stabilization of Z-type DNA duplexes. Furthermore, this comparison also suggests that the spermine(4+) tetracation has a higher affinity for Z-DNA than K$^+$., Published by Wiley-Blackwell, Oxford [u.a.]

A re-evaluation of m|F o| − D|F c| electron-density maps revealed that potential conformational transitions of 27% of DNA phosphates are found in previous crystallographic data. The analysis suggests that some of these unassigned densities correspond to ZI ↔ ZII or A/B → BI transitions., Conformational flexibility of DNA plays important roles in biological processes such as transcriptional regulation and DNA packaging etc. To understand the mechanisms of these processes, it is important to analyse when, where and how DNA shows conformational variations. Recent analyses have indicated that conventional refinement methods do not always provide accurate models of crystallographic heterogeneities and that some information on polymorphism has been overlooked in previous crystallographic studies. In the present study, the m|F o| − D|F c| electron-density maps of double-helical DNA crystal structures were calculated at a resolution equal to or better than 1.5 Å and potential conformational transitions were found in 27% of DNA phosphates. Detailed analyses of the m|F o| − D|F c| peaks indicated that some of these unassigned densities correspond to ZI ↔ ZII or A/B → BI conformational transitions. A relationship was also found between ZI/ZII transitions and metal coordination in Z-DNA from the detected peaks. The present study highlights that frequent transitions of phosphate backbones occur even in crystals and that some of these transitions are affected by the local molecular environment.

In this work, we have characterized the interactions of monospermine porphyrin derivative with calf thymus DNA (ct-DNA) and poly (dG-dC)2 in both B and Z conformation. By several spectroscopic techniques (UV-vis, electronic circular dichroism and resonance light scattering), the binding modes of monospermine porphyrin derivative with different DNA sequences have been elucidated. In the presence of ct-DNA, the porphyrin binds along the external double helix as well as in the presence of B conformation of poly (dG-dC)2 . Whilst when the Z form of the poly (dG-dC)2 is induced, a slight intercalation of the porphyrin between the basis has been detected.

Base pair mismatches can erroneously be incorporated in the DNA. An adenine pairing with another adenine is one of the eight possible mismatches. The atomistic insights about the structure and dynamics of an A…A mismatch in a DNA (unbound form) is not yet accessible to any experimental technique. Earlier molecular dynamics (MD) simulations have shown that A…A mismatch in the midst of 5′C A G/3′G A C, 5′G A C/3′C A G and 5′C A A/3′G A T (underline represents the mismatch) are highly dynamic in nature. By employing MD simulation, the influence of an A…A mismatch in the midst of 5′G A A/3′C A T, 5′G A G/3′C A C, 5′A A C/3′T A G, 5′A A G/3′T A C, 5′T A A/3′A A T, 5′T A T/3′A A A and 5′A A T/3′T A A sequences have been investigated here. The results indicate that irrespective of the flanking sequences, the mismatch samples a variety of transient conformations, including a B-Z junction. Further, circular dichroism studies have been carried out to explore the ability of these sequences to bind with hZαADAR1 which specifically recognizes B-Z junction/Z-DNA. The results indicate that hZαADAR1 could not lead to a complete B to Z transition in the above sequences. Notably, a complete transition to Z-form has been reported earlier for 5′G A C/3′C A G upon titrating with hZαADAR1. Intriguingly, 5′A A C/3′T A G, 5′A A G/3′T A C and 5′G A A/3′C A T exhibit a B-Z junction formation rather than a complete transition to Z-form, similar to the situation of 5′C A A/3′G A T. These indicate that although A…A mismatch could induce a local B-Z junction transiently, hZαADAR1 requires the presence of a G…C/C…G base pair adjacent to the A…A mismatch for the binding. Additionally, the extent of B-Z junction has enhanced upon binding with hZαADAR1 in the presence of the A…A mismatch (specifically when CG, CA, AC, GA and AG steps occur), but not in the presence of the canonical base pairs. These confirm the inclination of A…A mismatch towards the B-Z junction.

Herein, we report a hybrid polyoxometalate organic–inorganic compound, Na2[(HGMP)2Mo5O15]⋅7 H2O (1; where GMP=guanosine monophosphate), which spontaneously assembles into a structure with dimensions that are strikingly similar to those of the naturally occurring left-handed Z-form of DNA. The helical parameters in the crystal structure of the new compound, such as rise per turn and helical twist per dimer, are nearly identical to this DNA conformation, allowing a close comparison of the two structures. Solution circular dichroism studies show that compound 1 also forms extended secondary structures in solution. Gel electrophoresis studies demonstrate the formation of non-covalent adducts with natural plasmids. Thus we show a route by which simple hybrid inorganic–organic monomers, such as compound 1, can spontaneously assemble into a double helix without the need for a covalently connected linear sequence of nucleic acid base pairs.

High concentrations of Na+ or [Co(NH3)6]3+ can induce the B to Z conformational transition in alternating (dC-dG) oligo and polynucleotides. The use of short DNA oligomers (dC-dG)4 and (dm5C-dG)4 as models can allow a thermodynamic characterization of the transition. Both form right handed double helical structures (B-DNA) in standard phosphate buffer with 115 mM Na+ at 25 &deg, C. However, at 2.0 M Na+ or 200 &mu, M [Co(NH3)6]3+, (dm5C-dG)4 assumes a left handed double helical structure (Z-DNA) while the unmethylated (dC-dG)4 analogue remains right handed under those conditions. We have previously demonstrated that the enthalpy of the transition at 25 &deg, C for either inducer can be determined using isothermal titration calorimetry (ITC). Here, ITC is used to investigate the linkages between temperature, water activity and DNA conformation. We found that the determined enthalpy for each titration varied linearly with temperature allowing determination of the heat capacity change (&Delta, Cp) between the initial and final states. As expected, the &Delta, Cp values were dependent upon the cation (i.e., Na+ vs. [Co(NH3)6]3+) as well as the sequence of the DNA oligomer (i.e., methylated vs. unmethylated). Osmotic stress experiments were carried out to determine the gain or loss of water by the oligomer induced by the titration. The results are discussed in terms of solvent accessible surface areas, electrostatic interactions and the role of water.

Recognition of unusual left-handed Z-DNA by specific binding of small molecules is crucial for understanding biological functions in which this particular structure participates. Recent investigations indicate that zinc cationic porphyrin (ZnTMPyP4) is promising as a probe for recognizing Z-DNA due to its characteristic chiroptical properties upon binding with Z-DNA. However, binding mechanisms of the ZnTMPyP4/Z-DNA complex remain unclear. By employing time-resolved UV-visible absorption spectroscopy in conjunction with induced circular dichroism (ICD), UV-vis, and fluorescence measurements, we examined the binding interactions of ZnTMPyP4 towards B-DNA and Z-DNA. For the ZnTMPyP4/Z-DNA complex, two coexisting binding modes were identified as the electrostatic interaction between pyridyl groups and phosphate backbones, and the major groove binding by zinc(II) coordinating with the exposed guanine N7. The respective contribution of each mode is assessed, allowing a complete scenario of binding modes revealed for the ZnTMPyP4/Z-DNA. These interaction modes are quite different from those (intercalation and partial intercalation modes) for the ZnTMPyP4/B-DNA complex, thereby resulting in explicit differentiation between B-DNA and Z-DNA. Additionally, the binding interactions of planar TMPyP4 to DNA were also investigated as a comparison. It is shown that without available virtual orbitals to coordinate, TMPyP4 binds with Z-DNA solely in the intercalation mode, as with B-DNA, and the intercalation results in a structural transition from Z-DNA to B-ZNA. These results provide mechanistic insights for understanding ZnTMPyP4 as a probe of recognizing Z-DNA and afford a possible strategy for designing new porphyrin derivatives with available virtual orbitals for the discrimination of B-DNA and Z-DNA.

DNA oligomer duplexes containing alternating cytosines and guanines in their sequences tend to form left-handed helices of the Z-DNA type, with the sugar and phosphate backbone in a zigzag conformation and a helical repeat of two successive nucleotides. Z-DNA duplexes usually crystallize as hexagonally arranged parallel helical tubes, with various relative orientations and translation of neighboring duplexes. Four novel high-resolution crystal structures of d(CGCGCG)2duplexes are described here. They are characterized by a high degree of pseudosymmetry and/or twinning, with three or four independent duplexes differently oriented in a monoclinicP21lattice of hexagonal metric. The various twinning criteria give somewhat conflicting indications in these complicated cases of crystal pathology. The details of molecular packing in these crystal structures are compared with other known crystal forms of Z-DNA.

In this study, crystals of Z-DNA hexamer d(CGCGCG) complexed with MgCl2 and CaCl2 were obtained in the presence of high concentrations of alkaline earth salts (500mM) using a temperature control technique, and their crystal structures were determined at 1.3Å resolution. Mg(2+) and Ca(2+) cations in these structures tend to interact directly with phosphate groups of Z-DNA duplexes; however, they tend to form water-mediated interactions with Z-DNA in the presence of lower concentrations of alkaline earth salts. In these crystals, a DNA duplex was laid along its c-axis and interacted with its 6 neighboring DNA duplexes through coordination bonds of PO…(Mg(2+) or Ca(2+))…OP. A symmetrical hexagonal Z-DNA duplex assembly model may explain DNA condensation caused by alkaline earth salts. These structures offer insights into the functions of alkaline earth cations essential to the structures and assembly of Z-DNA duplexes.

The deoxyoligonucleotide d(BrU-G-C-G-C-G) was crystallised at pH 8.2 and its structure analysed by X-ray diffraction. The unit cell, of dimensions a = 17.94, b = 30.85, c = 49.94A contains four DNA duplexes in space group P2(1)2(1)2(1). The duplexes are in the Z conformation, with four Watson-Crick G.C base pairs and two BrU.G base pairs. The structure was refined to an R factor of 0.16 at a resolution of 2.2A with 64 solvent molecules located. The BrU.G base pair mismatch is of the wobble type, with both bases in the major tautomer form and hydrogen bonds linking 0-2 of BrU with N-1 of G and N3 of BrU with 0-6 of G. There is no indication of the presence of ionised base pairs, in spite of the high pH of crystallisation. The results are discussed in terms of the mutagenic properties of 5- bromouracil.

The self-complementary d(CGCGCG) hexanucleotide was synthesized with both D-2′-deoxyribose (the natural enantiomer) and L-2′-deoxyribose, and the two enantiomers were mixed in racemic (1:1) proportions and crystallized, producing a new crystal form withC2/csymmetry that diffracted X-rays to 0.78 Å resolution. The structure was solved by direct, dual-space and molecular-replacement methods and was refined to anRfactor of 13.86%. The asymmetric unit of the crystal contains one Z-DNA duplex and three Mg2+sites. The crystal structure is comprised of both left-handed (D-form) and right-handed (L-form) Z-DNA duplexes and shows an unexpectedly high degree of structural disorder, which is manifested by the presence of alternate conformations along the DNA backbone chains as well as at four nucleobases (including one base pair) modelled in double conformations. The crystal packing of the presented D/L-DNA–Mg2+structure exhibits novel DNA hydration patterns and an unusual arrangement of the DNA helices in the unit cell. The paper describes the structure in detail, concentrating on the mode of disorder, and compares the crystal packing of the racemic d(CGCGCG)2duplex with those of other homochiral and heterochiral Z-DNA structures.

It is often envisioned that cations might coordinate at specific sites of nucleic acids and play an important structural role, for instance in the transition between B-DNA and Z-DNA. However, nucleic acid models explicitly devoid of specific sites may also exhibit features previously considered as evidence for specific binding. Such is the case of the "composite cylinder" (or CC) model which spreads out localized features of DNA structure and charge by cylindrical averaging, while sustaining the main difference between the B and Z structures, namely the better immersion of the B-DNA phosphodiester charges in the solution. Here, we analyze the non-electrostatic component of the free-energy difference between B-DNA and Z-DNA. We also compute the composition of the counterion sheath in a wide range of mixed-salt solutions and of temperatures: in contrast with the large difference of composition between the B-DNA and Z-DNA forms, the temperature dependence of sheath composition, previously unknown, is very weak. In order to validate the model, the mixed-salt predictions should be compared to experiment. We design a procedure for future measurements of the sheath composition based on Anomalous Small-Angle X-ray Scattering and complemented by (31) P NMR. With due consideration for the kinetics of the B-Z transition and for the capacity of generating at will the B or Z form in a single sample, the 5'-d[T-(m(5) C-G)12 -T] 26-mer emerges as a most suitable oligonucleotide for this study. Finally, the application of the finite element method to the resolution of the Poisson-Boltzmann equation is described in detail. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 369-384, 2016.

The hexamer duplex d(CACGCG).d(CGCGTG) produces unusual hexagonal ring shaped crystals when crystallized in the presence of cobalt hexammine chloride. Observation of the growth of these crystals over a period of about 6 days indicated that this morphology resulted from radial dissolution of the crystal from the center of the well-formed hexagonal plates. X-ray diffraction studies were carried out for crystals of four stages in the ring crystal morphology. The structure of the DNA in all four crystals was the Z type DNA double helix. The packing mode of the Z DNA duplex in all four crystal structures was that of the ‘spermine’ form. The crystal morphology changes from hexagonal plates to hexagonal rings without any apparent change in the molecular structure of the oligonucleotide or helical packing of the duplexes.

Refinement of empirical force fields for nucleic acids requires their extensive testing using as wide range of systems as possible. However, finding unambiguous reference data is not easy. In this paper, we analyze four systems which we suggest should be included in standard portfolio of molecules to test nucleic acids force fields, namely, parallel and antiparallel stranded DNA guanine quadruplex stems, RNA quadruplex stem, and Z-DNA. We highlight parameters that should be monitored to assess the force field performance. The work is primarily based on 8.4 μs of 100-250 ns trajectories analyzed in detail followed by 9.6 μs of additional selected back up trajectories that were monitored to verify that the results of the initial analyses are correct. Four versions of the Cornell et al. AMBER force field are tested, including an entirely new parmχ(OL4) variant with χ dihedral specifically reparametrized for DNA molecules containing syn nucleotides. We test also different water models and ion conditions. While improvement for DNA quadruplexes is visible, the force fields still do not fully represent the intricate Z-DNA backbone conformation.

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.

DNA containing alternating purine and pyrimidine repeats has the potential to adopt the Z-DNA structure, one of the well-studied structures besides A- and B-DNA. Despite a number of molecular models that have been proposed to explain the mechanism for B→Z transition, there is continued discussion on the mechanism and physiological role of this transition. In this study, we have found that the bis(2-naphthyl)-maleimide-spermine conjugate (3c) exhibits a remarkable ability to cause the B→Z transition of d(CGCGCG) 2 at low salt concentrations. Using isothermal titration calorimetry (ITC) we show that the B→Z transition induced by 3c is both enthalpically and entropically favorable. The ligand might effect the dehydration of B-DNA, which leads to the B→ Z transition. Interestingly, an intermediate CD between the B and Z forms was observed in the pH-dependent transition in the presence of the ligand. The unique structure and characteristics of the ligand designed in this investigation will be useful for the study of Z-DNA.

The left-handed Z-DNA form of the short unmodified alternating guanine-cytosine oligonucleotides, 5'-(dGdC)(24) and 5'-(dGdC)(18), was selectively detected under physiological ionic strength and pH conditions using the anionic nickel(II) porphyrin, NiTPPS. No spectroscopic signal was observed for NiTPPS with any right-handed oligonucleotides under identical conditions. The 48mer 5'-(dGdC)(24) Z-form was detected at concentrations as low as 100nM. The binding of NiTPPS to the B- and Z-oligonucleotides was studied quantitatively by UV-vis absorption and circular dichroism spectroscopies. NiTPPS was found to be a universal DNA binder, with binding affinity and geometry depending on the ionic composition of the solution, rather than on the DNA helical twist. This is the first example of a successful spectroscopic detection of the Z-DNA of short unmodified oligonucleotides under physiological pH and ionic strength conditions.

Left-handed Z-DNA has fascinated biological scientists for decades by its extraordinary structure and potential involvement in biological phenomena. Despite its instability relative to B-DNA, Z-DNA is stabilized in vivo by negative supercoiling. A detailed understanding of Z-DNA formation is, however, still lacking. In this study, we have examined the B-Z transition in a short guanine/cytosine (GC) repeat in the presence of controlled tension and superhelicity via a hybrid technique of single-molecule FRET and magnetic tweezers. The hybrid scheme enabled us to identify the states of the specific GC region under mechanical control and trace conformational changes synchronously at local and global scales. Intriguingly, minute negative superhelicity can facilitate the B-Z transition at low tension, indicating that tension, as well as torsion, plays a pivotal role in the transition. Dynamic interconversions between the states at elevated temperatures yielded thermodynamic and kinetic constants of the transition. Our single-molecule studies shed light on the understanding of Z-DNA formation by highlighting the highly cooperative and dynamic nature of the B-Z transition.

The crystal structure of d(CACACG).d(CGTGTG) was solved to a resolution of 2.05 A in space group P2(1). The duplex assumes the left-handed Z-DNA structure. The presence of two A.T base pairs in the hexamer does not greatly affect the conformation. The most significant changes compared with the regular structure of Z-DNA are in the values of twist in the central portion of the helix. This variation, as well as others in the values of roll, inclination etc., follow the pattern observed previously in the structure of d(CGCACG).d(CGTGCG).

The energies of intra- and inter-strand stacking interactions in model d(GpC) and d(CpG) two-base-pair steps were estimated by MP2/aug-cc-pVDZ single point calculations corrected for basis superposition errors. The stacked two-nucleobase pairs were constructed using experimental values of base pair and base step parameters taken from Nucleic Acid Database (http://ndbserver.rutgers.edu/). Three distinct polymorphic forms were analysed, namely A-, B- and Z-DNA. The applied methodology enables statistical analysis of structural and energetic diversities. The structural relationships between polymorphic forms are quite complex and depend on the sequence of pairs. The variability of parameters such as shift and tilt is almost the same irrespective of the polymorphic form and sequence of steps analysed. In contrast, shift and twist distributions easily discriminate all three polymorphic forms of DNA. Interestingly, despite significant structural diversities, the energies of the most frequent energy ranges are comparable irrespective of the polymorphic form and base sequence. There was observed compensation of inter- and intra-strand interactions, especially for d(GpC) and d(CpG) steps found in A- and B-DNA. Thus, among many other roles, these pairs act as a kind of energetic buffer, balancing the double helix.

There are many great reports of polyamine stabilization of the Z-DNA by bridge conformation between neighboring, symmetry-related Z-DNA in the packing of crystals. However, polyamine binding to the minor groove of Z-DNA and stabilizing the Z-DNA structure has been rarely reported. We proved that the synthesized polyamines bind to the minor groove of Z-DNA and stabilize the conformation under various conditions, by X-ray crystallographic study. These polyamines consist of a polyamine nano wire structure. The modes of the polyamine interaction were changed under different conditions. It is the first example that the crystals consisted of metal free structure. This finding provides a basis for clarifying B-Z transition mechanics.

Several crystal structure analyses of complexes of synthetic polyamine compounds, including N(1)-(2-(2-aminoethylamino))ethyl)ethane-1,2-diamine PA(222) and N(1)-(2-(2-(2-aminoethylamino)ethylamino)ethyl)ethane-1,2-diamine PA(2222), and left-handed Z-DNA d(CGCGCG)(2) have been reported. However, until now, there have been no examples of naturally occurring polyamines bound to the minor groove of the left-handed Z-DNA of d(CGCGCG)(2) molecule. We have found that spermidine, a natural polyamine, is connected to the minor groove of left-handed Z-DNA of d(CGCGCG)(2) molecule in a crystalline complex grown at 10 degrees C. The electron density of the DNA molecule was clear enough to determine that the spermidine was connected in the minor groove of two symmetry related molecules of left-handed Z-DNA d(CGCGCG)(2). This is the first example that a spermidine molecule can form a bridge conformation between two symmetry related molecules of left-handed Z-DNA d(CGCGCG)(2) in the minor groove.

Typical CD spectrum of the right-handed poly[d(A-T)2] was reversed when trans-bis(N-methylpyrimidium-4-yl)diphenyl porphyrin (trans-BMPyP) was bound, suggesting that the helicity of the polynucleotide was reversed to the left-handed form. The formation of the left-handed Z-form poly[d(A-T)2] was confirmed by 31P NMR, in which a single 31P peak of B-form poly[d(A-T)2] was split into two peaks, which is similar to the conventional B-Z transition of poly[d(G-C)2] induced by the high ionic strength. The observed B-Z transition is unique for poly[d(A-T)2]. The other polynucleotides, including poly[d(G-C)2], poly(dG)·poly(dC) and poly(dA)·poly(dT) remained as the right-handed form in the presence of the same porphyrin. This observation suggests that the porphyrin array that was formed along the poly[d(A-T)2] provides a template to which left-handed poly[d(A-T)2] is associated with an electrostatic interaction.

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.

Recently, the stretched intermediate model was proposed for the B–Z deoxyribonucleic acid (DNA) transition based on simulation results carried out using the Stochastic Difference Equation (SDE) that showed unwinding and elongation of the oligomer during the transition. This model has proven to be successful in resolving the steric dilemma in short oligomers. However, extending the simulation method to larger DNA strands may prove to be computationally challenging. Such difficulty has led us to adopt a mean field approach using phenomenological interaction potentials to simulate the transition. Like the atomistic approach, the SDE simulations based on the mean field approach, also suggest the presence of a stretched intermediate during the transition.

The sequence d(TGCGCG).d(CGCGCA) crystallized in two crystal forms, orthorhombic and hexagonal, in the presence of cobalt hexammine chloride, a known inducer of the left-handed Z-form of DNA. The crystal structures have been solved and refined at 1.71 A resolution in space group P2(1)2(1)2(1) and 2.0 A resolution in space group P6(5). The orthorhombic structure contains one Z-DNA hexamer duplex, while the hexagonal structure contains two hexamer duplexes in the structure. Of the latter, one is situated on a crystallographic sixfold screw axis, leading to disorder. This paper reports the effects of sequence and crystal packing on the structure of Z-type DNA. The structures lend additional support to the authors' earlier conclusion that a stretch of four C.G base pairs is sufficient to nucleate and define the regular model of the left-handed helix based on the structure of d(CGCGCG)2.

We report here the crystal structure of the DNA hexamer duplex d(CGCGCA)*d(TGCGCG) at 1.71 A resolution. The crystals, in orthorhombic space group, were grown in the presence of cobalt hexammine, a known inducer of the left-handed Z form of DNA. The interaction of this ion with the DNA helix results in a change of the adenine base from the common amino tautomeric form to the imino tautomer. Consequently the A:T base pair is disrupted from the normal Watson-Crick base pairing to a 'wobble' like base pairing. This change is accommodated easily within the helix, and the helical parameters are those expected for Z-DNA. When the cobalt hexammine concentration is decreased slightly in the crystallization conditions, the duplex crystallizes in a different, hexagonal space group, with two hexamer duplexes in the asymmetric unit. One of these is situated on a crystallographic 6-fold screw axis, leading to disorder. The tautomeric shift is not observed in this space group. We show that the change in inter-helix interactions that lead to the two different space groups probably arise from the small decrease in ion concentration, and consequently disordered positions for the ion.

The crystal structure of DsrD from Desulfovibrio vulgaris Hildenborough has been determined at 1.2 A resolution. DsrD is in a dimeric form in the crystal, and five sulfate anions were located on the surface. The structure of DsrD comprises a winged-helix motif, which shows the highest structural homology to similar motifs found in Z-DNA binding proteins and some B-DNA binding proteins. The core structure of the molecule is constructed by intramolecular interactions of hydrophobic residues, which are well conserved in DNA binding proteins, suggesting that these proteins belong to the same superfamily on the basis of the structure. These results indicate a possible role of DsrD in transcription or translation of genes for enzymes catalyzing dissimilatory sulfite reduction.

The crystal structure of the heptamer rGd(CGCGCG) has been determined at 1.54 A resolution with R(work) and R(free) of 0.191 and 0.235, respectively. The crystal belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 17.96, b = 31.47, c = 44.73 A and two independent strands in the asymmetric unit. The chimera forms a Z-DNA hexamer duplex d(CGCGCG)(2), with the 5'-overhang rG invisible in the density. The replacement of rG in the 5' terminus of d(GCGCGCG) changes the reverse Hoogsteen G.G base pairing of the 5'-overhang dG (Pan et al., 1997). However, the replacement does not change the helix from Z-form to A-form or the water-structure motifs in the Z-DNA double-helical structure.

The crystal structure of a hexameric DNA fragment with the sequence d(TGCGCA)(2) has been solved and refined at 293 K at a resolution of 1.64 A. The molecule adopts a left-handed Z-type helical conformation which is common in alternating pyrimidine-purine sequences. The presence of A.T base pairs at the two terminals does not perturb the structure to any great degree. However, several sequence-specific microstructural changes are noticeable. The structure of the identical sequence determined at 120 K involving somewhat different crystallization conditions has been reported previously [Harper et al. (1998), Acta Cryst. D54, 1273-1284]. A comparison of the present structure with that at low temperature and with that of d(CGCGCG)(2) shows that the effect of the change in sequence is greater than the combined effect of changes in temperature and environment.

Figure 2. (A) The kex values of the G4b imino proton for the CA6ZαADAR1 complex determined at 25 C and (B) kex values of the G2b imino proton for the TA6-ZαADAR1 complex determined at 15 C as a function of the fZ. Black solid lines are the best fit to Eq. 1, where the kex data were weighted by the inverse of their variance. The grey lines indicate their upper and lower confidence limits (95% confidence level). Z-DNA contains nucleic acid bases in alternating antiand syn-conformations along the nucleotide chain and has only one groove that is similar to the minor groove of B-DNA. Z-DNA is in a higher energy conformation than B-DNA and is stabilized by negative supercoiling generated in vivo. Human ADAR1 has two left-handed Z-DNA binding domains at its NH2-terminus, Zα and Zβ, preferentially binds Z-DNA, rather than B-DNA, with high binding affinity. The co-crystal structure of the Zα domain of human ADAR1 (ZαADAR1) bound to Z-DNA revealed that one monomeric ZαADAR1 domain binds to one strand of double-stranded DNA and a second ZαADAR1 monomer binds to the opposite strand with two-fold symmetry with respect to the DNA helical axis. A structural study showed that ZαADAR1 binds to the Z-conformation of non-CG-repeat DNA duplexes through a common structural feature rather than by a specific sequence or structural alternations. A previous NMR study on a d(CGCGCG)2-ZαADAR1 complex suggests an active-mono B-Z transition mechanism (see Fig. 1) in which the ZαADAR1 protein first binds to B-DNA and then converts it to left-handed Z-DNA, a conformation that is then stabilized by the additional binding of a second ZαADAR1 molecule. Recently, we have reported NMR hydrogen exchange data of complexes between ZαADAR1 and the non-CG-repeat DNA duplexes, d(CACGTG)2 [referred to as CA6] or d(CGTACG)2 [referred to as TA6], with a variety of protein-to-DNA (P/N) molar ratios. The kex data for the G4b of the CA6-ZαADAR1 complex and for the G2b of the TA6-ZαADAR1 complex showed significant changes as the Z-DNA fraction (fZ) was increased (meaning that the P/N ratio increased) (see Fig. 2). These changes of the kex data can be explained by the presence of mixtures of two imino protons from B-form DNA (referred to as B) and B-DNA-ZαADAR1 complex (referred to as BP) in the imino peaks as given by Eq. 1

This work is part of our project aimed at characterizing metal-binding properties of left-handed Z-DNA helices. The three Cr3+ cations found in the asymmetric unit of the d(CGCGCG)2–Cr3+ crystal structure do not form direct coordination bonds with atoms of the Z-DNA molecule. Instead, the hydrated Cr3+ ions are engaged in outer-sphere interactions with phosphate groups and O6 and N7 guanine atoms of the DNA. The Cr3+(1) and Cr3+(2) ions have disordered coordination spheres occupied by six water molecules each. These partial-occupancy chromium cations are 2.354(15) Å apart and are bridged by three water molecules from their hydration spheres. The Cr3+(3) cation has distorted square pyramidal geometry. In addition to the high degree of disorder of the DNA backbone, alternate conformations are also observed for the deoxyribose and base moieties of the G2 nucleotide. Our work illuminates the question of conformational flexibility of Z-DNA and its interaction mode with transition-metal cations. Electronic supplementary material The online version of this article (doi:10.1007/s00775-015-1247-5) contains supplementary material, which is available to authorized users.

The Zα domains of human ADAR1 (Zα ADAR1 ) bind to Z-DNA via interaction mediated by the α3-core and β-hairpin. Five residues in the α3 helix and four residues in the β-hairpin play important roles in Zα function, forming direct or water-mediated hydrogen bonds with DNA backbone phosphates or interacting hydrophobically with DNA bases. To understand the roles of these residues during B–Z transition of duplex DNA, we performed NMR experiments on complexes of various Zα ADAR1 mutants with a 6-bp DNA duplex at various protein-to-DNA molar ratios. Our study suggests that single mutations at residues K169, N173, or Y177 cause unusual conformational changes in the hydrophobic faces of helices α1, α2, and α3, which dramatically decrease the Z-DNA binding affinity. 1D imino proton spectra and chemical shift perturbation showed that single mutations at residues K170, R174, T191, P192, P193, or W195 slightly affected the Z-DNA binding affinity. A hydrogen exchange study proved that the K170A- and R174A-Zα ADAR1 proteins could efficiently change B-DNA to left-handed Z-DNA via an active B–Z transition pathway, whereas the G2·C5 base pair was significantly destabilized compared to wild-type Zα ADAR1 .

Spermine and [Co(NH3)6]3+ are known to induce the B → Z transition in poly(dG-dC)·poly(dG-dC) DNA. In this paper gel electrophoresis shift assays using the long DNA poly(dG-dC)·poly(dG-dC) are developed to complement circular dichroism (CD) measurements of the conformation of the DNA in the presence of amines including spermine, spermidine, [Co(NH3)6]3+, (+)-[Co(en)3]3+ and (−)-[Co(en)3]3+ (en: ethylenediamine). CD is used to monitor the average conformation of the DNA and the gels show when there is a population of a given type of DNA in the solution. Spermidine is found to induce the transition at very low ionic strength, in contrast to literature wisdom. The cobalt amine-induced Z-DNA melts, with a broad transition, at a lower temperature than B-DNA. The electrophoresis data shows the presence of a DNA more flexible than B-DNA at low amine concentrations. This is ascribed to the existence of B/Z junctions. At higher amine concentrations, a DNA (Z-DNA) that is stiffer than B-DNA is observed. The Z-DNA induced by spermine is stiffer (runs more slowly on the gels) than that induced by the other amines. The spermine–DNA interaction is found to be very cooperative, whereas that of the cobalt amines is uncooperative. The kinetics of the B → Z transition with spermine at room temperature is very slow, whereas the cobalt amines induce it within seconds.

Alternating pyrimidine-purine sequences typically form Z-DNA, with the pyrimidines in the anti and purines in the syn conformations. The observation that dC and dT nucleotides can also adopt the syn conformation (i.e. the nucleotides are out-of-alternation) extends the range of sequences that can convert to this left-handed form of DNA. Here, we study the effects of placing two adjacent d(G*C) base pairs as opposed to a single d(G*C) base pair or two d(A*T) base pairs out-of-alternation by comparing the structure of d(m5CGGCm5CG)2with the previously published structures of d(m5CGGGm5CG)*d(m5CGCCm5CG) and d(m5CGATm5CG)2. A high buckle and loss of stacking interactions are observed as intrinsic properties of the out-of-alternation base pairs regardless of sequence and the context of the dinucleotide. From solution titrations, we find that the destabilizing effect of out-of-alternation d(G*C) base pairs are identical whether these base pairs are adjacent or isolated. We can therefore conclude that it is these intrinsic distortions in the structure of the base pairs and not neighboring effects that account for the inability of out-of-alternation base pairs to adopt the left-handed Z conformation.

The interaction of 1:2 copper(II) complexes of 1,10-phenanthroline (OP) and variously methyl-substituted phenanthrolines with calf thymus DNA has been investigated by viscometry and spectral and electrochemical techniques. Viscometry and competitive ethidium bromide (EthBr) emission studies reveal that substitutions at 4- and 4,7-positions confer the complex a reduced affinity for DNA via partial intercalative interaction of the middle ring of OP between the base pairs of DNA, while substitutions at 5- and 5,6-positions confer a weak affinity toward DNA. The tetramethyl substitution at 3,4,7,8-positions lead to an intermediate behavior for the complex. Circular dichroism spectral studies of the interaction disclose for the first time that, of all the complexes, the 5,6-dimethyl-OP complex is a unique and remarkable reagent in that it reversibly binds to DNA and effects the important conversion of right-handed B DNA to left-handed Z DNA even in the presence of EthBr, an allosteric effector of the B conformation of DNA. This novel conformational transition is unexpected of the low GC content of natural DNA. The ratios of the binding constants (K(+)/K(2+)) for DNA binding of the Cu(I) and Cu(II) forms of the redox active OP complexes rather than the Cu(II)/Cu(I) redox potentials (0.023 to -0.098 V vs SCE) are good measures of the substituent dependent DNA cleavage efficiency and rate. They also reveal that the Cu(I) form of OP and 4-methyl- and 5,6-dimethyl-substituted OP complexes displays enhanced affinity to bind but noncovalently to the minor groove of DNA. Attempts have been made to illustrate the cleavage rate and efficiency of nucleolytic reactions in the light of the relative binding constants of Cu(I) and Cu(II) forms of the OP complexes.

The salt-induced B- to Z-DNA conformational transition is a cooperative- and time-dependent process. From a modified form of the logistic equation which describes an equilibrium between two states we have deduced a kinetic function to quantify the degree of the B to Z transition of a synthetic (dG-dC) ⋅ (dG-dC) polynucleotide. This function was obtained by introduction of time as a variable in the logistic function so that the equilibrium constant, K, is replaced by a new constant K s , characteristic of the type of salt used. This constant is defined as the salt concentration needed to reach the B-Z transition-midpoint in the time unit. The equation fits the data obtained by circular dichroism (CD) for changes in molecular ellipticity of poly(dG-m5dC) ⋅ poly(dG-m5dC) and poly(dG-dC) ⋅ poly(dG-dC) incubated with various concentrations of mono-, di-, and trivalent salts at a constant temperature. The derived expression may be a very useful tool for studying the kinetics of the B- to Z-DNA transition.