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This article contains detailed synthetic protocols for the preparation of DNA oligonucleotides containing 8-trifluoromethyl-2'-deoxyguanosine (CF3 dG) and their application to observe Z-DNA structure in vitro and in living HeLa cells. First, using a catalytic system consisting of FeSO4 , H2 SO4 , and H2 O2 in DMSO, we achieved a one-step synthesis of CF3 dG through a radical reaction between deoxyguanosine (dG) and CF3 I, with a yield of 45%. We then obtained the 3'-phosphoramidite of CF3 dG through a routine three-step procedure. Next, we employed the CF3 dG phosphoramidite monomer in the synthesis of oligonucleotides on a solid-phase DNA synthesizer. Finally, we used the CF3 dG-modified DNA oligonucleotides to observe Z-DNA structure in vitro and in living HeLa cells through 19 F NMR spectroscopy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Synthesis of CF3 dG phosphoramidites Basic Protocol 2: Preparation of CF3 dG-modified DNA oligonucleotides Basic Protocol 3: Evaluation of CF3 dG stabilization of Z-DNA structure by CD spectroscopy Basic Protocol 4: Investigation of Z-DNA structure in vitro and in HeLa cells with CF3 dG-modified DNA oligonucleotides and 19 F NMR spectroscopy.

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.

Dimerization of a hydroxycarbazole produces an axially chiral biaryl, BICOL (2). One enantiomer (R)-2, is capable of enantioselective binding to different polymorphs of DNA. The biaryl (R)-2 was shown by fluorescence and circular dichroism to induce a shift of Z-DNA to B-DNA. The opposite enantiomer (S)-2 shows no specific binding. The significant difference in behaviour between the two enantiomers (S)-2 and (R)-2 is in line with molecular modelling studies which show two very different binding geometries between the enantiomers with each polymorph of DNA. Copyright © 2010 John Wiley & Sons, Ltd.

The Zα domain of human ADAR1 (Zα(ADAR1)) preferentially binds Z-DNA rather than B-DNA with high binding affinity. Zα(ADAR1) binds to the Z-conformation of both non-CG-repeat DNA duplexes and a d(CGCGCG)(2) duplex similarly. We performed NMR experiments on complexes between the Zα(ADAR1) and non-CG-repeat DNA duplexes, d(CACGTG)(2) or d(CGTACG)(2), with a variety of protein-DNA molar ratios. Comparison of these results with those from the analysis of d(CGCGCG)(2) in the previous study suggests that Zα(ADAR1) exhibits the sequence preference of d(CGCGCG)(2)≫d(CACGTG)(2)d(CGTACG)(2) through multiple sequence discrimination steps during the B-Z transition.

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 conformational transition from right-handed B-DNA to left-handed Z—DNA—the B-Z transition—has received increased attention recently because of its potential roles in biological systems and its applicability to bionanotechnology. Though the B-Z transition of poly(dG-dC) · poly(dG-dC) is inducible under high salt concentration conditions (over 4 M NaCl) or by addition of multivalent cations, such as hexaamminecobalt(III), no cationic polymer were known to induce the transition. In this study, it is shown by circular dichroism and UV spectroscopy that the cationic comb-type copolymer, poly(L-lysine)-grafi,dextran, but not Poly ( L -lysine) homopolymer or a basic peptide, induces the B-Z transition of poly(dG-dC) · poly(dG-dC). At a cationic amino group concentration of 10 -4 M the copolymer stabilizes Z-DNA. The transition pathway from the B to the Z form is different to that observed previously. We speculate that the cationic backbone of the copolymer, which reduces electrostatic repulsion among DNA phosphate groups, and the hydrophilic dextran chains, which reduce activity of water, cooperate to induce the B-Z transition. The copolymer specifically modified the micro-environment around DNA molecules to induce Z-DNA formation through stable and spontaneous inter-polyelectrolyte complex formation.

The interaction of berberine, palmatine, and coralyne with the B, Z, and H(L) form of poly[d(G-C)] was studied. Berberine and palmatine showed moderate binding to the B form, while coralyne showed higher binding, as revealed from spectroscopic and thermodynamic data. Berberine and coralyne binding to the B form was exothermic and enthalpy-driven, while palmatine showed exothermic binding which was favored by both negative enthalpy and negative entropy changes. Berberine and palmatine neither bind nor converted the Z-form structure to B form. Coralyne, on the other hand, exhibited a strong binding affinity to Z DNA structure that was enthalpy-driven. Berberine binding to the H(L) form was cooperative, exothermic, and favored by both negative enthalpy and negative entropy changes with the formation of an induced CD band. Palmatine showed weak binding, while coralyne showed a strong binding with the H(L) form. The structural differences in the isoquinoline alkaloids appear to influence the affinity and mode of interactions with these polymorphic DNA structures.

The stabilization of left-handed Z-DNA double strands by chemical modifications is especially of interest regarding investigations of its biological role. Incorporation of modified nucleotide building blocks in DNA allows recognition studies under physiological conditions without the need for a Z-DNA inducing environment. Approaches to enforce the Z-DNA double helix using guanosine derivatives are the introduction of sterically demanding groups at guanine C8 or of 2′-ribosyl substitutions determining the ribosyl conformation. 8-Bromo-2′-ethynyl-arabino-deoxyguanosine was synthesized as phosphoramidite building block. Incorporated in suitable oligonucleotides the potential to induce the Z-form DNA was investigated by CD spectroscopy. With a nucleotide containing the 8-bromo and the 2′-ethynyl group a synergistic effect of the two modifications was expected to provide stronger stabilization of Z-DNA. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

Charge transfer through DNA is of great interest because of the potential of DNA to be a building block for nanoelectronic sensors and devices. The photochemical reaction of 5-halouracil has been used for probing charge-transfer processes along DNA. We previously reported on unique charge transfer following photochemical reaction of 5-bromouracil within four-base π-stacks in Z-DNA. In this study, we incorporated a guanosine instead of a deoxyguanosine into Z-DNA, and found that electron transfer occurs in a different mechanism through four-base π-stacks.

It is shown that the phosphate-methylated DNA miniduplex d(CpG)2 adopts a left-handed Z-type structure, whereas the phosphate-methylated miniduplex d(GpC)2 is right-handed. Furthermore, it is shown that elongation of the systems to the tetramer level results in right-handed duplexes with rather low stability. The phosphate-methylated hexamer d(CpGpCpGpCpG) is present in the single-strand form. It is suggested that these results are of interest in the understanding of the exact role of salt cations in stabilizing the Z-DNA structure of natural duplexes d(CpG…CpG)2 in high salt solution, since the methylated phosphate groups in fact mimic the situation of complete phosphate-charge shielding. Therefore, it is concluded that exclusive cation-phosphate complexation in the d(CpG)2 segments stabilizes the Z structure. 31P-NMR experiments on the natural duplex d(CpGpCpGpCpG)2 at different concentrations of Mg2+ provided independent proof for the proposed model.

Important intrinsic characteristics of the rotating frame nuclear Overhauser effect spectroscopy (ROESY) experiment were found to be advantageous in DNA solution structure determination. In a ROESY experiment, the different mechanisms of relaxation result in different signs of cross peaks, enabling a clear distinction between H2' resonances and H2" resonances of the DNA sugar backbone. This method is of particular importance in crowded spectra, for purine resonances whose H2', H2" protons typically resonate closely, as well as in conditions where line broadening makes coupling constants in a correlated spectroscopy experiment impossible to determine. By observing the signs of cross peaks in the base proton to H2', H2" sugar proton region, the ROESY spectrum can be used to distinguish A-form, B-form, and Z-form DNA.

A novel competition dialysis method was used to study the structural selectivity of the nucleic acid binding of NB-506, a promising indolocarbazole anticancer agent. A pronounced preference for NB-506 binding to the DNA triplex poly [dA]:(poly[dT])2 was observed among potential binding to 12 different nucleic acid structures and sequences. Structures included in the assay ranged from single-stranded DNA, through a variety of right-handed DNA duplexes, to multistranded triplex and tetraplex forms. RNA and left-handed Z DNA were also included in the assay. The preferential binding to triplex was confirmed by UV melting experiments. The novel and unexpected structural selectivity shown by NB-506 may arise from a complementary shape between its extended aromatic ring system and the planar triplex stack.

The Z alpha domain of the human RNA editing enzyme double-stranded RNA deaminase I (ADAR1) binds to left-handed Z-DNA with high affinity. We found by analytical ultracentrifugation and CD spectroscopy that two Z alpha domains bind to one d(CG)3T4(CG)3 hairpin which contains a stem of six base pairs in the Z-DNA conformation. Both wild-type Z alpha and a C125S mutant show a mean dissociation constant of 30 nM as measured by surface plasmon resonance and analytical ultracentrifugation. Our data suggest that short (> or = 6 bp) segments of Z-DNA within a gene are able to recruit two ADAR1 enzymes to that particular site.

Nucleic acids control the expression of genes, and different conformations of DNA structure may regulate cell death. Left-handed Z-DNA, which is speculated to function as a transcriptional enhancer, may be directly influenced by the destructive effects of terminal differentiation. The nicking-denaturation of double-stranded Z-DNA could possibly initiate and enhance terminal differentiation within specific tissues.

The interaction of two DNA octamers, d(m5CG)4 and d(GGAATTCC), with the polyamines spermine4+ and spermidine3+, has been studied by means of 1H-nmr nuclear Overhauser effect (NOE) difference measurements. The experiments were performed at 10°C and for a polyamine charge to DNA charge (i.e., phosphate) ratio of 0.4, where the solution of d(m5CG)4 contains about 50% Z-form of the DNA. The results show that the polyamine intramolecular NOEs for the protons on the propyl chains are similarly negative with the two oligonucleotides, while those on the butyl chain show slightly more negative NOE with d(m5CG)4 than with d(GGAATTCC). The fully N-methylated analogues of spermine (Me10Spn4+) and spermidine (Me8Spd3+) as well as the diamines 1,3-diaminopropane (DAP2+) and 1,4-diaminobutane (putrescine2+) have been studied for the ability to transform d(m5CG)4 from the B- to the Z-form. 1H-nmr spectra showed the order spermine4+ > spermidine3+ > Me10Spn4+ > Me8Spd3+ > 1,3-diaminopropane2+ > putrescine2+, with spermine showing the largest relative amount of Z-DNA. 1H-nmr pulsed-gradient self-diffusion measurements of the triamines showed a large difference in the interaction of Spd and Me8Spd with the two different duplexes. With the same duplex (either of the two), however, no difference between Spd and Me8Spd can be seen. Within a two-state model this is interpreted as a larger fraction of bound polyamines with d(m5CG)4 than with d(GGAATTCC). © 1999 John Wiley & Sons, Inc. Biopoly 49: 41–53, 1999

Bovine zeta-crystallin has the ability to bind with different DNAs. Initially, this protein was named regulatory factor 36 (Kang et al. , 1985), but it has been shown to be an ocular lens zeta-crystallin (Jornvall et al. , 1993), which is considered an enzyme-crystallin (Rodakanaki et al. , 1989). The enzyme-linked immunosorbent assay (ELISA) was used to quantitate the binding of bovine zeta-crystallin to purified high molecular weight double-stranded (ds-) and single-stranded (ss-) DNA (bovine and synthetic DNA). ELISA quantitation was achieved by the addition of anti-zeta-crystallin antibodies to the DNA–zeta-crystallin complex, using a novel immunochemical avidin–biotin method. Zeta-crystallin shows much greater binding intensity for ss-DNA and for ds-Z-DNA than for ds-B-DNA. It also reacts slightly more with ds-Z-DNA than ss-DNA. Therefore, we speculate that zeta-crystallin may act as a transcriptional enhancer (outer lens cortex), possibly binding to Z-DNA regulatory elements within lens crystallin genes. It may also act to protect DNA from endogenous DNase activity and as a DNA unwinding (destabilizing) protein also involved with transcription, occurring in normal adult bovine lens nucleated secondary fiber cells.

The Z-DNA forming potentiality of native calf thymus DNA using spectrophotometric measurements and diethyl pyrocarbonate reactivity have been investigated. The [image omitted] value was evaluated from absorbance ratio for B- and Z-DNA and compared with the values obtained for native DNA. The results suggest that native DNA undergoes B- to Z-/Z-analogous transition to an extent of 20 per cent under constraint conditions. Possible Z-DNA formation was also characterized by hyper-reactivity of native DNA towards diethyl pyrocarbonate in aqueous solvent. The thermal melting profile of native and modified DNA and nuclease S1 digestibility data, in addition, reflects B- to Z-/Z-analogous transition in native DNA.

We review the effect of sequence on the structure of left-handed Z-DNA in single crystals. The various substituent groups that define a nucleotide base as guanine, cytosine, thymine, or adenine affect both the DNA conformation and the organization of solvent around the duplex. These are discussed in terms of their effect on the ability of sequences to adopt the unusual Z-DNA structure. In addition, the experimental and theoretical methods used to treat DNA hydration are discussed as they relate to the stability of Z-DNA. Finally, we argue that Z-DNA, as defined by the crystal conformation, is sufficient in itself to account for the physical properties of left-handed conformations observed in polymers and in genomic sequences. © 1997 John Wiley & Sons, Inc. Biopoly 44: 65–90, 1997

Native calf thymus DNA and poly(dA-dT).poly(dA-dT) were photo-adducted with 8-methoxypsoralen and characterized by thermal denaturation (Tm) and hydroxyapatite column chromatography. The data demonstrated the formation of interstrand photo-crosslinks. It has been shown by competition ELISA and band shift assays that crosslinked species of DNA-8-MOP and poly(dA-dT)-8-MOP photoadducts recognize previously defined monoclonal anti-Z-DNA antibody (Z22). The results indicate the possible presence of Z- or Z-like epitopes on nucleic acid-8-MOP crosslinks as Z22 antibody does not recognize other nucleic acid conformations. These studies also point out that conformational changes in DNA arising from the photo-addition could induce antibodies to DNA or could cause autoimmune disease.

This paper deals with the crystal structure of d(CG)3-spermidine complex. The DNA fragment, d(CG)3, was crystallized with N-(2-amino-propyl)-1,4-diamino-butane, PA(34), spermidine. The results of its X-ray crystallographic analysis showed many intermolecular contacts between d(CG)3 and spermidine, but the binding mode of spermidine to the d(CG)3 molecule is different from that of the d(CG)3 and N-(2-amino-ethyl)-1,4-diamino-butane [PA(24)]complex: a spermidine molecule bound to the d(CG)3 and its symmetrically related neighboring d(CG)3 molecules through the water molecules with hydrogen bonds, while one PA(24) molecule connected directly to one d(CG)3 molecule, but not to its neighboring d(CD)3 molecule. In the crystal, the d(CG)3 molecule was the left-handed Z-form, and three magnesium cations and a sodium cation were observed around the d(CG)3 moiety with different binding modes from the case of the d(CG)3-PA(24) complex.

The transcriptional control region of the Rouse sarcoma virus long terminal repeats (LTR) was shown to contain enhancer and promoter elements located within 200 base pairs upstream from the transcription initiation site [Cullen et al. (1985) Mol. Cell. Biol. 5, 438–447]. Deletion of these elements results in significant loss of LTR transcriptional activity. In the present paper it is shown that a short alternating purine-pyrimidine sequence can restore the constitutive activity of the Rouse sarcoma virus LTR in the absence of upstream elements when inserted in close proximity to the transcription initiator site. The possible molecular bases of this phenomena are discussed.

This unit describes basic protocols on using the non-B DNA Motif Search Tool (nBMST) to search for sequence motifs predicted to form alternative DNA conformations that differ from the canonical right-handed Watson-Crick double-helix, collectively known as non-B DNA and on using the associated PolyBrowse, a GBrowse (Stein et al., 2002) based genomic browser. The nBMST is a web-based resource that allows users to submit one or more DNA sequences to search for inverted repeats (cruciform DNA), mirror repeats (triplex DNA), direct/tandem repeats (slipped/hairpin structures), G4 motifs (tetraplex, G-quadruplex DNA), alternating purine-pyrimidine tracts (left-handed Z-DNA), and Aphased repeats (static bending). Basic protocol 1 illustrates different ways of submitting sequences, the required file input format, results comprising downloadable Generic Feature Format (GFF) files, static Portable Network Graphics (PNG) images, dynamic PolyBrowse link, and accessing documentation through the Help and Frequently Asked Questions (FAQs) pages. Basic Protocol 2 illustrates a brief overview of some of the PolyBrowse functionalities, particularly with reference to possible associations between predicted non-B DNA forming motifs and disease causing effects. The nBMST is versatile, simple to use, does not require bioinformatics skills, and can be applied to any type of DNA sequences, including viral and bacterial genomes, up to 20 megabytes (MB).

The Z form of poly [d(G-m5C)2], in presence of Mg2+ ion, is found to be transformed into B form upon interaction with 4',6-diamidino-2-phenylindole (DAPI). The Z --> B transformation is complete at a mixing ratio of about 0.07 DAPI per DNA base pairs, i.e., each DAPI molecule may be related to the conversion of 6-7 base pairs. An interaction between DAPI and poly [d(G-m5C)2] in its Z form at low drug: DNA ratios is suggested from optical dichroism and time-resolved luminescence anisotropy results. The spectroscopic behaviour of DAPI indicates that the Z conformation of DNA does not provide normal binding sites for DAPI, such as groove or intercalation sites, but that the initial association may be of external nature. (C) 1993 John Wiley & Sons, Inc.

When human U937 cells are placed in agarose microbeads and treated with a detergent, the cytoplasmic membrane is lysed and the nuclear membrane is permeabilized. However, the nuclei remain intact and maintain both replication and transcription. Biotin labeled monoclonal antibodies against Z-DNA have been diffused into this system and used to measure the amount of Z-DNA present in the nuclei. It has previously been shown that the amount of Z-DNA present decreases due to relaxation by topoisomerase I and increases as the level of transcription increases. Here we measure the formation of Z-DNA in the c-myc gene by crosslinking the antibodies to DNA using laser radiation at 266 nm for 10 ns. The crosslinked DNA is isolated by restriction digestion, separation of antibody labeled fractions through the biotin residue, and subsequent proteolysis to remove the crosslinked antibody. Three AluI restriction fragments of the c-myc gene are shown to form Z-DNA when the cell is transcribing c-myc. The Z-DNA forming segments are near the promoter regions of the gene. However, when U937 cells start to differentiate and transcription of the c-myc gene is down-regulated, the Z-DNA content goes to undetectable levels within 30-60 min.

The left-handed Z-DNA structure of an araC-containing (where araC stands for arabinosylcytosine) hexamer, (araC-dG)3, has been solved by x-ray diffraction analysis at 1.3 A resolution. This hexamer was crystallized in the hexagonal P6522 (a = b = 17.96 A, c = 43.22 A) space group in which the hexamers have statistically disordered packing arrangement along the 65 screw axis, yet the crystals diffract x-rays to high resolution. Its structure has been refined by the constrained least square refinement to a final R factor of 0.287 using 737 [> 3.0 σ(F)] observed reflections. The asymmetric unit of the unit cell contains only a dinucleotide, 5′-p(araC)p(dG). The overall conformation resembles that of the canonical Z-DNA, but with some differences in details. The O2′ hydroxyl groups of the araC residues form intramolecular hydrogen bonds with N2 of the 5′-guanine residues. In the deep groove of Z-DNA, these hydroxy groups replace the bridging water molecules that stabilize the guanine in the syn conformation. The results reinforce the earlier observation made by the structural analysis of another hexamer, d(CG[araC]GCG), with a mono-substitution of araC [M.-K. Teng, Y.-C. Liaw, G. A. van der Marel, J. H. van Boom, and A. H.-J. Wang (1989) Biochemistry, vol. 28, pp. 4923–4928]. These two structures show that araC residue can be incorporated readily into the Z structure and probably facilitates the B to Z transition, as supported by uv absorption spectroscopic studies in a number of araC-containing oligonucleotides. The potential biological roles of the araC-modified Z-DNA are discussed. © 1992 John Wiley & Sons, Inc.

In previous work, it was shown that poly [d(AC) · d(GT)] could be forced into the Z form by strong dehydrating conditions, provided EDTA was not present. Presumably multivalent impurities were also necessary for the transition. In order to gain control over the B to Z transition for this DNA, we carefully removed all divalent contaminants from the sample and asked the obvious question: What ions are necessary for the transition under dehydrating conditions? We systematically investigated the effect of various multivalent ions. The common contaminants Ca2+, Mg2+, and Fe3+ will not cause the transition, but Co2+ and Ni2+ facilitate the transition, undoubtedly because of their well-known propensity to bind to purine N7. Since the transition also depends on the synergistic dehydrating action of sodium perchlorate and ethanol, we include CD spectra for the independent variations of these two factors. In addition, vacuum-uv CD spectra for the A form and various B forms of poly [d (AC) · d (GT)] are presented for the first time.

The DNA fragment d(CG)3 was co-crystallized with N-(2-aminoethyl)-1,4-diaminobutane (PA(24)), a chemically synthesized polyamine. The complex crystal contained one polyamine, 3 magnesium cations and one sodium cation per duplex of d(CG)3, and well diffracted the X-ray intensities up to 1.0 Å resolution. The d(CG)3 took a left-handed Z-DNA conformation, and the PA(24) molecule electrostatically interacted with the phosphate groups of the d(CG)3 duplex.

Modeling, x-ray diffraction, and solution studies have contributed to the understanding of interactions between polyamines and nucleic acids. Polyamines stabilize a variety of unusual DNA structures and conformations in vitro, including both the left-handed Z and the right-handed A DNA. In addition, polyamines condense DNA and may be important in bending specific sequences. Investigations into the mechanisms of these effects provide support for both specific and nonspecific interactions between polyamines and DNA. Although exact relationships between the binding of polyamines and conformational changes in nucleic acids are still being clarified, polyamines remain important candidates for regulators of DNA conformation in vivo.

It was shown that the complex of osmium tetroxide with N,N,N',N'-tetramethylethylendiamine can be applied as a probe of DNA structure in the cell. This probe site-specifically recognized structural distortions at the B-Z junctions in plasmids pRW751 and pPK1 (containing (dC-dG)n. segments) in E. coli cells.

Polarized electronic absorption spectra of the (100) face of single crystals of the Z-form double helical duplex of d(m5CGUAm5CG) have been obtained from Kramers-Kronig analysis of reflection data. The c crystallographic axis is parallel to the helix axis and shows but weak absorption. The b axis is perpendicular to the helix axis and shows a structureless absorption band centered at 270 nm with an oscillator strength of 0.26. Calculations of the crystal spectra utilizing available transition moment data for the individual chromophores are carried through using the oriented gas model (no interbase interactions) and, again, employing all base-base interactions (point dipole) in the duplex. The calculated hypochromism of the 270 nm band is much less than the experimental value obtained from the crystal data. The crystal spectra appear to be representative of Z-form double helices of essentially infinite length and not of a collection of twelve base duplexes. No evidence for n pi* transitions polarized parallel to the helix axis is found.

Dielectric relaxation peak due to bound water was found around 100 MHz in poly(dG-dC).poly(dG-dC) and calf thymus DNA in water-ethanol mixtures with NaCl buffer. Relaxation time and strength show a transition for poly(dG-dC).poly(dG-dC) at an ethanol composition Cw = 0.45 (w/w) where the structural transition from B- to Z-DNA takes place. It has been suggested that the transition is caused by removal of the bound water molecules preferentially from the phosphate groups. If the bound water molecules are removed equally from the phosphate groups and the grooves, the structural transition from B to A takes place. By analogy with hydration of tropocollagen, it was found that 19 water molecules per one nucleotide are at least necessary to keep B-DNA. Thirteen molecules are bound to A-DNA and 9 molecules to Z-DNA. Stringlike multimers are proposed as available structures of the bound water.

Fifty years after the discovery of the double helical structure of DNA, the complete sequence of the human genome has been determined. All genetic information, which is necessary for life, is written in 30 billion base pairs of DNA. Many diseases, including cancer, hereditary and viral diseases, can be understood at the DNA sequence level. Local DNA conformations are thought to play an important role in biological processes such as gene expression. Therefore, DNA sequences and local DNA conformation are targets of novel drugs that would precisely switch certain genes on or off. Modified bases that perform various functions can also be incorporated into defined DNA sequences. Since DNA can be amplified by PCR and in other organisms, DNA becomes a promising unit for nanotechnology applications. In this review, we focus on the sequence and conformation-specific chemical reactions that occur in DNA, and the prospective uses of the chemical biology of DNA will be discussed.

Topological characteristics of DNA and specifically DNA supercoiling influence all major DNA transactions in living cells. DNA supercoiling induces the formation of unusual secondary structure by specific DNA repeats which can also affect DNA functioning. Keywords: supercoiling; knots; catenanes; Z-DNA; triplexes; cruciforms

The left-handed form of poly (dG-m5dC).poly(dG-m5dC) induced by heating the copolymer in the presence of magnesium and stabilized with polyarginine can be used to raise antibodies in rabbits. These antibodies are able to recognize the Z conformation of both methylated and nonmethylated forms of the copolymers. In the same experimental conditions, hypermethylated B DNA is not recognized by these antibodies.

The B and the Z forms of the DNA hexamers d(m5C-G)3 and d(br5C-G)3 were investigated by means of NMR spectroscopy. It is demonstrated that the low-salt form of d(m5C-G)3 is a B DNA structure. The form, which becomes increasingly predominant when increasing amounts of MgCl2 and/or methanol are added to the solution, has Z DNA characteristics. It is shown that the major geometrical features of the Z form of d(m5C-G)3 in the crystal structure are maintained in solution, with the dC residues S sugar conformation, gamma + and the base in the anti orientation and the dG residues N (except the 3'-terminal residue), gamma t and syn. Neither the Z form of the methylated nor that of the brominated compound resembles the Z' form, in which the deoxy guanosine sugar rings adopt a C1'-exo conformation. Substitution of m5C by br5C causes no perceptible conformational changes in either the B or in the Z forms.

Prokaryotic, eukaryotic and mitochondrial DNA sequences of total Length 300000 nucleotides have been analyzed to find out whether stretches of alternating purines and pyrimidines are unusual in terms of occurrence, composition and base sequence. Alternating runs longer than 5 nucleotides are significantly underrepresented in the natural sequences as compared to random ones. Octanucleotides are the most deficient, occurring at only 60% of the frequency expected in random sequences. An unexpectedly high proportion of these octamers consists of alternating tetramers with the repeat structure (PuPyPuPy)2or (PyPuPyPu)2. DNA stretches containing such sequences can potentially form a Sl nuclease sensitive slippage (staggered loop) structure, which might serve as a locally unstacked intermediate in the B- to Z-DNA conformational transition.

The ease with which a particular DNA segment adopts the left-handed Z-conformation depends largely on the sequence and on the degree of negative supercoiling to which it is subjected. We describe a computer program (Z-hunt) that is designed to search long sequences of naturally occurring DNA and retrieve those nucleotide combinations of up to 24 bp in length which show a strong propensity for Z-DNA formation. Incorporated into Z-hunt is a statistical mechanical model based on empirically determined energetic parameters for the B to Z transition accumulated to date. The Z-forming potential of a sequence is assessed by ranking its behavior as a function of negative superhelicity relative to the behavior of similar sized randomly generated nucleotide sequences assembled from over 80,000 combinations. The program makes it possible to compare directly the Z-forming potential of sequences with different base compositions and different sequence lengths. Using Z-hunt, we have analyzed the DNA sequences of the bacteriophage phi X174, plasmid pBR322, the animal virus SV40 and the replicative form of the eukaryotic adenovirus-2. The results are compared with those previously obtained by others from experiments designed to locate Z-DNA forming regions in these sequences using probes which show specificity for the left-handed DNA conformation.

The binding specificity of 16 sera from systemic lupus erythematosus (SLE) patients was studied by enzyme-linked immunosorbent assay (ELISA), using 4 native DNAs of different guanine-cytosine (G-C) content and a group of synthetic polynucleotides. All the SLE sera showed increased binding to poly(dA-dC).poly(dG-dT), compared with calf thymus DNA in the right-handed B conformation. No significant differences were noted in binding of selected SLE sera to the native DNAs that differed in G-C content or superhelicity of DNA. With poly(dG-dC).poly(dG-dC) and poly(dG-m5dC).poly(dG-m5dC), the majority of SLE sera showed a preferential binding to the salt-induced Z form, compared with the B form. In addition, an average twelve-fold increase was found in binding to Z-form brominated poly(dG-dC).poly(dG-dC) compared with B-form poly(dG-dC).poly(dG-dC), when the polymers were coated on the plates in 0.15M NaCl. The preferential binding of SLE sera to poly(dA-dC).poly(dG-dT) and to Z-DNA may be important in the formation of circulating immune complexes and subsequent vascular damage, or may provide a clue to the mechanism of production of anti-DNA antibodies in this disease.

Using an iterative approach, we have placed monovalent (“solvated”) and divalent (both solvated and “unsolvated”) ions around a 20 base pair sequence, (dC-dG)10, in standard B and ZI conformations. The molecule with its attendant ions in the various conformations is subjected to to energy minimization using the program AMBER. In the presence of solvated cations (both monovalent as well as divalent) the B form is more stable than the Z form. However, direct binding with the unsolvated divalent cations makes the Z form more stable. Groove-binding provides some insight into the facility with which the B to Z transition occurs with higher charged cations. In the presence of unsolvated divalent cations, the Z form binds more charges at the groove through more ligands, compared to the B form. The orientation around the CpG phosphates in the minor groove of the Z form is found ideal for ion binding. Detailed molecular models for the ion binding have been developed. In general, phosphate groups dominate the ion binding. Large perturbations are seen mostly in the angles that control the phosphate orientation.

Negative supercoiling of the plasmid pBR322 with or without an insert of (dG-dC)n induces the formation of Z-DNA as measured by the binding of antibodies specific for Z-DNA. Increasing the concentration of Na+ (or K+) is shown to inhibit the B to Z-DNA conversion. This may be due to the effect of the cation on the B-Z junction. Using the data for B to Z-DNA conversion of the (dG-dC)n inserts, we have estimated the free energy change per base pair as well as the energy of the B-Z junction. In pBR322, a 14-bp segment [CACGGGTGCGCATG] is believed to form Z-DNA at bacterial negative superhelical densities under salt conditions which are similar to those found in vivo.

—We have employed a damage-specific DNA binding protein from human cells as a probe for base damage in polymers irradiated with white light in the presence of methylene blue. Protein-recognizable damage is introduced only into guanine-containing polymers and quenching of damage introduction by H2O and sodium azide suggest the involvement of a singlet oxygen mechanism. Using poly d(G-m5C), we have demonstrated that the left-handed double helical Z conformation is much less susceptible to guanine photooxidation than is the usual B conformation. We speculate that this difference in reactivity may reflect steric hindrance at the purine C-4 position and could provide some insight into the initial steps of the reaction between singlet oxygen and guanine in nucleic acid polymers.