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The binding peculiarities of the water-soluble meso -tetra-(4N-hydroxyethylpyridyl) porphyrin (H 2 TOEtPyP4) and its Cu- and Co-derivatives (CuTOEtPyP4 and CoTOEtPyP4) with synthetic double-stranded alternating polynucleotide poly(dG-dC).poly(dG-dC) were investigated by UV/Vis absorption and circular dichroism (CD) methods. It was shown that the porphyrins with planar structure such as H 2 TOEtPyP4 and CuTOEtPyP4 interact with poly(dG-dC).poly(dG-dC) via intercalation at low relative concentrations ( r = [porphyrin]/[polynucleotide] ), while at high r - via intercalation and external binding modes. In the case of no planar porphyrin CoTOEtPyP4 complexation occurs only by external binding mode. The binding constant K b and the exclusion parameter n calculated for H 2 TOEtPyP4, CuTOEtPyP4 and CoTOEtPyP4 porphyrins with poly(dG-dC).poly(dG-dC) complexes was 1.50 x10 7 , M -1 ( n  = 1.76); 9.29 x10 7 , M -1 ( n  = 1.18); and 0.28 x10 7 , M -1 ( n  = 2.65) correspondingly. The values of binding parameters for each porphyrin-poly(dG-dC).poly(dG-dC) complexes demonstrated good agreement with the proposed binding models. Communicated by Ramaswamy H. Sarma.

Here we describe novel enzymatic procedures for the production of long (from tens of nanometers to microns) double-stranded poly(dG)-poly(dC), triple-helical poly(dG)-poly(dG)-poly(dC), and quadruple-helical G4 DNA. All these molecules are uniform in size and possess improved mechanical and electrical properties with respect to a canonical random sequence double-stranded DNA. They can potentially be used as elements in nanoelectronic devices and circuits.

To find their DNA target sites in complex solution environments containing excess heterogeneous DNA, sequence-specific DNA-binding proteins execute various translocation mechanisms known collectively as facilitated diffusion. For proteins harboring a single DNA contact surface, long-range translocation occurs by jumping between widely spaced DNA segments. We have configured biosensor-based surface plasmon resonance to directly measure the affinity and kinetics of this intersegmental jumping by the ETS-family transcription factor ETS variant 6 (ETV6). To isolate intersegmental target binding in a functionally defined manner, we pre-equilibrated ETV6 with excess salmon sperm DNA, a heterogeneous polymer, before exposing the nonspecifically bound protein to immobilized oligomeric DNA harboring a high-affinity ETV6 site. In this way, the mechanism of ETV6-target association could be toggled electrostatically through varying NaCl concentration in the bulk solution. Direct measurements of association and dissociation kinetics of the site-specific complex indicated that 1) freely diffusive binding by ETV6 proceeds through a nonspecific-like intermediate, 2) intersegmental jumping is rate-limited by dissociation from the nonspecific polymer, and 3) dissociation of the specific complex is independent of the history of complex formation. These results show that target searches by proteins with an ETS domain, such as ETV6, whose single DNA-binding domain cannot contact both source and destination sites simultaneously, are nonetheless strongly modulated by intersegmental jumping in heterogeneous site environments. Our findings establish biosensors as a general technique for directly and specifically measuring target site search by DNA-binding proteins via intersegmental translocation., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)

A new type of Förster Resonance Energy Transfer (FRET) system using highly emissive isomorphic nucleobase analogues is reported. The FRET pair consists of 2-aminothieno[3,4-d]pyrimidine G-mimic deoxyribonucleoside ( th dG) as an energy donor and 1,3-diaza-2-oxophenothiazine (tC) as an energy acceptor. The distance and orientation between donor and acceptor was controlled by systematic incorporation of th dG and tC into DNA sequences to investigate the FRET efficiencies. This is the first Watson-Crick base-pairable FRET pair to produce vivid colors. In addition, this nucleic acid-based FRET pair was used to monitor DNA conformation and achieved visualization of the B-Z transition., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A grand challenge that crosses synthetic chemistry and biology is the scalable production of functional analogues of biomacromolecules. We have focused our attention on the use of deoxynucleoside building blocks bearing non-natural bases to develop a synthetic methodology that allows for the construction of high molecular weight deoxynucleotide polymers. Our six-membered cyclic phosphoester ring-opening polymerization strategy is demonstrated, herein, by an initial preparation of novel polyphosphoesters, comprised of butenyl-functionalized deoxyribonucleoside repeat units, connected via 3',5'-backbone linkages. A thymidine-derived bicyclic monomer, 3',5'-cyclic 3-(3-butenyl) thymidine ethylphosphate, was synthesized in two steps directly from thymidine, via butenylation and diastereoselective cyclization promoted by N,N-dimethyl-4-aminopyridine. Computational modeling of the six-membered 3',5'-cyclic phosphoester ring derived from deoxyribose indicated strain energies at least 5.4 kcal/mol higher than those of the six-membered monocyclic phosphoester, 2-ethoxy-1,3,2-dioxaphosphinane 2-oxide. These calculations supported the hypothesis that the strained 3',5'-cyclic monomer can promote ring-opening polymerization to afford the resulting poly(3',5'-cyclic 3-(3-butenyl) thymidine ethylphosphate)s with low dispersities (Đ < 1.10). This advanced design combines the merits of natural product-derived materials and functional, degradable polymers to provide a new platform for functional, synthetically derived polydeoxyribonucleotide-analogue materials.

The synthesis of water-soluble SN38 derivatives is presented, and their stability in solutions used during drug development studies has been investigated. A preliminary study of mechanism of action of 9-aminomethyl SN38 is presented. Using NMR techniques, the interaction of the oligomer d(GCGATCGC) 2 is studied, showing that the terminal GC base pairs are the main site of interaction. Using pulsed field gradient spin echo and mass spectroscopy, evidence of a spontaneous alkylation reaction of the DNA oligomer with SN38 derivatives is presented. A proposed mechanism of reaction is suggested. Copyright © 2016 John Wiley & Sons, Ltd., (Copyright © 2016 John Wiley & Sons, Ltd.)

Surgical techniques in dental and maxillofacial surgery request fast bone tissue regeneration, so there is a significant need to improve therapy for bone regeneration. Several studies have recently underlined the importance of nucleotides and nucleosides to increase cell proliferation and activity; in particular, the ability of polydeoxyribonucleotide (PDRN) to induce growth and activity of human osteoblasts was demonstrated. Sodium-DNA is the deoxyribonucleic acid (DNA) extracted from the gonadic tissue of male sturgeon and then purified, depolymerized, and neutralized with sodium hydroxide. To date, there are no evidences about the use of Sodium-DNA for bone tissue regeneration. Consequently, our question is about the efficacy of Sodium-DNA in bone healing. For testing the role of Sodium-DNA in bone healing we used a rat calvarial defect model. Sodium-DNA at different concentrations used alone or in association with Fibrin and/or Bio-Oss was used for healing treatments and the bone healing process was evaluated by histomorphometric and immunohistochemical analyses. Our results suggested a positive effect of Sodium-DNA in bone regeneration, providing a useful protocol and a model for the future clinical evaluation of its osteogenic properties.

Doxorubicin and nogalamycin are antitumor antibiotics that interact with DNA via intercalation and threading mechanisms, respectively. Because the importance of water, particularly its impact on entropy changes, has been established in other biological processes, we investigated the role of water in these two drug-DNA binding events. We used the osmotic stress method to calculate the number of water molecules exchanged (Δnwater), and isothermal titration calorimetry to measure Kbinding, ΔH, and ΔS for two synthetic DNAs, poly(dA·dT) and poly(dG·dC), and calf thymus DNA (CT DNA). For nogalamycin, Δnwater<0 for CT DNA and poly(dG·dC). For doxorubicin, Δnwater>0 for CT DNA and Δnwater<0 for poly(dG·dC). For poly(dA·dT), Δnwater~0 with both drugs. Net enthalpy changes were always negative, but net entropy changes depended on the drug. The effect of water exchange on the overall sign of entropy change appears to be smaller than other contributions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Oxidatively generated DNA lesions are numerous and versatile, and have been the subject of intensive research since the discovery of 8-oxoguanine in 1984. Even for this prototypical lesion, the precise mechanism of formation remains elusive due to the inherent difficulties in characterizing high-energy intermediates. We have probed the stability of the guanine endoperoxide in B-DNA as a key intermediate and determined a unique activation free energy of around 6 kcal mol(-1) for the formation of the first C-O covalent bond upon the attack of singlet molecular oxygen ((1) O2 ) on the central guanine of a solvated 13 base-pair poly(dG-dC), described by means of quantum mechanics/molecular mechanics (QM/MM) simulations. The B-helix remains stable upon oxidation in spite of the bulky character of the guanine endoperoxide. Our modeling study has revealed the nature of the versatile (1) O2 attack in terms of free energy and shows a sensitivity to electrostatics and solvation as it involves a charge-separated intermediate., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The ground- and excited-state interactions of Pt(ii) meso-tetrakis(4-N-methylpyridyl)porphyrin (PtTMPyP4) with polynucleotides [poly(dG-dC)]2 and [poly(dA-dT)]2 have been investigated using UV/visible, circular dichroism, and steady-state and time-resolved emission spectroscopy. PtTMPyP4 intercalates into [poly(dG-dC)]2 with K∼ 10(6) M(-1). When bound to [poly(dG-dC)]2 in aerated solution there is a six-fold emission enhancement with 18 nm red-shift in emission maximum. Emission lifetimes are biexponential. In the presence of [poly(dA-dT)]2 at least two distinct groove-binding modes are observed, depending on the binding ratio. In [poly(dA-dT)]2 the emission intensity increases by a maximum factor of 17 with no shift in the emission spectrum. Three exponentials were required for lifetime fitting. The lower extent of emission enhancement in the presence of [poly(dG-dC)]2 suggests that a slow electron transfer may take place to guanine, which is significantly less efficient than that previously observed for PtTMPyP4 in the presence of guanosine 5'-monophosphate (GMP). The results are compared to those previously recorded with free base H2TMPyP4.

In 80-90% tumor cells, telomerase becomes active and stabilizes the length of telomeres. The formation and stabilization of G-quadruplexes formed from human telomeric sequences have been proved able to inhibit the activity of telomerase, thus human telomeric G-quadruplex structure has become a potential target for the development of cancer therapy. Hence, structure of G-quadruplex formed in K(+) solution has been an attractive hotspot for further studies. However, the exact structure of human telomeric G-quadruplex in K(+) is extremely controversial, this study provides information for the understanding of different G-quadruplexes. Here, we report that 22nt and 24nt human telomeric sequences form unimolecular hybrid-type mixed parallel/antiparallel G-quadruplex in K(+) solution elucidated utilizing Circular Dichroism, Differential Scanning Calorimetry, and gel electrophoresis. Moreover, individual configuration of these two sequences was speculated in this study. The detailed structure information of the G-quadruplex formed under physiologically relevant condition is necessary for structure-based rational drug design., (© 2016 John Wiley & Sons A/S.)

We report a molecular modeling study, coupled with spectroscopy experiments, on the behavior of two well known organic dyes, nile blue and nile red, when interacting with B-DNA. In particular, we evidence the presence of two competitive binding modes, for both drugs. However their subsequent photophysical behavior is different and only nile blue is able to induce DNA photosensitization via an electron transfer mechanism. Most notably, even in the case of nile blue, its sensitization capabilities strongly depend on the environment resulting in a single active binding mode: the minor groove. Fluorescence spectroscopy confirms the presence of competitive interaction modes for both sensitizers, while the sensitization via electron transfer, is possible only in the case of nile blue.

Transcription factor-DNA interactions are central to gene regulation. Many transcription factors regulate multiple target genes and can bind sequences that do not conform strictly to the consensus. To understand the structural mechanism utilized by the transcription regulators to bind diverse target sequences, we have employed the repressor AraR from Bacillus subtilis as a model system. AraR is known to bind to eight different operator sites in the bacterial genome. Although there are differences in the sequences of four of these operators, ORE1, ORX1, ORA1, and ORR3, the AraR-DNA binding domain (AraR-DBD) as well as full-length AraR unexpectedly binds to each of these sequences with similar affinities as measured by fluorescence anisotropy experiments. We have determined crystal structures of AraR-DBD in complex with two different natural operators ORE1 and ORX1 up to 2.07 and 1.97 Å resolution, respectively. These structures were compared with the previously reported structures of AraR-DBD bound to two other natural operators (ORA1 and ORR3). Interactions of two molecules of AraR-DBD with the symmetric operator, ORE1, are identical, but their interaction with the non-symmetric operator ORX1 results in breakdown of the symmetry in protein-DNA interactions. The novel interactions observed are accompanied by local conformational change in the DNA. ChIP-sequencing (ChIP-Seq) data on other transcription factors has shown that they can bind to diverse targets, and hence the plasticity exhibited by AraR may be a general phenomenon. The ability of transcription factors to form alternate interactions may be important for employment in new functions and evolution of novel regulatory circuits., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

This study investigated the interaction between polydeoxyribonucleotide (PDRN) and several ionic and nonionic isotonic agents, thickeners and a preservative that were employed as excipients in ophthalmic preparations. Interaction of each individual excipient and PDRN aqueous solution was evaluated by analyzing their rheological properties. Rheological properties of PDRN solutions were evaluated by dynamic oscillatory shear tests and values of elastic modulus (G'), viscous modulus (G″) and loss tangent (tan δ) were used to assess the relative changes in viscoelastic properties. At given concentrations, sodium chloride was found to show alteration in viscoelastic properties of PDRN solution while nonionic isotonic agents like d-glucose and d-sorbitol did not alter them. Similarly, nonionic water soluble polymers like polyvinylpyrrolidone (PVP) and hydroxypropyl methylcellulose (HPMC) also did not interact with PDRN to alter the viscoelastic properties. However, there were changes observed when carbopol 940 was used as a thickener. Therefore, PDRN was found to interact with ionic excipients and the interactions were negligible when nonionic materials were examined, which suggests that nonionic excipients are suitable to be formulated with PDRN.

Previously, we reported our investigations of the interaction between a cyclic naphthalene diimide derivative (cNDI 1) and double stranded DNA (dsDNA) (Bioorg. Med. Chem.2014, 22, 2593). Here, we report the synthesis of the novel cNDI 2, which has shorter linker chains than cNDI 1. We performed comparative investigations of the interactions of both cNDI 1 and cNDI 2 with different types of dsDNA, including analysis of their thermodynamics and kinetics. Interactions between the cNDIs and calf thymus DNA (CT-DNA), poly[d(A-T)]2, or poly[d(G-C)]2 were explored by physicochemical and biochemical methods, including UV-Vis spectroscopy, circular dichroism (CD) spectroscopy, stopped-flow kinetics, and a topoisomerase I assay. Upon addition of cNDIs to CT-DNA, the existence of an induced CD signal at approximately the wavelength of the naphthalene diimide chromophore and unwinding of the DNA duplex, as detected by the topoisomerase I assay, revealed that cNDIs bound to the DNA duplex. As indicated by the steric constraint in the formation of the complex, bis-threading intercalation was the more favorable binding mode. UV-Vis spectroscopic titration of the cNDIs with DNA duplexes showed affinities on the order of 10(5)-10(6)M(-1), with a stoichiometry of one cNDI molecule per four DNA base pairs. Thermodynamic parameters (ΔG, ΔH, and ΔS) based on the van't Hoff equation indicated that exothermic and entropy-dependent hydrophobic interactions played a major role in the reaction. Stopped-flow association and dissociation analysis showed that cNDI interactions with poly[d(G-C)]2 were more stable and had a slower dissociation rate than their interactions with poly[d(A-T)]2 and CT-DNA. Measurement of ionic strength indicated that electrostatic attraction is also an important component of the interaction between cNDIs and CT-DNA. Because of its longer linker chain, cNDI 1 showed higher binding selectivity, a more entropically favorable interaction, and much slower dissociation from dsDNA than cNDI 2., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

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 (31)P NMR, in which a single (31)P 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.

Deoxyribonucleic acid photosensitization, i.e. the photoinduced electron- or energy-transfer of chromophores interacting with DNA, is a crucial phenomenon that triggers important DNA lesions such as pyrimidine dimerization, even upon absorption of relatively low-energy radiation. Oxidative lesions may also be produced via the photoinduced production of reactive oxygen species. Aromatic ketones, and acetophenone in particular, are well known for their sensitization effects. In this contribution we model the structural and dynamical properties of the acetophenone/DNA aggregates as well as their spectroscopic and photophysical properties using high-level hybrid quantum mechanics/molecular mechanics methods. We show that the key steps of the photochemistry of acetophenone in gas phase are conserved in the macromolecular environment and thus an ultrafast singlet-triplet conversion of acetophenone is expected prior to the transfer to DNA., (© 2014 The American Society of Photobiology.)

DNA molecules have come under the spotlight as potential templates for the fabrication of nanoscale products, such as molecular-scale electronic or photonic devices. Herein, we report an enhanced approach for the synthesis of oligoblock copolymer-type DNA by using the Klenow fragment exonuclease minus of E. coli DNA polymerase I (KF(-) ) in a multi-step reaction with natural and unnatural nucleotides. First, we confirmed the applicability of unnatural nucleotides with 7-deaza-nucleosides-which was expected because they were non-metalized nucleotides-on the unique polymerization process known as the "strand-slippage model". Because the length of the DNA sequence could be controlled by tuning the reaction time, analogous to a living polymerization reaction on this process, stepwise polymerization provided DNA block copolymers with natural and unnatural bases. AFM images showed that this DNA block copolymer could be metalized sequence-selectively. This approach could expand the utility of DNA as a template., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: The visual compatibility of a solution of defibrotide (the only drug recommended for treatment and prophylaxis of hepatic venoocclusive disease) with solutions of various drugs commonly administered in bone marrow transplant procedures was studied., Methods: Solutions of 43 drug products in concentrations typically used in clinical practice were evaluated in 1:1 mixtures with defibrotide solution in glass tubes kept at room temperature. The evaluated products included antiinfectious, corticoid, sedative, analgesic, and cardiovascular agents widely used for hematopoietic stem cell transplantation and other marrow transplant procedures; in most cases, test solutions were prepared via dilution in or reconstitution with sterile water, 0.9% sodium chloride injection, or 5% dextrose injection. The mixtures were visually observed immediately after manual mixing and at specified time points (60, 150, and 240 minutes). Visual compatibility was defined as the absence of color change, haze, fibers, particles, gas generation, and precipitate formation. The effect of mixing order on visual compatibility was ascertained., Results: Of the 43 tested drug solutions, 36 were found to be visually compatible with the defibrotide solution over the entire four-hour study period. Solutions of 7 drugs (amikacin, furosemide, midazolam, mycophenolate mofetil, nicardipine, tobramycin, and vancomycin) were visually incompatible with defibrotide solution. In some cases, evidence of incompatibility was observed intermittently or was dependent on mixing order., Conclusion: Defibrotide solution was found to be visually compatible with solutions of 36 i.v. products that are likely to be coadministered with the drug in a bone marrow transplant unit. Seven drug solutions were visually incompatible with defibrotide solution., (Copyright © 2014 by the American Society of Health-System Pharmacists, Inc. All rights reserved.)

Although the subject of some scrutiny over the years, the mechanism of conduction in DNA has not yet been resolved, with competing theories suggesting either electronic and ionic conduction mechanisms. In this paper we use dielectrophoresis to determine the electrical properties of poly(dG)-poly(dC) (GC) and poly(dA)-poly(dT) (AT) DNA in solution. The molecules show different conduction mechanisms; GC DNA exhibits conduction primarily through the molecule, whereas in AT DNA conduction through the counterion cloud surrounding the molecule in solution is more significant.

Doxorubicin (DOX) is an important anthracycline antibiotic whose intricate features of binding to DNAs, not yet fully understood, have been the object of intense debate. The dimerization equilibrium has been studied at pH = 7.0, I = 2.5 mM, and T = 25 °C. A thermodynamic and kinetic study of the binding of doxorubicin to DNA, carried out by circular dichroism, viscometry, differential scanning calorimetry, fluorescence, isothermal titration calorimetry, and T-jump relaxation measurements, has enabled us to characterize for the first time two different types of calf thymus DNA (ctDNA)/DOX complexes: PD1 for C(DOX)/C(DNA) < 0.3, and PD2 for higher drug content. The nature of the PD1 complex is described better in light of the affinity of DOX with the synthetic copolymers [poly(dA-dT)]2 and [poly(dG-dC)]2. The formation of PD1 has been categorized kinetically as a two-step mechanism in which the fast step is the groove binding in the AT region, and the slow step is the intercalation into the GC region. This bifunctional nature provides a plausible explanation for the high PD1 constant obtained (K1 = 2.3 × 10(8) M(-1)). Moreover, the formation of an external aggregate complex ctDNA/DOX (PD2) at the expense of PD1, with K2 = 9.3 × 10(5) M(-1), has been evinced.

A parallel G-quadruplex-selective iridium(III) complex has been synthesized and employed as a luminescent probe in a label-free G-quadruplex-based detection assay for Ca(2+) ions in aqueous solution. In this assay, a guanine-rich oligonucleotide (G4, 5'-G4T4G4-3') initially exists in an antiparallel G-quadruplex conformation, resulting in a low luminescence signal. Upon incubation with Ca(2+) ions, the antiparallel G-quadruplex is induced into a parallel G-quadruplex conformation, which greatly enhances the luminescence emission of the iridium(III) probe. This method was highly sensitive for Ca(2+) ions with a limit of detection in the nanomolar range, and was selective for Ca(2+) over other metal ions., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A label-free G-quadruplex-based luminescent switch-on assay has been developed for the selective detection of micromolar histidine in aqueous solution. In this study, an iridium(III) complex was employed as a G-quadruplex-specific luminescent probe while a guanine-rich oligonucleotide (Pu27, 5'-TG4AG3TG4AG3TG4A2G2-3')/cupric ion (Cu(2+)) ensemble was employed as a recognition unit for histidine. The initial luminescence of the iridium(III) complex in the presence of G-quadruplex DNA is effectively quenched by Cu(2+) ions due to the Cu(2+)-mediated unfolding of the G-quadruplex motif. The addition of histidine sequesters Cu(2+) ions from the ensemble, thereby restoring the luminescence of the system. The assay could detect down to 1 μM of histidine in aqueous media, and also exhibited good selectivity for histidine over other amino acids with the use of the cysteine, masking agent N-ethylmaleimide. Furthermore, the application of the assay for the detection of histidine in diluted urine samples was demonstrated., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In the last decade, there has been an explosion in the use of G-quadruplex labels to detect various analytes, including DNA/RNA, proteins, metals and other metabolites. In this review, we focus on strategies for the detection of nucleic acids, using G-quadruplexes as detection labels or as enzyme labels that amplify detection signals. Methods to detect other analytes are briefly mentioned. We highlight various strategies, including split G-quadruplex, hemin-G-quadruplex conjugates, molecular beacon G-quadruplex or inhibited G-quadruplex probes. The tandem use of G-quadruplex labels with various DNA-modifying enzymes, such as polymerases (used for rolling circle amplification), exonucleases and endonucleases, is also discussed. Some of the detection modalities that are discussed in this review include fluorescence, colorimetric, chemiluminescence, and electrochemical methods., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The spermine conjugate of the cationic porphyrin ligand (1) selectively induced the B-Z transition of the [(dA-T)n]2 sequence at low salt concentrations. The [(dG-C)n]2 sequence was not transformed into the Z-form. The B-Z transition was induced via an intermediate DNA conformation, which was formed by the external binding and formation of an assembly of 1 onto B-DNA.

The interaction of mitoxantrone with alternating Poly(dG-dC).Poly(dG-dC) and Poly(dA-dT).Poly(dA-dT) duplex has been studied by absorption, fluorescence and Circular Dichroism (CD) spectroscopy at Drug to Phosphate base pair ratios D/P=20.0-0.04. Binding to GC polymer occurs in two distinct modes: partial stacking characterized by red shifts of 18-23nm at D/P=0.2-0.8 and external binding at D/P=1.0-20.0 whereas that to AT polymer occurs externally in the entire range of D/P. The binding constant and number of binding sites is 3.7×10(5)M(-1), 0.3 and 1.3× 10(4)M(-1), 1.5 in GC and AT polymers, respectively at low D/P ratios. CD binding isotherms show breakpoints at D/P=0.1, 0.5 and 0.25, 0.5 in GC and AT polymers, respectively. The intrinsic CD bands indicate that the distortions in GC polymer are significantly higher than that in AT polymer. Docking studies show partial insertion of mitoxantrone rings between to GC base pairs in alternating GC polymer. Side chains of mitoxantrone interact specifically with base pairs and DNA backbone. The studies are relevant to the understanding of suppression or inhibition of DNA cleavage on formation of ternary complex with topoisomerase-II enzyme and hence the anti cancer action., (Copyright © 2013 Elsevier B.V. All rights reserved.)

We report the chiroptical signature and binding interactions of cationic (meso-tetrakis(4-N-methylptridyl)porphyrin, 2HT4) and anionic (meso-tetrakis(4-sulfonatophenyl)porphyrin, 2HTPPS) porphyrins and their zinc(II) and nickel(II) derivatives (ZnT4, ZnTPPS, NiT4, and NiTPPS) with right-handed B-form and two forms of left-handed Z-form of alternating guanine-cytosine polydeoxynucleotide poly(dG-dC)2. NiTPPS is able to spectroscopically discriminate between spermine-induced Z-DNA and Co(III)-induced Z-DNA via new induced circular dichroism signal in the visible region of the electromagnetic spectrum., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The crystal structure of a putative HNH endonuclease, Gmet&#95;0936 protein from Geobacter metallireducens GS-15, has been determined at 2.6 Å resolution using single-wavelength anomalous dispersion method. The structure contains a two-stranded anti-parallel β-sheet that are surrounded by two helices on each face, and reveals a Zn ion bound in each monomer, coordinated by residues Cys38, Cys41, Cys73, and Cys76, which likely plays an important structural role in stabilizing the overall conformation. Structural homologs of Gmet&#95;0936 include Hpy99I endonuclease, phage T4 endonuclease VII, and other HNH endonucleases, with these enzymes sharing 15-20% amino acid sequence identity. An overlay of Gmet&#95;0936 and Hpy99I structures shows that most of the secondary structure elements, catalytic residues as well as the zinc binding site (zinc ribbon) are conserved. However, Gmet&#95;0936 lacks the N-terminal domain of Hpy99I, which mediates DNA binding as well as dimerization. Purified Gmet&#95;0936 forms dimers in solution and a dimer of the protein is observed in the crystal, but with a different mode of dimerization as compared to Hpy99I. Gmet&#95;0936 and its N77H variant show a weak DNA binding activity in a DNA mobility shift assay and a weak Mn²⁺-dependent nicking activity on supercoiled plasmids in low pH buffers. The preferred substrate appears to be acid and heat-treated DNA with AP sites, suggesting Gmet&#95;0936 may be a DNA repair enzyme.

Isoquinoline alkaloids and their analogs represent an important class of molecules for their broad range of clinical and pharmacological utility. These compounds are of current interest owing to their low toxicity and excellent chemo preventive properties. These alkaloids can play important role in stabilising the nucleic acid triple helices. The present study has focused on the interaction of five 9-O-(ω-amino) alkyl ether berberine analogs with the DNA triplex poly(dT)·poly(dA)*poly(dT) and the parent duplex poly(dA)·poly(dT) studied using various biophysical techniques. Scatchard analysis of the spectral data indicated that the analogs bind both to the duplex and triplex in a non-cooperative manner in contrast to the cooperative binding of berberine to the DNA triplex. Strong intercalative binding to the DNA triplex structure was revealed from ferrocyanide quenching, fluorescence polarization and viscosity results. Thermal melting studies demonstrated higher stabilization of the Hoogsteen base paired third strand of the DNA triplex compared to the Watson-Crick strand. Circular dichroism studies suggested a stronger perturbation of the DNA triplex conformation by the alkaloid analogs compared to the duplex. The binding was entropy-driven in each case and the entropy contribution to free energy increased as the length of the alkyl side chain increased. The analogs exhibited stronger binding affinity to the triple helical structure compared to the parent double helical structure.

The photoreduction of water-soluble cationic manganese(III) meso-tetrakis(1-methylpyridium-4-yl)porphyrin (Mn(III)(TMPyP)(4+)) bound to a synthetic polynucleotide, either poly[d(A-T)2] or poly[d(G-C)2], was examined by conventional absorption and circular dichroism (CD) spectroscopy, transient absorption, and transient Raman spectroscopy. Upon binding, Mn(III)(TMPyP)(4+) produced a positive CD signal for both polynucleotides, suggesting external binding. In the poly[d(A-T)2]-Mn(III)(TMPyP)(4+) adduct case, an interaction between the bound porphyrin was suggested. The transient absorption spectral features of Mn(III)(TMPyP)(4+) in the presence of poly[d(A-T)2] and poly[d(G-C)2] were similar to those of the photoreduced products, Mn(II)(TMPyP)(4+), whereas Mn(III)(TMPyP)(4+) in the absence of polynucleotides retained its oxidation state. This indicated that both poly[d(A-T)2] and poly[d(G-C)2] act as electron donors, resulting in photo-oxidized G and A bases. The transient Raman bands (ν2 and ν4) that were assigned to porphyrin macrocycles exhibited a large downshift of ~25 cm(-1), indicating the photoreduction of Mn(III) to Mn(II) porphyrins when bound to both polynucleotides. The transient Raman bands for pyridine were enhanced significantly, suggesting that the rotation of peripheral groups for binding with polynucleotides is the major change in the geometry expected in the photoreduction process. These photoinduced changes do not appear to be affected by the binding mode of porphyrin.

The transfection of DNA in gene therapy largely depends on the possibility of obtaining its condensation. The details of nanoparticle formation are essential for functioning, as mediated by the diverse elements containing molecular structure, ionic strength in mediums, and condensing motivator. Here, we report two kinds of DNA condensing agents based on simple cyclic/rigid polyamine molecules, having evaluated their structural effect on nanoparticle formation. The reversible condensation-dissociation process was achieved by ion-switching, attributing to a possible condensing mechanism-competitive building of external hydrogen bonds. Using poly[(dA-dT)2] and poly[(dG-dC)2] as substrates, respectively, circular dichroism (CD) signals clearly presented dissimilar interactions between polyamines and both rich sequences, implying potential preference for G-C sequence. The presence of divalent ion Zn(2+) as an efficient motivator accelerated the achievement of DNA condensation, and an accessible schematic model was depicted to explain the promotion in detail. In addition, by comparison with the behaviors of linear polyamines, differences between condensation and aggregation were explicitly elucidated in aspects of morphology and surface charges, as well as induced condition. The present work may have the potential to reveal the precise mechanism of DNA nanoparticle formation and, in particular, be applied to gene delivery as an efficient nonviral vector.

In the present study, the experimental data on the pH-induced formation of the i-motif structure in the nucleotide sequence 5'-CCTTTCCTTTTCCTTTCC-3' (25oC, pH 3.3-8.9) obtained by spectroscopic techniques, such as UV molecular absorption and circular dichroism, has been analysed using the chemometric soft modelling-based MCR-ALS approach and the hard modelling-based matrix method. Soft modelling using 2 or 3 spectral species correctly reproduced spectral variations observed experimentally. The use of hard chemical modelling enabled us to propose the equilibrium model, which describes spectral changes as functions of solution acidity. Additionally, the intrinsic protonation constant Kin, and the cooperativity parameter w have been calculated from the fitting procedure of the circular dichroism as well as molecular absorption spectra. The results indicated that folding was accompanied by a cooperative process, i.e. the enhancement of protonated structure stability upon protonation.

The synthesis, characterisation and solid state crystal structure of a cationic 4-amino-1,8-naphthalimide derivative (1) are described. The photophysical properties of 1 are shown to vary with the solvent polarity and H-bonding ability. The fluorescence of 1 is enhanced and blue-shifted in its 1:1 complex with 5'-adenosine-monophosphate while it is partially quenched and red-shifted in its complex with 5'-guanosine-monophosphate. Linear and circular dichroism measurements show that 1 binds to double-stranded DNA by intercalation. Comparative UV-visible and fluorescence studies with double stranded synthetic polynucleotides poly(dA-dT)(2) and poly(dG-dC)(2) show that 1 binds much more strongly to the AT polymer; 1 also has a strong preference for A-T rich sequences in natural DNA. Thermal denaturation measurements also reveal a much greater stabilisation of the double-stranded poly(dA-dT)(2) than of natural DNA., (This journal is © The Royal Society of Chemistry 2012)

PAX5 encodes a master regulator of B-cell development. It fuses to other genes associated with acute lymphoblastoid leukemia (ALL). These fusion products are potent dominant-negative (DN) inhibitors of wild-type PAX5, resulting in a blockade of B-cell differentiation. Here, we show that multimerization of PAX5 DNA-binding domain (DBD) is necessary and sufficient to cause extremely stable chromatin binding and DN activity. ALL-associated PAX5-C20S results from fusion of the N-terminal region of PAX5, including its paired DBD, to the C-terminus of C20orf112, a protein of unknown function. We report that PAX5-C20S is a tetramer, which interacts extraordinarily stably with chromatin as determined by Fluorescence Recovery After Photobleaching in living cells. Tetramerization, stable chromatin binding and DN activity all require a putative five-turn amphipathic α-helix at the C-terminus of C20orf112, and does not require potential corepressor binding peptides elsewhere in the sequence. In vitro, the monomeric PAX5 DBD and PAX5-C20S binds a PAX5-binding site with equal affinity when it is at the center of an oligonucleotide too short to bind to more than one PAX5 DBD. But, PAX5-C20S binds the same sequence with 10-fold higher affinity than the monomeric PAX5 DBD when it is in a long DNA molecule. We suggest that the increased affinity results from interactions of one or more of the additional DBDs with neighboring non-specific sites in a long DNA molecule, and that this can account for the increased stability of PAX5-C20S chromatin binding compared with wild-type PAX5, resulting in DN activity by competition for binding to PAX5-target sites. Consistent with this model, the ALL-associated PAX5 fused to ETV6 or the multimerization domain of ETV6 SAM results in stable chromatin binding and DN activity. In addition, PAX5 DBD fused to artificial dimerization, trimerization and tetramerization domains results in parallel increases in the stability of chromatin binding and DN activity. Our studies suggest that oncogenic fusion proteins that retain the DBD of the transcription factor (TF) and the multimerization sequence of the partner protein can act in a DN manner by multimerizing and binding avidly to gene targets, preventing the normal TF from binding and inducing expression of its target genes. Inhibition of this multimeriztion may provide a novel therapeutic approach for cancers with this or similar fusion proteins.

When the model polynucleotide poly(dG-dC)∙poly(dG-dC) [polyGC] is titrated with a strong acid (HCl) in unbuffered aqueous solutions containing the chlorides of the alkali metals in the concentration range 0.010 M-0.600 M, two transitions in the absorbance vs. pH plots are evidenced, characterized by the constants pK(a(₁)) and pK(a(₂)). The limiting values at infinite saline concentrations of these two constants, namely pK(∞)(a(₁)) and pK(∞)(a(₂)) obtained making use of the "one site saturation constant" equation or, in turn, of the double logarithmic plot: pK(a) vs. log([salt]⁻¹), exhibit a clear dependence on the nature of the cations. The effects of the different alkali cations on the pK(∞)(a) values follow the Hofmeister series. In fact, the pK(∞)(a(₁)) and the pK(∞)(a(₂)) values are smaller for Li+ and Na+ than for Rb+ and Cs+, with K+ at the border between the two, showing that the transitions require higher concentrations of protons to occur in the presence of high concentrations of the cosmotropic ions.

Efficient intercalation of a luminescent Ir(III) complex exclusively made of polypyridine ligands in natural and synthetic biopolymers is reported for the first time. The emission of the complex is largely enhanced in the presence of [poly(dA-dT)(2)] and strongly quenched in the presence of [poly(dG-dC)(2)]. By comparing the emission decays in DNA and in synthetic polynucleotides, it is proposed that the emission quenching of the title compound by guanine residues in DNA is no longer effective over a distance of four dA-dT base pairs., (© 2011 American Chemical Society)

The interesting structural, electronic, and optical properties of DNA provide fascinating opportunities for developing nanoscale smart materials by integrating DNA with opto-electronic components. In this article we demonstrate the electrostatic binding of an amine-terminated dithienylethene (DET) molecular switch to double-stranded synthetic polynucleotides. The DET switch can undergo photochemical ring-closure and opening reactions. Circular dichroism (CD) and UV-vis spectroscopy show that both the open, 1o, and the closed, 1c, forms of the switch bind to DNA. Upon addition of DNA to a solution of 1o or 1c, the UV-vis spectrum displays a hypochromic effect, indicative of an interaction between the switch and the DNA. The chirality of the DNA double-helix is transmitted to the switching unit which displays a well-defined CD signal upon supramolecular complexation to the DNA. Additionally, the CD signal of the DNA attenuates, demonstrating that both components of the complex mutually influence each other's structure; the DNA induces chirality in the switch, and the switch modifies the structure of the DNA. Modulation of the chiroptical properties of the complex is achieved by photochemically switching the DET between its ring open and closed isomers. A pH dependence study of the binding shows that when the pH is increased the switches lose their binding ability, indicating that electrostatic interactions between protonated amines and the negatively charged phosphate backbone are the dominant driving force for binding to the DNA. A comparison of poly(deoxyguanylic-deoxycytidylic) acid [poly(dGdC)(2)] polynucleotides with poly(deoxyadenylic-deoxythymidylic) acid [poly(dAdT)(2)] shows distinct differences in the CD spectra of the complexes.

The occurrence of DNA architectural proteins containing two functional domains derived from two different architectural proteins is an interesting emerging research theme in the field of nucleoid structure and function. Mycobacterium tuberculosis HupB, unlike Escherichia coli HU, is a two-domain protein that, in the N-terminal region, shows broad sequence homology with bacterial HU. The long C-terminal extension, on the other hand, contains seven PAKK/KAAK motifs, which are characteristic of the histone H1/H5 family of proteins. In this article, we describe several aspects of HupB function, in comparison with its truncated derivatives lacking either the C-terminus or N-terminus. We found that HupB binds a variety of DNA repair and replication intermediates with K(d) values in the nanomolar range. By contrast, the N-terminal fragment of M. tuberculosis HupB (HupB(MtbN)) showed diminished DNA-binding activity, with K(d) values in the micromolar range, and the C-terminal domain was completely devoid of DNA-binding activity. Unlike HupB(MtbN) , HupB was able to constrain DNA in negative supercoils and introduce negative superhelical turns into relaxed DNA. Similarly, HupB exerted a robust inhibitory effect on DNA strand exchange promoted by cognate and noncognate RecA proteins, whereas HupB(MtbN), even at a 50-fold molar excess, had no inhibitory effect. Considered together, these results suggest that synergy between the N-terminal and C-terminal domains of HupB is essential for its DNA-binding ability, and to modulate the topological features of DNA, which has implications for processes such as DNA compaction, gene regulation, homologous recombination, and DNA repair., (© 2011 The Authors Journal compilation © 2011 FEBS.)

The repair protein 8-oxo-7,8-dihydroguanine glycosylase (OGG1) initiates base excision repair (BER) in mammalian cells by removing the oxidized base 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Interestingly, OGG1 has been implicated in somatic expansion of the trinucleotide repeat (TNR) sequence CAG/CTG. Furthermore, a 'toxic oxidation cycle' has been proposed for age-dependent expansion in somatic cells. In this cycle, duplex TNR DNA is (1) oxidized by endogenous species; (2) BER is initiated by OGG1 and the DNA is further processed by AP endonuclease 1 (APE1); (3) a stem-loop hairpin forms during strand-displacement synthesis by polymerase β (pol β); (4) the hairpin is ligated and (5) incorporated into duplex DNA to generate an expanded CAG/CTG region. This expanded region is again subject to oxidation and the cycle continues. We reported previously that the hairpin adopted by TNR repeats contains a hot spot for oxidation. This finding prompted us to examine the possibility that the generation of a hairpin during a BER event exacerbates the toxic oxidation cycle due to accumulation of damage. Therefore, in this work we used mixed-sequence and TNR substrates containing a site-specific 8-oxoG lesion to define the kinetic parameters of human OGG1 (hOGG1) activity on duplex and hairpin substrates. We report that hOGG1 activity on TNR duplexes is indistinguishable from a mixed-sequence control. Thus, BER is initiated on TNR sequences as readily as non-repetitive DNA in order to start the toxic oxidation cycle. However, we find that for hairpin substrates hOGG1 has reduced affinity and excises 8-oxoG at a significantly slower rate as compared to duplexes. Therefore, 8-oxoG is expected to accumulate in the hairpin intermediate. This damage-containing hairpin can then be incorporated into duplex, resulting in an expanded TNR tract that now contains an oxidative lesion. Thus, the cycle restarts and the DNA can incrementally expand., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Patients taking the immunosuppressant and anticancer thiopurines 6-mercaptopurine, azathioprine or 6-thioguanine (6-TG), develop skin cancer at a very high frequency. Their DNA contains 6-TG which absorbs ultraviolet A (UVA) radiation, and their skin is UVA hypersensitive, consistent with the formation of DNA photodamage. Here we demonstrate that UVA irradiation of 6-TG-containing DNA causes DNA interstrand crosslinking. In synthetic duplex oligodeoxynucleotides, the interstrand crosslinks (ICLs) can form between closely opposed 6-TG bases and, in a less favoured reaction, between 6-TG and normal bases on the opposite strand. In vivo, UVA irradiation of cultured cells containing 6-TG-substituted DNA also causes ICL formation and induces the chromosome aberrations that are characteristically associated with this type of DNA lesion. 6-TG/UVA activates the Fanconi anemia (FA) pathway via monoubiquitination of the FANCD2 protein. Cells defective in the FA pathway or other factors involved in ICL processing, such as XPF and DNA Polζ, are all hypersensitive to killing by 6-TG/UVA-consistent with a significant contribution of photochemical ICLs to the cytotoxicity of this treatment. Our findings suggest that sunlight-exposed skin of thiopurine treated patients may experience chronic photochemical DNA damage that requires constant intervention of the FA pathway., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The transient species formed following excitation of fac-[Re(CO)(3)(F(2)dppz)(py)](+) (F(2)dppz = 11,12-difluorodipyrido[3,2-a:2',3'-c]phenazine) bound to double-stranded polynucleotides [poly(dA-dT)](2) or [poly(dG-dC)](2) have been studied by transient visible and infra-red spectroscopy in both the picosecond and nanosecond time domains. The latter technique has been used to monitor both the metal complex and the DNA by monitoring the regions 1900-2100 and 1500-1750 cm(-1) respectively. These data provide direct evidence for electron transfer from guanine to the excited state of the metal complex, which proceeds both on a sub-picosecond time scale and with a lifetime of 35 ps, possibly due to the involvement of two excited states. No electron transfer is found for the [poly(dA-dT)](2) complex, although characteristic changes are seen in the DNA-region TRIR consistent with changes in the binding of the bases in the intercalation site upon excitation of the dppz-complex., (This journal is © The Royal Society of Chemistry and Owner Societies 2011)

A method for fabricating DNA polymer brush arrays using photolithography and plasma etching followed by solid-phase enzymatic DNA amplification is reported. After attaching oligonucleotide primers to the surface of a glass coverslip, a thin layer of photoresist is spin-coated on the glass and patterned via photolithography to generate an array of posts in the resist. An oxygen-based plasma is then used to destroy the exposed oligonucleotide primers. The glass coverslip with the primer array is assembled into a microfluidic chip and DNA polymer brushes are synthesized on the oligonucleotide array by rolling-circle DNA amplification. We have demonstrated that the linear polymers can be rapidly synthesized in situ with a high degree of control over their density and length., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

RNA polymerases are essential enzymes which transcribe DNA into RNA. Here, we obtain mass spectra of the cellular forms of apo and holo eukaryotic RNA polymerase I and III, defining their composition under different solution conditions. By recombinant expression of subunits within the initiation heterotrimer of Pol III, we derive an interaction network and couple this data with ion mobility data to define topological restraints. Our data agree with available structural information and homology modeling and are generally consistent with yeast two hybrid data. Unexpectedly, elongation complexes of both Pol I and III destabilize the assemblies compared with their apo counterparts. Increasing the pH and ionic strength of apo and holo forms of Pol I and Pol III leads to formation of at least ten stable subcomplexes for both enzymes. Uniquely for Pol III many subcomplexes contain only one of the two largest catalytic subunits. We speculate that these stable subcomplexes represent putative intermediates in assembly pathways., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Using UV-vis absorption and circular dichroism (CD) spectroscopies, we explored the binding interactions of 3,3'-diethylthiatricarbocyanine iodide (Cy7) with polynucleotides of different sequences and helicity. CD showed to be a very diagnostic tool giving different spectroscopic chiroptical signatures for all explored DNA sequences upon Cy7 binding. Cy7 was able to spectroscopically discriminate between the right handed B-DNA of poly(dG-dC)(2) and its left handed Z-DNA counterpart induced by spermine or Co(III)hexamine via nearly opposite induced circular dichroic signal.

Here we describe novel procedures for production of DNA-based nanowires. This include synthesis and characterization of the one-to-one double-helical complex of poly(dG)-poly(dC), triple-helical poly(dG)-poly(dG)-poly(dC) and G4-DNA, which is a quadruple-helical form of DNA. All these types of DNA-based molecules were synthesized enzymatically using Klenow exo(-) fragment of DNA Polymerase I. All the above types of nanowires are characterized by a narrow-size distribution of molecules. The contour length of the molecules can be varied from tens to hundreds of nanometers. These structures possess improved conductive and mechanical properties with respect to a canonical random-sequenced DNA and can possibly be used as wire-like conducting or semiconducting nanostructures in the field of nanoelectronics.

Effects of Ni(2+) and Cd(2+) ions on thermally induced conformational transitions in the poly(dA)·poly(dT) polynucleotide duplex and poly(dA)·2poly(dT) triplex under near physiological ionic conditions were studied by measurement of UV absorption melting curves and static light scattering intensity. The diagrams of conformational transitions in poly(dA)-poly(dT)-Me(2+) systems were plotted. An aggregation in these polynucleotide systems arises at certain values of the metal ions concentration and the temperature after the polymer dissociation into single strands. The phenomenon is conditioned by the aggregation of poly(dA) via the interstrand cross-linking by the dication bridges. Unlike Ni(2+), Cd(2+) induces formation of very stable aggregates which did not disintegrate even upon cooling up to room temperature.

The sequence selectivity of the DNA binding of the phenazinium dyes phenosafranin and safranin O have been investigated with four sequence-specific deoxyribopolynucleotides from spectroscopic and calorimetric studies. The alternating guanine-cytosine sequence selectivity of the dyes has been revealed from binding affinity values, circular dichroism, thermal melting, competition dialysis, and calorimetric results. The binding affinities of both the dyes to the polynucleotides were of the order of 10(5) M(-1), but the values were higher for the guanine-cytosine polynucleotides over adenine-thymine ones. Phenosafranin had a higher binding affinity compared to safranin O. Isothermal titration calorimetric studies revealed that the binding reactions were exothermic and favored by negative enthalpy and predominantly large positive entropy contributions in all cases except poly(dA)·poly(dT) where the profile was anomalous. Although charged, nonpolyelectrolytic contribution was revealed to be dominant to the free energy of binding. The negative heat capacity values obtained from the temperature dependence of enthalpy changes, which were higher for phenosafranin compared to safranin O, suggested significant hydrophobic contribution to the binding process. In aggregate, the data presents evidence for the alternating guanine-cytosine base pair selectivity of these phenazinium dyes and a stronger binding of phenosafranin over safranin O.

The conditions under which different structures of left-handed DNA (poly(dG-me(5)dC)·poly(dG-me(5)dC)) can exist are investigated by spectrofluorometric, spectrophotometric, circular dichroism and calorimetric measurements and the kinetics of the transformations are analysed. The effects of temperature, salt and ethanol content on the transitions are also studied. The left-handed structure obtained by addition of either Mg(2+) ions or EtOH corresponds to Z-DNA, whereas the structure obtained using the mixture Mg(2+)/EtOH corresponds to the aggregate Z*-DNA. Upon addition of the fluorescent cyanine Thiazole Orange (TO), the transition Z → B immediately starts, whereas Z*-DNA retains its left-handed configuration in the presence of TO provided that the ratio [dye]/[polymer] ≤ 0.1. The equilibria and kinetics of the TO binding to Z*-DNA are investigated under the above conditions using the T-jump technique. The reaction mechanism consists of two series steps, the first one being characterized by the formation of an external electrostatic complex and the second corresponding to the dye penetration between the base pairs. A comparison with the B-DNA/TO system is drawn.