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The noncovalent interaction of meso-aryl-substituted thiacarbocyanine dyes I and II with dsDNA and ssDNA in aqueous solutions has been studied by spectral-fluorescent methods. Complexation with DNA is accompanied by both aggregation of the dyes and the formation of monomeric strongly fluorescent complexes. Experiments on molecular docking of dyes I and II with dsDNA confirm the previous assumption about the possibility of the formation of complexes of different types: intercalation between base pairs and in the grooves of the double helix of the biopolymer. The possibility of intercalation of the dyes in the complex is confirmed by experiments on thermal dissociation of dsDNA in the presence of dyes I and II, as well as experiments on the interaction of the dyes with ssDNA. An increase in the melting temperatures T m of dsDNA is obtained in the presence of I and II, similar to that observed for the classical intercalator ethidium bromide. The limits of detection and quantification of DNA, which are important for the use of the dyes as probes for DNA, have been determined. The primary photochemical processes of the dyes in complexes with ssDNA were studied by flash photolysis technique. Complexation with ssDNA hinders photoisomerization and creates favorable conditions for the dye triplet state formation. The decay kinetics of the triplet state of the dyes were monoexponential. The rate constant of quenching of the triplet state by air oxygen was estimated for dye I complexed with ssDNA and was found to be less than the diffusion-controlled limit. This is probably a consequence of the shielding effect of the complex on the triplet quenching process., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

This report presents a novel approach for detecting and visualizing small to trace amounts of DNA in a sample. By utilizing both the change in emission spectrum and change in fluorescence lifetime, there is a significant increase in detection sensitivity allowing for the imaging/visualizing of a picograms amount of DNA in a microliters volume. As in the previous reports, one of the oldest DNA intercalators, Ethidium Bromide (EtBr), is employed as a model system. With this new approach, it is feasible to visualize just a few hundred picograms of DNA without the need for prior DNA amplification. The sensitivity can later be largely improved by using an intercalator that exhibits a higher affinity to DNA and a larger fluorescence change upon binding to DNA ( e.g. , ethidium homodimer, YOYO, or Diamond nucleic acid dyes).

Fluorescent DNA-binding dyes are extensively employed as probe and biosensing in biological detection and imaging. Experiments and theoretical calculations of thiazole orange homodimeric (TOTO) dye binding to a single-strand DNA (ssDNA), poly(dG) n ( n = 2, 4, 6, 8), reveal that the n = 6 complex shows about 300-fold stronger fluorescence than n = 2, 4 and a slightly stronger one than n = 8 complexes, which is benefited from the length match between TOTO and poly(dG) 6 . The machine learning, based on molecular dynamics trajectories, indicates that TOTO is featured by the dihedral angle along its backbone and its end-to-end distance, in which the latter one defines the stretch and hairpin structures of TOTO, respectively. The time-dependent density functional theory calculations on the low-lying excited states show that the stretched TOTO with π-π end-stacking binding mode can bring about strong fluorescence with localized π-π * transitions. For the n = 2, 4, and 8 complexes, the linear scaling quantum mechanics calculations indicate that the dominant hairpin TOTO with intercalative binding modes have relatively larger binding energies, leading to fluorescence quenching by intramolecular charge transfer. Our results may provide an insight for modulating the DNA-dye binding modes to tune the degree of charge transfer and designing fluorescent probes for the recognition of specific DNA sequences.

Lack of specificity and subsequent therapeutic effectiveness of antimicrobial and antitumoral drugs is a common difficulty in therapy. The aim of this study is to investigate, both by experimental and computational methods, the antitumoral and antimicrobial properties of a series of synthesized imidazole-pyridine derivatives. Interaction with three targets was discussed: Dickerson-Drew dodecamer (PDB id 2ADU), G-quadruplex DNA string (PDB id 2F8U) and DNA strain in complex with dioxygenase (PDB id 3S5A). Docking energies were computed and represented graphically. On them, a QSAR model was developed in order to further investigate the structure-activity relationship. Results showed that synthesized compounds have antitumoral and antimicrobial properties. Computational results agreed with the experimental data., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

The double-strand break (DSB) is the most cytotoxic type of DNA damage and measurement of DSBs in cells is essential to understand their induction and repair. Pulsed-field gel electrophoresis (PFGE) allows for quantitative measurement of DSBs in a cell population generated by DNA damaging agents. PFGE has the capacity to separate DNA molecules from several hundred base pairs to over six million base pairs. In the method described here, molecules from five hundred thousand to three million base pairs are consolidated into a single band on the gel that is readily analyzed.

Two new 1,3,4-thiadiazole derivatives of ibuprofen and ciprofloxacin namely {(5-(1-(4-isobutylphenyl)ethyl)-1,3,4-thiadiazol-2-amine)} 1 and {(3-(5-amino-1,3,4-thiadiazol-2-yl)-1-cyclopropyl-6-fluoro-7-(piperazin-1-yl)quinolin-4(1H)-one)} 2 were synthesized and characterized by spectroscopic and elemental analysis. DFT and molecular docking were done initially for theoretical binding possibilities of the investigated compounds. In vitro DNA binding investigations were carried out with UV-visible spectroscopic, fluorescence spectroscopic, cyclic voltammetric (CV) experiments under physiological conditions of the stomach (4.7) and blood (7.4) pH and at normal body temperature (37 °C). Both theoretical and experimental results suggested spontaneous and significant intercalative binding of the compounds with DNA. Kinetic and thermodynamic parameters (K b , ΔG) were evaluated greater for compound 2 which showed comparatively more binding and more spontaneity of 2 than 1 to bind with DNA at both pH values. Binding site sizes were found greater (n > 1) and revealed the possibility of other sites for interactions along with intercalation. Overall results for DNA binding were found more significant for 2 at Stomach (4.7) pH. Viscometric studies further verified intercalation as a prominent binding mode for both compounds. IC 50 values obtained from human hepatocellular carcinoma (Huh-7) cell line studies revealed 2 as potent anticancer agent than 1 as value found 25.75 μM (lesser than 50 μM). Theoretical and experimental DNA binding studies showed good correlation with cancer cell (Huh-7) line activity of 1 and 2 and further suggested that these compounds could act as potential anti-cancer drug candidates., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The i-motif represents a paradigmatic example of the wide structural versatility of nucleic acids. In remarkable contrast to duplex DNA, i-motifs are four-stranded DNA structures held together by hemi- protonated and intercalated cytosine base pairs (C:C+). First observed 25 years ago, and considered by many as a mere structural oddity, interest in and discussion on the biological role of i-motifs have grown dramatically in recent years. In this review we focus on structural aspects of i-motif formation, the factors leading to its stabilization and recent studies describing the possible role of i-motifs in fundamental biological processes.

Surfactants have often been employed for the sequestration of drugs from DNA. However, for an effective sequestration, the concentration of the surfactant needs to be higher than its critical micellar concentration (CMC). Use of such high concentrations of the surfactant may limit its practical usage as a sequestering agent due to its cytotoxicity. In the present study we have shown that sodium dodecyl sulfate (SDS) itself at a concentration less than its CMC failed to sequester a drug from DNA. However, the sequestration power of SDS at sub-CMC concentration could be enhanced to a significant extent when incorporated into Pluronic polymer micelles in the form of supramolecular assemblies. Such a sequestration process was monitored through detailed photophysical properties of a model drug using steady-state and time-resolved fluorescence techniques. It has also been demonstrated that unlike a conventional surfactant, the sequestration of drugs by SDS-polymer supramolecular assemblies can be controlled by their compositions. Two Pluronic polymers with different compositions have been used to understand the effect of polymer composition on the sequestration process. It has been shown that with the increase in the length of the hydrophilic blocks of the polymer, the extent of sequestration decreases due to the decrease in the sequestering force exerted on the intercalated drug. Most importantly, our in vitro cell viability studies show that the toxicity of the SDS surfactant is reduced to a remarkable extent due to its incorporation into the polymer micelles.

We have used a combination of fluorescence microscopy experiments and Pruned Enriched Rosenbluth Method simulations of a discrete wormlike chain model to measure the mean extension and the variance in the mean extension of λ-DNA in 100 nm deep nanochannels with widths ranging from 100 nm to 1000 nm in discrete 100 nm steps. The mean extension is only weakly affected by the channel aspect ratio. In contrast, the fluctuations of the chain extension qualitatively differ between rectangular channels and square channels with the same cross-sectional area, owing to the "mixing" of different confinement regimes in the rectangular channels. The agreement between experiment and simulation is very good, using the extension due to intercalation as the only adjustable parameter.

5,6-Disubstituted phenanthridinium cations have a range of redox, fluorescence and biological properties. Some properties rely on phenanthridiniums intercalating into DNA, but the use of these cations as exomarkers for the reactive oxygen species (ROS), superoxide, and as inhibitors of acetylcholine esterase (AChE) do not require intercalation. A versatile modular synthesis of 5,6-disubstituted phenanthridiniums that introduces diversity by Suzuki–Miyaura coupling, imine formation and microwave-assisted cyclisation is presented. Computational modelling at the density functional theory (DFT) level reveals that the novel displacement of the aryl halide by an acyclic N-alkylimine proceeds by an S(N)Ar mechanism rather than electrocyclisation. It is found that the displacement of halide is concerted and there is no stable Meisenheimer intermediate, provided the calculations consistently use a polarisable solvent model and a diffuse basis set.

Tetracationic porphyrins of the TMPP (meso-tetra-(4-N-methylpyridyl)porphyrin) type, metalated with Cu(II) or with Au(III), were conjugated covalently to oligonucleotides. The Cu(I)-catalyzed cycloaddition (between an azide and an ethynyl group) and the Diels-Alder cycloaddition (between a furan and a maleimide functionality) were successfully applied as two alternative postsynthetic methods to modify the 2'-position of an internal uridine. Melting temperatures and UV/vis absorption properties were compared. CD measurements indicated that the type of conjugation chemistry determines the grade of intercalation of the attached and positively charged porphyrins.

The reactions between [Pd2{(C,N)-C6H4CH2NH(Et)}2(μ-X)2] (X = Cl or Br) and 1,2-bis(diphenylphosphino)ethane (dppe) in the 1:1 molar ratio resulted in the dppe-bridged Pd(II) complexes, [Pd2{(C,N)-C6H4CH2NH(Et)}2(μ-dppe)(Cl)2] (1) and [Pd2{(C,N)-C6H4CH2NH(Et)}2(μ-dppe)(Br)2] (2), respectively, which were characterized by elemental analyses, infrared (IR), (1)H- and (31)P{(1)H} NMR spectroscopy. The molecular structure of 1 was determined by single-crystal X-ray diffraction. In vitro cytotoxicity of 1, 2, dppe, PhCH2NH(Et) and cisplatin were carried out against four human tumor cell lines. The interactions of complexes towards DNA and protein are investigated. The results suggested that both complexes could interact with FS-DNA through the intercalation mode. Moreover, the reactivity towards BSA revealed that the microenvironment and the secondary structure of BSA were changed in the presence of Pd(II) complexes., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

The toxic interaction between 2-naphthylamine (2-NA) and herring sperm deoxyribonucleic acid (hs-DNA) has been thoroughly investigated by UV absorption, fluorescence, and circular dichroism (CD) spectroscopic methods. UV absorption result indicates that 2-NA may intercalate into the stack base pairs of DNA during the toxic interaction of 2-NA with DNA. A fluorescence quenching study shows that DNA quenches the intrinsic fluorescence of 2-NA via a static pathway. The studies on effects of ionic strength and anionic quenching rule out electrostatic and groove bindings as the dominant binding modes. Further studies on denatured DNA fluorescence quenching and thermal melting studies confirm that the dominant binding mode of 2-NA-DNA is intercalative binding. A CD spectral study shows that the binding interaction of 2-NA with DNA leads to the disorganization of the neat double-helical structure of hs-DNA., (© 2013 Wiley Periodicals, Inc.)

The ability of small molecules to perturb the natural structure and dynamics of nucleic acids is intriguing and has potential applications in cancer therapeutics. Intercalation is a special binding mode where the planar aromatic moiety of a small molecule is inserted between a pair of base pairs, causing structural changes in the DNA and leading to its functional arrest. Enormous progress has been made to understand the nature of the intercalation process since its idealistic conception five decades ago. However, the biological functions were detected even earlier. In this review, we focus mainly on the acridine and anthracycline types of drugs and provide a brief overview of the development in the field through various experimental methods that led to our present understanding of the subject. Subsequently, we discuss the molecular mechanism of the intercalation process, free-energy landscapes, and kinetics that was revealed recently through detailed and rigorous computational studies., (© 2013 Elsevier Inc. All rights reserved.)

A new Ru(II) complex of [Ru(bpy)2(Hmspip)]Cl2 {in which bpy=2,2'-bipyridine, Hmspip=2-(4-(methylsulfonyl)phenyl)-1H-imidazo[4,5-f][1,10]phenanthroline} have been synthesized and characterized. The ground- and excited-state acid-base properties of [Ru(bpy)2(Hmspip)]Cl2 and its parent complex of [Ru(bpy)2(Hpip)]Cl2 {Hpip=2-phenyl-1H-imidazo[4,5-f][1,10]phenanthroline} have been studied by UV-visible (UV-vis) and emission spectrophotometric pH titrations. [Ru(bpy)2(Hmspip)]Cl2 acts as a calf thymus DNA intercalators with a binding constant of 4.0×10(5) M(-1) in buffered 50 mM NaCl, as evidenced by UV-vis and luminescence titrations, steady-state emission quenching by [Fe(CN)6]4-, DNA competitive binding with ethidium bromide, reverse salt titrations and viscosity measurements., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We have synthesized fluorescent DNA duplexes featuring multiple thiazole orange (TO) intercalating dyes covalently attached to the DNA via a triazole linkage. The intercalating dyes stabilize the duplex against thermal denaturation and show bright fluorescence in the green region of the spectrum. The emission color can be changed to orange or red by addition of energy-accepting Cy3 or Cy5 dyes attached covalently to the DNA duplex. The dye-modified DNA duplexes were then attached to a secondary antibody for intracellular fluorescence imaging of centrosomes in Drosophila embryos. Bright fluorescent foci were observed at the centrosomes in both the donor (TO) and acceptor (Cy5) channels, because the energy transfer efficiency is moderate. Monitoring the Cy5 emission channel significantly minimized the background signal because of the large shift in emission wavelength allowed by energy transfer.

A synthetic methodology for the synthesis of various β-pyrrolic-functionalised porphyrins and their covalent attachment to 2'-deoxyuridine and DNA is described. Palladium(0)-catalysed Sonogashira and copper(I)-catalysed Huisgen 1,3-dipolar cycloaddition reactions were used to insert porphyrins into the structure of 2'-deoxyuridine and DNA. Insertion of a porphyrin into the middle of single-stranded CT oligonucleotides possessing a 5'-terminal run of four cytosines was shown to trigger the formation of pH- and temperature-dependent i-motif structures. Porphyrin insertion also led to the aggregation of single-stranded purine-pyrimidine sequences, which could be dissociated by heating at 90 °C for 5 min. Parallel triplexes and anti-parallel duplexes were formed in the presence of the appropriate complementary strand(s). Depending on the modification, porphyrins were placed in the major and minor grooves of duplexes and were used as bulged intercalating insertions in duplexes and triplexes. In general, the thermal stabilisation of parallel triplexes possessing porphyrin-modified triplex-forming oligonucleotide (TFO) strands was observed, whereas anti-parallel duplexes were destabilised. These results are compared and discussed on the basis of the results of molecular modelling calculations., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

With the expansion of super-resolution fluorescence microscopy methods, it is now possible to access the organization of cells and materials at the nanoscale by optical means. This review discusses recent progress in super-resolution imaging of isolated and cell DNA using single-molecule localization methods. A high labeling density of photoswitchable fluorophores is crucial for these techniques, which can be provided by sequence independent DNA stains in which photoblinking reactions can be induced. In particular, unsymmetrical cyanine intercalating dyes in combination with special buffers can be used to image isolated DNA with a spatial resolution of 30-40 nm. For super-resolution imaging of chromatin, cell permeant cyanine dyes that bind the minor groove of DNA have the potential to become a useful alternative to the labeling of histones and other DNA-associated proteins. Other recent developments that are interesting in this context such as high density labeling methods or new DNA probes with photoswitching functionalities are also surveyed. Progress in labeling, optics, and single-molecule localization algorithms is being rapid, and it is likely to provide real insight into DNA structuring in cells and materials., (Copyright © 2010 Wiley Periodicals, Inc.)

PicoGreen is a fluorescent probe that binds dsDNA and forms a highly luminescent complex when compared to the free dye in solution. This unique probe is widely used in DNA quantitation assays but has limited application in biophysical analysis of DNA and DNA-protein systems due to limited knowledge pertaining to its physical properties and characteristics of DNA binding. Here we have investigated PicoGreen binding to DNA to reveal the origin and mode of PicoGreen/DNA interactions, in particular the role of electrostatic and nonelectrostatic interactions in formation of the complex, as well as demonstrating minor groove binding specificity. Analysis of the fluorescence properties of free PicoGreen, the diffusion properties of PG/DNA complexes, and the excited-state lifetime changes upon DNA binding and change in solvent polarity, as well as the viscosity, reveal that quenching of PicoGreen in the free state results from its intramolecular dynamic fluctuations. On binding to DNA, intercalation and electrostatic interactions immobilize the dye molecule, resulting in a >1000-fold enhancement in its fluorescence. Based on the results of this study, a model of PicoGreen/DNA complex formation is proposed., (Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

A novel mediator-free biosensor was constructed by the co-intercalation of negatively charged DNA and positively charged hemoglobin (Hb) in the interlayer galleries of layered alpha-zirconium phosphate (alpha-ZrP) with the delamination-assembly procedure at pH 5.5. X-ray diffraction and field-emission scanning electron microscopy results revealed the featured layered structure for the re-assembled DNA/Hb/alpha-ZrP composite. Infrared spectroscopy and circular dichroism results confirmed the coexistence of Hb and DNA in the composite and the considerably retained protein conformation of intercalated Hb. The direct electron transfer of Hb was facilitated by the co-intercalation of DNA and Hb. Because of the synergistic effect of alpha-ZrP host and co-intercalated DNA guest, the DNA/Hb/alpha-ZrP modified electrode exhibited good electrocatalytic response to H(2)O(2) with higher sensitivity of 0.79 A M(-1) cm(-2) and lower detection of 4.28x10(-7) M in the linear range of 7.28x10(-7) to 9.71x10(-5) M. Furthermore, the electrocatalytic activity of Hb in the DNA/Hb/alpha-ZrP composite retained at high temperature (85 degrees C) or in the presence of organic solvent (CH(3)CN), which could be the protection of alpha-ZrP nanosheets., (Copyright 2010 Elsevier B.V. All rights reserved.)

Anticancer drugs that bind to DNA and inhibit DNA-processing enzymes represent an important class of anticancer drugs. Combilexin molecules, which combine DNA minor groove binding and intercalating functionalities, have the potential for increased DNA binding affinity and increased selectivity due to their dual mode of DNA binding. This study describes the synthesis of DNA minor groove binder netropsin analogs containing either one or two N-methylpyrrole carboxamide groups linked to DNA-intercalating anthrapyrazoles. Those hybrid molecules which had both two N-methylpyrrole groups and terminal (dimethylamino)alkyl side chains displayed submicromolar cytotoxicity towards K562 human leukemia cells. The combilexins were also evaluated for DNA binding by measuring the increase in DNA melting temperature, for DNA topoisomerase IIalpha-mediated double strand cleavage of DNA, for inhibition of DNA topoisomerase IIalpha decatenation activity, and for inhibition of DNA topoisomerase I relaxation of DNA. Several of the compounds stabilized the DNA-topoisomerase IIalpha covalent complex indicating that they acted as topoisomerase IIalpha poisons. Some of the combilexins had higher affinity for DNA than their parent anthrapyrazoles. In conclusion, a novel group of compounds combining DNA intercalating anthrapyrazole groups and minor groove binding netropsin analogs have been designed, synthesized and biologically evaluated as possible novel anticancer agents.

DNA hybridisation is an important tool for bioanalytical research and clinical diagnostics; conventional methods, however, require long incubation times and numerous washing steps, rendering the procedure time consuming and labour intensive. In this paper, we report on a rapid method for DNA hybridisation and isolation within a microfluidic device, where all reaction and washing steps are performed in continuous flow in an automated fashion within less than two minutes. Magnetic particles were used as a solid support and manipulated through laminar flow streams containing reagents and buffers by means of an external magnet. Thus, hybridisation, washing, intercalation, fluorescence detection and isolation were performed in continuous flow on the surface of the particles. Initially, the sensitivity of the system was investigated for a one-step DNA hybridisation of Alexa Fluor 555 labelled target DNA to a capture probe immobilised on the particle surface. Hybridisation and washing steps were performed in half a minute and target DNA was readily detected down to 20 nmol L(-1). Then a two-step assay, label-free DNA hybridisation followed by intercalation with PicoGreen was performed. All reaction and washing steps were carried out in continuous flow with a total assay time of about 1 min. This is a significant reduction in procedural time compared to conventional methods and opens the door for developing fully automated continuous flow integrated DNA analysis platforms., (2010 Elsevier B.V. All rights reserved.)

The interaction of DNR-D3 (daunorubicin derivative) synthesized in our laboratory with ctDNA was investigated by UV spectrum and fluorescence spectrum under physiological conditions (pH 7.4) for the first time. The red shifts and hypochromicities were observed from the absorption titration experiments. These results suggest that DNR-D3 was intercalated into the DNA base pairs. Through the fluorescence quenching data measured at different temperatures (20 degrees C, 30 degrees C and 37 degrees C), it is known that the quenching mechanism of fluorescence of DNR-D3 by ctDNA is a static quenching type. On the other hand, the binding constant, the number of binding sites and thermodynamic parameters were also obtained. These data also indicate that the binding mode of the interaction between DNR-D3 and ctDNA is intercalation. Additionally, the types of interaction force are mainly hydrogen bonding and electrostatic interaction, and the binding is exothermic enthalpy-entropy cooperative driven process. When the degree of fluorescence quenching of DNR-D3 is 50%, the ratio of the molar concentration of DNR-D3 to ctDNA is 7/25, which indicates that the DNR-D3 anthracycline was intercalated into the DNA base pairs and that DNR-D3 showed a strong anti-cancer activity. DNR-D3 is expected to become one of the drug candidates from our investigations.

Background: Topoisomerase II is critical for DNA replication, transcription and chromosome segregation and is a well validated target of anti-neoplastic drugs including the anthracyclines and epipodophyllotoxins. However, these drugs are limited by common tumor resistance mechanisms and side-effect profiles. Novel topoisomerase II-targeting agents may benefit patients who prove resistant to currently available topoisomerase II-targeting drugs or encounter unacceptable toxicities. Voreloxin is an anticancer quinolone derivative, a chemical scaffold not used previously for cancer treatment. Voreloxin is completing Phase 2 clinical trials in acute myeloid leukemia and platinum-resistant ovarian cancer. This study defined voreloxin's anticancer mechanism of action as a critical component of rational clinical development informed by translational research., Methods/principal Findings: Biochemical and cell-based studies established that voreloxin intercalates DNA and poisons topoisomerase II, causing DNA double-strand breaks, G2 arrest, and apoptosis. Voreloxin is differentiated both structurally and mechanistically from other topoisomerase II poisons currently in use as chemotherapeutics. In cell-based studies, voreloxin poisoned topoisomerase II and caused dose-dependent, site-selective DNA fragmentation analogous to that of quinolone antibacterials in prokaryotes; in contrast etoposide, the nonintercalating epipodophyllotoxin topoisomerase II poison, caused extensive DNA fragmentation. Etoposide's activity was highly dependent on topoisomerase II while voreloxin and the intercalating anthracycline topoisomerase II poison, doxorubicin, had comparable dependence on this enzyme for inducing G2 arrest. Mechanistic interrogation with voreloxin analogs revealed that intercalation is required for voreloxin's activity; a nonintercalating analog did not inhibit proliferation or induce G2 arrest, while an analog with enhanced intercalation was 9.5-fold more potent., Conclusions/significance: As a first-in-class anticancer quinolone derivative, voreloxin is a toposiomerase II-targeting agent with a unique mechanistic signature. A detailed understanding of voreloxin's molecular mechanism, in combination with its evolving clinical profile, may advance our understanding of structure-activity relationships to develop safer and more effective topoisomerase II-targeted therapies for the treatment of cancer.

A few novel anthracene-based cyclophanes CP-1, CP-2 and CP-3 were synthesized and their interactions with DNA were investigated employing photophysical and biophysical techniques. In methanol and acetonitrile, these systems exhibited optical properties characteristic of the anthracene chromophore. However, in the aqueous medium, the symmetric cyclophane CP-1 showed a dual emission having lambda(max) at 430 and 550 nm, due to the monomer and excimer, respectively. In contrast, the cyclophanes CP-2 and CP-3 in the aqueous medium showed structured anthracene absorption and emission spectra similar to those obtained in methanol and acetonitrile. DNA binding studies indicate that CP-1 undergoes efficient nonclassical partial intercalative interactions with DNA resulting in the exclusive formation of a sandwich-type excimer having enhanced emission intensity and lifetimes. The cyclophane CP-2 having one anthracene moiety exhibited nonclassical intercalative binding with DNA, albeit with less efficiency compared with CP-1. In contrast, CP-3, having sterically bulky viologen bridging group showed DNA electrostatic as well as groove binding interactions. These results demonstrate that the nature of the bridging unit plays an important role in the binding mode of the cyclophanes with DNA and in the formation of the novel sandwich-type excimer.

A SELEX approach has been developed in order to select oligonucleotides that bind double-stranded DNA in the presence of a triplex-stabilizing agent, and was applied to a target sequence containing an oligopurine-oligopyrimidine stretch. After only seven rounds of selection, the process led to the identification of oligonucleotides that were able to form triple helices within the antiparallel motif. Inspection of the selected sequences revealed that, contrary to GC base pair which were always recognized by guanines, recognition of AT base pair could be achieved by either adenine or thymine, depending on the sequence context. While thymines are strongly preferred for several positions, some others can accommodate the presence of adenines. These results contribute to set the rules for designing oligonucleotides that form stable triple helices in the presence of triplex-stabilizing agents at physiological pH. They set the basis for further experiments regarding extension of potential target sequences for triple-helix formation or recognition of ligand-DNA complexes.

The interaction between astragalin (AST) from lotus leaf and deoxyribonucleic acid (DNA) in Tris-HCl buffer (pH = 7.4) was investigated by the application of fluorescence spectroscopy and ultraviolet absorption spectroscopy, the effects of ionic strength and anion quencher KI on the fluorescence intensity of AST from lotus leaf and the system of AST-DNA were explored, and the competitive binding to DNA between AST from lotus leaf and Neutral Red(NR)dye was also studied. The results demonstrated that AST could bind to DNA and the formed complex quenched the intrinsic fluorescence of AST from lotus leaf through static quenching mechanism. The quenching rate constants of biomolecule(Kq)of the reaction of DNA with AST from lotus leaf were calculated to be 3.120 X 10(12) and 2.630 X 10(12) L x mol(-1) x s(-1) by Stern-Volumer equation, the corresponding binding constants (Kd) were computed to be 3.412 x 10(12) and 1.762 x 10(4) L x mol(-1) and the number of binding sites(n) was counted to be 1.007 and 0.962 between AST from lotus leaf and DNA at 298 and 308 K, respectively. When bound to DNA, the AST from lotus leaf showed hypochromic effect and red shift in the absorption spectra. It was also found that different ionic strength had little or no effect on the fluorescence intensity of AST and AST-DNA, but the fluorescence intensity of AST-DNA quenched by anionic quencher KI was much less than that of free AST. AST could be intercalated into DNA and displaced the NR from the NR-DNA complex. It was showed that AST from lotus leaf could combine with DNA in the mode of intercalation.

A novel fluorometric sensing of malachite green is proposed in this paper. The native double-stranded calf thymus DNA was used as sensing material. In the presence of native double-stranded calf thymus DNA, malachite green could interact with the DNA, which resulted in a strong fluorescence emission. The fluorescent intensity was linear with malachite green concentration in the range of 4.0 x 10(-10)-1.8 x 10(-7)g ml(-1) and the limit of detection was 2.0 x 10(-10)g ml(-1). Before fluorescence measurement, the only required operation is the mixing of two solutions. So, this method is rather simple and rapid. The method is very safe for the analyst. Furthermore, the mechanism for fluorescence enhancing of native double-stranded calf thymus DNA on MG was proposed based on a series of experiments. The results suggest that the interaction between MG and calf thymus DNA is intercalation in nature.

A novel copper (II) complex of Schiff base prepared through condensation between 2-formyl-17-deoxyestrone and d-glucosamine was synthesized and characterized. Fluorescence spectroscopy was conducted to assess their binding ability with CT-DNA. The results showed that the copper (II) complex could bind to DNA with a weak intercalative mode. The interaction between the copper (II) complex and DNA was also investigated by gel electrophoresis. Interestingly, we found that the complex could cleave plasmid DNA (pUC19) to nicked and linear forms through an oxidative mechanism without the use of exogenous agents.

A water soluble derivative (2) of topopyrones was selected for NMR studies directed to elucidate the mode of binding with specific oligonucleotides. Topopyrone 2 can intercalate into the CG base pairs, but the residence time into the double helix is very short and a fast chemical exchange averaging occurs at room temperature between the free and bound species. The equilibria involved become slow below room temperature, thus allowing to measure a mean lifetime of the complex of ca. 7 ms at 15 degrees C. Structural models of the complex with d(CGTACG)(2) were developed on the basis of DOSY, 2D NOESY and (31)P NMR experiments. Topopyrone 2 presents a strong tendency to self-associate. In the presence of oligonucleotide a certain number of ligand molecules are found to externally stack to the double-helix, in addition to a small fraction of the same ligand intercalated. The external binding to the ionic surface of the phosphoribose chains may thus represents the first step of the intercalation process.

DNA cleavage activity of quercetin zinc(II) complex has been studied, but little attention has been devoted to the relationship between antitumor activity of this complex and DNA-binding properties. DNA-binding properties of quercetin zinc(II) complex were studied using UV-vis spectra, fluorescence measurements, and viscosity measurements. The results obtained indicate that quercetin zinc(II) complex can intercalate into the stacked base pairs of DNA, and compete with the strong intercalator ethidium bromide for the intercalative binding sites with Stern-Volmer quenching constant, K(sq)=1.24. The complex was subjected to biological tests in vitro using three tumor cell lines (HepG2, SMMC7721, and A549), which showed significant cytotoxicity against three tumor cell lines. Moreover, Hoechst33258 staining showed HepG2 cells underwent the typical morphologic changes of apoptosis characterized by nuclear shrinkage, chromatin condensation, or fragmentation after exposure to the complex. In addition, Molecular modeling was performed to learn the complex could be preferentially bound to DNA in GC region. Our results suggest that antitumor activity of quercetin zinc(II) complex might be related to its intercalation into DNA.

Alkylating agents after formation of DNA adduct not only possess their harmful role on living cells but also can transfer this information to the next generation. Different techniques have been introduced to study the alkylated DNA, most of which are specific and designed for investigation of specific target DNA. But the exact differences between spectroscopic and functional properties of alkylated DNA are not seen in the literature. In the present study DNA was methylated using streptozotocin (STZ) by both in vitro and in vivo protocols, then methylated-DNA was investigated by various techniques. Our results show that (1) the binding of ethidium bromide as an intercalating dye decreases to methylated-DNA in comparison with normal DNA, (2) CD spectra of methylated-DNA show changes including a decrease in the positive band at 275 nm and a shift from 258 nm crossover to a longer wavelength, which is caused by reduction of water around it, due to the presence of additional hydrophobic methyl groups, (3) the stability of methylated-DNA against DTAB as a denaturant is decreased and (4) the enzyme-like activity of methylated-DNA in an electron transfer reaction is reduced. In conclusion, additional methyl groups not only protrude water around DNA, but also cause the loss of hydrogen bonding, loosening of conformation, preventing desired interactions and thus normal function of DNA.

This work is the first in-depth study of osmium binding to DNA and confirms the pharmacological activity of a new class of anticancer metallodrugs. We investigated the interactions between the potential biological target DNA and four osmium(II) arene complexes, of the type [(eta 6-arene)Os(LL)Cl]n+, where arene = biphenyl or p-cymene and LL = ethylenediamine, picolinate, or oxinate in an effort to understand their mechanism of action. Most notably we show that these complexes bind to DNA. DNA adducts of the OsII complexes that exhibit promising cytotoxic effects in ovarian tumor cell lines largely distort its conformation. The data are consistent with DNA binding of the complexes containing biphenyl as the arene ligand that involves combined coordination to guanine residues and noncovalent interactions between the arene ligand and DNA. The results also indicate both a mechanism of action and a detoxification mechanism for OsII arene compounds different from those of cisplatin.

Scanning electrochemical microscopy (SECM) has been employed in the imaging of DNA microarrays fabricated on gold substrates using methylene blue (MB) as a redox-active intercalator and ferrocyanide as the SECM mediator in solution. MB intercalated between base pairs of immobilized ds-DNA is electrochemically reduced via electron transfer from the underlying gold substrate, and the product is reoxidized in solution by SECM tip-generated ferricyanide. The resulting feedback current allows a heterogeneous electron-transfer rate constant for the MB-intercalated DNA to be deduced. Moreover, DNA microarray spots can be imaged at a detection level of 14 fmol/spot for ds-DNA consisting of 15 base pairs. Microarrays prepared using 20 microM DNA solutions are easily visualized, and the feasibility of detecting base pair mismatches is also demonstrated.

This research is an effort to further understand the physicochemical interaction between the novel drug, mitoxantrone (MTX) and its biologic receptor, DNA. The ultimate goal is to design drugs that interact more with DNA. Understanding the physicochemical properties of the drug as well as the mechanism by which it interacts with DNA, it should ultimately allow the rational design of novel anti-cancer or anti-viral drugs. Molecular modelling on the complex formed between MTX and DNA presented that this complex was indeed fully capable of participating in the formation of a stable intercalation site. Furthermore, the molecular geometries of MTX and the DNA bases (adenine, guanine, cytosine and thymine) were optimized with the aid of the B3LYP/6-31G* method. The properties of the isolated intercalator and its stacking interactions with the adenine...thymine (AT) and guanine...cytosine (GC) nucleic acid base pairs were studied with the DFTB method (density functional tight-binding), an approximate version of the DFT method, that was extended to cover the London dispersion energy. The B3LYP/6-31G* stabilization energies of the intercalator...base pair complexes were found 10.06 kcal/mol and 21.64 kcal/mol for AT...MTX and GC...MTX, respectively. It was concluded that the dispersion energy and the electrostatic interaction contributed to the stability of the intercalator.DNA base pair complexes. The results concluded from the comparison of the DFTB method and the Hartree-fock method point out that these methods show close results and support each other.

Forced intercalation probes (FIT-probes) are peptide nucleic acid-based probes in which the thiazole orange dye replaces a canonical nucleobase. FIT-probes are used in homogenous DNA detection. The analysis is based on sequence-specific binding of the FIT-probe with DNA. Binding of the FIT-probe places thiazole orange in the interior of the formed duplex. The intercalation of thiazole orange between nucleobases of the formed probe-target duplex restricts the torsional flexibility of the two heterocyclic ring systems. As a result, FIT probes show strong enhancements of fluorescence upon hybridization. A remarkable attenuation of fluorescence is observed when forcing thiazole orange to intercalate next to a mismatched base pair. This base specificity of fluorescence signaling, which adds to the specificity of probe-target recognition, allows the detection of single base mutations even at non-stringent hybridization conditions. The performance of FIT-probes in real-time PCR is demonstrated in an assay for the SNPtyping of human H-ras. FIT-probe was added at the start of a real-time amplification containing the wild-type (G,G)-allele, mutant (T,T)-allele or heterozygous (G,T)-allele of the human H-ras gene. The identity of the target DNA is determined in real time due to significant differences in signal intensities.

We report a novel method of identifying damage to DNA leading to the loss of intercalation sites. Thiazole orange (TO), an intercalating cyanine dye, fluoresces strongly when intercalated in DNA, but not free in solution. Upon UVC-induced damage to DNA, the change in TO fluorescence is greater than the change in any of the other spectral or biochemical indicators (absorbance, circular dichroism and agarose gel electrophoresis), thus providing a fast screening method to identify damage to DNA. The method is geared toward high levels of damage, such as those that may result during radiation treatment of food products.

Background: DNA melting curve analysis using double-stranded DNA-specific dyes such as SYTO9 produce complex and reproducible melting profiles, resulting in the detection of multiple melting peaks from a single amplicon and allowing the discrimination of different species. We compare the melting curves of several Naegleria and Cryptosporidium amplicons generated in vitro with in silico DNA melting simulations using the programs POLAND and MELTSIM., then test the utility of these programs for assay design using a genetic marker for toxin production in cyanobacteria., Results: The SYTO9 melting curve profiles of three species of Naegleria and two species of Cryptosporidium were similar to POLAND and MELTSIM melting simulations, excepting some differences in the relative peak heights and the absolute melting temperatures of these peaks. MELTSIM and POLAND were used to screen sequences from a putative toxin gene in two different species of cyanobacteria and identify regions exhibiting diagnostic melting profiles. For one of these diagnostic regions the POLAND and MELTSIM melting simulations were observed to be different, with POLAND more accurately predicting the melting curve generated in vitro. Upon further investigation of this region with MELTSIM, inconsistencies between the melting simulation for forward and reverse complement sequences were observed. The assay was used to accurately type twenty seven cyanobacterial DNA extracts in vitro., Conclusion: Whilst neither POLAND nor MELTSIM simulation programs were capable of exactly predicting DNA dissociation in the presence of an intercalating dye, the programs were successfully used as tools to identify regions where melting curve differences could be exploited for diagnostic melting curve assay design. Refinements in the simulation parameters would be required to account for the effect of the intercalating dye and salt concentrations used in real-time PCR. The agreement between the melting curve simulations for different species of Naegleria and Cryptosporidium and the complex melting profiles generated in vitro using SYTO9 verified that the complex melting profile of PCR amplicons was solely the result of DNA dissociation. Other data outputs from these simulations were also used to identify the melting domains that contributed to the observed melting peaks for each of the different PCR amplicons.

We report the volumetric and other thermodynamic properties of ethidium bromide (EB), propidium iodide (PI) and daunomycin (DAU) intercalating with poly(dA).poly(dT), poly[d(A-T)].poly[d(A-T)], and poly[d(G-C)].poly[d(G-C)], respectively, as well as minor groove binder Hoechst 33258 binding with poly[d(A-T)].poly[d(A-T)]. The data were obtained using fluorescence titration and hydrostatic pressure measurements. Our thermodynamic data are combined with enthalpies from literature reports to analyze the thermodynamic characteristics of the different interactions. The differences are interpreted based on three processes related to hydration: I. burial of non-polar hydrophobic solvent accessible surface, II. burial of polar surface and formation of solute-solute H-bonds, and III. disruption of "structural" hydration. Sequence dependent conformational changes may also be important when comparing ligand binding to different DNA sequences. We conclude that a combination of different thermodynamic parameters, especially volume change, is essential in order to understand the role of hydration in the energetics of DNA-ligand interactions.

The structures and related properties of the complex [Ru(phen)2(6-OH-dppz)]2+ (phen = 1,10-phenanthroline; dppz = dipyrido [3,2-a:2',3'-c]phenazine) in the ground state (S0), the first singlet excited state (S1), and the first triplet excited state (T1) have been studied using density functional theory (DFT), time-dependent (TD) DFT, Hartree-Fock (HF), and configuration interaction singles (CIS) methods. Three electronic absorption-spectral bands (1MLCT, 1LL, and 1LL) lying in the range of 250-550 nm in vacuo and in aqueous solution were theoretically calculated, simulated, and assigned with TDDFT method. In particular, the theoretical results show the following: (1) The positive charges of central Ru atom in the excited states (S1 and T1) are greatly increased relative to those in the ground state (S0), and thus the Ru atom in the excited states can be regarded as Ru(III). (2) The positive charges on the main ligand (6-OH-dppz) in the excited states are considerably reduced, and thus the interaction between the main ligand (intercalative ligand) and DNA base pairs is considerably weakened. (3) The geometric structures in excited states are also distorted, resulting in obvious increase in the coordination bond length. It is advantageous to the complex forming a high oxidizing center (i.e., Ru(III) ion). On the basis of these results, a theoretical explanation on photoinduced oxidation reduction mechanism of DNA photocleavage by [Ru(phen)2(6-OH-dppz)](2+) has been presented.

We report the 1.1-A resolution crystal structure of a bulky rhodium complex bound to two different DNA sites, mismatched and matched in the oligonucleotide 5'-(dCGGAAATTCCCG)2-3'. At the AC mismatch site, the structure reveals ligand insertion from the minor groove with ejection of both mismatched bases and elucidates how destabilized mispairs in DNA may be recognized. This unique binding mode contrasts with major groove intercalation, observed at a matched site, where doubling of the base pair rise accommodates stacking of the intercalator. Mass spectral analysis reveals different photocleavage products associated with the two binding modes in the crystal, with only products characteristic of mismatch binding in solution. This structure, illustrating two clearly distinct binding modes for a molecule with DNA, provides a rationale for the interrogation and detection of mismatches.

The interactions between plasmid DNA and cationic polymers are of interest for their potential biological applications. In this paper, the interactions of DNA with polyamidoamine (PAMAM) dendrimer were studied by fluorescence spectroscopy and stopped-flow technique. A rapid and reproducible fluorescent assay method had been developed for assessing PAMAM and DNA interactions using [Ru(phen)2 dppz]2+ as a probe. We further studied the kinetics of PAMAM binding to DNA and the reverse process of DNA dissociation from the complexes. The results indicated that DNA condensation was the rate-determining step during the complexation process, while DNA unfolding and expansion was the rate-determining step during the DNA dissociation process. At N/P ratios before reaching the thermodynamically most stable state, the complexes of DNA and PAMAM were incompact and could dissociate to some extent. And at N/P ratios above 2.0, DNA was fully condensed by PAMAM and dissociation was increasingly difficult. These results provided some useful instructions for self-assembling and disassembling of DNA as well as efficient gene delivery applications.

The hole transfer rates in the DNA/DNA B-form duplex and DNA/2'-OMeRNA A-form duplex were measured which occurred in the time range of approximately 100 micros. The hole transfer rates in the A-form duplexes were slower and more strongly dependent on the temperature compared to those in the B-form duplexes, suggesting that the A-form is more rigid than the B-form duplex in this time scale.

The affinities of polyamines consisting of ethylenediamine units equipped with either one or two terminal naphthyl-, anthryl-, or acridyl units towards PolyA.PolyU as an RNA model, and Poly(dA).Poly(dT) as a DNA model are screened by measuring the melting point changes (DeltaT(m)) of the double strands, and also partially by a fluorimetric binding assay using ethidium bromide. The larger aromatic moieties with long spacers between them allow bisintercalation; this leads to an increased preference for DNA in comparison to RNA, where ion pairing of the ammonium centers with the major RNA groove phosphates dominates. Allosteric affinity control by metalation is achieved e.g. with Cu(2+) ions, which induce conformational distortions within the chains. With anthryl- in contrast to naphthyl derivatives intercalation can be so strong that distortion of the ethylenediamine chain by metalation is not powerful enough. A particularly high concentration of positive charges is accessible with tripodal derivatives built up from ethylenediamine and propylenediamine units; in the absence of aryl parts, which interfere with the RNA groove preference, one observes the highest affinity difference known until today, reflected in a melting point ratio of DeltaT(m(RNA))/DeltaT(m(DNA)) = 40, whereas other synthetic ligands reach only a DeltaT(m(RNA))/DeltaT(m(DNA)) ratio of about 3.

Singlet molecular oxygen, a1Delta(g), can be detected from a single cell by its weak 1270 nm phosphorescence (a1Delta(g)-->X3Sigma(g)-) upon irradiation of the photosensitizer 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine (TMPyP) incorporated into the cell. The behavior of this sensitizer in a cell, and hence the behavior of the associated singlet oxygen phosphorescence signal, depends on the conditions under which the sample is exposed to light. Upon irradiation of a neuron freshly incubated with TMPyP, the intensity of TMPyP fluorescence initially increases and there is a concomitant increase in the singlet oxygen phosphorescence intensity from the cell. These results appear to reflect a photoinduced release of TMPyP bound to DNA in the nucleus of the cell, where TMPyP tends to localize, and the subsequent relocalization of TMPyP to a different microenvironment in the cell. Upon prolonged irradiation of the cell, TMPyP photobleaches and there is a corresponding decrease in the singlet oxygen phosphorescence intensity from the cell. The data reported herein provide insight into key factors that can influence photosensitized singlet oxygen experiments performed on biological samples.

Analysis of PCR fragments for applications, such as screening of nucleotide polymorphisms, detection of somatic mutations, or quantification of reverse-transcription PCR products, becomes central in clinical research as well as preventive testing, diagnostic screening, and pharmacogenomic genotyping. A variety of CE techniques, utilizing great potential of multicapillary-array sequencers, is now commonly applied in prevention, diagnosis, and treatment of a wide range of genetic diseases (cancer, cardiovascular, and neurodegenerative diseases, etc.). Costs of fluorescently labeled primers is often a major factor in large-scale projects requiring mutation analysis in hundreds or thousands of samples. In the present paper we introduce a simple approach of detecting unlabeled DNA fragments through intercalation without a need for adding intercalator to the separation polymer matrix. The dye is only added to the anode reservoir, and mixing with the separated DNA fragments takes place upon its migration opposite to the direction of the CE separation. Using two common intercalating dyes (ethidium bromide and SYBR Green II) we present this method as a tool for routine PCR detection and quantification.

The interactions between DNA and chitosans varying in fractional content of acetylated units (FA), degree of polymerization (DP), and degree of ionization were investigated by several techniques, including an ethidium bromide (EtBr) fluorescence assay, gel retardation, atomic force microscopy, and dynamic and electrophoretic light scattering. The charge density of the chitosan and the number of charges per chain were found to be the dominating factors for the structure and stability of DNA-chitosan complexes. All high molecular weight chitosans condensed DNA into physically stable polyplexes; however, the properties of the complexes were strongly dependent on FA, and thereby the charge density of chitosan. By employing fully charged oligomers of constant charge density, it was shown that the complexation of DNA and stability of the polyplexes is governed by the number of cationic residues per chain. A minimum of 6-9 positive charges appeared necessary to provide interaction strength comparable to that of polycations. In contrast, further increase in the number of charges above 9 did not increase the apparent binding affinity as judged from the EtBr displacement assay. The chitosan oligomers exhibited a pH-dependent interaction with DNA, reflecting the number of ionized amino groups. The complexation of DNA and the stability of oligomer-based polyplexes became reduced above pH 7.4. Such pH-dependent dissociation of polyplexes around the physiological pH is highly relevant in gene delivery applications and might be one of the reasons for the high transfection activity of oligomer-based polyplexes observed.

The designed cytosine-carbohydrate hybrid molecule selectively recognized and stabilized the bulged duplex DNA possessing the complementary bulged DNA base, guanine, while the nucleotide base itself did not exhibit any such ability. It was also found that the assistance of the carbohydrate to stabilize the interaction between the nucleotide base and the complementally bulge DNA base is very helpful for the selective recognition and stabilization of the single-bulged duplex DNA.

Ferrocenylnaphthalene diimide 1 can bind to double stranded DNA (dsDNA) by the threading intercalation mode and the resulting complex was stabilized further by beta- cyclodextrin (CD) by forming a supramolecular complex. These complex formation processes were studied by spectroscopic, viscometric, and electrochemical means in the absence or presence of beta-CD. Quantitative analysis by quartz crystal microbalance (QCM) and electrochemical experiments strongly suggested a 2:1 binding stoichiometry for beta-CD to 1 threading-intercalated to the dsDNA-immobilized electrode. Owing to this supramolecular complex formation, electrochemical DNA detection based on 1 was improved considerably.