Sample records for intrastrand dna g-quadruplexes

  1. Simultaneous G-Quadruplex DNA Logic. (United States)

    Bader, Antoine; Cockroft, Scott L


    A fundamental principle of digital computer operation is Boolean logic, where inputs and outputs are described by binary integer voltages. Similarly, inputs and outputs may be processed on the molecular level as exemplified by synthetic circuits that exploit the programmability of DNA base-pairing. Unlike modern computers, which execute large numbers of logic gates in parallel, most implementations of molecular logic have been limited to single computing tasks, or sensing applications. This work reports three G-quadruplex-based logic gates that operate simultaneously in a single reaction vessel. The gates respond to unique Boolean DNA inputs by undergoing topological conversion from duplex to G-quadruplex states that were resolved using a thioflavin T dye and gel electrophoresis. The modular, addressable, and label-free approach could be incorporated into DNA-based sensors, or used for resolving and debugging parallel processes in DNA computing applications. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Structure of a left-handed DNA G-quadruplex. (United States)

    Chung, Wan Jun; Heddi, Brahim; Schmitt, Emmanuelle; Lim, Kah Wai; Mechulam, Yves; Phan, Anh Tuân


    Aside from the well-known double helix, DNA can also adopt an alternative four-stranded structure known as G-quadruplex. Implications of such a structure in cellular processes, as well as its therapeutic and diagnostic applications, have been reported. The G-quadruplex structure is highly polymorphic, but so far, only right-handed helical forms have been observed. Here we present the NMR solution and X-ray crystal structures of a left-handed DNA G-quadruplex. The structure displays unprecedented features that can be exploited as unique recognition elements.

  3. G-Quadruplexes Involving Both Strands of Genomic DNA Are Highly Abundant and Colocalize with Functional Sites in the Human Genome.

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    Andrzej S Kudlicki

    Full Text Available The G-quadruplex is a non-canonical DNA structure biologically significant in DNA replication, transcription and telomere stability. To date, only G4s with all guanines originating from the same strand of DNA have been considered in the context of the human nuclear genome. Here, I discuss interstrand topological configurations of G-quadruplex DNA, consisting of guanines from both strands of genomic DNA; an algorithm is presented for predicting such structures. I have identified over 550,000 non-overlapping interstrand G-quadruplex forming sequences in the human genome--significantly more than intrastrand configurations. Functional analysis of interstrand G-quadruplex sites shows strong association with transcription initiation, the results are consistent with the XPB and XPD transcriptional helicases binding only to G-quadruplex DNA with interstrand topology. Interstrand quadruplexes are also enriched in origin of replication sites. Several topology classes of interstrand quadruplex-forming sequences are possible, and different topologies are enriched in different types of structural elements. The list of interstrand quadruplex forming sequences, and the computer program used for their prediction are available at the web address

  4. Identification of novel interactors of human telomeric G-quadruplex DNA. (United States)

    Pagano, Bruno; Margarucci, Luigi; Zizza, Pasquale; Amato, Jussara; Iaccarino, Nunzia; Cassiano, Chiara; Salvati, Erica; Novellino, Ettore; Biroccio, Annamaria; Casapullo, Agostino; Randazzo, Antonio


    A chemoproteomic-driven approach was used to investigate the interaction network between human telomeric G-quadruplex DNA and nuclear proteins. We identified novel G-quadruplex binding partners, able to recognize these DNA structures at chromosome ends, suggesting a possible, and so far unknown, role of these proteins in telomere functions.

  5. Effect of G-quadruplex polymorphism on the recognition of telomeric DNA by a metal complex.

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    Caterina Musetti

    Full Text Available The physiological role(s played by G-quadruplexes renders these 'non-canonical' DNA secondary structures interesting new targets for therapeutic intervention. In particular, the search for ligands for selective recognition and stabilization of G-quadruplex arrangements has led to a number of novel targeted agents. An interesting approach is represented by the use of metal-complexes, their binding to DNA being modulated by ligand and metal ion nature, and by complex stoichiometry. In this work we characterized thermodynamically and stereochemically the interactions of a Ni(II bis-phenanthroline derivative with telomeric G-quadruplex sequences using calorimetric, chiroptical and NMR techniques. We employed three strictly related sequences based on the human telomeric repeat, namely Tel22, Tel26 and wtTel26, which assume distinct conformations in potassium containing solutions. We were able to monitor specific enthalpy/entropy changes according to the structural features of the target telomeric sequence and to dissect the binding process into distinct events. Interestingly, temperature effects turned out to be prominent both in terms of binding stoichiometry and ΔH/ΔS contributions, while the final G-quadruplex-metal complex architecture tended to merge for the examined sequences. These results underline the critical choice of experimental conditions and DNA sequence for practical use of thermodynamic data in the rational development of effective G-quadruplex binders.

  6. Structural and functional analysis of DNA sequences with potential for forming G-quadruplex


    Luciana Souto Mofatto


    Resumo: Os G-quadruplexes são estruturas secundárias de DNA altamente organizadas, constituídas por sequências ricas em guaninas capazes de formar tétrades ligadas por pontes de hidrogênio. Essas sequências são capazes de modular a transcrição gênica e o splicing alternativo de éxons. Além disso, estudos também mostraram que os G-quadruplexes estão presentes na região promotora de oncogenes (como c-MYC) e nas regiões terminais dos telômeros, indicando que o G-quadruplex pode ser um possível a...

  7. Synthesis and Molecular Modeling of Thermally Stable DNA G-Quadruplexes with Anthraquinone Insertions

    DEFF Research Database (Denmark)

    Gouda, Alaa S.; Amine, Mahasen S.; Pedersen, Erik Bjerregaard


    Two new phosphoramidite building blocks for DNA synthesis were synthesized from 1,5- and 2,6-dihydroxyanthraquinones through alkylation with 3-bromo-1-propanol followed by DMT-protection. The novel synthesized 1,5- and 2,6-disubstituted anthraquinone monomers H15 and H26 are incorporated into a G......-quadruplex by single and double replacements of TGT and TT loops. Monomers H15 and H26 were found to destabilize G-quadruplex structures for all single replacements of TGT or TT loops. The largest destabilization was observed when H26 linker replaced a TT loop. In contrast, the presence of anthraquinone monomers...... anthraquinone-modified quadruplexes revealed no change of the antiparallel structure when compared with the wild type under potassium buffer conditions. The significantly increased thermostabilities were interpreted by molecular modeling of anthraquinone-modified G-quadruplexes....

  8. Putative DNA G-quadruplex formation within the promoters of Plasmodium falciparum var genes

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    Rowe J


    Full Text Available Abstract Background Guanine-rich nucleic acid sequences are capable of folding into an intramolecular four-stranded structure called a G-quadruplex. When found in gene promoter regions, G-quadruplexes can downregulate gene expression, possibly by blocking the transcriptional machinery. Here we have used a genome-wide bioinformatic approach to identify Putative G-Quadruplex Sequences (PQS in the Plasmodium falciparum genome, along with biophysical techniques to examine the physiological stability of P. falciparum PQS in vitro. Results We identified 63 PQS in the non-telomeric regions of the P. falciparum clone 3D7. Interestingly, 16 of these PQS occurred in the upstream region of a subset of the P. falciparum var genes (group B var genes. The var gene family encodes PfEMP1, the parasite's major variant antigen and adhesin expressed at the surface of infected erythrocytes, that plays a key role in malaria pathogenesis and immune evasion. The ability of the PQS found in the upstream regions of group B var genes (UpsB-Q to form stable G-quadruplex structures in vitro was confirmed using 1H NMR, circular dichroism, UV spectroscopy, and thermal denaturation experiments. Moreover, the synthetic compound BOQ1 that shows a higher affinity for DNA forming quadruplex rather than duplex structures was found to bind with high affinity to the UpsB-Q. Conclusion This is the first demonstration of non-telomeric PQS in the genome of P. falciparum that form stable G-quadruplexes under physiological conditions in vitro. These results allow the generation of a novel hypothesis that the G-quadruplex sequences in the upstream regions of var genes have the potential to play a role in the transcriptional control of this major virulence-associated multi-gene family.

  9. Guanine Bases in DNA G-Quadruplex Adopt Nonplanar Geometries Owing to Solvation and Base Pairing

    Czech Academy of Sciences Publication Activity Database

    Sychrovský, Vladimír; Sochorová Vokáčová, Zuzana; Trantírek, L.


    Roč. 116, č. 16 (2012), s. 4144-4151 ISSN 1089-5639 R&D Projects: GA ČR GAP205/10/0228 Institutional research plan: CEZ:AV0Z40550506 Keywords : pyramidalization * nonplanarity * G-quadruplex * DNA * solvent effect Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.771, year: 2012

  10. UvrD in Deinococcus radiodurans is optimized for processing G-quadruplex DNA

    International Nuclear Information System (INIS)

    Das, Anubrata; Misra, H.S.


    Deinococcus radiodurans R1 is a radiation resistant Gram-positive bacterium capable of tolerating very high doses of DNA-damaging agents such as gamma radiation (D10 ∼ 12kGy) desiccation (∼ 5% relative humidity), UVC radiation (D10 ∼ 800J/m 2 ) and hydrogen peroxide (40 mM). It achieves this by using a complex regulatory mechanism and novel proteins. Recently bioinformatic analysis showed several stretches of guanine runs in D.radiodurans genome, which could form G-quartets. The role of G-quartets in regulatory processes is well documented in various organisms. The presence of G -quartets in D. radiodurans means that there are regulatory or structural proteins which would bind to these elements. Several proteins are known to bind G-quartets. Finding the proteins which would bind to G4 DNA is difficult as no specific motifs are available for binding these elements. Also most of the known proteins that are shown to bind to G-quadruplex DNA are of eukaryotic nature. To overcome these challenges we defined a set of known G-quadruplex binding proteins and used a smith-waterman algorithm with our own scoring matrix to homologs of G-quadruplex binding proteins in D.radiodurans. Using bioinformatics analysis, we showed that UvrD (DR 1775) of D. radiodurans has ability to bind/translocate along G-quadruplex DNA, a novel feature in prokaryotes. The translocase activity of DR1775 is ATP specific and this ATPase activity is attenuated by ssDNA. Data supporting UvrD of D. radiodurans as a G-quadruplex DNA metabolizing proteins would be presented. (author)

  11. G-quadruplex DNA biosensor for sensitive visible detection of genetically modified food. (United States)

    Jiang, Xiaohua; Zhang, Huimin; Wu, Jun; Yang, Xiang; Shao, Jingwei; Lu, Yujing; Qiu, Bin; Lin, Zhenyu; Chen, Guonan


    In this paper, a novel label-free G-quadruplex DNAzyme sensor has been proposed for colorimetric identification of GMO using CaMV 35S promoter sequence as the target. The binary probes can fold into G-quadruplex structure in the presence of DNA-T (Target DNA) and then combine with hemin to form a DNAzyme resembling horseradish peroxidase. The detection system consists of two G-rich probes with 2:2 split mode by using the absorbance and color of ABTS(2-) as signal reporter. Upon the addition of a target sequence, two probes both hybridize with target and then their G-rich sequences combine to form a G-quadruplex DNAzyme, and the DNAzyme can catalyze the reaction of ABTS(2-) with H2O2. Then the linear range is from 0.05 to 0.5 μM while detection limit is 5nM. These results demonstrate that the proposed G-quadruplex DNAzyme method could be used as a simple, sensitive and cost-effective approach for assays of GMO. Copyright © 2014 Elsevier B.V. All rights reserved.

  12. G-Quadruplex DNA Motifs in the Malaria Parasite Plasmodium falciparum and Their Potential as Novel Antimalarial Drug Targets. (United States)

    Harris, Lynne M; Monsell, Katelyn R; Noulin, Florian; Famodimu, M Toyin; Smargiasso, Nicolas; Damblon, Christian; Horrocks, Paul; Merrick, Catherine J


    G-quadruplexes are DNA or RNA secondary structures that can be formed from guanine-rich nucleic acids. These four-stranded structures, composed of stacked quartets of guanine bases, can be highly stable and have been demonstrated to occur in vivo in the DNA of human cells and other systems, where they play important biological roles, influencing processes such as telomere maintenance, DNA replication and transcription, or, in the case of RNA G-quadruplexes, RNA translation and processing. We report for the first time that DNA G-quadruplexes can be detected in the nuclei of the malaria parasite Plasmodium falciparum , which has one of the most A/T-biased genomes sequenced and therefore possesses few guanine-rich sequences with the potential to form G-quadruplexes. We show that despite this paucity of putative G-quadruplex-forming sequences, P. falciparum parasites are sensitive to several G-quadruplex-stabilizing drugs, including quarfloxin, which previously reached phase 2 clinical trials as an anticancer drug. Quarfloxin has a rapid initial rate of kill and is active against ring stages as well as replicative stages of intraerythrocytic development. We show that several G-quadruplex-stabilizing drugs, including quarfloxin, can suppress the transcription of a G-quadruplex-containing reporter gene in P. falciparum but that quarfloxin does not appear to disrupt the transcription of rRNAs, which was proposed as its mode of action in both human cells and trypanosomes. These data suggest that quarfloxin has potential for repositioning as an antimalarial with a novel mode of action. Furthermore, G-quadruplex biology in P. falciparum may present a target for development of other new antimalarial drugs. Copyright © 2018 American Society for Microbiology.

  13. Targeting G-quadruplex DNA structures in the telomere and oncogene promoter regions by benzimidazole‒carbazole ligands. (United States)

    Kaulage, Mangesh H; Maji, Basudeb; Pasadi, Sanjeev; Ali, Asfa; Bhattacharya, Santanu; Muniyappa, K


    Recent studies support the idea that G-quadruplex structures in the promoter regions of oncogenes and telomere DNA can serve as potential therapeutic targets in the treatment of cancer. Accordingly, several different types of organic small molecules that stabilize G-quadruplex structures and inhibit telomerase activity have been discerned. Here, we describe the binding of benzimidazole-carbazole ligands to G-quadruplex structures formed in G-rich DNA sequences containing the promoter regions of human c-MYC, c-KIT1, c-KIT2, VEGF and BCL2 proto-oncogenes. The fluorescence spectroscopic data indicate that benzimidazole-carbazole ligands bind and stabilize the G-quadruplexes in the promoter region of oncogenes. The molecular docking studies provide insights into the mode and extent of binding of this class of ligands to the G-quadruplexes formed in oncogene promoters. The high stability of these G-quadruplex structures was validated by thermal denaturation and telomerase-catalyzed extension of the 3' end. Notably, benzimidazole-carbazole ligands suppress the expression of oncogenes in cancer cells in a dose-dependent manner. We anticipate that benzimidazole-carbazole ligands, by virtue of their ability to stabilize G-quadruplex structures in the promoter regions of oncogenes, might reduce the risk of cancer through the loss of function in the proteins encoded by these genes. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  14. Oligonucleotide Models of Telomeric DNA and RNA Form a Hybrid G-quadruplex Structure as a Potential Component of Telomeres* (United States)

    Xu, Yan; Ishizuka, Takumi; Yang, Jie; Ito, Kenichiro; Katada, Hitoshi; Komiyama, Makoto; Hayashi, Tetsuya


    Telomeric repeat-containing RNA, a non-coding RNA molecule, has recently been found in mammalian cells. The detailed structural features and functions of the telomeric RNA at human chromosome ends remain unclear, although this RNA molecule may be a key component of the telomere machinery. In this study, using model human telomeric DNA and RNA sequences, we demonstrated that human telomeric RNA and DNA oligonucleotides form a DNA-RNA G-quadruplex. We next employed chemistry-based oligonucleotide probes to mimic the naturally formed telomeric DNA-RNA G-quadruplexes in living cells, suggesting that the process of DNA-RNA G-quadruplex formation with oligonucleotide models of telomeric DNA and RNA could occur in cells. Furthermore, we investigated the possible roles of this DNA-RNA G-quadruplex. The formation of the DNA-RNA G-quadruplex causes a significant increase in the clonogenic capacity of cells and has an effect on inhibition of cellular senescence. Here, we have used a model system to provide evidence about the formation of G-quadruplex structures involving telomeric DNA and RNA sequences that have the potential to provide a protective capping structure for telomere ends. PMID:23012368

  15. Pyrrolobenzodiazepines (PBDs do not bind to DNA G-quadruplexes.

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    Khondaker M Rahman

    Full Text Available The pyrrolo[2,1-c][1,4] benzodiazepines (PBDs are a family of sequence-selective, minor-groove binding DNA-interactive agents that covalently attach to guanine residues. A recent publication in this journal (Raju et al, PloS One, 2012, 7, 4, e35920 reported that two PBD molecules were observed to bind with high affinity to the telomeric quadruplex of Tetrahymena glaucoma based on Electrospray Ionisation Mass Spectrometry (ESI-MS, Circular Dichroism, UV-Visible and Fluorescence spectroscopy data. This was a surprising result given the close 3-dimensional shape match between the structure of all PBD molecules and the minor groove of duplex DNA, and the completely different 3-dimensional structure of quadruplex DNA. Therefore, we evaluated the interaction of eight PBD molecules of diverse structure with a range of parallel, antiparallel and mixed DNA quadruplexes using DNA Thermal Denaturation, Circular Dichroism and Molecular Dynamics Simulations. Those PBD molecules without large C8-substitutents had an insignificant affinity for the eight quadruplex types, although those with large π-system-containing C8-substituents (as with the compounds evaluated by Raju and co-workers were found to interact to some extent. Our molecular dynamics simulations support the likelihood that molecules of this type, including those examined by Raju and co-workers, interact with quadruplex DNA through their C8-substituents rather than the PBD moiety itself. It is important for the literature to be clear on this matter, as the mechanism of action of these agents will be under close scrutiny in the near future due to the growing number of PBD-based agents entering the clinic as both single-agents and as components of antibody-drug conjugates (ADCs.

  16. Controlling the stoichiometry and strand polarity of a tetramolecular G-quadruplex structure by using a DNA origami frame (United States)

    Rajendran, Arivazhagan; Endo, Masayuki; Hidaka, Kumi; Lan Thao Tran, Phong; Mergny, Jean-Louis; Sugiyama, Hiroshi


    Guanine-rich oligonucleotides often show a strong tendency to form supramolecular architecture, the so-called G-quadruplex structure. Because of the biological significance, it is now considered to be one of the most important conformations of DNA. Here, we describe the direct visualization and single-molecule analysis of the formation of a tetramolecular G-quadruplex in KCl solution. The conformational changes were carried out by incorporating two duplex DNAs, with G–G mismatch repeats in the middle, inside a DNA origami frame and monitoring the topology change of the strands. In the absence of KCl, incorporated duplexes had no interaction and laid parallel to each other. Addition of KCl induced the formation of a G-quadruplex structure by stably binding the duplexes to each other in the middle. Such a quadruplex formation allowed the DNA synapsis without disturbing the duplex regions of the participating sequences, and resulted in an X-shaped structure that was monitored by atomic force microscopy. Further, the G-quadruplex formation in KCl solution and its disruption in KCl-free buffer were analyzed in real-time. The orientation of the G-quadruplex is often difficult to control and investigate using traditional biochemical methods. However, our method using DNA origami could successfully control the strand orientations, topology and stoichiometry of the G-quadruplex. PMID:23863846

  17. Biochemical techniques for the characterization of G-quadruplex structures: EMSA, DMS footprinting, and DNA polymerase stop assay. (United States)

    Sun, Daekyu; Hurley, Laurence H


    The proximal promoter region of many human growth-related genes contains a polypurine/polypyrimidine tract that serves as multiple binding sites for Sp1 or other transcription factors. These tracts often contain a guanine-rich sequence consisting of four runs of three or more contiguous guanines separated by one or more bases, corresponding to a general motif known for the formation of an intramolecular G-quadruplex. Recent results provide strong evidence that specific G-quadruplex structures form naturally within these polypurine/polypyrimidine tracts in many human promoter regions, raising the possibility that the transcriptional control of these genes can be modulated by G-quadruplex-interactive agents. In this chapter, we describe three general biochemical methodologies, electrophoretic mobility shift assay (EMSA), dimethylsulfate (DMS) footprinting, and the DNA polymerase stop assay, which can be useful for initial characterization of G-quadruplex structures formed by G-rich sequences.

  18. New insights from molecular dynamic simulation studies of the multiple binding modes of a ligand with G-quadruplex DNA (United States)

    Hou, Jin-Qiang; Chen, Shuo-Bin; Tan, Jia-Heng; Luo, Hai-Bin; Li, Ding; Gu, Lian-Quan; Huang, Zhi-Shu


    G-quadruplexes are higher-order DNA and RNA structures formed from guanine-rich sequences. These structures have recently emerged as a new class of potential molecular targets for anticancer drugs. An understanding of the three-dimensional interactions between small molecular ligands and their G-quadruplex targets in solution is crucial for rational drug design and the effective optimization of G-quadruplex ligands. Thus far, rational ligand design has been focused mainly on the G-quartet platform. It should be noted that small molecules can also bind to loop nucleotides, as observed in crystallography studies. Hence, it would be interesting to elucidate the mechanism underlying how ligands in distinct binding modes influence the flexibility of G-quadruplex. In the present study, based on a crystal structure analysis, the models of a tetra-substituted naphthalene diimide ligand bound to a telomeric G-quadruplex with different modes were built and simulated with a molecular dynamics simulation method. Based on a series of computational analyses, the structures, dynamics, and interactions of ligand-quadruplex complexes were studied. Our results suggest that the binding of the ligand to the loop is viable in aqueous solutions but dependent on the particular arrangement of the loop. The binding of the ligand to the loop enhances the flexibility of the G-quadruplex, while the binding of the ligand simultaneously to both the quartet and the loop diminishes its flexibility. These results add to our understanding of the effect of a ligand with different binding modes on G-quadruplex flexibility. Such an understanding will aid in the rational design of more selective and effective G-quadruplex binding ligands.

  19. Interaction of hnRNP A1 with telomere DNA G-quadruplex structures studied at the single molecule level

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    Krüger, Asger Christian; Raarup, Merete Krog; Nielsen, Morten Muhlig


    G-rich telomeric DNA sequences can form G-quadruplex structures. The heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) and a shortened derivative (UP1) are active in telomere length regulation, and it has been reported that UP1 can unwind G-quadruplex structures. Here, we investigate...... the interaction of hnRNP A1 with G-quadruplex DNA structures containing the human telomere repeat (TTAGGG) by gel retardation assays, ensemble fluorescence energy transfer (FRET) spectroscopy, and single molecule FRET microscopy. Our biochemical experiments show that hnRNP A1 binds well to the G...... to the previously reported crystal structures of UP1-telomere DNA complexes where the DNA oligo within the protein-DNA complex is in a fully open conformation....

  20. Volumetric contributions of loop regions of G-quadruplex DNA to the formation of the tertiary structure. (United States)

    Takahashi, Shuntaro; Sugimoto, Naoki


    DNA guanine-quadruplexes (G-quadruplexes) are unique DNA structures formed by guanine-rich sequences. The loop regions of G-quadruplexes play key roles in stability and topology of G-quadruplexes. Here, we investigated volumetric changes induced by pressure in the folding of the G-quadruplex formed by the thrombin binding aptamer (TBA) with mutations within the loop regions. The change of partial molar volume in the transition from coil to G-quadruplex, ∆V tr , of TBA with a mutation from T to A in the 5' most loop (TBA T3A) was 75.5cm 3 mol -1 , which was larger than that of TBA (54.6cm 3 mol -1 ). TBA with a G to T mutation in the central loop (TBA G8T) had thermal stability similar to TBA T3A but a smaller ∆V tr of 41.1cm 3 mol -1 . In the presence of poly(ethylene)glycol 200 (PEG200), ∆V tr values were 14.7cm 3 mol -1 for TBA T3A and 13.2cm 3 mol -1 for TBA G8T. These results suggest that the two mutations destabilize the G-quadruplex structure differently. Thus, volumetric data obtained using pressure-based thermodynamic analyses provides information about the dynamics of the loop regions and the roles of loops in the stabilities and folding of G-quadruplex structures. Copyright © 2017 Elsevier B.V. All rights reserved.

  1. Distinct differences in metal ion specificity of RNA and DNA G-quadruplexes. (United States)

    Guiset Miserachs, Helena; Donghi, Daniela; Börner, Richard; Johannsen, Silke; Sigel, Roland K O


    RNA G-quadruplexes, as their well-studied DNA analogs, require the presence of cations to fold and remain stable. This is the first comprehensive study on the interaction of RNA quadruplexes with metal ions. We investigated the formation and stability of two highly conserved and biologically relevant RNA quadruplex-forming sequences (24nt-TERRA and 18nt-NRAS) in the presence of several monovalent and divalent metal ions, namely Li + , Na + , K + , Rb + , Cs + , NH 4 + , Mg 2+ , Ca 2+ , Sr 2+ , and Ba 2+ . Circular dichroism was used to probe the influence of these metal ions on the folded fraction of the parallel G-quadruplexes, and UV thermal melting experiments allowed to assess the relative stability of the structures in each cationic condition. Our results show that the RNA quadruplexes are more stable than their DNA counterparts under the same buffer conditions. We have observed that the addition of mainly Na + , K + , Rb + , NH 4 + , as well as Sr 2+ and Ba 2+ in water, shifts the equilibrium to the folded quadruplex form, whereby the NRAS sequence responds stronger than TERRA. However, only K + and Sr 2+ lead to a significant increase in the stability of the folded structures, which is consistent with their coordination to the O6 atoms from the G-quartet guanosines. Compared to the respective DNA motives, dNRAS and htelo, the RNA sequences are not stabilized by Na + ions. Finally, the difference in response between NRAS and TERRA, as well as to the corresponding DNA sequences with respect to different metal ions, could potentially be exploited for selective targeting purposes.

  2. G-quadruplex and calf thymus DNA interaction of quaternized tetra and octa pyridyloxy substituted indium (III) phthalocyanines. (United States)

    Bağda, Efkan; Bağda, Esra; Durmuş, Mahmut


    The interactions of small molecules with G-quadruplex and double stranded DNA are important due to their potential biological and medical usages. In the present paper, the interactions of indium (III) phthalocyanines (quaternized 2,3,9,10,16,17,23,24-octakis-[(3-pyridyloxy) phthalocyaninato] chloroindium(III): OInPc and quaternized 2(3),9(10),16(17),23(24)-tetrakis-[(3-pyridyloxy) phthalocyaninato] chloroindium(III): TInPc) with hybrid G-quadruplex (Tel 21) and parallel G-quadruplexes (nucleolin, KRAS, c-MYC, vegf) were studied. The interactions of these phthalocyanines with ctDNA were also investigated. These interactions were measured by different spectroscopic techniques such as UV-Vis, fluorescence and circular dichroism. The UV-Vis spectroscopic data treated with Benesi-Hildebrand equation and Benesi-Hildebrand constants (K BH ) were calculated. These constants were found higher for octa peripheral pyridyloxy substituted phthalocyanine, OInPc. Besides, UV-Vis analysis showed that the interaction of G-quadruplexes with tetra peripheral pyridyloxy substituted phthalocyanine derivative (TInPc) resulted in removal of central indium (III) atom from the cavity of phthalocyanine macrocycle. The UV-Vis melting studies as well as fluorescence replacement techniques were also employed for clarification of mechanism. The binding mode of molecules with ct DNA was also supported with viscosity measurements. From the results, the stabilization and destabilization of G-quadruplex depending on the concentration of the OInPc and TInPc showed that these two indium (III) phthalocyanines have the potential of both the elucidation role of G-quadruplexes in gene expression and the usage in cancer therapy. Copyright © 2017. Published by Elsevier B.V.

  3. Effect of Pressure on Thermal Stability of G-Quadruplex DNA and Double-Stranded DNA Structures

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    Shuntaro Takahashi


    Full Text Available Pressure is a thermodynamic parameter that can induce structural changes in biomolecules due to a volumetric decrease. Although most proteins are denatured by pressure over 100 MPa because they have the large cavities inside their structures, the double-stranded structure of DNA is stabilized or destabilized only marginally depending on the sequence and salt conditions. The thermal stability of the G-quadruplex DNA structure, an important non-canonical structure that likely impacts gene expression in cells, remarkably decreases with increasing pressure. Volumetric analysis revealed that human telomeric DNA changed by more than 50 cm3 mol−1 during the transition from a random coil to a quadruplex form. This value is approximately ten times larger than that for duplex DNA under similar conditions. The volumetric analysis also suggested that the formation of G-quadruplex DNA involves significant hydration changes. The presence of a cosolute such as poly(ethylene glycol largely repressed the pressure effect on the stability of G-quadruplex due to alteration in stabilities of the interactions with hydrating water. This review discusses the importance of local perturbations of pressure on DNA structures involved in regulation of gene expression and highlights the potential for application of high-pressure chemistry in nucleic acid-based nanotechnology.

  4. A single-molecule force-spectroscopic study on stabilization of G-quadruplex DNA by a telomerase inhibitor. (United States)

    Funayama, Ryoto; Nakahara, Yoshio; Kado, Shinpei; Tanaka, Mutsuo; Kimura, Keiichi


    Single-molecule force spectroscopy was carried out using AFM force measurements for the purpose of direct observation of the stabilization of G-quadruplex DNA by a telomerase inhibitor, which is 5,10,15,20-tetrakis(N-methylpyridinium-4-yl)porphyrin tetrakis(p-toluenesulfonate) (TMPyP). In AFM force measurements, we used an AFM tip and an Au substrate modified chemically with terminal-biotinylated telomere DNA and streptavidin, respectively. The telomere DNA was fully stretched by the AFM tip based on the bridge formation between the AFM tip and the Au substrate through the streptavidin-biotin interaction. The force-extension curves, which reflected the stretching of a single DNA molecule, were distinguished from all of the curves, judging from the rupture force and the contour length. The selected curves were analyzed using a worm-like chain model, and one of the fitting parameters, persistence length (lp), was used as an index for the stabilization of the G-quadruplex structure. Consequently, the lp value was significantly increased by the addition of TMPyP under the experimental conditions where the G-quadruplex structure could be formed. On the other hand, the value was hardly changed by the addition of TMPyP under the conditions except the above. Furthermore, the methodology developed and demonstrated in this work was applied to evaluate the stabilization of G-quadruplex DNA by other telomerase inhibitors such as ethidium bromide and p-xylene-bis(N-pyridinium bromide).

  5. Target guided synthesis using DNA nano-templates for selectively assembling a G-quadruplex binding c-MYC inhibitor (United States)

    Panda, Deepanjan; Saha, Puja; Das, Tania; Dash, Jyotirmayee


    The development of small molecules is essential to modulate the cellular functions of biological targets in living system. Target Guided Synthesis (TGS) approaches have been used for the identification of potent small molecules for biological targets. We herein demonstrate an innovative example of TGS using DNA nano-templates that promote Huisgen cycloaddition from an array of azide and alkyne fragments. A G-quadruplex and a control duplex DNA nano-template have been prepared by assembling the DNA structures on gold-coated magnetic nanoparticles. The DNA nano-templates facilitate the regioselective formation of 1,4-substituted triazole products, which are easily isolated by magnetic decantation. The G-quadruplex nano-template can be easily recovered and reused for five reaction cycles. The major triazole product, generated by the G-quadruplex inhibits c-MYC expression by directly targeting the c-MYC promoter G-quadruplex. This work highlights that the nano-TGS approach may serve as a valuable strategy to generate target-selective ligands for drug discovery.

  6. Studies of G-quadruplex DNA structures at the single molecule level

    DEFF Research Database (Denmark)

    Kragh, Sofie Louise


    Folding of G-quaduplex structures adopted by the human telomeric repeat is here studied by single molecule FRET microscopy. This method allows for the investigation of G-quadruplex structures and their conformational dynamic. Telomeres are located at the ends of our chromosomes and end in a single...... with human telomeric repeat adopt several different G-quadruplex conformations in the presence of K+ ions. G-quadruplexes inhibit telomerase activity and are therefore potential targets for anti-cancer drugs, which can be small molecule ligands capable of stabilizing G-quadruplex structures. Understanding...... range. FRET spectroscopy can be performed on an ensemble of molecules, or on the single molecule level. In single molecule FRET experiments it is possible to follow the behaviour in time for each molecule independently, allowing insight into both dynamically and statistically heterogeneous molecular...

  7. Selective G-Quadruplex DNA Recognition by a New Class of Designed Cyanines

    Directory of Open Access Journals (Sweden)

    Markus W. Germann


    Full Text Available A variety of cyanines provide versatile and sensitive agents acting as DNA stains and sensors and have been structurally modified to bind in the DNA minor groove in a sequence dependent manner. Similarly, we are developing a new set of cyanines that have been designed to achieve highly selective binding to DNA G-quadruplexes with much weaker binding to DNA duplexes. A systematic set of structurally analogous trimethine cyanines has been synthesized and evaluated for quadruplex targeting. The results reveal that elevated quadruplex binding and specificity are highly sensitive to the polymethine chain length, heterocyclic structure and intrinsic charge of the compound. Biophysical experiments show that the compounds display significant selectivity for quadruplex binding with a higher preference for parallel stranded quadruplexes, such as cMYC. NMR studies revealed the primary binding through an end-stacking mode and SPR studies showed the strongest compounds have primary KD values below 100 nM that are nearly 100-fold weaker for duplexes. The high selectivity of these newly designed trimethine cyanines for quadruplexes as well as their ability to discriminate between different quadruplexes are extremely promising features to develop them as novel probes for targeting quadruplexes in vivo.

  8. Oncogene Expression Modulation in Cancer Cell Lines by DNA G-Quadruplex-Interactive Small Molecules. (United States)

    Francisco, Ana Paula; Paulo, Alexandra


    Nucleic acids are prone to structural polymorphism and a number of structures may be formed in addition to the well-known DNA double helix. Among these is a family of nucleic acid four-stranded structures known as G-quadruplexes (G4). These quadruplex structures can be formed by sequences containing repetitive guanine-rich tracks and the analysis of Non-B-DNA database indicated an enrichment of these sequences in genomic regions controlling cellular proliferation, such as for example in the promoter regions of c- MYC, k-RAS, c-KIT, HSP90 and VEGF among others. The broad concept of G4 targeting with small molecules is now generally accepted as a promising novel approach to anticancer therapy and several small molecules with antiproliferative activity in cancer cell lines have also been shown to stabilize these DNA structures, thus suggesting a potential application of G4-interactive small molecules as new anticancer drugs. Herein we review, by targeted oncogene and main chemical scaffold, those G4-interactive small molecules with reported gene expression modulatory activity in cancer cell lines. The data obtained so far are encouraging but further efforts are needed to validate G4 as drug targets and optimize the structure of G4- interactive small molecules into new anticancer drugs. Copyright© Bentham Science Publishers; For any queries, please email at

  9. Integration of G-quadruplex and DNA-templated Ag NCs for nonarithmetic information processing. (United States)

    Gao, Ru-Ru; Yao, Tian-Ming; Lv, Xiao-Yan; Zhu, Yan-Yan; Zhang, Yi-Wei; Shi, Shuo


    To create sophisticated molecular logic circuits from scratch, you may not believe how common the building blocks can be and how diverse and powerful such circuits can be when scaled up. Using the two simple building blocks of G-quadruplex and silver nanoclusters (Ag NCs), we experimentally construct a series of multifunctional, label-free, and multi-output logic circuits to perform nonarithmetic functions: a 1-to-2 decoder, a 4-to-2 encoder, an 8-to-3 encoder, dual transfer gates, a 2 : 1 multiplexer, and a 1 : 2 demultiplexer. Moreover, a parity checker which is capable of identifying odd and even numbers from natural numbers is constructed conceptually. Finally, a multi-valued logic gate (ternary inhibit gate) is readily achieved by taking this DNA/Ag NC system as a universal platform. All of the above logic circuits share the same building blocks, indicating the great prospects of the assembly of nanomaterials and DNA for biochemical logic devices. Considering its biocompatibility, the novel prototypes developed here may have potential applications in the fields of biological computers and medical diagnosis and serve as a promising proof of principle in the not-too-distant future.

  10. Ligand binding to telomeric G-quadruplex DNA investigated by funnel-metadynamics simulations. (United States)

    Moraca, Federica; Amato, Jussara; Ortuso, Francesco; Artese, Anna; Pagano, Bruno; Novellino, Ettore; Alcaro, Stefano; Parrinello, Michele; Limongelli, Vittorio


    G-quadruplexes (G4s) are higher-order DNA structures typically present at promoter regions of genes and telomeres. Here, the G4 formation decreases the replicative DNA at each cell cycle, finally leading to apoptosis. The ability to control this mitotic clock, particularly in cancer cells, is fascinating and passes through a rational understanding of the ligand/G4 interaction. We demonstrate that an accurate description of the ligand/G4 binding mechanism is possible using an innovative free-energy method called funnel-metadynamics (FM), which we have recently developed to investigate ligand/protein interaction. Using FM simulations, we have elucidated the binding mechanism of the anticancer alkaloid berberine to the human telomeric G4 ( d [AG 3 (T 2 AG 3 ) 3 ]), computing also the binding free-energy landscape. Two ligand binding modes have been identified as the lowest energy states. Furthermore, we have found prebinding sites, which are preparatory to reach the final binding mode. In our simulations, the ions and the water molecules have been explicitly represented and the energetic contribution of the solvent during ligand binding evaluated. Our theoretical results provide an accurate estimate of the absolute ligand/DNA binding free energy ([Formula: see text] = -10.3 ± 0.5 kcal/mol) that we validated through steady-state fluorescence binding assays. The good agreement between the theoretical and experimental value demonstrates that FM is a most powerful method to investigate ligand/DNA interaction and can be a useful tool for the rational design also of G4 ligands.

  11. Specific Stabilization of c-MYC and c-KIT G-Quadruplex DNA Structures by Indolylmethyleneindanone Scaffolds. (United States)

    Diveshkumar, K V; Sakrikar, Saaz; Rosu, Frédéric; Harikrishna, S; Gabelica, Valérie; Pradeepkumar, P I


    Stabilization of G-quadruplex DNA structures by small molecules has emerged as a promising strategy for the development of anticancer drugs. Since G-quadruplex structures can adopt various topologies, attaining specific stabilization of a G-quadruplex topology to halt a particular biological process is daunting. To achieve this, we have designed and synthesized simple structural scaffolds based on an indolylmethyleneindanone pharmacophore, which can specifically stabilize the parallel topology of promoter quadruplex DNAs (c-MYC, c-KIT1, and c-KIT2), when compared to various topologies of telomeric and duplex DNAs. The lead ligands (InEt2 and InPr2) are water-soluble and meet a number of desirable criteria for a small molecule drug. Highly specific induction and stabilization of the c-MYC and c-KIT quadruplex DNAs (ΔT1/2 up to 24 °C) over telomeric and duplex DNAs (ΔT1/2 ∼ 3.2 °C) by these ligands were further validated by isothermal titration calorimetry and electrospray ionization mass spectrometry experiments (Ka ∼ 10(5) to 10(6) M(-1)). Low IC50 (∼2 μM) values were emerged for these ligands from a Taq DNA polymerase stop assay with the c-MYC quadruplex forming template, whereas the telomeric DNA template showed IC50 values >120 μM. Molecular modeling and dynamics studies demonstrated the 5'- and 3'-end stacking modes for these ligands. Overall, these results demonstrate that among the >1000 quadruplex stabilizing ligands reported so far, the indolylmethyleneindanone scaffolds stand out in terms of target specificity and structural simplicity and therefore offer a new paradigm in topology specific G-quadruplex targeting for potential therapeutic and diagnostic applications.

  12. Ball with hair: modular functionalization of highly stable G-quadruplex DNA nano-scaffolds through N2-guanine modification. (United States)

    Lech, Christopher Jacques; Phan, Anh Tuân


    Functionalized nanoparticles have seen valuable applications, particularly in the delivery of therapeutic and diagnostic agents in biological systems. However, the manufacturing of such nano-scale systems with the consistency required for biological application can be challenging, as variation in size and shape have large influences in nanoparticle behavior in vivo. We report on the development of a versatile nano-scaffold based on the modular functionalization of a DNA G-quadruplex. DNA sequences are functionalized in a modular fashion using well-established phosphoramidite chemical synthesis with nucleotides containing modification of the amino (N2) position of the guanine base. In physiological conditions, these sequences fold into well-defined G-quadruplex structures. The resulting DNA nano-scaffolds are thermally stable, consistent in size, and functionalized in a manner that allows for control over the density and relative orientation of functional chemistries on the nano-scaffold surface. Various chemistries including small modifications (N2-methyl-guanine), bulky aromatic modifications (N2-benzyl-guanine), and long chain-like modifications (N2-6-amino-hexyl-guanine) are tested and are found to be generally compatible with G-quadruplex formation. Furthermore, these modifications stabilize the G-quadruplex scaffold by 2.0-13.3 °C per modification in the melting temperature, with concurrent modifications producing extremely stable nano-scaffolds. We demonstrate the potential of this approach by functionalizing nano-scaffolds for use within the biotin-avidin conjugation approach. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Intramolecular telomeric G-quadruplexes dramatically inhibit DNA synthesis by replicative and translesion polymerases, revealing their potential to lead to genetic change.

    Directory of Open Access Journals (Sweden)

    Deanna N Edwards

    Full Text Available Recent research indicates that hundreds of thousands of G-rich sequences within the human genome have the potential to form secondary structures known as G-quadruplexes. Telomeric regions, consisting of long arrays of TTAGGG/AATCCC repeats, are among the most likely areas in which these structures might form. Since G-quadruplexes assemble from certain G-rich single-stranded sequences, they might arise when duplex DNA is unwound such as during replication. Coincidentally, these bulky structures when present in the DNA template might also hinder the action of DNA polymerases. In this study, single-stranded telomeric templates with the potential to form G-quadruplexes were examined for their effects on a variety of replicative and translesion DNA polymerases from humans and lower organisms. Our results demonstrate that single-stranded templates containing four telomeric GGG runs fold into intramolecular G-quadruplex structures. These intramolecular G quadruplexes are somewhat dynamic in nature and stabilized by increasing KCl concentrations and decreasing temperatures. Furthermore, the presence of these intramolecular G-quadruplexes in the template dramatically inhibits DNA synthesis by various DNA polymerases, including the human polymerase δ employed during lagging strand replication of G-rich telomeric strands and several human translesion DNA polymerases potentially recruited to sites of replication blockage. Notably, misincorporation of nucleotides is observed when certain translesion polymerases are employed on substrates containing intramolecular G-quadruplexes, as is extension of the resulting mismatched base pairs upon dynamic unfolding of this secondary structure. These findings reveal the potential for blockage of DNA replication and genetic changes related to sequences capable of forming intramolecular G-quadruplexes.

  14. Multifunctional energy landscape for a DNA G-quadruplex: An evolved molecular switch

    Czech Academy of Sciences Publication Activity Database

    Cragnolini, T.; Chakraborty, D.; Šponer, Jiří; Derreumaux, P.; Pasquali, S.; Wales, D.J.


    Roč. 147, č. 15 (2017), č. článku 152715. ISSN 0021-9606 R&D Projects: GA ČR(CZ) GA16-13721S Institutional support: RVO:68081707 Keywords : telomeric g-quadruplex * gb1 hairpin peptide Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 2.965, year: 2016

  15. “Turn-off-on” fluorescent sensor for (N-methyl-4-pyridyl) porphyrin -DNA and G-quadruplex interactions based on ZnCdSe quantum dots

    Energy Technology Data Exchange (ETDEWEB)

    Zhao, Dan; Fan, Yao; Gao, Fang; Yang, Tian-ming, E-mail:


    As a new detection model, the reversible fluorescence “turn-off-on” sensor based on quantum dots (QDs) has already been successfully employed in the detections of many biochemical materials, especially in the researches on the interactions between anticancer drugs. The previous studies, however, mainly focused on simple-structured oligonucleotides and Calf thymus DNA. G-quadruplex, an important target for anti-cancer drug with special secondary structure, has been stimulating increasing research interests. In this paper, we report a new detection method based on the fluorescence “turn-off-on” model with water-soluble ZnCdSe QDs as the fluorescent probe, to analyze the interactions between anticancer drug (N-methyl-4-pyridyl) porphyrin (TMPyP) and nucleic acid, especially the G-quadruplex. The fluorescence of QDs can be quenched by TMPyP via photo-induced electron transfer and fluorescence resonance energy transfer, while on the other hand, the combination between TMPyP and G-quadruplex releases QDs from their quenchers and thus recovers the fluorescence. Most importantly, the fluorescence “turn-off-on” model has been employed, for the first time, to analyze the impacts of special factors on the interaction between TMPyP and G-quadruplex. The excellent selectivity of the system has been verified in the studies of the interactions between TMPyP and different DNAs (double-stranded DNA, single-stranded G-quadruplex, and different types of G-quadruplexes) in Na{sup +} or K{sup +}-containing buffer. - Highlights: • Reversible fluorescence sensor was firstly used on TMPyP and G-quadruplex study. • SsDNA and various G-quadruplexes were successfully recognized by fluorescence. • The new quantum dot is hypotoxicity and can be extensively applied.

  16. Interactions of DNA binding proteins with G-Quadruplex structures at the single molecule level (United States)

    Ray, Sujay

    Guanine-rich nucleic acid (DNA/RNA) sequences can form non-canonical secondary structures, known as G-quadruplex (GQ). Numerous in vivo and in vitro studies have demonstrated formation of these structures in telomeric and non-telomeric regions of the genome. Telomeric GQs protect the chromosome ends whereas non-telomeric GQs either act as road blocks or recognition sites for DNA metabolic machinery. These observations suggest the significance of these structures in regulation of different metabolic processes, such as replication and repair. GQs are typically thermodynamically more stable than the corresponding Watson-Crick base pairing formed by G-rich and C-rich strands, making protein activity a crucial factor for their destabilization. Inside the cell, GQs interact with different proteins and their enzymatic activity is the determining factor for their stability. We studied interactions of several proteins with GQs to understand the underlying principles of protein-GQ interactions using single-molecule FRET and other biophysical techniques. Replication Protein-A (RPA), a single stranded DNA (ssDNA) binding protein, is known to posses GQ unfolding activity. First, we compared the thermal stability of three potentially GQ-forming DNA sequences (PQS) to their stability against RPA-mediated unfolding. One of these sequences is the human telomeric repeat and the other two, located in the promoter region of tyrosine hydroxylase gene, are highly heterogeneous sequences that better represent PQS in the genome. The thermal stability of these structures do not necessarily correlate with their stability against protein-mediated unfolding. We conclude that thermal stability is not necessarily an adequate criterion for predicting the physiological viability of GQ structures. To determine the critical structural factors that influence protein-GQ interactions we studied two groups of GQ structures that have systematically varying loop lengths and number of G-tetrad layers. We

  17. Synthesis of New DNA G-Quadruplex Constructs with Anthraquinone Insertions and Their Anticoagulant Activity

    DEFF Research Database (Denmark)

    Gouda, Alaa S.; Amine, Mahasen S.; Pedersen, Erik Bjerregaard


    anthraquinone linker led to a significant stabilization of the G-quadruplex structure upon replacing a T in each of two neighboring lateral TT loops and a 26.28 increase in thermal melting temperature was found. CD Spectra of the modified quadruplexes confirmed anti-parallel conformations in all cases under...... potassium buffer conditions as previously observed for TBA. Although the majority of the anthraquinone modified TBA analogues showed a decrease in clotting times in a fibrinogen clotting assay when compared to TBA, modified aptamers containing a 1,8-disubstituted anthraquinone linker replacing G8 or T9...... in the TGT loop showed improved anticoagulant activities. Molecular modeling studies explained the increased thermal melting temperatures of anthraquinone-modified G-quadruplexes....

  18. G-quadruplex DNA sequences are evolutionarily conserved and associated with distinct genomic features in Saccharomyces cerevisiae. (United States)

    Capra, John A; Paeschke, Katrin; Singh, Mona; Zakian, Virginia A


    G-quadruplex DNA is a four-stranded DNA structure formed by non-Watson-Crick base pairing between stacked sets of four guanines. Many possible functions have been proposed for this structure, but its in vivo role in the cell is still largely unresolved. We carried out a genome-wide survey of the evolutionary conservation of regions with the potential to form G-quadruplex DNA structures (G4 DNA motifs) across seven yeast species. We found that G4 DNA motifs were significantly more conserved than expected by chance, and the nucleotide-level conservation patterns suggested that the motif conservation was the result of the formation of G4 DNA structures. We characterized the association of conserved and non-conserved G4 DNA motifs in Saccharomyces cerevisiae with more than 40 known genome features and gene classes. Our comprehensive, integrated evolutionary and functional analysis confirmed the previously observed associations of G4 DNA motifs with promoter regions and the rDNA, and it identified several previously unrecognized associations of G4 DNA motifs with genomic features, such as mitotic and meiotic double-strand break sites (DSBs). Conserved G4 DNA motifs maintained strong associations with promoters and the rDNA, but not with DSBs. We also performed the first analysis of G4 DNA motifs in the mitochondria, and surprisingly found a tenfold higher concentration of the motifs in the AT-rich yeast mitochondrial DNA than in nuclear DNA. The evolutionary conservation of the G4 DNA motif and its association with specific genome features supports the hypothesis that G4 DNA has in vivo functions that are under evolutionary constraint.

  19. G-quadruplex DNA sequences are evolutionarily conserved and associated with distinct genomic features in Saccharomyces cerevisiae.

    Directory of Open Access Journals (Sweden)

    John A Capra


    Full Text Available G-quadruplex DNA is a four-stranded DNA structure formed by non-Watson-Crick base pairing between stacked sets of four guanines. Many possible functions have been proposed for this structure, but its in vivo role in the cell is still largely unresolved. We carried out a genome-wide survey of the evolutionary conservation of regions with the potential to form G-quadruplex DNA structures (G4 DNA motifs across seven yeast species. We found that G4 DNA motifs were significantly more conserved than expected by chance, and the nucleotide-level conservation patterns suggested that the motif conservation was the result of the formation of G4 DNA structures. We characterized the association of conserved and non-conserved G4 DNA motifs in Saccharomyces cerevisiae with more than 40 known genome features and gene classes. Our comprehensive, integrated evolutionary and functional analysis confirmed the previously observed associations of G4 DNA motifs with promoter regions and the rDNA, and it identified several previously unrecognized associations of G4 DNA motifs with genomic features, such as mitotic and meiotic double-strand break sites (DSBs. Conserved G4 DNA motifs maintained strong associations with promoters and the rDNA, but not with DSBs. We also performed the first analysis of G4 DNA motifs in the mitochondria, and surprisingly found a tenfold higher concentration of the motifs in the AT-rich yeast mitochondrial DNA than in nuclear DNA. The evolutionary conservation of the G4 DNA motif and its association with specific genome features supports the hypothesis that G4 DNA has in vivo functions that are under evolutionary constraint.

  20. Spectroscopic studies on the interactions of 5-ethyl-6-phenyl-3,8-bis((3-aminoalkyl)propanamido)phenanthridin-5-ium derivatives with G-quadruplex DNA (United States)

    Yalçın, Ergin; Duyar, Halil; Ihmels, Heiko; Seferoğlu, Zeynel


    An improved microwave-induced synthesis of five ethidium derivatives (Ethidium derivatives, 2a-d) is presented. As the derivatives 2a-d have been proposed previously to be telomerase inhibitors, the binding interactions of these ethidium derivatives with G-quadruplex DNA were evaluated by means of photometric and fluorimetric titration, thermal DNA denaturation, CD and 1H NMR spectroscopy. In particular, the compound bearing 3,8-bis(pyrrolidin-1-yl)propanamido substituent 2a exhibits high selectivity for G-quadruplex DNA relative to duplex DNA.

  1. Identification, characterization and application of a G-quadruplex structured DNA aptamer against cancer biomarker protein anterior gradient homolog 2.

    Directory of Open Access Journals (Sweden)

    Jie Wu

    Full Text Available BACKGROUND: Anterior gradient homolog 2 (AGR2 is a functional protein with critical roles in a diverse range of biological systems, including vertebrate tissue development, inflammatory tissue injury responses, and cancer progression. Clinical studies have shown that the AGR2 protein is overexpressed in a wide range of human cancers, including carcinomas of the esophagus, pancreas, breast, prostate, and lung, making the protein as a potential cancer biomarker. However, the general biochemical functions of AGR2 in human cells remain undefined, and the signaling mechanisms that drive AGR2 to inhibit p53 are still not clearly illustrated. Therefore, it is of great interest to develop molecular probes specifically recognizing AGR2 for its detection and for the elucidation of AGR2-associated molecular mechanism. METHODOLOGY/PRINCIPAL FINDINGS: Through a bead-based and flow cytometry monitored SELEX technology, we have identified a group of DNA aptamers that can specifically bind to AGR2 with K(d values in the nanomolar range after 14 rounds of selections. Aptamer C14B was chosen to further study, due to its high binding affinity and specificity. The optimized and shortened C14B1 has special G-rich characteristics, and the G-rich region of this binding motif was further characterized to reveal an intramolecular parallel G-quadruplex by CD spectroscopy and UV spectroscopy. Our experiments confirmed that the stability of the G-quadruplex structure was strongly dependent on the nature of the monovalent ions and the formation of G-quadruplex structure was also important for the binding capacity of C14B1 to the target. Furthermore, we have designed a kind of allosteric molecule beacon (aMB probe for selective and sensitive detection of AGR2. CONCLUSION/SIGNIFICANCE: In this work, we have developed new aptamer probes for specific recognition of the AGR2. Structural study have identified that the binding motif of aptamer is an intramolecular parallel G-quadruplex

  2. Fluorescence detection of DNA, adenosine-5'-triphosphate (ATP), and telomerase activity by zinc(II)-protoporphyrin IX/G-quadruplex labels. (United States)

    Zhang, Zhanxia; Sharon, Etery; Freeman, Ronit; Liu, Xiaoqing; Willner, Itamar


    The zinc(II)-protoporphyrin IX (ZnPPIX) fluorophore binds to G-quadruplexes, and this results in the enhanced fluorescence of the fluorophore. This property enabled the development of DNA sensors, aptasensors, and a sensor following telomerase activity. The DNA sensor is based on the design of a hairpin structure that includes a "caged" inactive G-quadruplex sequence. Upon opening the hairpin by the analyte DNA, the resulting fluorescence of the ZnPPIX/G-quadruplex provides the readout signal for the sensing event (detection limit 5 nM). Addition of Exonuclease III to the system allows the recycling of the analyte and its amplified analysis (detection limit, 200 pM). The association of the ZnPPIX to G-quadruplex aptamer-substrate complexes allowed the detection of adenosine-5'-triphosphate (ATP, detection limit 10 μM). Finally, the association of ZnPPIX to the G-quadruplex repeat units of telomers allowed the detection of telomerase activity originating from 380 ± 20 cancer 293T cell extract.

  3. Label-free monitoring of DNA polymerase activity based on a thrombin-binding aptamer G-quadruplex. (United States)

    Wang, Jing; Liu, Haisheng; Ma, Changbei; Wang, Jun; Zhong, Linxiu; Wu, Kefeng


    We have developed a label-free assay for the detection of DNA polymerase activity based on a thrombin-binding aptamer (TBA) G-quadruplex. In the presence of DNA polymerase, the 3'-OH termini of the hairpin substrate are immediately elongated to replace the TBA, which can be recognized quickly by the ThT dye and results in an increase of fluorescence. This method is highly sensitive with a detection limit of 0.1 U/mL. It is simple and cost-effective without any requirement of labeling with a fluorophore-quencher pair. Furthermore, the proposed method can also be applied to analyze the inhibition of DNA polymerase, which clearly indicates that the proposed method can be applied for screening of potential DNA polymerase inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Identification of New Natural DNA G-Quadruplex Binders Selected by a Structure-Based Virtual Screening Approach

    Directory of Open Access Journals (Sweden)

    Stefano Alcaro


    Full Text Available The G-quadruplex DNA structures are mainly present at the terminal portion of telomeres and can be stabilized by ligands able to recognize them in a specific manner. The recognition process is usually related to the inhibition of the enzyme telomerase indirectly involved and over-expressed in a high percentage of human tumors. There are several ligands, characterized by different chemical structures, already reported in the literature for their ability to bind and stabilize the G-quadruplex structures. Using the structural and biological information available on these structures; we performed a high throughput in silico screening of commercially natural compounds databases by means of a structure-based approach followed by docking experiments against the human telomeric sequence d[AG3(T2AG33]. We identified 12 best hits characterized by different chemical scaffolds and conformational and physicochemical properties. All of them were associated to an improved theoretical binding affinity with respect to that of known selective G-binders. Among these hits there is a chalcone derivative; structurally very similar to the polyphenol butein; known to remarkably inhibit the telomerase activity.

  5. Linking of Antitumor trans NHC-Pt(II) Complexes to G-Quadruplex DNA Ligand for Telomeric Targeting. (United States)

    Betzer, Jean-François; Nuter, Frédérick; Chtchigrovsky, Mélanie; Hamon, Florian; Kellermann, Guillaume; Ali, Samar; Calméjane, Marie-Ange; Roque, Sylvain; Poupon, Joël; Cresteil, Thierry; Teulade-Fichou, Marie-Paule; Marinetti, Angela; Bombard, Sophie


    G-quadruplex structures (G4) are promising anticancerous targets. A great number of small molecules targeting these structures have already been identified through biophysical methods. In cellulo, some of them are able to target either telomeric DNA and/or some sequences involved in oncogene promotors, both resulting in cancer cell death. However, only a few of them are able to bind to these structures G4 irreversibly. Here we combine within the same molecule the G4-binding agent PDC (pyridodicarboxamide) with a N-heterocyclic carbene-platinum complex NHC-Pt already identified for its antitumor properties. The resulting conjugate platinum complex NHC-Pt-PDC stabilizes strongly G-quadruplex structures in vitro, with affinity slightly affected as compared to PDC. In addition, we show that the new conjugate binds preferentially and irreversibly the quadruplex form of the human telomeric sequence with a profile in a way different from that of NHC-Pt thereby indicating that the platination reaction is oriented by stacking of the PDC moiety onto the G4-structure. In cellulo, NHC-Pt-PDC induces a significant loss of TRF2 from telomeres that is considerably more important than the effect of its two components alone, PDC and NHC-Pt, respectively.

  6. A colorimetric platform for sensitively differentiating telomere DNA with different lengths, monitoring G-quadruplex and dsDNA based on silver nanoclusters and unmodified gold nanoparticles (United States)

    Qu, Fei; Chen, Zeqiu; You, Jinmao; Song, Cuihua


    Human telomere DNA plays a vital role in genome integrity control and carcinogenesis as an indication for extensive cell proliferation. Herein, silver nanoclusters (Ag NCs) templated by polymer and unmodified gold nanoparticles (Au NPs) are designed as a new colorimetric platform for sensitively differentiating telomere DNA with different lengths, monitoring G-quadruplex and dsDNA. Ag NCs can produce the aggregation of Au NPs, so the color of Au NPs changes to blue and the absorption peak moves to 700 nm. While the telomere DNA can protect Au NPs from aggregation, the color turns to red again and the absorption band blue shift. Benefiting from the obvious color change, we can differentiate the length of telomere DNA by naked eyes. As the length of telomere DNA is longer, the variation of color becomes more noticeable. The detection limits of telomere DNA containing 10, 22, 40, 64 bases are estimated to be 1.41, 1.21, 0.23 and 0.22 nM, respectively. On the other hand, when telomere DNA forms G-quadruplex in the presence of K+, or dsDNA with complementary sequence, both G-quadruplex and dsDNA can protect Au NPs better than the unfolded telomere DNA. Hence, a new colorimetric platform for monitoring structure conversion of DNA is established by Ag NCs-Au NPs system, and to prove this type of application, a selective K+ sensor is developed.

  7. Identification of G-quadruplex DNA/RNA binders: Structure-based virtual screening and biophysical characterization. (United States)

    Rocca, Roberta; Moraca, Federica; Costa, Giosuè; Nadai, Matteo; Scalabrin, Matteo; Talarico, Carmine; Distinto, Simona; Maccioni, Elias; Ortuso, Francesco; Artese, Anna; Alcaro, Stefano; Richter, Sara N


    Recent findings demonstrated that, in mammalian cells, telomere DNA (Tel) is transcribed into telomeric repeat-containing RNA (TERRA), which is involved in fundamental biological processes, thus representing a promising anticancer target. For this reason, the discovery of dual (as well as selective) Tel/TERRA G-quadruplex (G4) binders could represent an innovative strategy to enhance telomerase inhibition. Initially, docking simulations of known Tel and TERRA active ligands were performed on the 3D coordinates of bimolecular G4 Tel DNA (Tel 2 ) and TERRA (TERRA 2 ). Structure-based pharmacophore models were generated on the best complexes and employed for the virtual screening of ~257,000 natural compounds. The 20 best candidates were submitted to biophysical assays, which included circular dichroism and mass spectrometry at different K + concentrations. Three hits were here identified and characterized by biophysical assays. Compound 7 acts as dual Tel 2 /TERRA 2 G4-ligand at physiological KCl concentration, while hits 15 and 17 show preferential thermal stabilization for Tel 2 DNA. The different molecular recognition against the two targets was also discussed. Our successful results pave the way to further lead optimization to achieve both increased selectivity and stabilizing effect against TERRA and Tel DNA G4s. The current study combines for the first time molecular modelling and biophysical assays applied to bimolecular DNA and RNA G4s, leading to the identification of innovative ligand chemical scaffolds with a promising anticancer profile. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. G-quadruplex organic frameworks (United States)

    Wu, Yi-Lin; Horwitz, Noah E.; Chen, Kan-Sheng; Gomez-Gualdron, Diego A.; Luu, Norman S.; Ma, Lin; Wang, Timothy C.; Hersam, Mark C.; Hupp, Joseph T.; Farha, Omar K.; Snurr, Randall Q.; Wasielewski, Michael R.


    Two-dimensional covalent organic frameworks often π stack into crystalline solids that allow precise spatial positioning of molecular building blocks. Inspired by the hydrogen-bonded G-quadruplexes found frequently in guanine-rich DNA, here we show that this structural motif can be exploited to guide the self-assembly of naphthalene diimide and perylene diimide electron acceptors end-capped with two guanine electron donors into crystalline G-quadruplex-based organic frameworks, wherein the electron donors and acceptors form ordered, segregated π-stacked arrays. Time-resolved optical and electron paramagnetic resonance spectroscopies show that photogenerated holes and electrons in the frameworks have long lifetimes and display recombination kinetics typical of dissociated charge carriers. Moreover, the reduced acceptors form polarons in which the electron is shared over several molecules. The G-quadruplex frameworks also demonstrate potential as cathode materials in Li-ion batteries because of the favourable electron- and Li-ion-transporting capacity provided by the ordered rylene diimide arrays and G-quadruplex structures, respectively.

  9. Sites of instability in the human TCF3 (E2A gene adopt G-quadruplex DNA structures in vitro

    Directory of Open Access Journals (Sweden)

    Jonathan David Williams


    Full Text Available The formation of highly stable four-stranded DNA, called G-quadruplex (G4, promotes site-specific genome instability. G4 DNA structures fold from repetitive guanine sequences, and increasing experimental evidence connects G4 sequence motifs with specific gene rearrangements. The human TCF3 gene (also termed E2A is subject to genetic instability associated with severe disease, most notably a common translocation event t(1;19 associated with acute lymphoblastic leukemia. The sites of instability in TCF3 are not randomly distributed, but focused to certain sequences. We asked if G4 DNA formation could explain why TCF3 is prone to recombination and mutagenesis. Here we demonstrate that sequences surrounding the major t(1;19 break site and a region associated with copy number variations both contain G4 sequence motifs. The motifs identified readily adopt G4 DNA structures that are stable enough to interfere with DNA synthesis in physiological salt conditions in vitro. When introduced into the yeast genome, TCF3 G4 motifs promoted gross chromosomal rearrangements in a transcription-dependent manner. Our results provide a molecular rationale for the site-specific instability of human TCF3, suggesting that G4 DNA structures contribute to oncogenic DNA breaks and recombination.

  10. Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay (United States)

    Xing, Yun-Peng; Liu, Chun; Zhou, Xiao-Hong; Shi, Han-Chang


    This work was the first to report that the kanamycin-binding DNA aptamer (5'-TGG GGG TTG AGG CTA AGC CGA-3') can form stable parallel G-quadruplex DNA (G4-DNA) structures by themselves and that this phenomenon can be verified by nondenaturing polyacrylamide gel electrophoresis and circular dichroism spectroscopy. Based on these findings, we developed a novel label-free strategy for kanamycin detection based on the G4-DNA aptamer-based fluorescent intercalator displacement assay with thiazole orange (TO) as the fluorescence probe. In the proposed strategy, TO became strongly fluorescent upon binding to kanamycin-binding G4-DNA. However, the addition of kanamycin caused the displacement of TO from the G4-DNA-TO conjugate, thereby resulting in decreased fluorescent signal, which was inversely related to the kanamycin concentration. The detection limit of the proposed assay decreased to 59 nM with a linear working range of 0.1 μM to 20 μM for kanamycin. The cross-reactivity against six other antibiotics was negligible compared with the response to kanamycin. A satisfactory recovery of kanamycin in milk samples ranged from 80.1% to 98.0%, confirming the potential of this bioassay in the measurement of kanamycin in various applications. Our results also served as a good reference for developing similar fluorescent G4-DNA-based bioassays in the future.

  11. Templated Formation of Discrete RNA and DNA:RNA Hybrid G-Quadruplexes and Their Interactions with Targeting Ligands. (United States)

    Bonnat, Laureen; Dejeu, Jérôme; Bonnet, Hugues; Génnaro, Béatrice; Jarjayes, Olivier; Thomas, Fabrice; Lavergne, Thomas; Defrancq, Eric


    G-rich RNA and DNA oligonucleotides derived from the human telomeric sequence were assembled onto addressable cyclopeptide platforms through oxime ligations and copper-catalyzed azide-alkyne cycloaddition (CuAAc) reactions. The resulting conjugates were able to fold into highly stable RNA and DNA:RNA hybrid G-quadruplex (G4) architectures as demonstrated by UV, circular dichroism (CD), and NMR spectroscopic analysis. Whereas rationally designed parallel RNA and DNA:RNA hybrid G4 topologies could be obtained, we could not force the formation of an antiparallel RNA G4 structure, thus supporting the idea that this topology is strongly disfavored. The binding affinities of four representative G4 ligands toward the discrete RNA and DNA:RNA hybrid G4 topologies were compared to the one obtained with the corresponding DNA G4 structure. Surface plasmon resonance (SPR) binding analysis suggests that the accessibility to G4 recognition elements is different among the three structures and supports the idea that G4 ligands might be shaped to achieve structure selectivity in a biological context. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Microwave-assisted synthesis of ruthenium(II) complexes with alkynes as potential inhibitor by selectively recognizing c-myc G-quadruplex DNA. (United States)

    Zhang, Shuangyan; Wu, Qiong; Zhang, Hao; Wang, Qi; Wang, Xicheng; Mei, Wenjie; Wu, Xiaohui; Zheng, Wenjie


    Herein, two polypyridyl ruthenium(II) complexes with alkynes, [Ru(bpy) 2 L](ClO 4 ) 2 (L=p-TEPIP (1) and p-BEPIP (2); bpy=2,2'-bipyridine; p-TEPIP=2-(4-trimethylsilylpropargyl)-1H-imidazo[4,5f][1,10]phenanthroline; p-BEPIP=2-(4-phenyacetylenephenyl)-1H-imidazo[4,5f][1,10]phenanthroline) have been successfully achieved in yields of 32%-89% by a Sonogashira coupling reaction under microwave irradiation. We studied these complexes as potential stabilizers of c-myc G-quadruplex DNA. Observations revealed that both complexes could selectively bind to and stabilize c-myc G-quadruplex DNA with a constant of approximately 1.61±0.78 and 9.47±4.20×10 3 M -1 , respectively, as determined from ITC (isothermal ttitration calorimetry) experiments, FRET (fluorescence resonance energy ttransfer) assay and competitive FRET assay. Moreover, the melting point (T m ) of the c-myc G-quadruplex DNA increased in the presence of 1 and 2 ([Ru]=0.2μM) by approximately 9 and 19.9°C, respectively. It is noteworthy that the conformation of the c-myc G-quadruplex DNA appeared to change when titrated with 1 and 2, which was accompanied by a negative-induced CD (circular dichroism) signal that appeared at a wavelength of 295nm. Furthermore, the conformational change in c-myc G-quadruplex DNA induced by 1 and 2have also been confirmed by TEM (transmission electron microscopy) and AFM (atomic force microscopy). Consequently, the replication of c-myc DNA was blocked by 1 and 2, and especially by 2, as verified by PCR (polymerase chain reaction) -stop assay and Western-blot assay. Thus, these ruthenium(II) complexes can be developed as potential inhibitors in chemotherapy through their binding and stabilization of c-myc G-quadruplex DNA. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. A biochemical and biophysical model of G-quadruplex DNA recognition by positive coactivator of transcription 4. (United States)

    Griffin, Wezley C; Gao, Jun; Byrd, Alicia K; Chib, Shubeena; Raney, Kevin D


    DNA sequences that are guanine-rich have received considerable attention because of their potential to fold into a secondary, four-stranded DNA structure termed G-quadruplex (G4), which has been implicated in genomic instability and some human diseases. We have previously identified positive coactivator of transcription (PC4), a single-stranded DNA (ssDNA)-binding protein, as a novel G4 interactor. Here, to expand on these previous observations, we biochemically and biophysically characterized the interaction between PC4 and G4DNA. PC4 can bind alternative G4DNA topologies with a low nanomolar K d value of ∼2 nm, similar to that observed for ssDNA. In consideration of the different structural features between G4DNA and ssDNA, these binding data indicated that PC4 can interact with G4DNA in a manner distinct from ssDNA. The stoichiometry of the PC4-G4 complex was 1:1 for PC4 dimer:G4 substrate. PC4 did not enhance the rate of folding of G4DNA, and formation of the PC4-G4DNA complex did not result in unfolding of the G4DNA structure. We assembled a G4DNA structure flanked by duplex DNA. We find that PC4 can interact with this G4DNA, as well as the complementary C-rich strand. Molecular docking simulations and DNA footprinting experiments suggest a model where a PC4 dimer accommodates the DNA with one monomer on the G4 strand and the second monomer bound to the C-rich strand. Collectively, these data provide a novel mode of PC4 binding to a DNA secondary structure that remains within the framework of the model for binding to ssDNA. Additionally, consideration of the PC4-G4DNA interaction could provide insight into the biological functions of PC4, which remain incompletely understood. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Proximity hybridization triggered hemin/G-quadruplex formation for construction a label-free and signal-on electrochemical DNA sensor. (United States)

    Gao, Fenglei; Fan, Taotao; Wu, Jing; Liu, Sha; Du, Yan; Yao, Yao; Zhou, Fuyi; Zhang, Yu; Liao, Xianjiu; Geng, Deqin


    We describe a novel label-free and signal-on electrochemical DNA sensing platform via proximity hybridization triggered hemin/G-quadruplex formation based on the direct electron transfer of hemin. The thiolated modified G-DNA1 was first immobilized onto the Au electrode surface. In the presence of target DNA, Y-junction-structure ternary complex can be formed to trigger the proximity assembly of G-DNA1, hemin, and G-DNA2, which leads to the formation of hemin/G-quadruplex for generation an amplified electrochemical signal by differential pulse voltammetry. The observed signal gain was sufficient to achieve a demonstrated detection limit of 54 fM, with a wide linear dynamic range from 10 -13 to 10 -9 M and discriminated mismatched DNA from perfect matched target DNA with a high selectivity. Benefiting from the one step proximity dependent hemin/G-quadruplex formation, the assay method is extremely simple and can be carried out within 40min. The advantages of free of any label conjugation step, and versatility make it a promising candidate for point-of-care testing and commercial application. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Fuzzy logic sensing of G-quadruplex DNA and its cleavage reagents based on reduced graphene oxide. (United States)

    Huang, Wei Tao; Zhang, Jian Rong; Xie, Wan Yi; Shi, Yan; Luo, Hong Qun; Li, Nian Bing


    Herein, by combining the merits of nanotechnology and fuzzy logic theory, we develop a simple, label-free, and general strategy based on an organic dye-graphene hybrid system for fluorescence intelligent sensing of G-quadruplexes (G4) formation, hydroxyl radical (HO∙), and Fe(2+) in vitro. By exploiting acridine orange (AO) dyes-graphene as a nanofilter and nanoswitch and the ability of graphene to interact with DNA with different structures, our approach can efficiently distinguish, quantitatively detect target analytes. In vitro assays with G4DNA demonstrated increases in fluorescence intensity of the AO-rGO system with a linear range of 16-338 nM and a detection limit as low as 2.0 nM. The requenched fluorescence of the G4TBA-AO-rGO system has a non-linear response to Fenton reagent. But this requenching reduces the fluorescence intensity in a manner proportional to the logarithm to the base 10 of the concentration of Fenton reagent in the range of 0.1-100 μM and 100-2000 μM, respectively. Furthermore, we develop a novel and intelligent sensing method based on fuzzy logic which mimics human reasoning, solves complex and non-linear problems, and transforms the numerical output into the language description output for potential application in biochemical systems, environmental monitoring systems, and molecular-level fuzzy logic computing system. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Seven essential questions on G-quadruplexes. (United States)

    König, Sebastian L B; Evans, Amanda C; Huppert, Julian L


    The helical duplex architecture of DNA was discovered by Francis Crick and James Watson in 1951 and is well known and understood. However, nucleic acids can also adopt alternative structural conformations that are less familiar, although no less biologically relevant, such as the G-quadruplex. G-quadruplexes continue to be the subject of a rapidly expanding area of research, owing to their significant potential as therapeutic targets and their unique biophysical properties. This review begins by focusing on G-quadruplex structure, elucidating the intermolecular and intramolecular interactions underlying its formation and highlighting several substructural variants. A variety of methods used to characterize these structures are also outlined. The current state of G-quadruplex research is then addressed by proffering seven pertinent questions for discussion. This review concludes with an overview of possible directions for future research trajectories in this exciting and relevant field.

  17. Toward the design of a catalytic metallodrug: selective cleavage of G-quadruplex telomeric DNA by an anticancer copper-acridine-ATCUN complex. (United States)

    Yu, Zhen; Han, Menglu; Cowan, James A


    Telomeric DNA represents a novel target for the development of anticancer drugs. By application of a catalytic metallodrug strategy, a copper-acridine-ATCUN complex (CuGGHK-Acr) has been designed that targets G-quadruplex telomeric DNA. Both fluorescence solution assays and gel sequencing demonstrate the CuGGHK-Acr catalyst to selectively bind and cleave the G-quadruplex telomere sequence. The cleavage pathway has been mapped by matrix assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) experiments. CuGGHK-Acr promotes significant inhibition of cancer cell proliferation and shortening of telomere length. Both senescence and apoptosis are induced in the breast cancer cell line MCF7. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Architecture based on the integration of intermolecular G-quadruplex structure with sticky-end pairing and colorimetric detection of DNA hybridization. (United States)

    Li, Hongbo; Wu, Zai-Sheng; Shen, Zhifa; Shen, Guoli; Yu, Ruqin


    An interesting discovery is reported in that G-rich hairpin-based recognition probes can self-assemble into a nano-architecture based on the integration of an intermolecular G-quadruplex structure with the sticky-end pairing effect in the presence of target DNAs. Moreover, GNPs modified with partly complementary DNAs can intensively aggregate by hybridization-based intercalation between intermolecular G-quadruplexes, indicating an inspiring assembly mechanism and a powerful colorimetric DNA detection. The proposed intermolecular G-quadruplex-integrated sticky-end pairing assembly (called GISA)-based colorimetric system allows a specific and quantitative assay of p53 DNA with a linear range of more than two orders of magnitude and a detection limit of 0.2 nM, suggesting a considerably improved analytical performance. And more to the point, the discrimination of single-base mismatched target DNAs can be easily conducted via visual observation. The successful development of the present colorimetric system, especially the GISA-based aggregation mechanism of GNPs is different from traditional approaches, and offers a critical insight into the dependence of the GNP aggregation on the structural properties of oligonucleotides, opening a good way to design colorimetric sensing probes and DNA nanostructure.

  19. Long-range charge transport in single G-quadruplex DNA molecules

    DEFF Research Database (Denmark)

    Livshits, Gideon I.; Stern, Avigail; Rotem, Dvir


    DNA and DNA-based polymers are of interest in molecular electronics because of their versatile and programmable structures. However, transport measurements have produced a range of seemingly contradictory results due to differences in the measured molecules and experimental set-ups, and transport......DNA and DNA-based polymers are of interest in molecular electronics because of their versatile and programmable structures. However, transport measurements have produced a range of seemingly contradictory results due to differences in the measured molecules and experimental set......-ups, and transporting significant current through individual DNA-based molecules remains a considerable challenge. Here, we report reproducible charge transport in guanine-quadruplex (G4) DNA molecules adsorbed on a mica substrate. Currents ranging from tens of picoamperes to more than 100 pA were measured in the G4......-DNA over distances ranging from tens of nanometres to more than 100 nm. Our experimental results, combined with theoretical modelling, suggest that transport occurs via a thermally activated long-range hopping between multi-tetrad segments of DNA. These results could re-ignite interest in DNA...

  20. Structural and biophysical insight into dual site binding of the protoberberine alkaloid palmatine to parallel G-quadruplex DNA using NMR, fluorescence and circular dichroism spectroscopy. (United States)

    Padmapriya, Kumar; Barthwal, Ritu


    Plant derived small molecules, which interact with and stabilize G-quadruplex DNA, act as inhibitors of telomere elongation and oncogene expression in humans. The inhibition of telomerase enzyme has immense potential since it is over expressed in most cancer cells. Interaction of palmatine, an antitumor alkaloid, to parallel G-quadruplex DNA, [d(TTGGGGT)] 4 and [d(TTAGGGT)] 4 , has been investigated using Nuclear Magnetic Resonance (NMR), fluorescence and Circular Dichroism (CD) spectroscopy. Titrations were monitored by recording 1 H and 31 P NMR spectra and solution structure of palmatine-[d(TTGGGGT)] 4 complex was obtained by restrained Molecular Dynamics (rMD) simulations using distance restraints from 2D NOESY spectra. Thermal stabilization of DNA was determined by CD, 1 H NMR and Differential Scanning Calorimetry (DSC). Binding of palmatine induces 98% enhancement of fluorescence accompanied by blue shift ∼8 nm. CD spectral bands of DNA show minor changes. Diffusion NMR studies confirm formation of a stable complex. Proton NMR signals of palmatine shift upfield upon binding and NOE cross peaks of H10, H3, H28, 5OCH 3 protons with T2, A3/G3, G6 and T7 residues reveal dual recognition sites in both G-quadruplex DNA sequences, resulting in thermal stabilization of G-quadruplex by ∼13-17 °C. Restrained molecular dynamics simulations using NOE distance restraints for 2:1 palmatine-[d(TTGGGGT)] 4 complex reveal end-stacking of palmatine at G6pT7 step and groove binding along T2pG3 step. Binding to [d(TTAGGGT)] 4 takes place at T2pA3pG4 and G6pT7 steps. Structural features of molecular recognition of two different G-quadruplex DNA sequences by palmatine have relevance in rational drug development for anti-cancer therapy. Copyright © 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  1. Ruthenium(II) complexes as apoptosis inducers by stabilizing c-myc G-quadruplex DNA. (United States)

    Zhang, Zhao; Wu, Qiong; Wu, Xiao-Hui; Sun, Fen-Yong; Chen, Lan-Mei; Chen, Jin-Chan; Yang, Shu-Ling; Mei, Wen-Jie


    Two ruthenium(II) complexes, [Ru(L)2(p-tFMPIP)](ClO4)2 (L = bpy, 1; phen, 2; p-tFMPIP = 2-(4-(trifluoromethyphenyl)-1H-imidazo[4,5f][1,10] phenanthroline)), were prepared by microwave-assisted synthesis technology. The inhibitory activity evaluated by MTT assay shown that 2 can inhibit the growth of MDA-MB-231 cells with inhibitory activity (IC50) of 16.3 μM, which was related to the induction of apoptosis. Besides, 2 exhibit low toxicity against normal HAcat cells. The inhibitory growth activity of both complexes related to the induction of apoptosis was also confirmed. Furthermore, the studies on the interaction of both complexes with c-myc G4 DNA shown that 1 and 2 can stabilize the conformation of c-myc G4 DNA in groove binding mode, which has been rational explained by using DFT theoretical calculation methods. In a word, this type of ruthenium(II) complexes can act as potential apoptosis inducers with low toxicity in clinic by stabilizing c-myc G4 DNA. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

  2. Allelic Dropout During Polymerase Chain Reaction due to G-Quadruplex Structures and DNA Methylation Is Widespread at Imprinted Human Loci

    Directory of Open Access Journals (Sweden)

    Aaron J. Stevens


    Full Text Available Loss of one allele during polymerase chain reaction (PCR amplification of DNA, known as allelic dropout, can be caused by a variety of mechanisms. Allelic dropout during PCR may have profound implications for molecular diagnostic and research procedures that depend on PCR and assume biallelic amplification has occurred. Complete allelic dropout due to the combined effects of cytosine methylation and G-quadruplex formation was previously described for a differentially methylated region of the human imprinted gene, MEST. We now demonstrate that this parent-of-origin specific allelic dropout can potentially occur at several other genomic regions that display genomic imprinting and have propensity for G-quadruplex formation, including AIM1, BLCAP, DNMT1, PLAGL1, KCNQ1, and GRB10. These findings demonstrate that systematic allelic dropout during PCR is a general phenomenon for regions of the genome where differential allelic methylation and G-quadruplex motifs coincide, and suggest that great care must be taken to ensure biallelic amplification is occurring in such situations.

  3. DNA adducts of antitumor cisplatin preclude telomeric sequences from forming G quadruplexes

    Czech Academy of Sciences Publication Activity Database

    Heringová, Pavla; Kašpárková, Jana; Brabec, Viktor


    Roč. 14, č. 6 (2009), s. 959-968 ISSN 0949-8257 R&D Projects: GA MZd(CZ) NR8562; GA MŠk(CZ) LC06030; GA MŠk(CZ) ME08017; GA MŠk(CZ) OC08003; GA AV ČR(CZ) 1QS500040581; GA AV ČR(CZ) KAN200200651; GA AV ČR(CZ) IAA400040803 Grant - others:GA MŠk(CZ) OC09018 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : cisplatin * DNA quadruplex * telomere Subject RIV: BO - Biophysics Impact factor: 3.415, year: 2009

  4. Elevated levels of G-quadruplex formation in human stomach and liver cancer tissues. (United States)

    Biffi, Giulia; Tannahill, David; Miller, Jodi; Howat, William J; Balasubramanian, Shankar


    Four-stranded G-quadruplex DNA secondary structures have recently been visualized in the nuclei of human cultured cells. Here, we show that BG4, a G-quadruplex-specific antibody, can be used to stain DNA G-quadruplex structures in patient-derived tissues using immunohistochemistry. We observe a significantly elevated number of G-quadruplex-positive nuclei in human cancers of the liver and stomach as compared to background non-neoplastic tissue. Our results suggest that G-quadruplex formation can be detected and measured in patient-derived material and that elevated G-quadruplex formation may be a characteristic of some cancers.

  5. Plastic roles of phenylalanine and tyrosine residues of TLS/FUS in complex formation with the G-quadruplexes of telomeric DNA and TERRA. (United States)

    Kondo, Keiko; Mashima, Tsukasa; Oyoshi, Takanori; Yagi, Ryota; Kurokawa, Riki; Kobayashi, Naohiro; Nagata, Takashi; Katahira, Masato


    The length of a telomere is regulated via elongation and shortening processes. Telomeric DNA and telomeric repeat-containing RNA (TERRA), which both contain G-rich repeated sequences, form G-quadruplex structures. Previously, translocated in liposarcoma (TLS) protein, also known as fused in sarcoma (FUS) protein, was found to form a ternary complex with the G-quadruplex structures of telomeric DNA and TERRA. We then showed that the third RGG motif of TLS, the RGG3 domain, is responsible for the complex formation. However, the structural basis for their binding remains obscure. Here, NMR-based binding assaying revealed the interactions in the binary and ternary complexes of RGG3 with telomeric DNA or/and TERRA. In the ternary complex, tyrosine bound exclusively to TERRA, while phenylalanine bound exclusively to telomeric DNA. Thus, tyrosine and phenylalanine each play a central role in the recognition of TERRA and telomeric DNA, respectively. Surprisingly in the binary complexes, RGG3 used both tyrosine and phenylalanine residues to bind to either TERRA or telomeric DNA. We propose that the plastic roles of tyrosine and phenylalanine are important for RGG3 to efficiently form the ternary complex, and thereby regulate the telomere shortening.

  6. Electrochemical and AFM Characterization of G-Quadruplex Electrochemical Biosensors and Applications (United States)


    Guanine-rich DNA sequences are able to form G-quadruplexes, being involved in important biological processes and representing smart self-assembling nanomaterials that are increasingly used in DNA nanotechnology and biosensor technology. G-quadruplex electrochemical biosensors have received particular attention, since the electrochemical response is particularly sensitive to the DNA structural changes from single-stranded, double-stranded, or hairpin into a G-quadruplex configuration. Furthermore, the development of an increased number of G-quadruplex aptamers that combine the G-quadruplex stiffness and self-assembling versatility with the aptamer high specificity of binding to a variety of molecular targets allowed the construction of biosensors with increased selectivity and sensitivity. This review discusses the recent advances on the electrochemical characterization, design, and applications of G-quadruplex electrochemical biosensors in the evaluation of metal ions, G-quadruplex ligands, and other small organic molecules, proteins, and cells. The electrochemical and atomic force microscopy characterization of G-quadruplexes is presented. The incubation time and cations concentration dependence in controlling the G-quadruplex folding, stability, and nanostructures formation at carbon electrodes are discussed. Different G-quadruplex electrochemical biosensors design strategies, based on the DNA folding into a G-quadruplex, the use of G-quadruplex aptamers, or the use of hemin/G-quadruplex DNAzymes, are revisited. PMID:29666699

  7. Quantification of Chemical and Mechanical Effects on the Formation of the G-Quadruplex and i-Motif in Duplex DNA. (United States)

    Selvam, Sangeetha; Mandal, Shankar; Mao, Hanbin


    The formation of biologically significant tetraplex DNA species, such as G-quadruplexes and i-motifs, is affected by chemical (ions and pH) and mechanical [superhelicity (σ) and molecular crowding] factors. Because of the extremely challenging experimental conditions, the relative importance of these factors on tetraplex folding is unknown. In this work, we quantitatively evaluated the chemical and mechanical effects on the population dynamics of DNA tetraplexes in the insulin-linked polymorphic region using magneto-optical tweezers. By mechanically unfolding individual tetraplexes, we found that ions and pH have the largest effects on the formation of the G-quadruplex and i-motif, respectively. Interestingly, superhelicity has the second largest effect followed by molecular crowding conditions. While chemical effects are specific to tetraplex species, mechanical factors have generic influences. The predominant effect of chemical factors can be attributed to the fact that they directly change the stability of a specific tetraplex, whereas the mechanical factors, superhelicity in particular, reduce the stability of the competing species by changing the kinetics of the melting and annealing of the duplex DNA template in a nonspecific manner. The substantial dependence of tetraplexes on superhelicity provides strong support that DNA tetraplexes can serve as topological sensors to modulate fundamental cellular processes such as transcription.

  8. Plasmonic biosensor for label-free G-quadruplexes detection (United States)

    Qiu, Suyan; Zhao, Fusheng; Santos, Greggy M.; Shih, Wei-Chuan


    G-quadruplex, readily formed by the G-rich sequence, potentially distributes in over 40 % of all human genes, such as the telomeric DNA with the G-rich sequence found at the end of the chromosome. The G-quadruplex structure is supposed to possess a diverse set of critical functions in the mammalian genome for transcriptional regulation, DNA replication and genome stability. However, most of the currently available methods for G-quadruplex identification are restricted to fluorescence techniques susceptible to poor sensitivity. It is essential to propose methods with higher sensitivity to specifically recognize the G-quadruplexes. In this study, we demonstrate a label-free plasmonic biosensor for G-quadruplex detection by relying on the advantages of nanoporous gold (NPG) disks that provide high-density plasmonic hot spots, suitable for molecular recognition capability without the requirement for labeling processes.

  9. Xanthene and Xanthone Derivatives as G-Quadruplex Stabilizing Ligands

    Directory of Open Access Journals (Sweden)

    Alessandro Altieri


    Full Text Available Following previous studies on anthraquinone and acridine-based G-quadruplex ligands, here we present a study of similar aromatic cores, with the specific aim of increasing G-quadruplex binding and selectivity with respect to duplex DNA. Synthesized compounds include two and three-side chain xanthone and xanthene derivatives, as well as a dimeric “bridged” form. ESI and FRET measurements suggest that all the studied molecules are good G-quadruplex ligands, both at telomeres and on G-quadruplex forming sequences of oncogene promoters. The dimeric compound and the three-side chain xanthone derivative have been shown to represent the best compounds emerging from the different series of ligands presented here, having also high selectivity for G-quadruplex structures with respect to duplex DNA. Molecular modeling simulations are in broad agreement with the experimental data.

  10. Nonlinear optical and G-Quadruplex DNA stabilization properties of novel mixed ligand copper(II) complexes and coordination polymers: Synthesis, structural characterization and computational studies (United States)

    Rajasekhar, Bathula; Bodavarapu, Navya; Sridevi, M.; Thamizhselvi, G.; RizhaNazar, K.; Padmanaban, R.; Swu, Toka


    The present study reports the synthesis and evaluation of nonlinear optical property and G-Quadruplex DNA Stabilization of five novel copper(II) mixed ligand complexes. They were synthesized from copper(II) salt, 2,5- and 2,3- pyridinedicarboxylic acid, diethylenetriamine and amide based ligand (AL). The crystal structure of these complexes were determined through X-ray diffraction and supported by ESI-MAS, NMR, UV-Vis and FT-IR spectroscopic methods. Their nonlinear optical property was studied using Gaussian09 computer program. For structural optimization and nonlinear optical property, density functional theory (DFT) based B3LYP method was used with LANL2DZ basis set for metal ion and 6-31G∗ for C,H,N,O and Cl atoms. The present work reveals that pre-polarized Complex-2 showed higher β value (29.59 × 10-30e.s.u) as compared to that of neutral complex-1 (β = 0.276 × 10-30e.s.u.) which may be due to greater advantage of polarizability. Complex-2 is expected to be a potential material for optoelectronic and photonic technologies. Docking studies using AutodockVina revealed that complex-2 has higher binding energy for both G-Quadruplex DNA (-8.7 kcal/mol) and duplex DNA (-10.1 kcal/mol). It was also observed that structure plays an important role in binding efficiency.

  11. Microwave-Assisted Synthesis of Imidazo[4,5-f][1,10]phenanthroline Derivatives as Apoptosis Inducers in Chemotherapy by Stabilizing Bcl-2 G-quadruplex DNA

    Directory of Open Access Journals (Sweden)

    Li Li


    Full Text Available Herein, a series of imidazo[4,5-f][1,10] phenanthroline derivatives RPIP (PIP = imidazo [4,5-f][1,10] phenanthroline, R = NO2, 1; CF3, 2; Cl, 3; OH, 4 have been synthesized in yields of 82.3–94.7% at 100 °C under the irradiation of microwave. MTT assay has been utilized to evaluate the inhibitory activity (IC50 of these compounds against the growth of various tumor cells, and the results revealed that these compounds, especially 1, exhibited excellent inhibitory activity against the growth of A549 cells with IC50 of 15.03 μM. Moreover, it’s also confirmed that 1 can penetrate into the membrane of tumor cells and distribute in mitochondria when observed under microscopy, resulting apoptosis of tumor cells. The further studies showed that 1 can bind to bcl-2 G-quadruplex DNA, which demonstrated by the increase of melting point of bcl-2 G4 DNA in the presence of 1, as well as electronic titration and emission spectra. In a word, this kind of compound may develop as a potential apoptosis inducer in cancer chemotherapy via binding and stabilizing to the bcl-2 G-quadruplex DNA.

  12. A light-up probe targeting for Bcl-2 2345 G-quadruplex DNA with carbazole TO (United States)

    Gu, Yingchun; Lin, Dayong; Tang, Yalin; Fei, Xuening; Wang, Cuihong; Zhang, Baolian; Zhou, Jianguo


    As its significant role, the selective recognition of G-quadruplex with specific structures and functions is important in biological and medicinal chemistry. Carbazole derivatives have been reported as a kind of fluorescent probe with many excellent optical properties. In the present study, the fluorescence of the dye (carbazole TO) increased almost 70 fold in the presence of bcl-2 2345 G4 compared to that alone in aqueous buffer condition with almost no fluorescence and 10-30 fold than those in the presence of other DNAs. The binding study results by activity inhibition of G4/Hemin peroxidase experiment, NMR titration and molecular docking simulation showed the high affinity and selectivity to bcl-2 2345 G4 arises from its end-stacking interaction with G-quartet. It is said that a facile approach with excellent sensitive, good selectivity and quick response for bcl-2 2345 G-quadruplex was developed and may be used for antitumor recognition or antitumor agents.

  13. Transcriptional control by G-quadruplexes: In vivo roles and perspectives for specific intervention. (United States)

    Armas, Pablo; David, Aldana; Calcaterra, Nora B


    G-quadruplexes are non-canonical DNA secondary structures involved in several genomic and molecular processes. Here, we summarize the main G-quadruplex features and evidences proving the in vivo role on the transcriptional regulation of genes required for zebrafish embryonic development. We also discuss alternative strategies for specifically interfering G-quadruplex in vivo.

  14. DNA Sequences Proximal to Human Mitochondrial DNA Deletion Breakpoints Prevalent in Human Disease Form G-quadruplexes, a Class of DNA Structures Inefficiently Unwound by the Mitochondrial Replicative Twinkle Helicase* (United States)

    Bharti, Sanjay Kumar; Sommers, Joshua A.; Zhou, Jun; Kaplan, Daniel L.; Spelbrink, Johannes N.; Mergny, Jean-Louis; Brosh, Robert M.


    Mitochondrial DNA deletions are prominent in human genetic disorders, cancer, and aging. It is thought that stalling of the mitochondrial replication machinery during DNA synthesis is a prominent source of mitochondrial genome instability; however, the precise molecular determinants of defective mitochondrial replication are not well understood. In this work, we performed a computational analysis of the human mitochondrial genome using the “Pattern Finder” G-quadruplex (G4) predictor algorithm to assess whether G4-forming sequences reside in close proximity (within 20 base pairs) to known mitochondrial DNA deletion breakpoints. We then used this information to map G4P sequences with deletions characteristic of representative mitochondrial genetic disorders and also those identified in various cancers and aging. Circular dichroism and UV spectral analysis demonstrated that mitochondrial G-rich sequences near deletion breakpoints prevalent in human disease form G-quadruplex DNA structures. A biochemical analysis of purified recombinant human Twinkle protein (gene product of c10orf2) showed that the mitochondrial replicative helicase inefficiently unwinds well characterized intermolecular and intramolecular G-quadruplex DNA substrates, as well as a unimolecular G4 substrate derived from a mitochondrial sequence that nests a deletion breakpoint described in human renal cell carcinoma. Although G4 has been implicated in the initiation of mitochondrial DNA replication, our current findings suggest that mitochondrial G-quadruplexes are also likely to be a source of instability for the mitochondrial genome by perturbing the normal progression of the mitochondrial replication machinery, including DNA unwinding by Twinkle helicase. PMID:25193669

  15. Microwave-Assisted Synthesis of Arene Ru(II Complexes Induce Tumor Cell Apoptosis Through Selectively Binding and Stabilizing bcl-2 G-Quadruplex DNA

    Directory of Open Access Journals (Sweden)

    Yanhua Chen


    Full Text Available A series of arene Ru(II complexes coordinated with phenanthroimidazole derivatives, [(η6-C6H6Ru(lCl]Cl(1b L = p-ClPIP = 2-(4-Chlorophenylimidazole[4,5f] 1,10-phenanthroline; 2b L = m-ClPIP = 2-(3-Chlorophenylimidazole[4,5f] 1,10-phenanthroline; 3b L = p-NPIP = 2-(4-Nitrophenylimidazole[4,5f] 1,10-phenanthroline; 4b L = m-NPIP = 2-(3-Nitrophenyl imidazole [4,5f] 1,10-phenanthroline were synthesized in yields of 89.9%–92.7% under conditions of microwave irradiation heating for 30 min to liberate four arene Ru(II complexes (1b, 2b, 3b, 4b. The anti-tumor activity of 1b against various tumor cells was evaluated by MTT assay. The results indicated that this complex blocked the growth of human lung adenocarcinoma A549 cells with an IC50 of 16.59 μM. Flow cytometric analysis showed that apoptosis of A549 cells was observed following treatment with 1b. Furthermore, the in vitro DNA-binding behaviors that were confirmed by spectroscopy indicated that 1b could selectively bind and stabilize bcl-2 G-quadruplex DNA to induce apoptosis of A549 cells. Therefore, the synthesized 1b has impressive bcl-2 G-quadruplex DNA-binding and stabilizing activities with potential applications in cancer chemotherapy.

  16. Right-handed and left-handed G-quadruplexes have the same DNA sequence: distinct conformations induced by an organic small molecule and potassium. (United States)

    Fu, Boshi; Huang, Jinguo; Chen, Yuqi; Wang, Yafen; Xue, Tianrui; Xu, GuoHua; Wang, Shaoru; Zhou, Xiang


    Herein, we report two distinct G-quadruplex conformations of the same G-rich oligonucleotide, regulated by a small molecule. This is the first report in which both right- and left-handed G-quadruplex conformations have been obtained from the same sequence. We discriminated these two distinct conformations and investigated their kinetics and thermodynamics.

  17. Aminoglycosylation can enhance the G-quadruplex binding activity of epigallocatechin.

    Directory of Open Access Journals (Sweden)

    Li-Ping Bai

    Full Text Available With the aim of enhancing G-quadruplex binding activity, two new glucosaminosides (16, 18 of penta-methylated epigallocatechin were synthesized by chemical glycosylation. Subsequent ESI-TOF-MS analysis demonstrated that these two glucosaminoside derivatives exhibit much stronger binding activity to human telomeric DNA and RNA G-quadruplexes than their parent structure (i.e., methylated EGC (14 as well as natural epigallocatechin (EGC, 6. The DNA G-quadruplex binding activity of 16 and 18 is even more potent than strong G-quadruplex binder quercetin, which has a more planar structure. These two synthetic compounds also showed a higher binding strength to human telomeric RNA G-quadruplex than its DNA counterpart. Analysis of the structure-activity relationship revealed that the more basic compound, 16, has a higher binding capacity with DNA and RNA G-quadruplexes than its N-acetyl derivative, 18, suggesting the importance of the basicity of the aminoglycoside for G-quadruplex binding activity. Molecular docking simulation predicted that the aromatic ring of 16 π-stacks with the aromatic ring of guanine nucleotides, with the glucosamine moiety residing in the groove of G-quadruplex. This research indicates that glycosylation of natural products with aminosugar can significantly enhance their G-quadruplex binding activities, thus is an effective way to generate small molecules targeting G-quadruplexes in nucleic acids. In addition, this is the first report that green tea catechin can bind to nucleic acid G-quadruplex structures.

  18. “One Ring to Bind Them All”—Part I: The Efficiency of the Macrocyclic Scaffold for G-Quadruplex DNA Recognition

    Directory of Open Access Journals (Sweden)

    David Monchaud


    Full Text Available Macrocyclic scaffolds are particularly attractive for designing selective G-quadruplex ligands essentially because, on one hand, they show a poor affinity for the “standard” B-DNA conformation and, on the other hand, they fit nicely with the external G-quartets of quadruplexes. Stimulated by the pioneering studies on the cationic porphyrin TMPyP4 and the natural product telomestatin, follow-up studies have developed, rapidly leading to a large diversity of macrocyclic structures with remarkable-quadruplex binding properties and biological activities. In this review we summarize the current state of the art in detailing the three main categories of quadruplex-binding macrocycles described so far (telomestatin-like polyheteroarenes, porphyrins and derivatives, polyammonium cyclophanes, and in addressing both synthetic issues and biological aspects.

  19. Biological Function and Medicinal Research Significance of G-Quadruplex Interactive Proteins. (United States)

    Qiu, Jun; Wang, Mingxue; Zhang, Yan; Zeng, Ping; Ou, Tian-Miao; Tan, Jia-Heng; Huang, Shi-Liang; An, Lin-Kun; Wang, Honggen; Gu, Lian-Quan; Huang, Zhi-Shu; Li, Ding


    G-quadruplexes are four-stranded DNA structures formed from G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. Accumulating studies have revealed that G-quadruplex structures are formed in vivo and play important roles in biological processes such as DNA replication, transcription, recombination, epigenetic regulation, meiosis, antigenic variation, and maintenance of telomeres stability. Mounting evidence indicates that a variety of proteins are capable of binding selectively and tightly to G-quadruplex and play essential roles in G-quadruplex-mediated regulation processes. Some of these proteins promote the formation or/and stabilization of G-quadruplex, while some other proteins act to unwind G-quadruplex preferentially. From a drug discovery perspective, many of these G-quadruplex binding proteins and/or their complexes with G-quadruplexes are potential drug targets. Here, we present a general summary of reported G-quadruplex binding proteins and their biological functions, with focus on those of medicinal research significance. We elaborated the possibility for some of these G-quadruplex binding proteins and their complexes with G-quadruplexes as potential drug targets.

  20. A sensitive fluorescence anisotropy method for detection of lead (II) ion by a G-quadruplex-inducible DNA aptamer

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Dapeng [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085 (China); Yin, Lei; Meng, Zihui [School of Chemical Engineering and Environment, Beijing Institute of Technology, Beijing, 100081 (China); Yu, Anchi [Department of Chemistry, Renmin University of China, Beijing, 100872 (China); Guo, Lianghong [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085 (China); Wang, Hailin, E-mail: [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085 (China)


    Graphical abstract: -- Highlights: •A fluorescence anisotropy approach for detection of Pb{sup 2+} was developed. •The strategy was based on binding-induced allosteric conformational change of aptamer probe. •The sensing mechanism was established by testing the photoinduced electron transfer interaction. -- Abstract: Sensitive and selective detection of Pb{sup 2+} is of great importance to both human health and environmental protection. Here we propose a novel fluorescence anisotropy (FA) approach for sensing Pb{sup 2+} in homogeneous solution by a G-rich thrombin binding aptamer (TBA). The TBA labeled with 6-carboxytetramethylrhodamine (TMR) at the seventh thymine nucleotide was used as a fluorescent probe for signaling Pb{sup 2+}. It was found that the aptamer probe had a high FA in the absence of Pb{sup 2+}. This is because the rotation of TMR is restricted by intramolecular interaction with the adjacent guanine bases, which results in photoinduced electron transfer (PET). When the aptamer probe binds to Pb{sup 2+} to form G-quadruplex, the intramolecular interaction should be eliminated, resulting in faster rotation of the fluorophore TMR in solution. Therefore, FA of aptamer probe is expected to decrease significantly upon binding to Pb{sup 2+}. Indeed, we observed a decrease in FA of aptamer probe upon Pb{sup 2+} binding. Circular dichroism, fluorescence spectra, and fluorescence lifetime measurement were used to verify the reliability and reasonability of the sensing mechanism. By monitoring the FA change of the aptamer probe, we were able to real-time detect binding between the TBA probe and Pb{sup 2+}. Moreover, the aptamer probe was exploited as a recognition element for quantification of Pb{sup 2+} in homogeneous solution. The change in FA showed a linear response to Pb{sup 2+} from 10 nM to 2.0 μM, with 1.0 nM limit of detection. In addition, this sensing system exhibited good selectivity for Pb{sup 2+} over other metal ions. The method is simple

  1. Guanine base stacking in G-quadruplex nucleic acids (United States)

    Lech, Christopher Jacques; Heddi, Brahim; Phan, Anh Tuân


    G-quadruplexes constitute a class of nucleic acid structures defined by stacked guanine tetrads (or G-tetrads) with guanine bases from neighboring tetrads stacking with one another within the G-tetrad core. Individual G-quadruplexes can also stack with one another at their G-tetrad interface leading to higher-order structures as observed in telomeric repeat-containing DNA and RNA. In this study, we investigate how guanine base stacking influences the stability of G-quadruplexes and their stacked higher-order structures. A structural survey of the Protein Data Bank is conducted to characterize experimentally observed guanine base stacking geometries within the core of G-quadruplexes and at the interface between stacked G-quadruplex structures. We couple this survey with a systematic computational examination of stacked G-tetrad energy landscapes using quantum mechanical computations. Energy calculations of stacked G-tetrads reveal large energy differences of up to 12 kcal/mol between experimentally observed geometries at the interface of stacked G-quadruplexes. Energy landscapes are also computed using an AMBER molecular mechanics description of stacking energy and are shown to agree quite well with quantum mechanical calculated landscapes. Molecular dynamics simulations provide a structural explanation for the experimentally observed preference of parallel G-quadruplexes to stack in a 5′–5′ manner based on different accessible tetrad stacking modes at the stacking interfaces of 5′–5′ and 3′–3′ stacked G-quadruplexes. PMID:23268444

  2. Yeast Sub1 and human PC4 are G-quadruplex binding proteins that suppress genome instability at co-transcriptionally formed G4 DNA. (United States)

    Lopez, Christopher R; Singh, Shivani; Hambarde, Shashank; Griffin, Wezley C; Gao, Jun; Chib, Shubeena; Yu, Yang; Ira, Grzegorz; Raney, Kevin D; Kim, Nayun


    G-quadruplex or G4 DNA is a non-B secondary DNA structure consisting of a stacked array of guanine-quartets that can disrupt critical cellular functions such as replication and transcription. When sequences that can adopt Non-B structures including G4 DNA are located within actively transcribed genes, the reshaping of DNA topology necessary for transcription process stimulates secondary structure-formation thereby amplifying the potential for genome instability. Using a reporter assay designed to study G4-induced recombination in the context of an actively transcribed locus in Saccharomyces cerevisiae, we tested whether co-transcriptional activator Sub1, recently identified as a G4-binding factor, contributes to genome maintenance at G4-forming sequences. Our data indicate that, upon Sub1-disruption, genome instability linked to co-transcriptionally formed G4 DNA in Top1-deficient cells is significantly augmented and that its highly conserved DNA binding domain or the human homolog PC4 is sufficient to suppress G4-associated genome instability. We also show that Sub1 interacts specifically with co-transcriptionally formed G4 DNA in vivo and that yeast cells become highly sensitivity to G4-stabilizing chemical ligands by the loss of Sub1. Finally, we demonstrate the physical and genetic interaction of Sub1 with the G4-resolving helicase Pif1, suggesting a possible mechanism by which Sub1 suppresses instability at G4 DNA. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  3. Dual Recognition of Human Telomeric G-quadruplex by Neomycin-anthraquinone Conjugate (United States)

    Ranjan, Nihar; Davis, Erik; Xue, Liang


    The authors report the recognition of a G-quadruplex formed by four repeat human telomeric DNA with aminosugar intercalator conjugates. The recognition of G-quadruplex through dual binding mode ligands significantly increased the affinity of ligands for G-quadruplex. One such example is a neomycin-anthraquinone 2 which exhibited nanomolar affinity for the quadruplex, and the affinity of 2 is nearly 1000 fold higher for human telomeric G-quadruplex DNA than its constituent units, neomycin and anthraquinone. PMID:23698792

  4. G-quadruplex-based structural transitions in 15-mer DNA oligonucleotides varying in lengths of internal oligo(dG) stretches detected by voltammetric techniques

    Czech Academy of Sciences Publication Activity Database

    Vidláková, Pavlína; Pivoňková, Hana; Kejnovská, Iva; Trnková, L.; Vorlíčková, Michaela; Fojta, Miroslav; Havran, Luděk


    Roč. 407, č. 19 (2015), s. 5817-5826 ISSN 1618-2642 R&D Projects: GA ČR GAP206/12/2378 Institutional support: RVO:68081707 Keywords : Oligonucleotides * Electrochemical methods * G-quadruplex Subject RIV: BO - Biophysics Impact factor: 3.125, year: 2015

  5. Targeting G-quadruplex DNA Structures by EMICORON has a strong antitumor efficacy against advanced models of human colon cancer

    DEFF Research Database (Denmark)

    Porru, Manuela; Artuso, Simona; Salvati, Erica


    similar blood levels in humans. Moreover, EMICORON showed a marked therapeutic efficacy, as it inhibited the growth of patient-derived xenografts (PDX) and orthotopic colon cancer and strongly reduced the dissemination of tumor cells to lymph nodes, intestine, stomach, and liver. Finally, activation...... of human colon cancer that could adequately predict human clinical outcomes. Our results showed that EMICORON was well tolerated in mice, as no adverse effects were reported, and a low ratio of sensitivity across human and mouse bone marrow cells was observed, indicating a good potential for reaching...... of DNA damage and impairment of proliferation and angiogenesis are proved to be key determinants of EMICORON antitumoral activity. Altogether, our results, performed on advanced experimental models of human colon cancer that bridge the translational gap between preclinical and clinical studies...

  6. Identification of small molecules capable of regulating conformational changes of telomeric G-quadruplex (United States)

    Chen, Shuo-Bin; Liu, Guo-Cai; Gu, Lian-Quan; Huang, Zhi-Shu; Tan, Jia-Heng


    Design of small molecules targeted at human telomeric G-quadruplex DNA is an extremely active research area. Interestingly, the telomeric G-quadruplex is a highly polymorphic structure. Changes in its conformation upon small molecule binding may be a powerful method to achieve a desired biological effect. However, the rational development of small molecules capable of regulating conformational change of telomeric G-quadruplex structures is still challenging. In this study, we developed a reliable ligand-based pharmacophore model based on isaindigotone derivatives with conformational change activity toward telomeric G-quadruplex DNA. Furthermore, virtual screening of database was conducted using this pharmacophore model and benzopyranopyrimidine derivatives in the database were identified as a strong inducer of the telomeric G-quadruplex DNA conformation, transforming it from hybrid-type structure to parallel structure.

  7. Multimerization rules for G-quadruplexes

    Czech Academy of Sciences Publication Activity Database

    Kolesnikova, Sofia; Hubálek, Martin; Bednárová, Lucie; Cvačka, Josef; Curtis, Edward A.


    Roč. 45, č. 15 (2017), s. 8684-8696 ISSN 0305-1048 Institutional support: RVO:61388963 Keywords : tetramolecular G-quadruplexes * RNA G-quadruplexes * circular dichroism Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 10.162, year: 2016 https:// academic

  8. In silico identification of novel ligands for G-quadruplex in the c- MYC promoter (United States)

    Kang, Hyun-Jin; Park, Hyun-Ju


    G-quadruplex DNA formed in NHEIII1 region of oncogene promoter inhibits transcription of the genes. In this study, virtual screening combining pharmacophore-based search and structure-based docking screening was conducted to discover ligands binding to G-quadruplex in promoter region of c- MYC. Several hit ligands showed the selective PCR-arresting effects for oligonucleotide containing c- MYC G-quadruplex forming sequence. Among them, three hits selectively inhibited cell proliferation and decreased c- MYC mRNA level in Ramos cells, where NHEIII1 is included in translocated c- MYC gene for overexpression. Promoter assay using two kinds of constructs with wild-type and mutant sequences showed that interaction of these ligands with the G-quadruplex resulted in turning-off of the reporter gene. In conclusion, combined virtual screening methods were successfully used for discovery of selective c- MYC promoter G-quadruplex binders with anticancer activity.

  9. Superhelicity Constrains a Localized and R-Loop-Dependent Formation of G-Quadruplexes at the Upstream Region of Transcription. (United States)

    Zheng, Ke-Wei; He, Yi-de; Liu, Hong-He; Li, Xin-Min; Hao, Yu-Hua; Tan, Zheng


    Transcription induces formation of intramolecular G-quadruplex structures at the upstream region of a DNA duplex by an upward transmission of negative supercoiling through the DNA. Currently the regulation of such G-quadruplex formation remains unclear. Using plasmid as a model, we demonstrate that while it is the dynamic negative supercoiling generated by a moving RNA polymerase that triggers a formation of a G-quadruplex, the constitutional superhelicity determines the potential and range of the formation of a G-quadruplex by constraining the propagation of the negative supercoiling. G-quadruplex formation is maximal in negatively supercoiled and nearly abolished in relaxed plasmids while being moderate in nicked and linear ones. The formation of a G-quadruplex strongly correlates with the presence of an R-loop. Preventing R-loop formation virtually abolished G-quadruplex formation even in the negatively supercoiled plasmid. Enzymatic action and protein binding that manipulate supercoiling or its propagation all impact the formation of G-quadruplexes. Because chromosomes and plasmids in cells in their natural form are maintained in a supercoiled state, our findings reveal a physical basis that justifies the formation and regulation of G-quadruplexes in vivo. The structural features involved in G-quadruplex formation may all serve as potential targets in clinical and therapeutic applications.

  10. Direct visualization of nucleolar G-quadruplexes in live cells by using a fluorescent light-up probe. (United States)

    Zhang, Suge; Sun, Hongxia; Chen, Hongbo; Li, Qian; Guan, Aijiao; Wang, Lixia; Shi, Yunhua; Xu, Shujuan; Liu, Meirong; Tang, Yalin


    Direct detection of G-quadruplexes in human cells has become an important issue due to the vital role of G-quadruplex related to biological functions. Despite several probes have been developed for detection of the G-quadruplexes in cytoplasm or whole cells, the probe being used to monitor the nucleolar G-quadruplexes is still lacking. Formation of the nucleolar G-quadruplex structures was confirmed by using circular dichroism (CD) spectroscopy. The binding affinity and selectivity of Thioflavin T (ThT) towards various DNA/RNA motifs in solution and gel system were measured by using fluorescence spectroscopy and polyacrylamide gel electrophoresis (PAGE), respectively. G-quadruplex imaging in live cells was directly captured by using confocal laser scanning microscopy (CLSM). Formation of the rDNA and rRNA G-quadruplex structures is demonstrated in vitro. ThT is found to show much higher affinity and selectivity towards these G-quadruplex structures versus other nucleic acid motifs either in solution or in gel system. The nucleolar G-quadruplexes in living cells are visualized by using ThT as a fluorescent probe. G-quadruplex-ligand treatments in live cells lead to sharp decrease of ThT signal. The natural existence of the G-quadruplexes structure in the nucleoli of living cells is directly visualized by using ThT as an indicator. The research provides substantive evidence for formation of the rRNA G-quadruplex structures, and also offers an effective probe for direct visualization of the nucleolar G-quadruplexes in living cells. Copyright © 2018. Published by Elsevier B.V.

  11. Putative HIV and SIV G-Quadruplex Sequences in Coding and Noncoding Regions Can Form G-Quadruplexes

    Directory of Open Access Journals (Sweden)

    Petra Krafčíková


    Full Text Available The HIV virus is one of the most studied viruses in the world. This is especially true in terms of gene sequencing, and to date more than 9 thousand genomic sequences of HIV isolates have been sequenced and analyzed. In this study, a series of DNA sequences, which have the potential to form G-quadruplex structures, is analyzed. Several such sequences were found in various coding and noncoding virus domains, including the U3 LTR, tat, rev, env, and vpx regions. Interestingly, a homological sequence to the already well-known HIV integrase aptamer was identified in the minus-strand. The sequences derived from original isolates were analyzed using standard spectral and electrophoretic methods. In addition, a recently developed methodology is applied which uses induced circular dichroism spectral profiles of G-quadruplex-ligand (Thiazole Orange complexes to determine if G-rich sequences can adopt G-quadruplex structure. Targeting the G-quadruplexes or peptide domains corresponding to the G-rich coding sequence in HIV offers researchers attractive therapeutic targets which would be of particular use in the development of novel antiviral therapies. The analysis of G-rich regions can provide researchers with a path to find specific targets which could be of interest for specific types of virus.

  12. Role of electrostatic interactions in determining the G-quadruplex structures (United States)

    Lee, Jinkeong; Im, Haeri; Chong, Song-Ho; Ham, Sihyun


    We investigate the energetics of the antiparallel, hybrid and parallel type G-quadruplex structures of the human telomere DNA sequence. We find that both the conformational energy and solvation free energy of these structures are roughly inversely proportional to their radii of gyration. We rationalize this finding in terms of the dominance of the electrostatic contributions. We also show that the solvation free energy is more significant than the conformational energy in determining the G-quadruplex structures, which is in contrast to the canonical B-DNA structures. Our work will contribute to an understanding of the molecular mechanisms dictating various G-quadruplex topologies.

  13. A new cationic porphyrin derivative (TMPipEOPP with large side arm substituents: a highly selective G-quadruplex optical probe.

    Directory of Open Access Journals (Sweden)

    Li-Na Zhu

    Full Text Available The discovery of uncommon DNA structures and speculation about their potential functions in genes has brought attention to specific DNA structure recognition. G-quadruplexes are four-stranded nucleic acid structures formed by G-rich DNA (or RNA sequences. G-rich sequences with a high potential to form G-quadruplexes have been found in many important genomic regions. Porphyrin derivatives with cationic side arm substituents are important G-quadruplex-binding ligands. For example, 5,10,15,20-Tetrakis(N-methylpyridinium-4-yl-21H,23H-porphyrin (TMPyP4, interacts strongly with G-quadruplexes, but has poor selectivity for G-quadruplex versus duplex DNA. To increase the G-quadruplex recognition specificity, a new cationic porphyrin derivative, 5,10,15,20-tetra-{4-[2-(1-methyl-1-piperidinylethoxy]phenyl} porphyrin (TMPipEOPP, with large side arm substituents was synthesized, and the interactions between TMPipEOPP and different DNA structures were compared. The results show that G-quadruplexes cause large changes in the UV-Vis absorption and fluorescence spectra of TMPipEOPP, but duplex and single-stranded DNAs do not, indicating that TMPipEOPP can be developed as a highly specific optical probe for discriminating G-quadruplex from duplex and single-stranded DNA. Visual discrimination is also possible. Job plot and Scatchard analysis suggest that a complicated binding interaction occurs between TMPipEOPP and G-quadruplexes. At a low [G-quadruplex]/[TMPipEOPP] ratio, one G-quadruplex binds two TMPipEOPP molecules by end-stacking and outside binding modes. At a high [G-quadruplex]/[TMPipEOPP] ratio, two G-quadruplexes bind to one TMPipEOPP molecule in a sandwich-like end-stacking mode.

  14. Metal Cations in G-Quadruplex Folding and Stability (United States)

    Bhattacharyya, Debmalya; Mirihana Arachchilage, Gayan; Basu, Soumitra


    This review is focused on the structural and physico-chemical aspects of metal cation coordination to G-Quadruplexes (GQ) and their effects on GQ stability and conformation. G-Quadruplex structures are non-canonical secondary structures formed by both DNA and RNA. G-quadruplexes regulate a wide range of important biochemical processes. Besides the sequence requirements, the coordination of monovalent cations in the GQ is essential for its formation and determines the stability and polymorphism of GQ structures. The nature, location and dynamics of the cation coordination and their impact on the overall GQ stability are dependent on several factors such as the ionic radii, hydration energy and the bonding strength to the O6 of guanines. The intracellular monovalent cation concentration and the localized ion concentrations determine the formation of GQs and can potentially dictate their regulatory roles. A wide range of biochemical and biophysical studies on an array of GQ enabling sequences have generated at a minimum the knowledge base that allows us to often predict the stability of GQs in presence of the physiologically relevant metal ions, however, prediction of conformation of such GQs is still out of the realm.

  15. G-quadruplexes as novel cis-elements controlling transcription during embryonic development. (United States)

    David, Aldana P; Margarit, Ezequiel; Domizi, Pablo; Banchio, Claudia; Armas, Pablo; Calcaterra, Nora B


    G-quadruplexes are dynamic structures folded in G-rich single-stranded DNA regions. These structures have been recognized as a potential nucleic acid based mechanism for regulating multiple cellular processes such as replication, transcription and genomic maintenance. So far, their transcriptional role in vivo during vertebrate embryonic development has not yet been addressed. Here, we performed an in silico search to find conserved putative G-quadruplex sequences (PQSs) within proximal promoter regions of human, mouse and zebrafish developmental genes. Among the PQSs able to fold in vitro as G-quadruplex, those present in nog3, col2a1 and fzd5 promoters were selected for further studies. In cellulo studies revealed that the selected G-quadruplexes affected the transcription of luciferase controlled by the SV40 nonrelated promoter. G-quadruplex disruption in vivo by microinjection in zebrafish embryos of either small ligands or DNA oligonucleotides complementary to the selected PQSs resulted in lower transcription of the targeted genes. Moreover, zebrafish embryos and larvae phenotypes caused by the presence of complementary oligonucleotides fully resembled those ones reported for nog3, col2a1 and fzd5 morphants. To our knowledge, this is the first work revealing in vivo the role of conserved G-quadruplexes in the embryonic development, one of the most regulated processes of the vertebrates biology. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Folding equilibrium constants of telomere G-quadruplexes in free state or associated with proteins determined by isothermal differential hybridization. (United States)

    Wang, Quan; Ma, Li; Hao, Yu-Hua; Tan, Zheng


    Guanine rich (G-rich) nucleic acids form G-quadruplex structures that are implicated in many biological processes, pharmaceutical applications, and molecular machinery. The folding equilibrium constant (K(F)) of the G-quadruplex not only determines its stability and competition against duplex formation in genomic DNA but also defines its recognition by proteins and drugs and technical specifications. The K(F) is most conveniently derived from thermal melting analysis that has so far yielded extremely diversified results for the human telomere G-quadruplex. Melting analysis cannot be used for nucleic acids associated with proteins, thus has difficulty to study how protein association affects the folding equilibrium of G-quadruplex structure. In this work, we established an isothermal differential hybridization (IDH) method that is able to determine the K(F) of G-quadruplex, either alone or associated with proteins. Using this method, we studied the folding equilibrium of the core sequence G(3)(T(2)AG(3))(3) from vertebrate telomere in K(+) and Na(+) solutions and how it is affected by proteins associated at its adjacent regions. Our results show that the K(F) obtained for the free G-quadruplex is within 1 order of magnitude of most of those obtained by melting analysis and protein binding beside a G-quadruplex can dramatically destabilize the G-quadruplex.

  17. G-quadruplex induced chirality of methylazacalix[6]pyridine via unprecedented binding stoichiometry: en route to multiplex controlled molecular switch (United States)

    Guan, Ai-Jiao; Shen, Meng-Jie; Xiang, Jun-Feng; Zhang, En-Xuan; Li, Qian; Sun, Hong-Xia; Wang, Li-Xia; Xu, Guang-Zhi; Tang, Ya-Lin; Xu, Li-Jin; Gong, Han-Yuan


    Nucleic acid based molecular device is a developing research field which attracts great interests in material for building machinelike nanodevices. G-quadruplex, as a new type of DNA secondary structures, can be harnessed to construct molecular device owing to its rich structural polymorphism. Herein, we developed a switching system based on G-quadruplexes and methylazacalix[6]pyridine (MACP6). The induced circular dichroism (CD) signal of MACP6 was used to monitor the switch controlled by temperature or pH value. Furthermore, the CD titration, Job-plot, variable temperature CD and 1H-NMR experiments not only confirmed the binding mode between MACP6 and G-quadruplex, but also explained the difference switching effect of MACP6 and various G-quadruplexes. The established strategy has the potential to be used as the chiral probe for specific G-quadruplex recognition.

  18. Selection of G-quadruplex folding topology with LNA-modified human telomeric sequences in K+ solution

    DEFF Research Database (Denmark)

    Pradhan, Devranjan; Hansen, Lykke H; Vester, Birte


    G-rich nucleic acid oligomers can form G-quadruplexes built by G-tetrads stacked upon each other. Depending on the nucleotide sequence, G-quadruplexes fold mainly with two topologies: parallel, in which all G-tracts are oriented parallel to each other, or antiparallel, in which one or more G......-tracts are oriented antiparallel to the other G-tracts. In the former topology, all glycosidic bond angles conform to anti conformations, while in the latter topology they adopt both syn and anti conformations. It is of interest to understand the molecular forces that govern G-quadruplex folding. Here, we approach...... this problem by examining the impact of LNA (locked nucleic acid) modifications on the folding topology of the dimeric model system of the human telomere sequence. In solution, this DNA G-quadruplex forms a mixture of G-quadruplexes with antiparallel and parallel topologies. Using CD and NMR spectroscopies, we...

  19. GNG Motifs Can Replace a GGG Stretch during G-Quadruplex Formation in a Context Dependent Manner. (United States)

    Das, Kohal; Srivastava, Mrinal; Raghavan, Sathees C


    G-quadruplexes are one of the most commonly studied non-B DNA structures. Generally, these structures are formed using a minimum of 4, three guanine tracts, with connecting loops ranging from one to seven. Recent studies have reported deviation from this general convention. One such deviation is the involvement of bulges in the guanine tracts. In this study, guanines along with bulges, also referred to as GNG motifs have been extensively studied using recently reported HOX11 breakpoint fragile region I as a model template. By strategic mutagenesis approach we show that the contribution from continuous G-tracts may be dispensible during G-quadruplex formation when such motifs are flanked by GNGs. Importantly, the positioning and number of GNG/GNGNG can also influence the formation of G-quadruplexes. Further, we assessed three genomic regions from HIF1 alpha, VEGF and SHOX gene for G-quadruplex formation using GNG motifs. We show that HIF1 alpha sequence harbouring GNG motifs can fold into intramolecular G-quadruplex. In contrast, GNG motifs in mutant VEGF sequence could not participate in structure formation, suggesting that the usage of GNG is context dependent. Importantly, we show that when two continuous stretches of guanines are flanked by two independent GNG motifs in a naturally occurring sequence (SHOX), it can fold into an intramolecular G-quadruplex. Finally, we show the specific binding of G-quadruplex binding protein, Nucleolin and G-quadruplex antibody, BG4 to SHOX G-quadruplex. Overall, our study provides novel insights into the role of GNG motifs in G-quadruplex structure formation which may have both physiological and pathological implications.

  20. GNG Motifs Can Replace a GGG Stretch during G-Quadruplex Formation in a Context Dependent Manner.

    Directory of Open Access Journals (Sweden)

    Kohal Das

    Full Text Available G-quadruplexes are one of the most commonly studied non-B DNA structures. Generally, these structures are formed using a minimum of 4, three guanine tracts, with connecting loops ranging from one to seven. Recent studies have reported deviation from this general convention. One such deviation is the involvement of bulges in the guanine tracts. In this study, guanines along with bulges, also referred to as GNG motifs have been extensively studied using recently reported HOX11 breakpoint fragile region I as a model template. By strategic mutagenesis approach we show that the contribution from continuous G-tracts may be dispensible during G-quadruplex formation when such motifs are flanked by GNGs. Importantly, the positioning and number of GNG/GNGNG can also influence the formation of G-quadruplexes. Further, we assessed three genomic regions from HIF1 alpha, VEGF and SHOX gene for G-quadruplex formation using GNG motifs. We show that HIF1 alpha sequence harbouring GNG motifs can fold into intramolecular G-quadruplex. In contrast, GNG motifs in mutant VEGF sequence could not participate in structure formation, suggesting that the usage of GNG is context dependent. Importantly, we show that when two continuous stretches of guanines are flanked by two independent GNG motifs in a naturally occurring sequence (SHOX, it can fold into an intramolecular G-quadruplex. Finally, we show the specific binding of G-quadruplex binding protein, Nucleolin and G-quadruplex antibody, BG4 to SHOX G-quadruplex. Overall, our study provides novel insights into the role of GNG motifs in G-quadruplex structure formation which may have both physiological and pathological implications.

  1. Integration of G-quadruplex and DNA-templated Ag NCs for nonarithmetic information processing† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c7sc00361g Click here for additional data file. (United States)

    Gao, Ru-Ru; Lv, Xiao-Yan; Zhu, Yan-Yan; Zhang, Yi-Wei


    To create sophisticated molecular logic circuits from scratch, you may not believe how common the building blocks can be and how diverse and powerful such circuits can be when scaled up. Using the two simple building blocks of G-quadruplex and silver nanoclusters (Ag NCs), we experimentally construct a series of multifunctional, label-free, and multi-output logic circuits to perform nonarithmetic functions: a 1-to-2 decoder, a 4-to-2 encoder, an 8-to-3 encoder, dual transfer gates, a 2 : 1 multiplexer, and a 1 : 2 demultiplexer. Moreover, a parity checker which is capable of identifying odd and even numbers from natural numbers is constructed conceptually. Finally, a multi-valued logic gate (ternary inhibit gate) is readily achieved by taking this DNA/Ag NC system as a universal platform. All of the above logic circuits share the same building blocks, indicating the great prospects of the assembly of nanomaterials and DNA for biochemical logic devices. Considering its biocompatibility, the novel prototypes developed here may have potential applications in the fields of biological computers and medical diagnosis and serve as a promising proof of principle in the not-too-distant future. PMID:28626564

  2. Local epigenetic reprogramming induced by G-quadruplex ligands (United States)

    Guilbaud, Guillaume; Murat, Pierre; Recolin, Bénédicte; Campbell, Beth C.; Maiter, Ahmed; Sale, Julian E.; Balasubramanian, Shankar


    DNA and histone modifications regulate transcriptional activity and thus represent valuable targets to reprogram the activity of genes. Current epigenetic therapies target the machinery that regulates these modifications, leading to global transcriptional reprogramming with the potential for extensive undesired effects. Epigenetic information can also be modified as a consequence of disrupting processive DNA replication. Here, we demonstrate that impeding replication by small-molecule-mediated stabilization of G-quadruplex nucleic acid secondary structures triggers local epigenetic plasticity. We report the use of the BU-1 locus of chicken DT40 cells to screen for small molecules able to induce G-quadruplex-dependent transcriptional reprogramming. Further characterization of the top hit compound revealed its ability to induce a dose-dependent inactivation of BU-1 expression in two steps: the loss of H3K4me3 and then subsequent DNA cytosine methylation, changes that were heritable across cell divisions even after the compound was removed. Targeting DNA secondary structures thus represents a potentially new approach for locus-specific epigenetic reprogramming.

  3. Tetrazolylpyrene unnatural nucleoside as a human telomeric multimeric G-quadruplex selective switch-on fluorescent sensor. (United States)

    Bag, Subhendu Sekhar; Pradhan, Manoj Kumar; Talukdar, Sangita


    We report herein the specific sensing of dimeric H45 G-quadruplex DNA via a fluorescence light-up response using fluorescent tetrazolylpyrene nucleoside ( TzPy B Do ) as a probe. The strong binding of the probe via an intercalative stacking interaction inside the connecting loop of two G-quadruplex units of H45 and the discrimination to other monomeric and long DNA duplexes are accompanied by a drastic enhancement of the emission intensity without compromising the conformation and stability.

  4. Thermodynamic stability and energetics of DNA duplexes containing major intrastrand cross-links of second-generation antitumor dinuclear Pt(II) complexes. (United States)

    Florian, Jakub; Kasparkova, Jana; Farrell, Nicholas P; Brabec, Viktor


    The effects of major DNA intrastrand cross-links of antitumor dinuclear Pt(II) complexes [{trans-PtCl(NH(3))(2)}(2)-μ-{trans-(H(2)N(CH(2))(6)NH(2)(CH(2))(2)NH(2)(CH(2))(6)NH(2))}](4+) (1) and [{PtCl(DACH)}(2)-μ-{H(2)N(CH(2))(6)NH(2)(CH(2))(2)NH(2)(CH(2))(6)NH(2))}](4+) (2) (DACH is 1,2-diaminocyclohexane) on DNA stability were studied with emphasis on thermodynamic origins of that stability. Oligodeoxyribonucleotide duplexes containing the single 1,2, 1,3, or 1,5 intrastrand cross-links at guanine residues in the central TGGT, TGTGT, or TGTTTGT sequences, respectively, were prepared and analyzed by differential scanning calorimetry. The unfolding of the platinated duplexes was accompanied by unfavorable free energy terms. The efficiency of the cross-links to thermodynamically destabilize the duplex depended on the number of base pairs separating the platinated bases. The trend was 1,5→1,2→1,3 cross-link of 1 and 1,5→1,3→1,2 cross-link of 2. Interestingly, the results showed that the capability of the cross-links to reduce the thermodynamic stability of DNA (ΔG(298)(0)) correlated with the extent of conformational distortions induced in DNA by various types of intrastrand cross-links of 1 or 2 determined by chemical probes of DNA conformation. We also examined the efficiency of the mammalian nucleotide excision repair systems to remove from DNA the intrastrand cross-links of 1 or 2. The efficiency of the excinucleases to remove the cross-links from DNA depended on the length of the cross-link; the trend was identical to that observed for the efficiency of the intrastrand cross-links to thermodynamically destabilize the duplex. Thus, the results are consistent with the thesis that an important factor that determines the susceptibility of the intrastrand cross-links of dinuclear platinum complexes 1 and 2 to be removed from DNA by nucleotide excision repair is the efficiency of these lesions to thermodynamically destabilize DNA.

  5. Solution structure of a 2:1 quindoline-c-MYC G-quadruplex: insights into G-quadruplex-interactive small molecule drug design. (United States)

    Dai, Jixun; Carver, Megan; Hurley, Laurence H; Yang, Danzhou


    Unimolecular parallel-stranded G-quadruplex structures are found to be prevalent in gene promoters. The nuclease hypersensitivity element III(1) (NHE III(1)) of the c-MYC promoter can form transcriptionally active and silenced forms, and the formation of DNA G-quadruplex structures has been shown to be critical for c-MYC transcriptional silencing. The solution structure of a 2:1 quindoline-G-quadruplex complex has been solved and shows unexpected features, including the drug-induced reorientation of the flanking sequences to form a new binding pocket. While both 3' and 5' complexes show overall similar features, there are identifiable differences that emphasize the importance of both stacking and electronic interactions. For the first time, we describe the importance of the shape of the ligand as well as the two flanking bases in determining drug binding specificity. These structures provide important insights for the structure-based rational design of drugs that bind to unimolecular parallel G-quadruplexes commonly found in promoter elements.

  6. G-Quadruplexes: Prediction, Characterization, and Biological Application. (United States)

    Kwok, Chun Kit; Merrick, Catherine J


    Guanine (G)-rich sequences in nucleic acids can assemble into G-quadruplex structures that involve G-quartets linked by loop nucleotides. The structural and topological diversity of G-quadruplexes have attracted great attention for decades. Recent methodological advances have advanced the identification and characterization of G-quadruplexes in vivo as well as in vitro, and at a much higher resolution and throughput, which has greatly expanded our current understanding of G-quadruplex structure and function. Accumulating knowledge about the structural properties of G-quadruplexes has helped to design and develop a repertoire of molecular and chemical tools for biological applications. This review highlights how these exciting methods and findings have opened new doors to investigate the potential functions and applications of G-quadruplexes in basic and applied biosciences. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Stabilization of Telomere G-Quadruplexes Interferes with Human Herpesvirus 6A Chromosomal Integration. (United States)

    Gilbert-Girard, Shella; Gravel, Annie; Artusi, Sara; Richter, Sara N; Wallaschek, Nina; Kaufer, Benedikt B; Flamand, Louis


    Human herpesviruses 6A and 6B (HHV-6A/B) can integrate their genomes into the telomeres of human chromosomes using a mechanism that remains poorly understood. To achieve a better understanding of the HHV-6A/B integration mechanism, we made use of BRACO-19, a compound that stabilizes G-quadruplex secondary structures and prevents telomere elongation by the telomerase complex. First, we analyzed the folding of telomeric sequences into G-quadruplex structures and their binding to BRACO-19 using G-quadruplex-specific antibodies and surface plasmon resonance. Circular dichroism studies indicate that BRACO-19 modifies the conformation and greatly stabilizes the G-quadruplexes formed in G-rich telomeric DNA. Subsequently we assessed the effects of BRACO-19 on the HHV-6A initial phase of infection. Our results indicate that BRACO-19 does not affect entry of HHV-6A DNA into cells. We next investigated if stabilization of G-quadruplexes by BRACO-19 affected HHV-6A's ability to integrate its genome into host chromosomes. Incubation of telomerase-expressing cells with BRACO-19, such as HeLa and MCF-7, caused a significant reduction in the HHV-6A integration frequency ( P integration frequency in U2OS cells that lack telomerase activity and elongate their telomeres through alternative lengthening mechanisms. Our data suggest that the fluidity of telomeres is important for efficient chromosomal integration of HHV-6A and that interference with telomerase activity negatively affects the generation of cellular clones containing integrated HHV-6A. IMPORTANCE HHV-6A/B can integrate their genomes into the telomeres of infected cells. Telomeres consist of repeated hexanucleotides (TTAGGG) of various lengths (up to several kilobases) and end with a single-stranded 3' extension. To avoid recognition and induce a DNA damage response, the single-stranded overhang folds back on itself and forms a telomeric loop (T-loop) or adopts a tertiary structure, referred to as a G-quadruplex. In the

  8. Unexpected Position-Dependent Effects of Ribose G-Quartets in G-Quadruplexes

    Czech Academy of Sciences Publication Activity Database

    Zhou, J.; Amrane, S.; Rosu, F.; Salgado, G.; Bian, Y.; Tateishi-Karimata, H.; Largy, E.; Korkut, D. N.; Bourdoncle, A.; Miyoshi, D.; Zhang, J.; Ju, H.; Wang, W.; Sugimoto, N.; Gabelica, V.; Mergny, Jean-Louis


    Roč. 139, č. 23 (2017), s. 7768-7779 ISSN 0002-7863 Institutional support: RVO:68081707 Keywords : human telomeric rna * electrospray mass-spectrometry * molecular crowding conditions * dna g-quadruplexes Subject RIV: BO - Biophysics OBOR OECD: Electrochemistry (dry cells, batteries, fuel cells, corrosion metals, electrolysis) Impact factor: 13.858, year: 2016

  9. Mechanochemical properties of individual human telomeric RNA (TERRA) G-quadruplexes. (United States)

    Yangyuoru, Philip M; Zhang, Amy Y Q; Shi, Zhe; Koirala, Deepak; Balasubramanian, Shankar; Mao, Hanbin


    Potential functions: By following the unfolding and refolding of individual human RNA telomeric (TERRA) G-quadruplexes (GQs) in laser tweezers, the mechanical stability and transition kinetics of RNA GQs are obtained. Comparison between TERRA and DNA GQs suggests their different regulatory capacities for processes associated with human telomeres. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Xanthine and 8-oxoguanine in G-quadruplexes: formation of a G·G·X·O tetrad. (United States)

    Cheong, Vee Vee; Heddi, Brahim; Lech, Christopher Jacques; Phan, Anh Tuân


    G-quadruplexes are four-stranded structures built from stacked G-tetrads (G·G·G·G), which are planar cyclical assemblies of four guanine bases interacting through Hoogsteen hydrogen bonds. A G-quadruplex containing a single guanine analog substitution, such as 8-oxoguanine (O) or xanthine (X), would suffer from a loss of a Hoogsteen hydrogen bond within a G-tetrad and/or potential steric hindrance. We show that a proper arrangement of O and X bases can reestablish the hydrogen-bond pattern within a G·G·X·O tetrad. Rational incorporation of G·G·X·O tetrads in a (3+1) G-quadruplex demonstrated a similar folding topology and thermal stability to that of the unmodified G-quadruplex. pH titration conducted on X·O-modified G-quadruplexes indicated a protonation-deprotonation equilibrium of X with a pKa ∼6.7. The solution structure of a G-quadruplex containing a G·G·X·O tetrad was determined, displaying the same folding topology in both the protonated and deprotonated states. A G-quadruplex containing a deprotonated X·O pair was shown to exhibit a more electronegative groove compared to that of the unmodified one. These differences are likely to manifest in the electronic properties of G-quadruplexes and may have important implications for drug targeting and DNA-protein interactions. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. Altered biochemical specificity of G-quadruplexes with mutated tetrads

    Czech Academy of Sciences Publication Activity Database

    Švehlová, Kateřina; Lawrence, M. S.; Bednárová, Lucie; Curtis, Edward A.


    Roč. 44, č. 22 (2016), s. 10789-10803 ISSN 0305-1048 Institutional support: RVO:61388963 Keywords : G-quadruplex * G motif GTP aptamer * peroxidase deoxyribozyme Subject RIV: CE - Biochemistry Impact factor: 10.162, year: 2016

  12. Enantioselective targeting left-handed Z-G-quadruplex. (United States)

    Zhao, Andong; Zhao, Chuanqi; Ren, Jinsong; Qu, Xiaogang


    Herein, we report the first example where an M-enantiomer of a chiral metal complex can selectively stabilize a left-handed G-quadruplex, but its P-enantiomer cannot. The interactions between the chiral metal complexes and the left-handed G-quadruplex were evaluated by UV melting, circular dichroism, isothermal titration calorimetry, gel electrophoresis and NMR titrations.

  13. Biophysical Characterization of G-Quadruplex Recognition in the PITX1 mRNA by the Specificity Domain of the Helicase RHAU.

    Directory of Open Access Journals (Sweden)

    Emmanuel O Ariyo

    Full Text Available Nucleic acids rich in guanine are able to fold into unique structures known as G-quadruplexes. G-quadruplexes consist of four tracts of guanylates arranged in parallel or antiparallel strands that are aligned in stacked G-quartet planes. The structure is further stabilized by Hoogsteen hydrogen bonds and monovalent cations centered between the planes. RHAU (RNA helicase associated with AU-rich element is a member of the ATP-dependent DExH/D family of RNA helicases and can bind and resolve G-quadruplexes. RHAU contains a core helicase domain with an N-terminal extension that enables recognition and full binding affinity to RNA and DNA G-quadruplexes. PITX1, a member of the bicoid class of homeobox proteins, is a transcriptional activator active during development of vertebrates, chiefly in the anterior pituitary gland and several other organs. We have previously demonstrated that RHAU regulates PITX1 levels through interaction with G-quadruplexes at the 3'-end of the PITX1 mRNA. To understand the structural basis of G-quadruplex recognition by RHAU, we characterize a purified minimal PITX1 G-quadruplex using a variety of biophysical techniques including electrophoretic mobility shift assays, UV-VIS spectroscopy, circular dichroism, dynamic light scattering, small angle X-ray scattering and nuclear magnetic resonance spectroscopy. Our biophysical analysis provides evidence that the RNA G-quadruplex, but not its DNA counterpart, can adopt a parallel orientation, and that only the RNA can interact with N-terminal domain of RHAU via the tetrad face of the G-quadruplex. This work extends our insight into how the N-terminal region of RHAU recognizes parallel G-quadruplexes.

  14. Complicated behavior of G-quadruplexes and evaluating G-quadruplexes' ligands in various systems mimicking cellular circumstance

    Directory of Open Access Journals (Sweden)

    Shi-Ke Wang


    Full Text Available Environments surrounding G-rich sequences remarkably affect the conformations of these structures. A proper evaluation system mimicking the crowded environment in a cell with macromolecules should be developed to perform structural and functional studies on G-quadruplexes. In this study, the topology and stability of a G-quadruplex formed by human telomeric repeat sequences were investigated in a macromolecule-crowded environment created by polyethylene glycol 200 (PEG200, tumor cell extract, and Xenopus laevis egg extract. The interactions between small molecules and telomeric G-quadruplexes were also evaluated in the different systems. The results suggested that the actual behavior of G-quadruplex structures in cells extract is quite different from that in the PEG crowding system, and proteins or other factors in extracts might play a very important role in G-quadruplex structures.

  15. Thermodynamic fingerprints of ligand binding to human telomeric G-quadruplexes


    Bon?ina, Matja?; Podlipnik, ?rtomir; Piantanida, Ivo; Eilmes, Julita; Teulade-Fichou, Marie-Paule; Vesnaver, Gorazd; Lah, Jurij


    Thermodynamic studies of ligand binding to human telomere (ht) DNA quadruplexes, as a rule, neglect the involvement of various ht-DNA conformations in the binding process. Therefore, the thermodynamic driving forces and the mechanisms of ht-DNA G-quadruplex-ligand recognition remain poorly understood. In this work we characterize thermodynamically and structurally binding of netropsin (Net), dibenzotetraaza[14]annulene derivatives (DP77, DP78), cationic porphyrin (TMPyP4) and two bisquinolini...

  16. 5'-UTR G-quadruplex structures acting as translational repressors. (United States)

    Beaudoin, Jean-Denis; Perreault, Jean-Pierre


    Given that greater than 90% of the human genome is expressed, it is logical to assume that post-transcriptional regulatory mechanisms must be the primary means of controlling the flow of information from mRNA to protein. This report describes a robust approach that includes in silico, in vitro and in cellulo experiments permitting an in-depth evaluation of the impact of G-quadruplexes as translational repressors. Sequences including potential G-quadruplexes were selected within nine distinct genes encoding proteins involved in various biological processes. Their abilities to fold into G-quadruplex structures in vitro were evaluated using circular dichroism, thermal denaturation and the novel use of in-line probing. Six sequences were observed to fold into G-quadruplex structures in vitro, all of which exhibited translational inhibition in cellulo when linked to a reporter gene. Sequence analysis, direct mutagenesis and subsequent experiments were performed in order to define the rules governing the folding of G-quadruplexes. In addition, the impact of single-nucleotide polymorphism was shown to be important in the formation of G-quadruplexes located within the 5'-untranslated region of an mRNA. In light of these results, clearly the 5'-UTR G-quadruplexes represent a class of translational repressors that is broadly distributed in the cell.

  17. Effect of ATRX and G-Quadruplex Formation by the VNTR Sequence on α-Globin Gene Expression. (United States)

    Li, Yue; Syed, Junetha; Suzuki, Yuki; Asamitsu, Sefan; Shioda, Norifumi; Wada, Takahito; Sugiyama, Hiroshi


    ATR-X (α-thalassemia/mental retardation X-linked) syndrome is caused by mutations in chromatin remodeler ATRX. ATRX can bind the variable number of tandem repeats (VNTR) sequence in the promoter region of the α-globin gene cluster. The VNTR sequence, which contains the potential G-quadruplex-forming sequence CGC(GGGGCGGGG)n , is involved in the downregulation of α-globin expression. We investigated G-quadruplex and i-motif formation in single-stranded DNA and long double-stranded DNA. The promoter region without the VNTR sequence showed approximately twofold higher luciferase activity than the promoter region harboring the VNTR sequence. G-quadruplex stabilizers hemin and TMPyP4 reduced the luciferase activity, whereas expression of ATRX led to a recovery in reporter activity. Our results demonstrate that stable G-quadruplex formation by the VNTR sequence downregulates the expression of α-globin genes and that ATRX might bind to and resolve the G-quadruplex. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. RecQ-core of BLM unfolds telomeric G-quadruplex in the absence of ATP

    Czech Academy of Sciences Publication Activity Database

    Budhathoki, J.B.; Ray, S.; Urban, Václav; Janščák, Pavel; Jodh, J.G.; Balci, H.


    Roč. 42, č. 18 (2014), s. 11528-11545 ISSN 0305-1048 R&D Projects: GA ČR GA204/09/0565 Grant - others:U.S. National Science Foundation through the Physics Frontiers Center Program(US) 1430124 Institutional support: RVO:68378050 Keywords : single - stranded DNA * RECQ5 helicase * G-quadruplex structures Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 9.112, year: 2014

  19. Conformation of DNA GG intrastrand cross-link of antitumor oxaliplatin and its enantiomeric analog

    Czech Academy of Sciences Publication Activity Database

    Malina, Jaroslav; Nováková, Olga; Vojtíšková, Marie; Natile, G.; Brabec, Viktor


    Roč. 93, č. 11 (2007), s. 3950-3962 ISSN 0006-3495 R&D Projects: GA ČR(CZ) GA305/05/2030; GA ČR(CZ) GA203/06/1239; GA AV ČR(CZ) 1QS500040581; GA AV ČR(CZ) KAN200200651; GA MŠk(CZ) LC06030; GA AV ČR(CZ) KJB400040601 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : DNA * oxaliplatina * cancer Subject RIV: BO - Biophysics Impact factor: 4.627, year: 2007

  20. Cellular Detection of G-Quadruplexes by Optical Imaging Methods. (United States)

    Amor, Souheila; Yang, Sunny Y; Wong, Judy M Y; Monchaud, David


    G-quadruplexes (G4s) are higher-order nucleic acid structures that fold from guanine (G)-rich DNA and RNA strands. This field of research gains traction as a major chemical biology area since it aims at uncovering many key cellular mechanisms in which quadruplexes are involved. The wealth of knowledge acquired over the past three decades strongly supports pivotal roles of G4 in the regulation of gene expression at both transcriptional (DNA quadruplexes) and translational levels (RNA quadruplexes). Recent biochemical discoveries uncovered myriad of additional G4 actions: from chromosomal stability to the firing of replication origins, from telomere homeostasis to functional dysregulations underlying genetic diseases (including cancers and neurodegeneration). Here, we listed a repertoire of protocols that we have developed over the past years to visualize quadruplexes in cells. These achievements were made possible thanks to the discovery of a novel family of versatile quadruplex-selective fluorophores, the twice-as-smart quadruplex ligands named TASQ (for template-assembled synthetic G-quartet). The versatility of this probe allows for multiple imaging techniques in both fixed and live cells, including the use of the multiphoton microscopy, confocal microscopy, and real-time fluorescent image collection. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  1. Raman spectroscopy of DNA modified by intrastrand cross-links of antitumor cisplatin

    Czech Academy of Sciences Publication Activity Database

    Vrána, Oldřich; Mašek, Vlastimil; Dražan, Viktor; Brabec, Viktor


    Roč. 159, č. 1 (2007), s. 1-8 ISSN 1047-8477 R&D Projects: GA ČR(CZ) GA203/05/2032; GA ČR(CZ) GD204/03/H016; GA ČR(CZ) GA305/05/2030; GA AV ČR(CZ) 1QS500040581; GA AV ČR(CZ) KAN200200651; GA MŠk(CZ) LC06030; GA MŠk(CZ) 1P05OC070; GA MŠk(CZ) 1P05OC072 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : DNA * platinum * cancer Subject RIV: BO - Biophysics Impact factor: 3.677, year: 2007

  2. G-Quadruplex Forming Oligonucleotides as Anti-HIV Agents. (United States)

    Musumeci, Domenica; Riccardi, Claudia; Montesarchio, Daniela


    Though a variety of different non-canonical nucleic acids conformations have been recognized, G-quadruplex structures are probably the structural motifs most commonly found within known oligonucleotide-based aptamers. This could be ascribed to several factors, as their large conformational diversity, marked responsiveness of their folding/unfolding processes to external stimuli, high structural compactness and chemo-enzymatic and thermodynamic stability. A number of G-quadruplex-forming oligonucleotides having relevant in vitro anti-HIV activity have been discovered in the last two decades through either SELEX or rational design approaches. Improved aptamers have been obtained by chemical modifications of natural oligonucleotides, as terminal conjugations with large hydrophobic groups, replacement of phosphodiester linkages with phosphorothioate bonds or other surrogates, insertion of base-modified monomers, etc. In turn, detailed structural studies have elucidated the peculiar architectures adopted by many G-quadruplex-based aptamers and provided insight into their mechanism of action. An overview of the state-of-the-art knowledge of the relevance of putative G-quadruplex forming sequences within the viral genome and of the most studied G-quadruplex-forming aptamers, selectively targeting HIV proteins, is here presented.

  3. A triple stranded G-quadruplex formation in the promoter region of human myosin β(Myh7) gene. (United States)

    Singh, Anju; Kukreti, Shrikant


    Regulatory regions in human genome, enriched in guanine-rich DNA sequences have the propensity to fold into G-quadruplex structures. On exploring the genome for search of G-tracts, it was interesting to find that promoter of Human Myosin Gene (MYH7) contains a conserved 23-mer G-rich sequence (HM-23). Mutations in this gene are associated with familial cardiomyopathy. Enrichment of MYH7 gene in G-rich sequences could possibly play a critical role in its regulation. We used polyacrylamide gel electrophoresis (PAGE), UV-Thermal denaturation (UV-Tm) and Circular Dichroism (CD), to demonstrate the formation of a G-quadruplex by 23-mer G-rich sequence HM23 in promoter location of MYH7 gene. We observed that the wild G-rich sequence HM23 containing consecutive G 5 stretch in two stacks adopt G-quadruplexes of diverse molecularity by involvement of four-strand, three-strand and two-strands with same parallel topology. Interestingly, the mutated sequence in the absence of continuous G 5 stretch obstructs the formation of three-stranded G-quadruplex. We demonstrated that continuous G 5 stretch is mandatory for the formation of a unique three-stranded G-quadruplex. Presence of various transcription factors (TF) in vicinity of the sequence HM23 leave fair possibility of recognition by TF binding sites, and so modulate gene expression. These findings may add on our understanding about the effect of base change in the formation of varied structural species in similar solution condition. This study may give insight about structural polymorphism arising due to recognition of non-Watson-Crick G-quadruplex structures by cellular proteins and designing structure specific molecules.

  4. A G-quadruplex-binding compound showing anti-tumour activity in an in vivo model for pancreatic cancer

    Czech Academy of Sciences Publication Activity Database

    Ohnmacht, S.A.; Marchetti, C.; Gunaratnam, M.; Besser, R.J.; Haider, S.M.; Di Vita, G.; Lowe, H.L.; Mellinas-Gomez, M.; Diocou, S.; Robson, M.; Šponer, Jiří


    Roč. 5, JUN 2015 (2015) ISSN 2045-2322 R&D Projects: GA ČR(CZ) GAP208/11/1822; GA MŠk(CZ) ED1.1.00/02.0068 Institutional support: RVO:68081707 Keywords : NAPHTHALENE DIIMIDE LIGANDS * AMBER FORCE-FIELD * DNA G-QUADRUPLEX Subject RIV: BO - Biophysics Impact factor: 5.228, year: 2015

  5. 1,10-Phenanthroline platinum(II) complex: a simple molecule for efficient G-quadruplex stabilization. (United States)

    Wang, Jin-Tao; Zheng, Xiao-Hui; Xia, Qing; Mao, Zong-Wan; Ji, Liang-Nian; Wang, Kui


    Two simple complexes [Pt(phen)(2)](PF(6))(2) and [Pt(bpy)(2)](PF(6))(2) exhibit high stabilization potential for G-quadruplex DNA with [Pt(phen)(2)](PF(6))(2) possessing higher capability due to its greater planarity.

  6. The parallel G-quadruplex structure of vertebrate telomeric repeat sequences is not the preferred folding topology under physiological conditions

    Czech Academy of Sciences Publication Activity Database

    Hansel, R.; Loehr, F.; Foldynová-Trantírková, Silvie; Bamberg, E.; Trantírek, L.; Doetsch, V.


    Roč. 39, č. 13 (2011), 5768-5775 ISSN 0305-1048 Institutional research plan: CEZ:AV0Z60220518 Keywords : MOLECULAR CROWDING CONDITIONS * INTRAMOLECULAR G-QUADRUPLEX * CELL NMR-SPECTROSCOPY * C- MYC PROMOTER * K+ SOLUTION * DNA * STABILITY * PROTEIN * POLYMORPHISM * HYDRATION Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.026, year: 2011

  7. Delineation of G-Quadruplex Alkylation Sites Mediated by 3,6-Bis(1-methyl-4-vinylpyridinium iodide)carbazole-Aniline Mustard Conjugates. (United States)

    Chen, Chien-Han; Hu, Tsung-Hao; Huang, Tzu-Chiao; Chen, Ying-Lan; Chen, Yet-Ran; Cheng, Chien-Chung; Chen, Chao-Tsen


    A new G-quadruplex (G-4)-directing alkylating agent BMVC-C3M was designed and synthesized to integrate 3,6-bis(1-methyl-4-vinylpyridinium iodide)carbazole (BMVC) with aniline mustard. Various telomeric G-4 structures (hybrid-2 type and antiparallel) and an oncogene promoter, c-MYC (parallel), were constructed to react with BMVC-C3M, yielding 35 % alkylation yield toward G-4 DNA over other DNA categories (alkylation adducts by electrospray ionization mass spectroscopy (ESI-MS) revealed the stepwise DNA alkylation mechanism of aniline mustard for the first time. Furthermore, the monoalkylation sites and intrastrand cross-linking sites were determined and found to be dependent on G-4 topology based on the results of footprinting analysis in combination with mass spectroscopic techniques and in silico modeling. The results indicated that BMVC-C3M preferentially alkylated at A15 (H26), G12 (H24), and G2 (c-MYC), respectively, as monoalkylated adducts and formed A15-C3M-A21 (H26), G12-C3M-G4 (H24), and G2-C3M-G4/G17 (c-MYC), respectively, as cross-linked dialkylated adducts. Collectively, the stability and site-selective cross-linking capacity of BMVC-C3M provides a credible tool for the structural and functional characterization of G-4 DNAs in biological systems. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. A G-quadruplex-based Label-free Fluorometric Aptasensor for Adenosine Triphosphate Detection. (United States)

    Li, Li Juan; Tian, Xue; Kong, Xiang Juan; Chu, Xia


    A G-quadruplex-based, label-free fluorescence assay was demonstrated for the detection of adenosine triphosphate (ATP). A double-stranded DNA (dsDNA), hybridized by ATP-aptamer and its complementary sequence, was employed as a substrate for ATP binding. SYBR Green I (SG I) was a fluorescent probe and exonuclease III (Exo III) was a nuclease to digest the dsDNA. Consequently, in the absence of ATP, the dsDNA was inset with SG I and was digested by Exo III, resulting in a low background signal. In the presence of ATP, the aptamer in dsDNA folded into a G-quadruplex structure that resisted the digestion of Exo III. SG I was inserted into the structure, showing high fluorescence. Owing to a decrease of the background noise, a high signal-to-noise ratio could be obtained. This sensor can detect ATP with a concentration ranging from 50 μM to 5 mM, and possesses a capacity for the sensitive determination of other targets.

  9. Modular Assembly of Cell-targeting Devices Based on an Uncommon G-quadruplex Aptamer

    DEFF Research Database (Denmark)

    Opazo, Felipe; Eiden, Laura; Hansen, Line


    Aptamers are valuable tools that provide great potential to develop cost-effective diagnostics and therapies in the biomedical field. Here, we report a novel DNA aptamer that folds into an unconventional G-quadruplex structure able to recognize and enter specifically into human Burkitt's lymphoma...... cells. We further optimized this aptamer to a highly versatile and stable minimized version. The minimized aptamer can be easily equipped with different functionalities like quantum dots, organic dyes, or even a second different aptamer domain yielding a bi-paratopic aptamer. Although the target...

  10. Transposable elements and G-quadruplexes

    Czech Academy of Sciences Publication Activity Database

    Kejnovský, Eduard; Tokan, Viktor; Lexa, M.


    Roč. 23, č. 3 (2015), s. 615-623 ISSN 0967-3849 R&D Projects: GA ČR(CZ) GA15-02891S Institutional support: RVO:68081707 Keywords : TRINUCLEOTIDE REPEAT DNA * LTR RETROTRANSPOSONS * BINDING PROTEIN Subject RIV: BO - Biophysics Impact factor: 2.590, year: 2015

  11. Quinone methides tethered to naphthalene diimides as selective G-quadruplex alkylating agents. (United States)

    Di Antonio, Marco; Doria, Filippo; Richter, Sara N; Bertipaglia, Carolina; Mella, Mariella; Sissi, Claudia; Palumbo, Manlio; Freccero, Mauro


    We have developed novel G-quadruplex (G-4) ligand/alkylating hybrid structures, tethering the naphthalene diimide moiety to quaternary ammonium salts of Mannich bases, as quinone-methide precursors, activatable by mild thermal digestion (40 degrees C). The bis-substituted naphthalene diimides were efficiently synthesized, and their reactivity as activatable bis-alkylating agents was investigated in the presence of thiols and amines in aqueous buffered solutions. The electrophilic intermediate, quinone-methide, involved in the alkylation process was trapped, in the presence of ethyl vinyl ether, in a hetero Diels-Alder [4 + 2] cycloaddition reaction, yielding a substituted 2-ethoxychroman. The DNA recognition and alkylation properties of these new derivatives were investigated by gel electrophoresis, circular dichroism, and enzymatic assays. The alkylation process occurred preferentially on the G-4 structure in comparison to other DNA conformations. By dissecting reversible recognition and alkylation events, we found that the reversible process is a prerequisite to DNA alkylation, which in turn reinforces the G-quadruplex structural rearrangement.

  12. G-quadruplex-based aptamers against protein targets in therapy and diagnostics. (United States)

    Platella, Chiara; Riccardi, Claudia; Montesarchio, Daniela; Roviello, Giovanni N; Musumeci, Domenica


    Nucleic acid aptamers are single-stranded DNA or RNA molecules identified to recognize with high affinity specific targets including proteins, small molecules, ions, whole cells and even entire organisms, such as viruses or bacteria. They can be identified from combinatorial libraries of DNA or RNA oligonucleotides by SELEX technology, an in vitro iterative selection procedure consisting of binding (capture), partitioning and amplification steps. Remarkably, many of the aptamers selected against biologically relevant protein targets are G-rich sequences that can fold into stable G-quadruplex (G4) structures. Aiming at disseminating novel inspiring ideas within the scientific community in the field of G4-structures, the emphasis of this review is placed on: 1) recent advancements in SELEX technology for the efficient and rapid identification of new candidate aptamers (introduction of microfluidic systems and next generation sequencing); 2) recurrence of G4 structures in aptamers selected by SELEX against biologically relevant protein targets; 3) discovery of several G4-forming motifs in important regulatory regions of the human or viral genome bound by endogenous proteins, which per se can result into potential aptamers; 4) an updated overview of G4-based aptamers with therapeutic potential and 5) a discussion on the most attractive G4-based aptamers for diagnostic applications. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. A novel G-quadruplex motif in the Human MET promoter region. (United States)

    Yan, Jing; Zhao, Deming; Dong, Liping; Pan, Shuang; Hao, Fengjin; Guan, Yifu


    It is known that the guanine-rich strands in proto-oncogene promoters can fold into G-quadruplex structures to regulate gene expression. An intramolecular parallel G-quadruplex has been identified in MET promoter. It acts as a repressor in regulating MET expression. However, the full guanine-rich region in MET promoter forms a hybrid parallel/antiparallel G-quadruplex structure under physiological conditions, which means there are some antiparallel and hybrid parallel/antiparallel G-quadruplex structures in this region. In the present study, our data indicate that g3-5 truncation adopts an intramolecular hybrid parallel/antiparallel G-quadruplex under physiological conditions in vitro The g3-5 G-quadruplex structure significantly stops polymerization by Klenow fragment in K + buffer. Furthermore, the results of circular dichroism (CD) spectra and polymerase stop assay directly demonstrate that the G-quadruplex structure in g3-5 fragment can be stabilized by the G-quadruplex ligand TMPyP4 (5,10,15,20-tetra-(N-methyl-4-pyridyl) porphine). But the dual luciferase assay indicates TMPyP4 has no effect on the formation of g3-5 G-quadruplex in HepG2 cells. The findings in the present study will enrich our understanding of the G-quadruplex formation in proto-oncogene promoters and the mechanisms of gene expression regulation. © 2017 The Author(s).

  14. A Split G-Quadruplex and Graphene Oxide-Based Low-Background Platform for Fluorescence Authentication of Pseudostellaria heterophylla

    Directory of Open Access Journals (Sweden)

    Zhenzhu Zheng


    Full Text Available A label-free split G-quadruplex and graphene oxide (GO-based fluorescence platform has been designed to distinguish Pseudostellaria heterophylla (PH from its adulterants based on the differences in their nrDNA ITS sequences. Herein, GO has been first introduced to capture G-rich probes with 2:2 split mode and then decrease the background signal. As T-DNA exists, the probes leave the GO surface to form double-stranded structures followed by the formation of the overhanging G-rich sequence into a G-quadruplex structure, which combines quinaldine red specifically to produce a strong fluorescence signal. In addition, this strategy allows detection of T-DNA in a wide range of concentrations from 1.0 × 10−8 to 2.0 × 10−6 mol·L−1 with a detection limit of 7.8 × 10−9 mol·L−1. We hope that the split G-quadruplex/GO platform can be utilized to further develop gene identification sensors in Traditional Chinese Medicine or other analysis areas.

  15. Thermal stabilisation of RNA·RNA duplexes and G-quadruplexes by phosphorothiolate linkages. (United States)

    Piperakis, Michael M; Gaynor, James W; Fisher, Julie; Cosstick, Richard


    The effect of 3'-S-phosphorothiolate linkages on the stability of RNA·RNA duplexes and G-quadruplex structures has been studied. 3'-Thio-2'-deoxyuridine was incorporated into RNA duplexes and thermal melting studies revealed that the resulting 3'-S-phosphorothiolate linkages increased the stability of the duplex to thermal denaturation. Additionally, and contrary to expectation, a similar effect on duplex stability was observed when the same thionucleoside was incorporated into the RNA strand of a RNA·DNA duplex. A suitably protected derivative of 3'-thio-2'-deoxyguanosine was prepared using an oxidation-reduction strategy and this residue also increased the thermal stability the [d(TGGGGT)](4) G-quadruplex when positioned centrally. The results are discussed in terms of the influence that the sulfur atom has on the conformation of the furanose ring and imply that the previously noted high thermal stability of parallel RNA quadruplexes is not derived from H-bonding interactions of the 2'-hydroxyl group, but can be attributed to conformational effects.

  16. Discovery of selective ligands for telomeric RNA G-quadruplexes (TERRA) through 19F-NMR based fragment screening. (United States)

    Garavís, Miguel; López-Méndez, Blanca; Somoza, Alvaro; Oyarzabal, Julen; Dalvit, Claudio; Villasante, Alfredo; Campos-Olivas, Ramón; González, Carlos


    Telomeric repeat-containing RNA (TERRA) is a novel and very attractive antitumoral target. Here, we report the first successful application of (19)F-NMR fragment-based screening to identify chemically diverse compounds that bind to an RNA molecule such as TERRA. We have built a library of 355 fluorinated fragments, and checked their interaction with a long telomeric RNA as a target molecule. The screening resulted in the identification of 20 hits (hit rate of 5.6%). For a number of binders, their interaction with TERRA was confirmed by (19)F- and (1)H NMR as well as by CD melting experiments. We have also explored the selectivity of the ligands for RNA G-quadruplexes and found that some of the hits do not interact with other nucleic acids such as tRNA and duplex DNA and, most importantly, favor the propeller-like parallel conformation in telomeric DNA G-quadruplexes. This suggests a selective recognition of this particular quadruplex topology and that different ligands may recognize specific sites in propeller-like parallel G-quadruplexes. Such features make some of the resulting binders promising lead compounds for fragment based drug discovery.

  17. Phenanthroline-2,9-bistriazoles as selective G-quadruplex ligands

    DEFF Research Database (Denmark)

    Nielsen, Mads Corvinius; Larsen, Anders Foller; Abdikadir, Faisal Hussein


    G-quadruplex (G4) ligands are currently receiving considerable attention as potential anticancer therapeutics. A series of phenanthroline-2,9-bistriazoles carrying tethered positive end groups has been synthesized and evaluated as G4 stabilizers. The compounds were efficiently assembled by copper......(I)-catalyzed azide-alkyne cycloaddition (CuAAC) in CH2Cl2 and water in the presence of a complexing agent. Characterization of the target compounds on telomeric and c-KIT G4 sequences led to the identification of guanidinium-substituted compounds as potent G4 DNA ligands with high selectivity over duplex DNA....... The diisopropylguanidium ligands exhibited high selectivity for the proto-oncogenic sequence c-KIT over the human telomeric sequence in the surface plasmon resonance (SPR) assay, whereas the compounds appeared potent on both G4 structures in the FRET melting temperature assay. The phenanthroline-2,9-bistriazole ligands...

  18. Assaying the binding strength of G-quadruplex ligands using single-molecule TPM experiments. (United States)

    Liu, Shih-Wei; Chu, Jen-Fei; Tsai, Cheng-Ting; Fang, Hung-Chih; Chang, Ta-Chau; Li, Hung-Wen


    G-quadruplexes are stable secondary structures formed by Hoogsteen base pairing of guanine-rich single-stranded DNA sequences in the presence of monovalent cations (Na(+) or K(+)). Folded G-quadruplex (G4) structures in human telomeres have been proposed as a potential target for cancer therapy. In this study, we used single-molecule tethered particle motion (TPM) experiments to assay the binding strength of possible G4 ligands. We found that individual single-stranded DNA molecules containing the human telomeric sequence d[AGGG(TTAGGG)3] fluctuated between the folded and the unfolded states in a 10 mM Na(+) solution at 37 °C. The durations of folded and unfolded states were single-exponentially distributed, and in return the folding and unfolding rate constants were 1.68 ± 0.01 and 1.63 ± 0.03 (s(-1)), respectively. In the presence of G4 ligands, such as TMPyP4, DODCI, BMVC, and BMVPA, the unfolding rate constant decreased appreciably. In addition, combining the Cu(2+)-induced G4 unfolding and TPM assay, we showed that BMVC and TMPyP4 are better G4 stabilizers than DODCI. The capability of monitoring the fluctuation between the folded and the unfolded state of G4 DNA in real time allows the determination of both kinetic and thermodynamic parameters in a single measurement and offers a simple way to assay binding strength under various conditions. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

  19. Tracking the Dynamic Folding and Unfolding of RNA G-Quadruplexes in Live Cells. (United States)

    Chen, Xiu-Cai; Chen, Shuo-Bin; Dai, Jing; Yuan, Jia-Hao; Ou, Tian-Miao; Huang, Zhi-Shu; Tan, Jia-Heng


    Because of the absence of methods for tracking RNA G-quadruplex dynamics, especially the folding and unfolding of this attractive structure in live cells, understanding of the biological roles of RNA G-quadruplexes is so far limited. Herein, we report a new red-emitting fluorescent probe, QUMA-1, for the selective, continuous, and real-time visualization of RNA G-quadruplexes in live cells. The applications of QUMA-1 in several previously intractable applications, including live-cell imaging of the dynamic folding, unfolding, and movement of RNA G-quadruplexes and the visualization of the unwinding of RNA G-quadruplexes by RNA helicase have been demonstrated. Notably, our real-time results revealed the complexity of the dynamics of RNA G-quadruplexes in live cells. We anticipate that the further application of QUMA-1 in combination with appropriate biological and imaging methods to explore the dynamics of RNA G-quadruplexes will uncover more information about the biological roles of RNA G-quadruplexes. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Tetrasubstituted phenanthrolines as highly potent, water-soluble, and selective g-quadruplex ligands

    DEFF Research Database (Denmark)

    Larsen, Anders Foller; Nielsen, Mads Corvinius; Ulven, Trond


    Small molecules capable of stabilizing the G-quadruplex (G4) structure are of interest for the development of improved anticancer drugs. Novel 4,7-diamino-substituted 1,10-phenanthroline-2,9-dicarboxamides that represent hybrid structures of known phenanthroline-based ligands have been designed....... An efficient synthetic route to the compounds has been developed and their interactions with various G4 sequences have been evaluated by Förster resonance energy transfer (FRET) melting assays, fluorescent intercalator displacement (FID), electrospray ionization mass spectrometry (ESI-MS), and circular...... dichroism (CD) spectroscopy. The preferred compounds have high aqueous solubility and are strong and potent G4 binders with a high selectivity over duplex DNA; thus, they represent a significant improvement over the lead compounds. Two of the compounds are inhibitors of HeLa and HT1080 cell proliferation....

  1. Colorimetric detection of genetically modified organisms based on exonuclease III-assisted target recycling and hemin/G-quadruplex DNAzyme amplification. (United States)

    Zhang, Decai; Wang, Weijia; Dong, Qian; Huang, Yunxiu; Wen, Dongmei; Mu, Yuejing; Yuan, Yong


    An isothermal colorimetric method is described for amplified detection of the CaMV 35S promoter sequence in genetically modified organism (GMO). It is based on (a) target DNA-triggered unlabeled molecular beacon (UMB) termini binding, and (b) exonuclease III (Exo III)-assisted target recycling, and (c) hemin/G-quadruplex (DNAzyme) based signal amplification. The specific binding of target to the G-quadruplex sequence-locked UMB triggers the digestion of Exo III. This, in turn, releases an active G-quadruplex segment and target DNA for successive hybridization and cleavage. The Exo III impellent recycling of targets produces numerous G-quadruplex sequences. These further associate with hemin to form DNAzymes and hence will catalyze H 2 O 2 -mediated oxidation of the chromogenic enzyme substrate ABTS 2- causing the formation of a green colored product. This finding enables a sensitive colorimetric determination of GMO DNA (at an analytical wavelength of 420 nm) at concentrations as low as 0.23 nM. By taking advantage of isothermal incubation, this method does not require sophisticated equipment or complicated syntheses. Analyses can be performed within 90 min. The method also discriminates single base mismatches. In our perception, it has a wide scope in that it may be applied to the detection of many other GMOs. Graphical abstract An isothermal and sensitive colorimetric method is described for amplified detection of CaMV 35S promoter sequence in genetically modified organism (GMO). It is based on target DNA-triggered molecular beacon (UMB) termini-binding and exonuclease III assisted target recycling, and on hemin/G-quadruplex (DNAzyme) signal amplification.

  2. Use of alternative alkali chlorides in RT and PCR of polynucleotides containing G quadruplex structures. (United States)

    Ramos-Alemán, Fabiola; González-Jasso, Eva; Pless, Reynaldo C


    Several alkali chlorides were compared for their use in reverse transcription (RT) and PCR of different types of nucleic acid templates. On a test region of biological DNA incapable of forming G quadruplex (G4) structures, Taq DNA polymerase showed similar PCR performance with 50 mM KCl, CsCl, LiCl, and NaCl. In contrast, on a synthetic model polydeoxyribonucleotide prone to G4 formation, good PCR amplification was obtained with 50 mM CsCl, but little or none with LiCl or KCl. Similarly, in RT of a G4-prone model polyribonucleotide, MMLV reverse transcriptase produced a good yield with 50 mM CsCl, mediocre yields with LiCl or without added alkali chloride, and a poor yield with 50 mM KCl. The full RT-PCR assay starting from the G4-prone polyribonucleotide, showed good results with CsCl in both stages, poor results with LiCl, and no product formation with KCl. The model polynucleotides showed fast G quadruplex formation under PCR or RT conditions with 50 mM KCl, but not with CsCl or LiCl. The results argue for the use of CsCl instead of KCl for RT and PCR of G4-prone sequences. No advantage was observed when using the 7-deaza type nucleotide analog c 7 dGTP in PCR amplification of the G4-prone polydeoxyribonucleotide. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Symmetric cyanovinyl-pyridinium triphenylamine: a novel fluorescent switch-on probe for an antiparallel G-quadruplex. (United States)

    Lai, Han; Lai, Hai; Xiao, Yijie; Yan, Shengyong; Tian, Fangfang; Zhong, Cheng; Liu, Yi; Weng, Xiaocheng; Zhou, Xiang


    In this study, we present a fluorescent switch-on probe based on a cyanovinyl-pyridinium triphenylamine (CPT) derivative that exhibited a 190-fold increase in fluorescence upon binding to G-quadruplex-forming oligonucleotide 22AG. This probe showed specificity and selectivity towards an antiparallel G-quadruplex, indicating its promising potential in G-quadruplex imaging.

  4. Interaction of Pyrrolobenzodiazepine (PBD) Ligands with Parallel Intermolecular G-Quadruplex Complex Using Spectroscopy and ESI-MS (United States)

    Raju, Gajjela; Srinivas, Ragampeta; Santhosh Reddy, Vangala; Idris, Mohammed M.; Kamal, Ahmed; Nagesh, Narayana


    Studies on ligand interaction with quadruplex DNA, and their role in stabilizing the complex at concentration prevailing under physiological condition, has attained high interest. Electrospray ionization mass spectrometry (ESI-MS) and spectroscopic studies in solution were used to evaluate the interaction of PBD and TMPyP4 ligands, stoichiometry and selectivity to G-quadruplex DNA. Two synthetic ligands from PBD family, namely pyrene-linked pyrrolo[2,1-c][1,4]benzodiazepine hybrid (PBD1), mixed imine-amide pyrrolobenzodiazepine dimer (PBD2) and 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4) were studied. G-rich single-stranded oligonucleotide d(5′GGGGTTGGGG3′) designated as d(T2G8), from the telomeric region of Tetrahymena Glaucoma, was considered for the interaction with ligands. ESI-MS and spectroscopic methods viz., circular dichroism (CD), UV-Visible, and fluorescence were employed to investigate the G-quadruplex structures formed by d(T2G8) sequence and its interaction with PBD and TMPyP4 ligands. From ESI-MS spectra, it is evident that the majority of quadruplexes exist as d(T2G8)2 and d(T2G8)4 forms possessing two to ten cations in the centre, thereby stabilizing the complex. CD band of PBD1 and PBD2 showed hypo and hyperchromicity, on interaction with quadruplex DNA, indicating unfolding and stabilization of quadruplex DNA complex, respectively. UV-Visible and fluorescence experiments suggest that PBD1 bind externally where as PBD2 intercalate moderately and bind externally to G-quadruplex DNA. Further, melting experiments using SYBR Green indicate that PBD1 unfolds and PBD2 stabilizes the G-quadruplex complex. ITC experiments using d(T2G8) quadruplex with PBD ligands reveal that PBD1 and PBD2 prefer external/loop binding and external/intercalative binding to quadruplex DNA, respectively. From experimental results it is clear that the interaction of PBD2 and TMPyP4 impart higher stability to the quadruplex complex. PMID:22558271

  5. Morphological effects of G-quadruplex stabilization using a small molecule in zebrafish. (United States)

    Agarwal, Tani; Lalwani, Mukesh Kumar; Kumar, Santosh; Roy, Saumya; Chakraborty, Tushar Kanti; Sivasubbu, Sridhar; Maiti, Souvik


    Zebrafish (Danio rerio) embryos are transparent and advantageous for studying early developmental changes due to ex utero development, making them an appropriate model for studying gene expression changes as a result of molecular targeting. Zebrafish embryos were injected with a previously reported G-quadruplex selective ligand, and the phenotypic changes were recorded. We report marked discrepancies in the development of intersegmental vessels. In silico analysis determined that the putative G-quadruplex motif occur in the upstream promoter region of the Cdh5 (N-cadherin) gene. A real-time polymerase chain reaction-based investigation indicated that in zebrafish, CDH-2 (ZN-cad) was significantly downregulated in the ligand-treated embryos. Biophysical characterization of the interaction of the ligand with the G-quadruplex motif found in this promoter yielded strong binding and stabilization of the G-quadruplex with this ligand. Hence, we report for the first time the phenotypic impact of G-quadruplex targeting with a ligand in a vertebrate organism. This study has unveiled not only G-quadruplex targeting in non-human animal species but also the potential that G-quadruplexes can provide a ready tool for understanding the phenotypic effects of targeting certain important genes involved in differentiation and developmental processes in a living eukaryotic organism.

  6. Separation of the potential G-quadruplex ligands from the butanol extract of Zanthoxylum ailanthoides Sieb. & Zucc. by countercurrent chromatography and preparative high performance liquid chromatography. (United States)

    Han, Tian; Cao, Xueli; Xu, Jing; Pei, Hairun; Zhang, Hong; Tang, Yalin


    G-quadruplex DNA structure is considered to be a very attractive target for antitumor drug design due to its unique role in maintaining telomerase activities. Therefore, discovering ligands with high stability of G-quadruplex structure is of great interest. In this paper, pH-zone refining counter current chromatography (CCC) and preparative high performance liquid chromatography (HPLC) were employed for the separation of potent G-quadruplex ligands from the n-butanol fraction of the crude extract of Zanthoxylum ailanthoides, which is a traditional Chinese medicine recently found to display high inhibitory activity against several human cancer cells. The 75% aqueous ethanol extract of the stem bark of Z. ailanthoides and its fractions with petroleum ether, ethyl acetate and n-butanol displayed almost the same G-quadruplex stabilization ability. Here, pH-zone refining CCC was used for the separation of the alkaloids from the n-butanol fraction by a seldom used solvent system composed of dichloromethane-methanol-water (4:1:2.5) with 10mM TEA in the organic stationary phase as retainer and 10mM HCl in the aqueous mobile phase as eluter. Compounds I, II and III were obtained, with purity greater than 95%, in the quantities of 31.2, 94.0, and 26.4mg respectively from 300mg of lipophilic fraction within 80min, which were identified as three tetrahydroprotoberberines isolated for the first time in this plant. In addition, a phenylpropanoid glycoside compound IV (Syringin), an isoquinoline (Magnoflorine, V), and two lignin isomers (+)-lyoniresiol-3α-O-β-d-glucopyranoside (VI) and (-)-lyoniresinol -3α-O-β-D -glucopyranoside (VII) were isolated by traditional CCC together with preparative HPLC. Compounds IV, V, VI and VII were obtained, with purity greater than 95%, in the quantities of 4.0, 13.2, 6.7, and 6.5mg respectively from 960mg of hydrophilic fraction. Among the seven isolated compounds, tetrahydroprotoberberine I, II and III were found to display remarkable

  7. Thermodynamic fingerprints of ligand binding to human telomeric G-quadruplexes. (United States)

    Bončina, Matjaž; Podlipnik, Črtomir; Piantanida, Ivo; Eilmes, Julita; Teulade-Fichou, Marie-Paule; Vesnaver, Gorazd; Lah, Jurij


    Thermodynamic studies of ligand binding to human telomere (ht) DNA quadruplexes, as a rule, neglect the involvement of various ht-DNA conformations in the binding process. Therefore, the thermodynamic driving forces and the mechanisms of ht-DNA G-quadruplex-ligand recognition remain poorly understood. In this work we characterize thermodynamically and structurally binding of netropsin (Net), dibenzotetraaza[14]annulene derivatives (DP77, DP78), cationic porphyrin (TMPyP4) and two bisquinolinium ligands (Phen-DC3, 360A-Br) to the ht-DNA fragment (Tel22) AGGG(TTAGGG)3 using isothermal titration calorimetry, CD and fluorescence spectroscopy, gel electrophoresis and molecular modeling. By global thermodynamic analysis of experimental data we show that the driving forces characterized by contributions of specific interactions, changes in solvation and conformation differ significantly for binding of ligands with low quadruplex selectivity over duplexes (Net, DP77, DP78, TMPyP4; KTel22 ≈ KdsDNA). These contributions are in accordance with the observed structural features (changes) and suggest that upon binding Net, DP77, DP78 and TMPyP4 select hybrid-1 and/or hybrid-2 conformation while Phen-DC3 and 360A-Br induce the transition of hybrid-1 and hybrid-2 to the structure with characteristics of antiparallel or hybrid-3 type conformation. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  8. G-quadruplexes in pathogens: a common route to virulence control?

    Directory of Open Access Journals (Sweden)

    Lynne M Harris


    Full Text Available DNA can form several secondary structures besides the classic double helix: one that has received much attention in recent years is the G-quadruplex (G4. This is a stable four-stranded structure formed by the stacking of quartets of guanine bases. Recent work has convincingly shown that G4s can form in vivo as well as in vitro and can affect both replication and transcription of DNA. They also play important roles at G-rich telomeres. Now, a spate of exciting reports has begun to reveal roles for G4 structures in virulence processes in several important microbial pathogens of humans. Interestingly, these come from a range of kingdoms--bacteria and protozoa as well as viruses--and all facilitate immune evasion in different ways. In particular, roles for G4s have been posited in the antigenic variation systems of bacteria and protozoa, as well as in the silencing of at least two major human viruses, human immunodeficiency virus (HIV and Epstein-Barr virus (EBV. Although antigenic variation and the silencing of latent viruses are quite distinct from one another, both are routes to immune evasion and the maintenance of chronic infections. Thus, highly disparate pathogens can use G4 motifs to control DNA/RNA dynamics in ways that are relevant to common virulence phenotypes. This review explores the evidence for G4 biology in such processes across a range of important human pathogens.

  9. Characterization of a DNA damage-recognition protein mammalian cells that binds specifically to intrastrand d(GpG) and d(ApG) DNA adducts of the anticancer drug cisplatin

    International Nuclear Information System (INIS)

    Donahue, B.A.; Augot, M.; Bellon, S.F.; Treiber, D.K.; Toney, J.H.; Lippard, S.J.; Essigmann, J.M.


    A factor has been identified in extracts from human HeLa and hamster V79 cells that retards the electrophoretic mobility of several DNA restriction fragments modified with the antitumor drug cis-diamminedichloroplatinum(II) (cisplatin). Binding of the factor to cisplatin-modified DNA was sensitive to pretreatment with proteinase K, establishing that the factor is a protein. Gel mobility shifts were observed with probes containing as few as seven Pt atoms per kilobase of duplex DNA. By competition experiments the dissociation constant, K d , of the protein from cisplatin-modified DNA was estimated to be (1-20) x 10 -10 M. Protein binding is selective for DNA modified with cisplatin, [Pt(en)Cl 2 ] (en, ethylenediamine), and [Pt(dach)Cl 2 ] (dach, 1,2-diaminocyclohexane) but not with chemotherapeutically inactive trans-diamminedichloroplatinum(II) or monofunctionally coordinating [Pt(dien)Cl]Cl (dien, diethylenetriamine) complexes. The protein binds specifically to 1,2-intrastrand d(GpG) and d(ApG) cross-links formed by cisplatin. The apparent molecular weight of the protein is 91,000, as determined by sucrose gradient centrifugation of a preparation partially purified by ammonium sulfate fractionation. Binding of the protein to platinum-modified DNA does not require cofactors but is sensitive to treatment with 5 mM MnCl 2 , CdCl 2 , CoCl 2 , or ZnCl 2 and with 1 mM HgCl 2 . This protein, alone or in conjunction with other cellular constituents, could be of general importance in the initial stages of processing of mammalian DNA damaged by cisplatin or other genotoxic agents and may belong to a wider class of such cellular damage-recognition proteins (DRPs)

  10. Expression of Telomere-Associated Proteins is Interdependent to Stabilize Native Telomere Structure and Telomere Dysfunction by G-Quadruplex Ligand Causes TERRA Upregulation. (United States)

    Sadhukhan, Ratan; Chowdhury, Priyanka; Ghosh, Sourav; Ghosh, Utpal


    Telomere DNA can form specialized nucleoprotein structure with telomere-associated proteins to hide free DNA ends or G-quadruplex structures under certain conditions especially in presence of G-quadruplex ligand. Telomere DNA is transcribed to form non-coding telomere repeat-containing RNA (TERRA) whose biogenesis and function is poorly understood. Our aim was to find the role of telomere-associated proteins and telomere structures in TERRA transcription. We silenced four [two shelterin (TRF1, TRF2) and two non-shelterin (PARP-1, SLX4)] telomere-associated genes using siRNA and verified depletion in protein level. Knocking down of one gene modulated expression of other telomere-associated genes and increased TERRA from 10q, 15q, XpYp and XqYq chromosomes in A549 cells. Telomere was destabilized or damaged by G-quadruplex ligand pyridostatin (PDS) and bleomycin. Telomere dysfunction-induced foci (TIFs) were observed for each case of depletion of proteins, treatment with PDS or bleomycin. TERRA level was elevated by PDS and bleomycin treatment alone or in combination with depletion of telomere-associated proteins.

  11. Modular Assembly of Cell-targeting Devices Based on an Uncommon G-quadruplex Aptamer

    Directory of Open Access Journals (Sweden)

    Felipe Opazo


    Full Text Available Aptamers are valuable tools that provide great potential to develop cost-effective diagnostics and therapies in the biomedical field. Here, we report a novel DNA aptamer that folds into an unconventional G-quadruplex structure able to recognize and enter specifically into human Burkitt's lymphoma cells. We further optimized this aptamer to a highly versatile and stable minimized version. The minimized aptamer can be easily equipped with different functionalities like quantum dots, organic dyes, or even a second different aptamer domain yielding a bi-paratopic aptamer. Although the target molecule of the aptamer remains unknown, our microscopy and pharmacological studies revealed that the aptamer hijacks the clathrin-mediated endocytosis pathway for its cellular internalization. We conclude that this novel class of aptamers can be used as a modular tool to specifically deliver different cargoes into malignant cells. This work provides a thorough characterization of the aptamer and we expect that our strategy will pave the path for future therapeutic applications.

  12. Exonuclease-assisted multicolor aptasensor for visual detection of ochratoxin A based on G-quadruplex-hemin DNAzyme-mediated etching of gold nanorod. (United States)

    Yu, Xinhui; Lin, Yaohui; Wang, Xusheng; Xu, Liangjun; Wang, Zongwen; Fu, FengFu


    An exonuclease-assisted multicolor aptasensor was developed for the visual detection of ochratoxin A (OTA). It is based on the etching of gold nanorods (AuNRs) mediated by a G-quadruplex-hemin DNAzyme. A DNA sequence (AG4-OTA) was designed that comprises a hemin aptamer and an OTA aptamer. OTA binds to AG4-OTA to form an antiparallel G-quadruplex, which halts its digestion by exonuclease I (Exo I) from the 3'-end of AG4-OTA. Thus, the retained hemin aptamer can bind to hemin to form a G-quadruplex-hemin DNAzyme. This DNAzyme has peroxidase-like activity that catalyzes the oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) by H 2 O 2 to produce its diimine derivative (TMB 2+ ) in acidic solution. TMB 2+ can etch the AuNRs by oxidizing Au(0) into Au(I). This results in the generation of rainbow-like colors and provides a multicolor platform for the visual detection of OTA. The assay is based on the use of a single isolated aptamer and possesses obvious advantages such as multi-color visual inspection, relatively high sensitivity and accuracy. It can be used to detect as little as 30 nM concentrations of OTA by visual observation and even 10 nM concentrations by spectrophotometry. The method was successfully applied to the determination of OTA in spiked beer where it gave recoveries of 101-108%, with a relative standard deviation (RSD, n = 5) of <5%. Graphical abstract Schematic of an exonuclease-assisted multicolor bioassay based on the G-quadruplex-hemin DNAzyme-mediated etching of gold nanorods (AuNRs). It enables visual detection of ochratoxin A (OTA) with a detection limit of 30 nM.

  13. G-quadruplex RNA binding and recognition by the lysine-specific histone demethylase-1 enzyme. (United States)

    Hirschi, Alexander; Martin, William J; Luka, Zigmund; Loukachevitch, Lioudmila V; Reiter, Nicholas J


    Lysine-specific histone demethylase 1 (LSD1) is an essential epigenetic regulator in metazoans and requires the co-repressor element-1 silencing transcription factor (CoREST) to efficiently catalyze the removal of mono- and dimethyl functional groups from histone 3 at lysine positions 4 and 9 (H3K4/9). LSD1 interacts with over 60 regulatory proteins and also associates with lncRNAs (TERRA, HOTAIR), suggesting a regulatory role for RNA in LSD1 function. We report that a stacked, intramolecular G-quadruplex (GQ) forming TERRA RNA (GG[UUAGGG]8UUA) binds tightly to the functional LSD1-CoREST complex (Kd ≈ 96 nM), in contrast to a single GQ RNA unit ([UUAGGG]4U), a GQ DNA ([TTAGGG]4T), or an unstructured single-stranded RNA. Stabilization of a parallel-stranded GQ RNA structure by monovalent potassium ions (K(+)) is required for high affinity binding to the LSD1-CoREST complex. These data indicate that LSD1 can distinguish between RNA and DNA as well as structured versus unstructured nucleotide motifs. Further, cross-linking mass spectrometry identified the primary location of GQ RNA binding within the SWIRM/amine oxidase domain (AOD) of LSD1. An ssRNA binding region adjacent to this GQ binding site was also identified via X-ray crystallography. This RNA binding interface is consistent with kinetic assays, demonstrating that a GQ-forming RNA can serve as a noncompetitive inhibitor of LSD1-catalyzed demethylation. The identification of a GQ RNA binding site coupled with kinetic data suggests that structured RNAs can function as regulatory molecules in LSD1-mediated mechanisms. © 2016 Hirschi et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  14. Exploration of G-quadruplex function in c-Myb gene and its transcriptional regulation by topotecan. (United States)

    Li, Fangyuan; Zhou, Jiang; Xu, Ming; Yuan, Gu


    Our bioinformatics research shows that there are four G-rich sequences (S1-S4) in the upstream region of the transcription start site of c-Myb gene, and we have proved that these sequences have the ability to form G-quadruplex structures. This work mainly focuses on G-quadruplex function, recognition and transcription regulation in c-Myb gene, revealing a novel regulatory element in c-Myb proximal promoter region, and its transcription regulation by G-quadruplex binder. The research has identified that the enhancer effect in c-Myb transcription was primarily affected by the G-quadruplex formed by S1 sequence, and the up-regulation effect may due to the removal of repressive progress of MZF-1 by stabilizing G-quadruplex. Attentions were being paid to the development of G-quadruplex binders for selective recognition, and topotecan was found to have high binding affinity in vitro and could effectively affect the c-Myb transcription activities in cells. The regulation of G-quadruplex with binders in transcriptional, translational levels by Q-RT-PCR and western blot was in expectation of providing a strategy for gene expression modulation. In conclusion, our study revealed a G-quadruplex structure in c-Myb proximal promoter region, which was of great importance in the regulation of c-Myb function. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Sequence requirements and GTP-dependent formation of multimeric G-quadruplexes

    Czech Academy of Sciences Publication Activity Database

    Kolesnikova, Sofia; Hubálek, Martin; Bednárová, Lucie; Cvačka, Josef; Curtis, Edward A.


    Roč. 15, č. 1 (2017), s. 15-16 ISSN 2336-7202. [Mezioborové setkání mladých biologů, biochemiků a chemiků /17./. 30.05.2017-01.06.2017, Milovy] Institutional support: RVO:61388963 Keywords : G-quadruplex * GTP-dependent formation Subject RIV: CE - Biochemistry

  16. Structure of human telomeric RNA (TERRA): stacking of two G-quadruplex blocks in K(+) solution. (United States)

    Martadinata, Herry; Phan, Anh Tuân


    Telomeric repeat-containing RNAs (TERRA) are transcription products of the telomeres. Human TERRA sequences containing UUAGGG repeats can form parallel-stranded G-quadruplexes. The stacking interaction of such structures was shown to be important for ligand targeting and higher-order arrangement of G-quadruplexes in long TERRA sequences. Here we report on the first high-resolution structure of a stacked G-quadruplex formed by the 10-nucleotide human TERRA sequence r(GGGUUAGGGU) in potassium solution. This structure comprises two dimeric three-layer parallel-stranded G-quadruplex blocks, which stack on each other at their 5'-ends. The adenine in each UUA loop is nearly coplanar with the 5'-end G-tetrad forming an A·(G·G·G·G)·A hexad, thereby increasing the stacking contacts between the two blocks. Interestingly, this stacking and loop conformation is different from all structures previously reported for the free human TERRA but resembles the structure previously determined for a complex between a human TERRA sequence and an acridine ligand. This stacking conformation is a potential target for drugs that recognize or induce the stacking interface.

  17. Thioflavin T binds dimeric parallel-stranded GA-containing non-G-quadruplex DNAs: a general approach to lighting up double-stranded scaffolds. (United States)

    Liu, Shuangna; Peng, Pai; Wang, Huihui; Shi, Lili; Li, Tao


    A molecular rotor thioflavin T (ThT) is usually used as a fluorescent ligand specific for G-quadruplexes. Here, we demonstrate that ThT can tightly bind non-G-quadruplex DNAs with several GA motifs and dimerize them in a parallel double-stranded mode, accompanied by over 100-fold enhancement in the fluorescence emission of ThT. The introduction of reverse Watson-Crick T-A base pairs into these dimeric parallel-stranded DNA systems remarkably favors the binding of ThT into the pocket between G•G and A•A base pairs, where ThT is encapsulated thereby restricting its two rotary aromatic rings in the excited state. A similar mechanism is also demonstrated in antiparallel DNA duplexes where several motifs of two consecutive G•G wobble base pairs are incorporated and serve as the active pockets for ThT binding. The insight into the interactions of ThT with non-G-quadruplex DNAs allows us to introduce a new concept for constructing DNA-based sensors and devices. As proof-of-concept experiments, we design a DNA triplex containing GA motifs in its Hoogsteen hydrogen-bonded two parallel strands as a pH-driven nanoswitch and two GA-containing parallel duplexes as novel metal sensing platforms where C-C and T-T mismatches are included. This work may find further applications in biological systems (e.g. disease gene detection) where parallel duplex or triplex stretches are involved. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Tandem application of ligand-based virtual screening and G4-OAS assay to identify novel G-quadruplex-targeting chemotypes. (United States)

    Musumeci, Domenica; Amato, Jussara; Zizza, Pasquale; Platella, Chiara; Cosconati, Sandro; Cingolani, Chiara; Biroccio, Annamaria; Novellino, Ettore; Randazzo, Antonio; Giancola, Concetta; Pagano, Bruno; Montesarchio, Daniela


    G-quadruplex (G4) structures are key elements in the regulation of cancer cell proliferation and their targeting is deemed to be a promising strategy in anticancer therapy. A tandem application of ligand-based virtual screening (VS) calculations together with the experimental G-quadruplex on Oligo Affinity Support (G4-OAS) assay was employed to discover novel G4-targeting compounds. The interaction of the selected compounds with the investigated G4 in solution was analysed through a series of biophysical techniques and their biological activity investigated by immunofluorescence and MTT assays. A focused library of 60 small molecules, designed as putative G4 groove binders, was identified through the VS. The G4-OAS experimental screening led to the selection of 7 ligands effectively interacting with the G4-forming human telomeric DNA. Evaluation of the biological activity of the selected compounds showed that 3 ligands of this sub-library induced a marked telomere-localized DNA damage response in human tumour cells. The combined application of virtual and experimental screening tools proved to be a successful strategy to identify new bioactive chemotypes able to target the telomeric G4 DNA. These compounds may represent useful leads for the development of more potent and selective G4 ligands. Expanding the repertoire of the available G4-targeting chemotypes with improved physico-chemical features, in particular aiming at the discovery of novel, selective G4 telomeric ligands, can help in developing effective anti-cancer drugs with fewer side effects. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Probe development for detection of TERRA 1 intramolecular G-quadruplex formation using a fluorescent adenosine derivative. (United States)

    Kim, In Sun; Seo, Young Jun


    We developed a probing system to detect the intramolecular G-quadruplex of telomeric repeat-containing RNA (TERRA 1). We used a fluorescent adenosine derivative rA(py) as a fluorophore and incorporated it into the dangling position of the parallel-type G-quadruplex sequence of TERRA 1. The rA(py)-modified G-quadruplex structure exhibited a strong fluorescence emission signal, while the emission signals of the single-strand and duplex structures were much lower. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. p53 binds human telomeric G-quadruplex in vitro

    Czech Academy of Sciences Publication Activity Database

    Adámik, Matěj; Kejnovská, Iva; Bažantová, Pavla; Petr, Marek; Renčiuk, Daniel; Vorlíčková, Michaela; Brázdová, Marie


    Roč. 128, SEPT2016 (2016), s. 83-91 ISSN 0300-9084 R&D Projects: GA ČR GA13-36108S; GA ČR(CZ) GP14-33947P Institutional support: RVO:68081707 Keywords : crystal-structure * human-chromosomes * supercoiled dna Subject RIV: BO - Biophysics Impact factor: 3.112, year: 2016

  1. Sugar-modified G-quadruplexes: effects of LNA-, 2′F-RNA– and 2′F-ANA-guanosine chemistries on G-quadruplex structure and stability (United States)

    Li, Zhe; Lech, Christopher Jacques; Phan, Anh Tuân


    G-quadruplex-forming oligonucleotides containing modified nucleotide chemistries have demonstrated promising pharmaceutical potential. In this work, we systematically investigate the effects of sugar-modified guanosines on the structure and stability of a (4+0) parallel and a (3+1) hybrid G-quadruplex using over 60 modified sequences containing a single-position substitution of 2′-O-4′-C-methylene-guanosine (LNAG), 2′-deoxy-2′-fluoro-riboguanosine (FG) or 2′-deoxy-2′-fluoro-arabinoguanosine (FANAG). Our results are summarized in two parts: (I) Generally, LNAG substitutions into ‘anti’ position guanines within a guanine-tetrad lead to a more stable G-quadruplex, while substitutions into ‘syn’ positions disrupt the native G-quadruplex conformation. However, some interesting exceptions to this trend are observed. We discover that a LNAG modification upstream of a short propeller loop hinders G-quadruplex formation. (II) A single substitution of either FG or FANAG into a ‘syn’ position is powerful enough to perturb the (3+1) G-quadruplex. Substitution of either FG or FANAG into any ‘anti’ position is well tolerated in the two G-quadruplex scaffolds. FANAG substitutions to ‘anti’ positions are better tolerated than their FG counterparts. In both scaffolds, FANAG substitutions to the central tetrad layer are observed to be the most stabilizing. The observations reported herein on the effects of LNAG, FG and FANAG modifications on G-quadruplex structure and stability will enable the future design of pharmaceutically relevant oligonucleotides. PMID:24371274

  2. Effect of Monovalent Ion Parameters on Molecular Dynamics Simulations of G-Quadruplexes

    Czech Academy of Sciences Publication Activity Database

    Havrila, Marek; Stadlbauer, Petr; Islam, Barira; Otyepka, M.; Šponer, Jiří


    Roč. 13, č. 8 (2017), s. 3911-3926 ISSN 1549-9618 R&D Projects: GA MŠk EF15_003/0000477; GA ČR(CZ) GA16-13721S Institutional support: RVO:68081707 Keywords : telomeric g-quadruplex * amber force-field * nucleic-acid quadruplexes Subject RIV: CF - Physical ; Theoretical Chemistry OBOR OECD: Physical chemistry Impact factor: 5.245, year: 2016

  3. A mRNA-Responsive G-Quadruplex-Based Drug Release System

    Directory of Open Access Journals (Sweden)

    Hidenobu Yaku


    Full Text Available G-quadruplex-based drug delivery carriers (GDDCs were designed to capture and release a telomerase inhibitor in response to a target mRNA. Hybridization between a loop on the GDDC structure and the mRNA should cause the G-quadruplex structure of the GDDC to unfold and release the bound inhibitor, anionic copper(II phthalocyanine (CuAPC. As a proof of concept, GDDCs were designed with a 10-30-mer loop, which can hybridize with a target sequence in epidermal growth factor receptor (EGFR mRNA. Structural analysis using circular dichroism (CD spectroscopy showed that the GDDCs form a (3 + 1 type G-quadruplex structure in 100 mM KCl and 10 mM MgCl2 in the absence of the target RNA. Visible absorbance titration experiments showed that the GDDCs bind to CuAPC with Ka values of 1.5 × 105 to 5.9 × 105 M−1 (Kd values of 6.7 to 1.7 μM at 25 °C, depending on the loop length. Fluorescence titration further showed that the G-quadruplex structure unfolds upon binding to the target RNA with Ka values above 1.0 × 108 M−1 (Kd values below 0.01 μM at 25 °C. These results suggest the carrier can sense and bind to the target RNA, which should result in release of the bound drug. Finally, visible absorbance titration experiments demonstrated that the GDDC release CuAPC in response to the target RNA.

  4. Inverting the G-Tetrad Polarity of a G-Quadruplex by Using Xanthine and 8-Oxoguanine. (United States)

    Cheong, Vee Vee; Lech, Christopher Jacques; Heddi, Brahim; Phan, Anh Tuân


    G-quadruplexes are four-stranded nucleic acid structures that are built from consecutively stacked guanine tetrad (G-tetrad) assemblies. The simultaneous incorporation of two guanine base lesions, xanthine (X) and 8-oxoguanine (O), within a single G-tetrad of a G-quadruplex was recently shown to lead to the formation of a stable G⋅G⋅X⋅O tetrad. Herein, a judicious introduction of X and O into a human telomeric G-quadruplex-forming sequence is shown to reverse the hydrogen-bond polarity of the modified G-tetrad while preserving the original folding topology. The control exerted over G-tetrad polarity by joint X⋅O modification will be valuable for the design and programming of G-quadruplex structures and their properties. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Design, synthesis and evaluation of 4,7-diamino-1,10-phenanthroline G-quadruplex ligands

    DEFF Research Database (Denmark)

    Nielsen, Mads Corvinius; Borch, Jonas; Ulven, Trond


    the central ionic column. Introduction of positively charged side chains results in compounds with appreciable G-quadruplex stabilizing properties and high aqueous solubility, with the longer side chains giving more potent compounds. Ligands carrying guanidine side chains in general show higher quadruplex...... stabilizing activity and distinctly slower kinetic properties than their amino and dimethylamino analogues, possibly due to specific hydrogen bond interactions with the G-quadruplex loops....

  6. Behavior of the guanine base in G-quadruplexes probed by the fluorescent guanine analog, 6-methyl isozanthopterin

    Energy Technology Data Exchange (ETDEWEB)

    Han, Ji Hoon; Chitrapriya, Nataraj; Lee, Hyun Suk; Lee, Young Ae; Kim, Seog K. [Dept. of Chemistry, Yeungnam University, Gyeongsan (Korea, Republic of); Jung, Maeng Joon [Dept. of Chemistry, Kyungpook National University, Daegu (Korea, Republic of)


    In this study, circular dichroism (CD) spectrum and fluorescence techniques were used to examine the dynamic properties and microenvironment of the guanine base (G) at the central loop and at the middle of the G-stem of the G-quadruplex formed from the G{sub 3}T{sub 2}G{sub 3}TGTG{sub 3}T{sub 2}G{sub 3} sequence (G-quadruplex 1), in which the G base at the 10th and 13th position were replaced with a fluorescent G analog, 6-methyl isoxanthopterin (6MI) (G-quadruplex 2 and 3, respectively). For all G-quadruplexes, the CD spectrum revealed a positive band at 263 nm and a shoulder at 298 nm, and the thermal melting profiles were the sum of at least two sigmoidal curves. These observations indicated the presence of two conformers in the G-quadruplex. The fluorescence intensity of G-quadruplex 2 was greater than 3, as expected from the extent of stacking interaction, which is larger in the G(6MI)G sequence than the T(6MI)T sequence. The efficiency of fluorescence quenching by the polar acrylamide quencher and negatively charged I− quencher were larger for G-quadruplex 3, suggesting that 6MI in the G(6MI)G stem is exposed more to the aqueous environment compared to that in the T(6MI)T central loop. In the latter case, 6MI may direct to the center of the top G-quartet layer. The possibility of hydrogen bond formation between the carbonyl group of 6MI and the acrylamide of the G-quadruplex 3 was proposed.

  7. RNA G-quadruplex secondary structure promotes alternative splicing via the RNA-binding protein hnRNPF. (United States)

    Huang, Huilin; Zhang, Jing; Harvey, Samuel E; Hu, Xiaohui; Cheng, Chonghui


    It is generally thought that splicing factors regulate alternative splicing through binding to RNA consensus sequences. In addition to these linear motifs, RNA secondary structure is emerging as an important layer in splicing regulation. Here we demonstrate that RNA elements with G-quadruplex-forming capacity promote exon inclusion. Destroying G-quadruplex-forming capacity while keeping G tracts intact abrogates exon inclusion. Analysis of RNA-binding protein footprints revealed that G quadruplexes are enriched in heterogeneous nuclear ribonucleoprotein F (hnRNPF)-binding sites and near hnRNPF-regulated alternatively spliced exons in the human transcriptome. Moreover, hnRNPF regulates an epithelial-mesenchymal transition (EMT)-associated CD44 isoform switch in a G-quadruplex-dependent manner, which results in inhibition of EMT. Mining breast cancer TCGA (The Cancer Genome Atlas) data sets, we demonstrate that hnRNPF negatively correlates with an EMT gene signature and positively correlates with patient survival. These data suggest a critical role for RNA G quadruplexes in regulating alternative splicing. Modulation of G-quadruplex structural integrity may control cellular processes important for tumor progression. © 2017 Huang et al.; Published by Cold Spring Harbor Laboratory Press.

  8. G-Quadruplex Identification in the Genome of Protozoan Parasites Points to Naphthalene Diimide Ligands as New Antiparasitic Agents. (United States)

    Belmonte-Reche, Efres; Martínez-García, Marta; Guédin, Aurore; Zuffo, Michela; Arévalo-Ruiz, Matilde; Doria, Filippo; Campos-Salinas, Jenny; Maynadier, Marjorie; López-Rubio, José Juan; Freccero, Mauro; Mergny, Jean-Louis; Pérez-Victoria, José María; Morales, Juan Carlos


    G-quadruplexes (G4) are DNA secondary structures that take part in the regulation of gene expression. Putative G4 forming sequences (PQS) have been reported in mammals, yeast, bacteria, and viruses. Here, we present PQS searches on the genomes of T. brucei, L. major, and P. falciparum. We found telomeric sequences and new PQS motifs. Biophysical experiments showed that EBR1, a 29 nucleotide long highly repeated PQS in T. brucei, forms a stable G4 structure. G4 ligands based on carbohydrate conjugated naphthalene diimides (carb-NDIs) that bind G4's including hTel could bind EBR1 with selectivity versus dsDNA. These ligands showed important antiparasitic activity. IC 50 values were in the nanomolar range against T. brucei with high selectivity against MRC-5 human cells. Confocal microscopy confirmed these ligands localize in the nucleus and kinetoplast of T. brucei suggesting they can reach their potential G4 targets. Cytotoxicity and zebrafish toxicity studies revealed sugar conjugation reduces intrinsic toxicity of NDIs.

  9. The binding efficiency of RPA to telomeric G-strands folded into contiguous G-quadruplexes is independent of the number of G4 units. (United States)

    Lancrey, Astrid; Safa, Layal; Chatain, Jean; Delagoutte, Emmanuelle; Riou, Jean-François; Alberti, Patrizia; Saintomé, Carole


    Replication protein A (RPA) is a single-stranded DNA binding protein involved in replication and in telomere maintenance. During telomere replication, G-quadruplexes (G4) can accumulate on the lagging strand template and need to be resolved. It has been shown that human RPA is able to unfold a single G4. Nevertheless, the G-strand of human telomeres is prone to fold into higher-order structures formed by contiguous G-quadruplexes. To understand how RPA deals with these structures, we studied its interaction with telomeric G-strands folding into an increasing number of contiguous G4s. The aim of this study was to determine whether the efficiency of binding/unfolding of hRPA to telomeric G-strands depends on the number of G4 units. Our data show that the number n of contiguous G4 units (n ≥ 2) does not affect the efficiency of hRPA to coat transiently exposed single-stranded telomeric G-strands. This feature may be essential in preventing instability due to G4 structures during telomere replication. Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  10. Sequencing and G-quadruplex folding of the canine proto-oncogene KIT promoter region: might dog be used as a model for human disease?

    Directory of Open Access Journals (Sweden)

    Silvia Da Ros

    Full Text Available Downregulation of gene expression by induction of non-canonical DNA structures at promotorial level is a novel attractive anticancer strategy. In human, two guanine-rich sequences (h_kit1 and h_kit2 were identified in the promotorial region of oncogene KIT. Their stabilization into G-quadruplex structures can find applications in the treatment of leukemias, mastocytosis, gastrointestinal stromal tumor, and lung carcinomas which are often associated to c-kit mis-regulation. Also the most common skin cancer in domestic dog, mast cell tumor, is linked to a mutation and/or to an over-expression of c-kit, thus supporting dog as an excellent animal model. In order to assess if the G-quadruplex mediated mechanism of regulation of c-kit expression is conserved among the two species, herein we cloned and sequenced the canine KIT promoter region and we compared it with the human one in terms of sequence and conformational equilibria in physiologically relevant conditions. Our results evidenced a general conserved promotorial sequence between the two species. As experimentally confirmed, this grants that the conformational features of the canine kit1 sequence are substantially shared with the human one. Conversely, two isoforms of the kit2 sequences were identified in the analyzed dog population. In comparison with the human counterpart, both of them showed an altered distribution among several folded conformations.

  11. Topoisomerase I plays a critical role in suppressing genome instability at a highly transcribed G-quadruplex-forming sequence.

    Directory of Open Access Journals (Sweden)

    Puja Yadav


    Full Text Available G-quadruplex or G4 DNA is a non-B secondary DNA structure that comprises a stacked array of guanine-quartets. Cellular processes such as transcription and replication can be hindered by unresolved DNA secondary structures potentially endangering genome maintenance. As G4-forming sequences are highly frequent throughout eukaryotic genomes, it is important to define what factors contribute to a G4 motif becoming a hotspot of genome instability. Using a genetic assay in Saccharomyces cerevisiae, we previously demonstrated that a potential G4-forming sequence derived from a guanine-run containing immunoglobulin switch Mu (Sμ region becomes highly unstable when actively transcribed. Here we describe assays designed to survey spontaneous genome rearrangements initiated at the Sμ sequence in the context of large genomic areas. We demonstrate that, in the absence of Top1, a G4 DNA-forming sequence becomes a strong hotspot of gross chromosomal rearrangements and loss of heterozygosity associated with mitotic recombination within the ∼ 20 kb or ∼ 100 kb regions of yeast chromosome V or III, respectively. Transcription confers a critical strand bias since genome rearrangements at the G4-forming Sμ are elevated only when the guanine-runs are located on the non-transcribed strand. The direction of replication and transcription, when in a head-on orientation, further contribute to the elevated genome instability at a potential G4 DNA-forming sequence. The implications of our identification of Top1 as a critical factor in suppression of instability associated with potential G4 DNA-forming sequences are discussed.

  12. Design, Synthesis and Biological Evaluation of New Substituted Diquinolinyl-Pyridine Ligands as Anticancer Agents by Targeting G-Quadruplex. (United States)

    Das, Rabindra Nath; Chevret, Edith; Desplat, Vanessa; Rubio, Sandra; Mergny, Jean-Louis; Guillon, Jean


    G-quadruplexes (G4) are stacked non-canonical nucleic acid structures found in specific G-rich DNA or RNA sequences in the human genome. G4 structures are liable for various biological functions; transcription, translation, cell aging as well as diseases such as cancer. These structures are therefore considered as important targets for the development of anticancer agents. Small organic heterocyclic molecules are well known to target and stabilize G4 structures. In this article, we have designed and synthesized 2,6-di-(4-carbamoyl-2-quinolyl)pyridine derivatives and their ability to stabilize G4-structures have been determined through the FRET melting assay. It has been established that these ligands are selective for G4 over duplexes and show a preference for the parallel conformation. Next, telomerase inhibition ability has been assessed using three cell lines (K562, MyLa and MV-4-11) and telomerase activity is no longer detected at 0.1 μM concentration for the most potent ligand 1c . The most promising G4 ligands were also tested for antiproliferative activity against the two human myeloid leukaemia cell lines, HL60 and K562.

  13. Structure and possible function of a G-quadruplex in the long terminal repeat of the proviral HIV-1 genome. (United States)

    De Nicola, Beatrice; Lech, Christopher J; Heddi, Brahim; Regmi, Sagar; Frasson, Ilaria; Perrone, Rosalba; Richter, Sara N; Phan, Anh Tuân


    The long terminal repeat (LTR) of the proviral human immunodeficiency virus (HIV)-1 genome is integral to virus transcription and host cell infection. The guanine-rich U3 region within the LTR promoter, previously shown to form G-quadruplex structures, represents an attractive target to inhibit HIV transcription and replication. In this work, we report the structure of a biologically relevant G-quadruplex within the LTR promoter region of HIV-1. The guanine-rich sequence designated LTR-IV forms a well-defined structure in physiological cationic solution. The nuclear magnetic resonance (NMR) structure of this sequence reveals a parallel-stranded G-quadruplex containing a single-nucleotide thymine bulge, which participates in a conserved stacking interaction with a neighboring single-nucleotide adenine loop. Transcription analysis in a HIV-1 replication competent cell indicates that the LTR-IV region may act as a modulator of G-quadruplex formation in the LTR promoter. Consequently, the LTR-IV G-quadruplex structure presented within this work could represent a valuable target for the design of HIV therapeutics. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Nucleotide Pool Depletion Induces G-Quadruplex-Dependent Perturbation of Gene Expression

    Directory of Open Access Journals (Sweden)

    Charikleia Papadopoulou


    Full Text Available Nucleotide pool imbalance has been proposed to drive genetic instability in cancer. Here, we show that slowing replication forks by depleting nucleotide pools with hydroxyurea (HU can also give rise to both transient and permanent epigenetic instability of a reporter locus, BU-1, in DT40 cells. HU induces stochastic formation of Bu-1low variants in dividing cells, which have lost the H3K4me3 present in untreated cells. This instability is potentiated by an intragenic G quadruplex, which also promotes local H2Ax phosphorylation and transient heterochromatinization. Genome-wide, gene expression changes induced by HU significantly overlap with those resulting from loss of the G4-helicases FANCJ, WRN, and BLM. Thus, the effects of global replication stress induced by nucleotide pool depletion can be focused by local replication impediments caused by G quadruplex formation to induce epigenetic instability and changes in gene expression, a mechanism that may contribute to selectable transcriptional changes in cancer.

  15. Dual Binding of an Antibody and a Small Molecule Increases the Stability of TERRA G-Quadruplex. (United States)

    Yangyuoru, Philip M; Di Antonio, Marco; Ghimire, Chiran; Biffi, Giulia; Balasubramanian, Shankar; Mao, Hanbin


    In investigating the binding interactions between the human telomeric RNA (TERRA) G-quadruplex (GQ) and its ligands, it was found that the small molecule carboxypyridostatin (cPDS) and the GQ-selective antibody BG4 simultaneously bind the TERRA GQ. We previously showed that the overall binding affinity of BG4 for RNA GQs is not significantly affected in the presence of cPDS. However, single-molecule mechanical unfolding experiments revealed a population (48 %) with substantially increased mechanical and thermodynamic stability. Force-jump kinetic investigations suggested competitive binding of cPDS and BG4 to the TERRA GQ. Following this, the two bound ligands slowly rearrange, thereby leading to the minor population with increased stability. Given the relevance of G-quadruplexes in the regulation of biological processes, we anticipate that the unprecedented conformational rearrangement observed in the TERRA-GQ-ligand complex may inspire new strategies for the selective stabilization of G-quadruplexes in cells.

  16. TMPyP4 porphyrin distorts RNA G-quadruplex structures of the disease-associated r(GGGGCC)n repeat of the C9orf72 gene and blocks interaction of RNA-binding proteins. (United States)

    Zamiri, Bita; Reddy, Kaalak; Macgregor, Robert B; Pearson, Christopher E


    Certain DNA and RNA sequences can form G-quadruplexes, which can affect genetic instability, promoter activity, RNA splicing, RNA stability, and neurite mRNA localization. Amyotrophic lateral sclerosis and frontotemporal dementia can be caused by expansion of a (GGGGCC)n repeat in the C9orf72 gene. Mutant r(GGGGCC)n- and r(GGCCCC)n-containing transcripts aggregate in nuclear foci, possibly sequestering repeat-binding proteins such as ASF/SF2 and hnRNPA1, suggesting a toxic RNA pathogenesis, as occurs in myotonic dystrophy. Furthermore, the C9orf72 repeat RNA was recently demonstrated to undergo the noncanonical repeat-associated non-AUG translation (RAN translation) into pathologic dipeptide repeats in patient brains, a process that is thought to depend upon RNA structure. We previously demonstrated that the r(GGGGCC)n RNA forms repeat tract length-dependent G-quadruplex structures that bind the ASF/SF2 protein. Here we show that the cationic porphyrin (5,10,15,20-tetra(N-methyl-4-pyridyl) porphyrin (TMPyP4)), which can bind some G-quadruplex-forming sequences, can bind and distort the G-quadruplex formed by r(GGGGCC)8, and this ablates the interaction of either hnRNPA1 or ASF/SF2 with the repeat. These findings provide proof of concept that nucleic acid binding small molecules, such as TMPyP4, can distort the secondary structure of the C9orf72 repeat, which may beneficially disrupt protein interactions, which may ablate either protein sequestration and/or RAN translation into potentially toxic dipeptides. Disruption of secondary structure formation of the C9orf72 RNA repeats may be a viable therapeutic avenue, as well as a means to test the role of RNA structure upon RAN translation.

  17. G-quadruplex structure at intron 2 of TFE3 and its role in Xp11.2 translocation and splicing. (United States)

    Verma, Shiv Prakash; Das, Parimal


    Transcription Factor E3 (TFE3) translocation is found in a group of different type of cancers and most of the translocations are located in the 5' region of TFE3 which may be considered as Breakpoint Region (BR). In our In silico study by QGRS mapper and non BdB web servers we found a Potential G-quadruplex forming Sequence (PQS) in the intron 2 of TFE3 gene. In vitro G-quadruplex formation was shown by native PAGE in presence of Pyridostatin(PDS), which with inter molecular secondary structure caused reduced mobility to migrate slower. G-quadruplex formation was mapped at single base resolution by Sanger sequencing and Circular Dichroism showed the formation of parallel G-quadruplex. FRET analysis revealed increased and decreased formation of G-quadruplex in presence of PDS and antisense oligonucleotide respectively. PCR stop assay, transcriptional and translational inhibition by PQS showed stable G-quadruplex formation affecting the biological processes. TFE3 minigene splicing study showed the involvement of this G-quadruplex in TFE3 splicing too. Therefore, G-quadruplex is evident to be the reason behind TFE3 induced oncogenesis executed by translocation and also involved in the mRNA splicing. Copyright © 2017 Elsevier B.V. All rights reserved.

  18. Hsa-miR-1587 G-quadruplex formation and dimerization induced by NH4+, molecular crowding environment and jatrorrhizine derivatives. (United States)

    Tan, Wei; Yi, Long; Zhu, Zhentao; Zhang, Lulu; Zhou, Jiang; Yuan, Gu


    A guanine-rich human mature microRNA, miR-1587, was discovered to form stable intramolecular G-quadruplexes in the presence of K + , Na + and low concentration of NH 4 + (25mM) by electrospray ionization mass spectrometry (ESI-MS) combined with circular dichroism (CD) spectroscopy. Furthermore, under high concentration of NH 4 + (100mM) or molecular crowding environments, miR-1587 formed a dimeric G-quadruplex through 3'-to-3' stacking of two monomeric G-quadruplex subunits with one ammonium ion sandwiched between the interfaces. Specifically, two synthesized jatrorrhizine derivatives with terminal amine groups could also induce the dimerization of miR-1587 G-quadruplex and formed 1:1 and 2:1 complexes with the dimeric G-quadruplex. In contrast, jatrorrhizine could bind with the dimeric miR-1587 G-quadruplex, but could not induce dimerization of miR-1587 G-quadruplex. These results provide a new strategy to regulate the functions of miR-1587 through induction of G-quadruplex formation and dimerization. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Synthesis, Binding and Antiviral Properties of Potent Core-Extended Naphthalene Diimides Targeting the HIV-1 Long Terminal Repeat Promoter G-Quadruplexes. (United States)

    Perrone, Rosalba; Doria, Filippo; Butovskaya, Elena; Frasson, Ilaria; Botti, Silvia; Scalabrin, Matteo; Lago, Sara; Grande, Vincenzo; Nadai, Matteo; Freccero, Mauro; Richter, Sara N


    We have previously reported that stabilization of the G-quadruplex structures in the HIV-1 long terminal repeat (LTR) promoter suppresses viral transcription. Here we sought to develop new G-quadruplex ligands to be exploited as antiviral compounds by enhancing binding toward the viral G-quadruplex structures. We synthesized naphthalene diimide derivatives with a lateral expansion of the aromatic core. The new compounds were able to bind/stabilize the G-quadruplex to a high extent, and some of them displayed clear-cut selectivity toward the viral G-quadruplexes with respect to the human telomeric G-quadruplexes. This feature translated into low nanomolar anti-HIV-1 activity toward two viral strains and encouraging selectivity indexes. The selectivity depended on specific recognition of LTR loop residues; the mechanism of action was ascribed to inhibition of LTR promoter activity in cells. This is the first example of G-quadruplex ligands that show increased selectivity toward the viral G-quadruplexes and display remarkable antiviral activity.

  20. Structure of long human telomeric RNA (TERRA): G-quadruplexes formed by four and eight UUAGGG repeats are stable building blocks. (United States)

    Martadinata, Herry; Heddi, Brahim; Lim, Kah Wai; Phan, Anh Tuân


    The discovery of long RNA transcripts of telomeric repeats (TERRA) and their potential to form G-quadruplexes stimulated studies on the possible arrangements of G-quadruplexes along TERRA. Here we performed ribonuclease protection assay to investigate the structures formed by long human TERRA. We found that G-quadruplexes comprising four and eight UUAGGG repeats were most resistant to RNase T1 digestion, presumably with the former adopting an all-parallel-stranded propeller-type conformation and the latter forming a structure with two tandemly stacked G-quadruplex subunits each containing three G-tetrad layers. Molecular dynamics simulations of eight-repeat human TERRA sequences consisting of different stacking interfaces between the two G-quadruplex subunits, i.e., 5'-5', 3'-3', 3'-5', and 5'-3', demonstrated stacking feasibility for all but the 5'-3' arrangement. A continuous stacking of the loop bases from one G-quadruplex subunit to the next was observed for the 5'-5' stacking conformation. We also put forward other possible stacking arrangements that involve more than one linker connecting the two G-quadruplex subunits. On the basis of these results, we propose a "beads-on-a-string"-like arrangement along human TERRA, whereby each bead is made up of either four or eight UUAGGG repeats in a one- or two-block G-quadruplex arrangement, respectively. © 2011 American Chemical Society

  1. Extratelomeric Binding of the Telomere Binding Protein TRF2 at the PCGF3 Promoter Is G-Quadruplex Motif-Dependent. (United States)

    Purohit, Gunjan; Mukherjee, Ananda Kishor; Sharma, Shalu; Chowdhury, Shantanu


    Telomere repeat binding factor 2 (TRF2) is critical for the protection of chromosome ends. Mounting evidence suggests that TRF2 associates with extratelomeric sites and TRF2 functions may not be limited to telomeres. Here, we show that the PCGF3 promoter harbors a sequence capable of forming the DNA secondary structure G-quadruplex motif, which is required for binding of TRF2 at the PCGF3 promoter. We demonstrate that promoter binding by TRF2 mediates PCGF3 promoter activity, and both the N-terminal and C-terminal domains of TRF2 are necessary for promoter activity. Altogether, this shows for the first time that a telomere binding factor may regulate a component of the polycomb group of proteins.

  2. Enhanced anti-HIV-1 activity of G-quadruplexes comprising locked nucleic acids and intercalating nucleic acids

    DEFF Research Database (Denmark)

    Pedersen, Erik Bjerregaard; Nielsen, Jakob Toudahl; Nielsen, Claus


    Two G-quadruplex forming sequences, 50-TGGGAG and the 17-mer sequence T30177, which exhibit anti-HIV-1 activity on cell lines, were modified using either locked nucleic acids (LNA) or via insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (intercalating nucleic acid, INA) or (R)-1-O-[4-(1-pyrenylet......Two G-quadruplex forming sequences, 50-TGGGAG and the 17-mer sequence T30177, which exhibit anti-HIV-1 activity on cell lines, were modified using either locked nucleic acids (LNA) or via insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (intercalating nucleic acid, INA) or (R)-1-O-[4......-(1-pyrenylethynyl)phenylmethyl]glycerol (twisted intercalating nucleic acid, TINA). Incorporation of LNA or INA/TINA monomers provide as much as 8-fold improvement of anti-HIV-1 activity. We demonstrate for the first time a detailed analysis of the effect the incorporation of INA/TINA monomers in quadruplex forming...... of the antiviral QFOs in the present study formed more thermally stable G-quadruplexes and also high-order G-quadruplex structures which might be responsible for the increased antiviral activity observed....

  3. Chelerythrine down regulates expression of VEGFA, BCL2 and KRAS by arresting G-Quadruplex structures at their promoter regions (United States)

    Jana, Jagannath; Mondal, Soma; Bhattacharjee, Payel; Sengupta, Pallabi; Roychowdhury, Tanaya; Saha, Pranay; Kundu, Pallob; Chatterjee, Subhrangsu


    A putative anticancer plant alkaloid, Chelerythrine binds to G-quadruplexes at promoters of VEGFA, BCL2 and KRAS genes and down regulates their expression. The association of Chelerythrine to G-quadruplex at the promoters of these oncogenes were monitored using UV absorption spectroscopy, fluorescence anisotropy, circular dichroism spectroscopy, CD melting, isothermal titration calorimetry, molecular dynamics simulation and quantitative RT-PCR technique. The pronounced hypochromism accompanied by red shifts in UV absorption spectroscopy in conjunction with ethidium bromide displacement assay indicates end stacking mode of interaction of Chelerythrine with the corresponding G-quadruplex structures. An increase in fluorescence anisotropy and CD melting temperature of Chelerythrine-quadruplex complex revealed the formation of stable Chelerythrine-quadruplex complex. Isothermal titration calorimetry data confirmed that Chelerythrine-quadruplex complex formation is thermodynamically favourable. Results of quantative RT-PCR experiment in combination with luciferase assay showed that Chelerythrine treatment to MCF7 breast cancer cells effectively down regulated transcript level of all three genes, suggesting that Chelerythrine efficiently binds to in cellulo quadruplex motifs. MD simulation provides the molecular picture showing interaction between Chelerythrine and G-quadruplex. Binding of Chelerythrine with BCL2, VEGFA and KRAS genes involved in evasion, angiogenesis and self sufficiency of cancer cells provides a new insight for the development of future therapeutics against cancer.

  4. Study on the polymorphism of G-quadruplexes by reversed-phase HPLC and LC-MS. (United States)

    Qiao, Jun-Qin; Cao, Zhao-Ming; Liang, Chao; Chen, Hong-Juan; Zheng, Wei-Juan; Lian, Hong-Zhen


    Polymorphism is inherent for G-quadruplexes (G4s), and the different structural forms are important for the participation in different biological functions of telomeres. A lot of progress has been made in the exploration of G4 polymorphism. However, quick separation and reliable assessment methods for different conformations of G4 are still very few. In this work, the polymorphism of three sequences d[(G 3 T) 3 G 3 ], d[(G 3 C) 3 G 3 ] and d[(G 3 T) 4 ] annealed in six different solutions were investigated by means of reversed-phase high performance liquid chromatography (RP-HPLC), liquid chromatography-mass spectrometry (LC-MS), fluorescence spectroscopy, circular dichroism spectroscopy, together with native-polyacrylamide gel electrophoresis. Different G4 conformations of these three sequences can be separated clearly by RP-HPLC, and further characterized by on-line LC-MS analysis. It is revealed that high-order structures other than intramolecular quadruplexes were favored for d[(G 3 T) 3 G 3 ] and d[(G 3 C) 3 G 3 ] under the annealing conditions. However, flanking loop impeded d[(G 3 T) 4 ] to form higher-order structures than dimer. In addition, the nature and concentration of cation, as well as the annealing solution component, all have decent influence on the stability and relative ratios of various G4 building blocks. Based on the above findings, RP-HPLC and LC-MS combined with spectroscopic techniques can be used as a facile and powerful tool for quick separation and identification of G4s in solutions, and for effective assessment of DNA sequences and annealing environments on G4 polymorphism. The established protocol provides a novel strategy for evaluating G4 polymorphism, which will facilitate studies on quadruplex structures and their biophysical properties. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. The 3'-end region of the human PDGFR-β core promoter nuclease hypersensitive element forms a mixture of two unique end-insertion G-quadruplexes. (United States)

    Onel, Buket; Carver, Megan; Agrawal, Prashansa; Hurley, Laurence H; Yang, Danzhou


    While the most stable G-quadruplex formed in the human PDGFR-β promoter nuclease hypersensitive element (NHE) is the 5'-mid G-quadruplex, the 3'-end sequence that contains a 3'-GGA run forms a less stable G-quadruplex. Recently, the 3'-end G-quadruplex was found to be a transcriptional repressor and can be selectively targeted by a small molecule for PDGFR-β downregulation. We use 1D and 2D high-field NMR, in combination with Dimethylsulfate Footprinting, Circular Dichroism Spectroscopy, and Electrophoretic Mobility Shift Assay. We determine that the PDGFR-β extended 3'-end NHE sequence forms two novel end-insertion intramolecular G-quadruplexes that co-exist in equilibrium under physiological salt conditions. One G-quadruplex has a 3'-non-adjacent flanking guanine inserted into the 3'-external tetrad (3'-insertion-G4), and another has a 5'-non-adjacent flanking guanine inserted into the 5'-external tetrad (5'-insertion-G4). The two guanines in the GGA-run move up or down within the G-quadruplex to accommodate the inserted guanine. Each end-insertion G-quadruplex has a low thermal stability as compared to the 5'-mid G-quadruplex, but the selective stabilization of GSA1129 shifts the equilibrium toward the 3'-end G-quadruplex in the PDGFR-β NHE. An equilibrium mixture of two unique end-insertion intramolecular G-quadruplexes forms in the PDGFR-β NHE 3'-end sequence that contains a GGA-run and non-adjacent guanines in both the 3'- and 5'- flanking segments; the novel end-insertion structures of the 3'-end G-quadruplex are selectively stabilized by GSA1129. We show for the first time that an equilibrium mixture of two unusual end-insertion G-quadruplexes forms in a native promoter sequence and appears to be the molecular recognition for PDGFR-β downregulation. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. Formation of a unique cluster of G-quadruplex structures in the HIV-1 Nef coding region: implications for antiviral activity.

    Directory of Open Access Journals (Sweden)

    Rosalba Perrone

    Full Text Available G-quadruplexes are tetraplex structures of nucleic acids that can form in G-rich sequences. Their presence and functional role have been established in telomeres, oncogene promoters and coding regions of the human chromosome. In particular, they have been proposed to be directly involved in gene regulation at the level of transcription. Because the HIV-1 Nef protein is a fundamental factor for efficient viral replication, infectivity and pathogenesis in vitro and in vivo, we investigated G-quadruplex formation in the HIV-1 nef gene to assess the potential for viral inhibition through G-quadruplex stabilization. A comprehensive computational analysis of the nef coding region of available strains showed the presence of three conserved sequences that were uniquely clustered. Biophysical testing proved that G-quadruplex conformations were efficiently stabilized or induced by G-quadruplex ligands in all three sequences. Upon incubation with a G-quadruplex ligand, Nef expression was reduced in a reporter gene assay and Nef-dependent enhancement of HIV-1 infectivity was significantly repressed in an antiviral assay. These data constitute the first evidence of the possibility to regulate HIV-1 gene expression and infectivity through G-quadruplex targeting and therefore open a new avenue for viral treatment.

  7. Characterizing and controlling intrinsic biases of lambda exonuclease in nascent strand sequencing reveals phasing between nucleosomes and G-quadruplex motifs around a subset of human replication origins

    DEFF Research Database (Denmark)

    Foulk, M. S.; Urban, J. M.; Casella, Cinzia


    Nascent strand sequencing (NS-seq) is used to discover DNA replication origins genome-wide, allowing identification of features for their specification. NS-seq depends on the ability of lambda exonuclease (lambda-exo) to efficiently digest parental DNA while leaving RNA-primer protected nascent...... strands intact. We used genomics and biochemical approaches to determine if lambda-exo digests all parental DNA sequences equally. We report that lambda-exo does not efficiently digest G-quadruplex (G4) structures in a plasmid. Moreover, lambda-exo digestion of nonreplicating genomic DNA (LexoG0) enriches...... GC-rich DNA and G4 motifs genome-wide. We used LexoG0 data to control for nascent strand-independent lambda-exo biases in NSseq and validated this approach at the rDNA locus. The lambda-exo-controlled NS-seq peaks are not GC-rich, and only 35.5% overlap with 6.8% of all G4s, suggesting that G4s...

  8. Comprehensive identification of proteins binding to RNA G-quadruplex motifs in the 5' UTR of tumor-associated mRNAs. (United States)

    Serikawa, Tatsuo; Spanos, Christos; von Hacht, Annekathrin; Budisa, Nediljko; Rappsilber, Juri; Kurreck, Jens


    G-quadruplex structures in the 5' UTR of mRNAs are widely considered to suppress translation without affecting transcription. The current study describes the comprehensive analysis of proteins binding to four different G-quadruplex motifs located in mRNAs of the cancer-related genes Bcl-2, NRAS, MMP16, and ARPC2. Following metabolic labeling (Stable Isotope Labeling with Amino acids in Cell culture, SILAC) of proteins in the human cell line HEK293, G-quadruplex binding proteins were enriched by pull-down assays and identified by LC-orbitrap mass spectrometry. We found different patterns of interactions for the G-quadruplex motifs under investigation. While the G-quadruplexes in the mRNAs of NRAS and MMP16 specifically interacted with a small number of proteins, the Bcl-2 and ARPC2 G-quadruplexes exhibited a broad range of proteinaceous interaction partners with 99 and 82 candidate proteins identified in at least two replicates, respectively. The use of a control composed of samples from all G-quadruplex-forming sequences and their mutated controls ensured that the identified proteins are specific for RNA G-quadruplex structures and are not general RNA-binding proteins. Independent validation experiments based on pull-down assays and Western blotting confirmed the MS data. Among the interaction partners were many proteins known to bind to RNA, including multiple heterogenous nuclear ribonucleoproteins (hnRNPs). Several of the candidate proteins are likely to reflect stalling of the ribosome by RNA G-quadruplex structures. Interestingly, additional proteins were identified that have not previously been described to interact with RNA. Gene ontology analysis of the candidate proteins revealed that many interaction partners are known to be tumor related. The majority of the identified RNA G-quadruplex interacting proteins are thought to be involved in post-transcriptional processes, particularly in splicing. These findings indicate that protein-G-quadruplex interactions

  9. Asymmetric Cationic Porphyrin as a New G-Quadruplex Probe with Wash-Free Cancer-Targeted Imaging Ability Under Acidic Microenvironments. (United States)

    Zhang, Ran; Cheng, Meng; Zhang, Li-Ming; Zhu, Li-Na; Kong, De-Ming


    Porphyrins are promising candidates for nucleic acid G-quadruplex-specific optical recognition. We previously demonstrated that G-quadruplex recognition specificity of porphyrins could be improved by introducing bulky side arm substituents, but the enhanced protonation tendency limits their applications in some cases, such as under acidic conditions. Here, we demonstrated that the protonation tendency of porphyrin derivatives could be efficiently overcome by increasing molecular asymmetry. To validate this, an asymmetric, water-soluble, cationic porphyrin FA-TMPipEOPP (5-{4-[2-[[(2 E)-3-[3-methoxy-4-[2-(1-methyl-1-piperidinyl)ethoxy]phenyl]-1-oxo-2-propenyl]oxy]ethoxy]phenyl},10,15,20-tri{4-[2-(1-methyl-1-piperidinyl)ethoxy]-phenyl}porphyrin) was synthesized by introducing a ferulic acid (FA) unit at one side arm, and its structure was well-characterized. Unlike its symmetric counterpart TMPipEOPP that has a tendency to protonate under acidic conditions, FA-TMPipEOPP remained in the unprotonated monomeric form under the pH range of 2.0-8.0. Correspondingly, FA-TMPipEOPP showed better G-quadruplex recognition specificity than TMPipEOPP and thus might be used as a specific optical probe for colorimetric and fluorescent recognition of G-quadruplexes under acidic conditions. The feasibility was demonstrated by two proof-of-concept studies: probing structural competition between G-quadruplexes and duplexes and label-free and wash-free cancer cell-targeted bioimaging under an acidic tumor microenvironment. As G-quadruplex optical probes, FA-TMPipEOPP works well under acidic conditions, whereas TMPipEOPP works well under neutral conditions. This finding provides useful information for G-quadruplex probe research. That is, porphyrin-based G-quadruplex probes suitable for different pH conditions might be obtained by adjusting the molecular symmetry.

  10. Fragile X mental retardation protein recognizes a G quadruplex structure within the survival motor neuron domain containing 1 mRNA 5'-UTR. (United States)

    McAninch, Damian S; Heinaman, Ashley M; Lang, Cara N; Moss, Kathryn R; Bassell, Gary J; Rita Mihailescu, Mihaela; Evans, Timothy L


    G quadruplex structures have been predicted by bioinformatics to form in the 5'- and 3'-untranslated regions (UTRs) of several thousand mature mRNAs and are believed to play a role in translation regulation. Elucidation of these roles has primarily been focused on the 3'-UTR, with limited focus on characterizing the G quadruplex structures and functions in the 5'-UTR. Investigation of the affinity and specificity of RNA binding proteins for 5'-UTR G quadruplexes and the resulting regulatory effects have also been limited. Among the mRNAs predicted to form a G quadruplex structure within the 5'-UTR is the survival motor neuron domain containing 1 (SMNDC1) mRNA, encoding a protein that is critical to the spliceosome. Additionally, this mRNA has been identified as a potential target of the fragile X mental retardation protein (FMRP), whose loss of expression leads to fragile X syndrome. FMRP is an RNA binding protein involved in translation regulation that has been shown to bind mRNA targets that form G quadruplex structures. In this study we have used biophysical methods to investigate G quadruplex formation in the 5'-UTR of SMNDC1 mRNA and analyzed its interactions with FMRP. Our results show that SMNDC1 mRNA 5'-UTR forms an intramolecular, parallel G quadruplex structure comprised of three G quartet planes, which is bound specifically by FMRP both in vitro and in mouse brain lysates. These findings suggest a model by which FMRP might regulate the translation of a subset of its mRNA targets by recognizing the G quadruplex structure present in their 5'-UTR, and affecting their accessibility by the protein synthesis machinery.

  11. A new signal-on method for the detection of protein based on binding-induced strategy and photoinduced electron transfer between Ag nanoclusters and split G-quadruplex-hemin complexes

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Kai, E-mail:; Wang, Ke; Zhu, Xue; Xie, Minhao


    Proteins play important roles in biological and cellular processes. The levels of proteins can be useful biomarkers for cellular events or disease diagnosis, thus the method for sensitive and selective detection of proteins is imperative to proteins express, study, and clinical diagnosis. Herein, we report a “signal-on” platform for the assay of protein based on binding-induced strategy and photoinduced electron transfer between Ag nanoclusters and split G-quadruplex-hemin complexes. By using biotin as the affinity ligand, this simple protocol could sensitively detect streptavidin with a detection limit down to 10 pM. With the use of an antibody as the affinity ligand, a method for homogeneous fluorescence detection of Prostate Specific Antigen (PSA) was also proposed with a detection limit of 10 pM. The one-step and wash-free assay showed good selectivity. Its high sensitivity, acceptable accuracy, and satisfactory versatility of analytes led to various applications in bioanalysis. - Highlights: • AgNCs have great potential for application in biomedicine. • Binding of two affinity ligands can result in binding-induced DNA assemblies. • PET can be happened between DNA/AgNCs and G-quadruplex/hemin complexes. • A platform for the detection of proteins was proposed by using PET and binding-induced strategy.

  12. Development of an Efficient G-Quadruplex-Stabilised Thrombin-Binding Aptamer Containing a Three-Carbon Spacer Molecule

    DEFF Research Database (Denmark)

    Aaldering, Lukas J.; Poongavanam, Vasanthanathan; Langkjær, Niels


    -modified UNA (amino-UNA) on the structural dynamics and stability of TBA. All three modifications were incorporated at three different loop positions (T3, T7, T12) of the TBA G-quadruplex structure to result in a series of TBA variants and their stability was studied by thermal denaturation; folding...... was studied by circular dichroism spectroscopy and thrombin clotting time. The results showed that spacer-C3 introduction at the T7 loop position (TBA-SP7) significantly improved stability and thrombin clotting time while maintaining a similar binding affinity as TBA to thrombin. Detailed molecular modelling...

  13. High-resolution three-dimensional NMR structure of the KRAS proto-oncogene promoter reveals key features of a G-quadruplex involved in transcriptional regulation. (United States)

    Kerkour, Abdelaziz; Marquevielle, Julien; Ivashchenko, Stefaniia; Yatsunyk, Liliya A; Mergny, Jean-Louis; Salgado, Gilmar F


    Non-canonical base pairing within guanine-rich DNA and RNA sequences can produce G-quartets, whose stacking leads to the formation of a G-quadruplex (G4). G4s can coexist with canonical duplex DNA in the human genome and have been suggested to suppress gene transcription, and much attention has therefore focused on studying G4s in promotor regions of disease-related genes. For example, the human KRAS proto-oncogene contains a nuclease-hypersensitive element located upstream of the major transcription start site. The KRAS nuclease-hypersensitive element (NHE) region contains a G-rich element (22RT; 5'-AGGGCGGTGTGGGAATAGGGAA-3') and encompasses a Myc-associated zinc finger-binding site that regulates KRAS transcription. The NEH region therefore has been proposed as a target for new drugs that control KRAS transcription, which requires detailed knowledge of the NHE structure. In this study, we report a high-resolution NMR structure of the G-rich element within the KRAS NHE. We found that the G-rich element forms a parallel structure with three G-quartets connected by a four-nucleotide loop and two short one-nucleotide double-chain reversal loops. In addition, a thymine bulge is found between G8 and G9. The loops of different lengths and the presence of a bulge between the G-quartets are structural elements that potentially can be targeted by small chemical ligands that would further stabilize the structure and interfere or block transcriptional regulators such as Myc-associated zinc finger from accessing their binding sites on the KRAS promoter. In conclusion, our work suggests a possible new route for the development of anticancer agents that could suppress KRAS expression. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. A Role for the Fifth G-Track in G-Quadruplex Forming Oncogene Promoter Sequences during Oxidative Stress: Do These "Spare Tires" Have an Evolved Function? (United States)

    Fleming, Aaron M; Zhou, Jia; Wallace, Susan S; Burrows, Cynthia J


    Uncontrolled inflammation or oxidative stress generates electron-deficient species that oxidize the genome increasing its instability in cancer. The G-quadruplex (G4) sequences regulating the c-MYC , KRAS , VEGF , BCL-2 , HIF-1α , and RET oncogenes, as examples, are targets for oxidation at loop and 5'-core guanines (G) as showcased in this study by CO 3 •- oxidation of the VEGF G4. Products observed include 8-oxo-7,8-dihydroguanine (OG), spiroiminodihydantoin (Sp), and 5-guanidinohydantoin (Gh). Our previous studies found that OG and Gh, when present in the four G-tracks of the solved structure for VEGF and c-MY C, were not substrates for the base excision repair (BER) DNA glycosylases in biologically relevant KCl solutions. We now hypothesize that a fifth G-track found a few nucleotides distant from the G4 tracks involved in folding can act as a "spare tire," facilitating extrusion of a damaged G-run into a large loop that then becomes a substrate for BER. Thermodynamic, spectroscopic, and DMS footprinting studies verified the fifth domain replacing a damaged G-track with OG or Gh at a loop or core position in the VEGF G4. These new "spare tire"-containing strands with Gh in loops are now found to be substrates for initiation of BER with the NEIL1, NEIL2, and NEIL3 DNA glycosylases. The results support a hypothesis in which regulatory G4s carry a "spare-tire" fifth G-track for aiding in the repair process when these sequences are damaged by radical oxygen species, a feature observed in a large number of these sequences. Furthermore, formation and repair of oxidized bases in promoter regions may constitute an additional example of epigenetic modification, in this case of guanine bases, to regulate gene expression in which the G4 sequences act as sensors of oxidative stress.

  15. Cockayne syndrome group A and B proteins converge on transcription-linked resolution of non-B DNA

    DEFF Research Database (Denmark)

    Scheibye-Knudsen, Morten; Tseng, Anne; Jensen, Martin Borch


    of CSA or CSB in a neuroblastoma cell line converges on mitochondrial dysfunction caused by defects in ribosomal DNA transcription and activation of the DNA damage sensor poly-ADP ribose polymerase 1 (PARP1). Indeed, inhibition of ribosomal DNA transcription leads to mitochondrial dysfunction in a number...... to polymerase stalling at non-B DNA in a neuroblastoma cell line, in particular at G-quadruplex structures, and recombinant CSB can melt G-quadruplex structures. Indeed, stabilization of G-quadruplex structures activates PARP1 and leads to accelerated aging in Caenorhabditis elegans. In conclusion, this work...

  16. Disabling c-Myc in childhood medulloblastoma and atypical teratoid/rhabdoid tumor cells by the potent G-quadruplex interactive agent S2T1-6OTD. (United States)

    Shalaby, Tarek; von Bueren, André O; Hürlimann, Marie-Louise; Fiaschetti, Giulio; Castelletti, Deborah; Masayuki, Tera; Nagasawa, Kazuo; Arcaro, Alexandre; Jelesarov, Ilian; Shin-ya, Kazuo; Grotzer, Michael


    We investigated here the effects of S2T1-6OTD, a novel telomestatin derivative that is synthesized to target G-quadruplex-forming DNA sequences, on a representative panel of human medulloblastoma (MB) and atypical teratoid/rhabdoid (AT/RT) childhood brain cancer cell lines. S2T1-6OTD proved to be a potent c-Myc inhibitor through its high-affinity physical interaction with the G-quadruplex structure in the c-Myc promoter. Treatment with S2T1-6OTD reduced the mRNA and protein expressions of c-Myc and hTERT, which is transcriptionally regulated by c-Myc, and decreased the activities of both genes. In remarkable contrast to control cells, short-term (72-hour) treatment with S2T1-6OTD resulted in a dose- and time-dependent antiproliferative effect in all MB and AT/RT brain tumor cell lines tested (IC(50), 0.25-0.39 micromol/L). Under conditions where inhibition of both proliferation and c-Myc activity was observed, S2T1-6OTD treatment decreased the protein expression of the cell cycle activator cyclin-dependent kinase 2 and induced cell cycle arrest. Long-term treatment (5 weeks) with nontoxic concentrations of S2T1-6OTD resulted in a time-dependent (mainly c-Myc-dependent) telomere shortening. This was accompanied by cell growth arrest starting on day 28 followed by cell senescence and induction of apoptosis on day 35 in all of the five cell lines investigated. On in vivo animal testing, S2T1-6OTD may well represent a novel therapeutic strategy for childhood brain tumors.

  17. G-quadruplex recognition activities of E. Coli MutS

    Directory of Open Access Journals (Sweden)

    Ehrat Edward A


    Full Text Available Abstract Background Guanine quadruplex (G4 DNA is a four-stranded structure that contributes to genome instability and site-specific recombination. G4 DNA folds from sequences containing tandemly repetitive guanines, sequence motifs that are found throughout prokaryote and eukaryote genomes. While some cellular activities have been identified with binding or processing G4 DNA, the factors and pathways governing G4 DNA metabolism are largely undefined. Highly conserved mismatch repair factors have emerged as potential G4-responding complexes because, in addition to initiating heteroduplex correction, the human homologs bind non-B form DNA with high affinity. Moreover, the MutS homologs across species have the capacity to recognize a diverse range of DNA pairing variations and damage, suggesting a conserved ability to bind non-B form DNA. Results Here, we asked if E. coli MutS and a heteroduplex recognition mutant, MutS F36A, were capable of recognizing and responding to G4 DNA structures. We find by mobility shift assay that E. coli MutS binds to G4 DNA with high affinity better than binding to G-T heteroduplexes. In the same assay, MutS F36A failed to recognize G-T mismatched oligonucleotides, as expected, but retained an ability to bind to G4 DNA. Association with G4 DNA by MutS is not likely to activate the mismatch repair pathway because nucleotide binding did not promote release of MutS or MutS F36A from G4 DNA as it does for heteroduplexes. G4 recognition activities occur under physiological conditions, and we find that M13 phage harboring G4-capable DNA poorly infected a MutS deficient strain of E. coli compared to M13mp18, suggesting functional roles for mismatch repair factors in the cellular response to unstable genomic elements. Conclusions Taken together, our findings demonstrate that E. coli MutS has a binding activity specific for non-B form G4 DNA, but such binding appears independent of canonical heteroduplex repair activation.

  18. The SARS-unique domain (SUD of SARS coronavirus contains two macrodomains that bind G-quadruplexes.

    Directory of Open Access Journals (Sweden)

    Jinzhi Tan


    Full Text Available Since the outbreak of severe acute respiratory syndrome (SARS in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV, the non-structural proteins (Nsps, have been determined. However, within the large Nsp3 (1922 amino-acid residues, the structure and function of the so-called SARS-unique domain (SUD have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389-652 ("SUD(core" of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 A resolution, respectively revealed that SUD(core forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUD(core as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5-6 nucleotides, but more extended G-stretches are found in the 3'-nontranslated regions of mRNAs coding for certain host-cell proteins

  19. Insights into the Structure of Intrastrand Cross-Link DNA Lesion-Containing Oligonucleotides: G[8-5m]T and G[8-5]C from Molecular Dynamics Simulations

    Czech Academy of Sciences Publication Activity Database

    Dumont, E.; Dršata, Tomáš; Guerra, C. F.; Lankaš, Filip


    Roč. 54, č. 5 (2015), s. 1259-1267 ISSN 0006-2960 R&D Projects: GA ČR(CZ) GA14-21893S Institutional support: RVO:61388963 Keywords : hydrogen bonds * abasic sites * duplex DNA Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.876, year: 2015

  20. A G-quadruplex based fluorescent oligonucleotide turn-on probe towards iodides detection in real samples. (United States)

    Li, Qian; Li, Shuaihua; Chen, Xiu; Bian, Liujiao


    A basket-type G-quadruplex (GQ) fluorescent oligonucleotide (OND) probe is designed to detect iodides dependent on thymine-Hg(II)-thymine (T-Hg(II)-T) base pairs and the intrinsic fluorescence quenching capacity of GQ. In the presence of Hg(II) ions (Hg 2+ ), the two hexachloro-fluorescein-labeled ONDs form a hairpin structure and the fluorophores are dragged close to the GQ, leading to fluorescence quenching of the probe due to photoinduced electron transfer. Upon addition of iodide anions, Hg 2+ are extracted from T-Hg(II)-T complexes which attributes to the stronger binding with iodide anions, resulting in the fluorescence recovery. Through performing the fluorescence quenching and recovery processes, this probe developed a fluorescence turn-on sensor for iodide anions determination over a linear range of 20-200nmol/L with a limit of detection of 5nmol/L. The practical use of the turn-on technology was demonstrated by its application in determination of iodides in water, food, pharmaceutical products and biological samples. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Hybrid ligand-alkylating agents targeting telomeric G-quadruplex structures. (United States)

    Doria, Filippo; Nadai, Matteo; Folini, Marco; Di Antonio, Marco; Germani, Luca; Percivalle, Claudia; Sissi, Claudia; Zaffaroni, Nadia; Alcaro, Stefano; Artese, Anna; Richter, Sara N; Freccero, Mauro


    The synthesis, physico-chemical properties and biological effects of a new class of naphthalene diimides (NDIs) capable of reversibly binding telomeric DNA and alkylate it through an electrophilic quinone methide moiety (QM), are reported. FRET and circular dichroism assays showed a marked stabilization and selectivity towards telomeric G4 DNA folded in a hybrid topology. NDI-QMs' alkylating properties revealed a good reactivity on single nucleosides and selectivity towards telomeric G4. A selected NDI was able to significantly impair the growth of melanoma cells by causing telomere dysfunction and down-regulation of telomerase expression. These findings points to our hybrid ligand-alkylating NDIs as possible tools for the development of novel targeted anticancer therapies. This journal is © The Royal Society of Chemistry 2012

  2. A label-free luminescent switch-on assay for ATP using a G-quadruplex-selective iridium(III) complex. (United States)

    Leung, Ka-Ho; Lu, Lihua; Wang, Modi; Mak, Tsun-Yin; Chan, Daniel Shiu-Hin; Tang, Fung-Kit; Leung, Chung-Hang; Kwan, Hiu-Yee; Yu, Zhiling; Ma, Dik-Lung


    We report herein the G-quadruplex-selective property of a luminescent cyclometallated iridium(III) complex for the detection of adenosine-5'-triphosphate (ATP) in aqueous solution. The ATP-binding aptamer was employed as the ATP recognition unit, while the iridium(III) complex was used to monitor the formation of the G-quadruplex structure induced by ATP. The sensitivity and fold enhancement of the assay were higher than those of the previously reported assay using the organic dye crystal violet as a fluorescent probe. This label-free luminescent switch-on assay exhibits high sensitivity and selectivity towards ATP with a limit of detection of 2.5 µM.

  3. A label-free luminescent switch-on assay for ATP using a G-quadruplex-selective iridium(III complex.

    Directory of Open Access Journals (Sweden)

    Ka-Ho Leung

    Full Text Available We report herein the G-quadruplex-selective property of a luminescent cyclometallated iridium(III complex for the detection of adenosine-5'-triphosphate (ATP in aqueous solution. The ATP-binding aptamer was employed as the ATP recognition unit, while the iridium(III complex was used to monitor the formation of the G-quadruplex structure induced by ATP. The sensitivity and fold enhancement of the assay were higher than those of the previously reported assay using the organic dye crystal violet as a fluorescent probe. This label-free luminescent switch-on assay exhibits high sensitivity and selectivity towards ATP with a limit of detection of 2.5 µM.

  4. Clustered abasic lesions profoundly change the structure and stability of human telomeric G-quadruplexes

    Czech Academy of Sciences Publication Activity Database

    Kejnovská, Iva; Bednářová, Klára; Renčiuk, Daniel; Dvořáková, Zuzana; Školáková, Petra; Trantírek, L.; Fiala, R.; Vorlíčková, Michaela; Sagi, J.


    Roč. 45, č. 8 (2017), s. 4294-4305 ISSN 0305-1048 R&D Projects: GA MŠk EF15_003/0000477; GA ČR GAP205/12/0466; GA ČR GA13-28310S; GA ČR(CZ) GA15-06785S; GA MŠk(CZ) LQ1601 Institutional support: RVO:68081707 Keywords : dna-damage clusters * k+ solution * guanine quadruplexes Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 10.162, year: 2016

  5. 1,2-GG intrastrand cross-link of antitumor dinuclear bifunctional platinum compound with spermidine linker inhibits DNA polymerization more effectively than the cross-link of conventional cisplatin

    Czech Academy of Sciences Publication Activity Database

    Moriarity, B.; Nováková, Olga; Farrell, N.; Brabec, Viktor; Kašpárková, Jana


    Roč. 459, č. 2 (2007), s. 264-272 ISSN 0003-9861 R&D Projects: GA ČR(CZ) GA305/05/2030; GA ČR(CZ) GA203/06/1239; GA MZd(CZ) NR8562; GA MŠk(CZ) LC06030; GA AV ČR(CZ) 1QS500040581; GA AV ČR(CZ) KAN200200651 Institutional research plan: CEZ:AV0Z50040702 Keywords : cancer * DNA * platinum Subject RIV: BO - Biophysics Impact factor: 2.578, year: 2007

  6. Quinolino[3,4-b]quinoxalines and pyridazino[4,3-c]quinoline derivatives: Synthesis, inhibition of topoisomerase IIα, G-quadruplex binding and cytotoxic properties. (United States)

    Palluotto, Fausta; Sosic, Alice; Pinato, Odra; Zoidis, Grigoris; Catto, Marco; Sissi, Claudia; Gatto, Barbara; Carotti, Angelo


    The quinoline motif fused with other heterocyclic systems plays an important role in the field of anticancer drug development. An extensive series of tetracyclic quinolino[3,4-b]quinoxalines N-5 or C-6 substituted with basic side chain and a limited number of tricyclic pyridazino[4,3-c]quinolines N-6 substituted were designed, synthesized and evaluated for topoisomerase IIα (Topo IIα) inhibitory activity, ability to bind and stabilize G-quadruplex structures and cytotoxic properties against two human cancer cell lines (HeLa and MCF-7). Almost all of the tested agents showed a high activity as Topo IIα inhibitors and G-quadruplex stabilizers. Among all the derivatives studied, the quinolino[3,4-b]quinoxalines 11 and 23, N-5 and C-6 substituted respectively, stand out as the most promising compounds. Derivative 11 resulted a selective binder to selected G-quadruplex sequences, while derivative 23 displayed the most interesting Topo IIα inhibitory activity (IC50 = 5.14 μM); both showed high cytotoxic activity (IC50 HeLa = 2.04 μM and 2.32 μM, respectively). Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  7. Human telomeric G-quadruplex formation and highly selective fluorescence detection of toxic strontium ions. (United States)

    Qu, Konggang; Zhao, Chuanqi; Ren, Jinsong; Qu, Xiaogang


    Strontium ions play important roles in biological systems. The inhalation of strontium can cause severe respiratory difficulties, anaphylactic reaction and extreme tachycardia. Strontium can replace calcium in organisms, inhibit normal calcium absorption and induce strontium "rickets" in childhood. Thus, the development of sensitive and selective methods for the determination of trace amounts of Sr(2+) in aqueous media is of considerable importance for environmental and human health protection. A number of methodologies, such as X-ray energy dispersive spectrometry, inductively coupled argon plasma atomic emission spectroscopy (ICP-AES), atomic absorption spectrometry (AAS) and instrumental thermal neutron activation analysis, have been reported. However, these methods are somewhat complex, costly, time consuming and, especially, need special instruments. Thus, the design of convenient and inexpensive approaches for the sensitive and selective detection of Sr(2+) with rapid, easy manipulation is in ever-increasing demand. To the best of our knowledge, using DNA conformational change to detect Sr(2+) has not yet been reported. Herein we utilized thiazole orange (TO) as a signal reporter to devise a simple Sr(2+) detection assay based on Sr(2+) induced human telomeric DNA conformational change in the presence of SWNTs. The limit of detection is 10 nM Sr(2+) (0.87 μg L(-1)), far below 4 mg L(-1), the U.S. Federal threshold in drinking water defined by the U.S. EPA.

  8. Identifying the impact of G-quadruplexes on Affymetrix 3' arrays using cloud computing. (United States)

    Memon, Farhat N; Owen, Anne M; Sanchez-Graillet, Olivia; Upton, Graham J G; Harrison, Andrew P


    A tetramer quadruplex structure is formed by four parallel strands of DNA/ RNA containing runs of guanine. These quadruplexes are able to form because guanine can Hoogsteen hydrogen bond to other guanines, and a tetrad of guanines can form a stable arrangement. Recently we have discovered that probes on Affymetrix GeneChips that contain runs of guanine do not measure gene expression reliably. We associate this finding with the likelihood that quadruplexes are forming on the surface of GeneChips. In order to cope with the rapidly expanding size of GeneChip array datasets in the public domain, we are exploring the use of cloud computing to replicate our experiments on 3' arrays to look at the effect of the location of G-spots (runs of guanines). Cloud computing is a recently introduced high-performance solution that takes advantage of the computational infrastructure of large organisations such as Amazon and Google. We expect that cloud computing will become widely adopted because it enables bioinformaticians to avoid capital expenditure on expensive computing resources and to only pay a cloud computing provider for what is used. Moreover, as well as financial efficiency, cloud computing is an ecologically-friendly technology, it enables efficient data-sharing and we expect it to be faster for development purposes. Here we propose the advantageous use of cloud computing to perform a large data-mining analysis of public domain 3' arrays.

  9. Regulation of gene expression by the BLM helicase correlates with the presence of G-quadruplex DNA motifs

    DEFF Research Database (Denmark)

    Nguyen, Giang Huong; Tang, Weiliang; Robles, Ana I


    Bloom syndrome is a rare autosomal recessive disorder characterized by genetic instability and cancer predisposition, and caused by mutations in the gene encoding the Bloom syndrome, RecQ helicase-like (BLM) protein. To determine whether altered gene expression might be responsible for pathologic...

  10. Label-free and nicking enzyme-assisted fluorescence signal amplification for RNase H determination based on a G-quadruplexe/thioflavin T complex. (United States)

    Wu, Kefeng; Ma, Changbei; Deng, Zhiyi; Fang, Ning; Tang, Zhenwei; Zhu, Xingxing; Wang, Kemin


    In this paper, we describe a novel, label-free and nicking enzyme-assisted fluorescence signal amplification strategy that demonstrates to be cost efficient, sensitive, and unique for assaying the RNase H activity and inhibition based on G-quadruplex formation using a thioflavin T (ThT) dye. This novel assay method is able to detect RNase H with a detection limit of 0.03 U /mL and further exhibits a good linearity R 2 = 0.9923 at a concentration range of 0.03-1 U/mL under optimized conditions. Moreover, the inhibition effect of gentamycin on the RNase H activity is also studied. This strategy provides a potential tool for the biochemical enzyme analysis and inhibitor screening. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Investigation of spectral conversion of d(TTAGGG)4 and d(TTAGGG)13 upon potassium titration by a G-quadruplex recognizer BMVC molecule. (United States)

    Chang, Cheng-Chung; Chien, Chih-Wei; Lin, Yi-Hsueh; Kang, Chi-Chih; Chang, Ta-Chau


    We have introduced a G-quadruplex-binding ligand, 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC), to verify the major structure of d(T2AG3)4 (H24) in potassium solution and examine the structural conversion of H24 in sodium solution upon potassium titration. The studies of circular dichroism, induced circular dichroism, spectral titration and gel competition have allowed us to determine the binding mode and binding ratio of BMVC to the H24 in solution and eliminate the parallel form as the major G-quadruplex structure. Although the mixed-type form could not be eliminated as a main component, the basket and chair forms are more likely the main components of H24 in potassium solution. In addition, the circular dichroism spectra and the job plots reveal that a longer telomeric sequence d(T2AG3)13 (H78) could form two units of G4 structure both in sodium or potassium solutions. Of particular interest is that no appreciable change on the induced circular dichroism spectra of BMVC is found during the change of the circular dichroism patterns of H24 upon potassium titration. Considering similar spectral conversion detected for H24 and a long sequence H78 together with the G4 structure stabilized by BMVC, it is therefore unlikely that the rapid spectral conversion of H24 and H78 is due to structural change between different types of the G4 structures. With reference to the circular dichroism spectra of d(GAA)7 and d(GAAA)5, we suggest that the spectral conversion of H24 upon potassium titration is attributed to fast ion exchange resulting in different loop base interaction and various hydrogen bonding effects.

  12. A sensitive electrochemical aptasensor based on the co-catalysis of hemin/G-quadruplex, platinum nanoparticles and flower-like MnO2 nanosphere functionalized multi-walled carbon nanotubes. (United States)

    Xu, Wenju; Xue, Shuyan; Yi, Huayu; Jing, Pei; Chai, Yaqin; Yuan, Ruo


    In this work, a sensitive electrochemical aptasensor for the detection of thrombin (TB) is developed and demonstrated based on the co-catalysis of hemin/G-quadruplex, platinum nanoparticles (PtNPs) and flower-like MnO2 nanosphere functionalized multi-walled carbon nanotubes (MWCNT-MnO2).

  13. Glucose oxidase-initiated cascade catalysis for sensitive impedimetric aptasensor based on metal-organic frameworks functionalized with Pt nanoparticles and hemin/G-quadruplex as mimicking peroxidases. (United States)

    Zhou, Xingxing; Guo, Shijing; Gao, Jiaxi; Zhao, Jianmin; Xue, Shuyan; Xu, Wenju


    Based on cascade catalysis amplification driven by glucose oxidase (GOx), a sensitive electrochemical impedimetric aptasensor for protein (carcinoembryonic antigen, CEA as tested model) was proposed by using Cu-based metal-organic frameworks functionalized with Pt nanoparticles, aptamer, hemin and GOx (Pt@CuMOFs-hGq-GOx). CEA aptamer loaded onto Pt@CuMOFs was bound with hemin to form hemin@G-quadruplex (hGq) with mimicking peroxidase activity. Through sandwich-type reaction of target CEA and CEA aptamers (Apt1 and Apt2), the obtained Pt@CuMOFs-hGq-GOx as signal transduction probes (STPs) was captured to the modified electrode interface. When 3,3-diaminobenzidine (DAB) and glucose were introduced, the cascade reaction was initiated by GOx to catalyze the oxidation of glucose, in situ generating H 2 O 2 . Simultaneously, the decomposition of the generated H 2 O 2 was greatly promoted by Pt@CuMOFs and hGq as synergistic peroxide catalysts, accompanying with the significant oxidation process of DAB and the formation of nonconductive insoluble precipitates (IPs). As a result, the electron transfer in the resultant sensing interface was effectively hindered and the electrochemical impedimetric signal (EIS) was efficiently amplified. Thus, the high sensitivity of the proposed CEA aptasensor was successfully improved with 0.023pgmL -1 , which may be promising and potential in assaying certain clinical disease related to CEA. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Flower bud transcriptome analysis of Sapium sebiferum (Linn. Roxb. and primary investigation of drought induced flowering: pathway construction and G-quadruplex prediction based on transcriptome.

    Directory of Open Access Journals (Sweden)

    Minglei Yang

    Full Text Available Sapium sebiferum (Linn. Roxb. (Chinese Tallow Tree is a perennial woody tree and its seeds are rich in oil which hold great potential for biodiesel production. Despite a traditional woody oil plant, our understanding on S. sebiferum genetics and molecular biology remains scant. In this study, the first comprehensive transcriptome of S. sebiferum flower has been generated by sequencing and de novo assembly. A total of 149,342 unigenes were generated from raw reads, of which 24,289 unigenes were successfully matched to public database. A total of 61 MADS box genes and putative pathways involved in S. sebiferum flower development have been identified. Abiotic stress response network was also constructed in this work, where 2,686 unigenes are involved in the pathway. As for lipid biosynthesis, 161 unigenes have been identified in fatty acid (FA and triacylglycerol (TAG biosynthesis. Besides, the G-Quadruplexes in RNA of S. sebiferum also have been predicted. An interesting finding is that the stress-induced flowering was observed in S. sebiferum for the first time. According to the results of semi-quantitative PCR, expression tendencies of flowering-related genes, GA1, AP2 and CRY2, accorded with stress-related genes, such as GRX50435 and PRXⅡ39562. This transcriptome provides functional genomic information for further research of S. sebiferum, especially for the genetic engineering to shorten the juvenile period and improve yield by regulating flower development. It also offers a useful database for the research of other Euphorbiaceae family plants.

  15. DNA and RNA Quadruplex-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Václav Brázda


    Full Text Available Four-stranded DNA structures were structurally characterized in vitro by NMR, X-ray and Circular Dichroism spectroscopy in detail. Among the different types of quadruplexes (i-Motifs, minor groove quadruplexes, G-quadruplexes, etc., the best described are G-quadruplexes which are featured by Hoogsteen base-paring. Sequences with the potential to form quadruplexes are widely present in genome of all organisms. They are found often in repetitive sequences such as telomeric ones, and also in promoter regions and 5' non-coding sequences. Recently, many proteins with binding affinity to G-quadruplexes have been identified. One of the initially portrayed G-rich regions, the human telomeric sequence (TTAGGGn, is recognized by many proteins which can modulate telomerase activity. Sequences with the potential to form G-quadruplexes are often located in promoter regions of various oncogenes. The NHE III1 region of the c-MYC promoter has been shown to interact with nucleolin protein as well as other G-quadruplex-binding proteins. A number of G-rich sequences are also present in promoter region of estrogen receptor alpha. In addition to DNA quadruplexes, RNA quadruplexes, which are critical in translational regulation, have also been predicted and observed. For example, the RNA quadruplex formation in telomere-repeat-containing RNA is involved in interaction with TRF2 (telomere repeat binding factor 2 and plays key role in telomere regulation. All these fundamental examples suggest the importance of quadruplex structures in cell processes and their understanding may provide better insight into aging and disease development.

  16. Structural revisions of small molecules reported to cross-link G-quadruplex DNA in vivo reveal a repetitive assignment error in the literature

    Czech Academy of Sciences Publication Activity Database

    Reyes Gutierrez, Paul Eduardo; Kapal, Tomáš; Klepetářová, Blanka; Šaman, David; Pohl, Radek; Zawada, Zbigniew; Kužmová, Erika; Hájek, Miroslav; Teplý, Filip


    Roč. 6, Mar 23 (2016), č. článku 23499. ISSN 2045-2322 R&D Projects: GA ČR GA13-19213S; GA MŠk LO1302 Grant - others:AV ČR(CZ) M200551208 Institutional support: RVO:61388963 Keywords : 1,2-disubstituted benzimidazoles * chemical synthesis * promoter region Subject RIV: CC - Organic Chemistry Impact factor: 4.259, year: 2016

  17. Derivation of Reliable Geometries in QM Calculations of DNA Structures: Explicit Solvent QM/MM and Restrained Implicit Solvent QM Optimizations of G-Quadruplexes

    Czech Academy of Sciences Publication Activity Database

    Gkionis, Konstantinos; Kruse, Holger; Šponer, Jiří


    Roč. 12, č. 4 (2016), s. 2000-2016 ISSN 1549-9618 R&D Projects: GA ČR(CZ) GAP208/11/1822 Institutional support: RVO:68081707 Keywords : molecular- dynamics simulations * quantum-chemical computations * continuum solvation models Subject RIV: BO - Biophysics Impact factor: 5.245, year: 2016

  18. Real-Time Study of the Interaction between G-Rich DNA Oligonucleotides and Lead Ion on DNA Tetrahedron-Functionalized Sensing Platform by Dual Polarization Interferometry. (United States)

    Wang, Shuang; Lu, Shasha; Zhao, Jiahui; Huang, Jianshe; Yang, Xiurong


    G-quadruplex plays roles in numerous physiological and pathological processes of organisms. Due to the unique properties of G-quadruplex (e.g., forming G4/hemin complexes with catalytic activity and electron acceptability, binding with metal ions, proteins, fluorescent ligands, and so on), it has been widely applied in biosensing. But the formation process of G-quadruplex is not yet fully understood. Here, a DNA tetrahedron platform with higher reproducibility, regenerative ability, and time-saving building process was coupled with dual polarization interferometry technique for the real-time and label-free investigation of the specific interaction process of guanine-rich singled-stranded DNA (G-rich ssDNA) and Pb 2+ . The oriented immobilization of probes greatly decreased the spatial hindrance effect and improved the accessibility of the probes to the Pb 2+ ions. Through real-time monitoring of the whole formation process of the G-quadruplex, we speculated that the probes on the tetrahedron platform initially stood on the sensing surface with a random coil conformation, then the G-rich ssDNA preliminarily formed unstable G-quartets by H-bonding and cation binding, subsequently forming a completely folded and stable quadruplex structure through relatively slow strand rearrangements. On the basis of these studies, we also developed a novel sensing platform for the specific and sensitive determination of Pb 2+ and its chelating agent ethylenediaminetetraacetic acid. This study not only provides a proof-of-concept for conformational dynamics of G-quadruplex-related drugs and pathogenes, but also enriches the biosensor tools by combining nanomaterial with interfaces technique.

  19. DNA damage by reactive species: Mechanisms, mutation and repair

    Indian Academy of Sciences (India)

    DNA is continuously attacked by reactive species that can affect its structure and function severely. Structural modifications to DNA mainly arise from modifications in its bases that primarily occur due to their exposure to different reactive species. Apart from this, DNA strand break, inter- and intra-strand crosslinks and ...

  20. Bis-guanylhydrazone diimidazo[1,2-a:1,2-c]pyrimidine as a novel and specific G-quadruplex binding motif. (United States)

    Sparapani, Silvia; Bellini, Stefania; Gunaratnam, Mekala; Haider, Shozeb M; Andreani, Aldo; Rambaldi, Mirella; Locatelli, Alessandra; Morigi, Rita; Granaiola, Massimiliano; Varoli, Lucilla; Burnelli, Silvia; Leoni, Alberto; Neidle, Stephen


    A bis-guanylhydrazone derivative of diimidazo[1,2-a:1,2-c]pyrimidine has unexpectedly been found to be a potent stabiliser of several quadruplex DNAs, whereas there is no significant interaction with duplex DNA. Molecular modeling suggests that the guanylhydrazone groups play an active role in quadruplex binding.

  1. Coarse-Grained Simulations Complemented by Atomistic Molecular Dynamics Provide New Insights into Folding and Unfolding of Human Telomeric G-Quadruplexes

    Czech Academy of Sciences Publication Activity Database

    Stadlbauer, Petr; Mazzanti, L.; Cragnolini, T.; Wales, D.J.; Derreumaux, P.; Pasquali, C.; Sponer, Jiri


    Roč. 12, č. 12 (2016), s. 6077-6097 ISSN 1549-9618 R&D Projects: GA ČR(CZ) GA16-13721S Institutional support: RVO:68081707 Keywords : particle mesh ewald * amber force-field * free-energy profiles * binding dna aptamer Subject RIV: BO - Biophysics Impact factor: 5.245, year: 2016

  2. Electrochemical single-molecule conductivity of duplex and quadruplex DNA

    DEFF Research Database (Denmark)

    Zhang, Ling; Zhang, Jingdong; Ulstrup, Jens


    Photoinduced and electrochemical charge transport in DNA (oligonucleotides, OGNs) and the notions “hopping”, superexchange, polaron, and vibrationally gated charge transport have been in focus over more than two decades. In recent years mapping of electrochemical charge transport of pure and redox...... marked single- and double-strand OGNs has reached the single-molecule level based i.a. on electrochemical in situ scanning tunnelling microscopy (STM) and break-junction (B-J) STM. There are much fewer such reports on “non-canonical” OGN structures such as G-quadruplexes. We discuss first single......-molecule electrochemical conductivity of pure and redox marked duplex OGNs, and address next electrochemistry and electrochemical conductivity in the few reported monolayer and single-molecule G-quadruplex studies. Facile electrochemical electron transfer of iron protoporphyrin IX stacked onto three-quartet 12-guanine...

  3. Voltammetry and Molecular Assembly of G-quadruplex DNAzyme on Single-crystal Au(111)-electrode Surfaces – Hemin as an Electrochemical Intercalator

    DEFF Research Database (Denmark)

    Zhang, Ling; Ulstrup, Jens; Zhang, Jingdong


    ). The target DNZyme is formed from a single-strand OGN with 12 guanines and iron (III) porphyrin IX (hemin), and assembles on Au(111) by the mercapto alkyl linker. The DNAzyme monolayers exhibit a strong pair of redox peaks at 0.0 V (NHE) at pH 7 in acetate buffer, shifted positively by about 50 mV compared......DNA quadruplexes (qs’s) are a class of “non-canonical” oligonucleotides (OGNs) composed of stacked guanine (G) quartets generally stabilized by monovalent cations. Metal porphyrins selectively bind to G-qs complexes to form DNAzyme, which can exhibit peroxidase and other catalytic activity similar...... to free hemin physisorbed on the Au(111). The voltammetric signal of DNZyme is enhanced 15 times, indicative that hemin is strongly bound to the immobilized 12G-qs and in well-defined orientation favorable for interfacial ET with a rate constant of 6.0 s−1. The G-quartet structures with a size of 1.6 ± 0...

  4. Synthesis and Characterization of thermo/pH-responsive Supramolecular G-Quadruplexes for the Construction of Supramolecular Hacky Sacks for Biorelevant Applications (United States)

    Negron Rios, Luis M.

    The impact of size, shape, and distribution of lipophilic regions on the surfaces of nanoscopic objects that are amphiphilic or patchy (such as proteins) are yet to be fully understood. One of the reasons for this is the lack of an appropriate model systems in which to probe this question. Our group has previously reported 2'-deoxyguanosine (8ArG) derivatives that self-assemble in aqueous media into discrete supramolecular hexadecamers that show the lower critical solution temperature (LCST) phenomenon. The LCST phenomenon is a convenient and rigorous strategy to measure the hydrophobicity of a system. Although these SGQs are potentially attractive for biomedical applications like drug-delivery, the narrow window of physiological temperatures complicates their implementation. This moved us to redesign the constituent 8ArG subunits to incorporate imidazole moieties that would lead to pH-responsive SGQs, working isothermally. Upon reaching a threshold temperature (Lower Critical Solution Temperature, LCST) at pH 7, these dual-responsive SGQs further self-assemble to form nano/micro hydrogel globules that we called them supramolecular hacky sacks (SHS). However, we can isolate kinetically stable versions of these SHS by lowering the ionic strength of the medium (i.e., from the molar to the millimolar range) in a process that we term "fixing the SHS", in which these SHS maintain their integrity (size and shape) and stability without the requirement of crosslinking agents. After structural characterization and in vitro studies of SHS, we performed encapsulation studies of DOX, rhodamine, dsDNA (F26T), thrombin binding aptamer (TBA) and dextran (3 kDa) Texas Red conjugate. Then we performed in vivo studies of cell internalization and drug delivery with neuroblastoma SY-SH5Y. The performed studies will bring new approaches for the development of new biotechnology for fundamental applications and the emerging of novel therapeutic agents for biomedical applications.

  5. Evaluation of the Stability of DNA i-Motifs in the Nuclei of Living Mammalian Cells

    Czech Academy of Sciences Publication Activity Database

    Dzatko, S.; Krafčíková, M.; Haensel-Hertsch, R.; Fessl, T.; Fiala, R.; Loja, T.; Krafčík, D.; Mergny, Jean-Louis; Foldynova-Trantirkova, Silvie; Trantírek, L.


    Roč. 57, č. 8 (2018), s. 2165-2169 ISSN 1433-7851 R&D Projects: GA MŠk EF15_003/0000477 Institutional support: RVO:68081707 Keywords : g-quadruplex * telomeric dna * base-pairs * molecular switch Subject RIV: CG - Electrochemistry OBOR OECD: Electrochemistry (dry cells, batteries, fuel cells, corrosion metals, electrolysis) Impact factor: 11.994, year: 2016

  6. i-Motif of cytosine-rich human telomere DNA fragments containing natural base lesions

    Czech Academy of Sciences Publication Activity Database

    Dvořáková, Zuzana; Renčiuk, Daniel; Kejnovská, Iva; Školáková, Petra; Bednářová, Klára; Sagi, J.; Vorlíčková, Michaela


    Roč. 46, č. 4 (2018), s. 1624-1634 ISSN 1362-4962 R&D Projects: GA ČR(CZ) GA15-06785S; GA ČR GA17-12075S; GA ČR(CZ) GJ17-19170Y; GA MŠk EF15_003/0000477 Institutional support: RVO:68081707 Keywords : pair opening kinetics * g-quadruplex dna Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology

  7. New insights into transcription fidelity: thermal stability of non-canonical structures in template DNA regulates transcriptional arrest, pause, and slippage.

    Directory of Open Access Journals (Sweden)

    Hisae Tateishi-Karimata

    Full Text Available The thermal stability and topology of non-canonical structures of G-quadruplexes and hairpins in template DNA were investigated, and the effect of non-canonical structures on transcription fidelity was evaluated quantitatively. We designed ten template DNAs: A linear sequence that does not have significant higher-order structure, three sequences that form hairpin structures, and six sequences that form G-quadruplex structures with different stabilities. Templates with non-canonical structures induced the production of an arrested, a slipped, and a full-length transcript, whereas the linear sequence produced only a full-length transcript. The efficiency of production for run-off transcripts (full-length and slipped transcripts from templates that formed the non-canonical structures was lower than that from the linear. G-quadruplex structures were more effective inhibitors of full-length product formation than were hairpin structure even when the stability of the G-quadruplex in an aqueous solution was the same as that of the hairpin. We considered that intra-polymerase conditions may differentially affect the stability of non-canonical structures. The values of transcription efficiencies of run-off or arrest transcripts were correlated with stabilities of non-canonical structures in the intra-polymerase condition mimicked by 20 wt% polyethylene glycol (PEG. Transcriptional arrest was induced when the stability of the G-quadruplex structure (-ΔG°37 in the presence of 20 wt% PEG was more than 8.2 kcal mol(-1. Thus, values of stability in the presence of 20 wt% PEG are an important indicator of transcription perturbation. Our results further our understanding of the impact of template structure on the transcription process and may guide logical design of transcription-regulating drugs.

  8. Structural dynamics of possible late-stage intermediates in folding of quadruplex DNA studied by molecular simulations

    Czech Academy of Sciences Publication Activity Database

    Stadlbauer, Petr; Krepl, Miroslav; Cheatham III, T. E.; Koča, J.; Šponer, Jiří


    Roč. 41, č. 14 (2013), s. 7128-7143 ISSN 0305-1048 R&D Projects: GA ČR(CZ) GAP208/11/1822; GA MŠk(CZ) ED1.1.00/02.0068 Institutional support: RVO:68081707 Keywords : HUMAN TELOMERIC DNA * TETRAMOLECULAR G-QUADRUPLEXES * PARTICLE MESH EWALD Subject RIV: BO - Biophysics Impact factor: 8.808, year: 2013

  9. RNA Polymerase III Regulates Cytosolic RNA:DNA Hybrids and Intracellular MicroRNA Expression*


    Koo, Christine Xing'er; Kobiyama, Kouji; Shen, Yu J.; LeBert, Nina; Ahmad, Shandar; Khatoo, Muznah; Aoshi, Taiki; Gasser, Stephan; Ishii, Ken J.


    RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cy...

  10. Triplex-forming twisted intercalating nucleic acids (TINAs): design rules, stabilization of antiparallel DNA triplexes and inhibition of G-quartet-dependent self-association. (United States)

    Doluca, Osman; Boutorine, Alexandre S; Filichev, Vyacheslav V


    The majority of studies on DNA triple helices have been focused on pH-sensitive parallel triplexes with Hoogsteen CT-containing third strands that require protonation of cytosines. Reverse Hoogsteen GT/GA-containing antiparallel triplex-forming oligonucleotides (TFOs) do not require an acidic pH but their applicability in triplex technology is limited because of their tendency to form undesired highly stable aggregates such as G-quadruplexes. In this study, G-rich oligonucleotides containing 2-4 insertions of twisted intercalating nucleic acid(TINA) monomers are demonstrated to disrupt the formation of G-quadruplexes and form stable antiparallel triplexes with target DNA duplexes. The structure of TINA-incorporated oligonucleotides was optimized, the rules of their design were established and the optimal triplex-forming oligonucleotides were selected. These oligonucleotides show high affinity towards a 16 bp homopurine model sequence from the HIV-1 genome; dissociation constants as low as 160 nM are observed whereas the unmodified TFO does not show any triplex formation and instead forms an intermolecular G-quadruplex with T(m) exceeding 90°C in the presence of 50 mM NaCl. Here we present a set of rules that help to reach the full potential of TINATFOs and demonstrate the effect of TINA on the formation and stability of triple helical DNA.

  11. Exploring possible DNA structures in real-time polymerase kinetics using Pacific Biosciences sequencer data. (United States)

    Sawaya, Sterling; Boocock, James; Black, Michael A; Gemmell, Neil J


    Pausing of DNA polymerase can indicate the presence of a DNA structure that differs from the canonical double-helix. Here we detail a method to investigate how polymerase pausing in the Pacific Biosciences sequencer reads can be related to DNA sequences. The Pacific Biosciences sequencer uses optics to view a polymerase and its interaction with a single DNA molecule in real-time, offering a unique way to detect potential alternative DNA structures. We have developed a new way to examine polymerase kinetics data and relate it to the DNA sequence by using a wavelet transform of read information from the sequencer. We use this method to examine how polymerase kinetics are related to nucleotide base composition. We then examine tandem repeat sequences known for their ability to form different DNA structures: (CGG)n and (CG)n repeats which can, respectively, form G-quadruplex DNA and Z-DNA. We find pausing around the (CGG)n repeat that may indicate the presence of G-quadruplexes in some of the sequencer reads. The (CG)n repeat does not appear to cause polymerase pausing, but its kinetics signature nevertheless suggests the possibility that alternative nucleotide conformations may sometimes be present. We discuss the implications of using our method to discover DNA sequences capable of forming alternative structures. The analyses presented here can be reproduced on any Pacific Biosciences kinetics data for any DNA pattern of interest using an R package that we have made publicly available.

  12. The effect of single nucleotide polymorphisms in G-rich regions of high-risk human papillomaviruses on structural diversity of DNA. (United States)

    Marušič, Maja; Hošnjak, Lea; Krafčikova, Petra; Poljak, Mario; Viglasky, Viktor; Plavec, Janez


    Infection with high-risk human papillomaviruses (HPVs) can lead to development of cancer of the head and neck and anogenital regions. G-rich sequences found in genomes of high-risk HPVs can fold into non-canonical secondary structures that could serve as 3D motifs distinct from double-stranded DNA and present recognition sites for ligands and opportunity for gene expression modulation. Combination of UV, CD and NMR spectroscopy and PAGE electrophoresis were used as they offer complementary insights into structural changes of G-rich oligonucleotides. G-rich region of HPV16 is shown to preferentially form hairpin structures, while regions of HPV18, HPV52 and HPV58 fold into four-stranded DNA structures called G-quadruplexes. Single nucleotide polymorphisms found in G-rich sequences have been found to promote formation of hairpin structures of HPV16 and have affected number of species formed in G-rich region of HPV52, whereas they have exhibited minimal effect on the formation of HPV18 and HPV58 G-quadruplex structures. These structural changes were reflected in differences in apparent thermal stabilities. Potential of G-rich sequences as drug targets was evaluated based on the results of the current study. HPV16 and HPV18 are considered less appropriate targets due to several single nucleotide polymorphisms and low stability, respectively. On the other hand, HPV52 and HPV58 could be used for small-molecule mediated stabilization. G-rich sequences occurring in high-risk HPVs can fold into hairpin and G-quadruplex structures that could be potentially utilized as drug targets. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. The stability of DNA intrastrand cross-links of antitumor transplatin derivative containing non-bulky methylamine ligands

    Czech Academy of Sciences Publication Activity Database

    Frýbortová, Michaela; Nováková, Olga; Brabec, Viktor

    Roč. 19, č. 7 ( 2014 ), s. 1203-1208 ISSN 0949-8257 Institutional support: RVO:68081707 Keywords : Platinum drugs * Antisense technology * Anticancer Subject RIV: BO - Biophysics Impact factor: 2.538, year: 2014

  14. DNA G-Wire Formation Using an Artificial Peptide is Controlled by Protease Activity. (United States)

    Usui, Kenji; Okada, Arisa; Sakashita, Shungo; Shimooka, Masayuki; Tsuruoka, Takaaki; Nakano, Shu-Ichi; Miyoshi, Daisuke; Mashima, Tsukasa; Katahira, Masato; Hamada, Yoshio


    The development of a switching system for guanine nanowire (G-wire) formation by external signals is important for nanobiotechnological applications. Here, we demonstrate a DNA nanostructural switch (G-wire particles) using a designed peptide and a protease. The peptide consists of a PNA sequence for inducing DNA to form DNA-PNA hybrid G-quadruplex structures, and a protease substrate sequence acting as a switching module that is dependent on the activity of a particular protease. Micro-scale analyses via TEM and AFM showed that G-rich DNA alone forms G-wires in the presence of Ca 2+ , and that the peptide disrupted this formation, resulting in the formation of particles. The addition of the protease and digestion of the peptide regenerated the G-wires. Macro-scale analyses by DLS, zeta potential, CD, and gel filtration were in agreement with the microscopic observations. These results imply that the secondary structure change (DNA G-quadruplex DNA/PNA hybrid structure) induces a change in the well-formed nanostructure (G-wire particles). Our findings demonstrate a control system for forming DNA G-wire structures dependent on protease activity using designed peptides. Such systems hold promise for regulating the formation of nanowire for various applications, including electronic circuits for use in nanobiotechnologies.

  15. Tyramine Hydrochloride Based Label-Free System for Operating Various DNA Logic Gates and a DNA Caliper for Base Number Measurements. (United States)

    Fan, Daoqing; Zhu, Xiaoqing; Dong, Shaojun; Wang, Erkang


    DNA is believed to be a promising candidate for molecular logic computation, and the fluorogenic/colorimetric substrates of G-quadruplex DNAzyme (G4zyme) are broadly used as label-free output reporters of DNA logic circuits. Herein, for the first time, tyramine-HCl (a fluorogenic substrate of G4zyme) is applied to DNA logic computation and a series of label-free DNA-input logic gates, including elementary AND, OR, and INHIBIT logic gates, as well as a two to one encoder, are constructed. Furthermore, a DNA caliper that can measure the base number of target DNA as low as three bases is also fabricated. This DNA caliper can also perform concatenated AND-AND logic computation to fulfil the requirements of sophisticated logic computing. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. A tetrahelical DNA fold adopted by tandem repeats of alternating GGG and GCG tracts. (United States)

    Kocman, Vojč; Plavec, Janez


    DNA can form diverse higher-order structures, whose details are greatly dependent on nucleotide sequence. G-rich sequences containing four or more repeats of three guanines are expected to form G-quadruplexes. Here we show that DNA sequences with GGGAGCG repeats found in the regulatory region of the PLEKHG3 gene are capable of forming tetrahelical DNA structures that are distinct from G-quadruplexes. The d(GGGAGCGAGGGAGCG) sequence, VK1, forms a dimer. Two VK1 sequences connected by an adenine residue, VK2, fold into a monomer, which shares identical structural characteristics with the VK1 fold. Their four-stranded architectures are stabilized by four G-C, four G-A and six G-G base pairs. No G-quartets or Hoogsteen-type hydrogen-bonded guanine residues are present and the overall topology is conserved in the presence of Li(+), Na(+), K(+) and NH4(+) ions. Unique structural features include two edgewise loops on each side of the structure stabilized by three G-G base pairs in N1-carbonyl symmetric geometry.

  17. Enzyme-free colorimetric detection systems based on the DNA strand displacement competition reaction

    DEFF Research Database (Denmark)

    Zhang, Zhao; Birkedal, Victoria; Gothelf, Kurt Vesterager


    The strand displacement competition assay is based on the dynamic equilibrium of the competitive hybridization of two oligonucleotides (A and B) to a third oligonucleotide (S). In the presence of an analyte that binds to a specific affinity-moiety conjugated to strand B, the equilibrium shifts, w...... G-quadruplex DNAzyme for colorimetric readout of the detection of streptavidin by the naked eye. Finally, we integrate the whole G-quadruplex DNAzyme system in a single DNA strand and show that it is applicable to colorimetric detection......., which can be detected by a shift in the fluorescence resonance energy transfer signal between dyes attached to the DNA strands. In the present study we have integrated an ATP aptamer in the strand B and demonstrated the optical detection of ATP. Furthermore we explore a new readout method using a split......The strand displacement competition assay is based on the dynamic equilibrium of the competitive hybridization of two oligonucleotides (A and B) to a third oligonucleotide (S). In the presence of an analyte that binds to a specific affinity-moiety conjugated to strand B, the equilibrium shifts...

  18. Tetrahelical structural family adopted by AGCGA-rich regulatory DNA regions (United States)

    Kocman, Vojč; Plavec, Janez


    Here we describe AGCGA-quadruplexes, an unexpected addition to the well-known tetrahelical families, G-quadruplexes and i-motifs, that have been a focus of intense research due to their potential biological impact in G- and C-rich DNA regions, respectively. High-resolution structures determined by solution-state nuclear magnetic resonance (NMR) spectroscopy demonstrate that AGCGA-quadruplexes comprise four 5'-AGCGA-3' tracts and are stabilized by G-A and G-C base pairs forming GAGA- and GCGC-quartets, respectively. Residues in the core of the structure are connected with edge-type loops. Sequences of alternating 5'-AGCGA-3' and 5'-GGG-3' repeats could be expected to form G-quadruplexes, but are shown herein to form AGCGA-quadruplexes instead. Unique structural features of AGCGA-quadruplexes together with lower sensitivity to cation and pH variation imply their potential biological relevance in regulatory regions of genes responsible for basic cellular processes that are related to neurological disorders, cancer and abnormalities in bone and cartilage development.

  19. Addition of Bases to the 5'-end of Human Telomeric DNA: Influences on Thermal Stability and Energetics of Unfolding

    Directory of Open Access Journals (Sweden)

    Katherine L. Hayden


    Full Text Available Telomeric DNA has been intensely investigated for its role in chromosome protection, aging, cell death, and disease. In humans the telomeric tandem repeat (TTAGGGn is found at the ends of chromosomes and provides a novel target for the development of new drugs in the treatment of age related diseases such as cancer. These telomeric sequences show slight sequence variations from species to species; however, each contains repeats of 3 to 4 guanines allowing the G-rich strands to fold into compact and stable nuclease resistant conformations referred to as G-quadruplexes. The focus of this manuscript is to examine the effects of 5'-nucleotides flanking the human telomeric core sequence 5'-AGGG(TTAGGG 3-3' (h-Tel22. Our studies reveal that the addition of the 5'-flanking nucleotides (5'-T, and 5'-TT results in significant changes to the thermodynamic stability of the G-quadruplex structure. Our data indicate that the observed changes in stability are associated with changes in the number of bound waters resulting from the addition of 5'-flanking nucleotides to the h-Tel22 sequence as well as possible intermolecular interactions of the 5' overhang with the core structure.

  20. High-Resolution Profiling of Drosophila Replication Start Sites Reveals a DNA Shape and Chromatin Signature of Metazoan Origins

    Directory of Open Access Journals (Sweden)

    Federico Comoglio


    Full Text Available At every cell cycle, faithful inheritance of metazoan genomes requires the concerted activation of thousands of DNA replication origins. However, the genetic and chromatin features defining metazoan replication start sites remain largely unknown. Here, we delineate the origin repertoire of the Drosophila genome at high resolution. We address the role of origin-proximal G-quadruplexes and suggest that they transiently stall replication forks in vivo. We dissect the chromatin configuration of replication origins and identify a rich spatial organization of chromatin features at initiation sites. DNA shape and chromatin configurations, not strict sequence motifs, mark and predict origins in higher eukaryotes. We further examine the link between transcription and origin firing and reveal that modulation of origin activity across cell types is intimately linked to cell-type-specific transcriptional programs. Our study unravels conserved origin features and provides unique insights into the relationship among DNA topology, chromatin, transcription, and replication initiation across metazoa.

  1. Thermodynamic stability and energetics of DNA duplexes containing major intrastrand cross-links of second-generation antitumor dinuclear Pt-II complexes

    Czech Academy of Sciences Publication Activity Database

    Florian, Jakub; Kašpárková, Jana; Farell, N.P.; Brabec, Viktor


    Roč. 17, č. 2 (2012), s. 187-196 ISSN 0949-8257 R&D Projects: GA ČR(CZ) GD301/09/H004; GA ČR(CZ) GAP301/10/0598; GA MŠk(CZ) ME10066 Institutional support: RVO:68081707 Keywords : NUCLEOTIDE EXCISION-REPAIR * PLATINUM ANTICANCER DRUGS * GROUP DOMAIN PROTEINS Subject RIV: BO - Biophysics Impact factor: 3.353, year: 2012

  2. EGNAS: an exhaustive DNA sequence design algorithm

    Directory of Open Access Journals (Sweden)

    Kick Alfred


    Full Text Available Abstract Background The molecular recognition based on the complementary base pairing of deoxyribonucleic acid (DNA is the fundamental principle in the fields of genetics, DNA nanotechnology and DNA computing. We present an exhaustive DNA sequence design algorithm that allows to generate sets containing a maximum number of sequences with defined properties. EGNAS (Exhaustive Generation of Nucleic Acid Sequences offers the possibility of controlling both interstrand and intrastrand properties. The guanine-cytosine content can be adjusted. Sequences can be forced to start and end with guanine or cytosine. This option reduces the risk of “fraying” of DNA strands. It is possible to limit cross hybridizations of a defined length, and to adjust the uniqueness of sequences. Self-complementarity and hairpin structures of certain length can be avoided. Sequences and subsequences can optionally be forbidden. Furthermore, sequences can be designed to have minimum interactions with predefined strands and neighboring sequences. Results The algorithm is realized in a C++ program. TAG sequences can be generated and combined with primers for single-base extension reactions, which were described for multiplexed genotyping of single nucleotide polymorphisms. Thereby, possible foldback through intrastrand interaction of TAG-primer pairs can be limited. The design of sequences for specific attachment of molecular constructs to DNA origami is presented. Conclusions We developed a new software tool called EGNAS for the design of unique nucleic acid sequences. The presented exhaustive algorithm allows to generate greater sets of sequences than with previous software and equal constraints. EGNAS is freely available for noncommercial use at

  3. Genotoxicity of formaldehyde: Molecular basis of DNA damage and mutation

    Directory of Open Access Journals (Sweden)

    Masanobu eKawanishi


    Full Text Available Formaldehyde is commonly used in the chemical industry and is present in the environment, such as vehicle emissions, some building materials, food and tobacco smoke. It also occurs as a natural product in most organisms, the sources of which include a number of metabolic processes. It causes various acute and chronic adverse effects in humans if they inhale its fumes. Among the chronic effects on human health, we summarize data on genotoxicity and carcinogenicity in this review, and we particularly focus on the molecular mechanisms involved in the formaldehyde mutagenesis. Formaldehyde mainly induces N-hydroxymethyl mono-adducts on guanine, adenine and cytosine, and N-methylene crosslinks between adjacent purines in DNA. These crosslinks are types of DNA damage potentially fatal for cell survival if they are not removed by the nucleotide excision repair pathway. In the previous studies, we showed evidence that formaldehyde causes intra-strand crosslinks between purines in DNA using a unique method (Matsuda et al. Nucleic Acids Res. 26, 1769-1774,1998. Using shuttle vector plasmids, we also showed that formaldehyde as well as acetaldehyde induces tandem base substitutions, mainly at 5’-GG and 5’-GA sequences, which would arise from the intra-strand crosslinks. These mutation features are different from those of other aldehydes such as crotonaldehyde, acrolein, glyoxal and methylglyoxal. These findings provide molecular clues to improve our understanding of the genotoxicity and carcinogenicity of formaldehyde.

  4. DNA breaks and repair in interstitial telomere sequences: Influence of chromatin structure

    International Nuclear Information System (INIS)

    Revaud, D.


    Interstitial Telomeric Sequences (ITS) are over-involved in spontaneous and radiationinduced chromosome aberrations in chinese hamster cells. We have performed a study to investigate the origin of their instability, spontaneously or after low doses irradiation. Our results demonstrate that ITS have a particular chromatin structure: short nucleotide repeat length, less compaction of the 30 nm chromatin fiber, presence of G-quadruplex structures. These features would modulate breaks production and would favour the recruitment of alternative DNA repair mechanisms, which are prone to produce chromosome aberrations. These pathways could be at the origin of chromosome aberrations in ITS whereas NHEJ and HR Double Strand Break repair pathways are rather required for a correct repair in these regions. (author)

  5. DNA structural features of eukaryotic TATA-containing and TATA-less promoters. (United States)

    Yella, Venkata Rajesh; Bansal, Manju


    Eukaryotic genes can be broadly classified as TATA-containing and TATA-less based on the presence of TATA box in their promoters. Experiments on both classes of genes have revealed a disparity in the regulation of gene expression and cellular functions between the two classes. In this study, we report characteristic differences in promoter sequences and associated structural properties of the two categories of genes in six different eukaryotes. We have analyzed three structural features, DNA duplex stability, bendability, and curvature along with the distribution of A-tracts, G-quadruplex motifs, and CpG islands. The structural feature analyses reveal that while the two classes of gene promoters are distinctly different from each other, the properties are also distinguishable across the six organisms.

  6. Repair capacity for platinum-DNA adducts determines the severity of cisplatin-induced peripheral neuropathy. (United States)

    Dzagnidze, Anna; Katsarava, Zaza; Makhalova, Julia; Liedert, Bernd; Yoon, Min-Suk; Kaube, Holger; Limmroth, Volker; Thomale, Juergen


    The pronounced neurotoxicity of the potent antitumor drug cisplatin frequently results in the onset of peripheral polyneuropathy (PNP), which is assumed to be initially triggered by platination products in the nuclear DNA of affected tissues. To further elucidate the molecular mechanisms, we analyzed in a mouse model the formation and processing of the main cisplatin-induced DNA adduct (guanine-guanine intrastrand cross-link) in distinct neuronal cell types by adduct-specific monoclonal antibodies. Comparison of the adduct kinetics in cisplatin-injected mice either proficient or deficient for nucleotide excision repair (NER) functions revealed the essential role of this DNA repair pathway in protecting differentiated cells of the nervous system from excessive formation of such lesions. Hence, chronic exposure to cisplatin resulted in an accelerated accumulation of unrepaired intrastrand cross-links in neuronal cells of mice with dysfunctional NER. The augmented adduct levels in dorsal root ganglion (DRG) cells of those animals coincided with an earlier onset of PNP-like functional disturbance of their sensory nervous system. Independently from the respective repair phenotype, the amount of persisting DNA cross-links in DRG neurons at a given cumulative dose was significantly correlated to the degree of sensory impairment as measured by electroneurography. Collectively, these findings suggest a new model for the processing of cisplatin adducts in primary neuronal cells and accentuate the crucial role of effectual DNA repair capacity in the target cells for the individual risk of therapy-induced PNP.

  7. RNA polymerase III regulates cytosolic RNA:DNA hybrids and intracellular microRNA expression. (United States)

    Koo, Christine Xing'er; Kobiyama, Kouji; Shen, Yu J; LeBert, Nina; Ahmad, Shandar; Khatoo, Muznah; Aoshi, Taiki; Gasser, Stephan; Ishii, Ken J


    RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cytosolic RNA:DNA hybrids. Cytosolic RNA:DNA hybrids bind to several components of the microRNA (miRNA) machinery-related proteins, including AGO2 and DDX17. Furthermore, we identified miRNAs that are specifically regulated by Pol III, providing a potential link between RNA:DNA hybrids and the miRNA machinery. One of the target genes, exportin-1, is shown to regulate cytosolic RNA:DNA hybrids. Taken together, we reveal previously unknown mechanism by which Pol III regulates the presence of cytosolic RNA:DNA hybrids and miRNA biogenesis in various human cells. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. RNA Polymerase III Regulates Cytosolic RNA:DNA Hybrids and Intracellular MicroRNA Expression* (United States)

    Koo, Christine Xing'er; Kobiyama, Kouji; Shen, Yu J.; LeBert, Nina; Ahmad, Shandar; Khatoo, Muznah; Aoshi, Taiki; Gasser, Stephan; Ishii, Ken J.


    RNA:DNA hybrids form in the nuclei and mitochondria of cells as transcription-induced R-loops or G-quadruplexes, but exist only in the cytosol of virus-infected cells. Little is known about the existence of RNA:DNA hybrids in the cytosol of virus-free cells, in particular cancer or transformed cells. Here, we show that cytosolic RNA:DNA hybrids are present in various human cell lines, including transformed cells. Inhibition of RNA polymerase III (Pol III), but not DNA polymerase, abrogated cytosolic RNA:DNA hybrids. Cytosolic RNA:DNA hybrids bind to several components of the microRNA (miRNA) machinery-related proteins, including AGO2 and DDX17. Furthermore, we identified miRNAs that are specifically regulated by Pol III, providing a potential link between RNA:DNA hybrids and the miRNA machinery. One of the target genes, exportin-1, is shown to regulate cytosolic RNA:DNA hybrids. Taken together, we reveal previously unknown mechanism by which Pol III regulates the presence of cytosolic RNA:DNA hybrids and miRNA biogenesis in various human cells. PMID:25623070

  9. Structural basis for IL-1α recognition by a modified DNA aptamer that specifically inhibits IL-1α signaling

    Energy Technology Data Exchange (ETDEWEB)

    Ren, Xiaoming; Gelinas, Amy D.; von Carlowitz, Ira; Janjic, Nebojsa; Pyle, Anna Marie (Yale); (SomaLogic)


    IL-1α is an essential cytokine that contributes to inflammatory responses and is implicated in various forms of pathogenesis and cancer. Here we report a naphthyl modified DNA aptamer that specifically binds IL-1α and inhibits its signaling pathway. By solving the crystal structure of the IL-1α/aptamer, we provide a high-resolution structure of this critical cytokine and we reveal its functional interaction interface with high-affinity ligands. The non-helical aptamer, which represents a highly compact nucleic acid structure, contains a wealth of new conformational features, including an unknown form of G-quadruplex. The IL-1α/aptamer interface is composed of unusual polar and hydrophobic elements, along with an elaborate hydrogen bonding network that is mediated by sodium ion. IL-1α uses the same interface to interact with both the aptamer and its cognate receptor IL-1RI, thereby suggesting a novel route to immunomodulatory therapeutics.

  10. Concatenated logic circuits based on a three-way DNA junction: a keypad-lock security system with visible readout and an automatic reset function. (United States)

    Chen, Junhua; Zhou, Shungui; Wen, Junlin


    Concatenated logic circuits operating as a biocomputing keypad-lock security system with an automatic reset function have been successfully constructed on the basis of toehold-mediated strand displacement and three-way-DNA-junction architecture. In comparison with previously reported keypad locks, the distinctive advantage of the proposed security system is that it can be reset and cycled spontaneously a large number of times without an external stimulus, thus making practical applications possible. By the use of a split-G-quadruplex DNAzyme as the signal reporter, the output of the keypad lock can be recognized readily by the naked eye. The "lock" is opened only when the inputs are introduced in an exact order. This requirement provides defense against illegal invasion to protect information at the molecular scale. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Aryl-Substituted Ruthenium(II) Complexes: A Strategy for Enhanced Photocleavage and Efficient DNA Binding. (United States)

    Abreu, Felipe Diógenes; Paulo, Tercio de F; Gehlen, Marcelo H; Ando, Rômulo A; Lopes, Luiz G F; Gondim, Ana Cláudia S; Vasconcelos, Mayron A; Teixeira, Edson H; Sousa, Eduardo Henrique Silva; de Carvalho, Idalina Maria Moreira


    Ruthenium polypyridine complexes have shown promise as agents for photodynamic therapy (PDT) and tools for molecular biology (chromophore-assisted light inactivation). To accomplish these tasks, it is important to have at least target selectivity and great reactive oxygen species (ROS) photogeneration: two properties that are not easily found in the same molecule. To prepare such new agents, we synthesized two new ruthenium complexes that combine an efficient DNA binding moiety (dppz ligand) together with naphthyl-modified (1) and anthracenyl-modified (2) bipyridine as a strong ROS generator bound to a ruthenium complex. The compounds were fully characterized and their photophysical and photochemical properties investigated. Compound 2 showed one of the highest quantum yields for singlet oxygen production ever reported (Φ Δ = 0.96), along with very high DNA binding (log K b = 6.78). Such photochemical behavior could be ascribed to the lower triplet state involving the anthracenyl-modified bipyridine, which is associated with easier oxygen quenching. In addition, the compounds exhibited moderate selectivity toward G-quadruplex DNA and binding to the minor groove of DNA, most likely driven by the pendant ligands. Interestingly, they also showed DNA photocleavage activity even upon exposure to a yellow light-emitting diode (LED). Regarding their biological activity, the compounds exhibited an exciting antibacterial action, particularly against Gram-positive bacteria, which was enhanced upon blue LED irradiation. Altogether, these results showed that our strategy succeeded in producing light-triggered DNA binding agents with pharmacological and biotechnological potential.

  12. Ultraviolet irradiation produces cytotoxic synergy and increased DNA interstrand crosslinking with cis- and trans-diamminedichloroplatinum(II)

    International Nuclear Information System (INIS)

    Swinnen, L.J.; Erickson, L.C.


    The excision-repair mechanism responsible for the removal of UV-induced thymine dimers may also play a role in the repair of cis-diamminedichloroplatinum(II) (cis-DDP)-induced DNA adducts in both bacteria and mammalian cells. It was hypothesized that UV dimers and cis-DDP adducts, when present simultaneously, might compete for a common repair system. Colony survival assays were performed in HT-29 human colon carcinoma cells exposed either to cis-DDP alone or to cis-DDP immediately followed by UV exposure. Progressively greater cytotoxic synergy with both increasing UV dose and cis-DDP dose was observed, to a point of saturation beyond which further toxicity was purely additive. An approximate doubling in DNA crosslink frequency, relative to cis-DDP alone, was found in cells exposed to cis-DDP plus UV. Since cis-DDP produces both inter- and intrastrand DNA crosslinks similar studies were performed with trans-DDP, which is incapable of producing intrastrand crosslinks, but does produce interstrand crosslinks. Cytotoxic synergy and increased interstrand crosslinking again resulted from the addition of UV exposure, but not to the same extent as seen with cis-DDP. (author)

  13. DNA breaks and repair in interstitial telomere sequences: Influence of chromatin structure; Etude des cassures de l'ADN et des mecanismes de reparation dans les sequences telomeriques interstitielles: Influence de la structure chromatinienne

    Energy Technology Data Exchange (ETDEWEB)

    Revaud, D.


    Interstitial Telomeric Sequences (ITS) are over-involved in spontaneous and radiationinduced chromosome aberrations in chinese hamster cells. We have performed a study to investigate the origin of their instability, spontaneously or after low doses irradiation. Our results demonstrate that ITS have a particular chromatin structure: short nucleotide repeat length, less compaction of the 30 nm chromatin fiber, presence of G-quadruplex structures. These features would modulate breaks production and would favour the recruitment of alternative DNA repair mechanisms, which are prone to produce chromosome aberrations. These pathways could be at the origin of chromosome aberrations in ITS whereas NHEJ and HR Double Strand Break repair pathways are rather required for a correct repair in these regions. (author)

  14. A first-in-class and a fished out anticancer platinum compound: cis-[PtCl2(NH3)2] and cis-[PtI2(NH3)2] compared for their reactivity towards DNA model systems. (United States)

    Musumeci, Domenica; Platella, Chiara; Riccardi, Claudia; Merlino, Antonello; Marzo, Tiziano; Massai, Lara; Messori, Luigi; Montesarchio, Daniela


    Contrary to what was believed for many years, cis-PtI2(NH3)2, the diiodido analogue of cisplatin, displays high in vitro antiproliferative activity toward a set of tumour cell lines, overcoming resistance to cisplatin in a platinum-resistant cancer cell line. In the context of a general reappraisal of iodinated Pt(ii) derivatives, aiming at a more systematic evaluation of their chemical and biological profiles, here we report on the reactivity of cis-PtI2(NH3)2 with selected DNA model systems, in single, double strand or G-quadruplex form, using cisplatin as a control. A combined approach has been exploited in this study, including circular dichroism (CD), UV-visible spectroscopy and electrospray mass spectrometry (ESI-MS) analyses. The data reveal that cis-PtI2(NH3)2 shows an overall reactivity towards the investigated oligonucleotides significantly higher than cisplatin.

  15. Transcriptional blockages in a cell-free system by sequence-selective DNA alkylating agents. (United States)

    Ferguson, L R; Liu, A P; Denny, W A; Cullinane, C; Talarico, T; Phillips, D R


    There is considerable interest in DNA sequence-selective DNA-binding drugs as potential inhibitors of gene expression. Five compounds with distinctly different base pair specificities were compared in their effects on the formation and elongation of the transcription complex from the lac UV5 promoter in a cell-free system. All were tested at drug levels which killed 90% of cells in a clonogenic survival assay. Cisplatin, a selective alkylator at purine residues, inhibited transcription, decreasing the full-length transcript, and causing blockage at a number of GG or AG sequences, making it probable that intrastrand crosslinks are the blocking lesions. A cyclopropylindoline known to be an A-specific alkylator also inhibited transcription, with blocks at adenines. The aniline mustard chlorambucil, that targets primarily G but also A sequences, was also effective in blocking the formation of full-length transcripts. It produced transcription blocks either at, or one base prior to, AA or GG sequences, suggesting that intrastrand crosslinks could again be involved. The non-alkylating DNA minor groove binder Hoechst 33342 (a bisbenzimidazole) blocked formation of the full-length transcript, but without creating specific blockage sites. A bisbenzimidazole-linked aniline mustard analogue was a more effective transcription inhibitor than either chlorambucil or Hoechst 33342, with different blockage sites occurring immediately as compared with 2 h after incubation. The blockages were either immediately prior to AA or GG residues, or four to five base pairs prior to such sites, a pattern not predicted from in vitro DNA-binding studies. Minor groove DNA-binding ligands are of particular interest as inhibitors of gene expression, since they have the potential ability to bind selectively to long sequences of DNA. The results suggest that the bisbenzimidazole-linked mustard does cause alkylation and transcription blockage at novel DNA sites. in addition to sites characteristic of

  16. A DNA origami nanorobot controlled by nucleic acid hybridization. (United States)

    Torelli, Emanuela; Marini, Monica; Palmano, Sabrina; Piantanida, Luca; Polano, Cesare; Scarpellini, Alice; Lazzarino, Marco; Firrao, Giuseppe


    A prototype for a DNA origami nanorobot is designed, produced, and tested. The cylindrical nanorobot (diameter of 14 nm and length of 48 nm) with a switchable flap, is able to respond to an external stimulus and reacts by a physical switch from a disarmed to an armed configuration able to deliver a cellular compatible message. In the tested design the robot weapon is a nucleic acid fully contained in the inner of the tube and linked to a single point of the internal face of the flap. Upon actuation the nanorobot moves the flap extracting the nucleic acid that assembles into a hemin/G-quadruplex horseradish peroxidase mimicking DNAzyme catalyzing a colorimetric reaction or chemiluminescence generation. The actuation switch is triggered by an external nucleic acid (target) that interacts with a complementary nucleic acid that is beard externally by the nanorobot (probe). Hybridization of probe and target produces a localized structural change that results in flap opening. The flap movement is studied on a two-dimensional prototype origami using Förster resonance energy transfer and is shown to be triggered by a variety of targets, including natural RNAs. The nanorobot has potential for in vivo biosensing and intelligent delivery of biological activators. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. A DNA origami nanorobot controlled by nucleic acid hybridization

    KAUST Repository

    Torelli, Emanuela


    A prototype for a DNA origami nanorobot is designed, produced, and tested. The cylindrical nanorobot (diameter of 14 nm and length of 48 nm) with a switchable flap, is able to respond to an external stimulus and reacts by a physical switch from a disarmed to an armed configuration able to deliver a cellular compatible message. In the tested design the robot weapon is a nucleic acid fully contained in the inner of the tube and linked to a single point of the internal face of the flap. Upon actuation the nanorobot moves the flap extracting the nucleic acid that assembles into a hemin/G-quadruplex horseradish peroxidase mimicking DNAzyme catalyzing a colorimetric reaction or chemiluminescence generation. The actuation switch is triggered by an external nucleic acid (target) that interacts with a complementary nucleic acid that is beard externally by the nanorobot (probe). Hybridization of probe and target produces a localized structural change that results in flap opening. The flap movement is studied on a two-dimensional prototype origami using Förster resonance energy transfer and is shown to be triggered by a variety of targets, including natural RNAs. The nanorobot has potential for in vivo biosensing and intelligent delivery of biological activators. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Perturbed soliton excitations in inhomogeneous DNA

    International Nuclear Information System (INIS)

    Daniel, M.; Vasumathi, V.


    We study nonlinear dynamics of inhomogeneous DNA double helical chain under dynamic plane-base rotator model by considering angular rotation of bases in a plane normal to the helical axis. The DNA dynamics in this case is found to be governed by a perturbed sine-Gordon equation when taking into account the interstrand hydrogen bonding energy and intrastrand inhomogeneous stacking energy and making an analogy with the Heisenberg model of the Hamiltonian for an inhomogeneous anisotropic spin ladder with ferromagnetic legs and antiferromagentic rung coupling. In the homogeneous limit the dynamics is governed by the kink-antikink soliton of the sine-Gordon equation which represents the formation of open state configuration in DNA double helix. The effect of inhomogeneity in stacking energy in the form of localized and periodic variations on the formation of open states in DNA is studied under perturbation. The perturbed soliton is obtained using a multiple scale soliton perturbation theory by solving the associated linear eigen value problem and constructing the complete set of eigen functions. The inhomogeneity in stacking energy is found to modulate the width and speed of the soliton depending on the nature of inhomogeneity. Also it introduces fluctuations in the form of train of pulses or periodic oscillation in the open state configuration (author)

  19. RecBCD (Exonuclease V) is inhibited by DNA adducts produced by cisplatin and ultraviolet light. (United States)

    Leung, Wai Y; Chung, Long H; Kava, Hieronimus W; Murray, Vincent


    The presence of adducts on the DNA double-helix can have major consequences for the efficient functioning of DNA repair enzymes. E. coli RecBCD (exonuclease V) is involved in recombinational repair of double-strand breaks that are caused by defective DNA replication, DNA damaging agents and other factors. The holoenzyme possesses a bipolar helicase activity which helps unwind DNA from both 3'- and 5'-directions and is coupled with a potent exonuclease activity that is also capable of digesting DNA from both 3'- and 5'-ends. In this study, DNA sequences were damaged with cisplatin or UV followed by RecBCD treatment. DNA damaging agents such as cisplatin and UV induce the formation of intrastrand adducts in the DNA template. It was demonstrated that RecBCD degradation was inhibited by either cisplatin-damaged or UV-damaged DNA sequences. This is the first occasion that RecBCD has been demonstrated to be inhibited by DNA adducts induced by cisplatin or UV. In addition, we quantified the amounts of DNA remaining after RecBCD treatment and observed that the level of inhibition was concentration and dose dependent. A DNA-targeted 9-aminoacridinecarboxamide cisplatin analogue was also found to inhibit RecBCD activity. Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.

  20. TERRA mimicking ssRNAs prevail over the DNA substrate for telomerase in vitro due to interactions with the alternative binding site. (United States)

    Azhibek, Dulat; Skvortsov, Dmitry; Andreeva, Anna; Zatsepin, Timofei; Arutyunyan, Alexandr; Zvereva, Maria; Dontsova, Olga


    Telomerase is a key component of the telomere length maintenance system in the majority of eukaryotes. Telomerase displays maximal activity in stem and cancer cells with high proliferative potential. In humans, telomerase activity is regulated by various mechanisms, including the interaction with telomere ssDNA overhangs that contain a repetitive G-rich sequence, and with noncoding RNA, Telomeric repeat-containing RNA (TERRA), that contains the same sequence. So these nucleic acids can compete for telomerase RNA templates in the cell. In this study, we have investigated the ability of different model substrates mimicking telomere DNA overhangs and TERRA RNA to compete for telomerase in vitro through a previously developed telomerase inhibitor assay. We have shown in this study that RNA oligonucleotides are better competitors for telomerase that DNA ones as RNA also use an alternative binding site on telomerase, and the presence of 2'-OH groups is significant in these interactions. In contrast to DNA, the possibility of forming intramolecular G-quadruplex structures has a minor effect for RNA binding to telomerase. Taking together our data, we propose that TERRA RNA binds better to telomerase compared with its native substrate - the 3'-end of telomere DNA overhang. As a result, some specific factor may exist that participates in switching telomerase from TERRA to the 3'-end of DNA for telomere elongation at the distinct period of a cell cycle in vivo. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

  1. Self-entanglement of long linear DNA vectors using transient non-B-DNA attachment points: a new concept for improvement of non-viral therapeutic gene delivery. (United States)

    Tolmachov, Oleg E


    The cell-specific and long-term expression of therapeutic transgenes often requires a full array of native gene control elements including distal enhancers, regulatory introns and chromatin organisation sequences. The delivery of such extended gene expression modules to human cells can be accomplished with non-viral high-molecular-weight DNA vectors, in particular with several classes of linear DNA vectors. All high-molecular-weight DNA vectors are susceptible to damage by shear stress, and while for some of the vectors the harmful impact of shear stress can be minimised through the transformation of the vectors to compact topological configurations by supercoiling and/or knotting, linear DNA vectors with terminal loops or covalently attached terminal proteins cannot be self-compacted in this way. In this case, the only available self-compacting option is self-entangling, which can be defined as the folding of single DNA molecules into a configuration with mutual restriction of molecular motion by the individual segments of bent DNA. A negatively charged phosphate backbone makes DNA self-repulsive, so it is reasonable to assume that a certain number of 'sticky points' dispersed within DNA could facilitate the entangling by bringing DNA segments into proximity and by interfering with the DNA slipping away from the entanglement. I propose that the spontaneous entanglement of vector DNA can be enhanced by the interlacing of the DNA with sites capable of mutual transient attachment through the formation of non-B-DNA forms, such as interacting cruciform structures, inter-segment triplexes, slipped-strand DNA, left-handed duplexes (Z-forms) or G-quadruplexes. It is expected that the non-B-DNA based entanglement of the linear DNA vectors would consist of the initial transient and co-operative non-B-DNA mediated binding events followed by tight self-ensnarement of the vector DNA. Once in the nucleoplasm of the target human cells, the DNA can be disentangled by type II

  2. DNA damage by reactive species: Mechanisms, mutation and repair

    Indian Academy of Sciences (India)


    N7]G (scheme 6g) and G[N7-N3]A. (scheme 6h) intra-strand crosslinks (Liu et al. 2002b;. Hegmans et al. 2004; Harrington et al. 2010). Scheme 6. Structures of different inter-strand (a,b) and intra-strand (c–h) crosslink products ...

  3. Low-Energy Electron-Induced Strand Breaks in Telomere-Derived DNA Sequences-Influence of DNA Sequence and Topology. (United States)

    Rackwitz, Jenny; Bald, Ilko


    During cancer radiation therapy high-energy radiation is used to reduce tumour tissue. The irradiation produces a shower of secondary low-energy (DNA very efficiently by dissociative electron attachment. Recently, it was suggested that low-energy electron-induced DNA strand breaks strongly depend on the specific DNA sequence with a high sensitivity of G-rich sequences. Here, we use DNA origami platforms to expose G-rich telomere sequences to low-energy (8.8 eV) electrons to determine absolute cross sections for strand breakage and to study the influence of sequence modifications and topology of telomeric DNA on the strand breakage. We find that the telomeric DNA 5'-(TTA GGG) 2 is more sensitive to low-energy electrons than an intermixed sequence 5'-(TGT GTG A) 2 confirming the unique electronic properties resulting from G-stacking. With increasing length of the oligonucleotide (i.e., going from 5'-(GGG ATT) 2 to 5'-(GGG ATT) 4 ), both the variety of topology and the electron-induced strand break cross sections increase. Addition of K + ions decreases the strand break cross section for all sequences that are able to fold G-quadruplexes or G-intermediates, whereas the strand break cross section for the intermixed sequence remains unchanged. These results indicate that telomeric DNA is rather sensitive towards low-energy electron-induced strand breakage suggesting significant telomere shortening that can also occur during cancer radiation therapy. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Thermodynamic properties of water molecules in the presence of cosolute depend on DNA structure: a study using grid inhomogeneous solvation theory (United States)

    Nakano, Miki; Tateishi-Karimata, Hisae; Tanaka, Shigenori; Tama, Florence; Miyashita, Osamu; Nakano, Shu-ichi; Sugimoto, Naoki


    In conditions that mimic those of the living cell, where various biomolecules and other components are present, DNA strands can adopt many structures in addition to the canonical B-form duplex. Previous studies in the presence of cosolutes that induce molecular crowding showed that thermal stabilities of DNA structures are associated with the properties of the water molecules around the DNAs. To understand how cosolutes, such as ethylene glycol, affect the thermal stability of DNA structures, we investigated the thermodynamic properties of water molecules around a hairpin duplex and a G-quadruplex using grid inhomogeneous solvation theory (GIST) with or without cosolutes. Our analysis indicated that (i) cosolutes increased the free energy of water molecules around DNA by disrupting water–water interactions, (ii) ethylene glycol more effectively disrupted water–water interactions around Watson–Crick base pairs than those around G-quartets or non-paired bases, (iii) due to the negative electrostatic potential there was a thicker hydration shell around G-quartets than around Watson–Crick-paired bases. Our findings suggest that the thermal stability of the hydration shell around DNAs is one factor that affects the thermal stabilities of DNA structures under the crowding conditions. PMID:26538600

  5. Exploring the Dynamics of Propeller Loops in Human Telomeric DNA Quadruplexes Using Atomistic Simulations (United States)


    We have carried out a series of extended unbiased molecular dynamics (MD) simulations (up to 10 μs long, ∼162 μs in total) complemented by replica-exchange with the collective variable tempering (RECT) approach for several human telomeric DNA G-quadruplex (GQ) topologies with TTA propeller loops. We used different AMBER DNA force-field variants and also processed simulations by Markov State Model (MSM) analysis. The slow conformational transitions in the propeller loops took place on a scale of a few μs, emphasizing the need for long simulations in studies of GQ dynamics. The propeller loops sampled similar ensembles for all GQ topologies and for all force-field dihedral-potential variants. The outcomes of standard and RECT simulations were consistent and captured similar spectrum of loop conformations. However, the most common crystallographic loop conformation was very unstable with all force-field versions. Although the loss of canonical γ-trans state of the first propeller loop nucleotide could be related to the indispensable bsc0 α/γ dihedral potential, even supporting this particular dihedral by a bias was insufficient to populate the experimentally dominant loop conformation. In conclusion, while our simulations were capable of providing a reasonable albeit not converged sampling of the TTA propeller loop conformational space, the force-field description still remained far from satisfactory. PMID:28475322

  6. Logic gates and antisense DNA devices operating on a translator nucleic Acid scaffold. (United States)

    Shlyahovsky, Bella; Li, Yang; Lioubashevski, Oleg; Elbaz, Johann; Willner, Itamar


    A series of logic gates, "AND", "OR", and "XOR", are designed using a DNA scaffold that includes four "footholds" on which the logic operations are activated. Two of the footholds represent input-recognition strands, and these are blocked by complementary nucleic acids, whereas the other two footholds are blocked by nucleic acids that include the horseradish peroxidase (HRP)-mimicking DNAzyme sequence. The logic gates are activated by either nucleic acid inputs that hybridize to the respective "footholds", or by low-molecular-weight inputs (adenosine monophosphate or cocaine) that yield the respective aptamer-substrate complexes. This results in the respective translocation of the blocking nucleic acids to the footholds carrying the HRP-mimicking DNAzyme sequence, and the concomitant release of the respective DNAzyme. The released product-strands then self-assemble into the hemin/G-quadruplex-HRP-mimicking DNAzyme that biocatalyzes the formation of a colored product and provides an output signal for the different logic gates. The principle of the logic operation is, then, implemented as a possible paradigm for future nanomedicine. The nucleic acid inputs that bind to the blocked footholds result in the translocation of the blocking nucleic acids to the respective footholds carrying the antithrombin aptamer. The released aptamer inhibits, then, the hydrolytic activity of thrombin. The system demonstrates the regulation of a biocatalytic reaction by a translator system activated on a DNA scaffold.

  7. Generation of Guanine – Thymidine Cross-links in DNA by Peroxynitrite/Carbon Dioxide (United States)

    Yun, Byeong Hwa; Geacintov, Nicholas E.; Shafirovich, Vladimir


    Nitrosoperoxycarbonate derived from the combination of carbon dioxide and peroxynitrite, is an important chemical mediator of inflammation. In aqueous solutions, it rapidly decomposes to the reactive species CO3•− and •NO2 radicals that are known to initiate the selective oxidation and nitration of guanine in DNA. We have previously demonstrated that the reactions of carbonate radical anions with guanine in 2′-deoxyoligoribonucleotides generate a previously unknown intrastrand cross-linked guanine-thymine product G*-T* with a covalent bond between the C8 (G*) and thymine N3 (T*) atoms (Crean et al., Nucleic Acids Res., 2008, 36, 742–755). In this work we demonstrate that G*-T* cross-linked products are also formed when peroxynitrite (0.1 mM) reacts with native DNA in aqueous solutions (pH 7.5–7.7) containing 25 mM carbon dioxide/bicarbonate, in addition to the well known nitration/oxidation products of guanine such as 8-nitroguanine (8-nitroG), 5-guanidino-4-nitroimidazole (NIm), 8-oxo-7,8-dehydroguanine (8-oxoG) and spiroiminodihydantoin (Sp). The yields of these products, after enzymatic digestion with P1 nuclease and alkaline phosphatase to the nucleotide level, and reversed phase HPLC separation, were compared with those obtained with the uniformly, isotopically labeled 15N,13C-labeled 2′-deoxy oligoribonucleotides 5′-dGpT and 5′-dGpCpT. The d(G*pT*) and d(G*-T*) cross-linked products derived from the di- and tri-oligonucleotides, respectively, were used as standards for identifying the analogous lesions in calf thymus DNA by isotope dilution LC-MS/MS methods in the selected reaction-monitoring mode. The Nim and 8nitroG are the major products formed (~ 0.05% each), and lesser amounts of 8-oxoG (~ 0.02%), and d(G*pT*) and d(G*-T*) enzymatic digestion products (~ 0.002% each) were found. It is shown that the formation of d(G*pT*) enzyme digestion product can arise only from intrastrand cross-links, whereas d(G*-T*) can arise from both

  8. Shedding lights on the flexible-armed porphyrins: Human telomeric G4 DNA interaction and cell photocytotoxicity research. (United States)

    Sun, Xiang-Yu; Zhao, Ping; Jin, Shu-Fang; Liu, Min-Chao; Wang, Xia-Hong; Huang, Yu-Min; Cheng, Zhen-Feng; Yan, Si-Qi; Li, Yan-Yu; Chen, Ya-Qing; Zhong, Yan-Mei


    DNA polymorphism exerts a fascination on a large scientific community. Without crystallographic structural data, clarification of the binding modes between G-quadruplex (G4) and ligand (complex) is a challenging job. In the present work, three porphyrin compounds with different flexible carbon chains (arms) were designed, synthesized and characterized. Their binding, folding and stabilizing abilities to human telomeric G4 DNA structures were comparatively researched. Positive charges at the end of the flexible carbon chains seem to be favorable for the DNA-porphyrin interactions, which were evidenced by the spectral results and further confirmed by the molecular docking calculations. Biological function analysis demonstrated that these porphyrins show no substantial inhibition to Hela, A549 and BEL 7402 cancer cell lines under dark while exhibit broad inhibition under visible light. This significantly enhanced photocytotoxicity relative to the dark control is an essential property of photochemotherapeutic agents. The feature of the flexible arms emerges as critical influencing factors in the cell photocytotoxicity. Moreover, an ROS-mediated mitochondrial dysfunction pathway was suggested for the cell apoptosis induced by these flexible-armed porphyrins. It is found that the porphyrins with positive charges located at the end of the flexible arms represent an exciting opportunity for photochemotherapeutic anti-cancer drug design. Copyright © 2017. Published by Elsevier B.V.

  9. RecQ-core of BLM unfolds telomeric G-quadruplex in the absence of ATP

    Czech Academy of Sciences Publication Activity Database

    Budhathoki, J.B.; Ray, S.; Urban, Václav; Janščák, Pavel; Yodh, J.G.; Balci, H.


    Roč. 42, č. 18 (2014), 11528–11545 ISSN 0305-1048 R&D Projects: GA ČR GA204/09/0565 Grant - others:U.S. National Science Foundation(US) 1430124 Institutional support: RVO:68378050 Keywords : Bloom helicase * ATP * Gquadruplex (GQ) structure * GQ destabilization Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 9.112, year: 2014

  10. Novel molecular targets for kRAS downregulation: promoter G-quadruplexes (United States)


    monitor the effect of G4 formation, rather than mutating the promoter sequence. We screened the Diversity Set II and III from the National Cancer...Bgl I (kRAS-500 plasmid) or Nhe I (all kRAS-324 plasmids) and HIND III cut sites. kRAS-324 was con- structed by Operon; the kRAS-500, 324 mt Near, and...molecular targets for breast cancer therapeutics, Molecules ( Basel , Switz.) 18 (2013) 15019–15034. [38] T.C. He, A.B. Sparks, C. Rago, H. Hermeking

  11. Targeting C-Myc Promoter: Helquats As Novel G-Quadruplex Stabilizing Ligands

    Czech Academy of Sciences Publication Activity Database

    Kužmová, Erika; Kozák, Jaroslav; Komárková, Veronika; Pytlík, R.; Teplý, Filip; Hájek, Miroslav


    Roč. 124, č. 21 (2014) ISSN 0006-4971. [Annual Meeting of the American Society of Hematology /56./. 06.12.2014-09.12.2014, San Francisco] Institutional support: RVO:61388963 Keywords : helquats * C-Myc * leukemia Subject RIV: CE - Biochemistry

  12. G-quadruplex formation in the Oct4 promoter positively regulates Oct4 expression

    Czech Academy of Sciences Publication Activity Database

    Renčiuk, Daniel; Ryneš, J.; Kejnovská, Iva; Foldynova-Trantirkova, S.; Andaeng, M.; Trantírek, L.; Vorlíčková, Michaela


    Roč. 1860, č. 2 (2017), s. 175-183 ISSN 1874-9399 R&D Projects: GA ČR(CZ) GP14-33947P; GA ČR GAP205/12/0466; GA ČR(CZ) GA15-06785S Institutional support: RVO:68081707 Keywords : linked polymorphic region * guanine quadruplexes * transcription factor Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 5.018, year: 2016

  13. Wild-type p53 binds to MYC promoter G-quadruplex

    Czech Academy of Sciences Publication Activity Database

    Petr, Marek; Helma, Robert; Polášková, Alena; Krejci, Aneta; Dvořáková, Zuzana; Kejnovská, Iva; Navrátilová, Lucie; Adámik, Matěj; Vorlíčková, Michaela; Brázdová, Marie


    Roč. 36, OCT2016 (2016), č. článku e00397. ISSN 0144-8463 R&D Projects: GA ČR GA13-36108S; GA ČR GAP205/12/0466 Institutional support: RVO:68081707 Keywords : nuclease-hypersensitive element * c-terminal domain * gene-expression Subject RIV: BO - Biophysics Impact factor: 2.906, year: 2016

  14. Bloom’s syndrome protein unfolding G-quadruplexes in two pathways (United States)

    Zhao, Zhen-Ye; Xu, Chun-Hua; Shi, Jing; Li, Jing-Hua; Ma, Jian-Bing; Jia, Qi; Ma, Dong-Fei; Li, Ming; Lu, Ying


    Not Available Project supported by the National Natural Science Foundation of China (Grant Nos. 11674382, 11574381, and 11574382) and the Key Research Program of Frontier Sciences, Chinese Academy of Sciences (Grant No. QYZDJ-SSW-SYS014).

  15. Conformations of Human Telomeric G-Quadruplex Studied Using a Nucleotide-Independent Nitroxide Label

    Czech Academy of Sciences Publication Activity Database

    Zhang, X.J.; Xu, C.X.; Di Felice, R.; Šponer, Jiří; Islam, B.; Stadlbauer, Petr; Ding, Y.; Mao, L.; Mao, Z.W.; Qin, P.Z.


    Roč. 55, č. 2 (2016), s. 360-372 ISSN 0006-2960 R&D Projects: GA ČR(CZ) GAP208/11/1822; GA MŠk(CZ) ED1.1.00/02.0068 Institutional support: RVO:68081707 Keywords : PARAMAGNETIC-RESONANCE SPECTROSCOPY * AMBER FORCE-FIELD * NUCLEIC- ACIDS Subject RIV: BO - Biophysics Impact factor: 2.938, year: 2016

  16. Lovastatin prevents cisplatin-induced activation of pro-apoptotic DNA damage response (DDR) of renal tubular epithelial cells. (United States)

    Krüger, Katharina; Ziegler, Verena; Hartmann, Christina; Henninger, Christian; Thomale, Jürgen; Schupp, Nicole; Fritz, Gerhard


    The platinating agent cisplatin (CisPt) is commonly used in the therapy of various types of solid tumors. The anticancer efficacy of CisPt largely depends on the formation of bivalent DNA intrastrand crosslinks, which stimulate mechanisms of the DNA damage response (DDR), thereby triggering checkpoint activation, gene expression and cell death. The clinically most relevant adverse effect associated with CisPt treatment is nephrotoxicity that results from damage to renal tubular epithelial cells. Here, we addressed the question whether the HMG-CoA-reductase inhibitor lovastatin affects the DDR of renal cells by employing rat renal proximal tubular epithelial (NRK-52E) cells as in vitro model. The data show that lovastatin has extensive inhibitory effects on CisPt-stimulated DDR of NRK-52E cells as reflected on the levels of phosphorylated ATM, Chk1, Chk2, p53 and Kap1. Mitigation of CisPt-induced DDR by lovastatin was independent of the formation of DNA damage as demonstrated by (i) the analysis of Pt-(GpG) intrastrand crosslink formation by Southwestern blot analyses and (ii) the generation of DNA strand breaks as analyzed on the level of nuclear γH2AX foci and employing the alkaline comet assay. Lovastatin protected NRK-52E cells from the cytotoxicity of high CisPt doses as shown by measuring cell viability, cellular impedance and flow cytometry-based analyses of cell death. Importantly, the statin also reduced the level of kidney DNA damage and apoptosis triggered by CisPt treatment of mice. The data show that the lipid-lowering drug lovastatin extensively counteracts pro-apoptotic signal mechanisms of the DDR of tubular epithelial cells following CisPt injury. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. Dynamic DNA binding, junction recognition and G4 melting activity underlie the telomeric and genome-wide roles of human CST. (United States)

    Bhattacharjee, Anukana; Wang, Yongyao; Diao, Jiajie; Price, Carolyn M


    Human CST (CTC1-STN1-TEN1) is a ssDNA-binding complex that helps resolve replication problems both at telomeres and genome-wide. CST resembles Replication Protein A (RPA) in that the two complexes harbor comparable arrays of OB-folds and have structurally similar small subunits. However, the overall architecture and functions of CST and RPA are distinct. Currently, the mechanism underlying CST action at diverse replication issues remains unclear. To clarify CST mechanism, we examined the capacity of CST to bind and resolve DNA structures found at sites of CST activity. We show that CST binds preferentially to ss-dsDNA junctions, an activity that can explain the incremental nature of telomeric C-strand synthesis following telomerase action. We also show that CST unfolds G-quadruplex structures, thus providing a mechanism for CST to facilitate replication through telomeres and other GC-rich regions. Finally, smFRET analysis indicates that CST binding to ssDNA is dynamic with CST complexes undergoing concentration-dependent self-displacement. These findings support an RPA-based model where dissociation and re-association of individual OB-folds allow CST to mediate loading and unloading of partner proteins to facilitate various aspects of telomere replication and genome-wide resolution of replication stress. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  18. Improved Inhibition of Telomerase by Short Twisted Intercalating Nucleic Acids under Molecular Crowding Conditions

    DEFF Research Database (Denmark)

    Agarwal, Tani; Pradhan, Devranjan; Géci, Imrich


    Human telomeric DNA has the ability to fold into a 4-stranded G-quadruplex structure. Several G-quadruplex ligands are known to stabilize the structure and thereby inhibit telomerase activity. Such ligands have demonstrated efficient telomerase inhibition in dilute conditions, but under molecular...

  19. Folding topology of a bimolecular DNA quadruplex containing a stable mini-hairpin motif within the diagonal loop. (United States)

    Balkwill, Graham D; Garner, Thomas P; Williams, Huw E L; Searle, Mark S


    We describe the NMR structural characterisation of a bimolecular anti-parallel DNA quadruplex d(G(3)ACGTAGTG(3))(2) containing an autonomously stable mini-hairpin motif inserted within the diagonal loop. A folding topology is identified that is different from that observed for the analogous d(G(3)T(4)G(3))(2) dimer with the two structures differing in the relative orientation of the diagonal loops. This appears to reflect specific base stacking interactions at the quadruplex-duplex interface that are not present in the structure with the T(4)-loop sequence. A truncated version of the bimolecular quadruplex d(G(2)ACGTAGTG(2))(2), with only two core G-tetrads, is less stable and forms a heterogeneous mixture of three 2-fold symmetric quadruplexes with different loop arrangements. We demonstrate that the nature of the loop sequence, its ability to form autonomously stable structure, the relative stabilities of the hairpin loop and core quadruplex, and the ability to form favourable stacking interactions between these two motifs are important factors in controlling DNA G-quadruplex topology.

  20. Phototoxic Activity and DNA Interactions of Water-Soluble Porphyrins and Their Rhenium(I) Conjugates. (United States)

    Mion, Giuliana; Gianferrara, Teresa; Bergamo, Alberta; Gasser, Gilles; Pierroz, Vanessa; Rubbiani, Riccardo; Vilar, Ramon; Leczkowska, Anna; Alessio, Enzo


    In the search for alternative photosensitizers for use in photodynamic therapy (PDT), herein we describe two new water-soluble porphyrins, a neutral fourfold-symmetric compound and a +3-charged tris-methylpyridinium derivative, in which either four or one [1,4,7]-triazacyclononane (TACN) units are connected to the porphyrin macrocycle through a hydrophilic linker; we also report their corresponding tetracationic Re(I) conjugates. The in vitro (photo)toxic effects of the compounds toward the human cell lines HeLa (cervical cancer), H460M2 (non-small-cell lung carcinoma), and HBL-100 (non-tumorigenic epithelial cells) are reported. Three of the compounds are not cytotoxic in the dark up to 100 μm, and the fourfold-symmetric couple revealed very good phototoxic indexes (PIs). The intracellular localization of all derivatives was studied in HeLa cells by confocal fluorescence microscopy. Although low nuclear localization was observed for some of them, it still prompted us to investigate their capacity to bind both quadruplex and duplex DNA; we observed significant selectivity in the tris-methylpyridinium derivatives for G-quadruplex interactions. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Cisplatin adducts on a GGG sequence within a DNA duplex studied by NMR spectroscopy and molecular dynamics simulations. (United States)

    Téletchéa, Stéphane; Skauge, Tormod; Sletten, Einar; Kozelka, Jirí


    The antitumor drug cisplatin(cis-[PtCl2(NH3)2]) reacts with cellular DNA to form GG intrastrand adducts between adjacent guanines as predominant lesions. GGG sites have been shown to be hotspots of platination. To study the structural perturbation induced by binding of cisplatin to two adjacent guanines of a GGG trinucleotide,we examined here the decanucleotide duplex d[(G1C2C3G*4 G*5 G6T7-C8G9C10).d(G11C12G13A14C15C16C17G18-G19C20)] (dsCG*G*G) intrastrand cross-linked at the G* guanines by cis-{Pt(NH3)2}2+ using NMR spectroscopy and molecular dynamics (MD) simulations.The NMR spectra of dsCG*G*G were found to be similar to those of previously characterized DNA duplexes cross-linked by cisplatin at apyG*G*X site (py=pyrimidine; X=C,T, A). This similarity of NMR spectra indicates that the base at the 3'-side of the G*G*-Pt cross-link does not affect the structure to a large extent. An unprecedented reversible isomerization between the duplex dsCG*G*G (bearing a G*4 G*5 -Pt chelate) and duplex dsGG*G*T (bearing a G*5 G*6 -Pt chelate)was observed, which yielded a 40:60 equilibrium between the two intrastrand GG-Pt cross-links. No formation of interstrand cross-links was observed.NMR spectroscopic data of dsCG*G*G indicated that the deoxyribose of the 5'-G* adopts an N-type conformation, and the cytidines C3, C15,and C16 have average phase angles intermediate between S and N. The NMR spectroscopic chemical shifts of dsGG*G*T showed some fundamental differences to those of pyG*G*-platinum adducts but were in agreement with the NMR spectra reported previously for the DNA duplexes crosslinked at an AG*G*C sequence by cisplatin or oxaliplatin. The presence of apurine instead of a pyrimidine at the 5'-side of the G*G* cross-link seems therefore to affect the structure of the XG* step significantly.

  2. Deficiency in DNA damage response, a new characteristic of cells infected with latent HIV-1. (United States)

    Piekna-Przybylska, Dorota; Sharma, Gaurav; Maggirwar, Sanjay B; Bambara, Robert A


    Viruses can interact with host cell molecules responsible for the recognition and repair of DNA lesions, resulting in dysfunctional DNA damage response (DDR). Cells with inefficient DDR are more vulnerable to therapeutic approaches that target DDR, thereby raising DNA damage to a threshold that triggers apoptosis. Here, we demonstrate that 2 Jurkat-derived cell lines with incorporated silent HIV-1 provirus show increases in DDR signaling that responds to formation of double strand DNA breaks (DSBs). We found that phosphorylation of histone H2AX on Ser139 (gamma-H2AX), a biomarker of DSBs, and phosphorylation of ATM at Ser1981, Chk2 at Thr68, and p53 at Ser15, part of signaling pathways associated with DSBs, are elevated in these cells. These results indicate a DDR defect even though the virus is latent. DDR-inducing agents, specifically high doses of nucleoside RT inhibitors (NRTIs), caused greater increases in gamma-H2AX levels in latently infected cells. Additionally, latently infected cells are more susceptible to long-term exposure to G-quadruplex stabilizing agents, and this effect is enhanced when the agent is combined with an inhibitor targeting DNA-PK, which is crucial for DSB repair and telomere maintenance. Moreover, exposing these cells to the cancer drug etoposide resulted in formation of DSBs at a higher rate than in un-infected cells. Similar effects of etoposide were also observed in population of primary memory T cells infected with latent HIV-1. Sensitivity to these agents highlights a unique vulnerability of latently infected cells, a new feature that could potentially be used in developing therapies to eliminate HIV-1 reservoirs.

  3. Mycobacterium tuberculosis DinG is a structure-specific helicase that unwinds G4 DNA: implications for targeting G4 DNA as a novel therapeutic approach. (United States)

    Thakur, Roshan Singh; Desingu, Ambika; Basavaraju, Shivakumar; Subramanya, Shreelakshmi; Rao, Desirazu N; Nagaraju, Ganesh


    The significance of G-quadruplexes and the helicases that resolve G4 structures in prokaryotes is poorly understood. The Mycobacterium tuberculosis genome is GC-rich and contains >10,000 sequences that have the potential to form G4 structures. In Escherichia coli, RecQ helicase unwinds G4 structures. However, RecQ is absent in M. tuberculosis, and the helicase that participates in G4 resolution in M. tuberculosis is obscure. Here, we show that M. tuberculosis DinG (MtDinG) exhibits high affinity for ssDNA and ssDNA translocation with a 5' → 3' polarity. Interestingly, MtDinG unwinds overhangs, flap structures, and forked duplexes but fails to unwind linear duplex DNA. Our data with DNase I footprinting provide mechanistic insights and suggest that MtDinG is a 5' → 3' polarity helicase. Notably, in contrast to E. coli DinG, MtDinG catalyzes unwinding of replication fork and Holliday junction structures. Strikingly, we find that MtDinG resolves intermolecular G4 structures. These data suggest that MtDinG is a multifunctional structure-specific helicase that unwinds model structures of DNA replication, repair, and recombination as well as G4 structures. We finally demonstrate that promoter sequences of M. tuberculosis PE_PGRS2, mce1R, and moeB1 genes contain G4 structures, implying that G4 structures may regulate gene expression in M. tuberculosis. We discuss these data and implicate targeting G4 structures and DinG helicase in M. tuberculosis could be a novel therapeutic strategy for culminating the infection with this pathogen. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  4. Lentivector Integration Sites in Ependymal Cells From a Model of Metachromatic Leukodystrophy: Non-B DNA as a New Factor Influencing Integration (United States)

    McAllister, Robert G; Liu, Jiahui; Woods, Matthew W; Tom, Sean K; Rupar, C Anthony; Barr, Stephen D


    The blood–brain barrier controls the passage of molecules from the blood into the central nervous system (CNS) and is a major challenge for treatment of neurological diseases. Metachromatic leukodystrophy is a neurodegenerative lysosomal storage disease caused by loss of arylsulfatase A (ARSA) activity. Gene therapy via intraventricular injection of a lentiviral vector is a potential approach to rapidly and permanently deliver therapeutic levels of ARSA to the CNS. We present the distribution of integration sites of a lentiviral vector encoding human ARSA (LV-ARSA) in murine brain choroid plexus and ependymal cells, administered via a single intracranial injection into the CNS. LV-ARSA did not exhibit a strong preference for integration in or near actively transcribed genes, but exhibited a strong preference for integration in or near satellite DNA. We identified several genomic hotspots for LV-ARSA integration and identified a consensus target site sequence characterized by two G-quadruplex-forming motifs flanking the integration site. In addition, our analysis identified several other non-B DNA motifs as new factors that potentially influence lentivirus integration, including human immunodeficiency virus type-1 in human cells. Together, our data demonstrate a clinically favorable integration site profile in the murine brain and identify non-B DNA as a potential new host factor that influences lentiviral integration in murine and human cells. PMID:25158091

  5. Radiation-produced electron migration along 5-bromouracil-substituted DNA in cells and in solutions

    International Nuclear Information System (INIS)

    Beach, C.M.


    Results of work by other investigators support the theory of charge migration in DNA. Charge transfer between nucleotides and electron and energy migration in solid state DNA have been detected, but no previous experiments have demonstrated charge migration in aqueous solutions of DNA or in DNA inside an E. coli cell. Such experiments were performed by substituting different amounts of 5-bromouracil (BU) for thymine in E. coli DNA and assaying for the amount of bromide given off from the reaction of bromouracil with hydrated electrons produced by ionizing radiation to form uracil-5-yl radicals and free bromide. By varying the amount of BU incorporated in the DNA, the average distance between the BU bases was varied, and because the number of BU/electron reactions was monitored by the amount of bromide released, the maximum average electron migration distance along the BU-DNA was estimated. Hydrated electrons, e/sub aq/, were shown to react with BU in BU-DNA with the resultant release of bromide with G(-BR - ) = 0.519 +- 0.062. OH radicals were half as reactive as e/sub aq/ toward producing bromide from BU-DNA. O 2 , which has been shown to transfer charge to BU in aqueous solution, did not transfer charge to BU-DNA. The CO 2 radical was shown to cause the release of bromide from BU-DNA at least as effectively as e/sub aq/. Charge migration was demonstrated, and the maximum average electron migration distance in aqueous solutions of BU-DNA was measured to be 8 to 10 base distances (assuming only intrastrand migration). Only 11% to 16% of the electrons produced attacked BU-DNA in aqueous solution, and only 1% resulted in bromide release from BU-DNA inside E. coli. Charge migration was demonstrated in BU-DNA inside E. coli., and the maximum average migration distance was measured to be 5 to 6 base distances

  6. Single-molecule TPM studies on the conversion of human telomeric DNA. (United States)

    Chu, Jen-Fei; Chang, Ta-Chau; Li, Hung-Wen


    Human telomere contains guanine-rich (G-rich) tandem repeats of single-stranded DNA sequences at its 3' tail. The G-rich sequences can be folded into various secondary structures, termed G-quadruplexes (G4s), by Hoogsteen basepairing in the presence of monovalent cations (such as Na+ and K+). We developed a single-molecule tethered particle motion (TPM) method to investigate the unfolding process of G4s in the human telomeric sequence AGGG(TTAGGG)3 in real time. The TPM method monitors the DNA tether length change caused by formation of the G4, thus allowing the unfolding process and structural conversion to be monitored at the single-molecule level. In the presence of its antisense sequence, the folded G4 structure can be disrupted and converted to the unfolded conformation, with apparent unfolding time constants of 82 s and 3152 s. We also observed that the stability of the G4 is greatly affected by different monovalent cations. The folding equilibrium constant of G4 is strongly dependent on the salt concentration, ranging from 1.75 at 5 mM Na+ to 3.40 at 15 mM Na+. Earlier spectral studies of Na+- and K+-folded states suggested that the spectral conversion between these two different folded structures may go through a structurally unfolded intermediate state. However, our single-molecule TPM experiments did not detect any totally unfolded intermediate within our experimental resolution when sodium-folded G4 DNA molecules were titrated with high-concentration, excess potassium ions. This observation suggests that a totally unfolding pathway is likely not the major pathway for spectral conversion on the timescale of minutes, and that interconversion among folded states can be achieved by the loop rearrangement. This study also demonstrates that TPM experiments can be used to study conformational changes in single-stranded DNA molecules. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  7. Differential repair of platinum-DNA adducts in human bladder and testicular tumor continuous cell lines

    International Nuclear Information System (INIS)

    Bedford, P.; Fichtinger-Schepman, A.M.; Shellard, S.A.; Walker, M.C.; Masters, J.R.; Hill, B.T.


    The formation and removal of four platinum-DNA adducts were immunochemically quantitated in cultured cells derived from a human bladder carcinoma cell line (RT112) and from two lines derived from germ cell tumors of the testis (833K and SUSA), following exposure in vitro to 16.7 microM (5 micrograms/ml) cisplatin. RT112 cells were least sensitive to the drug and were proficient in the repair of all four adducts, whereas SUSA cells, which were 5-fold more sensitive, were deficient in the repair of DNA-DNA intrastrand cross-links in the sequences pApG and pGpG. Despite expressing a similar sensitivity to SUSA cells, 833K cells were proficient in the repair of all four adducts, although less so than the RT112 bladder tumor cells. In addition, SUSA cells were unable to repair DNA-DNA interstrand cross-links whereas 50-85% of these lesions were removed in RT112 and 833K cells 24 h following drug exposure. It is possible that the inability of SuSa cells to repair platinated DNA may account for their hypersensitivity to cisplatin

  8. Genome-wide identification and characterisation of human DNA replication origins by initiation site sequencing (ini-seq). (United States)

    Langley, Alexander R; Gräf, Stefan; Smith, James C; Krude, Torsten


    Next-generation sequencing has enabled the genome-wide identification of human DNA replication origins. However, different approaches to mapping replication origins, namely (i) sequencing isolated small nascent DNA strands (SNS-seq); (ii) sequencing replication bubbles (bubble-seq) and (iii) sequencing Okazaki fragments (OK-seq), show only limited concordance. To address this controversy, we describe here an independent high-resolution origin mapping technique that we call initiation site sequencing (ini-seq). In this approach, newly replicated DNA is directly labelled with digoxigenin-dUTP near the sites of its initiation in a cell-free system. The labelled DNA is then immunoprecipitated and genomic locations are determined by DNA sequencing. Using this technique we identify >25,000 discrete origin sites at sub-kilobase resolution on the human genome, with high concordance between biological replicates. Most activated origins identified by ini-seq are found at transcriptional start sites and contain G-quadruplex (G4) motifs. They tend to cluster in early-replicating domains, providing a correlation between early replication timing and local density of activated origins. Origins identified by ini-seq show highest concordance with sites identified by SNS-seq, followed by OK-seq and bubble-seq. Furthermore, germline origins identified by positive nucleotide distribution skew jumps overlap with origins identified by ini-seq and OK-seq more frequently and more specifically than do sites identified by either SNS-seq or bubble-seq. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  9. Platinum-induced kidney damage: Unraveling the DNA damage response (DDR) of renal tubular epithelial and glomerular endothelial cells following platinum injury. (United States)

    Krüger, Katharina; Thomale, Jürgen; Stojanović, Nikolina; Osmak, Maja; Henninger, Christian; Bormann, Stefanie; Fritz, Gerhard


    Platinum compounds are potent anticancer drugs but also evoke considerable normal tissue damage. Here, we elucidate the molecular mechanisms contributing to the nephrotoxic effects of cisplatin. We comparatively investigated the stress responses of rat kidney tubular (NRK-52E) and glomerular cells (RGE) following treatment with cisplatin (CisPt), oxaliplatin (OxaliPt) and carboplatin (CarboPt). To this end, cell viability, apoptosis, cell cycle progression, DNA damage response (DDR) and repair of DNA adducts were investigated. CisPt reduced the viability of tubular NRK-52E and glomerular RGE cells most efficiently. Cytotoxicity evoked by CarboPt occurred with a delay, which might be related to a retarded formation of Pt-(GpG) intrastrand crosslinks. RGE cells were more sensitive towards all platinum compounds than NRK-52E cells. Platinum drugs efficiently induced caspase-mediated apoptosis in tubular cells, while RGE cells favored G2/M arrest when treated with equitoxic platinum doses. Mitotic index of NKR-52E and RGE cells was worst affected by OxaliPt. Activation of the DDR was strikingly agent- and cell type-specific. Most comprehensive and substantial stimulation of DDR mechanisms was provoked by CisPt. Repair of Pt-(GpG) intrastrand crosslinks was best in RGE, which was reflected by high mRNA expression of nucleotide excision repair (NER) factors. There are substantial differences regarding the cause of sensitivity and mechanisms of DDR between tubular and glomerular cells following platinum injury. CisPt is the most potent stimulator of the DDR. We hypothesize that specific DNA adducts and thereby forcefully activated pro-toxic DDR mechanisms contribute to the exceptionally high acute nephrotoxicity of CisPt. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. IFI16 Preferentially Binds to DNA with Quadruplex Structure and Enhances DNA Quadruplex Formation

    Czech Academy of Sciences Publication Activity Database

    Haroniková, Lucia; Coufal, Jan; Kejnovská, Iva; Jagelská, Eva; Fojta, Miroslav; Dvořáková, P.; Muller, P.; Vojtešek, B.; Brázda, Václav


    Roč. 11, č. 6 (2016) E-ISSN 1932-6203 R&D Projects: GA ČR GA15-21855S; GA ČR(CZ) GBP206/12/G151 Institutional support: RVO:68081707 Keywords : INTERFERON-INDUCIBLE PROTEIN * CIRCULAR-DICHROISM SPECTROSCOPY * RELEVANT G-QUADRUPLEX Subject RIV: BO - Biophysics Impact factor: 2.806, year: 2016

  11. DNA Domino-Based Nanoscale Logic Circuit: A Versatile Strategy for Ultrasensitive Multiplexed Analysis of Nucleic Acids. (United States)

    Ravan, Hadi; Amandadi, Mojdeh; Esmaeili-Mahani, Saeed


    In recent years, the analytical application of logical nanodevices has attracted much attention for making accurate decisions on molecular diagnosis. Herein, a DNA domino-based nanoscale logic circuit has been constructed by integrating three logic gates (AND-AND-YES) for simultaneous analysis of multiple nucleic acid biomarkers. In the first AND gate, a chimeric target DNA comprising of four biomarkers was hybridized to three biomarker-specific oligonucleotides (TRs) via their 5'-end regions and to a capture probe-magnetic microparticle. After harvesting the complex, 3' overhang regions of the TRs were labeled with three distinct monolayer double-stranded (ds) DNA-gold nanoparticles (DNA-AuNPs). Upon gleaning the complex and addition of initiator oligonucleotide, a series of toehold-mediated strand displacement reactions, which are reminiscent of a domino chain, spontaneously occurred between the confined dsDNAs on the nanoparticles' surface in the second AND gate. The output of the second gate entered into the last gate and triggered an exponential hairpin assembly to form four-way junction nanostructures. The resulting nanostructures bear split parts of DNAzyme at each end of the four arms which, in the presence of hemin, form catalytic hemin/G-quadruplex DNAzymes with peroxidase activity. The smart biosensor has exhibited a turn-on signal when all biomarkers are present in the sample. In fact, should any of the biomarkers be nonexistent, the signal remains turned-off. The biosensor can detect the biomarkers with a LOD value of 100 aM and a noticeable capability to discriminate single-nucleotide substitutions.

  12. Thermodynamic and spectroscopic investigations of TMPyP4 association with guanine- and cytosine-rich DNA and RNA repeats of C9orf72. (United States)

    Alniss, Hasan; Zamiri, Bita; Khalaj, Melisa; Pearson, Christopher E; Macgregor, Robert B


    An expansion of the hexanucleotide repeat (GGGGCC)n·(GGCCCC)n in the C9orf72 promoter has been shown to be the cause of Amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD). The C9orf72 repeat can form four-stranded structures; the cationic porphyrin (TMPyP4) binds and distorts these structures. Isothermal titration calorimetry (ITC), and circular dichroism (CD) were used to study the binding of TMPyP4 to the C-rich and G-rich DNA and RNA oligos containing the hexanucleotide repeat at pH 7.5 and 0.1 M K + . The CD spectra of G-rich DNA and RNA TMPyP4 complexes showed features of antiparallel and parallel G-quadruplexes, respectively. The shoulder at 260 nm in the CD spectrum becomes more intense upon formation of complexes between TMPyP4 and the C-rich DNA. The peak at 290 nm becomes more intense in the c-rich RNA molecules, suggesting induction of an i-motif structure. The ITC data showed that TMPyP4 binds at two independent sites for all DNA and RNA molecules. For DNA, the data are consistent with TMPyP4 stacking on the terminal tetrads and intercalation. For RNA, the thermodynamics of the two binding modes are consistent with groove binding and intercalation. In both cases, intercalation is the weaker binding mode. These findings are considered with respect to the structural differences of the folded DNA and RNA molecules and the energetics of the processes that drive site-specific recognition by TMPyP4; these data will be helpful in efforts to optimize the specificity and affinity of the binding of porphyrin-like molecules. Copyright © 2018 Elsevier Inc. All rights reserved.

  13. DNA-reactive protein monoepoxides induce cell death and mutagenesis in mammalian cells. (United States)

    Tretyakova, Natalia Y; Michaelson-Richie, Erin D; Gherezghiher, Teshome B; Kurtz, Jamie; Ming, Xun; Wickramaratne, Susith; Campion, Melissa; Kanugula, Sreenivas; Pegg, Anthony E; Campbell, Colin


    Although cytotoxic alkylating agents possessing two electrophilic reactive groups are thought to act by cross-linking cellular biomolecules, their exact mechanisms of action have not been established. In cells, these compounds form a mixture of DNA lesions, including nucleobase monoadducts, interstrand and intrastrand cross-links, and DNA-protein cross-links (DPCs). Interstrand DNA-DNA cross-links block replication and transcription by preventing DNA strand separation, contributing to toxicity and mutagenesis. In contrast, potential contributions of drug-induced DPCs are poorly understood. To gain insight into the biological consequences of DPC formation, we generated DNA-reactive protein reagents and examined their toxicity and mutagenesis in mammalian cells. Recombinant human O(6)-alkylguanine DNA alkyltransferase (AGT) protein or its variants (C145A and K125L) were treated with 1,2,3,4-diepoxybutane to yield proteins containing 2-hydroxy-3,4-epoxybutyl groups on cysteine residues. Gel shift and mass spectrometry experiments confirmed that epoxide-functionalized AGT proteins formed covalent DPC but no other types of nucleobase damage when incubated with duplex DNA. Introduction of purified AGT monoepoxides into mammalian cells via electroporation generated AGT-DNA cross-links and induced cell death and mutations at the hypoxanthine-guanine phosphoribosyltransferase gene. Smaller numbers of DPC lesions and reduced levels of cell death were observed when using protein monoepoxides generated from an AGT variant that fails to accumulate in the cell nucleus (K125L), suggesting that nuclear DNA damage is required for toxicity. Taken together, these results indicate that AGT protein monoepoxides produce cytotoxic and mutagenic DPC lesions within chromosomal DNA. More generally, these data suggest that covalent DPC lesions contribute to the cytotoxic and mutagenic effects of bis-electrophiles.

  14. Erythropoietin overrides the triggering effect of DNA platination products in a mouse model of Cisplatin-induced neuropathy

    Directory of Open Access Journals (Sweden)

    Egensperger Rupert


    Full Text Available Abstract Background Cisplatin mediates its antineoplastic activity by formation of distinct DNA intrastrand cross links. The clinical efficacy and desirable dose escalations of cisplatin are restricted by the accumulation of DNA lesions in dorsal root ganglion (DRG cells leading to sensory polyneuropathy (PNP. We investigated in a mouse model by which mechanism recombinant erythropoietin (rhEPO protects the peripheral nervous system from structural and functional damage caused by cisplatin treatment with special emphasis on DNA damage burden. Results A cumulative dose of 16 mg cisplatin/kg resulted in clear electrophysiological signs of neuropathy, which were significantly attenuated by concomitant erythropoietin (cisplatin 32,48 m/s ± 1,68 m/s; cisplatin + rhEPO 49,66 m/s ± 1,26 m/s; control 55,01 m/s ± 1,88 m/s; p Conclusion The protective effect of recombinant erythropoietin is not mediated by reducing the burden of DNA platination in the target cells, but it is likely to be due to a higher resistance of the target cells to the adverse effect of DNA damage. The increased frequency of intact mitochondria might also contribute to this protective role.

  15. Strong preference of BRCA1 protein to topologically constrained non-B DNA structures

    Czech Academy of Sciences Publication Activity Database

    Brázda, Václav; Haroniková, Lucia; Liao, J.C.C.; Fridrichova, Helena; Jagelská, Eva


    Roč. 17, JAN2016 (2016), č. článku 14. ISSN 1471-2199 R&D Projects: GA ČR GA15-21855S Institutional support: RVO:68081707 Keywords : double-strand breaks * g-quadruplexes * c-myc Subject RIV: BO - Biophysics Impact factor: 1.939, year: 2016

  16. Optofluidics-based DNA structure-competitive aptasensor for rapid on-site detection of lead(II) in an aquatic environment. (United States)

    Long, Feng; Zhu, Anna; Wang, Hongchen


    Lead ions (Pb(2+)), ubiquitous and one of the most toxic metallic pollutants, have attracted increasing attentions because of their various neurotoxic effects. Pb(2+) has been proven to induce a conformational change in G-quadruplex (G4) aptamers to form a stabilizing G4/Pb(2+) complex. Based on this principle, an innovative optofluidics-based DNA structure-competitive aptasensor was developed for Pb(2+) detection in an actual aquatic environment. The proposed sensing system has good characteristics, such as high sensitivity and selectivity, reusability, easy operation, rapidity, robustness, portability, use of a small sample volume, and cost effectiveness. A fluorescence-labeled G4 aptamer was utilized as a molecular probe. A DNA probe, a complementary strand of G4 aptamer, was immobilized onto the sensor surface. When the mixture of Pb(2+) solution and G4 aptamer was introduced into the optofluidic cell, Pb(2+) and the DNA probe bound competitively with the G4 aptamer. A high Pb(2+) concentration reduced the binding of the aptamer and the DNA probe; thus, a low-fluorescence signal was detected. A sensitive sensing response to Pb(2+) in the range of 1.0-300.0 nM with a low detection limit of 0.22 nM was exhibited under optimal conditions. The potential interference of the environmental sample matrix was assessed with spiked samples, and the recovery of Pb(2+) ranged from 80 to 105% with a relative standard deviation value of monitoring of other trace analytes. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Can the response to a platinum-based therapy be predicted by the DNA repair status in non-small cell lung cancer? (United States)

    Macerelli, Marianna; Ganzinelli, Monica; Gouedard, Cedric; Broggini, Massimo; Garassino, Marina Chiara; Linardou, Helena; Damia, Giovanna; Wiesmüller, Lisa


    Preclinical evidence has been accumulating on the impact of the DNA repair status on the sensitivity/resistance to anticancer agents in different tumor types, including lung cancer. The possibility to predict the response to therapy, and specifically to platinum agents, based on tumor specific DNA repair functionality would enable to tailor its use only in those patients with maximum chances to respond, avoiding the burden of toxicity in those ones with lesser chances. We here reviewed the clinical evidence on the prognostic role of DNA repair markers and/or functional assays in predicting the response to a platinum-based chemotherapy in lung cancer patients. Consequently, we focused on those proteins involved in pathways repairing platinum induced DNA inter-strand and intra-strand crosslinks. Most promising clinical trials targeting the nucleotide repair protein ERCC1 in non-small cell lung cancer later on suffered from serious drawbacks. Nevertheless, these results spurred a variety of preclinical studies on a multitude of alternative DNA repair markers. However so far, no one of the analyzed DNA repair markers can be considered a reliable and mature biomarker for selecting patients. We discuss the reasons for such failure which discloses novel strategies for the future. Copyright © 2016 Elsevier Ltd. All rights reserved.

  18. Ancient DNA

    DEFF Research Database (Denmark)

    Willerslev, Eske; Cooper, Alan


    ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair......ancient DNA, palaeontology, palaeoecology, archaeology, population genetics, DNA damage and repair...

  19. Circular Dichroism of G-Quadruplex: A Laboratory Experiment for the Study of Topology and Ligand Binding (United States)

    Carvalho, Josue´; Queiroz, João A.; Cruz, Carla


    Circular dichroism (CD) has emerged as one of the standard biophysical techniques for the study of guaninequadruplex (G4) folding, cation effect, and ligand binding. The utility of this technique is based on its robustness, ease of use, and requirement of only small quantities of nucleic acid. This experiment is also extendable to the classroom…

  20. G-Quadruplex Identification in the Genome of Protozoan Parasites Points to Naphthalene Diimide Ligands as New Antiparasitic Agents

    Czech Academy of Sciences Publication Activity Database

    Belmonte-Reche, E.; Martínez-García, M.; Guédin, A.; Zuffo, M.; Arevalo-Ruiz, M.; Doria, F.; Campos-Salinas, J.; Maynadier, M.; Lopez-Rubio, J.J.; Freccero, M.; Mergny, Jean-Louis; Maria Perez-Victoria, J.; Carlos Morales, J.


    Roč. 61, č. 3 (2018), s. 1231-1240 ISSN 0022-2623 R&D Projects: GA MŠk EF15_003/0000477 Institutional support: RVO:68081707 Keywords : terminal repeat promoter * plasmodium-falciparum Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 6.259, year: 2016

  1. Triplex DNA-binding proteins are associated with clinical outcomes revealed by proteomic measurements in patients with colorectal cancer

    Directory of Open Access Journals (Sweden)

    Nelson Laura D


    Full Text Available Abstract Background Tri- and tetra-nucleotide repeats in mammalian genomes can induce formation of alternative non-B DNA structures such as triplexes and guanine (G-quadruplexes. These structures can induce mutagenesis, chromosomal translocations and genomic instability. We wanted to determine if proteins that bind triplex DNA structures are quantitatively or qualitatively different between colorectal tumor and adjacent normal tissue and if this binding activity correlates with patient clinical characteristics. Methods Extracts from 63 human colorectal tumor and adjacent normal tissues were examined by gel shifts (EMSA for triplex DNA-binding proteins, which were correlated with clinicopathological tumor characteristics using the Mann-Whitney U, Spearman’s rho, Kaplan-Meier and Mantel-Cox log-rank tests. Biotinylated triplex DNA and streptavidin agarose affinity binding were used to purify triplex-binding proteins in RKO cells. Western blotting and reverse-phase protein array were used to measure protein expression in tissue extracts. Results Increased triplex DNA-binding activity in tumor extracts correlated significantly with lymphatic disease, metastasis, and reduced overall survival. We identified three multifunctional splicing factors with biotinylated triplex DNA affinity: U2AF65 in cytoplasmic extracts, and PSF and p54nrb in nuclear extracts. Super-shift EMSA with anti-U2AF65 antibodies produced a shifted band of the major EMSA H3 complex, identifying U2AF65 as the protein present in the major EMSA band. U2AF65 expression correlated significantly with EMSA H3 values in all extracts and was higher in extracts from Stage III/IV vs. Stage I/II colon tumors (p = 0.024. EMSA H3 values and U2AF65 expression also correlated significantly with GSK3 beta, beta-catenin, and NF- B p65 expression, whereas p54nrb and PSF expression correlated with c-Myc, cyclin D1, and CDK4. EMSA values and expression of all three splicing factors correlated

  2. 1,1’,1’’,1’’’-[Porphyrin-5,10,15,20-tetrayltetrakis(3,1-phenylenemethylene]tetraquinolinium Tetrabromide

    Directory of Open Access Journals (Sweden)

    Satoshi Fujii


    Full Text Available Cationic porphyrins interact strongly with guanine quadruplex DNA (G-quadruplex. We herein report the preparation of a cationic porphyrin bearing quinolinium side arms, 1,1’,1’’,1’’’-[porphyrin-5,10,15,20-tetrayltetrakis(3,1-phenylenemethylene]­tetra­quinolinium tetrabromide (mQu, as a potential G-quadruplex ligand.

  3. PCNA ubiquitination is important, but not essential for translesion DNA synthesis in mammalian cells.

    Directory of Open Access Journals (Sweden)

    Ayal Hendel


    Full Text Available Translesion DNA synthesis (TLS is a DNA damage tolerance mechanism in which specialized low-fidelity DNA polymerases bypass replication-blocking lesions, and it is usually associated with mutagenesis. In Saccharomyces cerevisiae a key event in TLS is the monoubiquitination of PCNA, which enables recruitment of the specialized polymerases to the damaged site through their ubiquitin-binding domain. In mammals, however, there is a debate on the requirement for ubiquitinated PCNA (PCNA-Ub in TLS. We show that UV-induced Rpa foci, indicative of single-stranded DNA (ssDNA regions caused by UV, accumulate faster and disappear more slowly in Pcna(K164R/K164R cells, which are resistant to PCNA ubiquitination, compared to Pcna(+/+ cells, consistent with a TLS defect. Direct analysis of TLS in these cells, using gapped plasmids with site-specific lesions, showed that TLS is strongly reduced across UV lesions and the cisplatin-induced intrastrand GG crosslink. A similar effect was obtained in cells lacking Rad18, the E3 ubiquitin ligase which monoubiquitinates PCNA. Consistently, cells lacking Usp1, the enzyme that de-ubiquitinates PCNA exhibited increased TLS across a UV lesion and the cisplatin adduct. In contrast, cells lacking the Rad5-homologs Shprh and Hltf, which polyubiquitinate PCNA, exhibited normal TLS. Knocking down the expression of the TLS genes Rev3L, PolH, or Rev1 in Pcna(K164R/K164R mouse embryo fibroblasts caused each an increased sensitivity to UV radiation, indicating the existence of TLS pathways that are independent of PCNA-Ub. Taken together these results indicate that PCNA-Ub is required for maximal TLS. However, TLS polymerases can be recruited to damaged DNA also in the absence of PCNA-Ub, and perform TLS, albeit at a significantly lower efficiency and altered mutagenic specificity.

  4. Inducible Alkylation of DNA by a Quinone Methide-Peptide Nucleic Acid Conjugate† (United States)

    Liu, Yang; Rokita, Steven E.


    The reversibility of alkylation by a quinone methide intermediate (QM) avoids the irreversible consumption that plagues most reagents based on covalent chemistry and allows for site specific reaction that is controlled by the thermodynamics rather than kinetics of target association. This characteristic was originally examined with an oligonucleotide QM conjugate but broad application depends on alternative derivatives that are compatible with a cellular environment. Now, a peptide nucleic acid (PNA) derivative has been constructed and shown to exhibit an equivalent ability to delivery the reactive QM in a controlled manner. This new conjugate demonstrates high selectivity for a complementary sequence of DNA even when challenged with an alternative sequence containing a single T/T mismatch. Alkylation of non-complementary sequences is only possible when a template strand is present to co-localize the conjugate and its target. For efficient alkylation in this example, a single-stranded region of the target is required adjacent to the QM conjugate. Most importantly, the intrastrand self adducts formed between the PNA and its attached QM remained active and reversible over more than eight days in aqueous solution prior to reaction with a chosen target added subsequently. PMID:22243337

  5. Unique C. elegans telomeric overhang structures reveal the evolutionarily conserved properties of telomeric DNA

    Czech Academy of Sciences Publication Activity Database

    Školáková, Petra; Foldynová-Trantírková, Silvie; Bednářová, Klára; Fiala, R.; Vorlíčková, Michaela; Trantírek, L.


    Roč. 43, č. 9 (2015), s. 4733-4745 ISSN 0305-1048 R&D Projects: GA ČR(CZ) GA13-28310S; GA ČR(CZ) GAP205/12/0466 Institutional support: RVO:68081707 ; RVO:60077344 Keywords : NUCLEASE HYPERSENSITIVE ELEMENT * G-QUADRUPLEX STRUCTURES * I-MOTIF Subject RIV: BO - Biophysics Impact factor: 9.202, year: 2015

  6. Studies on the synthesis, characterization, binding with DNA and activities of two cis-planaramineplatinum(II complexes of the form: cis-PtL(NH3Cl2 where L = 3-hydroxypyridine and 2,3-diaminopyridine

    Directory of Open Access Journals (Sweden)

    Tayyem Hasan


    Full Text Available Abstract Background Cis-planaramineplatinum(II complexes like their trans isomers are often found to be active against cancer cell lines. The present study deals with the synthesis, characterization and determination of activity of new cis-planaramineplatinum(II complexes. Results Two cis-planaramineplatinum(II complexes: cis-(3-hydroxypyridine(amminedichloroplatinum(II (code named AH3 and cis-(2,3-diaminopyridine(amminedichloroplatinum(II (code named AH7 have been prepared and characterised based on elemental analyses, IR, Raman, mass and 1H NMR spectral measurements. The interactions of the compounds with pBR322 plasmid DNA have been investigated and their activity against ovarian cancer cell lines: A2780, A2780cisR and A2780ZD047Rhave been determined. Like cisplatin, AH3 and AH7 are believed to form mainly monofunctional N7(G and bifunctional intrastrand N7(GN7(G adducts with DNA, causing a local distortion of a DNA strand. As a result, gel mobility of the DNA changes. Both AH3 and AH7 are found to be less active than cisplatin against the three cell lines with AH3 being the more active compound of the two. The higher activity of AH3 is in line with its lower molar conductivity value corresponding to a lower degree of dissociation. Conclusion The differences in activity of AH3, AH7 and cisplatin against the cell lines illustrate structure-activity relationship.

  7. Tribbles 2 mediates cisplatin sensitivity and DNA damage response in epithelial ovarian cancer. (United States)

    Kritsch, Daniel; Hoffmann, Franziska; Steinbach, Daniel; Jansen, Lars; Mary Photini, Stella; Gajda, Mieczyslaw; Mosig, Alexander S; Sonnemann, Jürgen; Peters, Sven; Melnikova, Margarita; Thomale, Jürgen; Dürst, Matthias; Runnebaum, Ingo B; Häfner, Norman


    Aim was to identify methylated genes with functional involvement in cisplatin-resistance development of epithelial ovarian cancer (EOC). Genome-wide analyses of hypermethylated CpG-islands in resistant cell lines in combination with qRT-PCR analyses were used to identify epigenetically silenced genes. EOC-Type-II tumors were analyzed for gene methylation and expression and TCGA data were interrogated in-silico. Experiments revealed 37 commonly hypermethylated genes in resistant cells of which Tribbles 2 (TRIB2) showed the most pronounced downregulation on mRNA level and was characterized further. TRIB2 showed a reactivation after 5'-Aza-Cytidine treatment in resistant cells but a cisplatin-dependent, prominent upregulation on mRNA level in sensitive cells, only. Re-expression in resistant A2780 cells increased the sensitivity to cisplatin and other DNA-damaging agents, but not taxanes. Contrary, knockdown of TRIB2 increased resistance to cisplatin in sensitive cells. TRIB2 was involved in the induction of a cisplatin-dependent cell cycle arrest and apoptosis by influencing p21 and survivin expression. An increased Pt-DNA-adduct formation in TRIB2 re-expressing cells did not translate in higher levels of dsDNA damage (yH2AX-foci). Thus, TRIB2 is potentially involved in the signal transduction from nucleotide excision repair of intrastrand cross links. Importantly, patient stratification of two homogenous cohorts of EOC-Type-II patients from Jena (n = 38) and the TCGA (n = 149) by TRIB2 mRNA expression consistently revealed a significantly decreased PFS for patients with low TRIB2 levels (log-rank p < 0.05). Tumors from resistant patients expressed the lowest levels of TRIB2. Downregulation of TRIB2 contributes to platin-resistance and TRIB2 expression should be validated as prognostic and predictive marker for EOC. © 2017 UICC.

  8. Sensitive fluorescent detection of DNA methyltransferase using nicking endonuclease-mediated multiple primers-like rolling circle amplification. (United States)

    Huang, Juan; Li, Xiao-Yu; Du, Yi-Chen; Zhang, Li-Na; Liu, Ke-Ke; Zhu, Li-Na; Kong, De-Ming


    Sensitive and reliable detection of DNA methyltransferase (MTase) is of great significance for both early tumor diagnosis and therapy. In this study, a simple, label-free and sensitive DNA MTase-sensing method was developed on the basis of a nicking endonuclease-mediated multiple primers-like rolling circle amplification (RCA) strategy. In this method, a dumbbell RCA template was prepared by blunt-end ligation of two molecules of hairpin DNA. In addition to the primer-binding sequence, the dumbbell template contained another three important parts: 5'-CCGG-3' sequences in double-stranded stems, nicking endonuclease recognition sites and C-rich sequences in single-stranded loops. The introduction of 5'-CCGG-3' sequences allows the dumbbell template to be destroyed by the restriction endonuclease, HpaII, but is not destroyed in the presence of the target MTase-M.SssI MTase. The introduction of nicking endonuclease recognition sites makes the M.SssI MTase-protected dumbbell template-mediated RCA proceed in a multiple primers-like exponential mode, thus providing the RCA with high amplification efficiency. The introduction of C-rich sequences may promote the folding of amplification products into a G-quadruplex structure, which is specifically recognized by the commercially available fluorescent probe thioflavin T. Improved RCA amplification efficiency and specific fluorescent recognition of RCA products provide the M.SssI MTase-sensing platform with high sensitivity. When a dumbbell template containing four nicking endonuclease sites is used, highly specific M.SssI MTase activity detection can be achieved in the range of 0.008-50U/mL with a detection limit as low as 0.0011U/mL. Simple experimental operation and mix-and-detection fluorescent sensing mode ensures that M.SssI MTase quantitation works well in a real-time RCA mode, thus further simplifying the sensing performance and making high throughput detection possible. The proposed MTase-sensing strategy was also

  9. Mitochondrial DNA. (United States)

    Wright, Russell G.; Bottino, Paul J.


    Provides background information for teachers on mitochondrial DNA, pointing out that it may have once been a free-living organism. Includes a ready-to-duplicate exercise titled "Using Microchondrial DNA to Measure Evolutionary Distance." (JN)

  10. Modeling DNA (United States)

    Robertson, Carol


    Deoxyribonucleic acid (DNA) is life's most amazing molecule. It carries the genetic instructions that almost every organism needs to develop and reproduce. In the human genome alone, there are some three billion DNA base pairs. The most difficult part of teaching DNA structure, however, may be getting students to visualize something as small as a…

  11. Dna Sequencing (United States)

    Tabor, Stanley; Richardson, Charles C.


    A method for sequencing a strand of DNA, including the steps off: providing the strand of DNA; annealing the strand with a primer able to hybridize to the strand to give an annealed mixture; incubating the mixture with four deoxyribonucleoside triphosphates, a DNA polymerase, and at least three deoxyribonucleoside triphosphates in different amounts, under conditions in favoring primer extension to form nucleic acid fragments complementory to the DNA to be sequenced; labelling the nucleic and fragments; separating them and determining the position of the deoxyribonucleoside triphosphates by differences in the intensity of the labels, thereby to determine the DNA sequence.

  12. DNA damage by reactive species: Mechanisms, mutation and repair

    Indian Academy of Sciences (India)


    Jun 25, 2012 ... 2008). In the LC-MS/MS study performed in both aerobic and anaerobic conditions, it has been further found that due to radical formation at the C5 site of cytosine, it can be covalently bonded to the C8 position of guanine of same strand to form G[C8-C5]C intra-strand crosslink product. (scheme 6d) (Box et ...

  13. DNA glue

    DEFF Research Database (Denmark)

    Filichev, Vyacheslav V; Astakhova, Irina V.; Malakhov, Andrei D.


    Significant alterations in thermal stability of parallel DNA triplexes and antiparallel duplexes were observed upon changing the attachment of ethynylpyrenes from para to ortho in the structure of phenylmethylglycerol inserted as a bulge into DNA (TINA). Insertions of two ortho-TINAs as a pseudo...

  14. DNA Vaccines

    Indian Academy of Sciences (India)

    DNA vaccine, immune response, antibodies, infectious diseases. GENERAL I ARTICLE. DNA Vaccines. P N Rangarajan. History of Vaccine Development. The year 1996 marked the 200th anniversary of the first vaccine developed against smallpox by Edward Jenner. In the now- famous 1796 experiment, Jenner scratched ...

  15. Hyperstretching DNA

    NARCIS (Netherlands)

    Schakenraad, Koen; Biebricher, Andreas S.; Sebregts, Maarten; Ten Bensel, Brian; Peterman, Erwin J.G.; Wuite, Gijs J L; Heller, Iddo; Storm, Cornelis; Van Der Schoot, Paul


    The three-dimensional structure of DNA is highly susceptible to changes by mechanical and biochemical cues in vivo and in vitro. In particular, large increases in base pair spacing compared to regular B-DNA are effected by mechanical (over)stretching and by intercalation of compounds that are widely

  16. Ion Binding to Quadruplex DNA Stems. Comparison of MM and QM Descriptions Reveals Sizable Polarization Effects Not Included in Contemporary Simulations

    Czech Academy of Sciences Publication Activity Database

    Gkionis, K.; Kruse, H.; Platts, J. A.; Mládek, Arnošt; Koča, J.; Šponer, Jiří


    Roč. 10, č. 3 (2014), s. 1326-1340 ISSN 1549-9618 R&D Projects: GA ČR(CZ) GAP208/11/1822; GA MŠk(CZ) ED1.1.00/02.0068 Institutional support: RVO:68081707 Keywords : MOLECULAR-DYNAMICS SIMULATIONS * GAUSSIAN-BASIS SETS * TETRAMOLECULAR G-QUADRUPLEXES Subject RIV: BO - Biophysics Impact factor: 5.498, year: 2014

  17. DNA probes

    International Nuclear Information System (INIS)

    Castelino, J.


    The creation of DNA probes for detection of specific nucleotide segments differs from ligand detection in that it is a chemical rather than an immunological reaction. Complementary DNA or RNA is used in place of the antibody and is labelled with 32 P. So far, DNA probes have been successfully employed in the diagnosis of inherited disorders, infectious diseases, and for identification of human oncogenes. The latest approach to the diagnosis of communicable and parasitic infections is based on the use of deoxyribonucleic acid (DNA) probes. The genetic information of all cells is encoded by DNA and DNA probe approach to identification of pathogens is unique because the focus of the method is the nucleic acid content of the organism rather than the products that the nucleic acid encodes. Since every properly classified species has some unique nucleotide sequences that distinguish it from every other species, each organism's genetic composition is in essence a finger print that can be used for its identification. In addition to this specificity, DNA probes offer other advantages in that pathogens may be identified directly in clinical specimens

  18. Selectivity of major isoquinoline alkaloids from Chelidonium majus towards telomeric G-quadruplex: A study using a transition-FRET (t-FRET) assay

    Czech Academy of Sciences Publication Activity Database

    Noureini, S.K.; Esmaeili, H.; Abachi, F.; Khiali, S.; Islam, Barira; Kuta, M.; Saboury, A.A.; Hoffillann, M.; Šponer, Jiří; Parkinson, G.; Haider, S.


    Roč. 1861, č. 8 (2017), s. 2020-2030 ISSN 0304-4165 R&D Projects: GA ČR(CZ) GA16-13721S Institutional support: RVO:68081707 Keywords : prostate-cancer cells * amber force-field * nucleic-acids Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 4.702, year: 2016

  19. Electrochemical and circular dichroism spectroscopic evidence of two types of interaction between [Ru(NH3)(6)](3+) and an elongated thrombin binding aptamer G-quadruplex

    Czech Academy of Sciences Publication Activity Database

    De Rache, A.; Kejnovská, Iva; Buess-Herman, C.; Doneux, T.


    Roč. 179, OCT 2015 (2015), s. 84-92 ISSN 0013-4686 Institutional support: RVO:68081707 Keywords : Biosensors * Thrombin binding aptamer * Hexaammineruthenium Subject RIV: BO - Biophysics Impact factor: 4.803, year: 2015

  20. Formation and temperature stability of G-quadruplex structures studied by electronic and vibrational dichroism spectroscopy combined with ab initio calculations

    Czech Academy of Sciences Publication Activity Database

    Nový, J.; Böhm, S.; Králová, Jarmila; Král, V.; Urbanová, M.


    Roč. 89, č. 2 (2008), s. 144-152 ISSN 0006-3525 Institutional research plan: CEZ:AV0Z50520514 Keywords : vibrational circular dichroism * quadruplex * G-rich nucleotide Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.823, year: 2008

  1. DNA data (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — Raw DNA chromatogram data produced by the ABI 373, 377, 3130 and 3730 automated sequencing machines in ABI format. These are from fish (primarily Sebastes spp.,...

  2. DNA methylation

    DEFF Research Database (Denmark)

    Williams, Kristine; Christensen, Jesper; Helin, Kristian


    DNA methylation is involved in key cellular processes, including X-chromosome inactivation, imprinting and transcriptional silencing of specific genes and repetitive elements. DNA methylation patterns are frequently perturbed in human diseases such as imprinting disorders and cancer. The recent...... discovery that the three members of the TET protein family can convert 5-methylcytosine (5mC) into 5-hydroxymethylcytosine (5hmC) has provided a potential mechanism leading to DNA demethylation. Moreover, the demonstration that TET2 is frequently mutated in haematopoietic tumours suggests that the TET...... proteins are important regulators of cellular identity. Here, we review the current knowledge regarding the function of the TET proteins, and discuss various mechanisms by which they contribute to transcriptional control. We propose that the TET proteins have an important role in regulating DNA methylation...

  3. DNA nanotechnology (United States)

    Seeman, Nadrian C.; Sleiman, Hanadi F.


    DNA is the molecule that stores and transmits genetic information in biological systems. The field of DNA nanotechnology takes this molecule out of its biological context and uses its information to assemble structural motifs and then to connect them together. This field has had a remarkable impact on nanoscience and nanotechnology, and has been revolutionary in our ability to control molecular self-assembly. In this Review, we summarize the approaches used to assemble DNA nanostructures and examine their emerging applications in areas such as biophysics, diagnostics, nanoparticle and protein assembly, biomolecule structure determination, drug delivery and synthetic biology. The introduction of orthogonal interactions into DNA nanostructures is discussed, and finally, a perspective on the future directions of this field is presented.

  4. DNA expressions - A formal notation for DNA

    NARCIS (Netherlands)

    Vliet, Rudy van


    We describe a formal notation for DNA molecules that may contain nicks and gaps. The resulting DNA expressions denote formal DNA molecules. Different DNA expressions may denote the same molecule. Such DNA expressions are called equivalent. We examine which DNA expressions are minimal, which

  5. What Is Mitochondrial DNA? (United States)

    ... DNA What is mitochondrial DNA? What is mitochondrial DNA? Although most DNA is packaged in chromosomes within ... proteins. For more information about mitochondria and mitochondrial DNA: Molecular Expressions, a web site from the Florida ...

  6. Chemical and biological studies of the major DNA adduct of cis-diamminedichloroplatinum(II), cis-[Pt(NH3)2/d(GpG)/], built into a specific site in a viral genome

    International Nuclear Information System (INIS)

    Naser, L.J.; Pinto, A.L.; Lippard, S.J.; Essigmann, J.M.


    A duplex Escherichia coli bacteriophage M13 genome was constructed containing a single cis-[Pt(NH 3 ) 2 /d(GpG)/] intrastrand cross-link, the major DNA adduct of the anticancer drug cis-diamminedichloroplatinum(II). The duplex dodecamer d(AGAAGGCCTAGA) x d(TCTAGGCCTTCT) was ligated into the HincII site of M13mp18 to produce an insertion mutant containing a unique StuI restriction enzyme cleavage site. A genome with a 12-base gap in the minus strand was created by hybridizing HincII-linearized M13mp18 duplex DNA with the single-stranded circular DNA of the 12-base insertion mutant. Characterization by pH-dependent 1 H NMR spectroscopy established that platinum binds to the N7 positions of the adjacent guanosines. The platinated oligonucleotide was phosphorylated in the presence of [γ- 32 P]ATP with bacteriophage T4 polynucleotide kinase and incorporated into the 12-base gap of the heteroduplex, thus situating the adduct specifically within the StuI site in the minus strand of the genome. The site of incorporation of the dodecamer was mapped to the expected 36-base region delimited by the recognition sites of XbaI and HindIII. Gradient denaturing gel electrophoresis of a 289-base-pair fragment encompassing the site of adduction revealed that the presence of the cis-[Pt(NH 3 ) 2 /d)GpG)/] cross-link induces localized weakening of the DNA double helix. Comparative studies revealed no difference in survival between platinated and unmodified double-stranded genomes. In contrast, survival of the single-stranded platinated genome was only 10-12% that of the corresponding unmodified single-stranded genome, indicating that the solitary cis-[Pt(NH 3 ) 2 /d(GpG)/] cross-link is lethal to the single-stranded bacteriophage

  7. DNA nanotechnology


    Nadrian C Seeman


    Since Watson and Crick’s determination of its structure nearly 50 years ago, DNA has come to fill our lives in many areas, from genetic counseling to forensics, from genomics to gene therapy. These, and other ways in which DNA affects human activities, are related to its function as genetic material, not just our genetic material, but the genetic material of all living organisms. Here, we will ignore DNA’s biological role; rather, we will discuss how the properties that make it so successful ...

  8. DNA Investigations. (United States)

    Mayo, Ellen S.; Bertino, Anthony J.


    Presents a simulation activity that allow students to work through the exercise of DNA profiling and to grapple with some analytical and ethical questions involving a couple arranging with a surrogate mother to have a baby. Can be used to teach the principles of restriction enzyme digestion, gel electrophoresis, and probe hybridization. (MDH)

  9. Extended molecular dynamics of a c-kit promoter quadruplex

    Czech Academy of Sciences Publication Activity Database

    Islam, B.; Stadlbauer, Petr; Krepl, Miroslav; Koča, J.; Neidle, S.; Haider, S.; Šponer, Jiří


    Roč. 43, č. 18 (2015), s. 8673-8693 ISSN 0305-1048 R&D Projects: GA ČR(CZ) GAP208/11/1822 Institutional support: RVO:68081707 Keywords : TELOMERIC G-QUADRUPLEX * INTRAMOLECULAR DNA QUADRUPLEXES * GASTROINTESTINAL STROMAL TUMOR Subject RIV: BO - Biophysics Impact factor: 9.202, year: 2015

  10. Thermodynamic and biological evaluation of a thrombin binding aptamer modified with several unlocked nucleic acid (UNA) monomers and a 2′-C-piperazino-UNA monomer

    DEFF Research Database (Denmark)

    Jensen, Troels B.; Henriksen, Jonas Rosager; Rasmussen, Bjarne E.


    Thrombin binding aptamer is a DNA 15-mer which forms a G-quadruplex structure and possess promising anticoagulant properties due to specific interactions with thrombin. Herein we present the influence of a single 2′-C-piperazino-UNA residue and UNA residues incorporated in several positions on th...

  11. DNA Chip

    Indian Academy of Sciences (India)

    Imagine a world without identity cards; no I-cards for the college or office or bank account or anything! All you are carrying is a small (say, 2cm x 2cm) 'DNA-chip', which has the whole of your genetic profile on it. Your identity cannot get more authentic than that. Imagine a world where marriages are not decided by matching ...

  12. DNA repair

    International Nuclear Information System (INIS)

    Setlow, R.


    Some topics discussed are as follows: difficulty in extrapolating data from E. coli to mammalian systems; mutations caused by UV-induced changes in DNA; mutants deficient in excision repair; other postreplication mechanisms; kinds of excision repair systems; detection of repair by biochemical or biophysical means; human mutants deficient in repair; mutagenic effects of UV on XP cells; and detection of UV-repair defects among XP individuals

  13. Stool DNA Test (United States)

    ... The stool DNA test is a noninvasive laboratory test that identifies DNA changes in the cells of a stool sample. ... the presence of cancer. If a stool DNA test detects abnormal DNA, additional testing may be used to investigate the ...

  14. DNA Microarrays (United States)

    Nguyen, C.; Gidrol, X.

    Genomics has revolutionised biological and biomedical research. This revolution was predictable on the basis of its two driving forces: the ever increasing availability of genome sequences and the development of new technology able to exploit them. Up until now, technical limitations meant that molecular biology could only analyse one or two parameters per experiment, providing relatively little information compared with the great complexity of the systems under investigation. This gene by gene approach is inadequate to understand biological systems containing several thousand genes. It is essential to have an overall view of the DNA, RNA, and relevant proteins. A simple inventory of the genome is not sufficient to understand the functions of the genes, or indeed the way that cells and organisms work. For this purpose, functional studies based on whole genomes are needed. Among these new large-scale methods of molecular analysis, DNA microarrays provide a way of studying the genome and the transcriptome. The idea of integrating a large amount of data derived from a support with very small area has led biologists to call these chips, borrowing the term from the microelectronics industry. At the beginning of the 1990s, the development of DNA chips on nylon membranes [1, 2], then on glass [3] and silicon [4] supports, made it possible for the first time to carry out simultaneous measurements of the equilibrium concentration of all the messenger RNA (mRNA) or transcribed RNA in a cell. These microarrays offer a wide range of applications, in both fundamental and clinical research, providing a method for genome-wide characterisation of changes occurring within a cell or tissue, as for example in polymorphism studies, detection of mutations, and quantitative assays of gene copies. With regard to the transcriptome, it provides a way of characterising differentially expressed genes, profiling given biological states, and identifying regulatory channels.

  15. HPV DNA test (United States)

    ... HPV testing in women; Cervical cancer - HPV DNA test; Cancer of cervix - HPV DNA test ... The HPV DNA test may be done during a Pap smear . You lie on a table and place your feet in stirrups. The ...

  16. Synthesis of DNA (United States)

    Mariella, Jr., Raymond P.


    A method of synthesizing a desired double-stranded DNA of a predetermined length and of a predetermined sequence. Preselected sequence segments that will complete the desired double-stranded DNA are determined. Preselected segment sequences of DNA that will be used to complete the desired double-stranded DNA are provided. The preselected segment sequences of DNA are assembled to produce the desired double-stranded DNA.

  17. Structural Insight into the interaction of Flavonoids with Human Telomeric Sequence (United States)

    Tawani, Arpita; Kumar, Amit


    Flavonoids are a group of naturally available compounds that are an attractive source for drug discovery. Their potential to act as anti-tumourigenic and anti-proliferative agents has been reported previously but is not yet fully understood. Targeting human telomeric G-quadruplex DNA could be one of the mechanisms by which these flavonoids exert anticancer activity. We have performed detailed biophysical studies for the interaction of four representative flavonoids, Luteolin, Quercetin, Rutin and Genistein, with the human telomeric G-quadruplex sequence tetramolecular d-(T2AG3T) (Tel7). In addition, we used NMR spectroscopy to derive the first model for the complex formed between Quercetin and G-quadruplex sequence. The model showed that Quercetin stabilises the G-quadruplex structure and does not open the G-tetrad. It interacts with the telomeric sequence through π-stacking at two sites: between T1pT2 and between G6pT7. Based on our findings, we suggest that Quercetin could be a potent candidate for targeting the telomere and thus, act as a potent anti-cancer agent. PMID:26627543

  18. G4-DNA formation in the HRAS promoter and rational design of decoy oligonucleotides for cancer therapy.

    Directory of Open Access Journals (Sweden)

    Alexandro Membrino

    Full Text Available HRAS is a proto-oncogene involved in the tumorigenesis of urinary bladder cancer. In the HRAS promoter we identified two G-rich elements, hras-1 and hras-2, that fold, respectively, into an antiparallel and a parallel quadruplex (qhras-1, qhras-2. When we introduced in sequence hras-1 or hras-2 two point mutations that block quadruplex formation, transcription increased 5-fold, but when we stabilized the G-quadruplexes by guanidinium phthalocyanines, transcription decreased to 20% of control. By ChIP we found that sequence hras-1 is bound only by MAZ, while hras-2 is bound by MAZ and Sp1: two transcription factors recognizing guanine boxes. We also discovered by EMSA that recombinant MAZ-GST binds to both HRAS quadruplexes, while Sp1-GST only binds to qhras-1. The over-expression of MAZ and Sp1 synergistically activates HRAS transcription, while silencing each gene by RNAi results in a strong down-regulation of transcription. All these data indicate that the HRAS G-quadruplexes behave as transcription repressors. Finally, we designed decoy oligonucleotides mimicking the HRAS quadruplexes, bearing (R-1-O-[4-(1-Pyrenylethynyl phenylmethyl] glycerol and LNA modifications to increase their stability and nuclease resistance (G4-decoys. The G4-decoys repressed HRAS transcription and caused a strong antiproliferative effect, mediated by apoptosis, in T24 bladder cancer cells where HRAS is mutated.

  19. DNA Damage, DNA Repair, Aging, and Neurodegeneration (United States)

    Maynard, Scott; Fang, Evandro Fei; Scheibye-Knudsen, Morten; Croteau, Deborah L.; Bohr, Vilhelm A.


    Aging in mammals is accompanied by a progressive atrophy of tissues and organs, and stochastic damage accumulation to the macromolecules DNA, RNA, proteins, and lipids. The sequence of the human genome represents our genetic blueprint, and accumulating evidence suggests that loss of genomic maintenance may causally contribute to aging. Distinct evidence for a role of imperfect DNA repair in aging is that several premature aging syndromes have underlying genetic DNA repair defects. Accumulation of DNA damage may be particularly prevalent in the central nervous system owing to the low DNA repair capacity in postmitotic brain tissue. It is generally believed that the cumulative effects of the deleterious changes that occur in aging, mostly after the reproductive phase, contribute to species-specific rates of aging. In addition to nuclear DNA damage contributions to aging, there is also abundant evidence for a causative link between mitochondrial DNA damage and the major phenotypes associated with aging. Understanding the mechanistic basis for the association of DNA damage and DNA repair with aging and age-related diseases, such as neurodegeneration, would give insight into contravening age-related diseases and promoting a healthy life span. PMID:26385091

  20. ex vivo DNA assembly

    Directory of Open Access Journals (Sweden)

    Adam B Fisher


    Full Text Available Even with decreasing DNA synthesis costs there remains a need for inexpensive, rapid and reliable methods for assembling synthetic DNA into larger constructs or combinatorial libraries. Advances in cloning techniques have resulted in powerful in vitro and in vivo assembly of DNA. However, monetary and time costs have limited these approaches. Here, we report an ex vivo DNA assembly method that uses cellular lysates derived from a commonly used laboratory strain of Escherichia coli for joining double-stranded DNA with short end homologies embedded within inexpensive primers. This method concurrently shortens the time and decreases costs associated with current DNA assembly methods.

  1. Structure of a Stable G-Hairpin

    Czech Academy of Sciences Publication Activity Database

    Gajarský, M.; Zivkovic, M.L.; Stadlbauer, Petr; Pagano, B.; Fiala, R.; Amato, J.; Tomáška, L´.; Šponer, Jiří; Plavec, J.; Trantírek, L.


    Roč. 139, č. 10 (2017), s. 3591-3594 ISSN 0002-7863 R&D Projects: GA ČR GA13-28310S; GA ČR(CZ) GA16-13721S Institutional support: RVO:68081707 Keywords : g-quadruplex structures * human telomeric dna * single-stranded- dna * g-triplex Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 13.858, year: 2016

  2. DNA damage and autophagy

    International Nuclear Information System (INIS)

    Rodriguez-Rocha, Humberto; Garcia-Garcia, Aracely; Panayiotidis, Mihalis I.; Franco, Rodrigo


    Both exogenous and endogenous agents are a threat to DNA integrity. Exogenous environmental agents such as ultraviolet (UV) and ionizing radiation, genotoxic chemicals and endogenous byproducts of metabolism including reactive oxygen species can cause alterations in DNA structure (DNA damage). Unrepaired DNA damage has been linked to a variety of human disorders including cancer and neurodegenerative disease. Thus, efficient mechanisms to detect DNA lesions, signal their presence and promote their repair have been evolved in cells. If DNA is effectively repaired, DNA damage response is inactivated and normal cell functioning resumes. In contrast, when DNA lesions cannot be removed, chronic DNA damage triggers specific cell responses such as cell death and senescence. Recently, DNA damage has been shown to induce autophagy, a cellular catabolic process that maintains a balance between synthesis, degradation, and recycling of cellular components. But the exact mechanisms by which DNA damage triggers autophagy are unclear. More importantly, the role of autophagy in the DNA damage response and cellular fate is unknown. In this review we analyze evidence that supports a role for autophagy as an integral part of the DNA damage response.

  3. Subcloning of DNA fragments. (United States)

    Struhl, K


    The essence of recombinant DNA technology is the joining of two or more separate segments of DNA to generate a single DNA molecule that is capable of autonomous replication in a given host. The simplest constructions of hybrid DNA molecules involve the cloning of insert sequences into plasmid or bacteriophage cloning vectors. The insert sequences can derive from essentially any organism, and they may be isolated directly from the genome, from mRNA, or from previously cloned DNA segments (in which case, the procedure is termed subcloning). Alternatively, insert DNAs can be created directly by DNA synthesis. This unit provides protocols for the subcloning of DNA fragments and ligation of DNA fragments in gels.

  4. DNA tagged microparticles (United States)

    Farquar, George Roy; Leif, Roald N; Wheeler, Elizabeth


    A simulant that includes a carrier and DNA encapsulated in the carrier. Also a method of making a simulant including the steps of providing a carrier and encapsulating DNA in the carrier to produce the simulant.

  5. DNA computing models

    CERN Document Server

    Ignatova, Zoya; Zimmermann, Karl-Heinz


    In this excellent text, the reader is given a comprehensive introduction to the field of DNA computing. The book emphasizes computational methods to tackle central problems of DNA computing, such as controlling living cells, building patterns, and generating nanomachines.

  6. DNA Repair Systems

    Indian Academy of Sciences (India)

    exogenous damage). Endogenous damage ... of spontaneous DNA-damage due to endogenous factors. He es- timated that around 10,000 potentially mutagenic .... 3 –5 direction is defined as. 'upstream'. A single DNA strand is synthesized in a.

  7. Animal Mitochondrial DNA Replication (United States)

    Ciesielski, Grzegorz L.; Oliveira, Marcos T.; Kaguni, Laurie S.


    Recent advances in the field of mitochondrial DNA (mtDNA) replication highlight the diversity of both the mechanisms utilized and the structural and functional organization of the proteins at mtDNA replication fork, despite the simplicity of the animal mtDNA genome. DNA polymerase γ, mtDNA helicase and mitochondrial single-stranded DNA-binding protein- the key replisome proteins, have evolved distinct structural features and biochemical properties. These appear to be correlated with mtDNA genomic features in different metazoan taxa and with their modes of DNA replication, although a substantial integrative research is warranted to establish firmly these links. To date, several modes of mtDNA replication have been described for animals: rolling circle, theta, strand-displacement, and RITOLS/bootlace. Resolution of a continuing controversy relevant to mtDNA replication in mammals/vertebrates will have a direct impact on the mechanistic interpretation of mtDNA-related human diseases. Here we review these subjects, integrating earlier and recent data to provide a perspective on the major challenges for future research. PMID:27241933

  8. Replicating animal mitochondrial DNA

    Directory of Open Access Journals (Sweden)

    Emily A. McKinney


    Full Text Available The field of mitochondrial DNA (mtDNA replication has been experiencing incredible progress in recent years, and yet little is certain about the mechanism(s used by animal cells to replicate this plasmid-like genome. The long-standing strand-displacement model of mammalian mtDNA replication (for which single-stranded DNA intermediates are a hallmark has been intensively challenged by a new set of data, which suggests that replication proceeds via coupled leading-and lagging-strand synthesis (resembling bacterial genome replication and/or via long stretches of RNA intermediates laid on the mtDNA lagging-strand (the so called RITOLS. The set of proteins required for mtDNA replication is small and includes the catalytic and accessory subunits of DNA polymerase y, the mtDNA helicase Twinkle, the mitochondrial single-stranded DNA-binding protein, and the mitochondrial RNA polymerase (which most likely functions as the mtDNA primase. Mutations in the genes coding for the first three proteins are associated with human diseases and premature aging, justifying the research interest in the genetic, biochemical and structural properties of the mtDNA replication machinery. Here we summarize these properties and discuss the current models of mtDNA replication in animal cells.

  9. DNA: Structure and function

    DEFF Research Database (Denmark)

    Sinden, Richard R.; E. Pearson, Christopher; N. Potaman, Vladimir


    This chapter discusses the structure and function of DNA. DNA occupies a critical role in cells, because it is the source of all intrinsic genetic information. Chemically, DNA is a very stable molecule, a characteristic important for a macromolecule that may have to persist in an intact form...... for a long period of time before its information is accessed by the cell. Although DNA plays a critical role as an informational storage molecule, it is by no means as unexciting as a computer tape or disk drive. The structure of the DNA described by Watson and Crick in 1953 is a right handed helix of two...

  10. Fast phylogenetic DNA barcoding

    DEFF Research Database (Denmark)

    Terkelsen, Kasper Munch; Boomsma, Wouter Krogh; Willerslev, Eske


    We present a heuristic approach to the DNA assignment problem based on phylogenetic inferences using constrained neighbour joining and non-parametric bootstrapping. We show that this method performs as well as the more computationally intensive full Bayesian approach in an analysis of 500 insect...... DNA sequences obtained from GenBank. We also analyse a previously published dataset of environmental DNA sequences from soil from New Zealand and Siberia, and use these data to illustrate the fact that statistical approaches to the DNA assignment problem allow for more appropriate criteria...... for determining the taxonomic level at which a particular DNA sequence can be assigned....

  11. DNA Sequencing apparatus (United States)

    Tabor, Stanley; Richardson, Charles C.


    An automated DNA sequencing apparatus having a reactor for providing at least two series of DNA products formed from a single primer and a DNA strand, each DNA product of a series differing in molecular weight and having a chain terminating agent at one end; separating means for separating the DNA products to form a series bands, the intensity of substantially all nearby bands in a different series being different, band reading means for determining the position an This invention was made with government support including a grant from the U.S. Public Health Service, contract number AI-06045. The U.S. government has certain rights in the invention.

  12. DNA fragmentation in spermatozoa

    DEFF Research Database (Denmark)

    Rex, A S; Aagaard, J.; Fedder, J


    Sperm DNA Fragmentation has been extensively studied for more than a decade. In the 1940s the uniqueness of the spermatozoa protein complex which stabilizes the DNA was discovered. In the fifties and sixties, the association between unstable chromatin structure and subfertility was investigated....... In the seventies, the impact of induced DNA damage was investigated. In the 1980s the concept of sperm DNA fragmentation as related to infertility was introduced as well as the first DNA fragmentation test: the Sperm Chromatin Structure Assay (SCSA). The terminal deoxynucleotidyl transferase nick end labelling...... (TUNEL) test followed by others was introduced in the nineties. The association between DNA fragmentation in spermatozoa and pregnancy loss has been extensively investigated spurring the need for a therapeutic tool for these patients. This gave rise to an increased interest in the aetiology of DNA damage...

  13. Biophysics of DNA

    CERN Document Server

    Vologodskii, Alexander


    Surveying the last sixty years of research, this book describes the physical properties of DNA in the context of its biological functioning. It is designed to enable both students and researchers of molecular biology, biochemistry and physics to better understand the biophysics of DNA, addressing key questions and facilitating further research. The chapters integrate theoretical and experimental approaches, emphasising throughout the importance of a quantitative knowledge of physical properties in building and analysing models of DNA functioning. For example, the book shows how the relationship between DNA mechanical properties and the sequence specificity of DNA-protein binding can be analyzed quantitatively by using our current knowledge of the physical and structural properties of DNA. Theoretical models and experimental methods in the field are critically considered to enable the reader to engage effectively with the current scientific literature on the physical properties of DNA.

  14. Beyond DNA repair: DNA-PK function in cancer


    Goodwin, Jonathan F.; Knudsen, Karen E.


    The DNA-dependent protein kinase (DNA-PK) is a pivotal component of the DNA repair machinery that governs the response to DNA damage, serving to maintain genome integrity. However, the DNA-PK kinase component was initially isolated with transcriptional complexes, and recent findings have illuminated the impact of DNA-PK-mediated transcriptional regulation on tumor progression and therapeutic response. DNA-PK expression has also been correlated with poor outcome in selected tumor types, furthe...

  15. DNA replication and cancer

    DEFF Research Database (Denmark)

    Boyer, Anne-Sophie; Walter, David; Sørensen, Claus Storgaard


    A dividing cell has to duplicate its DNA precisely once during the cell cycle to preserve genome integrity avoiding the accumulation of genetic aberrations that promote diseases such as cancer. A large number of endogenous impacts can challenge DNA replication and cells harbor a battery of pathways...... to promote genome integrity during DNA replication. This includes suppressing new replication origin firing, stabilization of replicating forks, and the safe restart of forks to prevent any loss of genetic information. Here, we describe mechanisms by which oncogenes can interfere with DNA replication thereby...... causing DNA replication stress and genome instability. Further, we describe cellular and systemic responses to these insults with a focus on DNA replication restart pathways. Finally, we discuss the therapeutic potential of exploiting intrinsic replicative stress in cancer cells for targeted therapy....

  16. Detection and quantitation of single nucleotide polymorphisms, DNA sequence variations, DNA mutations, DNA damage and DNA mismatches (United States)

    McCutchen-Maloney, Sandra L.


    DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.

  17. DNA repair and cancer

    International Nuclear Information System (INIS)

    Rathore, Shakuntla; Joshi, Pankaj Kumar; Gaur, Sudha


    DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecule that encode it's genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many one million individual molecular lesions per day. Many of these lesions cause structural damage to the DNA molecule and can alter or eliminate the cell's ability to transcribe the gene that the affected DNA encodes. Other lesions include potentially harmful mutation in cell's genome which affect the survival of it's daughter cells after it undergoes mitosis. As a consequence, the DNA repair process is constantly active as it responds to damage in the DNA structure. Inherited mutation that affect DNA repair genes are strongly associated with high cancer risks in humans. Hereditary non polyposis colorectal cancer (HNPCC) is strongly associated with specific mutation in the DNA mismatch repair pathway. BRCA1, BRCA2 two famous mutation conferring a hugely increased risk of breast cancer on carrier, are both associated with a large number of DNA repair pathway, especially NHEJ and homologous recombination. Cancer therapy procedures such as chemotherapy and radiotherapy work by overwhelming the capacity of the cell to repair DNA damage, resulting in cell death. Cells that are most rapidly dividing most typically cancer cells are preferentially affected. The side effect is that other non-cancerous but rapidly dividing cells such as stem cells in the bone marrow are also affected. Modern cancer treatment attempt to localize the DNA damage to cells and tissue only associated with cancer, either by physical means (concentrating the therapeutic agent in the region of the tumor) or by biochemical means (exploiting a feature unique to cancer cells in the body). (author)

  18. DNA-based machines. (United States)

    Wang, Fuan; Willner, Bilha; Willner, Itamar


    The base sequence in nucleic acids encodes substantial structural and functional information into the biopolymer. This encoded information provides the basis for the tailoring and assembly of DNA machines. A DNA machine is defined as a molecular device that exhibits the following fundamental features. (1) It performs a fuel-driven mechanical process that mimics macroscopic machines. (2) The mechanical process requires an energy input, "fuel." (3) The mechanical operation is accompanied by an energy consumption process that leads to "waste products." (4) The cyclic operation of the DNA devices, involves the use of "fuel" and "anti-fuel" ingredients. A variety of DNA-based machines are described, including the construction of "tweezers," "walkers," "robots," "cranes," "transporters," "springs," "gears," and interlocked cyclic DNA structures acting as reconfigurable catenanes, rotaxanes, and rotors. Different "fuels", such as nucleic acid strands, pH (H⁺/OH⁻), metal ions, and light, are used to trigger the mechanical functions of the DNA devices. The operation of the devices in solution and on surfaces is described, and a variety of optical, electrical, and photoelectrochemical methods to follow the operations of the DNA machines are presented. We further address the possible applications of DNA machines and the future perspectives of molecular DNA devices. These include the application of DNA machines as functional structures for the construction of logic gates and computing, for the programmed organization of metallic nanoparticle structures and the control of plasmonic properties, and for controlling chemical transformations by DNA machines. We further discuss the future applications of DNA machines for intracellular sensing, controlling intracellular metabolic pathways, and the use of the functional nanostructures for drug delivery and medical applications.


    Directory of Open Access Journals (Sweden)

    Vaidhehi Narayan Nayak


    Full Text Available BACKGROUND Mitochondrion, the sausage-shaped organelle residing in the cytoplasm of all eukaryotic cells, apart from being the power house, represents endosymbiotic evolution of a free living organism to intracellular structure. Anthropologically, mitochondrial DNA is the fossilised source to trace the human ancestry particularly of maternal lineage. This article attempts to highlight the various biological functions of mitochondrial DNA (mtDNA with a note on its forensic application.

  20. Nonisotopic DNA probe techniques

    National Research Council Canada - National Science Library

    Kricka, Larry J


    The objective of this book is to bring together descriptions of the principal nonisotopic methods for DNA hybridization assays, together with experimental details of the methods, including labelling...


    Energy Technology Data Exchange (ETDEWEB)



    During this year we performed two major projects: I. We developed a detailed theoretical model which complements our experiments on surface DNA electrophoresis. We found that it was possible to enhance the separation of DNA chains by imposing a chemical nanoscale pattern on the surface. This approach utilized the surface interaction effect of the DNA chains with the substrate and is a refinement to our previous method in which DNA chains were separated on homogeneous flat surfaces. By introducing the nano-patterns on the surface, the conformational changes of DNA chains of different lengths can be amplified, which results in the different friction strengths with the substrate surface. Our results also show that, when compared to the DNA electrophoresis performed on homogeneous flat surfaces, nanopatterned surfaces offer a larger window in choosing different surface interactions to achieve separation. II. In collaboration with a large international manufacturer of skin care products we also embarked on a project involving photo toxicity of titanium dioxide nanoparticles, which are a key ingredient in sunscreen and cosmetic lotions. The results clearly implicated the nanoparticles in catalyzing damage to chromosomal DNA. We then used this knowledge to develop a polymer/anti-oxidant coating which prevented the photocatalytic reaction on DNA while still retaining the UV absorptive properties of the nanoparticles. The standard gel electrophoresis was not sufficient in determining the extent of the DNA damage. The conclusions of this study were based predominantly on analysis obtained with the surface electrophoresis method.

  2. DNA Repair Systems

    Indian Academy of Sciences (India)

    Thanks to the pioneering research work of Lindahl, Sancar, Modrich and their colleagues, we now have an holistic awareness of how DNA damage occurs and how the damage is rectified in bacteria as well as in higher organisms including human beings. A comprehensive understanding of DNA repair has proven crucial ...

  3. DNA-cell conjugates (United States)

    Hsiao, Shih-Chia; Francis, Matthew B.; Bertozzi, Carolyn; Mathies, Richard; Chandra, Ravi; Douglas, Erik; Twite, Amy; Toriello, Nicholas; Onoe, Hiroaki


    The present invention provides conjugates of DNA and cells by linking the DNA to a native functional group on the cell surface. The cells can be without cell walls or can have cell walls. The modified cells can be linked to a substrate surface and used in assay or bioreactors.

  4. DNA Repair Systems

    Indian Academy of Sciences (India)

    D N Rao is a professor at the. Department of Biochemistry,. Indian Institute of Science,. Bengaluru. His research work primarily focuses on. DNA interacting proteins in prokaryotes. This includes restriction-modification systems, DNA repair proteins from pathogenic bacteria and and proteins involved in horizontal gene ...

  5. Human Mitochondrial DNA Replication (United States)

    Holt, Ian J.; Reyes, Aurelio


    Elucidation of the process of DNA replication in mitochondria is in its infancy. For many years, maintenance of the mitochondrial genome was regarded as greatly simplified compared to the nucleus. Mammalian mitochondria were reported to lack all DNA repair systems, to eschew DNA recombination, and to possess but a single DNA polymerase, polymerase γ. Polγ was said to replicate mitochondrial DNA exclusively via one mechanism, involving only two priming events and a handful of proteins. In this “strand-displacement model,” leading strand DNA synthesis begins at a specific site and advances approximately two-thirds of the way around the molecule before DNA synthesis is initiated on the “lagging” strand. Although the displaced strand was long-held to be coated with protein, RNA has more recently been proposed in its place. Furthermore, mitochondrial DNA molecules with all the features of products of conventional bidirectional replication have been documented, suggesting that the process and regulation of replication in mitochondria is complex, as befits a genome that is a core factor in human health and longevity. PMID:23143808

  6. Dna fingerprinting - review paper


    Blundell, Renald


    Before the Polymerase Chain Reaction (PCR) was established, DNA fingerprinting technology has relied for years on Restriction Fragment Length Polymorphism (RFLP) and Variable Number of Tandom Repeats (VNTR) analysis, a very efficient technique but quite laborious and not suitable for high throughput mapping. Since its, development, PCR has provided a new and powerful tool for DNA fingerprinting.

  7. Characterization of muntjac DNA

    International Nuclear Information System (INIS)

    Davis, R.C.


    Sister chromatid exchange (SCE) in muntjac chromosomes is generally proportional to the chromosomal DNA content, but the SCE frequency is reduced in the heterochromatic neck region of the X chromosome. The physical properties of muntjac DNA and the kinetics of repair of UV damage in muntjac heterochromatin and euchromatin were examined and compared with the distribution of sister chromatid exchange

  8. Characterization of muntjac DNA

    Energy Technology Data Exchange (ETDEWEB)

    Davis, R.C.


    Sister chromatid exchange (SCE) in muntjac chromosomes is generally proportional to the chromosomal DNA content, but the SCE frequency is reduced in the heterochromatic neck region of the X chromosome. The physical properties of muntjac DNA and the kinetics of repair of UV damage in muntjac heterochromatin and euchromatin were examined and compared with the distribution of sister chromatid exchange.

  9. DNA Microarray Technology (United States)

    ... this page. En Español: Tecnología de micromatriz de ADN DNA Microarray Technology What is a DNA microarray? ... this page. En Español: Tecnología de micromatriz de ADN Get Email Updates Privacy Copyright Contact Accessibility Plug- ...

  10. DNA repair protocols

    DEFF Research Database (Denmark)

    Bjergbæk, Lotte

    In its 3rd edition, this Methods in Molecular Biology(TM) book covers the eukaryotic response to genomic insult including advanced protocols and standard techniques in the field of DNA repair. Offers expert guidance for DNA repair, recombination, and replication. Current knowledge of the mechanisms...... that regulate DNA repair has grown significantly over the past years with technology advances such as RNA interference, advanced proteomics and microscopy as well as high throughput screens. The third edition of DNA Repair Protocols covers various aspects of the eukaryotic response to genomic insult including...... recent advanced protocols as well as standard techniques used in the field of DNA repair. Both mammalian and non-mammalian model organisms are covered in the book, and many of the techniques can be applied with only minor modifications to other systems than the one described. Written in the highly...

  11. Whose DNA is this?

    DEFF Research Database (Denmark)

    Taroni, Franco; Biedermann, Alex; Vuille, Joëlle


    This communication seeks to draw the attention of researchers and practitioners dealing with forensic DNA profiling analyses to the following question: is a scientist's report, offering support to a hypothesis according to which a particular individual is the source of DNA detected during...... evoked during the international conference "The hidden side of DNA profiles. Artifacts, errors and uncertain evidence" held in Rome (April 27th to 28th, 2012). Indeed, despite the fact that this conference brought together some of the world's leading forensic DNA specialists, it appeared clearly...... talk considerably different languages. It thus is fundamental to address this issue of communication about results of forensic DNA analyses, and open a dialogue with practicing non-scientists at large who need to make meaningful use of scientific results to approach and help solve judicial cases...

  12. Racemic DNA crystallography. (United States)

    Mandal, Pradeep K; Collie, Gavin W; Kauffmann, Brice; Huc, Ivan


    Racemates increase the chances of crystallization by allowing molecular contacts to be formed in a greater number of ways. With the advent of protein synthesis, the production of protein racemates and racemic-protein crystallography are now possible. Curiously, racemic DNA crystallography had not been investigated despite the commercial availability of L- and D-deoxyribo-oligonucleotides. Here, we report a study into racemic DNA crystallography showing the strong propensity of racemic DNA mixtures to form racemic crystals. We describe racemic crystal structures of various DNA sequences and folded conformations, including duplexes, quadruplexes, and a four-way junction, showing that the advantages of racemic crystallography should extend to DNA. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Quantitive DNA Fiber Mapping

    Energy Technology Data Exchange (ETDEWEB)

    Lu, Chun-Mei; Wang, Mei; Greulich-Bode, Karin M.; Weier, Jingly F.; Weier, Heinz-Ulli G.


    Several hybridization-based methods used to delineate single copy or repeated DNA sequences in larger genomic intervals take advantage of the increased resolution and sensitivity of free chromatin, i.e., chromatin released from interphase cell nuclei. Quantitative DNA fiber mapping (QDFM) differs from the majority of these methods in that it applies FISH to purified, clonal DNA molecules which have been bound with at least one end to a solid substrate. The DNA molecules are then stretched by the action of a receding meniscus at the water-air interface resulting in DNA molecules stretched homogeneously to about 2.3 kb/{micro}m. When non-isotopically, multicolor-labeled probes are hybridized to these stretched DNA fibers, their respective binding sites are visualized in the fluorescence microscope, their relative distance can be measured and converted into kilobase pairs (kb). The QDFM technique has found useful applications ranging from the detection and delineation of deletions or overlap between linked clones to the construction of high-resolution physical maps to studies of stalled DNA replication and transcription.

  14. Regulating DNA Self-assembly by DNA-Surface Interactions. (United States)

    Liu, Longfei; Li, Yulin; Wang, Yong; Zheng, Jianwei; Mao, Chengde


    DNA self-assembly provides a powerful approach for preparation of nanostructures. It is often studied in bulk solution and involves only DNA-DNA interactions. When confined to surfaces, DNA-surface interactions become an additional, important factor to DNA self-assembly. However, the way in which DNA-surface interactions influence DNA self-assembly is not well studied. In this study, we showed that weak DNA-DNA interactions could be stabilized by DNA-surface interactions to allow large DNA nanostructures to form. In addition, the assembly can be conducted isothermally at room temperature in as little as 5 seconds. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. DNA Bending elasticity (United States)

    Sivak, David Alexander

    DNA bending elasticity on length scales of tens of basepairs is of critical importance in numerous biological contexts. Even the simplest models of DNA bending admit of few simple analytic results, thus there is a need for numerical methods to calculate experimental observables, such as distance distributions, forces, FRET efficiencies, and timescales of particular large-scale motions. We have implemented and helped develop a coarse-grained representation of DNA and various other covalently-linked groups that allows simple calculation of such observables for varied experimental systems. The simple freely-jointed chain (FJC) model and extremely coarse resolution proved useful in understanding DNA threading through nanopores, identifying steric occlusion by other parts of the chain as a prime culprit for slower capture as distance to the pore decreased. Enhanced sampling techniques of a finer resolution discrete wormlike chain (WLC) model permitted calculation of cyclization rates for small chains and identified the ramifications of a thermodynamically-sound treatment of thermal melts. Adding treatment of double-stranded DNA's helical nature and single-stranded DNA provided a model system that helped demonstrate the importance of statistical fluctuations in even highly-stressed DNA mini-loops, and allowed us to verify that even these constructs show no evidence of excitation-induced softening. Additional incorporation of salt-sensitivity to the model allowed us to calculate forces and FRET efficiencies for such mini-loops and their uncircularized precursors, thereby furthering the understanding of the nature of IHF binding and bending of its recognition sequence. Adding large volume-excluding spheres linked to the ends of the dsDNA permits calculation of distance distributions and thus small-angle X-ray scattering, whereby we demonstrated the validity of the WLC in describing bending fluctuations in DNA chains as short as 42 bp. We also make important connections

  16. DNA-PK assay

    Energy Technology Data Exchange (ETDEWEB)

    Anderson, Carl W.; Connelly, Margery A.


    The present invention provides a method for detecting DNA-activated protein kinase (DNA-PK) activity in a biological sample. The method includes contacting a biological sample with a detectably-labeled phosphate donor and a synthetic peptide substrate defined by the following features to provide specific recognition and phosphorylation by DNA-PK: (1) a phosphate-accepting amino acid pair which may include serine-glutamine (Ser-Gln) (SQ), threonine-glutamine (Thr-Gln) (TQ), glutamine-serine (Gln-Ser) (QS), or glutamine-threonine (Gln-Thr) (QT); (2) enhancer amino acids which may include glutamic acid or glutamine immediately adjacent at the amino- or carboxyl- side of the amino acid pair and forming an amino acid pair-enhancer unit; (3) a first spacer sequence at the amino terminus of the amino acid pair-enhancer unit; (4) a second spacer sequence at the carboxyl terminus of the amino acid pair-enhancer unit, which spacer sequences may include any combination of amino acids that does not provide a phosphorylation site consensus sequence motif; and, (5) a tag moiety, which may be an amino acid sequence or another chemical entity that permits separating the synthetic peptide from the phosphate donor. A compostion and a kit for the detection of DNA-PK activity are also provided. Methods for detecting DNA, protein phosphatases and substances that alter the activity of DNA-PK are also provided. The present invention also provides a method of monitoring protein kinase and DNA-PK activity in living cells. -A composition and a kit for monitoring protein kinase activity in vitro and a composition and a kit for monitoring DNA-PK activities in living cells are also provided. A method for identifying agents that alter protein kinase activity in vitro and a method for identifying agents that alter DNA-PK activity in living cells are also provided.

  17. Chimeric proteins for detection and quantitation of DNA mutations, DNA sequence variations, DNA damage and DNA mismatches (United States)

    McCutchen-Maloney, Sandra L.


    Chimeric proteins having both DNA mutation binding activity and nuclease activity are synthesized by recombinant technology. The proteins are of the general formula A-L-B and B-L-A where A is a peptide having DNA mutation binding activity, L is a linker and B is a peptide having nuclease activity. The chimeric proteins are useful for detection and identification of DNA sequence variations including DNA mutations (including DNA damage and mismatches) by binding to the DNA mutation and cutting the DNA once the DNA mutation is detected.

  18. DNA Mismatch Repair (United States)

    MARINUS, M. G.


    DNA mismatch repair functions to correct replication errors in newly synthesized DNA and to prevent recombination between related, but not identical (homeologous), DNA sequences. The mechanism of mismatch repair is best understood in Escherichia coli and is the main focus of this review. The early genetic studies of mismatch repair are described as a basis for the subsequent biochemical characterization of the system. The effects of mismatch repair on homologous and homeologous recombination are described. The relationship of mismatch repair to cell toxicity induced by various drugs is included. The VSP (Very Short Patch) repair system is described in detail. PMID:26442827

  19. "Artifactual" arsenate DNA

    DEFF Research Database (Denmark)

    Nielsen, Peter E


    The recent claim by Wolfe-Simon et al. that the Halomonas bacterial strain GFAJ-1 when grown in arsenate-containing medium with limiting phosphate is able to substitute phosphate with arsenate in biomolecules including nucleic acids and in particular DNA(1) arose much skepticism, primarily due...... to the very limited chemical stability of arsenate esters (see ref. 2 and references therein). A major part of the criticisms was concerned with the insufficient (bio)chemical evidence in the Wolfe-Simon study for the actual chemical incorporation of arsenate in DNA (and/or RNA). Redfield et al. now present...... evidence that the identification of arsenate DNA was artifactual....

  20. Apoptosis and DNA Methylation

    International Nuclear Information System (INIS)

    Meng, Huan X.; Hackett, James A.; Nestor, Colm; Dunican, Donncha S.; Madej, Monika; Reddington, James P.; Pennings, Sari; Harrison, David J.; Meehan, Richard R.


    Epigenetic mechanisms assist in maintaining gene expression patterns and cellular properties in developing and adult tissues. The molecular pathology of disease states frequently includes perturbation of DNA and histone methylation patterns, which can activate apoptotic pathways associated with maintenance of genome integrity. This perspective focuses on the pathways linking DNA methyltransferases and methyl-CpG binding proteins to apoptosis, and includes new bioinformatic analyses to characterize the evolutionary origin of two G/T mismatch-specific thymine DNA glycosylases, MBD4 and TDG

  1. DNA sequencing conference, 2

    Energy Technology Data Exchange (ETDEWEB)

    Cook-Deegan, R.M. [Georgetown Univ., Kennedy Inst. of Ethics, Washington, DC (United States); Venter, J.C. [National Inst. of Neurological Disorders and Strokes, Bethesda, MD (United States); Gilbert, W. [Harvard Univ., Cambridge, MA (United States); Mulligan, J. [Stanford Univ., CA (United States); Mansfield, B.K. [Oak Ridge National Lab., TN (United States)


    This conference focused on DNA sequencing, genetic linkage mapping, physical mapping, informatics and bioethics. Several were used to study this sequencing and mapping. This article also discusses computer hardware and software aiding in the mapping of genes.

  2. DNA fusion gene vaccines

    DEFF Research Database (Denmark)

    Holst, Peter Johannes; Bassi, Maria Rosaria; Thomsen, Allan Randrup


    DNA vaccines are versatile and safe, but limited immunogenicity has prevented their use in the clinical setting. Experimentally, immunogenicity may be enhanced by the use of new delivery technologies, by coadministration of cytokines and pathogen-associated molecular patterns, or by fusion...... of antigens into molecular domains that enhance antigen presentation. More specifically, the immunogenicity of DNA vaccines may benefit from increased protein synthesis, increased T-cell help and MHC class I presentation, and the addition of a range of specific cytokines and pathogen-associated molecular...... with these modifications, it is likely that the primary use of DNA vaccines may be as primers for viral-vectored vaccines, rather than as single agents. This review discusses the approaches used to enhance DNA vaccine immunogenicity, with a primary focus on fusion strategies that enhance antigen presentation....

  3. Close encounters with DNA (United States)

    Maffeo, C.; Yoo, J.; Comer, J.; Wells, D. B.; Luan, B.; Aksimentiev, A.


    Over the past ten years, the all-atom molecular dynamics method has grown in the scale of both systems and processes amenable to it and in its ability to make quantitative predictions about the behavior of experimental systems. The field of computational DNA research is no exception, witnessing a dramatic increase in the size of systems simulated with atomic resolution, the duration of individual simulations and the realism of the simulation outcomes. In this topical review, we describe the hallmark physical properties of DNA from the perspective of all-atom simulations. We demonstrate the amazing ability of such simulations to reveal the microscopic physical origins of experimentally observed phenomena and we review the frustrating limitations associated with imperfections of present atomic force fields and inadequate sampling. The review is focused on the following four physical properties of DNA: effective electric charge, response to an external mechanical force, interaction with other DNA molecules and behavior in an external electric field. PMID:25238560

  4. DNA from keratinous tissue

    DEFF Research Database (Denmark)

    Bengtsson, Camilla F.; Olsen, Maja E.; Brandt, Luise Ørsted


    Keratinous tissues such as nail, hair, horn, scales and feather have been used as a source of DNA for over 20 years. Particular benefits of such tissues include the ease with which they can be sampled, the relative stability of DNA in such tissues once sampled, and, in the context of ancient...... genetic analyses, the fact that sampling generally causes minimal visual damage to valuable specimens. Even when freshly sampled, however, the DNA quantity and quality in the fully keratinized parts of such tissues is extremely poor in comparison to other tissues such as blood and muscle – although little...... systematic research has been undertaken to characterize how such degradation may relate to sample source. In this review paper we present the current understanding of the quality and limitations of DNA in two key keratinous tissues, nail and hair. The findings indicate that although some fragments of nuclear...

  5. DNA fusion gene vaccines

    DEFF Research Database (Denmark)

    Holst, Peter Johannes; Bassi, Maria Rosaria; Thomsen, Allan Randrup


    DNA vaccines are versatile and safe, but limited immunogenicity has prevented their use in the clinical setting. Experimentally, immunogenicity may be enhanced by the use of new delivery technologies, by coadministration of cytokines and pathogen-associated molecular patterns, or by fusion...... of antigens into molecular domains that enhance antigen presentation. More specifically, the immunogenicity of DNA vaccines may benefit from increased protein synthesis, increased T-cell help and MHC class I presentation, and the addition of a range of specific cytokines and pathogen-associated molecular...... with viral-vectored vaccines, various synergistic components may need to be incorporated into DNA vaccines. From the perspective of the future clinical use of DNA vaccines, it has been suggested that antigen presentation should be improved and cytokine coadministration attempted. However, even...

  6. Gomphid DNA sequence data (United States)

    U.S. Environmental Protection Agency — DNA sequence data for several genetic loci. This dataset is not publicly accessible because: It's already publicly available on GenBank. It can be accessed through...

  7. DNA damage and carcinogenesis

    International Nuclear Information System (INIS)

    Stelow, R.B.


    Although cancer may arise as a result of many different types of molecular changes, there is little reason to doubt that changes to DNA are one of the more important ones in cancer initiation. Although DNA repair mechanisms seem able to eliminate a very large fraction of deleterious changes to DNA, we not only have little insight into the molecular mechanisms involved in such repair, but have a negligible amount of information to permit us to estimate the shape of dose response relations at low doses. The case of skin cancer is a special one, in that the average population is exposed to sufficient solar uv so that the effects of small increments in uv dose may be estimated. An approximate 85% reduction in DNA repair increases skin cancer incidence 10 4 fold

  8. Experimental DNA computing

    NARCIS (Netherlands)

    Henkel, Christiaan


    Because of their information storing and processing capabilities, nucleic acids are interesting building blocks for molecular scale computers. Potential applications of such DNA computers range from massively parallel computation to computational gene therapy. In this thesis, several implementations

  9. DNA Vaccines Against Anthrax

    National Research Council Canada - National Science Library

    Galloway, Darrell R; Baillie, Les


    DNA vaccination is vaccination at its simplest. Due to renewed interest in vaccination against anthrax and other biothreat agents, a genetic immunisation approach offers attractive possibilities for rapid, responsive vaccine development...

  10. DNA Sampling Hook (United States)

    National Oceanic and Atmospheric Administration, Department of Commerce — The DNA Sampling Hook is a significant improvement on a method of obtaining a tissue sample from a live fish in situ from an aquatic environment. A tissue sample...

  11. Retroviral DNA Integration (United States)


    The integration of a DNA copy of the viral RNA genome into host chromatin is the defining step of retroviral replication. This enzymatic process is catalyzed by the virus-encoded integrase protein, which is conserved among retroviruses and LTR-retrotransposons. Retroviral integration proceeds via two integrase activities: 3′-processing of the viral DNA ends, followed by the strand transfer of the processed ends into host cell chromosomal DNA. Herein we review the molecular mechanism of retroviral DNA integration, with an emphasis on reaction chemistries and architectures of the nucleoprotein complexes involved. We additionally discuss the latest advances on anti-integrase drug development for the treatment of AIDS and the utility of integrating retroviral vectors in gene therapy applications. PMID:27198982

  12. Radiation damage in DNA

    International Nuclear Information System (INIS)

    Lafleur, V.


    A number of experiments are described with the purpose to obtain a better insight in the chemical nature and the biological significance of radiation-induced damage in DNA, with some emphasis on the significance of alkali-labile sites. It is shown that not only reactions of OH radicals but also of H radicals introduce breaks and other inactivating damage in single-standed phiX174 DNA. It is found that phosphate buffer is very suitable for the study of the reactions of H radicals with DNA, as the H 2 PO 4 - ions convert the hydrated electrons into H radicals. The hydrated electron, which does react with DNA, does not cause a detectable inactivation. (Auth.)

  13. DNA-Origami

    DEFF Research Database (Denmark)

    Voigt, Niels Vinther; Tørring, Thomas; Gothelf, Kurt Vesterager


    DNA-nanostrukturer giver nye muligheder for studier af individuelle molekyler. Ved at udnytte DNAs unikke selvsamlende egenskaber kan man designe systemer, hvorpå der kan studeres kemiske reaktioner, fluoroforer og biiomolekyler på enkeltmolekyle-niveau....

  14. Patterning nanocrystals using DNA

    Energy Technology Data Exchange (ETDEWEB)

    Williams, Shara Carol [Univ. of California, Berkeley, CA (United States)


    One of the goals of nanotechnology is to enable programmed self-assembly of patterns made of various materials with nanometer-sized control. This dissertation describes the results of experiments templating arrangements of gold and semiconductor nanocrystals using 2'-deoxyribonucleic acid (DNA). Previously, simple DNA-templated linear arrangements of two and three nanocrystals structures have been made.[1] Here, we have sought to assemble larger and more complex nanostructures. Gold-DNA conjugates with 50 to 100 bases self-assembled into planned arrangements using strands of DNA containing complementary base sequences. We used two methods to increase the complexity of the arrangements: using branched synthetic doublers within the DNA covalent backbone to create discrete nanocrystal groupings, and incorporating the nanocrystals into a previously developed DNA lattice structure [2][3] that self-assembles from tiles made of DNA double-crossover molecules to create ordered nanoparticle arrays. In the first project, the introduction of a covalently-branched synthetic doubler reagent into the backbone of DNA strands created a branched DNA ''trimer.'' This DNA trimer templated various structures that contained groupings of three and four gold nanoparticles, giving promising, but inconclusive transmission electron microscopy (TEM) results. Due to the presence of a variety of possible structures in the reaction mixtures, and due to the difficulty of isolating the desired structures, the TEM and gel electrophoresis results for larger structures having four particles, and for structures containing both 5 and 10 nm gold nanoparticles were inconclusive. Better results may come from using optical detection methods, or from improved sample preparation. In the second project, we worked toward making two-dimensional ordered arrays of nanocrystals. We replicated and improved upon previous results for making DNA lattices, increasing the size of the lattices

  15. Celebrating DNA's Repair Crew. (United States)

    Kunkel, Thomas A


    This year, the Nobel Prize in Chemistry has been awarded to Tomas Lindahl, Aziz Sancar, and Paul Modrich for their seminal studies of the mechanisms by which cells from bacteria to man repair DNA damage that is generated by normal cellular metabolism and stress from the environment. These studies beautifully illustrate the remarkable power of DNA repair to influence life from evolution through disease susceptibility. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Das DNA-Puzzle (United States)

    Kirchner, Stefan

    Im Jahre 1953 wurde von James Watson und Francis Crick erstmalig der strukturelle Aufbau der sogenannten DNA (Desoxyribonukleinsäure) beschrieben, welche das Erbgut jedes Lebewesens enthält. Der wesentliche Teil des Erbguts wird dabei durch eine sehr lange Folge der vier Basen Adenin (A), Cytosin (C), Guanin (G) und Thymin (T) codiert. Seit einigen Jahren ist es möglich, die Folge der vier Basen zu einer gegebenen DNA zu bestimmen. Biologen bezeichnen diesen Vorgang als Sequenzierung.

  17. PDA: Pooled DNA analyzer

    Directory of Open Access Journals (Sweden)

    Lin Chin-Yu


    Full Text Available Abstract Background Association mapping using abundant single nucleotide polymorphisms is a powerful tool for identifying disease susceptibility genes for complex traits and exploring possible genetic diversity. Genotyping large numbers of SNPs individually is performed routinely but is cost prohibitive for large-scale genetic studies. DNA pooling is a reliable and cost-saving alternative genotyping method. However, no software has been developed for complete pooled-DNA analyses, including data standardization, allele frequency estimation, and single/multipoint DNA pooling association tests. This motivated the development of the software, 'PDA' (Pooled DNA Analyzer, to analyze pooled DNA data. Results We develop the software, PDA, for the analysis of pooled-DNA data. PDA is originally implemented with the MATLAB® language, but it can also be executed on a Windows system without installing the MATLAB®. PDA provides estimates of the coefficient of preferential amplification and allele frequency. PDA considers an extended single-point association test, which can compare allele frequencies between two DNA pools constructed under different experimental conditions. Moreover, PDA also provides novel chromosome-wide multipoint association tests based on p-value combinations and a sliding-window concept. This new multipoint testing procedure overcomes a computational bottleneck of conventional haplotype-oriented multipoint methods in DNA pooling analyses and can handle data sets having a large pool size and/or large numbers of polymorphic markers. All of the PDA functions are illustrated in the four bona fide examples. Conclusion PDA is simple to operate and does not require that users have a strong statistical background. The software is available at

  18. Variations in brain DNA

    Directory of Open Access Journals (Sweden)

    Jesus eAvila


    Full Text Available It is assumed that DNA sequences are conserved in the diverse cell types present in a multicellular organism like the human being. Thus, in order to compare the sequences in the genome of DNA from different individuals, nucleic acid is commonly isolated from a single tissue. In this regard, blood cells are widely used for this purpose because of their availability. Thus blood DNA has been used to study genetic familiar diseases that affect other tissues and organs, such as the liver, heart, and brain. While this approach is valid for the identification of familial diseases in which mutations are present in parental germinal cells and, therefore, in all the cells of a given organism, it is not suitable to identify sporadic diseases in which mutations might occur in specific somatic cells. This review addresses somatic DNA variations in different tissues or cells (mainly in the brain of single individuals and discusses whether the dogma of DNA invariance between cell types is indeed correct. We will also discuss how single nucleotide somatic variations arise, focusing on the presence of specific DNA mutations in the brain.

  19. Introduction to DNA methods

    International Nuclear Information System (INIS)

    Delincee, H.


    The purpose of this session is to discuss the various possibilities for detecting modifications in DNA after irradiation and whether these changes can be utilized as an indicator for the irradiation treatment of foods. The requirement to be fulfilled is that the method be able to distinguish irradiated food without the presence of a control sample, thus the measured response after irradiation must be large enough to supersede background levels from other treatments. Much work has been performed on the effects of radiation on DNA, particularly due to its importance in radiation biology. The main lesions of DNA as a result of irradiation are base damage, damage of the sugar moiety, single strand and double strand breaks. Crosslinking between bases also occurs, e.g. production of thymine dimers, or between DNA and protein. A valuable review on how to utilize these DNA changes for detection purposes has already appeared. Tables 1, 2 and 3 list the proposed methods of detecting changes in irradiated DNA, some identified products as examples for a possible irradiation indicator, in the case of immunoassay the substance used as antigen, and some selected literature references. In this short review, it is not intended to provide a complete literature survey

  20. Toward larger DNA origami. (United States)

    Marchi, Alexandria N; Saaem, Ishtiaq; Vogen, Briana N; Brown, Stanley; LaBean, Thomas H


    Structural DNA nanotechnology, and specifically scaffolded DNA origami, is rapidly developing as a versatile method for bottom-up fabrication of novel nanometer-scale materials and devices. However, lengths of conventional single-stranded scaffolds, for example, 7,249-nucleotide circular genomic DNA from the M13mp18 phage, limit the scales of these uniquely addressable structures. Additionally, increasing DNA origami size generates the cost burden of increased staple-strand synthesis. We addressed this 2-fold problem by developing the following methods: (1) production of the largest to-date biologically derived single-stranded scaffold using a λ/M13 hybrid virus to produce a 51 466-nucleotide DNA in a circular, single-stranded form and (2) inexpensive DNA synthesis via an inkjet-printing process on a chip embossed with functionalized micropillars made from cyclic olefin copolymer. We have experimentally demonstrated very efficient assembly of a 51-kilobasepair origami from the λ/M13 hybrid scaffold folded by chip-derived staple strands. In addition, we have demonstrated two-dimensional, asymmetric origami sheets with controlled global curvature such that they land on a substrate in predictable orientations that have been verified by atomic force microscopy.

  1. DNA Polymerase Gamma in Mitochondrial DNA Replication and Repair

    Directory of Open Access Journals (Sweden)

    William C. Copeland


    Full Text Available Mutations in mitochondrial DNA (mtDNA are associated with aging, and they can cause tissue degeneration and neuromuscular pathologies known as mitochondrial diseases. Because DNA polymerase γ (pol γ is the enzyme responsible for replication and repair of mitochondrial DNA, the burden of faithful duplication of mitochondrial DNA, both in preventing spontaneous errors and in DNA repair synthesis, falls on pol γ. Investigating the biological functions of pol γ and its inhibitors aids our understanding of the sources of mtDNA mutations. In animal cells, pol γ is composed of two subunits, a larger catalytic subunit of 125–140 kDa and second subunit of 35–55 kDa. The catalytic subunit contains DNA polymerase activity, 3’-5’ exonuclease activity, and a 5’-dRP lyase activity. The accessory subunit is required for highly processive DNA synthesis and increases the affinity of pol gamma to the DNA.

  2. Ribonucleotide triggered DNA damage and RNA-DNA damage responses. (United States)

    Wallace, Bret D; Williams, R Scott


    Research indicates that the transient contamination of DNA with ribonucleotides exceeds all other known types of DNA damage combined. The consequences of ribose incorporation into DNA, and the identity of protein factors operating in this RNA-DNA realm to protect genomic integrity from RNA-triggered events are emerging. Left unrepaired, the presence of ribonucleotides in genomic DNA impacts cellular proliferation and is associated with chromosome instability, gross chromosomal rearrangements, mutagenesis, and production of previously unrecognized forms of ribonucleotide-triggered DNA damage. Here, we highlight recent findings on the nature and structure of DNA damage arising from ribonucleotides in DNA, and the identification of cellular factors acting in an RNA-DNA damage response (RDDR) to counter RNA-triggered DNA damage.

  3. Target recycling amplification for label-free and sensitive colorimetric detection of adenosine triphosphate based on un-modified aptamers and DNAzymes. (United States)

    Gong, Xue; Li, Jinfu; Zhou, Wenjiao; Xiang, Yun; Yuan, Ruo; Chai, Yaqin


    Based on target recycling amplification, the development of a new label-free, simple and sensitive colorimetric detection method for ATP by using un-modified aptamers and DNAzymes is described. The association of the model target molecules (ATP) with the corresponding aptamers of the dsDNA probes leads to the release of the G-quadruplex sequences. The ATP-bound aptamers can be further degraded by Exonuclease III to release ATP, which can again bind the aptamers of the dsDNA probes to initiate the target recycling amplification process. Due to this target recycling amplification, the amount of the released G-quadruplex sequences is significantly enhanced. Subsequently, these G-quadruplex sequences bind hemin to form numerous peroxidase mimicking DNAzymes, which cause substantially intensified color change of the probe solution for highly sensitive colorimetric detection of ATP down to the sub-nanomolar (0.33nM) level. Our method is highly selective toward ATP against other control molecules and can be performed in one single homogeneous solution, which makes our sensing approach hold great potential for sensitive colorimetric detection of other small molecules and proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

  4. Supramolecular Complexes of DNA (United States)

    Zuber, G.; Scherman, D.

    Deoxyribose nucleic acid or DNA is a linear polymer in the form of a double strand, synthesised by sequential polymerisation of a large number of units chosen from among the nucleic bases called purines (adenosine A and guanosine G) and pyrimidines (cytosine C and thymidine T). DNA contains all the genetic information required for life. It exists in the form of a limited number (a few dozen) of very big molecules, called chromosomes. This genetic information is first of all transcribed. In this process, a restricted fragment of the DNA called a gene is copied in the form of ribonucleic acid, or RNA. This RNA is itself a polymer, but with a single strand in which the sequence of nucleic acids is schematically analogous to the sequence on one of the two strands of the transcribed DNA. Finally, this RNA is translated into a protein, yet another linear polymer. The proteins make up the main part of the active constituents ensuring the survival of the cell. Any loss of information, either by mutation or by deletion of the DNA, will cause an imbalance in the cell's metabolism that may in turn lead to incurable pathologies. Several strategies have been developed to reduce the consequences of such genetic deficiencies or, more generally, to act, by amplifying or suppressing them, on the mechanisms leading from the reading of the genetic information to the production of proteins: Strategies aiming to introduce synthetic DNA or RNA, which selectively block the expression of certain genes, are now being studied by an increasing number of research scientists and pharmacologists. They use antisense oligodeoxyribonucleotides or interfering oligoribonucleotides and they already have clinical applications. This kind of therapy is often called gene pharmacology. Other, more ambitious strategies aim to repair in situ mutated or incomplete DNA within the chromosomes themselves, by introducing short sequences of DNA or RNA which recognise and take the place of mutations. This is the

  5. Metric representation of DNA sequences. (United States)

    Wu, Z B


    A metric representation of DNA sequences is borrowed from symbolic dynamics. In view of this method, the pattern seen in the chaos game representation of DNA sequences is explained as the suppression of certain nucleotide strings in the DNA sequences. Frequencies of short nucleotide strings and suppression of the shortest ones in the DNA sequences can be determined by using the metric representation.

  6. Blood extracellular DNA after irradiation

    International Nuclear Information System (INIS)

    Vladimirov, V.G.; Tishchenko, L.I.; Surkova, E.A.; Vasil'eva, I.N.


    It has been shown that blood extracellular DNA of irradiated rats largely consists of the low-molecular DNA and its oligomers. Molecular masses of oligomers are multiple to molecular mass of monomer fragment with nucleosome size. The low-molecular DNA has linear form. The average content of GC-pairs in low-molecular DNA is higher than in total rat's DNA (48.5% against 41.5%). The low-molecular DNA is a part of complex containing RNA, acidic proteins and lipids. It is assumed that the formation of low-molecular DNA is a result of Ca/Mg - dependent nuclear endonuclease action

  7. DNA triplex structures in neurodegenerative disorder, Friedreich's ...

    Indian Academy of Sciences (India)

    canonical B-DNA structure or 'unusual' DNA structure. The unusual DNA structures like DNA-hairpin, cruciform, Z-DNA, triplex and tetraplex are represented as hotspots of chromosomal breaks, homologous recombination and gross ...

  8. Defects of mitochondrial DNA replication. (United States)

    Copeland, William C


    Mitochondrial DNA is replicated by DNA polymerase γ in concert with accessory proteins such as the mitochondrial DNA helicase, single-stranded DNA binding protein, topoisomerase, and initiating factors. Defects in mitochondrial DNA replication or nucleotide metabolism can cause mitochondrial genetic diseases due to mitochondrial DNA deletions, point mutations, or depletion, which ultimately cause loss of oxidative phosphorylation. These genetic diseases include mitochondrial DNA depletion syndromes such as Alpers or early infantile hepatocerebral syndromes, and mitochondrial DNA deletion disorders, such as progressive external ophthalmoplegia, ataxia-neuropathy, or mitochondrial neurogastrointestinal encephalomyopathy. This review focuses on our current knowledge of genetic defects of mitochondrial DNA replication (POLG, POLG2, C10orf2, and MGME1) that cause instability of mitochondrial DNA and mitochondrial disease. © The Author(s) 2014.

  9. Eukaryotic DNA Replicases

    KAUST Repository

    Zaher, Manal S.


    The current model of the eukaryotic DNA replication fork includes three replicative DNA polymerases, polymerase α/primase complex (Pol α), polymerase δ (Pol δ), and polymerase ε (Pol ε). The primase synthesizes 8–12 nucleotide RNA primers that are extended by the DNA polymerization activity of Pol α into 30–35 nucleotide RNA-DNA primers. Replication factor C (RFC) opens the polymerase clamp-like processivity factor, proliferating cell nuclear antigen (PCNA), and loads it onto the primer-template. Pol δ utilizes PCNA to mediate highly processive DNA synthesis, while Pol ε has intrinsic high processivity that is modestly stimulated by PCNA. Pol ε replicates the leading strand and Pol δ replicates the lagging strand in a division of labor that is not strict. The three polymerases are comprised of multiple subunits and share unifying features in their large catalytic and B subunits. The remaining subunits are evolutionarily not related and perform diverse functions. The catalytic subunits are members of family B, which are distinguished by their larger sizes due to inserts in their N- and C-terminal regions. The sizes of these inserts vary among the three polymerases, and their functions remain largely unknown. Strikingly, the quaternary structures of Pol α, Pol δ, and Pol ε are arranged similarly. The catalytic subunits adopt a globular structure that is linked via its conserved C-terminal region to the B subunit. The remaining subunits are linked to the catalytic and B subunits in a highly flexible manner.

  10. DNA repair synthesis in human fibroblasts requires DNA polymerase delta

    International Nuclear Information System (INIS)

    Nishida, C.; Reinhard, P.; Linn, S.


    When UV-irradiated cultured diploid human fibroblasts were permeabilized with Brij-58 then separated from soluble material by centrifugation, conservative DNA repair synthesis could be restored by a soluble factor obtained from the supernatant of similarly treated HeLa cells. Extensive purification of this factor yielded a 10.2 S, 220,000-dalton polypeptide with the DNA polymerase and 3'- to 5'-exonuclease activities reported for DNA polymerase delta II. Monoclonal antibody to KB cell DNA polymerase alpha, while binding to HeLa DNA polymerase alpha, did not bind to the HeLa DNA polymerase delta. Moreover, at micromolar concentrations N2-(p-n-butylphenyl)-2'-deoxyguanosine 5'-triphosphate (BuPdGTP) and 2-(p-n-butylanilino)-2'-deoxyadenosine 5'-triphosphate (BuAdATP) were potent inhibitors of DNA polymerase alpha, but did not inhibit the DNA polymerase delta. Neither purified DNA polymerase alpha nor beta could promote repair DNA synthesis in the permeabilized cells. Furthermore, under conditions which inhibited purified DNA polymerase alpha by greater than 90%, neither monoclonal antibodies to DNA polymerase alpha, BuPdGTP, nor BuAdATP was able to inhibit significantly the DNA repair synthesis mediated by the DNA polymerase delta. Thus, it appears that a major portion of DNA repair synthesis induced by UV irradiation might be catalyzed by DNA polymerase delta. When xeroderma pigmentosum human diploid fibroblasts were utilized, DNA repair synthesis dependent upon ultraviolet light could be restored by addition of both T4 endonuclease V and DNA polymerase delta, but not by addition of either one alone

  11. DNA methylation in obesity

    Directory of Open Access Journals (Sweden)

    Małgorzata Pokrywka


    Full Text Available The number of overweight and obese people is increasing at an alarming rate, especially in the developed and developing countries. Obesity is a major risk factor for diabetes, cardiovascular disease, and cancer, and in consequence for premature death. The development of obesity results from the interplay of both genetic and environmental factors, which include sedentary life style and abnormal eating habits. In the past few years a number of events accompanying obesity, affecting expression of genes which are not directly connected with the DNA base sequence (e.g. epigenetic changes, have been described. Epigenetic processes include DNA methylation, histone modifications such as acetylation, methylation, phosphorylation, ubiquitination, and sumoylation, as well as non-coding micro-RNA (miRNA synthesis. In this review, the known changes in the profile of DNA methylation as a factor affecting obesity and its complications are described.

  12. DNA Topoisomerases in Transcription

    DEFF Research Database (Denmark)

    Rødgaard, Morten Terpager


    This Ph.D. thesis summarizes the main results of my studies on the interplay between DNA topoisomerases and transcription. The work was performed from 2011 to 2015 at Aarhus University in the Laboratory of Genome Research, and was supervised by associate professor Anni H. Andersen. Most of the ex......This Ph.D. thesis summarizes the main results of my studies on the interplay between DNA topoisomerases and transcription. The work was performed from 2011 to 2015 at Aarhus University in the Laboratory of Genome Research, and was supervised by associate professor Anni H. Andersen. Most...... topoisomerase-DNA cleavage complex. The second study is an investigation of how topoisomerases influence gene regulation by keeping the genome in an optimal topological state....

  13. Duplication in DNA Sequences (United States)

    Ito, Masami; Kari, Lila; Kincaid, Zachary; Seki, Shinnosuke

    The duplication and repeat-deletion operations are the basis of a formal language theoretic model of errors that can occur during DNA replication. During DNA replication, subsequences of a strand of DNA may be copied several times (resulting in duplications) or skipped (resulting in repeat-deletions). As formal language operations, iterated duplication and repeat-deletion of words and languages have been well studied in the literature. However, little is known about single-step duplications and repeat-deletions. In this paper, we investigate several properties of these operations, including closure properties of language families in the Chomsky hierarchy and equations involving these operations. We also make progress toward a characterization of regular languages that are generated by duplicating a regular language.

  14. Principles of DNA architectonics: design of DNA-based nanoobjects

    International Nuclear Information System (INIS)

    Vinogradova, O A; Pyshnyi, D V


    The methods of preparation of monomeric DNA blocks that serve as key building units for the construction of complex DNA objects are described. Examples are given of the formation of DNA blocks based on native and modified oligonucleotide components using hydrogen bonding and nucleic acid-specific types of bonding and also some affinity interactions with RNA, proteins, ligands. The static discrete and periodic two- and three-dimensional DNA objects reported to date are described systematically. Methods used to prove the structures of DNA objects and the prospects for practical application of nanostructures based on DNA and its analogues in biology, medicine and biophysics are considered. The bibliography includes 195 references.

  15. DNA templated magnetic nanoparticles (United States)

    Kinsella, Joseph M.

    Recent discoveries in nanoscience are predicted to potentially revolutionize future technologies in an extensive number of fields. These developments are contingent upon discovering new and often unconventional methods to synthesize and control nanoscale components. Nature provides several examples of working nanotechnology such as the use of programmed self assembly to build and deconstruct complex molecular systems. We have adopted a method to control the one dimensional assembly of magnetic nanoparticles using DNA as a scaffold molecule. With this method we have demonstrated the ability to organize 5 nm particles into chains that stretch up to ˜20 mum in length. One advantage of using DNA compared is the ability of the molecule to interact with other biomolecules. After assembling particles onto DNA we have been able to cleave the molecule into smaller fragments using restriction enzymes. Using ligase enzymes we have re-connected these fragments, coated with either gold or iron oxide, to form long one-dimensional arrangements of the two different types of nanoparticles on a single molecular guide. We have also created a sensitive magnetic field sensor by incorporating magnetic nanoparticle coated DNA strands with microfabricated electrodes. The IV characteristics of the aligned nanoparticles are dependant on the magnitude of an externally applied magnetic field. This transport phenomenon known as tunneling magnetoresistance (TMR) shows room temperature resistance of our devices over 80% for cobalt ferrite coated DNA when a field of 20 kOe is applied. In comparison, studies using two dimensional nanoparticle films of irox oxides xii only exhibit a 35% MR effect. Confinement into one dimension using the DNA guide produces a TMR mechanism which produces significant increases in magnetoresistance. This property can be utilized for applications in magnetic field sensing, data storage, and logic elements.

  16. Electrochemical DNA sensor-based strategy for sensitive detection of DNA demethylation and DNA demethylase activity. (United States)

    Shen, Qingming; Fan, Mengxing; Yang, Yin; Zhang, Hui


    DNA demethylation and demethylase activity play important roles in DNA self-repair, and their detection is key to early diagnosis of fatal diseases. Herein, a facile electrochemical DNA (E-DNA) sensor was developed for the sensitive detection of DNA demethylation and demethylase activity based on an enzyme cleavage strategy. The thiol modified hemi-methylated hairpin probe DNA (pDNA) was self-assembled on a Au electrode surface through the formation of AuS bonds. The hemi-methylated pDNA served as the substrate of DNA demethylase (using methyl-CpG-binding domain protein 2 (MBD2) as an example). Following demethylation, the hairpin stem was then recognized and cleaved by BstUI endonuclease. The ferrocene carboxylic acid (FcA)-tagged pDNA strands were released into the buffer solution from the electrode surface, resulting in a significant decrease of electrochemical signal and providing a means to observe DNA demethylation. The activity of DNA demethylase was analyzed in the concentration ranging from 0.5 to 500 ng mL(-1) with a limit of detection as low as 0.17 ng mL(-1). With high specificity and sensitivity, rapid response, and low cost, this simple E-DNA sensor provides a unique platform for the sensitive detection of DNA demethylation, DNA demethylase activity, and related molecular diagnostics and drug screening. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. DNA nanomechanical devices for molecular biology and DNA nanotechnology (United States)

    Gu, Hongzhou

    The aim of nanotechnology is to put specific atomic and molecular species where we want them, when we want them there. Achieving such a dynamic and functional control could lead to molecular programming. Structural DNA nanotechnology offers a powerful route to this goal by combining stable branched DNA motifs with cohesive ends to produce objects, programmed nanomechanical devices and fixed or modified patterned lattices. In Chapter II, a two-armed nanorobotic device is built based on a DNA origami substrate. The arms face each other, ready to capture different DNA nanostructures into distinguishable nanopatterns. Combining with a simple error-correction protocol, we are able to achieve this goal in a nearly flawless fashion. In Chapter III, a DNA-based programmable assembly line is developed by combining three PX/JX2 cassettes and a novel DNA walker on a DNA origami substrate. This programmable assembly line can generate eight products by switching the cassettes to PX (ON) or JX2 (OFF) state when the DNA walker passes by. DNA nanomechanical devices hold the promise of controlling structure and performing exquisitely fine measurements on the molecular scale. Several DNA nanomechanical devices based on different biochemistry phenomena have been reported before. In Chapter IV, a scissors-based DNA device is built to measure the work that can be done by a DNA-bending protein (MutS) when it binds to DNA.

  18. DNA Nanotechnology for Cancer Therapy. (United States)

    Kumar, Vinit; Palazzolo, Stefano; Bayda, Samer; Corona, Giuseppe; Toffoli, Giuseppe; Rizzolio, Flavio


    DNA nanotechnology is an emerging and exciting field, and represents a forefront frontier for the biomedical field. The specificity of the interactions between complementary base pairs makes DNA an incredible building material for programmable and very versatile two- and three-dimensional nanostructures called DNA origami. Here, we analyze the DNA origami and DNA-based nanostructures as a drug delivery system. Besides their physical-chemical nature, we dissect the critical factors such as stability, loading capability, release and immunocompatibility, which mainly limit in vivo applications. Special attention was dedicated to highlighting the boundaries to be overcome to bring DNA nanostructures closer to the bedside of patients.

  19. DNA Nanotechnology for Cancer Therapy (United States)

    Kumar, Vinit; Palazzolo, Stefano; Bayda, Samer; Corona, Giuseppe; Toffoli, Giuseppe; Rizzolio, Flavio


    DNA nanotechnology is an emerging and exciting field, and represents a forefront frontier for the biomedical field. The specificity of the interactions between complementary base pairs makes DNA an incredible building material for programmable and very versatile two- and three-dimensional nanostructures called DNA origami. Here, we analyze the DNA origami and DNA-based nanostructures as a drug delivery system. Besides their physical-chemical nature, we dissect the critical factors such as stability, loading capability, release and immunocompatibility, which mainly limit in vivo applications. Special attention was dedicated to highlighting the boundaries to be overcome to bring DNA nanostructures closer to the bedside of patients. PMID:27022418

  20. Demonstration of a potent RET transcriptional inhibitor for the treatment of medullary thyroid carcinoma based on an ellipticine derivative. (United States)

    Kumarasamy, Vishnu Muthuraj; Sun, Daekyu


    Dominant-activating mutations in the RET (rearranged during transfection) proto-oncogene, which encodes a receptor tyrosine kinase, is often associated with the development of medullary thyroid carcinoma (MTC). The proximal promoter region of the RET gene consists of a guanine-rich sequence containing five runs of three consecutive guanine residues that serve as the binding site for transcriptional factors. As we have recently shown, this stretch of nucleotides in the promoter region is highly dynamic in nature and tend to form non-B DNA secondary structures called G-quadruplexes, which suppress the transcription of the RET gene. In the present study, ellipticine and its derivatives were identified as excellent RET G-quadruplex stabilizing agents. Circular dichroism (CD) spectroscopic studies revealed that the incorporation of a piperidine ring in an ellipticine derivative, NSC311153 improves its binding with the G-quadruplex structure and the stability induced by this compound is more potent than ellipticine. Furthermore, this compound also interfered with the transcriptional mechanism of the RET gene in an MTC derived cell line, TT cells and significantly decreased the endogenous RET protein expression. We demonstrated the specificity of NSC311153 by using papillary thyroid carcinoma (PTC) cells, the TPC1 cell line which lacks the G-quadruplex forming sequence in the promoter region due to chromosomal rearrangement. The RET downregulation selectively suppresses cell proliferation by inhibiting the intracellular Raf/MEK/ERK and PI3K/Akt/mTOR signaling pathways in the TT cells. In the present study, we also showed that the systemic administration of a water soluble NSC311153 analog in a mouse MTC xenograft model inhibited the tumor growth through RET downregulation.

  1. DNA polymerase having modified nucleotide binding site for DNA sequencing (United States)

    Tabor, Stanley; Richardson, Charles


    Modified gene encoding a modified DNA polymerase wherein the modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase.

  2. Cytotoxic, DNA binding, DNA cleavage and antibacterial studies of ...

    Indian Academy of Sciences (India)

    fluoroquinolone complexes. Mohan N ... DNA-binding properties of Ru complexes have been studied by means of absorption spectrophotometry and viscosity measurements as well as their HS DNA cleavage properties by means of agarose gel ...

  3. Cascaded strand displacement for non-enzymatic target recycling amplification and label-free electronic detection of microRNA from tumor cells

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Kai; Dou, Baoting; Yang, Jianmei; Yuan, Ruo; Xiang, Yun, E-mail:


    The monitoring of microRNA (miRNA) expression levels is of great importance in cancer diagnosis. In the present work, based on two cascaded toehold-mediated strand displacement reactions (TSDRs), we have developed a label- and enzyme-free target recycling signal amplification approach for sensitive electronic detection of miRNA-21 from human breast cancer cells. The junction probes containing the locked G-quadruplex forming sequences are self-assembled on the senor surface. The presence of the target miRNA-21 initiates the first TSDR and results in the disassembly of the junction probes and the release of the active G-quadruplex forming sequences. Subsequently, the DNA fuel strand triggers the second TSDR and leads to cyclic reuse of the target miRNA-21. The cascaded TSDRs thus generate many active G-quadruplex forming sequences on the sensor surface, which associate with hemin to produce significantly amplified current response for sensitive detection of miRNA-21 at 1.15 fM. The sensor is also selective and can be employed to monitor miRNA-21 from human breast cancer cells. - Highlights: • Amplified and sensitive detection of microRNA from tumor cells is achieved. • Signal amplification is realized by two cascaded strand displacement reactions. • The developed sensor is selective and label-free without involving any enzymes.

  4. Fungal DNA barcoding. (United States)

    Xu, Jianping


    Fungi are ubiquitous in both natural and human-made environments. They play important roles in the health of plants, animals, and humans, and in broad ecosystem functions. Thus, having an efficient species-level identification system could significantly enhance our ability to treat fungal diseases and to monitor the spatial and temporal patterns of fungal distributions and migrations. DNA barcoding is a potent approach for rapid identification of fungal specimens, generating novel species hypothesis, and guiding biodiversity and ecological studies. In this mini-review, I briefly summarize (i) the history of DNA sequence-based fungal identification; (ii) the emergence of the ITS region as the consensus primary fungal barcode; (iii) the use of the ITS barcodes to address a variety of issues on fungal diversity from local to global scales, including generating a large number of species hypothesis; and (iv) the problems with the ITS barcode region and the approaches to overcome these problems. Similar to DNA barcoding research on plants and animals, significant progress has been achieved over the last few years in terms of both the questions being addressed and the foundations being laid for future research endeavors. However, significant challenges remain. I suggest three broad areas of research to enhance the usefulness of fungal DNA barcoding to meet the current and future challenges: (i) develop a common set of primers and technologies that allow the amplification and sequencing of all fungi at both the primary and secondary barcode loci; (ii) compile a centralized reference database that includes all recognized fungal species as well as species hypothesis, and allows regular updates from the research community; and (iii) establish a consensus set of new species recognition criteria based on barcode DNA sequences that can be applied across the fungal kingdom.

  5. The dynamic interplay between DNA topoisomerases and DNA topology. (United States)

    Seol, Yeonee; Neuman, Keir C


    Topological properties of DNA influence its structure and biochemical interactions. Within the cell, DNA topology is constantly in flux. Transcription and other essential processes, including DNA replication and repair, not only alter the topology of the genome but also introduce additional complications associated with DNA knotting and catenation. These topological perturbations are counteracted by the action of topoisomerases, a specialized class of highly conserved and essential enzymes that actively regulate the topological state of the genome. This dynamic interplay among DNA topology, DNA processing enzymes, and DNA topoisomerases is a pervasive factor that influences DNA metabolism in vivo. Building on the extensive structural and biochemical characterization over the past four decades that has established the fundamental mechanistic basis of topoisomerase activity, scientists have begun to explore the unique roles played by DNA topology in modulating and influencing the activity of topoisomerases. In this review we survey established and emerging DNA topology-dependent protein-DNA interactions with a focus on in vitro measurements of the dynamic interplay between DNA topology and topoisomerase activity.

  6. Electrochemical DNA Sensors for Detection of DNA Damage

    Directory of Open Access Journals (Sweden)

    Ana Maria Oliveira Brett


    Full Text Available Electrochemical devices have received particular attention due to their rapiddetection and great sensitivity for the evaluation of DNA-hazard compounds interactionmechanisms. Several types of bioanalytical method use nucleic acids probes to detect DNAdamage. This article reviews current directions and strategies in the development andapplications of electrochemical DNA sensors for the detection of DNA damage.

  7. DNA Damage Induced Neuronal Death

    National Research Council Canada - National Science Library

    Kisby, Glen


    ... (nitrogen mustard or HN2) and the neurotoxic DNA-damaging agent methylazoxymethanol (MAM) using neuronal and astrocyte cell cultures from different brain regions of mice with perturbed DNA repair...

  8. The Dynamics of DNA Sequencing. (United States)

    Morvillo, Nancy


    Describes a paper-and-pencil activity that helps students understand DNA sequencing and expands student understanding of DNA structure, replication, and gel electrophoresis. Appropriate for advanced biology students who are familiar with the Sanger method. (DDR)

  9. An Introduction to DNA Fingerprinting. (United States)

    Hepfer, Carol Ely; And Others


    Provides background information on DNA fingerprinting, and describes exercises for introducing general biology students at the high school or college level to the methodology and applications of DNA fingerprinting. (PR)

  10. Esitleti kakskeelset luulekogu "Luule DNA"

    Index Scriptorium Estoniae


    Magrelli, Valerio. Luule DNA = Il DNA della poesia / tõlkinud [ja saatesõna:] Maarja Kangro ja Kalju Kruusa. Tallinn : Koma, 2006. Sisaldab autori teksti. Esitlus 24. jaan. Kirjanike majas Tallinnas

  11. Alterations of ultraviolet irradiated DNA

    International Nuclear Information System (INIS)

    Davila, C.; Garces, F.


    Thymine dimers production has been studied in several DNA- 3 H irradiated at various wave lenght of U.V. Light. The influence of dimers on the hydrodynamic and optic properties, thermal structural stability and transformant capacity of DNA have been studied too. At last the recognition and excision of dimers by the DNA-UV-Endonuclease and DNA-Polimerase-I was also studied. (author)

  12. Single-stranded DNA library preparation from highly degraded DNA using T4 DNA ligase. (United States)

    Gansauge, Marie-Theres; Gerber, Tobias; Glocke, Isabelle; Korlevic, Petra; Lippik, Laurin; Nagel, Sarah; Riehl, Lara Maria; Schmidt, Anna; Meyer, Matthias


    DNA library preparation for high-throughput sequencing of genomic DNA usually involves ligation of adapters to double-stranded DNA fragments. However, for highly degraded DNA, especially ancient DNA, library preparation has been found to be more efficient if each of the two DNA strands are converted into library molecules separately. We present a new method for single-stranded library preparation, ssDNA2.0, which is based on single-stranded DNA ligation with T4 DNA ligase utilizing a splinter oligonucleotide with a stretch of random bases hybridized to a 3΄ biotinylated donor oligonucleotide. A thorough evaluation of this ligation scheme shows that single-stranded DNA can be ligated to adapter oligonucleotides in higher concentration than with CircLigase (an RNA ligase that was previously chosen for end-to-end ligation in single-stranded library preparation) and that biases in ligation can be minimized when choosing splinters with 7 or 8 random nucleotides. We show that ssDNA2.0 tolerates higher quantities of input DNA than CircLigase-based library preparation, is less costly and better compatible with automation. We also provide an in-depth comparison of library preparation methods on degraded DNA from various sources. Most strikingly, we find that single-stranded library preparation increases library yields from tissues stored in formalin for many years by several orders of magnitude. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Small DNA circles as probes of DNA topology. (United States)

    Bates, Andrew D; Noy, Agnes; Piperakis, Michael M; Harris, Sarah A; Maxwell, Anthony


    Small DNA circles can occur in Nature, for example as protein-constrained loops, and can be synthesized by a number of methods. Such small circles provide tractable systems for the study of the structure, thermodynamics and molecular dynamics of closed-circular DNA. In the present article, we review the occurrence and synthesis of small DNA circles, and examine their utility in studying the properties of DNA and DNA-protein interactions. In particular, we highlight the analysis of small circles using atomistic simulations.

  14. Werner helicase wings DNA binding


    Hoadley, Kelly A.; Keck, James L.


    In this issue of Structure, Kitano et al. describe the structure of the DNA-bound winged-helix domain from the Werner helicase. This structure of a RecQ/DNA complex offers insights into the DNA unwinding mechanisms of RecQ family helicases.

  15. DNA Origami-Graphene Hybrid Nanopore for DNA Detection. (United States)

    Barati Farimani, Amir; Dibaeinia, Payam; Aluru, Narayana R


    DNA origami nanostructures can be used to functionalize solid-state nanopores for single molecule studies. In this study, we characterized a nanopore in a DNA origami-graphene heterostructure for DNA detection. The DNA origami nanopore is functionalized with a specific nucleotide type at the edge of the pore. Using extensive molecular dynamics (MD) simulations, we computed and analyzed the ionic conductivity of nanopores in heterostructures carpeted with one or two layers of DNA origami on graphene. We demonstrate that a nanopore in DNA origami-graphene gives rise to distinguishable dwell times for the four DNA base types, whereas for a nanopore in bare graphene, the dwell time is almost the same for all types of bases. The specific interactions (hydrogen bonds) between DNA origami and the translocating DNA strand yield different residence times and ionic currents. We also conclude that the speed of DNA translocation decreases due to the friction between the dangling bases at the pore mouth and the sequencing DNA strands.

  16. Processive DNA demethylation via DNA deaminase-induced lesion resolution.

    Directory of Open Access Journals (Sweden)

    Don-Marc Franchini

    Full Text Available Base modifications of cytosine are an important aspect of chromatin biology, as they can directly regulate gene expression, while DNA repair ensures that those modifications retain genome integrity. Here we characterize how cytosine DNA deaminase AID can initiate DNA demethylation. In vitro, AID initiated targeted DNA demethylation of methyl CpGs when in combination with DNA repair competent extracts. Mechanistically, this is achieved by inducing base alterations at or near methyl-cytosine, with the lesion being resolved either via single base substitution or a more efficient processive polymerase dependent repair. The biochemical findings are recapitulated in an in vivo transgenic targeting assay, and provide the genetic support of the molecular insight into DNA demethylation. This targeting approach supports the hypothesis that mCpG DNA demethylation can proceed via various pathways and mCpGs do not have to be targeted to be demethylated.

  17. Dine marker har DNA

    DEFF Research Database (Denmark)

    Eckholdt, Annette; Winding, Anne; Krogh, Paul Henning


    Ordet "biodiversitet" og at det er noget, vi skal have mere af, nævnes hyppigt. Men hvad er biodiversitet, og hvordan måles det? Agrologisk har bedt et par eksperter fra Aarhus Universitet forklare, hvordan et DNA-aftryk af jord og vand kan erstatte optællinger i felten og sige noget om biodivers......Ordet "biodiversitet" og at det er noget, vi skal have mere af, nævnes hyppigt. Men hvad er biodiversitet, og hvordan måles det? Agrologisk har bedt et par eksperter fra Aarhus Universitet forklare, hvordan et DNA-aftryk af jord og vand kan erstatte optællinger i felten og sige noget om...

  18. Fleet DNA (Presentation)

    Energy Technology Data Exchange (ETDEWEB)

    Walkokwicz, K.; Duran, A.


    The Fleet DNA project objectives include capturing and quantifying drive cycle and technology variation for the multitude of medium- and heavy-duty vocations; providing a common data storage warehouse for medium- and heavy-duty vehicle fleet data across DOE activities and laboratories; and integrating existing DOE tools, models, and analyses to provide data-driven decision making capabilities. Fleet DNA advantages include: for Government - providing in-use data for standard drive cycle development, R&D, tech targets, and rule making; for OEMs - real-world usage datasets provide concrete examples of customer use profiles; for fleets - vocational datasets help illustrate how to maximize return on technology investments; for Funding Agencies - ways are revealed to optimize the impact of financial incentive offers; and for researchers -a data source is provided for modeling and simulation.

  19. Extended DNA Tile Actuators

    DEFF Research Database (Denmark)

    Kristiansen, Martin; Kryger, Mille; Zhang, Zhao


    A dynamic linear DNA tile actuator is expanded to three new structures of higher complexity. The original DNA actuator was constructed from a central roller strand which hybridizes with two piston strands by forming two half-crossover junctions. A linear expansion of the actuator is obtained...... by fusing two actuators of different sequence designs with a third central roller strand. This structure spans 35 nm and its integrity was verified by PAGE analysis. Owing to sequence homology around the crossovers the actuator can obtain 12 different states. The states of the actuator are controlled...... by a lock strand inserted at one end of the actuator and monitored by Forster resonance energy transfer (FRET) spectroscopy between a fluorophore pair which is located at the other end of the actuator. Two other designs were made where the linear actuator monomer is expanded into two dimensions by forming...

  20. Geant4-DNA simulations using complex DNA geometries generated by the DnaFabric tool (United States)

    Meylan, S.; Vimont, U.; Incerti, S.; Clairand, I.; Villagrasa, C.


    Several DNA representations are used to study radio-induced complex DNA damages depending on the approach and the required level of granularity. Among all approaches, the mechanistic one requires the most resolved DNA models that can go down to atomistic DNA descriptions. The complexity of such DNA models make them hard to modify and adapt in order to take into account different biological conditions. The DnaFabric project was started to provide a tool to generate, visualise and modify such complex DNA models. In the current version of DnaFabric, the models can be exported to the Geant4 code to be used as targets in the Monte Carlo simulation. In this work, the project was used to generate two DNA fibre models corresponding to two DNA compaction levels representing the hetero and the euchromatin. The fibres were imported in a Geant4 application where computations were performed to estimate the influence of the DNA compaction on the amount of calculated DNA damage. The relative difference of the DNA damage computed in the two fibres for the same number of projectiles was found to be constant and equal to 1.3 for the considered primary particles (protons from 300 keV to 50 MeV). However, if only the tracks hitting the DNA target are taken into account, then the relative difference is more important for low energies and decreases to reach zero around 10 MeV. The computations were performed with models that contain up to 18,000 DNA nucleotide pairs. Nevertheless, DnaFabric will be extended to manipulate multi-scale models that go from the molecular to the cellular levels.

  1. Radiobiology with DNA ligands

    International Nuclear Information System (INIS)

    Weinreich, R.; Argentini, M.; Guenther, I.; Koziorowski, J.; Larsson, B.; Nievergelt-Egido, M.C.; Salt, C.; Wyer, L.; Dos Santos, D.F.; Hansen, H.J.


    The paper deals with the following topics: labelling of DNA ligands and other tumour-affinic compounds with 4.15-d 124 I, radiotoxicity of Hoechst 33258 and 33342 and of iodinated Hoechst 33258 in cell cultures, preparation of 76 Br-, 123 I-, and 221 At-labelled 5-halo-2'-deoxyuridine, chemical syntheses of boron derivatives of Hoechst 33258.III., Gadolinium neutron capture therapy

  2. Understanding the sequence preference of recurrent RNA building blocks using quantum chemistry: The intrastrand RNA dinucleotide platform

    Czech Academy of Sciences Publication Activity Database

    Mládek, Arnošt; Šponer, Judit E.; Kulhánek, P.; Lu, X.-J.; Olson, W.K.; Šponer, Jiří


    Roč. 8, č. 1 (2012), s. 335-347 ISSN 1549-9618 R&D Projects: GA AV ČR(CZ) IAA400040802; GA ČR(CZ) GAP208/10/2302; GA ČR(CZ) GA203/09/1476; GA ČR(CZ) GAP208/11/1822; GA ČR(CZ) GD203/09/H046 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : RNA dinucleotide platform * quantum-chemical calculation Subject RIV: BO - Biophysics Impact factor: 5.389, year: 2012

  3. Compressive Sensing DNA Microarrays

    Directory of Open Access Journals (Sweden)

    Sheikh Mona A


    Full Text Available Compressive sensing microarrays (CSMs are DNA-based sensors that operate using group testing and compressive sensing (CS principles. In contrast to conventional DNA microarrays, in which each genetic sensor is designed to respond to a single target, in a CSM, each sensor responds to a set of targets. We study the problem of designing CSMs that simultaneously account for both the constraints from CS theory and the biochemistry of probe-target DNA hybridization. An appropriate cross-hybridization model is proposed for CSMs, and several methods are developed for probe design and CS signal recovery based on the new model. Lab experiments suggest that in order to achieve accurate hybridization profiling, consensus probe sequences are required to have sequence homology of at least 80% with all targets to be detected. Furthermore, out-of-equilibrium datasets are usually as accurate as those obtained from equilibrium conditions. Consequently, one can use CSMs in applications in which only short hybridization times are allowed.

  4. Monitoring Biodiversity using Environmental DNA

    DEFF Research Database (Denmark)

    Thomsen, Philip Francis

    of mammalian DNA in leech guts is shown to persist for at least 4 months post feeding. Subsequently, it is shown that DNA from wild leeches, collected in tropical rainforest in Vietnam, contains DNA from wild mammals living in the area, representing poorly known, cryptic and threatened species. Finally......, a study tests the applicability of non-destructive DNA extraction from old and ancient insect remains. DNA is successfully retrieved, amplified and equenced from dried museum beetle specimens up to 188 years old, ermafrost-preserved macrofossils up to 26.000 years old and directly from 1800-3000 years old...

  5. Next generation DNA led technologies

    CERN Document Server

    Jyothsna, G; Kashyap, Amita


    This brief highlights advances in DNA technologies and their wider applications. DNA is the source of life and has been studied since a generation, but very little is known as yet. Several sophisticated technologies of the current era have laid their foundations on the principle of DNA based mechanisms. DNA based technologies are bringing a new revolution of Advanced Science and Technology. Forensic Investigation, Medical Diagnosis, Paternity Disputes, Individual Identity, Health insurance, Motor Insurance have incorporated the DNA testing and profiling technologies for settling the issues.

  6. Topoisomerase II minimizes DNA entanglements by proofreading DNA topology after DNA strand passage. (United States)

    Martínez-García, Belén; Fernández, Xavier; Díaz-Ingelmo, Ofelia; Rodríguez-Campos, Antonio; Manichanh, Chaysavanh; Roca, Joaquim


    By transporting one DNA double helix (T-segment) through a double-strand break in another (G-segment), topoisomerase II reduces fractions of DNA catenanes, knots and supercoils to below equilibrium values. How DNA segments are selected to simplify the equilibrium DNA topology is enigmatic, and the biological relevance of this activity is unclear. Here we examined the transit of the T-segment across the three gates of topoisomerase II (entry N-gate, DNA-gate and exit C-gate). Our experimental results uncovered that DNA transport probability is determined not only during the capture of a T-segment at the N-gate. When a captured T-segment has crossed the DNA-gate, it can backtrack to the N-gate instead of exiting by the C-gate. When such backtracking is precluded by locking the N-gate or by removing the C-gate, topoisomerase II no longer simplifies equilibrium DNA topology. Therefore, we conclude that the C-gate enables a post-DNA passage proofreading mechanism, which challenges the release of passed T-segments to either complete or cancel DNA transport. This proofreading activity not only clarifies how type-IIA topoisomerases simplify the equilibrium topology of DNA in free solution, but it may explain also why these enzymes are able to solve the topological constraints of intracellular DNA without randomly entangling adjacent chromosomal regions.

  7. Ancient DNA from marine mammals

    DEFF Research Database (Denmark)

    Foote, Andrew David; Hofreiter, Michael; Morin, Philip A.


    discuss studies recon- structing inter- and intra-specific phylogenies from aDNA sequences and discuss how aDNA sequences could be used to estimate mutation rates. Finally, we highlight some of the problems of aDNA studies on marine mammals, such as obtaining sufficient sample sizes and calibrating...... such as bone, tooth, baleen, skin, fur, whiskers and scrimshaw using ancient DNA (aDNA) approaches provide an oppor- tunity for investigating such changes over evolutionary and ecological timescales. Here, we review the application of aDNA techniques to the study of marine mammals. Most of the studies have...... focused on detecting changes in genetic diversity following periods of exploitation and environmental change. To date, these studies have shown that even small sample sizes can provide useful information on historical genetic diversity. Ancient DNA has also been used in investigations of changes...

  8. Mitogenomic analyses from ancient DNA

    DEFF Research Database (Denmark)

    Paijmans, Johanna L. A.; Gilbert, Tom; Hofreiter, Michael


    The analysis of ancient DNA is playing an increasingly important role in conservation genetic, phylogenetic and population genetic analyses, as it allows incorporating extinct species into DNA sequence trees and adds time depth to population genetics studies. For many years, these types of DNA...... analyses (whether using modern or ancient DNA) were largely restricted to the analysis of short fragments of the mitochondrial genome. However, due to many technological advances during the past decade, a growing number of studies have explored the power of complete mitochondrial genome sequences...... (mitogenomes). Such studies were initially limited to analyses of extant organisms, but developments in both DNA sequencing technologies and general methodological aspects related to working with degraded DNA have resulted in complete mitogenomes becoming increasingly popular for ancient DNA studies as well...

  9. Radiation damage of DNA. Model for direct ionization of DNA

    International Nuclear Information System (INIS)

    Kobayashi, Kazuo; Tagawa, Seiichi


    Current aspects of radiation damage of DNA, particularly induced by the direct effect of radiation, and author's method of pulse radiolysis are described in relation to behavior of ions formed by radiation and active principles to induce the strand break. In irradiation of DNA solution in water, the direct effect of radiation is derived from ionization of DNA itself and indirect one, from the reaction between DNA and radicals generated from water molecules and the former direct one has been scarcely investigated due to difficulty of experimental approach. Radicals generated in sugar moiety of DNA are shown important in the strand break by recent studies on crystalline DNA irradiated by X-ray, DNA solution by electron and photon beams, hydrated DNA by γ-ray and by high linear energy transfer (LET) ion. Author's pulse radiolysis studies have revealed behaviors of guanine and adenine radical cations in dynamics of DNA oxidation. Since reactions described are the model, the experimental approach is thought necessary for elucidation of the actually occurring DNA damage in living cells. (N.I.)

  10. Human DNA Ligase III Recognizes DNA Ends by Dynamic Switching Between Two DNA Bound States†


    Cotner-Gohara, Elizabeth; Kim, In-Kwon; Hammel, Michal; Tainer, John A.; Tomkinson, Alan E.; Ellenberger, Tom


    Human DNA ligase III has essential functions in nuclear and mitochondrial DNA replication and repair and contains a PARP-like zinc finger (ZnF) that increases DNA nick-joining and intermolecular DNA ligation. Yet, the bases for ligase III specificity and structural variation among human ligases are not understood. Here combined crystal structure and small angle x-ray scattering results reveal dynamic switching between two nick-binding components of ligase III: the ZnF-DNA binding domain (DBD)...

  11. Dynamics of DNA conformations and DNA-protein interaction

    DEFF Research Database (Denmark)

    Metzler, R.; Ambjörnsson, T.; Lomholt, Michael Andersen


    denaturation (bubble breathing), deriving its dynamic response to external physical parameters and the DNA sequence in terms of the bubble relaxation time spectrum and the autocorrelation function of bubble breathing. The interaction with binding proteins that selectively bind to the DNA single strand exposed......Optical tweezers, atomic force microscopes, patch clamping, or fluorescence techniques make it possible to study both the equilibrium conformations and dynamics of single DNA molecules as well as their interaction with binding proteins. In this paper we address the dynamics of local DNA...... in a denaturation bubble are shown to involve an interesting competition of time scales, varying between kinetic blocking of protein binding up to full binding protein-induced denaturation of the DNA. We will also address the potential to use DNA physics for the design of nanosensors. Finally, we report recent...

  12. DNA adducts as molecular dosimeters

    International Nuclear Information System (INIS)

    Lucier, G.W.


    There is compelling evidence that DNA adducts play an important role in the actions of many pulmonary carcinogens. During the last ten years sensitive methods (antibodies and 32 P-postlabeling) have been developed that permit detection of DNA adducts in tissues of animals or humans exposed to low levels of some genotoxic carcinogens. This capability has led to approaches designed to more reliably estimate the shape of the dose-response curve in the low dose region for a few carcinogens. Moreover, dosimetry comparisions can, in some cases, be made between animals and humans which help in judging the adequacy of animal models for human risk assessments. There are several points that need to be considered in the evaluation of DNA adducts as a molecular dosimeter. For example, DNA adduct formation is only one of many events that are needed for tumor development and some potent carcinogens do not form DNA adducts; i.e., TCDD. Other issues that need to be considered are DNA adduct heterogeneity, DNA repair, relationship of DNA adducts to somatic mutation and cell specificity in DNA adduct formation and persistence. Molecular epidemiology studies often require quantitation of adducts in cells such as lymphocytes which may or may not be reliable surrogates for adduct concentrations in target issues. In summary, accurate quantitation of low levels of DNA adducts may provide data useful in species to species extrapolation of risk including the development of more meaningful human monitoring programs

  13. Using DNA looping to measure sequence dependent DNA elasticity (United States)

    Kandinov, Alan; Raghunathan, Krishnan; Meiners, Jens-Christian


    We are using tethered particle motion (TPM) microscopy to observe protein-mediated DNA looping in the lactose repressor system in DNA constructs with varying AT / CG content. We use these data to determine the persistence length of the DNA as a function of its sequence content and compare the data to direct micromechanical measurements with constant-force axial optical tweezers. The data from the TPM experiments show a much smaller sequence effect on the persistence length than the optical tweezers experiments.

  14. The DNA Files

    Energy Technology Data Exchange (ETDEWEB)



    The DNA Files is a radio documentary which disseminates genetics information over public radio. The documentaries explore subjects which include the following: How genetics affects society. How human life began and how it evolved. Could new prenatal genetic tests hold the key to disease prevention later in life? Would a national genetic data base sacrifice individual privacy? and Should genes that may lead to the cure for cancer be privately owned? This report serves as a project update for the second quarter of 1998. It includes the spring/summer 1998 newsletter, the winter 1998 newsletter, the program clock, and the latest flyer.

  15. Adenovirus DNA Replication (United States)

    Hoeben, Rob C.; Uil, Taco G.


    Adenoviruses have attracted much attention as probes to study biological processes such as DNA replication, transcription, splicing, and cellular transformation. More recently these viruses have been used as gene-transfer vectors and oncolytic agents. On the other hand, adenoviruses are notorious pathogens in people with compromised immune functions. This article will briefly summarize the basic replication strategy of adenoviruses and the key proteins involved and will deal with the new developments since 2006. In addition, we will cover the development of antivirals that interfere with human adenovirus (HAdV) replication and the impact of HAdV on human disease. PMID:23388625

  16. Dynamics of DNA conformations and DNA-protein interaction

    DEFF Research Database (Denmark)

    Metzler, R.; Ambjörnsson, T.; Lomholt, Michael Andersen


    Optical tweezers, atomic force microscopes, patch clamping, or fluorescence techniques make it possible to study both the equilibrium conformations and dynamics of single DNA molecules as well as their interaction with binding proteins. In this paper we address the dynamics of local DNA...... denaturation (bubble breathing), deriving its dynamic response to external physical parameters and the DNA sequence in terms of the bubble relaxation time spectrum and the autocorrelation function of bubble breathing. The interaction with binding proteins that selectively bind to the DNA single strand exposed...

  17. Characterization of denaturation and renaturation of DNA for DNA hybridization

    Directory of Open Access Journals (Sweden)

    Xiaofang Wang


    Full Text Available Objectives The present study was designed to systematically characterize the denaturation and the renaturation of double stranded DNA (dsDNA, which is suitable for DNA hybridization. Methods A series of physical and chemical denaturation methods were implemented on well-defined 86-bp dsDNA fragment. The degree of each denaturation was measured and the most suitable denaturation method was determined. DNA renaturation tendency was also investigated for the suggested denaturation method. Results Heating, beads mill, and sonication bath did not show any denaturation for 30 minutes. However probe sonication fully denatured DNA in 5 minutes. 1 mol/L sodium hydroxide (alkaline treatment and 60% dimethyl sulfoxide (DMSO treatment fully denatured DNA in 2-5 minutes. Conclusions Among all the physical methods applied, the direct probe sonication was the most effective way to denature the DNA fragments. Among chemical methods, 60% DMSO was the most adequate denaturation method since it does not cause full renaturation during DNA hybridization.

  18. Recent progress in DNA origami technology. (United States)

    Endo, Masayuki; Sugiyama, Hiroshi


    DNA origami is an emerging technology for designing defined two-dimensional DNA nanostructures. In this review, we focus on and describe several types of DNA origami-related studies, as follows: (1) programmed DNA origami assembly, (2) DNA origami-templated molecular assembly, (3) design and construction of various three-dimensional DNA origami structures, (4) programmed functionalization of DNA origami and combination with top-down nanotechnology, (5) single molecular observation on a designed DNA origami, and (6) DNA nanomachines working on a DNA origami. © 2011 by John Wiley & Sons, Inc.

  19. Satellite DNA and related diseases

    Directory of Open Access Journals (Sweden)

    Rich J.


    Full Text Available Satellite DNA, also known as tandemly repeated DNA, consists of clusters of repeated sequences and represents a diverse class of highly repetitive elements. Satellite DNA can be divided into several classes according to the size of an individual repeat: microsatellites, minisatellites, midisatellites, and macrosatellites. Originally considered as «junk» DNA, satellite DNA has more recently been reconsidered as having various functions. Moreover, due to the repetitive nature of the composing elements, their presence in the genome is associated with high frequency mutations, epigenetic changes and modifications in gene expression patterns, with a potential to lead to human disease. Therefore, the satellite DNA study will be beneficial for developing a treatment of satellite-related diseases, such as FSHD, neurological, developmental disorders and cancers.

  20. DNA-based hybrid catalysis. (United States)

    Rioz-Martínez, Ana; Roelfes, Gerard


    In the past decade, DNA-based hybrid catalysis has merged as a promising novel approach to homogeneous (asymmetric) catalysis. A DNA hybrid catalysts comprises a transition metal complex that is covalently or supramolecularly bound to DNA. The chiral microenvironment and the second coordination sphere interactions provided by the DNA are key to achieve high enantioselectivities and, often, additional rate accelerations in catalysis. Nowadays, current efforts are focused on improved designs, understanding the origin of the enantioselectivity and DNA-induced rate accelerations, expanding the catalytic scope of the concept and further increasing the practicality of the method for applications in synthesis. Herein, the recent developments will be reviewed and the perspectives for the emerging field of DNA-based hybrid catalysis will be discussed. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. DNA vaccine: the miniature miracle

    Directory of Open Access Journals (Sweden)

    Karthik Kaliaperumal


    Full Text Available DNA, the essential part of the life is making way in to new vaccine technology. Plasmid vectors from the bacteria have revolutionized the world of vaccine design by its new technology – DNA vaccines. Small portion of the nucleotides from the pathogen held under the control of promoter in a plasmid vector can be used as a vaccine. DNA vaccines alleviate the odds of the other vaccines by having good hold on both the faces of the immunity. The key to the success of DNA vaccine lies in the route of administration of the vaccine which can be done in many ways. Prime boost strategy is an approach used to boost the action of DNA vaccine. To date there are only four DNA vaccine available in the market. [Vet World 2013; 6(4.000: 228-232

  2. Development of dengue DNA vaccines. (United States)

    Danko, Janine R; Beckett, Charmagne G; Porter, Kevin R


    Vaccination with plasmid DNA against infectious pathogens including dengue is an active area of investigation. By design, DNA vaccines are able to elicit both antibody responses and cellular immune responses capable of mediating long-term protection. Great technical improvements have been made in dengue DNA vaccine constructs and trials are underway to study these in the clinic. The scope of this review is to highlight the rich history of this vaccine platform and the work in dengue DNA vaccines accomplished by scientists at the Naval Medical Research Center. This work resulted in the only dengue DNA vaccine tested in a clinical trial to date. Additional advancements paving the road ahead in dengue DNA vaccine development are also discussed. Published by Elsevier Ltd.

  3. Monitoring Biodiversity using Environmental DNA

    DEFF Research Database (Denmark)

    Thomsen, Philip Francis

    DNA). Especially the advance in DNA sequencing technology has revolutionized this field and opened new frontiers in ecology, evolution and environmental sciences. Also, it is becoming a powerful tool for field biologist, with new and efficient methods for monitoring biodiversity. This thesis focuses on the use...... of eDNA in monitoring of biodiversity in different settings. First, it is shown that a diversity of rare freshwater animals – representing amphibians, fish, mammals, insects and crustaceans – can be detected based on eDNA obtained directly from 15 ml water samples of lakes, ponds and streams...... setting, showing that eDNA obtained directly from ½ l seawater samples can account for marine fish biodiversity using NGS. Promisingly, eDNA covered the fish diversity better than any of 9 methods, conventionally used in marine fish surveys. Additionally, it is shown that even short 100-bp. fish e...

  4. Human DNA ligase III bridges two DNA ends to promote specific intermolecular DNA end joining (United States)

    Kukshal, Vandna; Kim, In-Kwon; Hura, Gregory L.; Tomkinson, Alan E.; Tainer, John A.; Ellenberger, Tom


    Mammalian DNA ligase III (LigIII) functions in both nuclear and mitochondrial DNA metabolism. In the nucleus, LigIII has functional redundancy with DNA ligase I whereas LigIII is the only mitochondrial DNA ligase and is essential for the survival of cells dependent upon oxidative respiration. The unique LigIII zinc finger (ZnF) domain is not required for catalytic activity but senses DNA strand breaks and stimulates intermolecular ligation of two DNAs by an unknown mechanism. Consistent with this activity, LigIII acts in an alternative pathway of DNA double strand break repair that buttresses canonical non-homologous end joining (NHEJ) and is manifest in NHEJ-defective cancer cells, but how LigIII acts in joining intermolecular DNA ends versus nick ligation is unclear. To investigate how LigIII efficiently joins two DNAs, we developed a real-time, fluorescence-based assay of DNA bridging suitable for high-throughput screening. On a nicked duplex DNA substrate, the results reveal binding competition between the ZnF and the oligonucleotide/oligosaccharide-binding domain, one of three domains constituting the LigIII catalytic core. In contrast, these domains collaborate and are essential for formation of a DNA-bridging intermediate by adenylated LigIII that positions a pair of blunt-ended duplex DNAs for efficient and specific intermolecular ligation. PMID:26130724

  5. Human DNA Ligase III Recognizes DNA Ends by Dynamic Switching between Two DNA-Bound States

    Energy Technology Data Exchange (ETDEWEB)

    Cotner-Gohara, Elizabeth; Kim, In-Kwon; Hammel, Michal; Tainer, John A.; Tomkinson, Alan E.; Ellenberger, Tom (Scripps); (Maryland-MED); (WU-MED); (LBNL)


    Human DNA ligase III has essential functions in nuclear and mitochondrial DNA replication and repair and contains a PARP-like zinc finger (ZnF) that increases the extent of DNA nick joining and intermolecular DNA ligation, yet the bases for ligase III specificity and structural variation among human ligases are not understood. Here combined crystal structure and small-angle X-ray scattering results reveal dynamic switching between two nick-binding components of ligase III: the ZnF-DNA binding domain (DBD) forms a crescent-shaped surface used for DNA end recognition which switches to a ring formed by the nucleotidyl transferase (NTase) and OB-fold (OBD) domains for catalysis. Structural and mutational analyses indicate that high flexibility and distinct DNA binding domain features in ligase III assist both nick sensing and the transition from nick sensing by the ZnF to nick joining by the catalytic core. The collective results support a 'jackknife model' in which the ZnF loads ligase III onto nicked DNA and conformational changes deliver DNA into the active site. This work has implications for the biological specificity of DNA ligases and functions of PARP-like zinc fingers.

  6. Evolution of DNA sequencing. (United States)

    Tipu, Hamid Nawaz; Shabbir, Ambreen


    Sanger and coworkers introduced DNA sequencing in 1970s for the first time. It principally relied on termination of growing nucleotide chain when a dideoxythymidine triphosphate (ddTTP) was inserted in it. Detection of terminated sequences was done radiographically on Polyacrylamide Gel Electrophoresis (PAGE). Improvements that have evolved over time in original Sanger sequencing include replacement of radiography with fluorescence, use of separate fluorescent markers for each nucleotide, use of capillary electrophoresis instead of polyacrylamide gel electrophoresis and then introduction of capillary array electrophoresis. However, this technique suffered from few inherent limitations like decreased sensitivity for low level mutant alleles, complexities in analyzing highly polymorphic regions like Major Histocompatibility Complex (MHC) and high DNA concentrations required. Several Next Generation Sequencing (NGS) technologies have been introduced by Roche, Illumina and other commercial manufacturers that tend to overcome Sanger sequencing limitations and have been reviewed. Introduction of NGS in clinical research and medical diagnostics is expected to change entire diagnostic approach. These include study of cancer variants, detection of minimal residual disease, exome sequencing, detection of Single Nucleotide Polymorphisms (SNPs) and their disease association, epigenetic regulation of gene expression and sequencing of microorganisms genome.

  7. Biosensors for DNA sequence detection (United States)

    Vercoutere, Wenonah; Akeson, Mark


    DNA biosensors are being developed as alternatives to conventional DNA microarrays. These devices couple signal transduction directly to sequence recognition. Some of the most sensitive and functional technologies use fibre optics or electrochemical sensors in combination with DNA hybridization. In a shift from sequence recognition by hybridization, two emerging single-molecule techniques read sequence composition using zero-mode waveguides or electrical impedance in nanoscale pores.

  8. DNA methylation dynamics in neurogenesis (United States)

    Wang, Zhiqin; Tang, Beisha; He, Yuquan; Jin, Peng


    Neurogenesis is not limited to the embryonic stage, but continually proceeds in the adult brain throughout life. Epigenetic mechanisms, including DNA methylation, histone modification and noncoding RNA, play important roles in neurogenesis. For decades, DNA methylation was thought to be a stable modification, except for demethylation in the early embryo. In recent years, DNA methylation has proved to be dynamic during development. In this review, we summarize the latest understanding about DNA methylation dynamics in neurogenesis, including the roles of different methylation forms (5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine and 5-carboxylcytosine), as well as their ‘writers’, ‘readers’ and interactions with histone modifications. PMID:26950681

  9. Authenticity in ancient DNA studies

    DEFF Research Database (Denmark)

    Gilbert, M Thomas P; Willerslev, Eske


    Ancient DNA studies represent a powerful tool that can be used to obtain genetic insights into the past. However, despite the publication of large numbers of apparently successful ancient DNA studies, a number of problems exist with the field that are often ignored. Therefore, questions exist...... as to how reliable the conclusions of many of the published studies are. In this paper we outline first the problems associated with aDNA studies, and secondly present potential guidelines designed so as to enable non-specialist readers the opportunity to critically assess the quality of aDNA publications....

  10. Statistical Approaches for DNA Barcoding

    DEFF Research Database (Denmark)

    Nielsen, Rasmus; Matz, M.


    The use of DNA as a tool for species identification has become known as "DNA barcoding" (Floyd et al., 2002; Hebert et al., 2003; Remigio and Hebert, 2003). The basic idea is straightforward: a small amount of DNA is extracted from the specimen, amplified and sequenced. The gene region sequenced...... is chosen so that it is nearly identical among individuals of the same species, but different between species, and therefore its sequence, can serve as an identification tag for the species ("DNA barcode"). By matching the sequence obtained from an unidentified specimen ("query" sequence) to the database...

  11. DNA repair: keeping it together

    DEFF Research Database (Denmark)

    Lisby, Michael; Rothstein, Rodney


    A protein scaffold has been identified that holds a chromosome together in the event of a DNA double-strand break. This scaffold is dependent on Rad52 and the Rad50-Mre11-Xrs2 complex and withstands the pulling forces of the mitotic spindle during DNA damage checkpoint arrest.......A protein scaffold has been identified that holds a chromosome together in the event of a DNA double-strand break. This scaffold is dependent on Rad52 and the Rad50-Mre11-Xrs2 complex and withstands the pulling forces of the mitotic spindle during DNA damage checkpoint arrest....

  12. Normalizing cDNA libraries. (United States)

    Bogdanov, Ekaterina A; Shagina, Irina; Barsova, Ekaterina V; Kelmanson, Ilya; Shagin, Dmitry A; Lukyanov, Sergey A


    The characterization of rare messages in cDNA libraries is complicated by the substantial variations that exist in the abundance levels of different transcripts in cells and tissues. The equalization (normalization) of cDNA is a helpful approach for decreasing the prevalence of abundant transcripts, thereby facilitating the assessment of rare transcripts. This unit provides a method for duplex-specific nuclease (DSN)-based normalization, which allows for the fast and reliable equalization of cDNA, thereby facilitating the generation of normalized, full-length-enriched cDNA libraries, and enabling efficient RNA analyses. (c) 2010 by John Wiley & Sons, Inc.

  13. DNA nanotechnology-enabled biosensors. (United States)

    Chao, Jie; Zhu, Dan; Zhang, Yinan; Wang, Lianhui; Fan, Chunhai


    Biosensors employ biological molecules to recognize the target and utilize output elements which can translate the biorecognition event into electrical, optical or mass-sensitive signals to determine the quantities of the target. DNA-based biosensors, as a sub-field to biosensor, utilize DNA strands with short oligonucleotides as probes for target recognition. Although DNA-based biosensors have offered a promising alternative for fast, simple and cheap detection of target molecules, there still exist key challenges including poor stability and reproducibility that hinder their competition with the current gold standard for DNA assays. By exploiting the self-recognition properties of DNA molecules, researchers have dedicated to make versatile DNA nanostructures in a highly rigid, controllable and functionalized manner, which offers unprecedented opportunities for developing DNA-based biosensors. In this review, we will briefly introduce the recent advances on design and fabrication of static and dynamic DNA nanostructures, and summarize their applications for fabrication and functionalization of DNA-based biosensors. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Aging and DNA repair capability. [Review

    Energy Technology Data Exchange (ETDEWEB)

    Tice, R R


    A review of the literature on DNA repair processes in relation to aging is presented under the following headings: DNA repair processes; age-related occurrence of unrepaired DNA lesions; DNA repair capability as a function of age; tissue-specific DNA repair capability; acceleration of the aging process by exposure to DNA damaging agents; human genetic syndromes; and longevity and DNA repair processes. (HLW)

  15. A new DNA band display technology of microsatellite DNA ...

    African Journals Online (AJOL)

    This study proposes a new DNA band display technology of microsatellite DNA called Fluorescent Imaging Technology. In comparison with Silver Stain Technology, this technology is worth popularizing in the laboratory because of its high resolution, efficiency, simplicity and clear background. Key words: Microsatellite DNA ...

  16. Pea amyloplast DNA is qualitatively similar to pea chloroplast DNA (United States)

    Gaynor, J. J.


    Amyloplast DNA (apDNA), when subjected to digestion with restriction endonucleases, yields patterns nearly identical to that of DNA from mature pea chloroplasts (ctDNA). Southern transfers of apDNA and ctDNA, probed with the large subunit (LS) gene of ribulose-1,5-bisphosphate carboxylase (Rubisco), shows hybridization to the expected restriction fragments for both apDNA and ctDNA. However, Northern transfers of total RNA from chloroplasts and amyloplasts, probed again with the LS gene of Rubisco, shows that no detectable LS meggage is found in amyloplasts although LS expression in mature chloroplasts is high. Likewise, two dimensional polyacrylamide gel electrophoresis of etiolated gravisensitive pea tissue shows that both large and small subunits of Rubisco are conspicuously absent; however, in greening tissue these two constitute the major soluble proteins. These findings suggest that although the informational content of these two organelle types is equivalent, gene expression is quite different and is presumably under nuclear control.

  17. Dancing on DNA : Kinetic Aspects of Search Processes on DNA

    NARCIS (Netherlands)

    Tafvizi, Anahita; Mirny, Leonid A.; van Oijen, Antoine M.


    Recognition and binding of specific sites on DNA by proteins is central for many cellular functions such as transcription, replication, and recombination. In the search for its target site, the DNA-associated protein is facing both thermodynamic and kinetic difficulties. The thermodynamic challenge

  18. Clinical strains of acinetobacter classified by DNA-DNA hybridization

    International Nuclear Information System (INIS)

    Tjernberg, I.; Ursing, J.


    A collection of Acinetobacter strains consisting of 168 consecutive clinical strains and 30 type and reference strains was studied by DNA-DNA hybridization and a few phenotypic tests. The field strains could be allotted to 13 DNA groups. By means of reference strains ten of these could be identified with groups described by Bouvet and Grimont (1986), while three groups were new; they were given the numbers 13-15. The type strain of A. radioresistens- recently described by Nishimura et al. (1988) - was shown to be a member of DNA group 12, which comprised 31 clinical isolates. Of the 19 strains of A. junii, eight showed hemolytic acitivity on sheep and human blood agar and an additional four strains on human blood agar only. Strains of this species have previously been regarded as non-hemolytic. Reciprocal DNA pairing data for the reference strains of the DNA gropus were treated by UPGMA clustering. The reference strains for A. calcoaceticus, A. baumannii and DNA groups 3 and 13 formed a cluster with about 70% relatedness within the cluster. Other DNA groups joined at levels below 60%. (author)

  19. Clinical strains of acinetobacter classified by DNA-DNA hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Tjernberg, I.; Ursing, J. (Department of Medical Microbiology, University of Lund, Malmoe General Hospital, Malmoe (Sweden))


    A collection of Acinetobacter strains consisting of 168 consecutive clinical strains and 30 type and reference strains was studied by DNA-DNA hybridization and a few phenotypic tests. The field strains could be allotted to 13 DNA groups. By means of reference strains ten of these could be identified with groups described by Bouvet and Grimont (1986), while three groups were new; they were given the numbers 13-15. The type strain of A. radioresistens- recently described by Nishimura et al. (1988) - was shown to be a member of DNA group 12, which comprised 31 clinical isolates. Of the 19 strains of A. junii, eight showed hemolytic acitivity on sheep and human blood agar and an additional four strains on human blood agar only. Strains of this species have previously been regarded as non-hemolytic. Reciprocal DNA pairing data for the reference strains of the DNA gropus were treated by UPGMA clustering. The reference strains for A. calcoaceticus, A. baumannii and DNA groups 3 and 13 formed a cluster with about 70% relatedness within the cluster. Other DNA groups joined at levels below 60%. (author).

  20. DNA-Conjugated Organic Chromophores in DNA Stacking Interactions

    DEFF Research Database (Denmark)

    Filichev, Vyacheslav V.; Pedersen, Erik Bjerregaard


    Since the discovery of the intercalation of acridine derivatives into DNA (1961), chemists have synthesized many intercalators tethered to DNA. Advances in the chemical synthesis of modified nucleosides along with progress in oligonucleotide synthesis have made it possible to introduce organic ch...

  1. DNA-DNA hybridization determined in micro-wells using covalent attachment of DNA

    DEFF Research Database (Denmark)

    Christensen, H.; Angen, Øystein; Mutters, R.


    of results were a maximum of 15% standard deviation, calculated as a percentage of the mean for four replicate micro-wells, and that DNA similarities were not significantly different in at least two independent experiments. The relationship between DNA similarities obtained by the microwell method Cy......-DNA hybridizations to be carried out between all strains selected for a particular taxonomic study, in order to construct complete data matrices and improve species definition....

  2. Master equation approach to DNA breathing in heteropolymer DNA

    DEFF Research Database (Denmark)

    Ambjörnsson, Tobias; Banik, Suman K; Lomholt, Michael A


    After crossing an initial barrier to break the first base-pair (bp) in double-stranded DNA, the disruption of further bps is characterized by free energies up to a few k(B)T. Thermal motion within the DNA double strand therefore causes the opening of intermittent single-stranded denaturation zones......, the DNA bubbles. The unzipping and zipping dynamics of bps at the two zipper forks of a bubble, where the single strand of the denatured zone joins the still intact double strand, can be monitored by single molecule fluorescence or NMR methods. We here establish a dynamic description of this DNA breathing...... in a heteropolymer DNA with given sequence in terms of a master equation that governs the time evolution of the joint probability distribution for the bubble size and position along the sequence. The transfer coefficients are based on the Poland-Scheraga free energy model. We derive the autocorrelation function...

  3. DNA damage in neurodegenerative diseases

    International Nuclear Information System (INIS)

    Coppedè, Fabio; Migliore, Lucia


    Highlights: • Oxidative DNA damage is one of the earliest detectable events in the neurodegenerative process. • The mitochondrial DNA is more vulnerable to oxidative attack than the nuclear DNA. • Cytogenetic damage has been largely documented in Alzheimer's disease patients. • The question of whether DNA damage is cause or consequence of neurodegeneration is still open. • Increasing evidence links DNA damage and repair with epigenetic phenomena. - Abstract: Following the observation of increased oxidative DNA damage in nuclear and mitochondrial DNA extracted from post-mortem brain regions of patients affected by neurodegenerative diseases, the last years of the previous century and the first decade of the present one have been largely dedicated to the search of markers of DNA damage in neuronal samples and peripheral tissues of patients in early, intermediate or late stages of neurodegeneration. Those studies allowed to demonstrate that oxidative DNA damage is one of the earliest detectable events in neurodegeneration, but also revealed cytogenetic damage in neurodegenerative conditions, such as for example a tendency towards chromosome 21 malsegregation in Alzheimer's disease. As it happens for many neurodegenerative risk factors the question of whether DNA damage is cause or consequence of the neurodegenerative process is still open, and probably both is true. The research interest in markers of oxidative stress was shifted, in recent years, towards the search of epigenetic biomarkers of neurodegenerative disorders, following the accumulating evidence of a substantial contribution of epigenetic mechanisms to learning, memory processes, behavioural disorders and neurodegeneration. Increasing evidence is however linking DNA damage and repair with epigenetic phenomena, thereby opening the way to a very attractive and timely research topic in neurodegenerative diseases. We will address those issues in the context of Alzheimer's disease

  4. Effects of radiation on DNA

    International Nuclear Information System (INIS)

    Medina, V.F.O.


    Irradiation has been shown to depress DNA (deoxyribonucleic acid) synthesis resulting in deficient DNA synthesis. In one experiment, Hela S 3 cells completed the next division after a dose of 500 rads to 200 kw X-rays. Another experiment showed that the amount of DNA synthesized was dependent on the stage in the generation cycle at which the cells are irradiated (Giffites and Tolmach, 1975). DNA synthesis was measured by radioactive thymidine incorporation. The smallest deficiency (20-35%) after a dose of 500 rad X-ray was observed in Hela S 3 cells irradiated in early G 1 or early G 2 , while the greatest deficiency (55-70*) after 500 rad X-ray was found in cells irradiated at mitosis or at the Gsub(1)/S transition. Using velocity sedimentation in alkaline gradients of the DNA from hamster, Elkind, et al 1972, studied repair processes as a function of X-ray dose. DNA containing material released by alkaline lysis was found initially contained in a complex-containing lipid, the sedimentation of which was anomalous relative to denatured RNA from unirradated cells. Doses of X-rays small enough to be in the range that permits high survival (100-800 rads) speed the resolution of single-stranded DNA from this DNA complex, giving rise to a species having a number average molecular weight of 2 x 10 8 daltons. Larger doses greater than 1000 to 2000 rads resulted in a degradation of these DNA strands. Incubation after irradiation resulted in the rapid repair of damage, although the rate of repair of damage to the complex resulted in a reassociation of lipid and DNA. This evidence supports the possibility that a large DNA-membrane structure is a principal target of radiation

  5. DNA damage in neurodegenerative diseases

    Energy Technology Data Exchange (ETDEWEB)

    Coppedè, Fabio, E-mail:; Migliore, Lucia, E-mail:


    Highlights: • Oxidative DNA damage is one of the earliest detectable events in the neurodegenerative process. • The mitochondrial DNA is more vulnerable to oxidative attack than the nuclear DNA. • Cytogenetic damage has been largely documented in Alzheimer's disease patients. • The question of whether DNA damage is cause or consequence of neurodegeneration is still open. • Increasing evidence links DNA damage and repair with epigenetic phenomena. - Abstract: Following the observation of increased oxidative DNA damage in nuclear and mitochondrial DNA extracted from post-mortem brain regions of patients affected by neurodegenerative diseases, the last years of the previous century and the first decade of the present one have been largely dedicated to the search of markers of DNA damage in neuronal samples and peripheral tissues of patients in early, intermediate or late stages of neurodegeneration. Those studies allowed to demonstrate that oxidative DNA damage is one of the earliest detectable events in neurodegeneration, but also revealed cytogenetic damage in neurodegenerative conditions, such as for example a tendency towards chromosome 21 malsegregation in Alzheimer's disease. As it happens for many neurodegenerative risk factors the question of whether DNA damage is cause or consequence of the neurodegenerative process is still open, and probably both is true. The research interest in markers of oxidative stress was shifted, in recent years, towards the search of epigenetic biomarkers of neurodegenerative disorders, following the accumulating evidence of a substantial contribution of epigenetic mechanisms to learning, memory processes, behavioural disorders and neurodegeneration. Increasing evidence is however linking DNA damage and repair with epigenetic phenomena, thereby opening the way to a very attractive and timely research topic in neurodegenerative diseases. We will address those issues in the context of Alzheimer's disease

  6. Authentication of forensic DNA samples. (United States)

    Frumkin, Dan; Wasserstrom, Adam; Davidson, Ariane; Grafit, Arnon


    Over the past twenty years, DNA analysis has revolutionized forensic science, and has become a dominant tool in law enforcement. Today, DNA evidence is key to the conviction or exoneration of suspects of various types of crime, from theft to rape and murder. However, the disturbing possibility that DNA evidence can be faked has been overlooked. It turns out that standard molecular biology techniques such as PCR, molecular cloning, and recently developed whole genome amplification (WGA), enable anyone with basic equipment and know-how to produce practically unlimited amounts of in vitro synthesized (artificial) DNA with any desired genetic profile. This artificial DNA can then be applied to surfaces of objects or incorporated into genuine human tissues and planted in crime scenes. Here we show that the current forensic procedure fails to distinguish between such samples of blood, saliva, and touched surfaces with artificial DNA, and corresponding samples with in vivo generated (natural) DNA. Furthermore, genotyping of both artificial and natural samples with Profiler Plus((R)) yielded full profiles with no anomalies. In order to effectively deal with this problem, we developed an authentication assay, which distinguishes between natural and artificial DNA based on methylation analysis of a set of genomic loci: in natural DNA, some loci are methylated and others are unmethylated, while in artificial DNA all loci are unmethylated. The assay was tested on natural and artificial samples of blood, saliva, and touched surfaces, with complete success. Adopting an authentication assay for casework samples as part of the forensic procedure is necessary for maintaining the high credibility of DNA evidence in the judiciary system.

  7. DNA Movies and Panspermia

    Directory of Open Access Journals (Sweden)

    Victor Norris


    Full Text Available There are several ways that our species might try to send a message to another species separated from us by space and/or time. Synthetic biology might be used to write an epitaph to our species, or simply “Kilroy was here”, in the genome of a bacterium via the patterns of either (1 the codons to exploit Life's non-equilibrium character or (2 the bases themselves to exploit Life's quasi-equilibrium character. We suggest here how DNA movies might be designed using such patterns. We also suggest that a search for mechanisms to create and preserve such patterns might lead to a better understanding of modern cells. Finally, we argue that the cutting-edge microbiology and synthetic biology needed for the Kilroy project would put origin-of-life studies in the vanguard of research.

  8. DNA Extraction Techniques for Use in Education (United States)

    Hearn, R. P.; Arblaster, K. E.


    DNA extraction provides a hands-on introduction to DNA and enables students to gain real life experience and practical knowledge of DNA. Students gain a sense of ownership and are more enthusiastic when they use their own DNA. A cost effective, simple protocol for DNA extraction and visualization was devised. Buccal mucosal epithelia provide a…

  9. Disposable electrochemical DNA biosensor for environmental ...

    Indian Academy of Sciences (India)

    A simple procedure for the voltammetric detection of the DNA damage using a disposable electrochemical DNA biosensor is reported. The DNA biosensor is assembled by immobilizing the double stranded calf thymus DNA (dsDNA) on the surface of a disposable carbon screen-printed electrode. The interaction of ...

  10. Automated extraction of DNA from clothing

    DEFF Research Database (Denmark)

    Stangegaard, Michael; Hjort, Benjamin Benn; Nøhr Hansen, Thomas


    Presence of PCR inhibitors in extracted DNA may interfere with the subsequent quantification and short tandem repeat (STR) reactions used in forensic genetic DNA typing. We have compared three automated DNA extraction methods based on magnetic beads with a manual method with the aim of reducing...... the amount of PCR inhibitors in the DNA extracts and increasing the proportion of reportable DNA profiles....

  11. Replication of bacteriophage lambda DNA

    International Nuclear Information System (INIS)

    Tsurimoto, T.; Matsubara, K.


    In this paper results of studies on the mechanism of bacteriophage lambda replication using molecular biological and biochemical approaches are reported. The purification of the initiator proteins, O and P, and the role of the O and P proteins in the initiation of lambda DNA replication through interactions with specific DNA sequences are described. 47 references, 15 figures

  12. The journey of DNA repair. (United States)

    Saini, Natalie


    21 years ago, the DNA Repair Enzyme was declared "Molecule of the Year". Today, we are celebrating another "year of repair", with the 2015 Nobel Prize in Chemistry being awarded to Aziz Sancar, Tomas Lindahl and Paul Modrich for their collective work on the different DNA repair pathways.

  13. DNA damage by Auger emitters

    International Nuclear Information System (INIS)

    Martin, R.F.; d'Cunha, Glenn; Gibbs, Richard; Murray, Vincent; Pardee, Marshall; Allen, B.J.


    125 I atoms can be introduced at specific locations along a defined DNA target molecule, either by site-directed incorporation of an 125 I-labelled deoxynucleotide or by binding of an 125 I-labelled sequence-selective DNA ligand. After allowing accumulation of 125 I decay-induced damage to the DNA, application of DNA sequencing techniques enables positions of strand breaks to be located relative to the site of decay, at a resolution corresponding to the distance between adjacent nucleotides [0.34 nm]. Thus, DNA provides a molecular framework to analyse the extent of damage following [averaged] individual decay events. Results can be compared with energy deposition data generated by computer-simulation methods developed by Charlton et al. The DNA sequencing technique also provides information about the chemical nature of the termini of the DNA chains produced following Auger decay-induced damage. In addition to reviewing the application of this approach to the analysis of 125 I decay induced DNA damage, some more recent results obtained by using 67 Ga are also presented. (author)

  14. Aktionslæringens DNA

    DEFF Research Database (Denmark)

    Madsen, Benedicte

    Aktionslæringen DNA giver en række redskaber til læring i fællesskaber, uanset om der arbejdes med individuelle eller kollektive projekter i offentlig eller privat regi. Metoden danner modvægt til de mere individuelistiske traditioner inden for voksenpædagogikken. DNA-metaforen bruges bogen igennem...

  15. LEGO-like DNA Structures

    DEFF Research Database (Denmark)

    Gothelf, Kurt Vesterager


    -dimensional (3D) DNA structures by self-assembly of single-stranded DNA “bricks.” The method opens a new route to complex self-assembled (3D) nanostructures that may serve as addressable templates for placing guest molecules with high precision, with possible applications in biophysics, medicine...

  16. Bubble coalescence in breathing DNA

    DEFF Research Database (Denmark)

    Novotný, Tomas; Pedersen, Jonas Nyvold; Ambjörnsson, Tobias


    We investigate the coalescence of two DNA bubbles initially located at weak segments and separated by a more stable barrier region in a designed construct of double-stranded DNA. The characteristic time for bubble coalescence and the corresponding distribution are derived, as well as the distribu...

  17. DNA-based asymmetric catalysis

    NARCIS (Netherlands)

    Boersma, Arnold J.; Megens, Rik P.; Feringa, Ben L.; Roelfes, Gerard


    The unique chiral structure of DNA has been a source of inspiration for the development of a new class of bio-inspired catalysts. The novel concept of DNA-based asymmetric catalysis, which was introduced only five years ago, has been applied successfully in a variety of catalytic enantioselective

  18. Multiscale modelling of DNA mechanics

    Czech Academy of Sciences Publication Activity Database

    Dršata, Tomáš; Lankaš, Filip


    Roč. 27, č. 32 (2015), 323102/1-323102/12 ISSN 0953-8984 R&D Projects: GA ČR(CZ) GA14-21893S Institutional support: RVO:61388963 Keywords : DNA elasticity * DNA coarse-grained models * molecular dynamics simulations Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 2.209, year: 2015

  19. How adsorption influences DNA denaturation

    NARCIS (Netherlands)

    Allahverdyan, A.E.; Gevorkian, Z.S.; Hu, C.-K.; Nieuwenhuizen, T.M.


    The thermally induced denaturation of DNA in the presence of an attractive solid surface is studied. The two strands of DNA are modeled via two coupled flexible chains without volume interactions. If the two strands are adsorbed on the surface, the denaturation phase transition disappears. Instead,

  20. Effects of radiation on DNA

    International Nuclear Information System (INIS)

    Braddock, M.


    The hydroxyl radical (OH radical) is the most damaging radical produced by the effect of ionizing radiation in water. The rate of reaction of the OH radical with purified, native and isodisperse DNA has been determined as compared with calf thymus DNA. This has been achieved by direct observation of the rate of formation of the DNA-OH radical adduct, and by competition with SCN - . Results obtained from direct observation are consistent with calculations which have been performed using the encounter frequency model of Braams and Ebert. However, results obtained for OH radical with DNA derived from competition plots suggest a rate constant somewhat lower than that obtained from direct observation. The relative merits of both techniques are discussed. In order to study the effect of energy deposited directly in the DNA, dry films of purified plasmid DNA have been irradiated in a system where the indirect effects of radical interaction have been minimized. The present results indicate that with different molecular lengths of plasmid DNA, non-random breakage may occur, and that additional damage may be brought about at sites of previously existing damage. Differences in the sensitivity of plasmid DNA molecules of varying lengths to radiation induced double strand breaks have been demonstrated. (author)