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Sample records for putative dna g-quadruplex

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

    Directory of Open Access Journals (Sweden)

    Rowe J

    2009-08-01

    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.

  2. Single-Molecule FRET Study of DNA G-Quadruplex

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    The DNA G-quadruplex formed by the human telomeric sequence is a potential target for novel anticancer drugs. We have investigated an intramolecular DNA G-quadruplex using single-molecule fluorescence resonance energy transfer and shown that individual folded quadruplexes can be identified. The mean proximity ratio measured at the single-molecule level was consistent with ensemble measurement.

  3. [Conformational polymorphysm of G-rich fragments of DNA Alu-repeats. II. the putative role of G-quadruplex structures in genomic rearrangements].

    Science.gov (United States)

    Varizhuk, A M; Sekridova, A V; Tankevich, M V; Podgorsky, V S; Smirnov, I P; Pozmogova, G E

    2016-11-01

    Three evolutionary conserved sites of Alu repeats (PQS2, PQS3 and PQS4) were shown to form stable inter- and intramolecular G-quadruplexes (GQs) in vitro. Structures and topologies of these GQs were elucidated using spectral methods. Self-association of G-rich Alu fragments was studied. Dimeric GQ formation from two distal identical or different putative quadruplex sites - (PQS2)2, (PQS3)2 or PQS2-PQS3 - within one lengthy DNA strand was demonstrated by a FRET-based method. Oligomer PQS4 (folded into a parallel intramolecular GQ) was shown to form stacks of quadruplexes that are stabilized by stacking interactions of external G-tetrads (this was confirmed by DOSY NMR, AFM microscopy and differential CD spectroscopy). Comparative analysis of the properties of various GQs allowed us to put forward a hypothesis of two general mechanisms of intermolecular GQ-dependant genomic rearrangements: 1) formation of a dimeric GQs; 2) association of pre-folded intramolecular parallel GQs from different strands into GQ-stacks. Thus, the observed co-localization of G-rich motifs of Alu elements with double-strand break hotspots and rearrangement hotspots may be accounted for by the specific secondary structure of these motifs. At the same time, this is likely primarily due to high abundance of such G-rich Alu fragments in the genome.

  4. The G-quadruplex DNA stabilizing drug pyridostatin promotes DNA damage and downregulates transcription of Brca1 in neurons.

    Science.gov (United States)

    Moruno-Manchon, Jose F; Koellhoffer, Edward C; Gopakumar, Jayakrishnan; Hambarde, Shashank; Kim, Nayun; McCullough, Louise D; Tsvetkov, Andrey S

    2017-09-12

    The G-quadruplex is a non-canonical DNA secondary structure formed by four DNA strands containing multiple runs of guanines. G-quadruplexes play important roles in DNA recombination, replication, telomere maintenance, and regulation of transcription. Small molecules that stabilize the G-quadruplexes alter gene expression in cancer cells. Here, we hypothesized that the G-quadruplexes regulate transcription in neurons. We discovered that pyridostatin, a small molecule that specifically stabilizes G-quadruplex DNA complexes, induced neurotoxicity and promoted the formation of DNA double-strand breaks (DSBs) in cultured neurons. We also found that pyridostatin downregulated transcription of the Brca1 gene, a gene that is critical for DSB repair. Importantly, in an in vitro gel shift assay, we discovered that an antibody specific to the G-quadruplex structure binds to a synthetic oligonucleotide, which corresponds to the first putative G-quadruplex in the Brca1 gene promoter. Our results suggest that the G-quadruplex complexes regulate transcription in neurons. Studying the G-quadruplexes could represent a new avenue for neurodegeneration and brain aging research.

  5. Structure of a left-handed DNA G-quadruplex.

    Science.gov (United States)

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

    2015-03-03

    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.

  6. Computational and Experimental Characterization of Ribosomal DNA and RNA G-Quadruplexes

    Science.gov (United States)

    Cho, Samuel

    DNA G-quadruplexes in human telomeres and gene promoters are being extensively studied for their role in controlling the growth of cancer cells. Recent studies strongly suggest that guanine (G)-rich genes encoding pre-ribosomal RNA (pre-rRNA) are a potential anticancer target through the inhibition of RNA polymerase I (Pol I) in ribosome biogenesis. However, the structures of ribosomal G-quadruplexes at atomic resolution are unknown, and very little biophysical characterization has been performed on them to date. Here, we have modeled two putative rDNA G-quadruplex structures, NUC 19P and NUC 23P, which we observe via circular dichroism (CD) spectroscopy to adopt a predominantly parallel topology, and their counterpart rRNA. To validate and refine the putative ribosomal G-quadruplex structures, we performed all-atom molecular dynamics (MD) simulations using the CHARMM36 force field in the presence and absence of stabilizing K + or Na + ions. We optimized the CHARMM36 force field K + parameters to be more consistent with quantum mechanical calculations (and the polarizable Drude model force field) so that the K + ion is predominantly in the G-quadruplex channel. Our MD simulations show that the rDNA G-quadruplex have more well-defined, predominantly parallel-topology structures than rRNA and NUC 19P is more structured than NUC 23P, which features extended loops. Our study demonstrates that they are both potential targets for the design of novel chemotherapeutics.

  7. FANCJ promotes DNA synthesis through G-quadruplex structures

    NARCIS (Netherlands)

    Castillo Bosch, Pau; Segura-Bayona, Sandra; Koole, Wouter; van Heteren, Jane T; Dewar, James M; Tijsterman, Marcel; Knipscheer, Puck

    2014-01-01

    Our genome contains many G-rich sequences, which have the propensity to fold into stable secondary DNA structures called G4 or G-quadruplex structures. These structures have been implicated in cellular processes such as gene regulation and telomere maintenance. However, G4 sequences are prone to mut

  8. Homologous PNA Hybridization to Noncanonical DNA G-Quadruplexes.

    Science.gov (United States)

    Kormuth, Karen A; Woolford, John L; Armitage, Bruce A

    2016-03-29

    Potential guanine (G) quadruplex-forming sequences (QFSs) found throughout the genomes and transcriptomes of organisms have emerged as biologically relevant structures. These G-quadruplexes represent novel opportunities for gene regulation at the DNA and RNA levels. Recently, the definition of functional QFSs has been expanding to include a variety of unconventional motifs, including relatively long loop sequences (i.e., >7 nucleotides) separating adjacent G-tracts. We have identified a QFS within the 25S rDNA gene from Saccharomyces cerevisae that features a long loop separating the two 3'-most G-tracts. An oligonucleotide based on this sequence, QFS3, folds into a stable G-quadruplex in vitro. We have studied the interaction between QFS3 and several loop mutants with a small, homologous (G-rich) peptide nucleic acid (PNA) oligomer that is designed to form a DNA/PNA heteroquadruplex. The PNA successfully invades the DNA quadruplex target to form a stable heteroquadruplex, but with surprisingly high PNA:DNA ratios based on surface plasmon resonance and mass spectrometric results. A model for high stoichiometry PNA-DNA heteroquadruplexes is proposed, and the implications for quadruplex targeting by G-rich PNA are discussed.

  9. G-Quadruplexes Light up Localized DNA Circuits.

    Science.gov (United States)

    Mendoza, Oscar; Mergny, Jean-Louis; Aimé, Jean-Pierre; Elezgaray, Juan

    2016-01-13

    DNA circuits tethered to nanoplatforms can perform cascade reactions for signal amplification. One DNA single strand activates a strand-displacement cascade generating numerous outputs, and therefore amplifying the signal. These localized circuits present, however, an important limitation: the spontaneous activation of the cascade reaction. Current methods to stabilize these circuits employ combination of protective DNA strands, which need to be removed to activate the device. This protection-deprotection process generates an important amount of unwanted side reactions. This is indeed an important limitation for the large potential application of these amplification circuits. In the present work, G-quadruplex DNA structures were used to stabilize localized DNA circuits. This new protocol generates nanoplatforms that no longer requires protective-deprotective systems and is therefore completely neutral to the sample. In addition, cations such as Pb(2+) or Ca(2+) can be also employed to activate the device enlarging the potential applications from biosensors devices to metal detector sensors.

  10. Cyclic ferrocenylnaphthalene diimide derivative as a new class of G-quadruplex DNA binding ligand.

    Science.gov (United States)

    Islam, Md Monirul; Sato, Shinobu; Shinozaki, Shingo; Takenaka, Shigeori

    2017-01-15

    To identify an effective ligand that binds to a G-quadruplex structure but not a double-stranded DNA (dsDNA), a set of biophysical and biochemical experiments were carried out using newly synthesized cyclic ferrocenylnaphthalene diimide (cFNDI, 1) or the non-cyclic derivative (2) with various structures of G-quadruplex DNAs and dsDNA. Compound 1 bound strongly to G-quadruplexes DNAs (10(6)M(-1) order) with diminished binding to dsDNA (10(4)M(-1) order) in 100mM AcOH-AcOK buffer (pH 5.5) containing 100mM KCl. Interestingly, 1 showed an approximately 50-fold higher selectivity to mixed hybrid-type telomeric G-quadruplex DNA (K=3.4×10(6)M(-1) and a 2:1 stoichiometry) than dsDNA (K=7.5×10(4)M(-1)) did. Furthermore, 1 showed higher thermal stability to G-quadruplex DNAs than it did to dsDNA with a preference for c-kit and c-myc G-quadruplex DNAs over telomeric and thrombin binding aptamers. Additionally, 1 exhibited telomerase inhibitory activity with a half-maximal inhibitory concentration (IC50) of 0.4μM. Compound 2 showed a preference for G-quadruplex; however, the binding affinity magnitude and preference were improved in 1 because the former had a cyclic structure.

  11. Relations between the loop transposition of DNA G-quadruplex and the catalytic function of DNAzyme.

    Science.gov (United States)

    Cheng, Mingpan; Zhou, Jun; Jia, Guoqing; Ai, Xuanjun; Mergny, Jean-Louis; Li, Can

    2017-08-01

    The structures of DNA G-quadruplexes are essential for their functions in vivo and in vitro. Our present study revealed that sequential order of the three G-quadruplex loops, that is, loop transposition, could be a critical factor to determinate the G-quadruplex conformation and consequently improved the catalytic function of G-quadruplex based DNAzyme. In the presence of 100mM K(+), loop transposition induced one of the G-quadruplex isomers which shared identical loops but differed in the sequential order of loops into a hybrid topology while the others into predominately parallel topologies. (1)D NMR spectroscopy and mutation analysis suggested that the hydrogen bonding from loops residues with nucleotides in flanking sequences may be responsible for the stabilization of the different conformations. A well-known DNAzyme consisting of G-quadruplex and hemin (Ferriprotoporphyrin IX chloride) was chosen to test the catalytic function. We found that the loop transposition could enhance the reaction rate obviously by increasing the hemin binding affinity to G-quadruplex. These findings disclose the relations between the loop transposition, G-quadruplex conformation and catalytic function of DNAzyme. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. A G-Quadruplex/Hemin Complex with Switchable Peroxidase Activity by DNA Hybridization

    Institute of Scientific and Technical Information of China (English)

    邵从英; 鲁娜; 孙登明

    2012-01-01

    A heroin-binding DNA G-quadruplex (also known as a heroin aptamer or DNAzyme) has been previously re- ported to be able to enhance the peroxidase activity of heroin. In this work, we described a DNAzyme structure that had an effector-recognizing part appearing as a single stranded DNA linkage flanked by two split G-quadruplex halves. Hybridization of the single stranded part in the enzyme with a perfectly matched DNA strand (effector) formed a rigid DNA duplex between the two G-quadruplex halves and thus efficiently suppressed the enzymatic activity of the G-quadruplex/hemin complex, while the mismatched effector strand was not able to regulate the peroxidase activity effectively. With 2,2'-azinobis(3-ethylbenzthiazoline)-6-sulfonic acid (ABTS) as an oxidizable substrate, we were able to characterize the formation of the re-engineered G-quadruplex/hemin complex and verify its switchable peroxidase activity. Our results show that the split G-quadruplex is an especially useful module to design low-cost and label-free sensors toward various biologically or environmentally interesting targets.

  13. X-ray characterization of mesophases of human telomeric G-quadruplexes and other DNA analogues.

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    Yasar, Selcuk; Schimelman, Jacob B; Aksoyoglu, M Alphan; Steinmetz, Nicole F; French, Roger H; Parsegian, V Adrian; Podgornik, Rudolf

    2016-06-02

    Observed in the folds of guanine-rich oligonucleotides, non-canonical G-quadruplex structures are based on G-quartets formed by hydrogen bonding and cation-coordination of guanosines. In dilute 5'-guanosine monophosphate (GMP) solutions, G-quartets form by the self-assembly of four GMP nucleotides. We use x-ray diffraction to characterize the columnar liquid-crystalline mesophases in concentrated solutions of various model G-quadruplexes. We then probe the transitions between mesophases by varying the PEG solution osmotic pressure, thus mimicking in vivo molecular crowding conditions. Using the GMP-quadruplex, built by the stacking of G-quartets with no covalent linking between them, as the baseline, we report the liquid-crystalline phase behaviors of two other related G-quadruplexes: (i) the intramolecular parallel-stranded G-quadruplex formed by the 22-mer four-repeat human telomeric sequence AG3(TTAG3)3 and (ii) the intermolecular parallel-stranded G-quadruplex formed by the TG4T oligonucleotides. Finally, we compare the mesophases of the G-quadruplexes, under PEG-induced crowding conditions, with the corresponding mesophases of the canonical duplex and triplex DNA analogues.

  14. X-ray characterization of mesophases of human telomeric G-quadruplexes and other DNA analogues

    Science.gov (United States)

    Yasar, Selcuk; Schimelman, Jacob B.; Aksoyoglu, M. Alphan; Steinmetz, Nicole F.; French, Roger H.; Parsegian, V. Adrian; Podgornik, Rudolf

    2016-06-01

    Observed in the folds of guanine-rich oligonucleotides, non-canonical G-quadruplex structures are based on G-quartets formed by hydrogen bonding and cation-coordination of guanosines. In dilute 5‧-guanosine monophosphate (GMP) solutions, G-quartets form by the self-assembly of four GMP nucleotides. We use x-ray diffraction to characterize the columnar liquid-crystalline mesophases in concentrated solutions of various model G-quadruplexes. We then probe the transitions between mesophases by varying the PEG solution osmotic pressure, thus mimicking in vivo molecular crowding conditions. Using the GMP-quadruplex, built by the stacking of G-quartets with no covalent linking between them, as the baseline, we report the liquid-crystalline phase behaviors of two other related G-quadruplexes: (i) the intramolecular parallel-stranded G-quadruplex formed by the 22-mer four-repeat human telomeric sequence AG3(TTAG3)3 and (ii) the intermolecular parallel-stranded G-quadruplex formed by the TG4T oligonucleotides. Finally, we compare the mesophases of the G-quadruplexes, under PEG-induced crowding conditions, with the corresponding mesophases of the canonical duplex and triplex DNA analogues.

  15. G-quadruplex and i-motif are mutually exclusive in ILPR double-stranded DNA.

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    Dhakal, Soma; Yu, Zhongbo; Konik, Ryan; Cui, Yunxi; Koirala, Deepak; Mao, Hanbin

    2012-06-06

    G-quadruplex has demonstrated its biological functions in vivo. Although G-quadruplex in single-stranded DNA (ssDNA) has been well characterized, investigation of this species in double-stranded DNA (dsDNA) lags behind. Here we use chemical footprinting and laser-tweezers-based single-molecule approaches to demonstrate that a dsDNA fragment found in the insulin-linked polymorphic region (ILPR), 5'-(ACA GGGG TGT GGGG)2 TGT, can fold into a G-quadruplex at pH 7.4 with 100 mM K+, and an i-motif at pH 5.5 with 100 mM Li+. Surprisingly, under a condition that favors the formation of both G-quadruplex and i-motif (pH 5.5, 100 mM K+), a unique determination of change in the free energy of unfolding (ΔGunfold) by laser-tweezers experiments provides compelling evidence that only one species is present in each dsDNA. Under this condition, molecules containing G-quadruplex are more stable than those with i-motif. These two species have mechanical stabilities (rupture force≥17 pN) comparable to the stall force of RNA polymerases, which, from a mechanical perspective alone, could justify a regulatory mechanism for tetraplex structures in the expression of human insulin. Copyright © 2012 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  16. Selective recognition and stabilization of new ligands targeting the potassium form of the human telomeric G-quadruplex DNA

    Science.gov (United States)

    Lin, Yi-Hwa; Chuang, Show-Mei; Wu, Pei-Ching; Chen, Chun-Liang; Jeyachandran, Sivakamavalli; Lo, Shou-Chen; Huang, Hsu-Shan; Hou, Ming-Hon

    2016-08-01

    The development of a ligand that is capable of distinguishing among the wide variety of G-quadruplex structures and targeting telomeres to treat cancer is particularly challenging. In this study, the ability of two anthraquinone telomerase inhibitors (NSC749235 and NSC764638) to target telomeric G-quadruplex DNA was probed. We found that these ligands specifically target the potassium form of telomeric G-quadruplex DNA over the DNA counterpart. The characteristic interaction with the telomeric G-quadruplex DNA and the anticancer activities of these ligands were also explored. The results of this present work emphasize our understanding of the binding selectivity of anthraquinone derivatives to G-quadruplex DNA and assists in future drug development for G-quadruplex-specific ligands.

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

    Directory of Open Access Journals (Sweden)

    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.

  18. G-quadruplex enhanced fluorescence of DNA-silver nanoclusters and their application in bioimaging

    Science.gov (United States)

    Zhu, Jinbo; Zhang, Libing; Teng, Ye; Lou, Baohua; Jia, Xiaofang; Gu, Xiaoxiao; Wang, Erkang

    2015-07-01

    Guanine proximity based fluorescence enhanced DNA-templated silver nanoclusters (AgNCs) have been reported and applied for bioanalysis. Herein, we studied the G-quadruplex enhanced fluorescence of DNA-AgNCs and gained several significant conclusions, which will be helpful for the design of future probes. Our results demonstrate that a G-quadruplex can also effectively stimulate the fluorescence potential of AgNCs. The major contribution of the G-quadruplex is to provide guanine bases, and its special structure has no measurable impact. The DNA-templated AgNCs were further analysed by native polyacrylamide gel electrophoresis and the guanine proximity enhancement mechanism could be visually verified by this method. Moreover, the fluorescence emission of C3A (CCCA)4 stabilized AgNCs was found to be easily and effectively enhanced by G-quadruplexes, such as T30695, AS1411 and TBA, especially AS1411. Benefiting from the high brightness of AS1411 enhanced DNA-AgNCs and the specific binding affinity of AS1411 for nucleolin, the AS1411 enhanced AgNCs can stain cancer cells for bioimaging.Guanine proximity based fluorescence enhanced DNA-templated silver nanoclusters (AgNCs) have been reported and applied for bioanalysis. Herein, we studied the G-quadruplex enhanced fluorescence of DNA-AgNCs and gained several significant conclusions, which will be helpful for the design of future probes. Our results demonstrate that a G-quadruplex can also effectively stimulate the fluorescence potential of AgNCs. The major contribution of the G-quadruplex is to provide guanine bases, and its special structure has no measurable impact. The DNA-templated AgNCs were further analysed by native polyacrylamide gel electrophoresis and the guanine proximity enhancement mechanism could be visually verified by this method. Moreover, the fluorescence emission of C3A (CCCA)4 stabilized AgNCs was found to be easily and effectively enhanced by G-quadruplexes, such as T30695, AS1411 and TBA, especially

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

    OpenAIRE

    Luciana Souto Mofatto

    2013-01-01

    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...

  20. Discovery of the first dual G-triplex/G-quadruplex stabilizing compound: a new opportunity in the targeting of G-rich DNA structures?

    Science.gov (United States)

    Amato, Jussara; Pagano, Alessia; Cosconati, Sandro; Amendola, Giorgio; Fotticchia, Iolanda; Iaccarino, Nunzia; Marinello, Jessica; De Magis, Alessio; Capranico, Giovanni; Novellino, Ettore; Pagano, Bruno; Randazzo, Antonio

    2017-05-01

    Guanine-rich DNA motifs can form non-canonical structures known as G-quadruplexes, whose role in tumorigenic processes makes them attractive drug-target candidates for cancer therapy. Recent studies revealed that the folding and unfolding pathways of G-quadruplexes proceed through a quite stable intermediate named G-triplex. Virtual screening was employed to identify a small set of putative G-triplex ligands. The G-triplex stabilizing properties of these compounds were analyzed by CD melting assay. DSC, non-denaturing gel electrophoresis, NMR and molecular modeling studies were performed to investigate the interaction between the selected compound 1 and G-rich DNA structures. Cytotoxic activity of 1 was evaluated by MTT cell proliferation assay. The experiments led to the identification of a promising hit that was shown to bind preferentially to G-triplex and parallel-stranded G-quadruplexes over duplex and antiparallel G-quadruplexes. Molecular modeling results suggested a partial end-stacking of 1 to the external G-triad/G-tetrads as a binding mode. Biological assays showed that 1 is endowed with cytotoxic effect on human osteosarcoma cells. A tandem application of virtual screening along with the experimental investigation was employed to discover a G-triplex-targeting ligand. Experiments revealed that the selected compound actually acts as a dual G-triplex/G-quadruplex stabilizer, thus stimulating further studies aimed at its optimization. The discovery of molecules able to bind and stabilize G-triplex structures is highly appealing, but their transient state makes challenging their recognition. These findings suggest that the identification of ligands with dual G-triplex/G-quadruplex stabilizing properties may represent a new route for the design of anticancer agents targeting the G-rich DNA structures. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio. Copyright © 2016 Elsevier B

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

    OpenAIRE

    Germann, Markus W.; David Wilson, W.; Henary, Maged M.; Huynh, Hang T.; Stroeva, Ekaterina M.; Dost, Tyler L.; Rupesh Nanjunda; Leah Mickelson; Owens, Eric A.

    2013-01-01

    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 ...

  2. G-quadruplex DNA biosensor for sensitive visible detection of genetically modified food.

    Science.gov (United States)

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

    2014-10-01

    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.

  3. Anthracene-terpyridine metal complexes as new G-quadruplex DNA binders.

    Science.gov (United States)

    Gama, Sofia; Rodrigues, Inês; Mendes, Filipa; Santos, Isabel C; Gabano, Elisabetta; Klejevskaja, Beata; Gonzalez-Garcia, Jorge; Ravera, Mauro; Vilar, Ramon; Paulo, António

    2016-07-01

    The formation of quadruple-stranded DNA induced by planar metal complexes has particular interest in the development of novel anticancer drugs. This is especially relevant for the inhibition of telomerase, which plays an essential role in cancer cell immortalization and is overexpressed in ca. 85-90% of cancer cells. Moreover, G-quadruplexes also exist in other locations in the human genome, namely oncogene promoter regions, and it has been hypothesized that they play a regulatory role in gene transcription. Herein we report a series of new anthracene-containing terpyridine ligands and the corresponding Cu(II) and Pt(II) complexes, with different linkers between the anthracenyl moiety and the terpyridine chelating unit. The interaction of these ligands and metal complexes with different topologies of DNA was studied by several biophysical techniques. The Pt(II) and Cu(II) complexes tested showed affinity for quadruplex-forming sequences with a good selectivity over duplex DNA. Importantly, the free ligands do not have significant affinity for any of the DNA sequences used, which shows that the presence of the metal is essential for high affinity (and selectivity). This effect is more evident in the case of the Pt(II) complexes. Moreover, the presence of a longer linker between the chelating terpyridine unit and the anthracene moiety enhances the interaction with G-quadruplex-forming sequences. We further evaluated the ability of the Cu(II) complexes to interact with, and stabilize G-quadruplex containing regions in oncogene promoters via a polymerase stop assay. These studies indicated that the metal complexes are able to induce G-quadruplex formation and stop polymerase activity.

  4. Macrocyclic hexaoxazoles: Influence of aminoalkyl substituents on RNA and DNA G-quadruplex stabilization and cytotoxicity.

    Science.gov (United States)

    Satyanarayana, Mavurapu; Kim, Young-Ah; Rzuczek, Suzanne G; Pilch, Daniel S; Liu, Angela A; Liu, Leroy F; Rice, Joseph E; LaVoie, Edmond J

    2010-05-15

    A series of 24-membered macrocyclic hexaoxazoles containing one or two aminoalkyl substituents was synthesized and evaluated for cytotoxicity and for their ability to selectively stabilize G-quadruplex DNA and RNA. The most cytotoxic analog 4a, with IC(50) values of 25 and 130 nM using KB3-1 and RPMI 8402 cells, is efficacious in vivo in athymic nude mice with a human tumor xenograft from the breast cancer cell line MDA-MB-435.

  5. Native Electrospray Mass Spectrometry of DNA G-Quadruplexes in Potassium Solution

    OpenAIRE

    2014-01-01

    A commonly used electrolyte in electrospray mass spectrometry (ESI-MS) of biomolecules is ammonium acetate (NH4OAc). Although some nucleic acid structures such as duplexes require only proper physiological ionic strength (whatever the monovalent ions) to be properly folded in ESI-MS conditions, the folding of some other nucleic acid structures such as DNA G-quadruplexes also depends on direct binding of specific cations. Here, we developed ESI-MS compatible conditions that allow one to observ...

  6. Real time monitoring of Pb2+-induced formation of G-quadruplex DNA with LPFG sensor

    Science.gov (United States)

    Bagheri, Z.; Latifi, H.; Ranjbar, B.; Zibaii, M. I.; Behroodi, E.

    2015-09-01

    In this paper the potential of real time monitoring of AS1411 Guanine-quadruplex folding by using long period fiber grating (LPFG) biosensor was described. AS1411 ss-DNA folds into parallel bimolecular quadruplex in solution containing metal ions. While immobilized AS1411 folds, molecular crowding of sensing area will increase and cause change the refractive index (RI) around the sensor. In addition RI change is absolutely dependent on G-quadruplex factor concentration.

  7. Volumetric contributions of loop regions of G-quadruplex DNA to the formation of the tertiary structure.

    Science.gov (United States)

    Takahashi, Shuntaro; Sugimoto, Naoki

    2017-02-06

    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, ∆Vtr, 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 ∆Vtr of 41.1cm(3)mol(-1). In the presence of poly(ethylene)glycol 200 (PEG200), ∆Vtr 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.

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

    DEFF Research Database (Denmark)

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

    2010-01-01

    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......-quadruplex telomeric DNA. Ensemble and single molecule FRET measurements provide further insight into molecular conformation: the telomeric DNA overhang is found to be in a folded state in the absence of hnRNP A1 and to remain predominantly in a compact state when complexed with hnRNP A1. This finding is in contrast...

  9. Controlling the stoichiometry and strand polarity of a tetramolecular G-quadruplex structure by using a DNA origami frame

    Science.gov (United States)

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

    2013-01-01

    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

  10. Development of Fluorescent Protein Probes Specific for Parallel DNA and RNA G-Quadruplexes.

    Science.gov (United States)

    Dang, Dung Thanh; Phan, Anh Tuân

    2016-01-01

    We have developed fluorescent protein probes specific for parallel G-quadruplexes by attaching cyan fluorescent protein to the G-quadruplex-binding motif of the RNA helicase RHAU. Fluorescent probes containing RHAU peptide fragments of different lengths were constructed, and their binding to G-quadruplexes was characterized. The selective recognition and discrimination of G-quadruplex topologies by the fluorescent protein probes was easily detected by the naked eye or by conventional gel imaging.

  11. Identification of the DNA-Binding Domains of Human Replication Protein A That Recognize G-Quadruplex DNA

    Directory of Open Access Journals (Sweden)

    Aishwarya Prakash

    2011-01-01

    Full Text Available Replication protein A (RPA, a key player in DNA metabolism, has 6 single-stranded DNA-(ssDNA- binding domains (DBDs A-F. SELEX experiments with the DBDs-C, -D, and -E retrieve a 20-nt G-quadruplex forming sequence. Binding studies show that RPA-DE binds preferentially to the G-quadruplex DNA, a unique preference not observed with other RPA constructs. Circular dichroism experiments show that RPA-CDE-core can unfold the G-quadruplex while RPA-DE stabilizes it. Binding studies show that RPA-C binds pyrimidine- and purine-rich sequences similarly. This difference between RPA-C and RPA-DE binding was also indicated by the inability of RPA-CDE-core to unfold an oligonucleotide containing a TC-region 5′ to the G-quadruplex. Molecular modeling studies of RPA-DE and telomere-binding proteins Pot1 and Stn1 reveal structural similarities between the proteins and illuminate potential DNA-binding sites for RPA-DE and Stn1. These data indicate that DBDs of RPA have different ssDNA recognition properties.

  12. Synthesis and Evaluation of a Rationally Designed Click-Based Library for G-Quadruplex Selective DNA Photocleavage

    Directory of Open Access Journals (Sweden)

    Dominic McBrayer

    2015-09-01

    Full Text Available DNA containing repeating G-rich sequences can adopt higher-order structures known as G-quadruplexes (G4. These structures are believed to form within telomeres and the promoter regions of some genes, particularly in a number of proto-oncogenes, where they may play a role in regulating transcription. Alternatively, G4 DNA may act as a barrier to replication. To investigate these potential biological roles, probes that combine highly selective G4 DNA targeting with photocleavage activity can allow temporal detection of G4 DNA, providing opportunities to obtain novel insights about the biological roles of G4 DNA. We have designed, synthesized, and screened a small library of potential selective G-quadruplex DNA photocleavage agents incorporating the G-quadruplex targeting moiety of 360A with known photocleavage groups linked via “click” chemistry.

  13. Spectroscopic investigation on the interaction of copper porphyrazines and phthalocyanine with human telomeric G-quadruplex DNA.

    Science.gov (United States)

    Hassani, Leila; Hakimian, Fatemeh; Safaei, Elham

    2014-01-01

    The G-quadruplex DNA is a novel target for anticancer drug discovery and many scientific groups are investigating interaction of small molecules with G-quadruplex DNA to discover therapeutic agents for cancer. Here, interaction of a phthalocyanine (Cu(PcTs)) and two tetrapyridinoporphyrazines ([Cu(2,3-tmtppa)](4+) and [Cu(3,4-tmtppa)](4+)) with Na(+) and K(+) forms of human telomeric G-quadruplex DNA has been investigated by spectroscopic techniques. The results indicated that interaction of the cationic porphyrazines is remarkably stronger than the anionic phthalocyanine and they presumably bind to the G-quadruplex DNA through end-stacking. Fluorescent intercalator displacement assay implied the displacement ability of the complexes with thiazole orange. In addition, circular dichroism spectra of both quadruplex forms converge to the Na(+) isoform after binding to the porphyrazines. In conclusion, the porphyrazines as the complexes that bind to the G-quadruplex DNA, could be suitable candidates for further investigations about inhibition of telomerase enzyme. Copyright © 2013 Elsevier B.V. All rights reserved.

  14. Topology of a G-quadruplex DNA formed by C9orf72 hexanucleotide repeats associated with ALS and FTD.

    Science.gov (United States)

    Zhou, Bo; Liu, Changdong; Geng, Yanyan; Zhu, Guang

    2015-11-13

    Abnormal expansions of an intronic hexanucleotide GGGGCC (G4C2) repeat of the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Previous studies suggested that the C9orf72 hexanucleotide repeat expansion (HRE), either as DNA or the transcribed RNA, can fold into G-quadruplexes with distinct structures. These structural polymorphisms lead to abortive transcripts and contribute to the pathogenesis of ALS and FTD. Using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, we analyzed the structures of C9orf72 HRE DNA with various G4C2 repeats. They exhibited diverse G-quadruplex folds in potassium ions. Furthermore, we determined the topology of a G-quadruplex formed by d(G4C2)4. It favors a monomeric fold and forms a chair-type G-quadruplex with a four-layer antiparallel G-tetra core and three edgewise loops, which is distinct from known structures of chair-type G-quadruplexes. Our findings highlight the conformational heterogeneity of C9orf72 HRE DNA, and may lay the necessary structural basis for designing small molecules for the modulation of ALS/FTD pathogenesis.

  15. An isothermal titration and differential scanning calorimetry study of the G-quadruplex DNA-insulin interaction.

    Science.gov (United States)

    Timmer, Christine M; Michmerhuizen, Nicole L; Witte, Amanda B; Van Winkle, Margaret; Zhou, Dejian; Sinniah, Kumar

    2014-02-20

    The binding of insulin to the G-quadruplexes formed by the consensus sequence of the insulin-linked polymorphic region (ILPR) was investigated with differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC). The thermal denaturation temperature of insulin was increased by almost 4 °C upon binding to ILPR G-quadruplex DNA as determined by DSC. The thermodynamic parameters (K(D), ΔH, ΔG, and ΔS) of the insulin-G-quadruplex complex were further investigated by temperature-dependent ITC measurement over the range of 10-37 °C. The binding of insulin to the ILPR consensus sequence displays micromolar affinity in phosphate buffer at pH 7.4, which is mainly driven by entropic factors below 25 °C but by enthalpic terms above 30 °C. The interaction was also examined in several different buffers, and results showed that the observed ΔH is dependent on the ionization enthalpy of the buffer used. This indicates proton release upon the binding of G-quadruplex DNA to insulin. Additionally, the large negative change in heat capacity for this interaction may be associated with the dominant hydrophobicity of the amino acid sequence of insulin's β subunit, which is known to bind to the ILPR G-quadruplex DNA.

  16. Stabilizing G-quadruplex DNA by methylazacalix[n]pyridine through shape-complementary interaction.

    Science.gov (United States)

    Guan, Ai-Jiao; Shen, Meng-Jie; Zhang, En-Xuan; Li, Qian; Wang, Li-Xia; Xu, Li-Jin; Xiang, Jun-Feng; Tang, Ya-Lin

    2016-01-15

    It is found that G-quadruplexes have important functions in biological systems, such as gene expression. Molecules which can stabilize the G-quadruplex structure may have potential application in regulating the expression of gene. A series of methylazacalix[n]pyridine (n=4, 6, 7, 8, 9) has been tested to stabilize the intermolecular human telomeric G-quadruplex (T12 and H12), intramolecular TBA, c-kit and bcl-2 G-quadruplex by CD denaturation experiments. The results showed that only methylazacalix[6]pyridine (MACP6) can stabilize the intermolecular G-quadruplex formed from the 12bp human telomere. Further studies evidenced that the shape-complementary binding mode was what contributed to the interaction between MACP6 and T12 G-quadruplex.

  17. Measurement of the base number of DNA using a special calliper made of a split G-quadruplex.

    Science.gov (United States)

    Zhu, Jinbo; Zhang, Libing; Wang, Erkang

    2012-12-21

    A special DNA calliper made of a split G-quadruplex was constructed to measure the length of DNA in a single-digit base number range between two selected sequences. The enhanced fluorescence of PPIX changed with the distance between two G-rich segments.

  18. Charge conduction properties of a parallel-stranded DNA G-quadruplex: implications for chromosomal oxidative damage.

    Science.gov (United States)

    Huang, Yu Chuan; Cheng, Alan K H; Yu, Hua-Zhong; Sen, Dipankar

    2009-07-28

    The charge-flow properties and concomitant guanine damage patterns of a number of intermolecular and wholly parallel-stranded DNA G-quadruplexes were investigated. The DNA constructs were structurally well-defined and consisted of the G-quadruplex sandwiched and stacked between two Watson-Crick base-paired duplexes. Such duplex-quadruplex-duplex constructs were designed to minimize torsional stress as well as steric crowding at the duplex-quadruplex junctions. When anthraquinone was used to induce charge flow within the constructs, it was found that the quadruplex served both as a sink and as a moderately good conductor of electron holes, relative to DNA duplexes. Most strikingly, the quadruplex suffered very little charge-flow generated oxidative damage relative to guanines in the duplex regions and, indeed, to guanines in antiparallel quadruplexes reported in prior studies. It is likely that these differences result from a combination of steric and electronic factors. A biological conclusion that may be drawn from these data is that if, as anticipated, G-quadruplex structures form in vivo at the telomeres and other loci in eukaryotic chromosomes, their ability to serve as protective sinks against chromosomal oxidative damage may depend on their specific character and topology. From a separate perspective, our results on the conduction properties of duplex-quadruplex-duplex DNA composites suggest the utility of G-quadruplexes as junction modules in the construction of DNA-based biosensors and nanocircuitry.

  19. Target guided synthesis using DNA nano-templates for selectively assembling a G-quadruplex binding c-MYC inhibitor

    Science.gov (United States)

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

    2017-07-01

    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.

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

    DEFF Research Database (Denmark)

    Kragh, Sofie Louise

    2015-01-01

    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...... populations and thus providing more information than traditional ensemble experiments. Using single molecule FRET microscopy different aspects of G-quadruplex folding were investigated. We have obtained direct insight into G-quadruplex structural polymorphism both in K+ and Na+ solutions. Polymorphism have...... previously only been investigated in K+. Here, we observe significant polymorphism also in Na+. By investigating the dynamics of these conformational changes and comparing these findings with other experiments for G-quadruplexes with known topology we are able to identify different conformations and folding...

  1. Binding Studies of Natural Product Berberine with DNA G-Quadruplex

    Directory of Open Access Journals (Sweden)

    Nagendra K. Sharma

    2011-01-01

    Full Text Available Problem statement: The ends of chromosome had highly repetitive short G and C-rich sequences of DNA. These sequences were known to form stable tetraplex type of secondary structures which help to maintain gene integratity after cell divison. Approach: Any reagent which controls the random cell division would be useful to design anticancer drugs. Therefore a many natural and synthesized molecules which stabilized tetraplex structures are targeted as anticancer drug entities. Results: Among them, Berberine hydrochloride natural product and its analogues are well studies as G-quadruplex stabilizing agent. In this report, DNA sequence 5’-G3-C5-G3-3’ has been designed which has probability to form i-motif and G-qua druplex types of secondary structures. Herein we studied the interaction between this DNA strands and Berberine hydrochloride by 1H-NMR techniques and UV in two different PH (4.7 and 7.4 conditions. Conclusion/Recommendations: Our preliminary results showed that Berberine bind with this DNA strand in both pH conditions which is further supported by UV melting experiments. In future this sequence can be used as probe to screen out tetraplex binding natural products which help to generate new anticancer drugs.

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

    Directory of Open Access Journals (Sweden)

    Markus W. Germann

    2013-11-01

    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.

  3. Selective G-quadruplex DNA recognition by a new class of designed cyanines.

    Science.gov (United States)

    Nanjunda, Rupesh; Owens, Eric A; Mickelson, Leah; Dost, Tyler L; Stroeva, Ekaterina M; Huynh, Hang T; Germann, Markus W; Henary, Maged M; Wilson, W David

    2013-11-04

    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.

  4. A single molecule study of G-quadruplex and short duplex DNA structures

    Science.gov (United States)

    Roy, William A., Jr.

    Given that certain conditions are met, a single stranded DNA/RNA (ssDNA/RNA) structure called G-quadruplex (GQ) can form in regions throughout the genome, including at the telomeres and internal regions of the chromosomes. These structures serve various functions depending on the region in which they form which include protecting the chromosome ends, interfering with telomere elongation in cancer cells, and regulating transcription and translation level gene expression. Due to their high stability, various cellular mechanisms, such as GQ destabilizing proteins, are employed to unfold these structures during DNA replication or repair. Yet, their distinct layered structure has made GQs an attractive drug target in cancer treatment as GQ stabilizing molecules could inhibit telomerase dependent telomere elongation, a mechanism occurring in the majority of cancer cells to avoid senescence and apoptosis. However, proteins or small molecules interact with GQ that is under the influence of various cellular tension mechanisms, including the tension applied by other nearby molecules or the tension due to DNA structure within the chromatin context. Therefore, it is important to characterize the stability of various GQs and their response to interacting molecules when subjected to a tensile force. We employed a novel DNA-based nano tension generator that utilizes the elastic properties of circularized short double-stranded DNA (dsDNA) oligonucleotides to apply tension on the GQ. Since this is a completely new approach, the majority of this thesis was dedicated to proof-of-principle studies that demonstrated the feasibility and functionality of the method.

  5. Novel FeII and CoII Complexes of Natural Product Tryptanthrin: Synthesis and Binding with G-Quadruplex DNA

    Science.gov (United States)

    Zhong, Yi-ning; Zhang, Yan; Gu, Yun-qiong; Wu, Shi-yun; Shen, Wen-ying

    2016-01-01

    Tryptanthrin is one of the most important members of indoloquinoline alkaloids. We obtained this alkaloid from Isatis. Two novel FeII and CoII complexes of tryptanthrin were first synthesized. Single-crystal X-ray diffraction analyses show that these complexes display distorted four-coordinated tetrahedron geometry via two heterocyclic nitrogen and oxygen atoms from tryptanthrin ligand. Binding with G-quadruplex DNA properties revealed that both complexes were found to exhibit significant interaction with G-quadruplex DNA. This study may potentially serve as the basis of future rational design of metal-based drugs from natural products that target the G-quadruplex DNA. PMID:27698647

  6. Ligand binding to telomeric G-quadruplex DNA investigated by funnel-metadynamics simulations

    Science.gov (United States)

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

    2017-01-01

    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[AG3(T2AG3)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 (ΔGb0 = −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. PMID:28232513

  7. Structure, Properties, and Biological Relevance of the DNA and RNA G-Quadruplexes: Overview 50 Years after Their Discovery.

    Science.gov (United States)

    Dolinnaya, N G; Ogloblina, A M; Yakubovskaya, M G

    2016-12-01

    G-quadruplexes (G4s), which are known to have important roles in regulation of key biological processes in both normal and pathological cells, are the most actively studied non-canonical structures of nucleic acids. In this review, we summarize the results of studies published in recent years that change significantly scientific views on various aspects of our understanding of quadruplexes. Modern notions on the polymorphism of DNA quadruplexes, on factors affecting thermodynamics and kinetics of G4 folding-unfolding, on structural organization of multiquadruplex systems, and on conformational features of RNA G4s and hybrid DNA-RNA G4s are discussed. Here we report the data on location of G4 sequence motifs in the genomes of eukaryotes, bacteria, and viruses, characterize G4-specific small-molecule ligands and proteins, as well as the mechanisms of their interactions with quadruplexes. New information on the structure and stability of G4s in telomeric DNA and oncogene promoters is discussed as well as proof being provided on the occurrence of G-quadruplexes in cells. Prominence is given to novel experimental techniques (single molecule manipulations, optical and magnetic tweezers, original chemical approaches, G4 detection in situ, in-cell NMR spectroscopy) that facilitate breakthroughs in the investigation of the structure and functions of G-quadruplexes.

  8. CX-5461 is a DNA G-quadruplex stabilizer with selective lethality in BRCA1/2 deficient tumours

    Science.gov (United States)

    Xu, Hong; Di Antonio, Marco; McKinney, Steven; Mathew, Veena; Ho, Brandon; O'Neil, Nigel J.; Santos, Nancy Dos; Silvester, Jennifer; Wei, Vivien; Garcia, Jessica; Kabeer, Farhia; Lai, Daniel; Soriano, Priscilla; Banáth, Judit; Chiu, Derek S.; Yap, Damian; Le, Daniel D.; Ye, Frank B.; Zhang, Anni; Thu, Kelsie; Soong, John; Lin, Shu-chuan; Tsai, Angela Hsin Chin; Osako, Tomo; Algara, Teresa; Saunders, Darren N.; Wong, Jason; Xian, Jian; Bally, Marcel B.; Brenton, James D.; Brown, Grant W.; Shah, Sohrab P.; Cescon, David; Mak, Tak W.; Caldas, Carlos; Stirling, Peter C.; Hieter, Phil; Balasubramanian, Shankar; Aparicio, Samuel

    2017-01-01

    G-quadruplex DNAs form four-stranded helical structures and are proposed to play key roles in different cellular processes. Targeting G-quadruplex DNAs for cancer treatment is a very promising prospect. Here, we show that CX-5461 is a G-quadruplex stabilizer, with specific toxicity against BRCA deficiencies in cancer cells and polyclonal patient-derived xenograft models, including tumours resistant to PARP inhibition. Exposure to CX-5461, and its related drug CX-3543, blocks replication forks and induces ssDNA gaps or breaks. The BRCA and NHEJ pathways are required for the repair of CX-5461 and CX-3543-induced DNA damage and failure to do so leads to lethality. These data strengthen the concept of G4 targeting as a therapeutic approach, specifically for targeting HR and NHEJ deficient cancers and other tumours deficient for DNA damage repair. CX-5461 is now in advanced phase I clinical trial for patients with BRCA1/2 deficient tumours (Canadian trial, NCT02719977, opened May 2016). PMID:28211448

  9. Toward the design of new DNA G-quadruplex ligands through rational analysis of polymorphism and binding data.

    Science.gov (United States)

    Artese, Anna; Costa, Giosuè; Distinto, Simona; Moraca, Federica; Ortuso, Francesco; Parrotta, Lucia; Alcaro, Stefano

    2013-10-01

    Human telomeres play a key role in protecting chromosomal ends from fusion events; they are composed of d(TTAGGG) repeats, ranging in size from 3 to 15 kb. They form G-quadruplex DNA structures, stabilized by G-quartets in the presence of cations, and are involved in several biological processes. In particular, a telomere maintenance mechanism is provided by a specialized enzyme called telomerase, a reverse transcriptase able to add multiple copies of the 5'-GGTTAG-3' motif to the end of the G-strand of the telomere and which is over-expressed in the majority of cancer cells. The central cation has a crucial role in maintaining the stability of the structure. Based on its nature, it can be associated with different topological telomeric quadruplexes, which depend also on the orientation of the DNA strands and the syn/anti conformation of the guanines. Such a polymorphism, confirmed by the different structures deposited in the Protein Data Bank (PDB), prompted us to apply a computational protocol in order to investigate the conformational properties of a set of known G-quadruplex ligands and their molecular recognition against six different experimental models of the human telomeric sequence d[AG3(T2AG3)3]. The average AutoDock correlation between theoretical and experimental data yielded an r2 value equal to 0.882 among all the studied models. Such a result was always improved with respect to those of the single folds, with the exception of the parallel structure (r2 equal to 0.886), thus suggesting a key role of this G4 conformation in the stacking interaction network. Among the studied binders, a trisubstituted acridine and a dibenzophenanthroline derivative were well recognized by the parallel and the mixed G-quadruplex structures, allowing the identification of specific key contacts with DNA and the further design of more potent or target specific G-quadruplex ligands.

  10. 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.

  11. Molecular design of synthetic benzimidazoles for the switchover of the duplex to G-quadruplex DNA recognition.

    Science.gov (United States)

    Maji, Basudeb; Bhattacharya, Santanu

    2013-01-01

    Benzimidazole derivatives are well known for their antibacterial, antiviral, anticonvulsant, antihistaminic, anthelmintic and antidepressant activities. Benzimidazole's unique base-selective DNA recognition property has been studied widely. However, most of the early benzimidazole systems have been targeted towards the binding of duplex DNA. Here we have shown the evolution and progress of the design and synthesis of new benzimidazole systems towards selective recognition of the double-stranded DNA first. Then in order to achieve selective recognition of the G-quadruplex DNA and utilize their potential as future anti-cancer drug candidates, we have demonstrated their selective cytotoxicity towards the cancer cells and potent telomerase inhibition ability.

  12. Identification of G-quadruplex structures that possess transcriptional regulating functions in the Dele and Cdc6 CpG islands.

    Science.gov (United States)

    Bay, Daniyah H; Busch, Annika; Lisdat, Fred; Iida, Keisuke; Ikebukuro, Kazunori; Nagasawa, Kazuo; Karube, Isao; Yoshida, Wataru

    2017-06-27

    G-quadruplex is a DNA secondary structure that has been shown to play an important role in biological systems. In a previous study, we identified 1998 G-quadruplex-forming sequences using a mouse CpG islands DNA microarray with a fluorescent-labeled G-quadruplex ligand. Among these putative G-quadruplex-forming sequences, G-quadruplex formation was verified for 10 randomly selected sequences by CD spectroscopy and DMS footprinting analysis. In this study, the biological function of the 10 G-quadruplex-forming sequences in the transcriptional regulation has been analyzed using a reporter assay. When G-quadruplex-forming sequences from the Dele and Cdc6 genes have been cloned in reporter vectors carrying a minimal promoter and the luciferase gene, luciferase expression is activated. This has also been detected in experiments applying a promoterless reporter vector. Mutational analysis reveals that guanine bases, which form the G-tetrads, are important in the activation. In addition, the activation has been found to decrease by the telomestatin derivative L1H1-7OTD which can bind to the G-quadruplex DNA. When Dele and Cdc6 CpG islands, containing the G-quadruplex-forming sequence, have been cloned in the promoterless reporter vector, the luciferase expression is activated. Mutational analysis reveals that the expression level is decreased by mutation on Dele G-quadruplex; however, increased by mutation on Cdc6 G-quadruplex. Dele and Cdc6 G-quadruplex formation is significant in the transcriptional regulation. Dele and Cdc6 G-quadruplex DNA alone possess enhancer and promotor function. When studied in more complex CpG islands Dele G-quadruplex also demonstrates promotor activity, whereas Cdc6 G-quadruplex may possess a dual function of transcriptional regulation.

  13. “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: tmyang@mail.scuec.edu.cn

    2015-08-12

    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.

  14. Not All G-Quadruplexes are Created Equally: An Investigation of the Structural Polymorphism of the c-Myc G-Quadruplex-Forming Sequence and its Interaction with the Porphyrin meso-Tetra(N-methyl-4-pyridyl)porphine

    Science.gov (United States)

    Le, Huy T.; Miller, M. Clarke; Buscaglia, Robert; Dean, William L.; Holt, Patrick A.; Chaires, Jonathan B.; Trent, John O.

    2012-01-01

    G-quadruplexes, DNA tertiary structures highly localized to functionally important sites within the human genome, have emerged as important new drug targets. The putative G-quadruplex-forming sequence (Pu27) in the NHE-III1 promoter region of the c-Myc gene is of particular interest as stabilization of this G-quadruplex with TMPyP4 has been shown to repress c-Myc transcription. In this study, we examine the Pu27 G-quadruplex-forming sequence and its interaction with TMPyP4. We report that the Pu27 sequence exists as a heterogeneous mixture of monomeric and higher-order G-quadruplex species in vitro and that this mixture can be partially resolved by size exclusion chromatography (SEC) separation. Within this ensemble of configurations, the equilibrium can be altered by modifying the buffer composition, annealing procedure, and dialysis protocol thereby affecting the distribution of G-quadruplex species formed. TMPyP4 was found to bind preferentially to higher-order G-quadruplex species suggesting the possibility of stabilization of the junctions of the c-Myc G-quadruplex multimers by porphyrin end-stacking. We also examined four modified c-Myc sequences that have been previously reported and found a narrower distribution of quadruplex configurations compared to the parent Pu27 sequence. We could not definitively conclude whether these G-quadruplex structures were selected from the original ensemble or if they are new G-quadruplex structures. Since these sequences differ considerably from the wild-type promoter sequence, it is unclear whether their structures have any actual biological relevance. Additional studies are needed to examine how the polymorphic nature of G-quadruplexes affects the interpretation of in vitro data for c-Myc and other G-quadruplexes. The findings reported here demonstrate that experimental conditions contribute significantly to G-quadruplex formation and should be carefully considered, controlled, and reported in detail. PMID:23108607

  15. Design of a fluorescent DNA IMPLICATION logic gate and detection of Ag+ and cysteine with triphenylmethane dye/G-quadruplex complexes.

    Science.gov (United States)

    Guo, Jun-Hong; Kong, De-Ming; Shen, Han-Xi

    2010-10-15

    This paper describes the construction of a DNA IMPLICATION logic gate based on triphenylmethane (TPM) dye/G-quadruplex complexes, using Ag+ and cysteine (Cys) as the two inputs, and fluorescence intensity of the TPM dye as the output signal. Free triphenylmethane (TPM) dyes emit inherently low fluorescence signal, the formation of TPM dye/G-quadruplex complexes yielded greatly enhanced fluorescence signals from the dye, and the output signal of the gate was 1. The addition of Cys had no effect on the fluorescence signal, again yielding an output of 1. However, the addition of Ag+ instead of Cys greatly disrupted the G-quadruplex structure, causing a decrease in the fluorescence of the dye, and yielding an output signal of 0. The addition of Cys into the Ag+-quenched fluorescence system led to the release of Ag+ from G-quadruplex-forming DNAs, resulting in the reformation of G-quadruplex structures and the recovery of TMP dye fluorescence, the output signal of 1 was obtained again. Compared with previously published DNA logic gates, the gate operation described here was rapid and reversible, with a reliable, nondestructive readout and excellent digital behavior. In addition, the modulation of TPM dye/G-quadruplex complex fluorescence by Ag+ and Cys could be used to develop a simple, fast, label-free and highly specific homogenous sensing methods for Ag+ and Cys. Copyright © 2010 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2016-01-01

    G-quadruplexes are four-stranded nucleic acid structures that are implicated in the regulation of transcription, translation and replication. Genome regions enriched in putative G-quadruplex motifs include telomeres and gene promoters. Tumour suppressor p53 plays a critical role in regulatory pathways leading to cell cycle arrest, DNA repair and apoptosis. In addition to transcriptional regulation mediated via sequence-specific DNA binding, p53 can selectively bind various non-B DNA structures. In the present study, wild-type p53 (wtp53) binding to G-quadruplex formed by MYC promoter nuclease hypersensitive element (NHE) III1 region was investigated. Wtp53 binding to MYC G-quadruplex is comparable to interaction with specific p53 consensus sequence (p53CON). Apart from the full-length wtp53, its isolated C-terminal region (aa 320–393) as well, is capable of high-affinity MYC G-quadruplex binding, suggesting its critical role in this type of interaction. Moreover, wtp53 binds to MYC promoter region containing putative G-quadruplex motif in two wtp53-expressing cell lines. The results suggest that wtp53 binding to G-quadruplexes can take part in transcriptional regulation of its target genes. PMID:27634752

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

    Science.gov (United States)

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

    2016-10-01

    G-quadruplexes are four-stranded nucleic acid structures that are implicated in the regulation of transcription, translation and replication. Genome regions enriched in putative G-quadruplex motifs include telomeres and gene promoters. Tumour suppressor p53 plays a critical role in regulatory pathways leading to cell cycle arrest, DNA repair and apoptosis. In addition to transcriptional regulation mediated via sequence-specific DNA binding, p53 can selectively bind various non-B DNA structures. In the present study, wild-type p53 (wtp53) binding to G-quadruplex formed by MYC promoter nuclease hypersensitive element (NHE) III1 region was investigated. Wtp53 binding to MYC G-quadruplex is comparable to interaction with specific p53 consensus sequence (p53CON). Apart from the full-length wtp53, its isolated C-terminal region (aa 320-393) as well, is capable of high-affinity MYC G-quadruplex binding, suggesting its critical role in this type of interaction. Moreover, wtp53 binds to MYC promoter region containing putative G-quadruplex motif in two wtp53-expressing cell lines. The results suggest that wtp53 binding to G-quadruplexes can take part in transcriptional regulation of its target genes.

  18. 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

    2010-07-01

    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.

  19. Targeting human telomeric G-quadruplex DNA and inhibition of telomerase activity with [(dmb2Ru(obipRu(dmb2](4+.

    Directory of Open Access Journals (Sweden)

    Shuo Shi

    Full Text Available Inhibition of telomerase by inducing/stabilizing G-quadruplex formation is a promising strategy to design new anticancer drugs. We synthesized and characterized a new dinuclear complex [(dmb2Ru(obipRu(dmb2](4+ (dmb = 4,4'-dimethyl-2,2'-bipyridine, obip = (2-(2-pyridylimidazo[4,5-f][1,10]phenanthroline with high affinity for both antiparallel and mixed parallel / antiparallel G-quadruplex DNA. This complex can promote the formation and stabilize G-quadruplex DNA. Dialysis and TRAP experiments indicated that [(dmb2Ru(obipRu(dmb2](4+ acted as an excellent telomerase inhibitor due to its obvious selectivity for G-quadruplex DNA rather than double stranded DNA. In vitro co-culture experiments implied that [(dmb2Ru(obipRu(dmb2](4+ inhibited telomerase activity and hindered cancer cell proliferation without side effects to normal fibroblast cells. TUNEL assay indicated that inhibition of telomerase activity induced DNA cleavage further apoptosis in cancer cells. Therefore, Ru(II complex represents an exciting opportunity for anticancer drug design by specifically targeting cancer cell G-quadruplexes DNA.

  20. Mapping the affinity landscape of Thrombin-binding aptamers on 2'F-ANA/DNA chimeric G-Quadruplex microarrays.

    Science.gov (United States)

    Lietard, Jory; Abou Assi, Hala; Gómez-Pinto, Irene; González, Carlos; Somoza, Mark M; Damha, Masad J

    2017-01-18

    In situ fabricated nucleic acids microarrays are versatile and very high-throughput platforms for aptamer optimization and discovery, but the chemical space that can be probed against a given target has largely been confined to DNA, while RNA and non-natural nucleic acid microarrays are still an essentially uncharted territory. 2'-Fluoroarabinonucleic acid (2'F-ANA) is a prime candidate for such use in microarrays. Indeed, 2'F-ANA chemistry is readily amenable to photolithographic microarray synthesis and its potential in high affinity aptamers has been recently discovered. We thus synthesized the first microarrays containing 2'F-ANA and 2'F-ANA/DNA chimeric sequences to fully map the binding affinity landscape of the TBA1 thrombin-binding G-quadruplex aptamer containing all 32 768 possible DNA-to-2'F-ANA mutations. The resulting microarray was screened against thrombin to identify a series of promising 2'F-ANA-modified aptamer candidates with Kds significantly lower than that of the unmodified control and which were found to adopt highly stable, antiparallel-folded G-quadruplex structures. The solution structure of the TBA1 aptamer modified with 2'F-ANA at position T3 shows that fluorine substitution preorganizes the dinucleotide loop into the proper conformation for interaction with thrombin. Overall, our work strengthens the potential of 2'F-ANA in aptamer research and further expands non-genomic applications of nucleic acids microarrays.

  1. Classification of g-quadruplex DNA on the basis of the quadruplex twist angle and planarity of g-quartets.

    Science.gov (United States)

    Reshetnikov, R V; Kopylov, A M; Golovin, A V

    2010-10-01

    The present work is devoted to the analysis of the G-quadruplex DNA structure using the bioinformatics method. The interest towards quadruplex DNAs is determined by their involvement in the functioning of telomeres and onco-promoters as well as by the possibility to create on their basis aptamers and nanostructures. Here, we present an algorithm for a general analysis of the polymorphism of the G-quadruplex structure from the data bank PDB using original parameters. 74 structures were grouped according to the following parameters: the number of DNA strands, the number of G-quartets, and the location and orientation of the connecting loops. Two quantitative parameters were used to describe the quadruplex structure: the twist angle between two adjacent quartets (analogous to that for the complementary pair in the duplex DNA) and the quartet planarity (an original parameter). The distribution patterns of these values are specific for each group of quadruplex structures and are dependent upon the type of connecting loops used (diagonal, lateral or propeller). The tetramolecular loopless parallel quadruplex was used as a comparison template. The lateral loops introduce the strongest distortion into the structure of quadruplexes: the values of the twist angles are the lowest and are not typical for the other quadruplex groups. The loops of the diagonal type introduce much weaker deformation into quadruplexes; the structures with propeller loops are characterized by the optimum geometry of G-quartets. Hence, the correlation between the twist angle and the tension in the structure of quadruplex DNA is revealed.

  2. Amplified detection of DNA ligase and polynucleotide kinase/phosphatase on the basis of enrichment of catalytic G-quadruplex DNAzyme by rolling circle amplification.

    Science.gov (United States)

    Jiang, Hong-Xin; Kong, De-Ming; Shen, Han-Xi

    2014-05-15

    As two commonly used tool enzymes, DNA ligase and polynucleotide kinase/phosphatase (PNKP) play important roles in DNA metabolism. More and more studies show that regulation of their activity represents promising means for cancer therapy. To detect the activity of DNA ligase with high sensitivity and specificity, a G-quadruplex DNAzyme-based DNA ligase sensor was developed. In this sensor, the use of G-quadruplex DNAzyme eliminated the needs for any labeled oligonucleotide probes, thus making label-free detection possible. The introduction of rolling circle amplification (RCA) reaction could lead to the formation of multimeric G-quadruplexes containing thousands of G-quadruplex units, which can provide highly active hemin-binding sites, thus significantly improving the sensitivity of the sensor. The proposed sensor allowed specific detection of T4 DNA ligase with a detection limit of 0.0019 U/mL. By adding a PNKP-triggered 5'-phosphroylation step of the template DNA, the above sensing strategy could be easily extended to the design of PNKP sensor. The established sensor allowed specific detection of T4 PNKP with a detection limit of 0.0018 U/mL. In addition, these two sensors could also be used for the studies on inhibitors of these two enzymes.

  3. 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;

    2014-01-01

    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 pathological...... features of Bloom syndrome, we analyzed mRNA and microRNA (miRNA) expression in fibroblasts from individuals with Bloom syndrome and in BLM-depleted control fibroblasts. We identified mRNA and miRNA expression differences in Bloom syndrome patient and BLM-depleted cells. Differentially expressed m...... dysfunction, and other features observed in Bloom syndrome individuals. BLM binds to G-quadruplex (G4) DNA, and G4 motifs were enriched at transcription start sites (TSS) and especially within first introns (false discovery rate ≤ 0.001) of differentially expressed mRNAs in Bloom syndrome compared with normal...

  4. Conformational conversion of DNA G-quadruplex induced by a cationic porphyrin.

    Science.gov (United States)

    Zhang, Huijuan; Xiao, Xiao; Wang, Peng; Pang, Siping; Qu, Feng; Ai, Xicheng; Zhang, Jianping

    2009-09-15

    The interactions between cationic meso-tetrakis(4-(N-methylpyridiumyl))porphyrin (TMPyP4) and the G-quadruplex (G4) of human telomeric single-strand oligonucleotide d(TTAGGG)(2) (S12) have been investigated by means of circular dichroism (CD), UV-visible absorption and fluorescence spectroscopies. It is found that TMPyP4 can preferentially induce the conformational conversion of the G4 structure from the parallel type to the parallel/antiparallel mixture in the presence of K(+), and that it can directly induce the formation of antiparallel G4 structure from the single-strand oligonucleotide S12 in the absence of K(+). Furthermore, the comparable experiments of TMPyP4 with two single-strand oligonucleotides S6 d(TTAGGG) and S24 d(TAGGG(TTAGGG)(3)T) in the absence of K(+) show that TMPyP4 can also induce the formation of antiparallel G4 from S24 but not from S6, indicating that the end-loops of the G4 structure are the key factors for the formation of G4 induced by TMPyP4.

  5. Label-free ultrasensitive detection of telomerase activity via multiple telomeric hemin/G-quadruplex triggered polyaniline deposition and a DNA tetrahedron-structure regulated signal.

    Science.gov (United States)

    Liu, Yuanjian; Wei, Min; Liu, Xu; Wei, Wei; Zhao, Hongyu; Zhang, Yuanjian; Liu, Songqin

    2016-01-31

    Label-free detection of telomerase activity was done by using telomeric hemin/G-quadruplex triggered polyaniline deposition, not only on themselves but also on the DNA tetrahedron-structure (DTS). DTS size has a great impact on telomerase accessibility, reactivity and detection sensitivity. The method has been used to evaluate bladder cancer development.

  6. Label-free detection of kanamycin based on a G-quadruplex DNA aptamer-based fluorescent intercalator displacement assay

    Science.gov (United States)

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

    2015-01-01

    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.

  7. Cation Coordination Alters the Conformation of a Thrombin-Binding G-Quadruplex DNA Aptamer That Affects Inhibition of Thrombin.

    Science.gov (United States)

    Zavyalova, Elena; Tagiltsev, Grigory; Reshetnikov, Roman; Arutyunyan, Alexander; Kopylov, Alexey

    2016-10-01

    Thrombin-binding aptamers are promising anticoagulants. HD1 is a monomolecular antiparallel G-quadruplex with two G-quartets linked by three loops. Aptamer-thrombin interactions are mediated with two TT-loops that bind thrombin exosite I. Several cations were shown to be coordinated inside the G-quadruplex, including K(+), Na(+), NH4(+), Ba(2+), and Sr(2+); on the contrary, Mn(2+) was coordinated in the grooves, outside the G-quadruplex. K(+) or Na(+) coordination provides aptamer functional activity. The effect of other cations on aptamer functional activity has not yet been described, because of a lack of relevant tests. Interactions between aptamer HD1 and a series of cations were studied. A previously developed enzymatic method was applied to evaluate aptamer inhibitory activity. The structure-function correlation was studied using the characterization of G-quadruplex conformation by circular dichroism spectroscopy. K(+) coordination provided the well-known high inhibitory activity of the aptamer, whereas Na(+) coordination supported low activity. Although NH4(+) coordination yielded a typical antiparallel G-quadruplex, no inhibitory activity was shown; a similar effect was observed for Ba(2+) and Sr(2+) coordination. Mn(2+) coordination destabilized the G-quadruplex that drastically diminished aptamer inhibitory activity. Therefore, G-quadruplex existence per se is insufficient for aptamer inhibitory activity. To elicit the nature of these effects, we thoroughly analyzed nuclear magnetic resonance (NMR) and X-ray data on the structure of the HD1 G-quadruplex with various cations. The most reasonable explanation is that cation coordination changes the conformation of TT-loops, affecting thrombin binding and inhibition. HD1 counterparts, aptamers 31-TBA and NU172, behaved similarly with some distinctions. In 31-TBA, an additional duplex module stabilized antiparallel G-quadruplex conformation at high concentrations of divalent cations; whereas in NU172, a

  8. Templated Formation of Discrete RNA and DNA:RNA Hybrid G-Quadruplexes and Their Interactions with Targeting Ligands.

    Science.gov (United States)

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

    2016-02-24

    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.

  9. Sensitive pseudobienzyme electrocatalytic DNA biosensor for mercury(II) ion by using the autonomously assembled hemin/G-quadruplex DNAzyme nanowires for signal amplification

    Energy Technology Data Exchange (ETDEWEB)

    Yuan, Yali [Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); College of resources and environments, Southwest University, Chongqing 400715 (China); Gao, Min [Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Liu, Guangpeng [College of resources and environments, Southwest University, Chongqing 400715 (China); Chai, Yaqin [Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China); Wei, Shiqing, E-mail: sqwei@swu.edu.cn [College of resources and environments, Southwest University, Chongqing 400715 (China); Yuan, Ruo, E-mail: yuanruo@swu.edu.cn [Key Laboratory on Luminescence and Real-Time Analysis, Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715 (China)

    2014-02-06

    Graphical abstract: -- Highlights: •An ultrasensitive detection system for Hg{sup 2+} detection was presented. •The autonomously assembled hemin/G-quadruplex DNAzyme nanowires were employed. •The DNAzyme simultaneously served as an NADH oxidase and HRP-mimicking DNAzyme. •The DNAzyme nanowires served as carrier for loading substantial redox probe Thi. -- Abstract: Herein, a novel sensitive pseudobienzyme electrocatalytic DNA biosensor was proposed for mercury ion (Hg{sup 2+}) detection by using autonomously assembled hemin/G-quadruplex DNAzyme nanowires for signal amplification. Thiol functionalized capture DNA was firstly immobilized on a nano-Au modified glass carbon electrode (GCE). In presence of Hg{sup 2+}, the specific coordination between Hg{sup 2+} and T could result in the assembly of primer DNA on the electrode, which successfully triggered the HCR to form the hemin/G-quadruplex DNAzyme nanowires with substantial redox probe thionine (Thi). In the electrolyte of PBS containing NADH, the hemin/G-quadruplex nanowires firstly acted as an NADH oxidase to assist the concomitant formation of H{sub 2}O{sub 2} in the presence of dissolved O{sub 2}. Then, with the redox probe Thi as electron mediator, the hemin/G-quadruplex nanowires acted as an HRP-mimicking DNAzyme that quickly bioelectrocatalyzed the reduction of produced H{sub 2}O{sub 2}, which finally led to a dramatically amplified electrochemical signal. This method has demonstrated a high sensitivity of Hg{sup 2+} detection with the dynamic concentration range spanning from 1.0 ng L{sup −1} to 10 mg L{sup −1} Hg{sup 2+} and a detection limit of 0.5 ng L{sup −1} (2.5 pM) at the 3S{sub blank} level, and it also demonstrated excellent selectivity against other interferential metal ions.

  10. Hierarchical Self-Assembly of a Biomimetic Light-Harvesting Antenna Based on DNA G-Quadruplexes

    NARCIS (Netherlands)

    Oltra, Nuria Sancho; Browne, Wesley R.; Roelfes, Gerard

    2013-01-01

    A new modular approach to an artificial light-harvesting antenna system is presented. The approach involves the hierarchical self-assembly of porphyrin acceptor molecules to G-quadruplexes tethered to coumarin donor moieties.

  11. G-quadruplexes as novel cis-elements controlling transcription during embryonic development.

    Science.gov (United States)

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

    2016-05-19

    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.

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

    Science.gov (United States)

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

    2017-11-01

    Herein, two polypyridyl ruthenium(II) complexes with alkynes, [Ru(bpy)2L](ClO4)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 (Tm) 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. Fuzzy logic sensing of G-quadruplex DNA and its cleavage reagents based on reduced graphene oxide.

    Science.gov (United States)

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

    2014-07-15

    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.

  14. Formation of highly ordered multimers in G-quadruplexes.

    Science.gov (United States)

    Tóthová, Petra; Krafčíková, Petra; Víglaský, Viktor

    2014-11-18

    G-Rich DNA and RNA have a higher propensity to form G-quadruplex structures, but the presence of G-runs alone is not sufficient to prove that such sequences can form stable G-quadruplexes. While G-rich sequences are essential for G-quadruplex formation, not all G-rich sequences have the propensity to form G-quadruplex structures. In addition, monovalent metal ions, dehydrating agents, and loop sequences connecting the G-runs also play important roles in the topology of G-quadruplex folding. To date, no quantitative analysis of the CD spectra of G-quadruplexes in confrontation with the electrophoretic results has been performed. Therefore, in this study, we use information gained through the analysis of a series of well-known G-quadruplex-forming sequences to evaluate other less-studied sets of aptameric sequences. A simple and cost-effective methodology that can verify the formation of G-quadruplex motifs from oligomeric DNA sequences and a technique to determine the molecularity of these structures are also described. This methodology could be of great use in the prediction of G-quadruplex assembly, and the basic principles of our techniques can be extrapolated for any G-rich DNA sequences. This study also presents a model that can predict the multimerization of G-quadruplexes; the predictions offered by this model are shown to match the results obtained using circular dichroism.

  15. Finding a human telomere DNA-RNA hybrid G-quadruplex formed by human telomeric 6-mer RNA and 16-mer DNA using click chemistry: a protective structure for telomere end.

    Science.gov (United States)

    Xu, Yan; Suzuki, Yuta; Ishizuka, Takumi; Xiao, Chao-Da; Liu, Xiao; Hayashi, Tetsuya; Komiyama, Makoto

    2014-08-15

    Telomeric repeat-containing RNA is a non-coding RNA molecule newly found in mammalian cells. The telomere RNA has been found to localize to the telomere DNA, but how the newly discovered RNA molecule interacts with telomere DNA is less known. In this study, using the click chemistry we successfully found that a 6-mer human telomere RNA and 16-mer human telomere DNA sequence can form a DNA-RNA hybrid type G-quadruplex structure. Detection of the click-reaction products directly probes DNA-RNA G-quadruplex structures in a complicated solution, whereas traditional methods such as NMR and crystallography may not be suitable. Importantly, we found that formation of DNA-RNA G-quadruplex induced an exonuclease resistance for telomere DNA, indicating that such structures might be important for protecting telomeric DNA from enzyme digestion to avoid telomere DNA shortening. These results provide the direct evidence for formation of DNA-RNA hybrid G-quadruplex structure by human telomere DNA and RNA sequence, suggesting DNA-RNA hybrid G-quadruplex structure associated between telomere DNA and RNA may respond to chromosome end protection and/or present a valuable target for drug design.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    -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......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...

  17. Interactions between meso-tetrakis(4-(N-methylpyridiumyl))porphyrin TMPyP4 and DNA G-quadruplex of telomeric repeated sequence TTAGGG

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    The binding properties between meso-tetrakis(4-(N-methylpyridiumyl))porphyrin (TMPyP4) and the parallel DNA G-quadruplex (G4) of telomeric repeated sequence 5′-TTAGGG-3′ have been characterized by means of circular dichroism,steady-state absorption,steady-state fluorescence and picosecond time-resolved fluorescence spectroscopies. The binding constant and the saturated binding number were determined as 1.29×106 (mol/L)-1 and 3,respectively,according to steady-state absorption spec-troscopy. Based on the findings by the use of time-resolved fluorescence spectroscopic technique,it is deduced that TMPyP4 binds to a DNA G-quadruplex with both the thread-intercalating and end-stacking modes and at the saturated binding state,one TMPyP4 molecule intercalates into the intervals of G-tetrads while the other two stack to the ends of the DNA G-quadruplex.

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

    Science.gov (United States)

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

    2014-06-10

    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.

  19. Recognition of human telomeric G-quadruplex DNA by berberine analogs: effect of substitution at the 9 and 13 positions of the isoquinoline moiety.

    Science.gov (United States)

    Bhowmik, Debipreeta; Fiorillo, Gaetano; Lombardi, Paolo; Kumar, G Suresh

    2015-12-01

    G-quadruplex forming sequences are widely distributed in human genome and serve as novel targets for regulating gene expression and chromosomal maintenance. They offer unique targets for anticancer drug development. Here, the interaction of berberine (BC) and two of its analogs bearing substitution at 9 and 13-position with human telomeric G-quadruplex DNA sequence has been investigated by biophysical techniques. Both the analogs exhibited several-fold higher binding affinity than berberine. The Scatchard binding isotherms revealed non-cooperative binding. 9-ω-amino hexyl ether analog (BC1) showed highest affinity (1.8 × 10(6) M(-1)) while the affinity of the 13-phenylpropyl analog (BC2) was 1.09 × 10(6) M(-1). Comparative fluorescence quenching and polarization anisotropy of the emission spectra gave evidence for a stronger stacking interaction of the analogs compared to berberine. The thiazole orange displacement assay has clearly established that the analogs were more effective in displacing the end stacked dye in comparison to berberine. However, the binding of the analogs did not induce any major structural perturbation in the G-quadruplex structure, but led to higher thermal stability. Energetics of the binding indicated that the association of the analogs was exothermic and predominantly entropy driven phenomenon. Increasing the temperature resulted in weaker binding; the enthalpic contribution increased and the entropic contribution decreased. A small negative heat capacity change with significant enthalpy-entropy compensation established the involvement of multiple weak noncovalent interactions in the binding process. The 9-ω-amino hexyl ether analog stabilized the G-quadruplex structure better than the 13-phenyl alkyl analog.

  20. Circular Dichroism of DNA G-Quadruplexes: Combining Modeling and Spectroscopy To Unravel Complex Structures.

    Science.gov (United States)

    Gattuso, Hugo; Spinello, Angelo; Terenzi, Alessio; Assfeld, Xavier; Barone, Giampaolo; Monari, Antonio

    2016-03-31

    We report on the comparison between the computational and experimental determination of electronic circular dichroism spectra of different guanine quadruplexes obtained from human telomeric sequences. In particular the difference between parallel, antiparallel, and hybrid structures is evidenced, as well as the induction of transitions between the polymorphs depending on the solution environment. Extensive molecular dynamics simulations (MD) are used to probe the conformational space of the different quadruplexes, and subsequently state-of-the-art hybrid quantum mechanics/molecular mechanics (QM/MM) techniques coupled with excitonic semiempirical Hamiltonian are used to simulate the macromolecular induced circular dichroism. The coupling of spectroscopy and molecular simulation allows an efficient one-to-one mapping between structures and optical properties, offering a way to disentangle the rich, yet complicated, quantity of information embedded in circular dichroism spectra. We show that our methodology is robust and efficient and allows us to take into account subtle conformational changes. As such, it could be used as an efficient tool to investigate structural modification upon DNA/drug interactions.

  1. Lowering the overall charge on TMPyP4 improves its selectivity for G-quadruplex DNA.

    Science.gov (United States)

    Ruan, Thomas L; Davis, Supriya J; Powell, Barrett M; Harbeck, Cole P; Habdas, Jan; Habdas, Piotr; Yatsunyk, Liliya A

    2017-01-01

    Ligands that stabilize non-canonical DNA structures called G-quadruplexes (GQs) might have applications in medicine as anti-cancer agents, due to the involvement of GQ DNA in a variety of cancer-related biological processes. Five derivatives of 5,10,15,20-tetrakis(N-methyl-4-pyridyl)porphyrin (TMPyP4), where a N-methylpyridyl group was replaced with phenyl (4P3), 4-aminophenyl (PN3M), 4-phenylamidoproline (PL3M), or 4-carboxyphenyl (PC3M and P2C2M) were investigated for their interactions with human telomeric DNA (Tel22) using fluorescence resonance energy transfer (FRET) assay, and UV-visible and circular dichroism spectroscopies in K(+) buffer. The molecules are cationic or zwitterionic with an overall charge of 3+ (4P3, PN3M, and PL3M), 2+ (PC3M) or neutral (P2C2M). All porphyrins except P2C2M stabilize human telomeric DNA in FRET assays by ∼20 °C at 5 eq CD melting experiments suggest that 4P3 is the most stabilizing ligand with a stabilization temperature of 16.8 °C at 4 eq. Importantly, 4P3, PC3M and PL3M demonstrate excellent selectivity for quadruplexes, far superior to that of TMPyP4. Binding constants, determined using UV-vis titrations, correlate with charge: triply cationic 4P3, PN3M and PL3M display Ka of 5-9 μM(-1), doubly cationic PC3M displays Ka of 1 μM(-1), and neutral P2C2M displays weak-to-no binding. UV-vis data suggest that binding interactions are driven by electrostatic attractions and that the binding mode may be base-stacking (or end-stacking) judging by the high values of red shift (15-20 nm) and hypochromicity (40-50%). We conclude that lowering the charge on TMPyP4 to 3+ can achieve the desired balance between stabilizing ability, affinity, and high selectivity required for an excellent quadruplex ligand.

  2. Spectroscopic, biological, and molecular modeling studies on the interactions of [Fe(III)-meloxicam] with G-quadruplex DNA and investigation of its release from bovine serum albumin (BSA) nanoparticles.

    Science.gov (United States)

    Ebrahimi, Malihe; Khayamian, Taghi; Hadadzadeh, Hassan; Sayed Tabatabaei, Badraldin Ebrahim; Jannesari, Zahra; Khaksar, Ghazale

    2015-01-01

    The guanine-rich sequence, specifically in DNA, telomeric DNA, is a potential target of anticancer drugs. In this work, a mononuclear Fe(III) complex containing two meloxicam ligands was synthesized as a G-quadruplex stabilizer. The interaction between the Fe(III) complex and G-quadruplex with sequence of 5'-G3(T2AG3)3-3' (HTG21) was investigated using spectroscopic methods, molecular modeling, and polymerase chain reaction (PCR) assays. The spectroscopic methods of UV-vis, fluorescence, and circular dichroism showed that the metal complex can effectively induce and stabilize G-quadruplex structure in the G-rich 21-mer sequence. Also, the binding constant between the Fe(III) complex and G-quadruplex was measured by these methods and it was found to be 4.53(±0.30) × 10(5) M(-1)). The PCR stop assay indicated that the Fe(III) complex inhibits DNA amplification. The cell viability assay showed that the complex has significant antitumor activities against Hela cells. According to the UV-vis results, the interaction of the Fe(III) complex with duplex DNA is an order of magnitude lower than G-quadruplex. Furthermore, the release of the complex incorporated in bovine serum albumin nanoparticles was also investigated in physiological conditions. The release of the complex followed a bi-phasic release pattern with high and low releasing rates at the first and second phases, respectively. Also, in order to obtain the binding mode of the Fe(III) complex with G-quadruplex, molecular modeling was performed. The molecular docking results showed that the Fe(III) complex was docked to the end-stacked of the G-quadruplex with a π-π interaction, created between the meloxicam ligand and the guanine bases of the G-quadruplex.

  3. Elevated levels of G-quadruplex formation in human stomach and liver cancer tissues.

    Science.gov (United States)

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

    2014-01-01

    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.

  4. Conformation-dependent Formation of the G[8-5]U Intrastrand Cross-link in 5-Bromouracil-containing G-quadruplex DNA Induced by UVA Irradiation

    OpenAIRE

    Lin, Guangxin; ZHANG Jing; Zeng, Yu; Luo, Hai; Wang, Yinsheng

    2010-01-01

    G-quadruplex motifs are known to be present in telomeres of human and other organisms. Recent bioinformatic studies also revealed the widespread existence of these motifs in promoter regions of human genes. Treatment of cultured cells with 5-bromo-2’-deoxyuridine (BrdU) is known to result in the substitution of DNA thymidine with BrdU; such replacement has been shown to sensitize cells to killing induced by UV light. Our previous studies revealed that the exposure of BrdU-carrying duplex DNA ...

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

    Directory of Open Access Journals (Sweden)

    Alessandro Altieri

    2013-10-01

    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.

  6. Conformation-sensitive nucleoside analogues as topology-specific fluorescence turn-on probes for DNA and RNA G-quadruplexes

    Science.gov (United States)

    Tanpure, Arun A.; Srivatsan, Seergazhi G.

    2015-01-01

    Development of probes that can discriminate G-quadruplex (GQ) structures and indentify efficient GQ binders on the basis of topology and nucleic acid type is highly desired to advance GQ-directed therapeutic strategies. In this context, we describe the development of minimally perturbing and environment-sensitive pyrimidine nucleoside analogues, based on a 5-(benzofuran-2-yl)uracil core, as topology-specific fluorescence turn-on probes for human telomeric DNA and RNA GQs. The pyrimidine residues of one of the loop regions (TTA) of telomeric DNA and RNA GQ oligonucleotide (ON) sequences were replaced with 5-benzofuran-modified 2′-deoxyuridine and uridine analogues. Depending on the position of modification the fluorescent nucleoside analogues distinguish antiparallel, mixed parallel-antiparallel and parallel stranded DNA and RNA GQ topologies from corresponding duplexes with significant enhancement in fluorescence intensity and quantum yield. Further, these GQ sensors enabled the development of a simple fluorescence binding assay to quantify topology- and nucleic acid-specific binding of small molecule ligands to GQ structures. Together, our results demonstrate that these nucleoside analogues are useful GQ probes, which are anticipated to provide new opportunities to study and discover efficient G-quadruplex binders of therapeutic potential. PMID:26202965

  7. A Novel Ultrasensitive ECL Sensor for DNA Detection Based on Nicking Endonuclease-Assisted Target Recycling Amplification, Rolling Circle Amplification and Hemin/G-Quadruplex

    Directory of Open Access Journals (Sweden)

    Fukang Luo

    2015-01-01

    Full Text Available In this study, we describe a novel universal and highly sensitive strategy for the electrochemiluminescent (ECL detection of sequence specific DNA at the aM level based on Nt.BbvCI (a nicking endonuclease-assisted target recycling amplification (TRA, rolling circle amplification (RCA and hemin/G-quadruplex. The target DNAs can hybridize with self-assembled capture probes and assistant probes to form “Y” junction structures on the electrode surface, thus triggering the execution of a TRA reaction with the aid of Nt.BbvCI. Then, the RCA reaction and the addition of hemin result in the production of numerous hemin/G-quadruplex, which consume the dissolved oxygen in the detection buffer and result in a significant ECL quenching effect toward the O2/S2O82− system. The proposed strategy combines the amplification ability of TRA, RCA and the inherent high sensitivity of the ECL technique, thus enabling low aM (3.8 aM detection for sequence-specific DNA and a wide linear range from 10.0 aM to 1.0 pM. At the same time, this novel strategy shows high selectivity against single-base mismatch sequences, which makes our novel universal and highly sensitive method a powerful addition to specific DNA sequence detection.

  8. Enantioselective light switch effect of Δ- and Λ-[Ru(phenanthroline)2 dipyrido[3,2-a:2', 3'-c]phenazine](2+) bound to G-quadruplex DNA.

    Science.gov (United States)

    Park, Jin Ha; Lee, Hyun Suk; Jang, Myung Duk; Han, Sung Wook; Kim, Seog K; Lee, Young-Ae

    2017-07-04

    The interaction of Δ- and Λ-[Ru(phen)2DPPZ](2+) (DPPZ = dipyrido[3,2-a:2', 3'-c]phenazine, phen = phenanthroline) with a G-quadruplex formed from 5'-G2T2G2TGTG2T2G2-3'(15-mer) was investigated. The well-known enhancement of luminescence intensity (the 'light-switch' effect) was observed for the [Ru(phen)2DPPZ](2+) complexes upon formation of an adduct with the G-quadruplex. The emission intensity of the G-quadruplex-bound Λ-isomer was 3-fold larger than that of the Δ-isomer when bound to the G-quadruplex, which is opposite of the result observed in the case of double stranded DNA (dsDNA); the light switch effect is larger for the dsDNA-bound Δ-isomer. In the job plot of the G-quadruplex with Δ- and Λ-[Ru(phen)2DPPZ](2+), a major inflection point for the two isomers was observed at x ≈ .65, which suggests a binding stoichiometry of 2:1 for both enantiomers. When the G base at the 8th position was replaced with 6-methyl isoxanthopterin (6MI), a fluorescent guanine analog, the excited energy of 6-MI transferred to bound Δ- or Λ-[Ru(phen)2DPPZ](2+), which suggests that at least a part of both Ru(II) enantiomers is close to or in contact with the diagonal loop of the G-quadruplex. A luminescence quenching experiment using [Fe(CN)6](4-) for the G-quadruplex-bound Ru(II) complex revealed downward bending curves for both enantiomers in the Stern-Volmer plot, which suggests the presence of Ru(II) complexes that are both accessible and inaccessible to the quencher and may be related to the 2:1 binding stoichiometry.

  9. Macrocyclic G-quadruplex ligands

    DEFF Research Database (Denmark)

    Nielsen, M C; Ulven, Trond

    2010-01-01

    G-quadruplex stabilizing compounds have recently received increased interest due to their potential application as anticancer therapeutics. A significant number of structurally diverse G-quadruplex ligands have been developed. Some of the most potent and selective ligands currently known are macr...

  10. Cell-SELEX-based selection and characterization of a G-quadruplex DNA aptamer against mouse dendritic cells.

    Science.gov (United States)

    Moghadam, M; Sankian, M; Abnous, K; Varasteh, A; Taghdisi, S M; Mahmoudi, M; Ramezani, M; Gholizadeh, Z; Ganji, A

    2016-07-01

    Targeting of dendritic cells (DCs) by aptamers increases antigen capture and presentation to the immune system. Our aim was to produce aptamers against DC molecules using the cell-SELEX procedure. For this purpose, 18 rounds of cell-SELEX were performed on mouse macrophage J774A.1 and CT26 as target and control cells, respectively. The selected aptamers were truncated and their binding to mouse macrophages, and immature and mature DCs analyzed. Two macrophage-specific aptamers, Seq6 and Seq7, were identified. A truncated form of Seq7, Seq7-4, 33 nucleotides in length and containing the G-quadruplex, bound macrophages and immature DCs with KD values in the nanomolar range. We anticipate that Seq7-4 has potential as a therapeutic tool in targeting of mouse macrophages and immature DCs to efficiently improve different immunotherapy approaches. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. A platinum(II) complex of liriodenine from traditional Chinese medicine (TCM): Cell cycle arrest, cell apoptosis induction and telomerase inhibition activity via G-quadruplex DNA stabilization.

    Science.gov (United States)

    Li, Yu-Lan; Qin, Qi-Pin; Liu, Yan-Cheng; Chen, Zhen-Feng; Liang, Hong

    2014-08-01

    Liriodenine (L), an antitumor active ingredient from the traditional Chinese medicine (TCM), Zanthoxylum nitidum, afforded a platinum(II) complex (1) of L, cis-[PtCl2(L)(DMSO)], which previously reported for its in vitro antitumor activity and intercalative binding with DNA. In this study, complex 1 was further discussed for its antitumor mechanism and structure-activity relationship, comparing with L and cisplatin. Towards the most sensitive BEL-7404 human hepatoma cells, complex 1 significantly induced cell cycle arrest at both G2/M phase and S phase. It suggests that double helix DNA is not the simplex intracellular target for 1. On the other hand, the BEL-7404 cells incubated with 1 and stained by Hoechst 33258 and AO/EB showed typical cell apoptosis in dose-dependent manner. The BEL-7404 cells incubated with 1 and stained by JC-1 were also characteristic for cell apoptosis on the loss of mitochondrial membrane potential. Furthermore, the G-quadruplex DNA binding property of complex 1 was also investigated by spectroscopic analyses, fluorescent indicator displacement (FID) assay and fluorescence resonance energy transfer (FRET) assay. The results indicated that 1 stabilized the human telomeric G4-HTG21 DNA better than L. The telomerase inhibition ratio of 1 ((62.50±0.03)%), which was examined by telomerase polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA), was much higher than L ((21.77±0.01)%). It can be ascribed to the better G4-HTG21 DNA stabilization of 1 than L. The results suggested that the nuclei, mitochondria and telomerase via G-quadruplex DNA stabilization all should be key targets for the antitumor mechanism of 1, in which the central platinum(II) played a key role.

  12. Ebola virus derived G-quadruplexes: Thiazole orange interaction.

    Science.gov (United States)

    Krafčíková, Petra; Demkovičová, Erika; Víglaský, Viktor

    2016-12-13

    The Ebola and Marburg viruses are some of the deadliest viruses in the world. In this study a series of G-rich DNA sequences derived from these types of viruses which possess the potential to form G-quadruplex structures are analyzed. A set of DNA oligonucleotides derived from original viral isolates was used as a representative modeling sequence with which to demonstrate the influence of thiazole orange on circular dichroism (CD) spectral profiles. The results show the unique profile of the induced CD (ICD) signal in the visible region caused by interactions between the ligand and G-quadruplexes. This ligand was found to stabilize the G-quadruplex structure and can also induce topological changes and facilitate G-quadruplex multimerization. Thus, the ICD signatures can be used to determine whether specific unknown sequences can form G-quadruplex motifs. The viral sequences were analyzed using standard spectral and electrophoretic methods. In addition, the ability to target G-quadruplexes located in filoviruses offers researchers attractive therapeutic targets which would be of particular use in the development of novel antiviral therapies. This article is part of a Special Issue entitled "G-quadruplex" Guest Editor: Dr. Concetta Giancola and Dr. Daniela Montesarchio.

  13. 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

    2016-05-01

    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.

  14. Right-handed and left-handed G-quadruplexes have the same DNA sequence: distinct conformations induced by an organic small molecule and potassium.

    Science.gov (United States)

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

    2016-08-21

    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.

  15. “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

    2010-01-01

    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.

  16. 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.

  17. Exploring the Interactions of the Dietary Plant Flavonoids Fisetin and Naringenin with G-Quadruplex and Duplex DNA, Showing Contrasting Binding Behavior: Spectroscopic and Molecular Modeling Approaches.

    Science.gov (United States)

    Bhattacharjee, Snehasish; Chakraborty, Sandipan; Sengupta, Pradeep K; Bhowmik, Sudipta

    2016-09-01

    Guanine-rich sequences have the propensity to fold into a four-stranded DNA structure known as a G-quadruplex (G4). G4 forming sequences are abundant in the promoter region of several oncogenes and become a key target for anticancer drug binding. Here we have studied the interactions of two structurally similar dietary plant flavonoids fisetin and naringenin with G4 as well as double stranded (duplex) DNA by using different spectroscopic and modeling techniques. Our study demonstrates the differential binding ability of the two flavonoids with G4 and duplex DNA. Fisetin more strongly interacts with parallel G4 structure than duplex DNA, whereas naringenin shows stronger binding affinity to duplex rather than G4 DNA. Molecular docking results also corroborate our spectroscopic results, and it was found that both of the ligands are stacked externally in the G4 DNA structure. C-ring planarity of the flavonoid structure appears to be a crucial factor for preferential G4 DNA recognition of flavonoids. The goal of this study is to explore the critical effects of small differences in the structure of closely similar chemical classes of such small molecules (flavonoids) which lead to the contrasting binding properties with the two different forms of DNA. The resulting insights may be expected to facilitate the designing of the highly selective G4 DNA binders based on flavonoid scaffolds.

  18. Ru-indoloquinoline complex as a selective and effective human telomeric G-quadruplex binder

    Science.gov (United States)

    Yu, Hui-juan; Zhao, Ying; Mo, Wei-jie; Hao, Zhi-feng; Yu, Lin

    2014-11-01

    Indoloquinoline and its derivatives have been reported to be a kind of efficient G-quadruplex binder and have been found to interact preferentially to intramolecular G-quadruplex and inhibit telomerase activity in human K562 cells and SW620 cells. In contrast to indoloquinoline derivatives, much less is known about the metal complex based on indoloquinoline or its derivative. In this report, we studied the interaction of ruthenium complex [Ru(bpy)2(itatp)]2+ containing indoloquinoline moiety with human telomeric G-quadruplex DNA (Telo22) and c-myc G-quadruplex DNA (Pu27) by UV-visible (UV-Vis), fluorescence spectroscopy, fluorescent intercalator displacement (FID), thermal denaturation studies and CD spectroscopy. The results suggest that [Ru(bpy)2(itatp)]2+ displays a strong π-π stacking interaction with human telomeric G-quadruplex with a high binding constant (∼107 M-1), but just exhibits moderate binding affinity to c-myc G-quadruplex, thus showing significant selectivity to human telomeric G-quadruplex. The CD titration results indicate that [Ru(bpy)2(itatp)]2+ could effectively convert Telo22 into antiparallel G-quadruplex conformation, while in the c-myc G-quadruplex case, instead of promoting Pu27 to fold into G-quadruplex, [Ru(bpy)2(itatp)]2+ destroys the parallel G-quadruplex structure of Pu27.

  19. Exploiting Polydopamine Nanospheres to DNA Computing: A Simple, Enzyme-Free and G-Quadruplex-Free DNA Parity Generator/Checker for Error Detection during Data Transmission.

    Science.gov (United States)

    Fan, Daoqing; Wang, Erkang; Dong, Shaojun

    2017-01-18

    Molecular logic devices with various functions play an indispensable role in molecular data transmission/processing. However, during any kinds of data transmission, a constant and unavoidable circumstance is the appearance of bit errors, which have serious effects on the regular logic computation. Fortunately, these errors can be detected via plugging a parity generator (pG) at the transmitting terminal and a parity checker (pC) at the receiving terminal. Herein, taking advantage of the efficient adsorption/quenching ability of polydopamine nanospheres toward fluorophore-labeled single-stranded DNA, we explored this biocompatible nanomaterial to DNA logic computation and constructed the first simple, enzyme-free, and G-quadruplex-free DNA pG/pC for error detection through data transmission. Besides, graphene oxide (GO) was innovatively introduced as the "corrective element" to perform the output-correction function of pC. All the erroneous outputs were corrected to normal conditions completely, ensuring the regular operation of later logic computing. The total operation of this non-G4 pG/pC system (error checking/output-correction) could be completed within 1 h (about (1)/3 of previous G4 platform) in a simpler and more efficient way. Notably, the odd pG/pC with analogous functions was also achieved through negative logic conversion to the fabricated even one. Furthermore, the same system could also perform three-input concatenated logic computation (XOR-INHIBIT), enriching the complexity of PDs-based logic computation.

  20. New dimeric carbazole-benzimidazole mixed ligands for the stabilization of human telomeric G-quadruplex DNA and as telomerase inhibitors. A remarkable influence of the spacer.

    Science.gov (United States)

    Maji, Basudeb; Kumar, Krishan; Muniyappa, K; Bhattacharya, Santanu

    2015-08-14

    The development of G-quadruplex (G4) DNA binding small molecules has become an important strategy for selectively targeting cancer cells. Herein, we report the design and evolution of a new kind of carbazole-based benzimidazole dimers for their efficient telomerase inhibition activity. Spectroscopic titrations reveal the ligands high affinity toward the G4 DNA with significantly higher selectivity over duplex-DNA. The electrophoretic mobility shift assay shows that the ligands efficiently promote the formation of G4 DNA even at a lower concentration of the stabilizing K(+) ions. The TRAP-LIG assay demonstrates the ligand's potential telomerase inhibition activity and also establishes that the activity proceeds via G4 DNA stabilization. An efficient nuclear internalization of the ligands in several common cancer cells (HeLa, HT1080, and A549) also enabled differentiation between normal HFF cells in co-cultures of cancer and normal ones. The ligands induce significant apoptotic response and antiproliferative activity toward cancer cells selectively when compared to the normal cells.

  1. 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: hlwang@rcees.ac.cn [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085 (China)

    2014-02-17

    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

  2. Sequence, Stability, Structure of G-Quadruplexes and Their Drug Interactions

    Science.gov (United States)

    Chen, Yuwei; Yang, Danzhou

    2012-01-01

    Although DNA is most widely known to store and pass along genetic information, the discovery of G-quadruplex structures has illuminated a new role of DNA in biology. DNA G-quadruplexes are four-stranded globular nucleic acid secondary structures formed in specific G-rich sequences with biological significance, such as human telomeres and oncogene promoters. This review focuses on the unimolecular DNA G-quadruplexes, which can readily form in solution under physiological conditions and are considered to be most biologically relevant. Available structural data show a great conformational diversity of unimolecular G-quadruplexes, amenable to small molecule drug targeting. The relationship of sequence, structure, and stability of unimolecular DNA G-quadruplexes, as well as the recent progress on interactions with small molecule compounds and insights into rational design of G-quadruplex-interactive molecules, will be discussed. PMID:22956454

  3. Investigating actinomycin D binding to G-quadruplex, i-motif and double-stranded DNA in 27-nt segment of c-MYC gene promoter.

    Science.gov (United States)

    Niknezhad, Zhila; Hassani, Leila; Norouzi, Davood

    2016-01-01

    c-MYC DNA is an attractive target for drug design, especially for cancer chemotherapy. Around 90% of c-MYC transcription is controlled by NHE III1, whose 27-nt purine-rich strand has the ability to form G-quadruplex structure. In this investigation, interaction of ActD with 27-nt G-rich strand (G/c-MYC) and its equimolar mixture with the complementary sequence, (GC/c-MYC) as well as related C-rich oligonucleotide (C/c-MYC) was evaluated. Molecular dynamic simulations showed that phenoxazine and lactone rings of ActD come close to the outer G-tetrad nucleotides indicating that ActD binds through end-stacking to the quadruplex DNA. RMSD and RMSF revealed that fluctuation of the quadruplex DNA increases upon interaction with the drug. The results of spectrophotometry and spectrofluorometry indicated that ActD most probably binds to the c-MYC quadruplex and duplex DNA via end-stacking and intercalation, respectively and polarity of ActD environment decreases due to the interaction. It was also found that binding of ActD to the GC-rich DNA is stronger than the two other forms of DNA. Circular dichroism results showed that the type of the three forms of DNA structures doesn't change, but their compactness alters due to their interaction with ActD. Finally, it can be concluded that ActD binds differently to double stranded DNA, quadruplex DNA and i-motif. Copyright © 2015 Elsevier B.V. All rights reserved.

  4. Dual recognition of the human telomeric G-quadruplex by a neomycin-anthraquinone conjugate.

    Science.gov (United States)

    Ranjan, Nihar; Davis, Erik; Xue, Liang; Arya, Dev P

    2013-06-28

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

  5. G-Quadruplex Forming Oligonucleotides as Anti-HIV Agents.

    Science.gov (United States)

    Musumeci, Domenica; Riccardi, Claudia; Montesarchio, Daniela

    2015-09-22

    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.

  6. Dispersed dynamics of solvation in G-quadruplex DNA: comparison of dynamic Stokes shifts of probes in parallel and antiparallel quadruplex structures

    Science.gov (United States)

    Kiran Singh, Moirangthem; Shweta, Him; Sen, Sobhan

    2016-09-01

    G-quadruplex DNA (GqDNA) structures play an important role in many specific cellular functions and are promising anti-tumor targets for small molecules (ligands). Here, we measured the dynamic Stokes shift of a ligand (Hoechst) bound to parallel c-Myc (mPu22) GqDNA over five decades of time from 100 fs to 10 ns, and compared it with the previously reported dynamics of DAPI bound to antiparallel human telomeric (hTelo22) GqDNA (Pal et al 2015 J. Phys. Chem. Lett. 6 1754). Stokes shift data from fluorescence up-conversion and time-correlated single photon counting experiments was combined to cover the broad dynamic range. The results show that the solvation dynamics of Hoechst in parallel mPu22 GqDNA follow a power law relaxation, added to fast 2 ps exponential relaxation, from 100 fs to 10 ns, with only a subtle difference of power law exponents in the two ligand-GqDNA systems (0.06 in Hoechst-mPu22 compared to 0.16 in DAPI-hTelo22). We measured steady-state fluorescence spectra and time-resolved anisotropy decays which confirm the tight binding of Hoechst to parallel mPu22 with a binding constant of ~1  ×  105 M-1. The molecular docking of Hoechst in parallel GqDNA followed by a 50 ns molecular dynamics (MD) simulation on a Hoechst-GqDNA complex reveals that Hoechst binds to one of the outer G-tetrads by end-stacking near G13 and G4, which is different from the binding site of DAPI inside a groove of antiparallel hTelo22 GqDNA. Reconciling previous experimental and simulation results, we assign the 2 ps component to the hydration dynamics of only weakly perturbed water near mPu22 and the power law relaxation to the coupled motion of water and DNA (i.e. DNA backbone, unpaired bases and loops connecting G-tetrads) which come near the Hoechst inside parallel GqDNA.

  7. Colorimetric and fluorescence detection of G-quadruplex nucleic acids with a coumarin-benzothiazole probe.

    Science.gov (United States)

    Yan, Jin-wu; Tian, Yi-guang; Tan, Jia-heng; Huang, Zhi-shu

    2015-11-01

    A colorimetric and red-emitting fluorescent dual probe for G-quadruplexes was devised with a conjugated coumarin-benzothiazole scaffold. Its significant and distinct changes in both color and fluorescence enable the label-free and visual detection of G-quadruplex structures. In addition, this probe gives a distinct strong emission response to the nucleoli in fixed cells imaging, which might be attributed to the interaction between the probe and rDNA G-quadruplex based on the chromatin immunoprecipitation assay. All these results suggest its promising application prospects in the G-quadruplex research field.

  8. State-of-the-art methodologies for the discovery and characterization of DNA G-quadruplex binders.

    Science.gov (United States)

    Pagano, Bruno; Cosconati, Sandro; Gabelica, Valerie; Petraccone, Luigi; De Tito, Stefano; Marinelli, Luciana; La Pietra, Valeria; di Leva, Francesco Saverio; Lauri, Ilaria; Trotta, Roberta; Novellino, Ettore; Giancola, Concetta; Randazzo, Antonio

    2012-01-01

    Nowadays, the molecular basis of interaction between low molecular weight compounds and biological macromolecules is the subject of numerous investigations aimed at the rational design of molecules with specific therapeutic applications. In the last decades, it has been demonstrated that DNA quadruplexes play a critical role in several biological processes both at telomeric and gene promoting levels thus providing a great stride in the discovery of ligands able to interact with such a biologically relevant DNA conformation. So far, a number of experimental and computational approaches have been successfully employed in order to identify new ligands and to characterize their binding to the DNA. The main focus of this review is the description of these methodologies, placing a particular emphasis on computational methods, isothermal titration calorimetry (ITC), mass spectrometry (MS), nuclear magnetic resonance (NMR), circular dichroism (CD) and fluorescence spectroscopies.

  9. Differential scanning calorimetry to investigate G-quadruplexes structural stability.

    Science.gov (United States)

    Pagano, Bruno; Randazzo, Antonio; Fotticchia, Iolanda; Novellino, Ettore; Petraccone, Luigi; Giancola, Concetta

    2013-11-01

    Differential Scanning Calorimetry (DSC) is a straightforward methodology to characterize the energetics of thermally-induced transitions of DNA and other biological macromolecules. Therefore, DSC has been used to study the thermodynamic stability of several nucleic acids structures. G-quadruplexes are among the most important non-canonical nucleic acid architectures that are receiving great consideration. This article reports examples on the contribution of DSC to the knowledge of G-quadruplex structures. The selected case studies show the potential of this method in investigating the structure stability of G-quadruplex forming nucleic acids, and in providing information on their structural complexity. Indeed, DSC can determine thermodynamic parameters of G-quadruplex folding/unfolding processes, but it can also be useful to reveal the formation of multiple conformations or the presence of intermediate states along the unfolding pathway, and to evaluate the impact of chemical modifications on their structural stability. This article aims to show that DSC is an important complementary methodology to structural techniques, such as NMR and X-ray crystallography, in the study of G-quadruplex forming nucleic acids. Copyright © 2013 Elsevier Inc. All rights reserved.

  10. A label-free ultrasensitive fluorescence detection of viable Salmonella enteritidis using enzyme-induced cascade two-stage toehold strand-displacement-driven assembly of G-quadruplex DNA.

    Science.gov (United States)

    Zhang, Peng; Liu, Hui; Ma, Suzhen; Men, Shuai; Li, Qingzhou; Yang, Xin; Wang, Hongning; Zhang, Anyun

    2016-06-15

    The harm of Salmonella enteritidis (S. enteritidis ) to public health mainly by contaminating fresh food and water emphasizes the urgent need for rapid detection techniques to help control the spread of the pathogen. In this assay, an newly designed capture probe complex that contained specific S. enteritidis-aptamer and hybridized signal target sequence was used for viable S. enteritidis recognition directly. In the presence of the target S. enteritidis, single-stranded target sequences were liberated and initiated the replication-cleavage reaction, producing numerous G-quadruplex structures with a linker on the 3'-end. And then, the sensing system took innovative advantage of quadratic linker-induced strand-displacement for the first time to release target sequence in succession, leading to the cyclic reuse of the target sequences and cascade signal amplification, thereby achieving the successive production of G-quadruplex structures. The fluorescent dye, N-Methyl mesoporphyrin IX, binded to these G-quadruplex structures and generated significantly enhanced fluorescent signals to achieve highly sensitive detection of S. enteritidis down to 60 CFU/mL with a linear range from 10(2) to 10(7)CFU/mL. By coupling the cascade two-stage target sequences-recyclable toehold strand-displacement with aptamer-based target recognition successfully, it is the first report on a novel non-label, modification-free and DNA extraction-free ultrasensitive fluorescence biosensor for detecting viable S. enteritidis directly, which can discriminate from dead S. enteritidis.

  11. Irregular G-quadruplexes Found in the Untranslated Regions of Human mRNAs Influence Translation.

    Science.gov (United States)

    Bolduc, François; Garant, Jean-Michel; Allard, Félix; Perreault, Jean-Pierre

    2016-10-07

    G-quadruplex structures are composed of coplanar guanines and are found in both DNA and RNA. They are formed by the stacking of two or more G-quartets that are linked together by three loops. The current belief is that RNA G-quadruplexes include loops of l to 7 nucleotides in length, although recent evidence indicates that the central loop (loop 2) can be longer if loops 1 and 3 are limited to a single nucleotide each. With the objective of broadening the definition of irregular RNA G-quadruplexes, a bioinformatic search was performed to find potential G-quadruplexes located in the untranslated regions of human mRNAs (i.e. in the 5' and 3'-UTRs) that contain either a long loop 1 or 3 of up to 40 nucleotides in length. RNA molecules including the potential sequences were then synthesized and examined in vitro by in-line probing for the formation of G-quadruplex structures. The sequences that adopted a G-quadruplex structure were cloned into a luciferase dual vector and examined for their ability to modulate translation in cellulo Some irregular G-quadruplexes were observed to either promote or repress translation regardless of the position or the size of the long loop they possessed. Even if the composition of a RNA G-quadruplex is not quite completely understood, the results presented in this report clearly demonstrate that what defines a RNA G-quadruplex is much broader than what we previously believed.

  12. Solution NMR Structure of a Ligand/Hybrid-2-G-Quadruplex Complex Reveals Rearrangements that Affect Ligand Binding.

    Science.gov (United States)

    Wirmer-Bartoschek, Julia; Bendel, Lars Erik; Jonker, Hendrik R A; Grün, J Tassilo; Papi, Francesco; Bazzicalupi, Carla; Messori, Luigi; Gratteri, Paola; Schwalbe, Harald

    2017-06-12

    Telomeric G-quadruplexes have recently emerged as drug targets in cancer research. Herein, we present the first NMR structure of a telomeric DNA G-quadruplex that adopts the biologically relevant hybrid-2 conformation in a ligand-bound state. We solved the complex with a metalorganic gold(III) ligand that stabilizes G-quadruplexes. Analysis of the free and bound structures reveals structural changes in the capping region of the G-quadruplex. The ligand is sandwiched between one terminal G-tetrad and a flanking nucleotide. This complex structure involves a major structural rearrangement compared to the free G-quadruplex structure as observed for other G-quadruplexes in different conformations, invalidating simple docking approaches to ligand-G-quadruplex structure determination. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Studies on non-covalent interaction of coumarin attached pyrimidine and 1-methyl indole 1,2,3 triazole analogues with intermolecular telomeric G-quadruplex DNA using ESI-MS and spectroscopy.

    Science.gov (United States)

    Raju, G; Srinivas, R; Reddy, M Damoder; Reddy, Ch Raji; Nagesh, N

    2014-01-01

    In the present study, electrospray ionization mass spectrometry (ESI-MS) and spectroscopy have been used to evaluate the non-covalent interaction, stoichiometry, and selectivity of two synthetic coumarin-attached nucleoside and non-nucleoside 1,2,3-triazoles, namely, (1-(5-(hydroxymethyl)-4-(4-((2-oxo-2H-chromen-4-yloxy)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-furan-2-yl)5-methyl pyrimidine-2,4(1H,3H)-dione (Tr1) and 4-((1-((-1-methyl-1H-indol-2-yl)methyl)-1H-1,2,3-triazol-4-yl)methoxy)-2H-chromen-2-one (Tr2) with two different human telomeric intermolecular G-quadruplex DNA structures formed by d(T2AG3) and d(T2AG3)2 sequences. ESI-MS studies indicate that Tr1 specifically interacts with four-stranded intermolecular parallel quadruplex complex, whereas Tr2 interacts with two hairpin as well as four-stranded intermolecular parallel quadruplex complexes. UV-Visible spectroscopic studies suggest that Tr1 and Tr2 interact with G-quadruplex structure and unwind them. Job plots show that stoichiometry of ligand:quadruplex DNA is 1:1. Circular dichroism (CD) studies of G-quadruplex DNA and Tr1/Tr2 ligands manifest that they unfold DNA on interaction. Fluorescence studies demonstrate that ligand molecules intercalate between the two stacks of quadruplex DNA and non-radiative energy transfer occurs between the excited ligand molecules (donor) and quadruplex DNA (acceptor), resulting in enhancement of fluorescence emission intensity. Thus, these studies suggest that nucleoside and non-nucleoside ligands efficiently interact with d(T2AG3) and d(T2AG3)2 G-quadruplex DNA but the interaction is not alike with all kinds of quadruplex DNA, this is probably due to the variation in the pharmacophores and structure of the ligand molecules.

  14. Single-molecule choreography between telomere proteins and G quadruplexes.

    Science.gov (United States)

    Hopfner, Karl-Peter

    2014-06-10

    Telomeric DNA binds proteins to protect chromosome ends, but it also adopts G quadruplex (GQ) structures. Two new studies by Hwang and colleagues (in this issue of Structure) and Ray and colleagues (published elsewhere) use single molecule imaging to reveal how GQs affect the binding of different telomere associated proteins. The data suggest that GQs play important roles in regulating accessibility of telomeres.

  15. p53 binds human telomeric G-quadruplex in vitro.

    Science.gov (United States)

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

    2016-01-01

    The tumor suppressor protein p53 is a key factor in genome stability and one of the most studied of DNA binding proteins. This is the first study on the interaction of wild-type p53 with guanine quadruplexes formed by the human telomere sequence. Using electromobility shift assay and ELISA, we show that p53 binding to telomeric G-quadruplexes increases with the number of telomeric repeats. Further, p53 strongly favors G-quadruplexes folded in potassium over those formed in sodium, thus indicating the telomeric G-quadruplex conformational selectivity of p53. The presence of the quadruplex-stabilizing ligand, N-methyl mesoporphyrin IX (NMM), increases p53 recognition of G-quadruplexes in potassium. Using deletion mutants and selective p53 core domain oxidation, both p53 DNA binding domains are shown to be crucial for telomeric G-quadruplex recognition.

  16. Interactions of daidzin with intramolecular G-quadruplex.

    Science.gov (United States)

    Li, Wei; Zhang, Ming; Zhang, Jin-li; Li, Hui-qing; Zhang, Xiao-chen; Sun, Qian; Qiu, Chun-mei

    2006-09-04

    The potential interaction of daidzin, an ingredient of soy isoflavones, with human telomeric antiparallel G-quadruplex dAG(3)(T(2)AG(3))(3) was studied using ESI-MS, PAGE, CD and molecular simulation. Experimental studies indicated that daidzin molecules interacted with dAG(3)(T(2)AG(3))(3) and formed DNA-daidzin complex with the stoichiometric ratio of 1:1 and 1:2. The transition temperature of the G-quadruplex increased at higher ratio of daidzin to DNA. Under molecular crowding conditions the interactions between daidzin and the G-quadruplex become much stronger. Combining computational simulation and experimental results, it is demonstrated that the dAG(3)(T(2)AG(3))(3)/daidzin complex with a stoichiometric ratio of 1:1 is stabilized through the pi-pi conjugacy interactions and hydrogen bondings between daidzin and the bases of G-quadruplex. This work provides guidance not only on exploring the molecular anti-cancer mechanism of dietary isoflavones, but also searching small natural products as promising anticancer candidates that can inhibit telomerase activity.

  17. The binding modes of carbazole derivatives with telomere G-quadruplex

    Science.gov (United States)

    Zhang, Xiu-feng; Zhang, Hui-juan; Xiang, Jun-feng; Li, Qian; Yang, Qian-fan; Shang, Qian; Zhang, Yan-xia; Tang, Ya-lin

    2010-10-01

    It is reported that carbazole derivatives can stabilize G-quadruplex DNA structure formed by human telomeric sequence, and therefore, they have the potential to serve as anti-cancer agents. In this present study, in order to further explore the binding mode between carbazole derivatives and G-quadruplex formed by human telomeric sequence, two carbazole iodides (BMVEC, MVEC) molecules were synthesized and used to investigate the interaction with the human telomeric parallel and antiparallel G-quadruplex structures by NMR, CD and molecular modeling study. Interestingly, it is the pivotal the cationic charge pendant groups of pyridinium rings of carbazole that plays an essential role in the stabilizing and binding mode of the human telomeric sequences G-quadruplex structure. It was found that BMVEC with two cationic charge pendant groups of pyridinium rings of 9-ethylcarbazole cannot only stabilize parallel G-quadruple of Hum6 by groove binding and G-tetrad stacking modes and antiparallel G-quadruplex of Hum22 by groove binding, but also induce the formation of mixed G-quadruplex of Hum22. While MVEC with one cationic charge pendant groups of pyridinium ring only can bind with the parallel G-quadruplex of Hum6 by the stacking onto the G4 G-tetrad and could not interact with the G-quadruplex of Hum22.

  18. In silico identification of novel ligands for G-quadruplex in the c-MYC promoter.

    Science.gov (United States)

    Kang, Hyun-Jin; Park, Hyun-Ju

    2015-04-01

    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.

  19. Interaction of KRAS G-quadruplex with natural polyphenols: A spectroscopic analysis with molecular modeling.

    Science.gov (United States)

    Pattanayak, Rudradip; Basak, Pijush; Sen, Srikanta; Bhattacharyya, Maitree

    2016-08-01

    Researchers are endeavoring to find out new therapeutics for curing cancer and G-quadruplex DNA has already been identified as a prospective one in this venture. Stabilizing G-quadruplex structures of telomere has emerged to be an important strategy in this context. Mutation in KRAS is mostly responsible for pancreatic, lung and colon cancer. In this present study we explored binding and conformational behaviour of G-quadruplex with different ligands by utilizing several biophysical techniques. Natural polyphenols like Curcumin and Ellagic acid were observed to bind with the G-quadruplex and enhance the melting temperature significantly indicating higher stability. UV-vis spectroscopy confirms formation of G quadruplex-ligand complex for both the compounds with specific binding affinity. Fluorimetric studies revealed that Ellagic acid had stronger binding affinity, 1.10×10(5)M(-1) compared to Curcumin, 1.6×10(4)M(-1) towards G-quadruplex. Interestingly, Curcumin provides greater stability by stacking on the top of the quadruplex structure with the help of the loops compared to Ellagic acid as is evident by docking studies. The keto form of curcumin showed stronger affinity than the enol form. We have developed a general model to estimate the influence of the ligands towards stabilizing the G-quadruplex subsequently characterizing the binding profile to enlighten prospective therapeutics.

  20. Accurate high-throughput identification of parallel G-quadruplex topology by a new tetraaryl-substituted imidazole.

    Science.gov (United States)

    Hu, Ming-Hao; Chen, Shuo-Bin; Wang, Yu-Qing; Zeng, You-Mei; Ou, Tian-Miao; Li, Ding; Gu, Lian-Quan; Huang, Zhi-Shu; Tan, Jia-Heng

    2016-09-15

    G-quadruplex nucleic acids are four-stranded DNA or RNA secondary structures that are formed in guanine-rich sequences. These structures exhibit extensive structural polymorphism and play a pivotal role in the control of a variety of cellular processes. To date, diverse approaches for high-throughput identification of G-quadruplex structures have been successfully developed, but high-throughput methods for further characterization of their topologies are still lacking. In this study, we report a new tetra-arylimidazole probe psIZCM-1, which was found to display significant and distinctive changes in both the absorption and the fluorescence spectra in the presence of parallel G-quadruplexes but show insignificant changes upon interactions with anti-parallel G-quadruplexes or other non-quadruplex oligonucleotides. In view of this dual-output feature, we used psIZCM-1 to identify the parallel G-quadruplexes from a large set of 314 oligonucleotides (including 300 G-quadruplex-forming oligonucleotides and 14 non-quadruplex oligonucleotides) via a microplate reader and accordingly established a high-throughput method for the characterization of parallel G-quadruplex topologies. The accuracy of this method was greater than 95%, which was much higher than that of the commercial probe NMM. To make the approach more practical, we further combined psIZCM-1 with another G-quadruplex probe IZCM-7 to realize the high-throughput classification of parallel, anti-parallel G-quadruplexes and non-quadruplex structures.

  1. Fluorescence intercalator displacement assay for screening G4 ligands towards a variety of G-quadruplex structures.

    Science.gov (United States)

    Tran, Phong Lan Thao; Largy, Eric; Hamon, Florian; Teulade-Fichou, Marie-Paule; Mergny, Jean-Louis

    2011-08-01

    The potential formation of G-quadruplexes in many regions of the genome makes them an attractive target for drug design. A large number of small molecules synthesized in recent years display an ability to selectively target and stabilize G-quadruplexes. To screen for G4 ligands, we modified a G4-FID (G-quadruplex Fluorescent Intercalator Displacement) assay. This test is based on the displacement of an "on/off" fluorescence probe, Thiazole Orange (TO), from quadruplex or duplex DNA matrices by increasing amounts of a putative ligand. Selectivity measurements can easily be achieved by comparing the ability of the ligand to displace TO from various quadruplex and duplex structures. G4-FID requires neither modified oligonucleotides nor specific equipment and is an isothermal experiment. This test was adapted for high throughput screening onto 96-well plates allowing the comparison of more than twenty different structures. Fifteen different known G4 ligands belonging to different families were tested. Most compounds showed a good G4 vs duplex selectivity but exhibited little, if any, specificity for one quadruplex sequence over the others. The quest for the "perfect" specific G4 ligand is not over yet! Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  2. G-Quadruplexes as Potential Therapeutic Targets for Embryonal Tumors

    Directory of Open Access Journals (Sweden)

    Michael Grotzer

    2013-10-01

    Full Text Available Embryonal tumors include a heterogeneous group of highly malignant neoplasms that primarily affect infants and children and are characterized by a high rate of mortality and treatment-related morbidity, hence improved therapies are clearly needed. G-quadruplexes are special secondary structures adopted in guanine (G-rich DNA sequences that are often present in biologically important regions, e.g. at the end of telomeres and in the regulatory regions of oncogenes such as MYC. Owing to the significant roles that both telomeres and MYC play in cancer cell biology, G-quadruplexes have been viewed as emerging therapeutic targets in oncology and as tools for novel anticancer drug design. Several compounds that target these structures have shown promising anticancer activity in tumor xenograft models and some of them have entered Phase II clinical trials. In this review we examine approaches to DNA targeted cancer therapy, summarize the recent developments of G-quadruplex ligands as anticancer drugs and speculate on the future direction of such structures as a potential novel therapeutic strategy for embryonal tumors of the nervous system.

  3. 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

    2011-01-01

    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...

  4. Metal Cations in G-Quadruplex Folding and Stability

    Science.gov (United States)

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

    2016-01-01

    This review is focused on the structural and physicochemical 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 the presence of the physiologically relevant metal ions, however, prediction of conformation of such GQs is still out of the realm. PMID:27668212

  5. Metal Cations in G-Quadruplex Folding and Stability

    Science.gov (United States)

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

    2016-09-01

    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.

  6. Metal Cations in G-Quadruplex Folding and Stability

    Directory of Open Access Journals (Sweden)

    Debmalya Bhattacharyya

    2016-09-01

    Full Text Available 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.

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

    Science.gov (United States)

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

    2017-07-15

    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 G-quadruplex. In the current study, we have examined the effects of a G-quadruplex binding and stabilizing agent, BRACO-19, on HHV-6A chromosomal integration. By stabilizing G-quadruplex structures, BRACO-19 affects the ability of the telomerase complex to elongate telomeres. Our results indicate that BRACO-19 reduces the number of clones harboring integrated HHV-6A. This study is the first of its kind and suggests that telomerase activity is essential to restore a functional telomere of adequate length following HHV-6A integration. Copyright © 2017 American Society for Microbiology.

  8. General cell-binding activity of intramolecular G-quadruplexes with parallel structure.

    Science.gov (United States)

    Chang, Tianjun; Qi, Cui; Meng, Jie; Zhang, Nan; Bing, Tao; Yang, Xianda; Cao, Zehui; Shangguan, Dihua

    2013-01-01

    G-quadruplexes (G4s) are four-stranded nucleic acid structures adopted by some repetitive guanine-rich sequences. Putative G-quadruplex-forming sequences (PQSs) are highly prevalent in human genome. Recently some G4s have been reported to have cancer-selective antiproliferative activity. A G4 DNA, AS1411, is currently in phase II clinical trials as an anticancer agent, which is reported to bind tumor cells by targeting surface nucleolin. AS1411 also has been extensively investigated as a target-recognition element for cancer cell specific drug delivery or cancer cell imaging. Here we show that, in addition to AS1411, intramolecular G4s with parallel structure (including PQSs in genes) have general binding activity to many cell lines with different affinity. The binding of these G4s compete with each other, and their targets are certain cellular surface proteins. The tested G4s exhibit enhanced cellular uptake than non-G4 sequences. This uptake may be through the endosome/lysosome pathway, but it is independent of cellular binding of the G4s. The tested G4s also show selective antiproliferative activity that is independent of their cellular binding. Our findings provide new insight into the molecular recognition of G4s by cells; offer new clues for understanding the functions of G4s in vivo, and may extend the potential applications of G4s.

  9. General cell-binding activity of intramolecular G-quadruplexes with parallel structure.

    Directory of Open Access Journals (Sweden)

    Tianjun Chang

    Full Text Available G-quadruplexes (G4s are four-stranded nucleic acid structures adopted by some repetitive guanine-rich sequences. Putative G-quadruplex-forming sequences (PQSs are highly prevalent in human genome. Recently some G4s have been reported to have cancer-selective antiproliferative activity. A G4 DNA, AS1411, is currently in phase II clinical trials as an anticancer agent, which is reported to bind tumor cells by targeting surface nucleolin. AS1411 also has been extensively investigated as a target-recognition element for cancer cell specific drug delivery or cancer cell imaging. Here we show that, in addition to AS1411, intramolecular G4s with parallel structure (including PQSs in genes have general binding activity to many cell lines with different affinity. The binding of these G4s compete with each other, and their targets are certain cellular surface proteins. The tested G4s exhibit enhanced cellular uptake than non-G4 sequences. This uptake may be through the endosome/lysosome pathway, but it is independent of cellular binding of the G4s. The tested G4s also show selective antiproliferative activity that is independent of their cellular binding. Our findings provide new insight into the molecular recognition of G4s by cells; offer new clues for understanding the functions of G4s in vivo, and may extend the potential applications of G4s.

  10. Ruthenium Complex “Light Switches” that are Selective for Different G-Quadruplex Structures

    Science.gov (United States)

    Wachter, Erin; Moyá, Diego; Parkin, Sean

    2015-01-01

    Recognition and regulation of G-quadruplex nucleic acid structures is an important goal for the development of chemical tools and medicinal agents. The addition of a bromo substituent to the dipyridylphenazine (dppz) ligands in the photophysical “light switch”, [Ru(bpy)2dppz]2+, and the photochemical “light switch”, [Ru(bpy)2dmdppz]2+, creates compounds with increased selectivity for an intermolecular parallel G-quadruplex and the mixed-hybrid G-quadruplex, respectively. When [Ru(bpy)2dppz-Br]2+ and [Ru(bpy)2dmdppz-Br]2+ are incubated with the G-quadruplexes, they have a stabilizing effect on the DNA structures. Activation of [Ru(bpy)2dmdppz-Br]2+ with light results in covalent adduct formation with the DNA. These complexes demonstrate that subtle chemical modifications of RuII complexes can alter G-quadruplex selectivity, and could be useful for the rational design of in vivo G-quadruplex probes. PMID:26560887

  11. Human telomeric sequence forms a hybrid-type intramolecular G-quadruplex structure with mixed parallel/antiparallel strands in potassium solution

    OpenAIRE

    Ambrus, Attila; Chen, Ding; Dai, Jixun; Bialis, Tiffanie; Jones, Roger A.; Yang, Danzhou

    2006-01-01

    Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG). The formation and stabilization of DNA G-quadruplexes in the human telomeric sequence have been shown to inhibit the activity of telomerase, thus the telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. However, knowledge of the intact human telomeric G-quadruplex structure(s) formed under physiological conditions is a prerequisite for structure-based rational drug ...

  12. Assembly of supramolecular DNA complexes containing both G-quadruplexes and i-motifs by enhancing the G-repeat-bearing capacity of i-motifs

    Science.gov (United States)

    Cao, Yanwei; Gao, Shang; Yan, Yuting; Bruist, Michael F.; Wang, Bing; Guo, Xinhua

    2017-01-01

    The single-step assembly of supramolecular complexes containing both i-motifs and G-quadruplexes (G4s) is demonstrated. This can be achieved because the formation of four-stranded i-motifs appears to be little affected by certain terminal residues: a five-cytosine tetrameric i-motif can bear ten-base flanking residues. However, things become complex when different lengths of guanine-repeats are added at the 3′ or 5′ ends of the cytosine-repeats. Here, a series of oligomers d(XGiXC5X) and d(XC5XGiX) (X = A, T or none; i < 5) are designed to study the impact of G-repeats on the formation of tetrameric i-motifs. Our data demonstrate that tetramolecular i-motif structure can tolerate specific flanking G-repeats. Assemblies of these oligonucleotides are polymorphic, but may be controlled by solution pH and counter ion species. Importantly, we find that the sequences d(TGiAC5) can form the tetrameric i-motif in large quantities. This leads to the design of two oligonucleotides d(TG4AC7) and d(TGBrGGBrGAC7) that self-assemble to form quadruplex supramolecules under certain conditions. d(TG4AC7) forms supramolecules under acidic conditions in the presence of K+ that are mainly V-shaped or ring-like containing parallel G4s and antiparallel i-motifs. d(TGBrGGBrGAC7) forms long linear quadruplex wires under acidic conditions in the presence of Na+ that consist of both antiparallel G4s and i-motifs. PMID:27899568

  13. Phenyl 1,2,3-triazole-thymidine ligands stabilize G-quadruplex DNA, inhibit DNA synthesis and potentially reduce tumor cell proliferation over 3'-azido deoxythymidine.

    Science.gov (United States)

    Mahesh Kumar, Jerald; Idris, Mohammed M; Srinivas, Gunda; Vinay Kumar, Pallerla; Meghah, Vuppalapaty; Kavitha, Mitta; Reddy, Chada Raji; Mainkar, Prathama S; Pal, Biswajit; Chandrasekar, Srivari; Nagesh, Narayana

    2013-01-01

    Triazoles are known for their non-toxicity, higher stability and therapeutic activity. Few nucleoside (L1, L2 and L3) and non-nucleoside 1,2,3-triazoles (L4-L14) were synthesised using click chemistry and they were screened for tumor cell cytotoxicity and proliferation. Among these triazole ligands studied, nucleoside ligands exhibited higher potential than non-nucleoside ligands. The nucleoside triazole analogues, 3'-Phenyl-1,2,3- triazole-thymidine (L2) and 3'-4-Chlorophenyl-1,2,3-triazole-thymidine (L3), demonstrated higher cytotoxicity in tumor cells than in normal cells. The IC₅₀ value for L3 was lowest (50 µM) among the ligands studied. L3 terminated cell cycle at S, G2/M phases and enhanced sub-G1 populations, manifesting induction of apoptosis in tumor cells. Confocal studies indicated that nucleoside triazole ligands (L2/L3) cause higher DNA fragmentation than other ligands. Preclinical experiments with tumor-induced mice showed greater reduction in tumor size with L3. In vitro DNA synthesis reaction with L3 exhibited higher DNA synthesis inhibition with quadruplex forming DNA (QF DNA) than non quadruplex forming DNA (NQF DNA). T(m) of quadruplex DNA increased in the presence of L3, indicating its ability to enhance stability of quadruplex DNA at elevated temperature and the results indicate that it had higher affinity towards quadruplex DNA than the other forms of DNA (like dsDNA and ssDNA). From western blot experiment, it was noticed that telomerase expression levels in the tissues of tumor-induced mice were found to be reduced on L3 treatment. Microcalorimetry results emphasise that two nucleoside triazole ligands (L2/L3) interact with quadruplex DNA with significantly higher affinity (K(d)≈10⁻⁷ M). Interestingly the addition of an electronegative moiety to the phenyl group of L2 enhanced its anti-proliferative activity. Though IC₅₀ values are not significantly low with L3, the studies on series of synthetic 1,2,3-triazole ligands are

  14. Phenyl 1,2,3-triazole-thymidine ligands stabilize G-quadruplex DNA, inhibit DNA synthesis and potentially reduce tumor cell proliferation over 3'-azido deoxythymidine.

    Directory of Open Access Journals (Sweden)

    Jerald Mahesh Kumar

    Full Text Available Triazoles are known for their non-toxicity, higher stability and therapeutic activity. Few nucleoside (L1, L2 and L3 and non-nucleoside 1,2,3-triazoles (L4-L14 were synthesised using click chemistry and they were screened for tumor cell cytotoxicity and proliferation. Among these triazole ligands studied, nucleoside ligands exhibited higher potential than non-nucleoside ligands. The nucleoside triazole analogues, 3'-Phenyl-1,2,3- triazole-thymidine (L2 and 3'-4-Chlorophenyl-1,2,3-triazole-thymidine (L3, demonstrated higher cytotoxicity in tumor cells than in normal cells. The IC₅₀ value for L3 was lowest (50 µM among the ligands studied. L3 terminated cell cycle at S, G2/M phases and enhanced sub-G1 populations, manifesting induction of apoptosis in tumor cells. Confocal studies indicated that nucleoside triazole ligands (L2/L3 cause higher DNA fragmentation than other ligands. Preclinical experiments with tumor-induced mice showed greater reduction in tumor size with L3. In vitro DNA synthesis reaction with L3 exhibited higher DNA synthesis inhibition with quadruplex forming DNA (QF DNA than non quadruplex forming DNA (NQF DNA. T(m of quadruplex DNA increased in the presence of L3, indicating its ability to enhance stability of quadruplex DNA at elevated temperature and the results indicate that it had higher affinity towards quadruplex DNA than the other forms of DNA (like dsDNA and ssDNA. From western blot experiment, it was noticed that telomerase expression levels in the tissues of tumor-induced mice were found to be reduced on L3 treatment. Microcalorimetry results emphasise that two nucleoside triazole ligands (L2/L3 interact with quadruplex DNA with significantly higher affinity (K(d≈10⁻⁷ M. Interestingly the addition of an electronegative moiety to the phenyl group of L2 enhanced its anti-proliferative activity. Though IC₅₀ values are not significantly low with L3, the studies on series of synthetic 1,2,3-triazole ligands

  15. Xanthine and 8-oxoguanine in G-quadruplexes: formation of a G·G·X·O tetrad.

    Science.gov (United States)

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

    2015-12-02

    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.

  16. A tutorial review for employing enzymes for the construction of G-quadruplex-based sensing platforms.

    Science.gov (United States)

    Ma, Dik-Lung; Wang, Wanhe; Mao, Zhifeng; Yang, Chao; Chen, Xiu-Ping; Lu, Jin-Jian; Han, Quan-Bin; Leung, Chung-Hang

    2016-03-24

    With rapid advances in the field of DNA chemistry, nucleic acids and DNA-modifying enzymes have recently emerged as versatile components for the construction of oligonucleotide-based sensors. Meanwhile, the G-quadruplex motif has been widely employed for the development of DNA-based assays due to its diverse structural variety. In this tutorial, we introduce the principles of G-quadruplex-based sensing and the use of DNA-modifying enzymes for sensor platform development. We also highlight recent studies of the application of DNA-modifying enzymes for the development of G-quadruplex-based luminescent detection platforms with a view towards how those enzymes play an important role in sensitivity enhancement.

  17. Mechanistic insight into the interaction of BLM helicase with intra-strand G-quadruplex structures

    DEFF Research Database (Denmark)

    Chatterjee, Sujoy; Zagelbaum, Jennifer; Savitsky, Pavel;

    2014-01-01

    Bloom syndrome is an autosomal recessive disorder caused by mutations in the RecQ family helicase BLM that is associated with growth retardation and predisposition to cancer. BLM helicase has a high specificity for non-canonical G-quadruplex (G4) DNA structures, which are formed by G-rich DNA...

  18. G-Quadruplexes: Prediction, Characterization, and Biological Application.

    Science.gov (United States)

    Kwok, Chun Kit; Merrick, Catherine J

    2017-07-26

    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.

  19. Novel molecular targets for kRAS downregulation: promoter G-quadruplexes

    Science.gov (United States)

    2016-11-01

    quadruplex DNA and down-regulation of oncogene c-myc by quindoline derivatives. Journal of medicinal chemistry 50, 1465–1474 (2007). 9 Brown , R. V., Danford...KCl, the DMS cleavage pat - tern for the induced G-quadruplex in the WT sequence revealed a Fig. 2. Predominant G4 isoforms formedwithin the near kRAS...41 (2013) 4049–4064. [37] R.V. Brown , V.C. Gaerig, T. Simmons, T.A. Brooks, Helping Eve overcome ADAM: G-quadruplexes in the ADAM-15 promoter as new

  20. G-Quadruplex DNAzyme Molecular Beacon for Amplified Colorimetric Biosensing of Pseudostellaria heterophylla

    Directory of Open Access Journals (Sweden)

    Juan Hu

    2013-01-01

    Full Text Available With an internal transcribed spacer of 18 S, 5.8 S and 26 S nuclear ribosomal DNA (nrDNA ITS as DNA marker, we report a colorimetric approach for authentication of Pseudostellaria heterophylla (PH and its counterfeit species based on the differentiation of the nrDNA ITS sequence. The assay possesses an unlabelled G-quadruplex DNAzyme molecular beacon (MB probe, employing complementary sequence as biorecognition element and 1:1:1:1 split G-quadruplex halves as reporter. In the absence of target DNA (T-DNA, the probe can shape intermolecular G-quadruplex structures capable of binding hemin to form G-quadruplex-hemin DNAzyme and catalyze the oxidation of ABTS2− to blue-green ABTS•− by H2O2. In the presence of T-DNA, T-DNA can hybridize with the complementary sequence to form a duplex structure, hindering the formation of the G-quadruplex structure and resulting in the loss of the catalytic activity. Consequently, a UV-Vis absorption signal decrease is observed in the ABTS2−-H2O2 system. The “turn-off” assay allows the detection of T-DNA from 1.0 × 10−9 to 3.0 × 10−7 mol·L−1 (R2 = 0.9906, with a low detection limit of 3.1 × 10−10 mol·L−1. The present study provides a sensitive and selective method and may serve as a foundation of utilizing the DNAzyme MB sensor for identifying traditional Chinese medicines.

  1. Blocking the binding of WT1 to bcl-2 promoter by G-quadruplex ligand SYUIQ-FM05

    Directory of Open Access Journals (Sweden)

    Yun-Xia Xiong

    2016-03-01

    Full Text Available At present, wt1, a Wilms’ tumor suppressor gene, is recognized as a critical regulator of tumorigenesis and a potential therapeutic target. WT1 shows the ability to regulate the transcription of bcl-2 by binding to a GC-rich region in the promoter, which can then fold into a special DNA secondary structure called the G-quadruplex. This function merits the exploration of the effect of a G-quadruplex ligand on the binding and subsequent regulation of WT1 on the bcl-2 promoter. In the present study, WT1 was found to bind to the double strand containing the G-quadruplex-forming sequence of the bcl-2 promoter. However, the G-quadruplex ligand SYUIQ-FM05 effectively blocked this binding by interacting with the GC-rich sequence. Our new findings are significant in the exploration of new strategies to block WT1's transcriptional regulation for cancer-cell treatment.

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

    DEFF Research Database (Denmark)

    Opazo, Felipe; Eiden, Laura; Hansen, Line

    2015-01-01

    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...

  3. Fluorescent Dansyl-Guanosine Conjugates that Bind c-MYC Promoter G-Quadruplex and Downregulate c-MYC Expression.

    Science.gov (United States)

    Pavan Kumar, Y; Saha, Puja; Saha, Dhurjhoti; Bessi, Irene; Schwalbe, Harald; Chowdhury, Shantanu; Dash, Jyotirmayee

    2016-03-02

    The four-stranded G-quadruplex present in the c-MYC P1 promoter has been shown to play a pivotal role in the regulation of c-MYC transcription. Small-molecule compounds capable of inhibiting the c-MYC promoter activity by stabilising the c-MYC G-quadruplex could potentially be used as anticancer agents. In this context, here we report the synthesis of dansyl-guanosine conjugates through one-pot modular click reactions. The dansyl-guanosine conjugates can selectively detect c-MYC G-quadruplex over other biologically relevant quadruplexes and duplex DNA and can be useful as staining reagents for selective visualisation of c-MYC G-quadruplex over duplex DNA by gel electrophoresis. NMR spectroscopic titrations revealed the preferential binding sites of these dansyl ligands to the c-MYC G-quadruplex. A dual luciferase assay and qRT-PCR revealed that a dansyl-bisguanosine ligand represses the c-MYC expression, possibly by stabilising the c-MYC G-quadruplex. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Science.gov (United States)

    Gkionis, Konstantinos; Kruse, Holger; Šponer, Jiří

    2016-04-12

    Modern dispersion-corrected DFT methods have made it possible to perform reliable QM studies on complete nucleic acid (NA) building blocks having hundreds of atoms. Such calculations, although still limited to investigations of potential energy surfaces, enhance the portfolio of computational methods applicable to NAs and offer considerably more accurate intrinsic descriptions of NAs than standard MM. However, in practice such calculations are hampered by the use of implicit solvent environments and truncation of the systems. Conventional QM optimizations are spoiled by spurious intramolecular interactions and severe structural deformations. Here we compare two approaches designed to suppress such artifacts: partially restrained continuum solvent QM and explicit solvent QM/MM optimizations. We report geometry relaxations of a set of diverse double-quartet guanine quadruplex (GQ) DNA stems. Both methods provide neat structures without major artifacts. However, each one also has distinct weaknesses. In restrained optimizations, all errors in the target geometries (i.e., low-resolution X-ray and NMR structures) are transferred to the optimized geometries. In QM/MM, the initial solvent configuration causes some heterogeneity in the geometries. Nevertheless, both approaches represent a decisive step forward compared to conventional optimizations. We refine earlier computations that revealed sizable differences in the relative energies of GQ stems computed with AMBER MM and QM. We also explore the dependence of the QM/MM results on the applied computational protocol.

  5. Enantioselective targeting left-handed Z-G-quadruplex.

    Science.gov (United States)

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

    2016-01-25

    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.

  6. Spectroscopic studies of the interaction between methylene blue and G-quadruplex

    Institute of Scientific and Technical Information of China (English)

    SUN Hongxia; XIANG Junfeng; ZHANG Yazhou; XU Guangzhi; XU Lianghua; TANG Yalin

    2006-01-01

    G-rich single-stranded DNA (5'-TTAG-GG-3') adopted a G-quadruplex structure in buffer containing potassium ions. The spectroscopic feature and the interaction between methylene blue and G-quadruplex have been investigated by circular dichroism, and nuclear magnetic resonance spectroscopy. The UV-Vis absorption and fluorescence spectral results show that the fluorescence behavior of MB by single-stranded DNA fits Stern-Volmer static quenching equation very well and they formed 1:1 complexes; dimeric G-quadruplexes were bound to MB with 1:1 or 2:1, and their equilibrium constants were 1.047×105 and 8.79×104 L/mol, respectively.Based on the above results and 1H-NMR spectral data, one may conclude that MB stacked either the terminal tetrads to form 1:1 complexes or between two terminal tetrads of G-quadruplexes to form 1:2 sandwich complexes with G-qudruplexes.

  7. 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.

  8. Pentose phosphate pathway function affects tolerance to the G-quadruplex binder TMPyP4.

    Science.gov (United States)

    Andrew, Elizabeth J; Merchan, Stephanie; Lawless, Conor; Banks, A Peter; Wilkinson, Darren J; Lydall, David

    2013-01-01

    G-quadruplexes form in guanine-rich regions of DNA and the presence of these structures at telomeres prevents the activity of telomerase in vitro. Ligands such as the cationic porphyrin TMPyP4 stabilise G-quadruplexes and are therefore under investigation for their potential use as anti-cancer drugs. In order to investigate the mechanism of action of TMPyP4 in vivo, we carried out a genome-wide screen in the budding yeast Saccharomyces cerevisiae. We found that deletion of key pentose phosphate pathway (PPP) genes increased the sensitivity of yeast to the presence of TMPyP4. The PPP plays an important role in the oxidative stress response and sensitivity to TMPyP4 also increased when genes involved in the oxidative stress response, CCS1 and YAP1, were deleted. For comparison we also report genome wide-screens using hydrogen peroxide, which causes oxidative stress, RHPS4, another G-quadruplex binder and hydroxyurea, an S phase poison. We found that a number of TMPyP4-sensitive strains are also sensitive to hydrogen peroxide in a genome-wide screen. Overall our results suggest that treatment with TMPyP4 results in light-dependent oxidative stress response in budding yeast, and that this, rather than G-quadruplex binding, is the major route to cytotoxicity. Our results have implications for the usefulness and mechanism of action of TMPyP4.

  9. Unfolding thermodynamics of intramolecular G-quadruplexes: base sequence contributions of the loops.

    Science.gov (United States)

    Olsen, Chris M; Lee, Hui-Ting; Marky, Luis A

    2009-03-05

    G-quadruplexes are a highly studied DNA motif with a potential role in a variety of cellular processes and more recently are considered novel targets for drug therapy in aging and anticancer research. In this work, we have investigated the thermodynamic contributions of the loops on the stable formation of G-quadruplexes. Specifically, we use a combination of UV, circular dichroism (CD) and fluorescence spectroscopies, and differential scanning calorimetry (DSC) to determine thermodynamic profiles, including the differential binding of ions and water, for the unfolding of the thrombin aptamer: d(GGT2GGTGTGGT2GG) that is referred to as G2. The sequences in italics, TGT and T2, are known to form loops. Other sequences examined contained base substitutions in the TGT loop (TAT, TCT, TTT, TAPT, and UUU), in the T2 loops (T4, U2), or in both loops (UGU and U2, UUU and U2). The CD spectra of all molecules show a positive band centered at 292 nm, which corresponds to the "chair" conformation. The UV and DSC melting curves of each G-quadruplex show monophasic transitions with transition temperatures (T(M)s) that remained constant with increasing strand concentration, confirming their intramolecular formation. These G-quadruplexes unfold with T(M)s in the range from 43.2 to 56.5 degrees C and endothermic enthalpies from 22.9 to 37.2 kcal/mol. Subtracting the contribution of a G-quartet stack from each experimental profile indicated that the presence of the loops stabilize each G-quadruplex by favorable enthalpy contributions, larger differential binding of K+ ions (0.1-0.6 mol K+/ mol), and a variable uptake/release of water molecules (-6 to 8 mol H2O/mol). The thermodynamic contributions for these specific base substitutions are discussed in terms of loop stacking (base-base stacking within the loops) and their hydration effects.

  10. Synthesis of New Benzimidazole and Benzothiazole Disulfide Metal Complexes as G-quadruplex Binding Ligands.

    Science.gov (United States)

    Saour, Kawkab; Lafta, Dunya

    2016-01-01

    Compounds that can bind and stabilize non-canonical DNA structures are named quadruplex and are of interest in anticancer drug design due to their selective inhibitions of telomerase and consequent effects on cell proliferation. In this study, we report novel Co/Cu [II] complex compounds as G-quadruplex DNA binding ligands. The results from the preliminary assay indicated that the introduction of a positively charged 6-membered tail to the aromatic terminal group of benzimidazole significantly enhanced the binding affinity with the quadruplex and exhibited anti-telomerase activity. These derivatives showed significant selectivities for the telomeric quadruplex over duplex nucleic acids. The stabilization of non-canonical forms estimated with the FRET DNA technology using different sequences, such as F21T, c-kit1 and c-kit2, in cancer cell lines were assessed. Three members of this family showed to be very selective in stabilizing one particular G-quadruplex.

  11. Direct Fluorescent Detection of Blood Potassium by Ion-Selective Formation of Intermolecular G-Quadruplex and Ligand Binding.

    Science.gov (United States)

    Yang, Le; Qing, Zhihe; Liu, Changhui; Tang, Qiao; Li, Jishan; Yang, Sheng; Zheng, Jing; Yang, Ronghua; Tan, Weihong

    2016-09-20

    G-quadruplex analogues have been widely used as molecular tools for detection of potassium ion (K(+)). However, interference from a higher concentration of sodium ion (Na(+)), enzymatic degradation of the oligonucleotide, and background absorption and fluorescence of blood samples have all limited the use of G-quadruplex for direct detection of K(+) in blood samples. Here, we reported, for the first time, an intermolecular G-quadruplex-based assay capable of direct fluorescent detection of blood K(+). Increased stringency of intermolecular G-quadruplex formation based on our screened G-rich oligonucleotide (5'-TGAGGGA GGGG-3') provided the necessary selectivity for K(+) against Na(+) at physiological ion level. To increase long-term stability of oligonucleotide in blood, the screened oligonucleotide was modified with an inverted thymine nucleotide whose 3'-terminus was connected to the 3'-terminus of the upstream nucleotide, acting as a blocking group to greatly improve antinuclease stability. Lastly, to avoid interference from background absorption and autofluorescence of blood, a G-quadruplex-binding, two-photon-excited ligand, EBMVC-B, was synthesized and chosen as the fluorescence reporter. Thus, based on selective K(+) ion-induced formation of intermolecular G-quadruplex and EBMVC-B binding, this approach could linearly respond to K(+) from 0.5 to 10 mM, which matches quite well with the physiologically relevant concentration of blood K(+). Moreover, the system was highly selective for K(+) against other metal ions, including Na(+), Ca(2+), Mg(2+), Zn(2+) common in blood. The practical application was demonstrated by direct detection of K(+) from real blood samples by two-photon fluorescence technology. To the best of our knowledge, this is the first attempt to exploit molecular G-quadruplex-based fluorescent sensing for direct assay of blood target. As such, we expect that it will promote the design and practical application of similar DNA-based sensors in

  12. Cyclic dinucleotide detection with riboswitch-G-quadruplex hybrid.

    Science.gov (United States)

    Tsuji, Genichiro; Sintim, Herman O

    2016-03-01

    A cyclic dinucleotide riboswitch has been fused with a G-quadruplex motif to produce a conditional riboswitch-peroxidase-mimicking sensor that oxidizes both colorimetric and fluorogenic substrates in the presence of c-di-GMP. We find that signal-to-noise ratio could be improved by using a two-, not three-, floor split G-quadruplex for this conditional peroxidase-mimicking riboswitch.

  13. Effect of ATRX and G-Quadruplex Formation by the VNTR Sequence on α-Globin Gene Expression.

    Science.gov (United States)

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

    2016-05-17

    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.

  14. Using G-quadruplex/hemin to "switch-on" the cathodic photocurrent of p-type PbS quantum dots: toward a versatile platform for photoelectrochemical aptasensing.

    Science.gov (United States)

    Wang, Guang-Li; Shu, Jun-Xian; Dong, Yu-Ming; Wu, Xiu-Ming; Zhao, Wei-Wei; Xu, Jing-Juan; Chen, Hong-Yuan

    2015-03-03

    We present a novel photoelectrochemical (PEC) biosensing platform by taking advantage of the phenomenon that hemin intercalated in G-quadruplex "switched-on" the cathode photocurrent of p-type PbS quantum dots (QDs). Photoinduced electron transfer between PbS QDs and G-quadruplex/hemin(III) complexes with the subsequent catalytic oxygen reduction by the reduced G-quadruplex/hemin(II) led to an obvious enhancement in the cathodic photocurrent of the PbS QDs. For the detection process, in the presence of hemin, the specific recognition of the targets with the sensing sequence would trigger the formation of a stable G-quadruplex/hemin complex, which will result in reduced charge recombination and hence amplified photocurrent intensity of the PbS QDs. By using different target sequences, the developed system made possible a novel, label-free "switch-on" PEC aptasensor toward versatile biomolecular targets such as DNA and thrombin. Especially, with ambient oxygen to regenerate G-quadruplex/hemin(II) to G-quadruplex/hemin(III), this substrate-free strategy not only promoted the photoelectric effect and thus the enhanced sensitivity of the system, but also avoided the addition of supplementary substrates of G-quadruplex/hemin such as H2O2 and organic substances.

  15. Targeting BRCA1 and BRCA2 Deficiencies with G-Quadruplex-Interacting Compounds

    OpenAIRE

    Zimmer, J.; Tacconi, EM; Folio, C; Badie, S; Porru, M.; Klare, K; Tumiati, M; Markkanen, E; Halder, S.; Ryan, A; Jackson, SP; Ramadan, K; Kuznetsov, SG; Biroccio, A.; Sale, JE

    2015-01-01

    This is the final version of the article. It was first available from Elsevier via http://dx.doi.org/10.1016/j.molcel.2015.12.004 G-quadruplex (G4)-forming genomic sequences, including telomeres, represent natural replication fork barriers. Stalled replication forks can be stabilized and restarted by homologous recombination (HR), which also repairs DNA double-strand breaks (DSBs) arising at collapsed forks. We have previously shown that HR facilitates telomere replication. Here, we demons...

  16. G-rich VEGF aptamer with locked and unlocked nucleic acid modifications exhibits a unique G-quadruplex fold

    DEFF Research Database (Denmark)

    Marusic, Maja; Veedu, Rakesh N; Wengel, Jesper

    2013-01-01

    The formation of a single G-quadruplex structure adopted by a promising 25 nt G-rich vascular endothelial growth factor aptamer in a K(+) rich environment was facilitated by locked nucleic acid modifications. An unprecedented all parallel-stranded monomeric G-quadruplex with three G-quartet planes...... residues contribute to thermal stabilization of the adopted structure and formation of structurally pre-organized intermediates that facilitate folding into a single G-quadruplex. Understanding the impact of chemical modifications on folding, thermal stability and structural polymorphism of G...... exhibits several unique structural features. Five consecutive guanine residues are all involved in G-quartet formation and occupy positions in adjacent DNA strands, which are bridged with a no-residue propeller-type loop. A two-residue D-shaped loop facilitates inclusion of an isolated guanine residue...

  17. Spectral properties and thermal stability of AS1411 G-quadruplex.

    Science.gov (United States)

    Bagheri, Zeinab; Ranjbar, Bijan; Latifi, Hamid; Zibaii, Mohammad Ismail; Moghadam, Tahereh Tohidi; Azizi, Azade

    2015-01-01

    G-quadruplexes are supramolecular structures of G-rich nucleic acid, formed by non-canonical base pairing in the presence of specific environmental inducers. These structures have been vastly considered in diagnostic and therapeutic applications. However, detailed information on structure, optical properties and thermal stability of G-quadruplex potent oligonucleotides is scarce. Herein, optical properties and thermodynamic stability of AS1411 quadruplex is reported for various concentrations of potassium and lead ions. Circular dichroism showed that AS1411 ss-DNA folds into parallel conformation in the presence of metal ions and molecular crowding condition. UV-vis spectroscopy indicated formation of quadruplex and fluorescent spectroscopy revealed intercalation of PicoGreen in its structure, with enhancement of emission intensity upon increment of metal ion concentration. This investigation also proposes high-throughput and reliable analysis of AS1411 quadruplex's thermal stability by real-time PCR technique, which can be further applied for other quadruplex structures.

  18. Circular dichroism spectroscopic investigation of double-decker phthalocyanine with G-Quadruplex as promising telomerase inhibitor

    Science.gov (United States)

    Baǧda, Efkan; Baǧda, Esra; Yabaş, Ebru

    2017-01-01

    In the present study, interaction of a double-decker phthalocyanine with two G-quadruplex DNA, Tel 21 and cMYC, was investigated. To the best of our knowledge, this is the first study about G-quadruplex-double decker phthalocyanine interaction. The spectrophotometric titration method was used for binding constant calculations. From the binding constants, it can be said that double-decker phthalocyanine more likely to bind Tel 21 rather than cMYC. The conformational changes upon binding were monitored via circular dichroism spectroscopy. The ethidium bromide replacement assay was investigated spectrofluorometrically.

  19. Disordering of human telomeric G-quadruplex with novel antiproliferative anthrathiophenedione.

    Directory of Open Access Journals (Sweden)

    Dmitry Kaluzhny

    Full Text Available Linear heteroareneanthracenediones have been shown to interfere with DNA functions, thereby causing death of human tumor cells and their drug resistant counterparts. Here we report the interaction of our novel antiproliferative agent 4,11-bis[(2-{[acetimido]amino}ethylamino]anthra[2,3-b]thiophene-5,10-dione with telomeric DNA structures studied by isothermal titration calorimetry, circular dichroism and UV absorption spectroscopy. New compound demonstrated a high affinity (K(ass∼10⁶ M⁻¹ for human telomeric antiparallel quadruplex d(TTAGGG₄ and duplex d(TTAGGG₄∶d(CCCTAA₄. Importantly, a ∼100-fold higher affinity was determined for the ligand binding to an unordered oligonucleotide d(TTAGGG TTAGAG TTAGGG TTAGGG unable to form quadruplex structures. Moreover, in the presence of Na+ the compound caused dramatic conformational perturbation of the telomeric G-quadruplex, namely, almost complete disordering of G-quartets. Disorganization of a portion of G-quartets in the presence of K+ was also detected. Molecular dynamics simulations were performed to illustrate how the binding of one molecule of the ligand might disrupt the G-quartet adjacent to the diagonal loop of telomeric G-quadruplex. Our results provide evidence for a non-trivial mode of alteration of G-quadruplex structure by tentative antiproliferative drugs.

  20. Factors influencing the performance of G-quadruplex DNAzyme-based sensors.

    Science.gov (United States)

    Kong, De-Ming

    2013-12-15

    G-quadruplex DNAzymes are peroxidase-like complexes formed by nucleic acid G-quadruplexes and hemin. Compared with natural enzymes, G-quadruplex DNAzyme offers many advantages, thus making it a promising tool in the design of biosensors and chemical sensors. Many biosensors and chemical sensors based on G-quadruplex DNAzymes have been reported. A number of factors may affect the performance of G-quadruplex DNAzyme-based sensors. Here we focus on some aspects to be taken into account when designing a G-quadruplex DNAzyme-based sensor. These include the G-quadruplex-forming G-rich sequence, solution components, the reaction substrate, and enrichment strategy for G-quadruplex DNAzyme. We also provide an outlook for further research on G-quadruplex DNAzyme-based sensors.

  1. NMR structure of dual site binding of mitoxantrone dimer to opposite grooves of parallel stranded G-quadruplex [d-(TTGGGGT)]4.

    Science.gov (United States)

    Pradeep, Tarikere Palakshan; Barthwal, Ritu

    2016-01-01

    The formation of complex between anti-cancer drug mitoxantrone (MTX) and tetra-molecular parallel G-quadruplex DNA [d-(TTGGGGT)]4 has been studied by solution state one and two dimensional NMR spectroscopy. Mitoxantrone forms a head-to-tail dimer and binds at two opposite grooves of the G-quadruplex. The Job's method of continuous variation and thermal melting studies independently ascertain binding stoichiometry of 4:1 in mitoxantrone:DNA complex. The existence of only four guanine NH peaks corresponding to the four G-quartets during the course of titration shows that C4 symmetry of G-quadruplex is intact upon binding of mitoxantrone. The specific inter molecular short distance contacts between protons of two mitoxantrone molecules of dimer, that is, ring A protons with ring C and side chain methylene protons, confirms the formation of mitoxantrone head-to-tail dimer. The observed 38 Nuclear Overhauser Enhancement (NOE) cross peaks between MTX and G-quadruplex DNA indicate formation of a well-defined complex. The three dimensional structure of 4:1 mitoxantrone:[d-(TTGGGGT)]4 complex computed by using experimental distance restraints followed by restrained Molecular Dynamics (rMD) simulations envisages the critical knowledge of specific molecular interactions within ligand-G-quadruplex complex. The findings are of direct interest in development of anti-cancer therapeutic drug based on G-quadruplex stabilization, resulting in telomerase inhibition.

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

    Science.gov (United States)

    Singh, Anju; Kukreti, Shrikant

    2017-09-19

    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 G5 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 G5 stretch obstructs the formation of three-stranded G-quadruplex. We demonstrated that continuous G5 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.

  3. Structural Dynamics of Human Telomeric G-Quadruplex Loops Studied by Molecular Dynamics Simulations

    Science.gov (United States)

    Zhu, Hong; Xiao, Shiyan; Liang, Haojun

    2013-01-01

    Loops which are linkers connecting G-strands and supporting the G-tetrad core in G-quadruplex are important for biological roles of G-quadruplexes. TTA loop is a common sequence which mainly resides in human telomeric DNA (hTel) G-quadruplex. A series of molecular dynamics (MD) simulations were carried out to investigate the structural dynamics of TTA loops. We found that (1) the TA base pair formed in TTA loops are very stable, the occupied of all hydrogen bonds are more than 0.95. (2) The TA base pair makes the adjacent G-quartet more stable than others. (3) For the edgewise loop and the diagonal loop, most loop bases are stacking with others, only few bases have considerable freedom. (4) The stabilities of these stacking structures are distinct. Part of the loops, especially TA base pairs, and bases stacking with the G-quartet, maintain certain stable conformations in the simulation, but other parts, like TT and TA stacking structures, are not stable enough. For the first time, spontaneous conformational switches of TTA edgewise loops were observed in our long time MD simulations. (5) For double chain reversal loop, it is really hard to maintain a stable conformation in the long time simulation under present force fields (parm99 and parmbsc0), as it has multiple conformations with similar free energies. PMID:23951152

  4. Structural dynamics of human telomeric G-quadruplex loops studied by molecular dynamics simulations.

    Directory of Open Access Journals (Sweden)

    Hong Zhu

    Full Text Available Loops which are linkers connecting G-strands and supporting the G-tetrad core in G-quadruplex are important for biological roles of G-quadruplexes. TTA loop is a common sequence which mainly resides in human telomeric DNA (hTel G-quadruplex. A series of molecular dynamics (MD simulations were carried out to investigate the structural dynamics of TTA loops. We found that (1 the TA base pair formed in TTA loops are very stable, the occupied of all hydrogen bonds are more than 0.95. (2 The TA base pair makes the adjacent G-quartet more stable than others. (3 For the edgewise loop and the diagonal loop, most loop bases are stacking with others, only few bases have considerable freedom. (4 The stabilities of these stacking structures are distinct. Part of the loops, especially TA base pairs, and bases stacking with the G-quartet, maintain certain stable conformations in the simulation, but other parts, like TT and TA stacking structures, are not stable enough. For the first time, spontaneous conformational switches of TTA edgewise loops were observed in our long time MD simulations. (5 For double chain reversal loop, it is really hard to maintain a stable conformation in the long time simulation under present force fields (parm99 and parmbsc0, as it has multiple conformations with similar free energies.

  5. 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

    NARCIS (Netherlands)

    Bharti, S.K.; Sommers, J.A.; Zhou, J.; Kaplan, D.L.; Spelbrink, J.N.; Mergny, J.L.; Brosh, R.M., Jr.

    2014-01-01

    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

  6. Chelerythrine down regulates expression of VEGFA, BCL2 and KRAS by arresting G-Quadruplex structures at their promoter regions

    Science.gov (United States)

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

    2017-01-01

    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.

  7. Synthesis and G-Quadruplex-Binding Properties of Defined Acridine Oligomers

    Directory of Open Access Journals (Sweden)

    Rubén Ferreira

    2010-01-01

    Full Text Available The synthesis of oligomers containing two or three acridine units linked through 2-aminoethylglycine using solid-phase methodology is described. Subsequent studies on cell viability showed that these compounds are not cytotoxic. Binding to several DNA structures was studied by competitive dialysis, which showed a clear affinity for DNA sequences that form G-quadruplexes and parallel triplexes. The fluorescence spectra of acridine oligomers were affected strongly upon binding to DNA. These spectral changes were used to calculate the binding constants (K. Log K were found to be in the order of 4–6.

  8. 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

    NARCIS (Netherlands)

    Bharti, S.K.; Sommers, J.A.; Zhou, J.; Kaplan, D.L.; Spelbrink, J.N.; Mergny, J.L.; Brosh, R.M., Jr.

    2014-01-01

    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 mit

  9. A library screening approach identifies naturally occurring RNA sequences for a G-quadruplex binding ligand.

    Science.gov (United States)

    Mirihana Arachchilage, Gayan; Morris, Mark J; Basu, Soumitra

    2014-02-07

    An RNA G-quadruplex library was synthesised and screened against kanamycin A as the ligand. Naturally occurring G-quadruplex forming sequences that differentially bind to kanamycin A were identified and characterized. This provides a simple and effective strategy for identification of potential intracellular G-quadruplex targets for a ligand.

  10. A label-free G-quadruplex-based luminescent switch-on assay for the selective detection of histidine.

    Science.gov (United States)

    He, Hong-Zhang; Wang, Modi; Chan, Daniel Shiu-Hin; Leung, Chung-Hang; Qiu, Jian-Wen; Ma, Dik-Lung

    2013-12-15

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

  11. Formation and stabilization of the telomeric antiparallel G-quadruplex and inhibition of telomerase by novel benzothioxanthene derivatives with anti-tumor activity

    Science.gov (United States)

    Zhang, Wen; Chen, Min; Ling Wu, Yan; Tanaka, Yoshimasa; Juan Ji, Yan; Lin Zhang, Su; He Wei, Chuan; Xu, Yan

    2015-09-01

    G-quadruplexes formed in telomeric DNA sequences at human chromosome ends can be a novel target for the development of therapeutics for the treatment of cancer patients. Herein, we examined the ability of six novel benzothioxanthene derivatives S1-S6 to induce the formation of and stabilize an antiparallel G-quadruplex by EMSA, UV-melting and CD techniques and the influence of S1-S6 on A549 and SGC7901 cells through real-time cell analysis, wound healing, trap assay methods. Results show that six compounds could differentially induce 26 nt G-rich oligonucleotides to form the G-quadruplex with high selectivity vs C-rich DNA, mutated DNA and double-stranded DNA, stabilize it with high affinity, promote apoptosis and inhibit mobility and telomerase activity of A549 cells and SGC7901 cells. Especially, S1, S3, S4 displayed stronger abilities, of which S3 was the most optimal with the maximum ΔTm value being up to 29.8 °C for G-quadruplex, the minimum IC50 value being 0.53 μM and the maximum cell inhibitory rate being up to 97.2%. This study suggests that this type of compounds that induce the formation of and stabilize the telomeric antiparallel G-quadruplex, and consequently inhibit telomerase activity, leading to cell apoptosis, can be screened for the discovery of novel antitumor therapeutics.

  12. Polymeric membrane neutral phenol-sensitive electrodes for potentiometric G-quadruplex/hemin DNAzyme-based biosensing.

    Science.gov (United States)

    Wang, Xuewei; Ding, Zhaofeng; Ren, Qingwei; Qin, Wei

    2013-02-05

    The first potentiometric transducer for G-quadruplex/hemin DNAzyme-based biosensing has been developed by using potential responses of electrically neutral oligomeric phenols on polymeric membrane electrodes. In the presence of G-quadruplex/hemin DNAzyme and H(2)O(2), monomeric phenols (e.g., phenol, methylphenols, and methoxyphenols) can be condensed into oligomeric phenols. Because both substrates and products are nonionic under optimal pH conditions, these reactions are traditionally not considered in designing potentiometric biosensing schemes. However, in this paper, the electrically neutral oligomeric phenols have been found to induce highly sensitive potential responses on quaternary ammonium salt-doped polymeric membrane electrodes owing to their high lipophilicities. In contrast, the potential responses to monomeric phenolic substrates are rather low. Thus, the G-quadruplex/hemin DNAzyme-catalyzed oxidative coupling of monomeric phenols can induce large potential signals, and the catalytic activities of DNAzymes can be probed. A comparison of potential responses induced by peroxidations of 13 monomeric phenols indicates that p-methoxyphenol is the most efficient substrate for potentiometric detection of G-quadruplex/hemin DNAzymes. Finally, two label-free and separation-free potentiometric DNA assay protocols based on the G-quadruplex/hemin DNAzyme have been developed with sensitivities higher than those of colorimetric and fluorometric methods. Coupled with other features such as reliable instrumentation, low cost, ease of miniaturization, and resistance to color and turbid interferences, the proposed polymeric membrane-based potentiometric sensor promises to be a competitive transducer for peroxidase-mimicking DNAzyme-involved biosensing.

  13. Ring-closing metathesis for the synthesis of a highly G-quadruplex selective macrocyclic hexaoxazole having enhanced cytotoxic potency.

    Science.gov (United States)

    Satyanarayana, Mavurapu; Rzuczek, Suzanne G; Lavoie, Edmond J; Pilch, Daniel S; Liu, Angela; Liu, Leroy F; Rice, Joseph E

    2008-07-01

    The synthesis of a 24-membered macrocyclic hexaoxazole via ring-closing metathesis is described. The target compound selectively stabilizes G-quadruplex DNA with no detectable stabilization of duplex DNA. An MTT cytotoxicity assay indicated that this unsaturated macrocyclic hexaoxazole exhibits significant cytotoxicity toward P388, RPMI 8402, and KB3-1 cell lines with IC50 values of 45, 25, and 38 nM, respectively.

  14. Design and study of telomerase inhibitors based on G-quadruplex ligands

    Directory of Open Access Journals (Sweden)

    Negrutska V. V.

    2013-05-01

    Full Text Available In this review we have summarized the results of our recent research on telomerase inhibitors and G-quadruplex DNA ligands. A series of potential enzyme inhibitors were synthesized and studied. These compounds were based on tricyclic heteroaromatic systems (thiazolobenzimidazoles phenazines, acridones, amino-substituted cyanines and natural and synthetic porphyrins and their metalocomplexes. A number of compounds, including cyanines and especially porphyrin derivatives and conjugates, were found to efficiently inhibit telomerase at low micromolar concentrations in the in vitro TRAP assay. Porphyrins demonstrated antiproliferative activity in tumor cell cultures at micro- and nanomolar concentrations. Spectral-fluorescent and electrophoretic experiments were performed to investigate the interaction of ligands with duplex and quadruplex DNA, and in many cases binding mode was established. Convenient G-octet model of G-quadruplex was developed to study the ligand-target binding using quantum-chemical methods. QM/MM hybrid approach ONIUM2 was employed to model the interaction of small molecules with Tel22 quadruplex DNA.

  15. Assaying the binding strength of G-quadruplex ligands using single-molecule TPM experiments.

    Science.gov (United States)

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

    2013-05-15

    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.

  16. Synthesis and evaluation of 7-substituted-5,6-dihydrobenzo[c]acridine derivatives as new c-KIT promoter G-quadruplex binding ligands.

    Science.gov (United States)

    Guo, Qian-Liang; Su, Hua-Fei; Wang, Ning; Liao, Sheng-Rong; Lu, Yu-Ting; Ou, Tian-Miao; Tan, Jia-Heng; Li, Ding; Huang, Zhi-Shu

    2017-04-21

    It has been shown that treatment of cancer cells with c-KIT G-quadruplex binding ligands can reduce their c-KIT expression levels thus inhibiting cell proliferation and inducing cell apoptosis. Herein, a series of new 7-substituted-5,6-dihydrobenzo[c]acridine derivatives were designed and synthesized. Subsequent biophysical evaluation demonstrated that the derivatives could effectively bind to and stabilize c-KIT G-quadruplex with good selectivity against duplex DNA. It was found that 12-N-methylated derivatives with a positive charge introduced at 12-position of 5,6-dihydrobenzo[c]acridine ring had similar binding affinity but lower stabilizing ability to c-KIT G-quadruplex DNA, compared with those of nonmethylated derivatives. Further molecular modeling studies showed possible binding modes of G-quadruplex with the ligands. RT-PCR assay and Western blot showed that compound 2b suppressed transcription and translation of c-KIT gene in K562 cells, which was consistent with the property of an effective G-quadruplex binding ligand targeting c-KIT oncogene promoter. Further biological evaluation showed that compound 2b could induce apoptosis through activation of the caspase-3 cascade pathway.

  17. Water-Soluble Ruthenium(II) Complexes with Chiral 4-(2,3-Dihydroxypropyl)-formamide Oxoaporphine (FOA): In Vitro and in Vivo Anticancer Activity by Stabilization of G-Quadruplex DNA, Inhibition of Telomerase Activity, and Induction of Tumor Cell Apoptosis.

    Science.gov (United States)

    Chen, Zhen-Feng; Qin, Qi-Pin; Qin, Jiao-Lan; Zhou, Jie; Li, Yu-Lan; Li, Nan; Liu, Yan-Cheng; Liang, Hong

    2015-06-11

    Three water-soluble ruthenium(II) complexes with chiral 4-(2,3-dihydroxypropyl)-formamide oxoaporphine (FOA) were synthesized and characterized. It was found that these ruthenium(II) complexes exhibited considerable in vitro anticancer activities and that they were the effective stabilizers of telomeric and G-quadruplex-DNA (G4-DNA) in promoter of c-myc, which acted as a telomerase inhibitor targeting G4-DNA and induced cell senescence and apoptosis. Interestingly, the in vitro anticancer activity of 6 (LC-003) was higher than those of 4 (LC-001) and 5 (LC-002), more selective for BEL-7404 cells than for normal HL-7702 cells, and preferred to activate caspases-3/9. The different biological behaviors of the ruthenium complexes could be correlated with the chiral nature of 4-(2,3-dihydroxypropyl)-formamide oxoaporphine. More significantly, 6 exhibited effective inhibitory on tumor growth in BEL-7402 xenograft mouse model and higher in vivo safety than cisplatin. These mechanistic insights indicate that 6 displays low toxicity and can be a novel anticancer drug candidate.

  18. The G-quadruplex augments translation in the 5' untranslated region of transforming growth factor β2.

    Science.gov (United States)

    Agarwala, Prachi; Pandey, Satyaprakash; Mapa, Koyeli; Maiti, Souvik

    2013-03-05

    Transforming growth factor β2 (TGFβ2) is a versatile cytokine with a prominent role in cell migration, invasion, cellular development, and immunomodulation. TGFβ2 promotes the malignancy of tumors by inducing epithelial-mesenchymal transition, angiogenesis, and immunosuppression. As it is well-documented that nucleic acid secondary structure can regulate gene expression, we assessed whether any secondary motif regulates its expression at the post-transcriptional level. Bioinformatics analysis predicts an existence of a 23-nucleotide putative G-quadruplex sequence (PG4) in the 5' untranslated region (UTR) of TGFβ2 mRNA. The ability of this stretch of sequence to form a highly stable, intramolecular parallel quadruplex was demonstrated using ultraviolet and circular dichroism spectroscopy. Footprinting studies further validated its existence in the presence of a neighboring nucleotide sequence. Following structural characterization, we evaluated the biological relevance of this secondary motif using a dual luciferase assay. Although PG4 inhibits the expression of the reporter gene, its presence in the context of the entire 5' UTR sequence interestingly enhances gene expression. Mutation or removal of the G-quadruplex sequence from the 5' UTR of the gene diminished the level of expression of this gene at the translational level. Thus, here we highlight an activating role of the G-quadruplex in modulating gene expression of TGFβ2 at the translational level and its potential to be used as a target for the development of therapeutics against cancer.

  19. Simple and fast screening of G-quadruplex ligands with electrochemical detection system.

    Science.gov (United States)

    Fan, Qiongxuan; Li, Chao; Tao, Yaqin; Mao, Xiaoxia; Li, Genxi

    2016-11-01

    Small molecules that may facilitate and stabilize the formation of G-quadruplexes can be used for cancer treatments, because the G-quadruplex structure can inhibit the activity of telomerase, an enzyme over-expressed in many cancer cells. Therefore, there is considerable interest in developing a simple and high-performance method for screening small molecules binding to G-quadruplex. Here, we have designed a simple electrochemical approach to screen such ligands based on the fact that the formation and stabilization of G-quadruplex by ligand may inhibit electron transfer of redox species to electrode surface. As a proof-of-concept study, two types of classical G-quadruplex ligands, TMPyP4 and BRACO-19, are studied in this work, which demonstrates that this method is fast and robust and it may be applied to screen G-quadruplex ligands for anticancer drugs testing and design in the future.

  20. A Toolbox for Predicting G-Quadruplex Formation and Stability

    Directory of Open Access Journals (Sweden)

    Han Min Wong

    2010-01-01

    Full Text Available G-quadruplexes are four stranded nucleic acid structures formed around a core of guanines, arranged in squares with mutual hydrogen bonding. Many of these structures are highly thermally stable, especially in the presence of monovalent cations, such as those found under physiological conditions. Understanding of their physiological roles is expanding rapidly, and they have been implicated in regulating gene transcription and translation among other functions. We have built a community-focused website to act as a repository for the information that is now being developed. At its core, this site has a detailed database (QuadDB of predicted G-quadruplexes in the human and other genomes, together with the predictive algorithm used to identify them. We also provide a QuadPredict server, which predicts thermal stability and acts as a repository for experimental data from all researchers. There are also a number of other data sources with computational predictions. We anticipate that the wide availability of this information will be of use both to researchers already active in this exciting field and to those who wish to investigate a particular gene hypothesis.

  1. A toolbox for predicting g-quadruplex formation and stability.

    Science.gov (United States)

    Wong, Han Min; Stegle, Oliver; Rodgers, Simon; Huppert, Julian Leon

    2010-01-01

    G-quadruplexes are four stranded nucleic acid structures formed around a core of guanines, arranged in squares with mutual hydrogen bonding. Many of these structures are highly thermally stable, especially in the presence of monovalent cations, such as those found under physiological conditions. Understanding of their physiological roles is expanding rapidly, and they have been implicated in regulating gene transcription and translation among other functions. We have built a community-focused website to act as a repository for the information that is now being developed. At its core, this site has a detailed database (QuadDB) of predicted G-quadruplexes in the human and other genomes, together with the predictive algorithm used to identify them. We also provide a QuadPredict server, which predicts thermal stability and acts as a repository for experimental data from all researchers. There are also a number of other data sources with computational predictions. We anticipate that the wide availability of this information will be of use both to researchers already active in this exciting field and to those who wish to investigate a particular gene hypothesis.

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

    DEFF Research Database (Denmark)

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

    2014-01-01

    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...

  3. Topological effects of charge transfer in telomere G-quadruplex: Mechanism on telomerase activation and inhibition

    CERN Document Server

    Wang, Xin

    2015-01-01

    We explore charge transfer in the telomere G-Quadruplex (TG4) DNA theoretically by the nonequilibrium Green's function method, and reveal the topological effect of charge transport in TG4 DNA. The consecutive TG4(CTG4) is semiconducting with 0.2 ~ 0.3eV energy gap. Charges transfers favorably in the consecutive TG4, but are trapped in the non-consecutive TG4 (NCTG4). The global conductance is inversely proportional to the local conductance for NCTG4. The topological structure transition from NCTG4 to CTG4 induces abruptly ~ 3nA charge current, which provide a microscopic clue to understand the telomerase activated or inhibited by TG4. Our findings reveal the fundamental property of charge transfer in TG4 and its relationship with the topological structure of TG4.

  4. Topological Effects of Charge Transfer in Telomere G-Quadruplex Mechanism on Telomerase Activation and Inhibition

    Science.gov (United States)

    Wang, Xin; Liang, Shi-Dong

    2013-02-01

    We explore the charge transfer in the telomere G-Quadruplex (TG4) DNA theoretically by the nonequilibrium Green's function method, and reveal the topological effect of the charge transport in TG4 DNA. The consecutive TG4 (CTG4) is semiconducting with 0.2 0.3 eV energy gap. Charges transfer favorably in the CTG4, but are trapped in the nonconsecutive TG4 (NCTG4). The global conductance is inversely proportional to the local conductance for NCTG4. The topological structure transition from NCTG4 to CTG4 induces abruptly 3nA charge current, which provide a microscopic clue to understand the telomerase activated or inhibited by TG4. Our findings reveal the fundamental property of charge transfer in TG4 and its relationship with the topological structure of TG4.

  5. Platinum(II) phenanthroimidazole G-quadruplex ligand induces selective telomere shortening in A549 cancer cells.

    Science.gov (United States)

    Mancini, Johanna; Rousseau, Philippe; Castor, Katherine J; Sleiman, Hanadi F; Autexier, Chantal

    2016-02-01

    Telomere maintenance, achieved by the binding of protective shelterin capping proteins to telomeres and by either telomerase or a recombination-based alternative lengthening of telomere (ALT) mechanism, is critical for cell proliferation and survival. Extensive telomere shortening or loss of telomere integrity activates DNA damage checkpoints, leading to cell senescence or death. Although telomerase upregulation is an attractive target for anti-cancer therapy, the lag associated with telomere shortening and the potential activation of ALT pose a challenge. An alternative approach is to modify telomere interactions with binding proteins (telomere uncapping). G-quadruplex ligands stabilize structures generated from single-stranded G-rich 3'-telomere end (G-quadruplex) folding, which in principle, cannot be elongated by telomerase, thus leading to telomere shortening. Ligands can also mediate rapid anti-proliferative effects by telomere uncapping. We previously reported that the G-quadruplex ligand, phenylphenanthroimidazole ethylenediamine platinum(II) (PIP), inhibits telomerase activity in vitro[47]. In the current study, a long-term seeding assay showed that PIP significantly inhibited the seeding capacity of A549 lung cancer cells and to a lesser extent primary MRC5 fibroblast cells. Importantly, treatment with PIP caused a significant dose- and time-dependent decrease in average telomere length of A549 but not MRC5 cells. Moreover, cell cycle analysis revealed a significant increase in G1 arrest upon treatment of A549 cells, but not MRC5 cells. Both apoptosis and cellular senescence may contribute to the anti-proliferative effects of PIP. Our studies validate the development of novel and specific therapeutic ligands targeting telomeric G-quadruplex structures in cancer cells.

  6. The human mitochondrial transcription factor A is a versatile G-quadruplex binding protein

    Science.gov (United States)

    Lyonnais, Sébastien; Tarrés-Soler, Aleix; Rubio-Cosials, Anna; Cuppari, Anna; Brito, Reicy; Jaumot, Joaquim; Gargallo, Raimundo; Vilaseca, Marta; Silva, Cristina; Granzhan, Anton; Teulade-Fichou, Marie-Paule; Eritja, Ramon; Solà, Maria

    2017-01-01

    The ability of the guanine-rich strand of the human mitochondrial DNA (mtDNA) to form G-quadruplex structures (G4s) has been recently highlighted, suggesting potential functions in mtDNA replication initiation and mtDNA stability. G4 structures in mtDNA raise the question of their recognition by factors associated with the mitochondrial nucleoid. The mitochondrial transcription factor A (TFAM), a high-mobility group (HMG)-box protein, is the major binding protein of human mtDNA and plays a critical role in its expression and maintenance. HMG-box proteins are pleiotropic sensors of DNA structural alterations. Thus, we investigated and uncovered a surprising ability of TFAM to bind to DNA or RNA G4 with great versatility, showing an affinity similar than to double-stranded DNA. The recognition of G4s by endogenous TFAM was detected in mitochondrial extracts by pull-down experiments using a G4-DNA from the mtDNA conserved sequence block II (CSBII). Biochemical characterization shows that TFAM binding to G4 depends on both the G-quartets core and flanking single-stranded overhangs. Additionally, it shows a structure-specific binding mode that differs from B-DNA, including G4-dependent TFAM multimerization. These TFAM-G4 interactions suggest functional recognition of G4s in the mitochondria. PMID:28276514

  7. Effects of monovalent cations on folding kinetics of G-quadruplexes.

    Science.gov (United States)

    You, Jing; Li, Hui; Lu, Xi-Ming; Li, Wei; Wang, Peng-Ye; Dou, Shuo-Xing; Xi, Xu-Guang

    2017-08-31

    G-quadruplexes are special structures existing at the ends of human telomeres, the folding kinetics of which are essential for their functions, such as in the maintenance of genome stability and the protection of chromosome ends. In the present study, we investigated the folding kinetics of G-quadruplex in different monovalent cation environments and determined the detailed kinetic parameters for Na(+)- and K(+)-induced G-quadruplex folding, and for its structural transition from the basket-type Na(+) form to the hybrid-type K(+) form. More interestingly, although Li(+) was often used in previous studies of G-quadruplex folding as a control ion supposed to have no effect, we have found that Li(+) can actually influence the folding kinetics of both Na(+)- and K(+)-induced G-quadruplexes significantly and in different ways, by changing the folding fraction of Na(+)-induced G-quadruplexes and greatly increasing the folding rates of K(+)-induced G-quadruplexes. The present study may shed new light on the roles of monovalent cations in G-quadruplex folding and should be useful for further studies of the underlying folding mechanism. © 2017 The Author(s).

  8. Effects of metal ions and cosolutes on G-quadruplex topology.

    Science.gov (United States)

    Fujii, Taiga; Podbevšek, Peter; Plavec, Janez; Sugimoto, Naoki

    2017-01-01

    Topologies of G-quadruplexes depend on oligonucleotide sequences and on environmental factors, and the diversity of G-quadruplex topologies complicates investigation of functions of these nucleic acid structures. To investigate how metal ions and cosolutes regulate topologies of G-quadruplexes, we stabilized the antiparallel conformation by insertion of 2'-deoxyxanthosine and 8-oxo-2'-deoxyguanosine into selected positions of an oligonucleotide. Thermodynamic analyses of the oligonucleotide revealed that Na(+) stabilized the antiparallel G-quadruplex, whereas K(+) destabilized this topology. This result suggests that metal ions selectively stabilize G-quadruplex topologies with cavities between G-quartet planes of certain sizes. In the presence of KCl in 20wt% poly(ethylene glycol) with average molecular weight of 200, the antiparallel basket-type G-quadruplex conformation was not stabilized compared with the dilute condition. In the presence of NaCl, the cosolute did stabilize the G-quadruplex with respect to the dilute condition. The presented data show that metal ions and cosolutes regulate topologies of G-quadruplexes through mechanisms that depend on sizes of metal ion cavities and hydration states.

  9. Stabilization of c-myc G-Quadruplex DNA, inhibition of telomerase activity, disruption of mitochondrial functions and tumor cell apoptosis by platinum(II) complex with 9-amino-oxoisoaporphine.

    Science.gov (United States)

    Qin, Jiao-Lan; Qin, Qi-Pin; Wei, Zu-Zhuang; Yu, Yan-Cheng; Meng, Ting; Wu, Chen-Xuan; Liang, Yue-Lan; Liang, Hong; Chen, Zhen-Feng

    2016-11-29

    [Pd(L)(DMSO)Cl2] (1) and [Pt(L)(DMSO)Cl2] (2) with 9-amino-oxoisoaporphine (L), were synthesized and characterized. 1 and 2 are more selectively cytotoxic to Hep-G2 cells versus normal liver cells (HL-7702). Various experiments showed that 2 acted as telomerase inhibitors targeting G4-DNA and triggered cell apoptosis by interacting with c-myc G4-DNA. Furthermore, 2 significantly induced cell cycle arrest at both G2/M and S phase, which leading to the down-regulation of cdc25 A, cyclin D, cyclin B, cyclin A and CDK2 and the up-regulation of p53, p27, p21,chk1 and chk2. In addition, 2 also caused mitochondrial dysfunction. Taken together, we found that 2 exerted its cytotoxic activity mainly via inhibiting telomerase by interaction with c-myc G4-DNA and disruption of mitochondrial function.

  10. RNA G-quadruplexes are globally unfolded in eukaryotic cells and depleted in bacteria

    Science.gov (United States)

    Guo, Junjie U.; Bartel, David P.

    2017-01-01

    In vitro, some RNAs can form stable four-stranded structures known as G-quadruplexes. Although RNA G-quadruplexes have been implicated in post-transcriptional gene regulation and diseases, direct evidence for their formation in cells has been lacking. Here, we identified thousands of mammalian RNA regions that can fold into G-quadruplexes in vitro, but in contrast to previous assumptions, these regions were overwhelmingly unfolded in cells. Model RNA G-quadruplexes that were unfolded in eukaryotic cells were folded when ectopically expressed in Escherichia coli; however, they impaired translation and growth, which helps explain why we detected few G-quadruplex–forming regions in bacterial transcriptomes. Our results suggest that eukaryotes have a robust machinery that globally unfolds RNA G-quadruplexes, whereas some bacteria have instead undergone evolutionary depletion of G-quadruplex–forming sequences. PMID:27708011

  11. Targeting C-myc G-Quadruplex: Dual Recognition by Aminosugar-Bisbenzimidazoles with Varying Linker Lengths

    Directory of Open Access Journals (Sweden)

    Nihar Ranjan

    2013-11-01

    Full Text Available G-quadruplexes are therapeutically important biological targets. In this report, we present biophysical studies of neomycin-Hoechst 33258 conjugates binding to a G-quadruplex derived from the C-myc promoter sequence. Our studies indicate that conjugation of neomycin to a G-quadruplex binder, Hoechst 33258, enhances its binding. The enhancement in G-quadruplex binding of these conjugates varies with the length and composition of the linkers joining the neomycin and Hoechst 33258 units.

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

    Science.gov (United States)

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

    2012-01-01

    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

  13. Mechanistic insight into the interaction of BLM helicase with intra-strand G-quadruplex structures

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    Chatterjee, Sujoy; Zagelbaum, Jennifer; Savitsky, Pavel; Sturzenegger, Andreas; Huttner, Diana; Janscak, Pavel; Hickson, Ian D.; Gileadi, Opher; Rothenberg, Eli

    2014-11-01

    Bloom syndrome is an autosomal recessive disorder caused by mutations in the RecQ family helicase BLM that is associated with growth retardation and predisposition to cancer. BLM helicase has a high specificity for non-canonical G-quadruplex (G4) DNA structures, which are formed by G-rich DNA strands and play an important role in the maintenance of genomic integrity. Here we used single-molecule FRET to define the mechanism of interaction of BLM helicase with intra-stranded G4 structures. We show that the activity of BLM is substrate dependent, and highly regulated by a short-strand DNA (ssDNA) segment that separates the G4 motif from double-stranded DNA. We demonstrate cooperativity between the RQC and HRDC domains of BLM during binding and unfolding of the G4 structure, where the RQC domain interaction with G4 is stabilized by HRDC binding to ssDNA. We present a model that proposes a unique role for G4 structures in modulating the activity of DNA processing enzymes.

  14. 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.

    Science.gov (United States)

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

    2017-07-21

    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

  15. Development of a universal colorimetric indicator for G-quadruplex structures by the fusion of thiazole orange and isaindigotone skeleton.

    Science.gov (United States)

    Yan, Jin-Wu; Ye, Wen-Jie; Chen, Shuo-Bin; Wu, Wei-Bin; Hou, Jin-Qiang; Ou, Tian-Miao; Tan, Jia-Heng; Li, Ding; Gu, Lian-Quan; Huang, Zhi-Shu

    2012-08-07

    The rapid and convenient method for identification of all kinds of G-quadruplex is highly desirable. In the present study, a novel colorimetric indicator for a vast variety of G-quadruplex was designed and synthesized on the basis of thiazole orange and isaindigotone skeleton. Its distinct color change enables label-free visual detection of G-quadruplexes, which is due to the disassembly of dye H-aggregates to monomers. This specific detection of G-quadruplex arises from its end-stacking interaction with G-quartet. On the basis of this universal indicator, a facile approach for large-scale identification of G-quadruplex was developed.

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

    Science.gov (United States)

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

    2015-12-02

    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.

  17. Reprogramming the mechanism of action of chlorambucil by coupling to a G-quadruplex ligand.

    Science.gov (United States)

    Di Antonio, Marco; McLuckie, Keith I E; Balasubramanian, Shankar

    2014-04-23

    The nitrogen mustard Chlorambucil (Chl) generates covalent adducts with double-helical DNA and inhibits cell proliferation. Among these adducts, interstrand cross-links (ICLs) are the most toxic, as they stall replication by generating DNA double strand breaks (DSBs). Conversely, intrastrand cross-links generated by Chl are efficiently repaired by a dedicated Nucleotide Excision Repair (NER) enzyme. We synthesized a novel cross-linking agent that combines Chl with the G-quadruplex (G4) ligand PDS (PDS-Chl). We demonstrated that PDS-Chl alkylates G4 structures at low μM concentrations, without reactivity toward double- or single-stranded DNA. Since intramolecular G4s arise from a single DNA strand, we reasoned that preferential alkylation of such structures might prevent the generation of ICLs, while favoring intrastrand cross-links. We observed that PDS-Chl selectively impairs growth in cells genetically deficient in NER, but did not show any sensitivity to the repair gene BRCA2, involved in double-stranded break repair. Our findings suggest that G4 targeting of this clinically important alkylating agent alters the overall mechanism of action. These insights may inspire new opportunities for intervention in diseases specifically characterized by genetic impairment of NER, such as skin and testicular cancers.

  18. The yeast Pif1 helicase prevents genomic instability caused by G-quadruplex-forming CEB1 sequences in vivo.

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    Cyril Ribeyre

    2009-05-01

    Full Text Available In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4 secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Delta cells. Hence, we conclude that CEB1 instability in pif1Delta cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences.

  19. G-quadruplex DNAzymes-induced highly selective and sensitive colorimetric sensing of free heme in rat brain.

    Science.gov (United States)

    Li, Ruimin; Jiang, Qin; Cheng, Hanjun; Zhang, Guoqiang; Zhen, Mingming; Chen, Daiqin; Ge, Jiechao; Mao, Lanqun; Wang, Chunru; Shu, Chunying

    2014-04-21

    Direct selective determination of free heme in the cerebral system is of great significance due to the crucial roles of free heme in physiological and pathological processes. In this work, a G-quadruplex DNAzymes-induced highly sensitive and selective colorimetric sensing of free heme in rat brain is established. Initially, the conformation of an 18-base G-rich DNA sequence, PS2.M (5'-GTGGGTAGGGCGGGTTGG-3'), in the presence of K(+), changes from a random coil to a "parallel" G-quadruplex structure, which can bind free heme in the cerebral system with high affinity through π-π stacking. The resulted heme/G-quadruplex complex exhibits high peroxidase-like activity, which can be used to catalyze the oxidation of colorless ABTS(2-) to green ABTS˙(-) by H2O2. The concentration of heme can be evaluated by the naked eye and determined by UV-vis spectroscopy. The signal output showed a linear relationship for heme within the concentration range from 1 to 120 nM with a detection limit of 0.637 nM. The assay demonstrated here was highly selective and free from the interference of physiologically important species such as dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), ascorbate acid (AA), cysteine, uric acid (UA), glucose and lactate in the cerebral system. The basal dialysate level of free heme in the microdialysate from the striatum of adult male Sprague-Dawley rats was determined to be 32.8 ± 19.5 nM (n = 3). The analytic protocol possesses many advantages, including theoretical simplicity, low-cost technical and instrumental demands, and responsible detection of heme in rat brain microdialysate.

  20. Conformation and Stability of Intramolecular Telomeric G-Quadruplexes: Sequence Effects in the Loops

    Science.gov (United States)

    Sattin, Giovanna; Artese, Anna; Nadai, Matteo; Costa, Giosuè; Parrotta, Lucia; Alcaro, Stefano; Palumbo, Manlio; Richter, Sara N.

    2013-01-01

    Telomeres are guanine-rich sequences that protect the ends of chromosomes. These regions can fold into G-quadruplex structures and their stabilization by G-quadruplex ligands has been employed as an anticancer strategy. Genetic analysis in human telomeres revealed extensive allelic variation restricted to loop bases, indicating that the variant telomeric sequences maintain the ability to fold into G-quadruplex. To assess the effect of mutations in loop bases on G-quadruplex folding and stability, we performed a comprehensive analysis of mutant telomeric sequences by spectroscopic techniques, molecular dynamics simulations and gel electrophoresis. We found that when the first position in the loop was mutated from T to C or A the resulting structure adopted a less stable antiparallel topology; when the second position was mutated to C or A, lower thermal stability and no evident conformational change were observed; in contrast, substitution of the third position from A to C induced a more stable and original hybrid conformation, while mutation to T did not significantly affect G-quadruplex topology and stability. Our results indicate that allelic variations generate G-quadruplex telomeric structures with variable conformation and stability. This aspect needs to be taken into account when designing new potential anticancer molecules. PMID:24367632

  1. Conformation and stability of intramolecular telomeric G-quadruplexes: sequence effects in the loops.

    Directory of Open Access Journals (Sweden)

    Giovanna Sattin

    Full Text Available Telomeres are guanine-rich sequences that protect the ends of chromosomes. These regions can fold into G-quadruplex structures and their stabilization by G-quadruplex ligands has been employed as an anticancer strategy. Genetic analysis in human telomeres revealed extensive allelic variation restricted to loop bases, indicating that the variant telomeric sequences maintain the ability to fold into G-quadruplex. To assess the effect of mutations in loop bases on G-quadruplex folding and stability, we performed a comprehensive analysis of mutant telomeric sequences by spectroscopic techniques, molecular dynamics simulations and gel electrophoresis. We found that when the first position in the loop was mutated from T to C or A the resulting structure adopted a less stable antiparallel topology; when the second position was mutated to C or A, lower thermal stability and no evident conformational change were observed; in contrast, substitution of the third position from A to C induced a more stable and original hybrid conformation, while mutation to T did not significantly affect G-quadruplex topology and stability. Our results indicate that allelic variations generate G-quadruplex telomeric structures with variable conformation and stability. This aspect needs to be taken into account when designing new potential anticancer molecules.

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

    Science.gov (United States)

    Harris, Lynne M; Merrick, Catherine J

    2015-02-01

    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.

  3. G Quadruplex in Plants: A Ubiquitous Regulatory Element and Its Biological Relevance

    Directory of Open Access Journals (Sweden)

    Vikas Yadav

    2017-07-01

    Full Text Available G quadruplexes (G4 are higher-order DNA and RNA secondary structures formed by G-rich sequences that are built around tetrads of hydrogen-bonded guanine bases. Potential G4 quadruplex sequences have been identified in G-rich eukaryotic non-telomeric and telomeric genomic regions. Upon function, G4 formation is known to involve in chromatin remodeling, gene regulation and has been associated with genomic instability, genetic diseases and cancer progression. The natural role and biological validation of G4 structures is starting to be explored, and is of particular interest for the therapeutic interventions for human diseases. However, the existence and physiological role of G4 DNA and G4 RNA in plants species have not been much investigated yet and therefore, is of great interest for the development of improved crop varieties for sustainable agriculture. In this context, several recent studies suggests that these highly diverse G4 structures in plants can be employed to regulate expression of genes involved in several pathophysiological conditions including stress response to biotic and abiotic stresses as well as DNA damage. In the current review, we summarize the recent findings regarding the emerging functional significance of G4 structures in plants and discuss their potential value in the development of improved crop varieties.

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

    Directory of Open Access Journals (Sweden)

    Lynne M Harris

    2015-02-01

    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.

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

    Science.gov (United States)

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

    2014-01-24

    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 were thus identified as potent G4 ligands with high selectivity over duplex DNA, and preliminary results indicate that the scaffold may form basis for the development of subtype-specific G4 ligands.

  6. Label-free logic modules and two-layer cascade based on stem-loop probes containing a G-quadruplex domain.

    Science.gov (United States)

    Guo, Yahui; Cheng, Junjie; Wang, Jine; Zhou, Xiaodong; Hu, Jiming; Pei, Renjun

    2014-09-01

    A simple, versatile, and label-free DNA computing strategy was designed by using toehold-mediated strand displacement and stem-loop probes. A full set of logic gates (YES, NOT, OR, NAND, AND, INHIBIT, NOR, XOR, XNOR) and a two-layer logic cascade were constructed. The probes contain a G-quadruplex domain, which was blocked or unfolded through inputs initiating strand displacement and the obviously distinguishable light-up fluorescent signal of G-quadruplex/NMM complex was used as the output readout. The inputs are the disease-specific nucleotide sequences with potential for clinic diagnosis. The developed versatile computing system based on our label-free and modular strategy might be adapted in multi-target diagnosis through DNA hybridization and aptamer-target interaction. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Screening of a chemical library by HT-G4-FID for discovery of selective G-quadruplex binders.

    Science.gov (United States)

    Largy, Eric; Saettel, Nicolas; Hamon, Florian; Dubruille, Sylvie; Teulade-Fichou, Marie-Paule

    2012-01-01

    Due to the lack of structural guidelines about G-quadruplex ligands, rational design cannot be the only approach to discover potent G4-ligands. As a complementary approach, screening of chemical library may provide interesting scaffolds known as hits provided that specific tools are available. In this work, the Institut Curie-CNRS chemical library was firstly screened by chemoinformatics methods. Similarity estimations by comparison with reference compounds (Phen-DC3, 360A, MMQ12) provided a set of molecules, which were then evaluated by high-throughput G4-FID (HT-G4-FID) against various G-quadruplex DNA. A full investigation of the most interesting molecules, using the HT-G4-FID assay and molecular modeling, supplied an interesting structure-activity relationship confirming the efficiency of this general approach. Overall, we demonstrated that HT-G4-FID coupled with screening of chemical libraries is a powerful tool to identify new G4-DNA binding scaffolds.

  8. Label-free fluorescent sensor for lead ion detection based on lead(II)-stabilized G-quadruplex formation.

    Science.gov (United States)

    Zhan, Shenshan; Wu, Yuangen; Luo, Yanfang; Liu, Le; He, Lan; Xing, Haibo; Zhou, Pei

    2014-10-01

    A label-free fluorescent DNA sensor for the detection of lead ions (Pb(2+)) based on lead(II)-stabilized G-quadruplex formation is proposed in this article. A guanine (G)-rich oligonucleotide, T30695, was used as a recognition probe, and a DNA intercalator, SYBR Green I (SG), was used as a signal reporter. In the absence of Pb(2+), the SG intercalated with the single-stranded random-coil T30695 and emitted strong fluorescence. While in the presence of Pb(2+), the random-coil T30695 would fold into a G-quadruplex structure and the SG could barely show weak fluorescence, and the fluorescence intensity was inversely proportional to the involving amount of Pb(2+). Based on this, a selective lead ion sensor with a limit of detection of 3.79 ppb (parts per billion) and a detection range from 0 to 600 ppb was constructed. Because detection for real samples was also demonstrated to be reliable, this simple, low-cost, sensitive, and selective sensor holds good potential for Pb(2+) detection in real environmental samples.

  9. A series of logic gates based on electrochemical reduction of Pb2+ in self-assembled G-quadruplex on the gold electrode.

    Science.gov (United States)

    Zhai, Wei; Du, Chunyan; Li, Xiaohong

    2014-02-28

    Direct reduction of Pb(2+) in self-assembled G-quadruplex on the gold electrode was first observed, which was applied in constructing a series of simple and reversible logic gates, such as one-input, two-input and three-input logic gates. Importantly, the largest scale of reversibility among two-input logic gates was achieved based on the reciprocal transformations of DNA.

  10. Microfluidic-based G-quadruplex ligand displacement assay for alkaloid anticancer drug screening.

    Science.gov (United States)

    Shen, Haihui; Zhang, Bo; Xu, Huiyan; Sun, Yue; Wu, Qiwang; Shen, Hong; Liu, Yingchun

    2017-02-05

    Some natural heterocyclic alkaloids containing planar group show potential to complex with specific promoter region of protooncogene for stabilizing the G-quadruplex (G4) structure which nowadays promises to be a target in anticancer drug design. However, in view of the polymorphic characteristics and structural complexity of heterocyclic alkaloids, it is desirable to develop high-throughput and low-consumption approach for anticancer drug screening. In this paper, an intensive study on alkaloid ligand/G4 DNA interaction has been conducted, demonstrating that the end-stacking interaction is the favorable binding mode between the oncogene-related Pu22 G4 DNA and the heterocyclic alkaloid ligand. Based on structural feasibility and energy minimization, a ligand displacement assay for screening alkaloid ligand in stabilizing the oncogene target G4 has been developed, which also helps to facilitate the assessment of drug specificity. Coupled with microfluidic-based DNAzyme-catalytic chemiluminescence detection, the approach showed the advantages of high sensitivity, high throughput with low sample and reagent consumptions.

  11. Label-free and ratiometric detection of nuclei acids based on graphene quantum dots utilizing cascade amplification by nicking endonuclease and catalytic G-quadruplex DNAzyme.

    Science.gov (United States)

    Wang, Guang-Li; Fang, Xin; Wu, Xiu-Ming; Hu, Xue-Lian; Li, Zai-Jun

    2016-07-15

    Herein, we report a ratiometric fluorescence assay based on graphene quantum dots (GQDs) for the ultrasensitive DNA detection by coupling the nicking endonuclease assisted target recycling and the G-quadruplex/hemin DNAzyme biocatalysis for cascade signal amplifications. With o-phenylenediamine acted as the substrate of G-quadruplex/hemin DNAzyme, whose oxidization product (that is, 2,3-diaminophenazine, DAP) quenched the fluorescence intensity of GQDs (at 460nm) obviously, accompanied with the emergence of a new emission of DAP (at 564nm). The ratiometric signal variations at the emission wavelengths of 564 and 460nm (I564/I460) were utilized for label-free, sensitive, and selective detection of target DNA. Utilizing the nicking endonuclease assisted target recycling and the G-quadruplex/hemin DNAzyme biocatalysis for amplified cascade generation of DAP, the proposed bioassay exhibited high sensitivity toward target DNA with a detection limit of 30fM. The method also had additional advantages such as facile preparation and easy operation.

  12. Nemorubicin and doxorubicin bind the G-quadruplex sequences of the human telomeres and of the c-MYC promoter element Pu22.

    Science.gov (United States)

    Scaglioni, Leonardo; Mondelli, Rosanna; Artali, Roberto; Sirtori, Federico Riccardi; Mazzini, Stefania

    2016-06-01

    Intra-molecular G-quadruplex structures are present in the guanine rich regions of human telomeres and were found to be prevalent in gene promoters. More recently, the targeting of c-MYC transcriptional control has been suggested, because the over expression of the c-MYC oncogene is one of the most common aberration found in a wide range of human tumors. The interaction of nemorubicin and doxorubicin with DNA G-quadruplex structures has been studied by NMR, ESI-MS and molecular modelling, in order to obtain further information about the complex and the multiple mechanisms of action of these drugs. Nemorubicin intercalates between A3 and G4 of d(TTAGGGT)4 and form cap-complex at the G6pT7 site. The presence of the adenine in this sequence is important for the stabilization of the complex, as was shown by the interaction with d(TTGGGTT)4 and d(TTTGGGT)4, which form only a 1:1 complex. The interaction of doxorubicin with d(TTAGGGT)4 is similar, but the complex appears less stable. Nemorubicin also binds with high efficiency the c-MYC G-quadruplex sequence Pu22, to form a very well defined complex. Two nemorubicin molecules bind to the 3'-end and to the 5'-end, forming an additional plane of stacking over each external G-tetrad. The wild type c-MYCPu22 sequence forms with nemorubicin the same complex. Nemorubicin and doxorubicin, not only intercalate into the duplex DNA, but also result in significant ligands for G-quadruplex DNA segments, stabilizing their structure; this may in part explain the multiple mechanisms of action of their antitumor activity. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Label-free thioflavin T/G-quadruplex-based real-time strand displacement amplification for biosensing applications.

    Science.gov (United States)

    Du, Yi-Chen; Zhu, Li-Na; Kong, De-Ming

    2016-12-15

    To promote application of strand-displacement amplification (SDA) techniques in biosensing, a label-free, real-time monitoring strategy for isothermal nucleic acid amplification reactions was designed. G-quadruplex structures were introduced into SDA products using specific recognition of G-quadruplexes by the fluorogenic dye thioflavin T. Performance was good for real-time monitoring of traditional SDA by a linear-amplification mechanism and for exponential cross-triggered SDA amplification. The strategy worked on a commercial real-time PCR instrument, making it suitable for biosensing platforms. As examples, two highly sensitive and specific biosensors were designed for analysis of the activity of uracil-DNA glycosylase (UDG) and the restriction endonuclease EcoRI. Detection limits were 6×10(-5)U/mL for UDG and 0.016U/mL for EcoRI. Detection of corresponding targets in complex matrices such as cell lysates or human serum was also demonstrated. Compared to traditional end-point detection methods, real-time SDA-based approaches have the advantages of simple, fast operation; high sensitivity; low risk of carryover contamination; and very high throughput. The introduction of real-time monitoring strategies may promote application of SDA reactions in biosensor design.

  14. Exploring the binding of d(GGGT)4 to the HIV-1 integrase: An approach to investigate G-quadruplex aptamer/target protein interactions.

    Science.gov (United States)

    Esposito, Veronica; Pirone, Luciano; Mayol, Luciano; Pedone, Emilia; Virgilio, Antonella; Galeone, Aldo

    2016-08-01

    The aptamer d(GGGT)4 (T30923 or T30695) forms a 5'-5' dimer of two stacked parallel G-quadruplexes, each characterized by three G-tetrads and three single-thymidine reversed-chain loops. This aptamer has been reported to exhibit anti-HIV activity by targeting the HIV integrase, a viral enzyme responsible for the integration of viral DNA into the host-cell genome. However, information concerning the aptamer/target interaction is still rather limited. In this communication we report microscale thermophoresis investigations on the interaction between the HIV-1 integrase and d(GGGT)4 aptamer analogues containing abasic sites singly replacing thymidines in the original sequence. This approach has allowed the identification of which part of the aptamer G-quadruplex structure is mainly involved in the interaction with the protein.

  15. Selective Targeting of G-Quadruplex Structures by a Benzothiazole-Based Binding Motif.

    Science.gov (United States)

    Buchholz, Ina; Karg, Beatrice; Dickerhoff, Jonathan; Sievers-Engler, Adrian; Lämmerhofer, Michael; Weisz, Klaus

    2017-03-09

    A benzothiazole derivative was identified as potent ligand for DNA G-quadruplex structures. Fluorescence titrations revealed selective binding to quadruplexes of different topologies including parallel, antiparallel and (3+1) hybrid structures. The parallel c-MYC sequence was found to constitute the preferred target with dissociation constants in the micromolar range. Binding of the benzothiazole-based ligand to c-MYC was structurally and thermodynamically characterized in detail by employing a comprehensive set of spectroscopic and calorimetric techniques. Job plot analyses and mass spectral data indicate non-cooperative ligand binding to form 1:1 and 2:1 complex stoichiometries. Whereas stacking interactions are suggested by optical methods, NMR chemical shift perturbations also indicate significant rearrangements of both 5'- and 3'-flanking sequences upon ligand binding. Additional isothermal calorimetry studies yield a thermodynamic profile of the ligand-quadruplex association and reveal enthalpic contributions to be the major driving force for binding. The structural and thermodynamic information obtained in the present work provides the basis for the rational development of benzothiazole derivatives as promising quadruplex binding agents.

  16. RHPS4 G-quadruplex ligand induces anti-proliferative effects in brain tumor cells.

    Science.gov (United States)

    Lagah, Sunil; Tan, I-Li; Radhakrishnan, Priya; Hirst, Robert A; Ward, Jennifer H; O'Callaghan, Chris; Smith, Stuart J; Stevens, Malcolm F G; Grundy, Richard G; Rahman, Ruman

    2014-01-01

    Telomeric 3' overhangs can fold into a four-stranded DNA structure termed G-quadruplex (G4), a formation which inhibits telomerase. As telomerase activation is crucial for telomere maintenance in most cancer cells, several classes of G4 ligands have been designed to directly disrupt telomeric structure. We exposed brain tumor cells to the G4 ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) and investigated proliferation, cell cycle dynamics, telomere length, telomerase activity and activated c-Myc levels. Although all cell lines tested were sensitive to RHPS4, PFSK-1 central nervous system primitive neuroectodermal cells, DAOY medulloblastoma cells and U87 glioblastoma cells exhibited up to 30-fold increased sensitivity compared to KNS42 glioblastoma, C6 glioma and Res196 ependymoma cells. An increased proportion of S-phase cells were observed in medulloblastoma and high grade glioma cells whilst CNS PNET cells showed an increased proportion of G1-phase cells. RHPS4-induced phenotypes were concomitant with telomerase inhibition, manifested in a telomere length-independent manner and not associated with activated c-Myc levels. However, anti-proliferative effects were also observed in normal neural/endothelial cells in vitro and ex vivo. This study warrants in vivo validation of RHPS4 and alternative G4 ligands as potential anti-cancer agents for brain tumors but highlights the consideration of dose-limiting tissue toxicities.

  17. Aven recognition of RNA G-quadruplexes regulates translation of the mixed lineage leukemia protooncogenes.

    Science.gov (United States)

    Thandapani, Palaniraja; Song, Jingwen; Gandin, Valentina; Cai, Yutian; Rouleau, Samuel G; Garant, Jean-Michel; Boisvert, Francois-Michel; Yu, Zhenbao; Perreault, Jean-Pierre; Topisirovic, Ivan; Richard, Stéphane

    2015-08-12

    G-quadruplexes (G4) are extremely stable secondary structures forming stacks of guanine tetrads. DNA G4 structures have been extensively studied, however, less is known about G4 motifs in mRNAs, especially in their coding sequences. Herein, we show that Aven stimulates the mRNA translation of the mixed lineage leukemia (MLL) proto-oncogene in an arginine methylation-dependent manner. The Aven RGG/RG motif bound G4 structures within the coding regions of the MLL1 and MLL4 mRNAs increasing their polysomal association and translation, resulting in the induction of transcription of leukemic genes. The DHX36 RNA helicase associated with the Aven complex and was required for optimal translation of G4 mRNAs. Depletion of Aven led to a decrease in synthesis of MLL1 and MLL4 proteins resulting in reduced proliferation of leukemic cells. These findings identify an Aven-centered complex that stimulates the translation of G4 harboring mRNAs, thereby promoting survival of leukemic cells.

  18. Hemin/G-quadruplex structure and activity alteration induced by magnesium cations.

    Science.gov (United States)

    Kosman, J; Juskowiak, B

    2016-04-01

    The influence of metal cations on G-quadruplex structure and peroxidase-mimicking DNAzyme activity was investigated. Experiments revealed a significant role of magnesium ion, which in the presence of potassium cation influenced DNAzyme activity. This ability has been associated with alteration of G-quadruplex topology and consequently affinity to bind hemin molecule. It has been demonstrated that G-quadruplex based on PS2.M sequence under these conditions formed parallel topology, which exhibited lower activity than that observed in standard potassium-containing solution. On the other hand DNAzyme/magnesium ion system based on telomeric sequence, which did not undergo significant structural changes, exhibited higher peroxidase activity upon magnesium ion addition. In both cases, the stabilization effect of magnesium cations on G-quadruplex structure was observed. The mechanism of DNAzyme activity alteration by magnesium ion can be explained by its influence on the pKa value of DNAzyme. Magnesium ion decreased pKa for PS2.M based system but increased it for telomeric DNAzyme. Magnesium cation effect on G-quadruplex structure as well as DNAzyme activity is particularly important since this ion is one of the most common metal cations in biological samples.

  19. Fully integrated graphene electronic biosensor for label-free detection of lead (II) ion based on G-quadruplex structure-switching.

    Science.gov (United States)

    Li, Yijun; Wang, Cheng; Zhu, Yibo; Zhou, Xiaohong; Xiang, Yu; He, Miao; Zeng, Siyu

    2017-03-15

    This work presents a fully integrated graphene field-effect transistor (GFET) biosensor for the label-free detection of lead ions (Pb(2+)) in aqueous-media, which first implements the G-quadruplex structure-switching biosensing principle in graphene nanoelectronics. We experimentally illustrate the biomolecular interplay that G-rich DNA single-strands with one-end confined on graphene surface can specifically interact with Pb(2+) ions and switch into G-quadruplex structures. Since the structure-switching of electrically charged DNA strands can disrupt the charge distribution in the vicinity of graphene surface, the carrier equilibrium in graphene sheet might be altered, and manifested by the conductivity variation of GFET. The experimental data and theoretical analysis show that our devices are capable of the label-free and specific quantification of Pb(2+) with a detection limit down to 163.7ng/L. These results first verify the signaling principle competency of G-quadruplex structure-switching in graphene electronic biosensors. Combining with the advantages of the compact device structure and convenient electrical signal, a label-free GFET biosensor for Pb(2+) monitoring is enabled with promising application potential.

  20. Label-free detection of Cu(2+ and Hg(2+ ions using reconstructed Cu(2+-specific DNAzyme and G-quadruplex DNAzyme.

    Directory of Open Access Journals (Sweden)

    Hui Li

    Full Text Available Label-free metal ion detection methods were developed. To achieve these, a reconstructed Cu(2+-specific DNA-cleaving DNAzyme (Cu(2+-specific DNAzyme with an intramolecular stem-loop structure was used. G-quadruplex-forming G-rich sequence(s, linked at the ends of double-helix stem of an intramolecular stem-loop structure, was partly caged in an intramolecular duplex or formed a split G-quadruplex. Cu(2+-triggered DNA cleavage at a specific site decreased the stability of the double-helix stem, resulting in the formation or destruction of G-quadruplex DNAzyme that can effectively catalyze the 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS-H2O2 reaction. Based on these, two label-free, cost-effective and simple Cu(2+ sensors were designed. These two sensors followed different detection modes: 'turn-on' and 'turn-off'. As for the 'turn-on' sensor, the intramolecular stem-loop structure ensured a low background signal, and the co-amplification of detection signal by dual DNAzymes (Cu(2+-specific DNAzyme and G-quadruplex DNAzyme provided a high sensitivity. This sensor enabled the selective detection of aqueous Cu(2+ with a detection limit of 3.9 nM. Visual detection was possible. Although the 'turn-off' sensor gave lower detection sensitivity than the 'turn-on' one, the characteristics of cost-effectiveness and ease of operation made it an important implement to reduce the possibility of pseudo-positive or pseudo-negative results. Combining the ability of Hg(2+ ion to stabilize T-T base mismatch, above dual DNAzymes-based strategy was further used for Hg(2+ sensor design. The proposed sensor allowed the specific detection of Hg(2+ ion with a detection of 4.8 nM. Visual detection was also possible.

  1. Conformation Selective Antibody Enables Genome Profiling and Leads to Discovery of Parallel G-Quadruplex in Human Telomeres.

    Science.gov (United States)

    Liu, Hui-Yun; Zhao, Qi; Zhang, Tian-Peng; Wu, Yue; Xiong, Yun-Xia; Wang, Shi-Ke; Ge, Yuan-Long; He, Jin-Hui; Lv, Peng; Ou, Tian-Miao; Tan, Jia-Heng; Li, Ding; Gu, Lian-Quan; Ren, Jian; Zhao, Yong; Huang, Zhi-Shu

    2016-10-20

    G-quadruplexes are specialized secondary structures in nucleic acids that possess significant conformational polymorphisms. The precise G-quadruplex conformations in vivo and their relevance to biological functions remain controversial and unclear, especially for telomeric G-quadruplexes. Here, we report a novel single-chain variable fragment (scFv) antibody, D1, with high binding selectivity for parallel G-quadruplexes in vitro and in vivo. Genome-wide chromatin immunoprecipitation using D1 and deep-sequencing revealed the consensus sequence for parallel G-quadruplex formation, which is characterized by G-rich sequence with a short loop size (G-quadruplex was identified and its formation was regulated by small molecular ligands targeting and telomere replication. Together, parallel G-quadruplex specific antibody D1 was found to be a valuable tool for determination of G-quadruplex and its conformation, which will prompt further studies on the structure of G-quadruplex and its biological implication in vivo.

  2. Binding-induced and label-free colorimetric method for protein detection based on autonomous assembly of hemin/G-quadruplex DNAzyme amplification strategy.

    Science.gov (United States)

    Wu, Hao; Zhang, Kai; Liu, Yaling; Wang, Hongyong; Wu, Jun; Zhu, Feifan; Zou, Pei

    2015-02-15

    In this work, a new binding-induced and label-free colorimetric method for protein detection has been developed on the basis of an autonomous assembly of hemin/G-quadruplex DNAzyme amplification strategy. The system consists of two proximity probes carrying two aptamer sequences as recognition elements for target, and two hairpin structures include three-fourths and one-fourth of the G-quadruplex sequences in inactive configuration as functional elements. In the presence of target protein, two proximity probes bind to the protein simultaneously, forming a stable DNA-protein complex. Then the complex triggers an autonomous cross-opening of the two functional hairpin structures, leading to the formation of numerous hemin/G-quadruplex DNAzymes. The resulting DNAzymes catalyze the oxidation of colorless 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS(2-)) to the green-colored ABTS(•-) with the presence of H2O2, thus providing the amplified colorimetric detection of target. Using human α-thrombin as the protein target, this binding-induced DNAzyme amplification colorimetric method affords high sensitivity with a detection limit of 1.9 pM. Furthermore, this method might be further extended to sensitive detection of other proteins by simply replacing recognition elements of proximity probes.

  3. Study of STAT3 G-quadruplex folding patterns by CD spectroscopy and molecular modeling

    Institute of Scientific and Technical Information of China (English)

    Sen Lin; Ming Xu; Gu Yuan

    2012-01-01

    The G-quadruplexes formed from G-rich strands in the telomere and oncogene-promoter regions are regarded as new promising targets in the cancer therapy.A G-quadruplex in the downstream flanking region of the signal transducer and activator of transcription 3 (STAT3) gene was explored.Its folding patterns were proposed to be 3∶2∶2 and 3∶3∶1 loop isomers by the mutation analysis by CD spectroscopy.The structures were constructed and refined by molecular modeling method.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    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. ...

  5. RHPS4 G-quadruplex ligand induces anti-proliferative effects in brain tumor cells.

    Directory of Open Access Journals (Sweden)

    Sunil Lagah

    Full Text Available BACKGROUND: Telomeric 3' overhangs can fold into a four-stranded DNA structure termed G-quadruplex (G4, a formation which inhibits telomerase. As telomerase activation is crucial for telomere maintenance in most cancer cells, several classes of G4 ligands have been designed to directly disrupt telomeric structure. METHODS: We exposed brain tumor cells to the G4 ligand 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4 and investigated proliferation, cell cycle dynamics, telomere length, telomerase activity and activated c-Myc levels. RESULTS: Although all cell lines tested were sensitive to RHPS4, PFSK-1 central nervous system primitive neuroectodermal cells, DAOY medulloblastoma cells and U87 glioblastoma cells exhibited up to 30-fold increased sensitivity compared to KNS42 glioblastoma, C6 glioma and Res196 ependymoma cells. An increased proportion of S-phase cells were observed in medulloblastoma and high grade glioma cells whilst CNS PNET cells showed an increased proportion of G1-phase cells. RHPS4-induced phenotypes were concomitant with telomerase inhibition, manifested in a telomere length-independent manner and not associated with activated c-Myc levels. However, anti-proliferative effects were also observed in normal neural/endothelial cells in vitro and ex vivo. CONCLUSION: This study warrants in vivo validation of RHPS4 and alternative G4 ligands as potential anti-cancer agents for brain tumors but highlights the consideration of dose-limiting tissue toxicities.

  6. Novel application of fluorescence coupled capillary electrophoresis to resolve the interaction between the G-quadruplex aptamer and thrombin.

    Science.gov (United States)

    Wang, Jianhao; Gu, Yaqin; Liu, Li; Wang, Cheli; Wang, Jianpeng; Ding, Shumin; Li, Jinping; Qiu, Lin; Jiang, Pengju

    2017-08-01

    The dynamic binding status between the thrombin and its G-quadruplex aptamers and the stability of its interaction partners were probed using our previously established fluorescence-coupled capillary electrophoresis method. A 29-nucleic acid thrombin binding aptamer was chosen as a model to study its binding affinity with the thrombin ligand. First, the effects of the cations on the formation of G-quadruplex from unstructured 29-nucleic acid thrombin binding aptamer were examined. Second, the rapid binding kinetics between the thrombin and 6-carboxyfluorescein labeled G-quadruplex aptamer was measured. Third, the stability of G-quadruplex aptamer-thrombin complex was also examined in the presence of the interfering species. Remarkably, it was found that the complementary strand of 29-nucleic acid thrombin binding aptamer could compete with G-quadruplex aptamer and thus disassociated the G-quadruplex structure into an unstructured aptamer. These data suggest that our in-house established fluorescence-coupled capillary electrophoresis assay could be applied to binding studies of the G-quadruplex aptamers, thrombin, and their ligands, while overcoming the complicated and costly approaches currently available. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Directory of Open Access Journals (Sweden)

    Hidenobu Yaku

    2015-04-01

    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.

  8. 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)

    2017-02-15

    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.

  9. Investigation of G-quadruplex formation in the FGFR2 promoter region and its transcriptional regulation by liensinine.

    Science.gov (United States)

    Zhang, Lulu; Tan, Wei; Zhou, Jiang; Xu, Ming; Yuan, Gu

    2017-04-01

    Fibroblast growth factor receptor 2 (FGFR2) is overexpressed in breast cancer tissues and cells, and has been shown to be a susceptibility factor for breast cancer. In this study, we found that the G-rich sequences in the FGFR2 promoter region can form G-quadruplexes, which could be the target and inhibitor of the FGFR2 gene. Initially, the formation of G-quadruplexes was confirmed by ESI-MS and CD, and DMS footprinting experiments gave the folding pattern of the G-quadruplexes. After luciferase reporter assays revealed that the G-quadruplex could inhibit the activity of the FGFR2 promoter, MS and SPR showed binding affinity and selectivity of the ligand. Then cell culture experiments and ChIP assay showed the bioactivity of the ligand. We found that three G-rich sequences (S1-S3) in the FGFR2 promoter region can form G-quadruplex structures. And a natural alkaloid, liensinine, was found to bind to the S1 G-quadruplex with relative high affinity and selectivity. Cell culture experiments showed that liensinine inhibits FGFR2 activity at both the transcriptional and translational levels. Moreover, chromatin immunoprecipitation assay (ChIP) results showed that liensinine blocks the binding of E2F1 at the transcription factor binding site (TFBS) in the S1 sequence, which is the mechanism through which liensinine inhibits the FGFR2 gene. A natural alkaloid was discovered to selectively bind to the S1 G-quadruplex with relative high affinity, and therefore inhibited FGFR2 transcription and translation. Our discovery offers a useful strategy to inhibit FGFR2 transcription, i.e., targeting the G-quadruplex with a natural alkaloid. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Spectroscopic investigation of the interaction between G-quadruplex of KRAS promoter sequence and three isoquinoline alkaloids

    Science.gov (United States)

    Wen, Li-Na; Xie, Meng-Xia

    2017-01-01

    KRAS promoter can form G-quadruplex structure and regulate gene transcription. The drugs which can bind with G-quadruplex of KRAS promoter may be potential remedy for treatment of cancers associated with KRAS mutation. The interaction mechanism between the G-quadruplex of KRAS promoter and three isoquinoline alkaloids (jatrorrhizine, berberine and sanguinarine) has been investigated by UV-visible, fluorescence and circular dichroism spectroscopic methods. The results showed that the three alkaloids can form complexes with G-quadruplex KRAS promoter with the molecular ratio of 1:1, and the binding constants were (0.90 ± 0.16) × 106 L mol- 1, (0.93 ± 0.21) × 106 L mol- 1 and (1.16 ± 0.45) × 106 L mol- 1 for jatrorrhizine, berberine and sanguinarine. The absorption spectra, KI quenching and fluorescence anisotropy and polarization studies suggested jatrorrhizine and berberine interacted with G-quadruplex by not only end-stacking binding mode but also grooves or loops binding mode, while sanguinarine by end-stacking binding mode. Sanguinarine was more beneficial to maintain the stability and parallel conformation of KRAS promoter G-quadruplex. MTT assay was performed to evaluate antiproliferation effects of the three isoquinoline alkaloids on SW620 cells, and the antiproliferation effects of the three alkaloids were sanguinarine > berberine > jatrorrhizine. All the three alkaloids can bind with KRAS promoter G-quadruplex, and sanguinarine had the better binding property and antiproliferation effects on SW620 cells. The results obtained are meaningful to explore potential reagents targeting the parallel G-quadruplex structure of KRAS promoter for gene theraphy of colorectal carcinomas.

  11. 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

    2009-01-01

    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....

  12. 金属配合物与G-四链体作用的研究进展%Interaction of metal complexes with G-quadruplex

    Institute of Scientific and Technical Information of China (English)

    孙静; 陈嘉曦; 陈伙炎; 毛宗万

    2012-01-01

    The activity of telomerase can be prohibited by the secondary structure of the telomere DNA-G-quadruplex formed by the single chain in G-rich field, resulting in the apoptosis in tumor cells. Compound which can induce the formation or stabilize the structure of G-quadruplex might be potential anticancer drug. The relative ease of synthesis, the variable structure and the positively charged center enable the metal complexes to in-teract with the grooves and loops of the quadruplex and negatively charged phosphates backbone. Therefore, many metal complexes have been designed and synthesized. This paper reviews the discoveries in the interaction of G-quadruplex with metal complexes in the past years.%端粒DNA形成的独特二级结构G-四链体对端粒酶的活性有明显抑制作用,能达到促进肿瘤细胞凋亡的作用.研究表明,能够诱使端粒DNA形成G-四链体及可以稳定这种结构的药物均可能成为有效的化疗药物.金属配合物由于其合成路径简单、结构多变、金属中心所携带的正电荷有利于与G-四链体的沟区、loop环以及带负电荷的磷酸骨架作用,增强其稳定性,因此越来越多的过渡金属配合物被设计用来研究与G-四链体作用.该文综述了近年来金属配合物与G-四链体DNA相互作用方面的研究进展.

  13. Fragile X mental retardation protein recognition of G quadruplex structure per se is sufficient for high affinity binding to RNA.

    Science.gov (United States)

    Bole, Medhavi; Menon, Lakshmi; Mihailescu, Mihaela-Rita

    2008-12-01

    Fragile X syndrome, the most common form of inherited mental retardation is caused by the expansion of a CGG trinucleotide repeat in the fragile X mental retardation 1 (fmr1) gene. The abnormal expansion of the CGG repeat causes hypermethylation and subsequent silencing of the fmr1 gene, resulting in the loss of the fragile X mental retardation protein (FMRP). FMRP has been shown to use its arginine-glycine-glycine rich region (RGG box) to bind to messenger RNAs that form G quadruplex structures. Several studies reported that the G quadruplex RNA recognition alone is not sufficient for FMRP RGG box binding and that an additional stem and/or a G quadruplex-stem junction region may also be important in recognition. In this study we have used biophysical methods such as fluorescence, UV, CD and NMR spectroscopy to demonstrate that the recognition of the RNA G quadruplex structure per se, in the absence of a stem region, is sufficient for the FMRP high affinity and specific binding. These findings indicate that the presence of a stem structure in some of the FMRP G quadruplex forming mRNAs is not a requirement for protein recognition as previously believed, but rather for the proper formation of the correct RNA G quadruplex structure recognized by FMRP.

  14. Visual detection and differentiation of Classic Swine Fever Virus strains using nucleic acid sequence-based amplification (NASBA) and G-quadruplex DNAzyme assay

    Science.gov (United States)

    Lu, Xiaolu; Shi, Xueyao; Wu, Gege; Wu, Tiantian; Qin, Rui; Wang, Yi

    2017-01-01

    The split G-quadruplex DNAzyme has emerged as a valuable tool for visual DNA detection. Here, we successfully integrated colorimetric split G-quadruplex DNAzyme assay with nucleic acid sequence-based amplification to generate a novel detection approach, allowing visual and rapid detection for the RNA of Shimen and HCLV strains of Classic Swine Fever Virus (CSFV). CSFV is a RNA virus that causes a highly contagious disease in domestic pigs and wild boar. With this method, we were able to detect as little as 10 copies/ml of CSF viral RNA within 3 h in serum samples taken from the field. No interference was encountered in the amplification and detection of Classic Swine Fever Virus in the presence of non-target RNA or DNA. Moreover, Shimen and HCLV strains of Classic Swine Fever Virus could be easily differentiated using the NASBA-DNAzyme system. These findings indicate the NASBA-DNAzyme system is a rapid and practical technique for detecting and discriminating CSFV strains and may be applied to the detection of other RNA viruses. PMID:28287135

  15. A novel photoelectrochemical sensor based on photocathode of PbS quantum dots utilizing catalase mimetics of bio-bar-coded platinum nanoparticles/G-quadruplex/hemin for signal amplification.

    Science.gov (United States)

    Wang, Guang-Li; Liu, Kang-Li; Shu, Jun-Xian; Gu, Tian-Tian; Wu, Xiu-Ming; Dong, Yu-Ming; Li, Zai-Jun

    2015-07-15

    Photocathode based on p-type PbS quantum dots (QDs) combing a novel signal amplification strategy utilizing catalase (CAT) mimetics was designed and utilized for sensitive photoelectrochemical (PEC) detection of DNA. The bio-bar-coded Pt nanoparticles (NPs)/G-quadruplex/hemin exhibited high CAT-like activity following the Michaelis-Menten model for decomposing H2O2 to water and oxygen, whose activity even slightly exceeded that of natural CAT. The bio-bar-code as a catalytic label was conjugated onto the surface of PbS QDs modified electrodes through the formed sandwich-type structure due to DNA hybridization. Oxygen in situ generated by the CAT mimetics of the bio-bar-code of Pt NPs/G-quadruplex/hemin acted as an efficient electron acceptor of illuminated PbS QDs, promoting charge separation and enhancing cathodic photocurrent. Under optimal conditions, the developed PEC biosensor for target DNA exhibited a dynamic range of 0.2pmol/L to 1.0nmol/L with a low detection limit of 0.08pmol/L. The high sensitivity of the method was resulted from the sensitive response of PbS QDs to oxygen and the highly efficient CAT-like catalytic activity of the bio-bar-coded Pt NPs/G-quadruplex/hemin.

  16. Colorimetric Strategy for Highly Sensitive and Selective Simultaneous Detection of Histidine and Cysteine Based on G-Quadruplex-Cu(II) Metalloenzyme.

    Science.gov (United States)

    Wu, Changtong; Fan, Daoqing; Zhou, Chunyang; Liu, Yaqing; Wang, Erkang

    2016-03-01

    In this present work, we proposed a colorimetric strategy for simultaneous detection of histidine and cysteine based on G-quadruplex-Cu(II) metalloenzyme for the first time. Because of the adding of histidine or cysteine, the formation of G-quadruplex-Cu(II) metalloenzyme will be disturbed, thus the catalytic activity to TMB-H2O2 reaction is inversely proportional to the concentration of histidine or cysteine. With this strategy, the limit of detection in experimental measurement for histidine and cysteine is 10 nM and 5 nM, respectively, which are both lower than previous colorimetric arrays. With the help of NEM, cysteine is alkylated and the reaction between Cu(2+) is inhibited, so the selectivity can also be guaranteed. The cost is quite low since the developed array is label free and enzyme free by using low-cost DNA and Cu(2+). More importantly, the colorimetric detection operation is very simple without any further modification process.

  17. 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.

  18. Sequencing and G-Quadruplex Folding of the Canine Proto-Oncogene KIT Promoter Region: Might Dog Be Used as a Model for Human Disease?

    Science.gov (United States)

    Da Ros, Silvia; Zorzan, Eleonora; Giantin, Mery; Zorro Shahidian, Lara; Palumbo, Manlio; Dacasto, Mauro; Sissi, Claudia

    2014-01-01

    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. PMID:25084283

  19. A new application of click chemistry in situ: development of fluorescent probe for specific G-quadruplex topology.

    Science.gov (United States)

    Hu, Ming-Hao; Chen, Xiao; Chen, Shuo-Bin; Ou, Tian-Miao; Yao, Meicun; Gu, Lian-Quan; Huang, Zhi-Shu; Tan, Jia-Heng

    2015-11-25

    Target-guided synthesis is an approach to drug discovery that allows the target to self-assemble its own binding agents. So far, target-guided synthesis and especially in situ click chemistry have attracted extensive attention and have led to the identification of highly potent inhibitors for proteins. In this study, we expand the application of in situ click chemistry and present a procedure using this approach to identify selective fluorescent probes for a specific topology of G-quadruplex nucleic acids, the parallel G-quadruplexes. On this basis, compound 15 assembled by triarylimidazole scaffold and carboxyl side chain was a positive hit, demonstrating highly potential in the sensitive and selective detection of parallel G-quadruplexes. Such selective fluorescence response can be rationalized in terms of different binding affinities between 15 and G-quadruplexes. Our work accordingly represents a new development towards the application of in situ click chemistry to develop selective fluorescent probes and may also shed light on the search for probes for a specific G-quadruplex topology.

  20. The cellular protein hnRNP A2/B1 enhances HIV-1 transcription by unfolding LTR promoter G-quadruplexes

    Science.gov (United States)

    Scalabrin, Matteo; Frasson, Ilaria; Ruggiero, Emanuela; Perrone, Rosalba; Tosoni, Elena; Lago, Sara; Tassinari, Martina; Palù, Giorgio; Richter, Sara N.

    2017-01-01

    G-quadruplexes are four-stranded conformations of nucleic acids that act as cellular epigenetic regulators. A dynamic G-quadruplex forming region in the HIV-1 LTR promoter represses HIV-1 transcription when in the folded conformation. This activity is enhanced by nucleolin, which induces and stabilizes the HIV-1 LTR G-quadruplexes. In this work by a combined pull-down/mass spectrometry approach, we consistently found hnRNP A2/B1 as an additional LTR-G-quadruplex interacting protein. Surface plasmon resonance confirmed G-quadruplex specificity over linear sequences and fluorescence resonance energy transfer analysis indicated that hnRNP A2/B1 is able to efficiently unfold the LTR G-quadruplexes. Evaluation of the thermal stability of the LTR G-quadruplexes in different-length oligonucleotides showed that the protein is fit to be most active in the LTR full-length environment. When hnRNP A2/B1 was silenced in cells, LTR activity decreased, indicating that the protein acts as a HIV-1 transcription activator. Our data highlight a tightly regulated control of transcription based on G-quadruplex folding/unfolding, which depends on interacting cellular proteins. These findings provide a deeper understanding of the viral transcription mechanism and may pave the way to the development of drugs effective against the integrated HIV-1, present both in actively and latently infected cells.

  1. G-quadruplex fluorescence sensing by core-extended naphthalene diimides.

    Science.gov (United States)

    Zuffo, Michela; Doria, Filippo; Botti, Silvia; Bergamaschi, Greta; Freccero, Mauro

    2017-05-01

    Fluorescent sensing of G-quadruplex nucleic acids (G4s) is an effective strategy to elucidate their role in vitro and in vivo. Small molecule ligands have often been exploited, producing an emission light up upon binding. Naphthalene diimides (NDIs), although potent G4 binders exhibiting red-NIR fluorophores, have only been marginally exploited, as they are usually quenched upon binding. Contrary, aggregating core-extended naphthalene diimides (cex-NDIs) proved to be effective probes. We prepared a library of eighteen cex-NDIs by organic synthesis, characterising their aggregation-dependent absorption and emission properties. Absorption and emission titrations, fluorescent intercalator displacement assay (FID) and circular dichroism (CD) analysis were performed to elucidate their behavior as G4 fluorescent sensors, selectivity and binding mode. cex-NDIs aggregate under aqueous solvents and as a result, their fluorescence is mostly quenched under physiological conditions. Upon G4 binding, they disaggregate into binding monomers, producing a fluorescent light-up with anti-parallel and hybrid G4s. Contrary, with parallel G4s a light-off was recorded. For the formers a groove-like interaction was inferred by ICD signals, while for the latter an end-stacking interaction mode was hypothesized by G4-FID data. cex-NDIs G4 sensing mechanism works via a induced disaggregation. The emission response depends on the G4 topology, which dictates the prevailing -groove or end-stacking- binding mode. This study highlights the potential of cex-NDIs as G4 fluorescent probes. Besides being readily synthesized and conveniently emitting above 600nm, they light-up upon binding to anti-parallel and hybrid G4, complementing a number of other probes' selectivity for the parallel topology. 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.

  2. Rotation of Guanine Amino Groups in G-Quadruplexes: A Probe for Local Structure and Ligand Binding.

    Science.gov (United States)

    Adrian, Michael; Winnerdy, Fernaldo Richtia; Heddi, Brahim; Phan, Anh Tuân

    2017-08-22

    Nucleic acids are dynamic molecules whose functions may depend on their conformational fluctuations and local motions. In particular, amino groups are dynamic components of nucleic acids that participate in the formation of various secondary structures such as G-quadruplexes. Here, we present a cost-efficient NMR method to quantify the rotational dynamics of guanine amino groups in G-quadruplex nucleic acids. An isolated spectrum of amino protons from a specific tetrad-bound guanine can be extracted from the nuclear Overhauser effect spectroscopy spectrum based on the close proximity between the intra-residue imino and amino protons. We apply the method in different structural contexts of G-quadruplexes and their complexes. Our results highlight the role of stacking and hydrogen-bond interactions in restraining amino-group rotation. The measurement of the rotation rate of individual amino groups could give insight into the dynamic processes occurring at specific locations within G-quadruplex nucleic acids, providing valuable probes for local structure, dynamics, and ligand binding. Copyright © 2017 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  3. G-quadruplex based Exo III-assisted signal amplification aptasensor for the colorimetric detection of adenosine.

    Science.gov (United States)

    Xu, Lei; Shen, Xin; Li, Bingzhi; Zhu, Chunhong; Zhou, Xuemin

    2017-08-08

    Adenosine is an endogenous nucleotide pivotally involved in nucleic acid and energy metabolism. Its excessive existence may indicate tumorigenesis, typically lung cancer. Encouraged by its significance as the clinical biomarker, sensitive assay methods towards adenosine have been popularized, with high cost and tedious procedures as the inevitable defects. Herein, we report a label-free aptamer-based exonuclease III (Exo III) amplification colorimetric aptasensor for the highly sensitive and cost-effective detection of adenosine. The strategy employed two unlabeled hairpin DNA oligonucleotides (HP1 and HP2), where HP1 contained the aptamer towards adenosine and HP2 embedded the guanine-rich sequence (GRS). In the presence of adenosine, hairpin HP1 could form specific binding with adenosine and trigger the unfolding of HP1's hairpin structure. The resulting adenosine-HP1 complex could hybridize with HP2, generating the Exo III recognition site. After Exo III-assisted degradation, the GRS was released from HP2, and the adenosine-HP1 was released back to the solution to combine another HP2, inducing the cycling amplification. After multiple circulations, the released ample GRSs were induced to form G-quadruplex, further catalyzing the oxidation of TMB, yielding a color change which was finally mirrored in the absorbance change. On the contrary, the absence of adenosine failed to unfold HP1, remaining color unchanged eventually. Thanks to the amplification strategy, the limit of detection was lowered to 17 nM with a broad linear range from 50 nM to 6 μM. The proposed method was successfully applied to the detection of adenosine in biological samples and satisfying recoveries were acquired. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Antiproliferative Activity of G-quadruplex Nucleic Acids%具抗肿瘤活性的G-四链体核酸研究进展

    Institute of Scientific and Technical Information of China (English)

    常天俊; 龚红梅; 李卫国

    2012-01-01

    G-四链体(G-quadruplex,G4)是由富含串联重复的鸟嘌呤碱基(G)的DNA或RNA链折叠形成的一种特殊的核酸二级结构.可形成G4结构的核酸序列在基因组和人端粒中广泛存在,对生理和病理过程起重要的调节作用.近年来研究发现,一些化学合成的G4核酸具有选择性的抗肿瘤增殖活性;其中AS1411是一个26个碱基的G4序列核酸,目前已作为抗癌药物进入二期临床研究.对G4核酸的结构和功能,抗癌活性及分子机理研究进行综合评述,并简要介绍G4核酸在相关领域的应用研究.%G-quadruplexes(G4s) are four-stranded nucleic acid structures adopted by some repetitive guanine-rich sequences.G4 sequences are highly prevalent in human genome and telomere.Recently,some synthetic G4s have been reported to have cancer-selective antiproliferative activity.AS1411,a 26-mer G4 DNA,is currently in phase II clinical trials as an anticancer agent.The structures,functions,antiproliferative activities and mechanisms,and the applications of G4s are reviewed.

  5. The Truncated Human Telomeric Sequence forms a Hybrid-Type Intramolecular Mixed Parallel/antiparallel G-quadruplex Structure in K(+) Solution.

    Science.gov (United States)

    Liu, Yuxia; Cheng, Dengfeng; Ge, Min; Lin, Weizhen

    2016-07-01

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

  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. RNA G-quadruplexes cause eIF4A-dependent oncogene translation in cancer

    Science.gov (United States)

    Wolfe, Andrew L.; Singh, Kamini; Zhong, Yi; Drewe, Philipp; Rajasekhar, Vinagolu K.; Sanghvi, Viraj R.; Mavrakis, Konstantinos J.; Jiang, Man; Roderick, Justine E.; van der Meulen, Joni; Schatz, Jonathan H.; Rodrigo, Christina M.; Zhao, Chunying; Rondou, Pieter; de Stanchina, Elisa; Teruya-Feldstein, Julie; Kelliher, Michelle A.; Speleman, Frank; Porco, John A.; Pelletier, Jerry; Rätsch, Gunnar; Wendel, Hans-Guido

    2014-09-01

    The translational control of oncoprotein expression is implicated in many cancers. Here we report an eIF4A RNA helicase-dependent mechanism of translational control that contributes to oncogenesis and underlies the anticancer effects of silvestrol and related compounds. For example, eIF4A promotes T-cell acute lymphoblastic leukaemia development in vivo and is required for leukaemia maintenance. Accordingly, inhibition of eIF4A with silvestrol has powerful therapeutic effects against murine and human leukaemic cells in vitro and in vivo. We use transcriptome-scale ribosome footprinting to identify the hallmarks of eIF4A-dependent transcripts. These include 5' untranslated region (UTR) sequences such as the 12-nucleotide guanine quartet (CGG)4 motif that can form RNA G-quadruplex structures. Notably, among the most eIF4A-dependent and silvestrol-sensitive transcripts are a number of oncogenes, superenhancer-associated transcription factors, and epigenetic regulators. Hence, the 5' UTRs of select cancer genes harbour a targetable requirement for the eIF4A RNA helicase.

  8. A single molecule study of a fluorescently labeled telomestatin derivative and G-quadruplex interactions

    Science.gov (United States)

    Maleki, Parastoo; Ma, Yue; Iida, Keisuke; Nagasawa, Kazuo; Balci, Hamza

    2017-01-01

    The potential use of G-quadruplex (GQ) stabilizing small molecules as anti-cancer drugs has created a flurry of activity on various aspects of these molecules. Telomestatin and oxazole telomestatin derivatives (OTD) are some of the most prominent of such molecules, yet the underlying dynamics of their interactions with GQ and the extent of heterogeneities in these interactions are not known. We performed single molecule measurements to study binding kinetics, rotational freedom, and dwell time distributions of a Cy5-labeled OTD (L1Cy5–7OTD) as it interacted with several different GQ structures. Our measurements show that L1Cy5–7OTD dwells on more stable GQ for longer times and binds to such GQ with higher frequency. The dwell times showed a broad distribution, but were longer than a minute for a significant fraction of molecules (characteristic dwell time τ = 192 ± 15 s and τ = 98 ± 15 s for the more and less stable GQ, respectively). In addition, L1Cy5–7OTD might be able to bind to GQ in at least two different primary orientations and occasionally transition between these orientations. The dwell time in one of these orientations was significantly longer than that in the other one, suggesting different stabilities for different binding orientations. PMID:27899628

  9. Improved in vitro efficacy of gold nanoconstructs by increased loading of G-quadruplex aptamer.

    Science.gov (United States)

    Dam, Duncan Hieu M; Lee, Raymond C; Odom, Teri W

    2014-05-14

    This paper describes how in vitro efficacy of aptamer-loaded gold nanostars (Apt-AuNS) can be enhanced by the increased loading of a G-quadruplex homodimer AS1411 (Apt) on the AuNS surface. In a low pH buffer environment, the loading density of Apt on AuNS was increased up to 2.5 times that obtained using the conventional salt-aging process. These highly loaded AuNS nanoconstructs (*Apt-AuNS) were taken up in pancreatic cancer and fibrosarcoma cells ca. 2 times more and at faster rates compared to Apt-AuNS. When a similar number of AuNS carriers was internalized by the cancer cells, the amount of AS1411 delivered via *Apt-AuNS was effectively double that of Apt-AuNS, and *Apt-AuNS resulted in an average of 42% increase in cell death. These results suggest that increasing the loading density on AuNS could provide a simple means to improve uptake as well as in vitro efficacy of the nanoconstructs in cancer cells.

  10. 2'-F-ANA-guanosine and 2'-F-guanosine as powerful tools for structural manipulation of G-quadruplexes.

    Science.gov (United States)

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

    2012-12-04

    Here we demonstrate the applicability of 2'-F-ANA-guanosine and 2'-F-guanosine as powerful tools for manipulating G-quadruplex folding by anti-position-favoring substitutions. A single guanine to 2'-F-ANA-guanine substitution can favor a single (3+1) hybrid conformation from a mixture of conformers. Rational substitutions of either type of 2'-F-modified nucleotide enable conformational switching from a (3+1) hybrid to a parallel folding topology.

  11. 基于DNA G-四链体识别的抗肿瘤分子筛选及结构设计研究进展%Progress on G-quadruplex as targets in anticancer drug structure screening and rational design(in Chinese)

    Institute of Scientific and Technical Information of China (English)

    张秀凤; 向俊锋; 田明月; 唐亚林

    2009-01-01

    以生物靶分子为基础进行抗肿瘤药物先导化合物的筛选是抗肿瘤药物研究的热点之一.DNA G-四链体结构的发现和现代分子生物学技术对其与癌症关系的揭示,为目前抗肿瘤药物研发提供了一个新的契机.能够诱导DNA形成G-四链体结构或者与G-四链体特异性结合并使之稳定的化合物有望抑制肿瘤细胞的生长,从而达到抗癌的作用.以G-四链体为抗癌药物作用靶点对化合物进行筛选和结构设计是目前化学家和生物学家的关注点.本文旨在针对靶向G-四链体的抗肿瘤分子筛选、结构设计以及抗肿瘤药物开发三个方面的最新研究进展进行综述.首先,介绍两种基于G-四链体对其配体结构特异性识别而对化合物进行结构筛选的研究方法:基于核磁共振进行抗肿瘤化合物筛选方法以及计算机虚拟筛选.其次,从化合物与G-四链体之间静电相互作用方面来进行G-四链体配体的结构设计,主要包括以下4种类型:(1)原位胺的质子化;(2)通过杂环芳香族化合物上的N-甲基化;(3)中心金属离子的存在;(4)不带电荷的化合物.最后,对目前基于G-四链体为抗肿瘤作用靶点、已经走向临床实验的CX-3543,AS1411两个抗肿瘤药物的开发与作用机制进行介绍.

  12. 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: zhangkai@jsinm.org; Wang, Ke; Zhu, Xue; Xie, Minhao

    2015-08-05

    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.

  13. Halogenated pentamethine cyanine dyes exhibiting high fidelity for G-quadruplex DNA.

    Science.gov (United States)

    Nanjunda, Rupesh; Owens, Eric A; Mickelson, Leah; Alyabyev, Sergey; Kilpatrick, Nancy; Wang, Siming; Henary, Maged; Wilson, W David

    2012-12-15

    Design and optimization of quadruplex-specific small molecules is developing into an attractive strategy for anti-cancer therapeutics with some promising candidates in clinical trials. A number of therapeutically favorable features of cyanine molecules can be effectively exploited to develop them as promising quadruplex-targeting agents. Herein, the design, synthesis and evaluation of a series of dimethylindolenine cyanine dyes with varying halogen substitutions are reported. Their interactions with telomeric and c-myc quadruplexes as well as a reference duplex sequence have been evaluated using thermal melting, biosensor-surface plasmon resonance, circular dichroism, isothermal titration calorimetry and mass spectrometry. Thermal melting analysis indicates that these ligands exhibit significant quadruplex stabilization and a very low duplex binding, with the dimethyl incorporation of paramount importance for decreased duplex affinity. Circular dichroism studies showed that the interaction of cyanines with quadruplex structures are primarily through stacking at one or both ends of the terminal tetrads with the two (trimethylammonium)propyl groups interacting in the accessible quadruplex grooves. Surface plasmon resonance and mass spectral studies shows the formation of an initial strong 1:1 complex followed by a significantly weaker secondary binding. Isothermal calorimetry studies show that the interaction of cyanines is predominantly entropy driven. In line with the design principles, this work provides new insights for further developing potent, highly selective cyanines as promising quadruplex-specific agents.

  14. G-quadruplex fluorescent probe-mediated real-time rolling circle amplification strategy for highly sensitive microRNA detection.

    Science.gov (United States)

    Jiang, Hong-Xin; Liang, Zhen-Zhen; Ma, Yan-Hong; Kong, De-Ming; Hong, Zhang-Yong

    2016-11-02

    Real-time PCR has revolutionized PCR from qualitative to quantitative. As an isothermal DNA amplification technique, rolling circular amplification (RCA) has been demonstrated to be a versatile tool in many fields. Development of a simple, highly sensitive, and specific strategy for real-time monitoring of RCA will increase its usefulness in many fields. The strategy reported here utilized the specific fluorescence response of thioflavin T (ThT) to G-quadruplexes formed by RCA products. Such a real-time monitoring strategy works well in both traditional RCA with linear amplification efficiency and modified RCA proceeded in an exponential manner, and can be readily performed in commercially available real-time PCR instruments, thereby achieving high-throughput detection and making the proposed technique more suitable for biosensing applications. As examples, real-time RCA-based sensing platforms were designed and successfully used for quantitation of microRNA over broad linear ranges (8 orders of magnitude) with a detection limit of 4 aM (or 0.12 zmol). The feasibility of microRNA analysis in human lung cancer cells was also demonstrated. This work provides a new method for real-time monitoring of RCA by using unique nucleic acid secondary structures and their specific fluorescent probes. It has the potential to be extended to other isothermal single-stranded DNA amplification techniques.

  15. The G-quadruplex-forming aptamer AS1411 potently inhibits HIV-1 attachment to the host cell

    OpenAIRE

    Perrone, Rosalba; Butovskaya, Elena; Lago, Sara; Garzino-Demo, Alfredo; Pannecouque, Christophe; Palù, Giorgio; Richter, Sara N.

    2016-01-01

    AS1411 is a G-rich aptamer that forms a stable G-quadruplex structure and displays antineoplastic properties both in vitro and in vivo. This oligonucleotide has undergone phase 2 clinical trials. The major molecular target of AS1411 is nucleolin (NCL), a multifunctional nucleolar protein also present in the cell membrane where it selectively mediates the binding and uptake of AS1411. Cell-surface NCL has been recognised as a low-affinity co-receptor for human immunodeficiency virus type 1 (HI...

  16. 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.

    Science.gov (United States)

    Foulk, Michael S; Urban, John M; Casella, Cinzia; Gerbi, Susan A

    2015-05-01

    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 (λ-exo) to efficiently digest parental DNA while leaving RNA-primer protected nascent strands intact. We used genomics and biochemical approaches to determine if λ-exo digests all parental DNA sequences equally. We report that λ-exo does not efficiently digest G-quadruplex (G4) structures in a plasmid. Moreover, λ-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 λ-exo biases in NS-seq and validated this approach at the rDNA locus. The λ-exo-controlled NS-seq peaks are not GC-rich, and only 35.5% overlap with 6.8% of all G4s, suggesting that G4s are not general determinants for origin specification but may play a role for a subset. Interestingly, we observed a periodic spacing of G4 motifs and nucleosomes around the peak summits, suggesting that G4s may position nucleosomes at this subset of origins. Finally, we demonstrate that use of Na(+) instead of K(+) in the λ-exo digestion buffer reduced the effect of G4s on λ-exo digestion and discuss ways to increase both the sensitivity and specificity of NS-seq.

  17. Electrochemical aptasensor based on the dual-amplification of G-quadruplex horseradish peroxidase-mimicking DNAzyme and blocking reagent-horseradish peroxidase.

    Science.gov (United States)

    Yuan, Yali; Gou, Xuxu; Yuan, Ruo; Chai, Yaqin; Zhuo, Ying; Mao, Li; Gan, Xianxue

    2011-06-15

    A simple electrochemical aptasensor for sensitive detection of thrombin was fabricated with G-quadruplex horseradish peroxidase-mimicking DNAzyme (hemin/G-quadruplex system) and blocking reagent-horseradish peroxidase as dual signal-amplification scheme. Gold nanoparticles (nano-Au) were firstly electrodeposited onto single wall nanotube (SWNT)-graphene modified electrode surface for the immobilization of electrochemical probe of nickel hexacyanoferrates nanoparticles (NiHCFNPs). Subsequently, another nano-Au layer was electrodeposited for further immobilization of thrombin aptamer (TBA), which later formed hemin/G-quadruplex system with hemin. Horseradish peroxidases (HRP) then served as blocking reagent to block possible remaining active sites and avoided the non-specific adsorption. In the presence of thrombin, the TBA binded to thrombin and the hemin released from the hemin/G-quadruplex electrocatalytic structure, increasing steric hindrance of the aptasensor and decomposing hemin/G-quadruplex electrocatalytic structure, which finally decreased the electrocatalytic efficiency of aptasensor toward H(2)O(2) in the presence of NiHCFNPs with a decreased electrochemical signal. On the basis of the synergistic amplifying action, a detection limit as low as 2 pM for thrombin was obtained.

  18. A facile label-free G-quadruplex based fluorescent aptasensor method for rapid detection of ATP

    Science.gov (United States)

    Liu, Haisheng; Ma, Changbei; Ning, Feng; Chen, Hanchun; He, Hailun; Wang, Kemin; Wang, Jun

    2017-03-01

    The present work demonstrates a simple, rapid and label-free ATP detection method using a fluorescent aptasensor that is based on G-quadruplex formation. In the absence of ATP, the Thioflavin T (ThT) dye binds to the G-rich ATP aptamer and forms an ATP aptamer/ThT G-quadruplex complex, which results in high fluorescence intensity. Upon addition of ATP, the ATP aptamer/ThT complex will be replaced by the formation of an ATP aptamer/ATP complex. During this process, separation of the ThT dye from the ATP aptamer/ThT complex decreases the fluorescence intensity of the reaction mixture dramatically. This fluorescence aptasensor is highly sensitive and rapid, with a detection limit of 18 nM and a total reaction time of only 10 min. Furthermore, this method is cost-effective and simple, removing the requirement for labeling the detection reagents with a fluorophore-quencher pair.

  19. 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

    2011-01-01

    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...

  20. 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.

  1. Selectivity of major isoquinoline alkaloids from Chelidonium majus towards telomeric G-quadruplex: A study using a transition-FRET (t-FRET) assay.

    Science.gov (United States)

    Noureini, Sakineh Kazemi; Esmaeili, Hosein; Abachi, Farzane; Khiali, Soraia; Islam, Barira; Kuta, Martyna; Saboury, Ali A; Hoffmann, Marcin; Sponer, Jiri; Parkinson, Gary; Haider, Shozeb

    2017-08-01

    Natural bioproducts are invaluable resources in drug discovery. Isoquinoline alkaloids of Chelidonium majus constitute a structurally diverse family of natural products that are of great interest, one of them being their selectivity for human telomeric G-quadruplex structure and telomerase inhibition. The study focuses on the mechanism of telomerase inhibition by stabilization of telomeric G-quadruplex structures by berberine, chelerythrine, chelidonine, sanguinarine and papaverine. Telomerase activity and mRNA levels of hTERT were estimated using quantitative telomere repeat amplification protocol (q-TRAP) and qPCR, in MCF-7 cells treated with different groups of alkaloids. The selectivity of the main isoquinoline alkaloids of Chelidonium majus towards telomeric G-quadruplex forming sequences were explored using a sensitive modified thermal FRET-melting measurement in the presence of the complementary oligonucleotide CT22. We assessed and monitored G-quadruplex topologies using circular dichroism (CD) methods, and compared spectra to previously well-characterized motifs, either alone or in the presence of the alkaloids. Molecular modeling was performed to rationalize ligand binding to the G-quadruplex structure. The results highlight strong inhibitory effects of chelerythrine, sanguinarine and berberine on telomerase activity, most likely through substrate sequestration. These isoquinoline alkaloids interacted strongly with telomeric sequence G-quadruplex. In comparison, chelidonine and papaverine had no significant interaction with the telomeric quadruplex, while they strongly inhibited telomerase at transcription level of hTERT. Altogether, all of the studied alkaloids showed various levels and mechanisms of telomerase inhibition. We report on a comparative study of anti-telomerase activity of the isoquinoline alkaloids of Chelidonium majus. Chelerythrine was most effective in inhibiting telomerase activity by substrate sequesteration through G-quadruplex

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

    Science.gov (United States)

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

    2017-05-12

    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.

  3. Ultrafast Electron Transfer in Complexes of Doxorubicin with Human Telomeric G-Quadruplexes and GC Duplexes Probed by Femtosecond Fluorescence Spectroscopy.

    Science.gov (United States)

    Changenet-Barret, Pascale; Gustavsson, Thomas; Markovitsi, Dimitra; Manet, Ilse

    2016-05-04

    Doxorubicin (DOX) is a natural anthracycline widely used in chemotherapy; its combined application as a chemotherapeutic and photodynamic agent has been recently proposed. In this context, understanding the photoinduced properties of DOX complexes with nucleic acids is crucial. Herein, the study of photoinduced electron transfer in DOX-DNA complexes by femtosecond fluorescence spectroscopy is reported. The behaviour of complexes with two model DNA structures, a G-quadruplex (G4) formed by the human telomeric sequence (Tel21) and a d(GC) duplex, is compared. The DOX affinity for these two sequences is similar. Although both 1:1 and 2:1 stoichiometries have been reported for DOX-G4 complexes, only 1:1 complexes form with the duplex. The steady-state absorption indicates a strong binding interaction with the duplex due to drug intercalation between the GC base pairs. In contrast, the interaction of DOX with Tel21 is much weaker and arises from drug binding on the G4 external faces at two independent binding sites. As observed for DOX-d(GC) complexes, fluorescence of the drug in the first binding site of Tel21 exhibits decays within a few picoseconds following a biphasic pattern; this is attributed to the existence of two drug conformations. The fluorescence of the drug in the second binding site of Tel21 shows slower decays within 150 ps. These timescales are consistent with electron transfer from the guanines to the excited drug, as favoured by the lower oxidation potential of the stacked guanines of G4 with respect to those in the duplex.

  4. A novel electrochemical aptasensor for highly sensitive detection of thrombin based on the autonomous assembly of hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme nanowires.

    Science.gov (United States)

    Xie, Shunbi; Chai, Yaqin; Yuan, Yali; Bai, Lijuan; Yuan, Ruo

    2014-06-17

    In this work, a new signal amplified strategy was constructed based on isothermal exponential amplification reaction (EXPAR) and hybridization chain reaction (HCR) generating the hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-mimicking DNAzyme) nanowires as signal output component for the sensitive detection of thrombin (TB). We employed EXPAR's ultra-high amplification efficiency to produce a large amount of two hairpin helper DNAs within a minutes. And then the resultant two hairpin helper DNAs could autonomously assemble the hemin/G-quadruplex HRP-mimicking DNAzymes nanowires as the redox-active reporter units on the electrode surface via hybridization chain reaction (HCR). The hemin/G-quadruplex structures simultaneously served as electron transfer medium and electrocatalyst to amplify the signal in the presence of H2O2. Specifically, only when the EXPAR reaction process has occurred, the HCR could be achieved and the hemin/G-quadruplex complexes could be formed on the surface of an electrode to give a detectable signal. The proposed strategy combines the amplification power of the EXPAR, HCR, and the inherent high sensitivity of the electrochemical detection. With such design, the proposed assay showed a good linear relationship within the range of 0.1 pM-50 nM with a detection limit of 33 fM (defined as S/N=3) for TB.

  5. Porous platinum nanotubes labeled with hemin/G-quadruplex based electrochemical aptasensor for sensitive thrombin analysis via the cascade signal amplification.

    Science.gov (United States)

    Sun, Aili; Qi, Qingan; Wang, Xuannian; Bie, Ping

    2014-07-15

    For the first time, a sensitive electrochemical aptasensor for thrombin (TB) was developed by using porous platinum nanotubes (PtNTs) labeled with hemin/G-quadruplex and glucose dehydrogenase (GDH) as labels. Porous PtNTs with large surface area exhibited the peroxidase-like activity. Coupling with GDH and hemin/G-quadruplex as NADH oxidase and HRP-mimicking DNAzyme, the cascade signal amplification was achieved by the following ways: in the presence of glucose and NAD(+) in the working buffer, GDH electrocatalyzed the oxidation of glucose with the production of NADH. Then, hemin/G-quadruplex as NADH oxidase catalyzed the oxidation of NADH to in situ generate H2O2. Based on the corporate electrocatalysis of PtNTs and hemin/G-quadruplex toward H2O2, the electrochemical signal was significantly amplified, allowing the detection limit of TB down to 0.15 pM level. Moreover, the proposed strategy was simple because the intercalated hemin offered the readout signal, avoiding the adding of additional redox mediator as signal donator. Such an electrochemical aptasensor is highly promising for sensitive detection of other proteins in clinical diagnostics.

  6. A novel electrochemical aptasensor for highly sensitive detection of thrombin based on the autonomous assembly of hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme nanowires

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Shunbi, E-mail: xieshunbi@163.com; Chai, Yaqin, E-mail: yaqinchai@swu.edu.cn; Yuan, Yali; Bai, Lijuan; Yuan, Ruo, E-mail: yuanruo@swu.edu.cn

    2014-06-01

    Highlights: • This assay is label-free, the signal can be read out by measuring the electrochemical signal of hemin. • The hemin/G-quadruplex HRP-DNAzyme nanowires were formed via EXPAR reaction and HCR. • The prepared aptasensor exhibited low detection limit and wide linear range to TB. - Abstract: In this work, a new signal amplified strategy was constructed based on isothermal exponential amplification reaction (EXPAR) and hybridization chain reaction (HCR) generating the hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme (HRP-mimicking DNAzyme) nanowires as signal output component for the sensitive detection of thrombin (TB). We employed EXPAR’s ultra-high amplification efficiency to produce a large amount of two hairpin helper DNAs within a minutes. And then the resultant two hairpin helper DNAs could autonomously assemble the hemin/G-quadruplex HRP-mimicking DNAzymes nanowires as the redox-active reporter units on the electrode surface via hybridization chain reaction (HCR). The hemin/G-quadruplex structures simultaneously served as electron transfer medium and electrocatalyst to amplify the signal in the presence of H{sub 2}O{sub 2}. Specifically, only when the EXPAR reaction process has occurred, the HCR could be achieved and the hemin/G-quadruplex complexes could be formed on the surface of an electrode to give a detectable signal. The proposed strategy combines the amplification power of the EXPAR, HCR, and the inherent high sensitivity of the electrochemical detection. With such design, the proposed assay showed a good linear relationship within the range of 0.1 pM–50 nM with a detection limit of 33 fM (defined as S/N = 3) for TB.

  7. G quadruplex RNA structures in PSD-95 mRNA: potential regulators of miR-125a seed binding site accessibility.

    Science.gov (United States)

    Stefanovic, Snezana; Bassell, Gary J; Mihailescu, Mihaela Rita

    2015-01-01

    Fragile X syndrome (FXS) is the most common inherited form of intellectual disability caused by the CGG trinucleotide expansion in the 3'-untranslated region of the FMR1 gene on the X chromosome, that silences the expression of the Fragile X mental retardation protein (FMRP). FMRP has been shown to bind to a G-rich region within the PSD-95 mRNA which encodes for the postsynaptic density protein 95 (PSD-95), and together with the microRNA miR-125a, to play an important role in the reversible inhibition of the PSD-95 mRNA translation in neurons. The loss of FMRP in Fmr1 KO mice disables this translation control in the production of the PSD-95 protein. Interestingly, the miR-125a binding site on PSD-95 mRNA is embedded in the G-rich region bound by FMRP and postulated to adopt one or more G quadruplex structures. In this study, we have used different biophysical techniques to validate and characterize the formation of parallel G quadruplex structures and binding of miR-125a to its complementary sequence located within the 3' UTR of PSD-95 mRNA. Our results indicate that the PSD-95 mRNA G-rich region folds into alternate G quadruplex conformations that coexist in equilibrium. miR-125a forms a stable complex with PSD-95 mRNA, as evident by characteristic Watson-Crick base-pairing that coexists with one of the G quadruplex forms, suggesting a novel mechanism for G quadruplex structures to regulate the access of miR-125a to its binding site. © 2014 Stefanovic et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

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

    Directory of Open Access Journals (Sweden)

    Ehrat Edward A

    2012-07-01

    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.

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

    Directory of Open Access Journals (Sweden)

    Jinzhi Tan

    2009-05-01

    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

  10. An ion-controlled four-color fluorescent telomeric switch on DNA origami structures

    Science.gov (United States)

    Olejko, L.; Cywiński, P. J.; Bald, I.

    2016-05-01

    The folding of single-stranded telomeric DNA into guanine (G) quadruplexes is a conformational change that plays a major role in sensing and drug targeting. The telomeric DNA can be placed on DNA origami nanostructures to make the folding process extremely selective for K+ ions even in the presence of high Na+ concentrations. Here, we demonstrate that the K+-selective G-quadruplex formation is reversible when using a cryptand to remove K+ from the G-quadruplex. We present a full characterization of the reversible switching between single-stranded telomeric DNA and G-quadruplex structures using Förster resonance energy transfer (FRET) between the dyes fluorescein (FAM) and cyanine3 (Cy3). When attached to the DNA origami platform, the G-quadruplex switch can be incorporated into more complex photonic networks, which is demonstrated for a three-color and a four-color FRET cascade from FAM over Cy3 and Cy5 to IRDye700 with G-quadruplex-Cy3 acting as a switchable transmitter.The folding of single-stranded telomeric DNA into guanine (G) quadruplexes is a conformational change that plays a major role in sensing and drug targeting. The telomeric DNA can be placed on DNA origami nanostructures to make the folding process extremely selective for K+ ions even in the presence of high Na+ concentrations. Here, we demonstrate that the K+-selective G-quadruplex formation is reversible when using a cryptand to remove K+ from the G-quadruplex. We present a full characterization of the reversible switching between single-stranded telomeric DNA and G-quadruplex structures using Förster resonance energy transfer (FRET) between the dyes fluorescein (FAM) and cyanine3 (Cy3). When attached to the DNA origami platform, the G-quadruplex switch can be incorporated into more complex photonic networks, which is demonstrated for a three-color and a four-color FRET cascade from FAM over Cy3 and Cy5 to IRDye700 with G-quadruplex-Cy3 acting as a switchable transmitter. Electronic supplementary

  11. The G-quadruplex-forming aptamer AS1411 potently inhibits HIV-1 attachment to the host cell.

    Science.gov (United States)

    Perrone, Rosalba; Butovskaya, Elena; Lago, Sara; Garzino-Demo, Alfredo; Pannecouque, Christophe; Palù, Giorgio; Richter, Sara N

    2016-04-01

    AS1411 is a G-rich aptamer that forms a stable G-quadruplex structure and displays antineoplastic properties both in vitro and in vivo. This oligonucleotide has undergone phase 2 clinical trials. The major molecular target of AS1411 is nucleolin (NCL), a multifunctional nucleolar protein also present in the cell membrane where it selectively mediates the binding and uptake of AS1411. Cell-surface NCL has been recognised as a low-affinity co-receptor for human immunodeficiency virus type 1 (HIV-1) anchorage on target cells. Here we assessed the anti-HIV-1 properties and underlying mechanism of action of AS1411. The antiviral activity of AS1411 was determined towards different HIV-1 strains, host cells and at various times post-infection. Acutely, persistently and latently infected cells were tested, including HIV-1-infected peripheral blood mononuclear cells from a healthy donor. Mechanistic studies to exclude modes of action other than virus binding via NCL were performed. AS1411 efficiently inhibited HIV-1 attachment/entry into the host cell. The aptamer displayed antiviral activity in the absence of cytotoxicity at the tested doses, therefore displaying a wide therapeutic window and favourable selectivity indexes. These findings, besides validating cell-surface-expressed NCL as an antiviral target, open the way for the possible use of AS1411 as a new potent and promisingly safe anti-HIV-1 agent.

  12. Novel Molecular Targets for kRAS Downregulation: Promoter G-Quadruplexes

    Science.gov (United States)

    2015-09-01

    B-DNA structures, and con- tains two nuclease hypersensitivity elements [9–11]. Such G/C-rich regions preferentially cluster around the...1× penicillin /streptomycin solution at 37 °C, in a humidified atmosphere containing 5% CO2, in exponential growth. For cellular viability assays...dium supplemented with 10% fetal bovine serum and 1× penicillin / streptomycin solution at 37 °C, in a humidified atmosphere containing 5% CO2, in

  13. Molecular genetics: DNA analysis of a putative dog clone.

    Science.gov (United States)

    Parker, Heidi G; Kruglyak, Leonid; Ostrander, Elaine A

    2006-03-09

    In August 2005, Lee et al. reported the first cloning of a domestic dog from adult somatic cells. This putative dog clone was the result of somatic-cell nuclear transfer from a fibroblast cell of a three-year-old male Afghan hound into a donor oocyte provided by a dog of mixed breed. In light of recent concerns regarding the creation of cloned human cell lines from the same institution, we have undertaken an independent test to determine the validity of the claims made by Lee et al..

  14. Biophysical characterization of G-quadruplex forming FMR1 mRNA and of its interactions with different fragile X mental retardation protein isoforms.

    Science.gov (United States)

    Blice-Baum, Anna C; Mihailescu, Mihaela-Rita

    2014-01-01

    Fragile X syndrome, the most common form of inherited mental impairment in humans, is caused by the absence of the fragile X mental retardation protein (FMRP) due to a CGG trinucleotide repeat expansion in the 5'-untranslated region (UTR) and subsequent translational silencing of the fragile x mental retardation-1 (FMR1) gene. FMRP, which is proposed to be involved in the translational regulation of specific neuronal messenger RNA (mRNA) targets, contains an arginine-glycine-glycine (RGG) box RNA binding domain that has been shown to bind with high affinity to G-quadruplex forming mRNA structures. FMRP undergoes alternative splicing, and the binding of FMRP to a proposed G-quadruplex structure in the coding region of its mRNA (named FBS) has been proposed to affect the mRNA splicing events at exon 15. In this study, we used biophysical methods to directly demonstrate the folding of FMR1 FBS into a secondary structure that contains two specific G-quadruplexes and analyze its interactions with several FMRP isoforms. Our results show that minor splice isoforms, ISO2 and ISO3, created by the usage of the second and third acceptor sites at exon 15, bind with higher affinity to FBS than FMRP ISO1, which is created by the usage of the first acceptor site. FMRP ISO2 and ISO3 cannot undergo phosphorylation, an FMRP post-translational modification shown to modulate the protein translation regulation. Thus, their expression has to be tightly regulated, and this might be accomplished by a feedback mechanism involving the FMRP interactions with the G-quadruplex structures formed within FMR1 mRNA.

  15. Fragile X mental retardation protein interactions with a G quadruplex structure in the 3'-untranslated region of NR2B mRNA.

    Science.gov (United States)

    Stefanovic, Snezana; DeMarco, Brett A; Underwood, Ayana; Williams, Kathryn R; Bassell, Gary J; Mihailescu, Mihaela Rita

    2015-12-01

    Fragile X syndrome, the most common cause of inherited intellectual disability, is caused by a trinucleotide CGG expansion in the 5'-untranslated region of the FMR1 gene, which leads to the loss of expression of the fragile X mental retardation protein (FMRP). FMRP, an RNA-binding protein that regulates the translation of specific mRNAs, has been shown to bind a subset of its mRNA targets by recognizing G quadruplex structures. It has been suggested that FMRP controls the local protein synthesis of several protein components of the post synaptic density (PSD) in response to specific cellular needs. We have previously shown that the interactions between FMRP and mRNAs of the PSD scaffold proteins PSD-95 and Shank1 are mediated via stable G-quadruplex structures formed within the 3'-untranslated regions of these mRNAs. In this study we used biophysical methods to show that a comparable G quadruplex structure forms in the 3'-untranslated region of the glutamate receptor subunit NR2B mRNA encoding for a subunit of N-methyl-d-aspartate (NMDA) receptors that is recognized specifically by FMRP, suggesting a common theme for FMRP recognition of its dendritic mRNA targets.

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

    Science.gov (United States)

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

    2016-11-10

    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).

  17. G-quadruplex structures and CpG methylation cause drop-out of the maternal allele in polymerase chain reaction amplification of the imprinted MEST gene promoter.

    Directory of Open Access Journals (Sweden)

    Aaron J Stevens

    Full Text Available We observed apparent non-Mendelian behaviour of alleles when genotyping a region in a CpG island at the 5' end of the maternally imprinted human MEST isoform. This region contains three single nucleotide polymorphisms (SNPs in total linkage disequilibrium, such that only two haplotypes occur in the human population. Only one haplotype was detectable in each subject, never both, despite the use of multiple primers and several genotyping methods. We observed that this region contains motifs capable of forming several G-quadruplex structures. Circular dichroism spectroscopy and native polyacrylamide gel electrophoresis confirmed that at least three G-quadruplexes form in vitro in the presence of potassium ions, and one of these structures has a Tm of greater than 99°C in polymerase chain reaction (PCR buffer. We demonstrate that it is the methylated maternal allele that is always lost during PCR amplification, and that formation of G-quadruplexes and presence of methylated cytosines both contributed to this phenomenon. This observed parent-of-origin specific allelic drop-out has important implications for analysis of imprinted genes in research and diagnostic settings.

  18. G-Quadruplex Structures and CpG Methylation Cause Drop-Out of the Maternal Allele in Polymerase Chain Reaction Amplification of the Imprinted MEST Gene Promoter

    Science.gov (United States)

    Cree, Simone L.; Gibb, Andrew; Miller, Allison L.; Doudney, Kit; Aitchison, Alan; Eccles, Michael R.; Joyce, Peter R.; Filichev, Vyacheslav V.; Kennedy, Martin A.

    2014-01-01

    We observed apparent non-Mendelian behaviour of alleles when genotyping a region in a CpG island at the 5′ end of the maternally imprinted human MEST isoform. This region contains three single nucleotide polymorphisms (SNPs) in total linkage disequilibrium, such that only two haplotypes occur in the human population. Only one haplotype was detectable in each subject, never both, despite the use of multiple primers and several genotyping methods. We observed that this region contains motifs capable of forming several G-quadruplex structures. Circular dichroism spectroscopy and native polyacrylamide gel electrophoresis confirmed that at least three G-quadruplexes form in vitro in the presence of potassium ions, and one of these structures has a Tm of greater than 99°C in polymerase chain reaction (PCR) buffer. We demonstrate that it is the methylated maternal allele that is always lost during PCR amplification, and that formation of G-quadruplexes and presence of methylated cytosines both contributed to this phenomenon. This observed parent-of-origin specific allelic drop-out has important implications for analysis of imprinted genes in research and diagnostic settings. PMID:25437198

  19. Identifying the impact of G-quadruplexes on Affymetrix 3' arrays using cloud computing.

    Science.gov (United States)

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

    2010-01-15

    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.

  20. A pseudo triple-enzyme electrochemical aptasensor based on the amplification of Pt–Pd nanowires and hemin/G-quadruplex

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Yingning; Chai, Yaqin, E-mail: yqchai@swu.edu.cn; Yuan, Yali; Yuan, Ruo, E-mail: yuanruo@swu.edu.cn

    2014-06-27

    Highlights: • Pt–Pd nanowires were combined with pseudo triple-enzyme electrochemical aptasensor. • Pt–Pd nanowires served as signal enhancer. • Labeling process, deactivation and spatial distribution of enzymes were solved. - Abstract: Our present work aimed at developing a pseudo triple-enzyme cascade electrocatalytic electrochemical aptasensor for determination of thrombin with the amplification of alcohol dehydrogenase (ADH)-Pt–Pd nanowires bionanocomposite and hemin/G-quadruplex structure that simultaneously acted as NADH oxidase and HRP-mimicking DNAzyme. With the addition of ethanol to the electrolyte, the ADH immobilized on the Pt–Pd nanowires catalyzed ethanol to acetaldehyde accompanied by NAD{sup +} being converted to NADH. Then the hemin/G-quadruplex firstly served as NADH oxidase, converting the produced NADH to NAD{sup +} with the concomitant local formation of high concentration of H{sub 2}O{sub 2}. Subsequently, the hemin/G-quadruplex acted as HRP-mimicking DNAzyme, bioelectrocatalyzing the produced H{sub 2}O{sub 2}. At the same time, the Pt–Pd nanowires employed in our strategy not only provided a large surface area for immobilizing thrombin binding aptamer (TBA) and ADH, but also served as HRP-mimicking DNAzyme which rapidly bioelectrocatalyzed the reduction of the produced H{sub 2}O{sub 2}. Thus, such a pseudo triple-enzyme cascade electrochemical aptasensor could greatly promote the electron transfer of hemin and resulted in the dramatic enhancement of electrochemical signal. As a result, a wide dynamic concentration linear range from 0.2 pM to 20 nM with a low detection limit of 0.067 pM for thrombin (TB) determination was obtained. The excellent performance indicated that our strategy was a promising way for ultrasensitive assays in electrochemical aptasensors.

  1. Analysis of the Fragile X mental retardation protein isoforms 1, 2 and 3 interactions with the G-quadruplex forming semaphorin 3F mRNA.

    Science.gov (United States)

    Evans, Timothy L; Blice-Baum, Anna C; Mihailescu, Mihaela-Rita

    2012-02-01

    Fragile X syndrome, the most prevalent inheritable mental retardation, is caused by the loss of fragile X mental retardation protein (FMRP) expression. FMRP is an RNA-binding protein with nucleo-cytoplasmic shuttle activity, proposed to act as a translation regulator of specific mRNAs in the brain. It has been shown that FMRP uses its arginine-glycine-glycine (RGG) box domain to bind a subset of mRNA targets that form a G-quadruplex structure. FMRP has also been shown to undergo the post-translational modifications of arginine methylation and phosphorylation, as well as alternative splicing, resulting in multiple isoforms. The alternative splice isoforms investigated in this study, isoform 1 (ISO1), isoform 2 (ISO2), and isoform 3 (ISO3), are created by the alternative splicing acceptor site at exon 15. FMRP ISO2 and ISO3 are truncated by 12 and 13 residues, respectively, relative to the longest FMRP isoform ISO1. These truncations, which are in the close proximity of the RGG box domain, preserve the integrity of the RGG box in all three isoforms, but eliminate the in vivo phosphorylation sites, present only on FMRP ISO1. We have expressed and purified recombinant FMRP ISO1, ISO2 and ISO3 in Escherichia coli, free of post-translational modifications, and by using fluorescence spectroscopy, we show that each FMRP isoform binds G-quadruplex RNA, albeit with different binding affinities, suggesting that naturally occurring sequence modifications in the proximity of the RGG box modulate its G-quadruplex RNA binding ability.

  2. Epigenetic suppression of human telomerase (hTERT) is mediated by the metastasis suppressor NME2 in a G-quadruplex dependent fashion.

    Science.gov (United States)

    Saha, Dhurjhoti; Singh, Ankita; Hussain, Tabish; Srivastava, Vivek; Sengupta, Suman; Kar, Anirban; Dhapola, Parashar; Dhople, Vishnu; Ummani, Ramesh; Chowdhury, Shantanu

    2017-07-17

    Transcriptional activation of the human telomerase reverse transcriptase (hTERT) gene, which remains repressed in adult somatic cells, is critical during tumorigenesis. Several transcription factors and the epigenetic state of the hTERT promoter are known to be important for tight control of hTERT in normal tissues, but the molecular mechanisms leading to hTERT reactivation in cancer are not well understood. Surprisingly, here we found occupancy of the metastasis suppressor nonmetastatic 2 (NME2) within hTERT core promoter in HT1080 fibrosarcoma cells and HCT116 colon cancer cells, and NME2 mediated transcriptional repression of hTERT in these cells. We also report that loss of NME2 results in upregulated hTERT expression. Mechanistically, additional results indicated that the RE1 silencing transcription factor (REST), lysine-specific histone demethylase 1 (LSD1) co-repressor complex associates with the hTERT promoter in a NME2-dependent way, and that this assembly is required for maintaining repressive chromatin at the hTERT promoter. Interestingly, a G-quadruplex motif at the hTERT promoter was essential for occupancy of NME2 and the REST repressor complex on the hTERT promoter. In light of this mechanistic insight, we studied the effects of G quadruplex binding ligands on hTERT expression and observed that several of these ligands repressed hTERT expression. Together, our results support a mechanism of hTERT epigenetic control involving a G quadruplex promoter motif, which potentially can be targeted by tailored small molecules. Copyright © 2017, The American Society for Biochemistry and Molecular Biology.

  3. G-Quadruplex in the NRF2 mRNA 5' Untranslated Region Regulates De Novo NRF2 Protein Translation under Oxidative Stress.

    Science.gov (United States)

    Lee, Sang C; Zhang, Jack; Strom, Josh; Yang, Danzhou; Dinh, Thai Nho; Kappeler, Kyle; Chen, Qin M

    2017-01-01

    Inhibition of protein synthesis serves as a general measure of cellular consequences of chemical stress. A few proteins are translated selectively and influence cell fate. How these proteins can bypass the general control of translation remains unknown. We found that low to mild doses of oxidants induce de novo translation of the NRF2 protein. Here we demonstrate the presence of a G-quadruplex structure in the 5' untranslated region (UTR) of NRF2 mRNA, as measured by circular dichroism, nuclear magnetic resonance, and dimethylsulfate footprinting analyses. Such a structure is important for 5'-UTR activity, since its removal by sequence mutation eliminated H2O2-induced activation of the NRF2 5' UTR. Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based proteomics revealed elongation factor 1 alpha (EF1a) as a protein binding to the G-quadruplex sequence. Cells responded to H2O2 treatment by increasing the EF1a protein association with NRF2 mRNA, as measured by RNA-protein interaction assays. The EF1a interaction with small and large subunits of ribosomes did not appear to change due to H2O2 treatment, nor did posttranslational modifications, as measured by two-dimensional (2-D) Western blot analysis. Since NRF2 encodes a transcription factor essential for protection against tissue injury, our data have revealed a novel mechanism of cellular defense involving de novo NRF2 protein translation governed by the EF1a interaction with the G-quadruplex in the NRF2 5' UTR during oxidative stress. Copyright © 2016 American Society for Microbiology.

  4. G-quadruplex (G4) motifs in the maize (Zea mays L.) genome are enriched at specific locations in thousands of genes coupled to energy status, hypoxia, low sugar, and nutrient deprivation.

    Science.gov (United States)

    Andorf, Carson M; Kopylov, Mykhailo; Dobbs, Drena; Koch, Karen E; Stroupe, M Elizabeth; Lawrence, Carolyn J; Bass, Hank W

    2014-12-20

    The G-quadruplex (G4) elements comprise a class of nucleic acid structures formed by stacking of guanine base quartets in a quadruple helix. This G4 DNA can form within or across single-stranded DNA molecules and is mutually exclusive with duplex B-form DNA. The reversibility and structural diversity of G4s make them highly versatile genetic structures, as demonstrated by their roles in various functions including telomere metabolism, genome maintenance, immunoglobulin gene diversification, transcription, and translation. Sequence motifs capable of forming G4 DNA are typically located in telomere repeat DNA and other non-telomeric genomic loci. To investigate their potential roles in a large-genome model plant species, we computationally identified 149,988 non-telomeric G4 motifs in maize (Zea mays L., B73 AGPv2), 29% of which were in non-repetitive genomic regions. G4 motif hotspots exhibited non-random enrichment in genes at two locations on the antisense strand, one in the 5' UTR and the other at the 5' end of the first intron. Several genic G4 motifs were shown to adopt sequence-specific and potassium-dependent G4 DNA structures in vitro. The G4 motifs were prevalent in key regulatory genes associated with hypoxia (group VII ERFs), oxidative stress (DJ-1/GATase1), and energy status (AMPK/SnRK) pathways. They also showed statistical enrichment for genes in metabolic pathways that function in glycolysis, sugar degradation, inositol metabolism, and base excision repair. Collectively, the maize G4 motifs may represent conditional regulatory elements that can aid in energy status gene responses. Such a network of elements could provide a mechanistic basis for linking energy status signals to gene regulation in maize, a model genetic system and major world crop species for feed, food, and fuel.

  5. A G-quadruplex-binding macrodomain within the "SARS-unique domain" is essential for the activity of the SARS-coronavirus replication-transcription complex.

    Science.gov (United States)

    Kusov, Yuri; Tan, Jinzhi; Alvarez, Enrique; Enjuanes, Luis; Hilgenfeld, Rolf

    2015-10-01

    The multi-domain non-structural protein 3 of SARS-coronavirus is a component of the viral replication/transcription complex (RTC). Among other domains, it contains three sequentially arranged macrodomains: the X domain and subdomains SUD-N as well as SUD-M within the "SARS-unique domain". The X domain was proposed to be an ADP-ribose-1"-phosphatase or a poly(ADP-ribose)-binding protein, whereas SUD-NM binds oligo(G)-nucleotides capable of forming G-quadruplexes. Here, we describe the application of a reverse genetic approach to assess the importance of these macrodomains for the activity of the SARS-CoV RTC. To this end, Renilla luciferase-encoding SARS-CoV replicons with selectively deleted macrodomains were constructed and their ability to modulate the RTC activity was examined. While the SUD-N and the X domains were found to be dispensable, the SUD-M domain was crucial for viral genome replication/transcription. Moreover, alanine replacement of charged amino-acid residues of the SUD-M domain, which are likely involved in G-quadruplex-binding, caused abrogation of RTC activity. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Identification of putative DnaN-binding motifs in plasmid replication initiation proteins.

    Science.gov (United States)

    Dalrymple, Brian P; Kongsuwan, Kritaya; Wijffels, Gene

    2007-01-01

    Recently the plasmid RK2 replication initiation protein, TrfA, has been shown to bind to the beta subunit of DNA Polymerase III (DnaN) via a short pentapeptide with the consensus QL[S/D]LF. A second consensus peptide, the hexapeptide QLxLxL, has also been demonstrated to mediate binding to DnaN. Here we describe the results of a comprehensive survey of replication initiation proteins encoded by bacterial plasmids to identify putative DnaN-binding sites. Both pentapeptide and hexapeptide motifs have been identified in a number of families of replication initiation proteins. The distribution of sites is sporadic and closely related families of proteins may differ in the presence, location, or type of putative DnaN-binding motif. Neither motif has been identified in replication initiation proteins encoded by plasmids that replicate via rolling circles or strand displacement. The results suggest that the recruitment of DnaN to the origin of replication of a replisome by plasmid replication initiation proteins is not generally required for plasmid replication, but that in some cases it may be beneficial for efficiency of replication initiation.

  7. Discovery of widespread GTP-binding motifs in genomic DNA and RNA.

    Science.gov (United States)

    Curtis, Edward A; Liu, David R

    2013-04-18

    Biological RNAs that bind small molecules have been implicated in a variety of regulatory and catalytic processes. Inspired by these examples, we used in vitro selection to search a pool of genome-encoded RNA fragments for naturally occurring GTP aptamers. Several aptamer classes were identified, including one (the "G motif") with a G-quadruplex structure. Further analysis revealed that most RNA and DNA G-quadruplexes bind GTP. The G motif is abundant in eukaryotes, and the human genome contains ~75,000 examples with dissociation constants comparable to the GTP concentration of a eukaryotic cell (~300 μM). G-quadruplexes play roles in diverse cellular processes, and our findings raise the possibility that GTP may play a role in the function of these elements. Consistent with this possibility, the sequence requirements of several classes of regulatory G-quadruplexes parallel those of GTP binding.

  8. Identification of Putative Coffee Rust Mycoparasites via Single-Molecule DNA Sequencing of Infected Pustules.

    Science.gov (United States)

    James, Timothy Y; Marino, John A; Perfecto, Ivette; Vandermeer, John

    2015-11-13

    The interaction of crop pests with their natural enemies is a fundament to their control. Natural enemies of fungal pathogens of crops are poorly known relative to those of insect pests, despite the diversity of fungal pathogens and their economic importance. Currently, many regions across Latin America are experiencing unprecedented epidemics of coffee rust (Hemileia vastatrix). Identification of natural enemies of coffee rust could aid in developing management strategies or in pinpointing species that could be used for biocontrol. In the present study, we characterized fungal communities associated with coffee rust lesions by single-molecule DNA sequencing of fungal rRNA gene bar codes from leaf discs (≈28 mm(2)) containing rust lesions and control discs with no rust lesions. The leaf disc communities were hyperdiverse in terms of fungi, with up to 69 operational taxonomic units (putative species) per control disc, and the diversity was only slightly reduced in rust-infected discs, with up to 63 putative species. However, geography had a greater influence on the fungal community than whether the disc was infected by coffee rust. Through comparisons between control and rust-infected leaf discs, as well as taxonomic criteria, we identified 15 putative mycoparasitic fungi. These fungi are concentrated in the fungal family Cordycipitaceae and the order Tremellales. These data emphasize the complexity of diverse fungi of unknown ecological function within a leaf that might influence plant disease epidemics or lead to the development of species for biocontrol of fungal disease.

  9. Computational study of putative residues involved in DNA synthesis fidelity checking in Thermus aquaticus DNA polymerase I.

    Science.gov (United States)

    Elias, Angela A; Cisneros, G Andrés

    2014-01-01

    A fidelity-checking site for DNA polymerase I has been proposed based on recent single-molecule Förster resonance energy transfer studies. The checking site is believed to ensure proper base pairing of the newly inserted nucleotide. Computational studies have been utilized to predict residues involved in this putative checking site on the Klenow and Bacillus fragments. Here, we employ energy decomposition analysis, electrostatic free energy response, and noncovalent interaction plots to identify the residues involved in the hypothesized checking site in the homologous Klenow fragment from Thermus aquaticus (Klentaq). Our results indicate multiple protein residues that show altered interactions for three mispairs compared to the correctly paired DNA dimer. Many of these residues are also conserved along A family polymerases.

  10. Season of conception in rural gambia affects DNA methylation at putative human metastable epialleles.

    Directory of Open Access Journals (Sweden)

    Robert A Waterland

    Full Text Available Throughout most of the mammalian genome, genetically regulated developmental programming establishes diverse yet predictable epigenetic states across differentiated cells and tissues. At metastable epialleles (MEs, conversely, epigenotype is established stochastically in the early embryo then maintained in differentiated lineages, resulting in dramatic and systemic interindividual variation in epigenetic regulation. In the mouse, maternal nutrition affects this process, with permanent phenotypic consequences for the offspring. MEs have not previously been identified in humans. Here, using an innovative 2-tissue parallel epigenomic screen, we identified putative MEs in the human genome. In autopsy samples, we showed that DNA methylation at these loci is highly correlated across tissues representing all 3 embryonic germ layer lineages. Monozygotic twin pairs exhibited substantial discordance in DNA methylation at these loci, suggesting that their epigenetic state is established stochastically. We then tested for persistent epigenetic effects of periconceptional nutrition in rural Gambians, who experience dramatic seasonal fluctuations in nutritional status. DNA methylation at MEs was elevated in individuals conceived during the nutritionally challenged rainy season, providing the first evidence of a permanent, systemic effect of periconceptional environment on human epigenotype. At MEs, epigenetic regulation in internal organs and tissues varies among individuals and can be deduced from peripheral blood DNA. MEs should therefore facilitate an improved understanding of the role of interindividual epigenetic variation in human disease.

  11. Zuotin, a putative Z-DNA binding protein in Saccharomyces cerevisiae

    Science.gov (United States)

    Zhang, S.; Lockshin, C.; Herbert, A.; Winter, E.; Rich, A.

    1992-01-01

    A putative Z-DNA binding protein, named zuotin, was purified from a yeast nuclear extract by means of a Z-DNA binding assay using [32P]poly(dG-m5dC) and [32P]oligo(dG-Br5dC)22 in the presence of B-DNA competitor. Poly(dG-Br5dC) in the Z-form competed well for the binding of a zuotin containing fraction, but salmon sperm DNA, poly(dG-dC) and poly(dA-dT) were not effective. Negatively supercoiled plasmid pUC19 did not compete, whereas an otherwise identical plasmid pUC19(CG), which contained a (dG-dC)7 segment in the Z-form was an excellent competitor. A Southwestern blot using [32P]poly(dG-m5dC) as a probe in the presence of MgCl2 identified a protein having a molecular weight of 51 kDa. The 51 kDa zuotin was partially sequenced at the N-terminal and the gene, ZUO1, was cloned, sequenced and expressed in Escherichia coli; the expressed zuotin showed similar Z-DNA binding activity, but with lower affinity than zuotin that had been partially purified from yeast. Zuotin was deduced to have a number of potential phosphorylation sites including two CDC28 (homologous to the human and Schizosaccharomyces pombe cdc2) phosphorylation sites. The hexapeptide motif KYHPDK was found in zuotin as well as in several yeast proteins, DnaJ of E.coli, csp29 and csp32 proteins of Drosophila and the small t and large T antigens of the polyoma virus. A 60 amino acid segment of zuotin has similarity to several histone H1 sequences. Disruption of ZUO1 in yeast resulted in a slow growth phenotype.

  12. High-throughput identification of telomere-binding ligands based on the fluorescence regulation of DNA-copper nanoparticles.

    Science.gov (United States)

    Yang, Luzhu; Wang, Yanjun; Li, Baoxin; Jin, Yan

    2017-01-15

    Formation of the G-quadruplex in the human telomeric DNA is an effective way to inhibit telomerase activity. Therefore, screening ligands of G-quadruplex has potential applications in the treatment of cancer by inhibit telomerase activity. Although several techniques have been explored for screening of telomeric G-quadruplexes ligands, high-throughput screening method for fast screening telomere-binding ligands from the large compound library is still urgently needed. Herein, a label-free fluorescence strategy has been proposed for high-throughput screening telomere-binding ligands by using DNA-copper nanoparticles (DNA-CuNPs) as a signal probe. In the absence of ligands, human telomeric DNA (GDNA) hybridized with its complementary DNA (cDNA) to form double stranded DNA (dsDNA) which can act as an efficient template for the formation of DNA-CuNPs, leading to the high fluorescence of DNA-CuNPs. In the presence of ligands, GDNA folded into G-quadruplex. Single-strdanded cDNA does not support the formation of DNA-CuNP, resulting in low fluorescence of DNA-CuNPs. Therefore, telomere-binding ligands can be high-throughput screened by monitoring the change in the fluorescence of DNA-CuNPs. Thirteen traditional chinese medicines were screened. Circular dichroism (CD) measurements demonstrated that the selected ligands could induce single-stranded telomeric DNA to form G-quadruplex. The telomere repeat amplification protocol (TRAP) assay demonstrated that the selected ligands can effectively inhibit telomerase activity. Therefore, it offers a cost-effective, label-free and reliable high-throughput way to identify G-quadruplex ligands, which holds great potential in discovering telomerase-targeted anticancer drugs. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Ultrasensitive colorimetric detection of circulating tumor DNA using hybridization chain reaction and the pivot of triplex DNA

    Science.gov (United States)

    Li, Ruimin; Zou, Li; Luo, Yanwei; Zhang, Manjun; Ling, Liansheng

    2017-03-01

    This work presents an amplified colorimetric biosensor for circulating tumor DNA (ctDNA), which associates the hybridization chain reaction (HCR) amplification with G-Quadruplex DNAzymes activity through triplex DNA formation. In the presence of ctDNA, HCR occurs. The resulting HCR products are specially recognized by one sequence to include one GGG repeat and the other containing three GGG repeats, through the synergetic effect of triplex DNA and asymmetrically split G-Quadruplex forming. Such design takes advantage of the amplification property of HCR and the high peroxidase-like catalytic activity of asymmetrically split G-Quadruplex DNAzymes by means of triplex DNA formation, which produces color signals in the presence of ctDNA. Nevertheless, in the absence of ctDNA, no HCR happens. Thus, no triplex DNA and G-Quadruplex structure is formed, producing a negligible background. The colorimetric sensing platform is successfully applied in complex biological environments such as human blood plasma for ctDNA detection, with a detection limit corresponding to 0.1 pM. This study unambiguously uses triplex DNA forming as the pivot to integrate nucleic acid amplification and DNAzymes for producing a highly sensitive signal with low background.

  14. Putative DNA-dependent RNA polymerase in Mitochondrial Plasmid of Paramecium caudatum Stock GT704

    Directory of Open Access Journals (Sweden)

    Trina Ekawati Tallei

    2015-10-01

    Full Text Available Mitochondria of Paramecium caudatum stock GT704 has a set of four kinds of linear plasmids with sizes of 8.2, 4.1, 2.8 and 1.4 kb. The plasmids of 8.2 and 2.8 kb exist as dimers consisting of 4.1- and 1.4-kb monomers, respectively. The plasmid 2.8 kb, designated as pGT704-2.8, contains an open reading frame encodes for putative DNA-dependent RNA polymerase (RNAP. This study reveals that this RNAP belongs to superfamily of DNA/RNA polymerase and family of T7/T3 single chain RNA polymerase and those of mitochondrial plasmid of fungi belonging to Basidiomycota and Ascomycota. It is suggested that RNAP of pGT704-2.8 can perform transcription without transcription factor as promoter recognition. Given that only two motifs were found, it could not be ascertained whether this RNAP has a full function independently or integrated with mtDNA in carrying out its function.

  15. 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.

    Science.gov (United States)

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

    2017-12-15

    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 H2O2. Simultaneously, the decomposition of the generated H2O2 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.

  16. Small-angle X-ray scattering study of self-assembling lipophilic guanines in organic solvents: G-quadruplex formation and cation effects in cyclohexane.

    Science.gov (United States)

    Gonnelli, A; Ortore, M G; Baldassarri, E J; Spada, G P; Pieraccini, S; Perone, R C; Funari, S S; Mariani, P

    2013-01-31

    Lipophilic guanilic derivatives (lipoGs) dissolved in organic solvents can undergo different self-assembly pathways based on different H-bonded motifs, e.g., the cyclic discrete G-quartet, which forms in the presence of alkali-metal ions, and the "infinite" tape-like G-ribbon observed in the absence of ions. Using in-solution small-angle X-ray scattering, we analyzed a series of lipoGs dissolved in cyclohexane in the presence of different salts. The formation of G-quartet based supramolecular aggregates has been confirmed, evidencing the coexistence equilibrium of octamers and noncovalent molecular nanowires (the so-called G-quadruplexes). By global fitting the scattering data, the concentration of the two kinds of particles as well as the nanowire length have been derived as a function of temperature for the different compounds and salts. The thermodynamic parameters show that the self-assembly aggregation process is enthalpy driven, while the observed enthalpy-entropy compensation suggests that similar stacking interactions control the self-assembly of the different compounds. However, the strength of the stacking interactions, and then the nanowire stability, depends on the nature of templating cations and on their capacity to fill the central cavity of quadruplexes, with the order Sr(+) < Na(+) ≲ K(+).

  17. Screening of candidate G-quadruplex ligands for the human c-KIT promotorial region and their effects in multiple in-vitro models

    Science.gov (United States)

    Zorzan, Eleonora; Ros, Silvia Da; Musetti, Caterina; Shahidian, Lara Zorro; Ramos Coelho, Nuno Filipe; Bonsembiante, Federico; Létard, Sébastien; Gelain, Maria Elena; Palumbo, Manlio; Dubreuil, Patrice; Giantin, Mery; Sissi, Claudia; Dacasto, Mauro

    2016-01-01

    Stabilization of G-quadruplex (G4) structures in promoters is a novel promising strategy to regulate gene expression at transcriptional and translational levels. c-KIT proto-oncogene encodes for a tyrosine kinase receptor. It is involved in several physiological processes, but it is also dysregulated in many diseases, including cancer. Two G-rich sequences able to fold into G4, have been identified in c-KIT proximal promoter, thus representing suitable targets for anticancer intervention. Herein, we screened an “in house” library of compounds for the recognition of these G4 elements and we identified three promising ligands. Their G4-binding properties were analyzed and related to their antiproliferative, transcriptional and post-transcriptional effects in MCF7 and HGC27 cell lines. Besides c-KIT, the transcriptional analysis covered a panel of oncogenes known to possess G4 in their promoters. From these studies, an anthraquinone derivative (AQ1) was found to efficiently downregulate c-KIT mRNA and protein in both cell lines. The targeted activity of AQ1 was confirmed using c-KIT–dependent cell lines that present either c-KIT mutations or promoter engineered (i.e., α155, HMC1.2 and ROSA cells). Present results indicate AQ1 as a promising compound for the target therapy of c-KIT-dependent tumors, worth of further and in depth molecular investigations. PMID:26942875

  18. Rapid three-dimensional microfluidic mixer for high viscosity solutions to unravel earlier folding kinetics of G-quadruplex under molecular crowding conditions.

    Science.gov (United States)

    Liu, Chao; Li, Ying; Li, Yiwei; Chen, Peng; Feng, Xiaojun; Du, Wei; Liu, Bi-Feng

    2016-01-01

    Rapid mixing of highly viscous solutions is a great challenge, which helps to analyze the reaction kinetics in viscous liquid phase, particularly to discover the folding kinetics of macromolecules under molecular crowding conditions mimicking the conditions inside cells. Here, we demonstrated a novel microfluidic mixer based on Dean flows with three-dimensional (3D) microchannel configuration for fast mixing of high-viscosity fluids. The main structure contained three consecutive subunits, each consisting of a "U"-type channel followed by a chamber with different width and height. Thus, the two solutions injected from the two inlets would undergo a mixing in the first "U"-type channel due to the Dean flow effect, and simultaneous vortices expansions in both horizontal and vertical directions in the following chamber. Numerical simulations and experimental characterizations confirmed that the micromixer could achieve a mixing time of 122.4μs for solutions with viscosities about 33.6 times that of pure water. It was the fastest micromixer for high viscosity solutions compared with previous reports. With this highly efficient 3D microfluidic mixer, we further characterized the early folding kinetics of human telomere G-quadruplex under molecular crowding conditions, and unravelled a new folding process within 550μs.

  19. Solution and Solid-State Analysis of Binding of 13-Substituted Berberine Analogues to Human Telomeric G-quadruplexes.

    Science.gov (United States)

    Ferraroni, Marta; Bazzicalupi, Carla; Papi, Francesco; Fiorillo, Gaetano; Guamán-Ortiz, Luis Miguel; Nocentini, Alessio; Scovassi, Anna Ivana; Lombardi, Paolo; Gratteri, Paola

    2016-04-05

    The interaction between 13-phenylalkyl and 13-diphenylalkyl berberine derivatives (NAX) and human telomeric DNA G4 structures has been investigated by both spectroscopic and crystallographic methods. NAX042 and NAX053 are the best compounds improving the performance of the natural precursor berberine. This finding is in agreement with the X-ray diffraction result for the NAX053-Tel12 adduct, showing the ligand which interacts via π-stacking, sandwiched at the interface of two symmetry-related quadruplex units, with its benzhydryl group contributing to the overall stability of the adduct by means of additional π-stacking interactions with the DNA residues. The berberine derivatives were also investigated for their cytotoxic activity towards a panel of human cancer cell lines. Compounds NAX042 and NAX053 affect the viability of cancer cell lines in a dose-dependent manner.

  20. Steady-State and Time-Resolved Studies into the Origin of the Intrinsic Fluorescence of G-Quadruplexes.

    Science.gov (United States)

    Sherlock, Madeline E; Rumble, Christopher A; Kwok, Chun Kit; Breffke, Jens; Maroncelli, Mark; Bevilacqua, Philip C

    2016-06-16

    Stretches of guanines in DNA and RNA can fold into guanine quadruplex structures (GQSs). These structures protect telomeres in DNA and regulate gene expression in RNA. GQSs have an intrinsic fluorescence that is sensitive to different parameters, including loop sequence and length. However, the dependence of GQS fluorescence on solution and sequence parameters and the origin of this fluorescence are poorly understood. Herein we examine effects of dangling nucleotides and cosolute conditions on GQS fluorescence using both steady-state and time-resolved fluorescence spectroscopy. The quantum yield of dGGGTGGGTGGGTGGG, termed "dG3T", is found to be modest at ∼2 × 10(-3). Nevertheless, dG3T and its variants are significantly brighter than the common nucleic acid fluorophore 2-aminopurine (2AP) largely due to their sizable extinction coefficients. Dangling 5'-end nucleotides generally reduce emission and blue-shift the resultant spectrum, whereas dangling 3'-end nucleotides slightly enhance fluorescence, particularly on the red side of the emission band. Time-resolved fluorescence decays are broadly distributed in time and require three exponential components for accurate fits. Time-resolved emission spectra suggest the presence of two emitting populations centered at ∼330 and ∼390 nm, with the redder component being a well-defined long-lived (∼1 ns) entity. Insights into GQS fluorescence obtained here should be useful in designing brighter intrinsic RNA and DNA quadruplexes for use in label-free biotechnological applications.

  1. Insights into how nucleotide supplements enhance the peroxidase-mimicking DNAzyme activity of the G-quadruplex/hemin system.

    Science.gov (United States)

    Stefan, Loic; Denat, Franck; Monchaud, David

    2012-09-01

    Since the initial discovery of the catalytic capability of short DNA fragments, this peculiar enzyme-like property (termed DNAzyme) has continued to garner much interest in the scientific community because of the virtually unlimited applications in developing new molecular devices. Alongside the exponential rise in the number of DNAzyme applications in the last past years, the search for convenient ways to improve its overall efficiency has only started to emerge. Credence has been lent to this strategy by the recent demonstration that the quadruplex-based DNAzyme proficiency can be enhanced by ATP supplements. Herein, we have made a further leap along this path, trying first of all to decipher the actual DNAzyme catalytic cycle (to gain insights into the steps ATP may influence), and subsequently investigating in detail the influence of all the parameters that govern the catalytic efficiency. We have extended this study to other nucleotides and quadruplexes, thus demonstrating the versatility and broad applicability of such an approach. The defined exquisitely efficient DNAzyme protocols were exploited to highlight the enticing advantages of this method via a 96-well plate experiment that enables the detection of nanomolar DNA concentrations in real-time with the naked-eye (see movie as Supplementary Data).

  2. Pharmacodynamics of the G-quadruplex-stabilizing telomerase inhibitor 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) in vitro: activity in human tumor cells correlates with telomere length and can be enhanced, or antagonized, with cytotoxic agents.

    Science.gov (United States)

    Cookson, Jennifer C; Dai, Fangping; Smith, Victoria; Heald, Robert A; Laughton, Charles A; Stevens, Malcolm F G; Burger, Angelika M

    2005-12-01

    Telomeric integrity is required to maintain the replicative ability of cancer cells and is a target for the G-quadruplex-stabilizing drug 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4). We report a senescent-like growth arrest in MCF-7 breast cancer cells, within 14 to 17 days, and a reduction in telomere length (from 5.2 kilobases (kb) to 4.7 and 4.3 kb after 17 days of treatment at 0.5 and 1 microM, respectively). These effects occurred at noncytotoxic drug concentrations (doses < 1 microM over a 14-day exposure) compatible with long-term drug dosing. The telomere length of cancer cells influences their sensitivity to growth inhibition by RHPS4: mutant (mt) human telomerase reverse transcriptase (hTERT)-expressing MCF-7 cells [short telomere restriction fragment (TRF) length, 1.9 kb; IC50, 0.2 microM] were 10 times more sensitive to RHPS4 compared with wild-type (wt) hTERT-expressing, vector-transfected control cells (longer TRF-length 5.2 kb; IC50 2 microM) in the 5 day SRB assay. This relationship was corroborated in a panel of 36 human tumor xenografts grown in vitro showing a positive correlation between telomere length and growth inhibitory potency of RHPS4 (15-day clonogenic assay, r = 0.75). These observations are consistent with loss of the protective capping status of telomeres mediated by RHPS4 G-quadruplex-stabilization, thus leading to greater susceptibility of cells with shorter telomeres. In combination studies, paclitaxel (Taxol), doxorubicin (Adriamycin), and the experimental therapeutic agent 17-(allylamino)-17-demethoxygeldanamycin, which inhibits the 90-kDa heat shock protein, conferred enhanced sensitivity in RHPS4 treated MCF-7 cells, whereas the DNA-interactive temozolomide and cisplatin antagonized the action of RHPS4. Our results support the combined use of certain classes of cytotoxic anticancer agents with RHPS4 to enhance potential clinical benefit.

  3. Mitochondrial DNA analysis of the putative heart of Louis XVII, son of Louis XVI and Marie-Antoinette.

    Science.gov (United States)

    Jehaes, E; Pfeiffer, H; Toprak, K; Decorte, R; Brinkmann, B; Cassiman, J J

    2001-03-01

    According to official historiography, the 10-year-old Louis XVII died in the Temple of Paris on June 8, 1795. However, public rumour spread the theory that Louis XVII escaped and that his descendants would be alive today. One such putative 'Louis XVII' was Carl Wilhelm Naundorff, who died in 1845 in Delft (the Netherlands). Comparative mitochondrial DNA (mtDNA) analysis gave evidence that his remains could not be identified as those of Louis XVII. In the present study, mtDNA analysis was performed on the heart of the young boy who died in the prison of Paris in 1795. In order to obtain the strongest evidence possible, two laboratories independently analysed the heart. The results showed that the consensus mtDNA sequence of the heart was identical to that of the maternal relatives of Louis XVII.

  4. Absence of Yersinia pestis-specific DNA in human teeth from five European excavations of putative plague victims.

    Science.gov (United States)

    Gilbert, M Thomas P; Cuccui, Jon; White, William; Lynnerup, Niels; Titball, Richard W; Cooper, Alan; Prentice, Michael B

    2004-02-01

    This study reports the results of a collaborative study undertaken by two independent research groups to (a) confirm recent PCR-based detection of Yersinia pestis DNA in human teeth from medieval plague victims in France, and (b) to extend these observations over five different European burial sites believed to contain plague victims dating from the late 13th to 17th centuries. Several different sets of primers were used, including those previously documented to yield positive results on ancient DNA extracts. No Y. pestis DNA could be amplified from DNA extracted from 108 teeth belonging to 61 individuals, despite the amplification of numerous other bacterial DNA sequences. Several methods of extracting dentine prior to the DNA extraction were also compared. PCR for bacterial 16S rDNA indicated the presence of multiple bacterial species in 23 out of 27 teeth DNA extracts where dentine was extracted using previously described methods. In comparison, positive results were obtained from only five out of 44 teeth DNA extracts for which a novel contamination-minimizing embedding technique was used. Therefore, high levels of environmental bacterial DNA are present in DNA extracts where previously described methods of tooth manipulation are used. To conclude, the absence of Y. pestis-specific DNA in an exhaustive search using specimens from multiple putative European plague burial sites does not allow us to confirm the identification of Y. pestis as the aetiological agent of the Black Death and subsequent plagues. In addition, the utility of the published tooth-based ancient DNA technique used to diagnose fatal bacteraemias in historical epidemics still awaits independent corroboration.

  5. DNA and RNA Quadruplex-Binding Proteins

    Directory of Open Access Journals (Sweden)

    Václav Brázda

    2014-09-01

    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.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    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....

  7. Biological activity of the G-quadruplex ligand RHPS4 (3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate) is associated with telomere capping alteration.

    Science.gov (United States)

    Leonetti, Carlo; Amodei, Sarah; D'Angelo, Carmen; Rizzo, Angela; Benassi, Barbara; Antonelli, Anna; Elli, Raffaella; Stevens, Malcolm F G; D'Incalci, Maurizio; Zupi, Gabriella; Biroccio, Annamaria

    2004-11-01

    This study had two goals: 1) to evaluate the biological effect of the novel pentacyclic acridine 3,11-difluoro-6,8,13-trimethyl-8H-quino[4,3,2-kl]acridinium methosulfate (RHPS4) on human melanoma lines possessing long telomeres, and 2) to elucidate the relationship between G-quadruplex-based telomerase inhibitor-induced cellular effects and telomere length/dysfunction. The cellular pharmacological effects of RHPS4 have been evaluated by treating melanoma lines with increasing concentrations of RHPS4. A dose-dependent inhibition of cell proliferation was observed in all the lines during short-term treatment. Flow cytometric analysis demonstrated that RHPS4 induced a dose-dependent accumulation of cells in the S-G(2)/M phase of cell cycle. The RHPS4-induced cell cycle alteration was irreversible even at low doses, and the cells died from apoptosis. At high RHPS4 concentration, apoptosis was accompanied by the induction of a senescence phenotype: large cell size, vacuolated cytoplasm, and beta-galactosidase activity. The short-term biological activity of RHPS4 was not caused by telomere shortening, but it was associated with telomere dysfunction, in terms of presence of telomeric fusions, polynucleated cells, and typical images of telophase bridge. In conclusion, our results demonstrate that the G-quadruplex ligand RHPS4 can function in a telomere length-independent manner through its ability to cause telomere-capping alteration.

  8. A putative resistant DNA marker for wool yellowing susceptibility in sheep

    Directory of Open Access Journals (Sweden)

    Benavides M.V.

    2000-01-01

    Full Text Available An Australian Merino flock was screened for low (resistant and high (susceptible yellow predictive colour (YPC breeding values in order to compare extreme individuals using the differential display of mRNA technique. One differentially expressed cDNA band was visualised only in the resistant group. This band showed no identity with the DNA sequences of public databases; however, they showed short homologies with three database sequences related to transmembrane signalling functions. The use of these candidate genes as DNA markers needs to be confirmed against sheep with a wide range of susceptibility to wool yellowing to verify the results.

  9. Identification of putative human T cell receptor delta complementary DNA clones

    Energy Technology Data Exchange (ETDEWEB)

    Hata, S.; Brenner, M.B.; Krangel, M.S.

    1987-10-30

    A novel T cell receptor (TCR) subunit termed TCR delta, associated with TCY ..gamma.. and CD3 polypeptides, were recently found on a subpopulation of human T lymphocytes. T cell-specific complementary DNA clones present in a human TCR..gamma..delta T cell complementary DNA library were obtained and characterized in order to identify candidate clones encoding TCR delta. One cross-hybridizing group of clones detected transcripts that are expressed in lymphocytes bearing TCR ..gamma..delta but not in other T lymphocytes and are encoded by genes that are rearranged in TCR ..gamma..delta lymphocytes but deleted in other T lymphocytes. Their sequences indicate homology to the variable, joining, and constant elements of other TCR and immunoglobulin genes. These characteristics are strong evidence that the complementary DNA clones encode TCR delta.

  10. New function of exonuclease and highly sensitive label-free colorimetric DNA detection.

    Science.gov (United States)

    Li, Hongbo; Wang, Suqin; Wu, Zaisheng; Xu, Jianguo; Shen, Guoli; Yu, Ruqin

    2016-03-15

    Enzymatic manipulation and modulation of nucleic acids are a central part of cellular function, protection, and reproduction, while rapid and accurate detection of ultralow amount of nucleic acids remains a major challenge in molecular biology research and clinic diagnosis of genetic diseases. Herein, we reported that exonuclease III can degrade the G-quadruplex structure, indicating the new exonuclease's function. Basing on the function of exonuclease III, a novel G-quadruplex-hemin DNAzyme-based colorimetric detection of tumor suppressor gene p53 was successfully developed. Although only one oligonucleotide probe was involved, the sensing strategy could suppress the optical background and achieve an efficient G-quadruplex-hemin DNAzyme-based signal amplification. Specifically, a label-free functional nucleic acid probe (called THzyme probe) was designed via introducing target DNA probe-contained hairpin structure into G-quadruplex DNAzyme. Even if this probe can fold into G-quadruplex structure in the presence of hemin very different from the double-stranded DNA, it is easily degraded by exonuclease III. Thus, no change in UV-vis absorption intensity is detected in the absence of target DNA. However, the hybridization of target DNA can protect the integrity and catalytic activity of THzyme probe, producing the DNAzyme-amplified colorimetric signal. As a result, the p53 gene was able to be detected down to 1.0 pM (final concentration in the signal-generating solution: 50.0 fM) and mismatched target DNAs were easily distinguished. It is expected that this simple sensing methodology for DNA detection can find its utility in point-of-care applications. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. DNA and RNA from Uninfected Vertebrate Cells Contain Nucleotide Sequences Related to the Putative Transforming Gene of Avian Myelocytomatosis Virus

    Science.gov (United States)

    Sheiness, Diana; Bishop, J. Michael

    1979-01-01

    The avian carcinoma virus MC29 (MC29V) contains a sequence of approximately 1,500 nucleotides which may represent a gene responsible for tumorigenesis by MC29V. We present evidence that MC29V has acquired this nucleotide sequence from the DNA of its host. The host sequence which has been incorporated by MC29V is transcribed into RNA in uninfected chicken cells and thus probably encodes a cellular gene. We have prepared radioactive DNA complementary to the putative MC29V transforming gene (cDNAmc29) and have found that sequences homologous to cDNAmc29 are present in the genomes of several uninfected vertebrate species. The DNA of chicken, the natural host for MC29V, contains at least 90% of the sequences represented by cDNAmc29. DNAs from other animals show significant but decreasing amounts of complementarity to cDNAmc29 in accordance with their evolutionary divergence from chickens; the thermal stabilities of duplexes formed between cDNAmc29 and avian DNAs also reflect phylogenetic divergence. Sequences complementary to cDNAmc29 are transcribed into approximately 10 copies per cell of polyadenylated RNA in uninfected chicken fibroblasts. Thus, the vertebrate homolog of cDNAmc29 may be a gene which has been conserved throughout vertebrate evolution and which served as a progenitor for the putative transforming gene of MC29V. Recent experiments suggest that the putative transforming gene of avian erythroblastosis virus, like that of MC29V, may have arisen by incorporation of a host gene (Stehelin et al., personal communication). These findings for avian erythroblastosis virus and MC29V closely parallel previous results, suggesting a host origin for src (D. H. Spector, B. Baker, H. E. Varmus, and J. M. Bishop, Cell 13:381-386, 1978; D. H. Spector, K. Smith, T. Padgett, P. McCombe, D. Roulland-Dussoix, C. Moscovici, H. E. Varmus, and J. M. Bishop, Cell 13:371-379, 1978; D. H. Spector, H. E. Varmus, and J. M. Bishop, Proc. Natl. Acad. Sci. U.S.A. 75:4102-4106, 1978; D

  12. A Combined Computational and Experimental Study on the Structure-Regulation Relationships of Putative Mammalian DNA Replication Initiator GINS

    Institute of Scientific and Technical Information of China (English)

    Reiko Hayashi; Takako Arauchi; Moe Tategu; Yuya Goto; Kenichi Yoshida

    2006-01-01

    GINS, a heterotetramer of SLD5, PSF1, PSF2, and PSF3 proteins, is an emerging chromatin factor recognized to be involved in the initiation and elongation step of DNA replication. Although the yeast and Xenopus GINS genes are well documented, their orthologous genes in higher eukaryotes are not fully characterized.In this study, we report the genomic structure and transcriptional regulation of mammalian GINS genes. Serum stimulation increased the GINS Mrna levels in human cells. Reporter gene assay using putative GINS promoter sequences revealed that the expression of mammalian GINS is regulated by 17β-Estradiolstimulated estrogen receptor α, and human PSF3 acts as a gene responsive to transcription factor E2F1. The goal of this study is to present the current data so as to encourage further work in the field of GINS gene regulation and functions in mammalian cells.

  13. DNA methylation of miRNA coding sequences putatively associated with childhood obesity.

    Science.gov (United States)

    Mansego, M L; Garcia-Lacarte, M; Milagro, F I; Marti, A; Martinez, J A

    2017-02-01

    Epigenetic mechanisms may be involved in obesity onset and its consequences. The aim of the present study was to evaluate whether DNA methylation status in microRNA (miRNA) coding regions is associated with childhood obesity. DNA isolated from white blood cells of 24 children (identification sample: 12 obese and 12 non-obese) from the Grupo Navarro de Obesidad Infantil study was hybridized in a 450 K methylation microarray. Several CpGs whose DNA methylation levels were statistically different between obese and non-obese were validated by MassArray® in 95 children (validation sample) from the same study. Microarray analysis identified 16 differentially methylated CpGs between both groups (6 hypermethylated and 10 hypomethylated). DNA methylation levels in miR-1203, miR-412 and miR-216A coding regions significantly correlated with body mass index standard deviation score (BMI-SDS) and explained up to 40% of the variation of BMI-SDS. The network analysis identified 19 well-defined obesity-relevant biological pathways from the KEGG database. MassArray® validation identified three regions located in or near miR-1203, miR-412 and miR-216A coding regions differentially methylated between obese and non-obese children. The current work identified three CpG sites located in coding regions of three miRNAs (miR-1203, miR-412 and miR-216A) that were differentially methylated between obese and non-obese children, suggesting a role of miRNA epigenetic regulation in childhood obesity. © 2016 World Obesity Federation.

  14. Cloning, purification and preliminary crystallographic analysis of a putative DNA-binding membrane protein, YmfM, from Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ling; Sedelnikova, Svetlana E.; Baker, Patrick J.; Rice, David W., E-mail: d.rice@sheffield.ac.uk [Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield S10 2TN (United Kingdom)

    2008-07-01

    Truncation by the removal of the C-terminal hydrophobic transmembrane anchor has enabled the overexpression of a soluble domain of S. aureus YmfM in Escherichia coli, which has then been purified and subsequently crystallized. The Staphylococcus aureus protein YmfM contains a helix–turn–helix motif and is thought to be a putative DNA-binding protein which is associated with the membrane through a C-terminal hydrophobic transmembrane anchor. Truncation of the protein by the removal of this C-terminal hydrophobic segment has enabled the overexpression of a soluble domain of S. aureus YmfM (ΔYmfM) in Escherichia coli, which has been purified and subsequently crystallized. Crystals of ΔYmfM diffract to beyond 1.0 Å resolution and belong to one of the pair of enantiomorphic tetragonal space groups P4{sub 1}2{sub 1}2 or P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 45.5, c = 72.9 Å and one molecule in the asymmetric unit. The crystals of ΔYmfM have an unusually low V{sub M} of 1.6 Å{sup 3} Da{sup −1}, which is one of the lowest values observed for any protein to date. A full structure determination is under way in order to provide insights into the function of this protein.

  15. 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;

    2015-01-01

    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...... are not general determinants for origin specification but may play a role for a subset. Interestingly, we observed a periodic spacing of G4 motifs and nucleosomes around the peak summits, suggesting that G4s may position nucleosomes at this subset of origins. Finally, we demonstrate that use of Na+ instead of K...

  16. 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

    2016-01-01

    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 simila...

  17. Identification of a New G-Quadruplex Motif in the KRAS Promoter and Design of Pyrene-Modified G4-Decoys with Antiproliferative Activity in Pancreatic Cancer Cells

    DEFF Research Database (Denmark)

    Cogoi, Susanna; Paramasivam, Manikandan; Filitchev, Vyacheslav Viatcheslav;

    2009-01-01

    A new quadruplex motif located in the promoter of the human KRAS gene, within a nuclease hypersensitive element (NHE), has been characterized. Oligonucleotides mimicking this quadruplex are found to compete with a DNA-protein complex between NHE and a nuclear extract from pancreatic cancer cells...

  18. 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

    2016-01-01

    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 simila...

  19. Novel circular single-stranded DNA viruses identified in marine invertebrates reveal high sequence diversity and consistent predicted intrinsic disorder patterns within putative structural proteins

    Directory of Open Access Journals (Sweden)

    Karyna eRosario

    2015-07-01

    Full Text Available Viral metagenomics has recently revealed the ubiquitous and diverse nature of single-stranded DNA (ssDNA viruses that encode a conserved replication initiator protein (Rep in the marine environment. Although eukaryotic circular Rep-encoding ssDNA (CRESS-DNA viruses were originally thought to only infect plants and vertebrates, recent studies have identified these viruses in a number of invertebrates. To further explore CRESS-DNA viruses in the marine environment, this study surveyed CRESS-DNA viruses in various marine invertebrate species. A total of 27 novel CRESS-DNA genomes, with Reps that share less than 60.1% identity with previously reported viruses, were recovered from 21 invertebrate species, mainly crustaceans. Phylogenetic analysis based on the Rep revealed a novel clade of CRESS-DNA viruses that included approximately one third of the marine invertebrate associated viruses identified here and whose members may represent a novel family. Investigation of putative capsid proteins (Cap encoded within the eukaryotic CRESS-DNA viral genomes from this study and those in GenBank demonstrated conserved patterns of predicted intrinsically disordered regions (IDRs, which can be used to complement similarity-based searches to identify divergent structural proteins within novel genomes. Overall, this study expands our knowledge of CRESS-DNA viruses associated with invertebrates and explores a new tool to evaluate divergent structural proteins encoded by these viruses.

  20. Mitochondrial genome of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa): A linear DNA molecule encoding a putative DNA-dependent DNA polymerase.

    Science.gov (United States)

    Shao, Zhiyong; Graf, Shannon; Chaga, Oleg Y; Lavrov, Dennis V

    2006-10-15

    The 16,937-nuceotide sequence of the linear mitochondrial DNA (mt-DNA) molecule of the moon jelly Aurelia aurita (Cnidaria, Scyphozoa) - the first mtDNA sequence from the class Scypozoa and the first sequence of a linear mtDNA from Metazoa - has been determined. This sequence contains genes for 13 energy pathway proteins, small and large subunit rRNAs, and methionine and tryptophan tRNAs. In addition, two open reading frames of 324 and 969 base pairs in length have been found. The deduced amino-acid sequence of one of them, ORF969, displays extensive sequence similarity with the polymerase [but not the exonuclease] domain of family B DNA polymerases, and this ORF has been tentatively identified as dnab. This is the first report of dnab in animal mtDNA. The genes in A. aurita mtDNA are arranged in two clusters with opposite transcriptional polarities; transcription proceeding toward the ends of the molecule. The determined sequences at the ends of the molecule are nearly identical but inverted and lack any obvious potential secondary structures or telomere-like repeat elements. The acquisition of mitochondrial genomic data for the second class of Cnidaria allows us to reconstruct characteristic features of mitochondrial evolution in this animal phylum.

  1. Molecular Characterizations of a Novel Putative DNA-Binding Protein LvDBP23 in Marine Shrimp L. vannamei Tissues and Molting Stages

    OpenAIRE

    Yanisa Laoong-u-thai; Baoping Zhao; Amornrat Phongdara; Jinzeng Yang

    2011-01-01

    BACKGROUND: Litopenaeus Vannamei, well known as pacific white shrimp, is the most popular shrimp in the world shrimp market. Identification and characterization of shrimp muscle regulatory genes are not only important for shrimp genetic improvement, but also facilitate comparative genomic tools for understanding of muscle development and regeneration. METHODOLOGY/PRINCIPAL FINDINGS: A novel mRNA encoding for a putative DNA-binding protein LvDBP23 was identified from Litopenaeus vannamei abdom...

  2. Radical-induced purine lesion formation is dependent on DNA helical topology.

    Science.gov (United States)

    Terzidis, Michael A; Prisecaru, Andreea; Molphy, Zara; Barron, Niall; Randazzo, Antonio; Dumont, Elise; Krokidis, Marios G; Kellett, Andrew; Chatgilialoglu, Chryssostomos

    2016-11-01

    Herein we report the quantification of purine lesions arising from gamma-radiation sourced hydroxyl radicals (HO(•)) on tertiary dsDNA helical forms of supercoiled (SC), open circular (OC), and linear (L) conformation, along with single-stranded folded and non-folded sequences of guanine-rich DNA in selected G-quadruplex structures. We identify that DNA helical topology and folding plays major, and unexpected, roles in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA), along with tandem-type purine lesions 5',8-cyclo-2'-deoxyguanosine (5',8-cdG) and 5',8-cyclo-2'-deoxyadenosine (5',8-cdA). SC, OC, and L dsDNA conformers together with folded and non-folded G-quadruplexes d[TGGGGT]4 (TG4T), d[AGGG(TTAGGG)3] (Tel22), and the mutated tel24 d[TTGGG(TTAGGG)3A] (mutTel24) were exposed to HO(•) radicals and purine lesions were then quantified via stable isotope dilution LC-MS/MS analysis. Purine oxidation in dsDNA follows L > OC ≫ SC indicating greater damage towards the extended B-DNA topology. Conversely, G-quadruplex sequences were significantly more resistant toward purine oxidation in their unfolded states as compared with G-tetrad folded topologies; this effect is confirmed upon comparative analysis of Tel22 (∼50% solution folded) and mutTel24 (∼90% solution folded). In an effort to identify the accessibly of hydroxyl radicals to quadruplex purine nucleobases, G-quadruplex solvent cavities were then modeled at 1.33 Å with evidence suggesting that folded G-tetrads may act as potential oxidant traps to protect against chromosomal DNA damage.

  3. Cu(II)-vitamin D interaction leads to free radical-mediated cellular DNA damage: a novel putative mechanism for its selective cytotoxic action against malignant cells.

    Science.gov (United States)

    Rizvi, Asim; Chibber, Sandesh; Naseem, Imrana

    2015-03-01

    Vitamin D (vit D) is a known anticancer molecule, and cancer cells are reported to have elevated levels of Cu(II) ions. In this study, we show that interaction of vit D and Cu(II) leads to the formation of hydroxyl free radicals, superoxide anion and hydrogen peroxide, which causes severe oxidative stress, selectively in malignant cells. We show that the production of these reactive oxygen species causes cellular DNA fragmentation which may cause cell death. A novel putative chemical mechanism explaining how vit D causes cell death by DNA damage, selectively in malignant cells, is proposed.

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

    Science.gov (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. Enzyme-free colorimetric detection systems based on the DNA strand displacement competition reaction

    Science.gov (United States)

    Zhang, Z.; Birkedal, V.; Gothelf, K. V.

    2016-05-01

    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, 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 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.

  6. Dual amplified and ultrasensitive electrochemical detection of mutant DNA Biomarkers based on nuclease-assisted target recycling and rolling circle amplifications.

    Science.gov (United States)

    Wang, Qiong; Yang, Cuiyun; Xiang, Yun; Yuan, Ruo; Chai, Yaqin

    2014-05-15

    Based on nicking endonuclease (NEase)-assisted target recycling and rolling circle amplification (RCA) for in situ generation of numerous G-quadruplex/hemin complexes, we developed a new, dual amplified and ultrasensitive electrochemical biosensor for mutant human p53 gene. The target mutant DNA hybridizes with the loop portion of a dithiol-modified hairpin probe (HP) self-assembled on a gold sensing electrode and forms nicking site for the NEase, which cleaves the HP and releases the target DNA. The released target DNA again hybridizes with the intact HP and initiates the DNA recycling process with the assistance of the NEase, leading to the cleavage of a large number of the HPs and the generation of numerous primers for RCA. With rationally designed, G-quadruplex complementary sequence-encoded RCA circular template, subsequent RCA results in the formation of long DNA sequences with massive tandem-repeat G-quadruplex sequences, which further associate with hemin and generate significantly amplified current response for highly sensitive DNA detection down to 0.25 fM. The developed method also exhibits high specificity for the target DNA against single-base mismatched sequence. With the ultrahigh sensitivity feature induced by the dual signal amplification, the proposed method can thus offer new opportunities for the detection of trace amounts of DNA.

  7. Molecular cloning and sequencing of a cDNA encoding partial putative molt-inhibiting hormone from Penaeus chinensis

    Science.gov (United States)

    Wang, Zai-Zhao; Xiang, Jian-Hai

    2002-09-01

    Total RNA was extracted from eyestalks of shrimp Penaeus chinensis. Eyestalk cDNA was obtained from total RNA by reverse transcription. Reverse transcriptase-polymerase chain reaction (RT-PCR) was initiated using eyestalk cDNA and degenerate primers designed from the amino acid sequence of molt-inhibiting hormone from shrimp Penaeus japonicus. A specific cDNA was obtained and cloned into a T vector for sequencing. The cDNA consisted of 201 base pairs and encoding for a peptide of 67 amino acid residues. The peptide of P. chinensis had the highest identity with molt-inhibiting hormones of P. japonicus. The cDNA could be a partial gene of molt-inhibiting hormones from P. chinensis. This paper reports for the first time cDNA encoding for neuropeptide of P. chinensis.

  8. MOLECULAR CLONING AND SEQUENCING OF A cDNA ENCODING PARTIAL PUTATIVE MOLT-INHIBITING HORMONE FROM PENAEUS CHINENSIS

    Institute of Scientific and Technical Information of China (English)

    王在照; 相建海

    2002-01-01

    Total RNA was extracted from eyestalks of shrimp Penaeus chinensis. Eyestalk cDNA was obtained from total RNA by reverse transcription. Reverse transcriptase-polymer ase chain reaction (RT-PCR) was initiated using eyestalk cDNA and degenerate primers designed from the amino acid sequence of molt-inhibiting hormone from shrimp Penaeus japonicus. A s pecific cDNA was obtained and cloned into a T vector for sequencing. The cDNA consisted of 201 ba se pairs and encoding for a peptide of 67 amino acid residues. The peptide of P. chinensis had the highest identity with molt-inhibiting hormones of P. japonicus. The cDNA could be a partial gene of molt-inhibiting hormones from P. chinensis. This paper reports for the first time cDNA encoding for neuropeptide of P. chinensis.

  9. MOLECULAR CLONING AND SEQUENCING OF A cDNA ENCODING PARTIAL PUTATIVE MOLT-INHIBITING HORMONE FROM PENAEUS CHINENSIS

    Institute of Scientific and Technical Information of China (English)

    王在照; 相建海

    2002-01-01

    Total RNA was extracted from eyestalks of shrimp Penaeue chinensis. Eyestalk cDNA was obtained from total RNA by reverse transcription. Reverse transcriptase-polymerase chain reaction (RT-PCR) was initiated using eyestalk cDNA and degenerate primers designed from the amino acid sequence of molt-inhibiting hormone from shrimp Penaeus japonicus. A specific cDNA was obtained and cloned into a T vector for sequencing. The cDNA consisted of 201 base pairs and encoding for a peptide of 67 amino acid residues. The peptide of P. chinensis had the highest identity with molt-inhibiting hormones of P. japonicus. The cDNA could be a partial gene of molt-inhibiting hormones from P. chinensis. This paper reports for the first time cDNA encoding for neuropeptide of P. chinensis.

  10. A DNA nanomachine based on rolling circle amplification-bridged two-stage exonuclease III-assisted recycling strategy for label-free multi-amplified biosensing of nucleic acid.

    Science.gov (United States)

    Xue, Qingwang; Lv, Yanqin; Cui, Hui; Gu, Xiaohong; Zhang, Shuqiu; Liu, Jifeng

    2015-01-26

    An autonomous DNA nanomachine based on rolling circle amplification (RCA)-bridged two-stage exonuclease III (Exo III)-induced recycling amplification (Exo III-RCA-Exo III) was developed for label-free and highly sensitive homogeneous multi-amplified detection of DNA combined with sensitive fluorescence detection technique. According to the configuration, the analysis of DNA is accomplished by recognizing the target to a unlabeled molecular beacon (UMB) that integrates target-binding and signal transducer within one multifunctional design, followed by the target-binding of UMB in duplex DNA removed stepwise by Exo III accompanied by the releasing of target DNA for the successive hybridization and cleavage process and autonomous generation of the primer that initiate RCA process with a rational designed padlock DNA. The RCA products containing thousands of repeated catalytic sequences catalytically hybridize with a hairpin reporter probe that includes a "caged" inactive G-quadruplex sequence (HGP) and were then detected by Exo III-assisted recycling amplification, liberating the active G-quadruplex and generating remarkable ZnPPIX/G-quadruplex fluorescence signals with the help of zinc(II)-protoporphyrin IX (ZnPPIX). The proposed strategy showed a wide dynamic range over 7 orders of magnitude with a low limit of detection of 0.51 aM. In addition, this designed protocol can discriminate mismatched DNA from perfectly matched target DNA, and holds a great potential for early diagnosis in gene-related diseases.

  11. Mitochondrial DNA analysis on remains of a putative son of Louis XVI, King of France and Marie-Antoinette.

    Science.gov (United States)

    Jehaes, E; Decorte, R; Peneau, A; Petrie, J H; Boiry, P A; Gilissen, A; Moisan, J P; Van den Berghe, H; Pascal, O; Cassiman, J J

    1998-01-01

    Carl Wilhelm Naundorff was buried in 1845 in Delft as Louis Charles, Duc de Normandie, 'Louis XVII'. However, the son of Louis XVI and Marie-Antoinette-Louis XVII--officially died in the Temple of Paris in 1795. In order to resolve the identity of Naundorff, mitochondrial DNA (mtDNA) D-loop sequences of his remains were compared with the sequences obtained from the hairs of two sisters of Marie-Antoinette, Marie-Antoinette herself, and with the sequences obtained from DNA samples of two living maternal relatives. The mtDNA sequence of a bone sample from Naundorff showed two nucleotide differences from the sequences of the three sisters and four differences from the sequences of living maternal relatives. Based on this evidence it becomes very unlikely that Naundroff is the son of Marie-Antoinette.

  12. Cloning and molecular characterization of cDNA encoding a mouse male-enhanced antigen-2 (Mea-2): a putative family of the Golgi autoantigen.

    Science.gov (United States)

    Kondo, M; Sutou, S

    1997-01-01

    The male-enhanced antigen-2 (Mea-2) gene was originally identified with a monoclonal histocompatibility Y (H-Y) antibody (mAb4VII). There is no report of the full length cDNA encode for Mea-2 product until this report. In this study, we isolated the full length mouse Mea-2 cDNA by screening a testis cDNA library with a PCR-amplified Mea-2 product, and direct PCR amplification of its upstream sequences from the cDNA library. The primary structure of the Mea-2 peptide, deduced from this nucleotide sequence, shows that it encode a 150 kDa protein, of 1325 amino acid residues, which contained five putative N-glycosylation sites and four leucine zipper motifs. A data bank search indicated that it has high homology with a human Golgi autoantigen (golgin-160) both in its nucleotides (78%) and amino acids sequence (83%). This suggests that Mea-2 gene product may encode a golgi structural protein. In situ hybridization analysis suggested that the Mea-2 gene is expressed in spermatids during spermatogenesis as already shown by Mea-1, suggesting that Mea-2 gene product as well as Mea-1 have also some role for spermatogenesis.

  13. Sequence selective naked-eye detection of DNA harnessing extension of oligonucleotide-modified nucleotides.

    Science.gov (United States)

    Verga, Daniela; Welter, Moritz; Marx, Andreas

    2016-02-01

    DNA polymerases can efficiently and sequence selectively incorporate oligonucleotide (ODN)-modified nucleotides and the incorporated oligonucleotide strand can be employed as primer in rolling circle amplification (RCA). The effective amplification of the DNA primer by Φ29 DNA polymerase allows the sequence-selective hybridisation of the amplified strand with a G-quadruplex DNA sequence that has horse radish peroxidase-like activity. Based on these findings we develop a system that allows DNA detection with single-base resolution by naked eye.

  14. Molecular cloning and characterization of cDNA encoding a putative stress-induced heat-shock protein from Camelus dromedarius.

    Science.gov (United States)

    Elrobh, Mohamed S; Alanazi, Mohammad S; Khan, Wajahatullah; Abduljaleel, Zainularifeen; Al-Amri, Abdullah; Bazzi, Mohammad D

    2011-01-01

    Heat shock proteins are ubiquitous, induced under a number of environmental and metabolic stresses, with highly conserved DNA sequences among mammalian species. Camelus dromedaries (the Arabian camel) domesticated under semi-desert environments, is well adapted to tolerate and survive against severe drought and high temperatures for extended periods. This is the first report of molecular cloning and characterization of full length cDNA of encoding a putative stress-induced heat shock HSPA6 protein (also called HSP70B') from Arabian camel. A full-length cDNA (2417 bp) was obtained by rapid amplification of cDNA ends (RACE) and cloned in pET-b expression vector. The sequence analysis of HSPA6 gene showed 1932 bp-long open reading frame encoding 643 amino acids. The complete cDNA sequence of the Arabian camel HSPA6 gene was submitted to NCBI GeneBank (accession number HQ214118.1). The BLAST analysis indicated that C. dromedaries HSPA6 gene nucleotides shared high similarity (77-91%) with heat shock gene nucleotide of other mammals. The deduced 643 amino acid sequences (accession number ADO12067.1) showed that the predicted protein has an estimated molecular weight of 70.5 kDa with a predicted isoelectric point (pI) of 6.0. The comparative analyses of camel HSPA6 protein sequences with other mammalian heat shock proteins (HSPs) showed high identity (80-94%). Predicted camel HSPA6 protein structure using Protein 3D structural analysis high similarities with human and mouse HSPs. Taken together, this study indicates that the cDNA sequences of HSPA6 gene and its amino acid and protein structure from the Arabian camel are highly conserved and have similarities with other mammalian species.

  15. Molecular characterizations of a novel putative DNA-binding protein LvDBP23 in marine shrimp L. vannamei tissues and molting stages.

    Directory of Open Access Journals (Sweden)

    Yanisa Laoong-u-thai

    Full Text Available BACKGROUND: Litopenaeus Vannamei, well known as pacific white shrimp, is the most popular shrimp in the world shrimp market. Identification and characterization of shrimp muscle regulatory genes are not only important for shrimp genetic improvement, but also facilitate comparative genomic tools for understanding of muscle development and regeneration. METHODOLOGY/PRINCIPAL FINDINGS: A novel mRNA encoding for a putative DNA-binding protein LvDBP23 was identified from Litopenaeus vannamei abdominal muscle cDNA library. The LvDBP23 cDNA contains 639 nucleotides of protein-coding sequence with deduced 212 amino acids of predicted molecular mass 23.32 kDa with glycine-rich domain at amino acid position 94-130. The mRNA sequence is successfully used for producing LvDBP23 recombinant protein in sf9 insect cell expression system. The expression of LvDBP23 mRNA is presented in abdominal muscle and swimming leg muscle, as well as other tissues including intestine, lymphoid and gill. The mRNA expression has the highest level in abdominal muscle in all tested tissues. LVDBP23 transcript during the molt cycle is highly expressed in the intermolt stage. In vitro nucleic acid-binding assays reveal that LvDBP23 protein can bind to both ssDNA and dsDNA, indicating its possible role of regulation of gene transcription. CONCLUSIONS/SIGNIFICANCE: We are the first to report a DNA-binding protein identified from the abdominal muscle tissue of marine shrimp L. Vannamei. Its high-level specific expression during the intermot stage suggests its role in the regulation of muscle buildup during the growth phase of shrimp molt cycle.

  16. Resveratrol mobilizes endogenous copper in human peripheral lymphocytes leading to oxidative DNA breakage: a putative mechanism for chemoprevention of cancer.

    Science.gov (United States)

    Hadi, S M; Ullah, M F; Azmi, A S; Ahmad, A; Shamim, U; Zubair, H; Khan, H Y

    2010-06-01

    Plant polyphenols are important components of human diet, and a number of them are considered to possess chemopreventive and therapeutic properties against cancer. They are recognized as naturally occurring anti-oxidants but also act as pro-oxidants catalyzing DNA degradation in the presence of metal ions such as copper. The plant polyphenol resveratrol confers resistance to plants against fungal agents and has been implicated as a cancer chemopreventive agent. Of particular interest is the observation that resveratrol has been found to induce apoptosis in cancer cell lines but not in normal cells. Over the last few years, we have shown that resveratrol is capable of causing DNA breakage in cells such as human lymphocytes. Such cellular DNA breakage is inhibited by copper specific chelators but not by iron and zinc chelating agents. Similar results are obtained by using permeabilized cells or with isolated nuclei, indicating that chromatin-bound copper is mobilized in this reaction. It is well established that tissue, cellular and serum copper levels are considerably elevated in various malignancies. Therefore, cancer cells may be more subject to electron transfer between copper ions and resveratrol to generate reactive oxygen species responsible for DNA cleavage. The results are in support of our hypothesis that anti-cancer mechanism of plant polyphenols involves mobilization of endogenous copper and the consequent pro-oxidant action. Such a mechanism better explains the anti-cancer effects of resveratrol, as it accounts for the preferential cytotoxicity towards cancer cells.

  17. 基于T-Hg2+-T及G四聚体自身熄灭能力的"Turn on"型单标记DNA荧光探针用于碘离子的检测%Single-labeled "Turn on" Fluorescent Oligonucleotide Probe for Iodide Detection Based on T-Hg2+-T and Inherent Quenching Ability of G-quadruplex

    Institute of Scientific and Technical Information of China (English)

    朱颖; 刘沛; 羊小海; 何磊良; 李清照; 王青; 王柯敏; 黄晋; 刘剑波

    2012-01-01

    A simple and "Turn on" type iodide anion assay method was developed, based on the inherent quenching ability of G-quadruplex and fidelity of the " thymine-Hg2+ -thymine" binding motif. A T-rich sequence was labeled with 6-carboxyfluorescein(FAM) at its 5'-end, nearby the 3'-end was a G-rich sequence which could form G-quadruplex structure instead of traditional quenchers. Upon addition of Hg2+, T-rich sequence folded into a hairpin structure which led the G-quadruplex near to the FAM, and the FAM was quenched by the G-quadruplex owning to photoinduced electron transfer between the dye and the G-quadruplex. After the addition of iodide, the fluorescence of FAM recovered considering that iodide could bind with Hg2+. The novel method for the determination of iodide was developed in linear range of 50—500 nmol/L, with the detection limit(3σ) of 30 nmol/L. This proposed method was highly selective and other anions have no interfering effects on the determination, which was successfully applied for analysis of real samples with the recovery from 92%-109% , and the RSD<4% (n=4).%利用G四聚体可以熄灭荧光的特性以及T-Hg2+-T的特殊结构,发展了一种简便的"Turn on”型碘离子检测新方法.设计了一条5'端标有荧光基团的富T序列,3'端采用能形成G四聚体的富G序列代替传统的熄灭基团.加入汞离子后,富T序列形成T-Hg2+-T机构发生折叠,G四聚体靠近荧光基团,发生光诱导电子转移,使荧光被熄灭.若加入碘离子,碘离子会与汞离子形成较稳定的配合物,汞离子从DNA上被竞争下来,探针的荧光得以恢复,且荧光强度与50 ~ 500 nmol/L的碘离子呈良好线性关系,检出限为30 nmol/L.本方法选择性好,10倍于碘离子浓度的其它常见阴离子干扰较小.检测自来水样中碘离子的回收率为92%~ 109%,相对标准偏差RSD<4%(n=4).

  18. G-Quadruplex DNAzyme Biosensor for Quantitative Detection of T4 Polynucleotide Kinase Activity by Using Split-to-Intact G-Quadruplex DNAzyme Conversion%基于"分段"-"完整"转化的G-四链体脱氧核糖核酸酶传感器用于多聚核苷酸激酶的检测

    Institute of Scientific and Technical Information of China (English)

    周璐; 程慧; 王金娥; 裴仁军

    2016-01-01

    采用"分段"转化为"完整"G-四链体脱氧核糖核酸酶的策略,构建了检测多聚核苷激酶(T4 PNK)的传感器.将形成G-四链体的富G序列PS5.M序列拆分成5 '和3'端均为羟基的两条链:链S1OH和链S2OH,即分别具有12个碱基的"分段"的PS5.M.在ATP存在下,T4 PNK酶可以将链S2OH的5 '端的羟基磷酸化,转化为S2P;在S1OH、S2P以及Helper链S—H存在下,T4 DNA连接酶(T4 DNA Ligase)将链S1OH和链S2P连接成"完整"的PS5.M序列.在体系中再加入核酸外切酶Ⅲ(ExoⅢ),从S—H的3'端剪切S-H,释放出PS5.M.在K+存在下,PS5.M与氯化血红素(Hemin)作用,形成具有类过氧化物酶活性的复合物,催化H2O2氧化2,2'-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐(ABTS)的反应,通过检测氧化产物在418 nm处的吸收值变化,实现对T4 PNK活性的定量检测.线性检测范围为0.02 ~ 3.0 U/mL,检出限为0.014 U/mL(S/N=3).对Hela细胞和HEK293细胞实际样本的T4 PNK活性进行了检测,平均回收率为95.6%~105.7%.

  19. Avocado cellulase: nucleotide sequence of a putative full-length cDNA clone and evidence for a small gene family.

    Science.gov (United States)

    Tucker, M L; Durbin, M L; Clegg, M T; Lewis, L N

    1987-05-01

    A cDNA library was prepared from ripe avocado fruit (Persea americana Mill. cv. Hass) and screened for clones hybridizing to a 600 bp cDNA clone (pAV5) coding for avocado fruit cellulase. This screening led to the isolation of a clone (pAV363) containing a 2021 nucleotide transcribed sequence and an approximately 150 nucleotide poly(A) tail. Hybridization of pAV363 to a northern blot shows that the length of the homologous message is approximately 2.2 kb. The nucleotide sequence of this putative full-length mRNA clone contains an open reading frame of 1482 nucleotides which codes for a polypeptide of 54.1 kD. The deduced amino acid composition compares favorably with the amino acid composition of native avocado cellulase determined by amino acid analysis. Southern blot analysis of Hind III and Eco RI endonuclease digested genomic DNA indicates a small family of cellulase genes.

  20. Reconstruction of putative DNA virus from endogenous rice tungro bacilliform virus-like sequences in the rice genome: implications for integration and evolution

    Directory of Open Access Journals (Sweden)

    Kishima Yuji

    2004-10-01

    Full Text Available Abstract Background Plant genomes contain various kinds of repetitive sequences such as transposable elements, microsatellites, tandem repeats and virus-like sequences. Most of them, with the exception of virus-like sequences, do not allow us to trace their origins nor to follow the process of their integration into the host genome. Recent discoveries of virus-like sequences in plant genomes led us to set the objective of elucidating the origin of the repetitive sequences. Endogenous rice tungro bacilliform virus (RTBV-like sequences (ERTBVs have been found throughout the rice genome. Here, we reconstructed putative virus structures from RTBV-like sequences in the rice genome and characterized to understand evolutionary implication, integration manner and involvements of endogenous virus segments in the corresponding disease response. Results We have collected ERTBVs from the rice genomes. They contain rearranged structures and no intact ORFs. The identified ERTBV segments were shown to be phylogenetically divided into three clusters. For each phylogenetic cluster, we were able to make a consensus alignment for a circular virus-like structure carrying two complete ORFs. Comparisons of DNA and amino acid sequences suggested the closely relationship between ERTBV and RTBV. The Oryza AA-genome species vary in the ERTBV copy number. The species carrying low-copy-number of ERTBV segments have been reported to be extremely susceptible to RTBV. The DNA methylation state of the ERTBV sequences was correlated with their copy number in the genome. Conclusions These ERTBV segments are unlikely to have functional potential as a virus. However, these sequences facilitate to establish putative virus that provided information underlying virus integration and evolutionary relationship with existing virus. Comparison of ERTBV among the Oryza AA-genome species allowed us to speculate a possible role of endogenous virus segments against its related disease.

  1. Comparison and Validation of Putative Pathogenicity-Related Genes Identified by T-DNA Insertional Mutagenesis and Microarray Expression Profiling in Magnaporthe oryzae

    Science.gov (United States)

    Wáng, Ying; Tan, Qi; Gao, Ying Nv; Li, Yan

    2017-01-01

    High-throughput technologies of functional genomics such as T-DNA insertional mutagenesis and microarray expression profiling have been employed to identify genes related to pathogenicity in Magnaporthe oryzae. However, validation of the functions of individual genes identified by these high-throughput approaches is laborious. In this study, we compared two published lists of genes putatively related to pathogenicity in M. oryzae identified by T-DNA insertional mutagenesis (comprising 1024 genes) and microarray expression profiling (comprising 236 genes), respectively, and then validated the functions of some overlapped genes between the two lists by knocking them out using the method of target gene replacement. Surprisingly, only 13 genes were overlapped between the two lists, and none of the four genes selected from the overlapped genes exhibited visible phenotypic changes on vegetative growth, asexual reproduction, and infection ability in their knockout mutants. Our results suggest that both of the lists might contain large proportions of unrelated genes to pathogenicity and therefore comparing the two gene lists is hardly helpful for the identification of genes that are more likely to be involved in pathogenicity as we initially expected.

  2. Cytomolecular analysis of ribosomal DNA evolution in a natural allotetraploid Brachypodium hybridum and its putative ancestors – dissecting complex repetitive structure of intergenic spacers

    Directory of Open Access Journals (Sweden)

    Natalia Borowska-Zuchowska

    2016-10-01

    Full Text Available Nucleolar dominance is an epigenetic phenomenon associated with nuclear 35S rRNA genes and consists in selective suppression of gene loci inherited from one of the progenitors in the allopolyploid. Our understanding of the exact mechanisms that determine this process is still fragmentary, especially in case of the grass species. This study aimed to shed some light on the molecular basis of this genome-specific inactivation of 35S rDNA loci in an allotetraploid Brachypodium hybridum (2n=30, which arose from the interspecific hybridization between two diploid ancestors that were very similar to modern B. distachyon (2n=10 and B. stacei (2n=20. Using fluorescence in situ hybridization with 25S rDNA and chromosome-specific BAC clones as probes we revealed that the nucleolar dominance is present not only in meristematic root-tip cells but also in differentiated cell fraction of B. hybridum. Additionally, the intergenic spacers (IGSs from both of the putative ancestors and the allotetraploid were sequenced and analyzed. The presumptive transcription initiation sites, spacer promoters and repeated elements were identified within the IGSs. Two different length variants, 2.3 kb and 3.5 kb, of IGSs were identified in B. distachyon and B. stacei, respectively, however only the IGS that had originated from B. distachyon-like ancestor was present in the allotetraploid. The amplification pattern of B. hybridum IGSs suggests that some genetic changes occurred in inactive B. stacei-like rDNA loci during the evolution of the allotetraploid. We hypothesize that their preferential silencing is an effect of structural changes in the sequence rather than just the result of the sole inactivation at the epigenetic level.

  3. The effect of single nucleotide polymorphisms in G-rich regions of high-risk human papillomaviruses on structural diversity of DNA.

    Science.gov (United States)

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

    2017-05-01

    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.

  4. Molecular phylogenetics of the species-rich angiosperm genus Goniothalamus (Annonaceae) inferred from nine chloroplast DNA regions: Synapomorphies and putative correlated evolutionary changes in fruit and seed morphology.

    Science.gov (United States)

    Tang, Chin Cheung; Thomas, Daniel C; Saunders, Richard M K

    2015-11-01

    A phylogenetic study of the genus Goniothalamus (Annonaceae) is presented using maximum parsimony, maximum likelihood and Bayesian approaches, with 65 species sampled (48.5% of the genus) based on sequences of nine chloroplast DNA regions (11,214 aligned positions). The resultant phylogeny clearly indicates that Goniothalamus is monophyletic. Preliminary research initially focused on identifying synapomorphies and estimating the phylogenetic signal of selected morphological characters based on parsimony and likelihood ancestral character state reconstructions. This prescreening of characters enabled 40 to be selected for further study, and of these 15 are shown here to demonstrate significant phylogenetic signal and to provide clear synapomorphies for several infrageneric clades. Although floral structure in Goniothalamus is comparatively uniform, suggesting a common basic pattern of pollination ecology, fruit and seed morphology in the genus is very diverse and is presumably associated with different patterns of frugivory. The present study assesses correlations amongst fruit and seed characters which are putatively of functional importance with regard to frugivory and dispersal. One-way phylogenetic ANOVA indicates significant phylogenetically independent correlation between the following fruit and seed characters: fruits borne on older branches and/or on the main trunk have larger monocarps than fruits borne on young branches; and monocarps that contain seeds with a hairy testa are larger than those with glabrous seeds. We discuss fruit morphologies and potential explanations for the inferred correlations, and suggest that they may be the result of adaptation to different frugivores (birds, larger non-volant animal and primate seed dispersers, respectively).

  5. Interlocked DNA nanostructures controlled by a reversible logic circuit.

    Science.gov (United States)

    Li, Tao; Lohmann, Finn; Famulok, Michael

    2014-09-17

    DNA nanostructures constitute attractive devices for logic computing and nanomechanics. An emerging interest is to integrate these two fields and devise intelligent DNA nanorobots. Here we report a reversible logic circuit built on the programmable assembly of a double-stranded (ds) DNA [3]pseudocatenane that serves as a rigid scaffold to position two separate branched-out head-motifs, a bimolecular i-motif and a G-quadruplex. The G-quadruplex only forms when preceded by the assembly of the i-motif. The formation of the latter, in turn, requires acidic pH and unhindered mobility of the head-motif containing dsDNA nanorings with respect to the central ring to which they are interlocked, triggered by release oligodeoxynucleotides. We employ these features to convert the structural changes into Boolean operations with fluorescence labelling. The nanostructure behaves as a reversible logic circuit consisting of tandem YES and AND gates. Such reversible logic circuits integrated into functional nanodevices may guide future intelligent DNA nanorobots to manipulate cascade reactions in biological systems.

  6. Genomic diversity of Mycobacterium tuberculosis Beijing strains isolated in Tuscany, Italy, based on large sequence deletions, SNPs in putative DNA repair genes and MIRU-VNTR polymorphisms.

    Science.gov (United States)

    Garzelli, Carlo; Lari, Nicoletta; Rindi, Laura

    2016-03-01

    The Beijing genotype of Mycobacterium tuberculosis is cause of global concern as it is rapidly spreading worldwide, is considered hypervirulent, and is most often associated to massive spread of MDR/XDR TB, although these epidemiological or pathological properties have not been confirmed for all strains and in all geographic settings. In this paper, to gain new insights into the biogeographical heterogeneity of the Beijing family, we investigated a global sample of Beijing strains (22% from Italian-born, 78% from foreign-born patients) by determining large sequence polymorphism of regions RD105, RD181, RD150 and RD142, single nucleotide polymorphism of putative DNA repair genes mutT4 and mutT2 and MIRU-VNTR profiles based on 11 discriminative loci. We found that, although our sample of Beijing strains showed a considerable genomic heterogeneity, yielding both ancient and recent phylogenetic strains, the prevalent successful Beijing subsets were characterized by deletions of RD105 and RD181 and by one nucleotide substitution in one or both mutT genes. MIRU-VNTR analysis revealed 47 unique patterns and 9 clusters including a total of 33 isolates (41% of total isolates); the relatively high proportion of Italian-born Beijing TB patients, often occurring in mixed clusters, supports the possibility of an ongoing cross-transmission of the Beijing genotype to autochthonous population. High rates of extra-pulmonary localization and drug-resistance, particularly MDR, frequently reported for Beijing strains in other settings, were not observed in our survey.

  7. A selective and label-free strategy for rapid screening of telomere-binding Ligands via fluorescence regulation of DNA/silver nanocluster

    Science.gov (United States)

    Cheng, Rui; Xu, Jing; Zhang, Xiafei; Shi, Zhilu; Zhang, Qi; Jin, Yan

    2017-03-01

    Herein, the conformational switch of G-rich oligonucleotide (GDNA) demonstrated the obvious functional switch of GDNA which was found to significantly affect the fluorescence of the in-situ synthesized DNA/silver nanocluster (DNA-AgNC) in homogeneous solution. We envisioned that the allosteric interaction between GDNA and DNA-AgNC would be possible to be used for screening telomere-binding ligands. A unimolecular probe (12C5TG) is ingeniously designed consisting of three contiguous DNA elements: G-rich telomeric DNA (GDNA) as molecular recognition sequence, T-rich DNA as linker and C-rich DNA as template of DNA-AgNC. The quantum yield and stability of 12C5TG-AgNC is greatly improved because the nearby deoxyguanosines tended to protect DNA/AgNC against oxidation. However, in the presence of ligands, the formation of G-quadruplex obviously quenched the fluorescence of DNA-AgNC. By taking full advantage of intramolecular allosteric effect, telomere-binding ligands were selectively and label-free screened by using deoxyguanines and G-quadruplex as natural fluorescence enhancer and quencher of DNA-AgNC respectively. Therefore, the functional switching of G-rich structure offers a cost-effective, facile and reliable way to screen drugs, which holds a great potential in bioanalysis as well.

  8. A selective and label-free strategy for rapid screening of telomere-binding Ligands via fluorescence regulation of DNA/silver nanocluster

    Science.gov (United States)

    Cheng, Rui; Xu, Jing; Zhang, Xiafei; Shi, Zhilu; Zhang, Qi; Jin, Yan

    2017-01-01

    Herein, the conformational switch of G-rich oligonucleotide (GDNA) demonstrated the obvious functional switch of GDNA which was found to significantly affect the fluorescence of the in-situ synthesized DNA/silver nanocluster (DNA-AgNC) in homogeneous solution. We envisioned that the allosteric interaction between GDNA and DNA-AgNC would be possible to be used for screening telomere-binding ligands. A unimolecular probe (12C5TG) is ingeniously designed consisting of three contiguous DNA elements: G-rich telomeric DNA (GDNA) as molecular recognition sequence, T-rich DNA as linker and C-rich DNA as template of DNA-AgNC. The quantum yield and stability of 12C5TG-AgNC is greatly improved because the nearby deoxyguanosines tended to protect DNA/AgNC against oxidation. However, in the presence of ligands, the formation of G-quadruplex obviously quenched the fluorescence of DNA-AgNC. By taking full advantage of intramolecular allosteric effect, telomere-binding ligands were selectively and label-free screened by using deoxyguanines and G-quadruplex as natural fluorescence enhancer and quencher of DNA-AgNC respectively. Therefore, the functional switching of G-rich structure offers a cost-effective, facile and reliable way to screen drugs, which holds a great potential in bioanalysis as well. PMID:28262705

  9. Mono and trimethine cyanines Cyan 40 and Cyan 2 as probes for highly selective fluorescent detection of non-canonical DNA structures.

    Science.gov (United States)

    Kovalska, Vladyslava B; Losytskyy, Mykhaylo Yu; Yarmoluk, Sergiy M; Lubitz, Irit; Kotlyar, Alexander B

    2011-01-01

    Two of earlier reported dsDNA sensitive cyanine dyes-monomethine Cyan 40 and meso-substituted trimethine Cyan 2 were studied for their ability to interact with non-canonical DNA conformations. These dyes were characterized by spectral-luminescent methods in the presence of G-quadruplex, triplex and dsDNA motifs. We have demonstrated that Cyan 2 binds strongly and preferentially to triple- and quadruple-stranded DNA forms that results in a strong enhancement of the dye fluorescence, as compared to dsDNA, while Cyan 40 form fluorescent complexes preferentially only with the triplex form. Highly fluorescent complexes of Cyan 2 with DNA triplexes and G-quadruplexes and Cyan 40 with DNA triplexes are very stable and do not dissociate during gel electrophoresis, leading to preferential staining of the above DNA forms in gels. The data presented point to the intercalation mechanism of the Cyan 2 binding to G4-DNA, while the complexes of Cyan 40 and Cyan 2 with triplex DNA are believed to be formed via groove binding mode. The Cyan dyes can provide a highly sensitive method for detection and quantification of non-canonical structures in genome.

  10. An unusual gene arrangement for the putative chromosome replication origin and circadian expression of dnaN in Synechococcus sp. strain PCC 7942.

    Science.gov (United States)

    Liu, Y; Tsinoremas, N F

    1996-06-12

    In eubacteria, the clustering of DnaA boxes around the dnaN (beta subunit of DNA polymerase III) and dnaA genes usually defines the chromosome replication origin (oriC). In this study, the dnaN locus from the cyanobacterium Synechococcus sp. strain PCC 7942 was sequenced. The gene order in this region is cbbZp-dnaN-orf288-purL-purF which contrasts with other eubacteria. A cluster of eleven DnaA boxes (consensus sequence: TTTTCCACA) was found in the intergenic region between dnaN and cbbZp. We also found a 41-bp sequence within this region that is 80% identical to the proposed oriC of Streptomyces coelicolor. Therefore, we propose that this intergenic region may serve as an oriC in Synechococcus. Using bacterial luciferase as a reporter, we also showed that dnaN is rhythmically expressed, suggesting that DNA replication could be under circadian control in this organism.

  11. Lack of ligand-selective binding of the aryl hydrocarbon receptor to putative DNA binding sites regulating expression of Bax and paraoxonase 1 genes.

    Science.gov (United States)

    DeGroot, Danica E; Hayashi, Ai; Denison, Michael S

    2014-01-01

    The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates the biological and toxicological effects of structurally diverse chemicals through its ability to bind specific DNA recognition sites (dioxin responsive elements (DREs)), and activate transcription of adjacent genes. While the DRE has a highly conserved consensus sequence, it has been suggested that the nucleotide specificity of AhR DNA binding may be ligand-dependent. The upstream regulatory regions of the murine Bax and human paraoxonase 1 (PON1) genes reportedly contain unique DRE-like sequences that respond to AhRs activated by some ligands but not others. Given the significant implications of this observation to understanding the diversity in AhR responses and that of other ligand-dependent nuclear receptors, a combination of DNA binding, nuclear translocation and gene expression analysis was used to investigate the molecular mechanisms underlying these ligand-selective responses. Although known AhR agonists stimulated AhR nuclear translocation, DRE binding and gene expression, the ligand-selective DRE-like DNA elements identified in the Bax and PON1 upstream regulatory regions failed to bind ligand-activated AhR or confer AhR-responsiveness upon a reporter gene. These results argue against the reported ligand-selectivity of AhR DNA binding and suggest DNA binding by ligand activated AhR involves DRE-containing DNA.

  12. Thermostable and site-specific DNA binding of the gene product ORF56 from the Sulfolobus islandicus plasmid pRN1, a putative archael plasmid copy control protein

    Science.gov (United States)

    Lipps, Georg; Stegert, Mario; Krauss, Gerhard

    2001-01-01

    There is still a lack of information on the specific characteristics of DNA-binding proteins from hyperthermophiles. Here we report on the product of the gene orf56 from plasmid pRN1 of the acidophilic and thermophilic archaeon Sulfolobus islandicus. orf56 has not been characterised yet but low sequence similarily to several eubacterial plasmid-encoded genes suggests that this 6.5 kDa protein is a sequence-specific DNA-binding protein. The DNA-binding properties of ORF56, expressed in Escherichia coli, have been investigated by EMSA experiments and by fluorescence anisotropy measurements. Recombinant ORF56 binds to double-stranded DNA, specifically to an inverted repeat located within the promoter of orf56. Binding to this site could down-regulate transcription of the orf56 gene and also of the overlapping orf904 gene, encoding the putative initiator protein of plasmid replication. By gel filtration and chemical crosslinking we have shown that ORF56 is a dimeric protein. Stoichiometric fluorescence anisotropy titrations further indicate that ORF56 binds as a tetramer to the inverted repeat of its target binding site. CD spectroscopy points to a significant increase in ordered secondary structure of ORF56 upon binding DNA. ORF56 binds without apparent cooperativity to its target DNA with a dissociation constant in the nanomolar range. Quantitative analysis of binding isotherms performed at various salt concentrations and at different temperatures indicates that approximately seven ions are released upon complex formation and that complex formation is accompanied by a change in heat capacity of –6.2 kJ/mol. Furthermore, recombinant ORF56 proved to be highly thermostable and is able to bind DNA up to 85°C. PMID:11160922

  13. A putative role of Drep1 in apoptotic DNA fragmentation system in fly is mediated by direct interaction with Drep2 and Drep4.

    Science.gov (United States)

    Park, Ok Kyung; Park, Hyun Ho

    2013-04-01

    DNA fragmentation is common phenomenon for apoptotic cell death. DNA fragmentation factor, called DFF40 (CAD: mouse homologue), is a main nuclease for apoptotic DNA fragmentation. Nuclease activity of DFF40 is normally inhibited by DFF45 by tight interaction via CIDE domain without apoptotic stimuli. Once effector caspase is activated during apoptosis signaling, it cleave DFF45, allowing DFF40 to enter the nucleus and cleave chromosomal DNA. Unlike mammalian system, apoptotic DNA fragmentation in the fly might be controlled by four DFF-related proteins, known as Drep1, Drep2, Drep3 and Drep4. Although the function of Drep1 and Drep4 is well known as DFF45 and DFF40 homologues, respectively, the function of Drep2 and Drep3 is still unclear. DFF-related proteins contain a conserved CIDE domain of ~90 amino acid residues that is involved in protein-protein interaction. Here, we showed that Drep1 directly bind to Drep2 as well as Drep4 via CIDE domain. In addition, we found that the interaction of Drep2 and Drep4 to Drep1 was not competitive indicating that Drep2 and Drep4 bind different place of Drep1. All together, we suggest that Drep1 might be involved in apoptotic DNA fragmentation of fly system by direct interaction with Drep2 as well as Drep4.

  14. Ion-tuned DNA/Ag fluorescent nanoclusters as versatile logic device.

    Science.gov (United States)

    Li, Tao; Zhang, Libing; Ai, Jun; Dong, Shaojun; Wang, Erkang

    2011-08-23

    A novel kind of versatile logic device has been constructed utilizing ion-tuned DNA/Ag fluorescent nanoclusters, with K(+) and H(+) as two inputs. A well-chosen hairpin DNA with a poly-C loop serves as the template for synthesizing two species of Ag nanoclusters. Several G-tracts and C-tracts on its two terminals enable the hairpin DNA to convert into the G-quadruplex and/or i-motif structures upon input of K(+) and H(+). Such a structural change remarkably influences the spectral behaviors of Ag nanoclusters. In particular, different species of Ag nanoclusters have distinct fluorescence responses to the input of K(+) and H(+). These unique features of DNA/Ag nanoclusters enable multiple logic operations via multichannel fluorescence output, indicating the versatility as a molecular logic device. By altering the specific sequence of the hairpin DNA, more logic gates can be constructed utilizing Ag nanoclusters. © 2011 American Chemical Society

  15. Cascaded multiple amplification strategy for ultrasensitive detection of HIV/HCV virus DNA.

    Science.gov (United States)

    Wang, Kun; Fan, Daoqing; Liu, Yaqing; Dong, Shaojun

    2017-01-15

    Ultrasensitive detection of HIV and HCV virus DNA is of great importance for early accurate diagnostics and therapy of HIV virus-infected patients. Herein, to our best knowledge, it is the first to use DNA cascaded multiple amplification strategy for ultrasensitive detection of HIV virus DNA with G-quadruplex-specific fluorescent or colorimetric probes as signal carriers. The developed strategy also exhibited universal applicability for HCV virus DNA detection. After reaction for about 4h, high sensitivity and specificity can be achieved at both fluorescent and colorimetric strategies (limit of detection (LOD) of 10 fM and 0.5pM were reached for fluorescent and colorimetric detection, respectively). And the single-based mismatched DNA even can be distinguished by naked eyes. It is believed that the cascaded multiple amplification strategy presents a huge advance in sensing platform and potential application in future clinical diagnosis.

  16. Pro-oxidant DNA breakage induced by the interaction of L-DOPA with Cu(II): a putative mechanism of neurotoxicity.

    Science.gov (United States)

    Perveen, Asma; Khan, Husain Yar; Hadi, S M; Damanhouri, Ghazi A; Alharrasi, Ahmed; Tabrez, Shams; Ashraf, Ghulam Md

    2015-01-01

    There are reports in scientific literature that the concentration of copper ions in Parkinsonian brain is at a level that could promote oxidative DNA damage. The possibility of copper chelation by antioxidants excited us to explore the generation of reactive oxygen species (ROS) and DNA damage by the interaction of L-DOPA with Cu(II) ions. In the present manuscript, L-DOPA was tested for its ability to bind with Cu(II) and reduce it to Cu(I). The generation of ROS, such as superoxide anion (O(2)(-)) and hydroxyl radical (OH(•)), was also ascertained. As a result of L-DOPA and Cu(II) interaction, the generation of O(2)(-) was found to be increased in a time-dependent manner. Moreover, the formation of OH(•) was also found to be enhanced with increasing concentrations of L-DOPA. Furthermore, Comet assay results clearly showed significantly higher cellular DNA breakage in lymphocytes treated with L-DOPA and Cu(II) as compared to those that were treated with L-DOPA alone. However, such DNA degradation was inhibited to a significant extent by scavengers of ROS and neocuproine, a membrane permeable Cu(I)-specific sequestering agent. These findings demonstrate that L-DOPA exhibits a pro-oxidant activity in the presence of copper ions.

  17. Molecular cloning of the cDNA and chromosomal localization of the gene for a putative seven-transmembrane segment (7-TMS) receptor isolated from human spleen

    Energy Technology Data Exchange (ETDEWEB)

    Federsppiel, B.; Melhado, I.G.; Delaney, A.; Clark-Lewis, I. (Univ. of British Columbia, Vancouver (Canada)); Duncan, A.M.V. (Queens Univ., Kinston, Ontario (Canada)); Jirik, F.R. (Hospital for Sick Children, Toronto, Ontario (Canada))

    1993-06-01

    A family of proinflammatory cytokines sharing several structural features has been described and includes, for example, interleukin-8, monocyte chemoattractant protein-1, and melanocyte growth stimulatory activity. Recently, the receptors for interleukin-8 have been isolated and found to belong to the seven-transmembrane domain class of G protein-coupled receptors. As other members of this cytokine family likely interact with similar receptors, the polymerase chain reaction was employed to isolate related receptors from human peripheral blood adherent cells. Degenerate oligonucleotide primers based on the rabbit interleukin-8 receptor sequence were used. The corresponding full-length cDNA was isolated from a human spleen cDNA library. The predicted protein sequence of this clone, designated pBE1.3, was 93% identical to that of a cDNA isolated from bovine locus coeruleus, which apparently encodes a neuropeptide Y receptor, and also shows similarity with the interleukin-8 receptor and the human cytomegalovirus US28 sequences. The gene, designated D2S201E, was localized to human chromosome 2q21. By Northern blotting, transcripts hybridizing to this cDNA were present in a variety of tissues and cells, including those of hemopoietic origin. 32 refs., 5 figs.

  18. Nontargeted nucleotide analysis based on benzoylhistamine labeling-MALDI-TOF/TOF-MS: discovery of putative 6-oxo-thymine in DNA.

    Science.gov (United States)

    Wang, Poguang; Fisher, David; Rao, Anjana; Giese, Roger W

    2012-04-17

    A method for nontargeted analysis of modified nucleotides in DNA (and RNA) is reported based on labeling with benzoylhistamine (BH) followed by MALDI-MS. The method provides deoxynucleotide-specific detection, accurate measurement of molecular ions, high sensitivity, semiquantitation, and, to the extent studied to date, normalization of response within a factor of <3.

  19. Cu(II)-coumestrol interaction leads to ROS-mediated DNA damage and cell death: a putative mechanism for anticancer activity.

    Science.gov (United States)

    Zafar, Atif; Singh, Swarnendra; Naseem, Imrana

    2016-07-01

    Phytoestrogens have attracted considerable interest as natural alternatives to hormone replacement therapy and their potential as cancer therapeutic agents. Among phytoestrogens, coumestrol has shown multipharmacological properties such as antiinflammatory, neuroprotective, osteoblastic differentiation and anticancer. Though several studies have described anticancer effects of coumestrol, a clear underlying molecular mechanism has not been elucidated. Unlike normal cells, cancer cells contain elevated copper levels that play an integral role in angiogenesis. Copper is an important metal ion associated with the chromatin DNA, particularly with guanine. Thus, targeting copper in cancer cells can serve as effective anticancer strategy. Using human peripheral lymphocytes, we assessed lipid peroxidation, protein carbonylation, reactive oxygen species (ROS) generation, DNA damage and apoptosis by coumestrol in the presence of exogenously added Cu(II) in cells to simulate malignancy-like condition. Results showed that Cu(II)-coumestrol interaction leads to lipid peroxidation and protein carbonylation (markers of oxidative stress), DNA fragmentation and apoptosis in treated lymphocytes. Further, incubation of lymphocytes with ROS scavengers and membrane-permeant copper chelator, neocuproine, resulted in inhibition of DNA damage and apoptosis. This suggests that coumestrol engages in redox cycling of Cu(II) to generate ROS that leads to DNA fragmentation and apoptosis. In conclusion, this is the first report showing that coumestrol targets cellular copper to induce prooxidant death in malignant cells. We believe that such a prooxidant cytotoxic mechanism better explains the anticancer activity of coumestrol. These findings will provide significant insights into the development of new chemical molecules with better copper-chelating and prooxidant properties against cancer cells.

  20. Combining a Ru(II) "Building Block" and Rapid Screening Approach to Identify DNA Structure-Selective "Light Switch" Compounds.

    Science.gov (United States)

    Wachter, Erin; Moyá, Diego; Glazer, Edith C

    2017-02-13

    A chemically reactive Ru(II) "building block", able to undergo condensation reactions with substituted diamines, was utilized to create a small library of luminescent "light switch" dipyrido-[3,2-a:2',3'-c] phenazine (dppz) complexes. The impact of substituent identity, position, and the number of substituents on the light switch effect was investigated. An unbiased, parallel screening approach was used to evaluate the selectivity of the compounds for a variety of different biomolecules, including protein, nucleosides, single stranded DNA, duplex DNA, triplex DNA, and G-quadruplex DNA. Combining these two approaches allowed for the identification of hit molecules that showed different selectivities for biologically relevant DNA structures, particularly triplex and quadruplex DNA.

  1. Trichinella spiralis mtDNA: a nematode mitochondrial genome that encodes a putative ATP8 and normally structured tRNAS and has a gene arrangement relatable to those of coelomate metazoans.

    Science.gov (United States)

    Lavrov, D V; Brown, W M

    2001-01-01

    The complete mitochondrial DNA (mtDNA) of the nematode Trichinella spiralis has been amplified in four overlapping fragments and 16,656 bp of its sequence has been determined. This sequence contains the 37 genes typical of metazoan mtDNAs, including a putative atp8, which is absent from all other nematode mtDNAs examined. The genes are transcribed from both mtDNA strands and have an arrangement relatable to those of coelomate metazoans, but not to those of secernentean nematodes. All protein genes appear to initiate with ATN codons, typical for metazoans. Neither TTG nor GTT start codons, inferred for several genes of other nematodes, were found. The 22 T. spiralis tRNA genes fall into three categories: (i) those with the potential to form conventional "cloverleaf" secondary structures, (ii) those with TPsiC arm + variable arm replacement loops, and (iii) those with DHU-arm replacement loops. Mt-tRNA(R) has a 5'-UCG-3' anticodon, as in most other metazoans, instead of the very unusual 5'-ACG-3' present in the secernentean nematodes. The sequence also contains a large repeat region that is polymorphic in size at the population and/or individual level. PMID:11156984

  2. The Putative Metal Coordination Motif in the Endonuclease Domain of Human Parvovirus B19 NS1 Is Critical for NS1 Induced S Phase Arrest and DNA Damage

    Directory of Open Access Journals (Sweden)

    Violetta Kivovich, Leona Gilbert, Matti Vuento, Stanley J. Naides

    2012-01-01

    Full Text Available The non-structural proteins (NS of the parvovirus family are highly conserved multi-functional molecules that have been extensively characterized and shown to be integral to viral replication. Along with NTP-dependent helicase activity, these proteins carry within their sequences domains that allow them to bind DNA and act as nucleases in order to resolve the concatameric intermediates developed during viral replication. The parvovirus B19 NS1 protein contains sequence domains highly similar to those previously implicated in the above-described functions of NS proteins from adeno-associated virus (AAV, minute virus of mice (MVM and other non-human parvoviruses. Previous studies have shown that transient transfection of B19 NS1 into human liver carcinoma (HepG2 cells initiates the intrinsic apoptotic cascade, ultimately resulting in cell death. In an effort to elucidate the mechanism of mammalian cell demise in the presence of B19 NS1, we undertook a mutagenesis analysis of the protein's endonuclease domain. Our studies have shown that, unlike wild-type NS1, which induces an accumulation of DNA damage, S phase arrest and apoptosis in HepG2 cells, disruptions in the metal coordination motif of the B19 NS1 protein reduce its ability to induce DNA damage and to trigger S phase arrest and subsequent apoptosis. These studies support our hypothesis that, in the absence of replicating B19 genomes, NS1-induced host cell DNA damage is responsible for apoptotic cell death observed in parvoviral infection of non-permissive mammalian cells.

  3. The putative metal coordination motif in the endonuclease domain of human Parvovirus B19 NS1 is critical for NS1 induced S phase arrest and DNA damage.

    Science.gov (United States)

    Kivovich, Violetta; Gilbert, Leona; Vuento, Matti; Naides, Stanley J

    2012-01-01

    The non-structural proteins (NS) of the parvovirus family are highly conserved multi-functional molecules that have been extensively characterized and shown to be integral to viral replication. Along with NTP-dependent helicase activity, these proteins carry within their sequences domains that allow them to bind DNA and act as nucleases in order to resolve the concatameric intermediates developed during viral replication. The parvovirus B19 NS1 protein contains sequence domains highly similar to those previously implicated in the above-described functions of NS proteins from adeno-associated virus (AAV), minute virus of mice (MVM) and other non-human parvoviruses. Previous studies have shown that transient transfection of B19 NS1 into human liver carcinoma (HepG2) cells initiates the intrinsic apoptotic cascade, ultimately resulting in cell death. In an effort to elucidate the mechanism of mammalian cell demise in the presence of B19 NS1, we undertook a mutagenesis analysis of the protein's endonuclease domain. Our studies have shown that, unlike wild-type NS1, which induces an accumulation of DNA damage, S phase arrest and apoptosis in HepG2 cells, disruptions in the metal coordination motif of the B19 NS1 protein reduce its ability to induce DNA damage and to trigger S phase arrest and subsequent apoptosis. These studies support our hypothesis that, in the absence of replicating B19 genomes, NS1-induced host cell DNA damage is responsible for apoptotic cell death observed in parvoviral infection of non-permissive mammalian cells.

  4. New Evidences of Mitochondrial DNA Heteroplasmy by Putative Paternal Leakage between the Rock Partridge (Alectoris graeca) and the Chukar Partridge (Alectoris chukar)

    Science.gov (United States)

    Gandolfi, Andrea; Crestanello, Barbara; Fagotti, Anna; Simoncelli, Francesca; Chiesa, Stefania; Girardi, Matteo; Giovagnoli, Eleonora; Marangoni, Carla; Di Rosa, Ines

    2017-01-01

    The rock partridge, Alectoris graeca, is a polytypic species declining in Italy mostly due to anthropogenic causes, including the massive releases of the closely related allochthonous chukar partridge Alectoris chukar which produced the formation of hybrids. Molecular approaches are fundamental for the identification of evolutionary units in the perspective of conservation and management, and to correctly select individuals to be used in restocking campaigns. We analyzed a Cytochrome oxidase I (COI) fragment of contemporary and historical A. graeca and A. chukar samples, using duplicated analyses to confirm results and nuclear DNA microsatellites to exclude possible sample cross-contamination. In two contemporary specimens of A. graeca, collected from an anthropogenic hybrid zone, we found evidence of the presence of mtDNA heteroplasmy possibly associated to paternal leakage and suggesting hybridization with captive-bred exotic A. chukar. These results underline significant limitations in the reliability of mtDNA barcoding-based species identification and could have relevant evolutionary and ecological implications that should be accounted for when interpreting data aimed to support conservation actions. PMID:28114306

  5. Mitoxantrone and Analogues Bind and Stabilize i-Motif Forming DNA Sequences

    Science.gov (United States)

    Wright, Elisé P.; Day, Henry A.; Ibrahim, Ali M.; Kumar, Jeethendra; Boswell, Leo J. E.; Huguin, Camille; Stevenson, Clare E. M.; Pors, Klaus; Waller, Zoë A. E.

    2016-12-01

    There are hundreds of ligands which can interact with G-quadruplex DNA, yet very few which target i-motif. To appreciate an understanding between the dynamics between these structures and how they can be affected by intervention with small molecule ligands, more i-motif binding compounds are required. Herein we describe how the drug mitoxantrone can bind, induce folding of and stabilise i-motif forming DNA sequences, even at physiological pH. Additionally, mitoxantrone was found to bind i-motif forming sequences preferentially over double helical DNA. We also describe the stabilisation properties of analogues of mitoxantrone. This offers a new family of ligands with potential for use in experiments into the structure and function of i-motif forming DNA sequences.

  6. Cell-to-cell transformation in Escherichia coli: a novel type of natural transformation involving cell-derived DNA and a putative promoting pheromone.

    Directory of Open Access Journals (Sweden)

    Rika Etchuuya

    Full Text Available Escherichia coli is not assumed to be naturally transformable. However, several recent reports have shown that E. coli can express modest genetic competence in certain conditions that may arise in its environment. We have shown previously that spontaneous lateral transfer of non-conjugative plasmids occurs in a colony biofilm of mixed E. coli strains (a set of a donor strain harbouring a plasmid and a plasmid-free recipient strain. In this study, with high-frequency combinations of strains and a plasmid, we constructed the same lateral plasmid transfer system in liquid culture. Using this system, we demonstrated that this lateral plasmid transfer was DNase-sensitive, indicating that it is a kind of transformation in which DNase-accessible extracellular naked DNA is essential. However, this transformation did not occur with purified plasmid DNA and required a direct supply of plasmid from co-existing donor cells. Based on this feature, we have termed this transformation type as 'cell-to-cell transformation'. Analyses using medium conditioned with the high-frequency strain revealed that this strain released a certain factor(s that promoted cell-to-cell transformation and arrested growth of the other strains. This factor is heat-labile and protease-sensitive, and its roughly estimated molecular mass was between ∼9 kDa and ∼30 kDa, indicating that it is a polypeptide factor. Interestingly, this factor was effective even when the conditioned medium was diluted 10(-5-10(-6, suggesting that it acts like a pheromone with high bioactivity. Based on these results, we propose that cell-to-cell transformation is a novel natural transformation mechanism in E. coli that requires cell-derived DNA and is promoted by a peptide pheromone. This is the first evidence that suggests the existence of a peptide pheromone-regulated transformation mechanism in E. coli and in Gram-negative bacteria.

  7. Redox cycling of endogenous copper by ferulic acid leads to cellular DNA breakage and consequent cell death: A putative cancer chemotherapy mechanism.

    Science.gov (United States)

    Sarwar, Tarique; Zafaryab, Md; Husain, Mohammed Amir; Ishqi, Hassan Mubarak; Rehman, Sayeed Ur; Rizvi, M Moshahid Alam; Tabish, Mohammad

    2015-12-01

    Ferulic acid (FA) is a plant polyphenol showing diverse therapeutic effects against cancer, diabetes, cardiovascular and neurodegenerative diseases. FA is a known antioxidant at lower concentrations, however at higher concentrations or in the presence of metal ions such as copper, it may act as a pro-oxidant. It has been reported that copper levels are significantly raised in different malignancies. Cancer cells are under increased oxidative stress as compared to normal cells. Certain therapeutic substances like polyphenols can further increase this oxidative stress and kill cancer cells without affecting the proliferation of normal cells. Through various in vitro experiments we have shown that the pro-oxidant properties of FA are enhanced in the presence of copper. Comet assay demonstrated the ability of FA to cause oxidative DNA breakage in human peripheral lymphocytes which was ameliorated by specific copper-chelating agent such as neocuproine and scavengers of ROS. This suggested the mobilization of endogenous copper in ROS generation and consequent DNA damage. These results were further validated through cytotoxicity experiments involving different cell lines. Thus, we conclude that such a pro-oxidant mechanism involving endogenous copper better explains the anticancer activities of FA. This would be an alternate non-enzymatic, and copper-mediated pathway for the cytotoxic activities of FA where it can selectively target cancer cells with elevated levels of copper and ROS.

  8. Tetrahelical structural family adopted by AGCGA-rich regulatory DNA regions

    Science.gov (United States)

    Kocman, Vojč; Plavec, Janez

    2017-05-01

    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.

  9. "Use of Random Amplified Polymorphic DNA Polymerase Chain Reaction (RAPD-PCR and ITS2 PCR assays for differentiation of populations and putative sibling species of Anopheles fluviatilis (Diptera: Culicidae in Iran"

    Directory of Open Access Journals (Sweden)

    SR Naddaf Dezfouli

    2002-09-01

    Full Text Available Anopheles fluviatilis complex is known to be a vector of malaria in Iran. Since mosquitoes of this species cover a wide geographical range in Iran, they might have evolved into different separated populations. Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR assay was used to differentiate geographic populations of this species. DNA was extracted from individual mosquitoes from 8 localities in 4 south and southeast provinces and amplified in PCR reactions using 18 single primers of arbitrary nucleotide sequence. Results of RAPD-PCR showed that Kazeroun populations could simply be differentiated from other populations using a diagnostic fragment amplified with primer UBC-306. But other populations could not be differentiated either visually or by means of statistical analysis. Moreover ITS2 fragments of some selected specimens were amplified using a pair of universal primer and sequenced as a key standard for detection of putative sibling species. Sequence analysis of the ITS2 fragments revealed a very high (100% homology among the populations. These findings are crucial in epidemiological studies concerning relatedness of geographic populations and vector movement in the region. Results of RAPD-PCR and ITS2 analysis suggest that this taxon in Iran comprises of only one species with a low genetic variation among geographic populations.

  10. Construction and analysis of antennal cDNA library from rice striped stem borer, Chilo suppressalis (Walker) (Lepidoptera: Pyralidae), and expression profiles of putative odorant-binding protein and chemosensory protein genes.

    Science.gov (United States)

    Gong, Zhong-Jun; Liu, Su; Jiang, Yan-Dong; Zhou, Wen-Wu; Liang, Qing-Mei; Cheng, Jiaan; Zhang, Chuan-Xi; Zhu, Zeng-Rong; Gurr, Geoff M

    2015-05-01

    In this study, we constructed a high-quality cDNA library from the antennae of the Chilo suppressalis (Walker) (Lepidoptera: Pyralidae). A total of 1,235 colonies with inserts greater than 0.7 kb were sequenced and analyzed. Homology searching coupled with bioinformatics analysis identified 15 and 7 cDNA sequences, respectively, encoding putative odorant-binding proteins (OBPs) and chemosensory proteins (CSPs). A phylogenetic tree of CsupCSPs showed that each CsupCSP has orthologs in Manduca sexta and Bombyx mori with strong bootstrapping support. One CSP was either very specific or more related to the CSPs of another species than to conspecific CSP. The expression profiles of the OBPs and CSPs in different tissues were measured by real-time quantitative PCR. The results revealed that of the 11 OBP genes, the transcript levels of CsupOBP1, CsupOBP5, and CsupOBP7 were higher in both male and female antennae than those in other tissues. And CsupCSP7 was highly expressed in both male and female antennae. Based on these results, the possible physiological functions of CsupOBPs and CsupCSPs were discussed.

  11. Identification of a putative methylenetetrahydrofolate reductase by sequence analysis of a 6.8 kb DNA fragment of yeast chromosome VII.

    Science.gov (United States)

    Tizon, B; Rodríguez-Torres, M; Rodríguez-Belmonte, E; Cadahia, J L; Cerdan, E

    1996-09-01

    We report the sequence analysis of a 6.8 kb DNA fragment from Saccharomyces cerevisiae chromosome VII. This sequence contains five open reading frames (ORFs) greater than 100 amino acids. There is also an incomplete ORF flanking one of the extremes, G2868, which is the 3' end of the SCS3 gene (Hosaka et al., 1994). The translated sequence of ORF G2882 shows similarity to the human methylenetetrahydrofolate reductase (Goyette et al., 1994). ORF G2889 shows no significant homologies with the sequences compiled in databases. ORF G2893 corresponds to the gene SUP44, coding for the yeast ribosomal protein S4 (All-Robin et al., 1990). G2873 and G2896 are internal ORFs.

  12. Design and synthesis of new benzimidazole-carbazole conjugates for the stabilization of human telomeric DNA, telomerase inhibition, and their selective action on cancer cells.

    Science.gov (United States)

    Maji, Basudeb; Kumar, Krishan; Kaulage, Mangesh; Muniyappa, K; Bhattacharya, Santanu

    2014-08-28

    Cell-permeable small molecules that enhance the stability of the G-quadruplex (G4) DNA structures are currently among the most intensively pursued ligands for inhibition of the telomerase activity. Herein we report the design and syntheses of four novel benzimidazole-carbazole conjugates and demonstrate their high binding affinity to G4 DNA. S1 nuclease assay confirmed the ligand mediated G-quadruplex DNA protection. Additional evidence from Telomeric Repeat Amplification Protocol (TRAP-LIG) assay demonstrated efficient telomerase inhibition activity by the ligands. Two of the ligands showed IC50 values in the sub-micromolar range in the TRAP-LIG assay, which are the best among the benzimidazole derivatives reported so far. The ligands also exhibited cancer cell selective nuclear internalization, nuclear condensation, fragmentation, and eventually antiproliferative activity in long-term cell viability assays. Annexin V-FITC/PI staining assays confirm that the cell death induced by the ligands follows an apoptotic pathway. An insight into the mode of ligand binding was obtained from the molecular dynamics simulations.

  13. Dinuclear ruthenium complexes display loop isomer selectivity to c-MYC DNA G-quadriplex and exhibit anti-tumour activity.

    Science.gov (United States)

    Zheng, Chuping; Liu, Yanan; Liu, Ying; Qin, Xiuying; Zhou, Yanhui; Liu, Jie

    2016-03-01

    G-quadruplex DNA, especially the cellular-myelocytomatosis viral oncogene (c-MYC) is closely associated with cell-cycle regulation, proliferation of tumour cells. In this work, the interaction between the c-MYC and two dinuclear Ru(II) complexes [(bpy)2Ru(bpibp)Ru(bpy)2](ClO4)4 (compound 1) and [(phen)2Ru(bpibp)Ru(phen)2](ClO4)4 (compound 2) have been studied. The data from UV-Visible, PCR-stop and Fluorescence resonance energy transfer (FRET) showed that two complexes can stabilize the structure of G-quadruplex in the c-MYC promoter and targeting the G-quadruplex loop isomers. Interestingly, the complex 2 has a greater effect on the 1:2:1 and 2:1:1 loop isomers while the 1 prefers to the 1:2:1 isomers. The mechanism studies revealed that complexes can induce apoptosis in HepG2 cells by generating ROS metabolites, triggering mitochondrial membrane potential loss and down-regulation of P-Akt (Akt also known as protein kinase B), P-p44/42 MAP kinase protein (P-p44/42), and c-MYC. Taken together, these results suggested that the two dinuclear complexes may both be candidates as anti-tumour agents as they may reduce the c-MYC gene expression. {bpibp: 4, 4'-bis (1, 10-phenanthroline-[5, 6-d] imidazole-2-yl)-biphenyl, bpy: 2,2-bipyridine, phen: 1,10-phenanthroline}. Copyright © 2016. Published by Elsevier Inc.

  14. Cloning of a cDNA encoding a putative human very low density lipoprotein/Apolipoprotein E receptor and assignment of the gene to chromosome 9pter-p23[sup 6

    Energy Technology Data Exchange (ETDEWEB)

    Gafvels, M.E.; Strauss, J.F. III (Univ. of Pennyslvania, Philadelphia, PA (United States)); Caird, M.; Patterson, D. (Eleanor Roosevelt Institute, Denver, CO (United States)); Britt, D.; Jackson, C.L. (Brown Univ., Providence, RI (United States))

    1993-11-01

    The authors report the cloning of a 3656-bp cDNA encoding a putative human very low density lipoprotein (VLDL)/apolipoprotein E (ApoE) receptor. The gene encoding this protein was mapped to chromosome 9pter-p23. Northern analysis of human RNA identified cognate mRNAs of 6.0 and 3.8 kb with most abundant expression in heart and skeletal muscle, followed by kidney, placenta, pancreas, and brain. The pattern of expression generally paralleled that of lipoprotein lipase mRNA but differed from that of the low density lipoprotein (LDL) receptor and the low density lipoprotein receptor-related protein/[alpha][sub 2]-macroglobulin receptor (LRP), which are members of the same gene family. VLDL/ApoE receptor message was not detected in liver, whereas mRNAs for both LDL receptor and LRP were found in hepatic tissue. In mouse 3T3-L1 cells, VLDL/ApoE receptor mRNA was induced during the transformation of the cells into adipocytes. Expression was also detected in human choriocarcinoma cells, suggesting that at least part of the expression observed in placenta may be in trophoblasts, cells which would be exposed to maternal blood. Expression in brain may be related to high levels of ApoE expression in that organ, an observation of potential relevance to the recently hypothesized role for ApoE in late onset Alzheimer disease. The results suggest that the putative VLDL/ApoE receptor could play a role in the uptake of triglyceride-rich lipoprotein particles by specific organs including striated and cardiac muscle and adipose tissue and in the transport of maternal lipids across the placenta. The findings presented here, together with recent observations from other laboratories, bring up the possibility that a single gene, the VLDL/ApoE receptor, may play a role in the pathogenesis of certain forms of atherosclerosis, Alzheimer disease, and obesity.

  15. cDNA cloning, tissue distribution, and chromosomal localization of Ocp2, a gene encoding a putative transcription-associated factor predominantly expressed in the auditory organs

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Hong; Thalmann, I.; Thalmann, R. [Washington Univ., St. Louis, MO (United States)] [and others

    1995-06-10

    We report the cloning of the Ocp2 gene encoding OCP-II from a guinea pig organ-of-Corti cDNA library. The predicted open reading frame encodes a protein of 163 amino acids with an estimated molecular mass of 18.6 kDa. A homology search revealed that Ocp2 shares significant sequence similarity with p15, a sub-unit of transcription factor SIII that regulates the activity of the RNA polymerase II elongation complex. The Ocp2 messenger RNA is expressed abundantly in the cochlea while not significantly in any other tissues examined, including brain, eye, heart, intestine, kidney, liver, lung, thigh muscle, and testis, demonstrating that the expression of this gene may be restricted to auditory organs. A polyclonal antiserum was raised against the N-terminal region of OCP-II. Immunohistochemical staining of paraffin-embedded sections of the cochlea showed that OCP-II is localized abundantly in nonsensory cells in the organ of Corti; in addition, it was also detected, at a lower concentration, in vestibular sensory organs, as well as auditory and vestibular brain stem nuclei. The Ocp2 gene was mapped to mouse chromosome 4 as well as 11. Our results suggest that OCP-II may be involved in transcription regulation for the development or maintenance of specialized functions of the inner ear. 40 refs., 5 figs.

  16. 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

    2014-02-01

    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.

  17. Ultrafast Photodynamics in Diverse DNA Structures from A-tracts to Z-DNA

    Science.gov (United States)

    Kohler, Bern

    2009-03-01

    The vulnerability of the genome to UV photodamage has sustained interest in excited electronic states in DNA for over 50 years. Progress in understanding the nature and dynamics of electronic excitations in DNA has accelerated rapidly thanks in part to ultrafast spectroscopy. Most excitations in single DNA bases decay nonradiatively in hundreds of femtoseconds. Surprisingly, much longer-lived excited states are observed in femtosecond pump-probe experiments on single- and double-stranded DNAs. Localized charge transfer states are prominent in runs of adenine bases (A tracts). DNA is polymorphic and can adopt a range of structures beyond the iconic B-form double helix. The effect of helix conformation on excited-state dynamics has been studied in a double-stranded oligonucleotide that can be switched between B- and Z-forms. Experiments on G quadruplex structures and on i-motif DNA reveal that these forms have significantly slower relaxation than B-DNA. By altering π-π stacking and hydrogen bonding, structure profoundly affects the complex photoprocesses observed in DNA.

  18. A novel mutation in the putative DNA helicase XH2 is responsible for male-to-female sex reversal associated with an atypical form of the ATR-X syndrome

    Energy Technology Data Exchange (ETDEWEB)

    Ion, A.; Telvi, L.; Galacteros, F.; McElreavey, K. [Institut Pasteur, Paris (France)] [and others

    1996-06-01

    We describe a pedigree presenting X-linked severe mental retardation associated with multiple congenital abnormalities and 46,XY gonadal dysgenesis, leading in one family member to female gender assignment. Female carriers are unaffected. The dysmorphic features are similar to those described in the {alpha}-thalassemia and mental retardation (ATR-X) syndrome, although there is no clinical evidence of {alpha}-thalassemia in this family. In addition, the family had other clinical features not previously observed in the ATR-X syndrome, including partial optic-nerve atrophy and partial ocular albinism. Mutations in a putative DNA helicase, termed XH2, have been reported to give rise to the ATR-X syndrome. We screened the YCH2 gene for mutations in affected members of the family and identified a 4-bp deletion at an intron/exon boundary that removes an invariant 3{prime} splice-acceptor site. The mutation cosegregates with the syndrome. The genomic deletion causes missplicing of the pre-mRNA, which results in the loss of 8 bp of coding sequence, thereby generating a frameshift and a downstream premature stop codon. Our finding increases the range of clinical features associated with mutations in the XH2 gene. 17 refs., 4 figs., 2 tabs.

  19. Synthesis and the interaction of 2-(1H-pyrazol-4-yl)-1H-imidazo[4,5-f][1,10]phenanthrolines with telomeric DNA as lung cancer inhibitors.

    Science.gov (United States)

    Liu, Jiachun; Chen, Mei; Wang, Yanli; Zhao, Xiaoyin; Wang, Sijia; Wu, Yanling; Zhang, Wen

    2017-06-16

    A novel series of 2-(1H-pyrazol-4-yl)-1H-imidazo[4,5-f][1,10]phenanthrolines were designed, synthesized and evaluated for their antitumor activity against lung adenocarcinoma by CCK-8 assay, electrophoretic mobility shift assay (EMSA), UV-melting study, wound healing assay and docking study. These compounds showed good inhibitory activities against lung adenocarcinoma. Especially compound 12c exhibited potential antiproliferative activity against A549 cell line with the half maximal inhibitory concentration (IC50) value of 1.48 μM, which was a more potent inhibitor than cisplatin (IC50 = 12.08 μM) and leading compound 2 (IC50 = 1.69 μM), and the maximum cell inhibitory rate being up to 98.40%. Moreover, further experiments demonstrated that compounds 12a-d can strongly interact with telomeric DNA to stabilize G-quadruplex DNA with increased ΔTm values from 12.44 to 20.54 °C at a ratio of DNA to compound 1:10. These results implied that growth inhibition of A549 cells mediated by these phenanthroline derivatives is possibly positively correlated to the fact their interaction with telomeric G-quadruplexs. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  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

    2015-05-01

    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. High-resolution profiling of Drosophila replication start sites reveals a DNA shape and chromatin signature of metazoan origins.

    Science.gov (United States)

    Comoglio, Federico; Schlumpf, Tommy; Schmid, Virginia; Rohs, Remo; Beisel, Christian; Paro, Renato

    2015-05-05

    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.

  2. Synthesis and spectroscopic studies of the aminoglycoside (neomycin)--perylene conjugate binding to human telomeric DNA.

    Science.gov (United States)

    Xue, Liang; Ranjan, Nihar; Arya, Dev P

    2011-04-12

    Synthesis of a novel perylene-neomycin conjugate (3) and the properties of its binding to human telomeric G-quadruplex DNA, 5'-d[AG3(T2AG3)3] (4), are reported. Various spectroscopic techniques were employed to characterize the binding of conjugate 3 to 4. A competition dialysis assay revealed that 3 preferentially binds to 4, in the presence of other nucleic acids, including DNA, RNA, DNA-RNA hybrids, and other higher-order structures (single strands, duplexes, triplexes, other G-quadruplexes, and the i-motif). UV thermal denaturation studies showed that thermal stabilization of 4 increases as a function of the increasing concentration of 3. The fluorescence intercalator displacement (FID) assay displayed a significantly tighter binding of 3 with 4 as compared to its parent constituents [220-fold stronger than neomycin (1) and 4.5-fold stronger than perylene diamine (2), respectively]. The binding of 3 with 4 resulted in pronounced changes in the molar ellipticity of the DNA absorption region as confirmed by circular dichroism. The UV-vis absorption studies of the binding of 3 to 4 resulted in a red shift in the spectrum of 3 as well as a marked hypochromic change in the perylene absorption region, suggesting that the ligand-quadruplex interaction involves stacking of the perylene moiety. Docking studies suggest that the perylene moiety serves as a bridge that end stacks on 4, making contacts with two thymine bases in the loop, while the two neomycin moieties branch into the grooves of 4.

  3. A spectroscopic investigation of the interaction between c-MYC DNA and tetrapyridinoporphyrazinatozinc(II).

    Science.gov (United States)

    Hassani, Leila; Fazeli, Zahra; Safaei, Elham; Rastegar, Hossein; Akbari, Minoo

    2014-06-01

    The c-MYC gene plays an important role in the regulation of cell proliferation and growth and it is overexpressed in a wide variety of human cancers. Around 90% of c-MYC transcription is controlled by the nuclease-hypersensitive element III1 (NHE III1), whose 27-nt purine-rich strand has the ability to form a G-quadruplex structure under physiological conditions. Therefore, c-MYC DNA is an attractive target for drug design, especially for cancer chemotherapy. Here, the interaction of water-soluble tetrapyridinoporphyrazinatozinc(II) with 27-nt G-rich strand (G/c-MYC), its equimolar mixture with the complementary sequence (GC/c-MYC) and related C-rich oligonucleotide (C/c-MYC) is investigated. Circular dichroism (CD) measurements of the G-rich 27-mer oligonucleotide in 150 mM KCl, pH 7 demonstrate a spectral signature consistent with parallel G-quadruplex DNA. Furthermore, the CD spectrum of the GC rich oligonucleotide shows characteristics of both duplex and quadruplex structures. Absorption spectroscopy implies that the complex binding of G/c-MYC and GC/c-MYC is a two-step process; in the first step, a very small red shift and hypochromicity and in the second step, a large red shift and hyperchromicity are observed in the Q band. Emission spectra of zinc porphyrazine are quenched upon addition of three types of DNA. According to the results of spectroscopy, it can be concluded the dominant binding mode is probably, outside binding and end stacking.

  4. Self-Assembly of G-Rich Oligonucleotides Incorporating a 3'-3' Inversion of Polarity Site: A New Route Towards G-Wire DNA Nanostructures.

    Science.gov (United States)

    Oliviero, Giorgia; D'Errico, Stefano; Pinto, Brunella; Nici, Fabrizia; Dardano, Principia; Rea, Ilaria; De Stefano, Luca; Mayol, Luciano; Piccialli, Gennaro; Borbone, Nicola

    2017-08-01

    Obtaining DNA nanostructures with potential applications in drug discovery, diagnostics, and electronics in a simple and affordable way represents one of the hottest topics in nanotechnological and medical sciences. Herein, we report a novel strategy to obtain structurally homogeneous DNA G-wire nanostructures of known length, starting from the short unmodified G-rich oligonucleotide d(5'-CGGT-3'-3'-GGC-5') (1) incorporating a 3'-3' inversion of polarity site. The reported approach allowed us to obtain long G-wire assemblies through 5'-5' π-π stacking interactions in between the tetramolecular G-quadruplex building blocks that form when 1 is annealed in the presence of potassium ions. Our results expand the repertoire of synthetic methodologies to obtain new tailored DNA G-wire nanostructures.

  5. Site-specific control of N7-metal coordination in DNA by a fluorescent purine derivative.

    Science.gov (United States)

    Dumas, Anaëlle; Luedtke, Nathan W

    2012-01-01

    A synthetic strategy that utilizes O6-protected 8-bromoguanosine gives broad access to C8-guanine derivatives with phenyl, pyridine, thiophene, and furan substituents. The resulting 8-substituted 2'-deoxyguanosines are push-pull fluorophores that can exhibit environmentally sensitive quantum yields (Φ=0.001-0.72) due to excited-state proton-transfer reactions with bulk solvent. Changes in nucleoside fluorescence were used to characterize metal-binding affinity and specificity of 8-substituted 2'-deoxyguanosines. One derivative, 8-(2-pyridyl)-2'-deoxyguanosine (2PyG), exhibits selective binding of Cu(II), Ni(II), Cd(II), and Zn(II) through a bidentate effect provided by the N7 position of guanine and the 2-pyridyl nitrogen atom. Upon incorporation into DNA, 2-pyridine-modified guanine residues selectively bind to Cu(II) and Ni(II) with equilibrium dissociation constants (K(d)) that range from 25 to 850 nM; the affinities depend on the folded state of the oligonucleotide (duplex>G-quadruplex) as well as the identity of the metal ion (Cu>Ni≫Cd). These binding affinities are approximately 10 to 1 000 times higher than for unmodified metal binding sites in DNA, thereby providing site-specific control of metal localization in alternatively folded nucleic acids. Temperature-dependent circular-dichroism studies reveal metal-dependent stabilization of duplexes, but destabilization of G-quadruplex structures upon adding Cu(II) to 2PyG-modified oligonucleotides. These results demonstrate how the addition of a single pyridine group to the C8 position of guanine provides a powerful new tool for studying the effects of N7 metalation on the structure, stability, and electronic properties of nucleic acids.

  6. A double chain reversal loop and two diagonal loops define the architecture of a unimolecular DNA quadruplex containing a pair of stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads flanked by a G-(T-T) Triad and a T-T-T triple.

    Science.gov (United States)

    Kuryavyi, V; Majumdar, A; Shallop, A; Chernichenko, N; Skripkin, E; Jones, R; Patel, D J

    2001-06-29

    The architecture of G-G-G-G tetrad-aligned DNA quadruplexes in monovalent cation solution is dependent on the directionality of the four strands, which in turn are defined by loop connectivities and the guanine syn/anti distribution along individual strands and within individual G-G-G-G tetrads. The smallest unimolecular G-quadruplex belongs to the d(G2NnG2NnG2NnG2) family, which has the potential to form two stacked G-tetrads linked by Nn loop connectivities. Previous studies have focused on the thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2), where Nn was T2 for the first and third connecting loops and TGT for the middle connecting loop. This DNA aptamer in K(+) cation solution forms a unimolecular G-quadruplex stabilized by two stacked G(syn)-G(anti)-G(syn)-G(anti) tetrads, adjacent strands which are antiparallel to each other and edge-wise connecting T2, TGT and T2 loops. We now report on the NMR-based solution structure of the d(G2T4G2CAG2GT4G2T) sequence, which differs from the thrombin-binding DNA aptamer sequence in having longer first (T4) and third (GT4) loops and a shorter (CA) middle loop. This d(G2T4G2CAG2GT4G2T) sequence in Na(+) cation solution forms a unimolecular G-quadruplex stabilized by two stacked G(syn)-G(syn)-G(anti)-G(anti) tetrads, adjacent strands which have one parallel and one antiparallel neighbors and distinct non-edge-wise loop connectivities. Specifically, the longer first (T4) and third (GT4) loops are of the diagonal type while the shorter middle loop is of the double chain reversal type. In addition, the pair of stacked G-G-G-G tetrads are flanked on one side by a G-(T-T) triad and on the other side by a T-T-T triple. The distinct differences in strand directionalities, loop connectivities and syn/anti distribution within G-G-G-G tetrads between the thrombin-binding DNA aptamer d(G2T2G2TGTG2T2G2) quadruplex reported previously, and the d(G2T4G2CAG2GT4G2T) quadruplex reported here, reinforces the polymorphic nature of higher

  7. DNA origami nanorobot fiber optic genosensor to TMV.

    Science.gov (United States)

    Torelli, Emanuela; Manzano, Marisa; Srivastava, Sachin K; Marks, Robert S

    2018-01-15

    In the quest of greater sensitivity and specificity of diagnostic systems, one continually searches for alternative DNA hybridization methods, enabling greater versatility and where possible field-enabled detection of target analytes. We present, herein, a hybrid molecular self-assembled scaffolded DNA origami entity, intimately immobilized via capture probes linked to aminopropyltriethoxysilane, onto a glass optical fiber end-face transducer, thus producing a novel biosensor. Immobilized DNA nanorobots with a switchable flap can then be actuated by a specific target DNA present in a sample, by exposing a hemin/G-quadruplex DNAzyme, which then catalyzes the generation of chemiluminescence, once the specific fiber probes are immersed in a luminol-based solution. Integrating organic nanorobots to inorganic fiber optics creates a hybrid system that we demonstrate as a proof-of-principle can be utilized in specific DNA sequence detection. This system has potential applications in a wide range of fields, including point-of-care diagnostics or cellular in vivo biosensing when using ultrathin fiber optic probes for research purposes. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Molecular and cellular functions of the FANCJ DNA helicase defective in cancer and in Fanconi Anemia

    Directory of Open Access Journals (Sweden)

    Robert M. Brosh

    2014-10-01

    Full Text Available The FANCJ DNA helicase is mutated in hereditary breast and ovarian cancer as well as the progressive bone marrow failure disorder Fanconi anemia (FA. FANCJ is linked to cancer suppression and DNA double strand break (DSB repair through its direct interaction with the hereditary breast cancer associated gene product, BRCA1. FANCJ also operates in the FA pathway of interstrand cross-link (ICL repair and contributes to homologous recombination (HR. FANCJ collaborates with a number of DNA metabolizing proteins implicated in DNA damage detection and repair, and plays an important role in cell cycle checkpoint control. In addition to its role in the classical FA pathway, FANCJ is believed to have other functions that are centered on alleviating replication stress. FANCJ resolves G-quadruplex (G4 DNA structures that are known to affect cellular replication and transcription, and potentially plays a role in the preservation and functionality of chromosomal structures such as telomeres. Recent studies suggest that FANCJ helps to maintain chromatin structure and preserve epigenetic stability by facilitating smooth progression of the replication fork when it encounters DNA damage or an alternate DNA structure such as a G4. Ongoing studies suggest a prominent but still not well-understood role of FANCJ in transcriptional regulation, chromosomal structure and function, and DNA damage repair to maintain genomic stability. This review will synthesize our current understanding of the molecular and cellular functions of FANCJ that are critical for chromosomal integrity.

  9. Cloning of partial putative gonadotropin hormone receptor sequence from fish

    Indian Academy of Sciences (India)

    G Kumaresan; T Venugopal; A Vikas; T J Pandian; S M Athavan

    2000-03-01

    A search for the presence of mariner-like elements in the Labeo rohita genome by polymerase chain reaction led to the amplification of a partial DNA sequence coding for a putative transmembrane domain of gonadotropin hormone receptor. The amplified DNA sequence shows a high degree of homology to the available turkey and human luteinizing and follicle stimulating hormone receptor coding sequences. This is the first report on cloning such sequences of piscine origin.

  10. DNA

    Science.gov (United States)

    Stent, Gunther S.

    1970-01-01

    This history for molecular genetics and its explanation of DNA begins with an analysis of the Golden Jubilee essay papers, 1955. The paper ends stating that the higher nervous system is the one major frontier of biological inquiry which still offers some romance of research. (Author/VW)

  11. Label-free molecular beacon for real-time monitoring of DNA polymerase activity.

    Science.gov (United States)

    Ma, Changbei; Liu, Haisheng; Wang, Jun; Jin, Shunxin; Wang, Kemin

    2016-05-01

    Traditional methods for assaying DNA polymerase activity are discontinuous, time consuming, and laborious. Here, we report a new approach for label-free and real-time monitoring of DNA polymerase activity using a Thioflavin T (ThT) probe. In the presence of DNA polymerase, the DNA primer could be elongated through polymerase reaction to open MB1, leading to the release of the G-quartets. These then bind to ThT to form ThT/G-quadruplexes with an obvious fluorescence generation. It exhibits a satisfying detection result for the activity of DNA polymerase with a low detection limit of 0.05 unit/ml. In addition, no labeling with a fluorophore or a fluorophore-quencher pair is required; this method is fairly simple, fast, and low cost. Furthermore, the proposed method was also applied to assay the inhibition of DNA polymerase activity. This approach may offer potential applications in drug screening, clinical diagnostics, and some other related biomedical research.

  12. 人体端粒G4-D NA稳定剂的研究进展%Study of human telomere G4-DNA stabilizers

    Institute of Scientific and Technical Information of China (English)

    郑小辉; 刘海英; 夏立新; 毛宗万

    2016-01-01

    Telomere maintenance plays a critical role in cancer progression.Approximately 85% human cancer cells maintain their telomere length through activation of telomerase.Other 15%of cancers maintain telomere length independently of telom-erase by alternative lengthening of telomeres (ALT)pathway. Both events are equally important for telomere length mainte-nance of cancer cells.Human telomere consists of a series of G rich DNA sequences,which could form G-quadruplex.The for-mation of this structure can block the extension of telomeres by telomerase or ALT,resulting in cancer cell death.Thereby,G-quadruplex has been one of the focuses of anticancer therapy in recent years.This review focuses on the latest progress of G-quadruplex stabilizers.%端粒长度的维持在肿瘤细胞的永生化过程中起到至关重要的作用。约85%的人体肿瘤细胞通过端粒酶延伸端粒,从而获得持续的增殖能力。另外,15%的人体肿瘤细胞通过端粒替代延伸机制(alternative lengthening of telomeres, ALT)延伸端粒。这两种机制对于维持肿瘤细胞中端粒的长度具有同等重要的意义。人体端粒由富含鸟嘌呤(G)的DNA重复序列组成,该序列在特定的条件下可以形成G-四链体(G4)的结构。此结构的形成可以从根本上抑制端粒酶和ALT对端粒的延伸而达到抗肿瘤的目的。因此,人体端粒G4-DNA作为抗肿瘤靶点的研究是近年来抗肿瘤研究的重要前沿领域之一。该文重点综述人体端粒G4-DNA稳定剂研发的最新研究进展。

  13. Development of a high-throughput G4-FID assay for screening and evaluation of small molecules binding quadruplex nucleic acid structures.

    Science.gov (United States)

    Largy, Eric; Hamon, Florian; Teulade-Fichou, Marie-Paule

    2011-07-01

    G4-FID (G-quadruplex fluorescent intercalator displacement) is a simple and fast method that allows to evaluate the affinity of a compound for G-quadruplex DNA and its selectivity towards duplex DNA. This assay is based on the loss of fluorescence of thiazole orange (TO) upon competitive displacement from DNA by a putative ligand. We describe here the development of a high-throughput version of this assay performed in 96-well microplates, and fully transposable to 384-well microplates. The test was calibrated with a set of G-quadruplex ligands characterized for their ability to bind quadruplex within a large range of affinity. The comparison of the results obtained in microplates and in cuvettes was conducted indicating a full agreement. Additionally, the spectral range of the test was enlarged using two other fluorescent on/off probes whose absorption are red-shifted (TO-PRO-3) and blue-shifted (Hoechst 33258) as compared to that of TO. These labels enable to screen a large diversity of compounds with various optical properties, which was exemplified by evaluation of affinity and selectivity of the porphyrin TMPyP4 that could not be evaluated previously. Altogether, our study demonstrates that the HT-G4-FID assay offers the possibility to label a large variety of G-quadruplexes of biological interest and should enable screening of collections of putative G4-ligands of high structural diversity. It thus represents a powerful tool to bring into light new ligands able to discriminate between quadruplexes of different structures.

  14. 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.

    2009-06-15

    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)

  15. Spectroscopic detection of DNA quadruplexes by vibrational circular dichroism.

    Science.gov (United States)

    Andrushchenko, Valery; Tsankov, Dimiter; Krasteva, Maria; Wieser, Helmut; Bour, Petr

    2011-09-28

    The four-stranded G-quadruplex motif is a conformation frequently adopted by guanine-rich nucleic acids that plays an important role in biology, medicine, and nanotechnology. Although vibrational spectroscopy has been widely used to investigate nucleic acid structure, association of particular spectral features with the quadruplex structure has to date been ambiguous. In this work, experimental IR absorption and vibrational circular dichroism (VCD) spectra of the model quadruplex systems d(G)(8) and deoxyguanosine-5'-monophosphate (5'-dGMP) were analyzed using molecular dynamics (MD) and quantum-chemical modeling. The experimental spectra were unambiguously assigned to the quadruplex DNA arrangement, and several IR and VCD bands related to this structural motif were determined. Involvement of MD in the modeling was essential for realistic simulation of the spectra. The VCD signal was found to be more sensitive to dynamical structural variations than the IR signal. The combination of the spectroscopic techniques with multiscale simulations provides extended information about nucleic acid conformations and their dynamics.

  16. A DNA origami nanorobot controlled by nucleic acid hybridization

    KAUST Repository

    Torelli, Emanuela

    2014-03-20

    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. Multitriggered Shape-Memory Acrylamide-DNA Hydrogels.

    Science.gov (United States)

    Lu, Chun-Hua; Guo, Weiwei; Hu, Yuwei; Qi, Xiu-Juan; Willner, Itamar

    2015-12-23

    Acrylamide-acrylamide nucleic acids are cross-linked by two cooperative functional motives to form shaped acrylamide-DNA hydrogels. One of the cross-linking motives responds to an external trigger, leading to the dissociation of one of the stimuli-responsive bridges, and to the transition of the stiff shaped hydrogels into soft shapeless states, where the residual bridging units, due to the chains entanglement, provide an intrinsic memory for the reshaping of the hydrogels. Subjecting the shapeless states to counter stimuli restores the dissociated bridges, and regenerates the original shape of the hydrogels. By the cyclic dissociation and reassembly of the stimuli-responsive bridges, the reversible switchable transitions of the hydrogels between stiff shaped hydrogel structures and soft shapeless states are demonstrated. Shaped hydrogels bridged by K(+)-stabilized G-quadruplexes/duplex units, by i-motif/duplex units, or by two different duplex bridges are described. The cyclic transitions of the hydrogels between shaped and shapeless states are stimulated, in the presence of appropriate triggers and counter triggers (K(+) ion/crown ether; pH = 5.0/8.0; fuel/antifuel strands). The shape-memory hydrogels are integrated into shaped two-hydrogel or three-hydrogel hybrid structures. The cyclic programmed transitions of selective domains of the hybrid structures between shaped hydrogel and shapeless states are demonstrated. The possible applications of the shape-memory hydrogels for sensing, inscription of information, and controlled release of loads are discussed.

  18. A DNA origami nanorobot controlled by nucleic acid hybridization.

    Science.gov (United States)

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

    2014-07-23

    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.

  19. TA1 oncofetal rat liver cDNA and putative amino acid permease: temporal correlation with c-myc during acute CCl4 liver injury and variation of RNA levels in response to amino acids in hepatocyte cultures.

    Science.gov (United States)

    Shultz, V D; Campbell, W; Karr, S; Hixson, D C; Thompson, N L

    1999-01-01

    TA1 is a rat liver oncofetal cDNA and a member of an emerging family of evolutionarily conserved molecules with homology to amino acid transporters and permeases. The aim of these studies was to characterize the regulation and role of TA1 in acute rat liver injury by examining its relation to regeneration and metabolic stress. Following a single dose of CCl4, TA1 message was expressed 3-48 h. The major 3.3-kb TA1 transcript correlated temporally with c-myc expression. A novel 2.9-kb TA1 transcript was expressed more variably 24-48 h. TA1 protein was restricted to hepatocytes in G0 and G1 phases of the cell cycle. Relative to CCl4, a much smaller increase in TA1 was noted after partial hepatectomy and TA1 preceded the peak of c-myc expression. In vitro TA1 was not induced in hepatocytes by EGF or the acute-phase cytokines IL-6 and TNF-alpha, but was found to be modulated in response to amino acid availability. TA1 expression increased in media without arginine and glutamine and was repressed by total amino acid levels 5-fold over basal MEM. Together, these results contrast with the constitutive expression observed in transformed cells and suggest an adaptive role for TA1 during liver injury.

  20. The 28S-18S rDNA intergenic spacer from Crithidia fasciculata: repeated sequences, length heterogeneity, putative processing sites and potential interactions between U3 small nucleolar RNA and the ribosomal RNA precursor.

    Science.gov (United States)

    Schnare, M N; Collings, J C; Spencer, D F; Gray, M W

    2000-09-15

    In Crithidia fasciculata, the ribosomal RNA (rRNA) gene repeats range in size from approximately 11 to 12 kb. This length heterogeneity is localized to a region of the intergenic spacer (IGS) that contains tandemly repeated copies of a 19mer sequence. The IGS also contains four copies of an approximately 55 nt repeat that has an internal inverted repeat and is also present in the IGS of Leishmania species. We have mapped the C.fasciculata transcription initiation site as well as two other reverse transcriptase stop sites that may be analogous to the A0 and A' pre-rRNA processing sites within the 5' external transcribed spacer (ETS) of other eukaryotes. Features that could influence processing at these sites include two stretches of conserved primary sequence and three secondary structure elements present in the 5' ETS. We also characterized the C.fasciculata U3 snoRNA, which has the potential for base-pairing with pre-rRNA sequences. Finally, we demonstrate that biosynthesis of large subunit rRNA in both C. fasciculata and Trypanosoma brucei involves 3'-terminal addition of three A residues that are not present in the corresponding DNA sequences.

  1. An enhanced chemiluminescence resonance energy transfer system based on target recycling G-guadruplexes/hemin DNAzyme catalysis and its application in ultrasensitive detection of DNA.

    Science.gov (United States)

    Chen, Jia; Huang, Yong; Vdovenko, Marina; Sakharov, Ivan Yu; Su, Guifa; Zhao, Shulin

    2015-06-01

    An enhanced chemiluminescence resonance energy transfer (CRET) system based on target recycling G-guadruplexes/hemin DNAzyme catalysis was developed for ultrasensitive detection of DNA. CRET system consists of luminol as chemiluminescent donor, and fluorescein isothiocyanate (FITC) as acceptor. The sensitive detection was achieved by using the system consisted of G-riched DNA, blocker DNA, and the Nb.BbvCI biocatalyst. Upon addition of target DNA to the system, target DNA hybridizes with the quasi-circular DNA structure, and forms a DNA duplex. The formation of DNA duplex triggers selective enzymatic cleavage of quasi-circular DNA by Nb.BbvCI, resulting in the release of target DNA and two G-riched DNAzyme segments. Released target DNA then hybridizes with another quasi-circular DNA structure to initiate the cleavage of the quasi-circular DNA structure. Eventually, each target DNA can go through many cycles, resulting in the digestion of many quasi-circular DNA structures, generating many G-riched DNAzyme segments. G-riched DNAzyme segment products assemble with hemin to form stable hemin/G-quadruplexes that exhibit peroxidase-like activity which can catalyze the oxidation of luminol by H2O2 to produce CL signals. In the presence of FITC, CL of luminol can excite FITC molecules, and thus produced CRET between the luminol and FITC. This unique analysis strategy gives a detection limit down to 80 fM, which is at least four orders of magnitude lower than that of unamplified DNA detection methods.

  2. Single-molecule imaging of dynamic motions of biomolecules in DNA origami nanostructures using high-speed atomic force microscopy.

    Science.gov (United States)

    Endo, Masayuki; Sugiyama, Hiroshi

    2014-06-17

    direction of the reactions was controlled by introducing structural stress to the substrates. In addition, the movement of RNA polymerase and its reaction were visualized using a template dsDNA attached to the origami structure. To observe DNA structural changes, G-quadruplex formation and disruption, the switching behaviors of photoresponsive oligonucleotides, and B-Z transition were visualized using the DNA frame observation system. For the formation and disruption of G-quadruplex and double-helix DNA, the two dsDNA chains incorporated into the DNA frame could amplify the small structural change to the global structural change, which enabled the visualization of their association and dissociation by HS-AFM. The dynamic motion of the helical rotation induced by the B-Z transition was also directly imaged in the DNA frame. Furthermore, the stepwise motions of mobile DNA along the DNA track were visualized on the DNA origami surface. These target-orientated observation systems should contribute to the detailed analysis of biomolecule motions in real time and at molecular resolution.

  3. 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.

    Science.gov (United States)

    Tolmachov, Oleg E

    2012-05-01

    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

  4. Tropomyosin-1, A Putative Tumor-Suppressor and a Biomarker of Human Breast Cancer

    Science.gov (United States)

    2004-10-01

    cDNA. Lobular carcinoma - 2 A polyclonal pan-TM antibody that recognizes multiple TM Phyllodes tumor - 1 Not determined from the initial pathology...AD Award Number: DAMD17-98-1-8162 TITLE: Tropomyosin-1, A Putative Tumor -Suppressor and a Biomarker of Human Breast Cancer PRINCIPAL INVESTIGATOR...4. TITLE AND SUBTITLE 5. FUNDING NUMBERS Tropomyosin-l, A Putative Tumor -Suppressor and a Biomarker DAMD17-98-1-8162 of Human Breast Cancer 6. A UTHOR

  5. Structure and Stability of a Dimeric G-Quadruplex Formed by Cyclic Oligonucleotides

    Directory of Open Access Journals (Sweden)

    Joan Casals

    2010-01-01

    containing two copies of the human telomeric repeat. In the presence of sodium, NMR data are consistent with a dimeric structure of the molecule in which two cycles self-associate forming a quadruplex with three guanine tetrads connected by edgewise loops. The two macrocycles are arranged in a parallel way, and the dimeric structure exhibits a high melting temperature. These results indicate that cyclization of the phosphodiester chain does not prevent quadruplex formation, although it affects the global topology of the quadruplex.

  6. A putative helicase, the SUA5, PMR1, tRNALys1 genes and four open reading frames have been detected in the DNA sequence of an 8.8 kb fragment of the left arm of chromosome VII of Saccharomyces cerevisiae.

    Science.gov (United States)

    Klima, R; Coglievina, M; Zaccaria, P; Bertani, I; Bruschi, C V

    1996-09-01

    We report the sequence of an 8.8 kb segment of DNA from the left arm of chromosome VII of Saccharomyces cerevisiae. The sequence reveals seven open reading frames (ORFs) G1651, G1654, G1660, G1663, G1666, G1667 and G1669 greater than 100 amino acids in length and the tRNALys1 gene. ORF G1651 shows 100% identity with the ROK1 protein which is a putative RNA helicase of the 'DEAD box' protein family. ORF G1654 exhibits a motif highly conserved in ATP/GTP binding proteins generally referred to as 'P-loop'. From FastA analysis, G1660 and G1666 were found to be previously sequenced genes, respectively SUA5 and PMR1. The three other ORFs identified are partially (G1663) or completely (G1667 and G1669) overlapping with the PMR1 sequence on the complementary strand. This feature, together with their low codon adaptation indexes and the absence of significant homology with known proteins suggest that they do not correspond to real genes.

  7. Thermodynamic properties of water molecules in the presence of cosolute depend on DNA structure: a study using grid inhomogeneous solvation theory

    Science.gov (United States)

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

    2015-01-01

    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

  8. Arylsulfanyl groups - Suitable side chains for 5-substituted 1,10-phenanthroline and nickel complexes as G4 ligands and telomerase inhibitors.

    Science.gov (United States)

    Liu, Wanbo; Wang, Siwen; Dotsenko, Irina A; Samoshin, Vyacheslav V; Xue, Liang

    2017-08-01

    Guanine-rich DNA sequences can undergo self-assembly into unique G-quadruplex structures that interfere with the binding of proteins to the same DNA region. The formation of DNA G-quadruplexes requires monovalent cations (Na(+) and K(+)) or small molecules known as G-quadruplex (G4) ligands. Phenanthroline is a type of G4 ligand scaffold known for its coordination with metal ions to form complexes with a large aromatic surface area, which aptly stack with G-quartets. In this report, we have investigated the side chain effect on G-quadruplex recognition by evaluating a series of 5-substituted phenanthroline-based metal complexes (Phen-Ni) binding to telomeric G-quadruplex DNA. Results from biophysical methods including fluorescence and circular dichroism (CD) thermal denaturation, CD titration, and the fluorescent intercalator displacement (FID) assay suggest that several Phen-Ni complexes bind to G-quadruplex DNA with submicromolar (G4)DC50 values. Arylsulfanyl groups at the 5 position of 1,10-phenanthroline are the best side chains regarding binding affinity and selectivity towards G-quadruplex DNA. Most of the G-quadruplex binding Phen-Ni complexes can inhibit telomerase activity in vitro as indicated by the telomeric repeat amplification protocol (TRAP) assay and such inhibition is clearly concentration dependent. Our results here provide a guidance of utilizing 5-substituted phenanthroline derivatives as a viable and facile approach to design novel G4 ligands. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Shedding lights on the flexible-armed porphyrins: Human telomeric G4 DNA interaction and cell photocytotoxicity research.

    Science.gov (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

    2017-08-01

    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.

  10. DNA Replication Dynamics of the GGGGCC Repeat of the C9orf72 Gene.

    Science.gov (United States)

    Thys, Ryan Griffin; Wang, Yuh-Hwa

    2015-11-27

    DNA has the ability to form a variety of secondary structures in addition to the normal B-form DNA, including hairpins and quadruplexes. These structures are implicated in a number of neurological diseases and cancer. Expansion of a GGGGCC repeat located at C9orf72 is associated with familial amyotrophic lateral sclerosis and frontotemporal dementia. This repeat expands from two to 24 copies in normal individuals to several hundreds or thousands of repeats in individuals with the disease. Biochemical studies have demonstrated that as little as four repeats have the ability to form a stable DNA secondary structure known as a G-quadruplex. Quadruplex structures have the ability to disrupt normal DNA processes such as DNA replication and transcription. Here we examine the role of GGGGCC repeat length and orientation on DNA replication using an SV40 replication system in human cells. Replication through GGGGCC repeats leads to a decrease in overall replication efficiency and an increase in instability in a length-dependent manner. Both repeat expansions and contractions are observed, and replication orientation is found to influence the propensity for expansions or contractions. The presence of replication stress, such as low-dose aphidicolin, diminishes replication efficiency but has no effect on instability. Two-dimensional gel electrophoresis analysis demonstrates a replication stall with as few as 20 GGGGCC repeats. These results suggest that replication of the GGGGCC repeat at C9orf72 is perturbed by the presence of expanded repeats, which has the potential to result in further expansion, leading to disease.

  11. Tomato protoplast DNA transformation : physical linkage and recombination of exogenous DNA sequences

    NARCIS (Netherlands)

    Jongsma, Maarten; Koornneef, Maarten; Zabel, Pim; Hille, Jacques

    1987-01-01

    Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There

  12. A novel microfluidic mixer based on dual-hydrodynamic focusing for interrogating the kinetics of DNA-protein interaction.

    Science.gov (United States)

    Li, Ying; Xu, Fei; Liu, Chao; Xu, Youzhi; Feng, Xiaojun; Liu, Bi-Feng

    2013-08-21

    Kinetic measurement of biomacromolecular interaction plays a significant role in revealing the underlying mechanisms of cellular activities. Due to the small diffusion coefficient of biomacromolecules, it is difficult to resolve the rapid kinetic process with traditional analytical methods such as stopped-flow or laminar mixers. Here, we demonstrated a unique continuous-flow laminar mixer based on microfluidic dual-hydrodynamic focusing to characterize the kinetics of DNA-protein interactions. The time window of this mixer for kinetics observation could cover from sub-milliseconds to seconds, which made it possible to capture the folding process with a wide dynamic range. Moreover, the sample consumption was remarkably reduced to <0.55 μL min⁻¹, over 1000-fold saving in comparison to those reported previously. We further interrogated the interaction kinetics of G-quadruplex and the single-stranded DNA binding protein, indicating that this novel micromixer would be a useful approach for analyzing the interaction kinetics of biomacromolecules.

  13. Characterization of MNK1b DNA Aptamers That Inhibit Proliferation in MDA-MB231 Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Eva M García-Recio

    2016-01-01

    Full Text Available Elevated expression levels of eukaryotic initiation factor 4E (eIF4E promote cancer development and progression. MAP kinase interacting kinases (MNKs modulate the function of eIF4E through the phosphorylation that is necessary for oncogenic transformation. Therefore, pharmacologic MNK inhibitors may provide a nontoxic and effective anticancer strategy. MNK1b is a truncated isoform of MNK1a that is active in the absence of stimuli. Using in vitro selection, high-affinity DNA aptamers to MNK1b were selected from a library of ssDNA. Selection was monitored using the enzyme-linked oligonucleotide assay (ELONA, and the selected aptamer population was cloned and sequenced. Four groups of aptamers were identified, and the affinities of one representative for rMNK1b were determined using ELONA and quantitative polymerase chain reaction. Two aptamers, named apMNK2F and apMNK3R, had a lower Kd in the nmol/l range. The secondary structure of the selected aptamers was predicted using mFold, and the QGRS Mapper indicated the presence of potential G-quadruplex structures in both aptamers. The selected aptamers were highly specific against MNK1, showing higher affinity to MNK1b than to MNK1a. Interestingly, both aptamers were able to produce significant translation inhibition and prevent tumor cell proliferation and migration and colony formation in breast cancer cells. These results indicate that MNK1 aptamers have an attractive therapeutic potential.

  14. Structural varieties of selectively mixed G- and C-rich short DNA sequences studied with electrospray ionization mass spectrometry.

    Science.gov (United States)

    Cao, Yanwei; Gao, Shang; Li, Caijin; Yan, Yuting; Wang, Bing; Guo, Xinhua

    2016-10-01

    Short guanine(G)-repeat and cytosine(C)-repeat DNA strands can self-assemble to form four-stranded G-quadruplexes and i-motifs, respectively. Herein, G-rich and C-rich strands with non-G or non-C terminal bases and different lengths of G- or C-repeats are mixed selectively in pH 4.5 and 6.7 ammonium acetate buffer solutions and studied by electrospray ionization mass spectrometry (ESI-MS). Various strand associations corresponding to bi-, tri- and tetramolecular ions are observed in mass spectra, indicating that the formation of quadruplex structures is a random strand by strand association process. However, with increasing incubation time for the mixtures, initially associated hybrid tetramers will transform into self-assembled conformations, which is mainly driven by the structural stability. The melting temperature values of self-assembled quadruplexes suggest that the length of G-repeats or C-repeats shows more significant effect on the stability of quadruplex structures than that of terminal residues. Accordingly, we can obtain the self-associated tetrameric species generated from the mixtures of various homologous G- or C-strands efficiently by altering the length of G- or C-repeats. Our studies demonstrate that ESI-MS is a very direct, fast and sensitive tool to provide significant information on DNA strand associations and stoichiometric transitions, particularly for complex mixtures. Copyright © 2016 John Wiley & Sons, Ltd.

  15. Single-molecule TPM studies on the conversion of human telomeric DNA.

    Science.gov (United States)

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

    2010-04-21

    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.

  16. Label-free molecular beacons-based cascade amplification DNA machine for sensitive detection of telomerase activity.

    Science.gov (United States)

    Li, Kan; Wang, Lei; Xu, Xiaowen; Jiang, Wei

    2017-05-15

    Sensitive detection of telomerase activity is critical to cancer diagnosis, screening of anticancer drugs and evaluation of cancer therapy. Herein, a label-free molecular beacons-based DNA machine was developed for sensitive detection of telomerase activity. The DNA machine consisted of T7 exonuclease (T7 Exo), label-free recognition molecular beacon (RMB) and signal molecular beacon (SMB) with projecting 5'-terminuses, which can protect RMB and SMB from being digested by T7 Exo. Firstly, telomerase elongated telomerase substrate (TS) primer, generating a telomerase elongation production (TEP) with tandem repeats (TTAGGG)n. Next, TEP activated the DNA machine by hybridizing with RMB, unfolding RMB with a recessed 5'-terminus, making RMB deprotection from T7 Exo. Then T7 Exo-assisted cycling cleavage was incurred, releasing intact TEP and numerous DNA fragments (trigger DNA), which got recycling I. Subsequently, trigger DNA specifically opened SMB and was recycled by T7 Exo, liberating multiple G-quadruplex (G4) structures, which got recycling II. Finally, TEP and the liberative G4 structures strongly interacted with N-methyl-mesoporphyrin IX (NMM), yielding a significantly enhanced fluorescence together. In this way, per telomerase-mediated elongation event was efficiently converted into the greatly amplified fluorescence signals. Telomerase activity in crude HeLa cells extracts equivalent to 50 cells/mL was successfully measured with a linear range from 50 cells/mL to 2000 cells/mL. Besides, the strategy was also successfully used to assay the inhibition effect of a telomerase-inhibiting drug, demonstrating the strategy holds the potential to screen telomerase inhibitors.

  17. Molecular recognition of naphthalene diimide ligands by telomeric quadruplex-DNA: the importance of the protonation state and mediated hydrogen bonds.

    Science.gov (United States)

    Spinello, A; Barone, G; Grunenberg, J

    2016-01-28

    In depth Monte Carlo conformational scans in combination with molecular dynamics (MD) simulations and electronic structure calculations were applied in order to study the molecular recognition process between tetrasubstituted naphthalene diimide (ND) guests and G-quadruplex (G4) DNA receptors. ND guests are a promising class of telomere stabilizers due to which they are used in novel anticancer therapeutics. Though several ND guests have been studied experimentally in the past, the protonation state under physiological conditions is still unclear. Based on chemical intuition, in the case of N-methyl-piperazine substitution, different protonation states are possible and might play a crucial role in the molecular recognition process by G4-DNA. Depending on the proton concentration, different nitrogen atoms of the N-methyl-piperazine might (or might not) be protonated. This fact was considered in our simulation in terms of a case by case analysis, since the process of molecular recognition is determined by possible donor or acceptor positions. The results of our simulations show that the electrostatic interactions between the ND ligands and the G4 receptor are maximized in the case of the protonation of the terminal nitrogen atoms, forming compact ND G4 complexes inside the grooves. The influence of different protonation states in terms of the ability to form hydrogen bonds with the sugar-phosphate backbone, as well as the importance of mediated vs. direct hydrogen bonding, was analyzed in detail by MD and relaxed force constant (compliance constant) simulations.

  18. Carbon Nanomaterials and DNA: from Molecular Recognition to Applications.

    Science.gov (United States)

    Sun, Hanjun; Ren, Jinsong; Qu, Xiaogang

    2016-03-15

    DNA is polymorphic. Increasing evidence has indicated that many biologically important processes are related to DNA's conformational transition and assembly states. In particular, noncanonical DNA structures, such as the right-handed A-form, the left-handed Z-form, the triplex, the G-quadruplex, the i-motif, and so forth, have been specific targets for the diagnosis and therapy of human diseases. Meanwhile, they have been widely used in the construction of smart DNA nanomaterials and nanoarchitectures. As rising stars in materials science, the family of carbon nanomaterials (CNMs), including two-dimensional graphene, one-dimensional carbon nanotubes (CNTs), and zero-dimensional graphene or carbon quantum dots (GQDs or CQDs), interact with DNA and are able to regulate the conformational transitions of DNA. The interaction of DNA with CNMs not only opens new opportunities for specific molecular recognition, but it also expands the promising applications of CNMs from materials science to biotechnology and biomedicine. In this Account, we focus on our contributions to the field of interactions between CNMs and DNA in which we have explored their promising applications in nanodevices, sensing, materials synthesis, and biomedicine. For one-dimensional CNTs, two-dimensional graphene, and zero-dimensional GQDs and CQDs, the basic principles, binding modes, and applications of the interactions between CNMs and DNA are reviewed. We aim to give prominence to the important status of CNMs in the field of molecular recognition for DNA. First, we summarized our discovery of the interactions between single-walled carbon nanotubes (SWNTs) with duplex, triplex, and human telomeric i-motif DNA and their interesting applications. For example, SWNTs are the first chemical agents that can selectively stabilize human telomeric i-motif DNA and induce its formation under physiological conditions. On the basis of this principle, two types of nanodevices were designed. One was used for

  19. Sequence-specific fluorometric recognition of HIV-1 ds-DNA with zwitterionic zinc(II)-carboxylate polymers.

    Science.gov (United States)

    Sun, Bin; Zhao, Hai-Qing; Xie, Bao-Ping; Bai, Li-Ping; Jiang, Zhi-Hong; Chen, Jin-Xiang

    2017-11-01

    Four water-stable zwitterionic zinc-carboxylate polymers are prepared by reacting N-carboxymethyl-(3,5-dicarboxy)-pyridinium bromide (H3CmdcpBr) with zinc(II) nitrate in the presence of NaOH, through adjusting the solvents and ancillary ligands. With H2O as the solvent and the absence of an ancillary ligand, a two-dimensional (2D) polymer network [Zn(Cmdcp)(H2O)]n (1) is formed. In a mixed H2O/DMF solvent and with the presence of chelating ligands 2,2'-bipyridine (bipy), 1,10-phenanthroline (phen) and 2-(4-pyridyl)benzimidazole (pbz), a one-dimensional (1D) polymer of {[Zn2(Cmdcp)(bipy)2(H2O)5](NO3)2·3H2O}n (2), a mononuclear ionic species of [Zn(phen)(H2O)4][Cmdcp] (3), and a 2D polymer of {[Zn(Cmdcp)(pbz)][pbz]·7H2O}n (4) are accordingly formed. Compounds 1-4 are characterized by IR, elemental analyses and single crystal X-ray crystallography. Compound 2 strongly adsorbs single-stranded DNA (ss-DNA) probe (denoted as P-DNA) labeled with carboxyfluorescein (FAM) and quenches its fluorescence via a photo-induced electron transfer process. If, however, a double-stranded DNA (ds-DNA) of the human immunodeficiency virus 1 (HIV-1 ds-DNA) is further present, the P-DNA interacts with the major groove in HIV-1 ds-DNA via Hoogsteen hydrogen bonding to form a rigid triplex structure. This results in partial or complete fluorescence recovery depending on the concentration of HIV-1 ds-DNA. The findings are applied in fluorometric sensing of HIV-1 ds-DNA. The calibration plot is linear in the 0-60nM target DNA concentration range, with a 7.4nM detection limit (at a signal-to-noise ratio of 3). The assay is highly specific and not interfered by one base pair mutated for complementary target HIV-1 ds-DNA, complementary ss-DNA, single-base pair mutated for complementary ss-DNA, non-specific ss-DNA sequences, and higher-order dimeric G-quadruplexes. Copyright © 2017 Elsevier Inc. All rights reserved.

  20. Putative archaeal viruses from the mesopelagic ocean.

    Science.gov (United States)

    Vik, Dean R; Roux, Simon; Brum, Jennifer R; Bolduc, Ben; Emerson, Joanne B; Padilla, Cory C; Stewart, Frank J; Sullivan, Matthew B

    2017-01-01

    Oceanic viruses that infect bacteria, or phages, are known to modulate host diversity, metabolisms, and biogeochemical cycling, while the viruses that infect marine Archaea remain understudied despite the critical ecosystem roles played by their hosts. Here we introduce "MArVD", for Metagenomic Archaeal Virus Detector, an annotation tool designed to identify putative archaeal virus contigs in metagenomic datasets. MArVD is made publicly available through the online iVirus analytical platform. Benchmarking analysis of MArVD showed it to be >99% accurate and 100% sensitive in identifying the 127 known archaeal viruses among the 12,499 viruses in the VirSorter curated dataset. Application of MArVD to 10 viral metagenomes from two depth profiles in the Eastern Tropical North Pacific (ETNP) oxygen minimum zone revealed 43 new putative archaeal virus genomes and large genome fragments ranging in size from 10 to 31 kb. Network-based classifications, which were consistent with marker gene phylogenies where available, suggested that these putative archaeal virus contigs represented six novel candidate genera. Ecological analyses, via fragment recruitment and ordination, revealed that the diversity and relative abundances of these putative archaeal viruses were correlated with oxygen concentration and temperature along two OMZ-spanning depth profiles, presumably due to structuring of the host Archaea community. Peak viral diversity and abundances were found in surface waters, where Thermoplasmata 16S rRNA genes are prevalent, suggesting these archaea as hosts in the surface habitats. Together these findings provide a baseline for identifying archaeal viruses in sequence datasets, and an initial picture of the ecology of such viruses in non-extreme environments.

  1. DNA Domino-Based Nanoscale Logic Circuit: A Versatile Strategy for Ultrasensitive Multiplexed Analysis of Nucleic Acids.

    Science.gov (United States)

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

    2017-06-06

    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.

  2. Evaluation of antithrombotic activity of thrombin DNA aptamers by a murine thrombosis model.

    Directory of Open Access Journals (Sweden)

    Elena Zavyalova

    Full Text Available Aptamers are nucleic acid based molecular recognition elements with a high potential for the theranostics. Some of the aptamers are under development for therapeutic applications as promising antithrombotic agents; and G-quadruplex DNA aptamers, which directly inhibit the thrombin activity, are among them. RA-36, the 31-meric DNA aptamer, consists of two thrombin binding pharmacophores joined with the thymine linker. It has been shown earlier that RA-36 directly inhibits thrombin in the reaction of fibrinogen hydrolysis, and also it inhibits plasma and blood coagulation. Studies of both inhibitory and anticoagulation effects had indicated rather high species specificity of the aptamer. Further R&D of RA-36 requires exploring its efficiency in vivo. Therefore the development of a robust and adequate animal model for effective physiological studies of aptamers is in high current demand. This work is devoted to in vivo study of the antithrombotic effect of RA-36 aptamer. A murine model of thrombosis has been applied to reveal a lag and even prevention of thrombus formation when RA-36 was intravenous bolus injected in high doses of 1.4-7.1 µmol/kg (14-70 mg/kg. A comparative study of RA-36 aptamer and bivalirudin reveals that both direct thrombin inhibitors have similar antithrombotic effects for the murine model of thrombosis; though in vitro bivalirudin has anticoagulation activity several times higher compared to RA-36. The results indicate that both RA-36 aptamer and bivalirudin are direct thrombin inhibitors of different potency, but possible interactions of the thrombin-inhibitor complex with other components of blood coagulation cascade level the physiological effects for both inhibitors.

  3. Evaluation of antithrombotic activity of thrombin DNA aptamers by a murine thrombosis model.

    Science.gov (United States)

    Zavyalova, Elena; Samoylenkova, Nadezhda; Revishchin, Alexander; Golovin, Andrey; Pavlova, Galina; Kopylov, Alexey

    2014-01-01

    Aptamers are nucleic acid based molecular recognition elements with a high potential for the theranostics. Some of the aptamers are under development for therapeutic applications as promising antithrombotic agents; and G-quadruplex DNA aptamers, which directly inhibit the thrombin activity, are among them. RA-36, the 31-meric DNA aptamer, consists of two thrombin binding pharmacophores joined with the thymine linker. It has been shown earlier that RA-36 directly inhibits thrombin in the reaction of fibrinogen hydrolysis, and also it inhibits plasma and blood coagulation. Studies of both inhibitory and anticoagulation effects had indicated rather high species specificity of the aptamer. Further R&D of RA-36 requires exploring its efficiency in vivo. Therefore the development of a robust and adequate animal model for effective physiological studies of aptamers is in high current demand. This work is devoted to in vivo study of the antithrombotic effect of RA-36 aptamer. A murine model of thrombosis has been applied to reveal a lag and even prevention of thrombus formation when RA-36 was intravenous bolus injected in high doses of 1.4-7.1 µmol/kg (14-70 mg/kg). A comparative study of RA-36 aptamer and bivalirudin reveals that both direct thrombin inhibitors have similar antithrombotic effects for the murine model of thrombosis; though in vitro bivalirudin has anticoagulation activity several times higher compared to RA-36. The results indicate that both RA-36 aptamer and bivalirudin are direct thrombin inhibitors of different potency, but possible interactions of the thrombin-inhibitor complex with other components of blood coagulation cascade level the physiological effects for both inhibitors.

  4. Cryptic species in putative ancient asexual darwinulids (Crustacea, Ostracoda.

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    Isa Schön

    Full Text Available BACKGROUND: Fully asexually reproducing taxa lack outcrossing. Hence, the classic Biological Species Concept cannot be applied. METHODOLOGY/PRINCIPAL FINDINGS: We used DNA sequences from the mitochondrial COI gene and the nuclear ITS2 region to check species boundaries according to the evolutionary genetic (EG species concept in five morphospecies in the putative ancient asexual ostracod genera, Penthesilenula and Darwinula, from different continents. We applied two methods for detecting cryptic species, namely the K/θ method and the General Mixed Yule Coalescent model (GMYC. We could confirm the existence of species in all five darwinulid morphospecies and additional cryptic diversity in three morphospecies, namely in Penthesilenula brasiliensis, Darwinula stevensoni and in P. aotearoa. The number of cryptic species within one morphospecies varied between seven (P. brasiliensis, five to six (D. stevensoni and two (P. aotearoa, respectively, depending on the method used. Cryptic species mainly followed continental distributions. We also found evidence for coexistence at the local scale for Brazilian cryptic species of P. brasiliensis and P. aotearoa. Our ITS2 data confirmed that species exist in darwinulids but detected far less EG species, namely two to three cryptic species in P. brasiliensis and no cryptic species at all in the other darwinulid morphospecies. CONCLUSIONS/SIGNIFICANCE: Our results clearly demonstrate that both species and cryptic diversity can be recognized in putative ancient asexual ostracods using the EG species concept, and that COI data are more suitable than ITS2 for this purpose. The discovery of up to eight cryptic species within a single morphospecies will significantly increase estimates of biodiversity in this asexual ostracod group. Which factors, other than long-term geographic isolation, are important for speciation processes in these ancient asexuals remains to be investigated.

  5. Ten Putative Contributors to the Obesity Epidemic

    Science.gov (United States)

    McAllister, Emily J.; Dhurandhar, Nikhil V.; Keith, Scott W.; Aronne, Louis J.; Barger, Jamie; Baskin, Monica; Benca, Ruth M.; Biggio, Joseph; Boggiano, Mary M.; Eisenmann, Joe C.; Elobeid, Mai; Fontaine, Kevin R.; Gluckman, Peter; Hanlon, Erin C.; Katzmarzyk, Peter; Pietrobelli, Angelo; Redden, David T.; Ruden, Douglas M.; Wang, Chenxi; Waterland, Robert A.; Wright, Suzanne M.; Allison, David B.

    2010-01-01

    The obesity epidemic is a global issue and shows no signs of abating, while the cause of this epidemic remains unclear. Marketing practices of energy-dense foods and institutionally-driven declines in physical activity are the alleged perpetrators for the epidemic, despite a lack of solid evidence to demonstrate their causal role. While both may contribute to obesity, we call attention to their unquestioned dominance in program funding and public efforts to reduce obesity, and propose several alternative putative contributors that would benefit from equal consideration and attention. Evidence for microorganisms, epigenetics, increasing maternal age, greater fecundity among people with higher adiposity, assortative mating, sleep debt, endocrine disruptors, pharmaceutical iatrogenesis, reduction in variability of ambient temperatures, and intrauterine and intergenerational effects, as contributing factors to the obesity epidemic are reviewed herein. While the evidence is strong for some contributors such as pharmaceutical-induced weight gain, it is still emerging for other reviewed factors. Considering the role of such putative etiological factors of obesity may lead to comprehensive, cause specific, and effective strategies for prevention and treatment of this global epidemic. PMID:19960394

  6. Isolation of Single-Stranded DNA Aptamers That Distinguish Influenza Virus Hemagglutinin Subtype H1 from H5

    Science.gov (United States)

    Yim, Sanggyu; Jeong, Yong-Joo

    2015-01-01

    Surface protein hemagglutinin (HA) mediates the binding of influenza virus to host cell receptors containing sialic acid, facilitating the entry of the virus into host cells. Therefore, the HA protein is regarded as a suitable target for the development of influenza virus detection devices. In this study, we isolated single-stranded DNA (ssDNA) aptamers binding to the HA1 subunit of subtype H1 (H1-HA1), but not to the HA1 subunit of subtype H5 (H5-HA1), using a counter-systematic evolution of ligands by exponential enrichment (counter-SELEX) procedure. Enzyme-linked immunosorbent assay and surface plasmon resonance studies showed that the selected aptamers bind tightly to H1-HA1 with dissociation constants in the nanomolar range. Western blot analysis demonstrated that the aptamers were binding to H1-HA1 in a concentration-dependent manner, yet were not binding to H5-HA1. Interestingly, the selected aptamers contained G-rich sequences in the central random nucleotides region. Further biophysical analysis showed that the G-rich sequences formed a G-quadruplex structure, which is a distinctive structure compared to the starting ssDNA library. Using flow cytometry analysis, we found that the aptamers did not bind to the receptor-binding site of H1-HA1. These results indicate that the selected aptamers that distinguish H1-HA1 from H5-HA1 can be developed as unique probes for the detection of the H1 subtype of influenza virus. PMID:25901739

  7. DNA Fingerprinting Using PCR: A Practical Forensic Science Activity

    Science.gov (United States)

    Choi, Hyun-Jung; Ahn, Jung Hoon; Ko, Minsu

    2008-01-01

    This paper describes a forensic science simulation programme applicable for use in colleges. Students were asked to find a putative suspect by DNA fingerprinting using a simple protocol developed in this study. DNA samples were obtained from a hair root and a drop of blood, common sources of DNA in forensic science. The DNA fingerprinting protocol…

  8. DNA Fingerprinting Using PCR: A Practical Forensic Science Activity

    Science.gov (United States)

    Choi, Hyun-Jung; Ahn, Jung Hoon; Ko, Minsu

    2008-01-01

    This paper describes a forensic science simulation programme applicable for use in colleges. Students were asked to find a putative suspect by DNA fingerprinting using a simple protocol developed in this study. DNA samples were obtained from a hair root and a drop of blood, common sources of DNA in forensic science. The DNA fingerprinting protocol…

  9. Putative Nitrogen Sensing Systems in Higher Plants

    Institute of Scientific and Technical Information of China (English)

    Hon-Ming Lam; Ying Ann Chiao; Man-Wah Li; Yuk-Kwong Yung; Sang Ji

    2006-01-01

    Nitrogen (N) metabolism is essential for the biosynthesis of vital biomolecules. N status thus exerts profound effects on plant growth and development, and must be closely monitored. In bacteria and fungi, a few sophisticated N sensing systems have been extensively studied. In animals, the ability to receive amino acid signals has evolved to become an integral part of the nervous coordination system. In this review, we will summarize recent developments in the search for putative N sensing systems in higher plants based on homologous systems in bacteria, fungi, and animals. Apparently, although plants have separated and diversified from other organisms during the evolution process, striking similarities can be found in their N sensing systems compared with those of their counterparts; however, our understanding of these systems is still incomplete. Significant modifications of the N sensing systems (including cross-talk with other signal transduction pathways) in higher plants may be a strategy of adaptation to their unique mode of life.

  10. Putative respiratory chain of Porphyromonas gingivalis.

    Science.gov (United States)

    Meuric, Vincent; Rouillon, Astrid; Chandad, Fatiha; Bonnaure-Mallet, Martine

    2010-05-01

    The electron transfer chain in Porphyromonas gingivalis, or periodontopathogens, has not yet been characterized. P. gingivalis, a strict anaerobic bacteria and the second colonizer of the oral cavity, is considered to be a major causal agent involved in periodontal diseases. Primary colonizers create a favorable environment for P. gingivalis growth by decreasing oxygen pressure. Oxygen does not appear to be the final electron acceptor of the respiratory chain. Fumarate and cytochrome b have been implicated as major components of the respiratory activity. However, the P. gingivalis genome shows many other enzymes that could be implicated in aerobic or nitrite respiration. Using bioinformatic tools and literature studies of respiratory pathways, the ATP synthesis mechanism from the sodium cycle and nutrients metabolism, the putative respirasome of P. gingivalis has been proposed.

  11. Putative ancient microorganisms from amber nuggets.

    Science.gov (United States)

    Veiga-Crespo, Patricia; Blasco, Lucía; Poza, Margarita; Villa, Tomás G

    2007-06-01

    Evolutionary microbiology studies based on the isolation of ancient DNA and/or microbial samples are scarce due to the difficulty of finding well preserved biological specimens. However, amber is a fossil resin with natural preserving properties for microbial cells and DNA. The visualization by transmission electron microscopy of different microorganism-like specimens found in amber nuggets from both the Miocene and the Cretaceous periods was accompanied by studies of ancient DNA obtained from the nuggets. After the design of specific primers based on the present sequences of both genes in Saccharomyces cerevisiae, the ancestral AGP2 sequence from the Miocene, as well as the 18S rRNA from the Cretaceous, were amplified.

  12. Expression of putative expansin genes in phylloxera (Daktulosphaira vitifoliae Fitch) induced root galls of Vitis spp.

    Science.gov (United States)

    Lawo, N C; Griesser, M; Forneck, A

    Grape phylloxera (Daktulosphaira vitifoliae Fitch) is a serious global pest in viticulture. The insects are sedentary feeders and require a gall to feed and reproduce. The insects induce their feeding site within the meristematic zone of the root tip, where they stay attached, feeding both intra- and intercellularly, and causing damage by reducing plant vigour. Several changes in cell structure and composition, including increased cell division and tissue swelling close to the feeding site, cause an organoid gall called a nodosity to develop. Because alpha expansin genes are involved in cell enlargement and cell wall loosening in many plant tissues it may be anticipated that they are also involved in nodosity formation. To identify expansin genes in Vitis vinifera cv. Pinot noir, we mined for orthologues genes in a comparative analysis. Eleven putative expansin genes were identified and shown to be present in the rootstock Teleki 5C (V. berlandieri Planch. x V. riparia Michx.) using specific PCR followed by DNA sequencing. Expression analysis of young and mature nodosities and uninfested root tips were conducted via quantitative real time PCR (qRT-PCR). Up-regulation was measured for three putative expansin genes (VvEXPA15, -A17 and partly -A20) or down-regulation for three other putative genes (VvEXPA7, -A12, -A20) in nodosities. The present study clearly shows the involvement of putative expansin genes in the phylloxera-root interaction.

  13. Epigenetic regulation of putative tumor suppressor TGFBI in human leukemias

    Institute of Scientific and Technical Information of China (English)

    Fang Hongbo; Liu Jing; Guo Dan; Liu Peixiang; Zhao Yongliang

    2014-01-01

    Background Both in vitro and in vivo data have demonstrated the TGFBI gene functions as a putative tumor suppressor and is frequently downregulated in human tumors of different histological types.The hypermethylation of the TGFBI promoter,as one of the main regulatory mechanisms,is associated with TGFBI silencing.In this study,we used a methylation-specific PCR (MSP) method to evaluate the methylation status of the TGFBI promoter in human leukemias.Methods Real-time RT-PCR and methylation-specific PCR approaches were performed to define the TGFBI expression and promoter methylation in human leukemia call lines and clinical samples.Genomic DNA was isolated from peripheral blood mononuclear cells from leukemia patients,bisulfite-converted,and analyzed by the MSP method.Results Hypermethylation of the TGFBI promoter occurred in leukemia cell lines and demethylation treatment reexpressed TGFBI at a substantially increased level in most of leukemia cell lines tested.Furthermore,a much higher level of CpG island methylation and a significantly lower TGFBI expression were also identified in clinical leukemia samples.Conclusion The results suggest an important role of promoter methylation in regulating TGFBI expression in leukemia,which provides a useful diagnostic marker for clinical management of human leukemias.

  14. Expression and characterization of rice putative PAUSED gene

    Institute of Scientific and Technical Information of China (English)

    Chengguo Yao; Liangfa Ge; Wei Li; Botao Zhao; Chaoqun Li; Kangcheng Ruan; Hongxuan Lin; Youxin Jin

    2008-01-01

    In Arab idopsis, PA USED ( PSD ) encodes the ortholog of loslp/ exportin-t, which mediates the nuclear export of transfer RNA (tRNA) in yeast and mammals. However, in monocot plants such as rice, knowledge of the corresponding ortholog is limited, and its effects on growth development and productivity remain unknown. In this study, we verified a rice transfer-DNA insertional mutantpsd line and analyzed its phenotypes;the mutant displayed severe morphological defects including retarded development and low fertility compared with wild-type rice. Examining intronless tRNA-Tyr and intron-containing pre-tRNA-Ala expression levels in cytoplasmic and nuclear fraction with Northern blot analysis between wild -type and mutant leaf tissue suggested that rice PSD might be involved in tRNA export from the nucleus to the cytoplasm.Additionally, reverse transcription-polymerase chain reaction analysis revealed that PSD transcript was expressed throughout normal rice plant development, and subcellular localization assays showed that rice PSD protein was present in both the nucleus and cytoplasm. In summary, our data implied that the putative PSD gene might be indispensable for normal rice development and its function might be the same as that ofArabidopsis PSD.

  15. KLONING GEN PUTATIVE CLEAVAGE PROTEIN 1 (PCP-1 PADA UDANG VANAME (Litopenaeus vannamei YANG TERSERANG INFECTIOUS MYONECROSIS VIRUS

    Directory of Open Access Journals (Sweden)

    Hessy Novita

    2016-12-01

    Full Text Available Penanggulangan penyakit ikan dapat dilakukan dengan cara meningkatkan kekebalan tubuh ikan melalui program vaksinasi. Namun vaksinasi tidak tepat untuk udang, karena udang tidak mempunyai immunological memory seperti ikan. Oleh karena itu, perlindungan udang terhadap serangan penyakit viral dengan menggunakan RNA interference (RNAi. Teknologi RNAi digunakan untuk menghalangi (interfere proses replikasi infectious myonecrosis virus (IMNV pada udang vaname dengan cara menon-aktifkan gen putative cleavage protein 1 (PCP-1, yang berfungsi dalam pembentukan capsid dan proses transkripsi RNA IMNV. Penelitian ini bertujuan untuk melakukan kloning gen putative cleavage protein 1 dalam rangka perakitan teknologi RNAi untuk pengendalian penyakit IMNV pada udang vaname. Tahapan penelitian meliputi koleksi sampel, isolasi RNA, sintesis cDNA, amplifikasi PCR, purifikasi DNA, transformasi, isolasi plasmid, serta sekuensing dan analisis data. Hasil isolasi plasmid cDNA PCP-1 memperlihatkan semua koloni bakteri terseleksi ternyata membawa plasmid hasil insersi DNA gen PCP–1, hasil sekuen dengan nilai homologinya mencapai 100% dan 99% yang dibandingkan dengan sekuen di Genebank. Hasil penelitian menunjukkan bahwa kloning gen putative cleavage protein 1 (PCP-1 dari udang vaname yang terserang Infectious Myonecrosis Virus berhasil dikloning yang nantinya digunakan untuk perakitan RNAi. The prevention of fish diseases can be done by increasing of the fish immune through vaccination programs. However, the vaccination can not be done for the shrimp,due to the absence of  immunological memory. Therefore, the protection of shrimp against viral diseases was done by using of RNA interference (RNAi. RNAi technology is used to interfere infectious myonecrosis virus (IMNV replication process on white shrimp by disabling of putative cleavage protein 1 (PCP-1gene, which functions in capsid formation and RNA transcription process. The study was conducted to perform putative

  16. A dual-channel detection of mercuric ions using a label free G-quadruplex-based DNAzyme molecule.

    Science.gov (United States)

    Ma, Long; Liu, Haiyan; Wu, Guanrong; Sun, Nana; Meng, Lingpei; Li, Yuyin; Liu, Zhenxing; Diao, Aipo

    2016-06-20

    We have constructed a 'turn-off' and label free bio-sensor using a DNAzyme molecule. This facile bio-sensor is capable of selective detection of mercuric ions with a high sensitivity and satisfactory dynamic range. More importantly, it is able to generate both fluorescent and colourimetric signals for detection. This dual-channel bio-sensor is expected to afford high detection confidence and overcome false-positive readout especially when assaying complex biological samples.

  17. Luminescence detection of cysteine based on Ag⁺-mediated conformational change of terbium ion-promoted G-quadruplex.

    Science.gov (United States)

    Tan, Hongliang; Tang, Gonge; Ma, Chanjiao; Li, Qian

    2016-02-18

    In this work, we developed a simple and sensitive method for the detection of cysteine (Cys) by employing terbium ion (Tb(3+))-promoted G-qudraplex (G4/Tb) as a luminescent probe, which is based on Ag(+)-mediated conformational change of G4/Tb. Due to Ag(+) is able to compete with Tb(3+) to bind guanine at G4, the presence of Ag(+) can lead to the formation of G4/Tb-Ag(+) complex and disrupt the structure of G4/Tb. Meanwhile, the binding of Ag(+) with G4/Tb will also cause the alteration of the excited state of G4 and more efficient energy transfer from G4 to Tb(3+), enhancing the luminescence of G4/Tb. However, upon the addition of Cys, Ag(+) will be released from G4/Tb-Ag(+) complex because of the high affinity of Cys to Ag(+). This results in the re-formation of the conformation of G4/Tb and the decrease of the luminescence of G4/Tb. So, Ag(+)-enhanced luminescence of G4/Tb is associated with its conformational transformation. As a luminescent probe for Cys, G4/Tb not only shows excellent selectivity and sensitivity with a detection limit of 20 nM, but also possesses the features of simple preparation, easy reproducibility, and eliminating the interferences from background fluorescence. We envision that the presented strategy might provide new insight into the biosensing applications of lanthanide complex. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Luminescence detection of cysteine based on Ag{sup +}-mediated conformational change of terbium ion-promoted G-quadruplex

    Energy Technology Data Exchange (ETDEWEB)

    Tan, Hongliang, E-mail: hltan@jxnu.edu.cn; Tang, Gonge; Ma, Chanjiao; Li, Qian

    2016-02-18

    In this work, we developed a simple and sensitive method for the detection of cysteine (Cys) by employing terbium ion (Tb{sup 3+})-promoted G-qudraplex (G4/Tb) as a luminescent probe, which is based on Ag{sup +}-mediated conformational change of G4/Tb. Due to Ag{sup +} is able to compete with Tb{sup 3+} to bind guanine at G4, the presence of Ag{sup +} can lead to the formation of G4/Tb–Ag{sup +} complex and disrupt the structure of G4/Tb. Meanwhile, the binding of Ag{sup +} with G4/Tb will also cause the alteration of the excited state of G4 and more efficient energy transfer from G4 to Tb{sup 3+}, enhancing the luminescence of G4/Tb. However, upon the addition of Cys, Ag{sup +} will be released from G4/Tb–Ag{sup +} complex because of the high affinity of Cys to Ag{sup +}. This results in the re-formation of the conformation of G4/Tb and the decrease of the luminescence of G4/Tb. So, Ag{sup +}-enhanced luminescence of G4/Tb is associated with its conformational transformation. As a luminescent probe for Cys, G4/Tb not only shows excellent selectivity and sensitivity with a detection limit of 20 nM, but also possesses the features of simple preparation, easy reproducibility, and eliminating the interferences from background fluorescence. We envision that the presented strategy might provide new insight into the biosensing applications of lanthanide complex. - Highlights: • A G4/Tb-based luminescent probe for Cys detection was developed for the first time. • The binding and remove of Ag{sup +} mediate the luminescence of G4/Tb. • The luminescent ​sensor showed excellent selectivity and high sensitivity to Cys.

  19. The solution structure of ChaB, a putative membrane ion antiporter regulator from Escherichia coli

    Directory of Open Access Journals (Sweden)

    Iannuzzi Pietro

    2004-08-01

    Full Text Available Abstract Background ChaB is a putative regulator of ChaA, a Na+/H+ antiporter that also has Ca+/H+ activity in E. coli. ChaB contains a conserved 60-residue region of unknown function found in other bacteria, archaeabacteria and a series of baculoviral proteins. As part of a structural genomics project, the structure of ChaB was elucidated by NMR spectroscopy. Results The structure of ChaB is composed of 3 α-helices and a small sheet that pack tightly to form a fold that is found in the cyclin-box family of proteins. Conclusion ChaB is distinguished from its putative DNA binding sequence homologues by a highly charged flexible loop region that has weak affinity to Mg2+ and Ca2+ divalent metal ions.

  20. Putative bronchopulmonary flagellated protozoa in immunosuppressed patients.

    Science.gov (United States)

    Kilimcioglu, Ali Ahmet; Havlucu, Yavuz; Girginkardesler, Nogay; Celik, Pınar; Yereli, Kor; Özbilgin, Ahmet

    2014-01-01

    Flagellated protozoa that cause bronchopulmonary symptoms in humans are commonly neglected. These protozoal forms which were presumed to be "flagellated protozoa" have been previously identified in immunosuppressed patients in a number of studies, but have not been certainly classified so far. Since no human cases of bronchopulmonary flagellated protozoa were reported from Turkey, we aimed to investigate these putative protozoa in immunosuppressed patients who are particularly at risk of infectious diseases. Bronchoalveolar lavage fluid samples of 110 immunosuppressed adult patients who were admitted to the Department of Chest Diseases, Hafsa Sultan Hospital of Celal Bayar University, Manisa, Turkey, were examined in terms of parasites by light microscopy. Flagellated protozoal forms were detected in nine (8.2%) of 110 cases. Metronidazole (500 mg b.i.d. for 30 days) was given to all positive cases and a second bronchoscopy was performed at the end of the treatment, which revealed no parasites. In conclusion, immunosuppressed patients with bronchopulmonary symptoms should attentively be examined with regard to flagellated protozoa which can easily be misidentified as epithelial cells.

  1. The Biogeography of Putative Microbial Antibiotic Production.

    Directory of Open Access Journals (Sweden)

    Hélène Morlon

    Full Text Available Understanding patterns in the distribution and abundance of functional traits across a landscape is of fundamental importance to ecology. Mapping these distributions is particularly challenging for species-rich groups with sparse trait measurement coverage, such as flowering plants, insects, and microorganisms. Here, we use likelihood-based character reconstruction to infer and analyze the spatial distribution of unmeasured traits. We apply this framework to a microbial dataset comprised of 11,732 ketosynthase alpha gene sequences extracted from 144 soil samples from three continents to document the spatial distribution of putative microbial polyketide antibiotic production. Antibiotic production is a key competitive strategy for soil microbial survival and performance. Additionally, novel antibiotic discovery is highly relevant to human health, making natural antibiotic production by soil microorganisms a major target for bioprospecting. Our comparison of trait-based biogeographical patterns to patterns based on taxonomy and phylogeny is relevant to our basic understanding of microbial biogeography as well as the pressing need for new antibiotics.

  2. Mechanosensory neurons, cutaneous mechanoreceptors, and putative mechanoproteins.

    Science.gov (United States)

    Del Valle, M E; Cobo, T; Cobo, J L; Vega, J A

    2012-08-01

    The mammalian skin has developed sensory structures (mechanoreceptors) that are responsible for different modalities of mechanosensitivity like touch, vibration, and pressure sensation. These specialized sensory organs are anatomically and functionally connected to a special subset of sensory neurons called mechanosensory neurons, which electrophysiologically correspond with Aβ fibers. Although mechanosensory neurons and cutaneous mechanoreceptors are rather well known, the biology of the sense of touch still remains poorly understood. Basically, the process of mechanosensitivity requires the conversion of a mechanical stimulus into an electrical signal through the activation of ion channels that gate in response to mechanical stimuli. These ion channels belong primarily to the family of the degenerin/epithelium sodium channels, especially the subfamily acid-sensing ion channels, and to the family of transient receptor potential channels. This review compiles the current knowledge on the occurrence of putative mechanoproteins in mechanosensory neurons and mechanoreceptors, as well as the involvement of these proteins on the biology of touch. Furthermore, we include a section about what the knock-out mice for mechanoproteins are teaching us. Finally, the possibilities for mechanotransduction in mechanoreceptors, and the common involvement of the ion channels, extracellular membrane, and cytoskeleton, are revisited.

  3. Putative Enzymes of UV Photoproduct Repair

    Directory of Open Access Journals (Sweden)

    Cynthia J. Sakofsky

    2011-01-01

    Full Text Available In order to determine the biological relevance of two S. acidocaldarius proteins to the repair of UV photoproducts, the corresponding genes (Saci_1227 and Saci_1096 were disrupted, and the phenotypes of the resulting mutants were examined by various genetic assays. The disruption used integration by homologous recombination of a functional but heterologous pyrE gene, promoted by short sequences attached to both ends via PCR. The phenotypic analyses of the disruptants confirmed that ORF Saci_1227 encodes a DNA photolyase which functions in vivo, but they could not implicate ORF Saci_1096 in repair of UV- or other externally induced DNA damage despite its similarity to genes encoding UV damage endonucleases. The success of the gene-disruption strategy, which used 5′ extensions of PCR primers to target cassette integration, suggests potential advantages for routine construction of Sulfolobus strains.

  4. Ancient DNA

    DEFF Research Database (Denmark)

    Willerslev, Eske; Cooper, Alan

    2004-01-01

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

  5. 1,1’,1’’,1’’’-[Porphyrin-5,10,15,20-tetrayltetrakis(3,1-phenylenemethylene]tetraquinolinium Tetrabromide

    Directory of Open Access Journals (Sweden)

    Satoshi Fujii

    2010-11-01

    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.

  6. Putative modifier genes in mevalonate kinase deficiency.

    Science.gov (United States)

    Marcuzzi, Annalisa; Vozzi, Diego; Girardelli, Martina; Tricarico, Paola Maura; Knowles, Alessandra; Crovella, Sergio; Vuch, Josef; Tommasini, Alberto; Piscianz, Elisa; Bianco, Anna Monica

    2016-04-01

    Mevalonate kinase deficiency (MKD) is an autosomal recessive auto‑inflammatory disease, caused by impairment of the mevalonate pathway. Although the molecular mechanism remains to be elucidated, there is clinical evidence suggesting that other regulatory genes may be involved in determining the phenotype. The identification of novel target genes may explain non‑homogeneous genotype‑phenotype correlations, and provide evidence in support of the hypothesis that novel regulatory genes predispose or amplify deregulation of the mevalonate pathway in this orphan disease. In the present study, DNA samples were obtained from five patients with MKD, which were then analyzed using whole exome sequencing. A missense variation in the PEX11γ gene was observed in homozygosis in P2, possibly correlating with visual blurring. The UNG rare gene variant was detected in homozygosis in P5, without correlating with a specific clinical phenotype. A number of other variants were found in the five analyzed DNA samples from the MKD patients, however no correlation with the phenotype was established. The results of the presents study suggested that further analysis, using next generation sequencing approaches, is required on a larger sample size of patients with MKD, who share the same MVK mutations and exhibit 'extreme' clinical phenotypes. As MVK mutations may be associated with MKD, the identification of specific modifier genes may assist in providing an earlier diagnosis.

  7. A putative viral defence mechanism in archaeal cells

    Directory of Open Access Journals (Sweden)

    Reidun Lillestøl

    2006-01-01

    Full Text Available Clusters of regularly spaced direct repeats, separated by unconserved spacer sequences, are ubiquitous in archaeal chromosomes and occur in some plasmids. Some clusters constitute around 1% of chromosomal DNA. Similarly structured clusters, generally smaller, also occur in some bacterial chromosomes. Although early studies implicated these clusters in segregation/partition functions, recent evidence suggests that the spacer sequences derive from extrachromosomal elements, and, primarily, viruses. This has led to the proposal that the clusters provide a defence against viral propagation in cells, and that both the mode of inhibition of viral propagation and the mechanism of adding spacer-repeat units to clusters, are dependent on RNAs transcribed from the clusters. Moreover, the putative inhibitory apparatus (piRNA-based may be evolutionarily related to the interference RNA systems (siRNA and miRNA, which are common in eukarya. Here, we analyze all the current data on archaeal repeat clusters and provide some new insights into their diverse structures, transcriptional properties and mode of structural development. The results are consistent with larger cluster transcripts being processed at the centers of the repeat sequences and being further trimmed by exonucleases to yield a dominant, intracellular RNA species, which corresponds approximately to the size of a spacer. Furthermore, analysis of the extensive clusters of Sulfolobus solfataricus strains P1 and P2B provides support for the presence of a flanking sequence adjoining a cluster being a prerequisite for the incorporation of new spacer-repeat units, which occurs between the flanking sequence and the cluster. An archaeal database summarizing the data will be maintained at http://dac.molbio.ku.dk/dbs/SRSR/.

  8. A putative transglycosylase encoded by SCO4132 influences morphological differentiation and actinorhodin production in Streptomyces coelicolor

    Institute of Scientific and Technical Information of China (English)

    Pengfei Xie; Ana Zeng; Xiaoting Lv; Qiuxiang Cheng; Zhongjun Qin

    2013-01-01

    Here we report that tgdA,a novel gene encoding a putative transglycosylase,affects both the morphological differentiation and the yield of blue-pigmented compound actinorhodin in Streptomyces coelicolor.The tgdA null mutant displays sparse aerial hyphae and irregular spore chains frequently lacking chromosomal DNA.Elevated actinorhodin production coincides with the overexpression of actⅡ-orf4 in mutant.tgdA expression is temporally and developmentally regulated.The tgdA orthologs in Streptomyces avermilitis and Streptomyces lividans also affect differentiation.

  9. Studies of the silencing of Baculovirus DNA binding protein

    NARCIS (Netherlands)

    Quadt, I.; Lent, van J.W.M.; Knebel-Morsdorf, D.

    2007-01-01

    Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple

  10. Studies of the silencing of Baculovirus DNA binding protein

    NARCIS (Netherlands)

    Quadt, I.; Lent, van J.W.M.; Knebel-Morsdorf, D.

    2007-01-01

    Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovir

  11. 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

    2012-06-01

    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

  12. Tomato protoplast DNA transformation : physical linkage and recombination of exogenous DNA sequences

    NARCIS (Netherlands)

    Jongsma, Maarten; Koornneef, Maarten; Zabel, Pim; Hille, Jacques

    1987-01-01

    Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There w

  13. Tomato protoplast DNA transformation : physical linkage and recombination of exogenous DNA sequences

    NARCIS (Netherlands)

    Jongsma, Maarten; Koornneef, Maarten; Zabel, Pim; Hille, Jacques

    1987-01-01

    Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There w

  14. Identification and Characterization of Putative Integron-Like Elements of the Heavy-Metal-Hypertolerant Strains of Pseudomonas spp.

    Science.gov (United States)

    Ciok, Anna; Adamczuk, Marcin; Bartosik, Dariusz; Dziewit, Lukasz

    2016-11-28

    Pseudomonas strains isolated from the heavily contaminated Lubin copper mine and Zelazny Most post-flotation waste reservoir in Poland were screened for the presence of integrons. This analysis revealed that two strains carried homologous DNA regions composed of a gene encoding a DNA_BRE_C domain-containing tyrosine recombinase (with no significant sequence similarity to other integrases of integrons) plus a three-component array of putative integron gene cassettes. The predicted gene cassettes encode three putative polypeptides with homology to (i) transmembrane proteins, (ii) GCN5 family acetyltransferases, and (iii) hypothetical proteins of unknown function (homologous proteins are encoded by the gene cassettes of several class 1 integrons). Comparative sequence analyses identified three structural variants of these novel integron-like elements within the sequenced bacterial genomes. Analysis of their distribution revealed that they are found exclusively in strains of the genus Pseudomonas.

  15. The DNA sequence of a 7941 bp fragment of the left arm of chromosome VII of Saccharomyces cerevisiae contains four open reading frames including the multicopy suppressor gene of the pop2 mutation and a putative serine/threonine protein kinase gene.

    Science.gov (United States)

    Coglievina, M; Bertani, I; Klima, R; Zaccaria, P; Bruschi, C V

    1995-06-30

    We report the sequence of a 7941 bp DNA fragment from the left arm of chromosome VII of Saccharomyces cerevisiae which contains four open reading frames (ORFs) of greater than 100 amino acid residues. ORF biC834 shows 100% bp identity with the recently identified multicopy suppressor gene of the pop2 mutation (MPT5); its deduced protein product carries an eight-repeat domain region, homologous to that found in the hypothetical regulatory YGL023 protein of S. cerevisiae and the Pumilio protein of Drosophila. ORF biE560 protein exhibits patterns typical of serine/threonine protein kinases, with which it shares high degrees of homology.

  16. Identification of NDM-1 in a Putatively Novel Acinetobacter Species ("NB14") Closely Related to Acinetobacter pittii.

    Science.gov (United States)

    Espinal, Paula; Mosqueda, Noraida; Telli, Murat; van der Reijden, Tanny; Rolo, Dora; Fernández-Orth, Dietmar; Dijkshoorn, Lenie; Roca, Ignasi; Vila, Jordi

    2015-10-01

    In this study, we describe the molecular characterization of a plasmid-located blaNDM-1 harbored by an Acinetobacter clinical isolate recovered from a patient in Turkey that putatively constitutes a novel Acinetobacter species, as shown by its distinct ARDRA (amplified 16S ribosomal DNA restriction analysis) profile and molecular sequencing techniques. blaNDM-1 was carried by a conjugative plasmid widespread among non-baumannii Acinetobacter isolates, suggesting its potential for dissemination before reaching more clinically relevant Acinetobacter species.

  17. Putative Corneal Neuralgia Responding to Vitamin D Supplementation

    Directory of Open Access Journals (Sweden)

    Eric L. Singman

    2013-09-01

    Full Text Available A patient with putative corneal neuralgia was incidentally discovered to have hypovitaminosis D. Supplementation of vitamin D appears to have led to a resolution of the patient's pain, whereas other efforts to treat the patient were unsuccessful.

  18. Identification and characterization of a gene encoding a putative lysophosphatidyl acyltransferase from Arachis hypogaea

    Indian Academy of Sciences (India)

    Si-Long Chen; Jia-Quan Huang; Lei Yong; Yue-Ting Zhang; Xiao-Ping Ren; Yu-Ning Chen; Hui-Fang Jiang; Li-Ying Yan; Yu-Rong Li; Bo-Shou Liao

    2012-12-01

    Lysophosphatidyl acyltransferase (LPAT) is the important enzyme responsible for the acylation of lysophosphatidic acid (LPA), leading to the generation of phosphatidic acid (PA) in plant. Its encoding gene is an essential candidate for oil crops to improve oil composition and increase seed oil content through genetic engineering. In this study, a full-length AhLPAT4 gene was isolated via cDNA library screening and rapid amplification of cDNA ends (RACE); our data demonstrated that AhLPAT4 had 1631 nucleotides, encoding a putative 43.8 kDa protein with 383 amino acid residues. The deduced protein included a conserved acyltransferase domain and four motifs (I–IV) with putative LPA and acyl-CoA catalytic and binding sites. Bioinformatic analysis indicated that AhLPAT4 contained four transmembrane domains (TMDs), localized to the endoplasmic reticulum (ER) membrane; detailed analysis indicated that motif I and motifs II–III in AhLPAT4 were separated by the third TMD, which located on cytosolic and ER luminal side respectively, and hydrophobic residues on the surface of AhLPAT4 protein fold to form a hydrophobic tunnel to accommodate the acyl chain. Subcellular localization analysis confirmed that AhLPAT4 was a cytoplasm protein. Phylogenetic analysis revealed that AhLPAT4 had a high homology (63.7–78.3%) with putative LPAT4 proteins from Glycine max, Arabidopsis thaliana and Ricinus communis. AhLPAT4 was ubiquitously expressed in diverse tissues except in flower, which is almost undetectable. The expression analysis in different developmental stages in peanut seeds indicated that AhLPAT4 did not coincide with oil accumulation.

  19. Mutation analysis of novel human liver-related putative tumor suppressor gene in hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Cheng Liao; Tsai-Ping Li; Mu-Jun Zhao; Jing Zhao; Hai Song; Pascal Pineau; Agnès Marchio; Anne Dejean; Pierre Tiollais; Hong-Yang Wang

    2003-01-01

    AIM: To find the point mutations meaningful for inactivationof liver-related putative tumor suppressor gene (LPTS) gene,a human novel liver-related putative tumor suppressor geneand telomerase inhibitor in hepatocellular carcinoma.METHODS: The entire coding sequence of LPTS genewas examined for mutations by single strand conformationpolymorphism (SSCP) assay and PCR products directsequencing in 56 liver cancer cell lines, 7 ovarian cancerand 7 head & neck tumor cell lines and 70 pairs of HCCtissues samples. The cDNA fragment coding for the mostfrequent mutant protein was subcloned into GST fusionexpression vector. The product was expressed in E. coliand purified by glutathione-agarose column. Telomericrepeat amplification protocol (TRAP) assays wereperformed to study the effect of point mutation totelomerase inhibitory activity.RESULTS: SSCP gels showed the abnormal shifting bandsand DNA sequencing found that there were 5 differentmutations and/or polymorphisms in 12 tumor cell lineslocated at exon2, exon5 and exon7. The main alterationswere A(778)A/G and A(880)T in exon7. The change in siteof 778 could not be found in HCC tissue samples, while themutation in position 880 was seen in 7 (10 %) cases. Themutation in the site of 880 had no effect on telomeraseinhibitory activity.CONCLUSION: Alterations identified in this study arepolymorphisms of LPTS gene. LPTS mutations occur in HCCbut are infrequent and of little effect on the telomeraseinhibitory function of the protein. Epigenetics, such asmethylation, acetylation, may play the key role in inactivationof LPTS.

  20. Characterization of Putative cis-Regulatory Elements in Genes Preferentially Expressed in Arabidopsis Male Meiocytes

    Directory of Open Access Journals (Sweden)

    Junhua Li

    2014-01-01

    Full Text Available Meiosis is essential for plant reproduction because it is the process during which homologous chromosome pairing, synapsis, and meiotic recombination occur. The meiotic transcriptome is difficult to investigate because of the size of meiocytes and the confines of anther lobes. The recent development of isolation techniques has enabled the characterization of transcriptional profiles in male meiocytes of Arabidopsis. Gene expression in male meiocytes shows unique features. The direct interaction of transcription factors (TFs with DNA regulatory sequences forms the basis for the specificity of transcriptional regulation. Here, we identified putative cis-regulatory elements (CREs associated with male meiocyte-expressed genes using in silico tools. The upstream regions (1 kb of the top 50 genes preferentially expressed in Arabidopsis meiocytes possessed conserved motifs. These motifs are putative binding sites of TFs, some of which share common functions, such as roles in cell division. In combination with cell-type-specific analysis, our findings could be a substantial aid for the identification and experimental verification of the protein-DNA interactions for the specific TFs that drive gene expression in meiocytes.

  1. Identification and functional analysis of Penicillium digitatum genes putatively involved in virulence towards citrus fruit.

    Science.gov (United States)

    López-Pérez, Mario; Ballester, Ana-Rosa; González-Candelas, Luis

    2015-04-01

    The fungus Penicillium digitatum, the causal agent of green mould rot, is the most destructive post-harvest pathogen of citrus fruit in Mediterranean regions. In order to identify P. digitatum genes up-regulated during the infection of oranges that may constitute putative virulence factors, we followed a polymerase chain reaction (PCR)-based suppression subtractive hybridization and cDNA macroarray hybridization approach. The origin of expressed sequence tags (ESTs) was determined by comparison against the available genome sequences of both organisms. Genes coding for fungal proteases and plant cell wall-degrading enzymes represent the largest categories in the subtracted cDNA library. Northern blot analysis of a selection of P. digitatum genes, including those coding for proteases, cell wall-related enzymes, redox homoeostasis and detoxification processes, confirmed their up-regulation at varying time points during the infection process. Agrobacterium tumefaciens-mediated transformation was used to generate knockout mutants for two genes encoding a pectin lyase (Pnl1) and a naphthalene dioxygenase (Ndo1). Two independent P. digitatum Δndo1 mutants were as virulent as the wild-type. However, the two Δpnl1 mutants analysed were less virulent than the parental strain or an ectopic transformant. Together, these results provide a significant advance in our understanding of the putative determinants of the virulence mechanisms of P. digitatum.

  2. Draft Genome Sequence of Parabacteroides goldsteinii with Putative Novel Metallo-β-Lactamases Isolated from a Blood Culture from a Human Patient

    DEFF Research Database (Denmark)

    Krogh, Thøger Jensen; Agergaard, Charlotte Nielsen; Møller-Jensen, Jakob;

    2015-01-01

    Parabacteroides goldsteinii was isolated from a blood culture. Genomic DNA was sequenced using a MiSeq sequencer and assembled using the SPAdes genome assembler. The draft genome sequence was 6,851,868 bp, spanning 282 contigs of 5,253 coding sequences, 66 tRNAs, and 5 rRNAs. Several putative novel...

  3. Recombinational DNA repair is regulated by compartmentalization of DNA lesions at the nuclear pore complex

    DEFF Research Database (Denmark)

    Géli, Vincent; Lisby, Michael

    2015-01-01

    and colleagues shows that also physiological threats to genome integrity such as DNA secondary structure-forming triplet repeat sequences relocalize to the NPC during DNA replication. Mutants that fail to reposition the triplet repeat locus to the NPC cause repeat instability. Here, we review the types of DNA...... lesions that relocalize to the NPC, the putative mechanisms of relocalization, and the types of recombinational repair that are stimulated by the NPC, and present a model for NPC-facilitated repair....

  4. 烟草拟核小体组装蛋白cDNA的克隆、表达及其蛋白产物的纯化%Cloning and Expression of a Putative Tobacco Nucleosome Assembly Protein cDNA and Purification of Its Product

    Institute of Scientific and Technical Information of China (English)

    俞瑜; 董爱武; 朱炎; 曹凯鸣; 孙崇荣

    2002-01-01

    从烟草BY2细胞cDNA文库中筛选到核小体组装蛋白1(Nucleosome assembly protein 1, NAP 1)的cDNA,序列分析发现该NAP1的cDNA编码374个氨基酸,与已知植物来源的NAP1同源蛋白相比具有80%以上的同源性,而且一些具有重要功能的结构域是相当保守的,如核定位信号,核输出信号,酸性结构域及异戊烯化修饰位点,重要功能结构域的存在预示了NAP1蛋白潜在的多种功能. 为了深入研究NAP1的功能,应用大肠杆菌表达体系高效表达了烟草NAP1蛋白,并对表达产物进行了分离纯化.

  5. The gene corresponding to the putative Goodpasture antigen is present in Alport's syndrome.

    Science.gov (United States)

    Savige, J A

    1991-08-01

    Alport's syndrome is a heterogeneous group of inherited abnormalities of basement membranes that may result in progressive renal failure, defective hearing and lens abnormalities. The glomerular basement membrane (GBM) characteristically has areas of reduplication, lamellation and attenuation on electron microscopic examination. In the majority of affected males and some females, there is reduced or variable binding of serum from patients with anti-GBM disease (Goodpasture's syndrome) to these basement membranes. These sera contain antibodies directed against the Goodpasture antigen which has been thought to be located in the non-collagenous domain of the alpha3 chain of type IV collagen and is presumed to be important in cross-linking of the collagen molecules. The reduced staining for the Goodpasture antigen suggests that this structure is either absent or masked in Alport's syndrome. We have tested DNA from six unrelated individuals with Alport's syndrome. All had been transplanted for renal failure. The diagnosis of Alport's syndrome was made on the characteristic electron microscopic appearance of the renal basement membranes (n = 4), the presence of sensori-neural deafness (n = 4), a family history of Alport's syndrome (n = 5) and the presence of circulating inhibitable anti-GBM antibody activity post-transplant (n = 2). Oligonucleotides (20mers) corresponding to the 5' and 3' ends of the known 25 amino acid sequence for the putative Goodpasture antigen were used as primers for amplification of genomic DNA. The products were then blotted and probed with an intermediate 19-mer DNA. All Alport's patients contained a 75-bp fragment corresponding to the published peptide sequence for the non-collagenous domain of the alpha 3 chain of type IV collagen, suggesting that a large deletion of this region, the putative Goodpasture antigen, is unlikely to account for the defect in Alport's syndrome.

  6. Paternity testing that involves a DNA mixture.

    Science.gov (United States)

    Mortera, Julia; Vecchiotti, Carla; Zoppis, Silvia; Merigioli, Sara

    2016-07-01

    Here we analyse a complex disputed paternity case, where the DNA of the putative father was extracted from his corpse that had been inhumed for over 20 years. This DNA was contaminated and appears to be a mixture of at least two individuals. Furthermore, the mother's DNA was not available. The DNA mixture was analysed so as to predict the most probable genotypes of each contributor. The major contributor's profile was then used to compute the likelihood ratio for paternity. We also show how to take into account a dropout allele and the possibility of mutation in paternity testing.

  7. Isolation and characterization of two mitoviruses and a putative alphapartitivirus from Fusarium spp.

    Science.gov (United States)

    Osaki, Hideki; Sasaki, Atsuko; Nomiyama, Koji; Sekiguchi, Hiroyuki; Tomioka, Keisuke; Takehara, Toshiaki

    2015-06-01

    The filamentous fungus Fusarium spp. includes several important plant pathogens. We attempted to reveal presence of double-stranded (ds) RNAs in the genus. Thirty-seven Fusarium spp. at the MAFF collection were analyzed. In the strains of Fusarium coeruleum, Fusarium globosum and Fusarium solani f. sp. pisi, single dsRNA bands were detected. The strains of F. coeruleum and F. solani f. sp. pisi cause potato dry rot and mulberry twig blight, respectively. Sequence analyses revealed that dsRNAs in F. coeruleum and F. globosum consisted of 2423 and 2414 bp, respectively. Using the fungal mitochondrial translation table, the positive strands of these cDNAs were found to contain single open reading frames with the potential to encode a protein of putative 757 and 717 amino acids (molecular mass 88.5 and 84.0 kDa, respectively), similar to RNA-dependent RNA polymerases of members of the genus Mitovirus. These dsRNAs in F. coeruleum and F. globosum were assigned to the genus Mitovirus (family Narnaviridae), and these two mitoviruses were designated as Fusarium coeruleum mitovirus 1 and Fusarium globosum mitovirus 1. On the other hand, a positive strand of cDNA (1950 bp) from dsRNA in F. solani f. sp. pisi contained an ORF potentially encoding a putative RdRp of 608 amino acids (72.0 kDa). The putative RdRp was shown to be related to those of members of the genus of Alphapartitivirus (family Partitiviridae). We coined the name Fusarium solani partitivirus 2 for dsRNA in F. solani f. sp. pisi.

  8. Mixed Nipple Infections Caused by Variant of BPV3 and a Putative New Subtype of BPV in Cattle.

    Science.gov (United States)

    He, Z; Meng, Q; Qiao, J; Peng, Y; Xie, K; Liu, Y; Cai, X; Zhang, J; Chen, C

    2016-02-01

    Bovine papilloma is a chronic and proliferative skin and mucosal wart caused by Bovine papillomavirus (BPV). In June, 2013, a leaf-and flat-shaped wart disease was observed on the nipple skins in a cattle farm in Xinjiang. To diagnose the disease, we collected the diseased skins for pathological biopsy and DNA analysis by PCR amplification using a pair of degenerate primers FAP59 and FAP64. Sequencing and phylogenetic analysis showed that the infection was caused by a variant of BPV3 and putatively a new subtype of BPV (BPV/CHI-SW1, belonging to the Xi papillomavirus genus). This is the first report of mixed infection caused by variant of BPV3 and BPV (putatively new subtype) in China, and would be of importance for the molecular epidemiological study of the disease.

  9. A putative viral defence mechanism in archaeal cells

    DEFF Research Database (Denmark)

    Lillestøl, Reidun K; Redder, Peter; Garrett, Roger Antony

    2006-01-01

    in cells, and that both the mode of inhibition of viral propagation and the mechanism of adding spacer-repeat units to clusters, are dependent on RNAs transcribed from the clusters. Moreover, the putative inhibitory apparatus (piRNA-based) may be evolutionarily related to the interference RNA systems (si...

  10. Putative Lineage of Novel African Usutu Virus, Central Europe

    Centers for Disease Control (CDC) Podcasts

    2015-10-15

    Sarah Gregory reads an abridged version of "Putative Lineage of Novel African Usutu Virus, Central Europe.".  Created: 10/15/2015 by National Center for Emerging and Zoonotic Infectious Diseases (NCEZID).   Date Released: 10/15/2015.

  11. Putative golden proportions as predictors of facial esthetics in adolescents.

    NARCIS (Netherlands)

    Kiekens, R.M.A.; Kuijpers-Jagtman, A.M.; Hof, M.A. van 't; Hof, BE van 't; Maltha, J.C.

    2008-01-01

    INTRODUCTION: In orthodontics, facial esthetics is assumed to be related to golden proportions apparent in the ideal human face. The aim of the study was to analyze the putative relationship between facial esthetics and golden proportions in white adolescents. METHODS: Seventy-six adult laypeople

  12. Transcriptome-Based Examination of Putative Pollen Allergens of Rice(Oryza sativa ssp.japonica)

    Institute of Scientific and Technical Information of China (English)

    Scott D.Russell; Prem L Bhalla; Mohan B.Singh

    2008-01-01

    Pollen allergens are among the most abundantly transcribed and translated products in the Iife history of plants,and particularly grasses.To identify different pollen allergens in rice,putative allergens were identified in the rice genome and their expression characterized using the Affymetrix 57K rice GeneChip microarray.Among the most abundant pollen-specific candidate transcripts were Ory s 1 beta-expansin.Ory s 2,Ory s 7 EFhand,Ory s 11,Ory s 12 profilin A,Ory s 23,glycosyl hydrolase family 28(polygalacturonase).and FAD binding proteins.Highly expressed pollen proteins are frequently present in multiple copy numbers,sometimes with mirror images Iocated on nearby regions of the opposite DNA strand.Many of these are intronless and inserted as copies that retain nearly exact copies of their regulatory elements.Ory s 23 reflects low variability and high copy number,suggesting recent gene amplification.Some copies contain pseudogenes,which may reflect their origin through activity of retrotransposition;some putative allergenic sequences bear fusion products with repeat sequences of transposable elements(LTRs).The abundance of nearby repetitive sequences,activation of transposable elements.and high production of mRNA transcripts appear to coincide in pollen and may contribute to a syndrome in which highly transcribed proteins may be copied and inserted with streamlined features for translation,including grouping and removaI of introns.

  13. Transcriptome of Aphanomyces euteiches: new oomycete putative pathogenicity factors and metabolic pathways.

    Directory of Open Access Journals (Sweden)

    Elodie Gaulin

    Full Text Available Aphanomyces euteiches is an oomycete pathogen that causes seedling blight and root rot of legumes, such as alfalfa and pea. The genus Aphanomyces is phylogenically distinct from well-studied oomycetes such as Phytophthora sp., and contains species pathogenic on plants and aquatic animals. To provide the first foray into gene diversity of A. euteiches, two cDNA libraries were constructed using mRNA extracted from mycelium grown in an artificial liquid medium or in contact to plant roots. A unigene set of 7,977 sequences was obtained from 18,864 high-quality expressed sequenced tags (ESTs and characterized for potential functions. Comparisons with oomycete proteomes revealed major differences between the gene content of A. euteiches and those of Phytophthora species, leading to the identification of biosynthetic pathways absent in Phytophthora, of new putative pathogenicity genes and of expansion of gene families encoding extracellular proteins, notably different classes of proteases. Among the genes specific of A. euteiches are members of a new family of extracellular proteins putatively involved in adhesion, containing up to four protein domains similar to fungal cellulose binding domains. Comparison of A. euteiches sequences with proteomes of fully sequenced eukaryotic pathogens, including fungi, apicomplexa and trypanosomatids, allowed the identification of A. euteiches genes with close orthologs in these microorganisms but absent in other oomycetes sequenced so far, notably transporters and non-ribosomal peptide synthetases, and suggests the presence of a defense mechanism against oxidative stress which was initially characterized in the pathogenic trypanosomatids.

  14. Biochemical Characterization of Putative Adenylate Dimethylallyltransferase and Cytokinin Dehydrogenase from Nostoc sp. PCC 7120.

    Science.gov (United States)

    Frébortová, Jitka; Greplová, Marta; Seidl, Michael F; Heyl, Alexander; Frébort, Ivo

    2015-01-01

    Cytokinins, a class of phytohormones, are adenine derivatives common to many different organisms. In plants, these play a crucial role as regulators of plant development and the reaction to abiotic and biotic stress. Key enzymes in the cytokinin synthesis and degradation in modern land plants are the isopentyl transferases and the cytokinin dehydrogenases, respectively. Their encoding genes have been probably introduced into the plant lineage during the primary endosymbiosis. To shed light on the evolution of these proteins, the genes homologous to plant adenylate isopentenyl transferase and cytokinin dehydrogenase were amplified from the genomic DNA of cyanobacterium Nostoc sp. PCC 7120 and expressed in Escherichia coli. The putative isopentenyl transferase was shown to be functional in a biochemical assay. In contrast, no enzymatic activity was detected for the putative cytokinin dehydrogenase, even though the principal domains necessary for its function are present. Several mutant variants, in which conserved amino acids in land plant cytokinin dehydrogenases had been restored, were inactive. A combination of experimental data with phylogenetic analysis indicates that adenylate-type isopentenyl transferases might have evolved several times independently. While the Nostoc genome contains a gene coding for protein with characteristics of cytokinin dehydrogenase, the organism is not able to break down cytokinins in the way shown for land plants.

  15. Functional characterization of PaLAX1, a putative auxin permease, in heterologous plant systems.

    Science.gov (United States)

    Hoyerová, Klára; Perry, Lucie; Hand, Paul; Lanková, Martina; Kocábek, Tomás; May, Sean; Kottová, Jana; Paces, Jan; Napier, Richard; Zazímalová, Eva

    2008-03-01

    We have isolated the cDNA of the gene PaLAX1 from a wild cherry tree (Prunus avium). The gene and its product are highly similar in sequences to both the cDNAs and the corresponding protein products of AUX/LAX-type genes, coding for putative auxin influx carriers. We have prepared and characterized transformed Nicotiana tabacum and Arabidopsis thaliana plants carrying the gene PaLAX1. We have proved that constitutive overexpression of PaLAX1 is accompanied by changes in the content and distribution of free indole-3-acetic acid, the major endogenous auxin. The increase in free indole-3-acetic acid content in transgenic plants resulted in various phenotype changes, typical for the auxin-overproducing plants. The uptake of synthetic auxin, 2,4-dichlorophenoxyacetic acid, was 3 times higher in transgenic lines compared to the wild-type lines and the treatment with the auxin uptake inhibitor 1-naphthoxyacetic acid reverted the changes caused by the expression of PaLAX1. Moreover, the agravitropic response could be restored by expression of PaLAX1 in the mutant aux1 plants, which are deficient in auxin influx carrier activity. Based on our data, we have concluded that the product of the gene PaLAX1 promotes the uptake of auxin into cells, and, as a putative auxin influx carrier, it affects the content and distribution of free endogenous auxin in transgenic plants.

  16. EST Table: CK518826 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available rected DNA polymerase activity)|GO:0006278(RNA-dependent DNA replication) 10/09/29 61 %/161 aa ref|XP_002431503.1| enzyma...tic polyprotein; Endonuclease; Reverse transcriptase, putative [Pediculus humanus corporis] gb|EEB18765.1| enzyma

  17. EST Table: CK524730 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available rected DNA polymerase activity)|GO:0006278(RNA-dependent DNA replication) 10/09/29 61 %/149 aa ref|XP_002431503.1| enzyma...tic polyprotein; Endonuclease; Reverse transcriptase, putative [Pediculus humanus corporis] gb|EEB18765.1| enzyma

  18. EST Table: CK526515 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available 77(DNA binding)|GO:0015074(DNA integration) 10/09/29 46 %/144 aa ref|XP_002431503.1| enzymatic polyprotein; ...Endonuclease; Reverse transcriptase, putative [Pediculus humanus corporis] gb|EEB18765.1| enzymatic polyprot

  19. Genetic identification of putative remains of the famous astronomer Nicolaus Copernicus.

    Science.gov (United States)

    Bogdanowicz, Wiesław; Allen, Marie; Branicki, Wojciech; Lembring, Maria; Gajewska, Marta; Kupiec, Tomasz

    2009-07-28

    We report the results of mitochondrial and nuclear DNA analyses of skeletal remains exhumed in 2005 at Frombork Cathedral in Poland, that are thought to be those of Nicolaus Copernicus (1473-1543). The analyzed bone remains were found close to the altar Nicolaus Copernicus was responsible for during his tenure as priest. The mitochondrial DNA (mtDNA) profiles from 3 upper molars and the femurs were identical, suggesting that the remains originate from the same individual. Identical mtDNA profiles were also determined in 2 hairs discovered in a calendar now exhibited at Museum Gustavianum in Uppsala, Sweden. This calendar was the property of Nicolaus Copernicus for much of his life. These findings, together with anthropological data, support the identification of the human remains found in Frombork Cathedral as those of Nicolaus Copernicus. Up-to-now the particular mtDNA haplotype has been observed only 3 times in Germany and once in Denmark. Moreover, Y-chromosomal and autosomal short tandem repeat markers were analyzed in one of the tooth samples, that was much better preserved than other parts of the skeleton. Molecular sex determination revealed that the skeleton is from a male individual, and this result is consistent with morphological investigations. The minimal Y-chromosomal haplotype determined in the putative remains of Nicolaus Copernicus has been observed previously in many countries, including Austria, Germany, Poland, and the Czech Republic. Finally, an analysis of the SNP located in the HERC2 gene revealed the C/C genotype that is predominant in blue-eyed humans, suggesting that Copernicus may have had a light iris color.

  20. Structure determination and biochemical characterization of a putative HNH endonuclease from Geobacter metallireducens GS-15.

    Directory of Open Access Journals (Sweden)

    Shuang-yong Xu

    Full Text Available The crystal structure of a putative HNH endonuclease, Gmet_0936 protein from Geobacter metallireducens GS-15, has been determined at 2.6 Å resolution using single-wavelength anomalous dispersion method. The structure contains a two-stranded anti-parallel β-sheet that are surrounded by two helices on each face, and reveals a Zn ion bound in each monomer, coordinated by residues Cys38, Cys41, Cys73, and Cys76, which likely plays an important structural role in stabilizing the overall conformation. Structural homologs of Gmet_0936 include Hpy99I endonuclease, phage T4 endonuclease VII, and other HNH endonucleases, with these enzymes sharing 15-20% amino acid sequence identity. An overlay of Gmet_0936 and Hpy99I structures shows that most of the secondary structure elements, catalytic residues as well as the zinc binding site (zinc ribbon are conserved. However, Gmet_0936 lacks the N-terminal domain of Hpy99I, which mediates DNA binding as well as dimerization. Purified Gmet_0936 forms dimers in solution and a dimer of the protein is observed in the crystal, but with a different mode of dimerization as compared to Hpy99I. Gmet_0936 and its N77H variant show a weak DNA binding activity in a DNA mobility shift assay and a weak Mn²⁺-dependent nicking activity on supercoiled plasmids in low pH buffers. The preferred substrate appears to be acid and heat-treated DNA with AP sites, suggesting Gmet_0936 may be a DNA repair enzyme.

  1. Molecular mapping of the putative gonadoblastoma locus on the Y chromosome.

    Science.gov (United States)

    Salo, P; Kääriäinen, H; Petrovic, V; Peltomäki, P; Page, D C; de la Chapelle, A

    1995-11-01

    Based on the high incidence of gonadoblastoma in females with XY gonadal dysgenesis or 45,X/46,XY mosaicism, the existence of a susceptibility locus on the Y chromosome (GBY) has been postulated. We attempted to map GBY by making use of a recently developed dense map of Y-chromosomal sequence-tagged sites (STSs). In two female patients with gonadoblastoma, small marker chromosomes contained portions of the Y chromosome, and a single region of overlap could be defined extending from probe pDP97 in interval 4B, which contains the centromere, to marker sY182 in interval 5E of the proximal long arm. This interval is contained in a YAC contig that comprises approximately 4 Mb of DNA. Our findings confirm the previous localization of GBY and greatly refine it. The localization of GBY overlaps with the region to which a putative growth determinant, GCY, was recently assigned.

  2. Phylogenetic relationships within Pelargonium section Peristera (Geraniaceae) inferred from nrDNA and cpDNA sequence comparisons.

    NARCIS (Netherlands)

    Bakker, F.T.; Helbrugge, D.; Culham, A.; Gibby, M.

    1998-01-01

    Phylogenetic analysis of nrDNA ITS and tmL (UAA) 5' exon-tmF (GAA) chloroplast DNA sequences from 17 species of Pelargonium sect. Peristera, together with nine putative outgroups, suggests paraphyly for the section and a close relationship between the highly disjurmt South African and Australian spe

  3. Determination of cDNA and genomic DNA sequences of hevamine, a chitinase from the rubber tree Hevea brasiliensis

    NARCIS (Netherlands)

    Bokma, E; Spiering, M; Chow, KS; Mulder, PPMFA; Subroto, T; Beintema, JJ

    2001-01-01

    Hevamine is a chitinase from the rubber tree Hevea brasiliensis and belongs to the family 18 glycosyl hydrolases. This paper describes the cloning of hevamine DNA and cDNA sequences. Hevamine contains a signal peptide at the N-terminus and a putative vacuolar targeting sequence at the C-terminus whi

  4. Structural organization of DNA in chlorella viruses.

    Directory of Open Access Journals (Sweden)

    Timo Wulfmeyer

    Full Text Available Chlorella viruses have icosahedral capsids with an internal membrane enclosing their large dsDNA genomes and associated proteins. Their genomes are packaged in the particles with a predicted DNA density of ca. 0.2 bp nm(-3. Occasionally infection of an algal cell by an individual particle fails and the viral DNA is dynamically ejected from the capsid. This shows that the release of the DNA generates a force, which can aid in the transfer of the genome into the host in a successful infection. Imaging of ejected viral DNA indicates that it is intimately associated with proteins in a periodic fashion. The bulk of the protein particles detected by atomic force microscopy have a size of ∼60 kDa and two proteins (A278L and A282L of about this size are among 6 basic putative DNA binding proteins found in a proteomic analysis of DNA binding proteins packaged in the virion. A combination of fluorescence images of ejected DNA and a bioinformatics analysis of the DNA reveal periodic patterns in the viral DNA. The periodic distribution of GC rich regions in the genome provides potential binding sites for basic proteins. This DNA/protein aggregation could be responsible for the periodic concentration of fluorescently labeled DNA observed in ejected viral DNA. Collectively the data indicate that the large chlorella viruses have a DNA packaging strategy that differs from bacteriophages; it involves proteins and share similarities to that of chromatin structure in eukaryotes.

  5. Genetic profiling of putative breast cancer stem cells from malignant pleural effusions.

    Science.gov (United States)

    Tiran, Verena; Stanzer, Stefanie; Heitzer, Ellen; Meilinger, Michael; Rossmann, Christopher; Lax, Sigurd; Tsybrovskyy, Oleksiy; Dandachi, Nadia; Balic, Marija

    2017-01-01

    A common symptom during late stage breast cancer disease is pleural effusion, which is related to poor prognosis. Malignant cells can be detected in pleural effusions indicating metastatic spread from the primary tumor site. Pleural effusions have been shown to be a useful source for studying metastasis and for isolating cells with putative cancer stem cell (CSC) properties. For the present study, pleural effusion aspirates from 17 metastatic breast cancer patients were processed to propagate CSCs in vitro. Patient-derived aspirates were cultured under sphere forming conditions and isolated primary cultures were further sorted for cancer stem cell subpopulations ALDH1+ and CD44+CD24-/low. Additionally, sphere forming efficiency of CSC and non-CSC subpopulations was determined. In order to genetically characterize the different tumor subpopulations, DNA was isolated from pleural effusions before and after cell sorting, and compared with corresponding DNA copy number profiles from primary tumors or bone metastasis using low-coverage whole genome sequencing (SCNA-seq). In general, unsorted cells had a higher potential to form spheres when compared to CSC subpopulations. In most cases, cell sorting did not yield sufficient cells for copy number analysis. A total of five from nine analyzed unsorted pleura samples (55%) showed aberrant copy number profiles similar to the respective primary tumor. However, most sorted subpopulations showed a balanced profile indicating an insufficient amount of tumor cells and low sensitivity of the sequencing method. Finally, we were able to establish a long term cell culture from one pleural effusion sample, which was characterized in detail. In conclusion, we confirm that pleural effusions are a suitable source for enrichment of putative CSC. However, sequencing based molecular characterization is impeded due to insufficient sensitivity along with a high number of normal contaminating cells, which are masking genetic alterations of

  6. Putative melatonin receptors in a human biological clock

    Energy Technology Data Exchange (ETDEWEB)

    Reppert, S.M.; Weaver, D.R.; Rivkees, S.A.; Stopa, E.G.

    1988-10-07

    In vitro autoradiography with /sup 125/I-labeled melatonin was used to examine melatonin binding sites in human hypothalamus. Specific /sup 125/I-labeled melatonin binding was localized to the suprachiasmatic nuclei, the site of a putative biological clock, and was not apparent in other hypothalamic regions. Specific /sup 125/I-labeled melatonin binding was consistently found in the suprachiasmatic nuclei of hypothalami from adults and fetuses. Densitometric analysis of competition experiments with varying concentrations of melatonin showed monophasic competition curves, with comparable half-maximal inhibition values for the suprachiasmatic nuclei of adults (150 picomolar) and fetuses (110 picomolar). Micromolar concentrations of the melatonin agonist 6-chloromelatonin completely inhibited specific /sup 125/I-labeled melatonin binding, whereas the same concentrations of serotonin and norepinephrine caused only a partial reduction in specific binding. The results suggest that putative melatonin receptors are located in a human biological clock.

  7. Trypanosoma brucei: a putative RNA polymerase II promoter.

    Science.gov (United States)

    Bayele, Henry K

    2009-12-01

    RNA polymerase II (pol II) promoters are rare in the African trypanosome Trypanosoma brucei because gene regulation in the parasite is complex and polycistronic. Here, we describe a putative pol II promoter and its structure-function relationship. The promoter has features of an archetypal eukaryotic pol II promoter including putative canonical CCAAT and TATA boxes, and an initiator element. However, the spatial arrangement of these elements is only similar to yeast pol II promoters. Deletion mapping and transcription assays enabled delineation of a minimal promoter that could drive orientation-independent reporter gene expression suggesting that it may be a bidirectional promoter. In vitro transcription in a heterologous nuclear extract revealed that the promoter can be recognized by the basal eukaryotic transcription complex. This suggests that the transcription machinery in the parasite may be very similar to those of other eukaryotes.

  8. Chromosomal Abnormalities and Putative Susceptibility Genes in Autism Spectrum Disorders

    DEFF Research Database (Denmark)

    Nielsen, Mette Gilling

    Autism spectrum disorders (ASDs) is a heterogeneous group of neurodevelopmental disorders with a significant genetic component as shown by family and twin studies. However, only a few genes have repeatedly been shown to be involved in the development of ASDs. The aim of this study has been...... to identify possible ASD susceptibility genes. Genome screens in ASD patients suggest possible susceptibility gene regions on almost every chromosome. We identified four ASD patients with chromosomal rearrangements, two of which were familial rearrangements involving one of these putative susceptibility gene......) was performed for all four patients. By combination of these methods we identified several putative susceptibility genes for ASDs. Expression patterns were established for several of these genes by Quantitative PCR (Q-PCR) or in situ hybridization and one gene was sequenced in 157 ASD patients. Our results...

  9. A putative role for apelin in the etiology of obesity.

    Science.gov (United States)

    Rayalam, Srujana; Della-Fera, Mary Anne; Krieg, Paul A; Cox, Christopher M; Robins, Allan; Baile, Clifton A

    2008-04-11

    Apelin, the endogenous ligand of the G protein-coupled APJ receptor has been shown to promote tumor angiogenesis. However, the effect of apelin on inducing angiogenesis in adipose tissue has not been investigated. In this review, we propose a putative role for apelin in promoting angiogenesis in adipose tissue. We further propose that targeting adipose tissue vasculature by blocking apelin signaling with anti-apelin antibodies will lead not only to inhibition of angiogenesis in adipose tissue but also to decreased adiposity.

  10. Interindividual variation in DNA methylation at a putative POMC metastable epiallele is associated with obesity

    Science.gov (United States)

    The estimated heritability of human BMI is close to 75%, but identified genetic variants explain only a small fraction of interindividual body-weight variation. Inherited epigenetic variants identified in mouse models named "metastable epialleles" could in principle explain this "missing heritabilit...

  11. Studies of the silencing of Baculovirus DNA binding protein

    OpenAIRE

    Quadt, I.; Lent, van, J.W.M.; Knebel-Morsdorf, D.

    2007-01-01

    Baculovirus DNA binding protein (DBP) binds preferentially single-stranded DNA in vitro and colocalizes with viral DNA replication sites. Here, its putative role as viral replication factor has been addressed by RNA interference. Silencing of DBP in Autographa californica multiple nucleopolyhedrovirus-infected cells increased expression of LEF-3, LEF-4, and P35. In contrast, expression of the structural genes coding for P39 and polyhedrin was suppressed while expression of genes coding for P1...

  12. Isolation and Identification of Putative Oral Cancer Stem Cells

    Institute of Scientific and Technical Information of China (English)

    ZHANG Min; ZHAO Yan-Hua; TANG Xiao-Fei

    2011-01-01

    Objective: To isolate and characterize putative cancer stem cells in Tea8113 oral squmous cell carcinoma cell line. Methods: Putative cancer stem cells were isolated by limited dilution assay in Tea8113 cell line. Biological features of putative cancer stem cells were detected by MTT assay, flow cytometry, immunofluorescence, Colony Forming Efficiency assays, cell motility assay and in vivo tumor formation experiment. Results: Compared with untreated Tea8113 cells, the putative cancer stem cells proliferated more quickly and showed heteroploid cell cycle,higher G0/G1-arrested cells, higher CFE and higher