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Sample records for acid dna molecule

  1. Enzymatic DNA molecules

    NASA Technical Reports Server (NTRS)

    Joyce, Gerald F. (Inventor); Breaker, Ronald R. (Inventor)

    1998-01-01

    The present invention discloses deoxyribonucleic acid enzymes--catalytic or enzymatic DNA molecules--capable of cleaving nucleic acid sequences or molecules, particularly RNA, in a site-specific manner, as well as compositions including same. Methods of making and using the disclosed enzymes and compositions are also disclosed.

  2. [The effect of spermine on acid-base equilibrium in DNA molecule].

    PubMed

    Slonitskiĭ, S V; Kuptsov, V Iu

    1990-01-01

    The influence of spermine (Sp) on the acid-induced predenaturational and denaturational transitions in the DNA molecule structure has been studied by means of circular dichroism, spectrophotometric and viscometric titration at supporting electrolyte concentration 10 mM NaCl. The data available indicate that at [N]/[P] less than or equal to 0.60 (here [N] and [P] are molar concentrations of Sp nitrogen and DNA phosphours, respectively) the cooperative structural B----B(+)----S transitions are accompanied by the DNA double-helice winding. No competition for proton acceptor sites in the DNA molecule between H+ and Sp4+ cations has been observed when binding to neutral macromolecule. At 0.60 less than or equal to [N]/[P] less than or equal to 0.75 the displacement of the B----B(+)----S transitions midpoints to acidic pH region has been established. This is accompanied by DNA condensation and the appearance of differential scattering of circularly polarized light. The calculations carried out in the framework of the two-variable Manning theory have shown that the acid-induced reduction of the effective polyion charge density facilitates the Sp-induced DNA condensation. It has been shown that the acid-base equilibrium in the DNA molecule is determined by local [H+] in the 2-3 A hydrated monolayer of the macromolecule. An adequate estimation of [H+] can be obtained on the basis of the Poisson-Boltzman approach. The data obtained are consistent with recently proposed hypothesis of polyelectrolyte invariance of the acid-base equilibrium in the DNA molecule.

  3. Single molecule DNA interaction kinetics of retroviral nucleic acid chaperone proteins

    NASA Astrophysics Data System (ADS)

    Williams, Mark

    2010-03-01

    Retroviral nucleocapsid (NC) proteins are essential for several viral replication processes including specific genomic RNA packaging and reverse transcription. The nucleic acid chaperone activity of NC facilitates the latter process. In this study, we use single molecule biophysical methods to quantify the DNA interactions of wild type and mutant human immunodeficiency virus type 1 (HIV-1) NC and Gag and human T-cell leukemia virus type 1 (HTLV-1) NC. We find that the nucleic acid interaction properties of these proteins differ significantly, with HIV-1 NC showing rapid protein binding kinetics, significant duplex destabilization, and strong DNA aggregation, all properties that are critical components of nucleic acid chaperone activity. In contrast, HTLV-1 NC exhibits significant destabilization activity but extremely slow DNA interaction kinetics and poor aggregating capability, which explains why HTLV-1 NC is a poor nucleic acid chaperone. To understand these results, we developed a new single molecule method for quantifying protein dissociation kinetics, and applied this method to probe the DNA interactions of wild type and mutant HIV-1 and HTLV-1 NC. We find that mutations to aromatic and charged residues strongly alter the proteins' nucleic acid interaction kinetics. Finally, in contrast to HIV-1 NC, HIV-1 Gag, the nucleic acid packaging protein that contains NC as a domain, exhibits relatively slow binding kinetics, which may negatively impact its ability to act as a nucleic acid chaperone.

  4. Disentangling DNA molecules.

    PubMed

    Vologodskii, Alexander

    2016-09-01

    The widespread circular form of DNA molecules inside cells creates very serious topological problems during replication. Due to the helical structure of the double helix the parental strands of circular DNA form a link of very high order, and yet they have to be unlinked before the cell division. DNA topoisomerases, the enzymes that catalyze passing of one DNA segment through another, solve this problem in principle. However, it is very difficult to remove all entanglements between the replicated DNA molecules due to huge length of DNA comparing to the cell size. One strategy that nature uses to overcome this problem is to create the topoisomerases that can dramatically reduce the fraction of linked circular DNA molecules relative to the corresponding fraction at thermodynamic equilibrium. This striking property of the enzymes means that the enzymes that interact with DNA only locally can access their topology, a global property of circular DNA molecules. This review considers the experimental studies of the phenomenon and analyzes the theoretical models that have been suggested in attempts to explain it. We describe here how various models of enzyme action can be investigated computationally. There is no doubt at the moment that we understand basic principles governing enzyme action. Still, there are essential quantitative discrepancies between the experimental data and the theoretical predictions. We consider how these discrepancies can be overcome.

  5. Disentangling DNA molecules

    NASA Astrophysics Data System (ADS)

    Vologodskii, Alexander

    2016-09-01

    The widespread circular form of DNA molecules inside cells creates very serious topological problems during replication. Due to the helical structure of the double helix the parental strands of circular DNA form a link of very high order, and yet they have to be unlinked before the cell division. DNA topoisomerases, the enzymes that catalyze passing of one DNA segment through another, solve this problem in principle. However, it is very difficult to remove all entanglements between the replicated DNA molecules due to huge length of DNA comparing to the cell size. One strategy that nature uses to overcome this problem is to create the topoisomerases that can dramatically reduce the fraction of linked circular DNA molecules relative to the corresponding fraction at thermodynamic equilibrium. This striking property of the enzymes means that the enzymes that interact with DNA only locally can access their topology, a global property of circular DNA molecules. This review considers the experimental studies of the phenomenon and analyzes the theoretical models that have been suggested in attempts to explain it. We describe here how various models of enzyme action can be investigated computationally. There is no doubt at the moment that we understand basic principles governing enzyme action. Still, there are essential quantitative discrepancies between the experimental data and the theoretical predictions. We consider how these discrepancies can be overcome.

  6. Piezoresistivity in single DNA molecules

    PubMed Central

    Bruot, Christopher; Palma, Julio L.; Xiang, Limin; Mujica, Vladimiro; Ratner, Mark A.; Tao, Nongjian

    2015-01-01

    Piezoresistivity is a fundamental property of materials that has found many device applications. Here we report piezoresistivity in double helical DNA molecules. By studying the dependence of molecular conductance and piezoresistivity of single DNA molecules with different sequences and lengths, and performing molecular orbital calculations, we show that the piezoresistivity of DNA is caused by force-induced changes in the π–π electronic coupling between neighbouring bases, and in the activation energy of hole hopping. We describe the results in terms of thermal activated hopping model together with the ladder-based mechanical model for DNA proposed by de Gennes. PMID:26337293

  7. Autonomous DNA-Molecule Computing

    NASA Astrophysics Data System (ADS)

    Komiya, Ken; Rose, John A.; Yamamura, Masayuki

    DNA molecules autonomously change their forms from the single strand to the double helix by specific binding between complementary sequences according to the Watson-Crick base pairing rule. This paring rule allows us to control connections among molecules and to construct various structures by sequence design. Further, the motion of constructed structures can also be designed by considering sequential bindings. Recently, the feasibility to utilize the programmed DNA structural change for information processing was studied. In the present paper, we report an efficient synthetic chain reaction based on autonomous binding of DNA to realize a computing system, which enable us to implement computational intelligence in vitro.

  8. Electrical conduction through DNA molecule.

    PubMed

    Abdalla, S

    2011-09-01

    Several disorder parameters, inside the DNA molecule, lead to localization of charge carriers inside potential wells in the lowest unoccupied and highest occupied molecular orbits (LUMO and HOMO) which affects drastically the electrical conduction through the molecule, and demonstrates that the band carriers play an essential role in the conduction mechanism. So, a model is presented to shed light on the role of electrons of the LUMO in the electrical conduction through the DNA molecule. DC-, AC-conductivity and dielectric permittivity experimental data are well fitted with the presented model giving evidence that the free carriers in the LUMO and HOMO are responsible to make the DNA molecule conductor, insulator or semiconductor. The obtained results show that the localized charge carriers in the DNA molecule are characterized by four different types of relaxation phenomena which are thermally activated by corresponding four activation energies at 0.56 eV, 0.33 eV, 0.24 eV, and 0.05 eV respectively. Moreover, the calculations after the model, at room temperature, show that the time of the relaxation times of the current carriers are in the order of 5 × 10(-2)s, 1.74 × 10(-4)s, 5 × 10(-7)s, and 1.6 × 10(-10)s, respectively.

  9. Biophysical characterization of DNA binding from single molecule force measurements

    NASA Astrophysics Data System (ADS)

    Chaurasiya, Kathy R.; Paramanathan, Thayaparan; McCauley, Micah J.; Williams, Mark C.

    2010-09-01

    Single molecule force spectroscopy is a powerful method that uses the mechanical properties of DNA to explore DNA interactions. Here we describe how DNA stretching experiments quantitatively characterize the DNA binding of small molecules and proteins. Small molecules exhibit diverse DNA binding modes, including binding into the major and minor grooves and intercalation between base pairs of double-stranded DNA (dsDNA). Histones bind and package dsDNA, while other nuclear proteins such as high mobility group proteins bind to the backbone and bend dsDNA. Single-stranded DNA (ssDNA) binding proteins slide along dsDNA to locate and stabilize ssDNA during replication. Other proteins exhibit binding to both dsDNA and ssDNA. Nucleic acid chaperone proteins can switch rapidly between dsDNA and ssDNA binding modes, while DNA polymerases bind both forms of DNA with high affinity at distinct binding sites at the replication fork. Single molecule force measurements quantitatively characterize these DNA binding mechanisms, elucidating small molecule interactions and protein function.

  10. Biophysical characterization of DNA binding from single molecule force measurements

    PubMed Central

    Chaurasiya, Kathy R.; Paramanathan, Thayaparan; McCauley, Micah J.; Williams, Mark C.

    2010-01-01

    Single molecule force spectroscopy is a powerful method that uses the mechanical properties of DNA to explore DNA interactions. Here we describe how DNA stretching experiments quantitatively characterize the DNA binding of small molecules and proteins. Small molecules exhibit diverse DNA binding modes, including binding into the major and minor grooves and intercalation between base pairs of double-stranded DNA (dsDNA). Histones bind and package dsDNA, while other nuclear proteins such as high mobility group proteins bind to the backbone and bend dsDNA. Single-stranded DNA (ssDNA) binding proteins slide along dsDNA to locate and stabilize ssDNA during replication. Other proteins exhibit binding to both dsDNA and ssDNA. Nucleic acid chaperone proteins can switch rapidly between dsDNA and ssDNA binding modes, while DNA polymerases bind both forms of DNA with high affinity at distinct binding sites at the replication fork. Single molecule force measurements quantitatively characterize these DNA binding mechanisms, elucidating small molecule interactions and protein function. PMID:20576476

  11. Visualization of large elongated DNA molecules.

    PubMed

    Lee, Jinyong; Kim, Yongkyun; Lee, Seonghyun; Jo, Kyubong

    2015-09-01

    Long and linear DNA molecules are the mainstream single-molecule analytes for a variety of biochemical analysis within microfluidic devices, including functionalized surfaces and nanostructures. However, for biochemical analysis, large DNA molecules have to be unraveled, elongated, and visualized to obtain biochemical and genomic information. To date, elongated DNA molecules have been exploited in the development of a number of genome analysis systems as well as for the study of polymer physics due to the advantage of direct visualization of single DNA molecule. Moreover, each single DNA molecule provides individual information, which makes it useful for stochastic event analysis. Therefore, numerous studies of enzymatic random motions have been performed on a large elongated DNA molecule. In this review, we introduce mechanisms to elongate DNA molecules using microfluidics and nanostructures in the beginning. Secondly, we discuss how elongated DNA molecules have been utilized to obtain biochemical and genomic information by direct visualization of DNA molecules. Finally, we reviewed the approaches used to study the interaction of proteins and large DNA molecules. Although DNA-protein interactions have been investigated for many decades, it is noticeable that there have been significant achievements for the last five years. Therefore, we focus mainly on recent developments for monitoring enzymatic activity on large elongated DNA molecules.

  12. Deformation of DNA molecules by hydrodynamic focusing

    NASA Astrophysics Data System (ADS)

    Wong, Pak Kin; Lee, Yi-Kuen; Ho, Chih-Ming

    2003-12-01

    The motion of a DNA molecule in a solvent flow reflects the deformation of a nano/microscale flexible mass spring structure by the forces exerted by the fluid molecules. The dynamics of individual molecules can reveal both fundamental properties of the DNA and basic understanding of the complex rheological properties of long-chain molecules. In this study, we report the dynamics of isolated DNA molecules under homogeneous extensional flow. Hydrodynamic focusing generates homogeneous extensional flow with uniform velocity in the transverse direction. The deformation of individual DNA molecules in the flow was visualized with video fluorescence microscopy. A coil stretch transition was observed when the Deborah number (De) is larger than 0.8. With a sudden stopping of the flow, the DNA molecule relaxes and recoils. The longest relaxation time of T2 DNA was determined to be 0.63 s when scaling viscosity to 0.9 cP.

  13. Ultra-high-density 3D DNA arrays within nanoporous biocompatible membranes for single-molecule-level detection and purification of circulating nucleic acids

    NASA Astrophysics Data System (ADS)

    Aramesh, M.; Shimoni, O.; Fox, K.; Karle, T. J.; Lohrmann, A.; Ostrikov, K.; Prawer, S.; Cervenka, J.

    2015-03-01

    Extracellular nucleic acids freely circulating in blood and other physiologic fluids are important biomarkers for non-invasive diagnostics and early detection of cancer and other diseases, yet difficult to detect because they exist in very low concentrations and large volumes. Here we demonstrate a new broad-range sensor platform for ultrasensitive and selective detection of circulating DNA down to the single-molecule level. The biosensor is based on a chemically functionalized nanoporous diamond-like carbon (DLC) coated alumina membrane. The few nanometer-thick, yet perfect and continuous DLC-coating confers the chemical stability and biocompatibility of the sensor, allowing its direct application in biological conditions. The selective detection is based on complementary hybridization of a fluorescently-tagged circulating cancer oncomarker (a 21-mer nucleic acid) with covalently immobilized DNA on the surface of the membrane. The captured DNAs are detected in the nanoporous structure of the sensor using confocal scanning laser microscopy. The flow-through membrane sensor demonstrates broad-range sensitivity, spanning from 1015 molecules per cm2 down to single molecules, which is several orders of magnitude improvement compared to the flat DNA microarrays. Our study suggests that these flow-through type nanoporous sensors represent a new powerful platform for large volume sampling and ultrasensitive detection of different chemical biomarkers.Extracellular nucleic acids freely circulating in blood and other physiologic fluids are important biomarkers for non-invasive diagnostics and early detection of cancer and other diseases, yet difficult to detect because they exist in very low concentrations and large volumes. Here we demonstrate a new broad-range sensor platform for ultrasensitive and selective detection of circulating DNA down to the single-molecule level. The biosensor is based on a chemically functionalized nanoporous diamond-like carbon (DLC) coated

  14. Nucleic Acids as Information Molecules.

    ERIC Educational Resources Information Center

    McInerney, Joseph D.

    1996-01-01

    Presents an activity that aims at enabling students to recognize that DNA and RNA are information molecules whose function is to store, copy, and make available the information in biological systems, without feeling overwhelmed by the specialized vocabulary and the minutia of the central dogma. (JRH)

  15. Trapping and manipulating single molecules of DNA

    NASA Astrophysics Data System (ADS)

    Shon, Min Ju

    This thesis presents the development and application of nanoscale techniques to trap and manipulate biomolecules, with a focus on DNA. These methods combine single-molecule microscopy and nano- and micro-fabrication to study biophysical properties of DNA and proteins. The Dimple Machine is a lab-on-a-chip device that can isolate and confine a small number of molecules from a bulk solution. It traps molecules in nanofabricated chambers, or "dimples", and the trapped molecules are then studied on a fluorescence microscope at the single-molecule level. The sampling of bulk solution by dimples is representative, reproducible, and automated, enabling highthroughput single-molecule experiments. The device was applied to study hybridization of oligonucleotides, particularly in the context of reaction thermodynamics and kinetics in nanoconfinement. The DNA Pulley is a system to study protein binding and the local mechanical properties of DNA. A molecule of DNA is tethered to a surface on one end, and a superparamagnetic bead is attached to the other. A magnet pulls the DNA taut, and a silicon nitride knife with a nanoscale blade scans the DNA along its contour. Information on the local properties of the DNA is extracted by tracking the bead with nanometer precision in a white-light microscope. The system can detect proteins bound to DNA and localize their recognition sites, as shown with a model protein, EcoRI restriction enzyme. Progress on the measurements of nano-mechanical properties of DNA is included.

  16. The Arrangement of Information in DNA Molecules

    PubMed Central

    Thomas, Charles A.

    1966-01-01

    The anatomy of DNA molecules isolated from mature bacteriophage is reviewed. These molecules are linear, duplex DNA consisting mainly of uninterrupted polynucleotide chains. Certain phage (T5 and PB) contain four specifically located interruptions. While the nucleotide sequence of most of these molecules is unique (T5, T3, T7, λ), some are circular permutations of each other (T2, T4, P22). Partial degradation of these DNA molecules by exonuclease III predisposes some of them to form circles upon annealing, but indicating they are terminally redundant. PMID:5967428

  17. Mechanobiology of Short DNA Molecules: A Single Molecule Perspective

    NASA Astrophysics Data System (ADS)

    Raghunathan, Krishnan

    Mechanical properties of DNA are known to play a significant role in several biological processes like wrapping of DNA around histones and looping. Most of these cellular events occur on a DNA length scale of a few hundred basepairs. Single molecule methods have been highly successful in directly investigating heterogeneity in different biomolecular systems and serve as ideal tools to study the mechanical properties of DNA. However, their use in studying DNA of contour lengths less than a kilobase are fraught with experimental difficulties. The research presented in this thesis explores the behavior of short stretches of DNA (≤ 500bp) using existing and novel single molecule methods. We have quantified the variation in persistence lengths between sequences having different elasticity using a constant force axial optical tweezers. Our experiments have also revealed that this difference in persistence lengths manifests itself as a difference in looping lifetimes of lac repressor, in sequences having the aforementioned constructs as the intervening sequence between the operator sites. We have also developed a system to probe DNA dynamics in vivo. We have found that the active processes in the cell have distinct effects on dynamics of DNA and eliminating the active processes causes a 'phase transition' like behavior in the inside the cell. We are currently extending this technique to understand DNA dynamics inside bacterial systems. Our results provide vital insights into mechanical properties of DNA and the effect of athermal fluctuations on DNA dynamics.

  18. Sequence of a cDNA clone encoding the polysialic acid-rich and cytoplasmic domains of the neural cell adhesion molecule N-CAM.

    PubMed Central

    Hemperly, J J; Murray, B A; Edelman, G M; Cunningham, B A

    1986-01-01

    Purified fractions of the neural cell-adhesion molecule N-CAM from embryonic chicken brain contain two similar polypeptides (Mr, 160,000 and 130,000), each containing an amino-terminal external binding region, a carbohydrate-rich central region, and a carboxyl-terminal region that is associated with the cell. Previous studies indicate that the two polypeptides arise by alternative splicing of mRNAs transcribed from a single gene. We report here the 3556-nucleotide sequence of a cDNA clone (pEC208) that encodes 964 amino acids from the carbohydrate and cell-associated domains of the larger N-CAM polypeptide followed by 664 nucleotides of 3' untranslated sequence. The predicted protein sequence contains attachment sites for polysialic acid-containing oligosaccharides, four tandem homologous regions of polypeptide resembling those seen in the immunoglobulin superfamily, and a single hydrophobic sequence that appears to be the membrane-spanning segment. The cytoplasmic domain carboxyl terminal to this segment includes a block of approximately equal to 250 amino acids present in the larger but not in the smaller N-CAM polypeptide. We designate these the ld (large domain) polypeptide and the sd (small domain) polypeptide. The intracellular domains of the ld and sd polypeptides are likely to be critical for cell-surface modulation of N-CAM by interacting in a differential fashion with other intrinsic proteins or with the cytoskeleton. PMID:3458261

  19. Geranyl diphosphate synthase molecules, and nucleic acid molecules encoding same

    DOEpatents

    Croteau, Rodney Bruce; Burke, Charles Cullen

    2008-06-24

    In one aspect, the present invention provides isolated nucleic acid molecules that each encode a geranyl diphosphate synthase protein, wherein each isolated nucleic acid molecule hybridizes to a nucleic acid molecule consisting of the sequence set forth in SEQ ID NO:1 under conditions of 5.times.SSC at 45.degree. C. for one hour. The present invention also provides isolated geranyl diphosphate synthase proteins, and methods for altering the level of expression of geranyl diphosphate synthase protein in a host cell.

  20. Kinetic mechanism for DNA unwinding by multiple molecules of Dda helicase aligned on DNA.

    PubMed

    Eoff, Robert L; Raney, Kevin D

    2010-06-01

    Helicases catalyze the separation of double-stranded nucleic acids to form single-stranded intermediates. Using transient state kinetic methods, we have determined the kinetic properties of DNA unwinding under conditions that favor a monomeric form of the Dda helicase as well as conditions that allow multiple molecules to function on the same substrate. Multiple helicase molecules can align like a train on the DNA track. The number of base pairs unwound in a single binding event for Dda is increased from approximately 19 bp for the monomeric form to approximately 64 bp when as many as four Dda molecules are aligned on the same substrate, while the kinetic step size (3.2 +/- 0.7 bp) and unwinding rate (242 +/- 25 bp/s) appear to be independent of the number of Dda molecules present on a given substrate. The data support a model in which the helicase molecules bound to the same substrate move along the DNA track independently during DNA unwinding. The observed increase in processivity arises from the increased probability that at least one of the helicases will completely unwind the DNA prior to dissociation. These results are in contrast to previous reports in which multiple Dda molecules on the same track greatly enhanced the rate and amplitude for displacement of protein blocks on the track. Therefore, only when the progress of the lead molecule in the train is impeded by some type of block, such as a protein bound to DNA, do the trailing molecules interact with the lead molecule to overcome the block. The fact that trailing helicase molecules have little impact on the lead molecule in the train during routine DNA unwinding suggests that the trailing molecules are moving at rates similar to that of the lead molecule. This result implicates a step in the translocation mechanism as contributing greatly to the overall rate-limiting step for unwinding of duplex DNA.

  1. Fluorescently labeled circular DNA molecules for DNA topology and topoisomerases

    PubMed Central

    Gu, Maxwell; Berrido, Andrea; Gonzalez, Walter G.; Miksovska, Jaroslava; Chambers, Jeremy W.; Leng, Fenfei

    2016-01-01

    DNA topology plays essential roles in several fundamental biological processes, such as DNA replication, recombination, and transcription. Typically agarose gel electrophoresis is employed to study DNA topology. Since gel electrophoresis is time-consuming and labor intensive, it is desirable to develop other methods, such as fluorescence-based methods, for such studies. In this paper we report the synthesis of a type of unique fluorescence-labeled DNA molecules that can be used to study DNA topology and topoisomerases by fluorescence resonance energy transfer (FRET). Specifically, we inserted an 82 nt. synthetic DNA oligomer FL905 carrying a 42 nt. AT sequence with fluorescein and dabcyl labels into a gapped DNA molecule to generate relaxed and supercoiled pAB1_FL905. Since the fluorescence intensity of pAB1_FL905 is dependent on its supercoiling status, pAB1_FL905 is a powerful tool to study DNA topology and topoisomerases by FRET. pAB1_FL905 can also be developed into rapid and efficient high-throughput screening assays to identify inhibitors that target various DNA topoisomerases. PMID:27796331

  2. Internal Dynamics of Supercoiled DNA Molecules

    PubMed Central

    Kalkbrenner, Thomas; Arnold, Axel; Tans, Sander J.

    2009-01-01

    Abstract The intramolecular diffusive motion within supercoiled DNA molecules is of central importance for a wide array of gene regulation processes. It has recently been shown, using fluorescence correlation spectroscopy, that plasmid DNA exhibits unexpected acceleration of its internal diffusive motion upon supercoiling to intermediate density. Here, we present an independent study that shows a similar acceleration for fully supercoiled plasmid DNA. We have developed a method that allows fluorescent labeling of a 200-bp region, as well as efficient supercoiling by Escherichia coli gyrase. Compared to plain circular or linear DNA, the submicrosecond motion within the supercoiled molecules appears faster by up to an order of magnitude. The mean-square displacement as a function of time reveals an additional intermediate regime with a lowered scaling exponent compared to that of circular DNA. Although this unexpected behavior is not fully understood, it could be explained by conformational constraints of the DNA strand within the supercoiled topology in combination with an increased apparent persistence length. PMID:19527654

  3. Single-molecule visualization of ROS-induced DNA damage in large DNA molecules.

    PubMed

    Lee, Jinyong; Kim, Yongkyun; Lim, Sangyong; Jo, Kyubong

    2016-02-07

    We present a single molecule visualization approach for the quantitative analysis of reactive oxygen species (ROS) induced DNA damage, such as base oxidation and single stranded breaks in large DNA molecules. We utilized the Fenton reaction to generate DNA damage with subsequent enzymatic treatment using a mixture of three types of glycosylases to remove oxidized bases, and then fluorescent labeling on damaged lesions via nick translation. This single molecule analytical platform provided the capability to count one or two damaged sites per λ DNA molecule (48.5 kb), which were reliably dependent on the concentrations of hydrogen peroxide and ferrous ion at the micromolar level. More importantly, the labeled damaged sites that were visualized under a microscope provided positional information, which offered the capability of comparing DNA damaged sites with the in silico genomic map to reveal sequence specificity that GTGR is more sensitive to oxidative damage. Consequently, single DNA molecule analysis provides a sensitive analytical platform for ROS-induced DNA damage and suggests an interesting biochemical insight that the genome primarily active during the lysogenic cycle may have less probability for oxidative DNA damage.

  4. Methods And Devices For Characterizing Duplex Nucleic Acid Molecules

    DOEpatents

    Akeson, Mark; Vercoutere, Wenonah; Haussler, David; Winters-Hilt, Stephen

    2005-08-30

    Methods and devices are provided for characterizing a duplex nucleic acid, e.g., a duplex DNA molecule. In the subject methods, a fluid conducting medium that includes a duplex nucleic acid molecule is contacted with a nanopore under the influence of an applied electric field and the resulting changes in current through the nanopore caused by the duplex nucleic acid molecule are monitored. The observed changes in current through the nanopore are then employed as a set of data values to characterize the duplex nucleic acid, where the set of data values may be employed in raw form or manipulated, e.g., into a current blockade profile. Also provided are nanopore devices for practicing the subject methods, where the subject nanopore devices are characterized by the presence of an algorithm which directs a processing means to employ monitored changes in current through a nanopore to characterize a duplex nucleic acid molecule responsible for the current changes. The subject methods and devices find use in a variety of applications, including, among other applications, the identification of an analyte duplex DNA molecule in a sample, the specific base sequence at a single nulceotide polymorphism (SNP), and the sequencing of duplex DNA molecules.

  5. Reprogramming the assembly of unmodified DNA with a small molecule

    NASA Astrophysics Data System (ADS)

    Avakyan, Nicole; Greschner, Andrea A.; Aldaye, Faisal; Serpell, Christopher J.; Toader, Violeta; Petitjean, Anne; Sleiman, Hanadi F.

    2016-04-01

    The ability of DNA to store and encode information arises from base pairing of the four-letter nucleobase code to form a double helix. Expanding this DNA ‘alphabet’ by synthetic incorporation of new bases can introduce new functionalities and enable the formation of novel nucleic acid structures. However, reprogramming the self-assembly of existing nucleobases presents an alternative route to expand the structural space and functionality of nucleic acids. Here we report the discovery that a small molecule, cyanuric acid, with three thymine-like faces, reprogrammes the assembly of unmodified poly(adenine) (poly(A)) into stable, long and abundant fibres with a unique internal structure. Poly(A) DNA, RNA and peptide nucleic acid (PNA) all form these assemblies. Our studies are consistent with the association of adenine and cyanuric acid units into a hexameric rosette, which brings together poly(A) triplexes with a subsequent cooperative polymerization. Fundamentally, this study shows that small hydrogen-bonding molecules can be used to induce the assembly of nucleic acids in water, which leads to new structures from inexpensive and readily available materials.

  6. Quantitative detection of single DNA molecules on DNA tetrahedron decorated substrates.

    PubMed

    Wang, Zhenguang; Xue, Qingwang; Tian, Wenzhi; Wang, Lei; Jiang, Wei

    2012-10-07

    A single DNA molecule detection method on DNA tetrahedron decorated substrates has been developed. DNA tetrahedra were introduced onto substrates for both preventing nonspecific adsorption and sensitive recognition of single DNA molecules.

  7. Construction of DNA Hemicatenanes from Two Small Circular DNA Molecules

    PubMed Central

    Gaillard, Claire; Strauss, François

    2015-01-01

    DNA hemicatenanes, one of the simplest possible junctions between two double stranded DNA molecules, have frequently been mentioned in the literature for their possible function in DNA replication, recombination, repair, and organization in chromosomes. They have been little studied experimentally, however, due to the lack of an appropriate method for their preparation. Here we have designed a method to build hemicatenanes from two small circular DNA molecules. The method involves, first, the assembly of two linear single strands and their circularization to form a catenane of two single stranded circles, and, second, the addition and base-pairing of the two single stranded circles complementary to the first ones, followed by their annealing using DNA topoisomerase I. The product was purified by gel electrophoresis and characterized. The arrangement of strands was as expected for a hemicatenane and clearly distinct from a full catenane. In addition, each circle was unwound by an average of half a double helical turn, also in excellent agreement with the structure of a hemicatenane. It was also observed that hemicatenanes are quickly destabilized by a single cut on either of the two strands passing inside the junction, strongly suggesting that DNA strands are able to slide easily inside the hemicatenane. This method should make it possible to study the biochemical properties of hemicatenanes and to test some of the hypotheses that have been proposed about their function, including a possible role for this structure in the organization of complex genomes in loops and chromosomal domains. PMID:25799010

  8. Single-molecule study of DNA polymerization activity of HIV-1 reverse transcriptase on DNA templates.

    PubMed

    Kim, Sangjin; Schroeder, Charles M; Xie, X Sunney

    2010-02-05

    HIV-1 RT (human immunodeficiency virus-1 reverse transcriptase) is a multifunctional polymerase responsible for reverse transcription of the HIV genome, including DNA replication on both RNA and DNA templates. During reverse transcription in vivo, HIV-1 RT replicates through various secondary structures on RNA and single-stranded DNA (ssDNA) templates without the need for a nucleic acid unwinding protein, such as a helicase. In order to understand the mechanism of polymerization through secondary structures, we investigated the DNA polymerization activity of HIV-1 RT on long ssDNA templates using a multiplexed single-molecule DNA flow-stretching assay. We observed that HIV-1 RT performs fast primer extension DNA synthesis on single-stranded regions of DNA (18.7 nt/s) and switches its activity to slow strand displacement synthesis at DNA hairpin locations (2.3 nt/s). Furthermore, we found that the rate of strand displacement synthesis is dependent on the GC content in hairpin stems and template stretching force. This indicates that the strand displacement synthesis occurs through a mechanism that is neither completely active nor passive: that is, the opening of the DNA hairpin is driven by a combination of free energy released during dNTP (deoxyribonucleotide triphosphate) hydrolysis and thermal fraying of base pairs. Our experimental observations provide new insight into the interchanging modes of DNA replication by HIV-1 RT on long ssDNA templates.

  9. Dynamics of DNA molecules under gel electrophoresis

    SciTech Connect

    Kotaka, Tadao, Adachi, Shiro; Shikata, Toshiyuki

    1993-12-31

    Electrophoretic mobilities {mu} of double stranded linear DNAs were examined in agarose gels subjected to a biased sinusoidal field (BSF) that utilizes a sinusoidal field of strength E{sub s} and frequency f superposed on a steady bias field of strength E{sub b}. Under BSF with E{sub s} {much_gt} E{sub b}. DNA fragments with the size M > 20 kbp exhibited peculiar behavior which the authors called a pin down phenomenon in that the {mu} shows a minimum {mu}{sub p} at a particular f{sub p} (pin down frequency) specific to M, C{sub gel} and the field strengths. The dynamics of DNA molecules under such pin-down conditions were examined by direct observation via fluorescence microscopy as well as dynamic electric birefringence.

  10. Method for sequencing nucleic acid molecules

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2006-05-30

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  11. Method for sequencing nucleic acid molecules

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2006-06-06

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  12. SINGLE-MOLECULE STUDY OF DNA POLYMERIZATION ACTIVITY OF HIV-1 REVERSE TRANSCRIPTASE ON DNA TEMPLATES

    PubMed Central

    Kim, Sangjin; Schroeder, Charles M.; Xie, X. Sunney

    2009-01-01

    Human Immunodeficiency Virus-1 reverse transcriptase (HIV-1 RT) is a multifunctional polymerase responsible for reverse transcription of the HIV genome, including DNA replication on both RNA and DNA templates. During reverse transcription in vivo, HIV-1 RT replicates through various secondary structures on RNA and single-stranded DNA templates without the need for a nucleic acid unwinding protein, such as a helicase. In order to understand the mechanism of polymerization through secondary structures, we investigated the DNA polymerization activity of HIV-1 RT on long single-stranded DNA templates using a multiplexed single-molecule DNA flow-stretching assay. We observed that HIV-1 RT performs fast primer extension DNA synthesis on single-stranded regions of DNA (18.7 nt/s) and switches its activity to slow strand displacement synthesis at DNA hairpin locations (2.3 nt/s). Furthermore, we found that the rate of strand displacement synthesis is dependent on the GC content in hairpin stems and template stretching force. This indicates that the strand displacement synthesis occurs through a mechanism that is neither completely active nor passive, i.e. the opening of the DNA hairpin is driven by a combination of free energy released during dNTP hydrolysis and thermal fraying of base pairs. Our experimental observations provide new insight into the interchanging modes of DNA replication by HIV-1 RT on long single-stranded DNA templates. PMID:19968999

  13. Unfolding Dynamics of Single Collapsed DNA Molecules

    NASA Astrophysics Data System (ADS)

    Murayama, Y.; Wada, H.; Ishida, R.; Sano, M.

    We observed elastic responses of single DNA molecules and visualized them during the collapsing transition induced by trivalent cation, spermidine (SPD). The force-extension curves show worm-like behavior, force plateau, and stick-release responses depending on SPD concentration. The periodic stick-release responses may reflect the unraveling of toroidal condensates. At much higher SPD concentration, we observed re-elongation of a single collapsed DNA. For the visualization, a fluorescent dye, YOYO, was used. We observed bright spots in the fluorescence intensity profile of a collapsed DNA during stretching, which may correspond to the collapsed parts within the single DNA. The decrease of the intensity of the spots in stretching implies the mechanical unfolding of collapsed parts. Towards achieving a microscopic understanding of these experimental results, we also investigate the elastic properties of a highly charged polyelectrolyte (PE) chain by Brownian dynamics simulation method. In our dynamic simulation, a PE has a small intrinsic stiffness (i.e., the PE is semiflexible) to model the stiffness of DNA chain, and added multivalent counterions are explicitly taken into account. As the electrostatic coupling parameter (proportional to counterion valency) is increased, counterion condensation is observed, leading finally to the PE collapse through the discontinuous transition for a sufficiently large coupling parameter. Mechanical unfolding of a PE globule reveals its molecular elasticities including force plateau, in agreement with the experimental observations. A numerically deduced electrostatic condensation energy is compared to the experimental value. Charge ordering in the PE-counterion complex and its deformation by the external forcing are elucidated in conjunction with the PE elastic responses. Other dynamic effects such as the effect of a pulling speed are also discussed.

  14. DNA-psoralen interaction: A single molecule experiment

    NASA Astrophysics Data System (ADS)

    Rocha, M. S.; Viana, N. B.; Mesquita, O. N.

    2004-11-01

    By attaching one end of a single λ-DNA molecule to a microscope coverslip and the other end to a polystyrene microsphere trapped by an optical tweezers, we can study the entropic elasticity of the λ-DNA by measuring force versus extension as we stretch the molecule. This powerful method permits single molecule studies. We are particulary interested in the effects of the photosensitive drug psoralen on the elasticity of the DNA molecule. We have illuminated the sample with different light sources, studying how the different wavelengths affect the psoralen-DNA linkage. To do this, we measure the persistence length of individual DNA-psoralen complexes.

  15. Increased throughput single molecule detection of DNA

    NASA Astrophysics Data System (ADS)

    Gurjar, Rajan; Seetamraju, Madhavi; Kolodziejski, Noah; Myers, Richard; Staples, Christopher; Christian, James; Squillante, Michael R.; Entine, Gerald

    2007-09-01

    In this work, we present research in using confocal optical techniques with femtolitre focal volumes and obtain very high signal-to-noise and signal-to-background ratios for single molecule detection (SMD). We were able to achieve improved signal strength by using highly fluorescent quantum dots and nanopatterned substrates to obtain plasmon induced resonant fluorescence enhancement. A method to simultaneously using multiple excitation spots without the use of confocal apertures and an array of single photon sensitive Geiger mode avalanche photodiodes was used to increase the throughput of the detection system. Using this highly sensitive SMD system, we detect small quantities of synthetic DNA through hybridization eliminating the need of polymerase chain reaction.

  16. Improved DNA hybridization parameters by Twisted Intercalating Nucleic Acid (TINA).

    PubMed

    Schneider, Uffe Vest

    2012-01-01

    This thesis establishes oligonucleotide design rules and applications of a novel group of DNA stabilizing molecules collectively called Twisted Intercalating Nucleic Acid - TINA. Three peer-reviewed publications form the basis for the thesis. One publication describes an improved and rapid method for determination of DNA melting points and two publications describe the effects of positioning TINA molecules in parallel triplex helix and antiparallel duplex helix forming DNA structures. The third publication establishes that TINA molecules containing oligonucleotides improve an antiparallel duplex hybridization based capture assay's analytical sensitivity compared to conventionel DNA oligonucleotides. Clinical microbiology is traditionally based on pathogenic microorganisms' culture and serological tests. The introduction of DNA target amplification methods like PCR has improved the analytical sensitivity and total turn around time involved in clinical diagnostics of infections. Due to the relatively weak hybridization between the two strands of double stranded DNA, a number of nucleic acid stabilizing molecules have been developed to improve the sensitivity of DNA based diagnostics through superior binding properties. A short introduction is given to Watson-Crick and Hoogsteen based DNA binding and the derived DNA structures. A number of other nucleic acid stabilizing molecules are described. The stabilizing effect of TINA molecules on different DNA structures is discussed and considered in relation to other nucleic acid stabilizing molecules and in relation to future use of TINA containing oligonucleotides in clinical diagnostics and therapy. In conclusion, design of TINA modified oligonucleotides for antiparallel duplex helixes and parallel triplex helixes follows simple purpose dependent rules. TINA molecules are well suited for improving multiplex PCR assays and can be used as part of novel technologies. Future research should test whether combinations of TINA

  17. Organization of 'nanocrystal molecules' using DNA

    NASA Astrophysics Data System (ADS)

    Alivisatos, A. Paul; Johnsson, Kai P.; Peng, Xiaogang; Wilson, Troy E.; Loweth, Colin J.; Bruchez, Marcel P.; Schultz, Peter G.

    1996-08-01

    PATTERNING matter on the nanometre scale is an important objective of current materials chemistry and physics. It is driven by both the need to further miniaturize electronic components and the fact that at the nanometre scale, materials properties are strongly size-dependent and thus can be tuned sensitively1. In nanoscale crystals, quantum size effects and the large number of surface atoms influence the, chemical, electronic, magnetic and optical behaviour2-4. 'Top-down' (for example, lithographic) methods for nanoscale manipulation reach only to the upper end of the nanometre regime5; but whereas 'bottom-up' wet chemical techniques allow for the preparation of mono-disperse, defect-free crystallites just 1-10 nm in size6-10, ways to control the structure of nanocrystal assemblies are scarce. Here we describe a strategy for the synthesis of'nanocrystal molecules', in which discrete numbers of gold nanocrystals are organized into spatially defined structures based on Watson-Crick base-pairing interactions. We attach single-stranded DNA oligonucleotides of defined length and sequence to individual nanocrystals, and these assemble into dimers and trimers on addition of a complementary single-stranded DNA template. We anticipate that this approach should allow the construction of more complex two-and three-dimensional assemblies.

  18. Extraction of ultrashort DNA molecules from herbarium specimens.

    PubMed

    Gutaker, Rafal M; Reiter, Ella; Furtwängler, Anja; Schuenemann, Verena J; Burbano, Hernán A

    2017-02-01

    DNA extracted from herbarium specimens is highly fragmented; therefore, it is crucial to use extraction protocols that retrieve short DNA molecules. Improvements in extraction and DNA library preparation protocols for animal remains have allowed efficient retrieval of molecules shorter than 50 bp. Here, we applied these improvements to DNA extraction protocols for herbarium specimens and evaluated extraction performance by shotgun sequencing, which allows an accurate estimation of the distribution of DNA fragment lengths. Extraction with N-phenacylthiazolium bromide (PTB) buffer decreased median fragment length by 35% when compared with cetyl-trimethyl ammonium bromide (CTAB); modifying the binding conditions of DNA to silica allowed for an additional decrease of 10%. We did not observe a further decrease in length for single-stranded DNA (ssDNA) versus double-stranded DNA (dsDNA) library preparation methods. Our protocol enables the retrieval of ultrashort molecules from herbarium specimens, which will help to unlock the genetic information stored in herbaria.

  19. Quantum-Sequencing: Fast electronic single DNA molecule sequencing

    NASA Astrophysics Data System (ADS)

    Casamada Ribot, Josep; Chatterjee, Anushree; Nagpal, Prashant

    2014-03-01

    A major goal of third-generation sequencing technologies is to develop a fast, reliable, enzyme-free, high-throughput and cost-effective, single-molecule sequencing method. Here, we present the first demonstration of unique ``electronic fingerprint'' of all nucleotides (A, G, T, C), with single-molecule DNA sequencing, using Quantum-tunneling Sequencing (Q-Seq) at room temperature. We show that the electronic state of the nucleobases shift depending on the pH, with most distinct states identified at acidic pH. We also demonstrate identification of single nucleotide modifications (methylation here). Using these unique electronic fingerprints (or tunneling data), we report a partial sequence of beta lactamase (bla) gene, which encodes resistance to beta-lactam antibiotics, with over 95% success rate. These results highlight the potential of Q-Seq as a robust technique for next-generation sequencing.

  20. Detecting single DNA molecule interactions with optical microcavities (Presentation Recording)

    NASA Astrophysics Data System (ADS)

    Vollmer, Frank

    2015-09-01

    circular optical path, similar to an acoustic wave guided along the wall of St. Paul's Cathedral. These so called whispering gallery modes (WGM) propagate with little loss, so that even a whisper can be heard on the other side of the gallery. In the optical case, the light beam can travel many thousand times around the inside of the microsphere before being scattered or absorbed, thereby making numerous interactions with an analyte molecule, bound to microsphere from surrounding sample solution. The most part of the light intensity, however, remains inside the microsphere, just below the reflecting glass surface, resulting in a relatively weak interaction between the light and the bound molecule. To enhance this interaction further, we attach tiny 42 nm x 12 nm gold nanorods to the glass surface. When passing a nanorod, the lightwave induces oscillations of conduction electrons, resulting in so called plasmon resonance. These nanorod plasmons greatly enhance the light intensity on the nanorod, so that the interaction of the light with a molecule attached to the nanorod is also enhanced[4-6]. This enhanced interaction results in an increase in sensitivity by more than a factor of one thousand, putting our experiments of single DNA molecule detection within reach. For the specific detection of nucleic acids, we attach single-stranded DNA to the nanorod and immerse our device in a liquid solution. When a matching, i.e. complementary DNA fragment binds from solution to the "bait" on the nanorod, the enhanced interaction with the light results in an observable shift of the WGM wavelength. Since light propagates in a WGM only for a very precise resonance wavelength or frequency, this shift can be detected with great accuracy[3]. On our current biosensor platform, we detect wavelength shifts with an accuracy of less than one femtometer, resulting in an extremely high sensitivity for biosensing, which we leverage for the specific detection of single 8 mer oligonucleotides as well

  1. Using Synthetic Nanopores for Single-Molecule Analyses: Detecting SNPs, Trapping DNA Molecules, and the Prospects for Sequencing DNA

    ERIC Educational Resources Information Center

    Dimitrov, Valentin V.

    2009-01-01

    This work focuses on studying properties of DNA molecules and DNA-protein interactions using synthetic nanopores, and it examines the prospects of sequencing DNA using synthetic nanopores. We have developed a method for discriminating between alleles that uses a synthetic nanopore to measure the binding of a restriction enzyme to DNA. There exists…

  2. Structural changes of linear DNA molecules induced by cisplatin

    SciTech Connect

    Liu, Zhiguo; Liu, Ruisi; Zhou, Zhen; Zu, Yuangang; Xu, Fengjie

    2015-02-20

    Interaction between long DNA molecules and activated cisplatin is believed to be crucial to anticancer activity. However, the exact structural changes of long DNA molecules induced by cisplatin are still not very clear. In this study, structural changes of long linear double-stranded DNA (dsDNA) and short single-stranded DNA (ssDNA) induced by activated cisplatin have been investigated by atomic force microscopy (AFM). The results indicated that long DNA molecules gradually formed network structures, beads-on-string structures and their large aggregates. Electrostatic and coordination interactions were considered as the main driving forces producing these novel structures. An interesting finding in this study is the beads-on-string structures. Moreover, it is worth noting that the beads-on-string structures were linked into the networks, which can be ascribed to the strong DNA–DNA interactions. This study expands our knowledge of the interactions between DNA molecules and cisplatin. - Highlights: • We investigate structural changes of dsDNA and ssDNA induced by cisplatin. • AFM results indicated long dsDNA formed network, beads-on-string and aggregates. • ssDNA can form very similar structures as those of long linear dsDNA. • A possible formation process of theses novel structure is proposed.

  3. Featured Molecules: Ascorbic Acid and Methylene Blue

    NASA Astrophysics Data System (ADS)

    Coleman, William F.; Wildman, Randall J.

    2003-05-01

    The WebWare molecules of the month for May are featured in several articles in this issue. "Arsenic: Not So Evil After All?" discusses the pharmaceutical uses of methylene blue and its development as the first synthetic drug used against a specific disease. The JCE Classroom Activity "Out of the Blue" and the article "Greening the Blue Bottle" feature methylene blue and ascorbic acid as two key ingredients in the formulation of the blue bottle. You can also see a colorful example of these two molecules in action on the cover. "Sailing on the 'C': A Vitamin Titration with a Twist" describes an experiment to determine the vitamin C (ascorbic acid) content of citrus fruits and challenges students, as eighteenth-century sea captains, to decide the best fruit to take on a long voyage. Fully manipulable (Chime) versions of these and other molecules are available at Only@JCE Online.

  4. Did the Pre-RNA World Rest Upon DNA Molecules?

    NASA Technical Reports Server (NTRS)

    Lazcano, Antonio; Dworkin, Jason P.; Miller, Stanley L.

    2004-01-01

    The isolation of a DNA sequence that catalyzes the ligation of oligodeoxynucleotides via the formation of 3' - 5' phosphodiester linkage significance in selection experiments has been reported. Ball recently used this to discuss the possibility that natural DNA molecules may have formed in the primitive Earth leading to the origin of life. As noted by Ferris and Usher, if metabolic pathways evolved backwards, it could be argued that the biosynthesis of 2-deoxyribose from ribose suggests that RNA came from DNA. As summarized elsewhere, there are several properties of deoxyribose which could be interpreted to support the possibility that DNA-like molecules arose prior to the RNA world. For example, 2-deoxyribose is slightly more soluble than ribose (which may have been an advantage in a drying pool scenario), may have been more reactive under possible prebiotic conditions (it forms a nucleoside approx. 150 times faster than ribose with the alternative base urazole at 25 C), while it decomposes in solution (approximately 2.6 times more slowly than ribose at 100 C). Other advantages of DNA over RNA are that it has one fewer chiral center, has a greater stability at the 8.2 pH value of the current oceans, and does not has the 2'5' and 3'5' ambiguity in polymerizations. Yet, there is strong molecular biological and biochemical evidence that RNA was featured in the biology well before the last common ancestor. The presence of sugar acids, including both ribo- and deoxysugar acids, in the 4.6 Ga old Murchison meteorite suggest that both may have been available in the primitive Earth, derived from the accretion of extraterrestrial sources and/or from endogenous processes involving formaldehyde and its derivatives. However, the abiotic synthesis of deoxyribose, ribose, and other sugars from glyceraldehyde and acetaldehyde under alkaline conditions is inefficient and unespecific. Although sugars are labile compounds, the role of cyanamide or borate minerals in the

  5. How to determine local stretching and tension in a flow-stretched DNA molecule

    NASA Astrophysics Data System (ADS)

    Pedersen, Jonas N.; Marie, Rodolphe; Kristensen, Anders; Flyvbjerg, Henrik

    2016-04-01

    We determine the nonuniform stretching of and tension in a mega base pairs-long fragment of deoxyribonucleic acid (DNA) that is flow stretched in a nanofluidic chip. We use no markers, do not know the contour length of the DNA, and do not have the full DNA molecule inside our field of view. Instead, we analyze the transverse thermal motion of the DNA. Tension at the center of the DNA adds up to 16 pN, giving almost fully stretched DNA. This method was devised for optical mapping of DNA, specifically, DNA denaturation patterns. It may be useful also for other studies, e.g., DNA-protein interactions, specifically, their tension dependence. Generally, wherever long strands of DNA—e.g., native DNA extracted from human cells or bacteria—must be stretched with ease for inspection, this method applies.

  6. DNA micelles as nanoreactors: efficient DNA functionalization with hydrophobic organic molecules.

    PubMed

    Trinh, Tuan; Chidchob, Pongphak; Bazzi, Hassan S; Sleiman, Hanadi F

    2016-09-18

    We report a micelle-templated method to enhance the reactivity of DNA with highly hydrophobic molecules. Lipids, chromophores and polymers can be conjugated to DNA in high yield and under mild conditions. This method expands the range of DNA-templated reactions for DNA-encoded libraries, oligonucleotide and drug delivery, nanopore mimetics and DNA nanotechnology.

  7. DNA Mapping Using Microfluidic Stretching and Single-Molecule Detection of Fluorescent Site-Specific Tags

    PubMed Central

    Chan, Eugene Y.; Goncalves, Nuno M.; Haeusler, Rebecca A.; Hatch, Amie J.; Larson, Jonathan W.; Maletta, Anthony M.; Yantz, Gregory R.; Carstea, Eugene D.; Fuchs, Martin; Wong, Gordon G.; Gullans, Steven R.; Gilmanshin, Rudolf

    2004-01-01

    We have developed a rapid molecular mapping technology—Direct Linear Analysis (DLA)—on the basis of the analysis of individual DNA molecules bound with sequence-specific fluorescent tags. The apparatus includes a microfluidic device for stretching DNA molecules in elongational flow that is coupled to a multicolor detection system capable of single-fluorophore sensitivity. Double-stranded DNA molecules were tagged at sequence-specific motif sites with fluorescent bisPNA (Peptide Nucleic Acid) tags. The DNA molecules were then stretched in the microfluidic device and driven in a flow stream past confocal fluorescence detectors. DLA provided the spatial locations of multiple specific sequence motifs along individual DNA molecules, and thousands of individual molecules could be analyzed per minute. We validated this technology using the 48.5 kb λ phage genome with different 8-base and 7-base sequence motif tags. The distance between the sequence motifs was determined with an accuracy of ±0.8 kb, and these tags could be localized on the DNA with an accuracy of ±2 kb. Thus, DLA is a rapid mapping technology, suitable for analysis of long DNA molecules. PMID:15173119

  8. Visualization of DNA molecules in time during electrophoresis

    NASA Technical Reports Server (NTRS)

    Lubega, Seth

    1991-01-01

    For several years individual DNA molecules have been observed and photographed during agarose gel electrophoresis. The DNA molecule is clearly the largest molecule known. Nevertheless, the largest molecule is still too small to be seen using a microscope. A technique developed by Morikawa and Yanagida has made it possible to visualize individual DNA molecules. When these long molecules are labeled with appropriate fluorescence dyes and observed under a fluorescence microscope, although it is not possible to directly visualize the local ultrastructure of the molecules, yet because they are long light emitting chains, their microscopic dynamical behavior can be observed. This visualization works in the same principle that enables one to observe a star through a telescope because it emits light against a dark background. The dynamics of individual DNA molecules migrating through agarose matrix during electrophoresis have been described by Smith et al. (1989), Schwartz and Koval (1989), and Bustamante et al. (1990). DNA molecules during agarose gel electrophoresis advance lengthwise thorough the gel in an extended configuration. They display an extension-contraction motion and tend to bunch up in their leading ends as the 'heads' find new pores through the gel. From time to time they get hooked on obstacles in the gel to form U-shaped configurations before they resume their linear configuration.

  9. Localized single molecule isotherms of DNA molecules at confined liquid-solid interfaces.

    PubMed

    Liang, Heng; Cheng, Xiaoliang; Ma, Yinfa

    2009-03-15

    The study of dynamics and thermodynamics of single biological molecules at confined liquid-solid interfaces is crucially important, especially in the case of low-copy number molecules in a single cell. Using a high-throughput single molecule imaging system and Lagrangian coordinates of single molecule images, we discovered that the local equilibrium isotherms of single lambdaDNA molecules at a confined liquid-solid interface varied from a stair type for the regions of single or double molecular DNA to a mild "S" type for the regions of triple molecular DNA spots, which does not agree with the conventional equilibrium isotherms in the literature. Single molecule images in time sequence for different lambdaDNA concentrations were statistically analyzed by measuring preferential partitioning from shearing effects, which were used to measure the local velocity of DNA molecules by directly observing the migration of DNA fluorescence spots for the 12 continuous images. The local linear velocity of hydrodynamic flow was calculated by the Hagen-Poiseuille equation in different microregions with a local Lagrangian approach. The local single molecule isotherms for the tracked molecules in the regions of single, double, or triple molecular DNA layers within the laminar flows were obtained according to the average local velocities of both the stochastic molecule events and the corresponding local Poiseuille flows. A millisecond and microvolume approach to directly determine local single molecule isotherms at confined liquid-solid interfaces was established, and the microspace scale effects on the types of isotherms were discovered. This study may have significant impact on preparations of low-copy number proteins in a single cell, membrane separations, and other bioseparation studies.

  10. Small-molecule discovery from DNA-encoded chemical libraries.

    PubMed

    Kleiner, Ralph E; Dumelin, Christoph E; Liu, David R

    2011-12-01

    Researchers seeking to improve the efficiency and cost effectiveness of the bioactive small-molecule discovery process have recently embraced selection-based approaches, which in principle offer much higher throughput and simpler infrastructure requirements compared with traditional small-molecule screening methods. Since selection methods benefit greatly from an information-encoding molecule that can be readily amplified and decoded, several academic and industrial groups have turned to DNA as the basis for library encoding and, in some cases, library synthesis. The resulting DNA-encoded synthetic small-molecule libraries, integrated with the high sensitivity of PCR and the recent development of ultra high-throughput DNA sequencing technology, can be evaluated very rapidly for binding or bond formation with a target of interest while consuming minimal quantities of material and requiring only modest investments of time and equipment. In this tutorial review we describe the development of two classes of approaches for encoding chemical structures and reactivity with DNA: DNA-recorded library synthesis, in which encoding and library synthesis take place separately, and DNA-directed library synthesis, in which DNA both encodes and templates library synthesis. We also describe in vitro selection methods used to evaluate DNA-encoded libraries and summarize successful applications of these approaches to the discovery of bioactive small molecules and novel chemical reactivity.

  11. Visualizing helicases unwinding DNA at the single molecule level.

    PubMed

    Fili, Natali; Mashanov, Gregory I; Toseland, Christopher P; Batters, Christopher; Wallace, Mark I; Yeeles, Joseph T P; Dillingham, Mark S; Webb, Martin R; Molloy, Justin E

    2010-07-01

    DNA helicases are motor proteins that catalyze the unwinding of double-stranded DNA into single-stranded DNA using the free energy from ATP hydrolysis. Single molecule approaches enable us to address detailed mechanistic questions about how such enzymes move processively along DNA. Here, an optical method has been developed to follow the unwinding of multiple DNA molecules simultaneously in real time. This was achieved by measuring the accumulation of fluorescent single-stranded DNA-binding protein on the single-stranded DNA product of the helicase, using total internal reflection fluorescence microscopy. By immobilizing either the DNA or helicase, localized increase in fluorescence provides information about the rate of unwinding and the processivity of individual enzymes. In addition, it reveals details of the unwinding process, such as pauses and bursts of activity. The generic and versatile nature of the assay makes it applicable to a variety of DNA helicases and DNA templates. The method is an important addition to the single-molecule toolbox available for studying DNA processing enzymes.

  12. Single-molecule mechanochemical sensing using DNA origami nanostructures.

    PubMed

    Koirala, Deepak; Shrestha, Prakash; Emura, Tomoko; Hidaka, Kumi; Mandal, Shankar; Endo, Masayuki; Sugiyama, Hiroshi; Mao, Hanbin

    2014-07-28

    While single-molecule sensing offers the ultimate detection limit, its throughput is often restricted as sensing events are carried out one at a time in most cases. 2D and 3D DNA origami nanostructures are used as expanded single-molecule platforms in a new mechanochemical sensing strategy. As a proof of concept, six sensing probes are incorporated in a 7-tile DNA origami nanoassembly, wherein binding of a target molecule to any of these probes leads to mechanochemical rearrangement of the origami nanostructure, which is monitored in real time by optical tweezers. Using these platforms, 10 pM platelet-derived growth factor (PDGF) are detected within 10 minutes, while demonstrating multiplex sensing of the PDGF and a target DNA in the same solution. By tapping into the rapid development of versatile DNA origami nanostructures, this mechanochemical platform is anticipated to offer a long sought solution for single-molecule sensing with improved throughput.

  13. Partial sequencing of a single DNA molecule with a scanning tunnelling microscope

    NASA Astrophysics Data System (ADS)

    Tanaka, Hiroyuki; Kawai, Tomoji

    2009-08-01

    The scanning tunnelling microscope is capable of the real-space imaging and spectroscopy of molecules on an atomic scale. Numerous attempts have been made to use the scanning tunnelling microscope to sequence single DNA molecules, but difficulties in preparing samples of long-chain DNA molecules on surfaces, and problems in reproducing results have limited these experiments. Here, we report single-molecule DNA sequencing with a scanning tunnelling microscope by using an oblique pulse-injection method to deposit the molecules onto a copper surface. First, we show that guanine bases have a distinct electronic state that allows them to be distinguished from the other nucleic acid bases. Then, by comparing data on M13mp18, a single-stranded phage DNA, with a known base sequence, the `electronic fingerprint' of guanine bases in the DNA molecule is identified. These results show that it is possible to sequence individual guanine bases in real long-chain DNA molecules with high-resolution scanning tunnelling microscope imaging and spectroscopy.

  14. Immobilization of layered double hydroxides in the fluidic system for nanoextraction of specific DNA molecules

    NASA Astrophysics Data System (ADS)

    Chen, Jem-Kun; Chan, Chia-Hao; Chang, Feng-Chih

    2008-02-01

    The purpose of this study was to immobilize inorganic layered double hydroxides (LDHs) on the poly(methylmethacrylate) substrate as the media to extract the specific DNA molecules through fluidic system to enhance the efficiency of extract specific DNA molecules from extremely low concentration in sample solution. LDH immobilized through solvent swelling and plasma treatment on the polymer surface captured the specific DNA molecules lysed from Escherichia coli (E. coli) cells as the target DNA molecules with 2×10-4g/l of concentration in sample solution mixed biomacromolecules lysed from human blood. The encapsulated DNA molecules released through dissolving of LDHs by slight acid (pH=4-5) solution then amplified by polymerase chain reaction (PCR) process through the primers for E. coli cells. The DNA molecules amplified by PCR process were characterized by gel electrophoresis to recognize the existence of E. coli cells. The results show that immobilized LDHs could be regarded as the specific DNA detector for rapid disease diagnosis through fluidic system.

  15. Nanoconstructions Based on Spatially Ordered Nucleic Acid Molecules

    NASA Astrophysics Data System (ADS)

    Yevdokimov, Yu. M.

    Different strategies for the design of nanoconstructions whose building blocks are both linear molecules of double-stranded nucleic acids and nucleic acid molecules fixed in the spatial structure of particles of liquid-crystalline dispersions are described.

  16. Sequence-Specific Molecular Lithography on Single DNA Molecules

    NASA Astrophysics Data System (ADS)

    Keren, Kinneret; Krueger, Michael; Gilad, Rachel; Ben-Yoseph, Gdalyahu; Sivan, Uri; Braun, Erez

    2002-07-01

    Recent advances in the realization of individual molecular-scale electronic devices emphasize the need for novel tools and concepts capable of assembling such devices into large-scale functional circuits. We demonstrated sequence-specific molecular lithography on substrate DNA molecules by harnessing homologous recombination by RecA protein. In a sequence-specific manner, we patterned the coating of DNA with metal, localized labeled molecular objects and grew metal islands on specific sites along the DNA substrate, and generated molecularly accurate stable DNA junctions for patterning the DNA substrate connectivity. In our molecular lithography, the information encoded in the DNA molecules replaces the masks used in conventional microelectronics, and the RecA protein serves as the resist. The molecular lithography works with high resolution over a broad range of length scales from nanometers to many micrometers.

  17. Placing molecules with Bohr radius resolution using DNA origami

    NASA Astrophysics Data System (ADS)

    Funke, Jonas J.; Dietz, Hendrik

    2016-01-01

    Molecular self-assembly with nucleic acids can be used to fabricate discrete objects with defined sizes and arbitrary shapes. It relies on building blocks that are commensurate to those of biological macromolecular machines and should therefore be capable of delivering the atomic-scale placement accuracy known today only from natural and designed proteins. However, research in the field has predominantly focused on producing increasingly large and complex, but more coarsely defined, objects and placing them in an orderly manner on solid substrates. So far, few objects afford a design accuracy better than 5 nm, and the subnanometre scale has been reached only within the unit cells of designed DNA crystals. Here, we report a molecular positioning device made from a hinged DNA origami object in which the angle between the two structural units can be controlled with adjuster helices. To test the positioning capabilities of the device, we used photophysical and crosslinking assays that report the coordinate of interest directly with atomic resolution. Using this combination of placement and analysis, we rationally adjusted the average distance between fluorescent molecules and reactive groups from 1.5 to 9 nm in 123 discrete displacement steps. The smallest displacement step possible was 0.04 nm, which is slightly less than the Bohr radius. The fluctuation amplitudes in the distance coordinate were also small (±0.5 nm), and within a factor of two to three of the amplitudes found in protein structures.

  18. Arrays of Individual DNA Molecules on Nanopatterned Substrates

    PubMed Central

    Hager, Roland; Halilovic, Alma; Burns, Jonathan R.; Schäffler, Friedrich; Howorka, Stefan

    2017-01-01

    Arrays of individual molecules can combine the advantages of microarrays and single-molecule studies. They miniaturize assays to reduce sample and reagent consumption and increase throughput, and additionally uncover static and dynamic heterogeneity usually masked in molecular ensembles. However, realizing single-DNA arrays must tackle the challenge of capturing structurally highly dynamic strands onto defined substrate positions. Here, we create single-molecule arrays by electrostatically adhering single-stranded DNA of gene-like length onto positively charged carbon nanoislands. The nanosites are so small that only one molecule can bind per island. Undesired adsorption of DNA to the surrounding non-target areas is prevented via a surface-passivating film. Of further relevance, the DNA arrays are of tunable dimensions, and fabricated on optically transparent substrates that enable singe-molecule detection with fluorescence microscopy. The arrays are hence compatible with a wide range of bioanalytical, biophysical, and cell biological studies where individual DNA strands are either examined in isolation, or interact with other molecules or cells. PMID:28198806

  19. Arrays of Individual DNA Molecules on Nanopatterned Substrates

    NASA Astrophysics Data System (ADS)

    Hager, Roland; Halilovic, Alma; Burns, Jonathan R.; Schäffler, Friedrich; Howorka, Stefan

    2017-02-01

    Arrays of individual molecules can combine the advantages of microarrays and single-molecule studies. They miniaturize assays to reduce sample and reagent consumption and increase throughput, and additionally uncover static and dynamic heterogeneity usually masked in molecular ensembles. However, realizing single-DNA arrays must tackle the challenge of capturing structurally highly dynamic strands onto defined substrate positions. Here, we create single-molecule arrays by electrostatically adhering single-stranded DNA of gene-like length onto positively charged carbon nanoislands. The nanosites are so small that only one molecule can bind per island. Undesired adsorption of DNA to the surrounding non-target areas is prevented via a surface-passivating film. Of further relevance, the DNA arrays are of tunable dimensions, and fabricated on optically transparent substrates that enable singe-molecule detection with fluorescence microscopy. The arrays are hence compatible with a wide range of bioanalytical, biophysical, and cell biological studies where individual DNA strands are either examined in isolation, or interact with other molecules or cells.

  20. Recursive construction of perfect DNA molecules from imperfect oligonucleotides.

    PubMed

    Linshiz, Gregory; Yehezkel, Tuval Ben; Kaplan, Shai; Gronau, Ilan; Ravid, Sivan; Adar, Rivka; Shapiro, Ehud

    2008-01-01

    Making faultless complex objects from potentially faulty building blocks is a fundamental challenge in computer engineering, nanotechnology and synthetic biology. Here, we show for the first time how recursion can be used to address this challenge and demonstrate a recursive procedure that constructs error-free DNA molecules and their libraries from error-prone oligonucleotides. Divide and Conquer (D&C), the quintessential recursive problem-solving technique, is applied in silico to divide the target DNA sequence into overlapping oligonucleotides short enough to be synthesized directly, albeit with errors; error-prone oligonucleotides are recursively combined in vitro, forming error-prone DNA molecules; error-free fragments of these molecules are then identified, extracted and used as new, typically longer and more accurate, inputs to another iteration of the recursive construction procedure; the entire process repeats until an error-free target molecule is formed. Our recursive construction procedure surpasses existing methods for de novo DNA synthesis in speed, precision, amenability to automation, ease of combining synthetic and natural DNA fragments, and ability to construct designer DNA libraries. It thus provides a novel and robust foundation for the design and construction of synthetic biological molecules and organisms.

  1. Single-Molecule Denaturation Mapping of DNA in Nanofluidic Channels

    NASA Astrophysics Data System (ADS)

    Reisner, Walter; Larsen, Niels; Silahtaroglu, Asli; Kristensen, Anders; Tommerup, Niels; Tegenfeldt, Jonas O.; Flyvbjerg, Henrik

    2010-03-01

    Nanochannel based DNA stretching can serve as a platform for a new optical mapping technique based on measuring the pattern of partial melting along the extended molecules. We partially melt DNA extended in nanofluidic channels via a combination of local heating and added chemical denaturants. The melted molecules, imaged via a standard fluorescence videomicroscopy setup, exhibit a nonuniform fluorescence profile corresponding to a series of local dips and peaks in the intensity trace along the stretched molecule. We show that this barcode is consistent with the presence of locally melted regions along the molecule and can be explained by calculations of sequence-dependent melting probability. Specifically, we obtain experimental melting profiles for T4, T7, lambda-phage and bacterial artificial chromosome DNA (from human chromosome 12) and compare these profiles to theory. In addition, we demonstrate that the BAC melting profile can be used to align the BAC to its correct position on chromosome 12.

  2. Highly Parallel Translation of DNA Sequences into Small Molecules

    PubMed Central

    Weisinger, Rebecca M.; Wrenn, S. Jarrett; Harbury, Pehr B.

    2012-01-01

    A large body of in vitro evolution work establishes the utility of biopolymer libraries comprising 1010 to 1015 distinct molecules for the discovery of nanomolar-affinity ligands to proteins.[1], [2], [3], [4], [5] Small-molecule libraries of comparable complexity will likely provide nanomolar-affinity small-molecule ligands.[6], [7] Unlike biopolymers, small molecules can offer the advantages of cell permeability, low immunogenicity, metabolic stability, rapid diffusion and inexpensive mass production. It is thought that such desirable in vivo behavior is correlated with the physical properties of small molecules, specifically a limited number of hydrogen bond donors and acceptors, a defined range of hydrophobicity, and most importantly, molecular weights less than 500 Daltons.[8] Creating a collection of 1010 to 1015 small molecules that meet these criteria requires the use of hundreds to thousands of diversity elements per step in a combinatorial synthesis of three to five steps. With this goal in mind, we have reported a set of mesofluidic devices that enable DNA-programmed combinatorial chemistry in a highly parallel 384-well plate format. Here, we demonstrate that these devices can translate DNA genes encoding 384 diversity elements per coding position into corresponding small-molecule gene products. This robust and efficient procedure yields small molecule-DNA conjugates suitable for in vitro evolution experiments. PMID:22479303

  3. Streching of (DNA/functional molecules) complex between electrodes towards DNA molecular wire

    NASA Astrophysics Data System (ADS)

    Kobayashi, Norihisa; Nishizawa, Makoto; Inoue, Shintarou; Nakamura, Kazuki

    2009-08-01

    DNA/functional molecules such as (Ru(bpy)32+ complex, conducting polymer etc.) complex was prepared to study molecular structure and I-V characteristics towards DNA molecular wire. For example, Ru(bpy)32+ was associated with duplex of DNA by not only electrostatic interaction but also intercalation in the aqueous solution. Singlemolecular structure of DNA/Ru(bpy)32+ complex was analyzed with AFM. We found a network structure of DNA/Ru(bpy)32+ complex on the mica substrate, which is similar to native DNA. The height of DNA/Ru(bpy)32+ complex on the mica substrate was ranging from 0.8 to 1.6 nm, which was higher than the naked DNA (0.5-1.0 nm). This indicates that single-molecular DNA/Ru(bpy)32+ complex also connects to each other to form network structure on a mica substrate. In order to stretch DNA complex between electrodes, we employed high frequency and high electric field stretching method proposed by Washizu et al. We stretched and immobilized DNA single molecules between a pair of electrodes and its structures were analyzed with AFM technique. The I-V characteristics of DNA single molecules between electrodes were improved by the association of functional molecules with DNA. The molecular structure and I-V characteristics of DNA complex were discussed.

  4. Electrical characteristics and doping mechanism of DNA molecules doped with iodine solutions.

    PubMed

    Kim, Nam-Hoon; Lee, Woo Cheol; Roh, Yonghan

    2010-05-01

    This study examined the electrical characteristics of deoxyribonucleic acid (DNA) molecules doped with iodine solution and their chemical state changes before and after doping. The experiments were progressed in each lambda (A), poly(dA)-poly(dT) and poly(dG)-poly(dC) DNA under the same conditions. The authors prepared 20 nm gap Au/Ti electrodes fabricated by e-beam lithography. DNA solutions were dropped on the nano gap of the electrodes and DNA films were formed by drying in a vacuum. DNA films were doped with an iodine solution dissolved in methanol. The authors measured the electrical conductivity of DNA molecules as the number of iodine doping times in 10(-2) torr vacuum. As increase of the iodine solution doping number, the electrical conductivity of three sorts of DNA molecules was remarkably improved respectively. X-ray photoelectron spectroscopy (XPS) was performed to inspect the electrical conduction mechanism that holes on DNA nitrogen region were generated by transferring electrons to iodine molecules.

  5. Studying DNA Looping by Single-Molecule FRET

    PubMed Central

    Le, Tung T.; Kim, Harold D.

    2014-01-01

    Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA. PMID:24998459

  6. Single molecule study of a processivity clamp sliding on DNA

    SciTech Connect

    Laurence, T A; Kwon, Y; Johnson, A; Hollars, C; O?Donnell, M; Camarero, J A; Barsky, D

    2007-07-05

    Using solution based single molecule spectroscopy, we study the motion of the polIII {beta}-subunit DNA sliding clamp ('{beta}-clamp') on DNA. Present in all cellular (and some viral) forms of life, DNA sliding clamps attach to polymerases and allow rapid, processive replication of DNA. In the absence of other proteins, the DNA sliding clamps are thought to 'freely slide' along the DNA; however, the abundance of positively charged residues along the inner surface may create favorable electrostatic contact with the highly negatively charged DNA. We have performed single-molecule measurements on a fluorescently labeled {beta}-clamp loaded onto freely diffusing plasmids annealed with fluorescently labeled primers of up to 90 bases. We find that the diffusion constant for 1D diffusion of the {beta}-clamp on DNA satisfies D {le} 10{sup -14} cm{sup 2}/s, much slower than the frictionless limit of D = 10{sup -10} cm{sup 2}/s. We find that the {beta} clamp remains at the 3-foot end in the presence of E. coli single-stranded binding protein (SSB), which would allow for a sliding clamp to wait for binding of the DNA polymerase. Replacement of SSB with Human RP-A eliminates this interaction; free movement of sliding clamp and poor binding of clamp loader to the junction allows sliding clamp to accumulate on DNA. This result implies that the clamp not only acts as a tether, but also a placeholder.

  7. Developing DNA nanotechnology using single-molecule fluorescence.

    PubMed

    Tsukanov, Roman; Tomov, Toma E; Liber, Miran; Berger, Yaron; Nir, Eyal

    2014-06-17

    CONSPECTUS: An important effort in the DNA nanotechnology field is focused on the rational design and manufacture of molecular structures and dynamic devices made of DNA. As is the case for other technologies that deal with manipulation of matter, rational development requires high quality and informative feedback on the building blocks and final products. For DNA nanotechnology such feedback is typically provided by gel electrophoresis, atomic force microscopy (AFM), and transmission electron microscopy (TEM). These analytical tools provide excellent structural information; however, usually they do not provide high-resolution dynamic information. For the development of DNA-made dynamic devices such as machines, motors, robots, and computers this constitutes a major problem. Bulk-fluorescence techniques are capable of providing dynamic information, but because only ensemble averaged information is obtained, the technique may not adequately describe the dynamics in the context of complex DNA devices. The single-molecule fluorescence (SMF) technique offers a unique combination of capabilities that make it an excellent tool for guiding the development of DNA-made devices. The technique has been increasingly used in DNA nanotechnology, especially for the analysis of structure, dynamics, integrity, and operation of DNA-made devices; however, its capabilities are not yet sufficiently familiar to the community. The purpose of this Account is to demonstrate how different SMF tools can be utilized for the development of DNA devices and for structural dynamic investigation of biomolecules in general and DNA molecules in particular. Single-molecule diffusion-based Förster resonance energy transfer and alternating laser excitation (sm-FRET/ALEX) and immobilization-based total internal reflection fluorescence (TIRF) techniques are briefly described and demonstrated. To illustrate the many applications of SMF to DNA nanotechnology, examples of SMF studies of DNA hairpins and

  8. Single-Molecule Electrical Random Resequencing of DNA and RNA

    NASA Astrophysics Data System (ADS)

    Ohshiro, Takahito; Matsubara, Kazuki; Tsutsui, Makusu; Furuhashi, Masayuki; Taniguchi, Masateru; Kawai, Tomoji

    2012-07-01

    Two paradigm shifts in DNA sequencing technologies--from bulk to single molecules and from optical to electrical detection--are expected to realize label-free, low-cost DNA sequencing that does not require PCR amplification. It will lead to development of high-throughput third-generation sequencing technologies for personalized medicine. Although nanopore devices have been proposed as third-generation DNA-sequencing devices, a significant milestone in these technologies has been attained by demonstrating a novel technique for resequencing DNA using electrical signals. Here we report single-molecule electrical resequencing of DNA and RNA using a hybrid method of identifying single-base molecules via tunneling currents and random sequencing. Our method reads sequences of nine types of DNA oligomers. The complete sequence of 5'-UGAGGUA-3' from the let-7 microRNA family was also identified by creating a composite of overlapping fragment sequences, which was randomly determined using tunneling current conducted by single-base molecules as they passed between a pair of nanoelectrodes.

  9. Carrier molecules and extraction of circulating tumor DNA for next generation sequencing in colorectal cancer.

    PubMed

    Beránek, Martin; Sirák, Igor; Vošmik, Milan; Petera, Jiří; Drastíková, Monika; Palička, Vladimír

    The aims of the study were: i) to compare circulating tumor DNA (ctDNA) yields obtained by different manual extraction procedures, ii) to evaluate the addition of various carrier molecules into the plasma to improve ctDNA extraction recovery, and iii) to use next generation sequencing (NGS) technology to analyze KRAS, BRAF, and NRAS somatic mutations in ctDNA from patients with metastatic colorectal cancer. Venous blood was obtained from patients who suffered from metastatic colorectal carcinoma. For plasma ctDNA extraction, the following carriers were tested: carrier RNA, polyadenylic acid, glycogen, linear acrylamide, yeast tRNA, salmon sperm DNA, and herring sperm DNA. Each extract was characterized by quantitative real-time PCR and next generation sequencing. The addition of polyadenylic acid had a significant positive effect on the amount of ctDNA eluted. The sequencing data revealed five cases of ctDNA mutated in KRAS and one patient with a BRAF mutation. An agreement of 86% was found between tumor tissues and ctDNA. Testing somatic mutations in ctDNA seems to be a promising tool to monitor dynamically changing genotypes of tumor cells circulating in the body. The optimized process of ctDNA extraction should help to obtain more reliable sequencing data in patients with metastatic colorectal cancer.

  10. Nanofluidics and Single Molecule Detection for DNA analysis

    NASA Astrophysics Data System (ADS)

    Tegenfeldt, Jonas; Cao, Han; Austin, Robert H.; Cox, Edward C.; Tilghman, Shirley M.

    2002-03-01

    We present a device for high-resolution detection of fluorescent tags bound to DNA molecules. Submicron slits are defined in an aluminum film on a quartz wafer. Microfluidic channels are defined perpendicular to the slits. Fluorescently labeled DNA is passed through the microfluidic channels and is illuminated through the submicron slits. The resulting fluorescence is detected in using an APD. We are particularly interested in studying the pattern of transcription factors along single DNA molecules. We use the lac operon as a model system. Fusion proteins of lac-repressor and GFP have been made and imaged individually. To achieve reliable measurements of the positions of the transcription factors along the DNA, the DNA must be uniformly stretched. Previous devices relied on posts for stretching, resulting in poorly stretched DNA with highly disordered head and tail. Here we show that by forcing the DNA into channels that have a diameter close to or below the persistence length of the DNA (Lp=50nm), the DNA is forced into a stretched conformation along its entire length.

  11. Electrostatic Interaction of Long DNA Molecules with Solid State Surfaces

    NASA Astrophysics Data System (ADS)

    Li, Bingquan; Samuilov, Vladimir; Sokolov, Jonathan; Rafailovich, Miriam; Chu, Ben

    2004-03-01

    At low buffer concentration the electric charge of DNA molecules creates a strong electrostatic interaction and, as a result, a number of phenomena, such as the electro-hydrodynamic instability, partial adsorption at the buffer-semiconductor interface and stretching of DNA with the electric field. Long DNA molecules at the silicon substrate?buffer solution interface are very interesting objects for the electrical transport [1,2] and the mechanical properties, like entropic elasticity, studies. The system (DNA-substrate-electric field in the buffer solution) is very complicated. Due to the strong electrostatic interaction of DNA with the substrate, the image charge is generated, and the physical adsorption takes place. We have studied the S. Pombe genomic DNA of the order of 5 Mbp. Within a surface DNA is entropically partially recoiled due to electrostatic adsorption at a few points. While varying the direction of the low electric field the direction of the electroosmotic flow is changing and stretching the parts of DNA between the adsorption points. If the electric field is high enough, DNA is de-trapped and forms a compact coil. This behavior could be considered as an inverse mechanism of entropy trapping due to confined constrictions. In the case of the surface, DNA is recoiled and trapped in the stretched configuration in the deep energetic barrier by Si surface due to the strong electrostatic interaction. If the energy of the field is enough to overcome the barrier, DNA is detached. The Si surface could be considered as an analog of the entropic recoiling nanostructure. [1]. N. Pernodet, V. Samuilov, K. Shin, J. Sokolov, M.H. Rafailovich, D. Gersappe, B. Chu. DNA Electrophoresis on a Flat Surface, Physical Review Letters, 85 (2000) 5651-5654. [2] Y.-S. Seo, V.A. Samuilov, J. Sokolov, M. Rafailovich, B. Tinland, J. Kim, B. Chu. DNA separation at a liquid-solid interface, Electrophoresis, 23 (2002) 2618-2625.

  12. Long DNA Molecules at Liuid-Solid Interfaces

    NASA Astrophysics Data System (ADS)

    Seo, Young-Soo; Samuilov, Vladimir; Sokolov, John; Rafailovich, Miriam; Chu, Ben

    2003-03-01

    The electrical transport of long DNA molecules was studied using a newly developed method of electrophoresis on flat surfaces [1]. We have shown that a flat silicon substrate, without any surface features, can be used to fractionate DNA on a liquid-solid interface. We determine that the ability of a flat surface to separate DNA molecules results from the local friction between the surface and the adsorbed DNA segments. The mobility of lambda- DNA molecules on this surface was found to scale as the persistent length with the ionic strength of the buffer. This experimental result indicates that at high buffer concentration the separation mechanism of solid-liquid interface electrophoresis is expected to be due to surface friction rather than biased reptation [2]. At low buffer concentrations the adsorbed DNA move in the electrical field parallel to the surface, and also due the electroosmotic convection that drags the DNA chains and they are stretched . The electric double layer is responsible for a velocity profile of the electroosmotic flow. The net electrophoretic mobility of longer DNA, being trapped closer to the surface, is higher than of the shorter ones in the electric field, oriented along the surface. [1]. N. Pernodet, V. Samuilov, K. Shin, J. Sokolov, M.H. Rafailovich, D. Gersappe, B. Chu. DNA Electrophoresis on a Flat Surface, Physical Review Letters, 85 (2000) 5651-5654. [2] Y.-S. Seo, V.A. Samuilov, J. Sokolov, M. Rafailovich, B. Tinland, J. Kim, B. Chu. DNA separation at a liquid-solid interface, Electrophoresis, 23 (2002) 2618-2625.

  13. Single molecule conformational analysis of DNA G-quadruplexes

    PubMed Central

    Shirude, Pravin S.; Balasubramanian, Shankar

    2008-01-01

    Single molecule fluorescence resonance energy transfer (FRET) can be employed to study conformational heterogeneity and real-time dynamics of biological macromolecules. Here we present single molecule studies on human genomic DNA G-quadruplex sequences that occur in the telomeres and in the promoter of a proto-oncogene. The findings are discussed with respect to the proposed biological function(s) of such motifs in living cells. PMID:18295608

  14. Observation of HIV-1 Nucleocapsid Protein induced TAR DNA melting at the single molecule level

    NASA Astrophysics Data System (ADS)

    Cosa, Gonzalo; Harbron, Elizabeth; O'Connor, Donald; Musier-Forsyth, Karin; Barbara, Paul

    2003-03-01

    Reverse transcription of the HIV-1 RNA genome involves several nucleic acid rearrangement steps, and the HIV-1 nucleocapsid protein (NC) plays a key role in this process. NC is a nucleic acid chaperone protein, which facilitates the formation of the most stable nucleic acid structures. Single molecule fluorescence resonance energy transfer (SM-FRET) measurements enable us to observe the NC-induced conformational fluctuations of a transactivation response region (TAR) DNA hairpin, which is part of the initial product of reverse transcription known as minus-strand strong-stop DNA. SM-FRET studies show that the majority of conformational fluctuations of the fluorescently-labeled TAR DNA hairpin in the presence of NC occur in <100 ms. A single molecule explores a wide range of confomations unpon NC binding, with fluctuations encompassing as many as 40 bases in both arms of the hairpin. No conformational fluctuations are observed with the dye-labeled TAR DNA hairpin in the absence of NC or when a labeled TAR DNA hairpin variant lacking bulges and internal loops is analyzed in the presence of NC. This study represents the first real-time observation of NC-mediated nucleic acid conformational fluctuations, revealing new insights into NC's nucleic acid chaperone activity.

  15. Specific heat spectra of long-range correlated DNA molecules

    NASA Astrophysics Data System (ADS)

    Moreira, D. A.; Albuquerque, E. L.; Mauriz, P. W.; Vasconcelos, M. S.

    2006-11-01

    The specific heat spectra of long-range correlated DNA molecules is theoretically analyzed for a stacked array of single-stranded DNA made up from the nucleotides guanine G, adenine A, cytosine C and thymine T arranged in the Fibonacci and Rudin-Shapiro quasiperiodic sequences, with the aim to compare them with those related with a genomic DNA sequence. The energy spectra are calculated using the one-dimensional Schrödinger equation in a tight-binding approximation with the on-site energy exhibiting long-range disorder and nonrandom hopping amplitudes.

  16. Single Molecule Screening of Disease DNA Without Amplification

    SciTech Connect

    Lee, Ji-Young

    2006-01-01

    The potential of single molecule detection as an analysis tool in biological and medical fields is well recognized today. This fast evolving technique will provide fundamental sensitivity to pick up individual pathogen molecules, and therefore contribute to a more accurate diagnosis and a better chance for a complete cure. Many studies are being carried out to successfully apply this technique in real screening fields. In this dissertation, several attempts are shown that have been made to test and refine the application of the single molecule technique as a clinical screening method. A basic applicability was tested with a 100% target content sample, using electrophoretic mobility and multiple colors as identification tools. Both electrophoretic and spectral information of individual molecule were collected within a second, while the molecule travels along the flow in a capillary. Insertion of a transmission grating made the recording of the whole spectrum of a dye-stained molecule possible without adding complicated instrumental components. Collecting two kinds of information simultaneously and combining them allowed more thorough identification, up to 98.8% accuracy. Probing mRNA molecules with fluorescently labeled cDNA via hybridization was also carried out. The spectral differences among target, probe, and hybrid were interpreted in terms of dispersion distances after transmission grating, and used for the identification of each molecule. The probes were designed to have the least background when they are free, but have strong fluorescence after hybridization via fluorescence resonance energy transfer. The mRNA-cDNA hybrids were further imaged in whole blood, plasma, and saliva, to test how far a crude preparation can be tolerated. Imaging was possible with up to 50% of clear bio-matrix contents, suggesting a simple lysis and dilution would be sufficient for imaging for some cells. Real pathogen DNA of human papillomavirus (HPV) type-I6 in human genomic DNA

  17. Microfluidic-assisted analysis of replicating DNA molecules

    PubMed Central

    Sidorova, Julia M.; Li, Nianzhen; Schwartz, David C.; Folch, Albert; Monnat, Raymond J.

    2009-01-01

    Single molecule-based protocols have been gaining popularity as a way to visualize DNA replication at the global genomic and locus-specific levels. These protocols take advantage of the ability of many organisms to incorporate nucleoside analogs during DNA replication, together with a method for displaying stretched DNA on glass for immunostaining and microscopy. We describe here a microfluidic platform that can be used to stretch and capture labeled DNA molecules for replication analyses. This platform consists of parallel arrays of 3-sided, 3 or 4 μm high, variable width capillary channels fabricated from polymethyl siloxane (PDMS) by conventional soft lithography, and silane-modified glass coverslips to reversibly seal the open side of the channels. Capillary tension in these microchannels facilitates DNA loading, stretching and glass coverslip deposition from μL-scale DNA samples. The simplicity and extensibility of this platform should facilitate DNA replication analyses using small samples from a variety of biological and clinical sources. PMID:19444242

  18. Analysis of DNA interactions using single-molecule force spectroscopy.

    PubMed

    Ritzefeld, Markus; Walhorn, Volker; Anselmetti, Dario; Sewald, Norbert

    2013-06-01

    Protein-DNA interactions are involved in many biochemical pathways and determine the fate of the corresponding cell. Qualitative and quantitative investigations on these recognition and binding processes are of key importance for an improved understanding of biochemical processes and also for systems biology. This review article focusses on atomic force microscopy (AFM)-based single-molecule force spectroscopy and its application to the quantification of forces and binding mechanisms that lead to the formation of protein-DNA complexes. AFM and dynamic force spectroscopy are exciting tools that allow for quantitative analysis of biomolecular interactions. Besides an overview on the method and the most important immobilization approaches, the physical basics of the data evaluation is described. Recent applications of AFM-based force spectroscopy to investigate DNA intercalation, complexes involving DNA aptamers and peptide- and protein-DNA interactions are given.

  19. Confining individual DNA molecules in an axisymmetric entropy gradient

    NASA Astrophysics Data System (ADS)

    Peters, Robert D.; Dalnoki-Veress, Kari

    2010-03-01

    Many asymmetric and discontinuous confining environments have been used to study the properties of confined DNA. We have developed a unique method for studying DNA in micropipettes, resulting in a confining environment that is axisymmetric with a continuously changing entropy gradient. An applied electric field forces the chain into sub-micron confinement and fluorescence microscopy is used to track the effect of confinement on the entropy of individual DNA chains. Releasing the electric field, we probe the dynamics of the DNA chain in a continuously changing confinement, yielding a comprehensive study of the entropic force. This technique provides a novel method for studying the effect of polymer chain architecture on entropy. These architectures include knots in polymer chains, cyclic chains, or the presence of histones amongst DNA molecules.

  20. The properties and applications of single-molecule DNA sequencing

    PubMed Central

    2011-01-01

    Single-molecule sequencing enables DNA or RNA to be sequenced directly from biological samples, making it well-suited for diagnostic and clinical applications. Here we review the properties and applications of this rapidly evolving and promising technology. PMID:21349208

  1. Conductance of DNA molecules: Effects of decoherence and bonding

    NASA Astrophysics Data System (ADS)

    Zilly, Matías; Ujsághy, Orsolya; Wolf, Dietrich E.

    2010-09-01

    The influence of decoherence and bonding on the linear conductance of single double-stranded DNA molecules is examined by fitting a phenomenological statistical model developed recently [M. Zilly, O. Ujsághy, and D. E. Wolf, Eur. Phys. J. B 68, 237 (2009)10.1140/epjb/e2009-00101-0] to experimental results. The DNA molecule itself is described by a tight-binding ladder model with parameters obtained from published ab initio calculations [K. Senthilkumar, F. C. Grozema, C. F. Guerra, F. M. Bickelhaupt, F. D. Lewis, Y. A. Berlin, M. A. Ratner, and L. D. A. Siebbeles, J. Am. Chem. Soc. 127, 14894 (2005)10.1021/ja054257e]. The good agreement with the experiments on sequence and length dependence gives a hint on the nature of conduction in DNA and at the same time provides a crucial test of the model.

  2. Recursive construction and error correction of DNA molecules and libraries from synthetic and natural DNA.

    PubMed

    Yehezkel, Tuval Ben; Linshiz, Gregory; Kaplan, Shai; Gronau, Ilan; Ravid, Sivan; Adar, Rivka; Shapiro, Ehud

    2011-01-01

    Making error-free, custom DNA assemblies from potentially faulty building blocks is a fundamental challenge in synthetic biology. Here, we show how recursion can be used to address this challenge using a recursive procedure that constructs error-free DNA molecules and their libraries from error-prone synthetic oligonucleotides and naturally existing DNA. Specifically, we describe how divide and conquer (D&C), the quintessential recursive problem-solving technique, is applied in silico to divide target DNA sequences into overlapping, albeit error prone, oligonucleotides, and how recursive construction is applied in vitro to combine them to form error-prone DNA molecules. To correct DNA sequence errors, error-free fragments of these molecules are then identified, extracted, and used as new, typically longer and more accurate, inputs to another iteration of the recursive construction procedure; the entire process repeats until an error-free target molecule is formed. The method allows combining synthetic and natural DNA fragments into error-free designer DNA libraries, thus providing a foundation for the design and construction of complex synthetic DNA assemblies.

  3. Single Molecule Study of DNA Organization and Recombination

    NASA Astrophysics Data System (ADS)

    Xiao, Botao

    We have studied five projects related to DNA organization and recombination using mainly single molecule force-spectroscopy and statistical tools. First, HU is one of the most abundant DNA-organizing proteins in bacterial chromosomes and participates in gene regulation. We report experiments that study the dependence of DNA condensation by HU on force, salt and HU concentration. A first important result is that at physiological salt levels, HU only bends DNA, resolving a previous paradox of why a chromosome-compacting protein should have a DNA-stiffening function. A second major result is quantitative demonstration of strong dependencies of HU-DNA dissociation on both salt concentration and force. Second, we have used a thermodynamic Maxwell relation to count proteins driven off large DNAs by tension, an effect important to understanding DNA organization. Our results compare well with estimates of numbers of proteins HU and Fis in previous studies. We have also shown that a semi-flexible polymer model describes our HU experimental data well. The force-dependent binding suggests mechano-chemical mechanisms for gene regulation. Third, the elusive role of protein H1 in chromatin has been clarified with purified H1 and Xenopus extracts. We find that H1 compacts DNA by both bending and looping. Addition of H1 enhances chromatin formation and maintains the plasticity of the chromatin. Fourth, the topology and mechanics of DNA twisting are critical to DNA organization and recombination. We have systematically measured DNA extension as a function of linking number density from 0.08 to -2 with holding forces from 0.2 to 2.4 pN. Unlike previous proposals, the DNA extension decreases with negative linking number. Finally, DNA recombination is a dynamic process starting from enzyme-DNA binding. We report that the Int-DBD domain of lambda integrase binds to DNA without compaction at low Int-DBD concentration. High concentration of Int-DBD loops DNA below a threshold force

  4. Selective binding of single-stranded DNA-binding proteins onto DNA molecules adsorbed on single-walled carbon nanotubes.

    PubMed

    Nii, Daisuke; Hayashida, Takuya; Yamaguchi, Yuuki; Ikawa, Shukuko; Shibata, Takehiko; Umemura, Kazuo

    2014-09-01

    Single-stranded DNA-binding (SSB) proteins were treated with hybrids of DNA and single-walled carbon nanotubes (SWNTs) to examine the biological function of the DNA molecules adsorbed on the SWNT surface. When single-stranded DNA (ssDNA) was used for the hybridization, significant binding of the SSB molecules to the ssDNA-SWNT hybrids was observed by using atomic force microscopy (AFM) and agarose gel electrophoresis. When double-stranded DNA (dsDNA) was used, the SSB molecules did not bind to the dsDNA-SWNT hybrids in most of the conditions that we evaluated. A specifically modified electrophoresis procedure was used to monitor the locations of the DNA, SSB, and SWNT molecules. Our results clearly showed that ssDNA/dsDNA molecules on the SWNT surfaces retained their single-stranded/double-stranded structures.

  5. Ku stimulation of DNA ligase IV-dependent ligation requires inward movement along the DNA molecule.

    PubMed

    Kysela, Boris; Doherty, Aidan J; Chovanec, Miroslav; Stiff, Thomas; Ameer-Beg, Simon M; Vojnovic, Borivoj; Girard, Pierre-Marie; Jeggo, Penny A

    2003-06-20

    The DNA ligase IV.XRCC4 complex (LX) functions in DNA non-homologous-end joining, the main pathway for double-strand break repair in mammalian cells. We show that, in contrast to ligation by T4 ligase, the efficiency of LX ligation of double-stranded (ds) ends is critically dependent upon the length of the DNA substrate. The effect is specific for ds ligation, and LX/DNA binding is not influenced by the substrate length. Ku stimulates LX ligation at concentrations resulting in 1-2 Ku molecules bound per substrate, whereas multiply Ku-bound DNA molecules inhibit ds ligation. The combined footprint of DNA with Ku and LX bound is the sum of each individual footprint suggesting that the two complexes are located in tandem at the DNA end. Inhibition of Ku translocation by the presence of cis-platinum adducts on the DNA substrate severely inhibits ligation by LX. Fluorescence resonance energy transfer analysis using fluorophore-labeled Ku and DNA molecules showed that, as expected, Ku makes close contact with the DNA end and that addition of LX can disrupt this close contact. Finally, we show that recruitment of LX by Ku is impaired in an adenylation-defective mutant providing further evidence that LX interacts directly with the DNA end, possibly via the 5'-phosphate as shown for prokaryotic ligases. Taken together, our results suggest that, when LX binds to a Ku-bound DNA molecule, it causes inward translocation of Ku and that freedom to move inward on the DNA is essential to Ku stimulation of LX activity.

  6. Real-time DNA sequencing from single polymerase molecules.

    PubMed

    Eid, John; Fehr, Adrian; Gray, Jeremy; Luong, Khai; Lyle, John; Otto, Geoff; Peluso, Paul; Rank, David; Baybayan, Primo; Bettman, Brad; Bibillo, Arkadiusz; Bjornson, Keith; Chaudhuri, Bidhan; Christians, Frederick; Cicero, Ronald; Clark, Sonya; Dalal, Ravindra; Dewinter, Alex; Dixon, John; Foquet, Mathieu; Gaertner, Alfred; Hardenbol, Paul; Heiner, Cheryl; Hester, Kevin; Holden, David; Kearns, Gregory; Kong, Xiangxu; Kuse, Ronald; Lacroix, Yves; Lin, Steven; Lundquist, Paul; Ma, Congcong; Marks, Patrick; Maxham, Mark; Murphy, Devon; Park, Insil; Pham, Thang; Phillips, Michael; Roy, Joy; Sebra, Robert; Shen, Gene; Sorenson, Jon; Tomaney, Austin; Travers, Kevin; Trulson, Mark; Vieceli, John; Wegener, Jeffrey; Wu, Dawn; Yang, Alicia; Zaccarin, Denis; Zhao, Peter; Zhong, Frank; Korlach, Jonas; Turner, Stephen

    2009-01-02

    We present single-molecule, real-time sequencing data obtained from a DNA polymerase performing uninterrupted template-directed synthesis using four distinguishable fluorescently labeled deoxyribonucleoside triphosphates (dNTPs). We detected the temporal order of their enzymatic incorporation into a growing DNA strand with zero-mode waveguide nanostructure arrays, which provide optical observation volume confinement and enable parallel, simultaneous detection of thousands of single-molecule sequencing reactions. Conjugation of fluorophores to the terminal phosphate moiety of the dNTPs allows continuous observation of DNA synthesis over thousands of bases without steric hindrance. The data report directly on polymerase dynamics, revealing distinct polymerization states and pause sites corresponding to DNA secondary structure. Sequence data were aligned with the known reference sequence to assay biophysical parameters of polymerization for each template position. Consensus sequences were generated from the single-molecule reads at 15-fold coverage, showing a median accuracy of 99.3%, with no systematic error beyond fluorophore-dependent error rates.

  7. Recent Advances in Developing Small Molecules Targeting Nucleic Acid

    PubMed Central

    Wang, Maolin; Yu, Yuanyuan; Liang, Chao; Lu, Aiping; Zhang, Ge

    2016-01-01

    Nucleic acids participate in a large number of biological processes. However, current approaches for small molecules targeting protein are incompatible with nucleic acids. On the other hand, the lack of crystallization of nucleic acid is the limiting factor for nucleic acid drug design. Because of the improvements in crystallization in recent years, a great many structures of nucleic acids have been reported, providing basic information for nucleic acid drug discovery. This review focuses on the discovery and development of small molecules targeting nucleic acids. PMID:27248995

  8. Small Molecules, Inhibitors of DNA-PK, Targeting DNA Repair, and Beyond

    PubMed Central

    Davidson, David; Amrein, Lilian; Panasci, Lawrence; Aloyz, Raquel

    2012-01-01

    Many current chemotherapies function by damaging genomic DNA in rapidly dividing cells ultimately leading to cell death. This therapeutic approach differentially targets cancer cells that generally display rapid cell division compared to normal tissue cells. However, although these treatments are initially effective in arresting tumor growth and reducing tumor burden, resistance and disease progression eventually occur. A major mechanism underlying this resistance is increased levels of cellular DNA repair. Most cells have complex mechanisms in place to repair DNA damage that occurs due to environmental exposures or normal metabolic processes. These systems, initially overwhelmed when faced with chemotherapy induced DNA damage, become more efficient under constant selective pressure and as a result chemotherapies become less effective. Thus, inhibiting DNA repair pathways using target specific small molecule inhibitors may overcome cellular resistance to DNA damaging chemotherapies. Non-homologous end joining a major mechanism for the repair of double-strand breaks (DSB) in DNA is regulated in part by the serine/threonine kinase, DNA dependent protein kinase (DNA-PK). The DNA-PK holoenzyme acts as a scaffold protein tethering broken DNA ends and recruiting other repair molecules. It also has enzymatic activity that may be involved in DNA damage signaling. Because of its’ central role in repair of DSBs, DNA-PK has been the focus of a number of small molecule studies. In these studies specific DNA-PK inhibitors have shown efficacy in synergizing chemotherapies in vitro. However, compounds currently known to specifically inhibit DNA-PK are limited by poor pharmacokinetics: these compounds have poor solubility and have high metabolic lability in vivo leading to short serum half-lives. Future improvement in DNA-PK inhibition will likely be achieved by designing new molecules based on the recently reported crystallographic structure of DNA-PK. Computer based drug

  9. Detection of pathogenic DNA at the single-molecule level

    NASA Astrophysics Data System (ADS)

    Yahiatène, Idir; Klamp, Tobias; Schüttpelz, Mark; Sauer, Markus

    2011-03-01

    We demonstrate ultrasensitive detection of pathogenic DNA in a homogeneous assay at the single-molecule level applying two-color coincidence analysis. The target molecule we quantify is a 100 nucleotide long synthetic single-stranded oligonucleotide adapted from Streptococcus pneumoniae, a bacterium causing lower respiratory tract infections. Using spontaneous hybridization of two differently fluorescing Molecular Beacons we demonstrate a detection sensitivity of 100 fM (10-13M) in 30 seconds applying a simple microfluidic device with a 100 μm channel and confocal two-color fluorescence microscopy.

  10. Oriented and vectorial immobilization of linear M13 dsDNA between interdigitated electrodes--towards single molecule DNA nanostructures.

    PubMed

    Hölzel, Ralph; Gajovic-Eichelmann, Nenad; Bier, Frank F

    2003-05-01

    The ability to control molecules at a resolution well below that offered by photolithography has gained much interest recently. DNA is a promising candidate for this task since it offers excellent specificity in base-pairing combined with addressability at the nanometer scale. New applications in biosensing, e.g. interaction analysis at the single molecule level, or nanobiotechnology, e.g. ultradense DNA microarrays, have been devised that rely on stretched DNA bridges. The basic technology required is the ability to deposit spatially defined, stretched DNA-bridges between anchoring structures on surfaces. In this paper we present two techniques for spanning 2 microm long dsDNA bridges between neighboring interdigitated electrodes (IDEs). The extended DNA used was linearized M13 dsDNA (M13mp18 7231 bp, ca. 2.5 microm length), either unmodified, or with chemical modifications at both ends. The first approach is based on the dielectrophoretic (DEP) concentration and alignment of linearized wild-type dsDNA. IDEs with 1.7 microm spacing are driven with an AC voltage around 1 MHz leading to field strengths in the order of 1 MV m(-1). The dsDNA is polarized and linearized by the force field and accumulates in the gap between two neighboring electrodes. This process is reversible and was visualized by fluorescence staining of M13 DNA using PicoGreen, as intercalating dye. The resulting dsDNA bridges and their orientation are discernible under the fluorescence microscope using fluorescent particles of different color. The particles are tagged with sequence specific peptide nucleic acid (PNA) probes that bind to the DNA double strand at specific sites. The second approach is based on asymmetric electrochemical modification of a gold IDE with 2.0 microm spacings followed by spontaneous or stimulated deposition of a chemically modified M13-DNA. One side of the IDE was selectively coated with streptavidin by electropolymerization of a novel hydrophilic conductive polymer in

  11. Long DNA Molecules at Liquid-Solid Interfaces

    NASA Astrophysics Data System (ADS)

    Samuilov, Vladimir; Li, B.; Sokolov, J.; Rafailovich, M.; Chu, B.

    2006-03-01

    The electrophoresis of long DNA molecules was studied using a newly developed method of electrophoresis on flat surfaces [1] in the regime of strong electrostatic interaction. The mobility of lambda- DNA molecules on this surface was found to scale as the square root of the persistent length with the ionic strength at high buffer. This experimental result indicates that at high buffer concentration the separation mechanism of solid-liquid interface electrophoresis is expected to be due to surface friction rather than biased reptation [2-4]. At low buffer concentrations the DNA chains are stretched .The electric double layer is responsible for a velocity profile of the electroosmotic flow. The net electrophoretic mobility of longer DNA, being trapped closer to the surface as found to be higher then for the shorter ones in the electric field. [1]. N. Pernodet, V. Samuilov, K. Shin, et al. Physical Review Letters, 85 (2000) 5651-5654. [2] Y.-S. Seo, V.A. Samuilov, J. Sokolov, et al. Electrophoresis, 23 (2002) 2618-2625. [3] Y.-S. Seo, H.. Luo, V. A. Samuilov, et al. DNA Electrophoresis on nanopatterned surfaces, Nano Letters, 4, 2004, 659-664.

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

    PubMed

    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.

  13. Determination of the elastic properties of short ssDNA molecules by mechanically folding and unfolding DNA hairpins.

    PubMed

    Alemany, Anna; Ritort, Felix

    2014-12-01

    The characterization of elastic properties of biopolymers is crucial to understand many molecular reactions determined by conformational bending fluctuations of the polymer. Direct measurement of such elastic properties using single-molecule methods is usually hindered by the intrinsic tendency of such biopolymers to form high-order molecular structures. For example, single-stranded deoxyribonucleic acids (ssDNA) tend to form secondary structures such as local double helices that prevent the direct measurement of the ideal elastic response of the ssDNA. In this work, we show how to extract the ideal elastic response in the entropic regime of short ssDNA molecules by mechanically pulling two-state DNA hairpins of different contour lengths. This is achieved by measuring the force dependence of the molecular extension and stiffness on mechanically folding and unfolding the DNA hairpin. Both quantities are fit to the worm-like chain elastic model giving values for the persistence length and the interphosphate distance. This method can be used to unravel the elastic properties of short ssDNA and RNA sequences and, more generally, any biopolymer that can exhibit a cooperative two-state transition between mechanically folded and unfolded states (such as proteins).

  14. Simple horizontal magnetic tweezers for micromanipulation of single DNA molecules and DNA-protein complexes.

    PubMed

    McAndrew, Christopher P; Tyson, Christopher; Zischkau, Joseph; Mehl, Patrick; Tuma, Pamela L; Pegg, Ian L; Sarkar, Abhijit

    2016-01-01

    We report the development of a simple-to-implement magnetic force transducer that can apply a wide range of piconewton (pN) scale forces on single DNA molecules and DNA-protein complexes in the horizontal plane. The resulting low-noise force-extension data enable very high-resolution detection of changes in the DNA tether's extension: ~0.05 pN in force and <10 nm change in extension. We have also verified that we can manipulate DNA in near equilibrium conditions through the wide range of forces by ramping the force from low to high and back again, and observing minimal hysteresis in the molecule's force response. Using a calibration technique based on Stokes' drag law, we have confirmed our force measurements from DNA force-extension experiments obtained using the fluctuation-dissipation theorem applied to transverse fluctuations of the magnetic microsphere. We present data on the force-distance characteristics of a DNA molecule complexed with histones. The results illustrate how the tweezers can be used to study DNA binding proteins at the single molecule level.

  15. Single Molecule Measurements of Protelomerase TelK-DNA Complexes

    NASA Astrophysics Data System (ADS)

    Landry, Markita; Khafizov, Rustem; Huang, Wai Mun; Chemla, Yann

    2008-10-01

    Protein-DNA interactions lie at the heart of many essential cellular processes such as replication, recombination, and repair. Recent advances in optical ``tweezers'' have made it possible to resolve motions on the scale of a single base pair of DNA, 3.4å. High-resolution optical traps have the potential to reveal these interactions at their fundamental length scales and should reveal how certain proteins bind to DNA or recognize target sequences. Telomerases are enzymes that have been actively studied in various organisms because of their fundamental involvement with both cancer and aging^1. Protelomerase TelK is an enzyme responsible for forming closed DNA hairpin ends in linear DNA. TelK is not an ATP dependant enzyme, which is surprising given the degree of DNA distortion accomplished by the enzyme, and the large energy barrier intrinsic in DNA hairpin formation. Therefore, our focus is on TelK mutants lacking their c-terminal domain, and TelK YF mutants lacking their tyrosine active site amino acid. Preliminary data have shown remarkable differences in protein binding and unbinding forces caused by the removal of a single oxygen atom from a 73 kDa protein. Further measurements using high-resolution optical tweezers should provide fundamental insights into the nature and importance of the electrostatic interactions between TelK and its DNA substrate. 1. Shay, J. et al. Rad. Res. 155, 188 (2001) [1] Huang, W. et al. Mol. Cell. 27, 901 (2007).

  16. Visualizing Chemical Interaction Dynamics of Confined DNA Molecules

    NASA Astrophysics Data System (ADS)

    Henkin, Gilead; Berard, Daniel; Stabile, Frank; Leslie, Sabrina

    We present a novel nanofluidic approach to controllably introducing reagent molecules to interact with confined biopolymers and visualizing the reaction dynamics in real time. By dynamically deforming a flow cell using CLiC (Convex Lens-induced Confinement) microscopy, we are able to tune reaction chamber dimensions from micrometer to nanometer scales. We apply this gentle deformation to load and extend DNA polymers within embedded nanotopographies and visualize their interactions with other molecules in solution. Quantifying the change in configuration of polymers within embedded nanotopographies in response to binding/unbinding of reagent molecules provides new insights into their consequent change in physical properties. CLiC technology enables an ultra sensitive, massively parallel biochemical analysis platform which can acces a broader range of interaction parameters than existing devices.

  17. Single-Molecule Electronic Monitoring of DNA Polymerase Activity

    NASA Astrophysics Data System (ADS)

    Marushchak, Denys O.; Pugliese, Kaitlin M.; Turvey, Mackenzie W.; Choi, Yongki; Gul, O. Tolga; Olsen, Tivoli J.; Rajapakse, Arith J.; Weiss, Gregory A.; Collins, Philip G.

    Single-molecule techniques can reveal new spatial and kinetic details of the conformational changes occurring during enzymatic catalysis. Here, we investigate the activity of DNA polymerases using an electronic single-molecule technique based on carbon nanotube transistors. Single molecules of the Klenow fragment (KF) of polymerase I were conjugated to the transistors and then monitored via fluctuations in electrical conductance. Continuous, long-term monitoring recorded single KF molecules incorporating up to 10,000 new bases into single-stranded DNA templates. The duration of individual incorporation events was invariant across all analog and native nucleotides, indicating that the precise structure of different base pairs has no impact on the timing of incorporation. Despite similar timings, however, the signal magnitudes generated by certain analogs reveal alternate conformational states that do not occur with native nucleotides. The differences induced by these analogs suggest that the electronic technique is sensing KF's O-helix as it tests the stability of nascent base pairs.

  18. Dual-Colored DNA Comb Polymers for Single Molecule Rheology

    NASA Astrophysics Data System (ADS)

    Mai, Danielle; Marciel, Amanda; Schroeder, Charles

    2014-03-01

    We report the synthesis and characterization of branched biopolymers for single molecule rheology. In our work, we utilize a hybrid enzymatic-synthetic approach to graft ``short'' DNA branches to ``long'' DNA backbones, thereby producing macromolecular DNA comb polymers. The branches and backbones are synthesized via polymerase chain reaction with chemically modified deoxyribonucleotides (dNTPs): ``short'' branches consist of Cy5-labeled dNTPs and a terminal azide group, and ``long'' backbones contain dibenzylcyclooctyne-modified (DBCO) dNTPs. In this way, we utilize strain-promoted, copper-free cycloaddition ``click'' reactions for facile grafting of azide-terminated branches at DBCO sites along backbones. Copper-free click reactions are bio-orthogonal and nearly quantitative when carried out under mild conditions. Moreover, comb polymers can be labeled with an intercalating dye (e.g., YOYO) for dual-color fluorescence imaging. We characterized these materials using gel electrophoresis, HPLC, and optical microscopy, with atomic force microscopy in progress. Overall, DNA combs are suitable for single molecule dynamics, and in this way, our work holds the potential to improve our understanding of topologically complex polymer melts and solutions.

  19. DNA.

    ERIC Educational Resources Information Center

    Felsenfeld, Gary

    1985-01-01

    Structural form, bonding scheme, and chromatin structure of and gene-modification experiments with deoxyribonucleic acid (DNA) are described. Indicates that DNA's double helix is variable and also flexible as it interacts with regulatory and other molecules to transfer hereditary messages. (DH)

  20. Mutual inhibition of RecQ molecules in DNA unwinding.

    PubMed

    Pan, Bing-Yi; Dou, Shuo-Xing; Yang, Ye; Xu, Ya-Nan; Bugnard, Elisabeth; Ding, Xiu-Yan; Zhang, Lingyun; Wang, Peng-Ye; Li, Ming; Xi, Xu Guang

    2010-05-21

    Helicases make conformational changes and mechanical movements through hydrolysis of NTP to unwind duplex DNA (or RNA). Most helicases require a single-stranded overhang for loading onto the duplex DNA substrates. Some helicases have been observed to exhibit an enhanced unwinding efficiency with increasing length of the single-stranded DNA tail both by preventing reannealing of the unwound DNA and by compensating for premature dissociation of the leading monomers. Here we report a previously unknown mutual inhibition of neighboring monomers in DNA unwinding by the monomeric Escherichia coli RecQ helicase. With single molecule fluorescence resonance energy transfer microscopy, we observed that the unwinding initiation of RecQ at saturating concentrations was more delayed for a long rather than a short tailed DNA. In stopped-flow kinetic studies under both single and multiple turnover conditions, the unwinding efficiency decreased with increasing enzyme concentration for long tailed substrates. In addition, preincubation of RecQ and DNA in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate was observed to alleviate the inhibition. We propose that the mutual inhibition effect results from a forced closure of cleft between the two RecA-like domains of a leading monomer by a trailing one, hence the forward movements of both monomers are stalled by prohibition of ATP binding to the leading one. This effect represents direct evidence for the relative movements of the two RecA-like domains of RecQ in DNA unwinding. It may occur for all superfamily I and II helicases possessing two RecA-like domains.

  1. Single molecule studies of DNA packaging by bacteriophages

    NASA Astrophysics Data System (ADS)

    Fuller, Derek Nathan

    The DNA packaging dynamics of bacteriophages φ29, gamma, and T4 were studied at the single molecule level using a dual trap optical tweezers. Also, a method for producing long DNA molecules by PCR for optical tweezers studies of protein DNA interactions is presented and thoroughly characterized. This DNA preparation technique provided DNA samples for the φ29 and T4 studies. In the studies of φ29, the role of charge was investigated by varying the ionic conditions of the packaging buffer. Ionic conditions in which the DNA charge was highly screened due to divalent and trivalent cations showed the lowest resistance to packaging of the DNA to high density. This confirmed the importance of counterions in shielding the DNA interstrand repulsion when packaged to high density. While the ionic nature of the packaging buffer had a strong effect on packaging velocities, there was no clear trend between the counterion-screened charge of the DNA and the maximum packaging velocity. The packaging studies of lambda and T4 served as systems for comparative studies with φ29. Each system showed similarities to the φ29 system and unique differences. Both the lambda and T4 packaging motors were capable of generating forces in excess of 50 pN and showed remarkably high processivity, similar to φ29. However, dynamic structural transitions were observed with lambda that are not observed with φ29. The packaging of the lambda genome showed capsid expansion at approximately 30 percent of the genome packaged and capsid rupture at 90 percent of the genome packaged in the absence of capsid stabilizing protein gpD. Unique to the T4 packaging motor, packaging dynamics showed a remarkable amount of variability in velocities. This variability was seen both within individual packaging phages and from one phage to the next. This is possibly due to different conformational states of the packaging machinery. Additionally, lambda and T4 had average packaging velocities under minimal load of 600

  2. Adaptive resolution simulation of an atomistic DNA molecule in MARTINI salt solution

    NASA Astrophysics Data System (ADS)

    Zavadlav, J.; Podgornik, R.; Melo, M. N.; Marrink, S. J.; Praprotnik, M.

    2016-10-01

    We present a dual-resolution model of a deoxyribonucleic acid (DNA) molecule in a bathing solution, where we concurrently couple atomistic bundled water and ions with the coarse-grained MARTINI model of the solvent. We use our fine-grained salt solution model as a solvent in the inner shell surrounding the DNA molecule, whereas the solvent in the outer shell is modeled by the coarse-grained model. The solvent entities can exchange between the two domains and adapt their resolution accordingly. We critically asses the performance of our multiscale model in adaptive resolution simulations of an infinitely long DNA molecule, focusing on the structural characteristics of the solvent around DNA. Our analysis shows that the adaptive resolution scheme does not produce any noticeable artifacts in comparison to a reference system simulated in full detail. The effect of using a bundled-SPC model, required for multiscaling, compared to the standard free SPC model is also evaluated. Our multiscale approach opens the way for large scale applications of DNA and other biomolecules which require a large solvent reservoir to avoid boundary effects.

  3. Molding single DNA molecules in metals and sample preparation for electronic sequencing.

    PubMed

    Lund, John A; Parviz, Babak A

    2009-01-01

    We demonstrate the molding of single DNA molecules in 8 nm thin platinum molds. The molded structures have an apparent depth of 1 nm under STM imaging, and closely follow the contours of the DNA molecules. We have confirmed the presence of the embedded molecules and have verified the ability of this technique to scale down to single molecules. Additionally, we have utilized this method to perform electron tunneling analysis on embedded DNA molecules.

  4. Single-molecule dissection of stacking forces in DNA.

    PubMed

    Kilchherr, Fabian; Wachauf, Christian; Pelz, Benjamin; Rief, Matthias; Zacharias, Martin; Dietz, Hendrik

    2016-09-09

    We directly measured at the single-molecule level the forces and lifetimes of DNA base-pair stacking interactions for all stack sequence combinations. Our experimental approach combined dual-beam optical tweezers with DNA origami components to allow positioning of blunt-end DNA helices so that the weak stacking force could be isolated. Base-pair stack arrays that lacked a covalent backbone connection spontaneously dissociated at average rates ranging from 0.02 to 500 per second, depending on the sequence combination and stack array size. Forces in the range from 2 to 8 piconewtons that act along the helical direction only mildly accelerated the stochastic unstacking process. The free-energy increments per stack that we estimate from the measured forward and backward kinetic rates ranged from -0.8 to -3.4 kilocalories per mole, depending on the sequence combination. Our data contributes to understanding the mechanics of DNA processing in biology, and it is helpful for designing the kinetics of DNA-based nanoscale devices according to user specifications.

  5. A novel hybrid single molecule approach reveals spontaneous DNA motion in the nucleosome

    PubMed Central

    Wei, Sijie; Falk, Samantha J.; Black, Ben E.; Lee, Tae-Hee

    2015-01-01

    Structural dynamics of nucleic acid and protein is an important physical basis of their functions. These motions are often very difficult to synchronize and too fast to be clearly resolved with the currently available single molecule methods. Here we demonstrate a novel hybrid single molecule approach combining stochastic data analysis with fluorescence correlation that enables investigations of sub-ms unsynchronized structural dynamics of macromolecules. Based on the method, we report the first direct evidence of spontaneous DNA motions at the nucleosome termini. The nucleosome, comprising DNA and a histone core, is the fundamental packing unit of eukaryotic genes that must be accessed during various genome transactions. Spontaneous DNA opening at the nucleosome termini has long been hypothesized to enable gene access in the nucleosome, but has yet to be directly observed. Our approach reveals that DNA termini in the nucleosome open and close repeatedly at 0.1–1 ms−1. The kinetics depends on salt concentration and DNA–histone interactions but not much on DNA sequence, suggesting that this dynamics is universal and imposes the kinetic limit to gene access. These results clearly demonstrate that our method provides an efficient and robust means to investigate unsynchronized structural changes of DNA at a sub-ms time resolution. PMID:26013809

  6. Regulation of DNA Metabolism by DNA-Binding Proteins Probed by Single Molecule Spectroscopy

    DTIC Science & Technology

    2006-12-05

    denaturation The Watson - Crick double- helix is the thermodynamically stable configuration of a DNA molecule under physiological conditions (normal salt and...room/body temperature). This stability is effected (a) by Watson - Crick H-bonding, that is essential for the specificity of base pairing, i.e., for...guarantees the high level of fidelity during replication and transcription. (b) The second contribution to DNA helix stability comes from base-stacking

  7. Isolated menthone reductase and nucleic acid molecules encoding same

    DOEpatents

    Croteau, Rodney B; Davis, Edward M; Ringer, Kerry L

    2013-04-23

    The present invention provides isolated menthone reductase proteins, isolated nucleic acid molecules encoding menthone reductase proteins, methods for expressing and isolating menthone reductase proteins, and transgenic plants expressing elevated levels of menthone reductase protein.

  8. Effect of Aperiodicity on the Charge Transfer Through DNA Molecules

    NASA Astrophysics Data System (ADS)

    Ghosh, Angsula; Chaudhuri, Puspitapallab

    The effect of aperiodicity on the charge transfer process through DNA molecules is investigated using a tight-binding model. Single-stranded aperiodic Fibonacci polyGC and polyAT sequences along with aperiodic Rudin-Shapiro poly(GCAT) sequences are used in the study. Based on the tight-binding model, molecular orbital calculations of the DNA chains are performed and ionization potentials compared, as this might be relevant to understanding the charge transfer process. Charges migrate through the sequences in a multistep hopping process. Results for current conduction through aperiodic sequences are compared with those for the corresponding periodic sequences. We find that dinucleotide aperiodic Fibonacci sequences decrease the current while tetranucleotide aperiodic Rudin-Shapiro sequences increase the current when compared with the corresponding periodic sequences. The conductance in all cases decays exponentially as the sequence length increases.

  9. Dissolving Hydroxyolite: A DNA Molecule into Its Hydroxyapatite Mold.

    PubMed

    Bertran, Oscar; Revilla-López, Guillermo; Casanovas, Jordi; Del Valle, Luis J; Turon, Pau; Puiggalí, Jordi; Alemán, Carlos

    2016-05-04

    In spite of the clinical importance of hydroxyapatite (HAp), the mechanism that controls its dissolution in acidic environments remains unclear. Knowledge of such a process is highly desirable to provide better understanding of different pathologies, as for example osteoporosis, and of the HAp potential as vehicle for gene delivery to replace damaged DNA. In this work, the mechanism of dissolution in acid conditions of HAp nanoparticles encapsulating double-stranded DNA has been investigated at the atomistic level using computer simulations. For this purpose, four consecutive (multi-step) molecular dynamics simulations, involving different temperatures and proton transfer processes, have been carried out. Results are consistent with a polynuclear decalcification mechanism in which proton transfer processes, from the surface to the internal regions of the particle, play a crucial role. In addition, the DNA remains protected by the mineral mold and transferred proton from both temperature and chemicals. These results, which indicate that biomineralization imparts very effective protection to DNA, also have important implications in other biomedical fields, as for example in the design of artificial bones or in the fight against osteoporosis by promoting the fixation of Ca(2+) ions.

  10. The expression of acidic ribosomal phosphoproteins on the surface membrane of different tissues in autoimmune and normal mice which are the target molecules for anti-double-stranded DNA antibodies.

    PubMed Central

    Sun, K H; Liu, W T; Tang, S J; Tsai, C Y; Hsieh, S C; Wu, T H; Han, S H; Yu, C L

    1996-01-01

    Affinity-purified polyclonal anti-double-stranded DNA (anti-dsDNA) antibodies from patients with systemic lupus erythematosus (SLE) exert a cytostatic effect on cultured rat glomerular mesangial cells (MC). The cognate antigens expressed on the surface of MC have been proved to be acidic ribosomal phosphoproteins (P proteins) in our previous study. The mesangial cytostatic effect of anti-dsDNA antibodies is attributed to the cross-reactivity of the antibodies with membrane-expressed P proteins, but not to the effect of minute amounts of anti-ribosomal P proteins antibodies contained in the anti-dsDNA preparations. Immunofluorescence staining of the native cells demonstrated that anti-dsDNA antibodies bound to the surface of rat mesangial cells, rat brain astrocytes (RBA-1) and mouse fibroblasts (3T3). Anti-dsDNA antibodies also exert potent cytostatic effects on these cells in a dose-dependent manner. In addition, the plasma membranes of different cell lines and tissues from normal and autoimmune mice were isolated and probed by anti-dsDNA antibodies in Western blot analysis. We found the actively proliferating cells such as MC, RBA-1 and 3T3 may express both P0 (38,000 MW) and P1 (19,000 MW) on the surface membrane. In addition, the kidney, liver and spleen from either autoimmune MRL-lpr/lpr or BALB/c mice may constantly express P0 protein, but the expression of P1 is inconsistent. In contrast, brain and muscle from either mice failed to express P proteins on their surface. Unexpectedly, a high molecular weight substance (larger than 205,000 MW) with unknown nature appears in the membrane of brain and muscle tissues in both mice. Immunoprecipitation of the surface-biotinylated MC-lysate by anti-dsDNA antibodies further confirmed that P1 (19,000 MW) and P2 (17,000 MW) are really expressed on the cell surface. These results suggest that P proteins expressed on the surface of different tissues become the targets for anti-dsDNA antibodies mediating pleomorphic tissue

  11. Diffusion of isolated DNA molecules: dependence on length and topology.

    PubMed

    Robertson, Rae M; Laib, Stephan; Smith, Douglas E

    2006-05-09

    The conformation and dynamics of circular polymers is a subject of considerable theoretical and experimental interest. DNA is an important example because it occurs naturally in different topological states, including linear, relaxed circular, and supercoiled circular forms. A fundamental question is how the diffusion coefficients of isolated polymers scale with molecular length and how they vary for different topologies. Here, diffusion coefficients D for relaxed circular, supercoiled, and linear DNA molecules of length L ranging from approximately 6 to 290 kbp were measured by tracking the Brownian motion of single molecules. A topology-independent scaling law D approximately L(-nu) was observed with nu(L) = 0.571 +/- 0.014, nu(C) = 0.589 +/- 0.018, and nu(S) = 0.571 +/- 0.057 for linear, relaxed circular, and supercoiled DNA, respectively, in good agreement with the scaling exponent of nu congruent with 0.588 predicted by renormalization group theory for polymers with significant excluded volume interactions. Our findings thus provide evidence in support of several theories that predict an effective diameter of DNA much greater than the Debye screening length. In addition, the measured ratio D(Circular)/D(Linear) = 1.32 +/- 0.014 was closer to the value of 1.45 predicted by using renormalization group theory than the value of 1.18 predicted by classical Kirkwood hydrodynamic theory and agreed well with a value of 1.31 predicted when incorporating a recently proposed expression for the radius of gyration of circular polymers into the Zimm model.

  12. Mapping DNA polymerase errors by single-molecule sequencing

    PubMed Central

    Lee, David F.; Lu, Jenny; Chang, Seungwoo; Loparo, Joseph J.; Xie, Xiaoliang S.

    2016-01-01

    Genomic integrity is compromised by DNA polymerase replication errors, which occur in a sequence-dependent manner across the genome. Accurate and complete quantification of a DNA polymerase's error spectrum is challenging because errors are rare and difficult to detect. We report a high-throughput sequencing assay to map in vitro DNA replication errors at the single-molecule level. Unlike previous methods, our assay is able to rapidly detect a large number of polymerase errors at base resolution over any template substrate without quantification bias. To overcome the high error rate of high-throughput sequencing, our assay uses a barcoding strategy in which each replication product is tagged with a unique nucleotide sequence before amplification. This allows multiple sequencing reads of the same product to be compared so that sequencing errors can be found and removed. We demonstrate the ability of our assay to characterize the average error rate, error hotspots and lesion bypass fidelity of several DNA polymerases. PMID:27185891

  13. Hyperactivation of DNA-PK by double-strand break mimicking molecules disorganizes DNA damage response.

    PubMed

    Quanz, Maria; Chassoux, Danielle; Berthault, Nathalie; Agrario, Céline; Sun, Jian-Sheng; Dutreix, Marie

    2009-07-21

    Cellular response to DNA damage involves the coordinated activation of cell cycle checkpoints and DNA repair. The early steps of DNA damage recognition and signaling in mammalian cells are not yet fully understood. To investigate the regulation of the DNA damage response (DDR), we designed short and stabilized double stranded DNA molecules (Dbait) mimicking double-strand breaks. We compared the response induced by these molecules to the response induced by ionizing radiation. We show that stable 32-bp long Dbait, induce pan-nuclear phosphorylation of DDR components such as H2AX, Rpa32, Chk1, Chk2, Nbs1 and p53 in various cell lines. However, individual cell analyses reveal that differences exist in the cellular responses to Dbait compared to irradiation. Responses to Dbait: (i) are dependent only on DNA-PK kinase activity and not on ATM, (ii) result in a phosphorylation signal lasting several days and (iii) are distributed in the treated population in an "all-or-none" pattern, in a Dbait-concentration threshold dependant manner. Moreover, despite extensive phosphorylation of the DNA-PK downstream targets, Dbait treated cells continue to proliferate without showing cell cycle delay or apoptosis. Dbait treatment prior to irradiation impaired foci formation of Nbs1, 53BP1 and Rad51 at DNA damage sites and inhibited non-homologous end joining as well as homologous recombination. Together, our results suggest that the hyperactivation of DNA-PK is insufficient for complete execution of the DDR but induces a "false" DNA damage signaling that disorganizes the DNA repair system.

  14. Elastic Properties of Nucleic Acids by Single-Molecule Force Spectroscopy.

    PubMed

    Camunas-Soler, Joan; Ribezzi-Crivellari, Marco; Ritort, Felix

    2016-07-05

    We review the current knowledge on the use of single-molecule force spectroscopy techniques to extrapolate the elastic properties of nucleic acids. We emphasize the lesser-known elastic properties of single-stranded DNA. We discuss the importance of accurately determining the elastic response in pulling experiments, and we review the simplest models used to rationalize the experimental data as well as the experimental approaches used to pull single-stranded DNA. Applications used to investigate DNA conformational transitions and secondary structure formation are also highlighted. Finally, we provide an overview of the effects of salt and temperature and briefly discuss the effects of contour length and sequence dependence.

  15. Single-molecule kinetics and footprinting of DNA bis-intercalation: the paradigmatic case of Thiocoraline

    PubMed Central

    Camunas-Soler, Joan; Manosas, Maria; Frutos, Silvia; Tulla-Puche, Judit; Albericio, Fernando; Ritort, Felix

    2015-01-01

    DNA bis-intercalators are widely used in molecular biology with applications ranging from DNA imaging to anticancer pharmacology. Two fundamental aspects of these ligands are the lifetime of the bis-intercalated complexes and their sequence selectivity. Here, we perform single-molecule optical tweezers experiments with the peptide Thiocoraline showing, for the first time, that bis-intercalation is driven by a very slow off-rate that steeply decreases with applied force. This feature reveals the existence of a long-lived (minutes) mono-intercalated intermediate that contributes to the extremely long lifetime of the complex (hours). We further exploit this particularly slow kinetics to determine the thermodynamics of binding and persistence length of bis-intercalated DNA for a given fraction of bound ligand, a measurement inaccessible in previous studies of faster intercalating agents. We also develop a novel single-molecule footprinting technique based on DNA unzipping and determine the preferred binding sites of Thiocoraline with one base-pair resolution. This fast and radiolabelling-free footprinting technique provides direct access to the binding sites of small ligands to nucleic acids without the need of cleavage agents. Overall, our results provide new insights into the binding pathway of bis-intercalators and the reported selectivity might be of relevance for this and other anticancer drugs interfering with DNA replication and transcription in carcinogenic cell lines. PMID:25690887

  16. Single molecule analysis of Trypanosoma brucei DNA replication dynamics

    PubMed Central

    Calderano, Simone Guedes; Drosopoulos, William C.; Quaresma, Marina Mônaco; Marques, Catarina A.; Kosiyatrakul, Settapong; McCulloch, Richard; Schildkraut, Carl L.; Elias, Maria Carolina

    2015-01-01

    Eukaryotic genome duplication relies on origins of replication, distributed over multiple chromosomes, to initiate DNA replication. A recent genome-wide analysis of Trypanosoma brucei, the etiological agent of sleeping sickness, localized its replication origins to the boundaries of multigenic transcription units. To better understand genomic replication in this organism, we examined replication by single molecule analysis of replicated DNA. We determined the average speed of replication forks of procyclic and bloodstream form cells and we found that T. brucei DNA replication rate is similar to rates seen in other eukaryotes. We also analyzed the replication dynamics of a central region of chromosome 1 in procyclic forms. We present evidence for replication terminating within the central part of the chromosome and thus emanating from both sides, suggesting a previously unmapped origin toward the 5′ extremity of chromosome 1. Also, termination is not at a fixed location in chromosome 1, but is rather variable. Importantly, we found a replication origin located near an ORC1/CDC6 binding site that is detected after replicative stress induced by hydroxyurea treatment, suggesting it may be a dormant origin activated in response to replicative stress. Collectively, our findings support the existence of more replication origins in T. brucei than previously appreciated. PMID:25690894

  17. Multiplex single-molecule interaction profiling of DNA barcoded proteins

    PubMed Central

    Gu, Liangcai; Li, Chao; Aach, John; Hill, David E.; Vidal, Marc; Church, George M.

    2014-01-01

    In contrast with advances in massively parallel DNA sequencing1, high-throughput protein analyses2-4 are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule (SM) protein detection achieved using optical methods5 is limited by the number of spectrally nonoverlapping chromophores. Here, we introduce a single molecular interaction-sequencing (SMI-Seq) technology for parallel protein interaction profiling leveraging SM advantages. DNA barcodes are attached to proteins collectively via ribosome display6 or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide (PAA) thin film to construct a random SM array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies)7 and analyzed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimeter. Furthermore, protein interactions can be measured based on the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor (GPCR) and antibody binding profiling, were demonstrated. SMI-Seq enables “library vs. library” screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity. PMID:25252978

  18. Single molecule analysis of Trypanosoma brucei DNA replication dynamics.

    PubMed

    Calderano, Simone Guedes; Drosopoulos, William C; Quaresma, Marina Mônaco; Marques, Catarina A; Kosiyatrakul, Settapong; McCulloch, Richard; Schildkraut, Carl L; Elias, Maria Carolina

    2015-03-11

    Eukaryotic genome duplication relies on origins of replication, distributed over multiple chromosomes, to initiate DNA replication. A recent genome-wide analysis of Trypanosoma brucei, the etiological agent of sleeping sickness, localized its replication origins to the boundaries of multigenic transcription units. To better understand genomic replication in this organism, we examined replication by single molecule analysis of replicated DNA. We determined the average speed of replication forks of procyclic and bloodstream form cells and we found that T. brucei DNA replication rate is similar to rates seen in other eukaryotes. We also analyzed the replication dynamics of a central region of chromosome 1 in procyclic forms. We present evidence for replication terminating within the central part of the chromosome and thus emanating from both sides, suggesting a previously unmapped origin toward the 5' extremity of chromosome 1. Also, termination is not at a fixed location in chromosome 1, but is rather variable. Importantly, we found a replication origin located near an ORC1/CDC6 binding site that is detected after replicative stress induced by hydroxyurea treatment, suggesting it may be a dormant origin activated in response to replicative stress. Collectively, our findings support the existence of more replication origins in T. brucei than previously appreciated.

  19. Mechanisms of DNA disentangling by type II topoisomerases. Comment on "Disentangling DNA molecules" by Alexander Vologodskii

    NASA Astrophysics Data System (ADS)

    Yan, Jie

    2016-09-01

    In this article [1] Dr. Vologodskii presents a comprehensive discussion on the mechanisms by which the type II topoisomerases unknot/disentangle DNA molecules. It is motivated by a mysterious capability of the nanometer-size enzymes to keep the steady-state probability of DNA entanglement/knot almost two orders of magnitude below that expected from thermal equilibrium [2-5]. In spite of obvious functional advantages of the enzymes, it raises a question regarding how such high efficiency could be achieved. The off-equilibrium steady state distribution of DNA topology is powered by ATP consumption. However, it remains unclear how this energy is utilized to bias the distribution toward disentangled/unknotted topological states of DNA.

  20. Small circular DNA molecules act as rigid motifs to build DNA nanotubes.

    PubMed

    Zheng, Hongning; Xiao, Minyu; Yan, Qin; Ma, Yinzhou; Xiao, Shou-Jun

    2014-07-23

    Small circular DNA molecules with designed lengths, for example 64 and 96 nucleotides (nt), after hybridization with a few 32-nt staple strands respectively, can act as rigid motifs for the construction of DNA nanotubes with excellent uniformity in ring diameter. Unlike most native DNA nanotubes, which consist of longitudinal double helices, nanotubes assembled from circular DNAs are constructed from lateral double helices. Of the five types of DNA nanotubes designed here, four are built by alternating two different rings of the same ring size, while one is composed of all the same 96-nt rings. Nanotubes constructed from the same 96-nt rings are 10-100 times shorter than those constructed from two different 96-nt rings, because there are fewer hinge joints on the rings.

  1. One-end immobilization of individual DNA molecules on a functional hydrophobic glass surface.

    PubMed

    Matsuura, Shun-ichi; Kurita, Hirofumi; Nakano, Michihiko; Komatsu, Jun; Takashima, Kazunori; Katsura, Shinji; Mizuno, Akira

    2002-12-01

    We demonstrate an effective method for DNA immobilization on a hydrophobic glass surface. The new DNA immobilizing technique is extremely simple compared with conventional techniques that require heterobifunctional crosslinking reagent between DNA and substrate surface that are both modified chemically. In the first process, a coverslip was treated with dichlorodimethylsilane resulting in hydrophobic surface. lambda DNA molecules were ligated with 3'-terminus disulfide-modified 14 mer oligonucleotides at one cohesive end. After reduction of the disulfide to sulfhydryl (thiol) groups the resulting thiol-modified lambda DNA molecules were reacted on silanized coverslip. Fluorescent observation showed that the thiol-modified lambda DNA molecules were anchored specifically to the hydrophobic surface at one terminus, although non-specific binding of the DNA molecules was suppressed. It was observed that the one-end-attached DNA molecule was bound firmly to the surface and stretched reversibly in one direction when a d.c. electric field was applied.

  2. Pulsed IR heating studies of single-molecule DNA duplex dissociation kinetics and thermodynamics.

    PubMed

    Holmstrom, Erik D; Dupuis, Nicholas F; Nesbitt, David J

    2014-01-07

    Single-molecule fluorescence spectroscopy is a powerful technique that makes it possible to observe the conformational dynamics associated with biomolecular processes. The addition of precise temperature control to these experiments can yield valuable thermodynamic information about equilibrium and kinetic rate constants. To accomplish this, we have developed a microscopy technique based on infrared laser overtone/combination band absorption to heat small (≈10(-11) liter) volumes of water. Detailed experimental characterization of this technique reveals three major advantages over conventional stage heating methods: 1), a larger range of steady-state temperatures (20-100°C); 2), substantially superior spatial (≤20 μm) control; and 3), substantially superior temporal (≈1 ms) control. The flexibility and breadth of this spatial and temporally resolved laser-heating approach is demonstrated in single-molecule fluorescence assays designed to probe the dissociation of a 21 bp DNA duplex. These studies are used to support a kinetic model based on nucleic acid end fraying that describes dissociation for both short (<10 bp) and long (>10 bp) DNA duplexes. These measurements have been extended to explore temperature-dependent kinetics for the 21 bp construct, which permit determination of single-molecule activation enthalpies and entropies for DNA duplex dissociation.

  3. Observation of DNA Molecules Using Fluorescence Microscopy and Atomic Force Microscopy

    ERIC Educational Resources Information Center

    Ito, Takashi

    2008-01-01

    This article describes experiments for an undergraduate instrumental analysis laboratory that aim to observe individual double-stranded DNA (dsDNA) molecules using fluorescence microscopy and atomic force microscopy (AFM). dsDNA molecules are observed under several different conditions to discuss their chemical and physical properties. In…

  4. Oxidized fatty acids as inter-kingdom signaling molecules.

    PubMed

    Pohl, Carolina H; Kock, Johan L F

    2014-01-20

    Oxylipins or oxidized fatty acids are a group of molecules found to play a role in signaling in many different cell types. These fatty acid derivatives have ancient evolutionary origins as signaling molecules and are ideal candidates for inter-kingdom communication. This review discusses examples of the ability of organisms from different kingdoms to "listen" and respond to oxylipin signals during interactions. The interactions that will be looked at are signaling between animals and plants; between animals and fungi; between animals and bacteria and between plants and fungi. This will aid in understanding these interactions, which often have implications in ecology, agriculture as well as human and animal health.

  5. Single-molecule analysis of DNA cross-links using nanopore technology

    NASA Astrophysics Data System (ADS)

    Wolna, Anna H.

    The alpha-hemolysin (alpha-HL) protein ion channel is a potential next-generation sequencing platform that has been extensively used to study nucleic acids at a single-molecule level. After applying a potential across a lipid bilayer, the imbedded alpha-HL allows monitoring of the duration and current levels of DNA translocation and immobilization. Because this method does not require DNA amplification prior to sequencing, all the DNA damage present in the cell at any given time will be present during the sequencing experiment. The goal of this research is to determine if these damage sites give distinguishable current levels beyond those observed for the canonical nucleobases. Because DNA cross-links are one of the most prevalent types of DNA damage occurring in vivo, the blockage current levels were determined for thymine-dimers, guanine(C8)-thymine(N3) cross-links and platinum adducts. All of these cross-links give a different blockage current level compared to the undamaged strands when immobilized in the ion channel, and they all can easily translocate across the alpha-HL channel. Additionally, the alpha-HL nanopore technique presents a unique opportunity to study the effects of DNA cross-links, such as thymine-dimers, on the secondary structure of DNA G-quadruplexes folded from the human telomere sequence. Using this single-molecule nanopore technique we can detect subtle structural differences that cannot be easily addressed using conventional methods. The human telomere plays crucial roles in maintaining genome stability. In the presence of suitable cations, the repetitive 5'-TTAGGG human telomere sequence can fold into G-quadruplexes that adopt the hybrid fold in vivo. The telomere sequence is hypersensitive to UV-induced thymine-dimer (T=T) formation, and yet the presence of thymine dimers does not cause telomere shortening. The potential structural disruption and thermodynamic stability of the T=T-containing natural telomere sequences were studied to

  6. Physisorption of DNA molecules on chemically modified single-walled carbon nanotubes with and without sonication.

    PubMed

    Umemura, Kazuo; Ishibashi, Yu; Oura, Shusuke

    2016-09-01

    We investigated the physisorption phenomenon of single-stranded DNA (ssDNA) molecules onto two types of commercially available chemically functionalized single-walled carbon nanotubes (SWNTs) by atomic force microscopy (AFM) and agarose gel electrophoresis. We found that DNA molecules can adsorb on the water-soluble SWNT surfaces without sonication, although sonication treatment has been used for hybridization of DNA and SWNTs in many previous studies. Using our method, damage of DNA molecules by sonication can be avoided. On the other hand, the amount of DNA molecules adsorbed on SWNT surfaces increased when the samples were sonicated. This fact suggests that the sonication is effective not only at debundling of SWNTs, but also at assisting DNA adsorption. Furthermore, DNA adsorption was affected by the types of functionalized SWNTs. In the case of SWNTs functionalized with polyethylene glycol (PEG-SWNT), physisorption of ssDNA molecules was confirmed only by agarose-gel electrophoresis. In contrast, amino-terminated SWNTs (NH2-SWNTs) showed a change in the height distribution profile based on AFM observations. These results suggest that DNA molecules tended to adsorb to NH2-SWNT surfaces, although DNA molecules can also adsorb on PEG-SWNT surfaces. Our results revealed fundamental information for developing nanobiodevices using hybrids of DNA and SWNTs.

  7. Direct observation of λ-DNA molecule reversal movement within microfluidic channels under electric field with single molecule imaging technique

    NASA Astrophysics Data System (ADS)

    Fengyun, Yang; Kaige, Wang; Dan, Sun; Wei, Zhao; Hai-qing, Wang; Xin, He; Gui-ren, Wang; Jin-tao, Bai

    2016-07-01

    The electrodynamic characteristics of single DNA molecules moving within micro-/nano-fluidic channels are important in the design of biomedical chips and bimolecular sensors. In this study, the dynamic properties of λ-DNA molecules transferring along the microchannels driven by the external electrickinetic force were systemically investigated with the single molecule fluorescence imaging technique. The experimental results indicated that the velocity of DNA molecules was strictly dependent on the value of the applied electric field and the diameter of the channel. The larger the external electric field, the larger the velocity, and the more significant deformation of DNA molecules. More meaningfully, it was found that the moving directions of DNA molecules had two completely different directions: (i) along the direction of the external electric field, when the electric field intensity was smaller than a certain threshold value; (ii) opposite to the direction of the external electric field, when the electric field intensity was greater than the threshold electric field intensity. The reversal movement of DNA molecules was mainly determined by the competition between the electrophoresis force and the influence of electro-osmosis flow. These new findings will theoretically guide the practical application of fluidic channel sensors and lab-on-chips for precisely manipulating single DNA molecules. Project supported by the National Natural Science Foundation of China (Grant No. 61378083), the International Cooperation Foundation of the National Science and Technology Major Project of the Ministry of Science and Technology of China (Grant No. 2011DFA12220), the Major Research Plan of National Natural Science Foundation of China (Grant No. 91123030), and the Natural Science Foundation of Shaanxi Province of China (Grant Nos. 2010JS110 and 2013SZS03-Z01).

  8. Titantium Dioxide Nanoparticles Assembled by DNA Molecules Hybridization and Loading of DNA Interacting Proteins.

    PubMed

    Wu, Aiguo; Paunesku, Tatjana; Brown, Eric M B; Babbo, Angela; Cruz, Cecille; Aslam, Mohamed; Dravid, Vinayak; Woloschak, Gayle E

    2008-02-01

    This work demonstrates the assembly of TiO(2) nanoparticles with attached DNA oligonucleotides into a 3D mesh structure by allowing base pairing between oligonucleotides. A change of the ratio of DNA oligonucleotide molecules and TiO(2) nanoparticles regulates the size of the mesh as characterized by UV-visible light spectra, transmission electron microscopy and atomic force microscopy images. This type of 3D mesh, based on TiO(2)-DNA oligonucleotide nanoconjugates, can be used for studies of nanoparticle assemblies in material science, energy science related to dye-sensitized solar cells, environmental science as well as characterization of DNA interacting proteins in the field of molecular biology. As an example of one such assembly, proliferating cell nuclear antigen protein (PCNA) was cloned, its activity verified, and the protein was purified, loaded onto double strand DNA oligonucleotide-TiO(2) nanoconjugates, and imaged by atomic force microscopy. This type of approach may be used to sample and perhaps quantify and/or extract specific cellular proteins from complex cellular protein mixtures affinity based on their affinity for chosen DNA segments assembled into the 3D matrix.

  9. Mode of action of DNA-competitive small molecule inhibitors of tyrosyl DNA phosphodiesterase 2

    PubMed Central

    Hornyak, Peter; Askwith, Trevor; Walker, Sarah; Komulainen, Emilia; Paradowski, Michael; Pennicott, Lewis E.; Bartlett, Edward J.; Brissett, Nigel C.; Raoof, Ali; Watson, Mandy; Jordan, Allan M.; Ogilvie, Donald J.; Ward, Simon E.; Atack, John R.; Pearl, Laurence H.; Caldecott, Keith W.; Oliver, Antony W.

    2016-01-01

    Tyrosyl-DNA phosphodiesterase 2 (TDP2) is a 5′-tyrosyl DNA phosphodiesterase important for the repair of DNA adducts generated by non-productive (abortive) activity of topoisomerase II (TOP2). TDP2 facilitates therapeutic resistance to topoisomerase poisons, which are widely used in the treatment of a range of cancer types. Consequently, TDP2 is an interesting target for the development of small molecule inhibitors that could restore sensitivity to topoisomerase-directed therapies. Previous studies identified a class of deazaflavin-based molecules that showed inhibitory activity against TDP2 at therapeutically useful concentrations, but their mode of action was uncertain. We have confirmed that the deazaflavin series inhibits TDP2 enzyme activity in a fluorescence-based assay, suitable for high-throughput screen (HTS)-screening. We have gone on to determine crystal structures of these compounds bound to a ‘humanized’ form of murine TDP2. The structures reveal their novel mode of action as competitive ligands for the binding site of an incoming DNA substrate, and point the way to generating novel and potent inhibitors of TDP2. PMID:27099339

  10. Processive translocation and DNA unwinding by individual RecBCD enzyme molecules

    NASA Astrophysics Data System (ADS)

    Bianco, Piero R.; Brewer, Laurence R.; Corzett, Michele; Balhorn, Rod; Yeh, Yin; Kowalczykowski, Stephen C.; Baskin, Ronald J.

    2001-01-01

    RecBCD enzyme is a processive DNA helicase and nuclease that participates in the repair of chromosomal DNA through homologous recombination. We have visualized directly the movement of individual RecBCD enzymes on single molecules of double-stranded DNA (dsDNA). Detection involves the optical trapping of solitary, fluorescently tagged dsDNA molecules that are attached to polystyrene beads, and their visualization by fluorescence microscopy. Both helicase translocation and DNA unwinding are monitored by the displacement of fluorescent dye from the DNA by the enzyme. Here we show that unwinding is both continuous and processive, occurring at a maximum rate of 972 +/- 172 base pairs per second (0.30µms-1), with as many as 42,300base pairs of dsDNA unwound by a single RecBCD enzyme molecule. The mean behaviour of the individual RecBCD enzyme molecules corresponds to that observed in bulk solution.

  11. Crystal structure of a complex of a type IA DNA topoisomerase with a single-stranded DNA molecule

    SciTech Connect

    Changela, A.; Digate, R.J.; Mondragon, A.

    2010-03-05

    A variety of cellular processes, including DNA replication, transcription, and chromosome condensation, require enzymes that can regulate the ensuing topological changes occurring in DNA. Such enzymes - DNA topoisomerases - alter DNA topology by catalysing the cleavage of single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA), the passage of DNA through the resulting break, and the rejoining of the broken phosphodiester backbone. DNA topoisomerase III from Escherichia coli belongs to the type IA family of DNA topoisomerases, which transiently cleave ssDNA via formation of a covalent 5' phosphotyrosine intermediate. Here we report the crystal structure, at 2.05 {angstrom} resolution, of an inactive mutant of E. coli DNA topoisomerase III in a non-covalent complex with an 8-base ssDNA molecule. The enzyme undergoes a conformational change that allows the oligonucleotide to bind within a groove leading to the active site. We note that the ssDNA molecule adopts a conformation like that of B-DNA while bound to the enzyme. The position of the DNA within the realigned active site provides insight into the role of several highly conserved residues during catalysis. These findings confirm various aspects of the type IA topoisomerase mechanism while suggesting functional implications for other topoisomerases and proteins that perform DNA rearrangements.

  12. Sizing of single globular DNA molecules by using a circular acceleration technique with laser trapping.

    PubMed

    Hirano, Ken; Nagata, Hideya; Ishido, Tomomi; Tanaka, Yoshio; Baba, Yoshinobu; Ishikawa, Mitsuru

    2008-07-01

    We describe a method for in situ sizing individual huge DNA molecules by laser trapping. Single DNA molecules are reversibly transformed, without mechanical fragmentation of fragile huge-sized DNA, from their random coil state into their globular state induced by condensing agents poly(ethylene glycol) and Mg(2+). With the use of a globular DNA molecule folded by condensation, the critical velocity of the circularly accelerated single globular DNA molecule by laser trapping was found to be proportional to the size of the DNA. Yeast, Saccharomyces cerevisiae, chromosome III (285 kbp) was successfully sized (281 +/- 40 kbp) from a calibration curve scaled using lambda, T4, and yeast chromosome VI (48.5, 166, and 385 kbp, respectively). The use of critical velocity as a sizing parameter makes it possible to size single DNA molecules without prior conformational information, i.e., the radius of a single globular huge DNA molecule as a nanoparticle. A sized single globular DNA molecule could be trapped again for subsequent manipulation, such as transportation of it anywhere. We also investigated a possibility of reusing the globular DNA molecules condensed by PEG and Mg(2+) for PCR and found that PCR efficiency was not deteriorated in the presence of the condensation agents.

  13. Conformational dynamics of nucleic acid molecules studied by PELDOR spectroscopy with rigid spin labels

    NASA Astrophysics Data System (ADS)

    Prisner, T. F.; Marko, A.; Sigurdsson, S. Th.

    2015-03-01

    Nucleic acid molecules can adopt a variety of structures and exhibit a large degree of conformational flexibility to fulfill their various functions in cells. Here we describe the use of Pulsed Electron-Electron Double Resonance (PELDOR or DEER) to investigate nucleic acid molecules where two cytosine analogs have been incorporated as spin probes. Because these new types of spin labels are rigid and incorporated into double stranded DNA and RNA molecules, there is no additional flexibility of the spin label itself present. Therefore the magnetic dipole-dipole interaction between both spin labels encodes for the distance as well as for the mutual orientation between the spin labels. All of this information can be extracted by multi-frequency/multi-field PELDOR experiments, which gives very precise and valuable information about the structure and conformational flexibility of the nucleic acid molecules. We describe in detail our procedure to obtain the conformational ensembles and show the accuracy and limitations with test examples and application to double-stranded DNA.

  14. Conformational dynamics of nucleic acid molecules studied by PELDOR spectroscopy with rigid spin labels.

    PubMed

    Prisner, T F; Marko, A; Sigurdsson, S Th

    2015-03-01

    Nucleic acid molecules can adopt a variety of structures and exhibit a large degree of conformational flexibility to fulfill their various functions in cells. Here we describe the use of Pulsed Electron-Electron Double Resonance (PELDOR or DEER) to investigate nucleic acid molecules where two cytosine analogs have been incorporated as spin probes. Because these new types of spin labels are rigid and incorporated into double stranded DNA and RNA molecules, there is no additional flexibility of the spin label itself present. Therefore the magnetic dipole-dipole interaction between both spin labels encodes for the distance as well as for the mutual orientation between the spin labels. All of this information can be extracted by multi-frequency/multi-field PELDOR experiments, which gives very precise and valuable information about the structure and conformational flexibility of the nucleic acid molecules. We describe in detail our procedure to obtain the conformational ensembles and show the accuracy and limitations with test examples and application to double-stranded DNA.

  15. Rigid DNA beams for high-resolution single-molecule mechanics.

    PubMed

    Pfitzner, Emanuel; Wachauf, Christian; Kilchherr, Fabian; Pelz, Benjamin; Shih, William M; Rief, Matthias; Dietz, Hendrik

    2013-07-22

    Bridging the gap: Rigid DNA linkers (blue, see picture) between microspheres (green) for high-resolution single-molecule mechanical experiments were constructed using DNA origami. The resulting DNA helical bundles greatly reduce the noise generated in studies of conformation changes using optical tweezers and were applied to study small DNA secondary structures.

  16. Detection of a New Interstellar Molecule: Thiocyanic Acid HSCN

    NASA Astrophysics Data System (ADS)

    Halfen, D. T.; Ziurys, L. M.; Brünken, S.; Gottlieb, C. A.; McCarthy, M. C.; Thaddeus, P.

    2009-09-01

    A new interstellar molecule, HSCN (thiocyanic acid), an energetic isomer of the well-known species HNCS, has been detected toward Sgr B2(N) with the Arizona Radio Observatory 12 m telescope. Eight rotational transitions in the Ka = 0 ladder were observed in the 2 mm and 3 mm bands. Five consecutive transitions in the 3 mm band are unblended, but three in the 2 mm band are partially masked by lines of other molecules. The peak intensity of all eight transitions are well described by a rotational temperature that is in very good agreement with that of many other molecules in this source. The line width and radial velocity of HSCN match closely with those of the ground state isomer HNCS (isothiocyanic acid), HNCO (isocyanic acid), and HOCN (cyanic acid); preliminary maps indicate that all four molecules are similarly distributed in Sgr B2. Although HSCN is calculated to lie over 3000 K higher in energy than HNCS, its column density of 1.3 × 1013 cm-2 in Sgr B2(N) is only three times lower than that of HNCS. The fractional abundances of HSCN and HNCS relative to H2 are 4.5 × 10-12 and 1.1 × 10-11. By analogy with the isomeric pair HCN and HNC, these two sulfur-bearing isomers are plausibly formed from a common cation precursor.

  17. Hard and soft acids and bases: small molecules.

    PubMed

    Reed, James L

    2009-08-03

    The operational chemical hardness has been determined for the hydride, chloride, and fluoride derivatives of the anionic atomic bases of the second period. Of interest is the identification of the structure and associated processes that give rise to hard-soft behavior in small molecules. The Pearson Principle of Hard and Soft Acids and Bases has been taken to be the defining statement about hard-soft behavior and as a definition of chemical hardness. Similar to the case for atoms, the molecule's responding electrons have been identified as the structure giving rise to hard-soft behavior, and a relaxation described by a modified Slater model has been identified as the associated process. The responding electrons are the molecule's valence electrons that are not undergoing electron transfer in an acid-base interaction. However, it has been demonstrated that chemical hardness is a local property, and only those responding electrons that are associated with the base's binding atom directly impact chemical hardness.

  18. [Microspeciation of amphoteric molecules of unusual acid-base properties].

    PubMed

    Kóczián, Kristóf

    2007-01-01

    The phisico-chemical properties of bio- and drug molecules greatly influence their interactions in the body and strongly effect the mechanism of drug action. Among these properties, macroscopic and site-specific protonation constants are of crucial importance. Latter one is the tool to calculate the relative concentration of the various microspecies in the compartments of the body at different pH values, and also, it is the versatile parameter to improve the pharmacokinetic properties of a new molecule in a particular family of drugs. In the present thesis work, the microspeciation of three molecules of great pharmaceutical importance and unusual acid-base properties, were carried out. The microconstants of tenoxicam, the non-steroidal anti-inflammatory drug, were described, introducing a novel deductive method using Hammett constants. For this purpose, a total of 8 tenoxicam and piroxicam derivatives were synthesised. To the best of our knowledge, the log k(N)O microconstant of tenoxicam obtained thus is the lowest enolate basicity value, which, however, can be well explained by the effects of the intramolecular environment. The developed evaluation procedure is suitable for microconstant determination of compounds in other molecule families. Besides, prodrug-type compounds and analogues similar to the structures of selective COX-2 isoenzyme inhibitors were synthesised. The other two molecules studied, the 6-aminopenicillanic acid and 7-cephalosporanic acid, the core molecules of the two most important beta-lactam antibiotic-types were derivatised and investigated by 1D and 2D NMR techniques. The NMR-pH titration on the parent compounds and their ester derivatives, combined with in situ pH-measurements allowed the microspeciation of these easily decomposing molecules. One of the protonation constant of 7-ACA (log kN(O) = 4.12), to the best of our knowledge, is the least non-aromatic basic amino-site among the natural compounds.

  19. Elongation and migration of single DNA molecules in microchannels using oscillatory shear flows.

    PubMed

    Jo, Kyubong; Chen, Yeng-Long; de Pablo, Juan J; Schwartz, David C

    2009-08-21

    Much of modern biology relies on the strategic manipulation of molecules for creating ordered arrays prior to high throughput molecular analysis. Normally, DNA arrays involve deposition on surfaces, or confinement in nanochannels; however, we show that microfluidic devices can present stretched molecules within a controlled flow in ways complementing surface modalities, or extreme confinement conditions. Here we utilize pressure-driven oscillatory shear flows generated in microchannels as a new way of stretching DNA molecules for imaging "arrays" of individual DNA molecules. Fluid shear effects both stretch DNA molecules and cause them to migrate away from the walls becoming focused in the centerline of a channel. We show experimental findings confirming simulations using Brownian dynamics accounting for hydrodynamic interactions between molecules and channel-flow boundary conditions. Our findings characterize DNA elongation and migration phenomena as a function of molecular size, shear rate, oscillatory frequency with comparisons to computer simulation studies.

  20. Single molecule fluorescence studies of transition paths in DNA hairpin folding

    NASA Astrophysics Data System (ADS)

    Truex, Katherine; Chung, Hoi Sung; Louis, John; Eaton, William

    DNA hairpins are the simplest structures for investigating fundamental aspects of nucleic acid folding mechanisms. For two-state hairpins, all of the mechanistic information on how the hairpin folds is contained in the transition path (TP), the rare event in single molecule trajectories when the free energy barrier between folded and unfolded states is actually crossed. The only previous experimental study of TPs in nucleic acids used optical tweezer measurements and Szabo's analytical theory for diffusive barrier crossing to reconstruct the free energy surface for an indirect determination of average TP times (Neupane et al. PRL 2012). We used confocal single molecule FRET and maximum likelihood analysis of photon trajectories to determine an upper bound of 2.5 μs for the average TP time of a DNA hairpin (Truex et al., PRL 2015), compared to the value of 4 μs predicted by Neupane et al., providing an important test of energy landscape theory. Current experiments are aimed at eventually characterizing structural changes during TPs, which will provide a very demanding test of mechanisms predicted by both theoretical models and simulations.

  1. High-Throughput Universal DNA Curtain Arrays for Single-Molecule Fluorescence Imaging

    PubMed Central

    Gallardo, Ignacio F.; Pasupathy, Praveenkumar; Brown, Maxwell; Manhart, Carol M.; Neikirk, Dean P.; Alani, Eric; Finkelstein, Ilya J.

    2015-01-01

    Single-molecule studies of protein–DNA interactions have shed critical insights into the molecular mechanisms of nearly every aspect of DNA metabolism. The development of DNA curtains—a method for organizing arrays of DNA molecules on a fluid lipid bilayer—has greatly facilitated these studies by increasing the number of reactions that can be observed in a single experiment. However, the utility of DNA curtains is limited by the challenges associated with depositing nanometer-scale lipid diffusion barriers onto quartz microscope slides. Here, we describe a UV lithography-based method for large-scale fabrication of chromium (Cr) features and organization of DNA molecules at these features for high-throughput single-molecule studies. We demonstrate this approach by assembling 792 independent DNA arrays (containing >900 000 DNA molecules) within a single microfluidic flowcell. As a first proof of principle, we track the diffusion of Mlh1-Mlh3—a heterodimeric complex that participates in DNA mismatch repair and meiotic recombination. To further highlight the utility of this approach, we demonstrate a two-lane flowcell that facilitates concurrent experiments on different DNA substrates. Our technique greatly reduces the challenges associated with assembling DNA curtains and paves the way for the rapid acquisition of large statistical data sets from individual single-molecule experiments. PMID:26325477

  2. Exporters for Production of Amino Acids and Other Small Molecules.

    PubMed

    Eggeling, Lothar

    2016-11-11

    Microbes are talented catalysts to synthesize valuable small molecules in their cytosol. However, to make full use of their skills - and that of metabolic engineers - the export of intracellularly synthesized molecules to the culture medium has to be considered. This step is as essential as is each step for the synthesis of the favorite molecule of the metabolic engineer, but is frequently not taken into account. To export small molecules via the microbial cell envelope, a range of different types of carrier proteins is recognized to be involved, which are primary active carriers, secondary active carriers, or proteins increasing diffusion. Relevant export may require just one carrier as is the case with L-lysine export by Corynebacterium glutamicum or involve up to four carriers as known for L-cysteine excretion by Escherichia coli. Meanwhile carriers for a number of small molecules of biotechnological interest are recognized, like for production of peptides, nucleosides, diamines, organic acids, or biofuels. In addition to carriers involved in amino acid excretion, such carriers and their impact on product formation are described, as well as the relatedness of export carriers which may serve as a hint to identify further carriers required to improve product formation by engineering export.

  3. Effect of co-existing biologically relevant molecules and ions on DNA photocleavage caused by pyrene and its derivatives.

    PubMed

    Wang, Shuguang; Yu, Hongtao

    2005-04-01

    Inorganic ions, coenzymes, amino acids, and saccharides could co-exist with toxic environmental chemicals, such as polycyclic aromatic hydrocarbons (PAHs), in the cell. The presence of these co-existing chemicals can modulate the toxicity of the PAHs. One of the genotoxic effects by PAHs is light-induced cleavage, or photocleavage, of DNA. The effect of inorganic ions I-, Na+, Ca2+, Mg2+, Fe3+, Mn2+, Cu2+, and Zn2+ and biological molecules riboflavin, histidine, mannitol, nicotinamide adenine dinucleotide (NAD), glutathione, and glutamic acid on the DNA photocleavage by pyrene, 1-hydroxypyrene (1-HP), and 1-aminopyrene (1-AP), is studied. The non-transition metal ions Na+, Ca2+, and Mg2+, usually have very little inhibitory effects, while the transition metal ions Fe3+, Cu2+, and Zn2+ enhance, Mn2+ inhibits the DNA photocleavage. The effect by biological molecules is complex, depending on the photochemical reaction mechanisms of the compounds tested (1-AP, 1-HP and pyrene) and on the chemical nature of the added biological molecules. Riboflavin, histidine, and mannitol enhance DNA photocleavage by all three compounds, except that mannitol has no effect on the photocleavage of DNA by pyrene. Glutathione inhibits the DNA photocleavage by 1-AP and 1-HP, but has no effect on that by pyrene. NAD enhances the DNA photocleavage by 1-AP, but has no effect on that by 1-HP and pyrene. Glutamic acid enhances the DNA photocleavage by 1-AP and pyrene, but inhibits that by 1-HP. These results show that the co-existing chemicals may have a profound effect on the toxicity of PAHs, or possibly on the toxicity of many other chemicals. Therefore, if one studies the toxic effects of PAHs or other toxic chemicals, the effect of the co-existing chemicals or ions needs to be considered.

  4. Single-molecule surface studies of fibrinogen and DNA on semiconductors

    NASA Astrophysics Data System (ADS)

    Kong, Xianhua

    Understanding of protein adsorption onto non-biological substrates is of fundamental interest in science, but also has great potential technological applications in medical devices and biosensors. This study explores the non-specific interaction, at the single molecule level, of a blood protein and DNA with semiconductor surfaces through the use of a custom built, non rastering electron emission microscope and a scanning probe microscope. The specifics and history of electron emission are described as well as the equipment used in this study. The protein examined in this study is human plasma fibrinogen, which plays an important role in haemostatis and thrombosis, and deoxyribonucleic acid (DNA) is also studied. A novel technique for determining the photothreshold of biomolecules on single molecule level is developed and applied to fibrinogen molecules adsorbed on oxidized silicon surfaces, using photo-electron emission microscopy (PEEM). Three theoretical models are employed and compared to analyze the experimental photothreshold data. The non-specific adsorption of human plasma fibrinogen on oxidized p- and n- type silicon (100) surfaces is investigated to characterize both hydrophobic interactions and electrostatic forces. The experimental results indicate that hydrophobic interactions are one of the driving forces for protein adsorption and the electrostatic interactions also play a role in the height of the fibrinogen molecules adsorbed on the surface. PEEM images establish a photo threshold of 5.0 +/- 0.2 eV for fibrinogen on both n-type and p-type Si (100) surfaces. We suggest that the photothreshold results from surface state associated Fermi level (EF) pinning and there exists negative charge transfer from the adsorbed fibrinogen onto the p-type silicon substrates, while on n-type silicon substrates negative charge is transferred in the opposite direction. The adsorption of deoxyribonucleic acid (DNA) on mica and silicon is studied in liquid and ambient

  5. Dihedral angle preferences of DNA and RNA binding amino acid residues in proteins.

    PubMed

    Ponnuraj, Karthe; Saravanan, Konda Mani

    2017-04-01

    A protein can interact with DNA or RNA molecules to perform various cellular processes. Identifying or analyzing DNA/RNA binding site amino acid residues is important to understand molecular recognition process. It is quite possible to accurately model DNA/RNA binding amino acid residues in experimental protein-DNA/RNA complex by using the electron density map whereas, locating/modeling the binding site amino acid residues in the predicted three dimensional structures of DNA/RNA binding proteins is still a difficult task. Considering the above facts, in the present work, we have carried out a comprehensive analysis of dihedral angle preferences of DNA and RNA binding site amino acid residues by using a classical Ramachandran map. We have computed backbone dihedral angles of non-DNA/RNA binding residues and used as control dataset to make a comparative study. The dihedral angle preference of DNA and RNA binding site residues of twenty amino acid type is presented. Our analysis clearly revealed that the dihedral angles (φ, ψ) of DNA/RNA binding amino acid residues prefer to occupy (-89° to -60°, -59° to -30°) bins. The results presented in this paper will help to model/locate DNA/RNA binding amino acid residues with better accuracy.

  6. Molecular Combing of Single DNA Molecules on the 10 Megabase Scale

    PubMed Central

    Kaykov, Atanas; Taillefumier, Thibaud; Bensimon, Aaron; Nurse, Paul

    2016-01-01

    DNA combing allows the investigation of DNA replication on genomic single DNA molecules, but the lengths that can be analysed have been restricted to molecules of 200–500 kb. We have improved the DNA combing procedure so that DNA molecules can be analysed up to the length of entire chromosomes in fission yeast and up to 12 Mb fragments in human cells. Combing multi-Mb-scale DNA molecules revealed previously undetected origin clusters in fission yeast and shows that in human cells replication origins fire stochastically forming clusters of fired origins with an average size of 370 kb. We estimate that a single human cell forms around 3200 clusters at mid S-phase and fires approximately 100,000 origins to complete genome duplication. The procedure presented here will be adaptable to other organisms and experimental conditions. PMID:26781994

  7. History of gymnemic acid, a molecule that does not exist. .

    PubMed

    Zarrelli, Armando; Romanucci, Valeria; Gravante, Raffaele; Di Marino, Cinzia; Di Fabio, Giovanni

    2014-10-01

    In the literature there are hundreds of articles, the first dating back to 1866 and the last to 2014, on gymnemic acid, isolated from Gymnnema sylvestre, from its isolation to the determination of its biological activities. Gymnemic acid has a CAS number but its structure is not specified. Studies during the second half of the 1970s clearly demonstrated that what was being referred to as gymnemic acid is actually a very complex mixture of dozens of substances, belonging to different classes of natural compounds. This plant, whose infusions or complex mixtures of its metabolites are the basis for many formulas sold in pharmacies and by herbalists, has anti-diabetic and slimming effects. It is certainly misleading to talk about gymnemic acid as a specific molecule. There may be doubts about the exact composition of the products, and consequently about their origin and the claimed effects.

  8. DNA origami as biocompatible surface to match single-molecule and ensemble experiments

    PubMed Central

    Gietl, Andreas; Holzmeister, Phil; Grohmann, Dina; Tinnefeld, Philip

    2012-01-01

    Single-molecule experiments on immobilized molecules allow unique insights into the dynamics of molecular machines and enzymes as well as their interactions. The immobilization, however, can invoke perturbation to the activity of biomolecules causing incongruities between single molecule and ensemble measurements. Here we introduce the recently developed DNA origami as a platform to transfer ensemble assays to the immobilized single molecule level without changing the nano-environment of the biomolecules. The idea is a stepwise transfer of common functional assays first to the surface of a DNA origami, which can be checked at the ensemble level, and then to the microscope glass slide for single-molecule inquiry using the DNA origami as a transfer platform. We studied the structural flexibility of a DNA Holliday junction and the TATA-binding protein (TBP)-induced bending of DNA both on freely diffusing molecules and attached to the origami structure by fluorescence resonance energy transfer. This resulted in highly congruent data sets demonstrating that the DNA origami does not influence the functionality of the biomolecule. Single-molecule data collected from surface-immobilized biomolecule-loaded DNA origami are in very good agreement with data from solution measurements supporting the fact that the DNA origami can be used as biocompatible surface in many fluorescence-based measurements. PMID:22523083

  9. Model of DNA topology simplification has come full (supercoiled) circle after two decades of research. Comment on "Disentangling DNA molecules" by Alexander Vologodskii

    NASA Astrophysics Data System (ADS)

    Stasiak, Andrzej

    2016-09-01

    Being a geek of DNA topology, I remember very well the stir caused by 1997 Science paper showing that DNA topoisomerases have the ability to simplify DNA topology below the topological equilibrium values [1]. In their seminal experiments Rybenkov et al. [1] started with linear double-stranded DNA molecules with cohesive ends. The mutual cohesiveness of DNA ends was due to mutual complementarity of single-stranded extensions at both ends of linear double-stranded DNA molecules. When such DNA molecules were heated up and then slowly cooled down the single-stranded ends eventually annealed with each other causing DNA circularization. This experimental protocol permitted the authors to establish topological/thermodynamic equilibrium within samples of circularized DNA molecules. Among simple unknotted circles one also observed knotted and catenated DNA molecules. The fraction of knotted molecules in DNA samples at topological equilibrium was increasing with the length of DNA molecules undergoing slow circularization. The fraction of catenated molecules was increasing with the length and the concentration of the molecules undergoing slow circularization. Rybenkov et al. incubated then such equilibrated DNA samples with type II DNA topoisomerases, which pass DNA duplex regions through each other, and observed that as the result of it the fraction of knotted and catenated DNA molecules was dramatically decreased (up to 80-fold). This elegant experiment indicated for the first time that type II DNA topoisomerases acting on knotted or catenated DNA molecules have the ability to select among many potential sites of DNA-DNA passages these that result in DNA unknotting or decatenation. Without such a selection topoisomerases could only maintain the original topological equilibrium obtained during the slow cyclization. The big question was how DNA topoisomerases can be directed to do DNA-DNA passages that preferentially result in DNA unknotting and decatenation.

  10. Monitoring patterned enzymatic polymerization on DNA origami at single-molecule level.

    PubMed

    Okholm, A H; Aslan, H; Besenbacher, F; Dong, M; Kjems, J

    2015-07-07

    DNA origami has been used to orchestrate reactions with nano-precision using a variety of biomolecules. Here, the dynamics of albumin-assisted, localized single-molecule DNA polymerization by terminal deoxynucleotidyl transferase on a 2D DNA origami are monitored using AFM in liquid. Direct visualization of the surface activity revealed the mechanics of growth.

  11. Single-molecule imaging of DNA polymerase I (Klenow fragment) activity by atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Chao, J.; Zhang, P.; Wang, Q.; Wu, N.; Zhang, F.; Hu, J.; Fan, C. H.; Li, B.

    2016-03-01

    We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA.We report a DNA origami-facilitated single-molecule platform that exploits atomic force microscopy to study DNA replication. We imaged several functional activities of the Klenow fragment of E. coli DNA polymerase I (KF) including binding, moving, and dissociation from the template DNA. Upon completion of these actions, a double-stranded DNA molecule was formed. Furthermore, the direction of KF activities was captured and then confirmed by shifting the KF binding sites on the template DNA. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr06544e

  12. Sieving DNA molecules by length dependence in artificial nano-channel matrices

    NASA Astrophysics Data System (ADS)

    Wang, Chung-Hsuan; Hua Ho, Chia; Chou, Y. C.

    2013-01-01

    Nano-channel matrices are designed and fabricated for sieving DNA molecules by length. The length dependence is found to change with the size of the channels. Three regimes can be distinguished: (a) for the matrices with the size of the channels comparable to the persistence length (lp) of DNA molecules (45 nm), the mobility of DNA is found to decrease with the length of the molecules, similar to that found for the gel electrophoresis; (b) as the size of the nano-channel increases, the successful attacking frequency increases for the long molecules. The length-dependence of the mobility reverses; and (c) the Ogston mechanism holds for even larger channels. The short DNA molecules drift faster for the channels with diameter larger than 10 lp. Such a variety of the length dependence is observed for the first time in the electrophoresis in the artificial structures.

  13. Quantum dot binding to DNA: single-molecule imaging with atomic force microscopy.

    PubMed

    Li, Kungang; Zhang, Wen; Chen, Yongsheng

    2013-01-01

    The interaction between nanoparticles (NPs) and DNA is of significance for both application and implication research of NPs. In this study, a single-molecule imaging technique based on atomic force microscopy (AFM) was employed to probe the NP-DNA interactions with quantum dots (QDs) as model NPs. Reproducible high-quality images of single DNA molecules in air and in liquids were acquired on mica by optimizing sample preparation conditions. Furthermore, the binding of QDs to DNA was explored using AFM. The DNA concentration was found to be a key factor influencing AFM imaging quality. In air and liquids, the optimal DNA concentration for imaging DNA molecules was approximately 2.5 and 0.25 μg/mL, and that for imaging DNA binding with QDs was 0.5 and 0.25 μg/mL, respectively. In the presence of QDs, the DNA conformation was altered with the formation of DNA condensates. Finally, the fine conformation of QD-DNA binding sites was examined to analyze the binding mechanisms. This work will benefit investigations of NP-DNA interactions and the understanding of the structure of NP-DNA bioconjugates. See accompanying article by Wang DOI: 10.1002/biot.201200309.

  14. Preparation of multimilligram quantities of large, linear DNA molecules for structural studies.

    PubMed

    Muecke, Merlind; Samuels, Martin; Davey, Megan; Jeruzalmi, David

    2008-06-01

    We describe a method for preparing large, linear DNA molecules in amounts that are suitable for structural studies. The procedure employs self-primed DNA amplification on a starting molecule that consists of the sequence of interest flanked by the cohesive end sequences from bacteriophage lambda as well as endonuclease recognition sites. Amplification produces long polymers of DNA, tens of kilobases in length, which harbor many copies of the sequence of interest. Endonuclease digestion of these polymers, followed by chromatographic purification, yields high-quality preparations of the DNA molecule of interest. Reliance on the cohesive end sequences to initiate self-primed amplification effectively enables the synthesis of DNA molecules of interest with minimal restriction on length and sequence.

  15. Scanning a DNA Molecule for Bound Proteins Using Hybrid Magnetic and Optical Tweezers

    PubMed Central

    van Loenhout, Marijn T. J.; De Vlaminck, Iwijn; Flebus, Benedetta; den Blanken, Johan F.; Zweifel, Ludovit P.; Hooning, Koen M.; Kerssemakers, Jacob W. J.; Dekker, Cees

    2013-01-01

    The functional state of the genome is determined by its interactions with proteins that bind, modify, and move along the DNA. To determine the positions and binding strength of proteins localized on DNA we have developed a combined magnetic and optical tweezers apparatus that allows for both sensitive and label-free detection. A DNA loop, that acts as a scanning probe, is created by looping an optically trapped DNA tether around a DNA molecule that is held with magnetic tweezers. Upon scanning the loop along the λ-DNA molecule, EcoRI proteins were detected with ∼17 nm spatial resolution. An offset of 33±5 nm for the detected protein positions was found between back and forwards scans, corresponding to the size of the DNA loop and in agreement with theoretical estimates. At higher applied stretching forces, the scanning loop was able to remove bound proteins from the DNA, showing that the method is in principle also capable of measuring the binding strength of proteins to DNA with a force resolution of 0.1 pN/. The use of magnetic tweezers in this assay allows the facile preparation of many single-molecule tethers, which can be scanned one after the other, while it also allows for direct control of the supercoiling state of the DNA molecule, making it uniquely suitable to address the effects of torque on protein-DNA interactions. PMID:23755219

  16. Nanofabricated racks of aligned and anchored DNA substrates for single-molecule imaging.

    PubMed

    Gorman, Jason; Fazio, Teresa; Wang, Feng; Wind, Shalom; Greene, Eric C

    2010-01-19

    Single-molecule studies of biological macromolecules can benefit from new experimental platforms that facilitate experimental design and data acquisition. Here we develop new strategies to construct curtains of DNA in which the molecules are aligned with respect to one another and maintained in an extended configuration by anchoring both ends of the DNA to the surface of a microfluidic sample chamber that is otherwise coated with an inert lipid bilayer. This "double-tethered" DNA substrate configuration is established through the use of nanofabricated rack patterns comprised of two distinct functional elements: linear barriers to lipid diffusion that align DNA molecules anchored by one end to the bilayer and antibody-coated pentagons that provide immobile anchor points for the opposite ends of the DNA. These devices enable the alignment and anchoring of thousands of individual DNA molecules, which can then be visualized using total internal reflection fluorescence microscopy under conditions that do not require continuous application of buffer flow to stretch the DNA. This unique strategy offers the potential for studying protein-DNA interactions on large DNA substrates without compromising measurements through application of hydrodynamic force. We provide a proof-of-principle demonstration that double-tethered DNA curtains made with nanofabricated rack patterns can be used in a one-dimensional diffusion assay that monitors the motion of quantum dot-tagged proteins along DNA.

  17. Synergistic self-assembly of RNA and DNA molecules

    NASA Astrophysics Data System (ADS)

    Ko, Seung Hyeon; Su, Min; Zhang, Chuan; Ribbe, Alexander E.; Jiang, Wen; Mao, Chengde

    2010-12-01

    DNA has recently been used as a programmable 'smart' building block for the assembly of a wide range of nanostructures. It remains difficult, however, to construct DNA assemblies that are also functional. Incorporating RNA is a promising strategy to circumvent this issue as RNA is structurally related to DNA but exhibits rich chemical, structural and functional diversities. However, only a few examples of rationally designed RNA structures have been reported. Herein, we describe a simple, general strategy for the de novo design of nanostructures in which the self-assembly of RNA strands is programmed by DNA strands. To demonstrate the versatility of this approach, we have designed and constructed three different RNA-DNA hybrid branched nanomotifs (tiles), which readily assemble into one-dimensional nanofibres, extended two-dimensional arrays and a discrete three-dimensional object. The current strategy could enable the integration of the precise programmability of DNA with the rich functionality of RNA.

  18. Single-molecule elasticity of single-stranded DNA, a model flexible polyelectrolyte

    NASA Astrophysics Data System (ADS)

    McIntosh, Dustin B.

    Understanding the structure of unfolded, flexible polyelectrolytes is important for our comprehension of basic processes in molecular biology (e.g., RNA and protein folding) and our ability to exploit the polymers in technology (e.g., in self-assembled nanostructures). Here, we investigate the structure of single single-stranded DNA molecules and their interactions with ions using magnetic tweezers. Our data reveal that single-stranded DNA is not well-described by ideal polymer models such as the Worm-Like Chain. At low force, we report the first experimental observation of a nonlinear elastic regime revealing the relevance of long-range excluded volume effects. At high force, the extension scales as a logarithm in monovalent salt. Molecular dynamics simulations indicate that this logarithmic regime is the result of ion-stabilized wrinkles at short-length scales along the polymer backbone. Addition of divalent salt to the buffer results in enhanced elasticity indicating increased wrinkling or polymer ''wrapping" around the divalent ions. Using a thermodynamic identity, we are able to count ions as they are released into the bulk upon polymer elongation. We find that ssDNA releases significantly more ions than dsDNA. We posit that the recently termed ''Snake-Like Chain" model (Ullner, J. Phys. Chem B (2003)) for flexible polyelectrolytes may explain these observations. As a first step towards characterizing biologically relevant nucleic acid structures, we measure the effects of base-stacking on ssDNA elasticity. We find that base-stacking in poly(dA) significantly enhances the rigidity of the polymer as evidenced by the low-force elasticity. The unstacking transition of poly(dA) at high force reveals that the intrinsic electrostatic tension on the molecule varies significantly more weakly on salt concentration than predictions from mean-field models. Further, we provide a model-independent estimate of the free energy difference between stacked and unstacked nucleic

  19. Direct single-molecule observations of DNA unwinding by SV40 large tumor antigen under a negative DNA supercoil state.

    PubMed

    Takahashi, Shunsuke; Motooka, Shinya; Kawasaki, Shohei; Kurita, Hirofumi; Mizuno, Takeshi; Matsuura, Shun-Ichi; Hanaoka, Fumio; Mizuno, Akira; Oshige, Masahiko; Katsura, Shinji

    2017-01-05

    Superhelices, which are induced by the twisting and coiling of double-helical DNA in chromosomes, are thought to affect transcription, replication, and other DNA metabolic processes. In this study, we report the effects of negative supercoiling on the unwinding activity of simian virus 40 large tumor antigen (SV40 TAg) at a single-molecular level. The supercoiling density of linear DNA templates was controlled using magnetic tweezers and monitored using a fluorescent microscope in a flow cell. SV40 TAg-mediated DNA unwinding under relaxed and negative supercoil states was analyzed by the direct observation of both single- and double-stranded regions of single DNA molecules. Increased negative superhelicity stimulated SV40 TAg-mediated DNA unwinding more strongly than a relaxed state; furthermore, negative superhelicity was associated with an increased probability of SV40 TAg-mediated DNA unwinding. These results suggest that negative superhelicity helps to regulate the initiation of DNA replication.

  20. Single-stranded DNA scanning and deamination with Single molecule resolution

    NASA Astrophysics Data System (ADS)

    Rueda, David

    2012-04-01

    Over the past decade, single-molecule fluorescence resonance energy transfer spectroscopy (smFRET) has become an increasingly popular tool to study the structural dynamics of biopolymers, such as DNA, RNA and proteins. The most attractive aspect of single-molecule experiments is that, unlike ensemble-averaged techniques, they directly reveal the structural dynamics of individual molecules, which would otherwise be hidden in ensemble-averaged experiments. Here, we will present a novel single molecule assay to study, for the first time, scanning of an enzyme (APOBEC3G, involved in the defense against HIV) on single stranded DNA (ssDNA). We have investigated the ssDNA scanning and activity of Apo3G with smFRET. Our data show that Apo3G scans ssDNA randomly and bidirectionally with average excursion lengths of ˜ 10 å and ˜1 s-1 scanning rates. Apo3G quasi-localization is observed on highly reactive motifs located near the one end of the ssDNA. Motif-dependent ssDNA bending is also observed, where the bending is maximal for highly reactive targets located near the DNA end. Interestingly, both the Apo3G scanning and Apo3G-induced ssDNA bending is reduced with lowered ionic strength, indicating that Apo3G motion on ssDNA is facilitated by salt by reducing `electrostatic friction'. Although scanning is random, asymmetric catalytic orientation may be the reason for Apo3G directional activity.

  1. DNA molecule stretching through thermo-electrophoresis and thermal convection in a heated converging-diverging microchannel

    PubMed Central

    2013-01-01

    A novel DNA molecule stretching technique is developed and tested herein. Through a heated converging-diverging microchannel, thermal convection and thermophoresis induced by regional heating are shown to significantly elongate single DNA molecules; they are visualized via a confocal laser scanning microscopy. In addition, electrophoretic stretching is also implemented to examine the hybrid effect on the conformation and dynamics of single DNA molecules. The physical properties of the DNA molecules are secured via experimental measurements. PMID:23414121

  2. Recursive construction of perfect DNA molecules and libraries from imperfect oligonucleotides.

    PubMed

    Linshiz, Gregory; Yehezkel, Tuval Ben; Shapiro, Ehud

    2012-01-01

    Making faultless complex objects from potentially faulty building blocks is a fundamental challenge in computer engineering, nanotechnology, and synthetic biology. We developed an error-correcting recursive construction procedure that attempts to address this challenge. Making DNA molecules from synthetic oligonucleotides using the procedure described here surpasses existing methods for de novo DNA synthesis in speed, precision, and amenability to automation. It provides for the first time a unified DNA construction platform for combining synthetic and natural DNA fragments, for constructing designer DNA libraries, and for making the faultless long synthetic DNA building blocks needed for de novo genome construction.

  3. Automated High Resolution Optical Mapping Using Arrayed, Fluid-Fixed DNA Molecules

    NASA Astrophysics Data System (ADS)

    Jing, Junping; Reed, Jason; Huang, John; Hu, Xinghua; Clarke, Virginia; Edington, Joanne; Housman, Dan; Anantharaman, Thomas S.; Huff, Edward J.; Mishra, Bud; Porter, Brett; Shenker, Alexander; Wolfson, Estarose; Hiort, Catharina; Kantor, Ron; Aston, Christopher; Schwartz, David C.

    1998-07-01

    New mapping approaches construct ordered restriction maps from fluorescence microscope images of individual, endonuclease-digested DNA molecules. In optical mapping, molecules are elongated and fixed onto derivatized glass surfaces, preserving biochemical accessibility and fragment order after enzymatic digestion. Measurements of relative fluorescence intensity and apparent length determine the sizes of restriction fragments, enabling ordered map construction without electrophoretic analysis. The optical mapping system reported here is based on our physical characterization of an effect using fluid flows developed within tiny, evaporating droplets to elongate and fix DNA molecules onto derivatized surfaces. Such evaporation-driven molecular fixation produces well elongated molecules accessible to restriction endonucleases, and notably, DNA polymerase I. We then developed the robotic means to grid DNA spots in well defined arrays that are digested and analyzed in parallel. To effectively harness this effect for high-throughput genome mapping, we developed: (i) machine vision and automatic image acquisition techniques to work with fixed, digested molecules within gridded samples, and (ii) Bayesian inference approaches that are used to analyze machine vision data, automatically producing high-resolution restriction maps from images of individual DNA molecules. The aggregate significance of this work is the development of an integrated system for mapping small insert clones allowing biochemical data obtained from engineered ensembles of individual molecules to be automatically accumulated and analyzed for map construction. These approaches are sufficiently general for varied biochemical analyses of individual molecules using statistically meaningful population sizes.

  4. Endogenous molecules stimulating N-acylethanolamine-hydrolyzing acid amidase (NAAA).

    PubMed

    Tai, Tatsuya; Tsuboi, Kazuhito; Uyama, Toru; Masuda, Kim; Cravatt, Benjamin F; Houchi, Hitoshi; Ueda, Natsuo

    2012-05-16

    Fatty acid amide hydrolase (FAAH) plays the central role in the degradation of bioactive N-acylethanolamines such as the endocannabinoid arachidonoylethanolamide (anandamide) in brain and peripheral tissues. A lysosomal enzyme referred to as N-acylethanolamine-hydrolyzing acid amidase (NAAA) catalyzes the same reaction with preference to palmitoylethanolamide, an endogenous analgesic and neuroprotective substance, and is therefore expected as a potential target of therapeutic drugs. In the in vitro assays thus far performed, the maximal activity of NAAA was achieved in the presence of both nonionic detergent (Triton X-100 or Nonidet P-40) and the SH reagent dithiothreitol. However, endogenous molecules that might substitute for these synthetic compounds remain poorly understood. Here, we examined stimulatory effects of endogenous phospholipids and thiol compounds on recombinant NAAA. Among different phospholipids tested, choline- or ethanolamine-containing phospholipids showed potent effects, and 1 mM phosphatidylcholine increased NAAA activity by 6.6-fold. Concerning endogenous thiol compounds, dihydrolipoic acid at 0.1-1 mM was the most active, causing 8.5-9.0-fold stimulation. These results suggest that endogenous phospholipids and dihydrolipoic acid may contribute in keeping NAAA active in lysosomes. Even in the presence of phosphatidylcholine and dihydrolipoic acid, however, the preferential hydrolysis of palmitoylethanolamide was unaltered. We also investigated a possible compensatory induction of NAAA mRNA in brain and other tissues of FAAH-deficient mice. However, NAAA expression levels in all the tissues examined were not significantly altered from those in wild-type mice.

  5. Design of Sequence-Specific DNA Binding Molecules for DNA Methyltransferase Inhibition

    PubMed Central

    2015-01-01

    The CpG dyad, an important genomic feature in DNA methylation and transcriptional regulation, is an attractive target for small molecules. To assess the utility of minor groove binding oligomers for CpG recognition, we screened a small library of hairpin pyrrole-imidazole polyamides targeting the sequence 5′-CGCG-3′ and assessed their sequence specificity using an unbiased next-generation sequencing assay. Our findings indicate that hairpin polyamide of sequence PyImβIm-γ-PyImβIm (1), previously identified as a high affinity 5′-CGCG-3′ binder, favors 5′-GCGC-3′ in an unanticipated reverse binding orientation. Replacement of one β alanine with Py to afford PyImPyIm-γ-PyImβIm (3) restores the preference for 5′-CGCG-3′ binding in a forward orientation. The minor groove binding hairpin 3 inhibits DNA methyltransferase activity in the major groove at its target site more effectively than 1, providing a molecular basis for design of sequence-specific antagonists of CpG methylation. PMID:24502234

  6. Probe Microscopic Studies of DNA Molecules on Carbon Nanotubes

    PubMed Central

    Umemura, Kazuo; Izumi, Katsuki; Oura, Shusuke

    2016-01-01

    Hybrids of DNA and carbon nanotubes (CNTs) are promising nanobioconjugates for nanobiosensors, carriers for drug delivery, and other biological applications. In this review, nanoscopic characterization of DNA-CNT hybrids, in particular, characterization by scanning probe microscopy (SPM), is summarized. In many studies, topographical imaging by atomic force microscopy has been performed. However, some researchers have demonstrated advanced SPM operations in order to maximize its unique and valuable functions. Such sophisticated approaches are attractive and will have a significant impact on future studies of DNA-CNT hybrids.

  7. Drug-DNA interactions at single molecule level: A view with optical tweezers

    NASA Astrophysics Data System (ADS)

    Paramanathan, Thayaparan

    Studies of small molecule--DNA interactions are essential for developing new drugs for challenging diseases like cancer and HIV. The main idea behind developing these molecules is to target and inhibit the reproduction of the tumor cells and infected cells. We mechanically manipulate single DNA molecule using optical tweezers to investigate two molecules that have complex and multiple binding modes. Mononuclear ruthenium complexes have been extensively studied as a test for rational drug design. Potential drug candidates should have high affinity to DNA and slow dissociation kinetics. To achieve this, motifs of the ruthenium complexes are altered. Our collaborators designed a dumb-bell shaped binuclear ruthenium complex that can only intercalate DNA by threading through its bases. Studying the binding properties of this complex in bulk studies took hours. By mechanically manipulating a single DNA molecule held with optical tweezers, we lower the barrier to thread and make it fast compared to the bulk experiments. Stretching single DNA molecules with different concentration of drug molecules and holding it at a constant force allows the binding to reach equilibrium. By this we can obtain the equilibrium fractional ligand binding and length of DNA at saturated binding. Fitting these results yields quantitative measurements of the binding thermodynamics and kinetics of this complex process. The second complex discussed in this study is Actinomycin D (ActD), a well studied anti-cancer agent that is used as a prototype for developing new generations of drugs. However, the biophysical basis of its activity is still unclear. Because ActD is known to intercalate double stranded DNA (dsDNA), it was assumed to block replication by stabilizing dsDNA in front of the replication fork. However, recent studies have shown that ActD binds with even higher affinity to imperfect duplexes and some sequences of single stranded DNA (ssDNA). We directly measure the on and off rates by

  8. Single-molecule spectroscopy of amino acids and peptides by recognition tunnelling

    NASA Astrophysics Data System (ADS)

    Zhao, Yanan; Ashcroft, Brian; Zhang, Peiming; Liu, Hao; Sen, Suman; Song, Weisi; Im, Jongone; Gyarfas, Brett; Manna, Saikat; Biswas, Sovan; Borges, Chad; Lindsay, Stuart

    2014-06-01

    The human proteome has millions of protein variants due to alternative RNA splicing and post-translational modifications, and variants that are related to diseases are frequently present in minute concentrations. For DNA and RNA, low concentrations can be amplified using the polymerase chain reaction, but there is no such reaction for proteins. Therefore, the development of single-molecule protein sequencing is a critical step in the search for protein biomarkers. Here, we show that single amino acids can be identified by trapping the molecules between two electrodes that are coated with a layer of recognition molecules, then measuring the electron tunnelling current across the junction. A given molecule can bind in more than one way in the junction, and we therefore use a machine-learning algorithm to distinguish between the sets of electronic `fingerprints' associated with each binding motif. With this recognition tunnelling technique, we are able to identify D and L enantiomers, a methylated amino acid, isobaric isomers and short peptides. The results suggest that direct electronic sequencing of single proteins could be possible by sequentially measuring the products of processive exopeptidase digestion, or by using a molecular motor to pull proteins through a tunnel junction integrated with a nanopore.

  9. Single-molecule positioning in zeromode waveguides by DNA origami nanoadapters.

    PubMed

    Pibiri, Enrico; Holzmeister, Phil; Lalkens, Birka; Acuna, Guillermo P; Tinnefeld, Philip

    2014-06-11

    Nanotechnology is challenged by the need to connect top-down produced nanostructures with the bottom-up world of chemistry. A nanobiotechnological prime example is the positioning of single polymerase molecules in small holes in metal films, so-called zeromode waveguides (ZMWs), which is required for single-molecule real-time DNA sequencing. In this work, we present nanoadapters made of DNA (DNA origami) that match the size of the holes so that exactly one nanoadapter fits in each hole. By site-selective functionalization of the DNA origami nanoadapters, we placed single dye molecules in the ZMWs, thus optimizing the hole usage and improving the photophysical properties of dyes compared to stochastically immobilized molecules.

  10. Amino Acid Racemization and the Preservation of Ancient DNA

    NASA Technical Reports Server (NTRS)

    Poinar, Hendrik N.; Hoss, Matthias

    1996-01-01

    The extent of racemization of aspartic acid, alanine, and leucine provides criteria for assessing whether ancient tissue samples contain endogenous DNA. In samples in which the D/L ratio of aspartic acid exceeds 0.08, ancient DNA sequences could not be retrieved. Paleontological finds from which DNA sequences purportedly millions of years old have been reported show extensive racemization, and the amino acids present are mainly contaminates. An exception is the amino acids in some insects preserved in amber.

  11. Ultrasensitive nucleic acid sequence detection by single-molecule electrophoresis

    SciTech Connect

    Castro, A; Shera, E.B.

    1996-09-01

    This is the final report of a one-year laboratory-directed research and development project at Los Alamos National Laboratory. There has been considerable interest in the development of very sensitive clinical diagnostic techniques over the last few years. Many pathogenic agents are often present in extremely small concentrations in clinical samples, especially at the initial stages of infection, making their detection very difficult. This project sought to develop a new technique for the detection and accurate quantification of specific bacterial and viral nucleic acid sequences in clinical samples. The scheme involved the use of novel hybridization probes for the detection of nucleic acids combined with our recently developed technique of single-molecule electrophoresis. This project is directly relevant to the DOE`s Defense Programs strategic directions in the area of biological warfare counter-proliferation.

  12. [Superparamagnetic Cobalt Ferrite Nanoparticles "Blow up" Spatial Ordering of Double-stranded DNA Molecules].

    PubMed

    Yevdokimov, Yu M; Pershina, A G; Salyanov, V I; Magaeva, A A; Popenko, V I; Shtykova, E V; Dadinova, L A; Skuridin, S G

    2015-01-01

    The formation of cholesteric liquid-crystalline dispersions formed by double-stranded DNA molecules, handled by positively charged superparamagnetic cobalt ferrite nanoparticles, as well as action of these nanoparticles on DNA dispersion, are considered. The binding of magnetic nanoparticles to the linear double-stranded DNA in solution of high ionic strength (0.3 M NaCl) and subsequent phase exclusion of these complexes from polyethylene glycol-containing solutions lead to their inability to form dispersions, whose particles do possess the spatially twisted arrangement of neighboring double-stranded DNA molecules. The action of magnetic nanoparticles on DNA dispersion (one magnetic nanoparticle per one double-stranded DNA molecule) results in such "perturbation" of DNA structure at sites of magnetic nanoparticles binding that the regular spatial structure of DNA dispersion particles "blows up"; this process is accompanied by disappearance of both abnormal optical activity and characteristic Bragg maximum on the small-angle X-ray scattering curve. Allowing with the fact that the physicochemical properties of the DNA liquid-crystalline dispersion particles reflect features of spatial organization of these molecules in chromosomes of primitive organisms, it is possible, that the found effect can have the relevant biological consequences.

  13. Recent advances in small organic molecules as DNA intercalating agents: synthesis, activity, and modeling.

    PubMed

    Rescifina, Antonio; Zagni, Chiara; Varrica, Maria Giulia; Pistarà, Venerando; Corsaro, Antonino

    2014-03-03

    The interaction of small molecules with DNA plays an essential role in many biological processes. As DNA is often the target for majority of anticancer and antibiotic drugs, study about the interaction of drug and DNA has a key role in pharmacology. Moreover, understanding the interactions of small molecules with DNA is of prime significance in the rational design of more powerful and selective anticancer agents. Two of the most important and promising targets in cancer chemotherapy include DNA alkylating agents and DNA intercalators. For these last the DNA recognition is a critical step in their anti-tumor action and the intercalation is not only one kind of the interactions in DNA recognition but also a pivotal step of several clinically used anti-tumor drugs such as anthracyclines, acridines and anthraquinones. To push clinical cancer therapy, the discovery of new DNA intercalators has been considered a practical approach and a number of intercalators have been recently reported. The intercalative binding properties of such molecules can also be harnessed as diagnostic probes for DNA structure in addition to DNA-directed therapeutics. Moreover, the problem of intercalation site formation in the undistorted B-DNA of different length and sequence is matter of tremendous importance in molecular modeling studies and, nowadays, three models of DNA intercalation targets have been proposed that account for the binding features of intercalators. Finally, despite DNA being an important target for several drugs, most of the docking programs are validated only for proteins and their ligands. Therefore, a default protocol to identify DNA binding modes which uses a modified canonical DNA as receptor is needed.

  14. Single molecule FRET shows uniformity in TBP-induced DNA bending and heterogeneity in bending kinetics†

    PubMed Central

    Blair, Rebecca H.; Goodrich, James A.; Kugel, Jennifer F.

    2012-01-01

    TATA binding protein (TBP) is a key component of the eukaryotic RNA polymerase II (Pol II) transcription machinery that binds to TATA boxes located in the core promoter regions of many genes. Structural and biochemical studies have shown that when TBP binds DNA, it sharply bends the DNA. We used single-molecule FRET (smFRET) to study DNA bending by human TBP on consensus and mutant TATA boxes in the absence and presence of TFIIA. We found that the state of the bent DNA within populations of TBP/DNA complexes is homogeneous; partially bent intermediates were not observed. In contrast to previous ensemble studies, TBP was found to bend a mutant TATA box to the same extent as the consensus TATA box. Moreover, in the presence of TFIIA the extent of DNA bending was not significantly changed, although TFIIA did increase the fraction of DNA molecules bound by TBP. Analysis of the kinetics of DNA bending and unbending revealed that on the consensus TATA box two kinetically distinct populations of TBP/DNA complexes exist, however, the bent state of the DNA is the same in the two populations. Our smFRET studies reveal that human TBP bends DNA in a largely uniform manner under a variety of different conditions, which was unexpected given previous ensemble biochemical studies. Our new observations lead to us to revise the model for the mechanism of DNA binding by TBP and for how DNA bending is affected by TATA sequence and TFIIA. PMID:22934924

  15. Like-charge attraction and opposite-charge decomplexation between polymers and DNA molecules

    NASA Astrophysics Data System (ADS)

    Buyukdagli, Sahin

    2017-02-01

    We scrutinize the effect of polyvalent ions on polymer-DNA interactions. We extend a recently developed test-charge theory [S. Buyukdagli et al., Phys. Rev. E 94, 042502 (2016), 10.1103/PhysRevE.94.042502] to the case of a stiff polymer interacting with a DNA molecule in an electrolyte mixture. The theory accounts for one-loop level electrostatic correlation effects such as the ionic cloud deformation around the strongly charged DNA molecule as well as image-charge forces induced by the low DNA permittivity. Our model can reproduce and explain various characteristics of the experimental phase diagrams for polymer solutions. First, the addition of polyvalent cations to the electrolyte solution results in the attraction of the negatively charged polymer by the DNA molecule. The glue of the like-charge attraction is the enhanced shielding of the polymer charges by the dense counterion layer at the DNA surface. Second, through the shielding of the DNA-induced electrostatic potential, mono- and polyvalent cations of large concentration both suppress the like-charge attraction. Within the same formalism, we also predict a new opposite-charge repulsion effect between the DNA molecule and a positively charged polymer. In the presence of polyvalent anions such as sulfate or phosphate, their repulsion by the DNA charges leads to the charge screening deficiency of the region around the DNA molecule. This translates into a repulsive force that results in the decomplexation of the polymer from DNA. This opposite-charge repulsion phenomenon can be verified by current experiments and the underlying mechanism can be beneficial to gene therapeutic applications where the control over polymer-DNA interactions is the key factor.

  16. Probing Nucleosome Remodeling by Unzipping Single DNA Molecules

    NASA Astrophysics Data System (ADS)

    Wang, Michelle

    2006-03-01

    At the core of eukaryotic chromatin is the nucleosome, which consists of 147 bp of DNA wrapped 1.65 turns around an octamer of histone proteins. Even this lowest level of genomic compaction presents a strong barrier to DNA-binding cellular factors that are required for essential processes such as transcription, DNA replication, recombination and repair. Chromatin remodeling enzymes use the energy of ATP hydrolysis to regulate accessibility of the genetic code by altering chromatin structure. While remodeling enzymes have been the subject of extensive research in recent years, their precise mechanism remains unclear. In order to probe the structure of individual nucleosomes and their remodeling, we assembled a histone octamer onto a DNA segment containing a strong nucleosome positioning sequence. As the DNA double helix was unzipped through the nucleosome using a feedback-enhanced optical trap, the presence of the nucleosome was detected as a series of dramatic increases in the tension in the DNA, followed by sudden tension reductions. Analysis of the unzipping force throughout the disruption accurately revealed the spatial location and fine structure of the nucleosome to near base pair precision. Using this approach, we investigate how remodeling enzymes may alter the location and structure of a nucleosome.

  17. Selective enrichment of damaged DNA molecules for ancient genome sequencing

    PubMed Central

    2014-01-01

    Contamination by present-day human and microbial DNA is one of the major hindrances for large-scale genomic studies using ancient biological material. We describe a new molecular method, U selection, which exploits one of the most distinctive features of ancient DNA—the presence of deoxyuracils—for selective enrichment of endogenous DNA against a complex background of contamination during DNA library preparation. By applying the method to Neanderthal DNA extracts that are heavily contaminated with present-day human DNA, we show that the fraction of useful sequence information increases ∼10-fold and that the resulting sequences are more efficiently depleted of human contamination than when using purely computational approaches. Furthermore, we show that U selection can lead to a four- to fivefold increase in the proportion of endogenous DNA sequences relative to those of microbial contaminants in some samples. U selection may thus help to lower the costs for ancient genome sequencing of nonhuman samples also. PMID:25081630

  18. Topological events in single molecules of E. coli DNA confined in nanochannels.

    PubMed

    Reifenberger, Jeffrey G; Dorfman, Kevin D; Cao, Han

    2015-07-21

    We present experimental data concerning potential topological events such as folds, internal backfolds, and/or knots within long molecules of double-stranded DNA when they are stretched by confinement in a nanochannel. Genomic DNA from E. coli was labeled near the 'GCTCTTC' sequence with a fluorescently labeled dUTP analog and stained with the DNA intercalator YOYO. Individual long molecules of DNA were then linearized and imaged using methods based on the NanoChannel Array technology (Irys® System) available from BioNano Genomics. Data were collected on 189 153 molecules of length greater than 50 kilobases. A custom code was developed to search for abnormal intensity spikes in the YOYO backbone profile along the length of individual molecules. By correlating the YOYO intensity spikes with the aligned barcode pattern to the reference, we were able to correlate the bright intensity regions of YOYO with abnormal stretching in the molecule, which suggests these events were either a knot or a region of internal backfolding within the DNA. We interpret the results of our experiments involving molecules exceeding 50 kilobases in the context of existing simulation data for relatively short DNA, typically several kilobases. The frequency of these events is lower than the predictions from simulations, while the size of the events is larger than simulation predictions and often exceeds the molecular weight of the simulated molecules. We also identified DNA molecules that exhibit large, single folds as they enter the nanochannels. Overall, topological events occur at a low frequency (∼7% of all molecules) and pose an easily surmountable obstacle for the practice of genome mapping in nanochannels.

  19. DNA molecules sticking on a vicinal Si(111) surface observed by noncontact atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Arai, Toyoko; Tomitori, Masahiko; Saito, Masato; Tamiya, Eiichi

    2002-03-01

    The DNA molecules on a vicinal Si(111) substrate with steps of single and double bi-atomic layers are imaged by noncontact atomic force microscopy (nc-AFM) in ultrahigh vacuum. The water solution containing pBR322 plasmid DNA molecules digested by Cla I is dropped on the substrate in a pure nitrogen atmosphere in a glove box, which is connected to the introduction chamber of the AFM. The ends of DNA molecules are frequently folded and pinned at the steps on the substrate, and the DNA strings often lie along the step. The chemical and dipole interactions between the DNA and the semiconductor substrate seem to play an important role in folding, pinning and sticking on the Si(111) substrate.

  20. Effects of Tilt Angle, DNA Concentration, and Surface Potential on Directed Alignment of DNA Molecule for the Application to Nanodevices

    NASA Astrophysics Data System (ADS)

    Kim, Hyung Jin; Hong, Byungyou

    2013-03-01

    This paper reports an efficient approach to control both the density and direction of highly aligned DNA molecules and thus DNA-templated gold nanowires (AuNWs) on Si chips. We utilized tilting method to prepare stretched DNA structures on SiO2/Si substrate and found important parameters in the alignment process that tilt angle, DNA concentration, and surface potential are controlled the density and structure of DNA aligned on the surface. In additional, we also can be directly connected DNA-templated AuNWs between two terminal electrodes on Si chips. This method also describes a simple way to form singled, bundled and networked DNA arrays on Si substrates.

  1. Mechanisms of small molecule–DNA interactions probed by single-molecule force spectroscopy

    PubMed Central

    Almaqwashi, Ali A.; Paramanathan, Thayaparan; Rouzina, Ioulia; Williams, Mark C.

    2016-01-01

    There is a wide range of applications for non-covalent DNA binding ligands, and optimization of such interactions requires detailed understanding of the binding mechanisms. One important class of these ligands is that of intercalators, which bind DNA by inserting aromatic moieties between adjacent DNA base pairs. Characterizing the dynamic and equilibrium aspects of DNA-intercalator complex assembly may allow optimization of DNA binding for specific functions. Single-molecule force spectroscopy studies have recently revealed new details about the molecular mechanisms governing DNA intercalation. These studies can provide the binding kinetics and affinity as well as determining the magnitude of the double helix structural deformations during the dynamic assembly of DNA–ligand complexes. These results may in turn guide the rational design of intercalators synthesized for DNA-targeted drugs, optical probes, or integrated biological self-assembly processes. Herein, we survey the progress in experimental methods as well as the corresponding analysis framework for understanding single molecule DNA binding mechanisms. We discuss briefly minor and major groove binding ligands, and then focus on intercalators, which have been probed extensively with these methods. Conventional mono-intercalators and bis-intercalators are discussed, followed by unconventional DNA intercalation. We then consider the prospects for using these methods in optimizing conventional and unconventional DNA-intercalating small molecules. PMID:27085806

  2. Electronic density of states in sequence dependent DNA molecules

    NASA Astrophysics Data System (ADS)

    de Oliveira, B. P. W.; Albuquerque, E. L.; Vasconcelos, M. S.

    2006-09-01

    We report in this work a numerical study of the electronic density of states (DOS) in π-stacked arrays of DNA single-strand segments made up from the nucleotides guanine G, adenine A, cytosine C and thymine T, forming a Rudin-Shapiro (RS) as well as a Fibonacci (FB) polyGC quasiperiodic sequences. Both structures are constructed starting from a G nucleotide as seed and following their respective inflation rules. Our theoretical method uses Dyson's equation together with a transfer-matrix treatment, within an electronic tight-binding Hamiltonian model, suitable to describe the DNA segments modelled by the quasiperiodic chains. We compared the DOS spectra found for the quasiperiodic structure to those using a sequence of natural DNA, as part of the human chromosome Ch22, with a remarkable concordance, as far as the RS structure is concerned. The electronic spectrum shows several peaks, corresponding to localized states, as well as a striking self-similar aspect.

  3. Interactions of DNA binding proteins with G-Quadruplex structures at the single molecule level

    NASA Astrophysics Data System (ADS)

    Ray, Sujay

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

  4. Monitoring patterned enzymatic polymerization on DNA origami at single-molecule level

    NASA Astrophysics Data System (ADS)

    Okholm, A. H.; Aslan, H.; Besenbacher, F.; Dong, M.; Kjems, J.

    2015-06-01

    DNA origami has been used to orchestrate reactions with nano-precision using a variety of biomolecules. Here, the dynamics of albumin-assisted, localized single-molecule DNA polymerization by terminal deoxynucleotidyl transferase on a 2D DNA origami are monitored using AFM in liquid. Direct visualization of the surface activity revealed the mechanics of growth.DNA origami has been used to orchestrate reactions with nano-precision using a variety of biomolecules. Here, the dynamics of albumin-assisted, localized single-molecule DNA polymerization by terminal deoxynucleotidyl transferase on a 2D DNA origami are monitored using AFM in liquid. Direct visualization of the surface activity revealed the mechanics of growth. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr01945a

  5. Enzymatic Digestion of Single DNA Molecules Anchored on Nanogold-Modified Surfaces

    NASA Astrophysics Data System (ADS)

    Lü, Junhong; Ye, Ming; Duan, Na; Li, Bin

    2009-09-01

    To study enzyme-DNA interactions at single molecular level, both the attachment points and the immediate surroundings of surfaces must be carefully considered such that they do not compromise the structural information and biological properties of the sample under investigation. The present work demonstrates the feasibility of enzymatic digestion of single DNA molecules attached to nanoparticle-modified surfaces. With Nanogold linking DNA to the mica surface by electrostatic interactions, advantageous conditions with fewer effects on the length and topography of DNA are obtained, and an appropriate environment for the activities of DNA is created. We demonstrate that by using Dip-Pen Nanolithography, individual DNA molecules attached to modified mica surfaces can be efficiently digested by DNase I.

  6. Nanomechanical DNA origami 'single-molecule beacons' directly imaged by atomic force microscopy

    PubMed Central

    Kuzuya, Akinori; Sakai, Yusuke; Yamazaki, Takahiro; Xu, Yan; Komiyama, Makoto

    2011-01-01

    DNA origami involves the folding of long single-stranded DNA into designed structures with the aid of short staple strands; such structures may enable the development of useful nanomechanical DNA devices. Here we develop versatile sensing systems for a variety of chemical and biological targets at molecular resolution. We have designed functional nanomechanical DNA origami devices that can be used as 'single-molecule beacons', and function as pinching devices. Using 'DNA origami pliers' and 'DNA origami forceps', which consist of two levers ~170 nm long connected at a fulcrum, various single-molecule inorganic and organic targets ranging from metal ions to proteins can be visually detected using atomic force microscopy by a shape transition of the origami devices. Any detection mechanism suitable for the target of interest, pinching, zipping or unzipping, can be chosen and used orthogonally with differently shaped origami devices in the same mixture using a single platform. PMID:21863016

  7. High Electronic Conductance through Double-Helix DNA Molecules with Fullerene Anchoring Groups

    PubMed Central

    2017-01-01

    Determining the mechanism of charge transport through native DNA remains a challenge as different factors such as measuring conditions, molecule conformations, and choice of technique can significantly affect the final results. In this contribution, we have used a new approach to measure current flowing through isolated double-stranded DNA molecules, using fullerene groups to anchor the DNA to a gold substrate. Measurements were performed at room temperature in an inert environment using a conductive AFM technique. It is shown that the π-stacked B-DNA structure is conserved on depositing the DNA. As a result, currents in the nanoampere range were obtained for voltages ranging between ±1 V. These experimental results are supported by a theoretical model that suggests that a multistep hopping mechanism between delocalized domains is responsible for the long-range current flow through this specific type of DNA. PMID:28094940

  8. A 3D-DNA Molecule Made of PlayMais

    ERIC Educational Resources Information Center

    Caine, Massimo; Horié, Ninon; Zuchuat, Sandrine; Weber, Aurélia; Ducret, Verena; Linder, Patrick; Perron, Karl

    2015-01-01

    More than 60 years have passed since the work of Rosalind Franklin, James Watson, and Francis Crick led to the discovery of the 3D-DNA double-helix structure. Nowadays, due to the simple and elegant architecture of its double helix, the structure of DNA is widely known. The biological role of the DNA molecule (e.g., genetic information), however,…

  9. Endogenous Molecules Stimulating N-Acylethanolamine-Hydrolyzing Acid Amidase (NAAA)

    PubMed Central

    2012-01-01

    Fatty acid amide hydrolase (FAAH) plays the central role in the degradation of bioactive N-acylethanolamines such as the endocannabinoid arachidonoylethanolamide (anandamide) in brain and peripheral tissues. A lysosomal enzyme referred to as N-acylethanolamine-hydrolyzing acid amidase (NAAA) catalyzes the same reaction with preference to palmitoylethanolamide, an endogenous analgesic and neuroprotective substance, and is therefore expected as a potential target of therapeutic drugs. In the in vitro assays thus far performed, the maximal activity of NAAA was achieved in the presence of both nonionic detergent (Triton X-100 or Nonidet P-40) and the SH reagent dithiothreitol. However, endogenous molecules that might substitute for these synthetic compounds remain poorly understood. Here, we examined stimulatory effects of endogenous phospholipids and thiol compounds on recombinant NAAA. Among different phospholipids tested, choline- or ethanolamine-containing phospholipids showed potent effects, and 1 mM phosphatidylcholine increased NAAA activity by 6.6-fold. Concerning endogenous thiol compounds, dihydrolipoic acid at 0.1–1 mM was the most active, causing 8.5–9.0-fold stimulation. These results suggest that endogenous phospholipids and dihydrolipoic acid may contribute in keeping NAAA active in lysosomes. Even in the presence of phosphatidylcholine and dihydrolipoic acid, however, the preferential hydrolysis of palmitoylethanolamide was unaltered. We also investigated a possible compensatory induction of NAAA mRNA in brain and other tissues of FAAH-deficient mice. However, NAAA expression levels in all the tissues examined were not significantly altered from those in wild-type mice. PMID:22860206

  10. Molecular Dynamics Simulation of Multivalent-Ion Mediated Attraction between DNA Molecules

    NASA Astrophysics Data System (ADS)

    Dai, Liang; Mu, Yuguang; Nordenskiöld, Lars; van der Maarel, Johan R. C.

    2008-03-01

    All atom molecular dynamics simulations with explicit water were done to study the interaction between two parallel double-stranded DNA molecules in the presence of the multivalent counterions putrescine (2+), spermidine (3+), spermine (4+) and cobalt hexamine (3+). The inter-DNA interaction potential is obtained with the umbrella sampling technique. The attractive force is rationalized in terms of the formation of ion bridges, i.e., multivalent ions which are simultaneously bound to the two opposing DNA molecules. The lifetime of the ion bridges is short on the order of a few nanoseconds.

  11. Understanding the physics of DNA using nanoscale single-molecule manipulation

    NASA Astrophysics Data System (ADS)

    Frey, Eric W.; Gooding, Ashton A.; Wijeratne, Sitara; Kiang, Ching-Hwa

    2012-10-01

    Processes for decoding the genetic information in cells, including transcription, replication, recombination and repair, involve the deformation of DNA from its equilibrium structures such as bending, stretching, twisting, and unzipping of the double helix. Single-molecule manipulation techniques have made it possible to control DNA conformation and simultaneously detect the induced changes, revealing a rich variety of mechanically-induced conformational changes and thermodynamic states. These single-molecule techniques helped us to reveal the physics of DNA and the processes involved in the passing on of the genetic code.

  12. Molecular characterization of a new begomovirus infecting Sida cordifolia and its associated satellite DNA molecules.

    PubMed

    Guo, Xiaojian; Zhou, Xueping

    2006-12-01

    Two virus isolates Hn57 and Hn60 were obtained from Sida cordifolia showing mild upward leaf-curling symptoms in Hainan province of China. Comparison of partial sequences of DNA-A like molecule confirmed the existence of a single type of begomovirus. The complete nucleotide sequence of DNA-A of Hn57 was determined to be 2757 nucleotides, with a genomic organization typical of begomoviruses. Complete sequence comparison with other reported begomoviruses revealed that Hn57 DNA-A has the highest sequence identity (71.0%) with that of Tobacco leaf curl Yunnan virus. Consequently, Hn57 was considered to be a new begomovirus species, for which the name Sida leaf curl virus (SiLCV) is proposed. In addition to DNA-A molecule, two additional circular single-stranded satellite DNA molecules corresponding to DNAbeta and DNA1 were found to be associated with SiLCV isolates. Both DNAbeta and DNA1 were approximately half the size of their cognate genomic DNA. Sequence analysis shows that DNAbeta of Hn57 and Hn60 share 93.8% nucleotide sequence identity, and they have the highest sequence identity (58.5%) with DNAbeta associated with Ageratum leaf curl disease (AJ316027). The nucleotide sequence identity between DNA1 of Hn57 and that of Hn60 was 83.8%, they share 58.2-79.3% nucleotide sequence identities in comparison with other previously reported DNAl.

  13. Single-molecule studies reveal reciprocating of WRN helicase core along ssDNA during DNA unwinding.

    PubMed

    Wu, Wen-Qiang; Hou, Xi-Miao; Zhang, Bo; Fossé, Philippe; René, Brigitte; Mauffret, Olivier; Li, Ming; Dou, Shuo-Xing; Xi, Xu-Guang

    2017-03-07

    Werner syndrome is caused by mutations in the WRN gene encoding WRN helicase. A knowledge of WRN helicase's DNA unwinding mechanism in vitro is helpful for predicting its behaviors in vivo, and then understanding their biological functions. In the present study, for deeply understanding the DNA unwinding mechanism of WRN, we comprehensively characterized the DNA unwinding properties of chicken WRN helicase core in details, by taking advantages of single-molecule fluorescence resonance energy transfer (smFRET) method. We showed that WRN exhibits repetitive DNA unwinding and translocation behaviors on different DNA structures, including forked, overhanging and G-quadruplex-containing DNAs with an apparently limited unwinding processivity. It was further revealed that the repetitive behaviors were caused by reciprocating of WRN along the same ssDNA, rather than by complete dissociation from and rebinding to substrates or by strand switching. The present study sheds new light on the mechanism for WRN functioning.

  14. Single-molecule studies reveal reciprocating of WRN helicase core along ssDNA during DNA unwinding

    PubMed Central

    Wu, Wen-Qiang; Hou, Xi-Miao; Zhang, Bo; Fossé, Philippe; René, Brigitte; Mauffret, Olivier; Li, Ming; Dou, Shuo-Xing; Xi, Xu-Guang

    2017-01-01

    Werner syndrome is caused by mutations in the WRN gene encoding WRN helicase. A knowledge of WRN helicase’s DNA unwinding mechanism in vitro is helpful for predicting its behaviors in vivo, and then understanding their biological functions. In the present study, for deeply understanding the DNA unwinding mechanism of WRN, we comprehensively characterized the DNA unwinding properties of chicken WRN helicase core in details, by taking advantages of single-molecule fluorescence resonance energy transfer (smFRET) method. We showed that WRN exhibits repetitive DNA unwinding and translocation behaviors on different DNA structures, including forked, overhanging and G-quadruplex-containing DNAs with an apparently limited unwinding processivity. It was further revealed that the repetitive behaviors were caused by reciprocating of WRN along the same ssDNA, rather than by complete dissociation from and rebinding to substrates or by strand switching. The present study sheds new light on the mechanism for WRN functioning. PMID:28266653

  15. Nucleic acid chemistry in the organic phase: from functionalized oligonucleotides to DNA side chain polymers.

    PubMed

    Liu, Kai; Zheng, Lifei; Liu, Qing; de Vries, Jan Willem; Gerasimov, Jennifer Y; Herrmann, Andreas

    2014-10-08

    DNA-incorporating hydrophobic moieties can be synthesized by either solid-phase or solution-phase coupling. On a solid support the DNA is protected, and hydrophobic units are usually attached employing phosphoramidite chemistry involving a DNA synthesizer. On the other hand, solution coupling in aqueous medium results in low yields due to the solvent incompatibility of DNA and hydrophobic compounds. Hence, the development of a general coupling method for producing amphiphilic DNA conjugates with high yield in solution remains a major challenge. Here, we report an organic-phase coupling strategy for nucleic acid modification and polymerization by introducing a hydrophobic DNA-surfactant complex as a reactive scaffold. A remarkable range of amphiphile-DNA structures (DNA-pyrene, DNA-triphenylphosphine, DNA-hydrocarbon, and DNA block copolymers) and a series of new brush-type DNA side-chain homopolymers with high DNA grafting density are produced efficiently. We believe that this method is an important breakthrough in developing a generalized approach to synthesizing functional DNA molecules for self-assembly and related technological applications.

  16. DNA compaction by the bacteriophage protein Cox studied on the single DNA molecule level using nanofluidic channels.

    PubMed

    Frykholm, Karolin; Berntsson, Ronnie Per-Arne; Claesson, Magnus; de Battice, Laura; Odegrip, Richard; Stenmark, Pål; Westerlund, Fredrik

    2016-09-06

    The Cox protein from bacteriophage P2 forms oligomeric filaments and it has been proposed that DNA can be wound up around these filaments, similar to how histones condense DNA. We here use fluorescence microscopy to study single DNA-Cox complexes in nanofluidic channels and compare how the Cox homologs from phages P2 and WΦ affect DNA. By measuring the extension of nanoconfined DNA in absence and presence of Cox we show that the protein compacts DNA and that the binding is highly cooperative, in agreement with the model of a Cox filament around which DNA is wrapped. Furthermore, comparing microscopy images for the wild-type P2 Cox protein and two mutants allows us to discriminate between compaction due to filament formation and compaction by monomeric Cox. P2 and WΦ Cox have similar effects on the physical properties of DNA and the subtle, but significant, differences in DNA binding are due to differences in binding affinity rather than binding mode. The presented work highlights the use of single DNA molecule studies to confirm structural predictions from X-ray crystallography. It also shows how a small protein by oligomerization can have great impact on the organization of DNA and thereby fulfill multiple regulatory functions.

  17. Nanomechanical recognition measurements of individual DNA molecules reveal epigenetic methylation patterns

    NASA Astrophysics Data System (ADS)

    Zhu, Rong; Howorka, Stefan; Pröll, Johannes; Kienberger, Ferry; Preiner, Johannes; Hesse, Jan; Ebner, Andreas; Pastushenko, Vassili Ph.; Gruber, Hermann J.; Hinterdorfer, Peter

    2010-11-01

    Atomic force microscopy (AFM) is a powerful tool for analysing the shapes of individual molecules and the forces acting on them. AFM-based force spectroscopy provides insights into the structural and energetic dynamics of biomolecules by probing the interactions within individual molecules, or between a surface-bound molecule and a cantilever that carries a complementary binding partner. Here, we show that an AFM cantilever with an antibody tether can measure the distances between 5-methylcytidine bases in individual DNA strands with a resolution of 4 Å, thereby revealing the DNA methylation pattern, which has an important role in the epigenetic control of gene expression. The antibody is able to bind two 5-methylcytidine bases of a surface-immobilized DNA strand, and retracting the cantilever results in a unique rupture signature reflecting the spacing between two tagged bases. This nanomechanical approach might also allow related chemical patterns to be retrieved from biopolymers at the single-molecule level.

  18. Trapping of a single DNA molecule using nanoplasmonic structures for biosensor applications

    PubMed Central

    Kim, Jung-Dae; Lee, Yong-Gu

    2014-01-01

    Conventional optical trapping using a tightly focused beam is not suitable for trapping particles that are smaller than the diffraction limit because of the increasing need of the incident laser power that could produce permanent thermal damages. One of the current solutions to this problem is to intensify the local field enhancement by using nanoplasmonic structures without increasing the laser power. Nanoplasmonic tweezers have been used for various small molecules but there is no known report of trapping a single DNA molecule. In this paper, we present the trapping of a single DNA molecule using a nanohole created on a gold substrate. Furthermore, we show that the DNA of different lengths can be differentiated through the measurement of scattering signals leading to possible new DNA sensor applications. PMID:25136478

  19. Assembly of recombinant nucleosomes on nanofabricated DNA curtains for single-molecule imaging.

    PubMed

    Lee, Ja Yil; Greene, Eric C

    2011-01-01

    Eukaryotic chromosomes are highly packed into chromatin, the basic unit of which is the nucleosome. The presence of nucleosomes and the resulting organization of the genome into higher-order chromatin structures has profound consequences for virtually all aspects of DNA metabolism, including DNA transcription, repair, and chromosome segregation. We have developed novel approaches for nanofabricating "DNA curtains" for high-throughput single-molecule imaging, and we have begun adapting these new research tools in an effort to begin studying chromatin biology at the single-molecule level. In this protocol, we describe procedures for assembly and real-time single-molecule visualization of DNA curtains bound by reconstituted nucleosomes made from recombinant histones.

  20. Replacing the Nucleobases in DNA with Designer Molecules

    DTIC Science & Technology

    2007-11-02

    electrophiles or especially strong nucleophiles, and they generally do not change protonation near neutral pH. They offer (to the first approximation) only four...DNA bases can impart important biological activity, such as replacing the methyl group of thymine with fluorine , or the oxygen of guanine with sulfur...nucleobases that lack hydrogen- bonding functionality. The design involves replacing oxygen with fluorine and nitrogen with carbon, and keeping aromaticity

  1. Escape of a knot from a DNA molecule in flow

    NASA Astrophysics Data System (ADS)

    Renner, Benjamin; Doyle, Patrick

    2014-03-01

    Macroscale knots are an everyday occurrence when trying to unravel an unorganized flexible string (e.g. an iPhone cord taken out of your pocket). In nature, knots are found in proteins and viral capsid DNA, and the properties imbued by their topologies are thought to have biological significance. Unlike their macroscale counterparts, thermal fluctuations greatly influence the dynamics of polymer knots. Here, we use Brownian Dynamics simulations to study knot diffusion along a linear polymer chain. The model is parameterized to dsDNA, a model polymer used in previous simulation and experimental studies of knot dynamics. We have used this model to study the process of knot escape and transport along a dsDNA strand extended by an elongational flow. For a range of knot topologies and flow strengths, we show scalings that result in collapse of the data onto a master curve. We show a topologically mediated mode of transport coincides with observed differences in rates of knot transport, and we provide a simple mechanistic explanation for its effect. We anticipate these results will build on the growing body of fundamental studies of knotted polymers and inform future experimental study. This work is supported by the Singapore-MIT Alliance for Research and Technology (SMART) and National Science Foundation (NSF) grant CBET-0852235.

  2. DNA molecule provides a computing machine with both data and fuel.

    PubMed

    Benenson, Yaakov; Adar, Rivka; Paz-Elizur, Tamar; Livneh, Zvi; Shapiro, Ehud

    2003-03-04

    The unique properties of DNA make it a fundamental building block in the fields of supramolecular chemistry, nanotechnology, nano-circuits, molecular switches, molecular devices, and molecular computing. In our recently introduced autonomous molecular automaton, DNA molecules serve as input, output, and software, and the hardware consists of DNA restriction and ligation enzymes using ATP as fuel. In addition to information, DNA stores energy, available on hybridization of complementary strands or hydrolysis of its phosphodiester backbone. Here we show that a single DNA molecule can provide both the input data and all of the necessary fuel for a molecular automaton. Each computational step of the automaton consists of a reversible software molecule input molecule hybridization followed by an irreversible software-directed cleavage of the input molecule, which drives the computation forward by increasing entropy and releasing heat. The cleavage uses a hitherto unknown capability of the restriction enzyme FokI, which serves as the hardware, to operate on a noncovalent software input hybrid. In the previous automaton, software input ligation consumed one software molecule and two ATP molecules per step. As ligation is not performed in this automaton, a fixed amount of software and hardware molecules can, in principle, process any input molecule of any length without external energy supply. Our experiments demonstrate 3 x 10(12) automata per microl performing 6.6 x 10(10) transitions per second per microl with transition fidelity of 99.9%, dissipating about 5 x 10(-9) W microl as heat at ambient temperature.

  3. The conductive properties of single DNA molecules studied by torsion tunneling atomic force microscopy.

    PubMed

    Wang, W; Niu, D X; Jiang, C R; Yang, X J

    2014-01-17

    The conductive properties of single natural λ-DNA molecules are studied by torsion tunneling atomic force microscopy (TR-TUNA). The currents both parallel to and perpendicular to the DNA chains are investigated, but only weak or even no current signals are detected by TR-TUNA. To improve the conductance of DNA molecules, silver and copper metallized DNAs are fabricated and their conductivities are checked by TR-TUNA. It is found that for both Cu- and Ag-DNAs, the conductivity perpendicular to the DNA chain is enhanced significantly as the metal clusters are attached to the DNA chains. But parallel to the chain the electrical transport is still weak, most probably due to the 'beads-on-a-string' constructions of metallized DNAs.

  4. The conductive properties of single DNA molecules studied by torsion tunneling atomic force microscopy

    NASA Astrophysics Data System (ADS)

    Wang, W.; Niu, D. X.; Jiang, C. R.; Yang, X. J.

    2014-01-01

    The conductive properties of single natural λ-DNA molecules are studied by torsion tunneling atomic force microscopy (TR-TUNA). The currents both parallel to and perpendicular to the DNA chains are investigated, but only weak or even no current signals are detected by TR-TUNA. To improve the conductance of DNA molecules, silver and copper metallized DNAs are fabricated and their conductivities are checked by TR-TUNA. It is found that for both Cu- and Ag-DNAs, the conductivity perpendicular to the DNA chain is enhanced significantly as the metal clusters are attached to the DNA chains. But parallel to the chain the electrical transport is still weak, most probably due to the ‘beads-on-a-string’ constructions of metallized DNAs.

  5. Laser microbeam - kinetic studies combined with molecule - structures reveal mechanisms of DNA repair

    NASA Astrophysics Data System (ADS)

    Altenberg, B.; Greulich, K. O.

    2011-10-01

    Kinetic studies on double strand DNA damages induced by a laser microbeam have allowed a precise definition of the temporal order of recruitment of repair molecules. The order is KU70 / KU80 - XRCC4 --NBS1 -- RAD51. These kinetic studies are now complemented by studies on molecular structures of the repair proteins, using the program YASARA which does not only give molecular structures but also physicochemical details on forces involved in binding processes. It turns out that the earliest of these repair proteins, the KU70 / KU80 heterodimer, has a hole with high DNA affinity. The next molecule, XRCC4, has a body with two arms, the latter with extremely high DNA affinity at their ends and a binding site for Ligase 4. Together with the laser microbeam results this provides a detailed view on the early steps of DNA double strand break repair. The sequence of DNA repair events is presented as a movie.

  6. Discovery of small-molecule interleukin-2 inhibitors from a DNA-encoded chemical library.

    PubMed

    Leimbacher, Markus; Zhang, Yixin; Mannocci, Luca; Stravs, Michael; Geppert, Tim; Scheuermann, Jörg; Schneider, Gisbert; Neri, Dario

    2012-06-18

    Libraries of chemical compounds individually coupled to encoding DNA tags (DNA-encoded chemical libraries) hold promise to facilitate exceptionally efficient ligand discovery. We constructed a high-quality DNA-encoded chemical library comprising 30,000 drug-like compounds; this was screened in 170 different affinity capture experiments. High-throughput sequencing allowed the evaluation of 120 million DNA codes for a systematic analysis of selection strategies and statistically robust identification of binding molecules. Selections performed against the tumor-associated antigen carbonic anhydrase IX (CA IX) and the pro-inflammatory cytokine interleukin-2 (IL-2) yielded potent inhibitors with exquisite target specificity. The binding mode of the revealed pharmacophore against IL-2 was confirmed by molecular docking. Our findings suggest that DNA-encoded chemical libraries allow the facile identification of drug-like ligands principally to any protein of choice, including molecules capable of disrupting high-affinity protein-protein interactions.

  7. Thermoelectric effect and its dependence on molecular length and sequence in single DNA molecules

    PubMed Central

    Li, Yueqi; Xiang, Limin; Palma, Julio L.; Asai, Yoshihiro; Tao, Nongjian

    2016-01-01

    Studying the thermoelectric effect in DNA is important for unravelling charge transport mechanisms and for developing relevant applications of DNA molecules. Here we report a study of the thermoelectric effect in single DNA molecules. By varying the molecular length and sequence, we tune the charge transport in DNA to either a hopping- or tunnelling-dominated regimes. The thermoelectric effect is small and insensitive to the molecular length in the hopping regime. In contrast, the thermoelectric effect is large and sensitive to the length in the tunnelling regime. These findings indicate that one may control the thermoelectric effect in DNA by varying its sequence and length. We describe the experimental results in terms of hopping and tunnelling charge transport models. PMID:27079152

  8. Guiding the design of synthetic DNA-binding molecules with massively parallel sequencing.

    PubMed

    Meier, Jordan L; Yu, Abigail S; Korf, Ian; Segal, David J; Dervan, Peter B

    2012-10-24

    Genomic applications of DNA-binding molecules require an unbiased knowledge of their high affinity sites. We report the high-throughput analysis of pyrrole-imidazole polyamide DNA-binding specificity in a 10(12)-member DNA sequence library using affinity purification coupled with massively parallel sequencing. We find that even within this broad context, the canonical pairing rules are remarkably predictive of polyamide DNA-binding specificity. However, this approach also allows identification of unanticipated high affinity DNA-binding sites in the reverse orientation for polyamides containing β/Im pairs. These insights allow the redesign of hairpin polyamides with different turn units capable of distinguishing 5'-WCGCGW-3' from 5'-WGCGCW-3'. Overall, this study displays the power of high-throughput methods to aid the optimal targeting of sequence-specific minor groove binding molecules, an essential underpinning for biological and nanotechnological applications.

  9. Condensations of single DNA molecules induced by heptaplatin and its chiral isomer

    SciTech Connect

    Zhang, Hong-Yan; Liu, Yu-Ru; Li, Wei; Li, Hui; Dou, Shuo-Xing; Xie, Ping; Wang, Wei-Chi; Wang, Peng-Ye

    2014-08-15

    Heptaplatin is a third-generation platinum antitumor drug. It has a chiral isomer. We studied the interactions between the two isomers and DNA by using magnetic tweezers and atomic force microscopy (AFM) to investigate the effect of chiralities of the isomers on the interactions. We found that the extension curves and average condensation rates of DNA molecules incubated with heptaplatin were nearly the same as those incubated with its chiral isomer. In addition, the structures of DNA molecules incubated with heptaplatin were also similar to those incubated with its chiral isomer. These results indicate the difference in chirality of the two isomers does not induce different interactions of the isomers with DNA. Our study may facilitate the understanding of interactions of platinum complexes with DNA and the design of new antitumor platinum complexes.

  10. Condensations of single DNA molecules induced by heptaplatin and its chiral isomer

    NASA Astrophysics Data System (ADS)

    Zhang, Hong-Yan; Liu, Yu-Ru; Li, Wei; Li, Hui; Dou, Shuo-Xing; Xie, Ping; Wang, Wei-Chi; Wang, Peng-Ye

    2014-08-01

    Heptaplatin is a third-generation platinum antitumor drug. It has a chiral isomer. We studied the interactions between the two isomers and DNA by using magnetic tweezers and atomic force microscopy (AFM) to investigate the effect of chiralities of the isomers on the interactions. We found that the extension curves and average condensation rates of DNA molecules incubated with heptaplatin were nearly the same as those incubated with its chiral isomer. In addition, the structures of DNA molecules incubated with heptaplatin were also similar to those incubated with its chiral isomer. These results indicate the difference in chirality of the two isomers does not induce different interactions of the isomers with DNA. Our study may facilitate the understanding of interactions of platinum complexes with DNA and the design of new antitumor platinum complexes.

  11. Discrimination of Single Base Pair Differences Among Individual DNA Molecules Using a Nanopore

    NASA Technical Reports Server (NTRS)

    Vercoutere, Wenonah; DeGuzman, Veronica

    2003-01-01

    The protein toxin alpha-hemolysin form nanometer scale channels across lipid membranes. Our lab uses a single channel in an artificial lipid bilayer in a patch clamp device to capture and examine individual DNA molecules. This nanopore detector used with a support vector machine (SVM) can analyze DNA hairpin molecules on the millisecond time scale. We distinguish duplex stem length, base pair mismatches, loop length, and single base pair differences. The residual current fluxes also reveal structural molecular dynamics elements. DNA end-fraying (terminal base pair dissociation) can be observed as near full blockades, or spikes, in current. This technique can be used to investigate other biological processes dependent on DNA end-fraying, such as the processing of HIV DNA by HIV integrase.

  12. Efficient vaccine against pandemic influenza: combining DNA vaccination and targeted delivery to MHC class II molecules.

    PubMed

    Grødeland, Gunnveig; Bogen, Bjarne

    2015-06-01

    There are two major limitations to vaccine preparedness in the event of devastating influenza pandemics: the time needed to generate a vaccine and rapid generation of sufficient amounts. DNA vaccination could represent a solution to these problems, but efficacy needs to be enhanced. In a separate line of research, it has been established that targeting of vaccine molecules to antigen-presenting cells enhances immune responses. We have combined the two principles by constructing DNA vaccines that encode bivalent fusion proteins; these target hemagglutinin to MHC class II molecules on antigen-presenting cells. Such DNA vaccines rapidly induce hemagglutinin-specific antibodies and T cell responses in immunized mice. Responses are long-lasting and protect mice against challenge with influenza virus. In a pandemic situation, targeted DNA vaccines could be produced and tested within a month. The novel DNA vaccines could represent a solution to pandemic preparedness in the advent of novel influenza pandemics.

  13. Single DNA molecule jamming and history-dependent dynamics during motor-driven viral packaging

    PubMed Central

    Keller, Nicholas; Grimes, Shelley; Jardine, Paul J.; Smith, Douglas E.

    2016-01-01

    In many viruses molecular motors forcibly pack single DNA molecules to near-crystalline density into ~50–100 nm prohead shells1, 2. Unexpectedly, we found that packaging frequently stalls in conditions that induce net attractive DNA-DNA interactions3. Here, we present findings suggesting that this stalling occurs because the DNA undergoes a nonequilibrium jamming transition analogous to that observed in many soft-matter systems, such as colloidal and granular systems4–8. Experiments in which conditions are changed during packaging to switch DNA-DNA interactions between purely repulsive and net attractive reveal strongly history-dependent dynamics. An abrupt deceleration is usually observed before stalling, indicating that a transition in DNA conformation causes an abrupt increase in resistance. Our findings suggest that the concept of jamming can be extended to a single polymer molecule. However, compared with macroscopic samples of colloidal particles5 we find that single DNA molecules jam over a much larger range of densities. We attribute this difference to the nanoscale system size, consistent with theoretical predictions for jamming of attractive athermal particles.9, 10 PMID:27540410

  14. Single DNA molecule jamming and history-dependent dynamics during motor-driven viral packaging.

    PubMed

    Keller, Nicholas; Grimes, Shelley; Jardine, Paul J; Smith, Douglas E

    2016-08-01

    In many viruses molecular motors forcibly pack single DNA molecules to near-crystalline density into ~50-100 nm prohead shells(1, 2). Unexpectedly, we found that packaging frequently stalls in conditions that induce net attractive DNA-DNA interactions(3). Here, we present findings suggesting that this stalling occurs because the DNA undergoes a nonequilibrium jamming transition analogous to that observed in many soft-matter systems, such as colloidal and granular systems(4-8). Experiments in which conditions are changed during packaging to switch DNA-DNA interactions between purely repulsive and net attractive reveal strongly history-dependent dynamics. An abrupt deceleration is usually observed before stalling, indicating that a transition in DNA conformation causes an abrupt increase in resistance. Our findings suggest that the concept of jamming can be extended to a single polymer molecule. However, compared with macroscopic samples of colloidal particles(5) we find that single DNA molecules jam over a much larger range of densities. We attribute this difference to the nanoscale system size, consistent with theoretical predictions for jamming of attractive athermal particles.(9, 10).

  15. Single DNA molecule jamming and history-dependent dynamics during motor-driven viral packaging

    NASA Astrophysics Data System (ADS)

    Keller, Nicholas; Grimes, Shelley; Jardine, Paul J.; Smith, Douglas E.

    2016-08-01

    In many viruses, molecular motors forcibly pack single DNA molecules to near-crystalline density into ~50-100 nm prohead shells. Unexpectedly, we found that packaging frequently stalls in conditions that induce net attractive DNA-DNA interactions. Here, we present findings suggesting that this stalling occurs because the DNA undergoes a nonequilibrium jamming transition analogous to that observed in many soft-matter systems, such as colloidal and granular systems. Experiments in which conditions are changed during packaging to switch DNA-DNA interactions between purely repulsive and net attractive reveal strongly history-dependent dynamics. An abrupt deceleration is usually observed before stalling, indicating that a transition in DNA conformation causes an abrupt increase in resistance. Our findings suggest that the concept of jamming can be extended to a single polymer molecule. However, compared with macroscopic samples of colloidal particles we find that single DNA molecules jam over a much larger range of densities. We attribute this difference to the nanoscale system size, consistent with theoretical predictions for jamming of attractive athermal particles.

  16. Single-molecule analysis of DNA uncoiling by a type II topoisomerase

    NASA Astrophysics Data System (ADS)

    Strick, Terence R.; Croquette, Vincent; Bensimon, David

    2000-04-01

    Type II DNA topoisomerases are ubiquitous ATP-dependent enzymes capable of transporting a DNA through a transient double-strand break in a second DNA segment. This enables them to untangle DNA and relax the interwound supercoils (plectonemes) that arise in twisted DNA. In vivo, they are responsible for untangling replicated chromosomes and their absence at mitosis or meiosis ultimately causes cell death. Here we describe a micromanipulation experiment in which we follow in real time a single Drosophila melanogaster topoisomerase II acting on a linear DNA molecule which is mechanically stretched and supercoiled. By monitoring the DNA's extension in the presence of ATP, we directly observe the relaxation of two supercoils during a single catalytic turnover. By controlling the force pulling on the molecule, we determine the variation of the reaction rate with the applied stress. Finally, in the absence of ATP, we observe the clamping of a DNA crossover by a single topoisomerase on at least two different timescales (configurations). These results show that single molecule experiments are a powerful new tool for the study of topoisomerases.

  17. Single molecule FRET analysis of the 11 discrete steps of a DNA actuator.

    PubMed

    Hildebrandt, Lasse L; Preus, Søren; Zhang, Zhao; Voigt, Niels V; Gothelf, Kurt V; Birkedal, Victoria

    2014-06-25

    DNA hybridization allows the design and assembly of dynamic DNA-based molecular devices. Such structures usually accomplish their function by the addition of fuel strands that drive the structure from one conformation to a new one or by internal changes in DNA hybridization. We report here on the performance and robustness of one of these devices by the detailed study of a dynamic DNA actuator. The DNA actuator was chosen as a model system, as it is the device with most discrete states to date. It is able to reversibly slide between 11 different states and can in principle function both autonomously and nonautonomously. The 11 states of the actuator were investigated by single molecule Förster Resonance Energy Transfer (smFRET) microscopy to obtain information on the static and dynamic heterogeneities of the device. Our results show that the DNA actuator can be effectively locked in several conformations with the help of well-designed DNA lock strands. However, the device also shows pronounced static and dynamic heterogeneities both in the unlocked and locked modes, and we suggest possible structural models. Our study allows for the direct visualization of the conformational diversity and movement of the dynamic DNA-based device and shows that complex DNA-based devices are inherently heterogeneous. Our results also demonstrate that single molecule techniques are a powerful tool for structural dynamics studies and provide a stringent test for the performance of molecular devices made out of DNA.

  18. DNA condensation by protamine and arginine-rich peptides: analysis of toroid stability using single DNA molecules.

    PubMed

    Balhorn, R; Brewer, L; Corzett, M

    2000-06-01

    Both somatic cells and sperm have been shown to take up exogenous DNA, but the frequency of its integration is usually low. Scanning probe microscopy studies of sperm chromatin and synthetic DNA-protamine complexes indicate that the coiling of DNA into toroidal subunits, a process initiated in the maturing spermatid to prepare its genome for delivery into the egg, can be mimicked by simply adding protamine to DNA in vitro. The increased resistance of DNA-protamine complexes to nuclease digestion and their structural similarity to native sperm chromatin suggest that the packaging of DNA by protamine might offer a new approach for improving the efficiency of DNA uptake by sperm. Decondensation experiments performed with individual DNA molecules have provided a direct measure of the stability of toroids produced using salmon protamine and smaller arginine-rich peptides. These experiments show that the arginine content of protamine-related sequences can have a dramatic effect on their rate of dissociation from DNA. This technique and the information it provides can be used to identify protamine analogs that can be bound to DNA to increase the efficiency of its uptake by sperm and other cells.

  19. DNA compaction by the bacteriophage protein Cox studied on the single DNA molecule level using nanofluidic channels

    PubMed Central

    Frykholm, Karolin; Berntsson, Ronnie Per-Arne; Claesson, Magnus; de Battice, Laura; Odegrip, Richard; Stenmark, Pål; Westerlund, Fredrik

    2016-01-01

    The Cox protein from bacteriophage P2 forms oligomeric filaments and it has been proposed that DNA can be wound up around these filaments, similar to how histones condense DNA. We here use fluorescence microscopy to study single DNA–Cox complexes in nanofluidic channels and compare how the Cox homologs from phages P2 and WΦ affect DNA. By measuring the extension of nanoconfined DNA in absence and presence of Cox we show that the protein compacts DNA and that the binding is highly cooperative, in agreement with the model of a Cox filament around which DNA is wrapped. Furthermore, comparing microscopy images for the wild-type P2 Cox protein and two mutants allows us to discriminate between compaction due to filament formation and compaction by monomeric Cox. P2 and WΦ Cox have similar effects on the physical properties of DNA and the subtle, but significant, differences in DNA binding are due to differences in binding affinity rather than binding mode. The presented work highlights the use of single DNA molecule studies to confirm structural predictions from X-ray crystallography. It also shows how a small protein by oligomerization can have great impact on the organization of DNA and thereby fulfill multiple regulatory functions. PMID:27131370

  20. Chirality of Sulforhodamine Dye Molecules Incorporated in DNA Thin Films

    DTIC Science & Technology

    2008-11-13

    of the aqueous solution, but is soluble in simple organic solvents. Thin film formation by spin coating is markedly easier with organic solvents...filter. Solutions of 2.5, 5, 10, 15, 20, and 25 wt % SRh to DNA/CTMA were prepared. Thin films produced by spin coating were typically 1.5 m thick...The spin - coating process started by com- pletely flooding the substrate, followed by spreading the so- lution for 8 s at 300 rpm, a spin of 1 min at

  1. Nitrous acid induced damage in T7 DNA and phage

    SciTech Connect

    Scearce, L.M.; Masker, W.E.

    1986-05-01

    The response of bacteriophage T7 to nitrous acid damage was investigated. The T7 system allows in vitro mimicry of most aspects of in vivo DNA metabolism. Nitrous acid is of special interest since it has been previously shown to induce deletions and point mutations as well as novel adducts in DNA. T7 phage was exposed to 56 mM nitrous acid at pH 4.6 in vivo, causing a time dependent 98% decrease in survival for each 10 min duration of exposure to nitrous acid. These studies were extended to include examination of pure T7 DNA exposed in vitro to nitrous acid conditions identical to those used in the in vivo survival studies. The treated DNA was dialyzed to remove the nitrous acid and the DNA was encapsulated into empty phage heads. These in vitro packaged phage showed a survival curve analogous to the in vivo system. There was no change in survival when either in vitro or in vivo exposed phage were grown on wild type E. coli or on E. coli strains deficient in DNA repair due to mutations in DNA polymerase I, exonuclease III or a uvrA mutation. Survival was not increased when nitrous acid treated T7 were grown on E. coli induced for SOS repair. In vitro replication of nitrous acid treated DNA showed a time dependent decrease in the total amount of DNA synthesized.

  2. Nonlinear charge transport in the helicoidal DNA molecule.

    PubMed

    Dang Koko, A; Tabi, C B; Ekobena Fouda, H P; Mohamadou, A; Kofané, T C

    2012-12-01

    Charge transport in the twist-opening model of DNA is explored via the modulational instability of a plane wave. The dynamics of charge is shown to be governed, in the adiabatic approximation, by a modified discrete nonlinear Schrödinger equation with next-nearest neighbor interactions. The linear stability analysis is performed on the latter and manifestations of the modulational instability are discussed according to the value of the parameter α, which measures hopping interaction correction. In so doing, increasing α leads to a reduction of the instability domain and, therefore, increases our chances of choosing appropriate values of parameters that could give rise to pattern formation in the twist-opening model. Our analytical predictions are verified numerically, where the generic equations for the radial and torsional dynamics are directly integrated. The impact of charge migration on the above degrees of freedom is discussed for different values of α. Soliton-like and localized structures are observed and thus confirm our analytical predictions. We also find that polaronic structures, as known in DNA charge transport, are generated through modulational instability, and hence reinforces the robustness of polaron in the model we study.

  3. Single-molecule studies of high-mobility group B architectural DNA bending proteins.

    PubMed

    Murugesapillai, Divakaran; McCauley, Micah J; Maher, L James; Williams, Mark C

    2017-02-01

    Protein-DNA interactions can be characterized and quantified using single molecule methods such as optical tweezers, magnetic tweezers, atomic force microscopy, and fluorescence imaging. In this review, we discuss studies that characterize the binding of high-mobility group B (HMGB) architectural proteins to single DNA molecules. We show how these studies are able to extract quantitative information regarding equilibrium binding as well as non-equilibrium binding kinetics. HMGB proteins play critical but poorly understood roles in cellular function. These roles vary from the maintenance of chromatin structure and facilitation of ribosomal RNA transcription (yeast high-mobility group 1 protein) to regulatory and packaging roles (human mitochondrial transcription factor A). We describe how these HMGB proteins bind, bend, bridge, loop and compact DNA to perform these functions. We also describe how single molecule experiments observe multiple rates for dissociation of HMGB proteins from DNA, while only one rate is observed in bulk experiments. The measured single-molecule kinetics reveals a local, microscopic mechanism by which HMGB proteins alter DNA flexibility, along with a second, much slower macroscopic rate that describes the complete dissociation of the protein from DNA.

  4. Directly observing the motion of DNA molecules near solid-state nanopores.

    PubMed

    Ando, Genki; Hyun, Changbae; Li, Jiali; Mitsui, Toshiyuki

    2012-11-27

    We investigate the diffusion and the drift motion of λ DNA molecules near solid-state nanopores prior to their translocation through the nanopores using fluorescence microscopy. The radial dependence of the electric field near a nanopore generated by an applied voltage in ionic solution can be estimated quantitatively in 3D by analyzing the motion of negatively charged DNA molecules. We find that the electric field is approximately spherically symmetric around the nanopore under the conditions investigated. In addition, DNA clogging at the nanopore was directly observed. Surprisingly, the probability of the clogging event increases with increasing external bias voltage. We also find that DNA molecules clogging the nanopore reduce the electric field amplitude at the nanopore membrane surface. To better understand these experimental results, analytical method with Ohm's law and computer simulation with Poisson and Nernst-Planck (PNP) equations are used to calculate the electric field near the nanopore. These results are of great interest in both experimental and theoretical considerations of the motion of DNA molecules near voltage-biased nanopores. These findings will also contribute to the development of solid-state nanopore-based DNA sensing devices.

  5. Antigen Targeting to Human HLA Class II Molecules Increases Efficacy of DNA Vaccination

    PubMed Central

    Fredriksen, Agnete Brunsvik; Løset, Geir Åge; Vikse, Elisabeth; Fugger, Lars

    2016-01-01

    It has been difficult to translate promising results from DNA vaccination in mice to larger animals and humans. Previously, DNA vaccines encoding proteins that target Ag to MHC class II (MHC-II) molecules on APCs have been shown to induce rapid, enhanced, and long-lasting Ag-specific Ab titers in mice. In this study, we describe two novel DNA vaccines that as proteins target HLA class II (HLA-II) molecules. These vaccine proteins cross-react with MHC-II molecules in several species of larger mammals. When tested in ferrets and pigs, a single DNA delivery with low doses of the HLA-II–targeted vaccines resulted in rapid and increased Ab responses. Importantly, painless intradermal jet delivery of DNA was as effective as delivery by needle injection followed by electroporation. As an indication that the vaccines could also be useful for human application, HLA-II–targeted vaccine proteins were found to increase human CD4+ T cell responses by a factor of ×103 in vitro. Thus, targeting of Ag to MHC-II molecules may represent an attractive strategy for increasing efficacy of DNA vaccines in larger animals and humans. PMID:27671110

  6. Development of Single-Molecule DNA Sequencing Platform Based on Single-Molecule Electrical Conductance

    DTIC Science & Technology

    2015-05-25

    indicate that they are nearly harmless to cultured cells (17-20). The bio-distribution of injected GNPs has shown a size-dependent accumulation in liver ...Animals” of National Chiao Tung University. Four-week-old male BALB/C mice were housed at 22±2 C with a 12-h light/dark cycle and fed standard...Conference, Protein Folding Dynamics, 5-10 Jan, 2014, Hotel Galvez in Galveston TX, United States Guewha Steven Huang , Single-Molecule Kinetics of

  7. Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array

    PubMed Central

    Fuller, Carl W.; Kumar, Shiv; Porel, Mintu; Chien, Minchen; Bibillo, Arek; Stranges, P. Benjamin; Dorwart, Michael; Tao, Chuanjuan; Li, Zengmin; Guo, Wenjing; Shi, Shundi; Korenblum, Daniel; Trans, Andrew; Aguirre, Anne; Liu, Edward; Harada, Eric T.; Pollard, James; Bhat, Ashwini; Cech, Cynthia; Yang, Alexander; Arnold, Cleoma; Palla, Mirkó; Hovis, Jennifer; Chen, Roger; Morozova, Irina; Kalachikov, Sergey; Russo, James J.; Kasianowicz, John J.; Davis, Randy; Roever, Stefan; Church, George M.; Ju, Jingyue

    2016-01-01

    DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5′-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods. PMID:27091962

  8. Real-time single-molecule electronic DNA sequencing by synthesis using polymer-tagged nucleotides on a nanopore array.

    PubMed

    Fuller, Carl W; Kumar, Shiv; Porel, Mintu; Chien, Minchen; Bibillo, Arek; Stranges, P Benjamin; Dorwart, Michael; Tao, Chuanjuan; Li, Zengmin; Guo, Wenjing; Shi, Shundi; Korenblum, Daniel; Trans, Andrew; Aguirre, Anne; Liu, Edward; Harada, Eric T; Pollard, James; Bhat, Ashwini; Cech, Cynthia; Yang, Alexander; Arnold, Cleoma; Palla, Mirkó; Hovis, Jennifer; Chen, Roger; Morozova, Irina; Kalachikov, Sergey; Russo, James J; Kasianowicz, John J; Davis, Randy; Roever, Stefan; Church, George M; Ju, Jingyue

    2016-05-10

    DNA sequencing by synthesis (SBS) offers a robust platform to decipher nucleic acid sequences. Recently, we reported a single-molecule nanopore-based SBS strategy that accurately distinguishes four bases by electronically detecting and differentiating four different polymer tags attached to the 5'-phosphate of the nucleotides during their incorporation into a growing DNA strand catalyzed by DNA polymerase. Further developing this approach, we report here the use of nucleotides tagged at the terminal phosphate with oligonucleotide-based polymers to perform nanopore SBS on an α-hemolysin nanopore array platform. We designed and synthesized several polymer-tagged nucleotides using tags that produce different electrical current blockade levels and verified they are active substrates for DNA polymerase. A highly processive DNA polymerase was conjugated to the nanopore, and the conjugates were complexed with primer/template DNA and inserted into lipid bilayers over individually addressable electrodes of the nanopore chip. When an incoming complementary-tagged nucleotide forms a tight ternary complex with the primer/template and polymerase, the tag enters the pore, and the current blockade level is measured. The levels displayed by the four nucleotides tagged with four different polymers captured in the nanopore in such ternary complexes were clearly distinguishable and sequence-specific, enabling continuous sequence determination during the polymerase reaction. Thus, real-time single-molecule electronic DNA sequencing data with single-base resolution were obtained. The use of these polymer-tagged nucleotides, combined with polymerase tethering to nanopores and multiplexed nanopore sensors, should lead to new high-throughput sequencing methods.

  9. A unified sensor architecture for isothermal detection of double-stranded DNA, oligonucleotides, and small molecules.

    PubMed

    Brown, Carl W; Lakin, Matthew R; Fabry-Wood, Aurora; Horwitz, Eli K; Baker, Nicholas A; Stefanovic, Darko; Graves, Steven W

    2015-03-23

    Pathogen detection is an important problem in many areas of medicine and agriculture, which can involve genomic or transcriptomic signatures or small-molecule metabolites. We report a unified, DNA-based sensor architecture capable of isothermal detection of double-stranded DNA targets, single-stranded oligonucleotides, and small molecules. Each sensor contains independent target detection and reporter modules, enabling rapid design. We detected gene variants on plasmids by using a straightforward isothermal denaturation protocol. The sensors were highly specific, even with a randomized DNA background. We achieved a limit of detection of ∼15 pM for single-stranded targets and ∼5 nM for targets on denatured plasmids. By incorporating a blocked aptamer sequence, we also detected small molecules using the same sensor architecture. This work provides a starting point for multiplexed detection of multi-strain pathogens, and disease states caused by genetic variants (e.g., sickle cell anemia).

  10. Rapid prototyping of multichannel microfluidic devices for single-molecule DNA curtain imaging.

    PubMed

    Robison, Aaron D; Finkelstein, Ilya J

    2014-05-06

    Single-molecule imaging and manipulation of biochemical reactions continues to reveal numerous biological insights. To facilitate these studies, we have developed and implemented a high-throughput approach to organize and image hundreds of individual DNA molecules at aligned diffusion barriers. Nonetheless, obtaining statistically relevant data sets under a variety of reaction conditions remains challenging. Here, we present a method for integrating high-throughput single-molecule "DNA curtain" imaging with poly(dimethylsiloxane) (PDMS)-based microfluidics. Our benchtop fabrication method can be accomplished in minutes with common tools found in all molecular biology laboratories. We demonstrate the utility of this approach by simultaneous imaging of two independent biochemical reaction conditions in a laminar flow device. In addition, five different reaction conditions can be observed concurrently in a passive linear gradient generator. Combining rapid microfluidic fabrication with high-throughput DNA curtains greatly expands our capability to interrogate complex biological reactions.

  11. Single-molecule analysis of DNA replication in Xenopus egg extracts.

    PubMed

    Yardimci, Hasan; Loveland, Anna B; van Oijen, Antoine M; Walter, Johannes C

    2012-06-01

    The recent advent in single-molecule imaging and manipulation methods has made a significant impact on the understanding of molecular mechanisms underlying many essential cellular processes. Single-molecule techniques such as electron microscopy and DNA fiber assays have been employed to study the duplication of genome in eukaryotes. Here, we describe a single-molecule assay that allows replication of DNA attached to the functionalized surface of a microfluidic flow cell in a soluble Xenopus leavis egg extract replication system and subsequent visualization of replication products via fluorescence microscopy. We also explain a method for detection of replication proteins, through fluorescently labeled antibodies, on partially replicated DNA immobilized at both ends to the surface.

  12. Molecular dynamics simulations of G-DNA and perspectives on the simulation of nucleic acid structures

    PubMed Central

    šponer, Jiří; Cang, Xiaohui; Cheatham, Thomas E.

    2013-01-01

    The article reviews the application of biomolecular simulation methods to understand the structure, dynamics and interactions of nucleic acids with a focus on explicit solvent molecular dynamics simulations of guanine quadruplex (G-DNA and G-RNA) molecules. While primarily dealing with these exciting and highly relevant four-stranded systems, where recent and past simulations have provided several interesting results and novel insight into G-DNA structure, the review provides some general perspectives on the applicability of the simulation techniques to nucleic acids. PMID:22525788

  13. DNA Physical Mapping via the Controlled Translocation of Single Molecules through a 5-10nm Silicon Nitride Nanopore

    NASA Astrophysics Data System (ADS)

    Stein, Derek; Reisner, Walter; Jiang, Zhijun; Hagerty, Nick; Wood, Charles; Chan, Jason

    2009-03-01

    The ability to map the binding position of sequence-specific markers, including transcription-factors, protein-nucleic acids (PNAs) or deactivated restriction enzymes, along a single DNA molecule in a nanofluidic device would be of key importance for the life-sciences. Such markers could give an indication of the active genes at particular stage in a cell's transcriptional cycle, pinpoint the location of mutations or even provide a DNA barcode that could aid in genomics applications. We have developed a setup consisting of a 5-10 nm nanopore in a 20nm thick silicon nitride film coupled to an optical tweezer setup. The translocation of DNA across the nanopore can be detected via blockades in the electrical current through the pore. By anchoring one end of the translocating DNA to an optically trapped microsphere, we hope to stretch out the molecule in the nanopore and control the translocation speed, enabling us to slowly scan across the genome and detect changes in the baseline current due to the presence of bound markers.

  14. A New Direct Single-Molecule Observation Method for DNA Synthesis Reaction Using Fluorescent Replication Protein A

    PubMed Central

    Takahashi, Shunsuke; Kawasaki, Shohei; Miyata, Hidefumi; Kurita, Hirofumi; Mizuno, Takeshi; Matsuura, Shun-ichi; Mizuno, Akira; Oshige, Masahiko; Katsura, Shinji

    2014-01-01

    Using a single-stranded region tracing system, single-molecule DNA synthesis reactions were directly observed in microflow channels. The direct single-molecule observations of DNA synthesis were labeled with a fusion protein consisting of the ssDNA-binding domain of a 70-kDa subunit of replication protein A and enhanced yellow fluorescent protein (RPA-YFP). Our method was suitable for measurement of DNA synthesis reaction rates with control of the ssλDNA form as stretched ssλDNA (+flow) and random coiled ssλDNA (−flow) via buffer flow. Sequentially captured photographs demonstrated that the synthesized region of an ssλDNA molecule monotonously increased with the reaction time. The DNA synthesis reaction rate of random coiled ssλDNA (−flow) was nearly the same as that measured in a previous ensemble molecule experiment (52 vs. 50 bases/s). This suggested that the random coiled form of DNA (−flow) reflected the DNA form in the bulk experiment in the case of DNA synthesis reactions. In addition, the DNA synthesis reaction rate of stretched ssλDNA (+flow) was approximately 75% higher than that of random coiled ssλDNA (−flow) (91 vs. 52 bases/s). The DNA synthesis reaction rate of the Klenow fragment (3′-5′exo–) was promoted by DNA stretching with buffer flow. PMID:24625741

  15. DNA unwinding heterogeneity by RecBCD results from static molecules able to equilibrate.

    PubMed

    Liu, Bian; Baskin, Ronald J; Kowalczykowski, Stephen C

    2013-08-22

    Single-molecule studies can overcome the complications of asynchrony and ensemble-averaging in bulk-phase measurements, provide mechanistic insights into molecular activities, and reveal interesting variations between individual molecules. The application of these techniques to the RecBCD helicase of Escherichia coli has resolved some long-standing discrepancies, and has provided otherwise unattainable mechanistic insights into its enzymatic behaviour. Enigmatically, the DNA unwinding rates of individual enzyme molecules are seen to vary considerably, but the origin of this heterogeneity remains unknown. Here we investigate the physical basis for this behaviour. Although any individual RecBCD molecule unwound DNA at a constant rate for an average of approximately 30,000 steps, we discover that transiently halting a single enzyme-DNA complex by depleting Mg(2+)-ATP could change the subsequent rates of DNA unwinding by that enzyme after reintroduction to ligand. The proportion of molecules that changed rate increased exponentially with the duration of the interruption, with a half-life of approximately 1 second, suggesting that a conformational change occurred during the time that the molecule was arrested. The velocity after pausing an individual molecule was any velocity found in the starting distribution of the ensemble. We suggest that substrate binding stabilizes the enzyme in one of many equilibrium conformational sub-states that determine the rate-limiting translocation behaviour of each RecBCD molecule. Each stabilized sub-state can persist for the duration (approximately 1 minute) of processive unwinding of a DNA molecule, comprising tens of thousands of catalytic steps, each of which is much faster than the time needed for the conformational change required to alter kinetic behaviour. This ligand-dependent stabilization of rate-defining conformational sub-states results in seemingly static molecule-to-molecule variation in RecBCD helicase activity

  16. Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones.

    PubMed

    Rodriguez-Manzano, Jesus; Karymov, Mikhail A; Begolo, Stefano; Selck, David A; Zhukov, Dmitriy V; Jue, Erik; Ismagilov, Rustem F

    2016-03-22

    Digital single-molecule technologies are expanding diagnostic capabilities, enabling the ultrasensitive quantification of targets, such as viral load in HIV and hepatitis C infections, by directly counting single molecules. Replacing fluorescent readout with a robust visual readout that can be captured by any unmodified cell phone camera will facilitate the global distribution of diagnostic tests, including in limited-resource settings where the need is greatest. This paper describes a methodology for developing a visual readout system for digital single-molecule amplification of RNA and DNA by (i) selecting colorimetric amplification-indicator dyes that are compatible with the spectral sensitivity of standard mobile phones, and (ii) identifying an optimal ratiometric image-process for a selected dye to achieve a readout that is robust to lighting conditions and camera hardware and provides unambiguous quantitative results, even for colorblind users. We also include an analysis of the limitations of this methodology, and provide a microfluidic approach that can be applied to expand dynamic range and improve reaction performance, allowing ultrasensitive, quantitative measurements at volumes as low as 5 nL. We validate this methodology using SlipChip-based digital single-molecule isothermal amplification with λDNA as a model and hepatitis C viral RNA as a clinically relevant target. The innovative combination of isothermal amplification chemistry in the presence of a judiciously chosen indicator dye and ratiometric image processing with SlipChip technology allowed the sequence-specific visual readout of single nucleic acid molecules in nanoliter volumes with an unmodified cell phone camera. When paired with devices that integrate sample preparation and nucleic acid amplification, this hardware-agnostic approach will increase the affordability and the distribution of quantitative diagnostic and environmental tests.

  17. Reading Out Single-Molecule Digital RNA and DNA Isothermal Amplification in Nanoliter Volumes with Unmodified Camera Phones

    PubMed Central

    2016-01-01

    Digital single-molecule technologies are expanding diagnostic capabilities, enabling the ultrasensitive quantification of targets, such as viral load in HIV and hepatitis C infections, by directly counting single molecules. Replacing fluorescent readout with a robust visual readout that can be captured by any unmodified cell phone camera will facilitate the global distribution of diagnostic tests, including in limited-resource settings where the need is greatest. This paper describes a methodology for developing a visual readout system for digital single-molecule amplification of RNA and DNA by (i) selecting colorimetric amplification-indicator dyes that are compatible with the spectral sensitivity of standard mobile phones, and (ii) identifying an optimal ratiometric image-process for a selected dye to achieve a readout that is robust to lighting conditions and camera hardware and provides unambiguous quantitative results, even for colorblind users. We also include an analysis of the limitations of this methodology, and provide a microfluidic approach that can be applied to expand dynamic range and improve reaction performance, allowing ultrasensitive, quantitative measurements at volumes as low as 5 nL. We validate this methodology using SlipChip-based digital single-molecule isothermal amplification with λDNA as a model and hepatitis C viral RNA as a clinically relevant target. The innovative combination of isothermal amplification chemistry in the presence of a judiciously chosen indicator dye and ratiometric image processing with SlipChip technology allowed the sequence-specific visual readout of single nucleic acid molecules in nanoliter volumes with an unmodified cell phone camera. When paired with devices that integrate sample preparation and nucleic acid amplification, this hardware-agnostic approach will increase the affordability and the distribution of quantitative diagnostic and environmental tests. PMID:26900709

  18. Contactless experiments on individual DNA molecules show no evidence for molecular wire behavior

    PubMed Central

    Gómez-Navarro, C.; Moreno-Herrero, F.; de Pablo, P. J.; Colchero, J.; Gómez-Herrero, J.; Baró, A. M.

    2002-01-01

    A fundamental requirement for a molecule to be considered a molecular wire (MW) is the ability to transport electrical charge with a reasonably low resistance. We have carried out two experiments that measure first, the charge transfer from an electrode to the molecule, and second, the dielectric response of the MW. The latter experiment requires no contacts to either end of the molecule. From our experiments we conclude that adsorbed individual DNA molecules have a resistivity similar to mica, glass, and silicon oxide substrates. Therefore adsorbed DNA is not a conductor, and it should not be considered as a viable candidate for MW applications. Parallel studies on other nanowires, including single-walled carbon nanotubes, showed conductivity as expected. PMID:12070346

  19. Studying the organization of DNA repair by single-cell and single-molecule imaging

    PubMed Central

    Uphoff, Stephan; Kapanidis, Achillefs N.

    2014-01-01

    DNA repair safeguards the genome against a diversity of DNA damaging agents. Although the mechanisms of many repair proteins have been examined separately in vitro, far less is known about the coordinated function of the whole repair machinery in vivo. Furthermore, single-cell studies indicate that DNA damage responses generate substantial variation in repair activities across cells. This review focuses on fluorescence imaging methods that offer a quantitative description of DNA repair in single cells by measuring protein concentrations, diffusion characteristics, localizations, interactions, and enzymatic rates. Emerging single-molecule and super-resolution microscopy methods now permit direct visualization of individual proteins and DNA repair events in vivo. We expect much can be learned about the organization of DNA repair by linking cell heterogeneity to mechanistic observations at the molecular level. PMID:24629485

  20. Method for patterning stretched DNA molecules on mica surfaces by soft lithography.

    PubMed

    Gad, M; Sugiyama, Shigeru; Ohtani, Toshio

    2003-12-01

    Lambda DNA was stretched and patterned on mica surface using soft lithography. A highly diluted solution of amino propyl trimethoxy silane in hexane was deposited on a line patterned polydimethylsiloxane (PDMS) stamp. The functionalized stamp was then used to pick up DNA by molecular combing while the line patterns are parallel to the liquid surface. The stamp was then microcontact printed on freshly cleaved mica. We successfully obtained stretched DNA pattern on mica surface. DNA was found to be stretched in patterns perpendicular to those carved on the stamp. The stretched DNA population was large enough to be used for molecular biology mapping studies. Furthermore, the possibility of locating stretched DNA molecules in the desired position by stamping makes this method a good candidate for assembling non-semiconductor molecular devices.

  1. An unnatural base pair system for efficient PCR amplification and functionalization of DNA molecules

    PubMed Central

    Kimoto, Michiko; Kawai, Rie; Mitsui, Tsuneo; Yokoyama, Shigeyuki; Hirao, Ichiro

    2009-01-01

    Toward the expansion of the genetic alphabet, we present an unnatural base pair system for efficient PCR amplification, enabling the site-specific incorporation of extra functional components into DNA. This system can be applied to conventional PCR protocols employing DNA templates containing unnatural bases, natural and unnatural base triphosphates, and a 3′→5′ exonuclease-proficient DNA polymerase. For highly faithful and efficient PCR amplification involving the unnatural base pairing, we identified the natural-base sequences surrounding the unnatural bases in DNA templates by an in vitro selection technique, using a DNA library containing the unnatural base. The system facilitates the site-specific incorporation of a variety of modified unnatural bases, linked with functional groups of interest, into amplified DNA. DNA fragments (0.15 amol) containing the unnatural base pair can be amplified 107-fold by 30 cycles of PCR, with <1% total mutation rate of the unnatural base pair site. Using the system, we demonstrated efficient PCR amplification and functionalization of DNA fragments for the extremely sensitive detection of zeptomol-scale target DNA molecules from mixtures with excess amounts (pmol scale) of foreign DNA species. This unnatural base pair system will be applicable to a wide range of DNA/RNA-based technologies. PMID:19073696

  2. The origins of life -- the 'protein interaction world' hypothesis: protein interactions were the first form of self-reproducing life and nucleic acids evolved later as memory molecules.

    PubMed

    Andras, Peter; Andras, Csaba

    2005-01-01

    The 'protein interaction world' (PIW) hypothesis of the origins of life assumes that life emerged as a self-reproducing and expanding system of protein interactions. In mainstream molecular biology, 'replication' refers to the material copying of molecules such as nucleic acids. However, PIW is conceptualized as an abstract communication system constituted by the interactions between proteins, in which 'replication' happens at the level of self-reproduction of these interactions between proteins. Densely concentrated peptide interaction systems may have reproduced and expanded as 'protocell' vesicles surrounded by lipid bi-layer membranes. Protocells led to the emergence of proto-RNA molecules of greater chemical stability which served as chemically differentiated 'memories' of peptide interaction states, thereby facilitating the reproduction and expansion of protocells. Simplification-driven expansion led to the selection of biotic amino acids and the reduction of the typical RNA alphabet to the four usual bases (A, C, G and U). Dense interactions between RNA molecules led to the emergence of the RNA interaction subsystem of the cell, and to the emergence of 'memories' of RNA interactions in the form of DNA molecules with greater chemical stability. The expansion of DNA molecule interactions led to the dense clustering and encapsulation of DNA molecules within the cell nucleus. RNA molecules therefore serve as memories of protein interactions and DNA molecules are memories of RNA interactions. We believe that the PIW hypothesis is more evolutionarily plausible than the mainstream RNA world hypothesis, and has greater explanatory power.

  3. Molecular threading: mechanical extraction, stretching and placement of DNA molecules from a liquid-air interface.

    PubMed

    Payne, Andrew C; Andregg, Michael; Kemmish, Kent; Hamalainen, Mark; Bowell, Charlotte; Bleloch, Andrew; Klejwa, Nathan; Lehrach, Wolfgang; Schatz, Ken; Stark, Heather; Marblestone, Adam; Church, George; Own, Christopher S; Andregg, William

    2013-01-01

    We present "molecular threading", a surface independent tip-based method for stretching and depositing single and double-stranded DNA molecules. DNA is stretched into air at a liquid-air interface, and can be subsequently deposited onto a dry substrate isolated from solution. The design of an apparatus used for molecular threading is presented, and fluorescence and electron microscopies are used to characterize the angular distribution, straightness, and reproducibility of stretched DNA deposited in arrays onto elastomeric surfaces and thin membranes. Molecular threading demonstrates high straightness and uniformity over length scales from nanometers to micrometers, and represents an alternative to existing DNA deposition and linearization methods. These results point towards scalable and high-throughput precision manipulation of single-molecule polymers.

  4. Macromolecular crowding induced elongation and compaction of single DNA molecules confined in a nanochannel

    PubMed Central

    Zhang, Ce; Shao, Pei Ge; van Kan, Jeroen A.; van der Maarel, Johan R. C.

    2009-01-01

    The effect of dextran nanoparticles on the conformation and compaction of single DNA molecules confined in a nanochannel was investigated with fluorescence microscopy. It was observed that the DNA molecules elongate and eventually condense into a compact form with increasing volume fraction of the crowding agent. Under crowded conditions, the channel diameter is effectively reduced, which is interpreted in terms of depletion in DNA segment density in the interfacial region next to the channel wall. Confinement in a nanochannel also facilitates compaction with a neutral crowding agent at low ionic strength. The threshold volume fraction for condensation is proportional to the size of the nanoparticle, due to depletion induced attraction between DNA segments. We found that the effect of crowding is not only related to the colligative properties of the agent and that confinement is also important. It is the interplay between anisotropic confinement and osmotic pressure which gives the elongated conformation and the possibility for condensation at low ionic strength. PMID:19805352

  5. Macromolecular crowding induced elongation and compaction of single DNA molecules confined in a nanochannel.

    PubMed

    Zhang, Ce; Shao, Pei Ge; van Kan, Jeroen A; van der Maarel, Johan R C

    2009-09-29

    The effect of dextran nanoparticles on the conformation and compaction of single DNA molecules confined in a nanochannel was investigated with fluorescence microscopy. It was observed that the DNA molecules elongate and eventually condense into a compact form with increasing volume fraction of the crowding agent. Under crowded conditions, the channel diameter is effectively reduced, which is interpreted in terms of depletion in DNA segment density in the interfacial region next to the channel wall. Confinement in a nanochannel also facilitates compaction with a neutral crowding agent at low ionic strength. The threshold volume fraction for condensation is proportional to the size of the nanoparticle, due to depletion induced attraction between DNA segments. We found that the effect of crowding is not only related to the colligative properties of the agent and that confinement is also important. It is the interplay between anisotropic confinement and osmotic pressure which gives the elongated conformation and the possibility for condensation at low ionic strength.

  6. A renormalization approach to describe charge transport in quasiperiodic dangling backbone ladder (DBL)-DNA molecules

    NASA Astrophysics Data System (ADS)

    Sarmento, R. G.; Fulco, U. L.; Albuquerque, E. L.; Caetano, E. W. S.; Freire, V. N.

    2011-10-01

    We study the charge transport properties of a dangling backbone ladder (DBL)-DNA molecule focusing on a quasiperiodic arrangement of its constituent nucleotides forming a Rudin-Shapiro (RS) and Fibonacci (FB) Poly (CG) sequences, as well as a natural DNA sequence (Ch22) for the sake of comparison. Making use of a one-step renormalization process, the DBL-DNA molecule is modeled in terms of a one-dimensional tight-binding Hamiltonian to investigate its transmissivity and current-voltage (I-V) profiles. Beyond the semiconductor I-V characteristics, a striking similarity between the electronic transport properties of the RS quasiperiodic structure and the natural DNA sequence was found.

  7. A DNA Crystal Designed to Contain Two Molecules per Asymmetric Unit

    SciTech Connect

    T Wang; R Sha; J Birktoft; J Zheng; C Mao; N Seeman

    2011-12-31

    We describe the self-assembly of a DNA crystal that contains two tensegrity triangle molecules per asymmetric unit. We have used X-ray crystallography to determine its crystal structure. In addition, we have demonstrated control over the colors of the crystals by attaching either Cy3 dye (pink) or Cy5 dye (blue-green) to the components of the crystal, yielding crystals of corresponding colors. Attaching the pair of dyes to the pair of molecules yields a purple crystal.

  8. Complete nucleotide sequence of a subviral DNA molecule of porcine circovirus type 2.

    PubMed

    Wen, Han

    2016-07-01

    Porcine circovirus type 2 (PCV2) is a member of the genus Circovirus in the family Circoviridae. Most subgenomic molecules of PCV2 have been mapped. Here, the first full-length sequence of a subviral molecule of PCV2 (CH-IVT12) containing a reverse complement sequence of the PCV2 genome was determined by sequencing DNA extracted from PK15 cells infected with PCV2. The circular CH-IVT12 DNA consists of 1136 nucleotides and contains one major open reading frame.

  9. Structure-based DNA-targeting strategies with small molecule ligands for drug discovery.

    PubMed

    Sheng, Jia; Gan, Jianhua; Huang, Zhen

    2013-09-01

    Nucleic acids are the molecular targets of many clinical anticancer drugs. However, compared with proteins, nucleic acids have traditionally attracted much less attention as drug targets in structure-based drug design, partially because limited structural information of nucleic acids complexed with potential drugs is available. Over the past several years, enormous progresses in nucleic acid crystallization, heavy-atom derivatization, phasing, and structural biology have been made. Many complicated nucleic acid structures have been determined, providing new insights into the molecular functions and interactions of nucleic acids, especially DNAs complexed with small molecule ligands. Thus, opportunities have been created to further discover nucleic acid-targeting drugs for disease treatments. This review focuses on the structure studies of DNAs complexed with small molecule ligands for discovering lead compounds, drug candidates, and/or therapeutics.

  10. Structure-Based DNA-Targeting Strategies with Small Molecule Ligands for Drug Discovery

    PubMed Central

    Sheng, Jia; Gan, Jianhua; Huang, Zhen

    2014-01-01

    Nucleic acids are the molecular targets of many clinical anticancer drugs. However, compared with proteins, nucleic acids have traditionally attracted much less attention as drug targets in structure-based drug design, partially because limited structural information of nucleic acids complexed with potential drugs is available. Over the past several years, enormous progresses in nucleic acid crystallization, heavy-atom derivatization, phasing, and structural biology have been made. Many complicated nucleic acid structures have been determined, providing new insights into the molecular functions and interactions of nucleic acids, especially DNAs complexed with small molecule ligands. Thus, opportunities have been created to further discover nucleic acid-targeting drugs for disease treatments. This review focuses on the structure studies of DNAs complexed with small molecule ligands for discovering lead compounds, drug candidates, and/or therapeutics. PMID:23633219

  11. Combining single-molecule manipulation and imaging for the study of protein-DNA interactions.

    PubMed

    Monico, Carina; Belcastro, Gionata; Vanzi, Francesco; Pavone, Francesco S; Capitanio, Marco

    2014-08-27

    The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

  12. Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

    PubMed Central

    Monico, Carina; Belcastro, Gionata; Vanzi, Francesco; Pavone, Francesco S.; Capitanio, Marco

    2014-01-01

    The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable. PMID:25226304

  13. True single-molecule DNA sequencing of a pleistocene horse bone

    PubMed Central

    Orlando, Ludovic; Ginolhac, Aurelien; Raghavan, Maanasa; Vilstrup, Julia; Rasmussen, Morten; Magnussen, Kim; Steinmann, Kathleen E.; Kapranov, Philipp; Thompson, John F.; Zazula, Grant; Froese, Duane; Moltke, Ida; Shapiro, Beth; Hofreiter, Michael; Al-Rasheid, Khaled A.S.; Gilbert, M. Thomas P.; Willerslev, Eske

    2011-01-01

    Second-generation sequencing platforms have revolutionized the field of ancient DNA, opening access to complete genomes of past individuals and extinct species. However, these platforms are dependent on library construction and amplification steps that may result in sequences that do not reflect the original DNA template composition. This is particularly true for ancient DNA, where templates have undergone extensive damage post-mortem. Here, we report the results of the first “true single molecule sequencing” of ancient DNA. We generated 115.9 Mb and 76.9 Mb of DNA sequences from a permafrost-preserved Pleistocene horse bone using the Helicos HeliScope and Illumina GAIIx platforms, respectively. We find that the percentage of endogenous DNA sequences derived from the horse is higher among the Helicos data than Illumina data. This result indicates that the molecular biology tools used to generate sequencing libraries of ancient DNA molecules, as required for second-generation sequencing, introduce biases into the data that reduce the efficiency of the sequencing process and limit our ability to fully explore the molecular complexity of ancient DNA extracts. We demonstrate that simple modifications to the standard Helicos DNA template preparation protocol further increase the proportion of horse DNA for this sample by threefold. Comparison of Helicos-specific biases and sequence errors in modern DNA with those in ancient DNA also reveals extensive cytosine deamination damage at the 3′ ends of ancient templates, indicating the presence of 3′-sequence overhangs. Our results suggest that paleogenomes could be sequenced in an unprecedented manner by combining current second- and third-generation sequencing approaches. PMID:21803858

  14. Interaction between cationic agents and small interfering RNA and DNA molecules

    NASA Astrophysics Data System (ADS)

    Unksov, I. N.; Slita, A. V.; Petrova, A. V.; Pereviazko, I.; Bakulev, V. M.; Rolich, V. I.; Bondarenko, A. B.; Kasyanenko, N. A.

    2016-11-01

    Azobenzene containing surfactant AzoTAB was used for investigation of binding in cationic- agent + nucleic acid in NaCl salt aqueous solutions. Two nucleic acids, macromolecular DNA and small interfering RNA, were examined upon the interaction with the surfactant. For DNA the interaction was studied using spectral methods and the methods of viscometry and flow birefringence measurement. For siRNA the possibility of surfactant-based delivery was checked in vitro.

  15. How topoisomerase IV can efficiently unknot and decatenate negatively supercoiled DNA molecules without causing their torsional relaxation.

    PubMed

    Rawdon, Eric J; Dorier, Julien; Racko, Dusan; Millett, Kenneth C; Stasiak, Andrzej

    2016-06-02

    Freshly replicated DNA molecules initially form multiply interlinked right-handed catenanes. In bacteria, these catenated molecules become supercoiled by DNA gyrase before they undergo a complete decatenation by topoisomerase IV (Topo IV). Topo IV is also involved in the unknotting of supercoiled DNA molecules. Using Metropolis Monte Carlo simulations, we investigate the shapes of supercoiled DNA molecules that are either knotted or catenated. We are especially interested in understanding how Topo IV can unknot right-handed knots and decatenate right-handed catenanes without acting on right-handed plectonemes in negatively supercoiled DNA molecules. To this end, we investigate how the topological consequences of intersegmental passages depend on the geometry of the DNA-DNA juxtapositions at which these passages occur. We observe that there are interesting differences between the geometries of DNA-DNA juxtapositions in the interwound portions and in the knotted or catenated portions of the studied molecules. In particular, in negatively supercoiled, multiply interlinked, right-handed catenanes, we detect specific regions where DNA segments belonging to two freshly replicated sister DNA molecules form left-handed crossings. We propose that, due to its geometrical preference to act on left-handed crossings, Topo IV can specifically unknot supercoiled DNA, as well as decatenate postreplicative catenanes, without causing their torsional relaxation.

  16. How topoisomerase IV can efficiently unknot and decatenate negatively supercoiled DNA molecules without causing their torsional relaxation

    PubMed Central

    Rawdon, Eric J.; Dorier, Julien; Racko, Dusan; Millett, Kenneth C.; Stasiak, Andrzej

    2016-01-01

    Freshly replicated DNA molecules initially form multiply interlinked right-handed catenanes. In bacteria, these catenated molecules become supercoiled by DNA gyrase before they undergo a complete decatenation by topoisomerase IV (Topo IV). Topo IV is also involved in the unknotting of supercoiled DNA molecules. Using Metropolis Monte Carlo simulations, we investigate the shapes of supercoiled DNA molecules that are either knotted or catenated. We are especially interested in understanding how Topo IV can unknot right-handed knots and decatenate right-handed catenanes without acting on right-handed plectonemes in negatively supercoiled DNA molecules. To this end, we investigate how the topological consequences of intersegmental passages depend on the geometry of the DNA-DNA juxtapositions at which these passages occur. We observe that there are interesting differences between the geometries of DNA-DNA juxtapositions in the interwound portions and in the knotted or catenated portions of the studied molecules. In particular, in negatively supercoiled, multiply interlinked, right-handed catenanes, we detect specific regions where DNA segments belonging to two freshly replicated sister DNA molecules form left-handed crossings. We propose that, due to its geometrical preference to act on left-handed crossings, Topo IV can specifically unknot supercoiled DNA, as well as decatenate postreplicative catenanes, without causing their torsional relaxation. PMID:27106058

  17. Measuring p53 Binding to Single DNA Molecules in a Nanofluidic Device

    NASA Astrophysics Data System (ADS)

    Whelsky, Amber; Gonzalez, Nicholas, Jr.; Gal, Susannah; Levy, Stephen

    2012-02-01

    Protein-DNA binding is central to several important cellular processes, for instance, the transfer of genetic information into proteins. The p53 protein plays a central role in regulating several major cell cycle pathways, in part by binding to well-characterized DNA sequences in the promoters of specific genes. Recent studies show that the most common mutation to the protein occurs in the region responsible for its binding to DNA. We have fabricated slit-like nanofluidic devices that allow us to trap and stretch single molecules of DNA containing a known recognition sequence of p53. We use fluorescent microscopy to observe the diffusion of a single p53 protein as it searches for its DNA recognition site. We measure the reaction rates of binding to selected DNA sequences as well as the one-dimensional, non-sequence specific diffusion of p53 along a stretched DNA molecule as a function of salt concentration. The mechanism of facilitated diffusion attempts to explain how proteins seem able to find their DNA target sequences much more quickly than would be expected from three-dimensional diffusion alone. We compare the observed search mechanism used by normal and mutated p53 from cancer cells to predictions based on this theory.

  18. Dynamics of topological events within single molecules of DNA confined in nanochannels

    NASA Astrophysics Data System (ADS)

    Reifenberger, Jeffrey; Dorfman, Kevin; Cao, Han

    Genome mapping in nanochannels offers the ability to search for large genomic rearrangements within individual molecules of DNA often missed by sequencing techniques. This method labels DNA at specific sequence motifs such as `GCTCTTC' with a cy3-like fluorophore and then stains the backbone of dsDNA with an intercalating dye. DNA is electrophoretically loaded into an array of nanofluidic channels and linearized in physically confined narrow conduits fabricated on the silicon chip. The fluorescently labeled sequence motifs, unique to long genomic regions, are optically imaged and digitized reflecting structural changes that can occur within cancer. However, some molecules of DNA confined within the ~42 nm wide nanochannels contain topological structures: knots, S-folds, and end-folds that could appear as false genomic rearrangements. We present a technique in which thousands of molecules of E. coli DNA are sequentially imaged in the nanochannels during several minutes allowing for topological events like diffusion of knots, unfolding at the ends, and spontaneous formation of S-folds to be measured. This technology will provide insights and a solution in error correction for making more accurate measurements. NIH R01-HG006851.

  19. Integrated view of genome structure and sequence of a single DNA molecule in a nanofluidic device

    PubMed Central

    Marie, Rodolphe; Pedersen, Jonas N.; Bauer, David L. V.; Rasmussen, Kristian H.; Yusuf, Mohammed; Volpi, Emanuela; Flyvbjerg, Henrik; Kristensen, Anders; Mir, Kalim U.

    2013-01-01

    We show how a bird’s-eye view of genomic structure can be obtained at ∼1-kb resolution from long (∼2 Mb) DNA molecules extracted from whole chromosomes in a nanofluidic laboratory-on-a-chip. We use an improved single-molecule denaturation mapping approach to detect repetitive elements and known as well as unique structural variation. Following its mapping, a molecule of interest was rescued from the chip; amplified and localized to a chromosome by FISH; and interrogated down to 1-bp resolution with a commercial sequencer, thereby reconciling haplotype-phased chromosome substructure with sequence. PMID:23479649

  20. A Selective Small Molecule DNA2 Inhibitor for Sensitization of Human Cancer Cells to Chemotherapy

    PubMed Central

    Liu, Wenpeng; Zhou, Mian; Li, Zhengke; Li, Hongzhi; Polaczek, Piotr; Dai, Huifang; Wu, Qiong; Liu, Changwei; Karanja, Kenneth K.; Popuri, Vencat; Shan, Shu-ou; Schlacher, Katharina; Zheng, Li; Campbell, Judith L.; Shen, Binghui

    2016-01-01

    Cancer cells frequently up-regulate DNA replication and repair proteins such as the multifunctional DNA2 nuclease/helicase, counteracting DNA damage due to replication stress and promoting survival. Therefore, we hypothesized that blocking both DNA replication and repair by inhibiting the bifunctional DNA2 could be a potent strategy to sensitize cancer cells to stresses from radiation or chemotherapeutic agents. We show that homozygous deletion of DNA2 sensitizes cells to ionizing radiation and camptothecin (CPT). Using a virtual high throughput screen, we identify 4-hydroxy-8-nitroquinoline-3-carboxylic acid (C5) as an effective and selective inhibitor of DNA2. Mutagenesis and biochemical analysis define the C5 binding pocket at a DNA-binding motif that is shared by the nuclease and helicase activities, consistent with structural studies that suggest that DNA binding to the helicase domain is necessary for nuclease activity. C5 targets the known functions of DNA2 in vivo: C5 inhibits resection at stalled forks as well as reducing recombination. C5 is an even more potent inhibitor of restart of stalled DNA replication forks and over-resection of nascent DNA in cells defective in replication fork protection, including BRCA2 and BOD1L. C5 sensitizes cells to CPT and synergizes with PARP inhibitors. PMID:27211550

  1. Improving the performance of true single molecule sequencing for ancient DNA

    PubMed Central

    2012-01-01

    Background Second-generation sequencing technologies have revolutionized our ability to recover genetic information from the past, allowing the characterization of the first complete genomes from past individuals and extinct species. Recently, third generation Helicos sequencing platforms, which perform true Single-Molecule DNA Sequencing (tSMS), have shown great potential for sequencing DNA molecules from Pleistocene fossils. Here, we aim at improving even further the performance of tSMS for ancient DNA by testing two novel tSMS template preparation methods for Pleistocene bone fossils, namely oligonucleotide spiking and treatment with DNA phosphatase. Results We found that a significantly larger fraction of the horse genome could be covered following oligonucleotide spiking however not reproducibly and at the cost of extra post-sequencing filtering procedures and skewed %GC content. In contrast, we showed that treating ancient DNA extracts with DNA phosphatase improved the amount of endogenous sequence information recovered per sequencing channel by up to 3.3-fold, while still providing molecular signatures of endogenous ancient DNA damage, including cytosine deamination and fragmentation by depurination. Additionally, we confirmed the existence of molecular preservation niches in large bone crystals from which DNA could be preferentially extracted. Conclusions We propose DNA phosphatase treatment as a mechanism to increase sequence coverage of ancient genomes when using Helicos tSMS as a sequencing platform. Together with mild denaturation temperatures that favor access to endogenous ancient templates over modern DNA contaminants, this simple preparation procedure can improve overall Helicos tSMS performance when damaged DNA templates are targeted. PMID:22574620

  2. Self-assembly of molecule-like nanoparticle clusters directed by DNA nanocages.

    PubMed

    Li, Yulin; Liu, Zhiyu; Yu, Guimei; Jiang, Wen; Mao, Chengde

    2015-04-08

    Analogous to the atom-molecule relationship, nanoparticle (NP) clusters (or NP-molecules) with defined compositions and directional bonds could potentially integrate the properties of the component individual NPs, leading to emergent properties. Despite extensive efforts in this direction, no general approach is available for assembly of such NP-molecules. Here we report a general method for building this type of structures by encapsulating NPs into self-assembled DNA polyhedral wireframe nanocages, which serve as guiding agents for further assembly. As a demonstration, a series of NP-molecules have been assembled and validated. Such NP-molecules will, we believe, pave a way to explore new nanomaterials with emergent functions/properties that are related to, but do not belong to the individual component nanoparticles.

  3. NALDB: nucleic acid ligand database for small molecules targeting nucleic acid.

    PubMed

    Kumar Mishra, Subodh; Kumar, Amit

    2016-01-01

    Nucleic acid ligand database (NALDB) is a unique database that provides detailed information about the experimental data of small molecules that were reported to target several types of nucleic acid structures. NALDB is the first ligand database that contains ligand information for all type of nucleic acid. NALDB contains more than 3500 ligand entries with detailed pharmacokinetic and pharmacodynamic information such as target name, target sequence, ligand 2D/3D structure, SMILES, molecular formula, molecular weight, net-formal charge, AlogP, number of rings, number of hydrogen bond donor and acceptor, potential energy along with their Ki, Kd, IC50 values. All these details at single platform would be helpful for the development and betterment of novel ligands targeting nucleic acids that could serve as a potential target in different diseases including cancers and neurological disorders. With maximum 255 conformers for each ligand entry, our database is a multi-conformer database and can facilitate the virtual screening process. NALDB provides powerful web-based search tools that make database searching efficient and simplified using option for text as well as for structure query. NALDB also provides multi-dimensional advanced search tool which can screen the database molecules on the basis of molecular properties of ligand provided by database users. A 3D structure visualization tool has also been included for 3D structure representation of ligands. NALDB offers an inclusive pharmacological information and the structurally flexible set of small molecules with their three-dimensional conformers that can accelerate the virtual screening and other modeling processes and eventually complement the nucleic acid-based drug discovery research. NALDB can be routinely updated and freely available on bsbe.iiti.ac.in/bsbe/naldb/HOME.php. Database URL: http://bsbe.iiti.ac.in/bsbe/naldb/HOME.php.

  4. NALDB: nucleic acid ligand database for small molecules targeting nucleic acid

    PubMed Central

    Kumar Mishra, Subodh; Kumar, Amit

    2016-01-01

    Nucleic acid ligand database (NALDB) is a unique database that provides detailed information about the experimental data of small molecules that were reported to target several types of nucleic acid structures. NALDB is the first ligand database that contains ligand information for all type of nucleic acid. NALDB contains more than 3500 ligand entries with detailed pharmacokinetic and pharmacodynamic information such as target name, target sequence, ligand 2D/3D structure, SMILES, molecular formula, molecular weight, net-formal charge, AlogP, number of rings, number of hydrogen bond donor and acceptor, potential energy along with their Ki, Kd, IC50 values. All these details at single platform would be helpful for the development and betterment of novel ligands targeting nucleic acids that could serve as a potential target in different diseases including cancers and neurological disorders. With maximum 255 conformers for each ligand entry, our database is a multi-conformer database and can facilitate the virtual screening process. NALDB provides powerful web-based search tools that make database searching efficient and simplified using option for text as well as for structure query. NALDB also provides multi-dimensional advanced search tool which can screen the database molecules on the basis of molecular properties of ligand provided by database users. A 3D structure visualization tool has also been included for 3D structure representation of ligands. NALDB offers an inclusive pharmacological information and the structurally flexible set of small molecules with their three-dimensional conformers that can accelerate the virtual screening and other modeling processes and eventually complement the nucleic acid-based drug discovery research. NALDB can be routinely updated and freely available on bsbe.iiti.ac.in/bsbe/naldb/HOME.php. Database URL: http://bsbe.iiti.ac.in/bsbe/naldb/HOME.php PMID:26896846

  5. Selection and characterization of single stranded DNA aptamers for the hormone abscisic Acid.

    PubMed

    Grozio, Alessia; Gonzalez, Victor M; Millo, Enrico; Sturla, Laura; Vigliarolo, Tiziana; Bagnasco, Luca; Guida, Lucrezia; D'Arrigo, Cristina; De Flora, Antonio; Salis, Annalisa; Martin, Elena M; Bellotti, Marta; Zocchi, Elena

    2013-10-01

    The hormone abscisic acid (ABA) is a small molecule involved in pivotal physiological functions in higher plants. Recently, ABA has been also identified as an endogenous hormone in mammals, regulating different cell functions including inflammatory processes, stem cell expansion, insulin release, and glucose uptake. Aptamers are short, single-stranded (ss) oligonucleotidesable to recognize target molecules with high affinity. The small size of the ABA molecule represented a challenge for aptamer development and the aim of this study was to develop specific anti-ABA DNA aptamers. Biotinylated abscisic acid (bio-ABA) was immobilized on streptavidin-coated magnetic beads. DNA aptamers against bio-ABA were selected with 7 iterative rounds of the systematic evolution of ligands by exponential enrichment method (SELEX), each round comprising incubation of the ABA-binding beads with the ssDNA sequences, DNA elution, electrophoresis, and polymerase chain reaction (PCR) amplification. The PCR product was cloned and sequenced. The binding affinity of several clones was determined using bio-ABA immobilized on streptavidin-coated plates. Aptamer 2 and aptamer 9 showed the highest binding affinity, with dissociation constants values of 0.98 ± 0.14 μM and 0.80 ± 0.07 μM, respectively. Aptamers 2 and 9 were also able to bind free, unmodified ABA and to discriminate between different ABA enantiomers and isomers. Our findings indicate that ssDNA aptamers can selectively bind ABA and could be used for the development of ABA quantitation assays.

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

    NASA Astrophysics Data System (ADS)

    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.

  7. Computational methods for the construction, editing, and error correction of DNA molecules and their libraries.

    PubMed

    Raz, Ofir; Ben Yehezkel, Tuval

    2015-01-01

    The field of synthetic biology is fueled by steady advances in our ability to produce designer genetic material on demand. This relatively new technological capability stems from advancements in DNA construction biochemistry as well as supporting computational technologies such as tools for specifying large DNA libraries, as well as planning and optimizing their actual physical construction. In particular, the design, planning, and construction of user specified, combinatorial DNA libraries are of increasing interest. Here we present some of the computational tools we have built over the past decade to support the multidisciplinary task of constructing DNA molecules and their libraries. These technologies encompass computational methods for [1] planning and optimizing the construction of DNA molecules and libraries, [2] the utilization of existing natural or synthetic fragments, [3] identification of shared fragments, [4] planning primers and overlaps, [5] minimizing the number of assembly steps required, and (6) correcting erroneous constructs. Other computational technologies that are important in the overall process of DNA construction, such as [1] computational tools for efficient specification and intuitive visualization of large DNA libraries (which aid in debugging library design pre-construction) and [2] automated liquid handling robotic programming [Linshiz et al., Mol Syst Biol 4:191, 2008; Shabi et al., Syst Synth Biol 4:227-236, 2010], which aid in the construction process itself, have been omitted due to length limitations.

  8. Theory and simulations of toroidal and rod-like structures in single-molecule DNA condensation.

    PubMed

    Cortini, Ruggero; Caré, Bertrand R; Victor, Jean-Marc; Barbi, Maria

    2015-03-14

    DNA condensation by multivalent cations plays a crucial role in genome packaging in viruses and sperm heads, and has been extensively studied using single-molecule experimental methods. In those experiments, the values of the critical condensation forces have been used to estimate the amplitude of the attractive DNA-DNA interactions. Here, to describe these experiments, we developed an analytical model and a rigid body Langevin dynamics assay to investigate the behavior of a polymer with self-interactions, in the presence of a traction force applied at its extremities. We model self-interactions using a pairwise attractive potential, thereby treating the counterions implicitly. The analytical model allows to accurately predict the equilibrium structures of toroidal and rod-like condensed structures, and the dependence of the critical condensation force on the DNA length. We find that the critical condensation force depends strongly on the length of the DNA, and finite-size effects are important for molecules of length up to 10(5)μm. Our Langevin dynamics simulations show that the force-extension behavior of the rod-like structures is very different from the toroidal ones, so that their presence in experiments should be easily detectable. In double-stranded DNA condensation experiments, the signature of the presence of rod-like structures was not unambiguously detected, suggesting that the polyamines used to condense DNA may protect it from bending sharply as needed in the rod-like structures.

  9. Lac Repressor Mediated DNA Looping: Monte Carlo Simulation of Constrained DNA Molecules Complemented with Current Experimental Results

    PubMed Central

    Biton, Yoav Y.; Kumar, Sandip; Dunlap, David; Swigon, David

    2014-01-01

    Tethered particle motion (TPM) experiments can be used to detect time-resolved loop formation in a single DNA molecule by measuring changes in the length of a DNA tether. Interpretation of such experiments is greatly aided by computer simulations of DNA looping which allow one to analyze the structure of the looped DNA and estimate DNA-protein binding constants specific for the loop formation process. We here present a new Monte Carlo scheme for accurate simulation of DNA configurations subject to geometric constraints and apply this method to Lac repressor mediated DNA looping, comparing the simulation results with new experimental data obtained by the TPM technique. Our simulations, taking into account the details of attachment of DNA ends and fluctuations of the looped subsegment of the DNA, reveal the origin of the double-peaked distribution of RMS values observed by TPM experiments by showing that the average RMS value for anti-parallel loop types is smaller than that of parallel loop types. The simulations also reveal that the looping probabilities for the anti-parallel loop types are significantly higher than those of the parallel loop types, even for loops of length 600 and 900 base pairs, and that the correct proportion between the heights of the peaks in the distribution can only be attained when loops with flexible Lac repressor conformation are taken into account. Comparison of the in silico and in vitro results yields estimates for the dissociation constants characterizing the binding affinity between O1 and Oid DNA operators and the dimeric arms of the Lac repressor. PMID:24800809

  10. Nanoconstructions on the base of double-stranded DNA molecules and their optical properties

    NASA Astrophysics Data System (ADS)

    Skuridin, S. G.; Yevdokimov, Yu. M.; Chulkov, D. P.; Gusev, V. M.; Kompanets, O. N.; Vereschagin, F. V.

    2016-12-01

    Experimental results have been presented on studying optical properties of nanoconstructions formed of orientationally ordered neighbouring double-stranded DNA molecules in the structure of their liquid-crystalline phases and dispersion particles of these phases including ones cured with intercalators.

  11. Investigation of localization of DNA molecules using triangular metal electrodes with varying separation

    NASA Astrophysics Data System (ADS)

    Prasad, D. Nagendra; Ghonge, Sudarshan; Banerjee, Souri

    2016-04-01

    In this paper we investigate the effect of separation of triangular metal electrodes with both convex and concave geometries, on the localization of suspended DNA molecules under the combined effect of dielectrophoresis and AC electro-osmosis through simulations using COMSOL Multiphysics. Trapping points are realized within the electrodes which are found to vary with the separation of the electrodes.

  12. Topological events in single molecules of long genomic DNA confined in nanochannels

    NASA Astrophysics Data System (ADS)

    Reifenberger, Jeffrey; Dorfman, Kevin; Cao, Han

    2014-03-01

    ct- We present a rapid genome-wide analysis method based on new NanoChannel Array technology (IrysTM System) that confines and linearizes extremely long DNA molecules (100 to 1,000 kilobases) for direct image analysis at tens to hundred of gigabases per run. Genomic DNA is stained with YOYO and labeled specifically at the `GCTCTTC' sequence with fluorescent dyes allowing each molecule to be uniquely patterned and mapped to its corresponding reference. This high-throughput platform automates the imaging of such barcoded patterns on genomic DNA to identify wide spread structural variations in a genome. Here we describe a method to rule out possible topologically altered molecules in linear confinement by identifying possible topological events through a T-test looking for spikes in the fluorescence of the YOYO stained DNA backbone. These events are confirmed through aligning the marked individual molecules to a standard reference and measuring a distance differential between labels surrounding the suspected topological event compared to the reference. Such events could be flagged to distinguish from true structural variations.

  13. The Dynamics of Entangled DNA Networks using Single-Molecule Methods

    NASA Astrophysics Data System (ADS)

    Chapman, Cole David

    Single molecule experiments were performed on DNA, a model polymer, and entangled DNA networks to explore diffusion within complex polymeric fluids and their linear and non-linear viscoelasticity. DNA molecules of varying length and topology were prepared using biological methods. An ensemble of individual molecules were then fluorescently labeled and tracked in blends of entangled linear and circular DNA to examine the dependence of diffusion on polymer length, topology, and blend ratio. Diffusion was revealed to possess a non-monotonic dependence on the blend ratio, which we believe to be due to a second-order effect where the threading of circular polymers by their linear counterparts greatly slows the mobility of the system. Similar methods were used to examine the diffusive and conformational behavior of DNA within highly crowded environments, comparable to that experienced within the cell. A previously unseen gamma distributed elongation of the DNA in the presence of crowders, proposed to be due to entropic effects and crowder mobility, was observed. Additionally, linear viscoelastic properties of entangled DNA networks were explored using active microrheology. Plateau moduli values verified for the first time the predicted independence from polymer length. However, a clear bead-size dependence was observed for bead radii less than ~3x the tube radius, a newly discovered limit, above which microrheology results are within the continuum limit and may access the bulk properties of the fluid. Furthermore, the viscoelastic properties of entangled DNA in the non-linear regime, where the driven beads actively deform the network, were also examined. By rapidly driving a bead through the network utilizing optical tweezers, then removing the trap and tracking the bead's subsequent motion we are able to model the system as an over-damped harmonic oscillator and find the elasticity to be dominated by stress-dependent entanglements.

  14. Self-assembled nanowire arrays as three-dimensional nanopores for filtration of DNA molecules.

    PubMed

    Rahong, Sakon; Yasui, Takao; Yanagida, Takeshi; Nagashima, Kazuki; Kanai, Masaki; Meng, Gang; He, Yong; Zhuge, Fuwei; Kaji, Noritada; Kawai, Tomoji; Baba, Yoshinobu

    2015-01-01

    Molecular filtration and purification play important roles for biomolecule analysis. However, it is still necessary to improve efficiency and reduce the filtration time. Here, we show self-assembled nanowire arrays as three-dimensional (3D) nanopores embedded in a microfluidic channel for ultrafast DNA filtration. The 3D nanopore structure was formed by a vapor-liquid-solid (VLS) nanowire growth technique, which allowed us to control pore size of the filtration material by varying the number of growth cycles. λ DNA molecules (48.5 kbp) were filtrated from a mixture of T4 DNA (166 kbp) at the entrance of the 3D nanopore structure within 1 s under an applied electric field. Moreover, we observed single DNA molecule migration of T4 and λ DNA molecules to clarify the filtration mechanism. The 3D nanopore structure has simplicity of fabrication, flexibility of pore size control and reusability for biomolecule filtration. Consequently it is an excellent material for biomolecular filtration.

  15. Structure and Stability of Individual DNA or RNA Hairpin Molecules Captured in an Ion Channel

    NASA Astrophysics Data System (ADS)

    Akeson, Mark

    2002-03-01

    Nanoscale pores can be used to analyze individual DNA or RNA molecules. For example, a prototype device based on the alpha-hemolysin protein permits serial examination of hundreds to thousands of molecules per minute. It is routinely used to identify individual polynucleotide homopolymers, and to read segments within single DNA or RNA block copolymers as they thread through a narrow, trans-membrane pore formed by the protein (1.5 nm limiting aperture). In recent reports, we have shown that this device can also be used to examine structural details of individual DNA or RNA hairpins at single base-pair precision. This is achieved by capturing each hairpin in a vestibule of the channel leading to the trans-membrane pore. Under a 120 mV applied voltage, ionic current through the channel is gated by the RNA or DNA hairpin as it is perched in the vestibule, resulting in a dynamic current pattern that is exquisitely sensitive to sequence identity. In this presentation, I will explain the ion channel device and then describe structural details of the hairpin molecules that can be resolved by the instrument. These include: i) Watson-Crick base-pair identity at the hairpin stem terminus; ii) duplex fraying caused by single base pair mismatches internal to the hairpin stem; and iii) A form (RNA) versus B form (DNA) helix conformation. I will then discuss alternative mechanisms that can account for the discrete gating patterns that underlie the analysis.

  16. Replication of individual DNA molecules under electronic control using a protein nanopore

    NASA Astrophysics Data System (ADS)

    Olasagasti, Felix; Lieberman, Kate R.; Benner, Seico; Cherf, Gerald M.; Dahl, Joseph M.; Deamer, David W.; Akeson, Mark

    2010-11-01

    Nanopores can be used to analyse DNA by monitoring ion currents as individual strands are captured and driven through the pore in single file by an applied voltage. Here, we show that serial replication of individual DNA templates can be achieved by DNA polymerases held at the α-haemolysin nanopore orifice. Replication is blocked in the bulk phase, and is initiated only after the DNA is captured by the nanopore. We used this method, in concert with active voltage control, to observe DNA replication catalysed by bacteriophage T7 DNA polymerase (T7DNAP) and by the Klenow fragment of DNA polymerase I (KF). T7DNAP advanced on a DNA template against an 80-mV load applied across the nanopore, and single nucleotide additions were measured on the millisecond timescale for hundreds of individual DNA molecules in series. Replication by KF was not observed when this enzyme was held on top of the nanopore orifice at an applied potential of 80 mV. Sequential nucleotide additions by KF were observed upon applying controlled voltage reversals.

  17. Motion of a DNA Sliding Clamp Observed by Single Molecule Fluorescence Spectroscopy*S⃞

    PubMed Central

    Laurence, Ted A.; Kwon, Youngeun; Johnson, Aaron; Hollars, Christopher W.; O'Donnell, Mike; Camarero, Julio A.; Barsky, Daniel

    2008-01-01

    DNA sliding clamps attach to polymerases and slide along DNA to allow rapid, processive replication of DNA. These clamps contain many positively charged residues that could curtail the sliding due to attractive interactions with the negatively charged DNA. By single-molecule spectroscopy we have observed a fluorescently labeled sliding clamp (polymerase III β subunit or β clamp) loaded onto freely diffusing, single-stranded M13 circular DNA annealed with fluorescently labeled DNA oligomers of up to 90 bases. We find that the diffusion constant for the β clamp diffusing along DNA is on the order of 10–14 m2/s, at least 3 orders of magnitude less than that for diffusion through water alone. We also find evidence that the β clamp remains at the 3′ end in the presence of Escherichia coli single-stranded-binding protein. These results may imply that the clamp not only acts to hold the polymerase on the DNA but also prevents excessive drifting along the DNA. PMID:18556658

  18. Sequence specific sorting of DNA molecules with FACS using 3dPCR

    PubMed Central

    Sukovich, David J.; Lance, Shea T.; Abate, Adam R.

    2017-01-01

    Genetic heterogeneity is an important feature of many biological systems, but introduces technical challenges to their characterization. Even with the best modern instruments, only a small fraction of DNA molecules present in a sample can be read, and they are recovered in the form of short, hundred-base reads. In this paper, we introduce 3dPCR, a method to sort DNA molecules with sequence specificity. 3dPCR allows heterogeneous populations of DNA to be sorted to recover long targets for deep sequencing. It is valuable whenever a target sequence is rare in a mixed population, such as for characterizing mutations in heterogeneous cancer cell populations or identifying cells containing a specific genetic sequence or infected with a target virus. PMID:28051104

  19. Sequence specific sorting of DNA molecules with FACS using 3dPCR.

    PubMed

    Sukovich, David J; Lance, Shea T; Abate, Adam R

    2017-01-04

    Genetic heterogeneity is an important feature of many biological systems, but introduces technical challenges to their characterization. Even with the best modern instruments, only a small fraction of DNA molecules present in a sample can be read, and they are recovered in the form of short, hundred-base reads. In this paper, we introduce 3dPCR, a method to sort DNA molecules with sequence specificity. 3dPCR allows heterogeneous populations of DNA to be sorted to recover long targets for deep sequencing. It is valuable whenever a target sequence is rare in a mixed population, such as for characterizing mutations in heterogeneous cancer cell populations or identifying cells containing a specific genetic sequence or infected with a target virus.

  20. Single-molecule observation of DNA compaction by meiotic protein SYCP3

    PubMed Central

    Syrjänen, Johanna L; Heller, Iddo; Candelli, Andrea; Davies, Owen R; Peterman, Erwin J G; Wuite, Gijs J L; Pellegrini, Luca

    2017-01-01

    In a previous paper (Syrjänen et al., 2014), we reported the first structural characterisation of a synaptonemal complex (SC) protein, SYCP3, which led us to propose a model for its role in chromosome compaction during meiosis. As a component of the SC lateral element, SYCP3 has a critical role in defining the specific chromosome architecture required for correct meiotic progression. In the model, the reported compaction of chromosomal DNA caused by SYCP3 would result from its ability to bridge distant sites on a DNA molecule with the DNA-binding domains located at each end of its strut-like structure. Here, we describe a single-molecule assay based on optical tweezers, fluorescence microscopy and microfluidics that, in combination with bulk biochemical data, provides direct visual evidence for our proposed mechanism of SYCP3-mediated chromosome organisation. DOI: http://dx.doi.org/10.7554/eLife.22582.001 PMID:28287952

  1. Real-time single-molecule studies of the motions of DNA polymerase fingers illuminate DNA synthesis mechanisms.

    PubMed

    Evans, Geraint W; Hohlbein, Johannes; Craggs, Timothy; Aigrain, Louise; Kapanidis, Achillefs N

    2015-07-13

    DNA polymerases maintain genomic integrity by copying DNA with high fidelity. A conformational change important for fidelity is the motion of the polymerase fingers subdomain from an open to a closed conformation upon binding of a complementary nucleotide. We previously employed intra-protein single-molecule FRET on diffusing molecules to observe fingers conformations in polymerase-DNA complexes. Here, we used the same FRET ruler on surface-immobilized complexes to observe fingers-opening and closing of individual polymerase molecules in real time. Our results revealed the presence of intrinsic dynamics in the binary complex, characterized by slow fingers-closing and fast fingers-opening. When binary complexes were incubated with increasing concentrations of complementary nucleotide, the fingers-closing rate increased, strongly supporting an induced-fit model for nucleotide recognition. Meanwhile, the opening rate in ternary complexes with complementary nucleotide was 6 s(-1), much slower than either fingers closing or the rate-limiting step in the forward direction; this rate balance ensures that, after nucleotide binding and fingers-closing, nucleotide incorporation is overwhelmingly likely to occur. Our results for ternary complexes with a non-complementary dNTP confirmed the presence of a state corresponding to partially closed fingers and suggested a radically different rate balance regarding fingers transitions, which allows polymerase to achieve high fidelity.

  2. RAGE is a nucleic acid receptor that promotes inflammatory responses to DNA

    PubMed Central

    Sirois, Cherilyn M.; Jin, Tengchuan; Miller, Allison L.; Bertheloot, Damien; Nakamura, Hirotaka; Horvath, Gabor L.; Mian, Abubakar; Jiang, Jiansheng; Schrum, Jacob; Bossaller, Lukas; Pelka, Karin; Garbi, Natalio; Brewah, Yambasu; Tian, Jane; Chang, ChewShun; Chowdhury, Partha S.; Sims, Gary P.; Kolbeck, Roland; Coyle, Anthony J.; Humbles, Alison A.

    2013-01-01

    Recognition of DNA and RNA molecules derived from pathogens or self-antigen is one way the mammalian immune system senses infection and tissue damage. Activation of immune signaling receptors by nucleic acids is controlled by limiting the access of DNA and RNA to intracellular receptors, but the mechanisms by which endosome-resident receptors encounter nucleic acids from the extracellular space are largely undefined. In this study, we show that the receptor for advanced glycation end-products (RAGE) promoted DNA uptake into endosomes and lowered the immune recognition threshold for the activation of Toll-like receptor 9, the principal DNA-recognizing transmembrane signaling receptor. Structural analysis of RAGE–DNA complexes indicated that DNA interacted with dimers of the outermost RAGE extracellular domains, and could induce formation of higher-order receptor complexes. Furthermore, mice deficient in RAGE were unable to mount a typical inflammatory response to DNA in the lung, indicating that RAGE is important for the detection of nucleic acids in vivo. PMID:24081950

  3. Superresolution imaging of single DNA molecules using stochastic photoblinking of minor groove and intercalating dyes.

    PubMed

    Miller, Helen; Zhou, Zhaokun; Wollman, Adam J M; Leake, Mark C

    2015-10-15

    As proof-of-principle for generating superresolution structural information from DNA we applied a method of localization microscopy utilizing photoblinking comparing intercalating dye YOYO-1 against minor groove binding dye SYTO-13, using a bespoke multicolor single-molecule fluorescence microscope. We used a full-length ∼49 kbp λ DNA construct possessing oligo inserts at either terminus allowing conjugation of digoxigenin and biotin at opposite ends for tethering to a glass coverslip surface and paramagnetic microsphere respectively. We observed stochastic DNA-bound dye photoactivity consistent with dye photoblinking as opposed to binding/unbinding events, evidenced through both discrete simulations and continuum kinetics analysis. We analyzed dye photoblinking images of immobilized DNA molecules using superresolution reconstruction software from two existing packages, rainSTORM and QuickPALM, and compared the results against our own novel home-written software called ADEMS code. ADEMS code generated lateral localization precision values of 30-40 nm and 60-70 nm for YOYO-1 and SYTO-13 respectively at video-rate sampling, similar to rainSTORM, running more slowly than rainSTORM and QuickPALM algorithms but having a complementary capability over both in generating automated centroid distribution and cluster analyses. Our imaging system allows us to observe dynamic topological changes to single molecules of DNA in real-time, such as rapid molecular snapping events. This will facilitate visualization of fluorescently-labeled DNA molecules conjugated to a magnetic bead in future experiments involving newly developed magneto-optical tweezers combined with superresolution microscopy.

  4. Single-Molecule Measurements of Synthesis by DNA Polymerase with Base-Pair Resolution

    NASA Astrophysics Data System (ADS)

    Christian, Thomas; Romano, Louis; Rueda, David

    2010-03-01

    The catalytic mechanism of DNA polymerases involves multiple steps that precede and follow the transfer of a nucleotide to the 3'-hydroxyl of the growing DNA chain. Here we report a single-molecule approach to monitor the movement of E. coli DNA polymerase I (Klenow fragment) on a DNA template during DNA synthesis with single base-pair resolution. As each nucleotide is incorporated, the single-molecule F"orster resonance energy transfer intensity drops in discrete steps to values consistent with single nucleotide incorporations. Purines and pyrimidines are incorporated with comparable rates. A mismatched primer-template junction exhibits dynamics consistent with the primer moving into the exonuclease domain, which was used to determine the fraction of primer-termini bound to the exonuclease and polymerase sites. Most interestingly, we observe a structural change following the incorporation of a correctly paired nucleotide, consistent with transient movement of the polymerase past the pre-insertion site or a conformational change in the polymerase. This may represent a previously unobserved step in the mechanism of DNA synthesis that could be part of the proofreading process.

  5. Electrical conductance of DNA molecules with varied density of itinerant pi electrons.

    PubMed

    Gao, Xu-Tuan; Fu, Xue; Mei, Liang-Mo; Xie, Shi-Jie

    2006-06-21

    The electrical transport of DNA is closely related to the density of itinerant pi electrons because of the strong electron-lattice interaction. The resistivities of two typical DNA molecules [poly(dG)-poly(dC) and lambda-DNA] with varied densities of itinerant pi electrons are calculated. It is found that the dependence of the resistivity on the density of itinerant pi electrons is symmetrical about the half-filling state of itinerant pi electrons in poly(dG)-poly(dC). At the half-filling state, the Peierls phase transition takes place and poly(dG)-poly(dC) has a large resistivity. When the density of itinerant pi electrons departs far from the half-filling state, the resistivity of poly(dG)-poly(dC) becomes small. For lambda-DNA, there is no Peierls phase transition due to the aperiodicity of its base pair arrangement. The resistivity of poly(dG)-poly(dC) decreases with increasing length of the molecular chain, but the resistivity of lambda-DNA increases with increasing length. The conducting mechanisms for poly(dG)-poly(dC) and a few lambda-DNA molecules with varied densities of itinerant pi electrons are analyzed.

  6. Optoelectronic studies on heterocyclic bases of deoxyribonucleic acid for DNA photonics.

    PubMed

    El-Diasty, Fouad; Abdel-Wahab, Fathy

    2015-10-01

    The optoelectronics study of large molecules, particularly π-stacking molecules, such as DNA is really an extremely difficult task. We perform first electronic structure calculations on the heterocyclic bases of 2'-deoxyribonucleic acid based on Lorentz-Fresnel dispersion theory. In the UV-VIS range of spectrum, many of the optoelectronic parameters for DNA four bases namely adenine, guanine, cytosine and thymine are calculated and discussed. The results demonstrate that adenine has the highest hyperpolarizability, whereas thymine has the lowest hyperpolarizability. Cytosine has the lower average oscillator energy and the higher lattice energy. Thymine infers the most stable nucleic base with the lower phonon energy. Thymine also has the highest average oscillator energy and the lower lattice energy. Moreover, the four nucleic acid bases have large band gap energies less than 5 eV with a semiconducting behavior. Guanine shows the smallest band gap and the highest Fermi level energy, whereas adenine elucidates the highest band gap energy.

  7. High-throughput sequencing for the identification of binding molecules from DNA-encoded chemical libraries.

    PubMed

    Buller, Fabian; Steiner, Martina; Scheuermann, Jörg; Mannocci, Luca; Nissen, Ina; Kohler, Manuel; Beisel, Christian; Neri, Dario

    2010-07-15

    DNA-encoded chemical libraries are large collections of small organic molecules, individually coupled to DNA fragments that serve as amplifiable identification bar codes. The isolation of specific binders requires a quantitative analysis of the distribution of DNA fragments in the library before and after capture on an immobilized target protein of interest. Here, we show how Illumina sequencing can be applied to the analysis of DNA-encoded chemical libraries, yielding over 10 million DNA sequence tags per flow-lane. The technology can be used in a multiplex format, allowing the encoding and subsequent sequencing of multiple selections in the same experiment. The sequence distributions in DNA-encoded chemical library selections were found to be similar to the ones obtained using 454 technology, thus reinforcing the concept that DNA sequencing is an appropriate avenue for the decoding of library selections. The large number of sequences obtained with the Illumina method now enables the study of very large DNA-encoded chemical libraries (>500,000 compounds) and reduces decoding costs.

  8. Force and twist dependence of RepC nicking activity on torsionally-constrained DNA molecules

    PubMed Central

    Pastrana, Cesar L.; Carrasco, Carolina; Akhtar, Parvez; Leuba, Sanford H.; Khan, Saleem A.; Moreno-Herrero, Fernando

    2016-01-01

    Many bacterial plasmids replicate by an asymmetric rolling-circle mechanism that requires sequence-specific recognition for initiation, nicking of one of the template DNA strands and unwinding of the duplex prior to subsequent leading strand DNA synthesis. Nicking is performed by a replication-initiation protein (Rep) that directly binds to the plasmid double-stranded origin and remains covalently bound to its substrate 5′-end via a phosphotyrosine linkage. It has been proposed that the inverted DNA sequences at the nick site form a cruciform structure that facilitates DNA cleavage. However, the role of Rep proteins in the formation of this cruciform and the implication for its nicking and religation functions is unclear. Here, we have used magnetic tweezers to directly measure the DNA nicking and religation activities of RepC, the replication initiator protein of plasmid pT181, in plasmid sized and torsionally-constrained linear DNA molecules. Nicking by RepC occurred only in negatively supercoiled DNA and was force- and twist-dependent. Comparison with a type IB topoisomerase in similar experiments highlighted a relatively inefficient religation activity of RepC. Based on the structural modeling of RepC and on our experimental evidence, we propose a model where RepC nicking activity is passive and dependent upon the supercoiling degree of the DNA substrate. PMID:27488190

  9. When Maxwellian demon meets action at a distance. Comment on "Disentangling DNA molecules" by Alexander Vologodskii

    NASA Astrophysics Data System (ADS)

    Rybenkov, Valentin V.

    2016-09-01

    The ability of living systems to defy thermodynamics without explicitly violating it is a continued source of inspiration to many biophysicists. The story of type-2 DNA topoisomerases is a beautiful example from that book. DNA topoisomerases catalyze a concerted DNA cleavage-religation reaction, which is interjected by a strand passage event. This sequence of events results in a seemingly unhindered transfer of one piece of DNA through another upon their random collision. An obvious consequence of such transfer is a change in the topological state of the colliding DNAs; hence the name of the enzymes, topoisomerases. There are several classes of topoisomerases, which differ in how they capture the cleaved and transported DNA segments (which are often referred to as the gate and transfer segments; or the G- and T-segments, to be short). Type-2 topoisomerases have two cleavage-religation centers. They open a gate in double stranded DNA and transfer another piece of double stranded DNA through it [1]. And in doing so, they manage to collect information about the rest of the DNA and perform strand passage in a directional manner so as to take the molecule away from the thermodynamic equilibrium [2].

  10. Simultaneous single-molecule epigenetic imaging of DNA methylation and hydroxymethylation

    PubMed Central

    Song, Chun-Xiao; Brunger, Axel T.; Quake, Stephen R.

    2016-01-01

    The modifications 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) are the two major DNA epigenetic modifications in mammalian genomes and play crucial roles in development and pathogenesis. Little is known about the colocalization or potential correlation of these two modifications. Here we present an ultrasensitive single-molecule imaging technology capable of detecting and quantifying 5hmC and 5mC from trace amounts of DNA. We used this approach to perform single-molecule fluorescence resonance energy transfer (smFRET) experiments which measure the proximity between 5mC and 5hmC in the same DNA molecule. Our results reveal high levels of adjacent and opposing methylated and hydroxymethylated CpG sites (5hmC/5mCpGs) in mouse genomic DNA across multiple tissues. This identifies the previously undetectable and unappreciated 5hmC/5mCpGs as one of the major states for 5hmC in the mammalian genome and suggest that they could function in promoting gene expression. PMID:27035984

  11. Single-Molecule Counting of Point Mutations by Transient DNA Binding

    PubMed Central

    Su, Xin; Li, Lidan; Wang, Shanshan; Hao, Dandan; Wang, Lei; Yu, Changyuan

    2017-01-01

    High-confidence detection of point mutations is important for disease diagnosis and clinical practice. Hybridization probes are extensively used, but are hindered by their poor single-nucleotide selectivity. Shortening the length of DNA hybridization probes weakens the stability of the probe-target duplex, leading to transient binding between complementary sequences. The kinetics of probe-target binding events are highly dependent on the number of complementary base pairs. Here, we present a single-molecule assay for point mutation detection based on transient DNA binding and use of total internal reflection fluorescence microscopy. Statistical analysis of single-molecule kinetics enabled us to effectively discriminate between wild type DNA sequences and single-nucleotide variants at the single-molecule level. A higher single-nucleotide discrimination is achieved than in our previous work by optimizing the assay conditions, which is guided by statistical modeling of kinetics with a gamma distribution. The KRAS c.34 A mutation can be clearly differentiated from the wild type sequence (KRAS c.34 G) at a relative abundance as low as 0.01% mutant to WT. To demonstrate the feasibility of this method for analysis of clinically relevant biological samples, we used this technology to detect mutations in single-stranded DNA generated from asymmetric RT-PCR of mRNA from two cancer cell lines. PMID:28262827

  12. Single-Molecule Counting of Point Mutations by Transient DNA Binding

    NASA Astrophysics Data System (ADS)

    Su, Xin; Li, Lidan; Wang, Shanshan; Hao, Dandan; Wang, Lei; Yu, Changyuan

    2017-03-01

    High-confidence detection of point mutations is important for disease diagnosis and clinical practice. Hybridization probes are extensively used, but are hindered by their poor single-nucleotide selectivity. Shortening the length of DNA hybridization probes weakens the stability of the probe-target duplex, leading to transient binding between complementary sequences. The kinetics of probe-target binding events are highly dependent on the number of complementary base pairs. Here, we present a single-molecule assay for point mutation detection based on transient DNA binding and use of total internal reflection fluorescence microscopy. Statistical analysis of single-molecule kinetics enabled us to effectively discriminate between wild type DNA sequences and single-nucleotide variants at the single-molecule level. A higher single-nucleotide discrimination is achieved than in our previous work by optimizing the assay conditions, which is guided by statistical modeling of kinetics with a gamma distribution. The KRAS c.34 A mutation can be clearly differentiated from the wild type sequence (KRAS c.34 G) at a relative abundance as low as 0.01% mutant to WT. To demonstrate the feasibility of this method for analysis of clinically relevant biological samples, we used this technology to detect mutations in single-stranded DNA generated from asymmetric RT-PCR of mRNA from two cancer cell lines.

  13. A single-molecule approach to visualize the unwinding activity of DNA helicases.

    PubMed

    Fili, Natalia; Toseland, Christopher P; Dillingham, Mark S; Webb, Martin R; Molloy, Justin E

    2011-01-01

    Almost all aspects of DNA metabolism involve separation of double-stranded DNA catalyzed by helicases. Observation and measurement of the dynamics of these events at the single-molecule level provide important mechanistic details of helicase activity and give the opportunity to probe aspects that are not revealed in bulk solution measurements. The assay, presented here, provides information about helicase unwinding rates and processivity. Visualization is achieved by using a fluorescent single-stranded DNA-binding protein (SSB), which allows the time course of individual DNA unwinding events to be observed using total internal reflection fluorescence microscopy. Observation of a prototypical helicase, Bacillus subtilis AddAB, shows that the unwinding process consists of bursts of unwinding activity, interspersed with periods of pausing.

  14. Design, synthesis and biological activity of novel molecules designed to target PARP and DNA.

    PubMed

    Goodfellow, Elliot; Senhaji Mouhri, Zhor; Williams, Christopher; Jean-Claude, Bertrand J

    2017-02-01

    In order to enhance the cytotoxic potential of poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1 or 2 deficient tumours, we designed a series of molecules containing a 1,2,3-triazene moiety tethered to a PARP targeting scaffold. A cell-based selectivity assay involving a BRCA2-deficient Chinese hamster cell line and its corresponding BRCA2 wild type transfectant, was used to predict the PARP targeting potential of the latter agents. The results showed that adding a DNA damaging function to the PARP inhibitors decreased but did not abrogate the selective targeting of the BRCA2-deficient cells. The DNA damaging moiety augmented the potency in BRCA2 deficient cells by 2-20 fold. The most selective dual PARP-DNA targeting agent 14b was found to possess dual DNA and PARP targeting properties.

  15. Coil-stretch Transition of DNA Molecules in Slit-like Confinement

    PubMed Central

    Tang, Jing; Trahan, Daniel W.; Doyle, Patrick S.

    2010-01-01

    We experimentally investigate the influence of slit-like confinement on the coil-stretch transition of single DNA molecules in a homogeneous planar elongational electric field. We observe a more gradual coil-stretch transition characterized by two distinct critical strain rates for DNA in confinement, different from the unconfined case where a single critical strain rate exists. We postulate that the change in the coil-stretch transition is due to a modified spring law in confinement. We develop a dumbbell model to extract an effective spring law by following the relaxation of an initially stretched DNA. We then use this spring law and kinetic theory modeling to predict the extension and fluctuations of DNA in planar elongational fields. The model predicts that a two-stage coil-stretch transition emerges in confinement, in accord with experimental observations. PMID:21399708

  16. Digital quantification of rolling circle amplified single DNA molecules in a resistive pulse sensing nanopore.

    PubMed

    Kühnemund, M; Nilsson, M

    2015-05-15

    Novel portable, sensitive and selective DNA sensor methods for bio-sensing applications are required that can rival conventionally used non-portable and expensive fluorescence-based sensors. In this paper, rolling circle amplification (RCA) products are detected in solution and on magnetic particles using a resistive pulse sensing (RPS) nanopore. Low amounts of DNA molecules are detected by padlock probes which are circularized in a strictly target dependent ligation reaction. The DNA-padlock probe-complex is captured on magnetic particles by sequence specific capture oligonucleotides and amplified by a short RCA. Subsequent RPS analysis is used to identify individual particles with single attached RCA products from blank particles. This proof of concept opens up for a novel non-fluorescent digital DNA quantification method that can have many applications in bio-sensing and diagnostic approaches.

  17. Local thermodynamics of the water molecules around single- and double-stranded DNA studied by grid inhomogeneous solvation theory

    NASA Astrophysics Data System (ADS)

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

    2016-09-01

    Thermodynamic properties of water molecules around single- and double-stranded DNAs (ssDNAs and dsDNAs) with different sequences were investigated using grid inhomogeneous solvation theory. Free energies of water molecules solvating the minor groove of dsDNAs are lower than those near ssDNAs, while water molecules should be released during the formation of dsDNA. Free energies of water molecules around dsDNA are lower than those around ssDNA even in the second and third hydration shells. Our findings will help to clarify the role of water molecules in the formation of dsDNA from ssDNAs, thus facilitating the designs of drugs or nanomaterials using DNA.

  18. Coordinate Variation in Lengths of Deoxyribonucleic Acid Molecules and Head Lengths in Morphological Variants of Bacteriophage T4

    PubMed Central

    Mosig, Gisela; Carnighan, Janet Renshaw; Bibring, Jane Baxandall; Cole, Robert; Bock, Hans-Georg Otto; Bock, Susan

    1972-01-01

    We have investigated three classes of small bacteriophage T4 particles which differ from normal T4 particles in length of their deoxyribonucleic acid (DNA), in head length, in protein content, and in density. The different particles contain DNA molecules measuring 0.90, 0.77, or 0.67, respectively, of the normal T4 length. An additional class of viable particles contains DNA molecules of 1.1 unit length. These discrete differences in DNA length correspond to discrete differences in length (but not width) of the respective heads and are roughly proportional to the resulting differences in head volumes. The measured relative dimensions of the different heads fit best the relative dimensions predicted by a quasi-icosahedral model in which the smallest T4 head corresponds to an icosahedron with a triangulation number T = 21. The mid-portion of this structure is thought to be elongated by adding successive rows of gene 23 protein hexamers, the normal T4 head having three added rows. Different mutants produce small particles of the three classes in varying proportions, but no mutant produces exclusively particles of a single class. Particles of each class, with indistinguishable DNA content, show additional minor differences in protein content, as measured by differences in buoyant density and in the relative ratio of 32P to 35S. Images PMID:5025493

  19. Cyclic diguanylic acid behaves as a host molecule for planar intercalators.

    PubMed

    Liaw, Y C; Gao, Y G; Robinson, H; Sheldrick, G M; Sliedregt, L A; van der Marel, G A; van Boom, J H; Wang, A H

    1990-05-21

    Cyclic ribodiguanylic acid, c-(GpGp), is the endogenous effector regulator of cellulose synthase. Its three-dimensional structure from two different crystal forms (tetragonal and trigonal) has been determined by X-ray diffraction analysis at 1 A resolution. In both crystal forms, two independent c-(GpGp) molecules associate with each other to form a self-intercalated dimer. A hydrated cobalt ion is found to coordinate to two N7 atoms of adjacent guanines, forcing these two guanines to destack with a large dihedral angle (32 degrees), in the dimer of the tetragonal form. This metal coordination mechanism may be relevant to that of the anticancer drug cisplatin. Moreover, c-(GpGp) exhibits unusual spectral properties not seen in any other cyclic dinucleotide. It interacts with planar organic intercalator molecules in ways similar to double helical DNA. We propose a cage-like model consisting of a tetrameric c-(GpGp) aggregate in which a large cavity ('host') is generated to afford a binding site for certain planar intercalators ('guests').

  20. Design, synthesis and selection of DNA-encoded small-molecule libraries.

    PubMed

    Clark, Matthew A; Acharya, Raksha A; Arico-Muendel, Christopher C; Belyanskaya, Svetlana L; Benjamin, Dennis R; Carlson, Neil R; Centrella, Paolo A; Chiu, Cynthia H; Creaser, Steffen P; Cuozzo, John W; Davie, Christopher P; Ding, Yun; Franklin, G Joseph; Franzen, Kurt D; Gefter, Malcolm L; Hale, Steven P; Hansen, Nils J V; Israel, David I; Jiang, Jinwei; Kavarana, Malcolm J; Kelley, Michael S; Kollmann, Christopher S; Li, Fan; Lind, Kenneth; Mataruse, Sibongile; Medeiros, Patricia F; Messer, Jeffrey A; Myers, Paul; O'Keefe, Heather; Oliff, Matthew C; Rise, Cecil E; Satz, Alexander L; Skinner, Steven R; Svendsen, Jennifer L; Tang, Lujia; van Vloten, Kurt; Wagner, Richard W; Yao, Gang; Zhao, Baoguang; Morgan, Barry A

    2009-09-01

    Biochemical combinatorial techniques such as phage display, RNA display and oligonucleotide aptamers have proven to be reliable methods for generation of ligands to protein targets. Adapting these techniques to small synthetic molecules has been a long-sought goal. We report the synthesis and interrogation of an 800-million-member DNA-encoded library in which small molecules are covalently attached to an encoding oligonucleotide. The library was assembled by a combination of chemical and enzymatic synthesis, and interrogated by affinity selection. We describe methods for the selection and deconvolution of the chemical display library, and the discovery of inhibitors for two enzymes: Aurora A kinase and p38 MAP kinase.

  1. Stable Valence Anions of Nucleic Acid Bases and DNA Strand Breaks Induced by Low Energy Electrons

    SciTech Connect

    Rak, Janusz; Mazurkiewicz, Kamil; Kobylecka, Monika; Storoniak, Piotr; Haranczyk, Maciej; Dabkowska, Iwona; Bachorz, Rafal A.; Gutowski, Maciej S.; Radisic, Dunja; Stokes, Sarah T.; Eustis, Soren; Wang, Di; Li, Xiang; Ko, Yeon J.; Bowen, Kit H.

    2008-05-08

    The investigation of structures and properties of nucleic acids has fascinated and challenged researchers ever since the discovery of their relation to genes. Extensive studies have been carried out on these species to unravel the mystery behind the selection of these molecules as genetic material by nature and to explain various physico-chemical properties. However, a vast pool of information is yet to be discovered. DNA constituents, mainly aromatic purine and pyrimidine bases, absorb ultraviolet irradiation efficiently, but the absorbed energy is quickly released in the form of ultrafast nonradiative decays. Recently impressive progress has been made towards the understanding of photophysical and photochemical properties of DNA fragments.

  2. Topology simplification: Important biological phenomenon or evolutionary relic?. Comment on "Disentangling DNA molecules" by Alexander Vologodskii

    NASA Astrophysics Data System (ADS)

    Bates, Andrew D.; Maxwell, Anthony

    2016-09-01

    The review, Disentangling DNA molecules[1], gives an excellent technical description of the phenomenon of topology simplification (TS) by type IIA DNA topoisomerases (topos). In the 20 years since its discovery [2], this effect has attracted a good deal of attention, probably because of its apparently magical nature, and because it seemed to offer a solution to the conundrum that all type II topos rely on ATP hydrolysis, but only bacterial DNA gyrases were known to transduce the free energy of hydrolysis into torsion (supercoiling) in the DNA. It made good sense to think that the other enzymes are using the energy to reduce the level of supercoiling, knotting, and particularly decatenation (unlinking), below equilibrium, since the key activity of the non-supercoiling topos is the removal of links between daughter chromosomes [3]. As Vologodskii discusses [1], there have been a number of theoretical models developed to explain how the local effect of a type II topo can influence the global level of knotting and catenation in large DNA molecules, and he explains how features of two of the most successful models (bent G segment and hooked juxtapositions) may be combined to explain the magnitude of the effect and overcome a kinetic problem with the hooked juxtaposition model.

  3. Electromigration of single molecules of DNA in a crystalline array of 300-nm silica colloids.

    PubMed

    Zhang, Hui; Wirth, Mary J

    2005-03-01

    The velocities and conformations of single DNA chains were probed as they electromigrated at varying electric field strength through a crystalline array of silica colloids. An optically transparent film consisting of 300-nm silica colloids was formed on glass as a transparent crystalline layer of 7 mum thickness with an effective pore size of 45 nm. The behaviors of individual lambda-DNA molecules (48,502 base pairs) electromigrating through this material were observed to be analogous to the behaviors of long DNA chains in electrophoresis gels, including chain extension, hooking of chains around the matrix, and hernia formation. The electrophoretic mobility of lambda-DNA in this dense, narrow-pore material is surprisingly high: 1.8 cm2/Vs at 10 V/cm, which is at least as high as for much wider-pore gels. Imaging of the single molecules revealed that higher field strength caused increased chain extension and increased mobility, which reached an apparent plateau just above 2.0 cm2/Vs at 200 V/cm. Pulsed, crossed electric fields of 200 V/cm at 120 degrees to one another were applied to the material. The DNA chains were observed by imaging to electromigrate in an orderly fashion, and the migration rate was found to be length-dependent. The results indicate that these thin, robust, self-assembling inorganic materials are interesting as possible alternatives to polymeric gels for higher speed electrophoresis.

  4. Single-molecule study of DNA unlinking by eukaryotic and prokaryotic type-II topoisomerases

    NASA Astrophysics Data System (ADS)

    Charvin, G.; Bensimon, D.; Croquette, V.

    2003-08-01

    Type-II topoisomerases are responsible for untangling DNA during replication by removing supercoiled and interlinked DNA structures. Using a single-molecule micromanipulation setup, we follow the real-time decatenation of two mechanically braided DNA molecules by Drosophila melanogaster topoisomerase (Topo) II and Escherichia coli Topo IV. Although Topo II relaxes left-handed (L) and right-handed (R-) braids similarly at a rate of 2.9 s-1, Topo IV has a marked preference for L-braids, which it relaxes completely and processively at a rate of 2.4 s-1. However, Topo IV can unlink R-braids at about half that rate when they supercoil to form L-plectonemes. These results imply that the preferred substrate for unlinking by Topo IV has the symmetry of an L-crossing and shed new light on the decatenation of daughter strands during DNA replication, which are usually assumed to be linked in an R-braid. DNA replication

  5. Single Molecule Bioelectronics and Their Application to Amplification-Free Measurement of DNA Lengths.

    PubMed

    Gül, O Tolga; Pugliese, Kaitlin M; Choi, Yongki; Sims, Patrick C; Pan, Deng; Rajapakse, Arith J; Weiss, Gregory A; Collins, Philip G

    2016-06-24

    As biosensing devices shrink smaller and smaller, they approach a scale in which single molecule electronic sensing becomes possible. Here, we review the operation of single-enzyme transistors made using single-walled carbon nanotubes. These novel hybrid devices transduce the motions and catalytic activity of a single protein into an electronic signal for real-time monitoring of the protein's activity. Analysis of these electronic signals reveals new insights into enzyme function and proves the electronic technique to be complementary to other single-molecule methods based on fluorescence. As one example of the nanocircuit technique, we have studied the Klenow Fragment (KF) of DNA polymerase I as it catalytically processes single-stranded DNA templates. The fidelity of DNA polymerases makes them a key component in many DNA sequencing techniques, and here we demonstrate that KF nanocircuits readily resolve DNA polymerization with single-base sensitivity. Consequently, template lengths can be directly counted from electronic recordings of KF's base-by-base activity. After measuring as few as 20 copies, the template length can be determined with <1 base pair resolution, and different template lengths can be identified and enumerated in solutions containing template mixtures.

  6. Simultaneous Single-Molecule Force and Fluorescence Sampling of DNA Nanostructure Conformations Using Magnetic Tweezers.

    PubMed

    Kemmerich, Felix E; Swoboda, Marko; Kauert, Dominik J; Grieb, M Svea; Hahn, Steffen; Schwarz, Friedrich W; Seidel, Ralf; Schlierf, Michael

    2016-01-13

    We present a hybrid single-molecule technique combining magnetic tweezers and Förster resonance energy transfer (FRET) measurements. Through applying external forces to a paramagnetic sphere, we induce conformational changes in DNA nanostructures, which are detected in two output channels simultaneously. First, by tracking a magnetic bead with high spatial and temporal resolution, we observe overall DNA length changes along the force axis. Second, the measured FRET efficiency between two fluorescent probes monitors local conformational changes. The synchronized orthogonal readout in different observation channels will facilitate deciphering the complex mechanisms of biomolecular machines.

  7. Using Amino-Labeled Nucleotide Probes for Simultaneous Single Molecule RNA-DNA FISH

    PubMed Central

    Wu, Jun; Shao, Fangwei; Zhang, Li-Feng

    2014-01-01

    Using amino-labeled oligonucleotide probes, we established a simple, robust and low-noise method for simultaneous detection of RNA and DNA by fluorescence in situ hybridization, a highly useful tool to study the large pool of long non-coding RNAs being identified in the current research. With probes either chemically or biologically synthesized, we demonstrate that the method can be applied to study a wide range of RNA and DNA targets at the single-cell and single-molecule level in cellular contexts. PMID:25226542

  8. A Device for Performing Lateral Conductance Measurements on Individual Double-Stranded DNA Molecules

    PubMed Central

    Menard, Laurent D.; Mair, Chad E.; Woodson, Michael E.; Alarie, Jean Pierre; Ramsey, J. Michael

    2012-01-01

    A nanofluidic device is described that is capable of electrically monitoring the driven translocation of DNA molecules through a nanochannel. This is achieved by intersecting a long transport channel with a shorter orthogonal nanochannel. The ionic conductance of this transverse nanochannel is monitored while DNA is electrokinetically driven through the transport channel. When DNA passes the intersection, the transverse conductance is altered, resulting in a transient current response. In 1 M KCl solutions, this was found to be a current enhancement of 5–25%, relative to the baseline transverse ionic current. Two different device geometries were investigated. In one device, the DNA was detected after it was fully inserted into and translocating through the transport nanochannel. In the other device, the DNA was detected while it was in the process of entering the nanochannel. It was found that these two conditions are characterized by different transport dynamics. Simultaneous optical and electrical monitoring of DNA translocation confirmed that the transient events originated from DNA transport through the nanochannel intersection. PMID:22950784

  9. Identification and molecular characterization of begomovirus and associated satellite DNA molecules infecting Cyamopsis tetragonoloba.

    PubMed

    Kumar, J; Kumar, A; Roy, J K; Tuli, R; Khan, J A

    2010-08-01

    Monopartite begomoviruses comprise DNA-A as the main genome and associated satellite DNAs. Viral DNA extracted from guar (Cyamopsis tetragonoloba) showing leaf curl symptoms exhibited positive amplification of coat protein (CP) gene of DNA-A component, suggesting the presence of begomovirus. Full length DNA-A was amplified by primer pair re-designed from CP gene nucleotide sequence. The associated alphasatellite and betasatellite DNA molecules were amplified and sequenced, confirming the presence of monopartite begomovirus. Sequence comparisons showed 89% identity with other begomoviruses. The Neighbor-Joining tree based on full length DNA-A nucleotide sequence showed that the guar infecting begomovirus clustered separately from other known begomoviruses. The betasatellite shared a high (96%) nucleotide identity to Cotton leaf curl Multan betasatellites. The alphasatellite showed 91% nucleotide identity to alphasatellite associated with begomovirus infecting Okra. Recombination analyses showed three recombinant fragments in DNA-A, two in betasatellite, and four in alphasatellite. The results suggest that the begomovirus identified in this study was a new recombinant virus. Its name was proposed as Cyamopsis tetragonoloba leaf curl virus (CyTLCuV).

  10. Investigating hexameric helicases: Single-molecule studies of DnaB and T4 gp41

    NASA Astrophysics Data System (ADS)

    Saleh, Omar; Ribeck, Noah; Berezney, John

    2011-03-01

    Hexameric, ring-shaped motor proteins serve as replicative helicases in many systems. They function by encircling and translocating along ssDNA, denaturing dsDNA in advance of its motion by sterically occluding the complementary strand to the outside of the ring. We investigate the helicase activity of two such motors using single-molecule measurements with magnetic tweezers. First, we measure the activity of the E. coli helicase DnaB complexed with the tau subunit of the Pol III holoenzyme. Tau is known from bulk measurements to stimulate DnaB activity (Kim et al., Cell, 1996); we investigate the means of this stimulation. Second, we measure helicase activity of the T4 phage helicase gp41 in multiple tethered DNA geometries. Previous work on DnaB showed a dependence of helicase activity on DNA geometry (Ribeck et al., Biophys. J., 2010); here, we test gp41 for similar behavior to see whether it is a common characteristic of hexameric helicases.

  11. Ultrafast and Wide Range Analysis of DNA Molecules Using Rigid Network Structure of Solid Nanowires

    PubMed Central

    Rahong, Sakon; Yasui, Takao; Yanagida, Takeshi; Nagashima, Kazuki; Kanai, Masaki; Klamchuen, Annop; Meng, Gang; He, Yong; Zhuge, Fuwei; Kaji, Noritada; Kawai, Tomoji; Baba, Yoshinobu

    2014-01-01

    Analyzing sizes of DNA via electrophoresis using a gel has played an important role in the recent, rapid progress of biology and biotechnology. Although analyzing DNA over a wide range of sizes in a short time is desired, no existing electrophoresis methods have been able to fully satisfy these two requirements. Here we propose a novel method using a rigid 3D network structure composed of solid nanowires within a microchannel. This rigid network structure enables analysis of DNA under applied DC electric fields for a large DNA size range (100 bp–166 kbp) within 13 s, which are much wider and faster conditions than those of any existing methods. The network density is readily varied for the targeted DNA size range by tailoring the number of cycles of the nanowire growth only at the desired spatial position within the microchannel. The rigid dense 3D network structure with spatial density control plays an important role in determining the capability for analyzing DNA. Since the present method allows the spatial location and density of the nanostructure within the microchannels to be defined, this unique controllability offers a new strategy to develop an analytical method not only for DNA but also for other biological molecules. PMID:24918865

  12. Ultrafast and wide range analysis of DNA molecules using rigid network structure of solid nanowires.

    PubMed

    Rahong, Sakon; Yasui, Takao; Yanagida, Takeshi; Nagashima, Kazuki; Kanai, Masaki; Klamchuen, Annop; Meng, Gang; He, Yong; Zhuge, Fuwei; Kaji, Noritada; Kawai, Tomoji; Baba, Yoshinobu

    2014-06-11

    Analyzing sizes of DNA via electrophoresis using a gel has played an important role in the recent, rapid progress of biology and biotechnology. Although analyzing DNA over a wide range of sizes in a short time is desired, no existing electrophoresis methods have been able to fully satisfy these two requirements. Here we propose a novel method using a rigid 3D network structure composed of solid nanowires within a microchannel. This rigid network structure enables analysis of DNA under applied DC electric fields for a large DNA size range (100 bp-166 kbp) within 13 s, which are much wider and faster conditions than those of any existing methods. The network density is readily varied for the targeted DNA size range by tailoring the number of cycles of the nanowire growth only at the desired spatial position within the microchannel. The rigid dense 3D network structure with spatial density control plays an important role in determining the capability for analyzing DNA. Since the present method allows the spatial location and density of the nanostructure within the microchannels to be defined, this unique controllability offers a new strategy to develop an analytical method not only for DNA but also for other biological molecules.

  13. Adsorption of peptide nucleic acid and DNA decamers at electrically charged surfaces.

    PubMed Central

    Fojta, M; Vetterl, V; Tomschik, M; Jelen, F; Nielsen, P; Wang, J; Palecek, E

    1997-01-01

    Adsorption behavior of peptide nucleic acid (PNA) and DNA decamers (GTAGATCACT and the complementary sequence) on a mercury surface was studied by means of AC impedance measurements at a hanging mercury drop electrode. The nucleic acid was first attached to the electrode by adsorption from a 5-microliter drop of PNA (or DNA) solution, and the electrode with the adsorbed nucleic acid layer was then washed and immersed in the blank background electrolyte where the differential capacity C of the electrode double layer was measured as a function of the applied potential E. It was found that the adsorption behavior of the PNA with an electrically neutral backbone differs greatly from that of the DNA (with a negatively charged backbone), whereas the DNA-PNA hybrid shows intermediate behavior. At higher surface coverage PNA molecules associate at the surface, and the minimum value of C is shifted to negative potentials because of intermolecular interactions of PNA at the surface. Prolonged exposure of PNA to highly negative potentials does not result in PNA desorption, whereas almost all of the DNA is removed from the surface at these potentials. Adsorption of PNA decreases with increasing NaCl concentration in the range from 0 to 50 mM NaCl, in contrast to DNA, the adsorption of which increases under the same conditions. PMID:9129832

  14. Integrating DNA-strand-displacement circuitry with self-assembly of spherical nucleic acids.

    PubMed

    Yao, Dongbao; Song, Tingjie; Sun, Xianbao; Xiao, Shiyan; Huang, Fujian; Liang, Haojun

    2015-11-11

    Programmable and algorithmic behaviors of DNA molecules allow one to control the structures of DNA-assembled materials with nanometer precision and to construct complex networks with digital and analog behaviors. Here we developed a way of integrating a DNA-strand-displacement circuit with self-assembly of spherical nucleic acids, wherein a single DNA strand was used to initiate and catalyze the operation of upstream circuits to release a single strand that subsequently triggers self-assembly of spherical nucleic acids in downstream circuits, realizing a programmable kinetic control of self-assembly of spherical nucleic acids. Through utilizing this method, single-nucleotide polymorphisms or indels occurring at different positions of a sequence of oligonucleotide were unambiguously discriminated. We provide here a sophisticated way of combining the DNA-strand-displacement-based characteristic of DNA with the distinct assembly properties of inorganic nanoparticles, which may find broad potential applications in the fabrication of a wide range of complex multicomponent devices and architectures.

  15. Sequence-specific nucleic acid mobility using a reversible block copolymer gel matrix and DNA amphiphiles (lipid-DNA) in capillary and microfluidic electrophoretic separations.

    PubMed

    Wagler, Patrick; Minero, Gabriel Antonio S; Tangen, Uwe; de Vries, Jan Willem; Prusty, Deepak; Kwak, Minseok; Herrmann, Andreas; McCaskill, John S

    2015-10-01

    Reversible noncovalent but sequence-dependent attachment of DNA to gels is shown to allow programmable mobility processing of DNA populations. The covalent attachment of DNA oligomers to polyacrylamide gels using acrydite-modified oligonucleotides has enabled sequence-specific mobility assays for DNA in gel electrophoresis: sequences binding to the immobilized DNA are delayed in their migration. Such a system has been used for example to construct complex DNA filters facilitating DNA computations. However, these gels are formed irreversibly and the choice of immobilized sequences is made once off during fabrication. In this work, we demonstrate the reversible self-assembly of gels combined with amphiphilic DNA molecules, which exhibit hydrophobic hydrocarbon chains attached to the nucleobase. This amphiphilic DNA, which we term lipid-DNA, is synthesized in advance and is blended into a block copolymer gel to induce sequence-dependent DNA retention during electrophoresis. Furthermore, we demonstrate and characterize the programmable mobility shift of matching DNA in such reversible gels both in thin films and microchannels using microelectrode arrays. Such sequence selective separation may be employed to select nucleic acid sequences of similar length from a mixture via local electronics, a basic functionality that can be employed in novel electronic chemical cell designs and other DNA information-processing systems.

  16. Radical reaction in aqueous media injected by atmospheric pressure plasma jet and protective effect of antioxidant reagents evaluated by single-molecule DNA measurement

    NASA Astrophysics Data System (ADS)

    Kurita, Hirofumi; Shimizu, Mika; Sano, Kaori; Nakajima, Tomoko; Yasuda, Hachiro; Takashima, Kazunori; Mizuno, Akira

    2014-01-01

    Nonthermal atmospheric pressure plasma (NTP) has been extensively studied for biological and medical applications in recent years. Chemically active species are generated and injected into aqueous media subjected to plasma exposure. They play an important role in the activation and/or inactivation of biomolecules, such as nucleic acids, lipid, and proteins, in aqueous media. To evaluate the effect of NTP on these biomolecules at the molecular level, we have been considering large DNA molecules to be used as a biomarker. The single-molecule-based analysis of strand breakages induced by NTP, namely, the atmospheric pressure plasma jet (APPJ), on large DNA molecules has been performed. We demonstrated the facile estimation of the rate of double-strand breaks induced by the APPJ. Using this analysis, we have evaluated both the intensity of radical reaction and the protective effect on the reaction by adding antioxidant agents.

  17. Electrical Transport of Long DNA Molecules on Liuid-Solid Interfaces

    NASA Astrophysics Data System (ADS)

    Samuilov, Vladimir; Seo, Young-Soo; Sokolov, Jonathan; Rafailovich, Miriam; Chu, Benjamin

    2002-03-01

    The electrical transport properties of long DNA molecules were studied based upon a newly developed method of electrophoresis on flat surfaces [1]. The electrophoretic mobilities of DNA in the presence of Si surface were found to be approximately one order less than in free solution. The electropherogram peaks of 1 kb- and Hind III DNA ladders have been clearly identified. The experimental dependencies of the mobilities on molecular weight were found to be scaled with power law with the exponents of an opposite sign at 2 different buffer concentrations: negative for surface transport at 10 -2 M concentration of TBE buffer and positive at 10 -3 M. A novel mechanism responsible for DNA molecules separation in the presence of the surface at low buffer concentrations has been developed. The multi-ion system is governed by the Nernst-Planck equations (ion movement due to convection, migration and diffusion), in combination with the Poisson-Boltzmann equation. The discrepancy from charge neutrality that occurs in the diffuse double layer very close to the substrate is the driving force for the Navier-Stokes equation, which finally results in a liquid movement very close to the surface that is denoted as electro-osmosis. The adsorbed DNA move due to the electrical field parallel to the surface, and also due the electro-osmotic convection that drags the DNA chains if they are only partly adsorbed. The electric double layer is responsible for a velocity profile of the electroosmotic flow. The net electrophoretic mobility of longer DNA, being trapped closer to the surface, is higher than of the shorter ones in the electric field, oriented along the surface. The main features of the electro-hydrodynamic instability related to λ and T2 DNA molecules aggregation, observed in our system, are consistent with our model. This work was supported by NSF-MRSEC Program. [1]. N. Pernodet, V. Samuilov, K. Shin, J. Sokolov, M.H. Rafailovich, D. Gersappe, B. Chu, DNA Electrophoresis on a

  18. Nucleic Acid Engineering: RNA Following the Trail of DNA.

    PubMed

    Kim, Hyejin; Park, Yongkuk; Kim, Jieun; Jeong, Jaepil; Han, Sangwoo; Lee, Jae Sung; Lee, Jong Bum

    2016-02-08

    The self-assembly feature of the naturally occurring biopolymer, DNA, has fascinated researchers in the fields of materials science and bioengineering. With the improved understanding of the chemical and structural nature of DNA, DNA-based constructs have been designed and fabricated from two-dimensional arbitrary shapes to reconfigurable three-dimensional nanodevices. Although DNA has been used successfully as a building block in a finely organized and controlled manner, its applications need to be explored. Hence, with the myriad of biological functions, RNA has recently attracted considerable attention to further the application of nucleic acid-based structures. This Review categorizes different approaches of engineering nucleic acid-based structures and introduces the concepts, principles, and applications of each technique, focusing on how DNA engineering is applied as a guide to RNA engineering.

  19. DNA-templated nanoantennas for single-molecule detection at elevated concentrations.

    PubMed

    Acuna, Guillermo P; Holzmeister, Phil; Möller, Friederike M; Beater, Susanne; Lalkens, Birka; Tinnefeld, Philip

    2013-06-01

    The dynamic concentration range is one of the major limitations of single-molecule fluorescence techniques. We show how bottom-up nanoantennas enhance the fluorescence intensity in a reduced hotspot, ready for biological applications. We use self-assembled DNA origami structures as a breadboard where gold nanoparticle (NP) dimers are positioned with nanometer precision. A maximum of almost 100-fold intensity enhancement is obtained using 100-nm gold NPs within a gap of 23 nm between the particles. The results obtained are in good agreement with numerical simulations. Due to the intensity enhancement introduced by the nanoantenna, we are able to perform single-molecule measurements at concentrations as high as 500 nM, which represents an increment of 2 orders of magnitude compared to conventional measurements. The combination of metallic NPs with DNA origami structures with docking points for biological assays paves the way for the development of bottom-up inexpensive enhancement chambers for single-molecule measurements at high concentrations where processes like DNA sequencing occur.

  20. DNA-templated nanoantennas for single-molecule detection at elevated concentrations

    NASA Astrophysics Data System (ADS)

    Acuna, G. P.; Holzmeister, P.; Möller, F. M.; Beater, S.; Lalkens, B.; Tinnefeld, P.

    2013-02-01

    The dynamic concentration range is one of the major limitations of single-molecule fluorescence techniques. Here, we show how bottom-up nano-antennas enhance the fluorescence intensity in a reduced hot-spot, ready for biological applications. We use self-assembled DNA origami structures as a breadboard where gold nanoparticle dimers are positioned with nanometer precision. A maximum of almost 100fold intensity enhancement is obtained using 100 nm gold nanoparticles within a gap of 23 nm between the particles. The results obtained are in good agreement with numerical simulations. Due to the intensity enhancement introduced by the nano-antenna, we are able to perform single molecule measurements at concentrations as high as 500 nM which represents an increment of 2 orders of magnitude compared to conventional measurements. The combination of metallic nanoparticles with DNA origami structures with docking points for biological assays paves the way for the development of bottom-up inexpensive enhancement chambers for single molecule measurements at high concentrations where processes like DNA sequencing occur.

  1. DNA-templated nanoantennas for single-molecule detection at elevated concentrations

    NASA Astrophysics Data System (ADS)

    Acuna, Guillermo P.; Holzmeister, Phil; Möller, Friederike M.; Beater, Susanne; Lalkens, Birka; Tinnefeld, Philip

    2013-06-01

    The dynamic concentration range is one of the major limitations of single-molecule fluorescence techniques. We show how bottom-up nanoantennas enhance the fluorescence intensity in a reduced hotspot, ready for biological applications. We use self-assembled DNA origami structures as a breadboard where gold nanoparticle (NP) dimers are positioned with nanometer precision. A maximum of almost 100-fold intensity enhancement is obtained using 100-nm gold NPs within a gap of 23 nm between the particles. The results obtained are in good agreement with numerical simulations. Due to the intensity enhancement introduced by the nanoantenna, we are able to perform single-molecule measurements at concentrations as high as 500 nM, which represents an increment of 2 orders of magnitude compared to conventional measurements. The combination of metallic NPs with DNA origami structures with docking points for biological assays paves the way for the development of bottom-up inexpensive enhancement chambers for single-molecule measurements at high concentrations where processes like DNA sequencing occur.

  2. Single-molecule comparison of DNA Pol I activity with native and analog nucleotides

    NASA Astrophysics Data System (ADS)

    Gul, Osman; Olsen, Tivoli; Choi, Yongki; Corso, Brad; Weiss, Gregory; Collins, Philip

    2014-03-01

    DNA polymerases are critical enzymes for DNA replication, and because of their complex catalytic cycle they are excellent targets for investigation by single-molecule experimental techniques. Recently, we studied the Klenow fragment (KF) of DNA polymerase I using a label-free, electronic technique involving single KF molecules attached to carbon nanotube transistors. The electronic technique allowed long-duration monitoring of a single KF molecule while processing thousands of template strands. Processivity of up to 42 nucleotide bases was directly observed, and statistical analysis of the recordings determined key kinetic parameters for the enzyme's open and closed conformations. Subsequently, we have used the same technique to compare the incorporation of canonical nucleotides like dATP to analogs like 1-thio-2'-dATP. The analog had almost no affect on duration of the closed conformation, during which the nucleotide is incorporated. On the other hand, the analog increased the rate-limiting duration of the open conformation by almost 40%. We propose that the thiolated analog interferes with KF's recognition and binding, two key steps that determine its ensemble turnover rate.

  3. Expression and isolation of antimicrobial small molecules from soil DNA libraries.

    PubMed

    MacNeil, I A; Tiong, C L; Minor, C; August, P R; Grossman, T H; Loiacono, K A; Lynch, B A; Phillips, T; Narula, S; Sundaramoorthi, R; Tyler, A; Aldredge, T; Long, H; Gilman, M; Holt, D; Osburne, M S

    2001-04-01

    Natural products have been a critically important source of clinically relevant small molecule therapeutics. However, the discovery rate of novel structural classes of antimicrobial molecules has declined. Recently, increasing evidence has shown that the number of species cultivated from soil represents less than 1% of the total population, opening up the exciting possibility that these uncultured species may provide a large untapped pool from which novel natural products can be discovered. We have constructed and expressed in E. coli a BAC (bacterial artificial chromosome) library containing genomic fragments of DNA (5-120kb) isolated directly from soil organisms (S-DNA). Screening of the library resulted in the identification of several antimicrobial activities expressed by different recombinant clones. One clone (mg1.1) has been partially characterized and found to express several small molecules related to and including indirubin. These results show that genes involved in natural product synthesis can be cloned directly from S-DNA and expressed in a heterologous host, supporting the idea that this technology has the potential to provide novel natural products from the wealth of environmental microbial diversity and is a potentially important new tool for drug discovery.

  4. Multiplex single-molecule interaction profiling of DNA-barcoded proteins.

    PubMed

    Gu, Liangcai; Li, Chao; Aach, John; Hill, David E; Vidal, Marc; Church, George M

    2014-11-27

    In contrast with advances in massively parallel DNA sequencing, high-throughput protein analyses are often limited by ensemble measurements, individual analyte purification and hence compromised quality and cost-effectiveness. Single-molecule protein detection using optical methods is limited by the number of spectrally non-overlapping chromophores. Here we introduce a single-molecular-interaction sequencing (SMI-seq) technology for parallel protein interaction profiling leveraging single-molecule advantages. DNA barcodes are attached to proteins collectively via ribosome display or individually via enzymatic conjugation. Barcoded proteins are assayed en masse in aqueous solution and subsequently immobilized in a polyacrylamide thin film to construct a random single-molecule array, where barcoding DNAs are amplified into in situ polymerase colonies (polonies) and analysed by DNA sequencing. This method allows precise quantification of various proteins with a theoretical maximum array density of over one million polonies per square millimetre. Furthermore, protein interactions can be measured on the basis of the statistics of colocalized polonies arising from barcoding DNAs of interacting proteins. Two demanding applications, G-protein coupled receptor and antibody-binding profiling, are demonstrated. SMI-seq enables 'library versus library' screening in a one-pot assay, simultaneously interrogating molecular binding affinity and specificity.

  5. Therapeutic genome mutagenesis using synthetic donor DNA and triplex-forming molecules.

    PubMed

    Reza, Faisal; Glazer, Peter M

    2015-01-01

    Genome mutagenesis can be achieved in a variety of ways, though a select few are suitable for therapeutic settings. Among them, the harnessing of intracellular homologous recombination affords the safety and efficacy profile suitable for such settings. Recombinagenic donor DNA and mutagenic triplex-forming molecules co-opt this natural recombination phenomenon to enable the specific, heritable editing and targeting of the genome. Editing the genome is achieved by designing the sequence-specific recombinagenic donor DNA to have base mismatches, insertions, and deletions that will be incorporated into the genome when it is used as a template for recombination. Targeting the genome is similarly achieved by designing the sequence-specific mutagenic triplex-forming molecules to further recruit the recombination machinery thereby upregulating its activity with the recombinagenic donor DNA. This combination of extracellularly introduced, designed synthetic molecules and intercellularly ubiquitous, evolved natural machinery enables the mutagenesis of chromosomes and engineering of whole genomes with great fidelity while limiting nonspecific interactions. Herein, we demonstrate the harnessing of recombinagenic donor DNA and mutagenic triplex-forming molecular technology for potential therapeutic applications. These demonstrations involve, among others, utilizing this technology to correct genes so that they become physiologically functional, to induce dormant yet functional genes in place of non-functional counterparts, to place induced genes under regulatory elements, and to disrupt genes to abrogate a cellular vulnerability. Ancillary demonstrations of the design and synthesis of this recombinagenic and mutagenic molecular technology as well as their delivery and assayed interaction with duplex DNA reveal a potent technological platform for engineering specific changes into the living genome.

  6. Single-Molecule Studies of the Linker Histone H1 Binding to DNA and the Nucleosome.

    PubMed

    Yue, Hongjun; Fang, He; Wei, Sijie; Hayes, Jeffrey J; Lee, Tae-Hee

    2016-04-12

    Linker histone H1 regulates chromatin structure and gene expression. Investigating the dynamics and stoichiometry of binding of H1 to DNA and the nucleosome is crucial to elucidating its functions. Because of the abundant positive charges and the strong self-affinity of H1, quantitative in vitro studies of its binding to DNA and the nucleosome have generated results that vary widely and, therefore, should be interpreted in a system specific manner. We sought to overcome this limitation by developing a specially passivated microscope slide surface to monitor binding of H1 to DNA and the nucleosome at a single-molecule level. According to our measurements, the stoichiometry of binding of H1 to DNA and the nucleosome is very heterogeneous with a wide distribution whose averages are in reasonable agreement with previously published values. Our study also revealed that H1 does not dissociate from DNA or the nucleosome on a time scale of tens of minutes. We found that histone chaperone Nap1 readily dissociates H1 from DNA and superstoichiometrically bound H1 from the nucleosome, supporting a hypothesis whereby histone chaperones contribute to the regulation of the H1 profile in chromatin.

  7. Digitally encoded DNA nanostructures for multiplexed, single-molecule protein sensing with nanopores

    NASA Astrophysics Data System (ADS)

    Bell, Nicholas A. W.; Keyser, Ulrich F.

    2016-07-01

    The simultaneous detection of a large number of different analytes is important in bionanotechnology research and in diagnostic applications. Nanopore sensing is an attractive method in this regard as the approach can be integrated into small, portable device architectures, and there is significant potential for detecting multiple sub-populations in a sample. Here, we show that highly multiplexed sensing of single molecules can be achieved with solid-state nanopores by using digitally encoded DNA nanostructures. Based on the principles of DNA origami, we designed a library of DNA nanostructures in which each member contains a unique barcode; each bit in the barcode is signalled by the presence or absence of multiple DNA dumbbell hairpins. We show that a 3-bit barcode can be assigned with 94% accuracy by electrophoretically driving the DNA structures through a solid-state nanopore. Select members of the library were then functionalized to detect a single, specific antibody through antigen presentation at designed positions on the DNA. This allows us to simultaneously detect four different antibodies of the same isotype at nanomolar concentration levels.

  8. A single-molecule characterization of p53 search on DNA

    PubMed Central

    Tafvizi, Anahita; Huang, Fang; Fersht, Alan R.; Mirny, Leonid A.; van Oijen, Antoine M.

    2011-01-01

    The tumor suppressor p53 slides along DNA while searching for its cognate site. Central to this process is the basic C-terminal domain, whose regulatory role and its coordination with the core DNA-binding domain is highly debated. Here we use single-molecule techniques to characterize the search process and disentangle the roles played by these two DNA-binding domains in the search process. We demonstrate that the C-terminal domain is capable of rapid translocation, while the core domain is unable to slide and instead hops along DNA. These findings are integrated into a model, in which the C-terminal domain mediates fast sliding of p53, while the core domain samples DNA by frequent dissociation and reassociation, allowing for rapid scanning of long DNA regions. The model further proposes how modifications of the C-terminal domain can activate “latent” p53 and reconciles seemingly contradictory data on the action of different domains and their coordination. PMID:21178072

  9. Analysis of the fluctuations of a single-tethered, quantum-dot labeled DNA molecule in shear flow.

    PubMed

    Laube, K; Günther, K; Mertig, M

    2011-05-11

    A novel technique for analyzing the conformational fluctuations of a single, surface-tethered DNA molecule by fluorescence microscopy is reported. Attaching a nanometer-sized fluorescent quantum dot to the free end of a λ-phage DNA molecule allows us to study the fluctuations of a native DNA molecule without the mechanical properties being altered by fluorescent dye staining. We report on the investigation of single-tethered DNA in both the unperturbed and the shear flow induced stretched state. The dependence of the observed fractional extension and the magnitude of fluctuations on the shear rate can be qualitatively interpreted by Brochard's stem-and-flower model. The cyclic dynamics of a DNA molecule is directly observed in the shear flow experiment.

  10. Crown ether–electrolyte interactions permit nanopore detection of individual DNA abasic sites in single molecules

    PubMed Central

    An, Na; Fleming, Aaron M.; White, Henry S.; Burrows, Cynthia J.

    2012-01-01

    DNA abasic (AP) sites are one of the most frequent lesions in the genome and have a high mutagenic potential if unrepaired. After selective attachment of 2-aminomethyl-18-crown-6 (18c6), individual AP lesions are detected during electrophoretic translocation through the bacterial protein ion channel α-hemolysin (α-HL) embedded in a lipid bilayer. Interactions between 18c6 and Na+ produce characteristic pulse-like current amplitude signatures that allow the identification of individual AP sites in single molecules of homopolymeric or heteropolymeric DNA sequences. The bulky 18c6-cation complexes also dramatically slow the DNA motion to more easily recordable levels. Further, the behaviors of the AP-18c6 adduct are different with respect to the directionalities of DNA entering the protein channel, and they can be precisely manipulated by altering the cation (Li+, Na+ or K+) of the electrolyte. This method permits detection of multiple AP lesions per strand, which is unprecedented in other work. Additionally, insights into the thermodynamics and kinetics of 18c6-cation interactions at a single-molecule level are provided by the nanopore measurement. PMID:22711805

  11. Single-molecule FRET ruler based on rigid DNA origami blocks.

    PubMed

    Stein, Ingo H; Schüller, Verena; Böhm, Philip; Tinnefeld, Philip; Liedl, Tim

    2011-02-25

    Fluorescence resonance energy transfer (FRET) has become a work-horse for distance measurements on the nanometer scale and between single molecules. Recent model systems for the FRET distance dependence such as polyprolines and dsDNA suffered from limited persistence lengths and sample heterogeneity. We designed a series of rigid DNA origami blocks where each block is labeled with one donor and one acceptor at distances ranging between 2.5 and 14 nm. Since all dyes are attached in one plane to the top surface of the origami block, static effects of linker lengths cancel out in contrast to commonly used dsDNA. We used single-molecule spectroscopy to compare the origami-based ruler to dsDNA and found that the origami blocks directly yield the expected distance dependence of energy transfer since the influence of the linkers on the donor-acceptor distance is significantly reduced. Based on a simple geometric model for the inter-dye distances on the origami block, the Förster radius R(0) could directly be determined from the distance dependence of energy transfer yielding R(0)=5.3±0.3 nm for the Cy3-Cy5 pair.

  12. Oligonucleotide-directed self-assembly of proteins: semisynthetic DNA--streptavidin hybrid molecules as connectors for the generation of macroscopic arrays and the construction of supramolecular bioconjugates.

    PubMed Central

    Niemeyer, C M; Sano, T; Smith, C L; Cantor, C R

    1994-01-01

    Modified biomolecules were used for the non-covalent assembly of novel bioconjugates. Hybrid molecules were synthesized from short single-stranded DNA and streptavidin by chemical methods using a heterobispecific crosslinker. The covalent attachment of an oligonucleotide moiety to streptavidin provides a specific recognition domain for a complementary nucleic acid sequence, in addition to the four native biotin-binding sites. These bispecific binding capabilities allow the hybrid molecules to serve as versatile connectors in a variety of applications. Bifunctional constructs have been prepared from two complementary hybrid molecules, each previously conjugated to biotinylated immunoglobulin G or alkaline phosphatase. The use of nucleic acid sequences as a template for the formation of an array of proteins is further demonstrated on two size scales. A macroscopic DNA array on a microtiter plate has been transformed into a comparable protein chip. A nano-scale array was made by hybridizing DNA-tagged proteins to specific positions along a RNA or DNA sequence. The generation of supramolecular bioconjugates was shown by quantitative measurements and gel-retardation assays. Images PMID:7530841

  13. Nucleic acid sensing with enzyme-DNA binding protein conjugates cascade and simple DNA nanostructures.

    PubMed

    Aktas, Gülsen Betül; Skouridou, Vasso; Masip, Lluis

    2017-03-22

    A versatile and universal DNA sensing platform is presented based on enzyme-DNA binding protein tags conjugates and simple DNA nanostructures. Two enzyme conjugates were thus prepared, with horseradish peroxidase linked to the dimeric single-chain bacteriophage Cro repressor protein (HRP-scCro) and glucose oxidase linked to the dimeric headpiece domain of Escherichia coli LacI repressor protein (GOx-dHP), and used in conjunction with a hybrid ssDNA-dsDNA detection probe. This probe served as a simple DNA nanostructure allowing first for target recognition through its target-complementary single-stranded DNA (ssDNA) part and then for signal generation after conjugate binding on the double-stranded DNA (dsDNA) containing the specific binding sites for the dHP and scCro DNA binding proteins. The DNA binding proteins chosen in this work have different sequence specificity, high affinity, and lack of cross-reactivity. The proposed sensing system was validated for the detection of model target ssDNA from high-risk human papillomavirus (HPV16) and the limits of detection of 45, 26, and 21 pM were achieved using the probes with scCro/dHP DNA binding sites ratio of 1:1, 2:1, and 1:2, respectively. The performance of the platform in terms of limit of detection was comparable to direct HRP systems using target-specific oligonucleotide-HRP conjugates. The ratio of the two enzymes can be easily manipulated by changing the number of binding sites on the detection probe, offering further optimization possibilities of the signal generation step. Moreover, since the signal is obtained in the absence of externally added hydrogen peroxide, the described platform is compatible with paper-based assays for molecular diagnostics applications. Finally, just by changing the ssDNA part of the detection probe, this versatile nucleic acid platform can be used for the detection of different ssDNA target sequences or in a multiplex detection configuration without the need to change any of the

  14. DNA origami based Au-Ag-core-shell nanoparticle dimers with single-molecule SERS sensitivity

    NASA Astrophysics Data System (ADS)

    Prinz, J.; Heck, C.; Ellerik, L.; Merk, V.; Bald, I.

    2016-03-01

    DNA origami nanostructures are a versatile tool to arrange metal nanostructures and other chemical entities with nanometer precision. In this way gold nanoparticle dimers with defined distance can be constructed, which can be exploited as novel substrates for surface enhanced Raman scattering (SERS). We have optimized the size, composition and arrangement of Au/Ag nanoparticles to create intense SERS hot spots, with Raman enhancement up to 1010, which is sufficient to detect single molecules by Raman scattering. This is demonstrated using single dye molecules (TAMRA and Cy3) placed into the center of the nanoparticle dimers. In conjunction with the DNA origami nanostructures novel SERS substrates are created, which can in the future be applied to the SERS analysis of more complex biomolecular targets, whose position and conformation within the SERS hot spot can be precisely controlled.DNA origami nanostructures are a versatile tool to arrange metal nanostructures and other chemical entities with nanometer precision. In this way gold nanoparticle dimers with defined distance can be constructed, which can be exploited as novel substrates for surface enhanced Raman scattering (SERS). We have optimized the size, composition and arrangement of Au/Ag nanoparticles to create intense SERS hot spots, with Raman enhancement up to 1010, which is sufficient to detect single molecules by Raman scattering. This is demonstrated using single dye molecules (TAMRA and Cy3) placed into the center of the nanoparticle dimers. In conjunction with the DNA origami nanostructures novel SERS substrates are created, which can in the future be applied to the SERS analysis of more complex biomolecular targets, whose position and conformation within the SERS hot spot can be precisely controlled. Electronic supplementary information (ESI) available: Additional information about materials and methods, designs of DNA origami templates, height profiles, additional SERS spectra, assignment of DNA

  15. Small Molecule Inhibitors of 8-Oxoguanine DNA Glycosylase-1 (OGG1).

    PubMed

    Donley, Nathan; Jaruga, Pawel; Coskun, Erdem; Dizdaroglu, Miral; McCullough, Amanda K; Lloyd, R Stephen

    2015-10-16

    The DNA base excision repair (BER) pathway, which utilizes DNA glycosylases to initiate repair of specific DNA lesions, is the major pathway for the repair of DNA damage induced by oxidation, alkylation, and deamination. Early results from clinical trials suggest that inhibiting certain enzymes in the BER pathway can be a useful anticancer strategy when combined with certain DNA-damaging agents or tumor-specific genetic deficiencies. Despite this general validation of BER enzymes as drug targets, there are many enzymes that function in the BER pathway that have few, if any, specific inhibitors. There is a growing body of evidence that suggests inhibition of 8-oxoguanine DNA glycosylase-1 (OGG1) could be useful as a monotherapy or in combination therapy to treat certain types of cancer. To identify inhibitors of OGG1, a fluorescence-based screen was developed to analyze OGG1 activity in a high-throughput manner. From a primary screen of ∼50,000 molecules, 13 inhibitors were identified, 12 of which were hydrazides or acyl hydrazones. Five inhibitors with an IC50 value of less than 1 μM were chosen for further experimentation and verified using two additional biochemical assays. None of the five OGG1 inhibitors reduced DNA binding of OGG1 to a 7,8-dihydro-8-oxoguanine (8-oxo-Gua)-containing substrate, but all five inhibited Schiff base formation during OGG1-mediated catalysis. All of these inhibitors displayed a >100-fold selectivity for OGG1 relative to several other DNA glycosylases involved in repair of oxidatively damaged bases. These inhibitors represent the most potent and selective OGG1 inhibitors identified to date.

  16. The mitochondrial DNA molecule of Sumatran orangutan and a molecular proposal for two (Bornean and Sumatran) species of orangutan.

    PubMed

    Xu, X; Arnason, U

    1996-11-01

    The complete mitochondrial DNA (mtDNA) molecule of Sumatran orangutan, plus the complete mitochondrial control region of another Sumatran specimen and the control regions and five protein-coding genes of two specimens of Bornean orangutan were sequenced and compared with a previously reported complete mtDNA of Bornean orangutan. The two orangutans are presently separated at the subspecies level. Comparison with five different species pairs-namely, harbor seal/grey seal, horse/donkey, fin whale/blue whale, common chimpanzee/pygmy chimpanzee, and Homo/common chimpanzee-showed that the molecular difference between Sumatran and Bornean orangutan is much greater than that between the seals, and greater than that between the two chimpanzees, but similar to that between the horse and the donkey and the fin and blue whales. Considering their limited morphological distinction the comparison revealed unexpectedly great molecular difference between the two orangutans. The nucleotide difference between the orangutans is about 75% of that between Homo and the common chimpanzee, whereas the amino acid difference exceeds that between Homo and the common chimpanzee. On the basis of their molecular distinction we propose that the two orangutans should be recognized as different species, Pongo pygmaeus, Bornean orangutan, and P. abelii, Sumatran orangutan.

  17. Novel molecular beacon DNA probes for protein-nucleic acid interaction studies

    NASA Astrophysics Data System (ADS)

    Li, Jianwei J.; Perlette, John; Fang, Xiaohong; Kelley, Shannon; Tan, Weihong

    2000-03-01

    We report a novel approach to study protein-nucleic acid interactions by using molecular beacons (MBs). Molecular beacons are hairpin-shaped DNA oligonucleotide probes labeled with a fluorophore and a quencher, and can report the presence of target DNA/RNA sequences. MBs can also report the existence of single-stranded DNA binding proteins (SSB) through non-sequence specific binding. The interaction between SSB and MB has resulted in significant fluorescence restoration of the MB. The fluorescence enhancement brought by SSB and by complementary DNA is very comparable. The molar ratio of the binding between SSB and the molecular beacon is 1:1 with a binding constant of 2 X 107 M-1. Using the MB-SSB binding, we are able to determine SSB at 2 X 10-10 M with a conventional spectrometer. We have also applied MB DNA probes for the analysis of an enzyme lactic dehydrogenase (LDH), and for the investigation of its binding properties with ssDNA. The biding process between MB and different isoenzymes of LDH has been studied. We also show that there are significant differences in MB binding affinity to different proteins, which will enable selective binding studies of a variety of proteins. This new approach is potentially useful for protein-DNA/RNA interaction studies that require high sensitivity, speed and convenience. The results also open the possibility of using easily obtainable, custom designed, modified DNA molecules for studies of drug interactions and targeting. Our results demonstrate that MB can be effectively used for sensitive protein quantitation and for efficient protein-DNA interaction studies. MB has the signal transduction mechanism built within the molecule, and can thus be used for quick protein assay development and for real-time measurements.

  18. Interactions of carcinogens with DNA (deoxyribonucleic acid)

    SciTech Connect

    Broyde, S.; Shapiro, R.

    1989-10-01

    The principal goal of this research has been the determination of the conformational changes produced in DNA by the covalent binding of a carcinogenic aromatic amine, and the correlation of these changes with the mutations and carcinogenic effects initiated by the same substances. To this end, we have devised new synthetic methods for the preparation of oligonucleotides modified by derivatives af 4-aminobiphenyl and aniline. We have also performed potential energy minimization studies on the above substances and on single and double stranded DNA fragments bearing the above amines as well as acetylaminofluorene, aminofluorene, aminopyrene and the antibiotic mitomycin. Our computations have been carried out on DOE supercomputers using our program, DUPLEX. We have defined a number of novel structures for these modified DNAs, including Hoogsteen, wedge'' (see below) denatured, cross-linked and intercalated forms. Some suggestions have been made about the relation of these forms to mutagenesis. 7 refs.

  19. A new method for the covalent attachment of DNA to a surface for single-molecule studies.

    PubMed

    Schlingman, Daniel J; Mack, Andrew H; Mochrie, Simon G J; Regan, Lynne

    2011-03-01

    Attachments between DNA and a surface or bead are often necessary for single-molecule studies of DNA and DNA-protein interactions. In single-molecule mechanical studies using optical or magnetic tweezers, such attachments must be able to withstand the applied forces. Here we present a new method for covalently attaching DNA to a glass surface, which uses N-hydroxysuccinimide (NHS) modified PEG that is suitable for high-force single-molecule mechanical studies. A glass surface is coated with silane-PEG-NHS and DNA is covalently linked through a reaction between the NHS group and an amine modified nucleotide that has been incorporated into the DNA. After DNA attachment, non-reacted NHS groups are hydrolyzed leaving a PEG-covered surface which has the added benefit of reducing non-specific surface interactions. This method permits specific binding of the DNA to the surface through a covalent bond. At the DNA end not attached to the surface, we attach a streptavidin-coated polystyrene bead and measure force-versus-extension using an optical trap. We show that our method allows a tethered DNA molecule to be pulled through its overstretching transition (> 60pN) multiple times. We anticipate this simple yet powerful method will be useful for many researchers.

  20. Hydroxamic acid – A novel molecule for anticancer therapy

    PubMed Central

    Pal, Dilipkumar; Saha, Supriyo

    2012-01-01

    Hydroxamic acid is a potent moiety not only in the field of cancer therapy but also as a mutagenic agent. Among the various derivatives of hydroxamic acid, SAHA (Suberoylanilide Hydroxamic Acid) is considered as a potent anticancer agent. Scientists from the different corner synthesized different hydroxamic acid moieties with some straight chain oxazole, thiadiazole, biphenyl moieties in the terminal position. Acetylation and deacetylation of histones of the core proteins of nucleosomes in chromatin play an important role in the regulation of gene expression. The level of acetylation of histones is established and maintained by two classes of enzymes, histone acetyltransferase and histone deacetylases, which have been identified as transcriptional coactivators and transcriptional corepressors, respectively. There is increasing evidence that aberrant histone acetylation has been linked to various malignant diseases. Great efforts are currently underway for the design of more potent and less toxic candidates for the treatment of cancer. In recent years, hydroxamic acid derivatives have attracted increasing attention for their potential as highly efficacious in combating various etiological factors associated with cancer. Our main intention to draw an attention is that this single functional moiety has not only fit in the receptor but also create a diversified activity. PMID:22837956

  1. The new generation drug candidate molecules: Spectral, electrochemical, DNA-binding and anticancer activity properties

    NASA Astrophysics Data System (ADS)

    Gölcü, Ayşegül; Muslu, Harun; Kılıçaslan, Derya; Çeşme, Mustafa; Eren, Özge; Ataş, Fatma; Demirtaş, İbrahim

    2016-09-01

    The new generation drug candidate molecules [Cu(5-Fu)2Cl2H2O] (NGDCM1) and [Zn(5-Fu)2(CH3COO)2] (NGDCM2) were obtained from the reaction of copper(II) and zinc(II) salts with the anticancer drug 5-fluoracil (5-Fu). These compounds have been characterized by spectroscopic and analytical techniques. Thermal behavior of the compounds were also investigated. The electrochemical properties of the compounds have been investigated by cyclic voltammetry (CV) using glassy carbon electrode. The biological activity of the NGDCM1 and NGDCM2 has been evaluated by examining their ability to bind to fish sperm double strand DNA (FSdsDNA) with UV spectroscopy. UV studies of the interaction of the 5-Fu and metal derivatives with FSdsDNA have shown that these compounds can bind to FSdsDNA. The binding constants of the compounds with FSdsDNA have also been calculated. Thermal decomposition of the compounds lead to the formation of CuO and ZnO as final products. The effect of proliferation 5-Fu, NGDCM1 and NGDCM2 were examined on the HeLa cells using real-time cell analyzer with three different concentrations.

  2. Single-molecule measurements of trapped and migrating circular DNA during electrophoresis in agarose gels.

    PubMed

    Cole, Kenneth D; Gaigalas, Adolfas; Akerman, Björn

    2006-11-01

    The effect of agarose gel concentration and field strength on the electrophoretic trapping of open (relaxed) circular DNA was investigated using microscopic measurements of individual molecules stained with a fluorescent dye. Three open circles with sizes of 52.5, 115, and 220 kbp were trapped by the electric field (6 V/cm) and found to be predominately fixed and stretched at a single point in the gel. The length of the stretched circles did not significantly change with agarose concentration of the gels (mass fractions of 0.0025, 0.01, and 0.02). The relaxation kinetics of the trapped circles was also measured in the gels. The relaxation of the large open circles was found to be a slow process, taking several seconds. The velocity and average length of the 52.5 kbp open circles and 48.5 kbp linear DNA were measured during electrophoresis in the agarose gels. The velocity increased when the agarose concentrations were lowered, but the average length of the open-circle DNA (during electrophoresis) did not significantly change with agarose gel concentrations. The circles move through the gels by cycles of stretching and relaxation during electrophoresis. Linear dichroism was also used to investigate the trapping and alignment of the 52.5 kbp open circles. The results in this study provide information that can be used to improve electrophoretic separations of circular DNA, an important form of genetic material and commonly used to clone DNA.

  3. Beyond DNA origami: the unfolding prospects of nucleic acid nanotechnology.

    PubMed

    Michelotti, Nicole; Johnson-Buck, Alexander; Manzo, Anthony J; Walter, Nils G

    2012-01-01

    Nucleic acid nanotechnology exploits the programmable molecular recognition properties of natural and synthetic nucleic acids to assemble structures with nanometer-scale precision. In 2006, DNA origami transformed the field by providing a versatile platform for self-assembly of arbitrary shapes from one long DNA strand held in place by hundreds of short, site-specific (spatially addressable) DNA 'staples'. This revolutionary approach has led to the creation of a multitude of two-dimensional and three-dimensional scaffolds that form the basis for functional nanodevices. Not limited to nucleic acids, these nanodevices can incorporate other structural and functional materials, such as proteins and nanoparticles, making them broadly useful for current and future applications in emerging fields such as nanomedicine, nanoelectronics, and alternative energy.

  4. DNA-encoded chemical libraries: advancing beyond conventional small-molecule libraries.

    PubMed

    Franzini, Raphael M; Neri, Dario; Scheuermann, Jörg

    2014-04-15

    DNA-encoded chemical libraries (DECLs) represent a promising tool in drug discovery. DECL technology allows the synthesis and screening of chemical libraries of unprecedented size at moderate costs. In analogy to phage-display technology, where large antibody libraries are displayed on the surface of filamentous phage and are genetically encoded in the phage genome, DECLs feature the display of individual small organic chemical moieties on DNA fragments serving as amplifiable identification barcodes. The DNA-tag facilitates the synthesis and allows the simultaneous screening of very large sets of compounds (up to billions of molecules), because the hit compounds can easily be identified and quantified by PCR-amplification of the DNA-barcode followed by high-throughput DNA sequencing. Several approaches have been used to generate DECLs, differing both in the methods used for library encoding and for the combinatorial assembly of chemical moieties. For example, DECLs can be used for fragment-based drug discovery, displaying a single molecule on DNA or two chemical moieties at the extremities of complementary DNA strands. DECLs can vary substantially in the chemical structures and the library size. While ultralarge libraries containing billions of compounds have been reported containing four or more sets of building blocks, also smaller libraries have been shown to be efficient for ligand discovery. In general, it has been found that the overall library size is a poor predictor for library performance and that the number and diversity of the building blocks are rather important indicators. Smaller libraries consisting of two to three sets of building blocks better fulfill the criteria of drug-likeness and often have higher quality. In this Account, we present advances in the DECL field from proof-of-principle studies to practical applications for drug discovery, both in industry and in academia. DECL technology can yield specific binders to a variety of target

  5. Single Molecule Imaging of Transcription Factor Binding to DNA in Live Mammalian Cells

    PubMed Central

    Gebhardt, J Christof M; Suter, David M; Roy, Rahul; Zhao, Ziqing W; Chapman, Alec R; Basu, Srinjan; Maniatis, Tom; Xie, X Sunney

    2013-01-01

    Imaging single fluorescent proteins in living mammalian cells is challenging due to out-of-focus fluorescence excitation by common microscopy schemes. We report the development of a novel fluorescence microscopy method, reflected light sheet microscopy (RLSM), which allows selective plane illumination throughout the nucleus of living mammalian cells, for reducing out-of-focus fluorescence signal. Generation of a thin light sheet parallel to the imaging plane and close to the sample surface is achieved by reflecting an elliptical laser beam incident from the top by 45° with a small mirror. The thin light sheet allows for an increased signal-to-background ratio superior to previous illumination schemes and enables imaging of single fluorescent proteins with up to 100 Hz time resolution. We demonstrate the sensitivity of RLSM by measuring the DNA-bound fraction of glucocorticoid receptor (GR) and determine the residence times on DNA of various oligomerization states and mutants of GR and estrogen receptor (ER), enabling us to resolve different modes of DNA binding of GR. Finally, we demonstrate two-color single molecule imaging by observing the spatio-temporal co-localization of two different protein pairs. The combination of our single molecule measurements and statistical analysis reveals dynamic properties of transcription factors in live mammalian cells. PMID:23524394

  6. Unhooking dynamics of U-shaped DNA molecule undergoing gel electrophoresis.

    PubMed

    Song, L; Maestre, M F

    1991-08-01

    It has been found that DNA molecules are often hooked around obstacles in a U-shaped configuration in gel electrophoresis. To understand the dynamics of the unhooking of U-shaped DNA molecules undergoing gel electrophoresis, we have examined the length changes of the longer and shorter arms of the U-shape as a function of time. Two types of unhooking have been found. In one type, the length changes of both arms are expontential in time but with different time constants. In another type, the length changes of the shorter arm is exponential and that of the longer one is linear with time. The interpretation is that the extent of stretch of the spring-like DNA chain decreases as the length difference between the two arms increases during the unhooking processes, and that the frictions at the pivot point can be relatively large depending upon the local structure of the gel. The friction coefficient at the pivot point is estimated to be nu 0 = (2.98 +/- 1.42)x10(-5) g/sec.

  7. Model and computer simulations of the motion of DNA molecules during pulse field gel electrophoresis

    SciTech Connect

    Smith, S.B.; Bustamante, C. ); Heller, C. )

    1991-05-28

    A model is presented for the motion of individual molecules of DNA undergoing pulse field gel electrophoresis (PFGE). The molecule is represented by a chain of charged beads connected by entropic springs, and the gel is represented by a segmented tube surrounding the beads. This model differs from earlier reptation/tube models in that the tube is allowed to leak in certain places and the chain can double over and flow out of the side of the tube in kinks. It is found that these kinks often lead to the formation of U shapes, which are a major source of retardation in PFGE. The results of computer simulations using this model are compared with real DNA experimental results for the following cases: steady field motion as seen in fluorescence microscopy, mobility in steady fields, mobility in transverse field alternation gel electrophoresis (TFAGE), mobility in field inversion gel electrophoresis (FIGE), and linear dichroism (LD) of DNA in agarose gels during PFGE. Good agreement between the simulations and the experimental results is obtained.

  8. 2-Fatty acrylic acids: new highly derivatizable lipophilic platform molecules

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This paper reports the incorporation of an alpha-methylene unit into fatty acid skeletons. Since the new olefin is conjugated with the carboxylate, it is susceptible to 1,4- (Michael) additions. We have used multifunctional thiols and amines for additions at the methylene. The resulting products ...

  9. Surface functionalization of bioactive glasses with natural molecules of biological significance, Part I: Gallic acid as model molecule

    NASA Astrophysics Data System (ADS)

    Zhang, Xin; Ferraris, Sara; Prenesti, Enrico; Verné, Enrica

    2013-12-01

    Gallic acid (3,4,5-trihydroxybenzoic acid, GA) and its derivatives are a group of biomolecules (polyphenols) obtained from plants. They have effects which are potentially beneficial to heath, for example they are antioxidant, anticarcinogenic and antibacterial, as recently investigated in many fields such as medicine, food and plant sciences. The main drawbacks of these molecules are both low stability and bioavailability. In this research work the opportunity to graft GA to bioactive glasses is investigated, in order to deliver the undamaged biological molecule into the body, using the biomaterial surfaces as a localized carrier. GA was considered for functionalization since it is a good model molecule for polyphenols and presents several interesting biological activities, like antibacterial, antioxidant and anticarcinogenic properties. Two different silica based bioactive glasses (SCNA and CEL2), with different reactivity, were employed as substrates. UV photometry combined with the Folin&Ciocalteu reagent was adopted to test the concentration of GA in uptake solution after functionalization. This test verified how much GA consumption occurred with surface modification and it was also used on solid samples to test the presence of GA on functionalized glasses. XPS and SEM-EDS techniques were employed to characterize the modification of material surface properties and functional group composition before and after functionalization.

  10. Imaging of transverse electron transfer through a DNA molecule by simultaneous scanning tunneling and frequency-modulation atomic force microscopy.

    PubMed

    Maeda, Yasushi; Matsumoto, Takuya; Kawai, Tomoji

    2011-04-26

    Simultaneous scanning tunneling and frequency-modulation atomic force microscopy (STM/FM-AFM) was applied to a DNA molecule on a Cu(111) surface to analyze transverse electron transfer which is related to the electron transfer and the electrical contact in DNA-based devices. In particular, we demonstrated correlation mapping of the electron transfer parameters by visualizing specific combinations of the decay constant β and the contact conductance G. The results clearly revealed that electron transfer across the DNA strand varies spatially in the DNA molecule. This spatial variation is probably correlated with deformation of DNA on the surface. From quantitative analysis, the decay constant β was estimated to be 3.3 nm(-1), which is consistent with previously reported values for π-conjugated molecules.

  11. Stardust and the Molecules of Life (Why are the Amino Acids Left-Handed?)

    SciTech Connect

    Boyd, R N; Kajino, T; Onaka, T

    2010-04-02

    A mechanism for creating and selecting amino acid chirality is identified, and subsequent chemical replication and galactic mixing that would populate the galaxy with the predominant species will be described. This involves: (1) the spin of the {sup 14}N in the amino acids, or in precursor molecules from which amino acids might be formed, coupling to the chirality of the molecules; (2) the neutrinos emitted from the supernova, together with magnetic field from the nascent neutron star or black hole from the supernova selectively destroying one orientation of the {sup 14}N, thereby selecting the chirality associated with the other {sup 14}N orientation; (3) amplification by chemical evolution, by which the molecules replicate on a relatively short timescale; and (4) galactic mixing on a longer timescale mixing the selected molecules throughout the galaxy.

  12. Crystal Structure of Mycobacterium tuberculosis H37Rv AldR (Rv2779c), a Regulator of the ald Gene: DNA BINDING AND IDENTIFICATION OF SMALL MOLECULE INHIBITORS.

    PubMed

    Dey, Abhishek; Shree, Sonal; Pandey, Sarvesh Kumar; Tripathi, Rama Pati; Ramachandran, Ravishankar

    2016-06-03

    Here we report the crystal structure of M. tuberculosis AldR (Rv2779c) showing that the N-terminal DNA-binding domains are swapped, forming a dimer, and four dimers are assembled into an octamer through crystal symmetry. The C-terminal domain is involved in oligomeric interactions that stabilize the oligomer, and it contains the effector-binding sites. The latter sites are 30-60% larger compared with homologs like MtbFFRP (Rv3291c) and can consequently accommodate larger molecules. MtbAldR binds to the region upstream to the ald gene that is highly up-regulated in nutrient-starved tuberculosis models and codes for l-alanine dehydrogenase (MtbAld; Rv2780). Further, the MtbAldR-DNA complex is inhibited upon binding of Ala, Tyr, Trp and Asp to the protein. Studies involving a ligand-binding site G131T mutant show that the mutant forms a DNA complex that cannot be inhibited by adding the amino acids. Comparative studies suggest that binding of the amino acids changes the relative spatial disposition of the DNA-binding domains and thereby disrupt the protein-DNA complex. Finally, we identified small molecules, including a tetrahydroquinoline carbonitrile derivative (S010-0261), that inhibit the MtbAldR-DNA complex. The latter molecules represent the very first inhibitors of a feast/famine regulatory protein from any source and set the stage for exploring MtbAldR as a potential anti-tuberculosis target.

  13. Single molecule detection of double-stranded DNA in poly(methylmethacrylate) and polycarbonate microfluidic devices.

    PubMed

    Wabuyele, M B; Ford, S M; Stryjewski, W; Barrow, J; Soper, S A

    2001-10-01

    Single photon burst techniques were used to detect double-stranded DNA molecules in poly(methylmethacrylate) (PM MA) and polycarbonate (PC) microfluidic devices. A confocal epi-illumination detection system was constructed to monitor the fluorescence signature from single DNA molecules that were multiply labeled with the mono-intercalating dye, TOPRO-5, which possessed an absorption maximum at 765 nm allowing excitation with a solid-state diode laser and fluorescence monitoring in the near-infrared (IR). Near-IR excitation minimized autofluorescence produced from the polymer substrate, which was found to be significantly greater when excitation was provided in the visible range (488 nm). A solution containing lambda-DNA (48.5 kbp) was electrokinetically transported through the microfluidic devices at different applied voltages and solution pH values to investigate the effects of polymer substrate on the transport rate and detection efficiency of single molecular events. By applying an autocorrelation analysis to the data, we were able to obtain the molecular transit time of the individual molecules as they passed through the 7 microm laser beam. It was observed that the applied voltage for both devices affected the transport rate. However, solution pH did not alter the transit time for PM MA-based devices since the electroosmotic flow of PMMA was independent of solution pH. In addition, efforts were directed toward optimizing the sampling efficiency (number of molecules passing through the probe volume) by using either hydrodynamically focused flows from a sheath generated by electrokinetic pumping from side channels or reducing the channel width of the microfluidic device. Due to the low electroosmotic flows generated by both PMMA and PC, tight focusing of the sample stream was not possible. However, in PMMA devices, flow gating was observed by applying field strengths > -120 V/cm to the sheath flow channels. By narrowing the microchannel width, the number of

  14. The nature of the force-induced conformation transition of dsDNA studied by using single molecule force spectroscopy.

    PubMed

    Liu, Ningning; Bu, Tianjia; Song, Yu; Zhang, Wei; Li, Jinjing; Zhang, Wenke; Shen, Jiacong; Li, Hongbin

    2010-06-15

    Single-stranded DNA binding proteins (SSB) interact with single-stranded DNA (ssDNA) specifically. Taking advantage of this character, we have employed Bacillus subtilis SSB protein to investigate the nature of force-induced conformation transition of double-stranded DNA (dsDNA) by using AFM-based single molecule force spectroscopy (SMFS) technique. Our results show that, when a dsDNA is stretched beyond its contour length, the dsDNA is partially melted, producing some ssDNA segments which can be captured by SSB proteins. We have also systematically investigated the effects of stretching length, waiting time, and salt concentration on the conformation transition of dsDNA and SSB-ssDNA interactions, respectively. Furthermore, the effect of proflavine, a DNA intercalator, on the SSB-DNA interactions has been investigated, and the results indicate that the proflavine-saturated dsDNA can be stabilized to the extent that the dsDNA will no longer melt into ssDNA under the mechanical force even up to 150 pN, and no SSB-DNA interactions are detectable.

  15. Dissociative attachment reactions of electrons with strong acid molecules

    SciTech Connect

    Adams, N.G.; Smith, D.; Viggiano, A.A.; Paulson, J.F.; Henchman, M.J.

    1986-06-15

    Using the flowing afterglow/Langmuir probe (FALP) technique, we have determined (at variously 300 and 570 K) the dissociative attachment coefficients ..beta.. for the reactions of electrons with the common acids HNO/sub 3/ (producing NO/sup -//sub 2/) and H/sub 2/SO/sub 4/ (HSO/sup -//sub 4/), the superacids FSO/sub 3/H (FSO/sup -//sub 3/), CF/sub 3/SO/sub 3/H (CF/sub 3/SO/sup -//sub 3/), ClSO/sub 3/H (ClSO/sup -//sub 3/,Cl/sup -/), the acid anhydride (CF/sub 3/SO/sub 2/)/sub 2/O (CF/sub 3/SO/sup -//sub 3/), and the halogen halides HBr (Br/sup -/) and HI (I/sup -/). The anions formed in the reactions are those given in the parentheses. The reactions with HF and HCl were investigated, but did not occur at a measurable rate since they are very endothermic. Dissociative attachment is rapid for the common acids, the superacids, and the anhydride, the measured ..beta.. being appreciable fractions of the theoretical maximum ..beta.. for such reactions, ..beta../sub max/. The HI reaction is very fast ( ..beta..approx...beta../sub max/) but the HBr reaction occurs much more slowly because it is significantly endothermic. The data indicate that the extreme acidity of the (Bronsted-type) superacids has its equivalence in the very efficient gas-phase dissociative attachment which these species undergo when reacting with free electrons. The anions of the superacids generated in these reactions, notably FSO/sup -//sub 3/ and CF/sub 3/SO/sup -//sub 3/, are very stable (unreactive) implying exceptionally large electron affinities for the FSO/sub 3/ and CF/sub 3/SO/sub 3/ radicals.

  16. Studies of the dynamics of biological macromolecules using Au nanoparticle-DNA artificial molecules.

    PubMed

    Chen, Qian; Smith, Jessica M; Rasool, Haider I; Zettl, Alex; Alivisatos, A Paul

    2014-01-01

    The recent development of graphene liquid cells, a nanoscale version of liquid bubble wrap, is a breakthrough for in situ liquid phase electron microscopy (EM). Using ultrathin graphene sheets as the liquid sample container, graphene liquid cells have allowed the unprecedented atomic resolution observation of solution phase growth and dynamics of nanocrystals. Here we explore the potential of this technique to probe nanoscale structure and dynamics of biomolecules in situ, using artificial Au nanoparticle-DNA artificial molecules as model systems. The interactions of electrons with both the artificial molecules and the liquid environment have been demonstrated and discussed, revealing both the opportunities and challenges of using graphene liquid cell EM as a new method of bio-imaging.

  17. Another expert system rule inference based on DNA molecule logic gates

    NASA Astrophysics Data System (ADS)

    WÄ siewicz, Piotr

    2013-10-01

    With the help of silicon industry microfluidic processors were invented utilizing nano membrane valves, pumps and microreactors. These so called lab-on-a-chips combined together with molecular computing create molecular-systems-ona- chips. This work presents a new approach to implementation of molecular inference systems. It requires the unique representation of signals by DNA molecules. The main part of this work includes the concept of logic gates based on typical genetic engineering reactions. The presented method allows for constructing logic gates with many inputs and for executing them at the same quantity of elementary operations, regardless of a number of input signals. Every microreactor of the lab-on-a-chip performs one unique operation on input molecules and can be connected by dataflow output-input connections to other ones.

  18. Feasibility of Single Molecule DNA Sequencing using Surface-Enhanced Raman Scattering

    SciTech Connect

    Talley, C E; Reboredo, F; Chan, J; Lane, S M

    2006-02-03

    We have used a combined theoretical and experimental approach in order to assess the feasibility of using surface-enhanced Raman scattering (SERS) for DNA sequencing at the single molecule level. We have developed a numerical tool capable of calculating the E-field and resulting SERS enhancement factors for metallic structures of arbitrary size and shape. Measurements of the additional SERS enhancement by combining SERS with coherent antistokes Raman scattering (CARS) show that only modest increases in the signal are achievable due to thermal damage at higher laser powers. Finally, measurements of the SERS enhancement from nanoparticles coated with an insulating layer show that the SERS enhancement is decreased by as much as two orders of magnitude when the molecule is not in contact with the metal surface.

  19. Possible involvement of undissociated acid molecules in the acid response of the chorda tympani nerve of the rat.

    PubMed

    Ogiso, K; Shimizu, Y; Watanabe, K; Tonosaki, K

    2000-05-01

    To test whether undissociated acid is capable of exciting the chorda tympani nerves in rats, we have used buffered acid solutions as taste stimuli. These solutions were prepared by adding alkali to weak acids, such as acetic acid, so that the proportion of undissociated and dissociated acids was varied whereas keeping the total acid concentration constant. When acetic acid solutions, adjusted to wide ranges of pH by NaOH, were applied to the tongue, the response magnitude of the chorda tympani nerves was not varied systematically with pH changes. However, if the sodium effect was eliminated by amiloride or replacement of cation by potassium or Tris[hydroxymethyl]aminomethane; NH(2)C(CH(2)OH)(3) (Tris-base), the chorda tympani response was reduced systematically as pH increased. Similar results were obtained with citric acid and ascorbic acid. This pH-dependent change in taste nerve response to acid cannot be solely attributed to the proton gradient because the response magnitude induced by hydrogen itself, which was estimated from responses to strong acids, was much smaller than that by equi-pH acetic acid ( approximately 85%). Thus we cannot explain the pH-dependent responses of the chorda tympani nerves to weak acids unless effects of undissociated acid molecules are postulated. It is therefore concluded that undissociated acids in weak acid solutions can be a stimulant to taste receptor cells.

  20. Designing a Single-Molecule Biophysics Tool for Characterising DNA Damage for Techniques that Kill Infectious Pathogens Through DNA Damage Effects.

    PubMed

    Miller, Helen; Wollman, Adam J M; Leake, Mark C

    2016-01-01

    Antibiotics such as the quinolones and fluoroquinolones kill bacterial pathogens ultimately through DNA damage. They target the essential type IIA topoisomerases in bacteria by stabilising the normally transient double-strand break state which is created to modify the supercoiling state of the DNA. Here we discuss the development of these antibiotics and their method of action. Existing methods for DNA damage visualisation, such as the comet assay and immunofluorescence imaging can often only be analysed qualitatively and this analysis is subjective. We describe a putative single-molecule fluorescence technique for quantifying DNA damage via the total fluorescence intensity of a DNA origami tile fully saturated with an intercalating dye, along with the optical requirements for how to implement these into a light microscopy imaging system capable of single-molecule millisecond timescale imaging. This system promises significant improvements in reproducibility of the quantification of DNA damage over traditional techniques.

  1. Injection molded nanofluidic chips: fabrication method and functional tests using single-molecule DNA experiments.

    PubMed

    Utko, Pawel; Persson, Fredrik; Kristensen, Anders; Larsen, Niels B

    2011-01-21

    We demonstrate that fabrication of well-defined nanofluidic systems can be greatly simplified by injection molding of thermoplastic polymers. Chips featuring nanochannel arrays, microchannels and integrated interconnects are produced in a single processing step by injection molding. The resulting open channel structures are subsequently sealed by facile plasma-enhanced thermal bonding of a polymer film. This fast, inexpensive and industry-compatible method thus provides a single-use all-polymer platform for nanofluidic lab-on-a-chip applications. Its applicability for nanofluidics is demonstrated by DNA stretching experiments performed on individual double-stranded DNA molecules confined in the injection molded nanochannels. The obtained results are consistent with measurements performed in costly state-of-the-art silica nanochannels, for both straight and tapered channel geometries.

  2. The effects of geometry and stability of solid-state nanopores on detecting single DNA molecules

    NASA Astrophysics Data System (ADS)

    Rollings, Ryan; Graef, Edward; Walsh, Nathan; Nandivada, Santoshi; Benamara, Mourad; Li, Jiali

    2015-01-01

    In this work we use a combination of 3D-TEM tomography, energy filtered TEM, single molecule DNA translocation experiments, and numerical modeling to show a more precise relationship between nanopore shape and ionic conductance and show that changes in geometry while in solution can account for most deviations between predicted and measured conductance. We compare the structural stability of ion beam sculpted (IBS), IBS-annealed, and TEM drilled nanopores. We demonstrate that annealing can significantly improve the stability of IBS made pores. Furthermore, the methods developed in this work can be used to predict pore conductance and current drop amplitudes of DNA translocation events for a wide variety of pore geometries. We discuss that chemical dissolution is one mechanism of the geometry change for SiNx nanopores and show that small modification in fabrication procedure can significantly increase the stability of IBS nanopores.

  3. Visualization of DNA Double-Strand Break Repair at the Single-Molecule Level

    SciTech Connect

    Dynan, William S.; Li, Shuyi; Mernaugh, Raymond; Wragg, Stephanie; Takeda, Yoshihiko

    2003-03-27

    Exposure to low doses of ionizing radiation is universal. The signature injury from ionizing radiation exposure is induction of DNA double-strand breaks (DSBs). The first line of defense against DSBs is direct ligation of broken DNA ends via the nonhomologous end-joining pathway. Because even a relatively high environmental exposure induces only a few DSBs per cell, our current understanding of the response to this exposure is limited by the ability to measure DSB repair events reliably in situ at a single-molecule level. To address this need, we have taken advantage of biological amplification, measuring relocalization of proteins and detection of protein phosphorylation as a surrogate for detection of broken ends themselves. We describe the use of specific antibodies to investigate the kinetics and mechanism of repair of very small numbers of DSBs in human cells by the nonhomologous end-joining pathway.

  4. Synthesis of Small-Molecule/DNA Hybrids through On-Bead Amide-Coupling Approach.

    PubMed

    Okochi, Kenji D; Monfregola, Luca; Dickerson, Sarah Michelle; McCaffrey, Ryan; Domaille, Dylan W; Yu, Chao; Hafenstine, Glenn R; Jin, Yinghua; Cha, Jennifer N; Kuchta, Robert D; Caruthers, Marvin; Zhang, Wei

    2017-03-20

    Small molecule/DNA hybrids (SMDHs) have been considered as nanoscale building blocks for engineering 2D and 3D supramolecular DNA assembly. Herein, we report an efficient on-bead amide-coupling approach to prepare SMDHs with multiple oligodeoxynucleotide (ODN) strands. Our method is high yielding under mild and user-friendly conditions with various organic substrates and homo- or mixed-sequenced ODNs. Metal catalysts and moisture- and air-free conditions are not required. The products can be easily analyzed by LC-MS with accurate mass resolution. We also explored nanometer-sized shape-persistent macrocycles as novel multitopic organic linkers to prepare SMDHs. SMDHs bearing up to six ODNs were successfully prepared through the coupling of arylenethynylene macrocycles with ODNs, which were used to mediate the assembly of gold nanoparticles.

  5. Allosteric "beta-blocker" isolated from a DNA-encoded small molecule library.

    PubMed

    Ahn, Seungkirl; Kahsai, Alem W; Pani, Biswaranjan; Wang, Qin-Ting; Zhao, Shuai; Wall, Alissa L; Strachan, Ryan T; Staus, Dean P; Wingler, Laura M; Sun, Lillian D; Sinnaeve, Justine; Choi, Minjung; Cho, Ted; Xu, Thomas T; Hansen, Gwenn M; Burnett, Michael B; Lamerdin, Jane E; Bassoni, Daniel L; Gavino, Bryant J; Husemoen, Gitte; Olsen, Eva K; Franch, Thomas; Costanzi, Stefano; Chen, Xin; Lefkowitz, Robert J

    2017-02-14

    The β2-adrenergic receptor (β2AR) has been a model system for understanding regulatory mechanisms of G-protein-coupled receptor (GPCR) actions and plays a significant role in cardiovascular and pulmonary diseases. Because all known β-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human β2AR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 [([4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide], exhibiting a unique chemotype and low micromolar affinity for the β2AR. Binding of 15 to the receptor cooperatively enhances orthosteric inverse agonist binding while negatively modulating binding of orthosteric agonists. Studies with a specific antibody that binds to an intracellular region of the β2AR suggest that 15 binds in proximity to the G-protein binding site on the cytosolic surface of the β2AR. In cell-signaling studies, 15 inhibits cAMP production through the β2AR, but not that mediated by other Gs-coupled receptors. Compound 15 also similarly inhibits β-arrestin recruitment to the activated β2AR. This study presents an allosteric small-molecule ligand for the β2AR and introduces a broadly applicable method for screening DNA-encoded small-molecule libraries against purified GPCR targets. Importantly, such an approach could facilitate the discovery of GPCR drugs with tailored allosteric effects.

  6. Allosteric “beta-blocker” isolated from a DNA-encoded small molecule library

    PubMed Central

    Ahn, Seungkirl; Kahsai, Alem W.; Pani, Biswaranjan; Wang, Qin-Ting; Zhao, Shuai; Wall, Alissa L.; Strachan, Ryan T.; Staus, Dean P.; Wingler, Laura M.; Sun, Lillian D.; Sinnaeve, Justine; Choi, Minjung; Cho, Ted; Xu, Thomas T.; Hansen, Gwenn M.; Burnett, Michael B.; Lamerdin, Jane E.; Bassoni, Daniel L.; Gavino, Bryant J.; Husemoen, Gitte; Olsen, Eva K.; Franch, Thomas; Costanzi, Stefano; Chen, Xin; Lefkowitz, Robert J.

    2017-01-01

    The β2-adrenergic receptor (β2AR) has been a model system for understanding regulatory mechanisms of G-protein–coupled receptor (GPCR) actions and plays a significant role in cardiovascular and pulmonary diseases. Because all known β-adrenergic receptor drugs target the orthosteric binding site of the receptor, we set out to isolate allosteric ligands for this receptor by panning DNA-encoded small-molecule libraries comprising 190 million distinct compounds against purified human β2AR. Here, we report the discovery of a small-molecule negative allosteric modulator (antagonist), compound 15 [([4-((2S)-3-(((S)-3-(3-bromophenyl)-1-(methylamino)-1-oxopropan-2-yl)amino)-2-(2-cyclohexyl-2-phenylacetamido)-3-oxopropyl)benzamide], exhibiting a unique chemotype and low micromolar affinity for the β2AR. Binding of 15 to the receptor cooperatively enhances orthosteric inverse agonist binding while negatively modulating binding of orthosteric agonists. Studies with a specific antibody that binds to an intracellular region of the β2AR suggest that 15 binds in proximity to the G-protein binding site on the cytosolic surface of the β2AR. In cell-signaling studies, 15 inhibits cAMP production through the β2AR, but not that mediated by other Gs-coupled receptors. Compound 15 also similarly inhibits β-arrestin recruitment to the activated β2AR. This study presents an allosteric small-molecule ligand for the β2AR and introduces a broadly applicable method for screening DNA-encoded small-molecule libraries against purified GPCR targets. Importantly, such an approach could facilitate the discovery of GPCR drugs with tailored allosteric effects. PMID:28130548

  7. Analysis of single nucleic acid molecules in micro- and nano-fluidics.

    PubMed

    Friedrich, Sarah M; Zec, Helena C; Wang, Tza-Huei

    2016-03-07

    Nucleic acid analysis has enhanced our understanding of biological processes and disease progression, elucidated the association of genetic variants and disease, and led to the design and implementation of new treatment strategies. These diverse applications require analysis of a variety of characteristics of nucleic acid molecules: size or length, detection or quantification of specific sequences, mapping of the general sequence structure, full sequence identification, analysis of epigenetic modifications, and observation of interactions between nucleic acids and other biomolecules. Strategies that can detect rare or transient species, characterize population distributions, and analyze small sample volumes enable the collection of richer data from biosamples. Platforms that integrate micro- and nano-fluidic operations with high sensitivity single molecule detection facilitate manipulation and detection of individual nucleic acid molecules. In this review, we will highlight important milestones and recent advances in single molecule nucleic acid analysis in micro- and nano-fluidic platforms. We focus on assessment modalities for single nucleic acid molecules and highlight the role of micro- and nano-structures and fluidic manipulation. We will also briefly discuss future directions and the current limitations and obstacles impeding even faster progress toward these goals.

  8. Single molecule photobleaching (SMPB) technology for counting of RNA, DNA, protein and other molecules in nanoparticles and biological complexes by TIRF instrumentation.

    PubMed

    Zhang, Hui; Guo, Peixuan

    2014-05-15

    Direct counting of biomolecules within biological complexes or nanomachines is demanding. Single molecule counting using optical microscopy is challenging due to the diffraction limit. The single molecule photobleaching (SMPB) technology for direct counting developed by our team (Shu et al., 2007 [18]; Zhang et al., 2007 [19]) offers a simple and straightforward method to determine the stoichiometry of molecules or subunits within biocomplexes or nanomachines at nanometer scales. Stoichiometry is determined by real-time observation of the number of descending steps resulted from the photobleaching of individual fluorophore. This technology has now been used extensively for single molecule counting of protein, RNA, and other macromolecules in a variety of complexes or nanostructures. Here, we elucidate the SMPB technology, using the counting of RNA molecules within a bacteriophage phi29 DNA-packaging biomotor as an example. The method described here can be applied to the single molecule counting of other molecules in other systems. The construction of a concise, simple and economical single molecule total internal reflection fluorescence (TIRF) microscope combining prism-type and objective-type TIRF is described. The imaging system contains a deep-cooled sensitive EMCCD camera with single fluorophore detection sensitivity, a laser combiner for simultaneous dual-color excitation, and a Dual-View™ imager to split the multiple outcome signals to different detector channels based on their wavelengths. Methodology of the single molecule photobleaching assay used to elucidate the stoichiometry of RNA on phi29 DNA packaging motor and the mechanism of protein/RNA interaction are described. Different methods for single fluorophore labeling of RNA molecules are reviewed. The process of statistical modeling to reveal the true copy number of the biomolecules based on binomial distribution is also described.

  9. High-resolution atomic force microscopy of duplex and triplex DNA molecules

    NASA Astrophysics Data System (ADS)

    Klinov, Dmitry; Dwir, Benjamin; Kapon, Eli; Borovok, Natalia; Molotsky, Tatiana; Kotlyar, Alexander

    2007-06-01

    Double-stranded poly(dG)-poly(dC) and triple-stranded poly(dG)-poly(dG)-poly(dC) DNA were deposited on the modified surface of highly oriented pyrolitic graphite (HOPG) and visualized using atomic force microscopy with high-resolution (radius of ~1 nm) tips. The high resolution attained by this technique enabled us to detect single-stranded regions in double-stranded poly(dG)-poly(dC) and double-stranded and single-stranded regions in poly(dG)-poly(dG)-poly(dC) triplexes, as well as to resolve the helical pitch of the triplex molecules. We could also follow the reaction of G-strand extension in poly(dG)-poly(dC) by the Klenow exo- fragment of DNA polymerase I. This approach to molecular visualization could serve as a useful tool for the investigation of irregular structures in canonical DNA and other biopolymers, as well as studies of the molecular mechanisms of DNA replication and transcription.

  10. Separation of chromosomal DNA molecules from C.albicans by pulsed field gel electrophoresis.

    PubMed Central

    Snell, R G; Wilkins, R J

    1986-01-01

    Modifications have been made to standard pulse field gel electrophoresis (PFGE) systems to enable very large DNA molecules to be resolved. The single most important modification was to elevate the temperature of electrophoresis to 35 degrees C. This enabled the largest Saccharomyces cerevisiae chromosome to be reproducibly resolved. More impressively, it enabled the DNA of Candida albicans to be clearly resolved into six bands, a feat which was very difficult at lower temperatures. Even so, optimal resolution could only be obtained by carefully adjusting field voltages and switching times. The DNA from the two largest C. albicans chromosomes, which was estimated to be at least 5-10Mbp in size, ran somewhat anomalously, giving fuzzy bands which did not migrate in the direction of the average electric field. That the highest molecular weight band was a distinct chromosome was demonstrated by specific hybridisation to the C. albicans ADE2 gene probe. With further fine tuning, the PFGE system described here should be capable of resolving DNA from the smallest human chromosomes. Images PMID:3520483

  11. Single-molecule imaging of DNA curtains reveals intrinsic energy landscapes for nucleosome deposition

    PubMed Central

    Visnapuu, Mari-Liis; Greene, Eric C.

    2009-01-01

    Here we use single-molecule imaging to determine coarse-grained intrinsic energy landscapes for nucleosome deposition on model DNA substrates. Our results reveal distributions that are correlated with recent in silico predictions, reinforcing the hypothesis that DNA contains some intrinsic positioning information. We also show that cis-regulatory sequences in human DNA coincide with peaks in the intrinsic landscape, whereas valleys correspond to non-regulatory regions, and we present evidence arguing that nucleosome deposition in vertebrates is influenced by factors not accounted for by current theory. Finally, we demonstrate that intrinsic landscapes of nucleosomes containing the centromere-specific variant CenH3 are correlated with patterns observed for canonical nucleosomes, arguing that CenH3 does not alter sequence preferences of centromeric nucleosomes. However, the non-histone protein Scm3 alters the intrinsic landscape of CenH3-containing nucleosomes, enabling them to overcome the otherwise exclusionary effects of poly(dA–dT) tracts, which are enriched in centromeric DNA. PMID:19734899

  12. Single-Molecule Studies of the Temperature Dependence of Viral DNA Packaging Motors

    NASA Astrophysics Data System (ADS)

    White, Michael; Raymer, Dorian; Rickgauer, Peter; Fuller, Derek; Grimes, Shelley; Jardine, Paul; Anderson, Dwight; Smith, Doug

    2007-03-01

    A key step in the assembly of many viruses is the packaging of dsDNA into a preformed capsid by the action of a portal molecular motor complex. We have developed methods for directly measuring viral DNA translocation at the single molecule level using optical tweezers and applied these methods to study bacteriophages φ29, lambda, and T4. Our previous measurements with φ29 were performed at room temperature. Here we report that the rate of DNA translocation is strongly temperature dependent. Preliminary measurements indicate that the motor velocity increases ˜2-fold, to ˜250-300 bp/s when the temperature is increased from ˜20 to 30 degrees C. As the viral packaging motors are enzymes that catalyze ATP hydrolysis, such a trend with increasing temperature is to be expected, at least up to the point where the motor complex is thermally dissociated or denatured. However, the detailed form of the temperature dependence is difficult to quantify using standard bulk assay methods. We have installed a heating/cooling system in our optical tweezers instrument that allows us to precisely control the temperature in our sample chamber. This system allows us to systematically study the temperature dependence of the DNA translocation rate.

  13. Amending HIV Drugs: A Novel Small-Molecule Approach To Target Lupus Anti-DNA Antibodies.

    PubMed

    VanPatten, Sonya; Sun, Shan; He, Mingzhu; Cheng, Kai Fan; Altiti, Ahmad; Papatheodorou, Angelos; Kowal, Czeslawa; Jeganathan, Venkatesh; Crawford, James M; Bloom, Ona; Volpe, Bruce T; Grant, Christian; Meurice, Nathalie; Coleman, Thomas R; Diamond, Betty; Al-Abed, Yousef

    2016-10-13

    Systemic lupus erythematosus is an autoimmune disease that can affect numerous tissues and is characterized by the production of nuclear antigen-directed autoantibodies (e.g., anti-dsDNA). Using a combination of virtual and ELISA-based screens, we made the intriguing discovery that several HIV-protease inhibitors can function as decoy antigens to specifically inhibit the binding of anti-dsDNA antibodies to target antigens such as dsDNA and pentapeptide DWEYS. Computational modeling revealed that HIV-protease inhibitors comprised structural features present in DWEYS and predicted that analogues containing more flexible backbones would possess preferred binding characteristics. To address this, we reduced the internal amide backbone to improve flexibility, producing new small-molecule decoy antigens, which neutralize anti-dsDNA antibodies in vitro, in situ, and in vivo. Pharmacokinetic and SLE model studies demonstrated that peptidomimetic FISLE-412,1 a reduced HIV protease inhibitor analogue, was well-tolerated, altered serum reactivity to DWEYS, reduced glomeruli IgG deposition, preserved kidney histology, and delayed SLE onset in NZB/W F1 mice.

  14. Single-Molecule Studies of Nucleic Acid Interactions Using Nanopores

    NASA Astrophysics Data System (ADS)

    Wanunu, Meni; Soni, Gautam V.; Meller, Amit

    This chapter presents biophysical studies of single biopolymers using nanopores. Starting from the fundamental process of voltage-driven biopolymer translocation, the understanding of which is a prerequisite for virtually all nanopore applications, the chapter describes recent experiments that resolve nucleic acid structure and its interaction with enzymes, such as exonucleases and polymerases. It then outlines progress made with solid-state nanopores fabricated in ultrathin membranes and discusses experiments describing biopolymer dynamics in synthetic pores. The chapter concludes with a discussion on some of the main challenges facing nanopore technology, as well as on some of the future prospects associated with nanopore-based tools.

  15. Visualization of local DNA unwinding by Mre11/Rad50/Nbs1 using single-molecule FRET.

    PubMed

    Cannon, Brian; Kuhnlein, Jeffrey; Yang, Soo-Hyun; Cheng, Anita; Schindler, Detlev; Stark, Jeremy M; Russell, Rick; Paull, Tanya T

    2013-11-19

    The Mre11/Rad50/Nbs1 (MRN) complex initiates and coordinates DNA repair and signaling events at double-strand breaks. The interaction between MRN and DNA ends is critical for the recruitment of DNA-processing enzymes, end tethering, and activation of the ATM protein kinase. Here we visualized MRN binding to duplex DNA molecules using single-molecule FRET, and found that MRN unwinds 15-20 base pairs at the end of the duplex, holding the branched structure open for minutes at a time in an ATP-dependent reaction. A Rad50 catalytic domain mutant that is specifically deficient in this ATP-dependent opening is impaired in DNA end resection in vitro and in resection-dependent repair of breaks in human cells, demonstrating the importance of MRN-generated single strands in the repair of DNA breaks.

  16. Competitive Adsorption of Naphthenic Acids and Polyaromatic Molecules at a Toluene-Water Interface.

    PubMed

    Teklebrhan, Robel B; Jian, Cuiying; Choi, Phillip; Xu, Zhenghe; Sjöblom, Johan

    2016-12-22

    The early-stage competitive co-adsorption of interfacially active naphthenic acids (NAs) and polyaromatic (PA) molecules to a toluene-water interface from the bulk toluene phase was studied using molecular dynamics (MD) simulation. The NA molecules studied had the same polar functional group but different cycloaliphatic nonpolar tails, and a perylene bisimide (PBI)-based molecule was used as a representative PA compound. The results from our simulations suggest that the size and structural features of NA molecules greatly influence the interfacial activity of PA molecules and partitioning of NA molecules at the toluene-water interface. At low concentrations of PA (∼2.3 wt %) and NA (∼0.4 wt %) molecules, NA molecules containing large cycloaliphatic rings (e.g., four rings) or with a very long aliphatic tail (e.g., carbon chain length of 14) were observed to impede the migration of PA molecules to the interface, whereas small NA molecules containing two cycloaliphatic rings had little effect on the adsorption of PA molecules at the toluene-water interface. At high NA concentrations, the adsorption of PA molecules (∼5.75-17.25 wt %) was greatly hindered by the presence of small NA molecules (∼1.6-4.8 wt %) due to the solvation of PA nanoaggregates in the bulk. Adsorption mechanisms of PA and NA molecules at toluene-water interfaces were clarified through a detailed analysis on the interactions among different species in the system. The results obtained from this work provide insights into designing appropriate chemical demulsifiers or co-demulsifiers for breaking water-in-oil emulsions of great industrial applications.

  17. Small molecule-mediated duplex formation of nucleic acids with 'incompatible' backbones.

    PubMed

    Cafferty, Brian J; Musetti, Caterina; Kim, Keunsoo; Horowitz, Eric D; Krishnamurthy, Ramanarayanan; Hud, Nicholas V

    2016-04-07

    Proflavine, a known intercalator of DNA and RNA, promotes duplex formation by nucleic acids with natural and non-natural backbones that otherwise form duplexes with low thermal stability, and even some that show no sign of duplex formation in the absence of proflavine. These findings demonstrate the potential for intercalators to be used as cofactors for the assembly of rationally designed nucleic acid structures, and could provide fundamental insights regarding intercalation of natural nucleic acid duplexes.

  18. Translation of DNA into a Library of 13,000 Synthetic Small-Molecule Macrocycles Suitable for In Vitro Selection

    PubMed Central

    Tse, Brian N.; Snyder, Thomas M.; Shen, Yinghua; Liu, David R.

    2009-01-01

    DNA-templated organic synthesis enables the translation, selection, and amplification of DNA sequences encoding synthetic small-molecule libraries. Previously we described the DNA-templated multistep synthesis and model in vitro selection of a pilot library of 65 macrocycles. In this work we report several key developments that enable the DNA-templated synthesis of much larger (> 10,000-membered) small-molecule libraries. We developed and validated a capping-based approach to DNA-templated library synthesis that increases final product yields, simplifies the structure and preparation of reagents, and reduces the number of required manipulations. To expand the size and structural diversity of the macrocycle library, we augmented the number of building blocks in each DNA-templated step from four to 12, selected eight different starting scaffolds which result in four macrocycle ring sizes and two building-block orientations, and confirmed the ability of the 36 building blocks and eight scaffolds to generate DNA-templated macrocycle products. We computationally generated and experimentally validated an expanded set of codons sufficient to support 1,728 combinations of step 1, step 2, and step 3 building blocks. Finally, we developed new high-resolution LC/MS analysis methods to assess the quality of large DNA-templated small-molecule libraries. Integrating these four developments, we executed the translation of 13,824 DNA templates into their corresponding small-molecule macrocycles. Analysis of the resulting libraries is consistent with excellent (> 90%) representation of desired macrocycle products and a stringent test of sequence specificity suggests a high degree of sequence fidelity during translation. The quality and structural diversity of this expanded DNA-templated library provides a rich starting point for the discovery of functional synthetic small-molecule macrocycles. PMID:18956864

  19. Structure and Dynamics of Four-way DNA Junctions Dynamics Revealed by Single-Molecule AFM

    NASA Astrophysics Data System (ADS)

    Lyubchenko, Yuri

    2004-03-01

    For-way DNA junctions (Holliday junctions) are critical intermediates for homologous, site-specific recombination, DNA repair and replication. A wealth of structural information is available for immobile four-way junctions. However, these data cannot give the answer on the mechanism of branch migration, the major property of the Holliday junction. Two models for the mechanism of branch migration were suggested. According to the early model of Alberts-Meselson-Sigal, exchanging DNA strands around the junction remain parallel during branch migration. Kinetic studies of branch migration suggest an alternative model in which the junction adopts an extended conformation. We tested these models using a Holliday junction undergoing branch migration. Note that it was the first time when the dynamics of the four-way DNA junction capable of branch migration had been analyzed. We applied time-lapse atomic force microscopy (single molecule dynamics AFM) to image directly loosely bound DNA at liquid-surface interface. These experiments show that mobile Holliday junctions adopt an unfolded conformation during branch migration. This conformation of the junction remains unchanged until strand separation. The data obtained support the model for branch migration having the extended conformation of the Holliday junction. The analysis of the Holliday junctions dynamics at conditions limiting branch migration revealed a broad movement of the arms suggesting that the range of mobility of these junctions is much wider than detected before. Further applications of the time-lapse AFM approach in attempt to resolve the subpopulations of the junctions conformers and the prospects for analyses of dynamics of complex biological systems will be discussed.

  20. Single-molecule views of MutS on mismatched DNA

    PubMed Central

    Lee, Jong-Bong; Cho, Won-Ki; Park, Jonghyun; Jeon, Yongmoon; Kim, Daehyung; Lee, Seung Hwan; Fishel, Richard

    2014-01-01

    Base-pair mismatches that occur during DNA replication or recombination can reduce genetic stability or conversely increase genetic diversity. The genetics and biophysical mechanism of mismatch repair (MMR) has been extensively studied since its discovery nearly 50 years ago. MMR is a strand-specific excision-resynthesis reaction that is initiated by MutS homolog (MSH) binding to the mismatched nucleotides. The MSH mismatch-binding signal is then transmitted to the immediate downstream MutL homolog (MLH/PMS) MMR components and ultimately to a distant strand scission site where excision begins. The mechanism of signal transmission has been controversial for decades. We have utilized single molecule Forster Resonance Energy Transfer (smFRET), Fluorescence Tracking (smFT) and Polarization Total Internal Reflection Fluorescence (smP-TIRF) to examine the interactions and dynamic behaviors of single Thermus aquaticus MutS (TaqMutS) particles on mismatched DNA. We determined that Taq-MutS forms an incipient clamp to search for a mismatch in ∼1 s intervals by 1-dimensional (1D) thermal fluctuation-driven rotational diffusion while in continuous contact with the helical duplex DNA. When MutS encounters a mismatch it lingers for ∼3 s to exchange bound ADP for ATP (ADP → ATP exchange). ATP binding by TaqMutS induces an extremely stable clamp conformation (∼10 min) that slides off the mismatch and moves along the adjacent duplex DNA driven simply by 1D thermal diffusion. The ATP-bound sliding clamps rotate freely while in discontinuous contact with the DNA. The visualization of a train of MSH proteins suggests that dissociation of ATP-bound sliding clamps from the mismatch permits multiple mismatch-dependent loading events. These direct observations have provided critical clues into understanding the molecular mechanism of MSH proteins during MMR. PMID:24629484

  1. Kinetics of DNA Strand Displacement Systems with Locked Nucleic Acids.

    PubMed

    Olson, Xiaoping; Kotani, Shohei; Yurke, Bernard; Graugnard, Elton; Hughes, William L

    2017-03-30

    Locked nucleic acids (LNAs) are conformationally restricted RNA nucleotides. Their increased thermal stability and selectivity toward their complements make them well-suited for diagnostic and therapeutic applications. Although the structural and thermodynamic properties of LNA-LNA, LNA-RNA, and LNA-DNA hybridizations are known, the kinetic effects of incorporating LNA nucleotides into DNA strand displacement systems are not. Here, we thoroughly studied the strand displacement kinetics as a function of the number and position of LNA nucleotides in DNA oligonucleotides. When compared to that of an all-DNA control, with an identical sequence, the leakage rate constant was reduced more than 50-fold, to an undetectable level, and the invasion rate was preserved for a hybrid DNA/LNA system. The total performance enhancement ratio also increased more than 70-fold when calculating the ratio of the invading rate to the leakage rate constants for a hybrid system. The rational substitution of LNA nucleotides for DNA nucleotides preserves sequence space while improving the signal-to-noise ratio of strand displacement systems. Hybrid DNA/LNA systems offer great potential for high-performance chemical reaction networks that include catalyzed hairpin assemblies, hairpin chain reactions, motors, walkers, and seesaw gates.

  2. Acidity characterization of heterogeneous catalysts by solid-state NMR spectroscopy using probe molecules.

    PubMed

    Zheng, Anmin; Liu, Shang-Bin; Deng, Feng

    2013-01-01

    Characterization of the surface acidic properties of solid acid catalysts is a key issue in heterogeneous catalysis. Important acid features of solid acids, such as their type (Brønsted vs. Lewis acid), distribution and accessibility (internal vs. external sites), concentration (amount), and strength of acid sites are crucial factors dictating their reactivity and selectivity. This short review provides information on different solid-state NMR techniques used for acidity characterization of solid acid catalysts. In particular, different approaches using probe molecules containing a specific nucleus of interest, such as pyridine-d5, 2-(13)C-acetone, trimethylphosphine, and trimethylphosphine oxide, are compared. Incorporation of valuable information (such as the adsorption structure, deprotonation energy, and NMR parameters) from density functional theory (DFT) calculations can yield explicit correlations between the chemical shift of adsorbed probe molecules and the intrinsic acid strength of solid acids. Methods that combine experimental NMR data with DFT calculations can therefore provide both qualitative and quantitative information on acid sites.

  3. A candidate reference method for quantification of low concentrations of plasmid DNA by exhaustive counting of single DNA molecules in a flow stream

    NASA Astrophysics Data System (ADS)

    Yoo, Hee-Bong; Oh, Donggeun; Song, Jae Yong; Kawaharasaki, Mamoru; Hwang, Jeeseong; Yang, In Chul; Park, Sang-Ryoul

    2014-10-01

    This work demonstrates accurate measurement of the amount of substance concentration of low concentration plasmid DNA by counting individual DNA molecules using a high-sensitivity flow cytometric setup. Plasmid DNA is a widely used form of DNA, and its quantity often needs to be accurately determined. This work establishes a reference analytical method for direct quantification of low concentration plasmid DNA prepared as reference standards for polymerase chain reaction-based DNA quantification. The model plasmid DNA pBR322 (4361 bp) was stained with a fluorescent dye and was detected in a flow stream in a micro-fluidic channel with laser-induced fluorescence detection, for which the DNA flow was electro-hydrodynamically focused at the centre of the channel. 200 to 8000 DNA molecules in a ˜1 µL sample volume were counted within 2 min in an ‘exhaustive counting’ manner, which facilitated quantitation without calibration. The sample volume was measured and validated from the close agreement of the results of two independent measurement methods, gravimetric determination of water filling the capillary and graphical estimation of actual cross sectional area of the capillary tubing with the image of calibrated scanning electron microscopy. Within the given concentration range, an excellent measurement linearity (R2 = 0.999) was achieved with appropriate data processing for the correction of the events of double molecules (detection of double molecules opposed to single molecule detection assumed, which occurs due to their coincidental passing of the detection zone). The validity of the proposed method was confirmed from the close agreement with the results of quantitation of enzymatically released nucleotides using capillary electrophoresis.

  4. Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research

    PubMed Central

    Fujita, Toshitsugu; Fujii, Hodaka

    2015-01-01

    Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE) proteins and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR/Cas) system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing. PMID:26404236

  5. Fabrication of a new substrate for atomic force microscopic observation of DNA molecules from an ultrasmooth sapphire plate.

    PubMed

    Yoshida, K; Yoshimoto, M; Sasaki, K; Ohnishi, T; Ushiki, T; Hitomi, J; Yamamoto, S; Sigeno, M

    1998-04-01

    A new stable substrate applicable to the observation of DNA molecules by atomic force microscopy (AFM) was fabricated from a ultrasmooth sapphire (alpha-Al2O3 single crystal) plate. The atomically ultrasmooth sapphire as obtained by high-temperature annealing has hydrophobic surfaces and could not be used for the AFM observation of DNA. However, sapphire treated with Na3PO4 aqueous solution exhibited a hydrophilic character while maintaining a smooth surface structure. The surface of the wet-treated sapphire was found by x-ray photoelectron spectroscopy and AFM to be approximately 0.3 nm. The hydrophilic surface character of the ultrasmooth sapphire plate made it easy for DNA molecules to adhere to the plate. Circular molecules of the plasmid DNA could be imaged by AFM on the hydrophilic ultrasmooth sapphire plate.

  6. Fabrication of a new substrate for atomic force microscopic observation of DNA molecules from an ultrasmooth sapphire plate.

    PubMed Central

    Yoshida, K; Yoshimoto, M; Sasaki, K; Ohnishi, T; Ushiki, T; Hitomi, J; Yamamoto, S; Sigeno, M

    1998-01-01

    A new stable substrate applicable to the observation of DNA molecules by atomic force microscopy (AFM) was fabricated from a ultrasmooth sapphire (alpha-Al2O3 single crystal) plate. The atomically ultrasmooth sapphire as obtained by high-temperature annealing has hydrophobic surfaces and could not be used for the AFM observation of DNA. However, sapphire treated with Na3PO4 aqueous solution exhibited a hydrophilic character while maintaining a smooth surface structure. The surface of the wet-treated sapphire was found by x-ray photoelectron spectroscopy and AFM to be approximately 0.3 nm. The hydrophilic surface character of the ultrasmooth sapphire plate made it easy for DNA molecules to adhere to the plate. Circular molecules of the plasmid DNA could be imaged by AFM on the hydrophilic ultrasmooth sapphire plate. PMID:9545030

  7. Comparative Study on Single-Molecule Junctions of Alkane- and Benzene-Based Molecules with Carboxylic Acid/Aldehyde as the Anchoring Groups

    NASA Astrophysics Data System (ADS)

    Chen, Fang; Peng, Lin-Lu; Hong, Ze-Wen; Mao, Jin-Chuan; Zheng, Ju-Fang; Shao, Yong; Niu, Zhen-Jiang; Zhou, Xiao-Shun

    2016-08-01

    We have measured the alkane and benzene-based molecules with aldehyde and carboxylic acid as anchoring groups by using the electrochemical jump-to-contact scanning tunneling microscopy break junction (ECSTM-BJ) approach. The results show that molecule with benzene backbone has better peak shape and intensity than those with alkane backbone. Typically, high junction formation probability for same anchoring group (aldehyde and carboxylic acid) with benzene backbone is found, which contributes to the stronger attractive interaction between Cu and molecules with benzene backbone. The present work shows the import role of backbone in junction, which can guide the design molecule to form effective junction for studying molecular electronics.

  8. Single-molecule visualization of RecQ helicase reveals DNA melting, nucleation, and assembly are required for processive DNA unwinding.

    PubMed

    Rad, Behzad; Forget, Anthony L; Baskin, Ronald J; Kowalczykowski, Stephen C

    2015-12-15

    DNA helicases are motor proteins that unwind double-stranded DNA (dsDNA) to reveal single-stranded DNA (ssDNA) needed for many biological processes. The RecQ helicase is involved in repairing damage caused by DNA breaks and stalled replication forks via homologous recombination. Here, the helicase activity of RecQ was visualized on single molecules of DNA using a fluorescent sensor that directly detects ssDNA. By monitoring the formation and progression of individual unwinding forks, we observed that both the frequency of initiation and the rate of unwinding are highly dependent on RecQ concentration. We establish that unwinding forks can initiate internally by melting dsDNA and can proceed in both directions at up to 40-60 bp/s. The findings suggest that initiation requires a RecQ dimer, and that continued processive unwinding of several kilobases involves multiple monomers at the DNA unwinding fork. We propose a distinctive model wherein RecQ melts dsDNA internally to initiate unwinding and subsequently assembles at the fork into a distribution of multimeric species, each encompassing a broad distribution of rates, to unwind DNA. These studies define the species that promote resection of DNA, proofreading of homologous pairing, and migration of Holliday junctions, and they suggest that various functional forms of RecQ can be assembled that unwind at rates tailored to the diverse biological functions of RecQ helicase.

  9. Unraveling the complexity of the interactions of DNA nucleotides with gold by single molecule force spectroscopy

    NASA Astrophysics Data System (ADS)

    Bano, Fouzia; Sluysmans, Damien; Wislez, Arnaud; Duwez, Anne-Sophie

    2015-11-01

    Addressing the effect of different environmental factors on the adsorption of DNA to solid supports is critical for the development of robust miniaturized devices for applications ranging from biosensors to next generation molecular technology. Most of the time, thiol-based chemistry is used to anchor DNA on gold - a substrate commonly used in nanotechnology - and little is known about the direct interaction between DNA and gold. So far there have been no systematic studies on the direct adsorption behavior of the deoxyribonucleotides (i.e., a nitrogenous base, a deoxyribose sugar, and a phosphate group) and on the factors that govern the DNA-gold bond strength. Here, using single molecule force spectroscopy, we investigated the interaction of the four individual nucleotides, adenine, guanine, cytosine, and thymine, with gold. Experiments were performed in three salinity conditions and two surface dwell times to reveal the factors that influence nucleotide-Au bond strength. Force data show that, at physiological ionic strength, adenine-Au interactions are stronger, asymmetrical and independent of surface dwell time as compared to cytosine-Au and guanine-Au interactions. We suggest that in these conditions only adenine is able to chemisorb on gold. A decrease of the ionic strength significantly increases the bond strength for all nucleotides. We show that moderate ionic strength along with longer surface dwell period suggest weak chemisorption also for cytosine and guanine.Addressing the effect of different environmental factors on the adsorption of DNA to solid supports is critical for the development of robust miniaturized devices for applications ranging from biosensors to next generation molecular technology. Most of the time, thiol-based chemistry is used to anchor DNA on gold - a substrate commonly used in nanotechnology - and little is known about the direct interaction between DNA and gold. So far there have been no systematic studies on the direct

  10. Reconstructing multiple free energy pathways of DNA stretching from single molecule experiments.

    PubMed

    Frey, Eric W; Li, Jingqiang; Wijeratne, Sithara S; Kiang, Ching-Hwa

    2015-04-23

    Free energy landscapes provide information on the dynamics of proteins and nucleic acid folding. It has been demonstrated that such landscapes can be reconstructed from single molecule force measurement data using Jarzynski's equality, which requires only stretching data. However, when the process is reversible, the Crooks fluctuation theorem combines both stretch and relaxation force data for the analysis and can offer more rapid convergence of free energy estimates of different states. Here we demonstrate that, similar to Jarzynski's equality, the Crooks fluctuation theorem can be used to reconstruct the full free energy landscapes. In addition, when the free energy landscapes exhibit multiple folding pathways, one can use Jarzynski's equality to reconstruct individual free energy pathways if the experimental data show distinct work distributions. We applied the method to reconstruct the overstretching transition of poly(dA) to demonstrate that the nonequilibrium work theorem combined with single molecule force measurements provides a clear picture of the free energy landscapes.

  11. Dbl2 Regulates Rad51 and DNA Joint Molecule Metabolism to Ensure Proper Meiotic Chromosome Segregation

    PubMed Central

    Hyppa, Randy W.; Benko, Zsigmond; Misova, Ivana; Schleiffer, Alexander; Smith, Gerald R.; Gregan, Juraj

    2016-01-01

    To identify new proteins required for faithful meiotic chromosome segregation, we screened a Schizosaccharomyces pombe deletion mutant library and found that deletion of the dbl2 gene led to missegregation of chromosomes during meiosis. Analyses of both live and fixed cells showed that dbl2Δ mutant cells frequently failed to segregate homologous chromosomes to opposite poles during meiosis I. Removing Rec12 (Spo11 homolog) to eliminate meiotic DNA double-strand breaks (DSBs) suppressed the segregation defect in dbl2Δ cells, indicating that Dbl2 acts after the initiation of meiotic recombination. Analyses of DSBs and Holliday junctions revealed no significant defect in their formation or processing in dbl2Δ mutant cells, although some Rec12-dependent DNA joint molecules persisted late in meiosis. Failure to segregate chromosomes in the absence of Dbl2 correlated with persistent Rad51 foci, and deletion of rad51 or genes encoding Rad51 mediators also suppressed the segregation defect of dbl2Δ. Formation of foci of Fbh1, an F-box helicase that efficiently dismantles Rad51-DNA filaments, was impaired in dbl2Δ cells. Our results suggest that Dbl2 is a novel regulator of Fbh1 and thereby Rad51-dependent DSB repair required for proper meiotic chromosome segregation and viable sex cell formation. The wide conservation of these proteins suggests that our results apply to many species. PMID:27304859

  12. Laser-triggered release of encapsulated molecules from polylactic-co-glycolic acid microcapsules

    NASA Astrophysics Data System (ADS)

    Ariyasu, Kazumasa; Ishii, Atsuhiro; Umemoto, Taiga; Terakawa, Mitsuhiro

    2016-08-01

    The controlled release of encapsulated molecules from a microcapsule is a promising method of targeted drug delivery. Laser-triggered methods for the release of encapsulated molecules have the advantage of spatial and temporal controllability. In this study, we demonstrated the release of encapsulated molecules from biodegradable polymer-based microcapsules using near-infrared femtosecond laser pulses. The polylactic-co-glycolic acid microcapsules encapsulating fluorescein isothiocyanate-dextran molecules were fabricated using a dual-coaxial nozzle system. Irradiation of femtosecond laser pulses enhanced the release of the molecules from the microcapsules, which was accompanied by a decrease in the residual ratio of the microcapsules. The laser-induced modification of the surface of the shell of the microcapsules indicated the potential for sustained release as well as burst release.

  13. Separation of large DNA molecules by size exclusion chromatography-based microchip with on-chip concentration structure

    NASA Astrophysics Data System (ADS)

    Azuma, Naoki; Itoh, Shintaro; Fukuzawa, Kenji; Zhang, Hedong

    2016-06-01

    The separation of DNA molecules according to their size represents a fundamental bioanalytical procedure. Here, we report the development of a chip-sized device, consisting of micrometer-sized fence structures fabricated in a microchannel, for the separation of large DNA molecules (over 10 kbp) based on the principle of size exclusion chromatography (SEC). In order to achieve separation, two approaches were utilized: first, the DNA samples were concentrated immediately prior to separation using nanoslit structures, with the aim of improving the resolution. Second, a theoretical model of SEC-based separation was established and applied in order to predict the optimal voltage range for separation. In this study, we achieved separation of λ DNA (48.5 kbp) and T4 DNA (166 kbp) using the present SEC-based microchip.

  14. γ Sulphate PNA (PNA S): highly selective DNA binding molecule showing promising antigene activity.

    PubMed

    Avitabile, Concetta; Moggio, Loredana; Malgieri, Gaetano; Capasso, Domenica; Di Gaetano, Sonia; Saviano, Michele; Pedone, Carlo; Romanelli, Alessandra

    2012-01-01

    Peptide Nucleic Acids (PNAs), nucleic acid analogues showing high stability to enzyme degradation and strong affinity and specificity of binding toward DNA and RNA are widely investigated as tools to interfere in gene expression. Several studies have been focused on PNA analogues with modifications on the backbone and bases in the attempt to overcome solubility, uptake and aggregation issues. γ PNAs, PNA derivatives having a substituent in the γ position of the backbone show interesting properties in terms of secondary structure and affinity of binding toward complementary nucleic acids. In this paper we illustrate our results obtained on new analogues, bearing a sulphate in the γ position of the backbone, developed to be more DNA-like in terms of polarity and charge. The synthesis of monomers and oligomers is described. NMR studies on the conformational properties of monomers and studies on the secondary structure of single strands and triplexes are reported. Furthermore the hybrid stability and the effect of mismatches on the stability have also been investigated. Finally, the ability of the new analogue to work as antigene, interfering with the transcription of the ErbB2 gene on a human cell line overexpressing ErbB2 (SKBR3), assessed by FACS and qPCR, is described.

  15. Probing the Influence of Amino Acids on Photoluminescence from Carbon Nanotubes Suspended with DNA.

    PubMed

    Kurnosov, N V; Leontiev, V S; Karachevtsev, V A

    2016-11-01

    The quantitative analysis of amino acid levels in the human organism is required for the early clinical diagnosis of a variety of diseases. In this work the influence of 13 amino acid doping on the photoluminescence (PL) from the semiconducting single-walled carbon nanotubes (SWNTs) suspended with single-stranded DNA (ssDNA) in water has been studied. Amino acid doping leads to the PL enhancement and the strongest increase was found after cysteine doping of the nanotube suspension while addition of other amino acids yielded the significantly smaller effect. The emphasis of cysteine molecules is attributed to presence of the reactive thiol group that turns cysteine into reducing agent that passivates the p-defects on the nanotube sidewall and increases the PL intensity. The reasons of PL enhancement after doping with other amino acids are discussed. The response of nanotube PL to cysteine addition depends on the nanotube aqueous suspension preparation with tip or bath sonication treatment. The enhancement of the emission from different nanotube species after cysteine doping was analyzed too. It was shown that the increase of the carbon nanotube PL at addition of cysteine allows successful monitoring of the cysteine concentration in aqueous solution in the range of 50-1000 μM.

  16. DNA Tetrominoes: The Construction of DNA Nanostructures Using Self-Organised Heterogeneous Deoxyribonucleic Acids Shapes

    PubMed Central

    Ong, Hui San; Rahim, Mohd Syafiq; Firdaus-Raih, Mohd; Ramlan, Effirul Ikhwan

    2015-01-01

    The unique programmability of nucleic acids offers alternative in constructing excitable and functional nanostructures. This work introduces an autonomous protocol to construct DNA Tetris shapes (L-Shape, B-Shape, T-Shape and I-Shape) using modular DNA blocks. The protocol exploits the rich number of sequence combinations available from the nucleic acid alphabets, thus allowing for diversity to be applied in designing various DNA nanostructures. Instead of a deterministic set of sequences corresponding to a particular design, the protocol promotes a large pool of DNA shapes that can assemble to conform to any desired structures. By utilising evolutionary programming in the design stage, DNA blocks are subjected to processes such as sequence insertion, deletion and base shifting in order to enrich the diversity of the resulting shapes based on a set of cascading filters. The optimisation algorithm allows mutation to be exerted indefinitely on the candidate sequences until these sequences complied with all the four fitness criteria. Generated candidates from the protocol are in agreement with the filter cascades and thermodynamic simulation. Further validation using gel electrophoresis indicated the formation of the designed shapes. Thus, supporting the plausibility of constructing DNA nanostructures in a more hierarchical, modular, and interchangeable manner. PMID:26258940

  17. A New Physiological Role for the DNA Molecule as a Protector against Drying Stress in Desiccation-Tolerant Microorganisms

    PubMed Central

    García-Fontana, Cristina; Narváez-Reinaldo, Juan J.; Castillo, Francisco; González-López, Jesús; Luque, Irene; Manzanera, Maximino

    2016-01-01

    The DNA molecule is associated with the role of encoding information required to produce RNA which is translated into proteins needed by the cell. This encoding involves information transmission to offspring or to other organisms by horizontal transfer. However, despite the abundance of this molecule in both the cell and the environment, its physiological role seems to be restricted mainly to that of a coding and inheritance molecule. In this paper, we report a new physiological role for the DNA molecule as involved in protection against desiccation, in addition to its well-established main information transfer and other recently reported functions such as bio-film formation in eDNA form. Desiccation-tolerant microorganisms such as Microbacterium sp. 3J1 significantly upregulate genes involved in DNA synthesis to produce DNA as part of their defensive mechanisms to protect protein structures and functions from drying according to RNA-seq analysis. We have observed the intracellular overproduction of DNA in two desiccation-tolerant microorganisms, Microbacterium sp. 3J1 and Arthrobacter siccitolerans 4J27, in response to desiccation signals. In addition, this conclusion can be made from our observations that synthetic DNA protects two proteins from drying and when part of a xeroprotectant preparation, DNA from various organisms including desiccation-sensitive species, does the same. Removal of DNA by nuclease treatment results in absence of this additive protective effect. We validated this role in biochemical and biophysical assays in proteins and occurs in trans even with short, single chains of synthetically produced DNA. PMID:28066383

  18. Phenolic acids act as signaling molecules in plant-microbe symbioses

    PubMed Central

    Mandal, Santi M; Chakraborty, Dipjyoti

    2010-01-01

    Phenolic acids are the main polyphenols made by plants. These compounds have diverse functions and are immensely important in plant-microbe interactions/symbiosis. Phenolic compounds act as signaling molecules in the initiation of legumerhizobia symbioses, establishment of arbuscular mycorrhizal symbioses and can act as agents in plant defense. Flavonoids are a diverse class of polyphenolic compounds that have received considerable attention as signaling molecules involved in plant-microbe interactions compared to the more widely distributed, simple phenolic acids; hydroxybenzoic and hydroxycinnamic acids, which are both derived from the general phenylpropanoid pathway. This review describes the well-known roles attributed to phenolic compounds as nod gene inducers of legume-rhizobia symbioses, their roles in induction of the GmGin1 gene in fungus for establishment of arbuscular mycorrhizal symbiosis, their roles in inducing vir gene expression in Agrobacterium, and their roles as defense molecules operating against soil borne pathogens that could have great implications for rhizospheric microbial ecology. Amongst plant phenolics we have a lack of knowledge concerning the roles of phenolic acids as signaling molecules beyond the relatively well-defined roles of flavonoids. This may be addressed through the use of plant mutants defective in phenolic acids biosynthesis or knock down target genes in future investigations. PMID:20400851

  19. Structural analysis of mitochondrial DNA molecules from fungi and plants using moving pictures and pulsed-field gel electrophoresis.

    PubMed

    Bendich, A J

    1996-02-02

    The size and structure of mitochondrial DNA (mtDNA) molecules was investigated by conventional and pulsed-field gel electrophoresis (PFGE) and by analyzing moving pictures during electrophoresis of individual fluorescently labelled mtDNA molecules. Little or no mtDNA that migrated into the gel was found in circular form for fungi (Schizosaccharomyces pombe, Saccharomyces cerevisiae and Neurospora crassa) or plants (Brassica hirta, tobacco, voodoo lily and maize). Most mtDNA migrated as a smear of linear DNA sizes from about 50 to 100 or 250 kilobases (kb), depending on the species, irrespective of the size of the mitochondrial genome over a range of 0.06 to 570 kb. S. cerevisiae, B. hirta and tobacco also yielded a linear mtDNA fraction containing molecules > 1000 kb in size. About half the mtDNA remained in the well of the gel after PFGE. Moving pictures revealed that this well-bound (wb) mtDNA contained molecules larger than the genome size in linear form for all species (except N. crassa) and in multi-fibered, comet-like forms for most of the wb mtDNA of N. crassa and Sc. pombe. A minor amount of the wb mtDNA with visually interpretable structure was circular: circle sizes were both larger and smaller than the 80-kb genome of S. cerevisiae, larger than the 19-kb genome of Sc. pombe and smaller than the 208-kb and 570-kb genomes of B. hirta and maize, respectively. About 25 to 75% of the wb mtDNA from cultured tobacco cells was found in circles smaller than its genome size. Partial digestion of Sc. pombe mtDNA with restriction endonucleases that cleave once per genome revealed gel bands at about 38 kb and 19 kb with a smear of sizes between the bands and below the 19-kb band, suggesting a head-to-tail genomic concatemer as the most prominent form in extracted mtDNA. A pattern of bands with smears was also found for complete digests (with multiply cleaving enzymes) of mtDNA from Sc. pombe, S. cerevisiae and N. crassa, but bands without smears were found for

  20. [Physical mapping of Streptomyces coelicolor A3(2). V. Structural modifications of actinophage phiC43 DNA molecules].

    PubMed

    Sladkova, I A; Chinenova, T A; Pel'ts, L B; Rebentish, B A; Lomovskaia, N D

    1981-01-01

    As shown by genetical and physical methods, the total preparation of phiC43 phage obtained after spontaneous induction of the prophage from S. lividans 803 strain is a heterogenous population. The wild-type phage (phi C43 wt) is only represented in 5--10% of the population. The majority of phage variants are not able to establish the lysogenic state. The structure of DNA molecules of some phages from the total preparations was characterized by electron microscopy of DNA heteroduplexes. Molecules of phiC43 wt DNA appeared to be completely homologous to those of recently studied phiC62 phage, except for two small regions of approximately 0.3 kb in the central part. Phage variants defective in establishment of the lysogenic state were distributed to two groups. One of them consists of deletion variants, the other--deletion/insertion variants. Deletions in DNA molecule of all nonlysogenizing phage overlap. The region of overlapping seems to be responsible for establishment of the lysogenic state. In the same region, deletion of DNA molecules of mutant phiC311 yg2 has been located. Three deletion/insertion variants contain homologous foreign sequences of various length. It is likely that these insertions are fragments of the host chromosomal DNA.

  1. Selection and identification of malaria vaccine target molecule using bioinformatics and DNA vaccination.

    PubMed

    Shuaibu, M N; Kikuchi, M; Cherif, M S; Helegbe, G K; Yanagi, T; Hirayama, K

    2010-10-04

    Following a genome-wide search for a blood stage malaria DNA-based vaccine using web-based bioinformatic tools, 29 genes from the annotated Plasmodium yoelii genome sequence (www.PlasmoDB.org and www.tigr.org) were identified as encoding GPI-anchored proteins. Target genes were those with orthologues in P. falciparum, containing an N-terminal signal sequence containing hydrophobic amino acid stretch and signal P criteria, a transmembrane-like domain and GPI anchor motif. Focusing on the blood stage, we extracted mRNA from pRBCs, PCR-amplified 22 out of the 29 selected genes, and eventually cloned nine of these into a DNA vaccine plasmid, pVAX 200-DEST. Biojector-mediated delivery of the nine DNA vaccines was conducted using ShimaJET to C57BL/6 mice at a dose of 4 μg/mouse three times at an interval of 3 weeks. Two weeks after the second booster, immunized mice were challenged with P. y. yoelii 17XL-parasitized RBCs and the level of parasitaemia, protection and survival was assessed. Immunization with one gene (PY03470) resulted in 2-4 days of delayed onset and level of parasitaemia and was associated with increased survival compared to non-immunized mice. Antibody production was, however, low following DNA vaccination, as determined by immunofluorescence assay. Recombinant protein from this gene, GPI8p transamidase-related protein (rPyTAM) in PBS or emulsified with GERBU adjuvant was also used to immunize another set of C57BL/6 mice with 10-20 μg/mouse three times at 3-week interval. Higher antibody response was obtained as determined by ELISA with similar protective effects as observed after DNA vaccination.

  2. Flexibility of nucleic acids: From DNA to RNA

    NASA Astrophysics Data System (ADS)

    Lei, Bao; Xi, Zhang; Lei, Jin; Zhi-Jie, Tan

    2016-01-01

    The structural flexibility of nucleic acids plays a key role in many fundamental life processes, such as gene replication and expression, DNA-protein recognition, and gene regulation. To obtain a thorough understanding of nucleic acid flexibility, extensive studies have been performed using various experimental methods and theoretical models. In this review, we will introduce the progress that has been made in understanding the flexibility of nucleic acids including DNAs and RNAs, and will emphasize the experimental findings and the effects of salt, temperature, and sequence. Finally, we will discuss the major unanswered questions in understanding the flexibility of nucleic acids. Project supported by the National Basic Research Program of China (Grant No. 2011CB933600), the National Natural Science Foundation of China (Grant Nos. 11175132, 11575128, and 11374234), and the Program for New Century Excellent Talents, China (Grant No. NCET 08-0408).

  3. Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

    PubMed Central

    Gallardo, Ignacio F.; Zhou, Yi; Gong, Fade; Yang, Soo-Hyun; Wold, Marc S.; Miller, Kyle M.; Paull, Tanya T.

    2016-01-01

    Exonuclease 1 (Exo1) is a 5′→3′ exonuclease and 5′-flap endonuclease that plays a critical role in multiple eukaryotic DNA repair pathways. Exo1 processing at DNA nicks and double-strand breaks creates long stretches of single-stranded DNA, which are rapidly bound by replication protein A (RPA) and other single-stranded DNA binding proteins (SSBs). Here, we use single-molecule fluorescence imaging and quantitative cell biology approaches to reveal the interplay between Exo1 and SSBs. Both human and yeast Exo1 are processive nucleases on their own. RPA rapidly strips Exo1 from DNA, and this activity is dependent on at least three RPA-encoded single-stranded DNA binding domains. Furthermore, we show that ablation of RPA in human cells increases Exo1 recruitment to damage sites. In contrast, the sensor of single-stranded DNA complex 1—a recently identified human SSB that promotes DNA resection during homologous recombination—supports processive resection by Exo1. Although RPA rapidly turns over Exo1, multiple cycles of nuclease rebinding at the same DNA site can still support limited DNA processing. These results reveal the role of single-stranded DNA binding proteins in controlling Exo1-catalyzed resection with implications for how Exo1 is regulated during DNA repair in eukaryotic cells. PMID:26884156

  4. Stretching single DNA molecules to demonstrate high-force capabilities of holographic optical tweezers.

    PubMed

    Farré, Arnau; van der Horst, Astrid; Blab, Gerhard A; Downing, Benjamin P B; Forde, Nancy R

    2010-04-01

    The well calibrated force-extension behaviour of single double-stranded DNA molecules was used as a standard to investigate the performance of phase-only holographic optical tweezers at high forces. Specifically, the characteristic overstretch transition at 65 pN was found to appear where expected, demonstrating (1) that holographic optical trap calibration using thermal fluctuation methods is valid to high forces; (2) that the holographic optical traps are harmonic out to >250 nm of 2.1 mum particle displacement; and (3) that temporal modulations in traps induced by the spatial light modulator (SLM) do not affect the ability of optical traps to hold and steer particles against high forces. These studies demonstrate a new high-force capability for holographic optical traps achievable by SLM technologies.

  5. In Silico Single-Molecule Manipulation of DNA with Rigid Body Dynamics

    PubMed Central

    Carrivain, Pascal; Barbi, Maria; Victor, Jean-Marc

    2014-01-01

    We develop a new powerful method to reproduce in silico single-molecule manipulation experiments. We demonstrate that flexible polymers such as DNA can be simulated using rigid body dynamics thanks to an original implementation of Langevin dynamics in an open source library called Open Dynamics Engine. We moreover implement a global thermostat which accelerates the simulation sampling by two orders of magnitude. We reproduce force-extension as well as rotation-extension curves of reference experimental studies. Finally, we extend the model to simulations where the control parameter is no longer the torsional strain but instead the torque, and predict the expected behavior for this case which is particularly challenging theoretically and experimentally. PMID:24586127

  6. Syntheses of biodiesel precursors: sulfonic acid catalysts for condensation of biomass-derived platform molecules.

    PubMed

    Balakrishnan, Madhesan; Sacia, Eric R; Bell, Alexis T

    2014-04-01

    Synthesis of transportation fuel from lignocellulosic biomass is an attractive solution to the green alternative-energy problem. The production of biodiesel, in particular, involves the process of upgrading biomass-derived small molecules to diesel precursors containing a specific carbon range (C11 -C23). Herein, a carbon-upgrading process utilizing an acid-catalyzed condensation of furanic platform molecules from biomass is described. Various types of sulfonic acid catalysts have been evaluated for this process, including biphasic and solid supported catalysts. A silica-bound alkyl sulfonic acid catalyst has been developed for promoting carbon-carbon bond formation of biomass-derived carbonyl compounds with 2-methylfuran. This hydrophobic solid acid catalyst exhibits activity and selectivity that are comparable to those of a soluble acid catalyst. The catalyst can be readily recovered and recycled, possesses appreciable hydrolytic stability in the presence of water, and retains its acidity over multiple reaction cycles. Application of this catalyst to biomass-derived platform molecules led to the synthesis of a variety of furanic compounds, which are potential biodiesel precursors.

  7. A mushroom-derived amino acid, ergothioneine, is a potential inhibitor of inflammation-related DNA halogenation.

    PubMed

    Asahi, Takashi; Wu, Xiaohong; Shimoda, Hiroshi; Hisaka, Shinsuke; Harada, Etsuko; Kanno, Tomomi; Nakamura, Yoshimasa; Kato, Yoji; Osawa, Toshihiko

    2016-01-01

    Myeloperoxidase (MPO)-generated halogenating molecules, such as hypochlorous acid and hypobromous acid (HOBr), in inflammatory regions are postulated to contribute to disease progression. In this study, we showed that ergothioneine (EGT), derived from an edible mushroom, inhibited MPO activity as well as the formation of 8-bromo-2'-deoxyguanosine in vitro. The HOBr scavenging effect of EGT is higher than those of ascorbic acid and glutathione. We initially observed that the administration of Coprinus comatus, an edible mushroom containing a high amount of EGT, inhibited the UV-B-induced inflammatory responses and DNA halogenation, suggesting that EGT is a promising anti-inflammatory agent from mushrooms.

  8. Mechanistic pathway for controlled extraction of guest molecule bound to herring sperm DNA using α-cyclodextrin

    NASA Astrophysics Data System (ADS)

    Jaffer, S. Syed; Ghosh, Prasun; Purkayastha, Pradipta

    2011-05-01

    trans-2-[4-(Dimethylamino)styryl]benzothiazole (DMASBT) is known to have dual emitting states where the locally excited (LE) state is responsible for fluorescence in less polar environment and in polar milieu fluorescence is from the twisted intramolecular charge transfer (TICT) state. This compound also undergoes minor groove binding to herring sperm DNA (hsDNA) evidenced by the absorption spectra before and after the binding process and an effect on DMASBT fluorescence by an anionic quencher. The binding occurs efficiently in a 1:1 manner, i.e. one guest molecule binds to one site on the hsDNA. Instead of following the DNA twist, the aromatic part seems to project outward. Thus, the bound molecule can be successfully extracted out from the DNA in a controlled way by the hydrophobic cavity of α-cyclodextrin (α-CD). The extraction starts even with a low concentration of α-CD and increases as the concentration is increased. Absorption, steady-state and time resolved fluorescence spectroscopic methods have been employed to explore the mechanistic pathway of binding of DMASBT to hsDNA. The mechanistic approach toward controlled extraction of the guest molecules from hsDNA by α-CD is reported and is expected to serve a significant purpose in treatment of drug overdose.

  9. Nucleic Acid-Peptide Complex Phase Controlled by DNA Hybridization

    NASA Astrophysics Data System (ADS)

    Vieregg, Jeffrey; Lueckheide, Michael; Leon, Lorraine; Marciel, Amanda; Tirrell, Matthew

    When polyanions and polycations are mixed, counterion release drives formation of polymer-rich complexes that can either be solid (precipitates) or liquid (coacervates) depending on the properties of the polyelectrolytes. These complexes are important in many fields, from encapsulation of industrial polymers to membrane-free segregation of biomolecules such as nucleic acids and proteins. Condensation of long double-stranded DNA has been studied for several decades, but comparatively little attention has been paid to the polyelectrolyte behavior of oligonucleotides. We report here studies of DNA oligonucleotides (10 - 88 nt) complexed with polylysine (10 - 100 aa). Unexpectedly, we find that the phase of the resulting complexes is controlled by the hybridization state of the nucleic acid, with double-stranded DNA forming precipitates and single-stranded DNA forming coacervates. Stability increases with polyelectrolyte length and decreases with solution salt concentration, with complexes of the longer double-stranded polymers undergoing precipitate/coacervate/soluble transitions as ionic strength is increased. Mixing coacervates formed by complementary single-stranded oligonucleotides results in precipitate formation, raising the possibility of stimulus-responsive material design.

  10. Quantification of false positive reduction in nucleic acid purification on hemorrhagic fever DNA.

    SciTech Connect

    James, Conrad D.; Pohl, Kenneth Roy; Derzon, Mark Steven; McClain, Jaime; Achyuthan, Komandoor

    2006-11-01

    Columbia University has developed a sensitive highly multiplexed system for genetic identification of nucleic acid targets. The primary obstacle to implementing this technology is the high rate of false positives due to high levels of unbound reporters that remain within the system after hybridization. The ability to distinguish between free reporters and reporters bound to targets limits the use of this technology. We previously demonstrated a new electrokinetic method for binary separation of kb pair long DNA molecules and oligonucleotides. The purpose of this project 99864 is to take these previous demonstrations and further develop the technique and hardware for field use. Specifically, our objective was to implement separation in a heterogeneous sample (containing target DNA and background oligo), to perform the separation in a flow-based device, and to develop all of the components necessary for field testing a breadboard prototype system.

  11. Trimethylamine as a probe molecule to differentiate acid sites in Y-FAU zeolite: FTIR study.

    PubMed

    Sarria, Francisca Romero; Blasin-Aubé, Vanessa; Saussey, Jacques; Marie, Olivier; Daturi, Marco

    2006-07-06

    In heterogeneous catalysis acidity has a very important influence on activity and selectivity: correct determination of acidic properties is a base to improve industrial processes. The aim of this work was to study trimethylamine (TMA) as a probe molecule able to distinguish between the different Brønsted acid sites in zeolitic frameworks. Our work mainly focused on faujasite-type zeolites because the HY zeolite is one of the most used acidic catalysts in industrial processes. In this paper, typical IR bands assigned to TMA-protonated species (formed in supercages) are detected in the HY zeolite. TMA interacting by hydrogen bonding with the acid sites located in the sodalite units is also observed. The wavenumbers of some typical IR bands assigned to TMA-protonated species appear to depend on the acidic strength, and a complementary study with ZSM-5 and X-FAU samples confirms this proposition.

  12. Practical Calculation of Molecular Acidity with the Aid of a Reference Molecule

    SciTech Connect

    Burger, Steven K; Liu, Shubin; Ayers, Paul W

    2011-02-24

    A set of linear free energy models are presented for determining the pK{sub a} values of amines, alcohols, and carboxylic acids. Models are determined from a series of pK{sub a} predictors, taken both from traditional natural atomic orbital analysis (NAO) and from a novel approach introduced here of using a reference molecule: an ammonium ion for amines and a hydrogen sulfide molecule for alcohols and carboxylic acids. Using these reference molecules, we calculate the barrier to proton transfer and show that a number of properties associated with the transition state are correlated with the pK{sub a}. By considering 38 predictors, we obtain a four-variable model for amines and a three-variable model for oxygen-containing compounds. The model for amines is based on 145 compounds and has a root mean squared error (RMSE) of 0.45 and R{sup 2} = 0.98. The oxygen set has 48 molecules: RMSE = 0.26, and R{sup 2} = 0.993. Similar, linear, and multilinear models are constructed after separating the sets into chemically similar categories: alcohols, carboxylic acids, and primary, secondary, tertiary, and aromatic amines. This separation gives simpler models with relatively low RMSE values, where the most important predictor of the pK{sub a} is the difference in energy between transferring the proton from the reference molecular base to the conjugate acid from the data set.

  13. Quantitative single-molecule detection of protein based on DNA tetrahedron fluorescent nanolabels.

    PubMed

    Ding, Yongshun; Liu, Xingti; Zhu, Jing; Wang, Lei; Jiang, Wei

    2014-07-01

    A highly sensitive method for single-molecule quantitative detection of human IgG is presented by the employment of a new fluorescent nanolabel. In this method, fluorescent nanolabels were assembled by inserting SYBR Green I into DNA tetrahedron nanostructure. The bio-nanolabels were attached to the streptavidin-antihuman antibody by a specific reaction between biotin and streptavidin. The antibody was combined with the target antigen, human IgG, which was immobilized on the silanized glass subtrate surface. Finally, epi-fluorescence microscopy (EFM) coupled with an electron multiplying charge-coupled device was employed for fluorescence imaging. The fluorescent spots corresponding to single protein molecule on images were counted and further used for the quantitative detection. It was found that the new nanolabel shows good photostability, biocompatiblity and exhibits no blinking compared to traditional labels like fluorescence dyes and quantum dot (QDs). In addition, the number of fluorescence spots on the images has a linear relationship with the concentration of human IgG in the range of 3.0×10(-14) to 1.0×10(-12)mol L(-1). What is more, this method showed an excellent specificity and a low matrix effect.

  14. Breaking the Nanometer Barrier: Progress in Biological Nanoscience, Measured One Molecule at a Time (How a Biophysicist Watches DNA Transcription)

    SciTech Connect

    Block, Stephen

    2006-03-20

    A new field of scientific exploration - single molecule biophysics - is currently reshaping and redefining our understanding of the mechanochemistry of life. The development of laser-based optical traps, or 'optical tweezers,' has allowed for physiological assessments of such precision that bio-molecules can now be measured and studied one at a time. In this colloquium, Professor Block will present findings based on his group's construction of optical trapping instrumentation that has broken the nanometer barrier, allowing researchers to study single-molecule displacements on the Angstrom level. Focusing on RNA polymerase, the motor enzyme responsible for transcribing the genetic code contained in DNA, Block's group has been able to measure, in real time, the motion of a single molecule of RNA polymerase as it moves from base to base along the DNA template.

  15. Sulfate- and sialic acid-containing glycolipids inhibit DNA polymerase alpha activity.

    PubMed

    Simbulan, C M; Taki, T; Tamiya-Koizumi, K; Suzuki, M; Savoysky, E; Shoji, M; Yoshida, S

    1994-03-16

    The effects of various glycolipids on the activity of immunoaffinity-purified calf thymus DNA polymerase alpha were studied in vitro. Preincubation with sialic acid-containing glycolipids, such as sialosylparagloboside (SPG), GM3, GM1, and GD1a, and sulfatide (cerebroside sulfate ester, CSE) dose-dependently inhibited the activity of DNA polymerase alpha, while other glycolipids, as well as free sphingosine and ceramide did not. About 50% inhibition was achieved by preincubating the enzyme with 2.5 microM of CSE, 50 microM of SPG or GM3, and 80 microM of GM1. Inhibition was noncompetitive with both the DNA template and the substrate dTTP, as well as with the other dNTPs. Since the inhibition was largely reversed by the addition of 0.05% Nonidet P40, these glycolipids may interact with the hydrophobic region of the enzyme protein. Apparently, the sulfate moiety in CSE and the sialic acid moiety in gangliosides were essential for the inhibition since neither neutral glycolipids (i.e., glucosylceramide, galactosylceramide, lactosylceramide) nor asialo-gangliosides (GA1 and GA2) showed any inhibitory effect. Furthermore, the ceramide backbone was also found to be necessary for maximal inhibition since the inhibition was largely abolished by substituting the lipid backbone with cholesterol. Increasing the number of sialic acid moieties per molecule further enhanced the inhibition, while elongating the sugar chain diminished it. It was clearly shown that the N-acetyl residue of the sialic acid moiety is particularly essential for inhibition by both SPG and GM3 because the loss of this residue or substitution with a glycolyl residue completely negated their inhibitory effect on DNA polymerase alpha activity.

  16. Algebraic Statistics of Poincaré Recurrences in a DNA Molecule.

    PubMed

    Mazur, Alexey K; Shepelyansky, D L

    2015-10-30

    The statistics of Poincaré recurrences is studied for the base-pair breathing dynamics of an all-atom DNA molecule in a realistic aqueous environment with thousands of degrees of freedom. It is found that at least over five decades in time the decay of recurrences is described by an algebraic law with the Poincaré exponent close to β=1.2. This value is directly related to the correlation decay exponent ν=β-1, which is close to ν≈0.15 observed in the time resolved Stokes shift experiments. By applying the virial theorem we analyze the chaotic dynamics in polynomial potentials and demonstrate analytically that an exponent β=1.2 is obtained assuming the dominance of dipole-dipole interactions in the relevant DNA dynamics. Molecular dynamics simulations also reveal the presence of strong low frequency noise with the exponent η=1.6. We trace parallels with the chaotic dynamics of symplectic maps with a few degrees of freedom characterized by the Poincaré exponent β~1.5.

  17. Hairpin-bisulfite PCR: Assessing epigenetic methylation patterns on complementary strands of individual DNA molecules

    PubMed Central

    Laird, Charles D.; Pleasant, Nicole D.; Clark, Aaron D.; Sneeden, Jessica L.; Hassan, K. M. Anwarul; Manley, Nathan C.; Vary, Jay C.; Morgan, Todd; Hansen, R. Scott; Stöger, Reinhard

    2004-01-01

    Epigenetic inheritance, the transmission of gene expression states from parent to daughter cells, often involves methylation of DNA. In eukaryotes, cytosine methylation is a frequent component of epigenetic mechanisms. Failure to transmit faithfully a methylated or an unmethylated state of cytosine can lead to altered phenotypes in plants and animals. A central unresolved question in epigenetics concerns the mechanisms by which a locus maintains, or changes, its state of cytosine methylation. We developed “hairpin-bisulfite PCR” to analyze these mechanisms. This method reveals the extent of methylation symmetry between the complementary strands of individual DNA molecules. Using hairpin-bisulfite PCR, we determined the fidelity of methylation transmission in the CpG island of the FMR1 gene in human lymphocytes. For the hypermethylated CpG island of this gene, characteristic of inactive-X alleles, we estimate a maintenance methylation efficiency of ≈0.96 per site per cell division. For de novo methylation efficiency (Ed), remarkably different estimates were obtained for the hypermethylated CpG island (Ed = 0.17), compared with the hypomethylated island on the active-X chromosome (Ed < 0.01). These results clarify the mechanisms by which the alternative hypomethylated and hypermethylated states of CpG islands are stably maintained through many cell divisions. We also analyzed a region of human L1 transposable elements. These L1 data provide accurate methylation patterns for the complementary strand of each repeat sequence analyzed. Hairpin-bisulfite PCR will be a powerful tool in studying other processes for which genetic or epigenetic information differs on the two complementary strands of DNA. PMID:14673087

  18. Quantitative Single-Molecule Surface-Enhanced Raman Scattering by Optothermal Tuning of DNA Origami-Assembled Plasmonic Nanoantennas.

    PubMed

    Simoncelli, Sabrina; Roller, Eva-Maria; Urban, Patrick; Schreiber, Robert; Turberfield, Andrew J; Liedl, Tim; Lohmüller, Theobald

    2016-11-22

    DNA origami is a powerful approach for assembling plasmonic nanoparticle dimers and Raman dyes with high yields and excellent positioning control. Here we show how optothermal-induced shrinking of a DNA origami template can be employed to control the gap sizes between two 40 nm gold nanoparticles in a range from 1 to 2 nm. The high field confinement achieved with this optothermal approach was demonstrated by detection of surface-enhanced Raman spectroscopy (SERS) signals from single molecules that are precisely placed within the DNA origami template that spans the nanoparticle gap. By comparing the SERS intensity with respect to the field enhancement in the plasmonic hot-spot region, we found good agreement between measurement and theory. Our straightforward approach for the fabrication of addressable plasmonic nanosensors by DNA origami demonstrates a path toward future sensing applications with single-molecule resolution.

  19. DNA Origami Nanoantennas with over 5000-fold Fluorescence Enhancement and Single-Molecule Detection at 25 μM.

    PubMed

    Puchkova, Anastasiya; Vietz, Carolin; Pibiri, Enrico; Wünsch, Bettina; Sanz Paz, María; Acuna, Guillermo P; Tinnefeld, Philip

    2015-12-09

    Optical nanoantennas are known to focus freely propagating light and reversely to mediate the emission of a light source located at the nanoantenna hotspot. These effects were previously exploited for fluorescence enhancement and single-molecule detection at elevated concentrations. We present a new generation of self-assembled DNA origami based optical nanoantennas with improved robustness, reduced interparticle distance, and optimized quantum-yield improvement to achieve more than 5000-fold fluorescence enhancement and single-molecule detection at 25 μM background fluorophore concentration. Besides outperforming lithographic optical antennas, DNA origami nanoantennas are additionally capable of incorporating single emitters or biomolecular assays at the antenna hotspot.

  20. Sucrose gradient analysis: computer simulation and measurement of the parameters involved in the sedimentation of DNA molecules.

    PubMed

    Macchiato, M F; Grossi, G F; Gialanella, G C

    1977-01-01

    The Montecarlo method is used to computer simulate a random distribution of molecular lengths generated by inducing T4 DNA fragmentation through the decay of 32P atoms introduced in the molecule. Taking into account the experimental conditions we find that the value of alpha for alkali sucrose gradients is 0.46 +/- 0.02 and does not depend on the running time. Our findings also prove that the computer simulation can be utilized to analyze sedimentation profiles of DNA molecules fragmented in vivo.

  1. Transfer-printing of single DNA molecule arrays on graphene for high resolution electron imaging and analysis

    PubMed Central

    Cerf, Aline; Alava, Thomas; Barton, Robert A.; Craighead, Harold G.

    2011-01-01

    Graphene represents the ultimate substrate for high-resolution transmission electron microscopy, but the deposition of biological samples on this highly hydrophobic material has until now been a challenge. We present a reliable method for depositing ordered arrays of individual elongated DNA molecules on single-layer graphene substrates for high resolution electron beam imaging and electron energy loss spectroscopy analysis. This method is a necessary step towards the observation of single elongated DNA molecules with single base spatial resolution to directly read genetic and epigenetic information. PMID:21919532

  2. [Interactions of DNA bases with individual water molecules. Molecular mechanics and quantum mechanics computation results vs. experimental data].

    PubMed

    Gonzalez, E; Lino, J; Deriabina, A; Herrera, J N F; Poltev, V I

    2013-01-01

    To elucidate details of the DNA-water interactions we performed the calculations and systemaitic search for minima of interaction energy of the systems consisting of one of DNA bases and one or two water molecules. The results of calculations using two force fields of molecular mechanics (MM) and correlated ab initio method MP2/6-31G(d, p) of quantum mechanics (QM) have been compared with one another and with experimental data. The calculations demonstrated a qualitative agreement between geometry characteristics of the most of local energy minima obtained via different methods. The deepest minima revealed by MM and QM methods correspond to water molecule position between two neighbor hydrophilic centers of the base and to the formation by water molecule of hydrogen bonds with them. Nevertheless, the relative depth of some minima and peculiarities of mutual water-base positions in' these minima depend on the method used. The analysis revealed insignificance of some differences in the results of calculations performed via different methods and the importance of other ones for the description of DNA hydration. The calculations via MM methods enable us to reproduce quantitatively all the experimental data on the enthalpies of complex formation of single water molecule with the set of mono-, di-, and trimethylated bases, as well as on water molecule locations near base hydrophilic atoms in the crystals of DNA duplex fragments, while some of these data cannot be rationalized by QM calculations.

  3. Sustained Small Molecule Delivery from Injectable Hyaluronic Acid Hydrogels through Host-Guest Mediated Retention

    PubMed Central

    Mealy, Joshua E.; Rodell, Christopher B.; Burdick, Jason A.

    2015-01-01

    Self-assembled and injectable hydrogels have many beneficial properties for the local delivery of therapeutics; however, challenges still exist in the sustained release of small molecules from these highly hydrated networks. Host-guest chemistry between cyclodextrin and adamantane has been used to create supramolecular hydrogels from modified polymers. Beyond assembly, this chemistry may also provide increased drug retention and sustained release through the formation of inclusion complexes between drugs and cyclodextrin. Here, we engineered a two-component system from adamantane-modified and β-cyclodextrin (CD)-modified hyaluronic acid (HA), a natural component of the extracellular matrix, to produce hydrogels that are both injectable and able to sustain the release of small molecules. The conjugation of cyclodextrin to HA dramatically altered its affinity for hydrophobic small molecules, such as tryptophan. This interaction led to lower molecule diffusivity and the release of small molecules for up to 21 days with release profiles dependent on CD concentration and drug-CD affinity. There was significant attenuation of release from the supramolecular hydrogels (~20% release in 24h) when compared to hydrogels without CD (~90% release in 24h). The loading of small molecules also had no effect on hydrogel mechanics or self-assembly properties. Finally, to illustrate this controlled delivery approach with clinically used small molecule pharmaceuticals, we sustained the release of two widely used drugs (i.e., doxycycline and doxorubicin) from these hydrogels. PMID:26693019

  4. Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin)

    PubMed Central

    Erwin, Graham S.; Grieshop, Matthew P.; Bhimsaria, Devesh; Eguchi, Asuka; Rodríguez-Martínez, José A.; Ansari, Aseem Z.

    2016-01-01

    The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide–DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents. PMID:26863565

  5. Genome-wide Mapping of Drug-DNA Interactions in Cells with COSMIC (Crosslinking of Small Molecules to Isolate Chromatin).

    PubMed

    Erwin, Graham S; Grieshop, Matthew P; Bhimsaria, Devesh; Eguchi, Asuka; Rodríguez-Martínez, José A; Ansari, Aseem Z

    2016-01-20

    The genome is the target of some of the most effective chemotherapeutics, but most of these drugs lack DNA sequence specificity, which leads to dose-limiting toxicity and many adverse side effects. Targeting the genome with sequence-specific small molecules may enable molecules with increased therapeutic index and fewer off-target effects. N-methylpyrrole/N-methylimidazole polyamides are molecules that can be rationally designed to target specific DNA sequences with exquisite precision. And unlike most natural transcription factors, polyamides can bind to methylated and chromatinized DNA without a loss in affinity. The sequence specificity of polyamides has been extensively studied in vitro with cognate site identification (CSI) and with traditional biochemical and biophysical approaches, but the study of polyamide binding to genomic targets in cells remains elusive. Here we report a method, the crosslinking of small molecules to isolate chromatin (COSMIC), that identifies polyamide binding sites across the genome. COSMIC is similar to chromatin immunoprecipitation (ChIP), but differs in two important ways: (1) a photocrosslinker is employed to enable selective, temporally-controlled capture of polyamide binding events, and (2) the biotin affinity handle is used to purify polyamide-DNA conjugates under semi-denaturing conditions to decrease DNA that is non-covalently bound. COSMIC is a general strategy that can be used to reveal the genome-wide binding events of polyamides and other genome-targeting chemotherapeutic agents.

  6. Cyanobacteria Produce N-(2-Aminoethyl)Glycine, a Backbone for Peptide Nucleic Acids Which May Have Been the First Genetic Molecules for Life on Earth

    PubMed Central

    Banack, Sandra Anne; Metcalf, James S.; Jiang, Liying; Craighead, Derek; Ilag, Leopold L.; Cox, Paul Alan

    2012-01-01

    Prior to the evolution of DNA-based organisms on earth over 3.5 billion years ago it is hypothesized that RNA was the primary genetic molecule. Before RNA-based organisms arose, peptide nucleic acids may have been used to transmit genetic information by the earliest forms of life on earth. We discovered that cyanobacteria produce N-(2-aminoethyl)glycine (AEG), a backbone for peptide nucleic acids. We detected AEG in axenic strains of cyanobacteria with an average concentration of 1 µg/g. We also detected AEG in environmental samples of cyanobacteria as both a free or weakly bound molecule and a tightly bound form released by acid hydrolysis, at concentrations ranging from not detected to 34 µg/g. The production of AEG by diverse taxa of cyanobacteria suggests that AEG may be a primitive feature which arose early in the evolution of life on earth. PMID:23145061

  7. Cyanobacteria produce N-(2-aminoethyl)glycine, a backbone for peptide nucleic acids which may have been the first genetic molecules for life on Earth.

    PubMed

    Banack, Sandra Anne; Metcalf, James S; Jiang, Liying; Craighead, Derek; Ilag, Leopold L; Cox, Paul Alan

    2012-01-01

    Prior to the evolution of DNA-based organisms on earth over 3.5 billion years ago it is hypothesized that RNA was the primary genetic molecule. Before RNA-based organisms arose, peptide nucleic acids may have been used to transmit genetic information by the earliest forms of life on earth. We discovered that cyanobacteria produce N-(2-aminoethyl)glycine (AEG), a backbone for peptide nucleic acids. We detected AEG in axenic strains of cyanobacteria with an average concentration of 1 µg/g. We also detected AEG in environmental samples of cyanobacteria as both a free or weakly bound molecule and a tightly bound form released by acid hydrolysis, at concentrations ranging from not detected to 34 µg/g. The production of AEG by diverse taxa of cyanobacteria suggests that AEG may be a primitive feature which arose early in the evolution of life on earth.

  8. Label-free and dual-amplified detection of protein via small molecule-ligand linked DNA and a cooperative DNA machine.

    PubMed

    Li, Pei; Wang, Lei; Zhu, Jing; Wu, Yushu; Jiang, Wei

    2015-10-15

    Sensitive detection of protein is essential for both molecular diagnostics and biomedical research. Here, taking folate receptor as the model analyte, we developed a label-free and dual-amplified strategy via small molecular-ligand linked DNA and a cooperative DNA machine which could perform primary amplification and mediate secondary amplification simultaneously. Firstly, the specific binding of folate receptor to the small-molecule folate which linked to a trigger DNA could protect the trigger DNA from exonuclease I digestion, translating folate receptor detection into trigger DNA detection. Subsequently, trigger DNA initiated the DNA machine through hybridizing with the hairpin of the DNA machine, resulting in hairpin conformational change and stem open. The open stem further hybridized with a primer which initiated circular strand-displacement polymerization reaction; meanwhile the rolling circle amplification templates which were initially blocked in the DNA machine were liberated to mediate rolling circle amplification. In such a working model, the DNA machine achieved cooperatively controlling circular strand-displacement polymerization reaction and rolling circle amplification, realizing dual-amplification. Finally, the rolling circle amplification process synthesized a long repeated G-quadruplex sequence, which strongly interacted with N-methyl mesoporphyrin IX, bringing label-free fluorescence signal. This strategy could detect folate receptor as low as 0.23 pM. A recovery over 90% was obtained when folate receptor was detected in spiked human serum, demonstrating the feasibility of this detection strategy in biological samples.

  9. Investigation of pyridine carboxylic acids in CM2 carbonaceous chondrites: Potential precursor molecules for ancient coenzymes

    NASA Astrophysics Data System (ADS)

    Smith, Karen E.; Callahan, Michael P.; Gerakines, Perry A.; Dworkin, Jason P.; House, Christopher H.

    2014-07-01

    The distribution and abundances of pyridine carboxylic acids (including nicotinic acid) in eight CM2 carbonaceous chondrites (ALH 85013, DOM 03183, DOM 08003, EET 96016, LAP 02333, LAP 02336, LEW 85311, and WIS 91600) were investigated by liquid chromatography coupled to UV detection and high resolution Orbitrap mass spectrometry. We find that pyridine monocarboxylic acids are prevalent in CM2-type chondrites and their abundance negatively correlates with the degree of pre-terrestrial aqueous alteration that the meteorite parent body experienced. We also report the first detection of pyridine dicarboxylic acids in carbonaceous chondrites. Additionally, we carried out laboratory studies of proton-irradiated pyridine in carbon dioxide-rich ices (a 1:1 mixture) to serve as a model of the interstellar ice chemistry that may have led to the synthesis of pyridine carboxylic acids. Analysis of the irradiated ice residue shows that a comparable suite of pyridine mono- and dicarboxylic acids was produced, although aqueous alteration may still play a role in the synthesis (and ultimate yield) of these compounds in carbonaceous meteorites. Nicotinic acid is a precursor to nicotinamide adenine dinucleotide, a likely ancient molecule used in cellular metabolism in all of life, and its common occurrence in CM2 chondrites may indicate that meteorites may have been a source of molecules for the emergence of more complex coenzymes on the early Earth.

  10. Investigation of Pyridine Carboxylic Acids in CM2 Carbonaceous Chondrites: Potential Precursor Molecules for Ancient Coenzymes

    NASA Technical Reports Server (NTRS)

    Smith, Karen E.; Callahan, Michael P.; Gerakines, Perry A.; Dworkin, Jason P.; House, Christopher H.

    2014-01-01

    The distribution and abundances of pyridine carboxylic acids (including nicotinic acid) in eight CM2 carbonaceous chondrites (ALH 85013, DOM 03183, DOM 08003, EET 96016, LAP 02333, LAP 02336, LEW 85311, and WIS 91600) were investigated by liquid chromatography coupled to UV detection and high resolution Orbitrap mass spectrometry. We find that pyridine monocarboxylic acids are prevalent in CM2-type chondrites and their abundance negatively correlates with the degree of pre-terrestrial aqueous alteration that the meteorite parent body experienced. We also report the first detection of pyridine dicarboxylic acids in carbonaceous chondrites. Additionally, we carried out laboratory studies of proton-irradiated pyridine in carbon dioxide-rich ices (a 1:1 mixture) to serve as a model of the interstellar ice chemistry that may have led to the synthesis of pyridine carboxylic acids. Analysis of the irradiated ice residue shows that a comparable suite of pyridine mono- and dicarboxylic acids was produced, although aqueous alteration may still play a role in the synthesis (and ultimate yield) of these compounds in carbonaceous meteorites. Nicotinic acid is a precursor to nicotinamide adenine dinucleotide, a likely ancient molecule used in cellular metabolism in all of life, and its common occurrence in CM2 chondrites may indicate that meteorites may have been a source of molecules for the emergence of more complex coenzymes on the early Earth.

  11. Investigation of Pyridine Carboxylic Acids in CM2 Carbonaceous Chondrites: Potential Precursor Molecules for Ancient Coenzymes

    NASA Technical Reports Server (NTRS)

    Smith, Karen E.; Callahan, Michael P.; Gerakines, Perry A.; Dworkin, Jason P.; House, Christopher H.

    2014-01-01

    The distribution and abundances of pyridine carboxylic acids (including nicotinic acid) in eight CM2 carbonaceous chondrites (ALH 85013, DOM 03183, DOM 08003, EET 96016, LAP 02333, LAP 02336, LEW 85311, and WIS 91600) were investigated by liquid chromatography coupled to UV detection and high resolution Orbitrap mass spectrometry. We find that pyridine monocarboxylic acids are prevalent in CM2-type chondrites and their abundance negatively correlates with the degree of pre-terrestrial aqueous alteration that the meteorite parent body experienced. We lso report the first detection of pyridine dicarboxylic acids in carbonaceous chondrites. Additionally, we carried out laboratory studies of proton-irradiated pyridine in carbon dioxide-rich ices (a 1:1 mixture) to serve as a model of the interstellar ice chemistry that may have led to the synthesis of pyridine carboxylic acids. Analysis of the irradiated ice residue shows that a comparable suite of pyridine mono- and dicarboxylic acids was produced, although aqueous alteration may still play a role in the synthesis (and ultimate yield) of these compounds in carbonaceous meteorites. Nicotinic acid is a precursor to nicotinamide adenine dinucleotide, a likely ancient molecule used in cellular metabolism in all of life, and its common occurrence in CM2 chondrites may indicate that meteorites may have been a source of molecules for the emergence of more complex coenzymes on the early Earth.

  12. L-Ascorbic Acid: A Multifunctional Molecule Supporting Plant Growth and Development

    PubMed Central

    Gallie, Daniel R.

    2013-01-01

    L-Ascorbic acid (vitamin C) is as essential to plants as it is to animals. Ascorbic acid functions as a major redox buffer and as a cofactor for enzymes involved in regulating photosynthesis, hormone biosynthesis, and regenerating other antioxidants. Ascorbic acid regulates cell division and growth and is involved in signal transduction. In contrast to the single pathway responsible for ascorbic acid biosynthesis in animals, plants use multiple pathways to synthesize ascorbic acid, perhaps reflecting the importance of this molecule to plant health. Given the importance of ascorbic acid to human nutrition, several technologies have been developed to increase the ascorbic acid content of plants through the manipulation of biosynthetic or recycling pathways. This paper provides an overview of these approaches as well as the consequences that changes in ascorbic acid content have on plant growth and function. Discussed is the capacity of plants to tolerate changes in ascorbic acid content. The many functions that ascorbic acid serves in plants, however, will require highly targeted approaches to improve their nutritional quality without compromising their health. PMID:24278786

  13. Photodissociation of organic molecules in star-forming regions. II. Acetic acid

    NASA Astrophysics Data System (ADS)

    Pilling, S.; Santos, A. C. F.; Boechat-Roberty, H. M.

    2006-04-01

    Fragments from organic molecule dissociation (such as reactive ions and radicals) can form interstellar complex molecules like amino acids. The goal of this work is to experimentally study photoionization and photodissociation processes of acetic acid (CH3COOH), a glycine (NH2CH2COOH) precursor molecule, by soft X-ray photons. The measurements were taken at the Brazilian Synchrotron Light Laboratory (LNLS), employing soft X-ray photons from a toroidal grating monochromator (TGM) beamline (100-310 eV). Mass spectra were obtained using the photoelectron photoion coincidence (PEPICO) method. Kinetic energy distribution and abundances for each ionic fragment have been obtained from the analysis of the corresponding peak shapes in the mass spectra. Absolute photoionization and photodissociation cross sections were also determined. We have found, among the channels leading to ionization, that only 4-6% of CH3COOH survive the strong ionization field. CH3CO^+, COOH+ and CH3+ ions are the main fragments, and the presence of the former may indicate that the production-destruction process of acetic acid in hot molecular cores (HMCs) could decrease the H2O abundance since the net result of this process converts H2O into OH + H^+. The COOH+ ion plays an important role in ion-molecule reactions to form large biomolecules like glycine.

  14. Predicting Electrophoretic Mobility of Protein-Ligand Complexes for Ligands from DNA-Encoded Libraries of Small Molecules.

    PubMed

    Bao, Jiayin; Krylova, Svetlana M; Cherney, Leonid T; Hale, Robert L; Belyanskaya, Svetlana L; Chiu, Cynthia H; Shaginian, Alex; Arico-Muendel, Christopher C; Krylov, Sergey N

    2016-05-17

    Selection of target-binding ligands from DNA-encoded libraries of small molecules (DELSMs) is a rapidly developing approach in drug-lead discovery. Methods of kinetic capillary electrophoresis (KCE) may facilitate highly efficient homogeneous selection of ligands from DELSMs. However, KCE methods require accurate prediction of electrophoretic mobilities of protein-ligand complexes. Such prediction, in turn, requires a theory that would be applicable to DNA tags of different structures used in different DELSMs. Here we present such a theory. It utilizes a model of a globular protein connected, through a single point (small molecule), to a linear DNA tag containing a combination of alternating double-stranded and single-stranded DNA (dsDNA and ssDNA) regions of varying lengths. The theory links the unknown electrophoretic mobility of protein-DNA complex with experimentally determined electrophoretic mobilities of the protein and DNA. Mobility prediction was initially tested by using a protein interacting with 18 ligands of various combinations of dsDNA and ssDNA regions, which mimicked different DELSMs. For all studied ligands, deviation of the predicted mobility from the experimentally determined value was within 11%. Finally, the prediction was tested for two proteins and two ligands with a DNA tag identical to those of DELSM manufactured by GlaxoSmithKline. Deviation between the predicted and experimentally determined mobilities did not exceed 5%. These results confirm the accuracy and robustness of our model, which makes KCE methods one step closer to their practical use in selection of drug leads, and diagnostic probes from DELSMs.

  15. Electron microscopic studies of the interaction between a Bacillus subtilis alpha/beta-type small, acid-soluble spore protein with DNA: protein binding is cooperative, stiffens the DNA, and induces negative supercoiling.

    PubMed Central

    Griffith, J; Makhov, A; Santiago-Lara, L; Setlow, P

    1994-01-01

    DNA within spores of Bacillus subtilis is complexed with a group of alpha/beta-type small acid-soluble spore proteins (alpha/beta-type SASPs), which have almost identical primary sequences and DNA binding properties. Here electron microscopic and cyclization studies were carried out on alpha/beta-type SASP-DNA complexes. When an alpha/beta-type SASP was incubated with linear DNA, the protein bound cooperatively, forming a helical coating 6.6 +/- 0.4 nm wide with a 2.9 +/- 0.3 nm periodicity. alpha/beta-Type SASP binding to an 890-bp DNA was weakest at an (A+T)-rich region that was highly bent, but binding eliminated the bending. alpha/beta-Type SASP binding did not alter the rise per bp in DNA but greatly increased the DNA stiffness as measured by both electron microscopic and cyclization assays. Addition of alpha/beta-type SASPs to negatively supertwisted DNA led to protein binding without significant alteration of the plectonemically interwound appearance of the DNA. Addition of alpha/beta-type SASPs to relaxed or nicked circular DNA led to molecules that by electron microscopy appeared similar to supertwisted DNA. The introduction of negative supertwists in nicked circular DNA by alpha/beta-type SASPs was confirmed by ligation of these molecules followed by topoisomer analyses using agarose gel electrophoresis. Images PMID:8058784

  16. High-throughput sequencing allows the identification of binding molecules isolated from DNA-encoded chemical libraries.

    PubMed

    Mannocci, Luca; Zhang, Yixin; Scheuermann, Jörg; Leimbacher, Markus; De Bellis, Gianluca; Rizzi, Ermanno; Dumelin, Christoph; Melkko, Samu; Neri, Dario

    2008-11-18

    DNA encoding facilitates the construction and screening of large chemical libraries. Here, we describe general strategies for the stepwise coupling of coding DNA fragments to nascent organic molecules throughout individual reaction steps as well as the first implementation of high-throughput sequencing for the identification and relative quantification of the library members. The methodology was exemplified in the construction of a DNA-encoded chemical library containing 4,000 compounds and in the discovery of binders to streptavidin, matrix metalloproteinase 3, and polyclonal human IgG.

  17. A density functional and ab initio investigation of the p-aminobenzoic acid molecule

    NASA Astrophysics Data System (ADS)

    Lago, A. F.; Dávalos, J. Z.; de Brito, A. Naves

    2007-08-01

    The p-aminobenzoic acid (C 7H 7NO 2) molecule has been investigated at different levels of theory. DFT methods (B3LYP and PBE1PBE), second order Møller-Plesset perturbation theory (MP2) and composite ab initio methods (G3MP2 and CBS) have been employed, in conjunction with large basis sets. Important informations on the electronic structure and thermochemistry of this molecule have been extracted, and the performance of the density functional and ab initio methods has been evaluated, based on the comparison of the calculated and the available experimental data.

  18. Ultrasensitive non enzymatic multiple immunosensor for tumor markers detection by coupling DNA hybridization chain reaction with intercalated molecules.

    PubMed

    Guo, Jinjin; Wang, Junchun; Zhang, Junjun; Zhang, Wenjuan; Zhang, Yuzhong

    2017-04-15

    In this study, we tried coupling the small signal molecules that could intercalate into DNA double helix with hybridization chain reaction (HCR) technique to fabricate a multiple immunosensor. Doxorubicin hydrochloride (DXH) and methylene blue (MB) were used as signal molecules and alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA) were selected as model biomarkers. The immunosensor mainly consists of three parts as follows: First, two different primary antibodies (Ab1) immobilized on the surface of gold nanoparticles (Au NPs); Second, secondary antibodies (Ab2) conjugated with DNA primer; Third, long DNA concatemers from HCR were used as a carrier to intercalate amounts of signal molecules (DXH or MB). A sandwich immunocomplex was formed among primary antibodies, target biomarkers and secondary antibodies conjugated with DNA primer via specific recognition reaction. Afterwards, DNA concatemers intercalating amounts of DXH or MB were linked to DNA primer via DNA hybridization. Square wave voltammetry (SWV) was employed to record the response signals from electroactive molecules DXH and MB, and two distinguishable signals were obtained, which peak potentials were at about -0.30V (corresponding to MB) and -0.70V (corresponding to DXH, both vs SCE), respectively. The signal intensities of MB and DXH were linearly related to the logarithm of biomarkers concentration in the range of 0.05pgmL(-1)-25ngmL(-1), and the limit of detection were 0.03pgmL(-1) for CEA and 0.02pgmL(-1) for AFP (at S/N=3), respectively. Furthermore, the immunosensor exhibited a sensitive electrochemical response to biomarkers in human serum samples and the results obtained were in accordance with reference method, indicating the immunosensor can be applied to real sample analysis in clinic diagnosis.

  19. Interaction of photosensitive surfactant with DNA and poly acrylic acid

    SciTech Connect

    Zakrevskyy, Yuriy Paasche, Jens; Lomadze, Nino; Santer, Svetlana; Cywinski, Piotr; Cywinska, Magdalena; Reich, Oliver; Löhmannsröben, Hans-Gerd

    2014-01-28

    In this paper, we investigate interactions and phase transitions in polyelectrolyte-surfactant complexes formed between a cationic azobenzene-containing surfactant and two types of polyelectrolytes: natural (DNA) or synthetic (PAA: poly acrylic acid). The construction of a phase diagram allowed distancing between four major phases: extended coil conformation, colloidally stable compacted globules, colloidal instability range, and surfactant-stabilized compact state. Investigation on the complexes’ properties in different phases and under irradiation with UV light provides information about the role of the surfactant's hydrophobic trans isomers both in the formation and destruction of DNA and PAA globules as well as in their colloidal stabilization. The trans isomer shows much stronger affinity to the polyelectrolytes than the hydrophilic cis counterpart. There is no need for complete compensation of the polyelectrolyte charges to reach the complete compaction. On contrary to the findings previously reported in the literature, we demonstrate – for the first time – complete polyelectrolyte compaction which occurs already at 20% of DNA (and at 50% of PAA) charge compensation. The trans isomer plays the main role in the compaction. The aggregation between azobenzene units in the photosensitive surfactant is a driving force of this process. The decompaction can be realized during UV light irradiation and is strongly influenced by the interplay between surfactant-surfactant and surfactant-DNA interactions in the compacted globules.

  20. Dynamics and mass accommodation of HCl molecules on sulfuric acid-water surfaces.

    PubMed

    Behr, P; Scharfenort, U; Ataya, K; Zellner, R

    2009-09-28

    A molecular beam technique has been used to study the dynamics and mass accommodation of HCl molecules in collision with sulfuric acid-water surfaces. The experiments were performed by directing a nearly mono-energetic beam of HCl molecules onto a continuously renewed liquid film of 54-76 wt% sulfuric acid at temperatures between 213 K and 243 K. Deuterated sulfuric acid was used to separate sticking but non-reactive collisions from those that involved penetration through the phase boundary followed by dissociation and recombination with D+. The results indicate that the mass accommodation of HCl on sulfuric acid-water surfaces decreases sharply with increasing acidity over the concentration range 54-76 wt%. Using the capillary wave theory of mass accommodation this effect is explained by a change of the surface dynamics. Regarding the temperature dependence it is found that the mass accommodation of HCl increases with increasing temperature and is limited by the bulk phase viscosity and driven by the restoring forces of the surface tension. These findings imply that under atmospheric conditions the uptake of HCl from the gas phase depends crucially on the bulk phase parameters of the sulfuric acid aerosol.

  1. The DNA invertase Gin of phage Mu: formation of a covalent complex with DNA via a phosphoserine at amino acid position 9.

    PubMed Central

    Klippel, A; Mertens, G; Patschinsky, T; Kahmann, R

    1988-01-01

    The DNA invertase Gin encoded by bacteriophage Mu catalyses efficient site-specific recombination between inverted repeat sequences (IR) in vivo and in vitro in the presence of the host factor FIS and the recombinational enhancer. We demonstrate that Gin alone is able to introduce single strand breaks into duplex DNA fragments which contain the IR sequence. Strand cleavage is site-specific and can occur on either strand within the IR. Cleaved molecules contain Gin covalently attached to DNA. The covalent complex is formed through linkage of Gin to the 5' DNA phosphate at the site of the break via a phosphoserine. Extensive site-directed mutational analysis showed that all mutants altered at serine position 9 were completely recombination deficient in vivo and in vitro. The mutant proteins bind to DNA but lack topoisomerase activity and are unable to introduce nicks. This holds true even for a conservative amino acid substitution at position 9. We conclude that serine at position 9 is part of the catalytic domain of Gin. The intriguing finding that the DNA invertase Gin has the same catalytic center as the DNA resolvases that promote deletions without recombinational enhancer and host factor FIS is discussed. Images PMID:3042382

  2. Directly interrogating single quantum dot labelled UvrA2 molecules on DNA tightropes using an optically trapped nanoprobe

    PubMed Central

    Simons, Michelle; Pollard, Mark R.; Hughes, Craig D.; Ward, Andrew D.; Van Houten, Bennett; Towrie, Mike; Botchway, Stan W.; Parker, Anthony W.; Kad, Neil M.

    2015-01-01

    In this study we describe a new methodology to physically probe individual complexes formed between proteins and DNA. By combining nanoscale, high speed physical force measurement with sensitive fluorescence imaging we investigate the complex formed between the prokaryotic DNA repair protein UvrA2 and DNA. This approach uses a triangular, optically-trapped “nanoprobe” with a nanometer scale tip protruding from one vertex. By scanning this tip along a single DNA strand suspended between surface-bound micron-scale beads, quantum-dot tagged UvrA2 molecules bound to these ‘”DNA tightropes” can be mechanically interrogated. Encounters with UvrA2 led to deflections of the whole nanoprobe structure, which were converted to resistive force. A force histogram from all 144 detected interactions generated a bimodal distribution centered on 2.6 and 8.1 pN, possibly reflecting the asymmetry of UvrA2’s binding to DNA. These observations successfully demonstrate the use of a highly controllable purpose-designed and built synthetic nanoprobe combined with fluorescence imaging to study protein-DNA interactions at the single molecule level. PMID:26691010

  3. Probing radiation damage by alternated current conductivity as a method to characterize electron hopping conduction in DNA molecules

    SciTech Connect

    Gomes, Paulo J.; Coelho, Margarida; Antonio Ribeiro, Paulo; Raposo, Maria; Dionisio, Madalena

    2012-09-17

    Analysis of AC electrical conductivity of deoxyribonucleic acid (DNA) thin films, irradiated with ultraviolet (UV) light, revealed that electrical conduction arises from DNA chain electron hopping between base-pairs and phosphate groups. The hopping distance calculated from correlated barrier hopping model equals the distance between DNA base-pairs, which is consistent with the loss of conductivity with irradiation time arising from a decrease in phosphates groups. In the high frequency regime, at a given frequency, real part of conductivity strongly depends on irradiation time particularly for low dose levels suggesting the use of DNA based films for UV radiation sensors.

  4. Mitochondrial DNA in the sea urchin Arbacia lixula: nucleotide sequence differences between two polymorphic molecules indicate asymmetry of mutations.

    PubMed

    De Giorgi, C; De Luca, F; Saccone, C

    1991-07-22

    Two polymorphic forms of mitochondrial DNA (mtDNA) extracted from Arbacia lixula eggs were cloned and the nucleotide sequences of specific regions determined. A comparison of the sequences of the sense strand of the two molecules demonstrates that all the differences are transitions and only of the A----G type. A change such as G----A (or A----G) on the sense mtDNA strand results from either a direct G----A (or A----G) mutation on that strand or a C----T (or T----C) on the complementary strand. None of the C----T (or T----C) changes were detected on the sense strand, which implies that the A----G mutation bias on the sense strand is not reversed for the other strand. Our observation indicates the existence of mechanisms acting asymmetrically on the two mtDNA strands, possibly during mtDNA replication.

  5. A New Three-Dimensional Educational Model Kit for Building DNA and RNA Molecules: Development and Evaluation

    ERIC Educational Resources Information Center

    Beltramini, Leila Maria; Araujo, Ana Paula Ulian; de Oliveira, Tales Henrique Goncalves; dos Santos Abel, Luciano Douglas; da Silva, Aparecido Rodrigues; dos Santos, Neusa Fernandes

    2006-01-01

    International specialized literature focused on research in biology education is sadly scarce, especially regarding biochemical and molecular aspects. In this light, researchers from this Centre for Structural Molecular Biotechnology developed and evaluated a three-dimensional educational model named "Building Life Molecules DNA and RNA." The…

  6. Extracting physical chemistry from mechanics: a new approach to investigate DNA interactions with drugs and proteins in single molecule experiments.

    PubMed

    Rocha, M S

    2015-09-01

    In this review we focus on the idea of establishing connections between the mechanical properties of DNA-ligand complexes and the physical chemistry of DNA-ligand interactions. This type of connection is interesting because it opens the possibility of performing a robust characterization of such interactions by using only one experimental technique: single molecule stretching. Furthermore, it also opens new possibilities in comparing results obtained by very different approaches, in particular when comparing single molecule techniques to ensemble-averaging techniques. We start the manuscript reviewing important concepts of DNA mechanics, from the basic mechanical properties to the Worm-Like Chain model. Next we review the basic concepts of the physical chemistry of DNA-ligand interactions, revisiting the most important models used to analyze the binding data and discussing their binding isotherms. Then, we discuss the basic features of the single molecule techniques most used to stretch DNA-ligand complexes and to obtain "force × extension" data, from which the mechanical properties of the complexes can be determined. We also discuss the characteristics of the main types of interactions that can occur between DNA and ligands, from covalent binding to simple electrostatic driven interactions. Finally, we present a historical survey of the attempts to connect mechanics to physical chemistry for DNA-ligand systems, emphasizing a recently developed fitting approach useful to connect the persistence length of DNA-ligand complexes to the physicochemical properties of the interaction. Such an approach in principle can be used for any type of ligand, from drugs to proteins, even if multiple binding modes are present.

  7. Genome organization of Tobacco leaf curl Zimbabwe virus, a new, distinct monopartite begomovirus associated with subgenomic defective DNA molecules.

    PubMed

    Paximadis, M; Rey, M E

    2001-12-01

    The complete DNA A of the begomovirus Tobacco leaf curl Zimbabwe virus (TbLCZWV) was sequenced: it comprises 2767 nucleotides with six major open reading frames encoding proteins with molecular masses greater than 9 kDa. Full-length TbLCZWV DNA A tandem dimers, cloned in binary vectors (pBin19 and pBI121) and transformed into Agrobacterium tumefaciens, were systemically infectious upon agroinoculation of tobacco and tomato. Efforts to identify a DNA B component were unsuccessful. These findings suggest that TbLCZWV is a new member of the monopartite group of begomoviruses. Phylogenetic analysis identified TbLCZWV as a distinct begomovirus with its closest relative being Chayote mosaic virus. Abutting primer PCR amplified ca. 1300 bp molecules, and cloning and sequencing of two of these molecules revealed them to be subgenomic defective DNA molecules originating from TbLCZWV DNA A. Variable symptom severity associated with tobacco leaf curl disease and TbLCZWV is discussed.

  8. Identification of antisense nucleic acid hybridization sites in mRNA molecules with self-quenching fluorescent reporter molecules.

    PubMed

    Gifford, Lida K; Opalinska, Joanna B; Jordan, David; Pattanayak, Vikram; Greenham, Paul; Kalota, Anna; Robbins, Michelle; Vernovsky, Kathy; Rodriguez, Lesbeth C; Do, Bao T; Lu, Ponzy; Gewirtz, Alan M

    2005-02-17

    We describe a physical mRNA mapping strategy employing fluorescent self-quenching reporter molecules (SQRMs) that facilitates the identification of mRNA sequence accessible for hybridization with antisense nucleic acids in vitro and in vivo, real time. SQRMs are 20-30 base oligodeoxynucleotides with 5-6 bp complementary ends to which a 5' fluorophore and 3' quenching group are attached. Alone, the SQRM complementary ends form a stem that holds the fluorophore and quencher in contact. When the SQRM forms base pairs with its target, the structure separates the fluorophore from the quencher. This event can be reported by fluorescence emission when the fluorophore is excited. The stem-loop of the SQRM suggests that SQRM be made to target natural stem-loop structures formed during mRNA synthesis. The general utility of this method is demonstrated by SQRM identification of targetable sequence within c-myb and bcl-6 mRNA. Corresponding antisense oligonucleotides reduce these gene products in cells.

  9. Non-intercalative, deoxyribose binding of boric acid to calf thymus DNA.

    PubMed

    Ozdemir, Ayse; Gursaclı, Refiye Tekiner; Tekinay, Turgay

    2014-05-01

    The present study characterizes the effects of the boric acid binding on calf thymus DNA (ct-DNA) by spectroscopic and calorimetric methods. UV-Vis absorbance spectroscopy, circular dichroism (CD) spectroscopy, transmission electron microscopy (TEM), isothermal titration calorimetry (ITC), and Fourier transform infrared (FT-IR) spectroscopy were employed to characterize binding properties. Changes in the secondary structure of ct-DNA were determined by CD spectroscopy. Sizes and morphologies of boric acid-DNA complexes were determined by transmission electron microscopy (TEM). The kinetics of boric acid binding to calf thymus DNA (ct-DNA) was investigated by isothermal titration calorimetry (ITC). ITC results revealed that boric acid exhibits a moderate affinity to ct-DNA with a binding constant (K a) of 9.54 × 10(4) M(-1). FT-IR results revealed that boric acid binds to the deoxyribose sugar of DNA without disrupting the B-conformation at tested concentrations.

  10. Statics and Dynamics of Stretched Single DNA Molecules Tug-of-War at Micro-Nanofluidic Interfaces

    NASA Astrophysics Data System (ADS)

    Yeh, Jiawei; Taloni, Alessandro; Chen, Yeng-Long; Chou, Chia-Fu

    2011-03-01

    Understanding single molecule dynamics at micro-nanoscale interfaces has implications to polymer transport in biological processes, device design for single molecule analysis and biotechnological applications. We report our study on single DNA molecules straddling across a nanoslit, bridging two micro-nanofluidic interfaces, for both its tug-of-war behavior and confinement-induced entropic recoiling at varying length and height (h: 30 ~ 100 nm) of a nanoslit. From a modified worm-like chain model in the tug-of-war scenario and the scaling analysis in the entropic recoiling process, we demonstrate the entropic recoiling force is essentially constant, given the degree of confinement, irrespective of the DNA length inside the nanoslit and the slit length. The scaling exponents for the entropic force will also be discussed.

  11. DNA sequencing by a single molecule detection of labeled nucleotides sequentially cleaved from a single strand of DNA

    SciTech Connect

    Goodwin, P.M.; Schecker, J.A.; Wilkerson, C.W.; Hammond, M.L.; Ambrose, W.P.; Jett, J.H.; Martin, J.C.; Marrone, B.L.; Keller, R.A. ); Haces, A.; Shih, P.J.; Harding, J.D. )

    1993-01-01

    We are developing a laser-based technique for the rapid sequencing of large DNA fragments (several kb in size) at a rate of 100 to 1000 bases per second. Our approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA into a flowing sample stream, sequential cleavage of the end nucleotide from the DNA fragment with an exonuclease, and detection of the individual fluorescently labeled bases by laser-induced fluorescence.

  12. DNA sequencing by a single molecule detection of labeled nucleotides sequentially cleaved from a single strand of DNA

    SciTech Connect

    Goodwin, P.M.; Schecker, J.A.; Wilkerson, C.W.; Hammond, M.L.; Ambrose, W.P.; Jett, J.H.; Martin, J.C.; Marrone, B.L.; Keller, R.A.; Haces, A.; Shih, P.J.; Harding, J.D.

    1993-02-01

    We are developing a laser-based technique for the rapid sequencing of large DNA fragments (several kb in size) at a rate of 100 to 1000 bases per second. Our approach relies on fluorescent labeling of the bases in a single fragment of DNA, attachment of this labeled DNA fragment to a support, movement of the supported DNA into a flowing sample stream, sequential cleavage of the end nucleotide from the DNA fragment with an exonuclease, and detection of the individual fluorescently labeled bases by laser-induced fluorescence.

  13. Local conformational perturbations of the DNA molecule in the SG-model

    SciTech Connect

    Krasnobaeva, L. A.; Shapovalov, A. V.

    2015-11-17

    Within the formalism of the Fokker–Planck equation, the influence of nonstationary external force, random force, and dissipation effects on dynamics local conformational perturbations (kink) propagating along the DNA molecule is investigated. Such waves have an important role in the regulation of important biological processes in living systems at the molecular level. As a dynamic model of DNA was used a modified sine-Gordon equation, simulating the rotational oscillations of bases in one of the chains DNA. The equation of evolution of the kink momentum is obtained in the form of the stochastic differential equation in the Stratonovich sense within the framework of the well-known McLaughlin and Scott energy approach. The corresponding Fokker–Planck equation for the momentum distribution function coincides with the equation describing the Ornstein–Uhlenbek process with a regular nonstationary external force. The influence of the nonlinear stochastic effects on the kink dynamics is considered with the help of the Fokker– Planck nonlinear equation with the shift coefficient dependent on the first moment of the kink momentum distribution function. Expressions are derived for average value and variance of the momentum. Examples are considered which demonstrate the influence of the external regular and random forces on the evolution of the average value and variance of the kink momentum. Within the formalism of the Fokker–Planck equation, the influence of nonstationary external force, random force, and dissipation effects on the kink dynamics is investigated in the sine–Gordon model. The equation of evolution of the kink momentum is obtained in the form of the stochastic differential equation in the Stratonovich sense within the framework of the well-known McLaughlin and Scott energy approach. The corresponding Fokker–Planck equation for the momentum distribution function coincides with the equation describing the Ornstein–Uhlenbek process with a regular

  14. Sugar-assisted kinetic resolution of amino acids and amplification of enantiomeric excess of organic molecules.

    PubMed

    Córdova, Armando; Sundén, Henrik; Xu, Yongmei; Ibrahem, Ismail; Zou, Weibiao; Engqvist, Magnus

    2006-07-17

    The origins of biological homochirality have intrigued researchers since Pasteur's discovery of the optical activity of biomolecules. Herein, we propose and demonstrate a novel alternative for the evolution of homochirality that is not based on autocatalysis and forges a direct relationship between the chirality of sugars and amino acids. This process provides a mechanism in which a racemic mixture of an amino acid can catalyze the formation of an optically active organic molecule in the presence of a sugar product of low enantiomeric excess.

  15. Difficulties in Laboratory Studies and Astronomical Observations of Organic Molecules: Hydroxyacetone and Lactic Acid

    NASA Technical Reports Server (NTRS)

    Apponi, A. J.; Brewster, M. A.; Hoy, J.; Ziurys, L. M.

    2006-01-01

    For the past 35 years, radio astronomy has revealed a rich organic chemistry in the interstellar gas, which is exceptionally complex towards active star-forming regions. New solar systems condense out of this gas and may influence the evolution of life on newly formed planets. Much of the biologically important functionality is present among the some 130 gas-phase molecules found to date, including alcohols, aldehydes, ketones, acids, amines, amides and even the simplest sugar - glycolaldehyde. Still, many unidentified interstellar radio signals remain, and their identification relies on further laboratory study. The molecules hydroxyacetone and lactic acid are relatively small organic molecules, but possess rather complex rotational spectra owing to their high asymmetry. Hydroxyacetone is particularly problematic because it possess a very low barrier to internal rotation, and exhibits strong coupling of the free-rotor states with the overall rotation of the molecule. As in the case of acetamide, a full decomposition method was employed to order the resultant eigenstates onto normal asymmetric top eigenvectors.

  16. Wafer-scale integration of sacrificial nanofluidic chips for detecting and manipulating single DNA molecules

    PubMed Central

    Wang, Chao; Nam, Sung-Wook; Cotte, John M.; Jahnes, Christopher V.; Colgan, Evan G.; Bruce, Robert L.; Brink, Markus; Lofaro, Michael F.; Patel, Jyotica V.; Gignac, Lynne M.; Joseph, Eric A.; Rao, Satyavolu Papa; Stolovitzky, Gustavo; Polonsky, Stanislav; Lin, Qinghuang

    2017-01-01

    Wafer-scale fabrication of complex nanofluidic systems with integrated electronics is essential to realizing ubiquitous, compact, reliable, high-sensitivity and low-cost biomolecular sensors. Here we report a scalable fabrication strategy capable of producing nanofluidic chips with complex designs and down to single-digit nanometre dimensions over 200 mm wafer scale. Compatible with semiconductor industry standard complementary metal-oxide semiconductor logic circuit fabrication processes, this strategy extracts a patterned sacrificial silicon layer through hundreds of millions of nanoscale vent holes on each chip by gas-phase Xenon difluoride etching. Using single-molecule fluorescence imaging, we demonstrate these sacrificial nanofluidic chips can function to controllably and completely stretch lambda DNA in a two-dimensional nanofluidic network comprising channels and pillars. The flexible nanofluidic structure design, wafer-scale fabrication, single-digit nanometre channels, reliable fluidic sealing and low thermal budget make our strategy a potentially universal approach to integrating functional planar nanofluidic systems with logic circuits for lab-on-a-chip applications. PMID:28112157

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

    PubMed Central

    Marino, John A.; Perfecto, Ivette; Vandermeer, John

    2015-01-01

    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 mm2) 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. PMID:26567299

  18. Wafer-scale integration of sacrificial nanofluidic chips for detecting and manipulating single DNA molecules

    NASA Astrophysics Data System (ADS)

    Wang, Chao; Nam, Sung-Wook; Cotte, John M.; Jahnes, Christopher V.; Colgan, Evan G.; Bruce, Robert L.; Brink, Markus; Lofaro, Michael F.; Patel, Jyotica V.; Gignac, Lynne M.; Joseph, Eric A.; Rao, Satyavolu Papa; Stolovitzky, Gustavo; Polonsky, Stanislav; Lin, Qinghuang

    2017-01-01

    Wafer-scale fabrication of complex nanofluidic systems with integrated electronics is essential to realizing ubiquitous, compact, reliable, high-sensitivity and low-cost biomolecular sensors. Here we report a scalable fabrication strategy capable of producing nanofluidic chips with complex designs and down to single-digit nanometre dimensions over 200 mm wafer scale. Compatible with semiconductor industry standard complementary metal-oxide semiconductor logic circuit fabrication processes, this strategy extracts a patterned sacrificial silicon layer through hundreds of millions of nanoscale vent holes on each chip by gas-phase Xenon difluoride etching. Using single-molecule fluorescence imaging, we demonstrate these sacrificial nanofluidic chips can function to controllably and completely stretch lambda DNA in a two-dimensional nanofluidic network comprising channels and pillars. The flexible nanofluidic structure design, wafer-scale fabrication, single-digit nanometre channels, reliable fluidic sealing and low thermal budget make our strategy a potentially universal approach to integrating functional planar nanofluidic systems with logic circuits for lab-on-a-chip applications.

  19. Single-Molecule Force Spectroscopy of DNA-Based Reversible Polymer Bridges: Surface Robustness and Homogeneity

    PubMed Central

    Serpe, Michael J.; Whitehead, Jason R.; Rivera, Monica; Clark, Robert L.; Craig, Stephen L.

    2011-01-01

    Single-molecule force spectroscopy, as implemented in an atomic force microscope, provides a rarely-used method by which to monitor dynamic processes that occur near surfaces. Here, a methodology is presented and characterized that facilitates the study of polymer bridging across nanometer-sized gaps. The model system employed is that of DNA-based reversible polymers, and an automated procedure is introduced that allows the AFM tip-surface contact point to be automatically determined, and the distance d between opposing surfaces to be actively controlled. Using this methodology, the importance of several experimental parameters was systematically studied, e.g. the frequency of repeated tip/surface contacts, the area of the substrate surface sampled by the AFM, and the use of multiple AFM tips and substrates. Experiments revealed the surfaces to be robust throughout pulling experiments, so that multiple touches and pulls could be carried out on a single spot with no measurable affect on the results. Differences in observed bridging probabilities were observed, both on different spots on the same surface and, more dramatically, from one day to another. Data normalization via a reference measurement allows data from multiple days to be directly compared. PMID:21966095

  20. Wafer-scale integration of sacrificial nanofluidic chips for detecting and manipulating single DNA molecules.

    PubMed

    Wang, Chao; Nam, Sung-Wook; Cotte, John M; Jahnes, Christopher V; Colgan, Evan G; Bruce, Robert L; Brink, Markus; Lofaro, Michael F; Patel, Jyotica V; Gignac, Lynne M; Joseph, Eric A; Rao, Satyavolu Papa; Stolovitzky, Gustavo; Polonsky, Stanislav; Lin, Qinghuang

    2017-01-23

    Wafer-scale fabrication of complex nanofluidic systems with integrated electronics is essential to realizing ubiquitous, compact, reliable, high-sensitivity and low-cost biomolecular sensors. Here we report a scalable fabrication strategy capable of producing nanofluidic chips with complex designs and down to single-digit nanometre dimensions over 200 mm wafer scale. Compatible with semiconductor industry standard complementary metal-oxide semiconductor logic circuit fabrication processes, this strategy extracts a patterned sacrificial silicon layer through hundreds of millions of nanoscale vent holes on each chip by gas-phase Xenon difluoride etching. Using single-molecule fluorescence imaging, we demonstrate these sacrificial nanofluidic chips can function to controllably and completely stretch lambda DNA in a two-dimensional nanofluidic network comprising channels and pillars. The flexible nanofluidic structure design, wafer-scale fabrication, single-digit nanometre channels, reliable fluidic sealing and low thermal budget make our strategy a potentially universal approach to integrating functional planar nanofluidic systems with logic circuits for lab-on-a-chip applications.

  1. Sequential photochemical and microbial degradation of organic molecules bound to humic acid

    SciTech Connect

    Amador, J.A.; Zika, R.G. ); Alexander, M. )

    1989-11-01

    We studied the effects of photochemical processes on the mineralization by soil microorganisms of (2-{sup 14}C)glycine bound to soil humic acid. Microbial mineralization of these complexes in the dark increased inversely with the molecular weight of the complex molecules. Sunlight irradiation of glycine-humic acid complexes resulted in loss of absorbance in the UV range and an increase in the amount of {sup 14}C-labeled low-molecular-weight photoproducts and the rate and extent of mineralization. More than half of the radioactivity in the low-molecular-weight photoproducts appears to be associated with carboxylic acids. Microbial mineralization of the organic carbon increased with solar flux and was proportional to the loss of A{sub 330}. Mineralization was proportional to the percentage of the original complex that was converted to low-molecular-weight photoproducts. Only light at wavelengths below 380 nm had an effect on the molecular weight distribution of the products formed from the glycine-humic acid complexes and on the subsequent microbial mineralization. Our results indicate that photochemical processes generate low-molecular-weight, readily biodegradable molecules from high-molecular-weight complexes of glycine with humic acid.

  2. Nucleic acid molecules conferring enhanced ethanol tolerance and microorganisms having enhanced tolerance to ethanol

    DOEpatents

    Brown, Steven; Guss, Adam; Yang, Shihui; Karpinets, Tatiana; Lynd, Lee; Shao, Xiongjun

    2014-01-14

    The present invention provides isolated nucleic acid molecules which encode a mutant acetaldehyde-CoA/alcohol dehydrogenase or mutant alcohol dehydrogenase and confer enhanced tolerance to ethanol. The invention also provides related expression vectors, genetically engineered microorganisms having enhanced tolerance to ethanol, as well as methods of making and using such genetically modified microorganisms for production of biofuels based on fermentation of biomass materials.

  3. Effect of genomic long-range correlations on DNA persistence length: from theory to single molecule experiments.

    PubMed

    Moukhtar, Julien; Faivre-Moskalenko, Cendrine; Milani, Pascale; Audit, Benjamin; Vaillant, Cedric; Fontaine, Emeline; Mongelard, Fabien; Lavorel, Guillaume; St-Jean, Philippe; Bouvet, Philippe; Argoul, Françoise; Arneodo, Alain

    2010-04-22

    Sequence dependency of DNA intrinsic bending properties has been emphasized as a possible key ingredient to in vivo chromatin organization. We use atomic force microscopy (AFM) in air and liquid to image intrinsically straight (synthetic), uncorrelated (hepatitis C RNA virus) and persistent long-range correlated (human) DNA fragments in various ionic conditions such that the molecules freely equilibrate on the mica surface before being captured in a particular conformation. 2D thermodynamic equilibrium is experimentally verified by a detailed statistical analysis of the Gaussian nature of the DNA bend angle fluctuations. We show that the worm-like chain (WLC) model, commonly used to describe the average conformation of long semiflexible polymers, reproduces remarkably well the persistence length estimates for the first two molecules as consistently obtained from (i) mean square end-to-end distance measurement and (ii) mean projection of the end-to-end vector on the initial orientation. Whatever the operating conditions (air or liquid, concentration of metal cations Mg(2+) and/or Ni(2+)), the persistence length found for the uncorrelated viral DNA underestimates the value obtained for the straight DNA. We show that this systematic difference is the signature of the presence of an uncorrelated structural intrinsic disorder in the hepatitis C virus (HCV) DNA fragment that superimposes on local curvatures induced by thermal fluctuations and that only the entropic disorder depends upon experimental conditions. In contrast, the WLC model fails to describe the human DNA conformations. We use a mean-field extension of the WLC model to account for the presence of long-range correlations (LRC) in the intrinsic curvature disorder of human genomic DNA: the stronger the LRC, the smaller the persistence length. The comparison of AFM imaging of human DNA with LRC DNA simulations confirms that the rather small mean square end-to-end distance observed, particularly for G

  4. Investigating organic molecules responsible of auxin-like activity of humic acid fraction extracted from vermicompost.

    PubMed

    Scaglia, Barbara; Nunes, Ramom Rachide; Rezende, Maria Olímpia Oliveira; Tambone, Fulvia; Adani, Fabrizio

    2016-08-15

    This work studied the auxin-like activity of humic acids (HA) obtained from vermicomposts produced using leather wastes plus cattle dung at different maturation stages (fresh, stable and mature). Bioassays were performed by testing HA concentrations in the range of 100-6000mgcarbonL(-1). (13)C CPMAS-NMR and GC-MS instrumental methods were used to assess the effect of biological processes and starting organic mixtures on HA composition. Not all HAs showed IAA-like activity and in general, IAA-like activity increased with the length of the vermicomposting process. The presence of leather wastes was not necessary to produce the auxin-like activity of HA, since HA extracted from a mix of cattle manure and sawdust, where no leather waste was added, showed IAA-like activity as well. CPMAS (13)CNMR revealed that HAs were similar independently of the mix used and that the humification process involved the increasing concentration of pre-existing alkali soluble fractions in the biomass. GC/MS allowed the identification of the molecules involved in IAA-like effects: carboxylic acids and amino acids. The concentration of active molecules, rather than their simple presence in HA, determined the bio-stimulating effect, and a good linear regression between auxin-like activity and active stimulating molecules concentration was found (R(2)=-0.85; p<0.01, n=6).

  5. Sequence-selective single-molecule alkylation with a pyrrole-imidazole polyamide visualized in a DNA nanoscaffold.

    PubMed

    Yoshidome, Tomofumi; Endo, Masayuki; Kashiwazaki, Gengo; Hidaka, Kumi; Bando, Toshikazu; Sugiyama, Hiroshi

    2012-03-14

    We demonstrate a novel strategy for visualizing sequence-selective alkylation of target double-stranded DNA (dsDNA) using a synthetic pyrrole-imidazole (PI) polyamide in a designed DNA origami scaffold. Doubly functionalized PI polyamide was designed by introduction of an alkylating agent 1-(chloromethyl)-5-hydroxy-1,2-dihydro-3H-benz[e]indole (seco-CBI) and biotin for sequence-selective alkylation at the target sequence and subsequent streptavidin labeling, respectively. Selective alkylation of the target site in the substrate DNA was observed by analysis using sequencing gel electrophoresis. For the single-molecule observation of the alkylation by functionalized PI polyamide using atomic force microscopy (AFM), the target position in the dsDNA (∼200 base pairs) was alkylated and then visualized by labeling with streptavidin. Newly designed DNA origami scaffold named "five-well DNA frame" carrying five different dsDNA sequences in its cavities was used for the detailed analysis of the sequence-selectivity and alkylation. The 64-mer dsDNAs were introduced to five individual wells, in which target sequence AGTXCCA/TGGYACT (XY = AT, TA, GC, CG) was employed as fully matched (X = G) and one-base mismatched (X = A, T, C) sequences. The fully matched sequence was alkylated with 88% selectivity over other mismatched sequences. In addition, the PI polyamide failed to attach to the target sequence lacking the alkylation site after washing and streptavidin treatment. Therefore, the PI polyamide discriminated the one mismatched nucleotide at the single-molecule level, and alkylation anchored the PI polyamide to the target dsDNA.

  6. Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells.

    PubMed

    Valdiglesias, Vanessa; Méndez, Josefina; Pásaro, Eduardo; Cemeli, Eduardo; Anderson, Diana; Laffon, Blanca

    2010-07-07

    Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are mainly gastrointestinal, but the intoxication does not appear to be fatal. Despite this, this toxin presents a potential threat to human health even at concentrations too low to induce acute toxicity, since previous animal studies have shown that OA has very potent tumour promoting activity. However, its concrete action mechanism has not been described yet and the results reported with regard to OA cytotoxicity and genotoxicity are often contradictory. In the present study, the genotoxic and cytotoxic effects of OA on three different types of human cells (peripheral blood leukocytes, HepG2 hepatoma cells, and SHSY5Y neuroblastoma cells) were evaluated. Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction, and MTT test and Comet assay were performed in order to evaluate cytotoxicity and genotoxicity, respectively. The possible effects of OA on DNA repair were also studied by means of the DNA repair competence assay, using bleomycin as DNA damage inductor. Treatment with OA in absence of S9 fraction induced not statistically significant decrease in cell viability and significant increase in DNA damage in all cell types at the highest concentrations investigated. However, only SHSY5Y cells showed OA induced genotoxic and cytotoxic effects in presence of S9 fraction. Furthermore, we found that OA can induce modulations in DNA repair processes when exposure was performed prior to BLM treatment, in co-exposure, or during the subsequent DNA repair process.

  7. A DNA origami nanorobot controlled by nucleic acid hybridization.

    PubMed

    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.

  8. Internally labeled Cy3/Cy5 DNA constructs show greatly enhanced photo-stability in single-molecule FRET experiments

    PubMed Central

    Lee, Wonbae; von Hippel, Peter H.; Marcus, Andrew H.

    2014-01-01

    DNA constructs labeled with cyanine fluorescent dyes are important substrates for single-molecule (sm) studies of the functional activity of protein–DNA complexes. We previously studied the local DNA backbone fluctuations of replication fork and primer–template DNA constructs labeled with Cy3/Cy5 donor–acceptor Förster resonance energy transfer (FRET) chromophore pairs and showed that, contrary to dyes linked ‘externally’ to the bases with flexible tethers, direct ‘internal’ (and rigid) insertion of the chromophores into the sugar-phosphate backbones resulted in DNA constructs that could be used to study intrinsic and protein-induced DNA backbone fluctuations by both smFRET and sm Fluorescent Linear Dichroism (smFLD). Here we show that these rigidly inserted Cy3/Cy5 chromophores also exhibit two additional useful properties, showing both high photo-stability and minimal effects on the local thermodynamic stability of the DNA constructs. The increased photo-stability of the internal labels significantly reduces the proportion of false positive smFRET conversion ‘background’ signals, thereby simplifying interpretations of both smFRET and smFLD experiments, while the decreased effects of the internal probes on local thermodynamic stability also make fluctuations sensed by these probes more representative of the unperturbed DNA structure. We suggest that internal probe labeling may be useful in studies of many DNA–protein interaction systems. PMID:24627223

  9. Optical mapping of single-molecule human DNA in disposable, mass-produced all-polymer devices

    NASA Astrophysics Data System (ADS)

    Østergaard, Peter Friis; Lopacinska-Jørgensen, Joanna; Nyvold Pedersen, Jonas; Tommerup, Niels; Kristensen, Anders; Flyvbjerg, Henrik; Silahtaroglu, Asli; Marie, Rodolphe; Taboryski, Rafael

    2015-10-01

    We demonstrate all-polymer injection molded devices for optical mapping of denaturation-renaturation (DR) patterns on long, single DNA-molecules from the human genome. The devices have channels with ultra-low aspect ratio, only 110 nm deep while 20 μm wide, and are superior to the silica devices used previously in the field. With these polymer devices, we demonstrate on-chip recording of DR images of DNA-molecules stretched to more than 95% of their contour length. The stretching is done by opposing flows Marie et al (2013 Proc. Natl Acad. Sci. USA 110 4893-8). The performance is validated by mapping 20 out of 24 Mbp-long DNA fragments to the human reference genome. We optimized fabrication of the devices to a yield exceeding 95%. This permits a substantial economies-of-scale driven cost-reduction, leading to device costs as low as 3 USD per device, about a factor 70 lower than the cost of silica devices. This lowers the barrier to a wide use of DR mapping of native, megabase-size DNA molecules, which has a huge potential as a complementary method to next-generation sequencing.

  10. Circulating nucleic acids damage DNA of healthy cells by integrating into their genomes.

    PubMed

    Mittra, Indraneel; Khare, Naveen Kumar; Raghuram, Gorantla Venkata; Chaubal, Rohan; Khambatti, Fatema; Gupta, Deepika; Gaikwad, Ashwini; Prasannan, Preeti; Singh, Akshita; Iyer, Aishwarya; Singh, Ankita; Upadhyay, Pawan; Nair, Naveen Kumar; Mishra, Pradyumna Kumar; Dutt, Amit

    2015-03-01

    Whether nucleic acids that circulate in blood have any patho-physiological functions in the host have not been explored.We report here that far from being inert molecules, circulating nucleic acids have significant biological activities of their own that are deleterious to healthy cells of the body. Fragmented DNA and chromatin (DNAfs and Cfs) isolated from blood of cancer patients and healthy volunteers are readily taken up by a variety of cells in culture to be localized in their nuclei within a few minutes. The intra-nuclear DNAfs and Cfs associate themselves with host cell chromosomes to evoke a cellular DNA-damage-repair-response (DDR) followed by their incorporation into the host cell genomes. Whole genome sequencing detected the presence of tens of thousands of human sequence reads in the recipient mouse cells. Genomic incorporation of DNAfs and Cfs leads to dsDNA breaks and activation of apoptotic pathways in the treated cells. When injected intravenously into Balb/C mice, DNAfs and Cfs undergo genomic integration into cells of their vital organs resulting in activation of DDR and apoptotic proteins in the recipient cells. Cfs have significantly greater activity than DNAfs with respect to all parameters examined, while both DNAfs and Cfs isolated from cancer patients are more active than those from normal volunteers. All the above pathological actions of DNAfs and Cfs described above can be abrogated by concurrent treatment with DNase I and/or anti-histone antibody complexed nanoparticles both in vitro and in vivo. Taken together, our results suggest that circulating DNAfs and Cfs are physiological, continuously arising, endogenous DNA damaging agents with implications to ageing and a multitude of human pathologies including initiation of cancer.

  11. Limiting Dilution Bisulfite Pyrosequencing®: A Method for Methylation Analysis of Individual DNA Molecules in a Single or a Few Cells.

    PubMed

    Hajj, Nady El; Kuhtz, Juliane; Haaf, Thomas

    2015-01-01

    Bisulfite-based methods for DNA methylation analysis of small amounts of DNA from a limited number of cells are technologically challenging. Degradation of genomic DNA by bisulfite treatment, contamination with foreign DNA, and biases in the amplification of individual DNA molecules can generate results, which are not representative of the starting sample. Limiting dilution (LD) bisulfite Pyrosequencing(®) (BSP) is a relatively simple technique to ci